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Mechanisms of tolerance to self
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Mechanisms of tolerance to self Jacques FAP Miller* and Antony Bastent Several mechanisms exist to prevent lymphocytes from reacting against self-antigens. As T cells develop in the thymus and express antigen-specific receptors, those with high-affinity to self-antigens existing within the thymus are deleted. Low-affinity self-reactive T cells and T cells with receptors against antigens not represented intrathymically will mature and join the peripheral T cell pool. They may either ignore self-antigens expressed by tissues unable to activate T cells through a lack of the appropriate costimulator signals, or they may, under certain conditions, be deleted or rendered anergic and unable to respond. Likewise, B cells that express surface Ig receptors with high binding affinity to membrane-bound self-antigens present in the bone marrow will be rescued by receptor editing or will be deleted, whereas those of lower affinity will migrate to the periphery in either an anergic or indifferent state depending on the degree of receptor engagement by antigen. Once there, their ultimate fate is determined by the availability of T cell help.
Addresses *The Walter and Eliza Hall Institute of Medical Research, Post Office, The Royal Melbourne Hospital, Victoria 3050, Australia; e-mail: [email protected] '~The Centenary Institute of Cancer Medicine and Cell Biology, Locked Bag Number 6, Newtown, NSW 2042, Australia
of the lymphocyte at the time it encounters antigen. This was clearly pointed out by Lederberg in 1959 [5], in his modification of the clonal selection theory: contact of immature lymphocytes with antigen should lead to deletion or functional inactivation, whereas contact with mature cells should result in immunity. Formal experimental proof for this concept was obtained only in the late eighties and early nineties with the demonstration of the physical deletion (i.e. negative selection) of clones of developing T and B cells specifically reactive to self-antigens presented intrathymically [6,7] or within the bone marrow, respectively [8,9]. Other key factors influencing responsiveness relate to how the antigen is presented and include the nature of the cells that are presenting epitopes, the affinity of epitope binding, the production by APCs of costimulatory molecules and the secretion of nonepitope chemical products. It thus comes as no surprise that the few mature T cells that are present in neonatal mice can be activated by professional APCs [10 °] and that, given the right conditions, priming for both T h l and Th2 T cells can be induced in neonatal mice [11°,12°]. In fact it has been known for more than 20 years that, even at the level of thymocytes, the mature T cells in neonatal (one day old) mice are strongly reactive to foreign antigens [131.
Current Opinion in Immunology 1996, 8:815-821
© Current Biology Ltd ISSN 0952-7915 Abbreviations APC antigen-presenting cell HEL hen egg lysozyme IL interleukin mOVA membrane-bound OVA OVA ovalbumin RIP rat insulin promoter TCR specific T cell receptor Tg transgenic
Tolerance resulting from the inoculation of foreign mate~ rial may not always mimic self-tolerance and under certain circumstances can be an epiphenomenon reflecting the induction of various immunoregulatory feedback loops. For this reason, and because recent reviews have dealt with mechanisms of tolerance in general (e.g. [14,15]), in the present article we shall concentrate mainly, although not exclusively, on issues pertaining to tolerance to self-components.
Self-tolerance in the T cell repertoire Intrathymic selection
Introduction Although Burner suggested that antigen encountered in early life selectively deletes specific clones, whereas it activates them in later life [1], it has been realized for close to four decades that there is nothing intrinsically unique about the prenatal or neonatal period as far as tolerance is concerned. In fact, Burnet himself and his colleagues [2,3] failed to induce tolerance in chicks or mice, even after in o v o or in utero exposure, presumably due to rapid antigen clearance. By contrast, T and B cell tolerance to the immunogenic form of a protein could easily be induced in adult mice by inoculating the protein in deaggregated form [4]. One key factor in determining the nature of the response, whether tolerance or immunity, therefore is not the developmental stage of the individual but rather the state of differentiation
Both positive and negative selection involve the recognition of self-peptides, in association with self-MHC molecules, presented to differentiating T cells by thymic APCs. How then do signals initiated through the same T C R lead to these two opposing processes? Models to explain this conundrum fall into the following two major groups: those determined by the strength of the interaction between the T C R and its ligands [15]; and those dependent on changes in the conformation or topology of the T C R after it has bound different ligands [16]. In a recent study [17°], the outcome of thymic selection was correlated with the agonist or antagonist activity of M H C - p e p t i d e complexes for peripheral T cell activation, the agonist complexes having high-affinity for the T C R and the antagonist having low-affinity. These results are in agreement with kinetic discrimination
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models in which the rate of ligand dissociation from the T C R determines the agonist or antagonist properties of the ligand [18]. How the coreceptors, CD4 and CD8, might influence the outcome of some of these interactions has not been determined, however. To what extent intrathymic T cell deletion during negative selection involves the costimulatory molecules CD28, CTLA-4, B-7.1 or B-7.2 [14] is not clear. It does not appear to involve the CD95 molecule [19]. By contrast, interactions between CD40 and gp-39 (CD40 ligand) [20] provide costimulatory signals for thymic deletion and CD30 is an important coreceptor that produces a death signal during intrathymic T cell development [21].
