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Focus on Immune Evasion
Volume 3 No 11 November 2002
Welcome to the Nature Immunology Immune Evasion Web Focus
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The immune system has evolved a myriad of mechanisms to detect and eradicate pathogens and
tumors from the body. In spite of the excellent array of preventative measures at hand, everyone
has suffered microbial infections or knows an individual who has developed cancer. This is because
the most successful tumors and pathogens have coevolved with us and developed a panoply of
ploys to escape from both the innate and adaptive immune responses.
Our understanding of immune evasion strategies is continuing to grow, although much still remains
to be discovered. Nature Immunology feel that this is an ideal time to pause and take stock of how
far we’ve come and where we are heading. Thus, from 1 November 2002 until 1 February 2003, our
Focus on Immune Evasion, which examines recent progress in the field of immune escape, is free
to all registrants. We have deliberately avoided focusing on one system, but instead provide a
series of comprehensive reviews on microbial and tumor immune evasion. The juxtaposition of the
various mechanisms used in these different systems reveals many parallels. It is our hope that this
thorough analysis of immune escape will promote cross-fertilization between these areas and help
propel the field forward. Similar to our previous web focuses, we include a regularly updated
Round-up section that features recent papers on immune evasion, an annotated list of landmark or
classic articles on immune escape and links to major articles published by the Nature journals in
this area. These features can be reached through the navigational bar at the left.
It is obvious that researchers have barely scratched the surface of this rich field. We expect years of
exciting discoveries that will aid in our abilities to apply this knowledge. Better vaccines and
treatments, through the intelligent boosting of immune responses and specifically foiling of a
pathogen’s attacks, may finally enable us to gain the upper hand in our constant struggle with these
moving targets.
Focus on Immune Evasion
Overview
Nature Immunology 3, 987 - 989 (2002)
doi:10.1038/ni1102-987
© Nature America, Inc.
Volume 3 No 11 November 2002
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Immunology taught by Darwin
Rodney E. Phillips
The Peter Medawar Building for Pathogen Research, University of Oxford, South Parks Road, Oxford OX1 3SY, UK.
[email protected].
This Focus issue brings together the cornucopia of strategies that pathogens and tumors
utilize to avoid immune recognition. Here Rodney Phillips discusses some general principles
that emerge from this analysis.
Nothing in biology makes sense except in the light of evolution.
Theodisius Dobzhansky
We are still struggling to understand the implications of Charles Darwin's monumental
achievements. The most profound biological insight of the 19th century created a framework for
understanding variation in organisms that has yet to be fully realized. Before Darwin, biological
difference was arrayed before us in endless, unstructured confusion. Darwin made this variation a
highly legitimate subject for analysis by recorded observation but also by experimental science.
Nearly 150 years after the publication of the Origin of the Species1, we are still laboring to
understand evolution and selection in many biological systems. Twentieth century science gave us
tools, which, for the first time, allowed us to look directly at the biochemistry that enables genetic
variation2. But we are still often unable to explain the mechanisms that underlie clashes between
one organism and another.
Darwin envisaged selection operating over millennia, with a tempo dictated by brutish struggles in a
very competitive world. When complex organisms like humans come under threat from foreign
microorganisms and parasites or from within by malignant cellular change, a Darwinian framework
is essential if we are to understand these much-accelerated evolutionary encounters. The rapid
growth of microorganisms and the huge potential for mutation and recombination in RNA genomes
can produce high-speed versions of Darwin's original conception. If we define immunity as the
organism's sum capacity to resist attack, then with a tempo that can be as short as days or as long
as decades, this defensive resistance constitutes a formidable selection force on malignant cells or
pathogenic agents. Immune selection determines the repertoire of bacteria that colonize the skin,
the time it takes to die from HIV infection and contributes to a tumor-free youth in humans.
When assaulted by a new foreign organism, the immune system produces three basic outcomes:
early, complete expulsion of the foreigner; overwhelming infection with failure of control; or
persistence with potential long-term carriage or induction of disease. It is this last scenario that
allows the immune response to become a sustained selection force, although the dynamics of short
clashes can still select for evasive properties in some highly mutable pathogens3.
Burnet, inspired by Lewis Thomas, thought the adaptive immune response was involved in
continuous "surveillance" for malignant transformation in cells4. As reviewed by Schreiber and
colleagues in this issue of Nature Immunology, the concept was developed, refuted and has now
had a renaissance. A key prediction of the immune surveillance hypothesis is that breakthrough
tumors will have immune-evasion properties. Like microorganisms, tumors require adaptations that
allow evasion of the immune response before they can grow in an immunocompetent host.
What are the mechanisms of immune escape? The reviews contained in this Focus issue of Nature
Immunology describe immune-escape by pathogens and tumors. Some principles emerge from this
extensive catalog.
Dormancy
There is no better way to hide from immunity than to minimize antigen expression. The integrated
DNA form of a retrovirus, the minimal protein production of a herpesvirus and the quiescent forms of
mycobacteria all have this capacity. But the strategy has limits. Although some cells harboring HIV
proviruses may indeed express no viral proteins, others become highly activated and viral proteins
are readily processed for presentation to CD8+ killer T lymphocytes5. Successful immune evasion
under these selective conditions then depends on other strategies such as antigenic variation 6.
Thus microorganisms can have an array of immune-evasion strategies each designed for specific
settings.
Sequestration
Cellular and humoral forms of immunity are pervasive, although the "surveillance" does have blind
spots. Some organisms have evolved to occupy special niches where immunity may not penetrate
or is frustrated. Malarial parasites have an asexual phase within red blood cells. These complex
organisms pay the price of adaptation to life in a biochemically simple environment; the lack of
major histocompatibility complex (MHC) class I molecules on the red blood cell surface means that
the presence of the plasmodia will not be announced to the immune system in the form of antigenic
peptides bound to MHC. Other organisms, such as Mycobacterium tuberculosis, also live within
cells and show very low turnover. But how this is achieved is not understood. Genome comparisons
between M. tuberculosis and M. leprae provide a clue7. Less than half the genome of M. leprae
contains functional genes, but there are many pseudogenes with intact counterparts in M.
tuberculosis. Gene deletion and decay have eliminated many important metabolic pathways. The
potential evolutionary advantage of this is apparent: M. leprae and its metabolically "crippled" state
has a doubling time of approximately 14 days compared to a faster rate in M. tuberculosis. This
provides some hint as to why M. leprae has such a remarkably persistent, indolent lifestyle.
Gene loss in certain organisms seems to have led to an enhanced capacity for highly specialized
sequestration. Salmonella enterica Typhi has 145 fewer functional genes than its relative S.
enterica Typhimurium. Several of the lost genes are responsible for bacterial attachment; thus, loss
of a specific function may enhance the ability of a microorganism to colonize sites such as the gall
bladder, where persistence is favored. Many pathogens appear to enhance virulence by the
paradox of gene reduction8. This process of gene loss may produce an irreversible spiral if DNA
cannot be reacquired to complement the heavily diminished genome of such organisms ("Muller's
ratchet") and so lead to an evolutionary dead end9. This sort of evolutionary decline reflects the
"blindness" of adaptation.
Small changes in viral genomes, which alter receptor usage, may also enhance the capacity for
survival. HIV envelope polymorphisms, which allow binding to dendritic cells, may allow these cells
to be used as a "Trojan horse", ferrying the virus away from mucosal surfaces to lymph nodes10.
Failure of antigen display
Burnet's persuasive arguments in favor of immune surveillance were predicated on the existence of
a cellular immune response capable of sensing dangerous antigenic change within cells, wherever
they might lie in the body4. The vigor of allograft rejection was viewed by Thomas as a purely
artificial and contrived demonstration that histocompatibility was a safety mechanism to prevent
transmission of cancer11. How could these difficult concepts be reconciled? And what is the
evolutionary role of the adaptive immune system?
© Science Photo Library/Photo Researchers, Inc.
Engraving of Charles Robert Darwin (1809–1882).
Some of this uncertainty was clarified when Zinkernagel and Doherty defined the genetic
significance of MHC class I12. These highly polymorphic molecules dictated or "restricted" the
response of T cells to foreign antigen. Subsequently, Townsend, in a tour de force of experimental
biology, showed unequivocally that MHC class I acted as a molecular clasp, displaying on the cell
surface short peptides derived from viruses13, 14. We now know that the vast majority of MHC class I
molecules display peptides derived from self, that is, normal cellular proteins. Here was a major
clue concerning how the mutant, defective or parasitized cells could alert the policing lymphocytes
of the cellular immune response. When a virus harnesses intracellular machinery, a fraction of viral
protein is diverted into an active and elaborate process, which loads MHC class I with peptide.
Unless this pathway of "processing and presentation" is blocked or subverted, the killer lymphocyte
response will inevitably sense intracellular infection and destroy the parasitized cell.
In cancer, a more speculative extrapolation was that dangerous mutation would also be signaled to
the killer T cells when altered self-peptides arrived at the malignant cell surface bound to MHC class
I.
Reviews published in this issue of Nature Immunology provide overwhelming evidence that viruses,
which have paramount success in establishing chronic infection, have evolved remarkably
ingenious ways of subverting almost every part of the biochemistry of the MHC class I pathway. The
mechanism may be as simple as a protein sequence rendered indigestible by the peptide-liberating
proteasome, as with Epstein-Barr virus proteins, or the acquisition of proteins that interfere with
antigen processing at several levels, as with cytomegalovirus (CMV). Indeed, human CMV
represents amongst the best circumstantial evidence for the coevolution of pathogen and host 15.
Immune-surveillance theories imply that cancer cells might carry phenotypic evidence of immune
evasion. In this issue of Nature Immunology, Khong and Restifo review the impressive evidence
that tumor cells carry HLA class I deletions, which can arise through many mechanisms. They
summarize the mounting, if not overwhelming, evidence that encounters with tumors bearing Fas
can prove fatal to anticancer T cells. Evolutionary reasoning would argue that the acquisition of antiimmune cell weapons implies that T lymphocytes are a significant selection force in at least some
cancers.
The reduction of MHC class I density on cells is detected by a specialized class of lymphocytes with
the capacity to lyse these cells directly. These natural killer (NK) cells provide antiviral and
antitumor defense. Successful outgrowth of virus or tumor may necessitate ways of subverting this
cell population too. Strominger and colleagues review the mechanisms of escape from NK cell
surveillance.
Antigenic variation
Enormous genetic variation is the hallmark of many successful pathogens. Variation is also
characteristic of bacteria such as Staphylococcus aureus, which cause asymptomatic carriage as
well as disease16. High-throughput sequencing has begun to reveal how variable many
microorganisms really are. When subjected to intensive sequencing, some pathogens—such as
HIV and meningococcus—are so variable that new analytic tools are required to make some sense
of their genetic complexity. One masterly survey of epidemic meningitis identified nine "genoclouds"
(a frequent genotype plus its epidemiologically associated descendents) that have been responsible
for three pandemic waves of disease since the 1960s17. The authors argue convincingly that loci
under positive selection are highly antigenic and represent immune escape from herd immunity17.
Many of the variants identified are not fixed over the long term, presumably because of diminished
fitness as compared with the parental genotype17.
The concept that immunity may shape the detectable diversity in complex genotypes is not new, but
in the HIV field this has been highly controversial. One group has argued consistently that the
cellular immune response (or indeed any natural selection force) does not positively select for HIV
variants18. In this issue of Nature Immunology, Yewdell and Hill review recent evidence for retroviral
immune selection and emphasize the definitive studies that have described positive selection for
SIV antigenic variants as exerted by cytolytic T lymphocytes (CTLs)19. Another study provides
strong genetic evidence that HLA class I is closely linked to HIV polymorphism in a large cohort20.
After 12 years of controversy6, the current balance of evidence favors the view that CTLs exert
positive selection and so shape the HIV quasispecies in individuals and perhaps also in
populations20.
Vaccine-induced immunity has been linked with antigenic polymorphism in bacteria. In 1953 mass
pertussis vaccination was introduced in the Netherlands, but the disease has remained endemic
with occasional outbreaks, including a remarkable increase since 1996. Sequence comparisons
between the vaccine and recent isolates showed nonsynonymous mutations in a key antigencoding allele; this finding has been put forward as an explanation for poor vaccine performance in
the face of the new prevailing strains21. There is similar evidence for meningococcal vaccine
failure22 and for the vaccine-induced CD8+ response in the SIV model23.
Antigenic variation represents the most severe barrier to lasting successful preventative vaccination
in many infectious diseases, including, par excellence, HIV24.
Conclusion
Warm-blooded, long-lived vertebrate hosts such as humans have survived because of the
extraordinary capacity of the immune response to repel potentially harmful microorganisms. It is
clear that pathogens (and tumors) have highly evolved ways of subverting host immunity and that
although shaped (or "sculpted") by the immune response, the dangerous outgrowth often prevails.
In this century as we reflect upon the achievements of Watson and Crick, Burnet, Medawar,
Zinkernagel and Doherty, Townsend and the rest, it must humble modern biologists to think how far
we have to go before we master the challenges posed by Charles Darwin. Indeed we are still
learning to be his contemporaries.
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Price, D.A. et al. Positive selection of HIV-1 cytotoxic T lymphocyte escape variants during primary
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Burnet, F.M. The concept of immunological surveillance. Prog. Exp. Tumour Res. 13, 1-27
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Ho, D.D. et al. Rapid turnover of plasma virions and CD4 lymphocytes in HIV-1 infection. Nature
373, 123-126 (1995). | PubMed | ISI |
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Cole, S.T. et al. Massive gene decay in the leprosy bacillus. Nature 49, 1007-1011 (2001). | Article |
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Ochman, H. & Moran, N.A. Genes lost and genes found: evolution of bacterial pathogenesis and
symbiosis. Science 292, 1096-1098 (2001). | Article | PubMed | ISI |
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Andersson, D.I. & Hughes, D. Muller's ratchet decreases fitness of a DNA-based microbe. Proc.
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Baranowski, E., Ruiz-Jarabo, C.M. & Domingo, E. Evolution of cell recognition by viruses. Science
292, 1102-1105 (2001). | Article | PubMed | ISI |
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Thomas, L. On immunosurveillance in human cancer. Yale J. Biol. Med. 55, 329-333
(1982). | PubMed | ISI |
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Zinkernagel, R.M. & Doherty, P.C. Restriction of in vitro T cell-mediated cytotoxicity in lymphocytic
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(1974). | PubMed | ISI |
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Townsend, A.R., Rothbard, J., Gotch, F.M., Bahadur, G. & Wraith, D. The epitopes of influenza
nucleoprotein recognized by cytotoxic T lymphocytes can be defined with short synthetic peptides.
Cell 44, 959-968 (1986). | PubMed | ISI |
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Townsend, A. et al. Association of class I major histocompatibility heavy and light chains induced by
viral peptides. Nature 340, 443-448 (1989). | PubMed | ISI |
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Woolhouse, M.E.J., Webster, J.P., Domingo, E., Charlesworth, B. & Levine, B.R. Biological and
biomedical implications of the coevolution of pathogens and their hosts. Nature Genet. (in the
press, 2002).
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Day, N.P.J. et al. Link between virulence and ecological abundance in natural populations of
Staphylococcus aureus. Science 292, 114-116 (2001). | Article | PubMed | ISI |
17.
Zhu, P. et al. Fit genotypes and escape variants of subgroup III Neisseria meningitides during three
pandemics of epidemic meningitis. Proc. Natl. Acad. Sci. USA 98, 5234-5239
(2001). | Article | PubMed | ISI |
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Sala, M. & Wain-Hobson, S. Are RNA viruses adapting or merely changing? J. Mol. Evol. 51, 12-20
(2000). | PubMed | ISI |
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Allen, T.M. et al. Nature 407, 386-390 (2000). | Article | PubMed | ISI |
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Moore, C.B. et al. Evidence of HIV-1 adaptation to HLA-restricted immune responses at a
population level. Science 296, 1439-1443 (2002). | Article | PubMed | ISI |
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Van Loo, I.H.M. & Mooi, F.R. Changes in the Dutch Bordetella pertussis population in the first 20
years after the introduction of whole-cell vaccines. Microbiology 148, 2011-2018
(2002). | PubMed | ISI |
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Martin, S.L. et al. Effect of sequence variation in meningococcal pora outer membrane protein on
the effectiveness of a hexavalent pora outer membrane vesicle vaccine. Vaccine 18, 2476-2481
(2000). | Article | PubMed | ISI |
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Barouch, D.H. et al. Eventual AIDS vaccine failure in a rhesus monkey by viral escape from
cytotoxic T lymphocytes. Nature 415, 335-339 (2002). | Article | PubMed | ISI |
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Gaschen, B. et al. Diversity considerations in HIV-1 vaccine selection. Science 296, 2354-2360
(2002). | Article | PubMed | ISI |
Acknowledgments. I thank C. Bangham, M. Maiden, D. Watkins and P. Klenerman for lively
discussions over many years. The Peter Medawar Building for Pathogen Research was built by the
Wellcome Trust for the University of Oxford. Supported by the Wellcome Trust.
Reviews
Cancer immunoediting: from immunosurveillance to tumor escape
G P Dunn, A T Bruce, H Ikeda, L J Old & R D Schreiber
doi:10.1038/ni1102-991
p 991
| Abstract | Full text | PDF (787K) |
Natural selection of tumor variants in the generation of "tumor escape" phenotypes
H T Khong & N P Restifo
doi:10.1038/ni1102-999
p 999
| Abstract | Full text | PDF (720K) |
Viral evasion of natural killer cells
p 1006
J S Orange, M S Fassett, L A Koopman, J E Boyson & J L Strominger
doi:10.1038/ni1102-1006
| Abstract | Full text | PDF (283K) |
To kill or be killed: viral evasion of apoptosis
C A Benedict, P S Norris & C F Ware
doi:10.1038/ni1102-1013
p 1013
| Abstract | Full text | PDF (860K) |
Viral interference with antigen presentation
J W Yewdell & A B Hill
doi:10.1038/ni1102-1019
p 1019
| Abstract | Full text | PDF (696K) |
Chronic bacterial infections: living with unwanted guests
D Young, T Hussell & G Dougan
doi:10.1038/ni1102-1026
p 1026
| Abstract | Full text | PDF (1022K) |
Bacterial strategies for overcoming host innate and adaptive immune responses
M W Hornef, M J Wick, M Rhen & S Normark
doi:10.1038/ni1102-1033
p 1033
| Abstract | Full text | PDF (669K) |
Evasion of innate immunity by parasitic protozoa
D Sacks & A Sher
doi:10.1038/ni1102-1041
| Abstract | Full text | PDF (1006K) |
p 1041
Focus on Immune Evasion
Volume 3 No 11 November 2002
Review
Nature Immunology 3, 991 - 998 (2002)
doi:10.1038/ni1102-991
© Nature America, Inc.
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Cancer immunoediting: from immunosurveillance
to tumor escape
Gavin P. Dunn1, Allen T. Bruce1, Hiroaki Ikeda1, Lloyd J. Old2 & Robert D. Schreiber1
1
Department of Pathology and Immunology, Center for Immunology, Washington University School of Medicine, 660 South Euclid
Avenue, St. Louis, MO 63110, USA.
2 Ludwig Institute for Cancer Research, New York Branch at Memorial Sloan-Kettering Cancer Center, New York, NY 10021, USA.
Correspondence should be addressed to R D Schreiber [email protected]
The concept that the immune system can recognize and destroy nascent transformed cells
was originally embodied in the cancer immunosurveillance hypothesis of Burnet and
Thomas. This hypothesis was abandoned shortly afterwards because of the absence of
strong experimental evidence supporting the concept. New data, however, clearly show the
existence of cancer immunosurveillance and also indicate that it may function as a
component of a more general process of cancer immunoediting. This process is responsible
for both eliminating tumors and sculpting the immunogenic phenotypes of tumors that
eventually form in immunocompetent hosts. In this review, we will summarize the historical
and experimental basis of cancer immunoediting and discuss its dual roles in promoting
host protection against cancer and facilitating tumor escape from immune destruction.
Since the formalized introduction of the cancer immunosurveillance concept, the idea that the
immune system may have a protective role in tumor development has been hotly debated. Recent
work, however, has lent new support to the idea's central principle that the immune system can
indeed prevent tumor formation. At the same time, this work has shown that the immune system
also functions to promote or select tumor variants with reduced immunogenicity, thereby providing
developing tumors with a mechanism to escape immunologic detection and elimination. These
findings have led to the development of the cancer immunoediting hypothesis, a refinement of
cancer immunosurveillance that takes a broader view of immune system–tumor interactions by
acknowledging both the host-protecting and tumor-sculpting actions of the immune system on
developing tumors. Here we discuss first the history that led to formulation of the original cancer
immunosurveillance concept and then review the data that prompted the wide-scale abandonment
of the hypothesis. We continue with a summary of the data underlying the development of the
cancer immunoediting concept and present a unifying model that proposes the molecular and
cellular dynamics of the process. We conclude by highlighting the critical implications that
immunoediting may have on the development and treatment of cancer in humans.
The cancer immunosurveillance hypothesis
Paul Ehrlich was one of the first to conceive the idea that the immune system could repress a
potentially "overwhelming frequency" of carcinomas1. The idea of immune control of neoplastic
disease was not vigorously pursued, however, until the midpoint of the twentieth century. To a large
extent, the revisiting of the Ehrlich proposal had to await the maturation of the developing field of
immunology. In the 1950s, the work of Medawar and colleagues clarified the critical role for cellular
components of immunity in mediating allograft rejection2. This work cast doubt on data that were
used to argue for the existence of tumor antigens. Specifically, although it was generally accepted
that the immune response was capable of recognizing and destroying transplanted tumors derived
from noninbred strains of mice, it soon became clear that the underlying mechanism was one of
allograft rejection rather than tumor-specific rejection. With the availability of inbred strains of mice,
the idea that tumors were immunologically distinguishable from normal cells could be critically
tested. The demonstration that mice could be immunized against syngeneic transplants of tumors
induced by chemical carcinogens, viruses or other means established the existence of "tumorspecific antigens"3, 4 and provided a key cornerstone of the cancer immunosurveillance hypothesis.
Clearly, there could be no tumor immunosurveillance if there were no distinctive structures on tumor
cells that could be recognized by the immune system.
These emerging discoveries were incorporated into the formal hypothesis of "cancer
immunosurveillance" proposed by Sir Macfarlane Burnet and Lewis Thomas. In 1957, Burnet
stated5:
It is by no means inconceivable that small accumulations of tumour cells may develop and because
of their possession of new antigenic potentialities provoke an effective immunological reaction with
regression of the tumour and no clinical hint of its existence.
At about the same time, Thomas suggested that the primary function of cellular immunity was in fact
not to promote allograft rejection but rather to protect from neoplastic disease, thereby maintaining
tissue homeostasis in complex multicellular organisms6. These speculations formed the
evolutionary framework that ultimately resulted in the development of the immunosurveillance
concept, which was defined by Burnet as follows7, 8:
In large, long-lived animals, like most of the warm-blooded vertebrates, inheritable genetic changes
must be common in somatic cells and a proportion of these changes will represent a step toward
malignancy. It is an evolutionary necessity that there should be some mechanism for eliminating or
inactivating such potentially dangerous mutant cells and it is postulated that this mechanism is of
immunological character.
Both Burnet and Thomas speculated that lymphocytes acted as sentinels in recognizing and
eliminating continuously arising, nascent transformed cells7.
Challenging the immunosurveillance hypothesis
The introduction of the immunosurveillance hypothesis was rapidly followed by many experiments
aimed at testing its logical predictions—namely, that hosts with impaired immune systems would
exhibit increased incidences of spontaneous or chemically induced tumors. The earliest approaches
explored whether tumor development in mice was influenced by experimental immunosuppression
of the host9, 10. Whereas several groups used neonatal thymectomy to induce immunosuppression
in mice, they could not reach a consensus as to the effects of this treatment on the incidence of
either chemically induced10-13 or spontaneous tumors10, 14, 15. Other groups who compromised the
immune system of mice using either heterologous anti-lymphocyte serum or pharmacologic
methods obtained similar discordant results10. It also became clear that mice with induced
immunodeficiencies showed a high susceptibility to virally induced tumors and a greater tendency
to develop spontaneous lymphomas compared with immunocompetent mice. The prevailing view
about these observations, however, was that they reflected the greater susceptibility of
immunocompromised hosts to infectious agents, such as transforming viruses. The greater
frequency of lymphomas could also be ascribed to chronic antigenic stimulation from defective
control of bacterial or viral infections, resulting in increased lymphocyte proliferation, somatic
mutation and eventually the formation of lymphomas10, 16. As a group, these studies were
inconclusive and therefore failed either to prove or disprove the immunosurveillance hypothesis.
The identification and characterization of the athymic nude mouse17, 18 enabled researchers to study
tumor formation in a host with a genetic immunologic impairment. The work of Osias Stutman
represents one of the most extensive uses of nude mice to explore whether immunosurveillance
occurs in a physiologic setting. Stutman found that CBA/H strain nude mice did not form more
chemically induced tumors compared with their wild-type counterparts, nor did they show a
shortened tumor latency period after carcinogen injection. For example, in one experiment, nude
mice or immunocompetent mice that were heterozygous for the nude mutation were injected
subcutaneously with 0.1 mg of the chemical carcinogen methylcholanthrene (MCA) at birth and
were monitored for tumor incidence19. After 120 days, seven of 39 control mice formed tumors at
the injection site with a mean time to tumor appearance of 95 days. Of the nude mice tested, five of
27 formed tumors with a mean time to tumor appearance of 90 days. The similarity between
immunocompetent and nude mice was maintained in subsequent experiments that employed mice
of different ages, different doses of carcinogen and even in experiments where the observation
period was extended out to 420 days 20, 21. At least one other group corroborated these results 22. In
addition, Stutman showed that there were no statistically significant differences in the incidence of
spontaneous nonviral tumor formation between nude and wild-type mice23. These findings were
supported by a study of Rygaard and Povlsen that showed no differences in spontaneous tumor
formation in 10,800 nude mice over a study period of 3–7 months24, 25. The results using nude mice
were thus more conclusive than those based on experimental immunosuppression and failed to
uphold the central predictions of the immunosurveillance concept. Based on the limited
understanding of the immunologic defects in the nude mouse available at that time, these results
were highly convincing and thus led to the abandonment of the immunosurveillance hypothesis.
In hindsight, however, there are important caveats to these experiments. First, it is now clear that
nude mice are not completely immunocompromised. Although nude mice possess fewer T cells
than wild-type mice, they have detectable populations of functional
T cell receptor–bearing
lymphocytes26-28. Therefore, it is not possible to predict the functional effect of these T cells on
tumor formation when compared with wild-type controls. Second, the strain of mice used in the
Stutman experiments may have been highly sensitive to MCA-induced tumor formation. MCA
requires biotransformation from its pro-form into its carcinogenic form by the aryl hydrocarbon
hydroxylase enzyme system. It is now known that various inbred mouse strains produce distinctive
patterns of these enzyme isoforms with different specific activities 29. The CBA/H strain mouse used
in the Stutman studies expresses a form of the enzyme with a high specific activity30, and thus
cellular transformation induced by MCA might occur in these mice at a rate that overwhelms host
immunological defense mechanisms. Third, even though the study of Rygaard and Povlsen used
large numbers of nude mice, monitoring periods of 3–7 months were probably too short to see
spontaneous tumor formation in the face of fully functional intrinsic tumor suppressor systems (such
as p53). Last, these studies were carried out before the discovery of other lymphocyte populations
such as natural killer (NK) cells, which are not thymus dependent, and
may develop extrathymically31.
T cells, a subset of which
The lack of convincing support for the immunosurveillance concept left room for other theories on
the possible functions of immune cells during the development of neoplasia. For example, Prehn
proposed that the immune system can actually promote the growth of tumors. In this
"immunostimulation theory" 32, the presence of tumor rejection antigens (TRAs) on tumors is
explained by the argument that the immune cells that recognize these TRAs provide a positive
growth signal to the tumor cells. Although this theory was speculative rather than evidence based, it
reflected the mood that the proponents of the immunosurveillance theory were overstating their
case. Especially after the Stutman experiments, enthusiasm for the original immunosurveillance
concept began to wane. Thomas later noted, "The greatest trouble with the idea of
immunosurveillance is that it cannot be shown to exist in experimental animals"33.
During the ensuing years, little interest was paid to the possibility that the immune system could
prevent the development of nonvirally induced tumors. By all intents and purposes, the cancer
immunosurveillance concept was considered dead by 1978, and the field of tumor immunology
moved on to study other issues such as the definition and molecular nature of mouse and human
tumor antigens, the immune response to known tumor antigens and the development of
immunotherapeutic strategies to treat cancer. This view relegated immunosurveillance to the
historical dust bin and was clearly echoed in a major review that appeared in 2000, which listed the
six critical hurdles that a developing tumor must circumvent to grow and survive 34. No significant
mention was made of the natural immune response against tumors.
The renaissance of cancer immunosurveillance
Between the mid-1970s and 1990s, researchers made several experimental attempts to resurrect
the cancer immunosurveillance concept. The discovery of NK cells led to a considerable amount of
enthusiasm over the possibility that they functioned as the effectors of immunosurveillance 35. This
enthusiasm was dampened, however, when a precise definition and understanding of these cells
was difficult to obtain. Subsequently, others repeated the MCA induction experiments of Stutman
using nude mice on a BALB/c background36. When they injected nude and control mice with
different doses of MCA and monitored them for tumor development, nude mice formed more tumors
than controls. These data were limited, however, in their statistical power because of the small
experimental group sizes and the magnitude of the differences. Similar suggestive results were
obtained when tumor formation induced by MCA was compared between groups of wild-type
BALB/c mice and immunodeficient CB-17 severe-combined immunodeficiency (SCID) mice37. The
latter lack a functionally active subunit of the DNA-dependant protein kinase (DNA-PK) enzyme
(DNA-PKcs) that is required to generate rearranged antigen receptors in lymphocytes38, and thus
they are unable to develop antigen-specific immune responses. However, DNA-PKcs (and the intact
DNA-PK enzyme) is normally expressed in all cells and is important in repairing double-stranded
DNA breaks39, which includes those that can potentially be transforming. Thus, it was not possible
to interpret the basis for the increased tumor incidence observed in SCID mice because of an
inability to differentiate between tumor suppressor roles for the immune system or for the DNA-PK
enzyme itself.
Between 1994 and 1998, two key findings incited renewed interest in the process of cancer
immunosurveillance. First, endogenously produced interferon (IFN- ) was shown to protect the
host against the growth of transplanted tumors and the formation of primary chemically induced and
spontaneous tumors. Using tumor transplantation approaches, researchers found that immunogenic
fibrosarcomas grow faster and more efficiently in mice treated with neutralizing monoclonal
antibodies specific for IFN- 40. Overexpression of a dominant-negative mutant of the IFNreceptor subunit (IFNGR1) in sarcomas such as Meth A (BALB/c derived) or MCA-207 (C57BL/6
derived) completely ablated tumor sensitivity to this cytokine, and the tumors displayed enhanced
tumorigenicity and reduced immunogenicity when transplanted into naïve syngeneic hosts 40.
Experiments based on models of MCA-induced tumor formation showed that 129/SvEv mice that
were lacking either the IFN- receptor or signal transducer and activator of transcription 1 (STAT1,
the transcription factor that is important in mediating IFN- receptor signaling) were found to be
approximately 10–20 times more sensitive than wild-type mice to the tumor-inducing capacity of
MCA, developed more tumors than their wild-type counterparts and showed a shortened tumor
latency period41. Subsequent independent experiments using mice on a different genetic
background that lacked the gene encoding IFN- confirmed these results42. Similarly, mice lacking
the genes encoding the tumor suppressor p53 and the IFNGR1 subunit of the IFN- receptor
formed a wider spectrum of tumors as compared with IFN- -sensitive mice lacking only p5341. In
addition, IFN- -/- mice on a C57BL/6 background showed an increased incidence of disseminated
lymphomas despite the presence of a normal p53 tumor suppressor gene, and IFNBALB/c background showed a low incidence of lung adenocarcinomas43.
-/-
mice on a
The second key finding was the observation that C57BL/6 mice lacking perforin (perforin -/-) were
more prone to MCA-induced tumor formation compared with their wild-type counterparts. Perforin is
a component of the cytolytic granules of cytotoxic T cells and NK cells that is important in mediating
lymphocyte-dependent killing of many different target cells including tumor cells 44. After challenge
with MCA, perforin-/- mice developed significantly more tumors compared with perforin-sufficient
mice treated in the same manner42, 45, 46. Untreated perforin-/- mice also showed a high incidence of
spontaneous disseminated lymphomas, which was greater on a p53+/- background47, and a low
incidence of spontaneous lung adenocarcinomas43. Taken together, these observations showed
that components of the immune system were involved in controlling primary tumor development.
The definitive work supporting the existence of a cancer immunosurveillance process that is
dependent on both IFN- and lymphocytes came through the use of gene-targeted mice that lack
recombination activating gene 1 (RAG-1) or RAG-2. Like DNA-PK, these enzymes are involved in
the repair of double-stranded DNA breaks, but unlike DNA-PK, they are expressed exclusively in
the lymphoid compartment. Mice deficient in either of these genes fail to rearrange lymphocyte
antigen receptors and thus completely lack natural killer T (NKT), T and B cells 48. In contrast, DNA
repair mechanisms in the nonlymphoid compartment of these mice are completely normal.
For the first time, inbred strains of mice were available that carried genetically defined mutations
affecting specific tissues that led to an absence of lymphocytes bearing antigen receptors. These
mice enabled researchers to carry out carcinogenesis experiments that could unequivocally be
interpreted. After MCA injection, 129/SvEv RAG-2-/- mice developed sarcomas more rapidly and
with greater frequency than genetically matched wild-type controls49. After 160 days, 30 of 52 RAG2-/- mice formed tumors, compared with 11 of 57 wild-type mice. In addition, RAG-2-/- mice aged in a
specific pathogen free mouse facility formed far more spontaneous epithelial tumors than did wildtype mice housed in the same room 49 (and unpublished data). Specifically, 26 of 26 RAG-2-/- mice
aged 13–24 months developed spontaneous neoplasia, predominantly of the intestine; eight of
these mice had premalignant intestinal adenomas, 17 had intestinal adenocarcinomas and one had
both an intestinal adenoma and a lung adenocarcinoma. In contrast, only five of 20 wild-type mice
aged 13–24 months developed spontaneous neoplasia, which was predominantly benign. Three of
the wild-type mice developed adenomas of the Harderian gland, lung and intestine, respectively,
whereas the other two developed an adenocarcinoma of the Harderian gland and an endometrial
stromal carcinoma. Thus, lymphocytes in a mouse not only protect the host against formation of
primary sarcomas that are chemically induced but also prevent the development of spontaneous
epithelial tumors.
The overlap between the tumor suppressor pathways that depend on IFN- and lymphocytes was
defined by comparing tumor formation in 129/SvEv mice lacking IFN- responsiveness (IFNGR1
receptor-/- or STAT1-/- mice), lymphocytes (RAG-2-/- mice) or both RAG-2 and STAT1 (RkSk mice)49.
Each of the four lines of gene-targeted mice formed three times more chemically induced tumors
than syngeneic wild-type mice when injected with 0.1 mg of MCA. Because no significant
differences were detected between any of the groups of deficient mice, the researchers concluded
that the two extrinsic tumor suppressor mechanisms heavily overlapped. RkSk mice, however,
additionally developed spontaneous breast tumors that were not observed in wild-type or RAG-2-/mice, therefore demonstrating that the overlap between the two tumor suppressor pathways was
not complete. Similar findings were made in carcinogenesis experiments with mice that lacked
perforin, IFN- or both, in which a small increase was observed in tumor induction in the doubly
deficient mice compared with mice lacking only one of the two components 42.
