Immunological Tolerance Notes


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Immunological Tolerance Notes

  1. 1. PHCL 582 Immunological Tolerance Feb 27, 2006 TOLERANCE Introduction Tolerance refers to the specific immunological non-reactivity to an antigen resulting from a previous exposure to the same antigen. While the most important form of tolerance is non-reactivity to self antigens, it is possible to induce tolerance to non-self antigens. When an antigen induces tolerance, it is termed tolerogen. Induction of tolerance to non-self Tolerance can be induced to antigenic components on both soluble proteins and cells (tissues) by injecting these materials into animals. Induction of such a tolerance depends on a number of variables. Tolerance to tissues and cells Tolerance to tissue and cell antigens can be induced by injection of hemopoietic (stem) cells in neonatal or severely immunocompromised (by lethal irradiation or drug treatment) animals. Also, grafting of allogeneic bone marrow or thymus in early life results in tolerance to the donor type cells and tissues. Such animals are known as chimeras. These findings are of significant practical application in bone marrow grafting. Tolerance to soluble antigens A state of tolerance to a variety of T-dependent and T-independent antigens has been achieved in various experimental models. Based on these observations it is clear that a number of factors determine whether an antigen will stimulate an immune response or tolerance (Table 1). Table 1 Factors that determine induction of immune response or tolerance following challenge with antigen Factors that affect response Favor immune response Favor tolerance to Ag physical form of antigen large, aggregated, complex soluble, aggregate-free, relatively smaller, molecules; less complex molecules, Ag not processed by APC or processed by cell without class II MHC route of Ag administration sub-cutaneous or oral or sometimes intravenous intramuscular dose of antigen optimal dose very large (or sometime very small) dose age of responding animal older and immunologically Newborn (mice), immunologically immature mature differentiation state of cells fully differentiated cells; relatively undifferentiated: B cells with only memory T and memory B IgM (no IgD), T cells (e.g. cells in thymic cells cortex) Immunologic features of tolerance Tolerance is different from non-specific immunosuppression and immunodeficiency. It is an active antigen- dependent process in response to the antigen. Like immune response, tolerance is specific and like immunological memory, it can exist in T-cells, B cells or both and like immunological memory, tolerance at the T cell level is longer lasting than tolerance at the B cell level. Induction of tolerance in T cells is easier and requires relatively smaller amounts of tolerogen than tolerance in B cells. Maintenance of immunological tolerance requires persistence of antigen. Tolerance can be broken naturally (as in autoimmune diseases) or artificially (as shown in experimental animals, by x-irradiation, certain drug treatments and by exposure to cross reactive antigens). Tolerance may be induced to all epitopes or only some epitopes on an antigen and tolerance to a single antigen may exist at the B cell level or T cell level or at both levels. 1
  2. 2. PHCL 582 Immunological Tolerance Feb 27, 2006 Mechanism of tolerance induction The exact mechanism of induction and maintenance of tolerance is not fully understood. Experimental data, however, point to several possibilities. Clonal deletion: Functionally immature cells of a clone encountering antigen undergo a programmed cell death; for example, auto-reactive T-cells are eliminated in the thymus following interaction with self antigen during their differentiation (negative selection). Recent studies have shown that a variety of antigens are expressed in thymic epithelial cells. Likewise, differentiating early B cells become tolerant when they encounter cell-associated or soluble self antigen. B cells expressing only IgM (no IgD) on their surface when exposed to antigen are more prone to tolerance induction than immune response. Clonal deletion has been shown to occur also in the periphery. Clonal anergy: Auto-reactive T cells, when exposed to antigenic peptides which do not possess co-stimulatory molecules (B7-1 or B7-2), become anergic to the antigen. Also, B cells when exposed to large amounts of soluble antigen down regulate their surface IgM and become anergic. These cells also up-regulate the Fas molecules on their surface. An interaction of these B cells with Fas-ligand-bearing cells results in their death via apoptosis. Clonal ignorance: T cells reactive to self antigen not