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Transcript
CD8+ T Cell Response to Influenza
Virus
by
W
MG
Daniel Doty
HUSETTSIITUTE
OF TECHNOLOGY
SEP 17
B.S., Molecular and Cellular Biology (2002)
University of Arizona
;IRAFRIES_J
Submitted to the Department of Biology in partial fulfillment of the degree of
Master of Science in Biology
at the
Massachusetts Institute of Technology
September 2005
©2005 Massachusetts Institute of Technology. All rights reserved.
Signature of Author .:,.Department
of Biology
2 September, 2005
Certified by
Dr. Jianzhu Chen
Professor of Biology
Thesis advisor
Accepted by
Stephen P. Bell
Professor of Biology
Chair, Biology Graduate Committee
CD8+ T Cell Response to Influenza Virus
by
Daniel Doty
Submitted to the Department of Biology on 2 September 2005 in partial
fulfillment of the requirements for the degree of Master of Science in Biology
ABSTRACT
The flu is an extremely prevalent and potentially devastating disease, especially
dangerous to the very young, the elderly, and to people with compromised immune
systems. Influenza has a characteristic course of infection, and is often effectively
dispatched by the immune system. The cell-mediated lysis of infected cells is a
particularly important step in clearing the infection. Antigen specific CD8+ T
lymphocytes are selected and activated in the mediastinal lymph node, proliferate and
gain effector function, then migrate to the lungs, where they selectively destroy infected
cells. The CD8+ effector population pool undergoes a phase of contraction, when most
effector cells die. Those that survive become memory T cells, protecting the body from
subsequent influenza infections. The molecular and cellular interactions that comprise the
CD8+ cytotoxic T cell response to influenza virus are of particular interest because of
their implications for the prevention, treatment, and alleviation of the flu.
Thesis advisor: Jianzhu Chen
Title: Professor of Biology
9
Introduction
Influenza A virus is the cause of one of the most prevalent and harmful human
respiratory infections. Typical incarnations of the virus infect 10-20% of the population
of the United States, resulting in billions of dollars in lost production and health care
costs, as well as up to 40,000 deaths each year (1). Influenza became the agent of the
most devastating plague in human history when a particularly virulent strain killed an
estimated 40 million people worldwide in the winter of 1918 (2). No disease has ever
killed so many in so short a time.
Influenza is dangerous because it is highly contagious - the disease spreads
readily by aerosol - and because new, highly virulent strains periodically emerge (1). The
influenza genome is segmented, and as a result, flu can recombine very easily among
different strains. If a cell is infected by two different strains of flu, the progeny that bud
out from that cell can have an assortment of genes taken from both viruses. This can have
extremely dangerous consequences. Pigs, for instance, can become infected with both
avian and human strains of influenza. A new strain of flu virus may arise in concurrently
infected pigs that is able to infect humans, but appears completely foreign to any
established humoral immunity against ordinary strains of flu, a phenomenon called
antigenic shift. The emergence of such a "swine flu" in 1976 prompted the Ford
administration to attempt to vaccinate every American, an event that highlighted the
looming threat of such emergent strains of influenza (2). Other potential pandemics
occasionally arise, but they have thus far been quickly contained by organizations such as
the World Health Organization that are constantly vigilant against the emergence of
dangerous new flu strains.
In addition, the virus can escape from protective immunity by frequent mutation
in viral antigens; a phenomenon called antigenic drift. As opposed to chicken pox, for
example, where a person who contracts the disease is usually protected for a lifetime
from subsequent infection, a person who had the flu or was vaccinated last year is not
likely to be protected against the disease in this flu season. Flu vaccines must be
formulated every year against the strains that are predicted to be the most prevalent in the
coming flu season (1). The constant aggravation and potential threat of influenza make it
important to understand the virus and the protective immune response against flu
infection.
Cytotoxic CD8+ T cells are crucial to the normal clearance of flu infections.
