Hi
Having just subscribed to the immunology bionet and opeened it
for the first time, I loved finding a wonderful question from Chris
Thobur,about the 'real' function of the immune system. Here was his
question:
"When recently discussing the actual role that the immune system
plays, or rather what it does, I have become confused about my personal
understanding. In school I was always taught that "the role of the
immune system is to protect self from non-self." How then does
autoimmune disease occur? Why can neonatal tolerance be established?
Why can some people receive allo-transplants (solid organ or BMT) and
achieve tolerance over time? Most obviously, how can the immune system
be 'trained' to recognize foreign (non-self) antigens as self (as in
chronic infections)? I'm just throwing these things out for discussion
in hopes that this group can prove to be more useful than a catalog of
people asking for the 'reagent of the day."
Chris Thobur
The answer, of course, involves explaining the 'soul' of the immune
system, so here is my attempt. What followss is actually the first rough
draft of a paper that I have written to be sent to immunology today. I hope
it answers the question Chris asked and that it generates some more.
If anyone finds that there are parts that are difficult to understand,
please let me know so that I can correct them before sending it. It's
long (ten printed pages) so anyone who wants to read it should probably
print it out first. I apologize for sending it this way but I haven't
learned how to add 'attachments' yet.
Cheers!
Polly Matzinger
THE REAL FUNCTION OF THE IMMUNE SYSTEM
or
AN IMMUNE SYSTEM FOR THE THIRD MILLENIIUM
(pretty pompous title, need a better one)
or
TOLERANCE AND THE FOUR D's (danger, death,
destruction and distress)
As a student, I was taught that the immune system discriminates
between self and non-self. That it attacks foreign antigens and is
tolerant (except in disease states) of self and that it defines "self"
as the set of antigens present early in life, when the immune system is
developing . After years of having trouble with this view, I have now
abandoned it.
Although the old view explained most of the features of
immunity, it also left some real gaps. For example, if "self" is
learned during early fetal or neonatal life, what happens at puberty, or
during lactation? Though individuals change greatly at puberty, most
are not destroyed by their immune systems and the newly lactating
breast, generating large quantities of (new) milk proteins, is not
rejected. The changes that occur in 'self' are not the only problem
with the old view. Why do normal individuals contain natural antibodies
and/or T cells able to see stable self antigens, such as DNA (), tubulin
(), or myelin basic protein (), and, given that such autoantibodies and
T cells exist, why are autoimmune diseases rare? How are T cells made
tolerant of tissue-restricted MHC/peptide complexes that are not in the
thymus ()? Why are livers more easily transplanted than skin? Why are
tumours not rejected more often? As long as we continue in the old
beliefs, there are no good answers to these questions. However, Ephraim
fuchs and I have built a model of the immune system which supplies these
answers. In an earlier essay I described such an immune system in
detail and laid down the rules by which it operates to maintain self
tolerance. In this essay, I will describe it briefly and touch mainly
on the last two questions listed above, namely tumors and
transplantation.
Let's begin with the definition of "self" which, over the years,
has sometimes been extended from 'everything encoded by the genome' to
'everything under the skin', in order to include commensal genomes. It
has been narrowed to exclude the 'privileged' sites such as brain,
cornea and testes. It has been modified for T and B cells. For T
cells, Waldmann suggested that self consists only of MHC/peptide
complexes (2), and Zinkernagel et. al. reduced it further, arguing that
self consists only of peptides found in the thymus and that all other
tissues are "ignored" (3). For B cells, Cohn defined self as cell
surface and soluble molecules, proposing that"housekeeping" antibodies
to intracellular components might help clear cellular debris (4),
Mitchison defined it as those bodily proteins present at a concentration
above a certain threshold (5), Jerne excluded antibody idiotypes because
their individual concentrations are too low (6), and Coutinho and his
colleagues defined it in terms of an idiotype network, which they call a
"positive definition of self" (7).