Mechanisms preventing T cell autoimmunity
Although intrathymic negative selection of self-reactive T cells is generally considered to be the predominant mechanism for inducing self tolerance, there is ample evidence for the presence of autoreactive T cells in healthy individuals and animals (e.g. [22]). These cells may not have encountered their target autoantigen in the thymus or, if it was present intrathymically, they may have escaped negative selection because they expressed a T C R of too low an affinity for the self-peptide-MHC complex [17"]. Alternatively, the self-peptide may not have been able to produce a stable complex with M H C molecules in the thymus and thus could not be presented effectively [23]. To prevent autoimmunity, therefore, the autoaggressive potential of self-reactive T cell escapees must be held in check by a variety of mechanisms, some of which induce peripheral tolerance. These include indifference, deletion and immune regulation, as discussed here, and T C R and coreceptor downregulation and anergy, as reviewed elsewhere [24]. Because most naive T cells do not usually circulate through nonlymphoid tissues [25], unprimed autoreactive T cells should not inflict damage on target tissues [26]. This was clearly demonstrated in transgenic (Tg) mice expressing the class I molecule H-2K b in pancreatic islet 13 cells (rat insulin promoter [RIP]-Kb mice) and expressing in most of their CD8 ÷ T cells a Kb-specific T C R identifiable by a monoclonal clonotypic antibody. In the absence of intentional priming, the CD8 ÷ T cells ignored islet antigens. T h e mice were not tolerant of Kb as they rejected Kb-bearing skin grafts and most islets became infiltrated, resulting in specific destruction of 13 cells two weeks after such priming [27°]. Even if naive autoreactive T cells were to gain entry to a nonlymphoid tissue, as a result of local inflammation, they might still remain silent unless optimally stimulated by a strong immunogenic stimulus by professional APes, thus increasing their prect, rsor frequency. Indeed, one of the major factors that reverses indifference and leads to autoimmunity is an increase in the precursor frequency of autoreactive T cells [28",29"].
Induction of peripheral autoreactive T cell deletion
Antigen-induced proliferation of T cells is accompanied by the deletion of some of the activated cells; this is likely to represent a homeostatic mechanism that prevents unrestricted growth of these clones. Such a phenomenon, sometimes referred to as propriocidal regulation [30"], may also play a crucial role in preventing autoimmune reactions. Indeed, the failure to induce T cell apoptotic death is involved in the pathogenesis of autoimmunity in mice homozygous for the lpr gene and for the gld gene [31], and in mice in which the gene for CTLA-4 has been disrupted [32]. Highly relevant to the mechanism of preventing autoimmune responses against target nonlymphoid tissues cells is the question of which cell is responsible for inducing the deletion of activated T cells. Could any cell be involved or only professional APCs? T h e T cells of mice that are T g for the expression of the allogeneic M H C class I molecules H-2Kb, predominantly in the liver, and H-2Kb-specific TCR, in most of their CD8 ÷ T cells, showed signs of activation, then proliferated, caused damage to the hepatocytes, and eventually underwent apoptosis [33"]. In this and other T g models studied to date, it cannot be excluded that professional APCs were involved in activating T cells either because the APCs expressed the T g antigen aberrantly or acquired it in some way. This issue was resolved in another T g model in which ovalbumin (OVA)-specific T g CD8 ÷ T cells were injected into unirradiated T g RIP-mOVA mice, which express a membrane-bound form of OVA (mOVA) in pancreatic islet 13cells and in renal proximal tubular cells ([34°°]; C Kurts et al., unpublished data). In these studies, the nonlymphoid tissue-associated 'self' antigens, were presented, not by the tissue cells themselves, but by professional APCs in the context of MHC class I via a constitutive exogenous cross-over processing pathway. T h e CD8 + T cells were activated, proliferated (initially) and some of the progeny infiltrated the islets but had a curtailed lifespan. Their elimination may simply have reflected the operation of the normal homeostatic mechanism that regulates any CD8 ÷ T cell immune response, but as the tissue antigen persisted all the activated CD8 ÷ T cells may have eventually been doomed to die, particularly in the absence o f C D 4 ÷ T cell help. T h e results of these experiments provide a constitutive mechanism whereby T cells can be primed to self-antigens present in nonlymphoid tissues. T h e y therefore support the thesis that most naive T cells fail to migrate into nonlymphoid tissues [25] and argue against the recent suggestion that this is not true but that both naive and memory T cells migrate into both lymphoid and nonlymphoid organs [35]. T h e data also cast doubt on the physiological significance of the idea of naive T cells becoming anergized in vivo as a result of receiving only one signal from antigens expressed in tissues which lack the ability to generate a second signal such as costimulation.