Additional studies have used other inbred mouse lines with targeted disruptions in genes encoding
critical components of the immune system. They not only support the importance of immune system
control of tumor formation but also suggest the involvement of both the innate and adaptive immune
compartments in cancer immunosurveillance. Specifically, genetic, immunochemical or functional
ablations of NKT,
T cells, NK cells,
T cells, IFN- or interleukin 12 (IL-12) all lead to
increased susceptibility of the host to tumors (Table 1). One of these studies has shed light on the
specific subsets of lymphocytes expressing T cell receptors (TCR) that are important in tumor
surveillance. The relative contributions of
and
T cells in blocking primary tumor formation
cell-/-
were explored in
T
(lacking the TCR -chain) or
T cell-/- (lacking the TCR -chain)
50
mice . MCA treatment of either type of mouse increased the incidence of fibrosarcomas and
spindle cell carcinomas as compared with wild-type controls. These data showed that both T cell
subsets are critical for protecting the host in this particular model of tumor development. In an
initiation and promotion model of skin tumorigenesis induced by 7,12-dimethylbenzanthracene
(DMBA) and 12-O-tetradecanoylphorbol-13-acetate (TPA), however,
T cell-/- mice showed a
greater susceptibility to tumor formation and a higher incidence of papilloma-to-carcinoma
progression than wild-type mice, whereas
T cell-/- mice did not. This result suggests that
immunosurveillance may be a heterogeneous process requiring the actions of different immune
effectors in a manner that is dependent on the tumor's cell type of origin, mechanism of
transformation, anatomical localization and mechanism of immunologic recognition. In sum, the
large amount of recent data obtained by many independent groups overwhelmingly supports the
basic tenets of the cancer immunosurveillance concept as originally envisaged by Burnet and
Thomas—namely, that the unmanipulated immune system is capable of recognizing and eliminating
primary tumors and that lymphocytes and the cytokines they produce are important in this process.
High resolution image and legend (93K)
Cancer immunosurveillance in humans
If indeed cancer immunosurveillance exists in mice, does it exist in humans? The logical prediction
from the immunosurveillance hypothesis is that immunodeficient or immunosuppressed humans
should show greater incidences of cancer. Early follow-up studies of transplant patients who were
immunosuppressed51 and individuals with primary immunodeficiencies52 showed that they had a
significantly higher relative risk for cancer development. Based on long-term studies of patients, it is
clear that some of this higher risk was due to the development of tumors of viral origin 53. For
example, in the transplant registries from Cincinnatti53, Scandinavia54, and Australia and New
Zealand55, particularly high incidence ratios have been observed for non-Hodgkin's lymphoma,
Kaposi's sarcoma and carcinomas of the genitourinary and anogenital regions. Many of these
cancers have a viral etiology and are linked to infection with Epstein-Barr virus, human herpesvirus
8 and human papilloma virus, respectively56. These malignancies are now occurring with increasing
frequency in AIDS patients56. The increased incidence of virally induced tumors in
immunocompromised patients represents one immunosurveillance function that is not contested:
natural protection against infectious organisms. These data, however, do not invalidate the
Table 1. Enhance
possibility that the immune system protects humans against development of tumors of nonviral
origin.
Clearly, the difficulty in assessing cancer immunosurveillance in immunodeficient humans arises
from their greater susceptibility to endemic viruses and other pathogens as well as viral reactivation.
This can confound the likelihood of finding spontaneous tumors of nonviral origin, which develop
slowly because of the shortened life spans and other intercurrent medical problems of these
patients. Greater relative risk ratios have, however, been observed for a broad subset of tumors
with no apparent viral etiology. Approximately fourfold increases in the incidence of de novo
malignant melanoma after organ transplantation have been reported57, 58. A review of data
accumulated by the Cincinnati Transplant Tumor Registry from 1968 to 1995 found a twofold
greater risk in transplant patients for developing melanoma over that of the general population 59. In
addition, whereas only 0.3–0.4% of melanomas occur in the general pediatric population, the
occurrence in pediatric transplant patients was 4% 59. The same database showed that transplant
patients were three times more likely to develop non-Kaposi's sarcomas60. When neoplasia
occurrence was assessed in 608 cardiac transplant patients at the University of Pittsburgh between
1980 and 1993, the prevalence of lung tumors was 25-fold higher than in the general population61.
In Australia and New Zealand, the tracking of 925 patients who received cadaveric renal transplants
from 1965 to 1998 showed increased risk ratios for development of colon, pancreatic, lung and
endocrine tumors as well as malignant melanomas55. In addition, assessment of 5,692 renal
transplant patients from 1964 to 1982 in Finland, Denmark, Norway and Sweden showed higher
standardized cancer incidence ratios for colon, lung, bladder, kidney, ureter and endocrine tumors
as well as malignant melanomas as compared with the general population54. Thus, individuals with
severe deficits of immunity indeed have a higher probability of developing a variety of cancers with
no known viral etiology.
In addition to the supporting epidemiological data described above, there is accumulating evidence
showing a positive correlation between the presence of lymphocytes in a tumor and increased
patient survival. Some of the most convincing evidence comes from the study of cutaneous
melanomas. Three categories were established for tumor lymphocyte infiltration during the vertical
growth phase of cutaneous melanoma (brisk, nonbrisk and absent)62. Sorting more than 500
patients with primary melanoma who had 5-, 8- or 10-year follow-ups into these categories and
comparing their survival statistics showed that patients in the brisk tumor infiltrating lymphocyte
(TIL) response category survived one and one-half to three times longer than patients in the absent
TIL response group; patients in the nonbrisk response group had intermediate survival times62, 63.
Researchers obtained the same prognostic correlation when the presence of TILs in melanomas
that had metastasized to the lymph nodes was used as the criterion64. Similar correlations between
the presence of TILs and patient survival have also been made that involved more than 3,400
patients with cancer of the breast65, bladder66, colon67, 68, prostate69, ovary70 or rectum71 and for
neuroblastoma72. In some cases, the correlation has been refined to show that CD8+ T cells are the
relevant lymphocyte population that affect survival68.
In sum, the data obtained from both mouse and human studies provide strong support for the
existence and physiologic relevance of cancer immunosurveillance. These findings have rekindled
generalized interest in this process and have stimulated much work aimed at defining its
immunologic components. Thus, the new millennium has witnessed the resurrection of this old and
formerly controversial idea.
Immunologic sculpting during tumor development
Given the existence of cancer immunosurveillance, why then do cancers occur in
immunocompetent individuals? It has long been thought that the immune system functions during
tumor formation to select for tumor variants that are better suited to survive in an immunologically
intact environment, very much like it does with viruses, bacteria and parasites. Many studies have
shown the immunoselective effects of repassage of transplantable tumors through
immunocompetent hosts and the generation of tumor variants with reduced immunogenicity. Two
particularly informative studies used P815 mastocytomas73 and ultraviolet-induced 1591
fibrosarcomas74. In contrast, few studies have compared the immunogenic characteristics of tumors
originally generated in the presence or absence of a functional immune system. This issue was
difficult to study in the past because matched sets of tumors derived from isogenic immunodeficient
and control immunocompetent hosts were not generally available. A recent study, however, was
carried out using many MCA-induced sarcomas obtained from either RAG-2-/- or wild-type mice on a
pure 129/SvEv background49. When tumors isolated from wild-type or RAG-2-/- mice were
transplanted into RAG-2-/- recipients, they all grew with similar kinetics, which indicates that there
were no inherent growth differences between tumors raised in the presence or absence of an intact
immune system. In addition, 17 of 17 sarcomas originally isolated from wild-type mice were capable
of establishing progressively growing tumors when transplanted into naïve immunocompetent
129/SvEv hosts. In contrast, 8 of 20 tumors originally generated in RAG-2-/- mice were rejected
when transplanted into immunocompetent hosts, even when they were injected at a high cell
number. Thus, tumors formed in the absence of an intact immune system are, as a group, more
immunogenic than tumors that arise in immunocompetent hosts.
Other more limited experiments have reached similar conclusions. MCA-induced sarcomas derived
from nude75 or SCID mice37 were rejected more frequently than similar tumors derived from wildtype mice when transplanted into wild-type hosts. In addition, two MCA-induced sarcomas derived
from TCR J 281-/- mice, which lack a major population of NKT cells, grew more slowly when
transplanted into wild-type hosts than did sarcomas originally isolated from wild-type mice46. In
contrast, these tumors grew in a comparable manner when transplanted into TCR J 281-/recipients. Finally, lymphomas derived from perforin-/- mice grew avidly when transplanted into
perforin-/- mice, but most were rejected when transplanted into wild-type mice43.
Taken together, these results show that tumors are imprinted by the immunologic environment in
which they form. This imprinting process can often result in the generation of tumors that are better
able to withstand the tumor-suppressing actions of the immune system by eliminating tumor cells of
intrinsically high immunogenicity but leaving behind tumor variants of reduced immunogenicity (or
that have acquired other mechanisms to evade or suppress immune attack) that have a better
chance of surviving in the immunocompetent host. The alterations that must occur during the
immunologic sculpting of a developing tumor are probably facilitated by the inherent genetic
instability of tumors76. Some of the likely targets of the immunologic process that sculpts tumors are
genes encoding tumor antigens, components of the major histocompatibility complex (MHC)
pathways that process and present antigens or components of the IFN- receptor signaling
pathway. Tumors lacking antigen processing and presentation components are commonly found
(see Review by Khong and Restifo in this issue), and tumors have been identified that lack
functional expression of at least three components of the IFN- receptor signaling pathway41
(unpublished data). It is likely that immunologic sculpting of tumors occurs continuously, but the
major effects of this process are most prominent early when the tumor is perhaps histologically—but
not clinically—detectable. It follows, then, that the immunogenicity of most tumors that are clinically
apparent has already been modified to some degree by their interactions with the immune system.
Cancer immunoediting
Because the immune system exerts both host-protecting and tumor-sculpting effects on developing
tumors, the term cancer immunosurveillance may no longer be appropriate to accurately describe
the process because, in its original form, it was thought to function only to protect the host and to
act only at the earliest stages of cellular transformation. Rather, we have proposed the use of the
broader term "cancer immunoediting" to describe more appropriately the dual host-protecting and
tumor-sculpting actions of the immune system that not only prevent but also shape neoplastic
disease. We envisage the scope of this process to be very broad such that it can promote complete
elimination of some tumors, generate a nonprotective immune state to others or favor the
development of immunologic anergy, tolerance or indifference. We envision important roles for
components of both the innate and adaptive immune systems in this process.
Much work is needed to define the molecular and cellular dynamics of cancer immunoediting. A
proposed basic framework for this process is illustrated based on recent data from several groups
(Figs. 1 and 2). Although it was not possible to discuss in detail the contributions from each
laboratory that were used in the construction of these models because of space limitations, we have
incorporated key references in the model descriptions to acknowledge the work of these groups.
The models are presented in the hope that they will stimulate additional research aimed at
identifying the pathways that lead from cancer immunosurveillance to tumor escape.
High resolution image and legend (56K)
Figure 1. The three Es of cancer immunoediting.
Cancer immunoediting encompasses three process. (a) Elimination corresponds to
immunosurveillance. (b) Equilibrium represents the process by which the immune system iteratively
selects and/or promotes the generation of tumor cell variants with increasing capacities to survive
immune attack. (c) Escape is the process wherein the immunologically sculpted tumor expands in
an uncontrolled manner in the immunocompetent host. In a and b, developing tumor cells (blue),
tumor cell variants (red) and underlying stroma and nontransformed cells (gray) are shown; in c,
additional tumor variants (orange) that have formed as a result of the equilibrium process are
shown. Different lymphocyte populations are as marked. The small orange circles represent
cytokines and the white flashes represent cytotoxic activity of lymphocytes against tumor cells.
High resolution image and legend (128K)
Figure 2. A proposed model for the elimination
phase of the cancer immunoediting process.
(a) The initiation of the response in which lymphocytes that participate in innate immunity (NKT, NK
and
T cells) recognize transformed cells that have accumulated above a threshold that has yet to
be defined and are stimulated to produce IFN- . (b) The initial IFN- starts a cascade of innate
immune reactions that involve (i) the induction of chemokines, including the angiostatic chemokines
(CXCL10 (1P10), CXCL9 (MIG) AND CXCL11 (I-TAC)) that block neovascularization in the tumor
and that also effect the recruitment of NK cells, dendritic cells, macrophages and other immune
effector cells to the tumor site; (ii) an antiproliferative action of IFN- on the developing tumor and
(iii) the activation of cytocidal activity in macrophages and NK cells entering the tumor. These
events result in some tumor cell death by both immunologic and nonimmunologic mechanisms.
Dead tumor cells or tumor cell debris (blue squares) are ingested by dendritic cells and are
trafficked to the draining lymph node. (c) Tumor growth is kept in check by the cytocidal activities of
NK cells and activated macrophages while CD4+ and CD8+ T cells that are specific for tumor
antigens develop in the draining lymph node. (d) Tumor-specific CD4+ and CD8+ T cells home to
the tumor along a chemokine gradient where they recognize and destroy tumor cells expressing
distinctive tumor antigens. Tumor cells (blue); nontransformed cells (gray); dead tumor cells (white
to gray gradient circles surrounded by a dashed black line); lymphocytes, dendritic cells (DC) and
macrophages (Mac) are marked and colored appropriately.
We envisage cancer immunoediting as a result of three processes: elimination, equilibrium and
escape. We call these the three Es of cancer immunoediting (Fig. 1). Immunosurveillance occurs
during the elimination process, whereas the Darwinian selection of tumor variants occurs during the
equilibrium process. This in turn can ultimately lead to escape and the appearance of clinically
apparent tumors.
The elimination process encompasses the original concept of cancer immunosurveillance (Figs. 1a
and 2). As such, when it is successful in deleting the developing tumor, it represents the complete
editing process without progression to the subsequent phases. In the first phase of elimination (Fig.
2a), once solid tumors reach a certain size, they begin to grow invasively and require an enhanced
blood supply that arises as a consequence of the production of stromagenic and angiogenic
proteins77. Invasive growth causes minor disruptions within the surrounding tissue that induce
inflammatory signals leading to recruitment of cells of the innate immune system (NKT, NK,
T
cells, macrophages and dendritic cells) into the site50, 78, 79. Structures on the transformed cells
(either expressed as a result of the transformation process itself or induced by the ongoing but
limited inflammatory response) are recognized by infiltrating lymphocytes such as NKT, NK or
T
cells, which are then stimulated to produce IFN- 80-82. In the second phase (Fig. 2b), the IFN- that
was initially produced may induce a limited amount of tumor death by means of antiproliferative83
and apopotic84 mechanisms. However, it also induces the production of the chemokines CXCL10
(interferon-inducible protein-10, IP-10), CXCL9 (monokine induced by IFN- , MIG) and CXCL11
(interferon-inducible T cell chemoattractant, I-TAC) from the tumor cells themselves as well as
from surrounding normal host tissues85-87. At least some of these chemokines have potent
angiostatic capacities and thus block the formation of new blood vessels within the tumor, which
leads to even more tumor cell death88-91. Tumor cell debris formed as either a direct or indirect
consequence of IFN- production at the tumor is then ingested by local dendritic cells, which home
to draining lymph nodes. Chemokines produced during the escalating inflammatory process recruit
more NK cells and macrophages to the site. In the third phase (Fig. 2c), the tumor-infiltrating NK
cells and macrophages transactivate one another by reciprocal production of IFN- and IL-12, and
kill more of the tumor by mechanisms involving tumor necrosis factor–related apoptosis-inducing
ligand, perforin and reactive oxygen and nitrogen intermediates 46, 92-95. In the draining lymph node,
the newly immigrated dendritic cells induce tumor-specific CD4+ T helper cells expressing IFN(TH1 cells) that in turn facilitate the development of tumor-specific CD8+ T cells96-99. In the fourth
phase (Fig. 2d), tumor-specific CD4+ and CD8+ T cells home to the tumor site, where the cytolytic T
lymphocytes destroy the remaining antigen-bearing tumor cells whose immunogenicities have been
enhanced by exposure to locally produced IFN- 49.
In the equilibrium process (Fig. 1b), the host immune system and any tumor cell variant that has
survived the elimination process enter into a dynamic equilibrium. In this process, lymphocytes and
IFN- exert potent selection pressure on the tumor cells that is enough to contain, but not fully
extinguish, a tumor bed containing many genetically unstable and rapidly mutating tumor cells.
During this period of Darwinian selection, many of the original escape variants of the tumor cell are
destroyed, but new variants arise carrying different mutations that provide them with increased
resistance to immune attack. It is likely that equilibrium is the longest of the three processes and
may occur over a period of many years.
In the escape process (Fig. 1c), surviving tumor variants that have acquired insensitivity to
immunologic detection and/or elimination through genetic or epigenetic changes begin to expand in
an uncontrolled manner. This results in clinically observable malignant disease that, if left
unchecked, results in the death of the host.
Conclusions and implications
We have summarized here the events that led to the development of the concept of cancer
immunoediting and described the evolution of the concept from its birth in immunosurveillance to its
present form. The original formulation of the cancer immunosurveillance hypothesis by Burnet and
Thomas was indeed as prescient as it was powerful. Although it was based on an emerging
understanding of the existence of tumor antigens and the laws of transplantation immunity, it came
at a time when not enough was understood about mouse models of immunodeficiency. This led to
the unwitting use of imperfect animal models to critically test and prematurely abandon the concept.
The concept was resurrected nearly three decades later by the use of genetically defined mouse
models of complete immunodeficiency coupled with an enhanced understanding of the molecular
nature of tumor antigens. The incorporation of immunosurveillance into the cancer immunoediting
concept came about by the finding that the immune system not only protects the host against tumor
development but also can sculpt the immunogenic phenotype of a developing tumor. Thus, we now
recognize a process that has both positive and negative effects on host–tumor relationships.
Clearly, more work is needed to define the process on a molecular and cellular basis.
There are four immediate implications of the cancer immunoediting concept that directly apply to
human cancer. First, we may need to examine whether immunity plays a role in the development of
human cancers associated with known abnormalities in genes encoding such proteins as p53,
BRCA1, APC (adenomatous polyposis coli) Ras and others. Second, it will be important to reexamine the carcinogenic potential of many compounds that have never been tested in
experimental animals that are immunodeficient and thus may have been incorrectly labeled as
noncarcinogenic. Third, the relation between immunologic defects and increased incidences of
cancer associated with aging needs to be explored. Last, we need a way to assess the extent to
which a tumor has been edited.
Perhaps one of the most important contributions of the original immunosurveillance concept was
the furious experimentation that followed in its wake. In this respect, we hope for a similar fate for
the cancer immunoediting concept—new oeuvres framed by the three Es that test every prediction
of this hypothesis and, in eliciting a greater understanding of host–tumor interactions, will ultimately
have profound effects on our understanding and treatment of cancer.
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Acknowledgments. Supported by grants from the National Cancer Institute (CA43059 and
CA76464 to R. D. S.), the Cancer Research Institute (to R. D. S., H.I and A.B.), the Ludwig Institute
for Cancer Research (to R. D. S.), and the National Institute of Allergy and Infectious Diseases (to
R. D. S. and G. P. D.). We thank V. Shankaran, K. Sheehan, A. Dighe, D. Kaplan, R. Uppaluri, C.
Koebel, J. Bui, E. Stockert, E. Richards, M. White, C. Arthur and C. Brendel for their important roles
in developing the cancer immunoediting concept and for helpful comments during the preparation of
this manuscript.
Table 1. Enhanced susceptibility of immunodeficient mice to formation of
chemically induced and spontaneous tumors
Figure 1. The three Es of cancer immunoediting. Cancer immunoediting
encompasses three process. (a) Elimination corresponds to immunosurveillance.
(b) Equilibrium represents the process by which the immune system iteratively
selects and/or promotes the generation of tumor cell variants with increasing
capacities to survive immune attack. (c) Escape is the process wherein the
immunologically sculpted tumor expands in an uncontrolled manner in the
immunocompetent host. In a and b, developing tumor cells (blue), tumor cell
variants (red) and underlying stroma and nontransformed cells (gray) are shown; in
c, additional tumor variants (orange) that have formed as a result of the equilibrium
process are shown. Different lymphocyte populations are as marked. The small
orange circles represent cytokines and the white flashes represent cytotoxic activity
of lymphocytes against tumor cells.
Figure 2. A proposed model for the elimination phase of the cancer
immunoediting process. (a) The initiation of the response in which lymphocytes
that participate in innate immunity (NKT, NK and
T cells) recognize transformed
cells that have accumulated above a threshold that has yet to be defined and are
stimulated to produce IFN- . (b) The initial IFN- starts a cascade of innate
immune reactions that involve (i) the induction of chemokines, including the
angiostatic chemokines (CXCL10 (1P10), CXCL9 (MIG) AND CXCL11 (I-TAC)) that
block neovascularization in the tumor and that also effect the recruitment of NK
cells, dendritic cells, macrophages and other immune effector cells to the tumor
site; (ii) an antiproliferative action of IFN- on the developing tumor and (iii) the
activation of cytocidal activity in macrophages and NK cells entering the tumor.
These events result in some tumor cell death by both immunologic and
nonimmunologic mechanisms. Dead tumor cells or tumor cell debris (blue squares)
are ingested by dendritic cells and are trafficked to the draining lymph node. (c)
Tumor growth is kept in check by the cytocidal activities of NK cells and activated
macrophages while CD4+ and CD8+ T cells that are specific for tumor antigens
develop in the draining lymph node. (d) Tumor-specific CD4+ and CD8+ T cells
home to the tumor along a chemokine gradient where they recognize and destroy
tumor cells expressing distinctive tumor antigens. Tumor cells (blue);
nontransformed cells (gray); dead tumor cells (white to gray gradient circles
surrounded by a dashed black line); lymphocytes, dendritic cells (DC) and
macrophages (Mac) are marked and colored appropriately.
Focus on Immune Evasion
Volume 3 No 11 November 2002
Review
Nature Immunology 3, 999 - 1005 (2002)
doi:10.1038/ni1102-999
© Nature America, Inc.
<>
Natural selection of tumor variants in the
generation of "tumor escape" phenotypes
Hung T. Khong & Nicholas P. Restifo
National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA.
Correspondence should be addressed to H T Khong [email protected] or N P Restifo [email protected]
The idea that tumors must "escape" from immune recognition contains the implicit
assumption that tumors can be destroyed by immune responses either spontaneously or as
the result of immunotherapeutic intervention. Simply put, there is no need for tumor escape
without immunological pressure. Here, we review evidence supporting the immune escape
hypothesis and critically explore the mechanisms that may allow such escape to occur. We
discuss the idea that the central engine for generating immunoresistant tumor cell variants
is the genomic instability and dysregulation that is characteristic of the transformed
genome. "Natural selection" of heterogeneous tumor cells results in the survival and
proliferation of variants that happen to possess genetic and epigenetic traits that facilitate
their growth and immune evasion. Tumor escape variants are likely to emerge after
treatment with increasingly effective immunotherapies.
Progress in molecular and cellular immunology during the past two decades has advanced our
understanding of tumor-host interactions and opened extraordinary opportunities for the
development of antitumor immunotherapies. The identification of tumor antigens has allowed us, for
the first time, to tailor therapies to specific molecular targets expressed on tumor cells. In addition,
advances in recombinant biotechnology have enabled us to design and develop more effective
cancer vaccines for active immunization and cellular therapy for adoptive-transfer treatments.
However, consistently effective immunotherapy has not yet been developed for any type of
malignancy.
Although some maintain that the lack of broad success with current strategies is due to the
"escape" of tumor cells from the immunotherapies directed against them, it is also possible—and
even likely—that current immune-based treatments for malignancy are simply ineffective. For
example, current T cell–based immunotherapies may not be eliciting enough of the right kinds of T
cells in the right places at the right time to allow them to be consistently effective. To fully
understand the potential role played by tumor escape in the failure of immunotherapeutic
treatments in cancer patients, it is worth briefly discussing the basic principles of
immunosurveillance and its potential role in shaping tumor phenotypes.
Assumptions of the tumor escape hypothesis
It is not often explicitly stated that the tumor escape hypothesis assumes that if left unchecked, the
immune system would indeed spontaneously attack and destroy tumor. Simply put, immune escape
implies immune attack. A Review by Schreiber and colleagues in this issue examines the evidence
for immunosurveillance and attempts to resolve the long-standing controversy over its role in the
early stages of tumor development. Schreiber's group has demonstrated the existence of an
interferon- (IFN- )–dependent extrinsic tumor suppressor system in mice1. However, it is not clear
how large a role this plays in the immunosurveillance of tumors because only a minority of human
tumor cell lines examined in this study exhibited a permanent and selective IFN- insensitivity1.
Two studies have examined the potential roles of natural killer (NK) and
T cells bearing the
stimulatory lectin-like receptor NKG2D in immunosurveillance. The first study explored antitumor
immune responses elicited by tumors cells transduced with the NKG2D ligands, retinoic acid early
transcript 1b (Rae-1b) or H-602 and found that NK and/or CD8+ T cells mediated potent antitumor
immunity2. The study used NKG2D ligand–transduced cell lines that expressed high amounts of
Rae-1b or H-60; NKG2D ligand expression in most spontaneous tumors may be much lower.
Nevertheless, these findings demonstrated a possible mechanism of immunosurveillance that may
involve NK cells. A second study uncovered a possible role for NKG2D +
stages of tumor development3.
T cells at the very early
A note of caution is useful when interpreting these elegant mouse studies: there may be
fundamental differences between the development of tumors injected into a mouse or induced by
chemical carcinogens compared to naturally occurring spontaneous tumors in humans. For
example, the act of injection itself and the presence of dead tumor cells as well as tissue-culture
contaminants, such as fetal bovine serum in the inoculum, may induce local inflammatory reactions.
In tumors induced by painting chemical carcinogens onto the skin of highly susceptible mice, Rae1b and H-60 expression may be up-regulated, resulting in their rejection by
T cells. In addition,
whereas carcinogenesis in humans may be a slow process that occurs over several years, mouse
experiments use high doses of carcinogens, which cause transformation in just weeks or months. At
these doses, carcinogens may cause massive and rapid mutagenesis, resulting in greatly increased
number of neoantigens that may alert the immune system.
IFN- , perforin and tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) act as
effector molecules in immune surveillance for the prevention of tumor development. Mice that are
deficient in lymphocytes and/or in the IFN- signaling pathway have a much higher incidence of
carcinogen-induced sarcomas, lymphoma and spontaneous epithelial tumors 4, 5. Other studies have
shown that in perforin-deficient mice, there is a higher rate of spontaneous lymphoma and lung
adenocarcinoma4, 6. Upon transplantation into syngeneic wild-type mice, these lymphoma cells are
promptly rejected by cytotoxic T lymphocyte (CTL)-mediated activity. Inhibition of TRAIL (by
blocking antibody) promotes carcinogen-induced tumor development in mice; large numbers of
these tumors are TRAIL-sensitive, which is something rarely seen in tumors developed in wild-type
mice7.
Thus, evidence exists, in some models and under some conditions, that immunosurveillance can
play an active role in suppressing the growth of very early tumors. In this paradigm, when tumors do
successfully grow, they are thought of as having "escaped" from this immunosurveillance; these
early surveillance mechanisms are viewed as "shaping" the tumor's immunological phenotype.
Another model that explains why established tumors grow and do not undergo immune-mediated
rejection is that tumors function as immunologically normal tissue. In this paradigm, tumor cells
appear immunologically as healthy growing cells that do not send out danger signals to activate the
immune system because they express neither microbial immune-recognition patterns nor release
distress signals to alarm the innate immune cells8. Tumor histology and phenotype may determine
whether early tumors ultimately grow as a result of stealth and nonrecognition or as the result of
escape and immunological sculpting.
Later in the natural history of a tumor, during the progressive growth phase, tumors may become
more immune-activating for a variety of reasons (Fig. 1). Tumors can damage or disrupt
surrounding tissue or trigger a stress response when they outstrip their oxygen and nutrient
supplies. These processes can cause pH imbalance that results from metabolic disturbance,
generation of reactive oxygen species (ROS), up-regulation of stress protective factors—such as
heat shock proteins—and death by necrosis or apoptosis. Also, as tumors grow progressively,
dysregulated genetic and epigenetic events lead to the expression of large numbers of
neoantigens. One study estimated that the average malignancy contained more than 10,000
mutations9. All these factors may act as alarm signals to recruit and activate local innate immune
cells such as dendritic cells (DCs), macrophages, neutrophils and NK cells, which, in turn, activate
T cells to mount an adaptive immune response against tumors.
High resolution image and legend (58K)
tumor progression.
Figure 1. Activation versus suppression during
Immune activation during early tumor progression may be triggered by the expression of
neoantigens. In addition, the progressive growth of tumors may be associated with the invasion and
destruction of surrounding normal tissues. Other factors that may cause immune activation include
generation of heat shock proteins (Hsps), which result from cellular stress, and ROS such as OH and H2O2. Conversely, immune activation and function may be hampered by a lack of appropriate
costimulation, the presence of immunosuppressive cytokines (for example, VEGF, IL-10 and TGF), and the impact of immunoregulatory cells such as CD4+CD25+ T cells and NKT cells. As tumors
grow, these events occur in the presence of massive apoptosis and necrosis of tumor cells,
infiltrating immune cells and surrounding stromal cells. The fate of tumors may be dictated by the
net effect of immune activation and immune inhibition.
Whatever the roles of immunosurveillance, immune attack and immune escape in humans, it is
clear that once tumors are clinically detectable, spontaneous regression is exceedingly rare for the
vast majority of histologies. Once solid tumors become established, vascularized and clinically
detectable, it is simply not clear—given the available evidence—whether they need to "escape"
from immune recognition; this is because it is unclear whether most tumors elicit immune responses
that can cause tumor destruction8. Indeed, some observations contradict the idea that the immune
system spontaneously mounts lethal attacks against tumor cells. If cycles of immune pressure and
tumor escape were operative during tumor development, one might expect to observe (either by
examination or by imaging modalities) progressive tumor growth that was interspersed with one or
more periods of contraction. Ongoing immunological "shaping" or "sculpting" of tumors might be
expected to result in the destruction of sensitive cells followed by proliferation of immunoresistant
cells that would, in turn, form the bulk of a new tumor emerging from the bed of the old tumor (Fig.
2). However, solid tumors generally do not have growth curves with evidence of significant drops or
depressions: tumors simply grow, and then grow larger.
High resolution image and legend (38K)
Figure 2. Natural selection of tumor variants in
the generation of "tumor escape" phenotypes.
(a) Genomic instability gives rise to genetic diversity in tumors. Natural selection of tumor variants
occurs by differential propagation of tumor subclones in their microenvironment. (b) The same
concept also applies to tumor growth after effective immunotherapy.
On the other hand, it is also possible that selection is taking place at a cellular level, especially
during early tumor development, as discussed above. Ongoing selection of tumor variants on a
microscopic or cellular level would not necessarily result in gross or macroscopic changes, such as
those easily seen histologically or with commonly used imaging modalities. Similarly, a lack of
inflammation at the site of solid tumors may be interpreted by some to cast further doubt on the
hypotheses of spontaneous immune attack and immune escape. Although immune cells can be
observed in or around tumors, spontaneous local inflammation in an uninfected tumor is generally
not seen clinically or histologically. Again, however, it is possible that this inflammation is occurring
chronically and on a microscopic level. Thus, there is little direct evidence in mice or humans to
support the concept of tumor "shaping" or "sculpting" by the unmanipulated immune system once
tumors are vascularized and established. Nevertheless, these hypothesized processes may well
turn out to be important upon further investigation.
The natural selection of tumor cell variants
The setting of partially successful antitumor immunotherapy is one scenario that might be expected
to result in tumor escape. One of the most important factors that favors the survival and propagation
of evolving organisms is genetic diversity. It is interesting to view the survival of cancer cells in a
Darwinian light, where the survival of individual cancer cells is based on heritable variations and
differential survival associated with certain genotypes. Natural selection is a passive process by
which an organism has a survival advantage simply by possessing certain beneficial genes.
Genomic instability, an inherent property shared by all tumors, gives rise to genetic diversity, as
manifested by the great degree of heterogeneity seen in human tumors.
The law of natural selection is at work when cancer cells that possess genetic and epigenetic traits,
which are beneficial to their survival and/or proliferation, have a growth advantage over other cells.
The outcome of this passive process is determined by multiple factors in the tumor environment,
such as growth factors, nutrient supply and immune pressure. Genomic instability simply creates a
vast repertoire of tumor cells that will be selected according to environmental factors. Therefore,
terms such as "tumor evasion" or "tumor escape" are misnomers because evasion and escape
imply active acquisition of certain characteristics or phenotypes, rather than the differential
propagation of tumor subclones (Fig. 2a). The same concept also applies to tumor growth in the
face of an effective immunotherapy (Fig. 2b).
Loss or down-regulation of HLA class I antigens
It is certainly well documented that tumor cells can indeed lose HLA class I molecules through a
panoply of mechanisms. Descriptions of HLA loss causing immune escape have a great deal of
intuitive appeal, but are nevertheless correlative and indirect. There is little controlled evidence in
humans or animals that loss of major histocompatibility complex (MHC) class I molecules actually
leads to tumor escape or immunoresistance in the unmanipulated host. Indeed, loss of H-2
expression in transporter associated with antigen processing 1 (TAP-1)– or low-molecular weight
protein 2 (LMP-2)–deficient mice does not increase the onset or incidence of a variety of
spontaneous tumors10. On the other hand, some recurring tumors lose cell-surface MHC class I
both in mice with pre-existing immunity induced by immunization with B7-1–transfected tumor
cells11 and in humans after partial responses to T cell–based immunotherapy12. Decreased or
absent HLA class I expression is associated with invasive and metastatic lesions13. Total loss of
HLA class I expression is not uncommon in many tumors, including melanoma, colorectal
carcinoma and prostate adenocarcinoma14. In breast carcinoma, the frequency of total HLA class I
loss is >50%15. Thus, it is possible that in antigen-loss, HLA-loss and MHC class I processing–
defective variants, the loss or down-regulation of HLA class I antigens occurs as a result of
immunological "sculpting" of early tumor lesions (see the Review by Schreiber and colleagues in
this issue) or escape from immune attack later in tumor development.
Some mechanisms that can result in the loss or down-regulation of HLA class I expression are
shown (Fig. 3). The mechanisms that underlie total loss of HLA class I include mutations in one
copy of the 2-microglobulin gene in association with loss of heterozygosity (LOH) involving the
second allele16. The loss of 2-microglobulin has been observed in patients experiencing objective
partial responses after T cell–based immunotherapy12. We recently observed loss of 2microglobulin in tumor cells from two patients: one patient had an apparently complete response to
immunotherapy for nearly a decade; the other had experienced an extraordinary response after
adoptive transfer of tumor-specific T cells, then had a resurgence of tumor (unpublished
observations), although a causal relationship in such clinical scenarios is difficult to demonstrate
unequivocally. Other causes of total HLA class I down-regulation include defects in MHC genes and
in the antigen processing and transport pathway. Down-regulation of the proteosome multicatalytic
complex subunits LMP-2 and LMP-7 and of peptide transporters TAP-1 and TAP-2 have been
reported in tumor histologies that include small cell lung carcinoma17, non-small cell lung cancer
(NSCLC)18, prostate carcinoma19 and renal cell carcinoma20. In these situations, HLA class I can
often be up-regulated by treatment with IFN- .
High resolution image and legend (43K)
for HLA class I deficiency.