"Non-self" is equally difficult to define. There are plenty of
non-self structures that the immune system does not attack (e.g.
silicone, bone, peptides (depending on MHC type), solitary haptens, and
food) because, as Dresser pointed out years ago, they lack a curious
characteristic called "adjuvanticity"(). For example, though ovalbumin
is certainly "foreign" to a mouse and MBP is "self", both proteins
elicit responses if injected with adjuvant while neither does if
injected intravenously. Clearly self vs non-self matters less here than
adjuvanticity. Recently Janeway pointed out that few antigens are
particularly immunogenic. He suggested that "adjuvanticity" is a
property of evolutionarily distant antigens, like bacterial cell walls,
to which the immune system is poised to respond (8).
Ultimately all of the models boil down to the idea that the
immune system regards a certain subset of the body as self and a
particular fraction of the rest of the universe as foreign. In short,
it doesn't really discriminate self from non-self but some self from
some non-self. The immune system thus classifies antigens into four
categories.
1) Visible Self=bodily structures to which the immune
system is tolerant.
2) Invisible self= bodily structures to which it is not tolerant
but to which it does not normally respond.
3) Visible non-self= structures to which the immune system
normally responds.
4) Invisible non-self= non-immunogenic structures to which it
does not respond.
The problem with this kind of classification is that it is
virtually impossible to uncover the rules by which the immune system
could make the necessary distinctions. For example, Waldmann's idea
that self, for T cells, consists of common peptide/MHC complexes, begs
the question of what happens when, during the course of a normal life,
the peptides change. Zinkernagel's idea, that T cells are tolerant only
of antigens found in the thymus and "ignore" all other tissue antigens,
also runs into trouble. Since T cells respond to short peptides and
crossreact on non-identical but similar peptides, Zinkernagel's
"ignorant" T cells should often be activated by cross-reactive
environmental antigens and autoimmunity should be a frequent and
irrevocable event, rather than a rare occurance. Janeway's idea, that
the immune system responds to "infectious non-self" because it has a
phylogenetic memory of infectious organisms, may be partially right but
it does not cover such cases as graft rejection, or responses to many
viruses. Mitchison's and Jerne's view, that the immune system is
tolerant of "normal" concentrations of self antigens, cannot deal with
proteins whose concentrations change. In fact, though change is one of
the hallmarks of life itself, the self vs non-self models all suffer
from the idea that the immune system learns (either during evolution or
ontogeny) a static definition of self that it must then live with for
the life of the individual.
If we forget about self for the moment and step sideways to look
at the other side of the equations above, we find it possible to ask a
different question, namely "how does the immune system decide whether to
respond or not?" I have conjectured () that it might respond to danger,
not to non-self. Clearly, distinguishing dangerous from harmless
entities would be an efficient and evolutionarily sensible thing to do,
and it turns out that the task is not as difficult as it first might
seem. Among the many potential definitions for "dangerous", the one
with the most explanatory force and which leads to the simplest model
with the most predictive power is "anything that causes cell stress or
lytic cell death". Cell death is an integral part of living systems.
It occurs during ontogeny and in adult life. We find death in the
thymus, death in the bone marrow and blood, death in the brain, skin,
gut, liver. Literally, death everywhere. But this is normal,
programmed cell death. It usually involves apoptosis and the dying
cells are ordinarily shed to the outside environment or scavenged by
specialized cells. This sort of death does not appear dangerous to the
immune system. However, should a tissue be stressed or die abnormally,
the immune system is alerted and responds. Let's take an example of a
virus infection to see how it could work. part 1) Initiating the
response.
Danger is sensed by tissues themselves, which signal the professional
antigen presenting cells among them.