Mechanisms of tolerance to self Miller and Basten
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Similarly they are difficult to reconcile with the 'danger hypothesis' [36] as the cells were activated by shed self-antigen in a perfectly normal unstressed situation [34 °°] and they did infiltrate the islets, the extent of the infiltration (and hence, damage) being dependent on the precursor frequency (C Kurts et al., unpublished data). Under physiological conditions, the frequency of a given autoreactive T cell clone is likely to be so low that after activation the cells would not gain entry to healthy tissues in sufficient numbers to cause disease, but would be subjected to the normal homeostatic regulation described above. Furthermore, bona fide self-antigens may not be expressed as abundantly on nonlymphoid tissue cells as the mOVA in this model. Nonetheless, these results provide a plausible mechanism whereby infected cells and perhaps even malignant cells, that lack the molecular machinery for direct immune induction, could activate CD8 ÷ T cells via APCs specialized to process shed antigen and shunt it to the class I pathway. How then would CD8 ÷ T cells distinguish between the virus-specified antigen on virus-infected nonlymphoid tissue cells which they must kill and the self-antigen on normal tissue cells which must be kept intact? T h e answer may lie in the fact that, in contrast to the situation with pathogens, the CD4 ÷ T cell repertoire is tolerant of shed self-antigens ([34°°]; C Kurts et al., unpublished data) and T cell help is not available to allow sufficient expansion of the activated CD8 + T cells.
IL-10 by the CD4 ÷ T cells and the suggestion was made that pathogenic T h l - t y p e T cells had been diverted to protective Th2-type T cells [46]. In these T g N O D mice, the M H C class II transgenes are thought to have acted both intrathymically and extrathymically. T h e most likely explanation is that intrathymic expression of non-NOD class II genes is essential for the positive selection of class II-restricted immunoregulatory T cells, while peripheral expression is required to bring about the interaction of these cells in a tricellular complex with N O D autoantigen-specific T cells and APCs, so as to deviate the response down a nonpathogenetic pathway. Significantly, for protection to occur in the I-E T g N O D mice, the I-E transgene had to be expressed both intrathymically and on B cells [47], which would support the notion that autoantigen displayed by B cells may be required to activate protective Th2-type immunoregulatory T cells.
Immunoregulation and dominant T cell tolerance
S e l f t o l e r a n c e in t h e B cell r e p e r t o i r e The decision between immunity and tolerance
T-cell-dependent immunoregulation may be considered as a fail safe mechanism to inhibit activated autoreactive T cells [37]. It has been demonstrated in vivo in several experimental models in sheep [38] rats [39] and in T g and non-Tg mice [40,41]. CD4 ÷ T cells and Th2-type cytokines have been implicated in mediating immunoregulatory effects in most models of dominant tolerance. Thus, for example, anti-IgD-peptide complexes protected rats from the development of experimental allergic encephalomyelitis induced by immunization with myelin basic protein in complete Freund's adjuvant, and this protection was associated with a Th2-type cytokine response [42"]. Protective Th2 cells, presumably induced by peptide-presenting B cells [43], might have inhibited autoaggressive cells by secreting cytokines such as IL-4, IL-10, or transforming growth factor-[3 or by recruiting protective CD8 ÷ T cells. Transforming growth factor-[3 has indeed been shown to diminish T cell activation and inflammatory responses [441. T h e T g introduction of non-nonobese diabetic (NOD) M H C class II genes (H-2A or H-2E) into N O D mice has prevented these mice from developing spontaneous diabetes (for review see [45]). Protection was not achieved by deletion or by permanent silencing of all autoreactive T cell clones. Purified CD4÷ T cells from N O D mice expressing a normal I-Ab d molecule prevented diabetogenic T cells from transferring disease to non-Tg recipients. This was associated with the production of IL-4 and
How do immunoregulatory CD4 ÷ T cells arise in the normal animal? One possibility is that they would be selected by self-antigens expressed intrathymically. If such cells had low-affinity for these antigens, they could sneak through the censorship mechanism and build up a peripheral repertoire of immunoregulatory T cells responsible for controlling autoreactive T ceils by the mechanism of immune deviation.
