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Lederberg’s one-signal model (1959)
TIMELINE
Activation rules: the two-signal
theories of immune activation
Alan G. Baxter and Philip D. Hodgkin
Two-signal theories of lymphocyte activation
have evolved considerably over the past 35
years. In this article, we examine the
contemporary experimental observations and
theoretical concerns that have helped to
forge the most influential variants of the
theory. We also propose that more-rigorous
quantitative methods are required to sustain
theoretical development in the future.
By the 1960s, immunology had come of
age, and the main components of a cellular
recognition system had been assembled.
Lymphocytes recirculated around the body1
and responded to foreign molecules. Their
responses could involve the production of
specific antibodies2 or direct cellular attack3,4,
and the size of the response was, in part,
determined by the number of responding
cells5. Burnet’s clonal selection theory 6 provided a rationale for tolerance; the lymphocyte populations that were able to respond to
the body’s own tissues were depleted during
the window of prenatal, actively acquired
tolerance that had been described by
Billingham, Brent and Medawar7.
Box 1 | Lederberg’s nine postulates
• The stereospecific segment of each antibody globulin is determined by a unique sequence of
amino acids.
• The cell making a given antibody has a correspondingly unique sequence of nucleotides in a
segment of its chromosomal DNA — its ‘gene for globulin synthesis’.
• The genetic diversity of the precursors of antibody-forming cells arises from a high rate of
spontaneous mutation during their lifelong proliferation.
• This hypermutability consists of the random assembly of the DNA of the globulin gene during
certain stages of cellular proliferation.
• Each cell, as it begins to mature, spontaneously produces small amounts of the antibody
corresponding to its own genotype.
• The immature antibody-forming cell is hypersensitive to an antigen–antibody complex; it will
be suppressed if it encounters the homologous antigen at this time.
• The mature antibody-forming cell is reactive to an antigen–antibody complex; it will be
stimulated if it first encounters the homologous antigen at this time. The stimulation comprises
the acceleration of protein synthesis and the cytological maturation which mark the ‘plasma cell’.
• Mature cells proliferate extensively under antigenic stimulation but are genetically stable, and
therefore generate large clones genotypically pre-adapted to produce the homologous antibody.
• These clones tend to persist after the disappearance of the antigen, retaining their capacity to
react promptly to its later reintroduction.
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Lederberg’s nine postulates8, which were
drafted while visiting Burnet’s laboratory at
the Walter and Eliza Hall Institute, adequately summarized the state of immunological theory at that time (BOX 1). Together, these
postulates formed the basis of a one-signal
model of lymphocyte activation. This model
incorporated a temporal switch that governed the outcome of antigenic stimulation
— switching from suppression (Burnet suggested deletion) of immature lymphocytes to
activation of mature lymphocytes. The
model deviated significantly from Burnet’s
theory of immunological tolerance9, as modified by his clonal selection theory10, which
stated that lymphocytes were susceptible to
elimination only “in the late embryonic
period with the concomitant development of
immune tolerance”.
Talmage and Pearlman (1963)
Even at the time of its development, there
were experimental data that did not easily
fit Lederberg’s one-signal model. Hapten–
carrier phenomena had been studied ever
since Landsteiner11 originally divided antigens into two classes. The first class,‘carriers’,
were themselves immunogenic, and antibodies could be raised against them easily.
The second class, ‘haptens’, were not
immunogenic unless administered conjugated to a carrier, in which case antibodies
that were specific for both the hapten and
the carrier parts of the hybrid molecule
could be produced. Clearly, the structure of
the antigen was contributing to the outcome. Furthermore, Dresser12 subsequently
published experiments that indicated that
reactive immunocytes could be either tolerized or activated, depending on the physical
properties of the antigen.
An attempt to accommodate all of these
data within a theoretical framework was made
by Talmage and Pearlman13. They proposed
that although antigen alone could induce the
maturation of a lymphoid cell into a nondividing plasma cell, this resulted in minimal
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antibody production and led to tolerance, as
the antigen-reactive cell pool was now
depleted. By contrast, they suggested that
aggregates of antigen — perhaps associated
with complement — could deliver an additional nonspecific stimulus, and the combination of antigen-specific and -nonspecific
stimuli would trigger substantial clonal
expansion and significant production of
antibody. “Since it is unlikely that natural
proteins possess closely spaced identical
determinants,” they wrote, “the fixation of
complement probably requires the presence
of [pre-existing] antibody directed to two or
more different determinants”.
By 1968, studies of hybrid antigens had
produced other observations that were
inconsistent with Burnet’s and Lederberg’s
models. In 1967, Rajewsky and Rottlander14,
and Mitchison15 applied the concept of
hapten–carrier hybrids to a range of naturally occurring molecules, and they reported
that tolerance to an antigen could be broken
by immunization with that antigen conjugated to an immunogenic epitope. This
result provided evidence that cellular collaboration underpins many immune responses.
Mitchison16 proposed a model that involved
two or more receptors, but confessed,“we are
reluctant on general grounds to postulate more
than one specificity of receptor in an individual
antigen-sensitive cell … therefore cooperation
must occur prior to cell stimulation”.
A further important problem for one-signal
models was also emerging; like other dividing
mammalian cells17, lymphocytes presumably
continue to mutate at a basal rate, and there
was some indication that the generation of
high-affinity antibody might involve further
mutation. So, the specificity of responding
lymphocytes could drift towards self-reactivity,
which indicated the need for a tolerogenic
mechanism throughout the life of the clone.
would initiate cellular activation. They suggested specifically that the activating configuration of repeated determinants would be
the result of a second antibody (the carrier
antibody) binding to a second, independent
determinant on the antigen, which would produce a defined spatial distribution of bonds.