Focusing on CD8+ T cells is useful because they provide more general protection against
influenza than anti-influenza antibodies. Although antibodies can provide sterilizing
protection against the strain for which they are specific, CD8+ T cells can respond to
more conserved regions of the influenza proteome, providing greater protection against
infection with new strains of flu. It is likely, then, that vaccine strategies that induce both
humoral and cellular memory would provide much greater protection against flu infection
than the flu shot, which only stimulates an antibody response.
Initiation of infection
Influenza A is a single-stranded (-)sense RNA virus of the Orthomyxoviridae
subfamily. The genome is divided into eight segments, bound with nucleocapsid protein
(NP) into ribonucleoprotein complexes inside the capsid. Surrounding the capsid is a
lipid membrane envelope, which is studded with two major surface glycoproteins,
neuraminidase (NA) and haemagglutinin (HA), and lined with matrix protein (MP 1) (3).
This membrane makes influenza relatively vulnerable to deactivation by drying, heat,
detergents, and solvents.
Since NA and HA are exposed to the outside of the viral particle, they are the
proteins against which the immune system forms an antibody defense to flu (4). NA and
HA are highly mutable, and mutations in these genes can render antibodies that recognize
the unmutated form to be useless against the mutant virus. Antibodies are so specific to
spatial conformations that small genetic changes can have disproportionately large
antigenic consequences. This exerts substantial selective pressure to promote new strains
of flu (5).
Influenza spreads when mucous droplets containing virus particles are aerosolized
through sneezing or coughing of the infected individual and inhaled by a new host,
exposing the epithelial cells of the upper airway and lung to virus. The virus attaches to
the cell surface by binding HA to mucoproteins containing terminal N-acetyl neuraminic
acid groups. Neuraminidase can free the virus from these bonds, preventing the
sequestration of the virus by inappropriate cell types that will not promote replication.
Virus particles are endocytosed, and are engulfed within endosomes in the cytoplasm.
These endosomes are acidified and proteolysis of their contents begins. HA is cleaved by
metabolic proteases, causing a conformational change that activates a membrane fusion
domain, which then fuses the viral envelope with the membrane of the endosome. MP1
releases the nucleocapsid, spilling it into the cytoplasm (6). Nuclear targeting sequences
in the NPs translocate the viral genome into the nucleus (7). In the nucleus, viral RNA is
transcribed by the three polymerase polypeptides associated with each genome segment:
PBI, PB2, and PA. Viral proteins are then translated by the cell's own ribosomes (3).
Initiation of the innate immune response
Early in an infection, cytotoxic T cells are not yet activated to destroy infected
cells. The immune system must take steps, then, to slow infections until the adaptive
immune system is properly armed and deployed. An important early cellular response to
replication of many viruses is to produce Type I interferons alpha and beta (IFN-ca and
-s). These molecules serve several important functions as a warning signal from infected
cells. IFNs-u and -P are quickly produced, likely due to a strong reaction of host cells to
double-stranded RNA, which is not normally found in vertebrate cells, but is produced
during the influenza infectious life cycle (8 and 9). Type I interferons bind to interferon
receptors on nearby cells, as well as on the releasing cell, which induces a Jak-Stat signal
transduction pathway that activates the transcription of several genes with antiviral
functions (10). One of these genes activates an endoribonuclease that degrades viral
RNA. Another IFN-inducible gene phosphorylates eIF-2, a eukaryotic protein synthesis
initiation factor, which inhibits translation and thereby slows viral replication. Mx is
another interferon-inducible protein, which interferes with influenza virus replication by
forming a complex with NP and thereby blocking its activity in viral transcription. Mice
that lack the gene for Mx are much more susceptible to influenza infection than
genetically normal mice (11). Influenza partially suppresses early Type I IFN signaling
with the viral protein NS 1, which represses IFN synthesis by preventing activation of the
signaling pathway responsible for inducing transcription of IFN genes, the NF-KB
signaling pathway (12 and 8).