Figure 1A shows what a typical tissue (say skin) might look like
under normal, conditions. It consists of a mixture of actively growing
(and dying) skin cells and local antigen-presenting dendritic cells, in
this case Langerhans cells, which are essentially quiescent. When a
lytic virus infects the skin, the infected cells die by non-apoptotic
death, activating the Langerhans cells, which then capture the antigens
in their neighborhood, up-regulate MHC molecules, lose Fc receptors, and
travel to the local lymph node, where they present the captured antigens
to passing T cells. The signals sent by stressed or dying cells are not
known at the moment but they could be of several sorts. A dendritic
cell might become activated if a cell to which it is connected suddenly
dies, simply from the sudden loss of connection. It might have
receptors for a heat shock protein elaborated by stressed cells or for a
protein, normally found only on the inside of intact healthy cells, that
leaks out only if the cell dies lytically. There are a myriad of
potential ways in which a tissue could communicate danger to its local
APCs and I have no reason to choose any particular ones. It suffices
for the moment to postulate that such danger signals exist, that the
constant normal (apoptotic) cell death found in many tissues does not
elicit them, and that without them, the APCs remain quiescent and
intiate no immune responses. part 2) training the T cells
Since APCs cannot distinguish self from non-self (), they
capture normal skin antigens as well as viral antigens and present both
to passing T cells. In self-non-self models, T cells must be trained to
tell the difference between these foreign and self antigens. In an
immune system poised for danger, the T cells need only to distinguish
APCs from everything else. In a nutshell, if any T cell specific foran
APC antigen were deleted, and if APCs were the only cells able to
activate T cells, then the only T cells that would be activated during
an immune response would be those specific for new antigens presented by
APCs. To build such a T cell population, we need three basic laws and
one minor exception. the laws of lymphotics
The First law, taken from the Bretscher-Cohn and
Lafferty-Cunningham models (), assumes that resting T cells need two
signals to be activated; signal One from TCR binding to MHC/peptide and
signal Two (co-stimulation) from an APC. It states that T cells die if
they receive signal One without signal Two and become activated if they
receive both.
The Second law states that resting T cells can only receive
co-stimulatory signals from APCs . Interdigitating dendritic cells
(and, perhaps, macrophages) can serve as APCs for both virgin and
experienced T cells, and B cells can re-stimulate experienced but not
virgin T cells. Though other tissues might express surface MHC/peptide
complexes, they do not express the appropriate co-stimulatory signals
needed by T cells.
The Third law states that the activated effector stage only
lasts for a certain period of time. During this time, T cells do not
require co-stimulatory signals and can be triggered to function (eg.
help B cells or kill targets) by signal One alone. After a while, they
either die or return to a resting state from which they can only be
drawn again by the appropriate combination of signal One plus signal
Two.
Together, the first and second laws compel any mature T cell
that encounters a non-APC tissue (like skin) to die from the lack of a
second signal. The third law ensures that the first two apply to
experienced T cells. Add the idea that stressed or dying tissues signal
their local APCs, and a picture emerges of an extended immune system in
which every bodily tissue is deeply involved in its own protection.
Each tissue essentially has three functions. First it does its normal
job; e.g. it filters plasma or pumps blood. Second, by elaborating
stress signals, it alerts the rest of the immune system to the presence
of danger. Third, by displaying its own antigens in the absence of
co-stimulation, it induces tolerance to itself. deleting T cells in the
thymus:
There is a single necessary exception to the three laws. It
occurs in the thymus and is designed to delete T cells able to recognize
dendritic cells. Since thymic dendritic cells are perfectly capable of
providing co-stimulatory signals, we borrow from Lederberg () and
propose that thymocytes pass through a developmental stage in which they
are tolerizable but not yet activatable, a stage in which the pathways
for receipt of second signals are not hooked up. Thus, as an exception
to the second law, thymocytes at this stage would be unable to receive
second signals from any cell, including professional APCs. Any
thymocyte recognizing the normal surface MAP (MHC/antigen profile) of a
dendritic cell would thus be eliminated by the receipt of signal One
without signal Two.2
Having finished maturation, the remaining T cells exit into the
periphery and begin to circulate. Let's return to the hypothetical
virus infection to see how the system works. activating and tolerizing T
cells in the periphery
Among the virgin T cells circulating through the lymph node
draining the infected site, some are specific for viral peptides (V)
displayed by the activated Langerhans cell and others, having had no
opportunity to become tolerant of non-thymic tissue antigens, will
recognize peptides from the skin (Sk). Let's look first at the cells
specific for V.
Stimulated by signal One and Two from the activated Langerhans
cells, they multiply, become effector cells and circulate out of the
node looking for virus infected targets. Each killer destroys a few
infected cells and then reverts to a resting state and drains back into
the local lymph node, where, if the infection is still going on, it will
be re-activated. This cycle of activation into effector cell and
reversion to resting cell should continue until the infection is cleared
and there are no longer any activated APCs presenting the viral antigen.