Activation
The involvement of ‘carrier antibody’ in the
initiation of the second form of signal
explained the apparent requirement for at
least two antigenic epitopes for immunogenicity. Note that, in this model, the two signals are different, mutually exclusive signals
that achieve two different outcomes; there is
Paralysis
a Lederberg, 1959
h c
1
1
Mature
h c
Young
b Bretscher and Cohn, 1968
h c
2
1
h c
1
h c
1
h c
h c
c Bretscher and Cohn, 1970 ('old' model)
1
h c
2
h c
d Bretscher and Cohn, 1970 (modern synthesis)
Bretscher and Cohn (1968)
Bretscher and Cohn attempted to accommodate hapten–carrier phenomena within the
framework of self–non-self discrimination,
taking into account the problem that was presented by the constant threat of a clone
mutating towards self-reactivity. Their first
model, published in 1968 (REF. 18), proposed
that antigen receptors on the surface of
immunocytes were able to transmit two qualitatively different signals. Should a single free
determinant bind to a receptor, a specific signal would be generated that would induce
cellular paralysis (or death) of the lymphocyte. By contrast, binding to an appropriate
aggregation of two or more determinants
would induce a second type of signal that
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| JUNE 2002 | VOLUME 2
c h
1
B cell
T cell
2
c
Figure 1 | The evolution of Bretscher and Cohn’s associative recognition model. An immuneresponsive cell carries receptors (antibodies) on its surface (green). a | In Lederberg’s model8, the outcome
of antigenic stimulation (activation or paralysis of the immune-responsive cell) depended on the timing of
stimulation (mature or young immunocytes, respectively). By contrast, in Bretscher and Cohn’s models, the
outcome of stimulation depended on associative antigen recognition. b | In their original model18, activation
required that a carrier antibody bound the carrier portion (c) of a hybrid antigen, and that the responding
immunocyte bound the hapten portion (h). If carrier antibody was not bound, then an inhibitory signal was
generated. c | Two years later, Bretscher and Cohn20 replaced this system of two mutually exclusive signals
with a form of cellular calculus, in which a second signal was used to interpret the appropriate response to
antigenic stimulation. d | This model is now interpreted in terms of T-cell-dependent B-cell stimulation; a
carrier peptide (c) that is presented by an MHC molecule (blue) on the surface of a B cell triggers a T cell
through its antigen receptor (pink) to provide a second signal to the B cell.
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no logical summation of multiple signals.
Furthermore, tolerance induction is effective
throughout the life of the cell — which
ensures that clones that mutate to being selfreactive are deleted — because of the low
probability that a second self-antigen-specific
clone will be generated simultaneously to
provide the self-reactive carrier antibody.
Bretscher and Cohn briefly considered the
possibility that the carrier antibody could be
present on the surface of another antigenspecific cell, but did not think this probable
because,“the induction of an antigen-sensitive
cell would then require an antigen molecule to
interact simultaneously with two cells which
are presumably rare”.
The main problem with their theory was
the origin of the carrier antibody. Although
they proposed that the carrier antibody be of a
special class, there was no qualitative difference
described in the model between the haptenspecific antibody and the carrier antibody. The
lymphocyte that produced the carrier antibody would, therefore, presumably have the
same activation requirements as that producing the hapten-specific antibody. It would also
need its own carrier antibody to ensure stimulation instead of paralysis — forming a circular paradox that is now known as the ‘primer
problem’19. The only solution offered was that
“such carrier antibody may be acquired early
in life from maternal sources”.
Associative recognition (1970)
When Bretscher and Cohn redrafted their
two-signal model in 1970 (REF. 20), they began
the manuscript by summarizing their 1968
position — but with some interesting and
important changes. In 1968, the two signals
(either stimulatory or inhibitory) were mutually exclusive and both were mediated
through the surface-bound receptor of the
lymphocyte (FIG. 1). By 1970, the carrier antibody mediated its own ‘second’ signal. This
second signal was used to interpret the appropriate response to signal one, the haptenspecific signal (FIG. 1). The two signals were no
longer mutually exclusive, and the lymphocyte was now responsible for integrating
them. A form of cellular calculus was implied.
Another important modification was
influenced by the recent realization that there
were at least two types of lymphocyte; the
production of antibody had been associated
with the bursa of Fabricius in chickens21 and
the recognition of histocompatibility antigens
with the thymus22. It was known that the
lymphocytes that were produced by each of
these organs — with the bone marrow taking
the role of the bursa in mammals — were
synergistic for the production of antibody23,24.
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Figure 2 | Kevin J. Lafferty. Photograph courtesy
of Marc Fenning, Photography, John Curtin
School of Medical Research.
“The simplest interpretation of these results,”
wrote Bretscher and Cohn20,“[is that] the formation of carrier-antigen-sensitive cells is
thymus dependent, whereas humoral-antigensensitive cells are derived from the bone marrow”. Mitchison subsequently confirmed that
the helper activity of the carrier-antigensensitive cells was concentrated in lymphocytes of thymic origin25,26, which showed that
T-cell-dependent B-cell activation involved
the associative recognition of two distinct
antigenic determinants.
Bretscher and Cohn20 maintained their
earlier position that the induction of carrier
antibody required the same activation conditions as the induction of hapten-specific antibody, so invoking the ‘primer problem’. As
each antigen-specific receptor was associated
with a different type of lymphocyte, however,
this was no longer necessarily a component of
the model19,27, because different types of lymphocyte could reasonably be expected to have
different activation conditions.
Lafferty and Jones (1969)
By the late 1960s, the contribution of MHC
genes to tissue incompatibility — in particular,
transplant rejection and graft-versus-host disease — had emerged as an intriguing phenomenon. A particular focus of discussion was the
high frequency of lymphocytes that responded
to foreign tissues and, consequently, the
surprising vigour of the allogeneic response.
For practical reasons, Lafferty (FIG. 2) and
Jones28 chose to study graft-versus-host disease by inoculating the allantoic membrane of
viable chicken eggs with mature lymphocyte
preparations — either allogeneic or xenogeneic. This model had been validated previously by Simonsen29, and the resulting growth
of lymphocyte colonies was regarded widely
as being consistent with Burnet’s theory of
clonal selection. To their surprise, Lafferty and
Jones28 found that “as the genetic relationship
between donor and recipient becomes more
distinct, the degree of reactivity falls to an
undetectable level”. For example, chicken lymphocytes injected into a genetically distinct
chicken egg vigorously attacked the host cells.