zI
These early defenses are vital to the immune response to flu, but the adaptive
immune system is required to clear the infection. The cellular immune response is
dependent upon the presentation of viral antigens on host MHC molecules. A CD8+ T
cell will recognize its target cell when its T cell receptor (TCR) binds a particular
peptide:MHC complex. Proteins within the cytosol, viral and host alike, are regularly
broken down by the proteasome and replaced with newly synthesized proteins. The result
is a large pool of short peptide sequences, a subset of which are able to bind stably to
MHC class I molecules. Peptides are actively transported into the endoplasmic reticulum
by a heterodimer consisting of Transporters associated with Antigen Processing-I and -2
(TAP-1 and -2). The TAP transporter preferentially transports peptides that are likely to
bind MHC I. Peptides are assembled with MHC I in the endoplasmic reticulum and then
the complex is transported to the cell surface (4). Each cell displays on its surface, then, a
representation of the proteins produced within that cell; including viral proteins, if
present. Antigen specific CD8+ T cells use this display to recognize foreign proteins
within host cells.
Type I IFNs also aid in fighting viral infection by increasing the likelihood that an
infected cell will be destroyed, ending its potential as a virus factory. In order to avoid or
delay the cellular immune response, many viruses reduce MHC expression. Type I IFNs
induce upregulation of MHC class I, leading to more antigens being presented on the cell
surface (8 and 4). This increases the probability that an infected cell will be targeted and
destroyed by an effector CD8+ cytotoxic T cell, making T cells more efficient at clearing
infection. Natural killer cells (NK cells) are also activated by Type I interferons, and
these provide an important early defense against infection (4). NK cells can differentiate
between infected and uninfected cells and kill infected cells using the same molecular
tools as cytotoxic T cells, though NK cells are not antigen specific and require a separate
set of signals in order to lyse target cells. Killer Inhibitory Receptors on NK cells transmit
an inhibitory signal if they encounter MHC class I molecules on the cell surface (4).
Those cells that upregulate MHC I in response to Type I IFNs are protected from NK
cells, then, while infected cells that downregulate MHC I are more likely to be destroyed
by NK cells. NK cells further aid the immune response by secreting cytokine signals such
as IFN-a and the Type II interferon y (4).
CD8+ T cell activation
Up until this point in the infection, the adaptive immune system has not played
much of a role. CD8+ T cells are not activated at the site of infection. Nafve T cells are
not armed for lysis and will not mount a response against infection without help. CD8+ T
cells are first activated in peripheral lymphoid tissues. Dendritic cells take in antigen at
the site of infection in the lung and travel to the draining lymph node, which for the lung
is the mediastinal lymph node (MLN). Respiratory dendritic cells migrate rapidly to the
MLN during the first 24 hours after infection (13). Dendritic cells take up residence in the
lymph node and present the antigens they have gathered.
T cells migrate into the node through the afferent lymph duct and come in contact
with the antigen presenting dendritic cells. When a T cell and a dendritic cell come in
contact with one another, binding of adhesion molecules on their surfaces induce changes
in each other's cytoskeleton that leads to the formation of an immune synapse (14). This
increases the concentration of MHC and TCR molecules at the synapse, making antigen
presentation more effective. If a T cell recognizes antigen presented by a dendritic cell,
signaling of the T cell receptor (TCR) will create a conformation change in an adhesion
molecule on the surface of the T cell, LFA-1, that will allow it to bind more tightly to ICAM adhesion molecules on the dendritic cell (14 and 4). This increases the interactions
between these cells and improves the probability that a T cell will mount a response to
antigen presented by a dendritic cell. As the T cells flow through the dendritic cells, rare
antigen specific T cells bind their TCRs to peptide:MHC complexes and stay in the
lymph node, while those cells that are not specific for any of the antigens being presented
leave through the efferent lymph duct. This allows the lymph node to select for antigen
specific T cells as an affinity chromatography column may select for specific proteins.
Non-specific T cells enter the bloodstream and recirculate to other lymph nodes so that
they may one day find their antigens and participate in an immune response, should such
an antigen be presented.