represented in the thymus will mature and migrate to the periphery, but they may never encounter the appropriate antigen because it is sequestered in inaccessible tissues. Such cells may die out for lack of stimulus. Auto-reactive B cells that escape deletion may not find the antigen or the specific helper T-cells and hence not be activated and die out. Receptor editing: B cells which encounter large amounts of soluble antigen, as they do in the body, and bind to this antigen with very low affinity become activated to re-express their RAG1 and RAG2 genes. These genes cause them to undergo DNA recombination and change their specificity. Anti-idiotype antibody: Anti-idiotype antibodies produced experimentally have been demonstrated to inhibit immune response to specific antigens. Anti-idiotype antibodies are produced during the process of tolerization and such antibodies have been demonstrated in tolerant animals. These antibodies prevent the receptor from combining with antigen. Suppressor cells: Both low and high doses of antigen may induce suppressor T cells which can specifically suppress immune responses of both B and T cells, either directly or by production of cytokines, most importantly, TGF-beta and IL-10. Termination of tolerance Experimentally induced tolerance can be terminated by prolonged absence of exposure to the tolerogen, by treatments which severely damage the immune system (x-irradiation) or by immunization with cross reactive antigens. These observations are of significance in the conceptualization of autoimmune diseases. Tolerance Our own bodies produce some 100,000 different proteins and one of the longstanding conundrums of immunology has been to understand how the immune system produces a virtually universal repertoire while at the same time avoiding reacting to self. We can consider two different definitions of immunological tolerance. The strict definition might be that immunological tolerance occurs when an immunocompetent host fails to respond to an immunogenic challenge (ie. one that would produce a measurable response in some other, nontolerant host) with a specific antigen. A more operational definition might be that immunological tolerance occurs when an immunocompetent host fails to respond to the presence of a specific antigen. We have already seen that the adaptive immune system consists of two distinct clonally variable repertoires expressed by T and B lymphocytes respectively. Clearly the essentially random process which generates these repertoires will produce autoreactive cells in both lineages. How does the immune system prevent the activation of these cells? We can divide the mechanisms the immune system uses to ensure the absence of self-reactivity (autoimmunity) into two main types: • Central Tolerance - this occurs during lymphocyte development. • Peripheral Tolerance - occurs after lymphocytes leave the primary organs. Central Tolerance T cells 2
  3. 3. PHCL 582 Immunological Tolerance Feb 27, 2006 As we have already seen, during T cell development in the thymus the process of negative selection leads to the deletion of thymocytes whose T cell receptors have 'high affinity' for self. One aspect we did not consider was what other parameters influenced this selection event. The only one we need to discuss is antigen concentration. Remember that in the context of T cells, antigen means the complex of specific peptide and MHC so the density of any given peptide-MHC complex is determined by the abundance of the peptide and it's affinity for the particular MHC allele. The number of molecules of any given peptide-MHC complex on the surface of a thymic antigen presenting cell probably varies from 1-50,000. We can imagine that if there is only a mean of 1 complex/cell then even the highest affinity T cells might fail to be deleted whereas for the most abundant peptide-MHC complexes quite low affinity T cells would be deleted. Thus we would predict an inverse relationship between the threshold affinity of TCR for peptide-MHC required to produce deletion and the abundance of that peptide-MHC complex. Using transgenic mice and organ culture techniques it has been possible to provide evidence for this hypothesis. B cells During B cell development in the bone marrow when the complete antigen receptor (IgM) is first expressed on 'immature' B cells if those cells encounter their target antigen in a form which can cross-link their sIgM then such cells are programmed to die (deleted from the repertoire). This was discovered many years ago when it was shown that injection of a polyclonal anti-IgM from birth prevented the development of B cells, resulting in a 'B-less' mouse. The requirement for crosslinking means that the antigen has to be polyvalent, the most obvious example of this being cell-surface molecules. This has been directly demonstrated by using transgenic mice expressing rearranged Immunoglobulin genes specific for natural or artificial membrane bound molecules. Presumably other multivalent self antigens to which immature B cells are exposed also induce deletion of self reactive cells. Peripheral Tolerance 3