By contrast, grafted lymphocytes from a
pigeon gave a much diminished graft-versushost reaction, and no response was initiated
by the injection of sheep or mouse lymphocytes28. Lafferty and Jones concluded that the
reactivity of xenogeneic cells was always less
than that of allogeneic cells, a paradox that
had been reported previously, but not
explained adequately (reviewed in REF. 29).
Over the course of a series of experimental
papers with different collaborators, Lafferty
developed a new theory to explain allogeneic
interactions. He proposed that something
more than antigen was required to stimulate
an allograft response and that the interaction
of a lymphocyte with a histoincompatible target triggered some of the foreign antigenic
cells (the ‘stimulator’ cells) to produce a
strong proliferative stimulus. It was the need
for lymphocytes to recognize this second signal in addition to the antigenic difference,
Lafferty argued, that accounted for the
requirement for species compatibility and,
therefore, explained the paradox of alloreactivity (FIG. 3). Lafferty called this second signal
the ‘allogeneic stimulus’30.
The many unanswered questions that
were associated with this theory were a significant spur to further experiments. In the early
1970s, Lafferty and co-workers determined
that the cells that produced the allogeneic
stimulus were derived from the haematopoietic system and that they had to be metabolically active to stimulate lymphocytes28,30.
Also, they found that, once generated, activated cytotoxic T lymphocytes were able to
kill any cell that expressed the foreign antigen
— that is, once activated, the requirement for
the allogeneic stimulus was lost30. On the basis
of these results, Lafferty proposed that the
donor haematopoietic cells that are carried
within grafted tissue provided a potent activation signal for the host immunocytes, which
then attacked the main body of the graft.
These ‘passenger leukocytes’ were, therefore,
the main barrier to graft acceptance. He
found that these cells could be removed from
a graft by briefly culturing the tissues that
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were to be transplanted, and that thyroid tissue treated in this way could be grafted indefinitely across a histoincompatible barrier31. In
remarkable agreement with his theory,
Lafferty found that the injection of a small
number of spleen or peritoneal-exudate cells
from the donor could induce the rapid rejection of a previously tolerated graft32. This
sequence of experiments showed unequivocally that antigen-bearing stimulator cells,
and not antigen itself, were the main barrier
to allograft acceptance, and that activation of
the T-cell response in this case was the unique
property of these cells.
paper proposed that the concept of the stimulator cell should be extended from the consideration of allogeneic reactions to that of all
cellular immune responses.“We would modify
[Bretscher and Cohn’s] theory somewhat to
suggest that ‘signal two’ is provided by a stimulator cell … which is bound to the responsive cell … by means of an antigen bridge,”
they wrote. “Normal antigen induction now
has the same general form as an allogeneic
Lafferty and Cunningham (1975)
In 1975, Lafferty, together with a new collaborator, Cunningham, produced a remarkably
comprehensive manuscript entitled “A new
analysis of allogeneic interactions”33. This
a Theory of allogeneic interactions
b Preventing graft rejection
?
L
R
Activation
S
Allogeneic stimulus
?
NL
R
No activation
? or signal (1)
Graft
rejection
S
R
Allogeneic stimulus or signal (2)
c
Antigen
d
Ab
Ab
(1)
S
(1)
R
S
R'
(2)
Stimulator cell
(2)
Responder cell
e
R
Stimulator cell
Activated
responder cell
Responder cell
MHC forms
compound
with antigen (1)
T cell
Activated
T cell
S
MHC triggers
second signal
(2)
All activated T cells
are MHC restricted
Figure 3 | The development of the two-signal theory in Kevin Lafferty’s laboratory. a | The theory of allogeneic interactions. The immune-responsive cell (R)
interacts with a foreign lymphoid cell (L). This interaction leads to the provision of a potent allogeneic stimulus; the stimulus was proposed to be species specific,
thereby accounting for the fact that allogeneic interactions lead to more vigorous responses than xenogeneic ones. Whether the responsive cell had to receive an
antigenic signal was not known (as indicated by the question mark). However, it was clear that some foreign cells could be antigenic, but without providing an
allogeneic stimulus (non-lymphoid cells; NL). b | Lafferty proposed a model to explain the initiation of graft rejection. The panel shows graft tissue containing
resident donor cells (stimulatory cells; S) that are able to provide an allogeneic stimulus to the host immune system (responsive cells; R). Once activated, host
T cells could attack the main body of the graft, leading to graft rejection. This theory was consistent with all of the versions of Lafferty’s two-signal model.
c | In 1975, Lafferty and Cunningham identified a basic similarity between allogeneic interactions and normal lymphocyte activation. Their model of normal
lymphocyte activation used Bretscher and Cohn’s ‘signal one’ and ‘signal two’ terminology and borrowed the idea that the cell operated as a logical device,
requiring both signals before becoming activated. Signal two in this model was provided by the stimulatory cell after an antigen-dependent interaction with the
responsive cell. d | Lafferty and Cunningham’s equivalent scheme for allogeneic interaction, in which the stimulatory cell passively presented the antigen and also
responded to cellular interactions with a responsive cell by producing signal two. e | In 1977, Lafferty and Cunningham presented a version of their scheme that
accounted for the MHC restriction of T cells by placing control of the production of the second signal with MHC engagement on the stimulating-cell surface.
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PERSPECTIVES
interaction. In both cases, induction depends
not on a simple antigen–cell contact (signal
one), but on the coming together of a stimulator and a responder cell.”When the two cells
came together, the release of a second signal
(later termed a ‘co-stimulator’34) by the stimulator cell would complete the requirements
for activation (FIG. 3).