Figure 3. Molecular mechanisms responsible
Tumor antigens are processed in the proteosome and generate peptides that are transported by
TAP to the endoplasmic reticulum (ER); there they bind to certain HLA class I heavy chain in
association with 2-microglobulin. The peptide-HLA complexes are then transported through the
Golgi to the cell surface. Several different defects are associated with tumor "escape" from immune
recognition: (a) defects in components of the antigen-processing machinery (such as LMP-2 and
LMP-7) in the proteosome result in HLA class I down-regulation; (b) defects in peptide transporters
TAP-1 or TAP-2 cause HLA class I down-regulation; and (c) LOH on chromosome 6 causes HLA
class I haplotype loss. Defects in the transcriptional regulation of the HLA class I gene result in HLA
class I locus loss. Point mutations or gene deletions involving the HLA class I heavy chain result in
HLA class I allelic loss. (d) 2-microglobulin ( 2M) mutation or deletion results in total loss of HLA
class I.
Tumors can also express selective loss of HLA class I haplotype, locus or allele. HLA haplotype
loss can be due to LOH on chromosome 621. Several mechanisms are involved in locus downregulation, which is more frequent with HLA-B than HLA-A antigens. In melanomas, c-Myc
oncogene overexpression correlates with selective HLA-B locus down-regulation22. Loss of
transcription factor binding to locus-specific regulatory elements can induce HLA-B locus downregulation in colon carcinoma cells23. In melanoma, the gene products of the HLA-C locus are often
expressed poorly or not at all24. The defects underlying HLA class I allele–specific loss include
mutations in the genes encoding HLA class I heavy chain25.
Descriptions of partial or complete losses of HLA class I as mechanisms of immune escape often
fail to consider an increased susceptibility to NK cell lysis, which is a direct consequence of such a
loss26. Why do HLA class I loss tumor cell variants continue to grow and are not destroyed by NK
cells? NK cells express activating receptors, such as NKG2D, which bind to stress-induced ligands
(MICA and MICB) that can be up-regulated in a variety of tumors. Activation of NK cells through this
signaling pathway can overcome the inhibitory effect of HLA class I–binding receptors (KIRs)27, 28.
Thus, although HLA class I–negative tumors should be susceptible to NK killing, the loss or downregulation of MICA or MICB expression by actively growing tumors represents a potential escape
strategy, but one that has not yet been demonstrated in human tumors29.
Alternative explanations for why tumor cells that have lost HLA class I are not destroyed by NK cells
may be derived from the activation-inhibition model (Fig. 1). NK cells are rapidly activated in the
presence of stimulatory factors such as interleukin 12 (IL-12), IL-2, IL-15 or type 1 IFNs in response
to inflammatory conditions associated with microbial infection. In "sterile" environments, as seen
with tumors or transplantation, such stimulatory factors may not be readily available, and the crosstalk between DCs and resting NK cells that induces NK cell activation may not occur30. In addition, a
lack of expression of costimulatory molecules—such as B7-1 (also known as CD80), B7-2 (also
known as CD86), CD40 and CD70—by tumors may also hinder optimal NK cell activation via CD28
and CD27 costimulation pathways 31-33. It is possible that in some situations, tumors may produce
immunomodulatory cytokines, such as transforming growth factor- (TGF- ) or macrophage
migration inhibitory factor (MIF)34, which can directly inhibit NK cell activation and function.
Loss of tumor antigens and immunodominance
Loss of surface antigen expression can occur independently of the dysregulation of HLA class I
expression. It is well documented that tumor antigen expression is heterogeneous, even within the
same tumor. Decreased expression of melanoma-melanocyte differentiation antigens (MDAs) such
as gp100, melanoma antigen recognized by T cells 1 (MART1) and tyrosinase is associated with
disease progression35. In one study, cells were MART1+ in 100% of stage I lesions but only 75% of
stage IV lesions36. Decreased antigen expression has also been found in residual tumors after
peptide vaccination37, 38. With a gp100 peptide (209-2M) vaccine, there was a decrease in gp100
expression in tumors after ztreatment (47% versus 32%), whereas expression of MART1 was
unchanged (54% versus 54%)39. In addition, the amount of tumor antigen expressed may also be
important for recognition40. Again, these are correlative studies and fully controlled human studies
can be difficult or impossible to come by. It seems likely, however, that as T cell–based tumor
immunotherapy becomes stronger, escape mechanisms such as antigen loss are likely to become
more prominent.
The exact mechanisms that control the down-regulation of tumor antigens are not known in most
cases; however, the propagation of such antigen loss variants may be facilitated by epitope
immunodominance41. The phenomenon of immunodominance may be thought of as the preferential
immunodetection of one or a few epitopes among many expressed on a given target. The theory of
immunodominance, as it relates to tumor escape, predicts that one of the ways that antigen-loss
variants within a tumor are shielded from immune pressure is that the parental tumor cells that carry
the immunodominant epitope serve as a red flag for immune attack, thereby diverting attention from
the tumor variants. Once the parental cells are eliminated, a new hierarchy is established among
the variant subpopulations, and formerly immunorecessive epitopes become dominant 41. A tumor
variant that has lost the restricting HLA class I allele while retaining the immunodominant antigen
could cross-present this antigen to CD8+ CTLs by DCs and maintain an immunodominant response
to a "phantom" target at the expense of more appropriate and effective responses to other
antigens41.
Defective death receptor signaling
Two death receptor ligands that play a role in immune surveillance against tumor development are
Fas ligand (FasL) and TRAIL7, 42-44. Defective death receptor signaling is a mechanism that may
contribute to the survival and proliferation of tumor cells. Death receptors have cytoplasmic
sequences called "death domains" that are essential for the transmission of apoptotic signals via
the caspase cascade. For example, upon engagement of the death receptor Fas by its ligand, Fasassociated death domain (FADD) engages caspase-8, which, in turn, autoactivates itself and
cleaves downstream substrates such as caspase-3, caspase-6 and caspase-745. The caspase-8
inhibitor cellular FLICE-inhibitory protein (cFLIP) is expressed in various tumors. In these cases,
cFLIP may render tumor cells resistant to death receptor–mediated apoptosis46. Increased
expression of cFLIP by tumor cells may also contribute to immunoresistance to T cells in vivo47.
Down-regulation or loss of Fas expression in tumors may also contribute to their resistance to
apoptosis. Missense mutations and loss of the gene encoding Fas have been identified in multiple
myeloma48, non-Hodgkin's lymphoma (NHL)49 and melanoma50. These mutations disrupt Fas
signaling, resulting in a loss-of-function death receptor. In addition, mutations of the genes in the
proximal pathways downstream of Fas signaling have been also found in NSCLC and lymphoma 51,
52. These include inactivating mutations of FADD and caspase-10. Most were detected in metastatic
lesions of NSCLC. Besides defective death receptor signaling, tumors can also block CTL-mediated
cytotoxicity via the perforin pathway by overexpressing PI-9 (also known as SPI-6), a serine
protease inhibitor that inactivates granzyme B53.
In the case of TRAIL-mediated apoptosis, loss of expression of all TRAIL receptors by one of the
following causes: chromosomal loss; loss of caspase-8 by chromosomal loss or mutation; no
signaling from death-inducing signaling complex (DISC) due to FADD mutation, X-linked inhibitor of
apoptosis protein (XIAP) inhibition of caspase-3 and low second mitochondria–derived activator of
caspase or direct IAP-binding protein with low pI SMAC (also known as Diablo) release; and low
expression of death receptors by post-transcriptional regulation are associated with tumor
resistance to TRAIL-mediated apoptosis54. Thus in tumor cells, there may be defects at multiple
sites in the death receptor pathways that can favor tumor escape.
Lack of costimulation
As discussed above, most tumors seem to grow in a noninflammatory microenvironment that is not
conducive to immune activation. Histologically, tumors generally coexist innocuously with normal
tissues, apparently without giving or inducing immune-activating signals, especially during the early
stage of growth. Recognition of tumor antigens by DCs under these conditions will not lead to DC
activation and maturation. In addition, lack of expression of costimulatory molecules by tumor cells
may lead to T cell anergy55 and suboptimal activation of NK cells. In an experimental setting,
insertion of genes encoding B7-1, B7-2 or both into tumors generally increases the immunogenicity
of those tumors but does not necessarily lead to regression56.
Immunosuppressive cytokines
Activation or inhibition of T cells also depends on the presence or absence of cytokines in their
immediate microenvironment. Tumor cells produce a variety of cytokines and chemokines that can
negatively effect maturation and function of immune cells. Vascular endothelial growth factor
(VEGF) is a cytokine that is secreted by most tumors 57. In vitro studies show VEGF inhibits DC
differentiation and maturation through suppression of the transcription factor NF- B in hematopoietic
stem cells58. Immunohistochemical staining of gastric carcinoma tissues revealed an inverse
correlation between the density of DCs and VEGF expression. This finding was also associated with
poor prognosis59. In patients with lung, head and neck, and breast cancers, there was a decrease in
the function and number of mature DCs, which was associated with increased plasma
concentrations of VEGF60. Besides VEGF, increased concentrations of another cytokine, IL-10, are
frequently detected in the serum of patients with cancer. IL-10 can exert an inhibitory effect on DC
differentiation from stem cell precursors61. In addition, maturation and the functional status of DCs
are also compromised by IL-10. This cytokine also inhibits antigen presentation, IL-12 production
and induction of T helper type 1 responses in vivo62, 63. IL-10 also enhances spontaneous DC
apoptosis64 as well as susceptibility to autologous NK cell lysis 65. IL-10 may protect tumor cells from
CTLs by down-regulation of HLA classes I and II and ICAM-1 (intercellular adhesion molecule 1)66.
The loss of HLA class I expression could also be due to IL-10–mediated down-regulation of TAP1
and TAP2 proteins in tumor cells67, 68.
The proinflammatory factor prostaglandin E2 (PGE2) is another cytokine that is expressed by tumors
as a result of enhanced expression of the enzyme cyclooxygenase 2, which is the rate-limiting
enzyme for PGE2 synthesis, in multiple human tumors69-71. PGE2 increases the production of IL-10
by lymphocytes and macrophages and inhibits IL-12 production by macrophages72. High
concentrations of TGF- are also frequently found in cancer patients and are associated with
disease progression73 and poor responses to immunotherapy74. In addition to potentially being
produced by some tumor cell lines, TGF- may also be released by cells dying apoptotically75.
TGF- inhibits the activation, proliferation and activity of lymphocytes in vivo76. One point to
consider is that tumors may not necessarily produce these cytokines as escape mechanisms: the
hypothesized immunosuppressive functions may be mere side-effects of the angiogenic and growth
factor functions of these cytokines.
Apoptosis of activated T cells
One of the more controversial mechanisms of tumor escape is the expression of death receptor
ligands by tumor cells. A variety of cancer cells express functional FasL, which induces apoptosis of
Fas+-susceptible target cells. These include lung carcinoma77, melanoma78, colon carcinoma79 and
hepatocellular carcinoma80. The most recent study on this controversial topic asserts that FasL is
coexpressed with melanosomal and lysosomal markers in multivesicular bodies in human
melanoma cells81. Some of the data concerning FasL expression on tumor cells have been
questioned due to a lack of appropriate negative controls and technical concerns, such as the use
of nonspecific antibodies, non-intron–spanning polymerase chain reaction primers without proper
controls and functional assays that use the Jurkat cell line, which can themselves be induced to
express FasL82, 83. In our own work exploring FasL expression on human melanoma cells, we found
no evidence of significant expression either at the mRNA or protein level. In addition, there was no
killing of Fas+-susceptible targets by melanoma cells in controlled functional assays84. Another level
of complexity in the role of FasL may be that it is actually pro-inflammatory in some circumstances.
In fact, all well controlled in vivo experiments with membrane-bound FasL–transfected tumor cells
show accelerated rejection accompanied by neutrophil infiltration 85-87. However, the activity of FasL
may be modulated if it is processed to a secreted soluble form or if it is elaborated in the presence
or absence of certain cytokines within the tumor microenvironment. For example, TGF- can
regulate the proinflammatory effects of FasL88. The most crucial role of Fas-FasL interactions in the
tumor setting may be the induction of activation-induced cell death (AICD) of antitumor T cells.
Upon activation by tumor antigen recognition, T cells express high amounts of FasL, which induces
apoptosis of these T cells ("suicide") and between T cells ("fratricide")84, 89 (Fig. 4).
High resolution image and legend (55K)
Figure 4. Alternative models for the induction of
FasL-mediated T cell death after encounter with tumor cells.
(a) AICD of T cells after recognition of tumor cells. Tumor recognition leads to activation of T cells
and up-regulation of Fas and FasL on T cell surface, which results in the T cell killing of itself
("suicide") and of other T cells ("fratricide"). (b) Proposed tumor "counterattack" model. Tumor cells
express functional FasL and kill infiltrating Fas-expressing T cells via Fas-FasL binding, which leads
to tumor escape. However, ligation of Fas expressed on innate immune cells such as neutrophils,
macrophages and immature DCs by FasL expressed on tumor cells may also lead to release of
multiple proinflammatory cytokines and chemokines, setting the stage for tumor rejection.
Other TNF family ligands, such as TRAIL, are expressed by tumors, but there is no convincing
evidence that they play a major role in the induction of tumor-specific T cell apoptosis in human
cancers90. Tumor-associated B7-H1 ligand also induces T cell apoptosis91. However, this study
relied heavily on artificial models of B7-H1–transfected tumor cells that expressed high amounts of
B7-H1. It remains unclear whether the relatively small increases in specific T cell apoptosis over
background caused by endogenously expressed B7-H1 will be enough to enable human tumors to
evade immune recognition.
The role of suppressor T cells
Immunoregulatory CD4+ CD25+ T cells (also known as Treg, TH3 and TR1 cells) control key aspects
of immunological tolerance to self-antigens92, 93. Removal of these cells, which constitute 5–10% of
CD4+ T cells in humans and rodents, induces autoimmune disease in the ovaries, thyroid gland,
salivary glands and the mucosal linings of the stomach and small intestine and accelerate diabetes
in nonobsese diabetic mice. Depletion of CD4+CD25+ cells plus injection of an antibody capable of
blocking CTLA-4 (cytotoxic T lymphocyte antigen 4), enhanced reactivity to a known tumorassociated antigen (TAA)94. However, based on the available evidence from experimental mouse
tumor models, it seems that simply blocking or even eliminating T regulatory cell function will not be
enough to treat established tumors95. Identification of the glucocorticoid-induced TNF receptor
family–related gene (GITR, also known as TNFRSF18) expressed on T regulatory cells might afford
new therapeutic opportunities, as stimulation of GITR with an activating antibody reverses
CD4+CD25+ T cell–mediated suppression96, 97. Another possible therapeutic intervention could be
blockade of signaling through the molecular pair of TNF-related activation-induced cytokine
(TRANCE) and receptor-activator of NF- B (RANK), as these signals are involved in the generation
and activation of CD4+CD25+ T regulatory cells98. In addition to CD4+CD25+ T cells, NKT cells can
exert immunoinhibitory effects on tumor immunity. CD4+ NKT cells inhibit effective CTL-mediated
tumor rejection by IL-13 via the IL-4R–STAT6 (interleukin 4 receptor–signal transducers and
activators of transcription 6) pathway99. Another study has identified CD4+DX5+ NKT suppressor
cells that regulate the growth of ultraviolet-induced skin cancers and mediate antigen-specific
immune suppression100; thus, targeting NKT cells could be another therapeutic option. The
challenge for immunotherapists now is to discover how to use an understanding of T regulatory
function to enhance other immunotherapeutic interventions.
Conclusion
Despite recent progress in tumor immunobiology and technical advances in the field of tumor
immunotherapy, current immunotherapeutic strategies used in the treatment of patients with cancer
have not been successful in most cases. It is not clear whether tumor escape accounts for these
failures or whether lack of observed tumor regression is due to inadequacies of the
immunotherapies themselves.
The balance of immune activation and immune inhibition must favor the former if tumors are to
come under immune pressure. However, even in some instances where effective antitumor
responses can be achieved with immunotherapy, tumors often recur. It seems likely that as the
tumor immunologist's armamentarium becomes more powerful and immunotherapies become more
effective, the selection of tumor variants with immunoresistant phenotypes will be observed with
greater frequency. Tumor escape may ultimately thwart some dramatic responses to
immunotherapy. Indeed, some tumors that recur after successful immunotherapy often possess
immunoresistant phenotypes.
The challenge for the tumor immunologists now is to understand the mechanisms by which tumors
become refractory to immune modulation. The optimal immunotherapy strategy may be to achieve
three things concurrently: provision of appropriate immune activating signals, elimination of
inhibitory factors and avoidance of the emergence of immunoresistant phenotypes. The latter might
be achieved with a combination of modalities sustained for a long enough period of time to
complete tumor destruction.
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Figure 1. Activation versus suppression during tumor progression. Immune
activation during early tumor progression may be triggered by the expression of
neoantigens. In addition, the progressive growth of tumors may be associated with
the invasion and destruction of surrounding normal tissues. Other factors that may
cause immune activation include generation of heat shock proteins (Hsps), which
result from cellular stress, and ROS such as OH- and H2O2. Conversely, immune
activation and function may be hampered by a lack of appropriate costimulation,
the presence of immunosuppressive cytokines (for example, VEGF, IL-10 and
TGF- ), and the impact of immunoregulatory cells such as CD4+CD25+ T cells and
NKT cells. As tumors grow, these events occur in the presence of massive
apoptosis and necrosis of tumor cells, infiltrating immune cells and surrounding
stromal cells. The fate of tumors may be dictated by the net effect of immune
activation and immune inhibition.
Figure 2. Natural selection of tumor variants in the generation of "tumor
escape" phenotypes. (a) Genomic instability gives rise to genetic diversity in
tumors. Natural selection of tumor variants occurs by differential propagation of
tumor subclones in their microenvironment. (b) The same concept also applies to
tumor growth after effective immunotherapy.
Figure 3. Molecular mechanisms responsible for HLA class I deficiency.
Tumor antigens are processed in the proteosome and generate peptides that are
transported by TAP to the endoplasmic reticulum (ER); there they bind to certain
HLA class I heavy chain in association with 2-microglobulin. The peptide-HLA
complexes are then transported through the Golgi to the cell surface. Several
different defects are associated with tumor "escape" from immune recognition: (a)
defects in components of the antigen-processing machinery (such as LMP-2 and
LMP-7) in the proteosome result in HLA class I down-regulation; (b) defects in
peptide transporters TAP-1 or TAP-2 cause HLA class I down-regulation; and (c)
LOH on chromosome 6 causes HLA class I haplotype loss. Defects in the
transcriptional regulation of the HLA class I gene result in HLA class I locus loss.
Point mutations or gene deletions involving the HLA class I heavy chain result in
HLA class I allelic loss. (d) 2-microglobulin ( 2M) mutation or deletion results in
total loss of HLA class I.
Figure 4. Alternative models for the induction of FasL-mediated T cell death
after encounter with tumor cells. (a) AICD of T cells after recognition of tumor
cells. Tumor recognition leads to activation of T cells and up-regulation of Fas and
FasL on T cell surface, which results in the T cell killing of itself ("suicide") and of
other T cells ("fratricide"). (b) Proposed tumor "counterattack" model. Tumor cells
express functional FasL and kill infiltrating Fas-expressing T cells via Fas-FasL
binding, which leads to tumor escape. However, ligation of Fas expressed on
innate immune cells such as neutrophils, macrophages and immature DCs by FasL
expressed on tumor cells may also lead to release of multiple proinflammatory
cytokines and chemokines, setting the stage for tumor rejection.
Focus on Immune Evasion
Volume 3 No 11 November 2002
Review
Nature Immunology 3, 1006 - 1012 (2002)
doi:10.1038/ni1102-1006
© Nature America, Inc.
<>
Viral evasion of natural killer cells
Jordan S. Orange, Marlys S. Fassett, Louise A. Koopman, Jonathan E. Boyson &
Jack L. Strominger
Department of Molecular and Cellular Biology, Harvard University, Cambridge MA, USA.
Correspondence should be addressed to J L Strominger [email protected]
Viruses have evolved mechanisms to avoid the host immune system, including means of
escaping detection by both the innate and adaptive immune responses. Natural killer (NK)
cells are a central component of the innate immune system and are crucial in defense
against certain viruses. To attain a state of chronic infection, some successful viruses have
developed specific mechanisms to evade detection by and activation of NK cells. These NK
cell–specific evasion mechanisms fall into distinct mechanistic categories used in numerous
virus families.
NK cells are lymphocytes that do not undergo genetic recombination events to increase their affinity
for particular ligands, and are thus considered part of the innate immune system. They are capable
of mediating cytotoxic activity and of producing cytokines after ligation of a variety of germlineencoded receptors. NK cells mediate direct lysis of target cells by releasing cytotoxic granules
containing perforin and granzymes, or by binding to apoptosis-inducing receptors on the target cell.
They also secrete cytokines such as interferon- (IFN- ) and tumor necrosis factor- (TNF- )
during infection and inflammation. Several receptors that can activate NK cells have been identified,
including the human natural cytotoxicity receptors NKp30, NKp44, NKp461 and Ly49D and Ly49H in
the mouse2. Although the specificities of many NK cell–activating receptors are still unknown, some
recognize viral products; these include influenza hemagglutinin, recognized by NKp463, and murine
cytomegalovirus (MCMV) m157, recognized by Ly49H 4, 5. Other well known molecules can also
function as activation receptors in NK cells, including leukocyte function–associated antigen 1 (LFA1)6 and the CD2 family7. NK cell responses are also coordinated and modulated by cytokines,
including IFN- , IFN- , interleukin 2 (IL-2), IL-12, IL-15 and IL-188.
Because of the possible consequences of NK cell activation, normal host cells must be able to
readily and effectively inhibit NK cells. Various inhibitory receptors are consistently expressed by
subsets of NK cells, including killer-cell immunoglobulin-like receptors (KIR), immunoglobulin-like
inhibitory receptors (ILT) and the lectin-like heterodimer CD94-NKG2A1. These receptors bind to
host MHC class I molecules and transmit inhibitory signals to the NK cell through intracellular
tyrosine-based inhibitory motifs (ITIMs) contained in their cytoplasmic domains. Signaling via
coreceptors with inhibitory potential, such as CD81, may also silence NK cells. Thus, NK cells can
mediate powerful effector functions, but are effectively regulated by healthy host cells under normal
circumstances.
NK cells are activated during a wide variety of viral infections by virus-induced type I IFNs8. Studies
in animal models, however, have demonstrated that NK cells are required for clearance of only
certain viruses, including herpesviruses. The importance of NK cell defense against these viruses is
highlighted by the susceptibility of mice depleted of NK cells to experimental infection and by the
invasive or disseminated viral disease that is associated with naturally occurring NK cell
deficiencies in humans8-11. Many of these pathogens have effective means of avoiding the adaptive
immune response. In eluding T cells, however, these viruses might have increased their
susceptibility to NK cell–mediated defenses.
Members of the herpesvirus, papillomavirus, retrovirus, poxvirus and flavivirus families have
developed mechanisms to evade the NK cell response. These fall into five categories (Fig. 1):
expression of virally encoded MHC class I homologs; selective modulation of MHC class I protein
expression by viral proteins; virus-mediated inhibition of activating receptor function; production of
virally encoded cytokine-binding proteins or cytokine-receptor antagonists; and direct viral effects on
NK cells. The putative purpose of these mechanisms is to block NK cell activity. Examples of each
strategy (summarized in Table 1) are discussed below. Many other viruses probably use these
strategies, and additional viral products may affect NK cell functions. This discussion is limited to
viral infections and viral gene products known to affect the NK cell response.
High resolution image and legend (69K)
Figure 1. Viral mechanisms for evading NK cells.
The strategies by which viruses evade NK cells fall into five categories and are depicted in the
interaction between a virus infected target cell (left) and an NK cell (right). (a) NK cells can be
inhibited by a viral MHC class I homolog with structural similarity to endogenous host class I that
binds to inhibitory class I receptors on NK cells. (b) Viruses can inhibit expression of HLA-A and
HLA-B, resulting in a relative increase in HLA-C and HLA-E on the surface of the target cell; these
inhibit NK cells through the class I inhibitory receptors CD94-NKG2A and KIR, respectively.
Alternatively, viral gene expression can result in selectively increased expression of HLA-E, which
inhibits NK cells through CD94-NKG2A. (c) Virus-encoded proteins can function as cytokine binding
proteins that block the action of NK cell activating cytokines. In addition, viruses can produce
homologs, or increase host production of cytokines that inhibit NK cells. (d) NK cell activities can
also be avoided by decreased expression of NK cell–activating ligands in virus-infected target cells,
which prevent signal transduction via NK cell–activating receptors. To achieve the same end,
viruses can encode antagonists of the activating receptor–ligand interaction. (e) Viruses can also
directly inhibit NK cells by infecting them or using envelope proteins to ligate NK cell inhibitory
receptors. Proteins outlined in red are virally encoded. Each mechanism corresponds to the
similarly numbered section of the text where additional details and examples are provided.
High resolution image and legend (136K)
Table 1. Viral mechanisms of evading NK cell responses
Inhibition of NK cells by viral homologs of MHC class I
Virus-encoded homologs of cellular MHC class I genes represent the earliest recognized viral
mechanism for evading NK cells. Many viruses evade T cell recognition by down-regulating class I
molecules on the surface of the host cell (discussed below). In theory this leaves infected cells
susceptible to NK lysis owing to the reduced opportunity for class I molecules—human leukocyte
antigen C (HLA-C) and HLA-E—to engage NK cell inhibitory receptors. The discovery of a class I
homolog, UL18, in the genome of human cytomegalovirus (HCMV)12 has led to speculation that
these viral proteins might serve as NK cell decoys and ligate inhibitory receptors to block NK cell
cytotoxicity in the absence of host class I molecules.
Early studies in which HCMV UL18 was expressed in the HLA-A, HLA-B and HLA-C–deficient B cell
line 721.221 demonstrated a CD94-dependent inhibition of killing by various NK cell lines and
clones13. Subsequently, a principal ligand of the CD94-NKG2A inhibitory receptor was found to be
HLA-E, a class I protein that is expressed only after forming a complex with a peptide nonamer
derived mainly from the signal peptides of some of the class I proteins14. Further efforts to identify a
nonamer in the UL18 sequence that would form a complex with HLA-E have been unsuccessful15.
Thus, the mechanism underlying the reported inhibition of lysis of the human B cell line transfected
with UL18 remains unexplained. It was not due to inadvertent selection of HLA-E–expressing
721.221 cells because immunoprecipitation of class I molecules from the 721.221-UL18
transfectants yielded a heavily glycosylated -chain of the size of UL18 ( 66 kD) and not HLA-E
(44 kD)13. In a contrasting report, however, UL18-transfected fibroblasts showed increased
susceptibility to lysis by NK cell lines, and fibroblasts infected with wild-type HCMV were lysed more
efficiently than those infected with a UL18-deficient HCMV16. Alternatively, the inhibitory activity
mediated by UL18 may be due to its binding of ILT-2 (LIR-1), an inhibitory receptor expressed by B
cells, monocytes and a subset of NK cells17. Thus, the role of UL18 in evasion of NK cells is not
completely understood, but it may be an important mechanism by which certain subpopulations of
NK cells are inhibited.
Compelling data support the notion that the MCMV MHC class I homolog m144 is involved in the
inhibition of NK cells. An m144 deletion strain of MCMV is less virulent than the wild-type virus, and
this effect is reversed after in vivo depletion of NK cells18. Transfection of m144 into the Raji cell line
results in partial inhibition of antibody-dependent cellular cytotoxicity19. In addition, m144transfected RMA-S lymphoma cells injected into mice are tumorigenic, whereas untransfected
RMA-S tumors are rejected by NK cells20. Further investigation of m144 will benefit from
identification of the cognate inhibitory receptor. The structures of m144 and UL18 are considerably
different (for example, the latter binds peptides, the former does not), so that their receptors,
mechanisms and functions may be distinct.
A newly described viral class I homolog, MCMV m157, has both activating and inhibitory effects
upon NK cells, depending on the mouse strain from which the cells were derived. MCMV m157 is a
ligand for the NK-activating receptor Ly49H in MCMV-resistant C57BL/6 mice and is predicted to
share sequence and structural similarity with other nonclassical MHC class I genes4, 5. Because a
virus-encoded ligand that activates NK cells seems teleologically unsound from the standpoint of
the virus, it is notable that m157 binds the putative inhibitory receptor Ly49I on a subset of NK cells
in CMV-susceptible 129/J mice4. The significance of m157-induced activating and inhibitory
functions is unknown, particularly in noninbred mice, but their existence suggests constant evolution
in the host as well as the virus.
The dual specificity of MCMV m157 for NK-activating and NK-inhibitory receptors could provide new
insight about HCMV UL18. Like m157, UL18 might exert different effects upon NK cells by
interacting with distinct receptors in different in vitro or in vivo systems. In addition, the example of
m157 is a useful reminder that viral homologs of MHC class I function in a complex, dynamic
environment of viral and immune elements during in vivo infection.
Additional MHC class I homologs await characterization. Rat cytomegalovirus (RCMV) r144 has not
been assayed for direct effects upon NK cell cytotoxicity, but lower virus titers occur in the spleens
of rats infected with an r144-deleted strain of RCMV than in those infected with wild-type RCMV21.
This suggests that like other class I homologs, r144 may be a ligand for an NK cell inhibitory
receptor. Molluscum contagiosum virus (MCV) also encodes a class I homolog, MC080R, which is
retained in the endoplasmic reticulum and Golgi of transfected cells 22. Its effects on NK cell activity
are presently unknown. Most promising is the recent identification of ten class I homologs in the
MCMV genome, in addition to m144 and m1575. These genes were not detected earlier because
they have structural rather than sequence similarity to known class I molecules. Their discovery is
suggestive of the possibility that additional, undetected class I homologs might exist in other viral
genomes as well.
Selective modulation of MHC class I allele expression
A common feature of many viral infections is the virus-induced modulation of class I expression.
Viruses down-modulate class I molecules that are efficient at presenting viral peptides to CD8 +
cytotoxic T cells (CTLs), such as HLA-A and HLA-B, to evade CTL-mediated destruction. In
contrast, either HLA-C and HLA-E, the dominant ligands for NK cell–inhibitory receptors, are spared
from virus-induced clearance from the cell surface or their expression is specifically enhanced. The
selectivity of viral proteins for certain class I molecules appears to be indicative of a compromise
ensuring that class I expression is diminished only to an extent that will still allow efficient inhibition
of NK cells. Such a strategy appears to allow viruses to walk a fine line between the adaptive and
innate arms of the immune system. A large number of viral proteins participate in class I downregulation23. Here, the discussion will be limited to viral down-modulation of class I molecules as it
relates to NK cell responses.
Many viral proteins cause class I molecules to deviate from their normal progression from the
endoplasmic reticulum to the cell surface. At least four HCMV proteins, US2, US3, US6 and US11,
function in this manner. US2 and US11 show a certain degree of selectivity in their targeting of
class I molecules24, 25. In particular, two dominant inhibitory receptor ligands, HLA-C and HLA-E, are
resistant to either US2- or US11-mediated degradation, suggesting that virus-infected cells evade
NK cell activity by sparing the class I molecules least effective at presenting viral peptides to CTL
but most effective at inhibiting NK cells26, 27. In contrast, class I molecules are nonselectively downmodulated from the cell surface by US3 and US6, and these proteins can partially inhibit the cell
surface expression of HLA-C and HLA-G28. It is not clear whether the extent of down-modulation
mediated by US3 and US6 is sufficient to make infected cells susceptible to NK cell lysis. Notably,
none of these US proteins that down-regulate HLA expression inhibit the expression of the viral
encoded class I homolog UL18, further demonstrating a selective targeting for NK cell evasion 29.
MCMV possesses three genes, m04, m06 and m152, that selectively modulate class I expression;
these genes are not homologous to the HCMV US genes and use slightly different modulation
mechanisms. This indicates that HCMV and MCMV independently evolved systems to regulate
class I expression. Notably, although the m04–class I protein complex inhibits CTLs, it is expressed
on the cell surface where it could potentially inhibit NK cells 30.
A second mechanism whereby certain viruses modulate class I expression is by accelerating the
endocytosis of class I molecules from the cell surface. The nef gene, present in primate lentiviruses
such as HIV-1, HIV-2 and SIV, encodes a 27-kD protein that is expressed early after infection and
selectively down-modulates the expression of HLA-A and HLA-B, but not HLA-C or HLA-E31, 32.
Thus, HIV-infected target cells remain resistant to lysis by NK cells, and their resistance depends on
the failure of Nef to down-modulate HLA-C and HLA-E from the cell surface. Nef acts by inducing
the clathrin adaptor protein complex to recognize a tyrosine-based sorting motif in the cytoplasmic
tail of HLA-A and HLA-B32. The resistance of HLA-C to Nef-induced down-regulation is due to locusspecific tyrosine-to-cysteine and aspartic-acid-to-asparagine substitutions in the cytoplasmic tail31,
32. The SIV Nef protein also down-modulates monkey class I proteins via endocytosis but uses a
COOH-terminal region of SIV Nef that is not present in HIV Nef, suggesting once again that viruses
have repeatedly evolved multiple mechanisms to modulate class I expression 33.
Acceleration of class I molecule endocytosis is also a feature of Kaposi's sarcoma–associated
herpesvirus (KSHV). Two KSHV proteins, K3 and K5, induce the rapid endocytosis of class I
proteins from the cell surface34, 35. Whereas K3 down-modulates HLA-A, HLA-B, HLA-C and HLA-E,
K5 is more selective and down-modulates only HLA-A and HLA-B36. Because K5 has other
functions, it is difficult to evaluate the importance of HLA-C and HLA-E resistance to K5 endocytosis
with regard to NK cells36.
In addition to their specific down-modulation of class I alleles, viruses also up-regulate certain class
I alleles to evade NK cells. Previous work suggests that HCMV actively enhances the expression of
HLA-E, the ligand for the inhibitory CD94–NKG2A receptor complex15, 37. Cell surface expression of
HLA-E requires binding of a nonamer peptide derived from the signal sequence of most HLA
molecules38. The HCMV UL40 protein possesses a nonamer peptide homologous to HLA signal
sequences and thus can enhance cell surface expression of HLA-E15, 37. HLA-E binds the UL40
peptide in a TAP-independent manner, presumably bypassing the inhibitory effects of the HCMV
US6 protein. In HCMV-infected cells, UL40 is necessary to mediate resistance to NK cell lysis in a
CD94- and MHC class I–dependent manner39.
Additional investigation is required to test the effects of virus-induced MHC modulation on NK cell
resistance. One complicating feature is that the retention of HLA-C on the cell surface may actually
result in lysis of the virus-infected cells by NK cells possessing the cognate activating KIR but no
inhibitory KIR40, 41. Similarly, because HLA-E also binds to the CD94-NKG2C–activating receptor14,
UL40-induced up-regulation of HLA-E could potentially activate NK cells. Thus, it is likely that
selective HLA regulation contributes to viral evasion of NK cells, but the relative importance of this
mechanism is uncertain.
Virus-mediated inhibition of activating receptor function
In addition to receptors with potent inhibitory capabilities, NK cells have receptors whose ligation
can induce cytotoxicity, proliferation and cytokine production 1. Several such activating receptors
have been characterized, and some recognize putative ligands that include specific viral products 3-5.
An effective viral evasion strategy, therefore, would be to interfere with the process of activating
receptor ligation. Several means to this end have been identified.
The most commonly documented mechanism of interference with activating receptor function is
virus-mediated down-regulation of activating receptor ligands in infected cells. For example, certain
strains of HCMV increase the resistance of their infected host cells to NK cell cytotoxicity by downregulating LFA-342. This regulation, which is independent of the virus effects on class I molecules
discussed previously, presumably interferes with the binding of LFA-3 to the NK cell–activating
receptor, CD2. In addition, MCMV m152 encodes gp40, a protein that presumably inhibits surface
expression of the murine ligand for the NKG2D-activating receptor, H-6043. Cells infected with
m152-deleted MCMV have greater expression of H-60 and are more readily lysed by NK cells. An
additional example of host-cell ligand regulation as an evasion strategy is found in KSHV.