The cellular immune response is relatively slow to react to infection, but also very
potent. A large pool of activated nonspecific T cells could be damaging to tissues in the
body, and so a balance must be made between aggressive responsiveness and specificity.
Nafve cytotoxic CD8+ T cells require more than one signal in order to activate. These
signals are provided by dendritic cells in the draining lymph node, and not by other
tissues.
The first and most obvious signal is the TCR-peptide:MHC interaction. When
TCR binds peptide:MHC, receptor-associated kinases phosphorylate tyrosine-based
activation motifs (ITAMs) on the intracellular signaling region of the TCR complex,
which then allows a protein called Zap 70 to bind to this region (4 and 15). CD8 on the T
cell also binds to MHC, which brings CD8 and TCR in close proximity. CD8 associates
with a cytoplasmic tyrosine kinase called Lck, and binding of CD8 to MHC class I brings
Lck very close to the intracellular signaling region of the TCR complex, where it can
activate Zap 70. Zap 70 initiates several signal transduction cascades that lead to
transcriptional changes in the T cell that cause it to proliferate and to differentiate into an
effector T cell capable of recognizing and killing infected cells (4 and 15). In summary,
activation of Zap 70 mediates the transition from naive to effector T cell, but this requires
the binding of both TCR and CD8 to MHC on the target dendritic cell. This signal alone
is not sufficient for proliferation of a naive T cell, however. A costimulatory signal is
required for proliferation.
B7, a molecule present on the surface of dendritic cells, provides an important
costimulatory signal by binding to CD28 on the surface of the T cell. CD28 signaling
results in an increase of proliferation, cytotoxicity, and cytokine secretion (4 and 16).
CD28 is necessary for the synthesis and secretion interleukin-2 (IL-2), which is a
powerful T cell growth factor. T cells that do not receive a CD28 costimulatory signal do
not produce IL-2 or the IL-2 receptor and will become anergic or die (16). The
requirement of a costimulatory signal prevents T cells from becoming activated by
healthy cells, which present antigen, but lack B7. Other molecules have also been
described as costimulatory, either by promoting more efficient engagement of TCR or by
providing additional signals that augment cell survival, promote cell division, or induce
effector functions such as cytokine secretion. These costimulatory signals may reinforce
TCR signaling by sharing common pathways and signaling intermediates, or may
I;
comprise distinct signals that do not overlap. It seems, though, that several costimulatory
signals may be required to mount an effective CD8T T cell response (17).
Proliferation and Differentiation
T cells that are activated in the MLN proliferate quickly between days 3-4 post
infection (18). During this time, the population of antigen-specific T cells grows
extensively, and these cells also undergo extensive changes in gene expression. The
changes made to a CD8+ T cell during this phase transforms it from a rare, resting naive
cell into a large pool of effector clones.
T cells alter their complement of adhesion molecules in order to migrate properly
to the site of infection and target infected cells. Effector molecules lose L-selectin, which
naive cells use to home to lymph nodes (18 and 4). Expression of VLA molecules is
increased, which binds to V-CAM adhesion molecules expressed on inflamed endothelial
cell surfaces, and are required for effector T cell retention in the lungs (19). These
changes allow effector T cells to migrate to leave the lymph node and efficiently enter the
site of infection. Other adhesion molecules, such as LFA-i, are also upregulated that
increase interactions between T cells and potential target cells (20).
Effector CD8+ T cells also undergo changes that allow them to kill target cells
once they arrive at the site of infection. Effector molecules that contribute directly to
killing of target cells, such as perforin and granzyme, are greatly upregulated. Fas ligand
is expressed on the cell surface, which allows cytotoxic T cells to induce apoptosis in
target cells. CD8+ T cells also lose their requirement for costimulation, allowing them to
destroy virtually any target cell that presents the right peptide:MHC class I complex (4).