The experienced cells, or at least a selected few of them (), will now
recirculate as resting memory cells, waiting for the next encounter3 .
The autoreactive Sk specific T cells are also stimulated by the
activated APCs, but because Sk is everywhere on normal skin, they are in
a critically different situation from the killers specific for V. While
the virus specific killers accumulate at the infected site and return to
the original lymph node, where they are likely to meet a
virus-presenting APC, the Sk specific killers will distribute all over
the body. Each will kill a few skin cells and then drain to a local
lymph node. Unless that node is draining an infected site, the Sk
specific killers will not be reactivated. They will exit the node as
resting memory cells and recirculate from blood to tissues.
Encountering Sk again on normal skin cells, and thus receiving signal
One without signal Two, they will die. Any Sk specific cells returning
to the original infected area will also be deleted by normal skin cells
after the infection is cleared.
A critical point to remember here is that killer cells induce
apoptosis in their targets. Although the chromium release assay gives
the impression that targets die by membrane disruption, DNA
fragmentation actually occurs hours before the membranes disintegrate
4(). In vivo, these apoptotic cells are probably scavenged long before
their membranes fall apart. Consequently death induced by killer cells
does not signal local APCs to perpetuate the immune response. Only real
danger can do that.
Thus, although both autoreactive and virus specific killers
follow the laws of lymphotics, their fates differ because their antigens
are critically dissimilar. Autoantigens are continuously expressed by
healthy cells incapable of delivering signal Two. By sheer numbers and
persistence, they ensure that circulating autoreactive cells should
regularly be deleted. Of course the efficiency of deletion will depend
on the size of an organ and the rate at which lymphocytes circulate
through. Large organs, and those that have minimal blood-tissue
barriers, like the liver, should tolerize quite effectively whereas
small and/or well barricaded organs (brain, pancreas?) should induce
deletion at a much slower rate. part 4: lactation and puberty, tumors
and transplants
Life's changes: An organism in which tissues tolerize for
themselves has no problem with change. Take puberty. Though many
organs and their hormones are affected, there is no associated lytic
cell death and hence no activation of local APCs. Therefore any T cells
that recognise the changes will simply die because of the first law of
lymphotics. The newly lactating breast making casein and perhaps
expressing casein peptide/MHC complexes on its surface will not activate
casein-specific T cells, but, by offering signal One without signal Two,
it will delete them. These are examples of the constant conversation
going on between lymphocytes and other tissues. The tissues themselves,
offering their antigens in the absence of signal Two, need not be static
entities, stuck with an immune system tolerant only of yesterday's
'self'. They can afford to change.
Tumors: A newly arising tumor cell may express antigens not
expressed by its normal tissue mates, but this is not enough to alert
the immune system. There is no intrinsic difference between a rapidly
dividing tumor cell and a rapidly dividing hematopoietic cell, gut cell
or thymocyte. If it dies, it dies by apoptosis. It does not normally
produce stress proteins nor activate local APCs and there is no
particular reason why the immune system should be able to distinguish it
from any other rapidly dividing cell type unless it becomes infected,
stressed or otherwise necrotic. Consequently, as it grows, any tumor
unable to deliver signal Two should induce deletion of tumor specific T
cells.5
Sometimes, however, tumors spontaneously regress. The cause may
be simple. Should a melanoma, for example, be traumatized by viral,
bacterial or physical insult, the local APCs would become activated,
capture the tumor antigens (as well as normal melanocyte antigens) and
present them to passing T cells in the draining nodes. Any tumor
specific T cells that had not yet been deleted would become activated
and begin to destroy the tumor. If the melanoma were small or if the T
cells were repeatedly activated (e.g. by a chronic, remittent or long
lasting viral infection), the melanoma would be destroyed. In some
cases, the T cells might also destroy normal melanocytes expressing
similar antigens (), leading to the vitiligo occasionally found with
spontaneous melanoma regression. This view fits with the finding that T
cell clones isolated from such patients can kill melanoma cells taken
from HLA matched patients that have not rejected their tumors (),
showing again that it is not lack of antigen expression that prevents
tumor rejection.