Recent work on self-tolerance in B cells, similar to that in T cells, has been focused on the cellular and molecular events responsible for determining whether interaction with antigen leads to persistent activation and survival or to transient activation and death by apoptosis. In this context it is becoming increasingly apparent that anergy in B cells (and possibly T cells) may represent an intermediate step in the activation pathway when the final decision between survival and deletion remains in the balance [481. Experiments conducted in T g mice including the well defined hen egg lysozyme (HEL)/anti-HEL T g model indicate that B cells are susceptible to tolerance induction at multiple stages during their development [49°°]. In practical terms, such a scenario makes good sense given the predilection of B cells to undergo hypermutation in response to antigen and T cell signals within peripheral lymphoid tissues: it also highlights the complexity of the events involved in the decision making process between immunity and tolerance. As a consequence of this complexity, even now the molecular basis for the long established sensitivity of immature B cells to tolerance induction remains elusive. To cite one example, the recent demonstration of a selective deficiency in thefvn (andfgr) members of the src family of tyrosine kinases in immature B cells [50 ° ] needs to be reconciled with the equally convincing finding that lyn -/- mice spontaneously develop
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an autoimmune syndrome characterized by circulating autoantibodies and immune complex glomerulonephritis [51°,52°]. On the other hand, the relative importance of the various factors responsible for the decision between immunity and tolerance in mature B cells is becoming clearer at least at the cellular level, with a major role being attributable to the degree of receptor engagement and the availability of T cell help. Role of T cell help in B cell tolerance
Irrespective of the precise mechanism involved, the selective susceptibility of immature self-reactive B cells to tolerance induction is thought to be an intrinsic property of the cells themselves and according to Monroe, may not be influenced by the availability of T cell help [53°]. On the other hand, mature self-reactive B cells that have escaped negative selection in the bone marrow are clearly capable of receiving T cell help which highlights the potential importance of the presence or absence of extrinsic second signals in determining the fate of B cells following antigen stimulation. This prediction receives support from previous experiments demonstrating the reversability of B cell tolerance by gp-39 in vitro and by strong antigenic stimuli in vivo [54,55], and from more recent studies of the effects of idiotype-specific [56] or antigen-specific T cells [57 -°] on B cell tolerance. In the case of the latter, Ig Tg B cells labelled with the inttacytoplasmic dye, 5,6-carboxy-succinimidyl-fluorescein-ester, were transferred into self-antigen-expressing Tg recipients where they homed within 18 hours to the outer T cell zone. When T cell help was provided through recognition of a peptide on the B cell surface, the B cells migrated to the outer T cell zone and then proliferated throughout the follicles with the formation of germinal centres, proliferative foci and the production of autoantibodies. By contrast, antigen-stimulated B cells, which could not interact with the transferred T cells, died in the outer T cell zone within three days; in other words, T cell help was crucial to the decision between activation and tolerance. A similar conclusion has been reached by Finkelman et al. [58-°] using the surrogate antigen, anti-IgD.
Ig receptor engagement Experiments involving the transfer of Ig Tg B cells into H E L T g recipients or non-Tg recipients given exogenous H E L indicated that B cells, irrespective of their specificity for self-antigen or foreign antigen or their stage of maturation, homed to the outer T cell zone of the follicles in search of T cell help, provided they received an antigenic signal above a critical threshold [57°°]. When expressed in terms of receptor occupanc'~; the threshold required to direct the B cells to the outer T cell zone and to cause significant downregulation of surface Ig was shown to lie between 30% and 75% of surface Ig molecules [57"°,59"], and in the case of soluble self-antigen resulted in anergy. On the other hand, a substantially lower level of receptor occupancy on the B cells was associated with a failure in migration to the outer T cell zone and
corresponded to a state of indifference. By inference, exposure to multivalent membrane-bound self-antigen that causes deletion must involve a receptor occupancy in excess of the figure responsible for anergy. These findings, if taken in conjunction with previous studies on longevity of tolerant B cells, point to a close relationship between Ig receptor occupancy by self-antigen, tolerance induction and lifespan. Thus, the lifespans of deleting, anergic and indifferent B cells were very short, intermediate and normal respectively [48]. Moreover, anergy in B cells may simply be a form of delayed deletion. One of the intriguing aspects of the transfer system, in which anti-HEL Ig T g B cells were injected into antigen-expressing Tg hosts, was the finding that naive B cells transferred into tolerant double Tg recipients expressing both Ig and H E L transgenes, as opposed to single H E L Tg recipients, failed to home to the outer T cell zone and moved into the follicles where they survived for at least seven days [57°',60]. When the effective concentration of antigen was measured in the serum of the two types (double T g and single Tg) of recipients it was found to be below the threshold required to cause significant downregulation of surface