Signaling through the TCR becomes more sensitive, as expression of the tyrosine
phosphatase CD45 changes. CD45 de-phosphorylates the inhibitory tyrosine on a Srcfamily kinase that participates in phosphorylating TCR ITAMs: it turns on a protein that
promotes TCR signaling. A balance is present where a protein called Csk turns off this
kinase and CD45 turns it back on. Effector CD8+ T cells switch to a variant splicing of
the CD45 gene that associates with the TCR complex (15). This shifts the balance so that
more of the TCR associated tyrosine kinases are in a ready state. This makes it more
likely that the kinases required for TCR signaling will be ready when TCR binds
peptide:MHC, increasing the sensitivity of T cells for antigen recognition.
Effector function
Armed effector T cells leave the MLN and migrate to the lung at about day 5 after
infection with influenza (18). These T cells are guided to the lung by chemokines and by
cell adhesion molecules on the target tissue. By days 6-7, there is significant
accumulation of effector T cells in the lung (18). The appearance of armed effector CD8+
T cells in the lungs marks a significant reduction in viral load. Effector CD8+ T cells are
highly selective and efficient killers, making viral replication much more difficult.
When a CD8+ T cell comes in contact with a target cell, its TCR engages the
antigen displayed on class I MHC. CD8 also binds to MHC, bringing together the factors
needed for TCR signaling. Due to the changes made in differentiating to effector cells,
TCR signaling is much more sensitive than in naive T cells and can respond more readily
to lower levels of antigen. One of the results of Zap 70 activation in both naifve and
effector CD8+ T cells is the activation of phospholipase C-y and an increase in
7
intracellular Ca2+ due to IP3 signaling (4 and 15). In naYve T cells, this Ca 2+induces
activation of transcription factors that affect differentiation. In effector CD8+ T cells, this
calcium induces the release of lytic vesicles containing perforin and granzyme. Signals
within the T cell direct the release of these vesicles towards the target cell. These vesicles
maintain conditions that render these molecules inactive and harmless to the T cell. When
released, the effector molecules are exposed to conditions that allow them to destroy the
target cell (4). Perforin polymerizes to form pores in the membrane of the target cell.
Granzymes are serine proteases that are able to enter into the perforin pores and begin to
cleave proteins within the target cell. Accumulated damage from granzyme digestion and
compromised membrane integrity kill the target cell. Meanwhile, Fas ligand on the
surface of the CD8+ T cell binds to Fas on the target cell, activating the target's powerful
apoptotic pathway and inducing cell suicide (15 and 4).
Several cytokine signals are secreted by effector CD8+ T cells that help coordinate
the immune response. Effector CD8+ T cells produce tumor necrosis factor-c and -P
(TNF-a and -) and IFN-y (21). TNFa activates endothelial cells, quickly changing their
adhesion properties to allow recruitment of a variety of immune cells (9). IFN-y inhibits
viral replication and activates macrophages, as well as induces MHC production in host
cells. IFN-y has also been found to modulate the priming, recruitment, and apoptosis of
activated T cells (9 and 22).
Contraction and memory acquisition
Effector CD8+ T cells quickly route influenza infection, usually eliminating the
virus from the respiratory tract by about day 10 after infection (18). With the battle won,
the body no longer requires a large pool of armed effector CD8+ T cells. Most of the
effector T cells die by apoptosis by about 14 days after infection (18). Only about 5-10%
of the peak population remains. The surviving effector CD8+ T cells become long-lived
memory T cells (4). This process appears to be pre-programmed, and independent of the
magnitude of that peak population. The initial activation of CD8+ T cells seems to drive
them through a full program of cell division without the need for addition encounters
with antigen (23).
The mechanism that mediates this programmed contraction is largely unknown,
but evidence suggests a role for IFN-y. Experiments with mice deficient in IFN-y have
shown that even though both deficient and wild type groups have similar responses to
Listeria through the effector phase, they have diminished contraction of effector T cell
populations compared with wild type mice. This leads to a greater number of antigen
specific T cells 28 days after Listeria infection, compared with wild type mice (24).