In attempting to use their general scheme
to explain all types of immune activation,
Lafferty and Cunningham did not distinguish
between T and B cells. For this reason, the
requirement of Bretscher and Cohn’s theory
for associative recognition was not always satisfied. For example, although their scheme for
‘normal’ antigen-mediated immune induction involved antigen being presented to the
reactive lymphocyte by carrier antibody, the
constraint of associative recognition was
relaxed for allogeneic stimulation, for which
there was clearly only one antigen specificity
involved. This reintroduced the possibility of
peripheral tolerance being broken if an
immunocyte mutated towards self-reactivity.
“There is a risk of autoimmunity associated
with this mechanism of activation”, Lafferty
and Cunningham wrote.“If an immunocyte
were to spontaneously arise with receptors for
self-antigens, these receptors could mediate the
combination of this cell with a stimulator cell.
The result would be the induction of an
autoimmune clone.” Their solution was to
retain from Bretscher and Cohn the idea that
stimulation with signal one in the absence of
signal two would favour tolerance induction.
So, they argued,“in the self-environment, this
risk would be low because soluble antigen present in the tissue fluids would favour tolerance
induction by delivering signal one alone to the
potentially responsive cell”. As stimulator cells
are rare and do not present self-antigens exclusively, the large number of non-stimulating
cells should ensure a tolerogenic environment.
In fact, Lafferty and Cunningham borrowed a lot more from Bretscher and Cohn
than just the idea of two signals and a default
for tolerance. Their model had a marked formulaic similarity to Bretscher and Cohn’s
1970 model — the allogeneic stimulus was
converted from being a nonspecific inducer to
a participant in a logical operation, in which
the responding lymphocyte adapted its
response to signal one according to the presence or absence of signal two.“Our model was
derived from Bretscher and Cohn’s”, Lafferty
told us,“there’s no doubt about that”35.
MHC as a second signal (1977)
The discovery of MHC restriction by
Zinkernagel and Doherty36 was made just
down the corridor from Lafferty’s laboratory
NATURE REVIEWS | IMMUNOLOGY
at the John Curtin School in Canberra, and it
further linked alloreactivity with normal
T-cell behaviour in his thoughts. Lafferty and
Cunningham37 suggested a new version of
their general scheme, which was specific for
T cells and in which the MHC-encoded molecule was the trigger for the stimulator cell to
provide a co-stimulatory signal. As a consequence of this, only T cells that saw antigen in
the context of a self-MHC product (they
referred to it as a ‘compound’ or ‘interaction’
antigen) could ever induce the production of
a second signal. T cells that recognized antigenic differences, but not the framework of
the MHC product, would receive the first signal, but not the essential second signal. So, all
T cells that are capable of activation must be
restricted to a particular allelic MHC product.
This very elegant version of the two-signal
theory returned the site of control to the
responding lymphocyte and economically
explained allogeneic interactions, MHC
restriction, normal T-cell activation and the
need for co-stimulation in one theory37,38.
Jenkins and Schwartz (1987)
The discovery of T-cell growth factor (interleukin-2; IL-2) allowed the prolonged culture
of T-cell clones and the detailed exploration
of their requirements for activation. Although
this technique produced T cells with uniform
specificities, these cells were ‘antigen experienced’39 and were, therefore, likely to have
different activation requirements from naive
T cells30. Despite these concerns, a resting state
could be induced after a rapid phase of
growth, and Jenkins and Schwartz40 chose to
examine the proliferative responses of several
clones after restimulation with defined antigens that were presented in different ways.
They found that an unresponsive state could
be induced when antigen was presented by
killed or fixed antigen-presenting cells
(APCs), and that viable APCs were required
to provide an essential second signal for full
activation40. Antigen alone seemed to lead to
an inert state, which was later termed ‘anergy’,
in analogy to a similar state that had been
reported in B cells41.
These experiments provided important
confirmation that an antigenic stimulus in the
absence of co-stimulation could inactivate a
mature T cell. Although the demonstration
that this effect could be overcome by a nonantigen-dependent stimulus from a viable
APC was operationally identical to Lafferty
and Cunningham’s33 two-signal model,
Jenkins and Schwartz were more taken with
the similarities to Bretscher and Cohn’s 1970
model20. They wrote, “The model that we
have at the present time which, we feel, best
explains the results is a modification of the
model originally proposed by Bretscher and
Cohn”42. Cohn was unhappy with this interpretation of Jenkins and Schwartz’s model,
because it did not involve associative recognition — the crucial feature of his model.
“I mistook his concerns at the time for a
reluctance to abandon the T-cell receptor
model”, Schwartz wrote, “It was not until
several years later — after much harping by
Polly Matzinger — that I appreciated his real
reluctance stemmed from his difficulty in
accepting the non-antigen specificity of the
second signal”43.
Jenkins and Schwartz helped to popularize a hybrid form of the Bretscher and Cohn,
and Lafferty and Cunningham two-signal
models. Accepting that T-helper activation
did not require associative recognition solved
the primer problem, but at a terrible cost —
it was no longer clear how immunological
tolerance could be maintained. The hybrid
model had transferred the decision about
which cells to activate and, therefore, the
preservation of self-tolerance to the stimulatory cell. This solution brought its own problems, as Matzinger noted: “The upshot is that
the presence or absence of co-stimulation cannot account for self tolerance because the APC
does not distinguish self from non-self”39.
Janeway’s infectious non-self (1989)
Charles Janeway introduced the published proceedings of the 1989 Cold Spring Harbor
Symposium on Immune Recognition44 with a
manuscript that denounced as a fallacy
Landsteiner’s work on the specificity of
immunological reactions.“The Landsteinerian
fallacy”, he wrote,“is the idea that the immune
system has evolved to recognise equally all
nonself substances … I contend that [it] has
evolved specifically to recognise and respond
to infectious organisms, and that this involves
recognition not only of specific antigenic
determinants, but also of certain characteristics or patterns common on infectious organisms but absent from the host”. He pointed
out that for Landsteiner and others who were
working on hapten–carrier systems to raise
antibodies against innocuous antigens, these
antigens usually had to be mixed with adjuvants that contained killed bacteria, such
as Mycobacterium tuberculosis or Bordetella
pertussis.“I call this the immunologists’ dirty
little secret”, he wrote.