Ig or to direct the B cells to the outer T cell zone in the case of the double but not single Tg mice [57°°,59°]. In a related experiment, it was shown that anergic B cells in intact double Tg mice given zinc to increase the serum concentration of H E L and thereby the antigenic signal, could now home to the outer T cell zone. Moreover, Ig T g B cells pulsed with H E L in vitro also acquired the capacity to localize to the outer T cell zone on transfer into double Tg recipients (M Cook et al., unpublished data). In other words, the strength of the antigenic signal rather than either the functional state of the B cells (normal versus anergic [61°]) or the repertoire of specificities of the B cells in the follicle (polyclonal in single H E L Tg mice versus monoclonal in double Tg mice [60]) determines the positioning and ultimate fate of self-reactive B cells in this system. Presumably, the reason why B cells from double Tg mice are rendered tolerant, despite their failure to localize to the T cell zone, is that they are exposed to soluble self-antigen within the bone marrow at an immature stage in their development when the threshold of receptor occupancy and signalling required for tolerance induction is lower than in mature B cells. Role of B cells in T cell tolerance
B cells by virtue of their specific Ig receptors are highly efficient APCs for protein antigens and have been shown to cooperate effectively with CD4 + Th cells in antibody responses [62]. On the other hand, their role in the induction of peripheral T cell tolerance has been controversial. Among the reasons for this controversy is the inevitable presence of other APCs in addition to B cells in intact hosts and potential differences in their state of activation and that of cooperating T cells at the time of antigen encounter. Two strategies have
Mechanisms of tolerance to self Miller and Basten
been used in an attempt to resolve the problem. In one approach, gene-targeted B cell deficient ~ M T - / - mice were exposed to antigen in tolerogenic form. T h e results indicated that tolerance in the T cell compartment was as prolonged in mice lacking B cells as in B cell sufficient controls [63••,64°']. In other words, APCs other than B cells arc clearly capable of inducing class II-restricted T cell tolerance which is consistent with the great rarity of T cell autoimmunity in human subjects with X-linked agammaglobulinaemia. These findings, however, are not relevant to the reciprocal issue of the outcome of restricting antigen presentation to B cells. According to both Fuchs and Matzinger [65] and Eynon and Parker [66], the answer is tolerance not activation, although they and others have not reached a consensus on the activation state of the B cells involved. In order to re-examine this particular question, a different approach was used involving the transfer of anti-HEL [g T g B cells together with naive T C R T g T cells into severe combined immunodeficient recipients. When the conditions were such as to restrict antigen presentation exclusively to the B cells, the transferred T cells initially proliferated but were then rendered anergic on restimulation with antigen in vitro. By contrast, T cells underwent sustained activation if other APCs in addition to B cells were available to present antigen. Moreover, anergic B cells which express reduced levels of costimulatory molecules such as the CD80/86 complex induced less T cell proliferation and only partial tolerance (M Cook et al., unpublished data). These findings not only confirmed the toleragenic capacity of antigen-activated B cells for naive T cells, but also provided further evidence to suggest that the early stages of tolerance and immunity are remarkably similar. A similar conclusion concerning the toleragenicity of B cells was reached by Buhlmann et al. [67 ° ] who demonstrated allospecific T cell tolerance in recipients of resting allogeneic B cells in which CD40 signalling had been disrupted either genetically or by administration of anti-gp-39 antibody. Although these results confirm the potential therapeutic value of inhibiting CD40 signalling, their relevance to the role of B cells in T cell tolerance is less clear cut, because allogeneic B cells given on their own were not toleragenic. In addition to the explanation given here, namely the 'ease with which they acquire professional APC capacities upon transfer', it is possible that the B cell population may have been contaminated with very small numbers of other APCs [65].
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Sarzotti M, Robbins DS, Hoffman PM: Induction of protective CTL responses in newborn mice by a murine retrovirus. Science 1996, 271:1726-1729. A cytotoxic T lymphocyte response occurred in newborn mice inoculated with low doses of Cas-Br-M routine leukaemia virus, but when high doses of virus were injected, a nonprotective Th2-type response resulted. Thus, by reducing the dose of virus in proportion to the peripheral T cell number, neonatal mice were able to produce a protective CD8 + T cell response, again showing that toleragenicity is not related to the stage of maturity of the animal as a whole. 12. Forsthuber T, Yip HC, Lehmann PV: Induction of TH1 and TH2 • immunity in neonatal mice. Science 1996, 271:1728-1730. Newborn mice injected with protein in incomplete Freund's adjuvant produced a vigorous Th2 response when challenged with the protein in complete Freund's adjuvant. Interestingly, only spleen and not lymph node ceils, were responsible for this response presumably because T cell activation is associated with downregulation of L-selectin and this prevents re-entry into lymph nodes but allows accumulation in the spleen. 13.
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Miller JFAP, Heath WR: Self ignorance in the peripheral T cell pool. Immuno/Rev 1993, 133:131-150.