Transfection of target cells with the K5 gene of KSHV results in decreased expression of ICAM-1
and B7-2, both of which can serve as ligands for NK cell–activating receptors36, 44. The K5-mediated
reduction in the surface expression of these ligands results in target-cell escape from NK cell
cytotoxicity in certain in vitro NK cell systems36, but not in others44. K5 is an E2 ubiquitin ligase and
thus probably directs the ubiquitination of the NK cell–activating receptor ligands it targets,
ultimately resulting in their degradation34. As a group, the host-cell molecules targeted by viruses
have functions other than as NK cell–activating receptor ligands and thus these examples, although
relevant, are not entirely specific to NK cells. The discovery of ligands for activating receptors
specific to NK cells will probably lead to fruitful studies of viral regulation of their expression.
A second way that viruses may interfere with activating receptor function is by virus-induced
modification of the ligand on target-cell surfaces. Infection of certain target cells by HIV, human T
cell lymphotrophic virus I (HTLV-I) or HTLV-II can result in resistance to NK cell lysis associated
with sialylation of surface molecules45. Although target-cell binding is not impaired by infection,
chemical removal of sialic acid restores susceptibility to NK cell cytotoxicity. These receptor
modifications have not been specifically seen on known NK cell ligands, but it can be reasoned that
activating receptor ligands would be the most likely targets of virus-induced sialylation. It is to be
hoped that follow-up studies will reveal the viral proteins responsible for, and specific cellular targets
of, this evasion strategy.
A third mechanism for avoiding NK cell activation is antagonism of the activating receptor and
ligand interaction. An example is the interaction of an HCMV encoded protein with the NKG2D
receptor. NKG2D is an activating receptor expressed on NK cells and subsets of T cells that binds
to the UL16-binding protein (ULBP) family of glycosylphosphatidylinositol-linked receptors and MHC
class I–related molecules (MIC) in humans, as well as the retinoic acid–inducible early gene 1 (Rae1) protein family and H-60 minor histocompatibility antigen in mice46. Ligation of NKG2D results in
signal transduction via DAP10, leading to NK cell cytotoxicity46. HCMV encodes UL16, which in a
soluble form is capable of binding ULBP, thus blocking the interaction between the NKG2D
activating receptor complex and its cognate activating ligand47, 48. These data suggest a mechanism
by which UL16 might inhibit NK cell function in vivo.
A final means by which viruses interfere with activating receptor function is through the inhibition of
signaling induced by activating-receptor ligation. Although these mechanisms can potentially inhibit
many cells, at least one viral protein appears to have some specificity for NK cell activation. HIV-1
Tat can block the NK cell activation and function induced by ligation of LFA-1 on the NK cell
surface49, 50. Tat does not affect NK cell adhesion to the target cell, but specifically binds to an L-
type calcium channel on NK cells49. Its binding blocks calcium influx and subsequent induction of
the calcium-calmodulin kinase II in NK cells, which is required for cytotoxicity50. The search for viral
products that specifically block signaling for NK cell cytotoxicity will certainly become more
productive as the appreciation of these pathways in NK cells grows.
Evasion by modulation of cytokines or chemokines
Viruses may subvert NK cell responses through virus-encoded proteins that counteract or modulate
the interactions between cytokine or chemokine molecules and their cognate receptors. Numerous
poxviruses and herpesviruses encode homologs to known cytokines and chemokines with agonistic
or antagonistic function, or secreted proteins or receptors that bind with high affinity to cytokines
and chemokines51. Although several examples of this sort of molecular mimicry have been
described for different viruses, direct evidence for the involvement of NK cells in this strategy of
immune escape in vivo is scarce. Interference with anti-viral NK cell function could involve inhibition
or antagonism of cytokines such as IL-12, IL-18, TNF- , IL-1 , IL-1 and IL-15, which participate in
inducing NK cell IFN- production and cytotoxicity8. Alternatively, viruses could facilitate
overproduction or encode homologs of other cytokines (such as IL-1052) that have an inhibitory
effect upon NK cells. Among the chemokines, targets for viral modulation could involve those that
directly affect NK cell chemotaxis, including MIP-1 (CCL3), MIP-1 (CCL4), MCP-1 (CCL2), MCP2 (CCL8), MCP-3 (CCL7) and RANTES (CCL5)8, or other chemokines and chemokine receptors
involved in recruitment of leukocyte subsets that influence NK cell function.
Several virus-produced chemokine homologs have been described that might interfere with NK
cell–mediated defense. The putative CC-chemokine homolog m131-m129 (or MCK-2) of MCMV is
directly linked to NK cell evasion53, 54. Infection of mice with an m131-m129–deleted mutant virus
results in decreased viral burden in the spleen and liver. The increased clearance of this mutant
virus is negated by NK cell depletion, suggesting that m131-m129 inhibits NK cell–mediated viral
clearance. The exact mechanism underlying this function of m131-m129 is unclear. Another
chemokine homolog with possible relevance to NK cells is the broad-spectrum CC, CXC and CX3C
chemokine antagonist vMIP-II of KHSV, which could block chemotactic responses of monocytes to
RANTES, MIP-1 and MIP-1 55. In addition, the CCR8 antagonist vMIP-I binds to chemokine
receptors on NK cells56. Similarly, MCV also encodes a homolog, MC148, that is a narrow-spectrum
antagonist of MCP-157. Each of these chemokine antagonists could potentially interfere with NK cell
responses. In contrast, virus-encoded homologs of the cytokine IL-10, which function as agonists,
have been cloned from HCMV and Epstein-Barr virus (EBV)58, 59. These viral proteins may impair
NK cell activation by inhibiting production of type 1 cytokines as well as by directly acting upon NK
cells.
Specific interference with NK cell activation can also be mediated by cytokine-binding protein
homologs. A principal target is IL-18, which is central to NK cell production of IFN- . Ectromelia
poxvirus (EV) encodes a cytokine-binding protein with NK cell–specific effects60. The EV p13
protein is homologous to mammalian IL-18BP, binds murine IL-18 and inhibits IL-18 receptor
binding and activity. Mice infected with a p13-deleted mutant virus have markedly greater NK cell
cytotoxicity than those infected with wild-type EV, as a result of increased NK cell activation.
Compared to mice infected with p13-deleted virus, mice infected with wild-type EV have lower IFNproduction and higher IL-10 production, further highlighting the importance of this viral protein in
NK cell evasion60. MCV also encodes several gene products that likely modulate host immunity61, 62,
including a functional IL-18BP homolog, MC54L63. The evolution of these homologs points to the
importance of the NK cell IFN- pathway in the elimination of virus-infected cells, and shows the
importance of IL-18 function.
Binding and sequestration of IL-18 from its cognate receptor has also been suggested to be
involved in human papillomavirus (HPV)–related pathogenesis64, 65. The oncoproteins HPV16 E6
and E7 inhibit IL-18–induced IFN- production in PHA-stimulated PBMC and in an IL-12–stimulated
immortalized NK cell line by specific and competitive binding to IL-1865 or IL-18R 64.
Other proteins without homology to known cytokine or chemokine receptors or binding proteins that
can act as cytokine or chemokine antagonists include murine -herpesvirus 68 M3, which encodes
a secreted 44-kD broad-spectrum chemokine binding protein (hvCKBP), and a 35-kD soluble CCchemokine–specific binding protein (vCKBP) encoded by vaccinia virus. Both proteins block the
activity of several chemokines, including the NK cell chemoattractants MIP-1 66, 67, MCP-1 and
RANTES67, and thus function as inhibitors with potential NK cell specificity.
Direct viral effects on NK cells
Viruses can exert direct effects on NK cells. In particular, viruses can infect and inhibit or destroy
NK cells, or cause inhibition through presumably direct contact with NK cells. These mechanisms
should be particularly advantageous to viruses that attain chronically high systemic or organspecific titers.
Both HIV and HSV infect NK cells in vitro. HIV can infect cultured NK cells, and is found ex vivo in
NK cells from HIV-seropositive individuals68, 69. Although in vitro infection of NK cells with HIV does
not affect the overall lytic activity of NK cell cultures, it does greatly reduce viability68. Thus, HIV
may evade NK cell responses to some degree by direct infection and induction of cytopathic effects.
In contrast, HSV can spread from infected fibroblasts to cultured NK cells, and thus inhibits NK cell
cytotoxicity69. This direct infection of NK cells is unusual and suggests that HSV-mediated inhibition
of these cells may fall into the category of direct viral effects, although the mechanism is unknown.
It will be useful to ascertain whether HSV gene products can specifically interfere with intracellular
NK cell signals leading to activation in infected NK cells.
Interactions between virus particles and NK cells that do not result in infection may exert other
direct effects upon NK cells. One viral envelope protein has recently been implicated in inhibition of
NK cells. E2, the major envelope protein of hepatitis C virus (HCV), binds to CD81 (TAPA-1), which
induces a costimulatory signal in T cells70. In NK cells, however, ligation of CD81 by immobilized E2
or anti-CD81 inhibits NK cell cytotoxicity, IL-2–induced proliferation and IL-2–, IL-12– or IL-15–
induced IFN- production71, 72. In addition, CD81 ligation specifically inhibits ERK and MAPK
phosphorylation induced by CD16-mediated activation of NK cells71. These effects of E2 binding to
CD81 are specific to NK cells, as opposite activities are induced in T cells. Thus, E2 can mediate
specific inhibition of NK cells via an inhibitory receptor, and therefore direct viral binding to NK cells
may quell NK cell responses. This mechanism may be particularly useful to HCV during viremia and
in the liver, where viral titers are high and NK cell defense is important.
Conclusion
Viruses have evolved numerous mechanisms to evade immune responses. Although many are
used to avoid multiple immunologic effector cells, several are specifically directed at NK cells. Viral
evasion strategies targeting NK cells can be divided into five mechanistic categories: ligation of
inhibitory class I receptors on NK cells by virus-encoded MHC class I homologs; virus-induced
regulation of class I protein expression resulting in selective expression or up-regulation of class I
molecules that can bind NK cell class I inhibitory receptors; interference with NK cell–activating
receptor function caused by virus-mediated inhibition of the expression of the corresponding ligand,
of their signaling in infected host cells or of the production of virus encoded antagonists of NK cell–
activating receptors; inhibition of NK cell activation, trafficking, or both, resulting from viral
modulation of cytokine–chemokine networks or virus-encoded cytokine or chemokine homologs that
prevent NK cell function; and direct inhibitory effects of viruses on NK cells, including infection of NK
cells and ligation of non-class I NK cell inhibitory receptors by viral envelope proteins. These
multiple mechanisms highlight the importance of NK cells in defense against viral infections. In
particular, certain viruses, including herpesviruses, appear to be especially adept in their avoidance
of NK cells, consistent with the crucial role of NK cells in the control of these viruses. Further study
of viral evasion mechanisms will provide useful models to elucidate NK cell biology as well as to
provide therapeutic targets to enhance host advantage during infection.
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Figure 1. Viral mechanisms for evading NK cells. The strategies by which
viruses evade NK cells fall into five categories and are depicted in the interaction
between a virus infected target cell (left) and an NK cell (right). (a) NK cells can be
inhibited by a viral MHC class I homolog with structural similarity to endogenous
host class I that binds to inhibitory class I receptors on NK cells. (b) Viruses can
inhibit expression of HLA-A and HLA-B, resulting in a relative increase in HLA-C
and HLA-E on the surface of the target cell; these inhibit NK cells through the class
I inhibitory receptors CD94-NKG2A and KIR, respectively. Alternatively, viral gene
expression can result in selectively increased expression of HLA-E, which inhibits
NK cells through CD94-NKG2A. (c) Virus-encoded proteins can function as
cytokine binding proteins that block the action of NK cell activating cytokines. In
addition, viruses can produce homologs, or increase host production of cytokines
that inhibit NK cells. (d) NK cell activities can also be avoided by decreased
expression of NK cell–activating ligands in virus-infected target cells, which prevent
signal transduction via NK cell–activating receptors. To achieve the same end,
viruses can encode antagonists of the activating receptor–ligand interaction. (e)
Viruses can also directly inhibit NK cells by infecting them or using envelope
proteins to ligate NK cell inhibitory receptors. Proteins outlined in red are virally
encoded. Each mechanism corresponds to the similarly numbered section of the
text where additional details and examples are provided.
Table 1. Viral mechanisms of evading NK cell responses
Focus on Immune Evasion
Volume 3 No 11 November 2002
Review
Nature Immunology 3, 1013 - 1018 (2002)
doi:10.1038/ni1102-1013
© Nature America, Inc.
<>
To kill or be killed: viral evasion of apoptosis
Chris A. Benedict, Paula S. Norris & Carl F. Ware
Division of Molecular Immunology, La Jolla Institute for Allergy and Immunology, 10355 Science Center Drive, San Diego, CA 92121,
USA.
Correspondence should be addressed to C F Ware [email protected]
In the struggle between virus and host, control over the cell's death machinery is crucial for
survival. Viruses are obligatory intracellular parasites and, as such, must modulate
apoptotic pathways to control the lifespan of their host in order to complete their replication
cycle. Many of the counter-assaults mounted by the immune system incorporate activation
of the apoptotic pathway—particularly by members of the tumor necrosis factor cytokine
family—as a mechanism to restrict viral replication. Thus, apoptosis serves as a powerful
selective pressure for the virus to evade. However, for the host, success is harsh and
potentially costly, as apoptosis often contributes to pathogenesis. Here we examine some of
the molecular mechanisms by which viruses manipulate the apoptotic machinery to their
advantage and how we (as vertebrates) have evolved and learned to cope with viral evasion.
The apoptotic machinery
Apoptosis results from a collapse of cellular infrastructure through regulated internal proteolytic
digestion, which leads to cytoskeletal disintegration, metabolic derangement and genomic
fragmentation. Members of the cytosolic caspase family of proteinases (cysteine-based, aspartatedirected) form "the engine" of the apoptotic pathway1 (Fig. 1). The caspases (11 in total) represent
one of more than 20 distinct components involved in initiation, execution and regulatory phases of
the pathway, which indicates the extensive regulation this process has engendered over time. The
apoptotic machinery shows sensitivity to a variety of agents by coordinating signals initiated by both
internal sensors (intrinsic pathway, mitochondria-dependent) and external stimuli (extrinsic pathway,
death receptor–mediated). When triggered, internal sensors (for example, p53) can initiate
processes that result in the ultimate loss of mitochondrial integrity and apoptosis. Signals from the
internal sensors are propagated to the mitochondria via pro-apoptotic Bcl-2 subfamily members
(BH3 only), such as Bid, to oligomerize Bax and Bak, which are outer mitochondrial membrane
proteins that promote the release of cytochrome c. Cytochrome c oligomerizes Apaf1 and recruits
pro-caspase-9 (forming the apoptosome), which results in proteolytic conversion of pro-caspase-9
to an active enzyme. Caspase-9 then converts pro-caspase-3 to its active form, which, with other
executioner caspases, such as caspase-7, then cleaves key substrates in the cell to orchestrate the
cell's fatal collapse.
High resolution image and legend (67K)
Figure 1. Molecular pathways of apoptosis.
The intrinsic apoptotic pathway is initiated by internal sensors that monitor cellular stresses such as
viral infection via activation of BH3 domain–only members of the Bcl-2 family. Activated BH3-only
proteins are thought to mediate the assembly of pro-apoptotic members of the Bcl-2 family (Bax,
Bak, Bcl-rambo, Bok) into hetero-oligomeric "pores" in the outer membrane of the mitochondria; this
results in the release of factors such as cytochrome c, Smac (also known as Diablo) and Omi (also
known as HtrA2) into the cytoplasm. The loss of mitochondrial membrane integrity can be blocked
by the anti-apoptotic Bcl-2 family members Bcl-2, Bcl-xL, Bcl-w, Mcl-1 and Bcl-B. Release of
cytochrome c promotes formation of the apoptosome, which contains Apaf-1 and pro-caspase-9.
Autocatalytic activation of caspase-9 initiates the effector caspase cascade, which includes
caspase-2, -3, -6 and/or -7. Caspase activation is negatively regulated by the IAPs, which are
counter-balanced by the release of pro-apoptotic Smac and Omi from the mitochondria. The
extrinsic pathway of apoptosis is triggered by TNF family death ligands binding to their cognate
death receptors. Via their DDs, multimerized receptors interact with the DDs of adaptor proteins
such as FADD. These adaptor proteins also contain DEDs that facilitate their binding to procaspase-8 and/or pro-caspase-10 to form the DISC. As part of the DISC, the pro-caspases are
cleaved into their active forms and initiate the intrinsic pathway of apoptosis by cleaving Bid into tBid and activate the effector caspase cascade (caspase-3 is shown).
In contrast, the extrinsic pathway starts with members of the tumor necrosis factor (TNF)
superfamily of death receptors transmitting external signals provided by immune effector cells to the
virus-infected cell. Innate effector cells—for example, natural killer (NK) cells and dendritic cells
(DCs)—can mount rapid antiviral responses through direct detection of viral products by pattern
recognition receptors—such as binding of double-stranded RNA (dsRNA) by Toll-like receptor 3
(TLR3)—or by up-regulating death receptor ligands—for example, Fas ligand (FasL), TNF receptor–
related apoptosis-inducing ligand (TRAIL) and TNF2. Later during adaptive immunity, these same
death ligands are produced by antigen-specific cytotoxic T cells (CTLs).
The lethal program set in motion by these ligands mandates that their expression is transient and
highly regulated at the transcriptional and post-translational steps. Upon binding TNF-family
ligands, death receptors recruit adaptors and initiator caspases in a stepwise sequence based on
specific interaction domains and form the death-inducing signaling complex (DISC) at the plasma
membrane. In the case of Fas, the cytosolic adaptor FADD (Fas-associated death domain–
containing protein) contains two interaction motifs: a death domain (DD) that associates with the
homologous structure in Fas and a death effector domain (DED) that interacts with a homologous
DED in pro-caspase-8 or pro-caspase-10. The proximal positioning of pro-caspase-8 and procaspase-10 in the DISC is thought to lead to autocatalysis and conversion to an active enzyme.
Activated caspase-8 or caspase-10 can then directly convert pro-caspase-3 to its active form,
completing the initiation phase of the pathway. There is cross-talk between the death receptor
pathways and the mitochrondria-dependent arm through cleavage of the BH3-only protein Bid by
caspase-8. In addition, the cytotoxic granule–associated proteinase granzyme B can bypass
caspase-8 and, upon delivery by CTLs and NK effector cells, can directly cleave and activate
caspase-3. Caspase-3 and other executioner caspases cleave numerous substrates, such as ICAD
(inhibitor of caspase-activated DNase), which leads to genome fragmentation, collapse of the cell
and preparation of cellular remnants for phagocytosis. Thus, vertebrates have evolved several
distinct strategies to initiate apoptosis that lead to a common execution phase of the pathway.
All of the key pro-apoptotic components are preformed in the cell, enabling the pathway to respond
rapidly to apoptosis-inducing signals. In contrast, the cellular regulatory elements of the death
pathway often require new gene expression. The transcription factor NF- B controls several genes,
including those of the Bcl-2 and inhibitors of apoptosis (IAP) families, whose functions regulate
apoptosis and promote cell survival3. This is especially pertinent when a virus has compromised the
biosynthetic capacity of the host cell: death becomes the default outcome and, predictably, viral
counter-strategies have evolved that mimic the key cellular regulatory elements. The key regulatory
steps in the cell death pathway targeted by viral pathogens include control over the expression of
internal sensors, cytochrome c release by Bcl-2 family members, inhibition of caspases and
regulation of death receptor signaling. Indeed, the discovery of several of the key regulatory points
in the apoptotic pathway have emerged directly from studies of the death-escape mechanisms used
by viruses.
Blinding the sensors
The tumor suppressor p53 limits cellular proliferation by inducing apoptosis or cell cycle arrest in
response to cellular stresses and is intimately linked to cancer development. p53-deficient mice are
more prone to certain viral infections, indicating a role beyond tumor suppression4. Some of the
genes whose transcription is stimulated by p53 include those that encode the death receptors Fas
and TRAIL receptor 2 (TRAILR2), although the importance of their up-regulation by p53 in triggering
apoptosis is unclear. Additionally, p53 induces the transcription of genes such as Bax, Bak1, Bbc3
and Pmaip1 whose products are involved in death signal propagation through the mitochondria5-9.
p53 represses transcription of the anti-apoptotic protein Bcl-2, an antagonist of Bax and Bak 10.
Viruses can also disrupt apoptosis by inactivating p53. The SV40 large T antigen binds to p53 and
sequesters it in an inactive complex11, 12. The human papillomavirus E6 protein and adenovirus
E1B-55K protein, in concert with E4orf6, promote ubiquitination and degradation of p53, albeit via
different mechanisms13-16. Additionally, the pX protein encoded by hepatitis B virus complexes with
p53 and inhibits p53-mediated transcriptional activation as well as p53-dependent apoptosis17.
Mimicking Bcl-2
Viral encoded orthologs of the anti-apoptotic regulator Bcl-2 are a widely used immune evasion
strategy (Fig. 2). A pertinent example is adenovirus E1B-19K, which is similar both in sequence and
function to Bcl-218, 19. Unlike cell death signaled through cell-surface receptors, p53-mediated
apoptosis in response to adenovirus infection does not require the cleavage of Bid into t-Bid to
achieve Bax-Bak oligomerization and the subsequent release of cytochrome c. Instead, p53
stimulates a conformational change in Bax that is required for Bax-Bak interaction. These events
are blocked by adenovirus E1B-19K as a result of its binding to Bak and the abrogation of Bax-Bak
oligomerization20. E1B-19K also affects the apoptotic process signaled via TNF at the level of Bax
and Bak activation. Treatment of cells with TNF produces a death signal that results in the cleavage
of Bid to t-Bid followed by the recruitment of monomeric, pro-apoptotic Bax into a 500-kD protein
complex and the release of cytochrome c from the mitochondria. In the case of TNF-mediated
apoptosis, E1B-19K appears to interact primarily with Bax, inhibiting oligomerization and the
subsequent release of cytochrome c21.
High resolution image and legend (61K)
apoptotic pathway.
Figure 2. Viral regulation of the intrinsic
Cellular sensors, such as p53, that detect stress and initiate the apoptotic process are inactivated
by the proteins adenovirus E1B-55K and human papillomavirus E6. Several viruses encode
orthologs of Bcl-2 family proteins that antagonize their pro-apoptotic activity. Several viral strategies
target the caspases. The vIAPs contain conserved BIR domains and their expression may shift the
equilibrium of Smac to favor cell survival. Non-BIR–containing proteins, such as p35 and the
serpins (including CrmA), also inhibit caspase activation.
Oncogenic human herpesviruses use Bcl-2 orthologs to block mitochondrial release of cytochrome
c. This is also true for Epstein-Barr virus (EBV), which encodes two Bcl-2 orthologs (BHRF1 and
BALF-122, 23), and Kaposi's sarcoma–associated -herpesvirus (KSHV), which expresses KSbcl-224,
25. Mouse
-herpesvirus MHV-68, a virus that serves as a model for human EBV infection, encodes
a Bcl-2 ortholog (MHVBcl-2) that protects against TNF-mediated apoptosis in cell culture. In
addition, MHVBcl-2 is important for chronic infection, as demonstrated by the impaired virulence of
MHV-68 virus lacking the Bcl-2 ortholog in interferon- (IFN- )–deficient mice26. In contrast, the
human cytomegalovirus (CMV) UL37 gene product vMIA shares no sequence homology to Bcl-2;
however, it resides in the mitochondria and appears to be functionally similar to Bcl-2, as it
associates with the adenine nucleotide translocator and inhibits Fas-mediated apoptosis27.
Other viral proteins inhibit apoptosis by modulating Bcl-2 family members at the transcriptional level
or via post-translational modification. The human T cell leukemia virus type 1 (HTLV-1) Tax protein
transcriptionally activates the Bcl-xL promoter while repressing transcription of Bax28. HIV-1 Nef
mediates phosphorylation of the pro-apoptotic Bad protein, abrogating its activity and suppressing
apoptosis in T cells29. Similarly, the U(S)3 protein kinase encoded by herpes simplex virus 1 (HSV1) mediates a post-translational modification of Bad and blocks its cleavage and subsequent
activation of apoptosis30.
Caspase regulation
Caspases play a central role in apoptosis and are regulated in several ways 31-33. The enzymatic
activity of caspases is inhibited by a conserved family of inhibitor of apoptosis proteins (IAPs) 34, 35
that were originally defined in baculovirus based on the suppression of apoptosis and presence of a
zinc-binding motif called a BIR (baculoviral IAP repeat) 36. To date, eight cellular IAPs have been
identified that regulate both the effector and initiator caspases37. For example, XIAP, c-IAP1, c-IAP2
and ML-IAP (livin) target the initiator caspase, caspase-9, and the effector caspases, caspase-3
and caspase-7. XIAP, c-IAP1 and c-IAP2 contain three BIR domains, each with a different function.
The third BIR domain (BIR3) inhibits the activity of processed caspase-9, whereas the linker region
between BIR1 and BIR2 abrogates the activity of caspase-3 and caspase-7. An IAP-ortholog
strategy is used infrequently in mammalian viruses, with the exception of African swine fever virus,
which encodes a viral IAP (vIAP) that does not contribute to virulence 38. In mammals, the serpin
CrmA—which is derived from cowpox and is present in most poxviruses—also inhibits several
caspases, likely through covalent modification of caspase-8, and blocks or delays apoptosis in
response to TNF and Fas signaling or CTLs39-41. Also, CrmA inhibits caspase-1, a critical
processing enzyme for the inflammatory cytokine interleukin 1 (IL-1 ).
Modulation of TNFR signaling
Several members of the TNF receptor (TNFR) superfamily, including Fas, TNFR1 and TRAILR2,
are potent inducers of apoptosis42. Not surprisingly many viruses specifically target these cytokine
receptors (Fig. 3). Neutralization of TNF by soluble decoy receptors was one of the first-described
evasion tactics, as shown by the secreted TNFR2 ortholog expressed by Shope fibroma virus
(rabbit poxvirus)43. Several TNFR orthologs have been identified in the genomes of lepri- and
orthopoxviruses, including smallpox, which indicates its impact on the success of poxviruses 44. The
T2 protein of myxoma virus is a dimeric, high-affinity binding protein for TNF and virulence is
attenuated when it is deleted from the viral genome45. Interestingly, the TNFR decoys in vaccinia,
the attenuated form of smallpox, are mutated. Additionally, the poxviral TNFR ortholog T2 exists in
an intracellular form that is required to inhibit apoptosis of lymphocytes 46. Another TNFR ortholog is
found in avian leukocytosis virus, which encodes an ortholog of TRAILR2 that serves as a virus
entry factor47. Additionally, human CMV (but not mouse CMV) contains a TNFR ortholog encoded
by the UL144 orf, although its functional significance remains obscure 48.
High resolution image and legend (52K)
apoptotic pathway.
Figure 3. Viral regulation of the extrinsic
Poxviruses block signaling via TNF and lymphotoxin by producing soluble decoy receptors (for
example, myxoma virus T2 protein). vFLIPs contain two DEDs that interact with the homologous
DED of FADD and pro-caspase-8 or pro-caspase-10, blocking caspase activation and inhibiting
death receptor–induced apoptosis. The human CMV protein vICA binds and inhibits pro-caspase-8
activation, but lacks a DED. The EBV protein LMP1 self-aggregates and engages TRAF and
TRADD molecules, activating "anti-apoptotic" NF- B– and Jnk-dependent pathways. A
heterocomplex of proteins encoded by the E3 region of adenovirus (E3-10.4K, E3-14.5K and E36.7K) facilitates the removal of the death receptors Fas, TRAILR1 and TRAILR2 from the surface of
infected cells. Consequently, receptors accumulate in late endocytic compartments, and cells are
desensitized to killing.
Herpes simplex virus 1, through its envelope glycoprotein D, uses a TNFR family member,
herpesvirus entry mediator (HVEM), to gain access to the lymphoid compartment, where it can
induce apoptosis of T cells49 and block maturation of antigen-presenting DCs50. The major B cell–
transforming protein in EBV, LMP1, behaves like a constitutively activated CD40 by engaging
TRAFs and TNFR1-associated DD protein (TRADD), which are adaptors used by TNFR to activate
the transcription of anti-apoptotic genes through NF- B and c-Jun NH2-terminal kinase (Jnk)dependent signaling pathways. In a transgenic model, LMP1 expression prevents B cells from
localizing to the follicle, thus protecting cells harboring latent virus from interactions with T cells 51.
Whereas poxviruses inhibit TNF ligand-receptor interactions by deploying a soluble receptor smoke
screen, a distinct strategy is used by adenoviruses. The E3 region encodes several proteins that
sweep the cell surface clear of the death-inducing receptors Fas, TRAILR1 and TRAILR2. The E3
proteins responsible for modulating cell surface amounts of Fas are E3-10.4K and E3-14.5K52, 53,
which localize to various cellular membrane compartments, including the plasma membrane, as a
heteromeric complex54. However, the ability of adenovirus to modulate TRAILR2 absolutely requires
a third E3 protein, E3-6.7K, in addition to E3-10.4K and E3-14.5K55; this highlights the complexity of
the mechanisms used by adenoviruses to inhibit signaling by these death receptors. Upon E3induced down-regulation, death-inducing receptors accumulate in late endocytic compartments,
resulting in the desensitization of infected cells to killing by FasL and TRAIL53, 55.
Prevailing wisdom is that the E3-10.4K–E3-14.5K–E3-6.7K complex pirates the cellular endocytic
compartments that direct membrane protein trafficking. A similar strategy is used by the HIV Nef
protein, which down-modulates CD4 and major histocompatibility complex (MHC) class I from the
cell surface by cross-linking these proteins to the cellular endocytic machinery56. As a consequence
of Nef's actions, the membrane forms of TNF and the related cytokine LIGHT show sustained
expression on the surface of T cells, potentially contributing to the cytopathic effects of HIV on the T
cell compartment57. The substantial death of bystander (uninfected) lymphocytes during HIV
infection may stimulate lymphopoiesis, possibly by homeostatic control mechanisms, which could
provide a pool of dividing (but not HIV-specific) T cells for HIV genome replication58.
Viruses can down-modulate or enhance expression of death receptors and ligands to their own
advantage. The loss of death receptors may function to protect infected cells from cytolysis by CTLs
or NK cells, which express FasL or TRAIL upon activation59. In addition, this down-regulation may
help inhibit apoptosis mediated by neighboring cells that are induced to express death receptor
ligands upon infection. Induction of TRAIL has been observed in HCMV-50, 60 and reovirus-infected61
cells and of FasL by HSV in T cells and HCMV in DCs49, 50. It has even been proposed that this
induction of FasL and TRAIL is another viral immune evasion tactic, through the killing of infiltrating
host CTLs and DCs49, 50. IFNs induce TRAIL in various cell types60, 62, 63; this suggests that a
possible mechanism for up-regulation of TRAIL could be via production of IFNs by the virus-infected
cell.
Several viruses have evolved a different strategy for blocking death receptor signaling at the level of
DISC assembly; they do this through blockade of caspase-8 and caspase-10 processing. The viral
FLICE (caspase-8) inhibitory proteins (FLIPs) contain DEDs, but lack caspase activity, and are
present in the genomes of various -herpesviruses, including equine herpesvirus 2 (EHV-2),
herpesvirus saimiri (HVS), KSHV, bovine herpesvirus 4 (BHV-4) and moluscum contagiosum virus
(MCV)64, 65. The cellular ortholog of vFLIP was subsequently cloned after identification of the
vFLIPs65 and exists in both a 26-kD short (cFLIPS) and 55-kD long (cFLIPL) form generated by
alternative splicing. cFLIPS is essentially the cellular ortholog of vFLIP and encodes two DEDs,
whereas cFLIPL encodes an additional COOH-terminal domain with high homology to caspase-8
and caspase-10. Both isoforms of cFLIP are recruited to the DISC and inhibit death receptor–
induced apoptosis; however, their mechanisms of action are slightly different: cFLIP S completely
inhibits proteolytic processing of caspase-8 (similar to vFLIP) and cFLIPL allows partial caspase-8
processing66. The HCMV UL36 gene product vICA also associates with caspase-8 and blocks its
activation, but shows no sequence identity to its proposed cellular orthologs, the FLIPs67.
vFLIPs interact with adaptor proteins that regulate the expression of NF- B, including TNFRassociated factor 2 (TRAF2), receptor-interacting protein (RIP), NF- B–inducing kinase (NIK) and
the inhibitor of B-kinase 2 (IKK2)68, which indicates that the vFLIPs may also regulate activation of
transcription factors important in inhibiting apoptosis.
Death taxes
Cell death comes at a potentially large cost for the host. However, it is becoming appreciated that
IFN-dependent nonapoptotic mechanisms can result in the successful attenuation of viral spread 69.
IFNs play a critical role in mounting innate responses to viral infection. IFNs are typically considered
nonapoptotic but, in some circumstances, can promote apoptosis of virus-infected cells and are
especially potent when they act in concert with TNF-related ligands. IFNs signal via the Janus
protein kinase–signal transducers and activators of transcription pathway (Jak-STAT)70. Well
studied IFN-inducible signaling pathways include the RNA-dependent protein kinase (PKR)71 and
the 2',5-oligoadenylate and RNase-L systems72. Upon interaction with virus-derived dsRNA, PKR is
activated and can inhibit host-cell translation via phosphorylation of elongation initiation-factor 2
(eIF2 )73. More recently, it has been shown that PKR has additional activities, including the
modulation of NF- B74 and promotion of apoptosis by functional enhancement of the tumor
suppressor genes p53 and IFN response factor 1 (IRF-1)75. The regulation of NF- B by PKR places
this protein at a critical crossroad in the coordination of apoptotic signals, potentially through the
NF- B–dependent induction of FasL and IFN-induced TRAIL.
Several noncytolytic antiviral programs activated by IFNs can arrest the viral life cycle at different
steps, thereby attenuating virus spread and limiting the infection. However, IFNs and their response
genes are themselves targeted by many viruses76. Replication-competent HCMV specifically inhibits
induction of IFN- transcription by an unknown mechanism 77 that may involve inhibition of IRF-3
activation78. This blockade of IFN- transcription is overridden by signaling via TNFR1 or the
lymphotoxin receptor (LT R), but not by Fas or TRAIL receptors79. Induction of IFN- by these
two TRAF-adapting receptors is NF- B–dependent, but occurs only in HCMV-infected cells.
Therefore, host and virus factors cooperate to induce IFN- , establishing a state of détente in which
the host cell survives and the viral genome persists, but cannot produce new virions. This differs
from the case of hepatitis B, where IFN signaling results in protease-dependent clearance of the
viral genome69. These two examples highlight the virus- and cell type–specific issues often seen
with IFN's antiviral action.
The diversity of strategies used by viruses to modulate the apoptotic pathway is as varied as there
are viruses. In addition, for each virus, these strategies are integrated into a wider scheme that
manipulates other aspects of host defenses. For example, herpesviruses have large DNA genomes
that have accumulated an extensive repertoire of immune-evasion tactics that target both the
afferent (for example, antigen recognition) and effector phases (apoptosis) of host defenses80. The
cumulative effect of these mechanisms is thought to contribute to the ability of herpesviruses to
sustain life-long infection. As a cautionary note, nearly all the data gathered to date are on clinically
important viruses, which unfortunately often lack comparable animal models with which we can
assess the role of immune-modulatory genes in viral pathogenesis. Indeed, there are significant
differences in the molecular mechanisms used to modulate immune function by mouse CMV
compared with those of human CMV. It is also important to recognize that a particular viral evasion
strategy may act differently in a non-native host compared to in tissue culture, which also
contributes to the difficulty in defining mechanisms of action. Nonetheless, the elucidation of
evasion strategies directed at apoptotic mechanisms can provide deeper insight into the host-virus
relationship that hopefully will yield better vaccine strategies.