  4. 4. PHCL 582 Immunological Tolerance Feb 27, 2006 Because many of the proteins which the body uses are not expressed in thymus or serum, and in some cases not expressed until after the immune system has matured there need to be some mechanisms to prevent autoreactivity of lymphocytes after they have emigrated from the thymus/bone marrow. In fact there are several such mechanisms: • Ignorance • Suppression • Anergy • Split Tolerance There are a number of factors which affect which of these mechanisms operates. · Timing · Location · Co-stimulation · Dose Ignorance It can be shown that there are in fact both T cells and B cells specific for autoantigens present in circulation. In some cases these cells are quite capable of making a response but are unaware of the presence of their autoantigen. This arises for 2 reasons. The first is that the antigen may simply be present in too low concentration. Since all lymphocytes have a threshold for receptor occupancy which is required to trigger a reponse then very low concentrations of antigen (in the case of T cells these are very low, see below) will not be sensed. The second possibility is a more interesting one. Some antigens are sequestered from the immune system in locations which are not freely exposed to surveillance. These are termed immunologically privileged sites. Examples of such sites are the eye, CNS and testis. Split Tolerance This term simply means that some part of the immune system is tolerant and some other is not. The most frequent situation is that where T cell tolerance has been established but autoreactive B cells are still present. This arises because T and B cells have different thresholds for activation and therefore tolerance. In this situation the B cells are 'helpless'. That is to say, as we have already discussed for most antigens B cells require help from an antigen-specific T cell in order to make a response. Thus autoreactive B cells can be present without being able to become activated provided that there is no T cell help available. If T cell help is provided, for example by injecting the autoantigen chemically coupled to an immunogenic foreign carrier, then these B cells will mount a response. 4
  5. 5. PHCL 582 Immunological Tolerance Feb 27, 2006 Experiments of this kind indicate that it takes 100 -1000 × more antigen to tolerise B cells that T cells. As a result this type of split tolerance situation is reasonably common for self serum proteins. Anergy - T cells As mentioned in a previous lecture, naive T cells need co-stimulatory signals to become activated. The expression of these co-stimulatory molecules is restricted so that most tissue cells lack either B7.1/B7.2 or CD40 or both. Such cells also normally lack class II MHC molecules. Thus tissue cells normally present a spectrum of peptides from their endogenously synthesised proteins on self MHC class I in the absence of co- stimulation. Interaction of such cells with autoreactive T cells leads to the T cell becoming refractive to later encounter with the same antigen even when co-stimulation is present. This refractory state is termed anergy. 5
  6. 6. PHCL 582 Immunological Tolerance Feb 27, 2006 Anergy - B cells As implied from the section on split tolerance, there is a mechanism for tolerising B cells to soluble antigens if they are present at sufficiently high concentration. The general rules for this tolerance mechanism were worked out using mice transgenic for rearranged immunoglobulin molecules and a transgenic soluble protein whose concentration in serum could be regulated. It is apparent that the critical parameter is receptor (surface Ig) occupancy. When more than 5%* of the sIgM molecules are normally occupied by monomeric soluble antigen the B cell becomes anergic. This anergic state can be recognised in the case of B cells by the downregulation of surface IgM. Note the level of surface IgD remains unaffected and the precise explanation for why such B cells are refractory to stimulation even when T cell help is available is not known. Added Value * Actually the precise threshold level is not known. It has only been measured in one system and in that case 45% occupancy gave anergy, 5% did not 6