Although the innate immune system and
its pattern recognition of pathogens by
germline-encoded receptors had already been
recognized as being important for host
defence, Janeway 45 proposed that these receptors had the further role of being gatekeepers
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Timeline | The evolution of two-signal theories of immune activation
Landsteiner
distinguished
between carriers
and haptens.
1930
Burnet proposed that
discrimination
between self and
non-self is established
in early development.
1944
1944–1945
Medawar characterized
allograft rejection.
1949
1954
Basal rates of
somatic-cell
mutation were
measured by
Russell.
1956
Billingham, Brent and
Medawar supported Burnet’s
prediction of prenatally
acquired tolerance.
of the adaptive immune system. His model
incorporated Bretscher and Cohn’s associative
recognition model for T-cell-dependent B-cell
activation, but rejected it for CD4+ T-cell
activation because of the primer problem.
Accepting the need for a signal in addition to
antigenic stimulation, he adapted the Lafferty
and Cunningham model. Janeway proposed
that immunogenicity required both signalling through the antigen receptor and
a second signal that was induced on host
APCs by infectious agents as a result of pattern
recognition of the constituents of microorganisms. So, in Janeway’s model, Lafferty’s second
signal was not triggered by the T cell engaging
the APC, but was instead triggered by common
microbial products binding germline-encoded
receptors on (or within) the APC. He suggested that the antigen-nonspecific triggers
of APC activation could even operate before
the development of specific antigen recognition and might, therefore, be “viewed more as
positive initiators of immunity than as late
adaptations to avoid autoimmunity”.
This proposal accommodated the facts
that bacterial products do act as adjuvants
and that this effect is mediated by germlineencoded receptors. Furthermore, Janeway’s
hypothesis has been productive; it has had an
important role in the characterization of Tolllike molecules as pathogen receptors. Toll, a
transmembrane protein that was identified
originally as being required for dorsal–ventral
polarity in the Drosophila embryo, can also
activate the immune responses of Drosophila
haemocytes. Medzhitov, working in Janeway’s
laboratory, showed that homologues of Toll
are also important in vertebrate immune
responses to infection46. He showed that a
constitutively active mutant of Toll that was
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Gowans reported
the recirculation
of lymphocytes.
1957
1959
Burnet published
his clonal
selection theory.
1969–1974 Lafferty’s theory of
alloreactivity accounted for the low
immunogenicity of xenogeneic grafts.
Talmage and Pearlman suggested
that antigen aggregates produce
a second, nonspecific stimulus.
1961
Bretscher and Cohn’s two-signal
model proposed that there is
cooperative associative recognition
between T cells and B cells.
Hapten–carrier systems
revealed the dual specificity
of some immune responses.
Lederberg proposed that tolerance
could be induced in immature
lymphocytes at any time.
1963
Evidence accumulated
that tolerance requires
continuous exposure
to (self-)antigen.
1966
1966–1968
T-cell–B-cell
collaboration
was reported.
transfected into human cell lines induced the
activation of nuclear factor-κB (NF-κB) and
the expression of the inflammatory cytokines
IL-1, IL-6 and IL-8, as well as the expression
of the co-stimulatory molecule B7.1 (CD80).
So far, ten mammalian Toll-like receptors
have been identified, and their known bacterial
ligands include lipopolysaccharide47, peptidoglycan48 and flagellin49.
Paradoxically, although Bretscher and
Cohn’s associative recognition model
required two recognition signals of the same
type (antigenic), their model described a very
different outcome if only one of the two signals was received rather than both. Janeway’s
model required two very different signals —
one antigenic, one pattern recognition — yet
the outcome of failed pattern recognition was
the same in a host who possessed an adaptive
immune system as one who did not. One
might, therefore, argue that such a dual system of activation is needlessly redundant.
Janeway 45 recognized this problem, writing,
“A successful pathogen (could) simply avoid
these receptors and … thus induce tolerance
rather than immunity”. He proposed that
mature dendritic cells, which are characterized by poor antigen uptake but constitutive
co-stimulatory activity, could have evolved
specifically to deal with such pathogens.
Despite this, it remains the case that it is frequently possible for an effective immune
response to be generated against foreign cells
(for example allogeneic cells) without either
signalling from innate receptors or the
involvement of dendritic cells.
Matzinger’s danger hypothesis (1994)
Like Janeway, Matzinger39 accepted Bretscher
and Cohn’s associative recognition model for
1967
1968
1969
1970
Bretscher and Cohn’s carrier-antibody theory
linked detection of the number of foreign
determinants to activation or tolerance.
T-cell-dependent B-cell activation, as well as
the Lafferty and Cunningham two-signal
model for CD4+ T-cell activation. She also
placed the control of immune activation with
the APCs, which administered the second signal under appropriate circumstances. Where
she differed from Janeway was in terms of the
nature of the stimulus for a second signal.
Matzinger proposed that APC activation was
induced by various triggers that are associated
with host-cell damage, termed ‘danger signals’.
These stimuli were divided into two groups;
the first group encompassed exclusively intracellular components that are released when
cells are damaged (such as DNA, RNA and
mitochondria), whereas the second group contained ‘inducible alarm signals’, such as heatshock proteins and interferons. As pathogens,
by definition, induce tissue damage, the recognition of that damage could provide a crucial
validation for any immune response.
Another important difference between the
‘danger hypothesis’ and Janeway’s model
(which is sometimes known as the ‘stranger
hypothesis’) is that Matzinger strongly
emphasized the role of tolerance. Her model
predicted that any T cell that is stimulated
through its antigen receptors in the absence of
a second signal would be inactivated. So, selfreactive T cells that emigrate from the thymus
would be rendered harmless as soon as they
met healthy tissues.
The danger hypothesis neatly circumvents
the main problems that were identified in
Janeway’s model. For example, a successful
pathogen that had evolved to avoid triggering
damage receptors would probably have ceased
to damage tissues, and would no longer be a
pathogen, but a commensal. Furthermore, the
hypothesis is consistent with the observation
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PERSPECTIVES
Lafferty and Cunningham’s twosignal model generalized the role of
a stimulating-cell-derived second
signal in immune activation.