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Figure 1. Molecular pathways of apoptosis. The intrinsic apoptotic pathway is
initiated by internal sensors that monitor cellular stresses such as viral infection via
activation of BH3 domain–only members of the Bcl-2 family. Activated BH3-only
proteins are thought to mediate the assembly of pro-apoptotic members of the Bcl2 family (Bax, Bak, Bcl-rambo, Bok) into hetero-oligomeric "pores" in the outer
membrane of the mitochondria; this results in the release of factors such as
cytochrome c, Smac (also known as Diablo) and Omi (also known as HtrA2) into
the cytoplasm. The loss of mitochondrial membrane integrity can be blocked by the
anti-apoptotic Bcl-2 family members Bcl-2, Bcl-xL, Bcl-w, Mcl-1 and Bcl-B. Release
of cytochrome c promotes formation of the apoptosome, which contains Apaf-1 and
pro-caspase-9. Autocatalytic activation of caspase-9 initiates the effector caspase
cascade, which includes caspase-2, -3, -6 and/or -7. Caspase activation is
negatively regulated by the IAPs, which are counter-balanced by the release of proapoptotic Smac and Omi from the mitochondria. The extrinsic pathway of apoptosis
is triggered by TNF family death ligands binding to their cognate death receptors.
Via their DDs, multimerized receptors interact with the DDs of adaptor proteins
such as FADD. These adaptor proteins also contain DEDs that facilitate their
binding to pro-caspase-8 and/or pro-caspase-10 to form the DISC. As part of the
DISC, the pro-caspases are cleaved into their active forms and initiate the intrinsic
pathway of apoptosis by cleaving Bid into t-Bid and activate the effector caspase
cascade (caspase-3 is shown).
Figure 2. Viral regulation of the intrinsic apoptotic pathway. Cellular sensors,
such as p53, that detect stress and initiate the apoptotic process are inactivated by
the proteins adenovirus E1B-55K and human papillomavirus E6. Several viruses
encode orthologs of Bcl-2 family proteins that antagonize their pro-apoptotic
activity. Several viral strategies target the caspases. The vIAPs contain conserved
BIR domains and their expression may shift the equilibrium of Smac to favor cell
survival. Non-BIR–containing proteins, such as p35 and the serpins (including
CrmA), also inhibit caspase activation.
Figure 3. Viral regulation of the extrinsic apoptotic pathway. Poxviruses block
signaling via TNF and lymphotoxin by producing soluble decoy receptors (for
example, myxoma virus T2 protein). vFLIPs contain two DEDs that interact with the
homologous DED of FADD and pro-caspase-8 or pro-caspase-10, blocking
caspase activation and inhibiting death receptor–induced apoptosis. The human
CMV protein vICA binds and inhibits pro-caspase-8 activation, but lacks a DED.
The EBV protein LMP1 self-aggregates and engages TRAF and TRADD
molecules, activating "anti-apoptotic" NF- B– and Jnk-dependent pathways. A
heterocomplex of proteins encoded by the E3 region of adenovirus (E3-10.4K, E314.5K and E3-6.7K) facilitates the removal of the death receptors Fas, TRAILR1
and TRAILR2 from the surface of infected cells. Consequently, receptors
accumulate in late endocytic compartments, and cells are desensitized to killing.
Focus on Immune Evasion
Volume 3 No 11 November 2002
Review
Nature Immunology 3, 1019 - 1025 (2002)
doi:10.1038/ni1102-1019
© Nature America, Inc.
<>
Viral interference with antigen presentation
Jonathan W. Yewdell1 & Ann B. Hill2
1
2
Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, Bethesda, MD, USA.
Department of Molecular Microbiology and Immunology, Oregon Health and Science University, Portland, OR, USA.
Correspondence should be addressed to J W Yewdell [email protected]
CD8+ T cells play an important role in immunity to viruses. Just how important these cells
are is demonstrated by the evolution of viral strategies for blocking the generation or display
of peptide–major histocompatibility complex class I complexes on the surfaces of virusinfected cells. Here, we focus on viral interference with antigen presentation; in particular we
consider the importance (and difficulty) of establishing the evolutionary significance (that is,
the ability to enhance viral transmission) of viral gene products that interfere with antigen
presentation in vitro.
Viruses are the ultimate obligate intracellular parasites. Lacking virtually all the machinery
necessary for their own replication, they consist of a fragment of nucleic acid (encoding anywhere
from less than ten to several hundred proteins) enclosed in a protein or proteolipid shell with just
enough of the right polymerases to initiate gene expression ("bad news wrapped in protein",
according to Peter Medawar). For viruses to survive in nature, they must devise a means to be
transmitted between hosts. This, not dissemination within a host, is their sole evolutionary selection
factor, although dissemination is usually a prerequisite for transmission. At a bare minimum, viruses
must encode information to enable regeneration of their structural proteins. Because hosts are
generally hostile to sharing their resources with viruses, viruses need to be a bit cleverer than this.
Virus versus host: role of CD8+ T cells
Through various mechanisms, host cells may sense the presence of a virus and attempt to trigger
apoptosis to preclude viral replication. Viruses can counter these attempts (see Review by Ware
and colleagues in this issue). The presence of unusual nucleic acids produced in the early stages of
viral replication (for example, double-stranded RNA) triggers protein kinase R (PKR), which induces
an antiviral state in the cells and synthesis and release of interferons (IFNs). IFNs signal the
presence of virus to the cellular immune system, whose initial response consists of natural killer
(NK) cells. Some viruses have devised means for evading NK cell recognition (see Review by
Strominger and colleagues in this issue).
The second wave of the cellular immune counterattack consists of CD8 + T cells, which are activated
via the presentation of viral antigens by professional antigen-presenting cells (APCs). The
contribution of CD8+ T cells to antiviral immunity has been extensively demonstrated in mouse
model systems. Starting with experiments in the late 1960s, T cells (which were later shown to be
CD8+) were demonstrated to be responsible for the recovery of mice from acute mousepox
(ectromelia) infection1-3. Subsequently, we have learned that CD8+ T cells are important in mouse
immunity to many viruses. CD8+ T cells exert antiviral effects via the localized secretion of
molecules in close vicinity to the virus-infected APC (professional or not). Many CD8+ T cells kill
APCs by releasing perforin and granzymes (if APCs express Fas, engagement by Fas ligand on
CD8+ T cells can also induce lysis). In addition, nearly all CD8+ T cells secrete IFN- and tumor
necrosis factor- (TNF- ) (a small percentage of cells secrete IFN- only), which induces a potent
antiviral state in cells.
It is important to emphasize that CD8+ T cells are but one of many weapons deployed by the
immune system to combat viruses. NK cells, CD4+ T cells and antibody all participate in antiviral
immunity. Mice deficient in CD8+ T cell responses effectively handle infections with many viruses,
although they do succumb to others, such as ectromelia. Direct demonstration of the role of CD8+ T
cells in human antiviral immunity is more difficult to establish. On the one hand, adoptively
transferred virus-specific CD8+ T cells are effective against human cytomegalovirus (HCMV) 4, HIV15 and Epstein-Barr virus (EBV)6. On the other hand, T cell–deficient individuals seem to do quite
well with common viral infections. There is a single hereditary condition that selectively interferes
with CD8+ T cell induction: absence of functional transporter associated with antigen processing
(TAP) (described below). Curiously, afflicted individuals suffer from bacterial, not viral, infections 7.
These patients show only a partial reduction in CD8+ T cells, however, and the residual CD8+ T cell
activity may be sufficient to handle common viral infections. Evolutionary selection for individual
elements of the immune system may be punctuated: for example, the sporadic appearance of a
potentially lethal pathogen would be sufficient to maintain an immune effector mechanism that
reduces its lethality. It is plausible, for example, that variola virus (the agent of smallpox) would
display a greatly increased mortality rate in TAP-deficient individuals.
Perhaps the best evidence for the importance of CD8+ T cells in human immunity to viruses is the
lengths that some human viruses have gone to interfere with antigen presentation, which is the
principal focus of our review.
Antigen-presentation primer
To enhance nonspecialists' comprehension of the area of antigen processing and presentation we
shall briefly discuss how viral proteins are processed for recognition by virus-specific CD8+ T cells8,
9. The specificity of CD8+ T cells is conferred by the clonally restricted T cell receptor (TCR) that
interacts with residues from both major histocompatibility complex (MHC) class I molecules and an
oligopeptide (>90% are 8–11 amino acids in length) encoded by a viral gene. Peptides are
predominantly generated from the byproducts of proteasomal degradation (Fig. 1). Most of the
substrates consist of defective ribosomal products (DRiPs) that are degraded within 30 min of their
synthesis10. This process enables the rapid recognition of viral proteins early in infections, when
viral proteins represent a tiny fraction of total cell proteins. Peptides are transported into the
endoplasmic reticulum (ER) by the TAP protein. Here, MHC class I molecules are folded through
the concerted actions of general purpose molecular chaperones working with a dedicated
chaperone (tapasin) that tethers MHC class I to TAP. Upon peptide binding, MHC class I molecules
dissociate from TAP, leave the ER and make their way to the plasma membrane via the Golgi
complex.
High resolution image and legend (78K)
Figure 1. The classical MHC class I pathway is
depicted with reference to viral interfering proteins.
Oligopeptides (red circle) are derived from DRiPs through the action of proteasomes. Nascent MHC
class I molecules—consisting of a heavy chain and 2-microglobulin ( 2M)—bind to TAP via
tapasin. Peptide binding releases MHC class I to the cell surface. VIPRs interfere with this process
at multiple steps. (1) EBV EBNA-1 contains a sequence that renders it resistant to proteasomal
degradation. HCMV IE is phosphorylated by another viral protein, preventing generation of the
immunodominant peptide epitope. (2A) HSV ICP47 and a BHV1 protein bind to the cytosolic side of
TAP and prevent peptide translocation. (2B) HCMV US6 binds to TAP in the ER lumen and
prevents peptide translocation. (2C) Several VIPRs bind to MHC class I in the ER, retaining it
and/or interfering with the function of the peptide-loading complex. The proteins include AdE319K
(retains MHC class I and also prevents tapasin-mediated docking with TAP); HCMV US3 (binds
MHC class I but dissociates, the mechanism of MHC class I retention is not clear but may involve
repeated rebinding by newly synthesized US3). MCMV m4/gp34 forms extensive complexes with
MHC class I in the ER. (3) HCMV US2 and US11 and MHV-68 K3 bind MHC class I in the ER and
induce retrotranslocation for degradation by the proteasome. For US11, ubiquitylation of heavy
chain cytoplasmic tail, possibly after retrograde translocation, has been described. MHV-68 K3
ubiquitylates the tail before translocation. HIV-1 Vpu also induces degradation of newly synthesized
MHC class I—probably via retrograde translocation and proteasome degradation—although, unlike
US2 and US11, proteasome activity is required for retrograde translocation. (4) MCMV m152/gp40
causes MHC class I to be retained in the ER cis-Golgi complex intermediate compartment (ERGIC).
The fact that m152, itself a distant MHC class I homolog, can reduce cell surface expression of H60 as well as classical MHC class I (in an allomorph-specific manner) makes its poorly understood
mechanism of action of particular interest. Only the lumenal domain of m152/gp40 is required for
the retention of MHC class I. Binding of m152/gp40 to MHC class I has not been demonstrated, and
m152/gp40 itself has a default pathway of export to and degradation in the lysosome. (5) Several
VIPRs remove MHC class I either from the Golgi or the cell surface. MCMV m6/gp48 contains a
lysosomal targeting di-leucine motif in its tail; it binds directly to MHC class I and redirects it to the
lysosome. HIV and SIV Nef use the PACS-1 (phosphofurin acidic cluster sorting protein 1)–sorting
protein to remove MHC class I from the cell surface and sequester it in the trans-Golgi network.
KSHV K3 and K5 ubiquitylate the MHC class I tail and target MHC class I to lysosomes in a TG101dependent manner. K5 also targets ICAM-1 (intercellular adhesion molecule 1) and B7-2. (6)
MCMV m4/gp34 is found associated with MHC class I at the cell surface and inhibits CTL
recognition, but cause and effect have not been firmly linked.
As peptide-MHC (pMHC) class I complexes accumulate at the cell surface, they have a greater
chance of triggering activation by CD8+ T cells with a cognate receptor. The half-life of cell surface
complexes depends on the koff of the bound peptide; immunogenic peptides generally dissociate
with half-lives in the range of hours to hundreds of hours. There is no simple relationship between
complex stability or abundance and the magnitude of the cognate CD8 + T cell response11.
It is important to distinguish naïve CD8+ T cells from armed effector CD8+ T cells. Naïve CD8+ T
cells can only be activated by cells expressing the proper costimulatory molecules, that is, by
professional APCs. Dendritic cells (DCs) are believed to be the principal APCs for activating naïve
CD8+ T cells, although the evidence is largely circumstantial. Effector CD8 + T cells have no such
limitation and require only recognition of a cognate complex for activation, although expression of
adhesion molecules can decrease the number of complexes required. Effector CD8 + T cells
circulate "ready to kill" with preformed perforin and granzymes, but only express IFN- and TNFupon activation by interaction with a virus-infected APC.
Viral strategies for circumventing CD8+ T cells
In principal, viruses can thwart CD8+ T cell function by interfering with the activation of naïve cells,
CD8+ T cells trafficking to infected cells, antigen presentation to effector CD8+ T cells or CD8+ T
cell–mediated effects on virus-infected cells. For viruses that are capable of infecting CD8+ T cells,
destroying responding CD8+ T cells would seem to be a simple strategy. This mechanism has been
described in vitro (but not demonstrated in vivo) for herpes simplex virus (HSV)12. Viruses with this
ability are highly unusual, which is interesting, inasmuch as it suggests that hosts are capable of
modulating the antiviral state in a cell type–specific manner, with CD8+ T cells being particularly
difficult to infect. Blocking activation of naïve CD8+ T cells can be achieved by preventing APC
antigen presentation. Many viruses are capable of infecting DCs, and there have been a number of
reports that viruses interfere with DC-mediated CD8+ T cells activation in vitro13-16. This is an area,
however, with a potentially enormous gap between in vitro phenomenology and in vivo reality.
There is no certain relationship between DCs propagated ex vivo and immature DCs residing in the
tissue or activated DCs that have migrated to draining lymph nodes once activated by viral infection.
Viral infections generally result in a gross alteration of host cell functions over time. The conditions
of infection in vivo and in vitro can differ in terms of the number of viral particles that initiate the
infection. Even if a virus-induced decline in antigen presentation measured in vitro accurately
reflects the in vivo situation, there could be sufficient residual presentation to enable CD8 + T cell
activation in vivo. For example, whereas the antigen-presenting function of DCs in vitro is adversely
affected by infection with vaccinia virus (VV)15, VV-infected DCs can be visualized presenting
antigens to CD8+ T cells in lymph nodes; this provides the most direct evidence, to date, that DCs
function as APCs in viral infections17.
Numerous poxviruses and herpesviruses encode molecules with known or likely effects on
chemokine function. As chemokines direct trafficking of CD8+ T cells to the sites of inflammation,
these could influence the effectiveness of CD8+ T cells. A secreted chemokine ligand homolog,
murine herpes virus 68 (MHV-68) M3, has been implicated as affecting CD8+ T cell function18. Many
viruses induce an anti-apoptotic state that should hinder the cytolytic activity of CD8+ T cells.
Resistance to the effects of IFN- and TNF- is also a common viral strategy19, 20.
Viruses block the presentation of endogenous antigens by APCs by expressing VIPRs (viral
proteins interfering with antigen presentation, pronounced "viper", with an etymological nod to
herpesviruses). Numerous VIPRs have been described, and they target virtually all steps in the
antigen-processing and -presentation pathway (Fig. 1). This appears to be the principal means by
which viruses interfere with CD8+ T cell function, and it is certainly the best characterized. For these
reasons we will focus on VIPRs, but not their cell biology and mechanisms of function, which have
been the subject of detailed reviews19-21. Instead, we will deal primarily with the in vivo function of
VIPRs, a topic ripe for review.
Cross-priming: a hurdle to VIPR function
Viruses that block CD8+ T cell activation by expressing VIPRs in APCs face a potentially
insurmountable hurdle in cross-priming22. In this process, DCs (and macrophages) internalize
antigen from infected cells and reprocess it in the cytosol or endosomal compartment for
presentation on their own MHC class I molecules. The nature of the cross-priming material is
unknown at present, but it is likely to represent a mixture of phagocytosed intact dying cells and cell
debris containing intact and partially degraded proteins, some complexed with sundry molecular
chaperones. Because, by definition, cross-priming APCs are not infected, viruses can only impose
global restrictions on antigen presentation by releasing soluble inhibitory factors from infected cells.
In lieu of this, the best they can do is to evolve proteins that cannot be processed by cross-priming,
but even this appears to be rather difficult. EBV nuclear antigen 1 (EBNA-1) is not processed and
presented by infected cells due to its indigestibility by proteasomes23, yet EBNA-1–specific CD8+ T
cells appear to be generated in vivo by cross-priming24.
Cross-priming is a robust process that may have evolved in response to viral interference with CD8 +
T cell activation. There is now solid evidence for cross-priming in a number of viral systems25-28.
Ultimately, the most compelling evidence for the effectiveness of cross-priming may be the vigorous
responses to viruses that are loaded with genes encoding proteins capable of interfering with the
generation of pMHC class I complexes. A murine CMV (MCMV) determinant whose presentation
was completely abrogated by m152 in vitro was equally immunodominant in mice infected with wildtype virus or m152-deficient viruses29. This strongly suggests that cross-priming contributes to the
generation of CD8+ T cells specific for this determinant. Similarly, the enigma that CD8+ T cells
specific for immediate early protein (IE) are readily generated in HCMV-infected individuals when
antigen presentation of IE is efficiently prevented in HCMV-infected cells30 is probably explained by
cross-priming. Cross-priming can occur by either the classical cytosolic processing route or via the
alternative endosomal pathway whose details are just beginning to appear from the mists 31. An
important question regarding the endosomal route is the extent to which the peptides it generates
overlap with those generated by the cytosolic route, particularly because distinct proteases are
involved.
Herpesviruses: a VIPR catalog
Although known VIPRs are encoded by retroviruses and adenoviruses, herpesviruses are the clear
champions in the evolution of VIPRs. Herpesviruses encode proteins that interfere with virtually
every step of antigen processing and presentation (Fig. 1); indeed, individual family members
encode multiple VIPRs (HCMV encodes at least four). It seems worthwhile to pause to consider the
characteristics of this family of viruses that are so hell-bent on interfering with CD8+ T cell function.
Herpesviruses are large double-stranded DNA viruses that replicate their genomes with high fidelity
compared to RNA viruses (for example, HIV or influenza virus). Herpesviruses fall into three
subfamilies. -herpesviruses are neurotropic: HSV and varicella zoster (VZV, the cause of
chickenpox and shingles) are the prototypes. -herpesviruses are ubiquitous, highly speciesspecific and cause minimal or no disease in immunocompetent hosts. They include CMVs and
human herpesvirus 6 (HHV6) and HHV7. -herpesviruses fall into two subgroups: the 1 prototype
is EBV (the cause of infectious mononucleosis, Burkitt's lymphoma and nasopharyngeal carcinoma)
and the 2 viruses include Kaposi's sarcoma herpesvirus (KSHV) and the important model virus
MHV-68. Unique among the herpesviruses, -herpesviruses encode genes that are only expressed
during latency. They also encode oncogenes and are associated with malignancy.
Herpesviruses are the most ancient known mammalian viruses and are extremely successful in
evolutionary terms. Many family members infect a high percentage of individuals of their host
species for the lifetime of the individual. Although the cell types they infect are diverse,
herpesviruses share the ability to establish latent infection. True viral latency means that the virus
can exist in host cells without reproducing itself, in contrast to persistence, when basal viral
replication continues (the distinction is imperfect). Herpesviruses reactivate and replicate in a fully
immune host, which enables their transmission to children, the next generation of hosts.
Herpesviruses live on the edge: they depend on immune control for their host's (and hence their
own) survival, yet must impair immunity to avoid eradication. Severely immunocompromised
patients often succumb to reactivated herpesvirus infections (especially CMV). The stability of the
host-virus relationship shows that this is a robust and well-buffered equilibrium, only rarely does the
host or the virus gain the advantage. This equilibrium is generally maintained only when
herpesviruses infect their natural host. For example, the monkey virus herpes B virus causes an
unapparent infection in monkeys but kills 70% of the people who are infected.
Dissecting the complex relationship between herpesviruses and the immune system depends on
mouse models, where it is possible to genetically alter both virus and host. The best characterized
mouse herpesviruses are MCMV, a pathogen that is ubiquitous in wild mice, and MHV-68, originally
isolated from voles but nevertheless able to infect and establish latent infection in laboratory mice.
Immune control of MCMV is complex. Both type I IFN and IFN- and NK cells play major roles in
containing primary infection32, 33, yet acute infection is invariably fatal in the absence of B and T cell
responses. Antibodies, NK cells, CD8+ T cells and CD4+ T cells all contribute to preventing
dissemination of reactivated latent virus. Reactivation in antibody-deficient mice requires depleting
any two of these three effector cell populations34. For the 2 herpesvirus MHV-68 the situation is
complicated further by a biphasic acute infection and limited understanding of the genetic program
of latent infection. Again, all components of the immune response are involved in host defense 35.
After intranasal infection, MHV-68 replicates acutely in the lungs and disseminates to the spleen.
This first phase of infection is controlled primarily by type I IFN, with some help from CD4 + and
CD8+ T cells. A second (mononucleosis-like) phase of the infection ensues, characterized by a
massive CD4+ T cell–dependent splenic B cell expansion and increase in viral load. Various viral
transcripts can be detected, but infectious virus cannot be recovered, so virus at this stage is
considered latent. This phase is gradually controlled by antibodies and both T cell subsets. Finally,
a poorly characterized true latency is established, during which depleting both T cell subsets does
not lead to virus reactivation.
Acute viruses: VIPR-free?
Viruses that are transmitted via acute infections are not known to encode VIPRs. This cannot be
easily dismissed as evolutionary incompetence. HSV-encoded ICP47 is the "poster" protein for how
easy blocking presentation can be. This highly effective VIPR comprises only 87 amino acids (even
a 32-residue fragment is highly active). It would be trivial for even small viruses with severe nucleic
acid packaging constraints to encode something similar (this could easily be accommodated in an
overlapping reading frame, requiring no increase in genome size). This suggests that blocking
antigen presentation to CD8+ T cells is useful only under a highly restricted set of circumstances.
Perhaps blocking MHC class I expression greatly sensitizes cells for NK recognition, and the costs
of countering both CD8+ T cells and NK cells are just too steep for acute viruses. Or perhaps very
acute viruses are transmitted too rapidly for CD8+ T cells to have an impact on their evolution.
MHC class II VIPRs
Interference with the MHC class II antigen-processing pathway has been described for several
herpesviruses36-38. As with cross-priming of CD8+ T cells, MHC class II VIPRs are not expected to
block the presentation of exogenous antigens to CD4+ T cells, as—by definition—the APC is not
infected and will not therefore express the VIPR. Thus, as with MHC class I VIPRs, class II VIPRs
would have to act in vivo by interfering with CD4+ T cell–mediated clearance of virus-infected cells.
For viruses whose transmission entails infection of MHC class II–expressing cells, this is certainly
possible, but the physiological relevance of MHC class II VIPRs remains to be established.
Establishing VIPR function
Physiological relevance is, of course, the ultimate measure of VIPR function. Demonstrating that a
viral protein can interfere with some aspect of antigen presentation or CD8+ T cell activation in vitro
is not synonymous with equivalent in vivo function. There are examples of individual VIPRs that
interfere with the function of numerous host proteins in cultured cells, and it is possible (even likely)
that some of the interactions are not important (admittedly, disproving something is difficult). The
degree of skepticism regarding the relevance of in vitro findings should reflect the deviation from
natural circumstances, for example, when transfection is used to achieve expression that vastly
exceeds that seen in infected cells39. Such approaches reveal potential functions, but eventually
must be reinforced by evidence obtained from animal experiments.
We do not intend to demean the value of in vitro studies. Some VIPRs act with such efficiency in
cultured cells (interference with TAP function by HSV ICP47 protein is the best example), as to
make it exceedingly unlikely that the interaction is insignificant. Establishing VIPR function in vivo is
never easy. For human viruses, often the best that can be achieved is demonstrating interference
with CTL recognition of virus-infected cells in vitro. This has been shown for some VIPRs, notably
HIV Nef40 and HCMV pp6530. With few exceptions, human viruses do not naturally infect other
species, so it is simply not possible to rigorously study their VIPRs under natural circumstances.
VIPRs can demonstrate similar interactions with homologous targets from different species: for
example, AdE319K and HCMV US2 and US11 down-regulate mouse MHC class I, but HSV ICP47
does not. When a VIPR does function in mice, it may be possible to establish an in vivo function for
a given gene product from a human virus, but again caution is necessary. No matter how the VIPR
is expressed, the conditions of expression will differ from the natural situation in a manner that can
profoundly affect its function. Variables include the amount of expression, cell type and a lack of
other appropriate viral gene products. This basically limits thorough investigation of VIPRs to those
expressed by natural animal pathogens. Given the vast superiority of mice as an experimental
system, this puts the focus on mouse viruses, although the importance of the AIDS epidemic has
made the rhesus macaque model of SIV an important exception.
The basic rules for establishing the in vivo function of VIPRs (and other immunomodulatory
proteins) were spelled out in a report by Koszinowski and colleagues 41 (extending a previously
pioneered approach to demonstrating the function of a viral complement–interfering protein42). First,
disabling the gene reduces the fitness of the mutant virus in vivo without affecting its ability to
replicate in tissue culture. Ideally, fitness is defined by transmission. This is technically difficult to
determine (for some viruses, well nigh impossible), so viral titers are taken as a surrogate. Second,
reinserting the gene into the mutant virus (generating a "rescuant") restores reproductive fitness.
This step is needed to ensure that other alterations in the mutant virus inadvertently introduced
during its generation are not responsible for the phenotype. Small viruses can be completely
sequenced, but this remains impractical for herpesviruses and other large viruses. Third, the fitness
of the mutant virus is restored by interfering with CD8+ T cell function, either with an appropriate
genetically deficient mouse (TAP-deficient, for example) or by treating mice with a CD8+ T cell–
depleting antibody.
Just as Koch's postulates (KPI) for demonstrating the cause of transmissible diseases cannot
constitute proof (in the mathematical sense of the word) that a given agent is responsible for a
given disease, fulfilling Koszinowski's postulates (KPII) does not guarantee that a gene product
evolved to perform the proposed function. At the very least, the following caveats need to be
considered.
Even the simple proposition that removing a gene should reduce fitness and reveal its function is
problematical. If there is true redundancy in either the viral genes or the host defense mechanisms
(or in both) such an effect may be difficult to demonstrate. Multifunctional viral genes (which are
common) raise obvious problems. Here the researcher must attempt to make mutants in which the
putative immune evasion phenotype is lost while other functions are maintained (this presumes that
the other functions are known).
Excluding extraneous effects due to inadvertent genetic alterations is usually tidily dealt with by
making resucants, but even this can be misinterpreted. If disruption of the targeted gene disrupts
expression or control of an adjacent or unappreciated overlapping open-reading frame (ORF) that
contributes to the phenotype, restoring the original genetic sequence will also restore the
phenotype. Replacing the gene elsewhere in the genome is theoretically a better control, but rarely
done. An alternative is to make multiple independent mutants, disrupting the gene in different ways
(for example, mutating the promoter or the ORF) and showing that they have the same phenotype.
Equalizing the fitness of the mutant and wild-type virus by deleting the putatively targeted host
mechanism (so called "genetic complementation") is an intellectually appealing and essential part of
KPII. Yet interpreting such experiments can be fraught with hazards. The first comes from making
qualitative interpretations of an outcome that may result from an unrelated quantitative advantage.
That is, if the balance between virus and host is such that CD8+ T cells achieve good but imperfect
control, in theory, any (that is, VIPR-unrelated) reduction in viral fitness could tip the balance and
enable CD8+ T cells to achieve complete control. Another problem is that genes may have
unappreciated targets whose function contributes to the phenotype examined. The MCMV class I
homolog m144 is assumed to act as a ligand for inhibitory NK receptors. Indeed, deleting m144
reduced virus fitness, the rescuant showed wild-type fitness and the mutant and wild-type fitnesses
were equalized by NK cell depletion43. However, the appreciation that the ligand for the homologous
HCMV class I protein UL18 is in fact a leukocyte Ig-like receptor (LIR) expressed more on
macrophages than NK cells suggests other interpretations for the data. For example, m144 may
inhibit MCMV-induced macrophage or DC activation, reducing cytokine secretion and hence the NK
cell response. This scenario shows that our interpretation of KPII relies on current models through
whose smoky glass we interpret the data.
Despite these caveats, KPII remains the essential means for addressing the in vivo function of viral
gene products. We now have a handful of examples of its application. The first application of KPII to
establish the function of a VIPR was the seminal study that showed MCMV lacking m152 replicated
less efficiently than wild-type or rescuant virus 7 days after infection, a difference that was abolished
by depleting CD8+ T cells41. Although welcome as the first demonstration of VIPR function in vivo,
the real surprise of this article was just how modest the effect was (usually less than a tenfold
difference). This raises an important question: do VIPRs generally exert such a modest effect? Or is
there something that we are missing about m152? Indeed, m152 had an even more profound effect
on the activity on NK cells and hence on viral titers at day 3, due to its inhibition of the NK activating
ligand H-6044. So the "true" evolutionary function of m152 is difficult to establish. The most
important message from m152 may be that evolution favors complexity and that we need to
interpret experiments with this in mind.
MHV-68 lacking the M3 chemokine-binding protein replicated acutely in lung epithelium and initially
seeded the spleen to almost the same extent as wild-type virus. However, in contrast to wild-type
virus, the mutant virus did not cause B cell expansion in the spleen with its concomitant
amplification of latent viral load unless CD8+ T cells were depleted18. The interpretation favored by
the authors—that M3 prevents chemokine-driven CD8+ T cells trafficking and impairs their ability to
control latent virus—is reasonable. The difficulty in interpreting this type of experiment (see above)
is underscored by the fact that an independent M3 virus carrying a deletion had little, if any, latency
deficit45.
MHV-68 K3 interferes directly with CD8+ T cell recognition of infected cells46. The K3-deficient
mutant, like the M3-deficient mutant, was primarily impaired during the second mononucleosis-like
phase of infection, which was attributed to more efficient CD8+ T cell function47. These two mutants
suggest CD8+ T cells must be disabled for splenic amplification of the latent virus pool and,
therefore, that MHV-68 VIPRs are important for this phase. It is not known how closely infection of
mice mimics infection of the natural host (voles); consequently, the real contribution made by VIPRs
to the fitness (that is, transmission) of 2 herpesviruses may be difficult to determine in this model.
The role of MHC class I down-regulation by Nef has been investigated in the SIV model48. Three
rhesus macaques were infected with SIV with a Nef that was modified (Y223F) to disable its VIPR
activity without affecting its myriad other functions. Four weeks after infection, the majority of
viruses recovered from each animal had reverted to wild-type Nef. Although this suggests that MHC
class I down-regulation has a strong influence on viral fitness, further evidence is needed to fulfill
KPII. Does reversion occur in CD8+ T cell–depleted animals? Is SIV replication compromised by the
introduction of a mutation that is more difficult to revert?
These examples represent the vanguard of what we hope will be a serious long-term effort to
assess the in vivo function of VIPRs and other immunomodulatory proteins. Although the complexity
of the immune system makes such studies difficult, they should provide a bounty of findings that will
enliven the enterprise and deepen our insight into the general workings of the immune system.
Escape mutants
Viruses have another potential means for avoiding recognition by CD8+ T cells. RNA viruses have
extremely high mutation rates, which means that they exist in nature as a diverse genetic mix. Of
course, most mutations are harmful, and in the absence of selective pressure the average genetic
composition of the population tends to remain fairly stable, reflecting the optimal gene function.
However, these viruses have the potential to rapidly decrease the immunogenicity of their gene
products. This is not terribly difficult due to the number of filters in place that limit the potential
immunogenicity of peptides. Consequently, the chance that any given peptide in a protein will be
immunogenic with any given MHC class I allomorph is 1/200011.
Highly artificial model systems have shown that CD8+ T cells can select for antigenic escape
mutants in mice. Clear evidence has been presented for the selection of escape variants in SIV and
HIV infections49-51. Individual escape mutants may enable a virus to persist in one host and hence
improve transmission to others. MHC polymorphism, however, limits the advantage in a new host,
which is likely to possess an MHC allomorph that binds to different peptides. Thus, RNA viruses
profit from their ability to rapidly generate new variants per se, rather than generate any given
escape variant. Indeed, preventing escape variants is thought to be the primary evolutionary
selection factor for MHC polymorphism (ask any viral immunologist).
In theory, viruses could avoid MHC polymorphism by creating proteins that are antigenically
identical to the host. This would enable viral avoidance of antibody recognition as well. Despite the
high hopes of immunologists, if viruses exploit mimicry, they do so subtly and infrequently; viral
proteins are usually only distantly related to host proteins, even those with similar functions.
Horizons
We will end our review by considering what we have learned from our fascination with VIPRs and
what might be gleaned from further studies. Following the long tradition of viral gene products,
VIPRs have contributed to our understanding of basic cellular biological processes. An early
contribution came from the first-defined VIPR, AdE3-19K, which led to the identification of ERretention motifs in the cytosolic domains of membrane proteins 52. More recently, HCMV US2 and
US11 greatly facilitated studies in mammalian cells of retro-translocation of ER proteins into the
cytosol.
Overall, however, our understanding of the classical MHC class I pathway has enabled us to
understand the mechanistic actions of VIPRs rather than vice versa. VIPRs may yet teach us
something about antigen presentation. Two classes of VIPRs (HCMV US1153 and the K3 from
KSHV and MHV-6854) ubiquitylate MHC class I heavy chain cytosolic domains to target it for
destruction, either in the ER or a post-medial Golgi complex compartment. The lysine ubiquitylated
through the action of K3 is highly conserved among MHC class I allomorphs, which suggests that
this process has a normal counterpart, perhaps identifying MHC class I molecules lacking peptides
for internalization and degradation. No doubt this will soon be sorted out. VIPRs may also fulfill their
promise and become powerful tools for sorting out the contributions of direct and cross-presentation
in activating naïve virus-specific CD8+ T cells28.
Occurring simultaneously with a great leap in deciphering the molecular basis for NK cell
recognition, the study of herpesvirus immune evasion has played an important role in the expansion
of our concept of the extended family of MHC class I molecules and their cognate receptors. It is
likely that when the dust settles, the very definition of the MHC class I family and its role in
immunobiology will have to be reconsidered. The HCMV MHC class I homolog UL18 seemed tailor
made as an NK cell decoy, but identification of a cellular ligand led to the discovery of an entire new
family of killer cell inhibitory receptor (KIR)-like genes, the LIRs55. The LIRs also recognize classical
MHC class I gene products, but are expressed more by macrophages and B cells than by NK cells
or T cells. The functions of the HCMV LIR ligand56 and the LIRs expressed by myeloid cells are a
complete mystery.
Meanwhile, NK cell biologists have extended the concept of "class I–like" by identifying ligands for
the activating NK receptor NKG2D. An HCMV gene, UL16, played an important role in this story57.
Although homology of some of these ligands to class I molecules is so low as to strain credulity, it
seems sufficient for the MCMV VIPR m152, which targets both classical MHC class I and a gene
product that hardly looks like one (H-60)44. Extending the boundaries still further, the MHC class I
family has recently accepted another cohort of relatives, with the identification of another MCMV
gene, m157, as the ligand for the activating NK receptor Ly49H58, 59. m157 has distant secondary
structural homology to MHC class I, as do a handful of other MCMV genes, including m15259 (and
L. Lanier, personal communication). This story remains to be written, but it is clear that VIPRs are
providing crucial insights into both the identities and functions of members the far-flung MHC class I
family.