  7. 7. PHCL 582 Immunological Tolerance Feb 27, 2006 Suppression It is also clear that in some cases there are autoreactive T cells present which are capable of reacting to their cognate antigen as presented within the host [ ie. they are not anergic or ignorant], yet do not express this reactivity in the normal intact animal. These cells appear to be prevented from reacting by the presence of other T cells, a phenomenon which has been termed Dominant Regulation or Suppression. The mechanism is again obscure, but the following experiments make it likely that this form of tolerance is at least as important as anergy. 7
  8. 8. PHCL 582 Immunological Tolerance Feb 27, 2006 Timing Some 50 years ago Owen observed two types of non-identical twin cattle, those that had shared a haemopoietic system in utero were tolerant of blood cells from each other and those who had not, were not cross-tolerant. Burnet postulated that there was a temporal window of tolerance such that antigens encountered while the immune system was immature tolerised the relevant lymphocytes. Medewar subsequently investigated the effects of transferring haemopoietic cells from histoincompatible mice at different times after birth. He found that if the cells were transferred in the first few days of life (but not later) the recipient mouse acquired lifelong tolerance to the antigens of the donor. 8
  9. 9. PHCL 582 Immunological Tolerance Feb 27, 2006 We now understand a good deal about the mechanism of this acquired tolerance. In essence bone marrow stem cells establish chimaerism of the host. Some of these cells differentiate into antigen presenting cells and migrate to the thymus where they tolerise developing thymocytes by deletion (central tolerance). Lifelong chimaerism is needed to maintain tolerance and even a low level of chimaerism is sufficient. For the first few days of the mouse's life there are too few post-thymic T cells to sustain a response and these are tolerised either by peripheral deletion or some other mechanism. If the transfer is done later the number and maturity of the peripheral T cell pool is sufficient to destroy the donor stem cells before they can engraft. Even antigens which cannot establish chimaerism can establish a state of tolerance in neonatal mice, this is less permanent however. Timing is an important parameter on the tolerisability of the immune system. Experimental Tolerance If we could induce tolerance in adults, this would have obvious benefits in transplantation and the treatment of autoimmunity. Much effort has therefore been expended towards this goal. Indeed a number of regimes have been successfully applied under experimental conditions and with particular antigens. A clinically usable protocol is still elusive however. It will suffice here to give just two examples of tolerising conditions which have worked. Peptide sniffing (inhalation tolerance) If immunogenic (that is MHC binding) peptides are aerosolised and administered via the nostrils to mice, the animals can be rendered tolerant to immunogenic challenge with the same peptide (ie given subcutaneously in adjuvant). 9
  10. 10. PHCL 582 Immunological Tolerance Feb 27, 2006 Co-receptor blockade The use of monoclonal antibodies to any of the important co-receptors, CD4/8, B7, CD40 where a particular response is dependent on the blockaded co-receptor can lead to the development of tolerance. The Danger Hypothesis - Matzinger versus Medewar? Matzinger has proposed that there is not a special window for tolerance during neonatal life but that whether encounter with an antigen results in tolerance or an immune response is determined by whether the prevailing host environment promotes a response via nonspecific cues 'sensing' danger. She has further suggested that the controlled death process of apoptosis is critical in preventing autoimmunity when old or surplus cells are disposed of. The notion that the normal, default pathway of the immune system is tolerance rather than 10
  11. 11. PHCL 582 Immunological Tolerance Feb 27, 2006 response is not a new idea to immunologists - antigens usually fail to elicit a response unless given with adjuvants, whose purpose is probably to generate stimulatory cues (cytokines). Recent experiments have shown that not only can adults be tolerised under certain circumstances, but that neonates can make effective immune responses if the antigen is presented in sufficiently immunogenic form. I believe that the supposed conflict between Matzinger and Medewar is rather 'hyped up' and essentially a matter of detail. Neonatal T cells are not intrinsically tolerisable but the systemic neonatal environment does predispose to tolerance. Nevertheless, I think that her hypothesis has drawn the attention of a wider audience to current ideas about tolerance induction and the factors determining immune responsiveness. 11