A similar scenario describes the finding that an injection of
heavily irradiated tumor cells transfected with GMCSF can evoke
protection against the untransfected tumor (). This treatment has two
important features. First, though radiation death is usually apoptotic,
an injection of large numbers of dying cells may well overload local
scavenging capacities, allowing some of the tumour cells to disintegrate
before they can be cleared, and thus signalling local APCs. Second,
GMCSF enhances the well being of dendritic cells in vitro () and may do
the same in vivo. The combination of dying cells and APC-enhancing
cytokine would thus provoke activation of tumor specific T cells that
could now kill a certain number of living, untransfected tumor cells.
Soon, however, the killers would rest down and, in the absence of more
danger signals, would not be re-activated, explaining why the protection
generated by the transfected tumor can be overcome with a large enough
challenge of normal tumor cells (). A prediction would be that the
larger challenge doses could be dealt with by repeated immunization with
irradiated transfected tumor cells.
The same reasoning may also explain why tumor infiltrating
lymphocytes (TILs) work only in some cases. If TILs are removed from
the tumor, activated in vitro and re-injected along with a source of
IL-2, they may well return to the tumor and engage in a round of
destruction. If the tumor burden is small, this may be enough to
destroy it. However, if there are too many tumor cells for the injected
T cells to destroy in the first round, the T cells will go through their
natural cycle of resting down and waitng to be re-stimulated. Without a
source of activated APCs, they will remain in the resting state, now to
be tolerized by recognition of tumor antigens in the absence of signal
Two.
Direct trauma to the tumor itself is not the only way in which
specific immunization might occur. Activation by any crossreactive
antigen should be enough, and this suggests that getting rid of tumors
might be a simple matter of immunizing repeatedly with appropriate
antigens. The immunizations could merely be injections of disrupted
tumor cells in adjuvant. The injections might consist of tumor cells
that had been infected with a lytic virus or an inducible death gene,
with or without the addition of APC-enhancing cytokines. One could
paint visible surface tumors (repeatedly) with noxious substances,
calculated to kill off enough tumor cells to activate local APCs. It
might help to add carrier determinants to activate helper cells, or to
inject professional APCs fed with the tumor cells (or their antigens, if
known). In any case, repeated immunizations would be necessary. Though
a single immunization would initiate immunity, the response would soon
die down for lack of repeated stimulus. Even if some of the tumor were
destroyed by killers activated during the first immunization, this
apoptotic death would not maintain the response. The tumor meanwhile,
like any other tissue expressing signal One without signal Two, would
induce deletion of tumor-specific memory cells as they rested down. To
be effective, therefore, immunizations should be repeated until the last
vestiges of tumor are gone. In addition such immunizations should be
done early, while the tumor is small and has not had time to delete a
large number of tumor specific T killers, or alternatively they should
be held off for a while after removal of the main tumor mass in order to
give the thymus time to repopulate the periphery with new tumor specific
T cell populations.
Transplantation: Graft rejection is usually considered a
function of signal One; the grafted tissues are rejected because they
express foreign antigens. However, a great deal of evidence suggests
that signal Two is also critical here. For example, ovaries, kidneys,
thyroid and pancreas can all be successfully transplanted if they are
first carefully purged of passenger APCs (105,106 jacobs). If fresh
donor type APCs, are given at the time of transplantation or soon after,
the grafts are rapidly rejected, showing that their failure to act as
immunogens is not due to a lack of expressed antigen (Lafferty). More
importantly, the APC depleted grafts gradually induce tolerance to
themselves such that the recipient mice are eventually unable to reject
their grafts even when immunized with fresh APCs (). This tolerance is
not a generalized suppression because the mice are still able to respond
to donor type spleen cells, showing that the tolerance induced by
thyroid or pancreas, for example, does not extend to cover the MAPs of
other tissues such as spleen.