Confirmation of
associative
recognition in
T-cell-dependent
B-cell responses.
1971
Allogeneic stimulating
cells were shown to
activate graft rejection.
1975
1977
Lafferty and Cunningham’s MHC
triggering model explained
allogeneic interactions, MHC
restriction, normal T-cell activation
and the need for co-stimulation.
Jenkins and Schwartz
experimentally induced
T-cell unresponsiveness
with signal one alone.
Matzinger described her
danger hypothesis, in which
the second signal was
triggered by the detection of
tissue damage.
1987
1994
Janeway described his theory
of infectious non-self as a
second signal, in which the
second signal was triggered
by microbial products.
that foreign cells can generate effective
immune responses, because, although it is relatively easy to avoid bacterial contamination,
it is virtually impossible to avoid any cellular
damage during the preparation and systemic
administration of allogeneic cells.
Future developments
The most surprising recurring idea throughout these models is the concept that stimulation requires something else together with an
antigen signal. Matzinger called it her “First
law of lymphotics” (without any apparent
apology to Asimov): “Die if you receive signal
one in the absence of signal two”39. Janeway
wrote that “Co-stimulatory molecules are
absolutely essential to the activation of naive
T cells…”50. The problem with this idea is that
it isn’t quite true.
The tools of the molecular revolution have
greatly facilitated the identification of costimulators. An important second signal for
B cells has been identified as being provided
by CD40L, which is expressed on the helper
T-cell surface after activation. At this point,
the T-cell surface alone is strongly stimulatory
and will activate even naive B cells. So, B cells
can be activated by ‘signal two’ alone, although
the final outcome is determined by the net
contribution of several positive and negative
ligand interactions51. Clearly, although the
activation of T-cell-dependent B cells operationally conforms to associative recognition, it
does not fit a simple binary system whereby
both signals must be received simultaneously.
If anything, the status of two-signal theories in our understanding of T-cell behaviour
is even less clear. A series of co-stimulators has
been identified that includes cytokines such as
IL-1, IL-6 and IL-4, as well as cell-surface
NATURE REVIEWS | IMMUNOLOGY
1989
1997
Medzhitov reported
mammalian homologues of
Toll, which supported
Janeway’s theory of infectious
non-self as a second signal.
molecules that are expressed on APCs, such as
B7.1, B7.2 (CD86) and CD40. The term ‘costimulator’ has now acquired the pragmatic
definition of anything that can enhance proliferation in a T-cell-stimulation assay in
which signal one is relatively weak — for
example, in the presence of low concentrations of anti-CD3 antibody. In vivo, however,
it is clear from gene-knockout studies that
none of these molecules is obligatory as a
T-cell second signal.
We believe that two mindsets contribute
to these difficulties. The first is the level at
which these theories operate. Since the theories of Burnet, immunological models have
considered interactions primarily at the cellular level and have placed the decision-making
unit within a cell or a pair of cells. With the
exception of certain phenomena, such as the
role of immune deviation in determining the
class of immune responses, there has been
little discussion of population distributions of
cellular characteristics or of features of
immune responses that emerge only at the
level of the population, organ or whole organism. For example, T-cell growth is largely
dependent on autocrine factors and is, therefore, affected profoundly by the precursor frequency of reactive cells. So, the strength of the
response — even the distinction between self
and non-self — can differ without there being
any significant difference in the proliferative
signals that are received by individual
T cells52,53.
The second problem is that these theories
are qualitative. As a consequence, the experimental methods that are used to test them,
such as co-stimulation assays, are usually
interpreted in a black-and-white manner —
in terms of whether the particular molecule
was or was not a co-stimulator. The field is in
a cycle — the theories are qualitative, so the
experiments are. We maintain that predictive
power is greater for quantitative models, and
that progress will depend on new systems for
the quantitative description of cellular signal
integration and immunological outcomes.
For example, after an antigenic signal, costimulators decrease T-cell cycle time in an
additive fashion and, therefore, relatively
small changes in conditions contribute to
remarkably large differences in cell number53.
A theory of signal integration developed in
this way can account for the fact that no single
component is obligatory, as well as explain
why the many co-stimulation assays give such
dramatic differences in response.
We expect that by following a more quantitative course, our understanding of the
immune system can proceed to a richer level
that will accommodate a theory of immuneresponse class discrimination, as well as incorporate the many molecular contributors to
decision making. Although we should pay
due deference to the past attempts to formulate general principles from immensely complex data (TIMELINE), it is time to move on to a
new era.
Alan G. Baxter is at the Centenary Institute of
Cancer Medicine and Cell Biology, Locked bag #6,
Newtown, New South Wales 2042, Australia.
Philip D. Hodgkin is at the Walter and Eliza Hall
Institute, c/o Post Office, Royal Melbourne
Hospital, Parkville, Victoria 3050, Australia.
Correspondence to A.G.B.
e-mail: [email protected]
doi:10.1038/nri823
1.
Gowans, J. L. The recirculation of lymphocytes from
blood to lymph in the rat. J. Physiol. (Lond.) 146, 54–69
(1959).
2. Fagraeus, A. Antibody production in relation to
development of plasma cells: in vivo and in vitro
experiments. Acta Med. Scand. 130, 3–116 (1948).
3. Medawar, P. B. The behaviour and fate of skin autografts
and skin homografts in rabbits. J. Anat. 78, 176–199
(1944).
4. Medawar, P. B. A second study of the behaviour and fate
of skin homografts in rabbits. J. Anat. 79, 157–176
(1945).
5. Billingham, R. E., Brent, L., Medawar, P. B. &
Sparrow, E. M. Quantitative studies on tissue
transplantation immunity. II. The origin, strength and
duration of actively and adoptively acquired immunity.
Proc. R. Soc. Lond. B 143, 58–80 (1954).