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Heath WR, Karamalis F, Donoghue J, Miller JFAP: Autoimmunity caused by ignorant CD8 + T cells is transient and depends on avidity. J Immuno/1995, 155:2339-2349. Transgenic mice expressing H-2K b (K b) in pancreatic islet 13 cells were tolerant of Kb-bearing skin due to intrathymic expression of a few molecules of transgenic K b. To prevent thymus expression, RIP-K b mice were thymectomized, irradiated with 90OR, protected with bone marrow from Des-TCR transgenic mice (which expressed in most of their CD8 + T cells a Kb-specific TCR identifiable by a clonotypic antibody) and grafted with an irradiated (IO00R) thymus from nontransgenic syngeneic newborn donors. RIP-K b mice manipulated in this way had circulating Kb-specific CD8 + T cells with a high density of surface clonotype + TCR, but only two out of twenty-two mice showed very few CD8 + T cells in some of the 20 islets scored. In the absence of intentional priming, therefore, these T cells ignored islet antigens. The mice were not tolerant of Kb as they rejected Kb-bearing skin grafts and most islets were infiltrated two weeks after such priming. Analysis revealed that ~ cells were specifically destroyed in this response. Many manipulated mice rapidly lost weight and died. Hence, high density T cells, presumably those with high avidity for K b, were capable of inducing lethal diabetes after priming and in the absence of constitutive local IL-2 production. Von Herrath MG, Guerder S, Lewicki H, Flavell RA, Oldstone MAB: Coexpression of B7-1 and viral ("selF') transgenes in pancreatic [3 cells can break peripheral ignorance and lead to spontaneous autoimmune diabetes. Immunity 1995, 3:727-738. Tg mice expressing either B7-1 alone (RIP-B7-1) or only the glycoprotein (RIP-GP) or nucleoprotein (RIP-NP) of the lymphocytic choriomeningitis virus, in pancreatic islet ~ cells did not develop insulin-dependent diabetes mellitus. The RIP-NP, but not the RIP-GP mice, aberrantly expressed the transgene in the thymus. RIP-B7-1 x RIP-NP bigenic mice deleted most of their autoreactive CD8 + T cells intrathymically and failed to develop insulindependent diabetes mellitus, but did so within 14 days of lymphocytic choriomeningitis virus infection. On the other hand, RIP-B7-1 xRIP-GP bigenic mice developed insulin-dependent diabetes mellitus in the absence of viral infection. These results suggest a correlation between the development and frequency of insulin-dependent diabetes mellitus and the precursor frequency of autoreactive T cells.
33. •
Bertolino P, Heath WR, Hardy CL, Morahan G, Miller JFAP: Peripheral deletion of autoreactive CD8 + T cells in transgenic mice expressing H-2K b in the liver. Eur J Immuno11995, 25:1932-1942. B10.BR Tg mice expressing K b in hepatocytes under the control of the metallothionein promoter (MET-K b mice) were manipulated as the RIP-K b mice in [27"] to prevent thymus expression of the K b transgene. The livers were heavily infiltrated with lymphocytes by four weeks after thymus grafting and to a lesser extent by 20 weeks, many of the CD8 + T cells showing apoptosis. As the liver has great regenerative ability, continuous antigenic stimulation must have confronted the infiltrating T cells a,~d this was probably the reason for their deletion. It was thought unlikely that the immunogenic stimulus originated from radioresistant and persisting professional APCs and Kupffer cells which might have aberrantly expressed the K b transgene. 34. •-
Kurts C, Heath WR, Carbone F, Allison J, Miller JFAP, Kosaka H: Constitutive class I-restricted exogenous presentation of self antigens in vivo. J Exp Med 1996, in press. Two sets of Tg mice were used: mice of the RIP-mOVA line which expressed mOVA in the pancreatic islet [3 cells and the renal proximal tubular cells and mice of the OT-I line which produced OVA-specific CD8 + T cells. OT-I cells injected intravenously into nonirradiated RIP-mOVA mice (shown to be tolerant to OVA in the CD4 + T cell population), selectively homed to the draining lymph nodes of the kidneys and pancreas, expressed activation markers there and some entered the cell-cycle. Unilateral nephrectomy 7-14 days prior to OT-1 cell inoculation allowed the OT-I cells to home only to the regional lymph node of the remaining kidney and of the pancreas, but when nephrectomy was performed four hours before injecting the T cells, homing to the regional nodes on both sides occurred. The use of bone marrow chimeras (in which marrow ceils from bin1 or bin8 - the cells of which cannot present the OVA peptide to OT-I cells - or from syngeneic donors was given to protect irradiated C57BL/6 RIP-mOVA mice) proved that the OT-I cells were activated by OVA presented by short lived APCs of bone marrow origin in the draining lymph nodes of the OVA-expressing tissues and not in the tissues themselves. 35.
Westermann J, Pabst R: How organ-specific is the migration of 'naive' and 'memory' T cells? Immunol Today 1996, 17:278-282.
36.
Matzinger P: Tolerance, danger and the extended family. Annu Rev/mmunol 1994, 12:991-1045.
37.
Charlton B, Lafferty K: The T h l / T h 2 balance in autoimmunity. Curr Opin /mmuno11995, 7:793-798.
36.
McCullagh P: The significance of immune suppression in normal self tolerance./mmunol Rev 1996, 149:127-153.
39.
Saoudi A, Seddon B, Heath V, Fowell D, Mason D: The physiological role of regulatory T cells in the prevention of autoimmunity: the function of the thymus in the generation of the regulatory T cell subset. Immunol Rev 1996, 149:195-216.
40.
Liblau RS, Singer SNM, McDevitt HO: Thl and Th2 CD4 + T cells in the pathogenesis of organ-specific autoimmune diseases. /mmuno/Today 1995, 16:34-38.
41.
Kumar V, Sercarz EE: The involvement of T cell receptor peptide-specific regulatory CD4 + T cells in recovery from antigen-induced autoimmune disease. J Exp Med 1993, 178:909-916.