Experiments with mice lacking the IFN-y receptor have shown that these mice also
experience less apoptosis of effector T cells after infection with lymphocytic
choriomeningitis virus (25). IFN-y, then, appears to promote the apoptosis of effector T
cells.
One of the early determinants of memory appears to be expression of the IL-7
receptor u-chain. IL-7 is known to be an important cytokine for persistence and survival
of memory T cells (26). A fraction of CD8+ effector T cells upregulate IL-7Ru earlier in
an immune response than other memory markers, and these cells use the IL-7 survival
signal to outlast the contraction phase and become memory cells (27).
R
Memory CD8+ T cells remain in the body in order to mount a more effective
response should the individual become infected with the same pathogen. Memory cells
have two main advantages over nafve cells when mounting a secondary response. First,
there are more memory cells specific to influenza antigens. The memory pool for a
particular antigen is approximately 1 02-103 greater than the initial frequency (4). This
makes it more likely that a memory cell will recognize antigens from a pathogen that has
reinfected the host. Also, memory cells are intrinsically more sensitive to activation than
naive cells (28 and 29). This allows for a quicker, more vigorous response from the
memory cell. Since memory cells are better suited to fighting reinfection than naifve cells,
activation of naYve lymphocytes is suppressed during a secondary response.
Two populations of memory CD8 t cells arise from the pool of effector cells that
survive contraction. Effector memory cells (EM cells) stay in peripheral tissues and
express high levels of integrins and receptors for inflammatory cytokines (29). This
seems to allow EM cells to respond quickly at the site of infection. After influenza
infection, though, EM cells decline rapidly in the lung. This depletion correlates with a
loss of memory response to subsequent influenza infection (30). Central memory cells
(CM cells) are recirculated in lymphoid tissues, scanning for the reemergence of viral
antigens (31).
The pool of antigen specific memory cells appears to remain roughly constant
throughout life. Memory cells maintain this population by a low level of proliferation
(32). While naive CD8+ T cells require both TCR engagement and cytokine signaling to
survive, memory cells have gained the ability to survive without antigen, and require only
cytokine signals, such as IL-7 and IL- 15 (32). Experiments have shown that CD4+ helper
T cells are important for maintaining the memory population. Mice that lack CD4+ T
cells have largely normal CD8+ T cell responses to influenza, but have reduced antigen
specific memory pools (33 and 34). The reduced immunological memory of CD4+ T cell
deficient mice results in reduced protection against reinfection with influenza.
Perspectives on treatment and prevention of influenza
The acquisition and maintenance of memory cells is the goal of any vaccination.
The traditional influenza vaccine, the flu shot, uses a combination of killed flu virus
particles to induce an antibody response. The vaccine must be formulated in advance
using three flu strains predicted to be most likely to cause widespread infection (1 and
35). This does not provide protection against other strains, and must be reformulated
every year as new strains arise. There is currently a cold adapted live virus vaccine called
FluMist available on the market. This is a recombinant live virus that causes an
attenuated infection. The virus is cold adapted so that it will replicate in the nasal
mucosa, but can not reproduce in warmer areas such as the lungs (36). It is possible to
create other attenuated flu viruses, so similar live vaccines may be available in the future.
Other treatment options include antiviral drugs that directly inhibit influenza viral
replication. These drugs must be taken on a strict schedule to maintain therapeutic levels
in the body. Antiviral drugs are generally effective against influenza, but side effects and
concerns over patient compliance with taking the drugs as recommended limit their
practical widespread use. One possibility for treating flu is to inhibit translation of viral
genes by using RNA interference(RNAi). Double stranded RNA directs sequence
specific degradation of messenger RNA, preventing translation of proteins (37). This
0
strategy could be used to disrupt any of the influenza genes, each of which is important to
the life cycle of the virus. Short interfering RNA (siRNA) could be introduced to the
epithelial cells of the upper airways and lung, resulting in a disruption of viral replication
The main challenge is to develop an effective and safe system for delivering siRNA to
these cells, but RNAi could be an invaluable treatment tool in the case of a lethal flu
pandemic.
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