With the possible exception of MHV-68 K3, we lack a clear demonstration that VIPRs are necessary
for viral persistence via interfering with CD8+ T cell recognition of infected cells. The odds still favor
the possibility that this property is a selective factor driving VIPR evolution. However, our broadened
awareness of the biology of the extended MHC class I family and its ligands forces us to consider
broader hypotheses for the interest of viruses in MHC class I molecules. It would be an ironic twist
in the VIPR story if interference with antigen presentation to CD8+ T cells turns out to be only a
minor part of their complete job description. After all, snakes enjoy a varied diet.
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Figure 1. The classical MHC class I pathway is depicted with reference to viral
interfering proteins. Oligopeptides (red circle) are derived from DRiPs through the
action of proteasomes. Nascent MHC class I molecules—consisting of a heavy
chain and 2-microglobulin ( 2M)—bind to TAP via tapasin. Peptide binding
releases MHC class I to the cell surface. VIPRs interfere with this process at
multiple steps. (1) EBV EBNA-1 contains a sequence that renders it resistant to
proteasomal degradation23, 60. HCMV IE is phosphorylated by another viral protein,
preventing generation of the immunodominant peptide epitope30. (2A) HSV ICP4761,
62 and a BHV1 protein63 bind to the cytosolic side of TAP and prevent peptide
translocation. (2B) HCMV US6 binds to TAP in the ER lumen and prevents peptide
translocation64. (2C) Several VIPRs bind to MHC class I in the ER, retaining it
and/or interfering with the function of the peptide-loading complex. The proteins
include AdE319K (retains MHC class I and also prevents tapasin-mediated docking
with TAP65); HCMV US3 (binds MHC class I but dissociates, the mechanism of
MHC class I retention is not clear but may involve repeated rebinding by newly
synthesized US3)66, 67. MCMV m4/gp34 forms extensive complexes with MHC class
I in the ER68. (3) HCMV US2 and US11 and MHV-68 K3 bind MHC class I in the ER
and induce retrotranslocation for degradation by the proteasome69, 70. For US11,
ubiquitylation of heavy chain cytoplasmic tail, possibly after retrograde
translocation, has been described53. MHV-68 K3 ubiquitylates the tail before
translocation71. HIV-1 Vpu also induces degradation of newly synthesized MHC
class I72—probably via retrograde translocation and proteasome degradation—
although, unlike US2 and US11, proteasome activity is required for retrograde
translocation73. (4) MCMV m152/gp40 causes MHC class I to be retained in the ER
cis-Golgi complex intermediate compartment (ERGIC)74. The fact that m152, itself a
distant MHC class I homolog, can reduce cell surface expression of H-60 as well as
classical MHC class I (in an allomorph-specific manner75) makes its poorly
understood mechanism of action of particular interest. Only the lumenal domain of
m152/gp40 is required for the retention of MHC class I 76. Binding of m152/gp40 to
MHC class I has not been demonstrated, and m152/gp40 itself has a default
pathway of export to and degradation in the lysosome74. (5) Several VIPRs remove
MHC class I either from the Golgi or the cell surface. MCMV m6/gp48 contains a
lysosomal targeting di-leucine motif in its tail; it binds directly to MHC class I and
redirects it to the lysosome77. HIV and SIV Nef use the PACS-1 (phosphofurin
acidic cluster sorting protein 1)–sorting protein to remove MHC class I from the cell
surface and sequester it in the trans-Golgi network78. KSHV K3 and K5 ubiquitylate
the MHC class I tail and target MHC class I to lysosomes in a TG101-dependent
manner54. K5 also targets ICAM-1 (intercellular adhesion molecule 1) and B7-279.
(6) MCMV m4/gp34 is found associated with MHC class I at the cell surface 80 and
inhibits CTL recognition75, but cause and effect have not been firmly linked.
Focus on Immune Evasion
Volume 3 No 11 November 2002
Review
Nature Immunology 3, 1026 - 1032 (2002)
doi:10.1038/ni1102-1026
© Nature America, Inc.
<>
Chronic bacterial infections: living with unwanted
guests
Douglas Young, Tracy Hussell & Gordon Dougan
Centre for Molecular Microbiology and Infection, Imperial College of Science, Technology and Medicine, London SW7 2AZ, UK.
Correspondence should be addressed to G Dougan [email protected]
Some bacterial pathogens can establish life-long chronic infections in their hosts.
Persistence is normally established after an acute infection period involving activation of
both the innate and acquired immune systems. Bacteria have evolved specific pathogenic
mechanisms and harbor sets of genes that contribute to the establishment of a persistent
lifestyle that leads to chronic infection. Persistent bacterial infection may involve occupation
of a particular tissue type or organ or modification of the intracellular environment within
eukaryotic cells. Bacteria appear to adapt their immediate environment to favor survival and
may hijack essential immunoregulatory mechanisms designed to minimize immune
pathology or the inappropriate activation of immune effectors.
Most encounters with pathogenic bacteria lead to an acute period of subclinical infection and,
occasionally, clinical symptoms. If the infected host survives the encounter, the acute infection is
usually fully resolved by elimination of the invading bacteria. However, in a minority of cases, small
numbers of bacteria survive and cause persistent and sometimes life-long infections. The
persistence of bacteria in the face of innate and primed acquired immunity means that an immune
status quo has been established after the acute response. This situation is intriguing because we
know that the innate immune system has evolved Toll and other pattern-recognition receptors to
interact with common bacteria-specific macromolecules, such as lipopolysaccharide (LPS) 1 and
CpG-containing motifs. Although bacteria can modify the structure of some Toll-binding components
(for example, Salmonella and other bacteria will modify their lipid A composition under different
growth conditions2) they cannot live without these classes of macromolecules and, consequently,
mechanisms are likely to be operating during persistent infection to moderate or modify innate
immunity. Before discussing particular types of persistent bacterial infections, it is worth considering
some general mechanisms that could contribute to the persistent lifestyle (Table 1).
High resolution image and legend (90K)
Persistence and commensal bacteria
Sophisticated regulatory controls are built into the mammalian immune system to prevent the
activation of inappropriate immune responses and minimize the potential for immune damage 3.
Consequently, mounting an effective response to pathogens is compromised by the needs of the
host to prevent immune-mediated damage. This is most apparent at the mucosal surfaces of the
body. Commensal colonization provides a model for persistent infections and may provide insights
into how some microbes survive. Neonates are colonized by bacteria very shortly after birth and
there is a rapid onset of immune activation driven by the newly colonized microbes that leads to
both local and systemic humoral and cellular responses against these bacteria. The generation of
significant antibacterial responses to the normal flora may go on throughout the life of an individual.
Evidence suggests that commensals such as Bacteroides fragilis in the gut4 and Neisseria
meningitidis in the upper respiratory tract5 undergo significant antigenic variation in their surface
antigens, such as polysaccharide capsules, which is almost certainly immune-driven. Bacterial
colonization of the intestine is a required signal for the normal development and regulation of the
mucosal immune system, as germ-free animals show impaired development of Peyer's patches and
other mucosal immune tissues. Also, certain knockout mice that show defects in regulatory
cytokines (including interleukin 2 (IL-2)6 and IL-107, 8), key components of the signaling pathways9-11
or maintenance of mucosal integrity12, 13 will develop inflamed bowels if housed under normal
laboratory conditions, but will develop less bowel inflammation in a germ-free environment or when
receiving antibiotics14. Thus, the mucosal immune system consistently encounters a substantial
number of bacterial antigens. How, then, is the balance maintained between this exposure that
could drive immune activation, the need to prevent immunological damage and the requirement for
eliminating pathogens?
A simple explanation often put forward is that antigens from commensals are rapidly eliminated
from mucosa-associated tissues by local effectors15. The mucosal immune system has developed
specialized noninflammatory mechanisms for eliminating antigens. For example, dimeric
immunoglobulin A (IgA) is secreted by mucosal tissues and prevents pathogen entry by
agglutination (immune-exclusion). The T cell repertoire at resting mucosal sites in some species,
such as laboratory-housed mice, is skewed towards a T helper type 2 (T H2) phenotype16. The IL-4
and IL-5 produced by these T cells, together with transforming growth factor- (TGF- ), contribute
to classical IgA class switching. A T cell–independent IgA production mechanism that targets
commensal bacteria has also been identified17. Commensal bacteria reside mainly in the lumen of
the intestine behind a glycocalyx barrier that separates them from enterocytes. This location may
limit their potential to activate inflammatory responses18. In addition, epithelial cells and
intraepithelial lymphocytes may have a higher threshold for responding to cues from bacteria. The
down-regulation of receptors such as Toll-like receptor 4 (TLR4) on epithelial cells, gut
macrophages and T cells serve to limit the inappropriate response to commensal LPS 19, and this
may be true of other molecules that respond to bacterial components 20. The down-regulated,
antigen-specific responses by mucosal T cells, although most likely designed to prevent clonal
Table 1. Some m
expansion of T cells specific for dietary and commensal antigens, could also assist in the bacterial
persistence process. Pathogenic bacteria that can cross the glycocalyx and bind enterocytes or
epithelial cells directly, such as pathogenic Escherichia coli and Helicobacter pylori, can trigger IL-8
and chemokine production that leads to inflammation. This inflammation is often associated with the
recruitment of neutrophils and bacterial elimination, although in the case of H. pylori some bacteria
may be resident at sites in the stomach not available to the effector cells and will remain as
commensals or pathogens. In some H. pylori–infected individuals, this balance between
commensalism and binding leads to chronic gastritis or even, in some individuals, stomach cancer.
Evidence suggests that individuals become colonized for life by particular H. pylori strain types and
that only a percentage of these infected individuals will go on to manifest clinical disease. This
particular situation vividly illustrates the close relationship between colonization and disease at
mucosal surfaces.
Dysregulation of the TH1-TH2 balance at the mucosa can lead to disease. Induction of TH1
interferon- (IFN- )–secreting T cells at mucosal surfaces is associated with ulcerative colitis,
inflammatory bowel disease, gastritis, pneumonia and bronchiolitis 21. Mechanisms responsible for
the breakdown of mucosal immune homeostasis are unclear, but it is thought that genetically
susceptible individuals over-respond to antigens from commensal and possibly pathogenic bacteria.
Coupled with modified innate immune responses, it is likely that a balance involving a cascade of
signals including the action of regulatory T cells secreting IL-10 and/or TGF- 22 and the activation
state of dendritic and other antigen-presenting cells is critical15. Many Peyer's patch–associated
mucosal dendritic cells are of the myeloid lineage and a combination of the pattern of costimulatory
molecules expressed on their surface and their exposure to anti-inflammatory signals such as IL-10
and prostaglandin E2 may modify their ability to become activated or their migrational targeting 23. It
is clear that T cell activation is critical for controlling mucosal immune responses and specialized T
regulatory cells have been identified22, 24. Specific T cells are recruited to the mucosa through
mucosal homing addressins such as the 4 7 or E 7 integrins and the expression of specific
chemokine receptors such as CCR925. Such mechanisms clearly control the population of
lymphocytes resident at mucosal sites. It appears that a cascade of signals may be required to fully
activate mucosal immune responses. Even studies with simple mucosal adjuvants such as cholera
toxin or the E. coli heat-labile toxin suggest that multiple factors in addition to binding are needed
for full adjuvant activation26. This situation is even more complex in that the threshold needed to
activate mucosal cells may be markedly different between species. For example, cells from the
human Peyer's patch constitutively secrete IL-12 and show high activation of the signal transducers
and activators of transcription factor 4 (STAT4) pathway27. Although we do not know all the
mechanisms involved, it is clear that the normal flora is in some form of stasis with the mucosal
immune system thus limiting pathology.
S. enterica Typhi, adapted to systemic persistence
Many bacterial pathogens cause a localized and acute infection in the intestine. A typical example
is Salmonella enterica serovar Typhimurium, which causes invasive gastroenteritis and diarrhea in
humans and cattle. S. enterica Typhimurium bacteria do not normally pass beyond the mesenteric
lymph nodes in significant numbers except in immunocompromised individuals, such as those
infected with HIV. In contrast to S. enterica Typhimurium, S. enterica Typhi is a close relative that
routinely causes systemic infection (typhoid) that involves colonization of the reticuloendothelial
system, including spleen and bone marrow (Fig. 1). Certain individuals infected with S. enterica
Typhi become life-long carriers, periodically secreting high amounts of bacteria in their stools.
Others will relapse to typhoid disease with the same S. enterica Typhi strain several months after
the initial infection (1–5% of antibiotic-treated individuals), which suggests the presence of a
persistent reservoir of bacteria in these individuals28. Short-term persistence (lasting several
months) by S. enterica Typhi may involve colonization of immature immune cells in the bone
marrow29, whereas longer term carriage is associated with infection of the gall bladder from where
bacteria can be directly shed into the intestine via the bile duct. We know little about how bacteria
adapt from being gut pathogens to derivatives capable of persistence in deeper tissues. However,
the genomes of representative S. enterica Typhi30 and S. enterica Typhimurium31 have been
published and comparison reveals some differences. S. enterica Typhi and S. enterica Typhimurium
differ in about 10% of their genes. This difference also includes mutations in over 200 S. enterica
Typhi genes, 145 of which are apparently intact in S. enterica Typhimurium. These S. enterica
Typhi pseudogenes include mutations in 7 of 12 bacterial attachment factors (fimbrial operons)32 as
well as mutations in genes involved in fecal shedding33 or modifying the intracellular lifestyle34 of
Salmonella (for example, sopA, sopD2, sopE2, sseJ, cigR and misL). Pseudogene accumulation
has been associated with other pathogens such as Yersinia pestis (plague bacilli)35 that have
moved towards a systemic lifestyle as well as those that have an obligate intracellular lifestyle such
as Mycobacterium leprae36. In the case of S. enterica Typhi, the loss of multiple adhesive
determinants may preferentially target the pathogen to particular cell types such as dendritic cells 37
or CD18+ cells38 capable of delivering the bacteria to the systemic system while avoiding
nonspecific targeting to epithelial cells, which leads to local gut inflammation. S. enterica Typhi may
also modify the intracellular environment in a different manner to S. enterica Typhimurium (one
consequence of which is host restriction to humans). In a direct comparison of analogous
attenuated S. enterica Typhi– and S. enterica Typhimurium–based vaccines in human volunteers,
the S. enterica Typhi vaccine was less efficient in colonizing the bowel and inducing inflammatory
molecules, such as C-reactive protein, compared to S. enterica Typhimurium39. Poor colonization of
the bowel by S. enterica Typhi may normally be compensated for by gall bladder–shedding to
maintain transmission between hosts. Although the gall bladder is the principle site for life-long
colonization of humans, virtually nothing is known about the mechanisms of persistence.
High resolution image and legend (69K)
Figure 1. Schematic representation of a model
for persistence in human typhoid involving S. enterica Typhi.
On the left of the figure, the acute infection (human gastroenteritis) mediated by S. enterica
Typhimurium involves bacteria replicating freely in the intestine lumen and using multiple
attachment factors (represented by multicolored tips or fimbriae). S. enterica Typhimurium targets
both enterocytes and M cells for invasion, but is stopped at the mesenteric lymph nodes.
Neutrophils are quickly attracted to the invasion site and inflammation follows, leading to diarrhea.
S. enterica Typhi has dispensed with many attachment and shedding factors and may preferentially
target a limited number of host cell types that favor dissemination to deeper tissues. S. enterica
Typhi can persist in the bone marrow for extended periods and in the gall bladder for life. T, T cell;
B, B cell.
Although the genetics and immunology of S. enterica persistence has been studied in the mouse
and could shed light on other persistent Salmonella infections, for example in the chicken or cattle,
care should be taken in extrapolating data from the mouse model to human typhoid. Mutations that
affect long-term Salmonella persistence in the mouse have been identified40. Indeed, it is possible
to generate mutant S. enterica Typhimurium derivatives (such as aroA-purA double mutants) that
persist for many months, even in susceptible mouse strains 41. Sublethal infection can also be
established in genetically resistant mice and can be used to explore the immunology of persistence.
Early bacterial clearance is influenced by factors such as the LPS type of the bacteria and by
complement factors such as C1q42. Both antibodies and B cells also play critical roles in controlling
infection by preventing initial colonization43. In mice, S. enterica Typhimurium targets the M cells
and is subsequently taken into macrophages, polymorphs and dendritic cells. Salmonella bacteria
may ultimately reside in particular macrophage types such as the red pulp macrophages of the
spleen44. In the liver, Kupfer cells are initially targeted and infection foci are normally controlled by
granuloma formation. Bacterial growth rate in macrophages is regulated in mice by such factors as
Nramp1, although no evidence is available for a role of Nramp1 in human typhoid 45. Several
inflammatory cytokines such as IL-12, tumor necrosis factor(TNF) and IL-18 are also
key controlling factors. However, ultimate clearance of bacteria from tissues is dependent on T cell
activity with CD4+ T cells playing a central role46, 47. Mice deficient in T cells (nu/nu-/-) or
costimulatory molecules (CD28-/-) are more susceptible to S. enterica Typhimurium and T cell
receptor (TCR)–deficient mice clear infection poorly at later time points 48-50. CD8+ T cells play a less
important role46 and the contribution47 of
TCR T cells in Salmonella control has yet to be
completely resolved. The pivotal role played by CD4+ T cells in pathogen control is clearly observed
in major histocompatibility complex (MHC) class II–deficient mice, which show impaired clearance
of Salmonella in both mouse and human typhoid51.
Tuberculosis initial evasion
Mycobacteria are perhaps the first infectious agents to spring to mind in connection with chronic or
persistent infections. Tuberculosis infection is initiated when Mycobacterium tuberculosis bacteria
are inhaled deep into the alveoli of the lung, and persistence depends on the ability of the bacteria
to resist the antimicrobial activities of resident alveolar macrophages (Fig. 2). A key step for bacteria
to maintain persistence is interference with the process of phagosome maturation together with an
inherent ability to survive under adverse conditions. By subverting the normal program by which
phagosomes mature through the endocytic pathway, pathogenic mycobacteria are able to persist in
a nonacidified compartment with the characteristics of an early endosome52. The immature
phagosome continues to interact with recycling endosomes but excludes the vacuolar proton
ATPase and retains the small GTPase Rab5 in preference to Rab7, which is found on mature
phagosomes. Phagosomal arrest is a feature of live organisms; killed mycobacteria are rapidly
transferred to conventional acidified phagolysosomes. Mechanisms underlying phagosomal arrest
have yet to be identified and might include secretion of a low molecular weight metabolite by viable
bacteria, expression of an enzyme capable of modifying a host component involved in regulation of
endosomal interactions or modification of the phagosomal membrane by lipids present on the
mycobacterial outer surface. Mycobacteria characteristically express a complex assortment of lipids
and glycolipids53, with a major portion of the genome of M. tuberculosis encoding for protein
important in lipid metabolism 54. Lipid components—including the long-chain fatty dialcohol
phthiocerol dimycocerosate—form a waxy coating around the bacteria and make an important
contribution to their inherent resistance to desiccation and exposure to osmotic imbalance and
extremes of pH55. In combination with several enzymes capable of detoxifying active oxygen and
nitrogen radicals56, the inherent resistance conferred by the cell wall structure makes mycobacteria
a challenging target for the effector mechanisms of host macrophages. The ability of M. tuberculosis
to negotiate this first phase of infection is lost after the mutation of genes involved in nutrition 57 and
in cell wall structure55. During this initial stage of infection, mycobacteria spread from the lung and
have the ability to establish infection in most parts of the body.
High resolution image and legend (64K)
tuberculosis.
Figure 2. Persistent infection with M.
(a) Infecting mycobacteria taken up by alveolar macrophages in the lung resist killing by subverting
phagosome maturation and by the protective effect of their lipid-rich cell wall. (b) Inflammatory
signaling in response to mycobacterial components results in the recruitment of TH1 T cells. T cell–
mediated activation of macrophages enhances their ability to control mycobacteria. (c) Remaining
viable mycobacteria are sequestered within a granuloma made up of macrophages and a variety of
T cell subsets. (d) M. tuberculosis is able to persist in an asymptomatic form within the host over
many decades. It is likely that mycobacteria are held in a nondividing or slowly dividing state in
open lesions or in closed lesions walled-off by fibrosis. (e) Reduced immunity is associated with
reactivation disease. Lack of immunosurveillance may stimulate renewed replication of
mycobacteria in open lesions. For mycobacteria in closed lesions, liquefaction and breakdown in
the absence of immune surveillance will result in disease. Once the disease process is underway,
immunopathology contributes to tissue damage and ultimately efficient aerosol transmission to a
new host.
Containment by adaptive immune responses
The antimycobacterial activity of macrophages is enhanced by T cell stimulation. Phagosomal
arrest is reversed in macrophages that have been activated by IFN- before mycobacterial
infection58, and this interface between the innate and acquired arms of the immune response is
central to the control of mycobacterial disease. Genetic defects in the IL-12– or IFN- –activation
pathways severely compromise the ability to control mycobacterial infection in both humans59 and
mice60. Mycobacteria express a range of ligands that trigger signaling through pattern recognition
receptors on host phagocytes61, 62, resulting in NF- B–mediated release of pro-inflammatory signals
including TNF- and IL-12. This response may be reduced in the case of live M. tuberculosis63, but
infection provokes a potent TH1 cell–mediated immune response. Activation of the adaptive immune
response results in arrest of mycobacterial replication. The duration of the initial phase of bacterial
muliplication depends on previous exposure. Priming by Bacillus Calmette Guerin (BCG)
vaccination establishes a circulating pool of memory T cells that are rapidly recruited to the site of
infection, limiting the extent of initial replication. In contrast, the requirement for T cell priming
(presumably mediated by dendritic cells migrating to adjacent lymph nodes) in naïve hosts delays
the response, thus extending the initial phase of infection. CD4 + T cells recognizing antigens
presented on MHC class II molecules are central to this initial phase of bacterial containment,
whereas CD8+ T cells may assume greater importance at the later stages of infection 60. In addition,
T cells respond vigorously to phosphorylated ligands from mycobacteria 64 and may make a
further contribution to the pool of IFN- , as may T cells restricted by CD165. CD1-restricted cells
recognize lipid and glycolipid antigens that are exported from the mycobacterial phagosome 66 and
may be available, therefore, at an early stage of infection. Lipoproteins also leave the live
phagosome and, by gaining access to MHC class I processing pathways, may contribute to the set
of antigens available for recognition by CD8+ T cells67. In addition to IFN- –mediated activation of
macrophages, the cytotoxic function of T cells may contribute to control of the infection by releasing
mycobacteria from cells that are refractory to activation and perhaps also by delivery of
antimicrobial peptides68. The process of cell recruitment and activation results in sequestration of
the infection within a granuloma. The lesion may then be further walled-off by fibrosis. At this late
stage of the infection the balance seems to lie in favor of the host.
In addition to falling back on their inherent cell wall defense mechanisms, several strategies have
been suggested to contribute to mycobacterial survival in the face of this aggressive immune attack.
Mycobacteria can interfere with T cell recognition of infected macrophages by reducing antigen
presentation and by induction of suppressive cytokines. The expression of MHC class II molecules
is down-regulated in infected cells, for example, by mechanisms that include signaling through
TLRs69. High IL-6 secretion is associated with reduced T cell proliferation, and IL-10 and TGFmay play a role in suppressing T cell responses and macrophage activation. Sensitive assay
systems demonstrate the presence of T H2 cytokines, IL-4 and IL-5, during active tuberculosis, and
these may further contribute to suppression of macrophage activation 70. M. tuberculosis infection
has also been associated with a reduction in the ability of macrophages to respond to IFNsignaling71 and with promotion of apoptosis. In this complex set of responses, it is hard to
distinguish active microbe-mediated suppression from natural regulatory pathways associated with
the immune homeostasis that is required to prevent pathological consequences of chronic
macrophage activation.
Persistent infection in tuberculosis
Whether immunity to the initial infection is limited by pathogen-mediated suppression or by hostmediated regulation, it results in an opportunity for survival of a population of viable M. tuberculosis.
This incomplete elimination, seeding the potential for future reactivation disease, is central to the
pathogenesis of tuberculosis. Despite the obvious clinical importance, there are formidable gaps in
our understanding of the frequency, location and physiological status of persistent populations of M.
tuberculosis.
Some idea of the extent of persistent human infection can be obtained by post mortem examination
of tissues from asymptomatic individuals that died from causes unlinked to tuberculosis. Positive
culture rates of 40–50% have been recorded in lung samples from individuals who have lived in
high-incidence areas72. Old lesions characterized by extensive fibrosis and calcification yielded
positive cultures less frequently than more open lesions. One extensive study recorded the
presence of viable mycobacteria in areas of the lung free from granulomatous lesions 73. A recent
polymerase chain reaction–based study supported the conclusion that persistent M. tuberculosis is
not confined to histological lesions74. Studies of resected lung lesions from patients with active
tuberculosis also showed the existence of distinct bacterial populations. Mycobacteria in open
lesions were found to be actively replicating, whereas those in closed lesions were nondividing and
yielded cultures only after prolonged in vitro incubation75.
Experimental animal models have been used to analyze the biology of persistent infection. The
simplest model is based on the natural immune-mediated control of a low-dose infection, generally
in C57BL/6 mice. After the initial phase of mycobacterial multiplication, the bacterial load stabilizes
at around one-million organisms in the lung. This is maintained for approximately one year, with the
mice eventually succumbing to progressive pathology in the lung. The absence of substantial
accumulation of dead organisms suggests that the bacteria in this model are replicating either
slowly or not at all76, with a thermoresistant phenotype similar to that seen in stationary phase
cultures77. This model has been exploited in the characterization of mutant strains of M.
tuberculosis78, 79. Distinct from the auxotrophic and cell wall mutants discussed above that fail to
multiply during the initial phase of infection, a second class of mutants that lacks a functional
isocitrate gene matches the wild-type during the initial phase of infection, but show defective
persistence78. Isocitrate lyase is particularly important for the utilization of fatty acids as a carbon
source, and the behavior of the mutant suggests that lipids are a predominant source of nutrition
during persistent infection. A regulatory mutation that results in constitutive overexpression of heatshock proteins also shows a persistence defect. In this case it is proposed that the effect is
mediated through enhanced immune interactions79. A third class of mutant, defective in a twocomponent regulator resembling the phoPQ system of Salmonella, is restricted in its initial
replication but is able to persist in infected tissues80.
A second model for persistence involves infection of mice and incomplete treatment with isoniazid
and pyrazinamide. At the end of the treatment, no bacteria are detected by conventional
microbiological culture of tissue homogenates, although active infection becomes evident after
immunosuppression81. This system, referred to as the "Cornell" model, may mimic the phenotype
observed in closed lesions from human lungs and is of particular importance in investigating
mycobacterial populations that persist during chemotherapy.
A constant bacterial load during persistence could represent a nondividing population or a balance
between active replication and immune killing. From the fragmentary information available, it seems
likely that persistent M. tuberculosis divide only very slowly, if at all. The consequent production of
only low amounts of new antigen is probably crucial in ensuring that the immune system tolerates
the presence of the persistent infection. It is also likely that at least a portion of the persistent
bacteria is physically screened from immune surveillance within closed lesions. The regulatory
process involved in shutdown of mycobacterial cell division remains to be defined. The simplest
hypothesis is that this resembles entry into stationary phase as a result of nutrient deprivation within
the intracellular, or intra-granulomatous, microenvironment. Anoxia has been extensively
investigated as a potential physiological stimulus82, and the resulting transcriptional changes have
been characterized by whole genome microarray83. Of particular interest is the induction of
expression of a protein belonging to the -crystallin or low molecular weight heat shock protein
family. The -crystallin protein is a prominent target of B and T cell responses to tuberculosis and is
essential for mycobacterial survival in macrophages. An alternative hypothesis would be that
pathogenic mycobacteria have evolved a developmental program that generates some spore-like
form specifically adapted for persistence. Although unusual forms of mycobacteria have been
reported in studies of infected tissues, these observations have yet to find any counterpart in postgenomic analysis.
Reactivation disease in tuberculosis
It is estimated that one in ten individuals who mount an effective immune response to an initial
encounter with M. tuberculosis will nevertheless develop clinical tuberculosis in later life. Disease
may result from reactivation of the initial infection or from a subsequent reinfection; the relative
prevalence of the two pathways is unknown and probably differs between geographical areas.
Whether the source of bacteria is endogenous or exogenous, it remains to be explained why there
is a failure of an initially protective immune response.
This is most readily understood in the case of HIV infection. At the early stage of progression to
AIDS, reduced CD4 counts are associated with markedly enhanced susceptibility to tuberculosis;
individuals coinfected with HIV have a 10% annual risk of disease. Similarly, the reduced T cell
immunity that occurs during immune senescence in elderly populations and transient
immunosuppression caused by poor nutrition or alcohol consumption probably contributes to an
increased risk of tuberculosis. Therapy for autoimmune disease that reduces TNF- activity also
increases the risk of tuberculosis84. This occurs earlier in females than in males, suggesting an
endocrine influence. Other infections may also be an important trigger for disease in endemic
areas. Helminth infections that induce a strong systemic TH2 response might compromise TH1
antimycobacterial immunity, for example. Alternatively, repeated exposure to environmental
mycobacteria may alter the kinetics of T cell turnover, effectively exhausting the original
antimycobacterial memory pool. A third potential mechanism involves the induction of immune
tolerance as a result of prolonged exposure to antigen in the absence of an appropriate second
signal required for T cell activation. This is an attractive explanation for the highly specific TH1
anergy seen in lepromatous leprosy patients. Finally, it may be that an immune response sufficient
to control a low dose infection is simply overwhelmed by a subsequent high dose exposure. This
might occur as a result of intimate contact with a particularly infectious individual or from breakdown
of an initially contained lesion.
Most patients with active tuberculosis mount a strong immune response to mycobacterial antigens.
The response (assessed by T cell proliferation or IFN- production, for example) is generally lower
in patients than in healthy exposed individuals, but again it is predominantly T H1. Once immune
containment has been lost, however, the TH1 response contributes to pathology rather than to
protection85. As the immune system responds to challenge with an escalating concentration of
mycobacterial antigen, the resulting inflammatory response causes localized tissue destruction,
increasing the extent of mycobacterial multiplication and subsequent transmission. Thus, success of
M. tuberculosis as a pathogen depends on its ability to withstand initial attack by the immune
response and also on its ability to induce the immune-mediated tissue destruction required for
efficient transmission.
Elimination of persistent M. tuberculosis infection
Elimination of persistent infection represents an important strategy for control of the associated
diseases. Preventive therapy of individuals with latent M. tuberculosis infection significantly reduces
the risk of subsequent disease. Although this intervention is routinely applied in the United States, it
requires a robust infrastructure for prolonged administration of potentially toxic drugs, with a risk
that misuse will contribute to drug resistance. Drugs targeted specifically against the nonreplicating
or slowly replicating phenotypes implicated in persistence would enhance the feasibility of
widespread preventive therapy. Despite the obvious pitfalls in extrapolating from mice to humans,
the persistence mutants identified in animal models present potential leads for the development of
such interventions.
It is attractive to envisage an immune-based strategy for elimination of persistent infection. This
could be based on boosting T H1 T cell responses that are induced during the initial infection but
may wane with time or on artificial induction of additional responses directed to antigens that are
under-represented in the natural response. An alternative strategy would be to enhance existing T
cell responses, for example, by stimulating second signal responses in infected cells. A caveat
concerning immune control of latent tuberculosis is the need to avoid triggering of the
immunopathological manifestations characteristic of active disease.
Other issues
Modification of the intravacuole environment is a key feature of other persistent bacterial
pathogens, including Salmonella34, Brucella and Chlamydia species. A modified intracellular
environment could clearly favor persistence and evasion of immune responses through reduced
surface antigen presentation or the control of apoptotic pathways. Brucella species use a type IV
secretion system to avoid phagosomal fusion with the lysosomes and survive within an acidified
intracellular compartment86, 87. Brucella accumulates inside macrophages where the type IV
secretion system senses an increasing acidic environment and maintains a Brucella-adapted
compartment. Successful replication by Chlamydia pneumoniae also involves inhibition of
phagolysosomal fusion, although the mechanisms used by C. pneumoniae are unknown. With the
use of pH-sensitive probes it has been shown that C. pneumoniae circumvents the host endocytic
pathway and inhabits a nonacidic vacuole 88. This vacuole does not express the late endosomal
marker cation-independent mannose-6-phosphate receptor or lysosomal-associated membrane
protein 1 (LAMP-1) and LAMP-2 and CD63. A set of chlamydial proteins (Inc proteins) has been
mapped to the surface membrane containing chlamydial inclusions. It is thought that these proteins
may anchor the pathogen to the inclusion membrane, allowing it to occupy this distinct intracellular
environment. The intracellular growth rate of C. pneumoniae slows dramatically during chronic
infection, leading to attenuated production of new elementary bodies, appearance of
morphologically aberrant reticulate bodies and altered expression of several chlamydial genes.
Using infected HEp-2 cells, one group has shown that IFN- treatment reduced the expression of
genes involved in cell division but maintained the transcription of genes involved in chlamydial DNA
replication89. In order for C. pneumoniae to persist in the host, it would be an advantage for the
infected cell to be long-lived. C. pneumoniae is capable of interfering with host cell apoptosis
induced by staurosporine and CD95 death receptor–signaling90. This, together with inhibition of
phagolysosomal fusion, may in part explain the ability of these bacteria to cause chronic infections
in humans. For bacteria to survive long-term inside cells they must be able to adapt to a lifestyle
involving slow replication or even dormancy. At present, little is known about how bacterial growth
rate is regulated in vivo, although mycobacteria are well adapted to grow slowly.
Antigenic variation is also a strategy used by several bacterial pathogens to evade the humoral
immune system and persist in the host. Some bacteria such as Borrelia burgdorferi91 and Neisseria
gonorrhea92 encode sophisticated genetic systems and gene families to increase the diversity of
surface-located proteins in a similar manner to eukaryotic trypanosomes. Although the well
characterized antigens of M. tuberculosis show no sequence diversity, it is speculated that two large
gene families identified by genome analysis might provide a source of mycobacterial antigenic
variation54, 93. Unlike the related spirochete B. burgdorferi, Treponema pallidum—the etiological
agent of syphilis—apparently has a limited genetic ability to vary antigenic composition 94. The
mechanisms of T. pallidum persistence remain a mystery. Although several hypotheses have been
put forward to explain persistence, such as lack of expression of surface proteins, infection of
nonprofessional antigen-presenting cells95 or coating in host antigens, our understanding of the
immunology of T. pallidum persistence is almost nonexistent. Indeed, we know very little about the
detailed mechanisms of how bacteria evade immunosurveillance on a long-term basis in the host. If
we are to improve our therapeutic approaches to dealing with microbes such as M. tuberculosis, the
gathering of more knowledge would be useful.
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Acknowledgments. Supported by The Wellcome Trust (G. D. and D. Y.) and the Medical Research
Council (T. H.).
Table 1. Some mechanisms of increasing persistence used by pathogenic
bacteria
Figure 1. Schematic representation of a model for persistence in human
typhoid involving S. enterica Typhi. On the left of the figure, the acute infection
(human gastroenteritis) mediated by S. enterica Typhimurium involves bacteria
replicating freely in the intestine lumen and using multiple attachment factors
(represented by multicolored tips or fimbriae). S. enterica Typhimurium targets both
enterocytes and M cells for invasion, but is stopped at the mesenteric lymph nodes.