6. Burnet, F. M. The Clonal Selection Theory of Acquired
Immunity (Vanderbilt Univ. Press, Nashville, Tennessee,
1959).
7. Billingham, R. E., Brent, L. & Medawar, P. B. Actively
acquired tolerance of foreign cells. Nature 172, 603–606
(1953).
8. Lederberg, J. Genes and antibodies. Science 129,
1649–1653 (1959).
9. Burnet, F. M. & Fenner, F. The Production of Antibodies
(Macmillan, London, 1949).
10. Burnet, F. M. A modification of Jerne’s theory of antibody
production using the concept of clonal selection. Aust. J.
Sci. 20, 67–69 (1957).
11. Landsteiner, K. The Specificity of Serological Reactions
(Harvard Univ. Press, Cambridge, Massachusetts, 1945).
12. Dresser, D. W. Specific inhibition of antibody production.
II. Paralysis induced in adult mice by small quantities of
protein antigen. Immunology 5, 378–388 (1962).
VOLUME 2 | JUNE 2002 | 4 4 5
PERSPECTIVES
13. Talmage, D. W. & Pearlman, D. S. The antibody
response: a model based on antagonistic actions of
antigen. J. Theor. Biol. 5, 321–339 (1963).
14. Rajewsky, K. & Rottlander, E. Tolerance specificity and
the immune response to lactic dehydrogenase
isoenzymes. Cold Spring Harb. Symp. Quant. Biol. 32,
547–554 (1967).
15. Mitchison, N. A. Antigen recognition responsible for the
induction in vitro of the secondary response. Cold Spring
Harb. Symp. Quant. Biol. 32, 431–439 (1967).
16. Mitchison, N. A. Recognition of antigen by cells. Prog.
Biophys. Mol. Biol. 16, 3–14 (1966).
17. Russell, W. L. A high rate of somatic reversion in the
mouse. Genetics 41, 658 (1956).
18. Bretscher, P. A. & Cohn, M. Minimal model for the
mechanism of antibody induction and paralysis by
antigen. Nature 220, 444–448 (1968).
19. Bretscher, P. The control of humoral and associative
antibody synthesis. Transplant. Rev. 11, 217–267 (1972).
20. Bretscher, P. & Cohn, M. A theory of self–nonself
discrimination. Science 169, 1042–1049 (1970).
21. Glick, B., Chang, T. S. & Jaap, R. G. The bursa of
Fabricius and antibody production. Poult. Sci. 35,
224–226 (1956).
22. Szenberg, A. & Warner, N. L. Dissociation of
immunological responsiveness in fowls with a hormonally
arrested development of lymphoid tissue. Nature 194,
146–147 (1962).
23. Claman, H. N., Chaperon, E. A. & Triplett, R. F.
Thymus–marrow-cell combinations — synergism in
antibody production. Proc. Soc. Exp. Biol. Med. 122,
1167–1171 (1966).
24. Mitchell, G. F. & Miller, J. F. A. P. Immunological activity of
the thymus and thoracic-duct lymphocytes. Proc. Natl
Acad. Sci. USA 59, 296–303 (1968).
25. Mitchison, N. A. The carrier effect in the secondary
response to hapten–protein conjugates. II. Cellular
cooperation. Eur. J. Immunol. 1, 18–27 (1971).
26. Boak, J. L., Mitchison, N. A. & Pattison, P. H. The carrier
effect in the secondary response to hapten–protein
conjugates. III. The anatomical distribution of helper cells
and antibody-forming cell precursors. Eur. J. Immunol.
1, 63–65 (1971).
27. Bretscher, P. A. A two-step, two-signal model for the
primary activation of precursor helper T cells. Proc. Natl
Acad. Sci. USA 96, 185–190 (1999).
28. Lafferty, K. J. & Jones, M. A. S. Reactions of the graftversus-host (GVH) type. Aust. J. Exp. Biol. Med. Sci. 47,
17–54 (1969).
29. Simonsen, M. Graft-versus-host reactions. Their natural
history and applicability as tools of research. Prog. Allergy
6, 349–360 (1962).
30. Lafferty, K. J., Misko, I. S. & Cooley, M. A. Allogeneic
stimulation modulates the in vitro response of T cells to
transplantation antigen. Nature 249, 275–276 (1974).
31. Lafferty, K. J., Cooley, M. A., Woolnough, J. A. & Walker,
K. Z. Thyroid allograft immunogenicity is reduced after a
period in organ culture. Science 18, 259–261 (1975).
32. Talmage, D. W., Dart, G., Radovich, J. & Lafferty, K. J.
Activation of transplant immunity: effect of donor
leukocytes on thyroid allograft rejection. Science 191,
385–388 (1976).
33. Lafferty, K. J. & Cunningham, A. J. A new analysis of
allogeneic interactions. Aust. J. Exp. Biol. Med. Sci. 53,
27–42 (1975).
34. Lafferty, K. J., Warren, H. S., Woolnough, J. A. &
Talmage, D. W. Immunological induction of
T lymphocytes: role of antigen and the lymphocyte
costimulator. Blood Cells 4, 395–404 (1978).
35. Baxter, A. G. Germ Warfare: Breakthroughs in
Immunology (Allen and Unwin, Sydney, 2000).
36. Zinkernagel, R. M. & Doherty, P. C. Restriction of in vitro
T-cell-mediated cytotoxicity in lymphocytic
choriomeningitis within a syngeneic or semiallogeneic
system. Nature 248, 701–702 (1974).
37. Cunningham, A. J. & Lafferty, K. J. A simple conservative
explanation of the H-2 restriction of interactions between
lymphocytes. Scand. J. Immunol. 6, 1–6 (1977).
38. Lafferty, K. J., Andrus, L. & Prowse, S. J. Role of
lymphokine and antigen in the control of specific T-cell
responses. Immunol. Rev. 51, 280–314 (1980).
39. Matzinger, P. Tolerance, danger and the extended family.
Annu. Rev. Immunol. 12, 991–1045 (1994).