28. •
F6rster I, Hirose R, Arbeit JM, Clausen BE, Hanahan D: Limited capacity for tolerization of CD4 + T cells specific for a pancreatic 13cell neoantigen. Immunity 1995, 2:573-585. As in the previous study [28°], there is a correlation between the number of circulating autoreactive transgenic T cells specific for a known 13 cell transgenic antigen and heavy intraislet infiltration. 29. •
30. •
Lenardo MJ, Boehme SA, Chen L, Combadiere B, Fisher G, Freedman M, McFarland H, Pelfrey C, Zheng L: Autocrine feedback death and the regulation of mature T lymphocyte antigen responses. Intern Rev Immunol 1995, 13:115-134. A very thoughtful review relating to antigen-induced T cell death as an important regulatory mechanism in the peripheral immune system.
42. •-
Saoudi A, Simmonds S, Huitinga I, Mason D: Prevention of experimental allergic encephalomyelitis in rats by targeting autoantigen to B cells: evidence that the protective mechanism depends on changes in the cytokine response and migratory properties of the autoantigen-specific T cells. J Exp Med 1995, 182:335-344. Intravenous injection of anti-lgD-myelin basic protein conjugated was administered to Lewis rats one or two weeks before immunization with myelin basic protein in adjuvant and prevented the development of encephalomyelitis but not the production of myelin basic protein-specific antibodies. Lymph node cells from pretreated rats proliferated m vitro in response to myelin basic protein as vigorously as cells from nonpretreated controls, but produced less interferon-y and more RNA for IL-4 and IL-13. They were inferior in their ability to transfer disease after in vitro activation. Pretreated rats also showed greatly reduced levels of leukocyte infiltration in the central nervous system. Protection afforded by pretreatment thus appears to be mediated by T cells with a Th2-type cytokine response and these cells seem to have an intrinsic inability to ir'iltrate the noninflamed central nervous system.
Mechanisms of tolerance to self Miller and Basten
43.
Mason D: The role of B cells in the programming of T cells for IL-4 synthesis. J Exp Med 1996, 183:717-719.
44.
KehrlJH, Wakefield LM, Roberts AB, Jakolew S, Alvarez-Mon M, Derynck R, Spore MB, Fauci AS: Production of transforming growth factor b by human T lymphocytes and its potential role in the regulation of T cell growth. J Exp Mad 1986, 163:1037-1050.
45.
Slattery RM, Miller JFAP: Influence of T lymphocytes and major histocompaUbility complex class II genes on diabetes susceptibility in the NOD mouse. Curt Topics Microbiol Immunol 1 9 9 5 , 206:51-66.
46.
Singer SM, Tisch R, Yang XD, McDevitt HO: An Abd transgene prevents diabetes in nonobese diabetic mice by inducing regulatory T cells. Proc Nat/Acad Sci USA 1993, 90:9566-9570.
4"7.
B~hme J, Schuhbaur B, Kanagawa O, Benoist C, Mathis D: MHC-linked protection from diabetes dissociated from clonal deletion of T cells. Science 1987, 249:293-295.
48.
FulcherDA, Basten A: Reduced life span of anergic selfreactive B cells in a double transgenic model. J Exp Med 1994, 179:125-134.
49. o•
Goodnow CC, Cyster JG, Hartley SB, Bell SE, Cooke MP, Healy JJ, Akkaraju S, Rathmell JC, Pogue SL, Shokat KP: Self-tolerance checkpoints in B lymphocyte development. Advlmmuno11995, 59:279-369. A comprehensive overview of recent work on the cellular and molecular mechanisms underlying self-tolerance in the B cell repertoire. It makes the important point that tolerance can be imposed at multiple stages of the development of the B cell lineage and draws attention where appropriate to the 'clinical' consequences of a breakdown in self-tolerance at the various checkpoints. 50. •
Wechsler RJ, Monroe JG: Immature B lymphocytes are deficient in expression of the src-family kinases p59fYn and p55fgrl. J Immuno/1995, 154:1919-1929. See annotation [53"]. 51. Hibbs ML, Tarlinton DM, Armes J, Grail D, Hodgson G, Maglitto R, • Stacker SS, Dunn AR: Multiple defects in the immune system of lyn-deficient mice culminating in autoimmune disease. Ca// 1995, 83:301-311. See annotation [52"]. 52. •
NishizumiH, Taniuchi I, Yamanashi Y, Kitamura D, Ilic D, Mori S, Watanabe T, Yamamoto T: Impaired proliferation of peripheral B cells and indication of eutoimmune disease in lyn-deficient mice. Ce/11995, 3:549-560. This paper and [51 ",52"] shows that disruption of the src family protein-tyrosine kinase lyn leads to a profound disturbance in B cell function manifest by reduced B cell numbers, hyper-gammaglobulinaemiaand a lupus-like autoimmune syndrome. Interestingly, deliberate or spontaneous disruption of genes encoding other tyrosine kinases and phosphatases [e.g. PTP1C] involved in Ig receptor signalling also leads to immunodeficiency and autoimmunity, highlighting the complexity of the molecular events underlying B cell regulation. 53. Monroe JG: Tolerance sensitivity of immature-stage B cells. J • Immuno11996, 156:2657-2660. This paper and [50"] discuss the issue of the relative importance of the stage of maturation in induction of B cell tolerance and suggest that a selective deficiency in fyn and fgr kinases may define the immature (transitional) stage in B cell development, but see [52"]. 54.