Neutrophils are quickly attracted to the invasion site96 and inflammation follows39,
leading to diarrhea. S. enterica Typhi has dispensed with many attachment32 and
shedding factors33 and may preferentially target a limited number of host cell types
that favor dissemination to deeper tissues. S. enterica Typhi can persist in the bone
marrow for extended periods and in the gall bladder for life. T, T cell; B, B cell.
Figure 2. Persistent infection with M. tuberculosis. (a) Infecting mycobacteria
taken up by alveolar macrophages in the lung resist killing by subverting
phagosome maturation and by the protective effect of their lipid-rich cell wall. (b)
Inflammatory signaling in response to mycobacterial components results in the
recruitment of TH1 T cells. T cell–mediated activation of macrophages enhances
their ability to control mycobacteria. (c) Remaining viable mycobacteria are
sequestered within a granuloma made up of macrophages and a variety of T cell
subsets. (d) M. tuberculosis is able to persist in an asymptomatic form within the
host over many decades. It is likely that mycobacteria are held in a nondividing or
slowly dividing state in open lesions or in closed lesions walled-off by fibrosis. (e)
Reduced immunity is associated with reactivation disease. Lack of
immunosurveillance may stimulate renewed replication of mycobacteria in open
lesions. For mycobacteria in closed lesions, liquefaction and breakdown in the
absence of immune surveillance will result in disease. Once the disease process is
underway, immunopathology contributes to tissue damage and ultimately efficient
aerosol transmission to a new host.
Focus on Immune Evasion
Volume 3 No 11 November 2002
Review
Nature Immunology 3, 1033 - 1040 (2002)
doi:10.1038/ni1102-1033
© Nature America, Inc.
<>
Bacterial strategies for overcoming host innate and
adaptive immune responses
Mathias W. Hornef1, Mary Jo Wick2, Mikael Rhen1 & Staffan Normark1
1
2
Microbiology and Tumor Biology Center (MTC), Karolinska Institutet, Nobelsväg 16, SE-17177 Stockholm, Sweden.
Department of Clinical Immunology, University of Göteborg, Guldhedsgatan 10, SE-41346 Göteborg, Sweden.
Correspondence should be addressed to S Normark [email protected]
In higher organisms a variety of host defense mechanisms control the resident microflora
and, in most cases, effectively prevent invasive microbial disease. However, it appears that
microbial organisms have coevolved with their hosts to overcome protective host barriers
and, in selected cases, actually take advantage of innate host responses. Many microbial
pathogens avoid host recognition or dampen the subsequent immune activation through
sophisticated interactions with host responses, but some pathogens benefit from the
stimulation of inflammatory reactions. This review will describe the spectrum of strategies
used by microbes to avoid or provoke activation of the host's immune response as well as
our current understanding of the role this immunomodulatory interference plays during
microbial pathogenesis.
Like all other higher organisms, humans have evolved in the continuous presence of various
microbes. In fact, many body surfaces are densely populated by what we call the "normal
microflora", which is mainly constituted of a variety of commensal bacteria, such as Bacteroides
thetaiotamicron and Lactobacillus species. These bacteria are harmless and even beneficial under
normal circumstances, but may cause local or systemic inflammatory disease if the integrity of the
hosts' surface is disturbed. On the other hand, pathogenic bacteria are able to invade sterile body
sites, proliferate and cause substantial tissue damage or systemic inflammation, such as is seen
after infection with Shigella dysenteriae or Mycobacterium tuberculosis. The success of many
pathogens relies on their ability to circumvent, resist or counteract host defense mechanisms; yet
some bacteria also provoke activation of the immune system, which ultimately leads to disruption of
the epithelial barrier and bacterial invasion. Consequently, pathogens have provided many
examples of how to avoid and manipulate host responses.
In this review, we will discuss how pathogens mechanistically avoid recognition by the immune
system, and how they interfere—or possibly could interfere—with innate or adaptive immune
responses, should recognition occur. We will examine the various steps that take place during the
course of infection, starting with microbial attachment and colonization of host surfaces, which can
eventually lead to invasion of the epithelial cell layer and penetration to the subepithelial tissue, and
finally facilitate systemic dissemination of the microbes. We will discuss the strategies these
microbes use to modify or circumvent the host defense mechanisms that come into play during this
process, including recognition by surface immune receptors, secretion of antimicrobial effector
molecules, internalization and degradation by phagocytes and activation of the humoral as well as
the cellular immune systems.
Attachment to and colonization of body surfaces
Bacteria are excluded from the host tissue by anatomical barriers that consist of the skin and
mucous membranes. The integrity of the mucosal surfaces is protected by active removal of
bacteria, for example by the acid environment of the stomach, the ciliary movement in the upper
respiratory tract and the continuous flushing with urine of the lower urinary tract. Thus, motility and
attachment factors (so-called adhesins) found in most pathogenic bacteria are essential for
approaching cellular surfaces and withstanding mechanical removal. For example, lack of
expression of the major attachment factor toxin-coregulated pili (TCP) in Vibrio cholerae,
significantly reduces the severity of bacteria-induced diarrhea in humans1. Alternatively, the
secretion of bacterial toxins impairs protective functions and facilitates colonization. For example,
Bordetella pertussis, the agent that causes whooping cough, paralyses the ciliary clearance
function of the respiratory tract via the release of cell wall constituents that induce nitric oxide–
mediated ciliostasis2. Biofilm formation by the opportunistic pathogens Pseudomonas aeruginosa
and Staphylococcus epidermidis or the production of a protective bacterial extracellular matrix
("curli" surface fibers) by Escherichia coli shield bacteria from the hostile environment and might
facilitate resistance against the host surface protective mechanisms 3. Finally, the presence of an
extensive resident microflora represents yet another means of effectively protecting the host's
mucosal surfaces; this is illustrated by infection with the opportunistic pathogen Clostridium
difficile—the agent that causes pseudomembranous colitis—in patients undergoing antibiotic
treatment, which results in disturbance of the enteric microflora. Thus, colonization by pathogens in
the presence of a resident flora requires successful strategies that enable invading microbes to
successfully compete for nutritional and spatial resources and displace commensal organisms from
the microbial niche.
Evasion of immune recognition by mucosal surfaces
Besides acting as mechanical barriers, vulnerable mucosal membranes are covered with an array
of soluble opsonizing factors, such as antibodies, that immobilize and remove approaching bacteria.
Bacteria counter this with the proteolytic degradation of secretory immunoglobulin. This method of
evasion is used particularly by bacteria that colonize the upper respiratory tract; Haemophilus
influenzae—an important causative agent of respiratory tract infections—is one example of a
microbe that uses such mechanisms to prevent opsonization and Fc receptor–mediated
phagocytosis. Many types of epithelial cells have the intrinsic ability to sense the presence of
microbial organisms and respond specifically through the identification of conserved components of
these microbes. These microbial structures are termed pathogen-associated molecular patterns
(PAMPs) and include parts of the bacterial cell envelope, such as lipopolysaccharide (LPS),
peptidoglycan and bacterial DNA. Recognition of microbial structures by host cells relies on diverse
families of genome-encoded receptors that allow detection of infectious nonself particles and
provide signals that activate the defense mechanisms4. One group of membrane receptors, the tolllike receptors (TLRs), has attracted substantial attention due to their role in cellular signaling and
their importance during initiation of the adaptive immune response5. The most effective strategy for
avoiding innate recognition could involve steric shielding or modification of exposed PAMPs. In fact,
host-like bacterial capsular structures have long been recognized as important virulence factors.
Also, various LPS species from different commensal as well as pathogenic bacteria show some
variance in the capacity to induce cytokine synthesis. Multiple alterations in the structure of
Salmonella LPS decrease the microbe's potential to provoke innate immune responses 6. However,
due to the pivotal role played by most PAMPs in essential bacterial cell functions as well as
structure, major modifications might well decrease the viability and fitness of the bacterial intruders.
Bacterial flagellin, which is recognized by TLR5, might represent an exception; flagellin shows in
many Gram-negative bacteria as a result of phase and antigenic variation7. Also, although it is an
important virulence factor for many bacteria (for example V. cholerae), it appears that flagellar
expression is not an essential contributor to the pathogenicity of the prominent enteric pathogen
Salmonella enterica serovar Typhimurium 8.
The cellular process of pattern molecule recognition is only beginning to be understood. In addition
to the need for soluble as well as membrane-bound accessory proteins such as LPS-binding protein
(LBP), CD14 and MD-2, the cellular localization of a given TLR seems to be highly specific. For
example, TLR2 is situated on the plasma membrane of macrophages and stays bound to its
ligands—such as yeast zymosan—even after internalization in the phagosome9. In order for
hypomethylated CpG motifs—a characteristic feature of bacterial DNA—to be recognized,
endocytosis must occur so that the cell can signal through the intracellularly located receptor
TLR910. TLR4 is found on the surface of macrophages but in the Golgi apparatus of intestinal
epithelial cells, colocalized with its internalized ligand LPS11. These examples demonstrate the
complexity of TLR-mediated recognition processes, which involve ligand internalization, cell traffic
and fusion of subcellular compartments.
Although the exact relationship between ligand localization and TLR-mediated signaling has not
been determined, the possibility exists that microbes inhibit or delay recognition by interference with
membrane and vesicular trafficking. Alternatively, because expression of recognition receptors
seems to be organ-specific, recognition might be avoided through the selection of certain favorable
anatomical sites for colonization and invasion12.
In contrast to the avoidance of immune recognition, some microbial pathogens, under certain
conditions, enhance immune-activation and pro-inflammatory responses by producing maximally
stimulatory pattern molecules. For example, S. dysenteriae, which causes bacillary dysentery in
humans, contains two copies of the msbB gene; one of these genes is located on the virulence
plasmid. The msbB gene product is involved in the biosynthetic pathway of lipid A, the
immunostimulatory part of the LPS molecule. Deletion of the msbB gene in E. coli leads to the
production of hypoacylated lipid A with strongly decreased pro-inflammatory activity. The second
msbB gene encoded by Shigella might be used to ensure complete acylation of lipid A and
generate maximal stimulatory LPS. Cell activation is required to induce intestinal leukocyte
infiltration followed by disruption of the enteric mucosal layer, which facilitates bacterial invasion13.
As this example demonstrates, inflammation during the early course of infection might, under
certain conditions, be advantageous. In contrast, long-term microbial colonization requires that
cellular stimulation and activation of host defenses are avoided. This point is illustrated by
Helicobacter pylori, which colonizes the human gastric mucosa and causes chronic infections in a
large percentage of the human population. H. pylori activates stomach epithelial cells in a process
that is mainly dependent on proteins encoded by the CagA pathogenicity island 14. After prolonged
colonization, part of the bacterial population in the stomach tends to delete cag genes15. This may
reflect a need to reduce the inflammatory response as soon as microbial colonization is established.
In addition, a global modulation of virulence gene expression is associated with the transition from
acute to chronic infection of mice with S. enterica Typhimurium16. In contrast, isolates of P.
aeruginosa that chronically colonize the lungs of patients with the inherited disease cystic fibrosis
continue to produce highly stimulatory LPS17. Biofilm formation and low susceptibility to host
defense molecules (such as antimicrobial peptides and complement) might provide sufficient
protection to allow P. aeruginosa to persist in the face of ongoing inflammation, which enhances the
supply of nutrients.
Recognition via host receptor molecules eventually leads to the activation of signal transduction
cascades—including recruitment of adaptor molecules, tyrosine phosphorylation and activation of
transcription factors—and subsequent activation of defense responses such as chemokine release
and antimicrobial peptide production. An alternative immune-evasion strategy might interfere with
cellular signaling during the stages that follow actual recognition. However, regarding microbial
interference with TLR-induced signaling, only one example—that used by the vaccinia virus—has
been described18. Possibly, disruption of immediate TLR-mediated signaling in host cells requires a
pace that simply is not easily achieved. The alternative, then, would be to interfere with downstream
signaling events. Active suppression, at the molecular level, of an induced pro-inflammatory
immune response is demonstrated by S. enterica serovar Pullorum, the agent that causes fowl
typhoid. In contrast to the well studied serovar Typhimurium—which causes inflammatory
gastroenteritis in humans, and high secretion of pro-inflammatory cytokines in polarized human
intestinal epithelial cells—S. enterica Pullorum produces only a minimal cellular response. More
strikingly S. enterica Pullorum can suppress the pro-inflammatory activation of a subsequent
exposure to S. enterica Typhimurium through active inhibition of I B ubiquitination19. Inhibition of
cellular activation by commensal or pathogenic microbes may therefore represent a strategy with
which gastrointestinal mucosal tolerance to pro-inflammatory stimuli can be maintained and host
defenses avoided. Microbial strategies for the manipulation or avoidance of surface defense
mechanisms of the host epithelial barrier are illustrated (Fig. 1).
High resolution image and legend (75K)
epithelial defense mechanisms.
Figure 1. Strategies for bacterial escape from
Prevention of opsonization (1) is required to facilitate colonization of host surfaces (2). Toxin
secretion can paralyze the host's defenses (3) and disrupt its mucosal integrity (4). Microbial
recognition and host responses—such as the secretion of antimicrobial peptides (5) or chemokine
production (6)—can be impaired by modification of pattern molecule presentation or interference
with intracellular signaling or cell trafficking (8). Microbe-induced self-uptake (7) and escape from
the phagosome along with inhibition of intracellular recognition (9) or persistence in modified
endosomes (10) can then impede removal by host defense mechanisms. Green, host responses;
orange, bacterial components and interference with host defense strategies.
Resistance to antibacterial effectors on epithelial surfaces
In addition to their ability to attract professional immune cells, the epithelial body surfaces
themselves provide effective innate antimicrobial defense. A large variety of antimicrobial peptides
protect the inner and outer surfaces of most multicellular organisms against environmental microbial
pathogens. These locally secreted short peptides are highly resistant to enzymatic degradation and
show a net positive charge, which facilitates their binding to prokaryotic cell surfaces. Antimicrobial
peptide–induced bacterial killing involves attachment and integration of the peptide into the surface
of the invading prokaryote and subsequent disturbance of membrane integrity20. A whole spectrum
of adaptive mechanisms used by bacteria lowers susceptibility to antimicrobial peptides expressed
by the host. Although they are considered relatively resistant to enzymatic digestion, degradation of
at least some linear antimicrobial peptides by bacterial proteases has been reported 21, 22, and active
transport of peptides out of the bacterial cytoplasm also occurs 23. Some bacteria degrade
extracellular matrix, and the resulting fragments bind to antimicrobial peptides and abolish their
efficacy24. Bacterial membranes are much less susceptible to antimicrobial peptides than artificial
membranes25. This might be explained by the fact that the negatively charged membranes of many
bacteria are modified by the addition of positively charged residues. Staphylococcus aureus, the
dominant causative agent of purulent wound infections, modifies its principal membrane lipid,
phosphatidylglycerol, with lysine26 and adds D-alanine to teichoic acid27. Both changes reduce the
net negative charge of the membrane. Similarly, under certain circumstances Gram-negative
bacteria modify the structure of their LPS so they become less susceptible to antimicrobial killing.
For example, S. enterica Typhimurium can form hepta-acylated lipid A (via the addition of palmitate
by the bacterial protein PagP), add phosphate and phosphoethanolamine to the core
polysaccharide and modify lipid A phosphate groups with ethanolamine and aminoarabinose. These
alterations decrease the susceptibility of microbes to -helical antimicrobial peptides or the cyclic
polypeptide polymyxin6, 28.
Adaptation to antimicrobial peptides seems to play a critical role in microbial virulence, as 11 out of
12 S. enterica Typhimurium mutants with decreased susceptibility to host peptides, showed
reduced virulence in vivo 29. Also, the mechanism used by the virulence factor encoded by the S.
enterica Typhimurium mig14 gene was recently identified: mig-14 mutants showed enhanced
susceptibility to antimicrobial peptides30. Finally, S. aureus that was unable to modify
phosphatidylglycerol with L-lysin and thereby reduce its negatively charged surface membrane
showed attenuated virulence in mice26. Thus, bacteria have evolved a number of mechanisms in
order to adapt to surrounding antimicrobial peptides, and these mechanisms appear to be important
in the expression of full virulence. However, high concentrations of animicrobial peptides at
vulnerable body sites in vivo do nevertheless impede microbial colonization and growth.
Why do bacteria not attempt full resistance to antimicrobial peptides? One explanation may lie in
the high costs to microbial organisms that the development and expression of resistance engender.
The diverse and highly specific biological functions of the microbial membrane might preclude
modifications that allow resistance to the membrane-disturbing activity of antimicrobial peptides in
the interest of preserving the functional and structural integrity of the microbial cell 20. For example, a
strain of Streptococcus pyogenes that is resistant to the murine antimicrobial peptide Cramp shows
growth inhibition in enriched culture medium 31. Therefore, the importance of decreased
susceptibility to antimicrobial peptides may, in most cases, lie in the competition with resident
microbial organisms for nutrients and space rather than resistance to the hosts' immune defense.
Resistance-enhancing changes to LPS structure in Salmonella are tightly regulated by the PhoPPhoQ and PmrA-PmrB two-component signal transduction systems, which are central regulators of
bacterial virulence6, 32. The ability to monitor the environment and accordingly modify the cell wall
structure might allow the organism to adapt to specific requirements during infection and therefore
minimize the accompanying high metabolic costs.
Another explanation for the lack of emergence of resistance to antimicrobial peptides is the
simultaneous production of a variety of different peptides at most body sites. The simultaneous use
of different antimicrobial substances significantly impairs the development of microbial resistance,
as is illustrated by the modern multiple drug regimens prescribed to treat tuberculosis or HIV
infection. A recent genetic analysis has identified a large number of potential antimicrobial peptides
in vertebrates, which further increases the quantity and diversity of molecules identified to date 33.
Accordingly, gene-deletion strategies to prevent the expression of a single antimicrobial peptide
have so far failed to reveal a clear phenotype of enhanced susceptibility to infection. (The only
exception is a report on mice deficient in Cramp, the only murine member of the cathelicidin gene
family, which showed enhanced susceptibility to skin infection with S. pyogenes 31.) In contrast,
mice lacking the whole group of enteric -defensins—as a result of deletion of the proteolytic
enzyme matrilysin—showed an increased susceptibility to orally administered S. enterica
Typhimurium; this demonstrates the importance of enteric antimicrobial peptides in host defense 34.
A third reason for the lack of fully resistant bacteria may be the fact that the activity of antimicrobial
peptides seems to be highly regulated both at the transcriptional level and through enzymatic
processing and secretion35, 36. Consequently, interference with antimicrobial peptide regulation
seems to represent another microbial strategy for avoiding killing37, 38. In addition, the continuous
presence of high peptide concentrations is restricted to defined and particularly vulnerable body
sites such as the mouth, airways and intestinal crypts (where the intestinal epithelium regenerates)
as a means of controlling the distribution of the normal flora 39, 40. The situation in the intestinal
crypts illustrates this scenario well. These small, gland-like appendices (with a volume of 4–6 l)
contain high concentrations of antimicrobial peptides (estimated to be of the order of grams per liter)
that are produced by the Paneth cells at the lower end of the crypts and which effectively inhibit
bacterial entry into the crypts and protect the site of epithelial regeneration 20, 40. Diffusion into the
comparatively large intestinal lumen, absorption by the mucus overlying the epithelium and
consumption through the abundant intestinal microflora results in a peptide concentration below that
required to inhibit bacterial growth. Therefore, restricted secretion of antimicrobial peptides might
help to avoid the development of microbial resistance by minimizing the selective pressure on the
surrounding resident flora.
Strategies for invading and crossing the epithelium
Invasion of the epithelial layer provides protection from surface defense molecules. For example, S.
enterica Typhimurium invades epithelial cells using a mechanism by which it induces its own
uptake. The microbe uses a syringe-like transfer apparatus—termed a type III secretion system—to
transfer two bacterial products, SopE and SopE2, directly from the bacterial cytoplasm into the
eukaryotic host cell. Both proteins act as nucleotide exchange factors that activate central
regulators of the actin cytoskeleton, the small GTP-binding proteins CDC42 and Rac, and induce
subsequent engulfment of the bacterium 41, 42. However, activation of these proteins also stimulates
nuclear responses through the transcription factors NF- B and AP-1, ultimately leading to the
secretion of pro-inflammatory cytokines and attraction of professional phagocytes. This immune
stimulation is counteracted by yet another translocated bacterial protein, SptP, which quenches the
activated GTP-binding proteins involved and thereby limits cell activation42. Similarly, uropathogenic
E. coli invade the bladder epithelium and thereby avoid clearance by surface host defense
mechanisms43. Internalized Shigella flexneri—which has a similar clinical profile to S. dysenteriae—
produces and secretes IpaB, which mediates lysis of the phagosome and allows the bacterium to
escape into the cytoplasmic space44. Actin nucleation and polymerization initiated by the bacterial
protein IcsA—which is located at the rear pole of the bacterium—enables S. flexneri to move
through the cytoplasm and enter neighboring cells, facilitating microbe evasion of activated immune
reponses45. One might assume that the cytosolic location provides optimal protection from immune
recognition and response. However, even the cytosol seems to be equipped to detect the presence
of bacterial pattern molecules, such as LPS, mediated by members of the nonobese diabetic (NOD)
protein family leading to a pro-inflammatory cellular response46. Mutations in the human NOD2
gene, which are involved in cytosolic recognition of LPA, are associated with Crohns' disease, an
inflammatory bowel disease of unknown etiology47, 48.
Direct penetration of the skin is found with vector-born microbial diseases. In the case of Lyme
disease, the protective skin barrier is transversed by the bite of a tick. The tick translocates Borrelia
burgdorferi directly to the subepithelial space, where the bacteria initiate systemic infection.
Infection with the spirochete Leptospira in an example of active transcutaneous migration, as this
bacterium has the exceptional ability to actively penetrate the skin without the aid of any vector.
Other bacteria such as S. pyogenes or Clostridium perfringens, both prominent causative agents of
soft tissue infections, bypass the epithelial barrier via pre-existing injuries and use enzymatic
degradation of the host's extracellular matrix, toxin-mediated cell destruction or induction of
programmed cell death to spread themselves through intact tissue49. Yet another important mode of
entry, bypassing the intestinal epithelial barrier—used, for example, by Salmonella and Shigella—
occurs through a specialized cell type: the M cell. M cells overlay the Peyer's patches in the small
intestine and can translocate luminal antigens (and even intact bacteria) to the basolateral side of
the epithelia for uptake and recognition by the underlying cells of the immune system 50. However,
once they are beyond this entry point, bacteria must defend themselves against resident
professional immune cells.
Escape from phagocyte responses
Upon arrival at the subepithelial space, bacteria encounter locally resident as well as newly
infiltrated professional phagocytic cells that are attracted by the chemokine response of the
overlying epithelial cells. The strategies used by bacteria to overcome this additional defense
barrier are shown (Fig. 2) Phagocytes are equipped with a number of receptors that detect the
presence of invading microbes and bind opsonized microbial surfaces. Membrane-bound
scavenger receptors, lectins, Fc receptors and complement receptors as well as signaling through
TLRs may cooperate to determine the ultimate cellular response. This may lead to phagocyte
maturation, activation of antimicrobial substances and secretion of pro-inflammatory cytokines, as
well as phagocytosis and microbial degradation.
High resolution image and legend (60K)
Figure 2. Bacterial defense against phagocytes.
Bacterial defense strategies against phagocyte engulfment include the induction of programmed
cell death (1) as well as inhibition of uptake (2) by translocated effector proteins. Effector proteins
can also be used to down-regulate other host cell nuclear responses (3). Should phagocytosis
occur, bacteria can escape from the endosome into the host cell cytosol (4) or interfere with
endosomal trafficking as well as maturation of the phagosome (5) and the subcellular localization of
defense factors (6).
Consequently, bacteria use a variety of strategies to avoid engulfment and degradation by
phagocytes and facilitate proliferation and spread among host tissues 51. Examples are the inhibition
of phagocytosis by capsule formation or toxin-mediated cellular destruction and necrosis. In
contrast, induction of apoptosis avoids the release of pro-inflammatory signals49. Host-induced
apoptosis of lung epithelial cells during infection with P. aeruginosa plays an important role in
reducing leukocyte infiltration and maintaining the essential function of the lung: the oxygenation of
blood52. In contrast, Salmonella and Shigella both actively stimulate pro-apoptotic pathways in order
to paralyze phagocytic defense: SipB from S. enterica Typhimurium and the similar IpaB from S.
flexneri are translocated via a type III secretion apparatus into the host cytosol. These proteins bind
to caspase-1, which activates downstream caspases and induces apoptosis53, 54. The observation
that caspase-1–deficient mice are resistant to infection with wild-type Salmonella suggests that this
mechanism may contribute to the pathogenesis of this bacterium 55. Yersinia enterocolitica YopP
(like its homolog Yersinia pseudotuberculosis YopJ) can also inhibit anti-apoptotic signals via the
repression of NF- B activation as well as stimulation of pro-apoptotic signals through LPS-mediated
activation of the TLR4 pathway56.
Y. enterocolitica and Y. pseudotuberculosis—which both cause enterocolitis and abdominal
lymphadenitis—can inhibit phagocytosis by the translocation of bacterial mediators that specifically
disorganize the host cell cytoskeleton preventing bacterial uptake by macrophages and
polymorphonuclear leukocytes. Bacterial YopE RhoGAP activity promotes the disruption of actin
filaments by interaction with the Rho GTPases Rac, Rho and CDC42. YopH destabilizes focal
adhesion via dephosphorylation of the adapter protein p130Cas and inhibits phagocytosis that is
mediated by Fc receptors and complement receptors57, 58. Once internalization has occurred, some
bacteria—such as the food-born pathogen Listeria monocytogenes, which is responsible for serious
infections in immunocompromised individuals—manage to survive, persist and even proliferate in
host phagocytes. To avoid degradation in the phagolysosome, L. monocytogenes is able to escape
into the host cell cytosol by means of a bacterial toxin, listeriolysin, which disrupts the endosomal
membrane59. Other pathogens such as Salmonella are able to manipulate endosomal trafficking
and recruit defense factors to the maturing vacuole60. S. enterica Typhimurium, for example, is able
to reduce the recruitment of NADPH oxidase and inducible nitric oxide synthase (iNOS) to the
vacuole through interference with vacuolar trafficking, thereby preventing oxygen radical production
and bacterial killing in macrophages61-63. The fact that many different Salmonella mutants that are
able to down-regulate host iNOS activity could be isolated in a screen of macrophage-adapted
bacteria suggests that Salmonella use several strategies for interfering with the host NO
response60. And like many other bacteria, Salmonella is able to detoxify oxygen radicals
enzymatically64. M. tuberculosis inhibits phagosomal maturation by depleting H+ ATPase molecules
from the vacuolar membrane65. This leads to reduced acidification and allows intracellular survival
and growth.
Resistance to humoral defense mechanisms
Successful escape by microbes from internalization by phagocytes opens the way for systemic
spread in the host via the blood or lymph vessels. However, the limited supply of essential nutrients
such as iron requires a high degree of adaptation to this environment. This is illustrated by the
example of Yersinia, which carries genes that encode high-affinity uptake systems for ferric iron. In
addition, bacteria will encounter humoral defenses. Soluble factors such as the C-reactive protein
(CRP), mannan-binding lectin (MBL) and serum amyloid protein (SAP) are produced by the liver
and function as opsonins. CRP and MBL also act as alternative recognition molecules for the
antibody-independent activation of complement by binding to C1q, the activator of the classical
complement activation pathway. Both S. pyogenes and Streptococcus pneumoniae possess
surface structures that bind the complement regulatory component factor H66, 67. Factor H binding
consequently promotes complement factor I–mediated degradation of C3b deposited on the
bacterial surface and inhibits the release of chemotactic molecules, such as C5a and C3a, as well
as formation of the membrane attack complex. Additionally, certain bacteria express proteases that
degrade C1q, C3, C4 and C5-C968. As we mentioned before, intracellular persistence and
proliferation, such as that seen with S. enterica Typhimurium, represents an opposite yet similarly
effective strategy for avoiding the limited growth factors as well as soluble humoral defense
molecules.
Bacterial interference with cytokine secretion
The innate immune system is clearly critical in the early control of bacterial replication and
successful eradication of an infection. It is also linked to the adaptive immune response, which
helps clear the infection and builds specific immunity with a memory component. Activation of the
adaptive response occurs through cytokine secretion, antigenic processing and presentation as well
as proliferation and differentiation of effector cells. Secretion of cytokines—particularly by effector T
cells—killing of cells harboring intracellular pathogens by cells with cytolytic activity—such as CD8+
T cells—and antibody production by B cells all then contribute to controlling bacterial infections.
Examples of the strategies bacteria use to deal with this complex defense network are shown (Fig.
3).
High resolution image and legend (57K)
the adaptive immune response.
Figure 3. Bacterial defense strategies against
Strategies include the induction of immunosuppressive cytokines, such as IL-10, IL-6 and TGF(1); inhibition of pro-inflammatory cytokine production; and surface expression of costimulatory
molecules such as CD86 (2) by antigen presenting cells (APC). Interference with bacterial uptake
(3), phagosome maturation (4) and antigen processing (5) as well as MHC class I and II expression
(6) also lead to diminished antigen presentation. Inhibiting tyrosine phosphorylation of the T and B
cell receptors (7) and activating the inhibitory CEACAM1 receptor on T cells (8) further decreases
effector cell function. Certain bacteria can also induce regulatory T cells (formerly called suppressor
T cells) that dampen the immune response (9) or induce T cell apoptosis by enhancing FasL
expression on T cells (10).
The production of pro-inflammatory cytokines such as tumor necrosis factor- (TNF- ), interleukin
1 (IL-1), IL-8 and IL-12 by host cells upon sensing bacterial products is crucial in the innate and
adaptive immune responses to infection. These cytokines play a role in enhancing the bactericidal
capacity of phagocytes, recruiting additional innate cell populations to sites of infection, inducing
dendritic cell maturation and directing the ensuing specific immune response to the invading
microbes. Some bacterial pathogens have evolved mechanisms for modulating cytokine production
by host cells, which modifies the host's subsequent immune response.
Mycobacteria provide a good example of bacterial manipulation of the cytokine response. These
bacteria can induce the production of anti-inflammatory cytokines, which dampen the immune
response. Mycobacteria-infected macrophages produce IL-6, which inhibits T cell activation69, as
well as the potent immunosuppressive cytokines IL-1070 and transforming growth factor- (TGF)71. IL-10 is immunosuppressive in several ways72, including the inhibition of macrophage activation
and production of reactive oxygen and nitrogen intermediates, suppression of inflammatory cytokine
production as well as down-regulation of the production of molecules important in triggering specific
immunity (for example the major histocompatibility complex (MHC) class II antigen presentation
complex and the costimulatory molecule CD86). Mycobacterium-induced production of
immunosuppressive cytokines may also contribute to the generation of regulatory T cells, also
called T suppressor cells, that down-regulate immune activation. For example, aerosol treatment of
mice with killed Mycobacterium vaccae induces regulatory T cells that prevent airway inflammation
in an IL-10 and TGF- –dependent manner73. Similarly, B. pertussis exploits IL-10 in order to downregulate the host immune response. Bordetella filamentous hemagglutinin (FHA) induces IL-10
production by dendritic cells. This induces naïve T cells to develop into regulatory cells that
suppress interferon- (IFN- ) production by antigen-specific T cells74. Also the LcrV protein
produced by Y. enterocolitica induces macrophages to secrete IL-10, which, in turn, suppresses
TNF- production75. Thus, bacterial exploitation of host cell capacity to produce
immunosuppressive cytokines, particularly IL-10, provides an effective means for invading microbes
to modulate host defense mechanisms and evade immune recognition.
Certain bacteria have evolved mechanisms for interfering with the signal transduction pathways
important in regulating expression of cytokines and other proteins involved in inflammation. For
example, YopP from Y. enterocolitica and YopJ from Y. pseudotuberculosis inhibit NF- B and
MAPK (mitogen-activated protein kinase) signal transduction pathways 76-78. Thus, Yersinia avoids
the detrimental effects of pro-inflammatory cytokines secretion by suppressing TNF, IL-1 and IL-8
production. In contrast to Yersinia inhibition of NF- B activation, the intracellular pathogen L.
monocytogenes activates this transcription factor as a potential means of increasing its
pathogenicity79, 80. Listeria-mediated NF- B activation of endothelial cells results in increased
expression of the adhesion molecules intercellular adhesion molecule 1 (ICAM-1) and E-selectin,
and secretion of IL-8 and macrophage chemoattractant protein 1 (MCP-1)80. This attracts circulating
phagocytes and promotes diapedesis and tissue infiltration. This "Trojan horse" mechanism directs
Listeria-infected phagocytes to the subendothelial space, facilitating tissue spread and bacterial
dissemination.
Bacterial interference with antigen presentation
Interfering with antigen processing and presentation is another strategy used by bacterial
pathogens to prevent stimulation of an adaptive immune response. For example, S. enterica
Typhimurium mutants that constitutively express the phoP-phoQ regulatory locus, which is
important for survival in macrophages and bacterial virulence, are inefficiently processed by
macrophages for MHC class II presentation81, 82. Similarly, the vacuolating toxin VacA produced by
H. pylori diminishes the capacity of antigen-presenting cells to degrade internalized antigens83.
Also, M. tuberculosis shows several strategies for suppressing antigen presentation and T cell
activation, including inhibition of phagosomal maturation (see above) and sequestration of
mycobacterial antigens from molecules required for T cell stimulation, such as MHC class II
presentation84-86. Mycobacteria also down-regulate surface expression of MHC class II and CD1
and interfere with the presentation of antigens by MHC class II molecules 87-90. Mycobacteria inhibit
the transcription of IFN- –responsive genes, including the master regulator for MHC class II
expression, the class II transactivator (CIITA)89, 91. As MHC class II expression by resting
macrophages is very low and IFN- is a potent inducer of MHC class II on these cells, the capacity
of Mycobacteria to inhibit MHC class II expression by interfering with IFN- –mediated signaling
pathways provides a potent means for dampening critical CD4+ T cell responses to this bacterium.
Chlamydia trachomatis, a sexually transmitted pathogen that causes urogenital tract and ocular
infections, also inhibits surface expression of MHC molecules on infected cells 92, 93. Like
Mycobacteria, C. trachomatis inhibits IFN- –inducible MHC class II expression by interfering with
CIITA activation. The mechanism used by Chlamydia to inhibit activation of CIITA involves
degrading the upstream stimulatory factor 1 (USF-1) which is required for IFN- –mediated CIITA
induction and, thus, IFN- –inducible MHC class II expression92. In addition, C. trachomatis
suppresses both constitutive and IFN- –inducible MHC class I expression on infected cells by
degrading the transcription factor regulatory factor X 5 (RFX5) in addition to degrading USF-193. As
regulatory factor RFX5, a key component of the RFX transcription complex, is required for both
constitutive and IFN- –inducible MHC class I expression and the RFX complex is required for MHC
class II transcription94, the ability of C. trachomatis to degrade these transcription factors provides
an effective means of blocking adaptive immunity.
Inhibiting T and B cell effector functions
Some bacteria interfere with the capacity of T and B cells—the effector cells of the adaptive immune
system—to carry out their functions. For example, H. pylori Cag pathogenicity island-encoded
genes induce Fas ligand (FasL) expression on T cells and mediate apoptosis 95. YopH from Y.
pseudotuberculosis also suppresses antigen-specific T cell activation and IL-2 production by
inhibiting tyrosine phosphorylation of components of the T cell receptor 96. YopH also inhibits
tyrosine phosphorylation of components of the B cell receptor and suppresses up-regulation of the
costimulatory molecule CD86 after B cell receptor engagement with antigen96. The YopHdependent inhibition of signaling cascades associated with antigen receptor engagement is an
additional immune evasion strategy to the above-mentioned capacity of YopH to impair bacterial
internalization97.