Livers seem to be especially extraordinary, since they are often
accepted even without APC depletion (107,calne pigs). Though a
rejection crisis is initiated, it wanes after a few weeks and eventually
dies out altogether, leaving the recipients tolerant (107). This is
tough to explain from a self-non-self viewpoint. Why should liver
grafts, replete with APCs, be tolerogenic when skin or heart grafts are
rejected? The reason is that livers are big, they do not have strong
vascular barriers, and they regenerate; consequently they are a large
and easily accessible source of signal One. The process begins when
APCs in the liver become activated by the surgical trauma, home to the
local (recipient) lymph node and activate donor specific T cells which
then migrate to the organ and begin killing all donor cells, including
hepatocytes, endothelial cells and APCs. After a while, all the bone
marrow-derived APCs will have died, but the regenerating liver lasts
longer, continues to offer signal One without signal Two and, rather
quickly because of its size, deletes all the relevant T cells. Should
any hematopoietic stem cells remain, they can now take up residence,
creating the chimerism that is often (but not always) seen in recipients
of liver grafts ().
An apparant exception to the idea that tissues induce tolerance
to themselves comes from transplant recipients who suddenly stop taking
Cyclosporin A and reject their grafts, even though they may have had
them for over 20 years! According to the model, these patients should
be tolerant. The problem here is not the model but the drug. Since CsA
blocks signal One not signal Two (), the first law of lymphotics cannot
operate and no deletion can occur. This is nicely illustrated by an
experiment in which rats were given allogeneic livers under a two week
course of CsA and, at various times later, were immunized with
donor-type skin (). Skin grafts given 0-4 weeks after CsA withdrawal
were rapidly rejected and also stimulated rejection of the livers. By
eight weeks, the livers had tolerized for themselves and, though the
skin grafts were rejected, the livers were not6. The authors determined
that there were three stages of reactivity. In the first, during CsA
treatment, alloreactive T cells were unable either to respond or be
tolerized. In the second "transitional" stage, when the drug was
withdrawn, the T cells were slowly tolerized unless activated by a new
source of APCs. In the third stage, the animal had become solidly
tolerant of the graft. They wondered whether the transitional stage was
delayed by the CsA treatment. Today, we would answer yes. Tissues
cannot induce tolerance to themselves in the presence of a signal One
blocker. This may also be why irradiated rodents whose immune systems
are allowed to regenerate under the cover of CsA suffer autoimmune
symptoms when the drug is discontinued ().
The solution, of course is to find drugs that block signal Two
without obstructing signal One, and some steps have already been made in
this direction. For example, in two back to back studies, soluble CTLA4
evoked long lasting tolerance of xenogeneic islet grafts (). It also
inhibited antibody responses to KLH and SRBC though, to the perplexity
of the authors, not permanently (). I would suggest that the treatment
had the same effect in both cases. It blocked signal Two and stopped
the immune response. In the case of the islets, the grafted tissue
stayed in place, continually tolerizing for itself by expressing its
antigens in the absence of signal Two. However, neither KLH nor SRBC
persist for long in mice and, in their case, a temporary ablation could
not generate long lasting tolerance.
I am less certain of protocols in which antibodies to adhesion
molecules are used (). They run the risk of inhibiting signal One, in
which case, like CsA, they may stop rejection but nevertheless not allow
for the induction of tolerance. An optimal combination might be to give
blockers of signal Two along with a source of stem cells in addition to
the grafted organ. In this case, the organ itself should tolerize any
mature T cells and the stem cells should generate the necessary
chimerism to induce tolerance of newly maturing T cells in the thymus, a
protocol occasionally mimicked by liver grafts, accounting for their
success. coda
Although the danger model does eventually produce a rough
definition of self, the difference from that made by self-non-self
discrimination models is more than just semantics. An organism in which
the availability of second signals governs immunity and tolerance needs
no static definition of self. Its immune system is not a separate army
protecting (and regulating) the rest of the organs of the body, but an
extended, highly interactive network making its decisions on the basis
of input from all bodily tissues. This is a flexible immune system that
changes as the organism changes, that welcomes the presence of useful
commensal organisms and allows the passage of harmless opportunistic
ones. In short this is an immune system that exists in harmony with
both its internal and its external environment.