40. Jenkins, M. K. & Schwartz, R. H. Antigen presentation by
chemically modified splenocytes induces antigen-specific
T-cell unresponsiveness in vitro and in vivo. J. Exp. Med.
165, 302–319 (1987).
41. Nossal, G. J. & Pike, B. L. Clonal anergy: persistence in
tolerant mice of antigen-binding B lymphocytes
incapable of responding to antigen or mitogen. Proc. Natl
Acad. Sci. USA 77, 1602–1606 (1980).
446
| JUNE 2002 | VOLUME 2
42. Jenkins, M. K., Pardoll, D. M., Mizuguchi, J., Quill, H. &
Schwartz, R. H. T-cell unresponsiveness in vivo and
in vitro: fine specificity of induction and molecular
characterisation of the unresponsive state. Immunol. Rev.
95, 113–135 (1987).
43. Schwartz, R. H. In Immunology: the Making of a Modern
Science (eds Gallagher, R. B., Gilder, J., Nossal, G. J. V.
& Salvatore, G.) 95–101 (Academic, London, 1995).
44. Janeway, C. A. Approaching the asymptote? Evolution
and revolution in immunology. Cold Spring Harb. Symp.
Quant. Biol. 54, 1–13 (1989).
45. Janeway, C. A. The immune system evolved to
discriminate infectious nonself from noninfectious self.
Immunol. Today 13, 11–16 (1992).
46. Medzhitov, R., Preton-Hurlburt, P. & Janeway, C. A.
A human homologue of the Drosophila Toll protein signals
activation of adaptive immunity. Nature 388, 394–397
(1997).
47. Yang, R. B. et al. Toll-like receptor-2 mediates
lipopolysaccharide-induced cellular signalling. Nature
395, 284–288 (1998).
48. Yoshimura, A. et al. Cutting edge: recognition of
Gram-positive bacterial cell wall components by the
innate immune system occurs via Toll-like receptor 2.
J. Immunol. 163, 1–5 (1999).
49. Hayashi, F. et al. The innate immune response to
bacterial flagellin is mediated by Toll-like receptor 5.
Nature 410, 1099–1102 (2001).
50. Janeway, C. A. Presidential address to the American
Association of Immunologists. J. Immunol. 161, 539–544
(1998).
51. Rathmell, J. C., Townsend, S. E., Xu, J. C., Flavell, R. A.
& Goodnow, C. C. Expansion or elimination of B cells in
vivo: dual roles for CD40- and Fas (CD95)-ligands
modulated by the B-cell antigen receptor. Cell 87,
319–329 (1996).
52. Waldmann, H., Qin, S. & Cobbold, S. Monoclonal
antibodies as agents to reinduce tolerance in immunity.
J. Autoimmun. 5, 93–102 (1992).
53. Gett, A. V. & Hodgkin, P. D. A cellular calculus for signal
integration by T cells. Nature Immunol. 1, 239–244
(2000).
Acknowledgements
We are grateful to the following people for their recollections, suggestions, input and advice: A. Basten, J. F. A. P. Miller, K. Lafferty
(deceased), C. Simeonovic, B. Fazekas and C. Jolly. A.G.B. is the
recipient of an interim fellowship from the Australian National
Health and Medical Research Council (NHMRC). P.D.H. is the
recipient of a senior fellowship from the NHMRC. We dedicate this
article to the memory of Kevin Lafferty. His remarkable contributions to the study of T-cell activation and alloreactivity will be long
remembered, and his forceful advocacy for reason in scientific
endeavour will be sadly missed.
Online links
DATABASES
The following terms in this article are linked online to:
Entrez: http://www.ncbi.nlm.nih.gov/Entrez/
Bordetella pertussis | Mycobacterium tuberculosis
LocusLink: http://www.ncbi.nlm.nih.gov/LocusLink/
CD3 | CD40 | CD40L | CD80 | CD86 | IL-1 | IL-2 | IL-4 | IL-6 |
IL-8 | NF-κB | Toll | Toll-like receptors
FURTHER INFORMATION
Bright Sparcs Biography — Frank Macfarlane Burnet:
http://www.asap.unimelb.edu.au/bsparcs/biogs/P000279b.htm
NOBEL e-MUSEUM — Karl Landsteiner:
http://www.nobel.se/medicine/laureates/1930/landsteinerbio.html
NOBEL e-MUSEUM — Peter C. Doherty and Rolf M.
Zinkernagel (Nobel Prize in Physiology or Medicine 1996):
http://www.nobel.se/medicine/laureates/1996/
Access to this interactive links box is free online.
OPINION
The future of antigen-specific
immunotherapy of allergy
Rudolf Valenta
More than 25% of the population in
industrialized countries suffers from
immunoglobulin-E-mediated allergies.
The antigen-specific immunotherapy that is
in use at present involves the administration
of allergen extracts to patients with the aim
to cure allergic symptoms. However, the
risk of therapy-induced side effects limits its
broad application. Recent work indicates
that the epitope complexity of natural
allergen extracts can be recreated using
recombinant allergens, and hypoallergenic
derivatives of these can be engineered to
increase treatment safety. It is proposed
that these modified molecules will improve
the current practice of specific
immunotherapy and form a basis for
prophylactic vaccination.
Ninety years ago, long before the immunopathological mechanisms that underlie type I
(immunoglobulin-E-dependent) allergy were
understood, Leonard Noon immunized patients suffering from pollen-induced hayfever
with subcutaneous injections of pollen
extracts1. Although this was based on the
erroneous belief that seasonal hayfever might
be caused by a grass-pollen toxin, successful
outcomes were recorded and induced protection was found to last for at least one year
after the treatment was discontinued.
Since then, many of the underlying
immunological and molecular mechanisms,
as well as environmental factors, that influence the development of allergy have been
analysed2. Type I allergy is a classical IgEmediated disease, which, as shown by a landmark experiment by Prausnitz and Küstner
in 1921, requires at least three components: a
disease-eliciting antigen (allergen); a transferable serum factor that discriminates allergic patients from healthy individuals (IgE);
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