Another bacterium that exploits the host receptor signal transduction machinery in order to
modulate immunity is Neisseria gonorrhoeae, a sexually transmitted pathogen that causes
urogenital infection. One class of the multiallelic, phase-variable Neisseria OPA proteins—which
bind various ligands and mediate uptake by host cells—binds members of the CD66 receptor
family, also known as carcinoembryonic antigen–related cellular adhesion molecule (CEACAM)
family. CECAM1 is the only CEACAM molecule expressed on human lymphocytes, and the
presence of an immunoreceptor tyrosine-based inhibitory motif (ITIM) in its cytoplasmic tail
highlights its role as a coinhibitory receptor on CEACAM1+ cells. N. gonorroheae expressing a
CEACAM1-binding OPA protein inhibits the activation and proliferation of CD4+ T cells stimulated
by ligation of the T cell receptor98. This inhibitory effect was associated with increased recruitment
of two tyrosine phosphatases, SHP-1 and SHP-2, that are critical to the inhibitory function of ITIMcontaining receptors98. Thus, the capacity of N. gonorrohea to inhibit T cell activation in addition to
antigenic variation of surface proteins including OPAs99 likely contributes to the poor specific
immune response observed in Neisseria-infected individuals.
Our current knowledge of the strategies used by bacteria to interfere with innate and adaptive
immunity and escape host defenses is largely incomplete. Nevertheless, the diverse disciplines of
immunology, microbiology, infectious diseases and cell biology have contributed much to the
exciting progress we have made in our knowledge over recent years. These studies have also
revealed the complex interplay between microbial pathogens and higher organisms. However,
when the role played by the host's normal microbial flora is included in the analysis, the complexity
of bacteria-host interactions is even greater. How does the host differentiate between its responses
to pathogens and commensals? One explanation is that the mucosal linings are tolerant to
microbes at locations colonized by the normal flora, and that innate responses are induced only
after bacterial intrusion beyond these barriers. However, even in the absence of pathogenic
microorganisms, host defense mechanisms are required to maintain the integrity of the anatomical
barrier against the resident microbial flora.
The need for continuous vigilance is illustrated by mice that are deficient in the production of
bactericidal oxygen and nitrogen intermediates (gp91phox-/-NOS2-/-). Such mice spontaneously
develop massive abscesses that are caused by the normal flora of the intestine, respiratory tract
and skin100. On the other hand, down-regulation of pro-inflammatory responses as well as
enhancement of the intestinal barrier function appear to represent important functions of the normal
microbial flora. The intestinal commensal Bacteroides thetaiotamicron protects host cells from
complement-mediated cytotoxicity via the up-regulation of DAF (decay-accelerating factor), a
central regulator of complement deposition on nucleated cells, and simultaneous enhancement of
cutaneous repair and barrier functions101. Therefore, one might assume that commensals, like
pathogens, express factors that directly or indirectly interfere with immune defense. However, the
extent to which commensal microbes apply similar strategies remains an important question that
needs to be addressed. Another important factor that should be taken into account is the
heterogeneity of the host population: the genetic polymorphisms of receptor or effector molecules,
and also the diversity of environmental conditions including the constitution of the resident
microflora. Upcoming studies will undoubtedly reveal further surprising details of the intriguing
relationship between bacteria and host immunity, and will hopefully provide us with the knowledge
to improve the prevention and treatment of infectious diseases in the near future.
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Acknowledgments. Supported by grants from the Deutsche Forschungsgemeinschaft and the
Karolinska Institutet (to M. W. H.); the Swedish Medical Research Council (to M.-J. W., M. R. and S.
N.), the Foundation for Strategic Research (to M. R.) and the Swedish Cancer Foundation (to S.
N.).
Figure 1. Strategies for bacterial escape from epithelial defense mechanisms.
Prevention of opsonization (1) is required to facilitate colonization of host surfaces
(2). Toxin secretion can paralyze the host's defenses (3) and disrupt its mucosal
integrity (4). Microbial recognition and host responses—such as the secretion of
antimicrobial peptides (5) or chemokine production (6)—can be impaired by
modification of pattern molecule presentation or interference with intracellular
signaling or cell trafficking (8). Microbe-induced self-uptake (7) and escape from the
phagosome along with inhibition of intracellular recognition (9) or persistence in
modified endosomes (10) can then impede removal by host defense mechanisms.
Green, host responses; orange, bacterial components and interference with host
defense strategies.
Figure 2. Bacterial defense against phagocytes. Bacterial defense strategies
against phagocyte engulfment include the induction of programmed cell death (1)
as well as inhibition of uptake (2) by translocated effector proteins. Effector proteins
can also be used to down-regulate other host cell nuclear responses (3). Should
phagocytosis occur, bacteria can escape from the endosome into the host cell
cytosol (4) or interfere with endosomal trafficking as well as maturation of the
phagosome (5) and the subcellular localization of defense factors (6).
Figure 3. Bacterial defense strategies against the adaptive immune response.
Strategies include the induction of immunosuppressive cytokines, such as IL-10, IL6 and TGF- (1); inhibition of pro-inflammatory cytokine production; and surface
expression of costimulatory molecules such as CD86 (2) by antigen presenting
cells (APC). Interference with bacterial uptake (3), phagosome maturation (4) and
antigen processing (5) as well as MHC class I and II expression (6) also lead to
diminished antigen presentation. Inhibiting tyrosine phosphorylation of the T and B
cell receptors (7) and activating the inhibitory CEACAM1 receptor on T cells (8)
further decreases effector cell function. Certain bacteria can also induce regulatory
T cells (formerly called suppressor T cells) that dampen the immune response (9)
or induce T cell apoptosis by enhancing FasL expression on T cells (10).
Focus on Immune Evasion
Volume 3 No 11 November 2002
Review
Nature Immunology 3, 1041 - 1047 (2002)
doi:10.1038/ni1102-1041
© Nature America, Inc.
<>
Evasion of innate immunity by parasitic protozoa
David Sacks & Alan Sher
Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD
20892, USA.
Correspondence should be addressed to D Sacks [email protected]
Parasitic protozoa are a major cause of global infectious disease. These eukaryotic
pathogens have evolved with the vertebrate immune system and typically produce longlasting chronic infections. A critical step in their host interaction is the evasion of innate
immune defenses. The ability to avoid attack by humoral effector mechanisms, such as
complement lysis, is of particular importance to extracellular parasites, whereas intracellular
protozoa must resist killing by lysosomal enzymes and toxic metabolites. They do so by
remodeling the phagosomal compartments in which they reside and by interfering with
signaling pathways that lead to cellular activation. In addition, there is growing evidence that
protozoan pathogens modify the antigen-presenting and immunoregulatory functions of
dendritic cells, a process that facilitates their evasion of both innate and adaptive immunity.
Parasitic protozoa are unicellular eukaryotic pathogens that dwell inside host cells and/or in
extracellular fluids. These organisms typically produce long-lasting chronic infections in order to
maximize their opportunities for successful transmission by contact with their intermediate hosts or
release into the environment. Their success as parasites depends on a series of intricate and highly
evolved host adaptations that enable them to evade destruction by the immune system.
Protozoan infections are a major global health problem, affecting over half a billion people world
wide. Several of the diseases they induce (such as malaria, African trypanosomiasis and visceral
leishmaniasis) represent major causes of mortality and morbidity in tropical countries and, as such,
are important impediments to economic development. No standardized vaccines exist for
preventing any of the protozoan infections of humans, a situation attributed both to the complexity
of the pathogens involved and their sophisticated strategies for evading host immune responses 1.
Protozoan parasites are highly adept at escaping the effects of adaptive humoral and cellular
immunity. Perhaps the simplest solution for evading antibody responses is the adoption of an
intracellular life style, as is done by Leishmania, Trypanosoma cruzi, Toxoplasma gondii and the
liver and blood stages of malaria. Another major strategy—antigenic variation—protects
extracellular protozoa such as African trypanosomes2 and Giardia3 and even malaria parasites4,
which express their antigens on the surface of infected erythrocytes, from immune recognition. In
addition, there is abundant evidence that protozoan infections actively regulate adaptive T cell
responses, resulting in suppressed effector functions 1. A striking example of this phenomenon is the
recent demonstration that Leishmania major actively induces interleukin 10 (IL-10)–producing
CD25+ T regulatory cells to prevent complete clearance of the parasite5.
Although parasitic protozoa have provided some of the best studied paradigms of evasion of
antibody- and T cell–mediated immunity by pathogens, a series of equally important adaptations
occur during the initial establishment of infection, when parasitic invaders confront the innate
immune system. The innate host defenses that the major blood and tissue protozoan pathogens of
humans must penetrate, modify or avoid vary according to their mode of entry and the primary host
tissue encountered (Table 1). These innate defenses include the epithelial barrier of the skin, the
alternative complement cascade and other lytic serum components, lysosomal hydrolases and toxic
oxygen metabolites of mononuclear phagocytes and the antigen-presentation and
immunoregulatory functions of dendritic cells (DCs), which provide a crucial link with the adaptive
immune response6. That protozoa have evolved specific mechanisms to evade these defenses is
dramatically shown by the rapid destruction of preinfective vector-derived life cycle stages when
artificially inoculated into mammalian hosts. These developmental adaptations, which enable blood
and tissue protozoa to "run the gauntlet" of the vertebrate innate response, are the focus of our
review.
High resolution image and legend (31K)
humans
Table 1. The major protozoan pathogens of
Evasion of humoral innate defenses
Innate resistance to protozoa is mediated, in part, by pre-existing soluble factors that can potentially
recognize and destroy invading parasites or target them for killing by effector cells. The alternative
pathway of complement activation provides a first line of defense against extracellular parasites that
must be subverted for infection to proceed. For example, whereas the epimastigote stage of T. cruzi
found in insect vectors is susceptible to the alternative complement pathway, infective metacyclic
and blood-stream trypomastigotes are resistant7. In this case, evasion of complement appears to be
due to trypomastigote expression of a 160-kD glycoprotein (gp160), which is a homolog of the host
complement-regulatory protein decay-accelerating factor (DAF)8. Like DAF, gp160 can bind to C3b
and C4b and inhibit the uptake of subsequent members of the complement cascade, thus
preventing convertase formation and lysis of the parasite. Importantly, whereas complementsensitive epimastigotes fail to express gp160, epimastigotes transfected with gp160 are resistant to
complement-mediated lysis9.
Another interesting strategy is deployed by Leishmania species, for which the infective insect stage
is transiently exposed to potentially lethal serum components after inoculation by pool-feeding
phlebotomine vectors. Leishmania evade complement-mediated lysis while using complement
activation as a mechanism for targeting host cells. When insect-stage procyclic promastigotes
develop into infective metacyclic forms, their membrane is altered to prevent insertion of the lytic
C5b-C9 membrane attack complex (MAC)10. This correlates with their expression of a modified
surface lipophosphoglycan (LPG) that is approximately twice as long as the form on procyclic
promastigotes, and which may act as a barrier for insertion of the MAC into the parasite surface
membrane11. A further developmental change that occurs during generation of metacyclics is
enhanced synthesis of the surface proteinase gp63, which can cleave C3b to the inactive iC3b
form, thus preventing deposition of the lytic C5b-C9 complex12. However, iC3b also opsonizes the
parasites for phagocytosis through the complement receptors CR3 and CR1, thereby targeting the
parasite to the macrophage, its host cell of choice13. Conclusive evidence that LPG and gp63 are
virulence factors has emerged from studies with L. major null-mutants that do not express these
molecules; in each case these mutants were highly sensitive to complement-mediated lysis and
showed reduced virulence in BALB/c mice14, 15.
In addition to complement, protozoa must evade other soluble mediators of innate immunity in order
to establish a foothold in the host. Perhaps the best studied are the primate-specific trypanosome
lysis factors (TLFs)16, which contribute to the resistance of humans to infection with Trypanosoma
brucei, an important pathogen of livestock. Biochemical analysis of the activity present in human
serum revealed that high-density lipoproteins are part of the substance that mediates cytolysis of
the parasite, and initial studies demonstrated that this complex, TLF1, is composed of several
common apolipoproteins as well as a haptoglobin-related protein (Hpr)17, 18. A second cytolytic
complex, TLF2, has also been identified that shares many of the components of TLF1 but contains
a distinct immunoglobulin M component and a lower lipid content19. Although the final lytic event
has not been delineated, TLF must undergo receptor-mediated uptake and enter an intracellular
acidic compartment for cytotoxicity to occur18. Whereas T. brucei is sensitive to TLF-mediated
killing, the species that infect humans—T. brucei gambiense and T. brucei rhodesiense—are both
refractory. This resistance is associated with a block in TLF endocytosis and retention of the
complex in a structure known as the flagellar pocket16. The refractoriness of certain T. brucei
rhodensiese strains to lysis also correlates with the expression of a serum resistance–associated
(SRA) gene that is equivalent to truncated variant surface glycoprotein20. Importantly, transfection of
SRA from T. brucei rhodesiense into T. brucei confers resistance to lysis by human serum; this
argues that its expression may have been a critical step in the adaptation of an ancestral parasite
for infection of primates21. Interestingly, antibodies raised against a homolog of SRA localize to the
flagellar pocket, suggesting a possible role for the protein in TLF retention22.
Remodeling host-cell compartments
Parasitic protozoa that are adapted to an intracellular lifestyle must resist the antimicrobial
mechanisms that can be induced in phagocytic and even nonphagoctyic host cells. Insofar as the
acidified hydrolytic environment of host cell lysosomes represents the heart of the defensive
machinery of many nucleated cells, the ability of acid-labile protease-sensitive parasites to avoid or
modify this compartment is likely to be one key to their survival (Fig. 1). T. gondii resides in a
phagosome that restricts its fusion with host endosomes and lysosomes. Toxoplasma actively
penetrates both phagocytic and nonphagocytic cells, propelled by an actin-myosin–dependent
gliding motility23. In the process, it establishes a nonfusigenic compartment, called the
parasitophorous vacuole (PV), that lacks integral membrane proteins of host cell origin, but is
extensively modified by secreted parasite proteins24, 25. This remodeling seems to be crucial to the
inhibition of PV acidification and lysosome fusion, as these events proceed normally after uptake of
dead or opsonized parasites, which are internalized via classical receptor-mediated phagocytosis.
High resolution image and legend (64K)
Figure 1. Remodeling of macrophage
intracellular compartments by parasitic protozoa.
T. cruzi trypomastigotes enter the macrophage by inducing the recruitment of lysosomes to the
plasma membrane; they only transiently reside in the parasitophorous vacuole before escape into
the cytoplasm via secretion of a pore-forming molecule, termed Tc-TOX (yellow). T. gondii
tachyzoites actively invade the cell and remodel a parasitophorous vacuole membrane (blue) that
contains secreted parasite proteins but excludes host proteins that would normally promote
phagosome maturation, thereby preventing lysosome fusion. Leishmania metacyclic promastigotes
are taken up by receptor-mediated phagocytosis; phagosome maturation may be transiently
inhibited by LPG (green), which becomes incorporated into the phagosome membrane. The
replicating amastigote stage ultimately resides within a phagolysosome where they survive via
production of cell-surface and secreted glycoconjugates, including GIPLS and
proteophosphoglycan (PPG) (green).
T. cruzi trypomastigotes also actively invade mammalian cells but, unlike T. gondii, their cell entry is
dependent not on the propulsive properties of the parasite, but on their ability to subvert a Ca 2+regulated lysosomal exocytic pathway23. The membrane of the parasitophorous vacuole is,
therefore, derived from the membrane of lysosomes, and the vacuole itself remains acidic and
potentially fusigenic with other lysosomes. T. cruzi growth and development cannot be sustained
within this compartment; it depends instead on the ability of the parasite to escape rapidly into the
cytosol. Exit from the vacuole is mediated by a parasite-secreted molecule, Tc-TOX, which has
membrane pore-forming activity at acidic pH and is facilitated by a trans-sialidase present on the
trypomastigote surface26, 27. Interestingly, T. cruzi is unable to invade cells lacking transforming
growth factor- (TGF- ) receptors I or II28; this suggests that triggering this signaling pathway,
perhaps by some parasite TNF- homolog, might be needed to deactivate host cells during the
early stages of infection.
Leishmania parasites lack the machinery necessary for active invasion and are confined instead to
professional phagocytes, mainly macrophages, with some exceptions (for example, fibroblasts, DCs
and neutrophils)29. The uptake of Leishmania by macrophages proceeds via conventional receptormediated phagocytosis that involves a diversity of opsonic or pattern-recognition receptors (for
example, CR3, CR1 and mannose fucose receptors)30 that are used depending on the species and
stage of parasite and the presence or absence of fresh serum. Leishmania metacyclic
promastigotes and amastigotes do not seem to remodel the phagosome in a major way because it
rapidly fuses (within 30 min) with late endosomes or lysosomes 31, generating a parasitophorous
vacuole that maintains an acidic pH and hydrolytic activity. Using macrophage cell lines and
unselected promastigotes, researchers have shown that LPG-repeating units can transiently inhibit
phagosome maturation and that this delay may be necessary to allow sufficient time for
promastigotes to differentiate into more hydrolase-resistant amastigotes32, 33. These findings are
consistent with the attenuation of L. major promastigote survival within macrophages after infection
with targeted null mutants lacking LPG14. The general significance of the transient inhibition of
phagosome-endosome fusion has been questioned, however, for two reasons. First, Leishmania
mexicana promastigote mutants deficient in LPG or other phosphoglycan-containing molecules
survive equally well as wild-type organisms within macrophages34, and second, there is an absence
of direct data to indicate that mature promastigotes are any less hydrolase-resistant than
amastigotes. That amastigotes are remarkably robust is clear: the parasite does not escape from
the vacuole, but manages to withstand the low pH and onslaught of acid hydrolases, presumably by
producing an abundance of cell surface and secreted glyconjugates that protect the cell from
proteolytic damage. As the L. mexicana phosphoglycan–deficient mutants were able to survive
normally, it is likely that this species displays different or redundant virulence determinants. It is
possible, for example, that the huge parasitophorous vacuole that is formed after infection with
parasites of the L. mexicana complex might effectively dilute the hydrolases to which the parasite is
exposed within the vacuole and obviate the need for certain surface or secreted glycans.
Inhibition of host cell signaling pathways
Macrophages possess primary defense mechanisms—including activation of macrophage oxidative
metabolism and synthesis and release of arachidonic acid metabolites—that are induced by the
attachment and engulfment of microbial agents. The major source of reactive oxygen intermediates
(ROIs) in macrophages is the NADPH oxidase, a multicomponent enzyme that catalyzes the
transfer of electrons from NADPH to molecular oxygen, resulting in the production of superoxide
and hydrogen peroxide35. It is generally believed that activation of protein kinase C (PKC) and
protein tyrosine kinases (PTKs) are the two critical events involved in regulating phagocyte
functions in response to a variety of extracellular stimuli. In vitro studies involving human cells have
shown that macrophage functions, including phagocytosis and ROI generation, are severely
impaired after uptake of an insoluble degraded host hemoglobin, called hemozoin, generated during
blood-stage malarial infection36 (Fig. 2). These dysfunctions were attributed to inhibition of PKC
translocation and activation in the hemozoin-loaded cells37. Early studies suggested that
Leishmania parasites also avoid triggering the oxidative burst by actively inhibiting PKC activation in
macrophages38, 39. Inhibition of PKC-mediated protein phosphorylation was observed with purified
LPG40, which might act either as competitive inhibitor of the PKC activator DAG and/or by altering
the physical properties of the bilayer to inhibit PKC membrane translocation. These findings are
again consistent with impaired intracellular survival of the L. major LPG–deficient mutant compared
to the mutant in which LPG expression was restored, although effects on specific signaling events
were not directly compared14.
High resolution image and legend (75K)
pathways.
Figure 2. Inhibition of macrophage signaling
Inhibition of pathways by malaria-generated hemozoin or infection with Leishmania or T. gondii.
Hemozoin and Leishmania impair the oxidative burst associated with phagocytosis by inhibiting the
PKC activation required for assembly of the NADPH oxidase complex in its active form. Leishmania
parasites also inhibit IFN- – or CD40L-induced PTK-dependent signaling involved in IL-12
production by activation of the cellular phosphatase SHP-1 that inhibits Jak2 and STAT1
phosphorylation (P). Toxoplasma inhibits LPS-induced cytokine responses by inhibiting nuclear
translocation of NF- B and possibly phosphorylated STAT1.
In addition to their innate microbicidal responses, macrophages can initiate the host activation
cascade by presenting antigens and costimulatory molecules and by providing regulatory cytokines
to T cells. A number of studies suggest mechanisms by which intracellular protozoa can interfere
with the immune-initiation functions of macrophages. One of the more consistent and striking
dysfunctions observed in macrophages infected with protozoan parasites is their inability to produce
IL-12, which—as the main physiological inducer of interferon- (IFN- ) and T helper type 1 (TH1)
cell differentiation—is an essential cytokine for the development of acquired resistance to most
intracellular pathogens. In addition, excess IL-12 production can lead to severe tissue damage and
even mortality, a consequence that may also be detrimental to the parasite in blocking its
transmission cycle. It is no wonder then that protozoa have evolved a series of intricate and often
redundant mechanisms for regulating IL-12 production by antigen-presenting cells, especially
macrophages, which are the major potential source of the cytokine stimulated during early infection.
Infectious stages of Leishmania do not merely avoid IL-12 induction, they actively and selectively
inhibit it, leaving other pro-inflammatory cytokine or chemokine response pathways relatively
intact41. Whereas the glycophosphatidylinositol (GPI) anchors of many parasites are potent
macrophage activators, Leishmania LPG and glycoinositol phospholipid (GIPL) inhibit IL-12p40
transcription while failing to suppress TNF gene expression42. Although the receptors and signaling
pathways involved in this selective inhibition of IL-12 synthesis have yet to be identified,
suppression of NF- B activity does not appear to be involved. Because many IL-12 agonists that are
inhibited in Leishmania-infected macrophages—including lipopolysaccharide (LPS), CD40 ligand
(CD40L) and especially IFN- —signal primarily through protein-tyrosine kinases, the observation
that Janus kinase–signal transducers and activators of transcription (Jak-STAT) signaling pathways
are also inhibited in Leishmania-infected cells seems especially relevant to the defective IL-12
response43. Defective phosphorylation of Jak2 is attributed to rapid activation of a cytoplasmic
protein tyrosine phosphatase (PTP), SHP-144 (Fig. 2). SHP-1 is necessary for L. major survival
insofar as SHP-1–deficient motheaten mice do not produce lesions and their macrophages fail to
support infection in vitro45. Because specific ligation of the macrophage receptors CR1 and CR3
also leads to selective inhibition of IL-12 production—which, at least in the case of CR3, is
associated with impaired tyrosine phosphorylation of STAT146—it is likely that host cell PTPs are
rapidly induced by CR3 ligation during attachment and uptake of serum-exposed C3-opsonized
parasites.
Given that Jak-STAT signaling pathways are involved in a broad range of macrophage responses,
including induction of most pro-inflammatory cytokines, it is difficult to understand how Leishmania
infection maintains such a selective effect on IL-12. One possible explanation lies in two
observations: first, STAT1 regulates IFN consensus-binding protein (ICSBP) induction and—of the
cytokines assayed—only activation of the IL-12p40 promoter appears to require ICSBP, and
second, ICSBP knock-out mice (on resistant background) are susceptible to L. major infection47.
The involvement of redundant STAT1-independent induction pathways for tumor necrosis factor(TNF- ), IL-1 and inducible nitric oxide synthase (iNOS) has been described (see below) and
suggests a basis for the maintenance of these responses in Leishmania-infected cells. A similar
mechanism may account for the down-regulation of IL-12p40 gene expression in mouse
macrophages after uptake of Plasmodium berghei–infected erythrocytes48, which appear to be
selective for IL-12 also and do not appear to be due to impaired NF- B activation.
The NF- B family of transcription factors is an evolutionarily conserved group of proteins that are
important in the regulation of numerous genes involved in innate and adaptive immunity—including
those encoding IL-12, IFN- , TNF- , iNOS and adhesion molecules—as well as those involved in
cell proliferation and survival. Studies in T. gondii49, 50 have demonstrated the ability of intracellular
protozoa to actively interfere with the NF- B–activation pathway in macrophages. In T. gondii–
infected cells, despite rapid I B phosphorylation and degradation, NF- B fails to translocate to the
nucleus. In contrast to Leishmania, T. gondii–induced response defects are more generalized and
include both IL-12 and TNF- . With the use of virulent strains of T. gondii, the inhibition of NF- B
translocation in macrophages and its effect on iNOS expression was shown to be dependent on
HS70 expression by the parasite that might impede nuclear transport by competing for access to
nuclear pore complexes51. Disrupted nuclear transport of phosphorylated STAT has also been
reported for T. gondii–infected macrophages and is thought to inhibit Jak-STAT–dependent MHC
class II expression51 (Fig. 2).
In addition to being directly suppressed by the parasite within infected cells, macrophage IL-12
production and effector functions can also be held in check by the down-modulatory cytokines IL-10
and TGF- , which can themselves be up-regulated by the parasites or their products. Striking
evidence in support of the importance of IL-10–dependent down-regulation of IL-12 production for
both host and parasite comes from studies in IL-10–deficient mice infected with either T. gondii or T.
cruzi. These animals display dysregulated IL-12 production and die of cytokine-associated tissue
damage while showing decreased parasite burdens 52, 53. Similarly, low amounts of active TGFproduction by splenic mononuclear cells are associated with a lethal outcome in rodent malaria, and
anti–TGF- treatment transformed a normally resolving infection into a lethal one due to
overproduction of pathogenic pro-inflammatory cytokines54. With respect to Leishmania, there is as
yet no evidence that deficient production of these anti-inflammatory mediators is responsible for
severe clinical outcomes. In contrast, there is ample evidence to suggest that overproduction of
these cytokines contribute to uncontrolled parasite growth and nonhealing infections. Both IL-10
and TGF- are produced by murine macrophages after Leishmania infection in vitro, and both
promote intramacrophage replication and are important factors for determining in vivo
susceptibility55, 56.
Finally, parasitic protozoa have evolved strategies to down-modulate signaling pathways leading to
host cell apoptosis, thereby prolonging the life of the host cell and their own intracellular survival 57.
Induced apoptotic pathways in macrophages infected with either Leishmania donovani
promastigotes58 or T. gondii tachyzoites59 were strongly inhibited, perhaps by parasite induced upregulation of Bcl-2 homologs. Interestingly, the pro-apoptotic effects that T. cruzi infection has on
both CD4+ and CD8+ T cells indirectly enhanced parasite growth, as the binding of apoptotic
lymphocytes to vitronectin receptors on macrophages triggered TGF- production and a burst of
parasite replication in infected cells60.
Manipulation of DC function
To the extent that pathogenic protozoa have learned to condition their initial encounter with the
innate immune system as a means of manipulating the subsequent adaptive immune response, it
has been important to extend these observations to effects on "professional" antigen-presenting
cells. In contrast to primary targets of infection, such as macrophages, DCs may be temporally
removed from encounter with parasites or their products and may respond distinctively. For
example, whereas IL-12 production is actively suppressed in macrophages infected with L. major or
T. gondii, DCs actively produce IL-12p40 in response to the same parasites, both in vitro and in
vivo61-65. It should be noted that in each case the parasite infection alone was not sufficient for high
IL-12p70 production in vitro and that endogenous agonists, such as CD40L or IFN- , were
necessary as costimuli. The different outcomes of protozoan encounter on IL-12 production by
macrophages versus DCs is consistent with the delayed, albeit effective, immune response to these
infections that is ultimately achieved. Apart from the obvious parasite advantage that delayed onset
of immunity brings to bear, suppression of IL-12 production by infected macrophages may again
represent a coadaptation that dampens their local activation while protecting the host from cytokine
shock.
For those parasitic infections associated with T cell control mechanisms that are not simply delayed,
but are persistently compromised, impaired maturation and function of appropriate DC subsets
might be more expected (Table 2). A particularly interesting example is the inhibition of DC
maturation by the malaria parasite Plasmodium falciparum66. Malaria-infected erythrocytes bind to
the surface of myeloid DCs in vitro and markedly suppress the normal up-regulation of major
histocompatibility complex (MHC) class II molecules, adhesion molecules (for example, ICAM-1)
and costimulatory molecules (CD83 and CD86) on the cells after stimulation with LPS. The resulting
DCs were severely impaired in their capacity to induce allogenic as well as antigen-specific primary
and secondary T cell responses67. The receptors on the surface of DCs mediating this inhibitory
effect are CD36 and CD51, and their artificial ligation mimicked the suppression induced by infected
erythrocytes68. Interestingly, the same receptors are also involved in the recognition of apoptotic
cells and, when incubated with DCs, such cells trigger reduced secretion of IL-12 and increased
production of IL-10 by these antigen-presenting cells. The major parasite ligand for CD36 binding in
infected erythrocytes is thought to be a conserved domain of P. falciparum erythrocyte membrane
protein 1 (PfEMP1), a molecule that undergoes antigenic variation. Thus, a nonadherent parasite
line, which does not express PfEMP1 antigens on the red cell surface, failed to inhibit DC
maturation.
High resolution image and legend (36K) Table 2. Impairment of DC function by parasitic
protozoa
The relevance of these observations to acute disease is indicated by the finding that the percentage
of HLA-DR+ DCs was significantly lower in children with severe or mild malaria compared to healthy
controls68. At present there is no direct evidence that impaired DC maturation in children with acute
malaria is CD36-mediated or that this event is at all necessary for successful malaria infection.
Nevertheless, it is intriguing that infected erythrocytes with high CD36 binding affinity are more
frequently observed in isolates from patients with mild as opposed to severe disease 69, 70. It
suggests that adhesion of infected erythrocytes to CD36 may suppress the pro-inflammatory
response to the parasite, an outcome that would favor both host and parasite.
Infection of monocyte-derived human DCs with T. cruzi trypomastigotes also inhibits LPS-induced
secretion of pro-inflammatory cytokines—including IL-12 and TNF- —as well as HLA-DR and
CD40 up-regulation71. Similar effects were observed when human DCs were exposed to T. cruzi–
derived GIPL or to only the ceramide portion of the compound72, although nonphysiological high
concentrations of both molecules were used in this in vitro study.
With respect to Leishmania, the spontaneous migration of murine splenic DCs or Langerhans cells
(LCs) was inhibited by L. major promastigote–conditioned media or L. major LPGs, respectively;
this suggested that their ability to transport antigen to or within lymphoid tissue might be impaired
during infection73, 74. These effects need to be interpreted, however, in the context of other studies
(referred to above). These studies involved infection of murine DCs with viable L. major
promastigotes or amastigotes; the infected cells up-regulated MHC class II and costimulatory
molecules, produced IL-12p40 and down regulated E-cadherin, which indicated that they were
competent for antigen transport and presentation61, 75. Uptake of Leishmania amazonensis
amastigotes or metacyclic promastigotes by mouse bone marrow–derived DCs (BMDCs) also
induced up-regulated expression of MHC class II, CD40, CD80 and CD86 but, in contrast to L.
major, the infected DCs produced IL-4 and no IL-1276. This suggested that the DCs were
conditioned by the parasite to prime the pathogenic T H2 cells that dominate the response in vivo.
Studies involving yet other Leishmania species suggest a more passive strategy for immune
evasion, in which their interactions with antigen-presenting cells appear to proceed in a relatively
silent fashion. Uptake of L. mexicana by mouse BMDCs was not sufficient to activate them in vitro,
although their maturation response to other stimuli was not impaired77. A similar species restriction
in Leishmania-induced DC activation was observed after uptake of metacyclic promastigotes by
human myeloid DCs, which were efficiently primed for CD40L-induced ILp70 secretion by L. major,
but not by L. tropica or L. donovani78. These observations may be significant because whereas L.
major produces self-limiting infections that are controlled in the skin, the other species mentioned
are capable of disseminating to the viscera or to other cutaneous sites. Given the well described
polymorphisms in the surface and secreted glycan structures that are expressed by different
Leishmania species79, it will be interesting to determine whether they account—at least in part—for
the differences in innate response patterns and the spectrum of clinical disease.
From the above discussion it would appear that parasites carefully regulate the induction of IL-12
from DCs during early infection as a means of determining the character of the adaptive immune
response that eventually decides their fate in the host. In addition, there is emerging evidence for
the existence of distinct parasite-triggered pathways for regulating DC IL-12 production once it has
been initiated. One such mechanism has been identified by studies of IL-12 synthesis by murine
splenic DCs after injection of a soluble extract of T. gondii tachyzoites (STAg). The response was
short-lived and could not be recalled by a second injection of STAg for a period of 1 week after
initial priming80. This "paralysis" of the DC IL-12 response induced by STAg does not require IL-10
but instead appears to depend on the induction of lipoxin A4 (LXA4), a product of arachadonic
metabolism81. This eicosonoid appears to function by suppressing CCR5 expression, a chemokine
receptor involved in IL-12 stimulation by STAg. The same mechanism of IL-12 suppression is likely
to operate during natural infection because T. gondii–infected mice with a defect in an enzyme (5LO) required for LXA4 show excess IL-12 production and, in common with infected IL-10–deficient
mice53, succumb to cytokine shock and show decreased parasite burdens (J. Aliberti et al.,
unpublished data). Taken together, these findings suggest that once IL-12 production by DCs has
been induced and TH1 responses initiated, synthesis of the cytokine is actively down-regulated,
probably to the joint benefit of both host and parasite. Whether analogous mechanisms suppress
the IL-12 responses of DCs to other protozoa remains to be determined.
Concluding comments
In learning to evade innate host defenses, protozoan parasites appear to have mastered the
intricacies of both cell biology and cellular immunology. As such, they can teach us important
lessons about both fields of biology as well as their points of intersection. In particular, the
strategies used by these pathogens for remodeling cellular compartments offer insights into the
dynamics of host membranes and intracellular trafficking. At the same time there is much to be
learned about host-signaling pathways and immune-response initiation and polarization by studying
the mechanisms used by protozoa to suppress or divert immune responses. Because innate
defenses (for example, lysis by complement or TLF) have the potential to block infection, the
mechanisms used to evade them are potential "Achilles' heels", and offer newly identified and
largely unexploited targets for vaccine and/or pharmacologic intervention.
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Figure 1. Remodeling of macrophage intracellular compartments by parasitic
protozoa. T. cruzi trypomastigotes enter the macrophage by inducing the
recruitment of lysosomes to the plasma membrane; they only transiently reside in
the parasitophorous vacuole before escape into the cytoplasm via secretion of a
pore-forming molecule, termed Tc-TOX (yellow). T. gondii tachyzoites actively
invade the cell and remodel a parasitophorous vacuole membrane (blue) that
contains secreted parasite proteins but excludes host proteins that would normally
promote phagosome maturation, thereby preventing lysosome fusion. Leishmania
metacyclic promastigotes are taken up by receptor-mediated phagocytosis;
phagosome maturation may be transiently inhibited by LPG (green), which
becomes incorporated into the phagosome membrane. The replicating amastigote
stage ultimately resides within a phagolysosome where they survive via production
of cell-surface and secreted glycoconjugates, including GIPLS and
proteophosphoglycan (PPG) (green).
Figure 2. Inhibition of macrophage signaling pathways. Inhibition of pathways
by malaria-generated hemozoin or infection with Leishmania or T. gondii.
Hemozoin and Leishmania impair the oxidative burst associated with phagocytosis
by inhibiting the PKC activation required for assembly of the NADPH oxidase
complex in its active form. Leishmania parasites also inhibit IFN- – or CD40Linduced PTK-dependent signaling involved in IL-12 production by activation of the
cellular phosphatase SHP-1 that inhibits Jak2 and STAT1 phosphorylation (P).
Toxoplasma inhibits LPS-induced cytokine responses by inhibiting nuclear
translocation of NF- B and possibly phosphorylated STAT1.
Table 2. Impairment of DC function by parasitic protozoa
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