56.
Eris JM, Basten A, Brink R, Doherty K, Kehry MR, Hodgkin PD: Anergic self-reactive B cells present antigen and respond normally to CD40 dependent signals but ere defective in antigen receptor mediated functions. Proc Nat/Acad Sci USA 1994, 91:4392-4396. Cooke MP, Heath AW, Shokat KM, Zeng Y, Finkelman FD, Linsley PS, Howard M, Goodnow CC: Immunoglobulin signal trensduction guides the specificity of B celI-T cell interactions end is blocked in tolerant self-reactive B cells. J Exp Mad 1994, 179:425-438.
56.
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Caulfield MJ, Stank• D: T-cell dependent response to immune complexes abrogates B-cell unresponsiveness to pneumococcal cell wall polysaccharide. Immunology 1995, 86:331-335.
57. o•
Fulcher DA, Lyons AB, Korn SL, Cook MC, Kileda C, Parish C, Fazekasde St.Groth B, Basten A: The fate of self-reactive B cells depends primarily on the degree of antigen receptor engagement and availability of T cell help. J Exp Med 1996, 183:2313-2328. Utilises the well defined HEL/anti-HEL transgenic model to analyze the relative importance of various extrinsic factors in induction of self-tolerance in the B cell repertoire. By labelling B cells with a novel intracytoplasmic dye, 5,6-carboxy-succinimidyl-fiuorescein-ester, it was possible to track them in tolerant hosts. The results indicated that the absence of T cell help and the strength of the antigenic signal play a major role in determining whether or not self-reactive B cells become tolerant. 58. o•
FinkelmanFD, Holmes JM, Dukhanina OI, Morris SC: Crosslinking of membrane immunoglobulin D, in the absence of T cell help, kills mature B cells in vivo. J Exp Mad 1995, 181:515-525. Utilises this group's neat in vivo system of T cell and complement depletion to study the role of T cell help in determining the fate of antigen stimulated B cells. The results are in agreement with those described in [57"'] for specific antigens. 59. •
Fulcher DA, Basten A: B-cell activation versus tolerance-the central role of immunoglobulin receptor engagement and T-cell help. Int Rev Immuno11996, in press. Provides a quantitative estimate of the relationship between the receptor occupancy, B cell homing to the T cell zone and lifespan in the HEL/anti-HEL transgenic model mentioned in [49"'] and [57"']. 60.
Cyster JG, Hartley SB, Goodnow CC: Competition for follicular niches excludes self-reactive cells from the recirculeting B-cell repertoire. Nature 1994, 371:389-395.
61. •
Cyster JG, Goodnow CC: Antigen-induced exclusion from follicles and anergy ere separate end complementary processes that influence peripheral B-cell fate. Immunity 1995, 3:691-701. This paper suggests that the functional state of self-reactive B cells and the repertoire of B cells within follicles (polyclonal versus monoclonal) are important but discrete factors involved in determining the outcome of interactions between B cells and antigen. This conclusion differs from that drawn in [57"] and [59"]. 62.
Hodgkin PD, Basten A: B-cell activation, tolerance end antigenpresenting function. Curt Opin/mmuno/1995, 7:121-129.
63. •.
Phillips JA, Romball CG, Hobbs MV, Ernst DN, Shultz L, Weigle WO: CD4 + T cell activation and tolerance induction in B-cell knockout mice. J Exp Med 1996, 183:1339-1344. See annotation [64"']. 64. ••
Vella AT, Scherer MT, Shultz L, Kappler JW, Marrack P: B cells are not essential for peripheral T cell tolerance. Proc Nat/Acad Sci USA 1996, 93:951-955. This paper and [63"'] utilised B-cell knockout mice [I~MT-/-] to demonstrate that T cell tolerance to soluble antigen, peptide and superantigen could be induced by APCs other than B cells. It would be interesting to know whether the thymus played any role in this form of B cell independent tolerance. 65.
FuchsEJ, Matzinger P: B cells turn off virgin but not memory T cells. Science 1992, 2 5 8 : 1 1 5 6 - 1 1 5 9 .
66.
EynonEE, Parker DC: Small B-cells as antigen-presenting cells in the induction of tolerance to soluble protein antigens. J Exp Med 1992, 175:131-138.
67. •
BuhlmannJE, Foy TM, Aruffo A, Crassi KM, Ledbetter JA, Green WR, Xu JC, Shultz LD, Roopesian D, Flavell RA et al.: In the absence of a CD40 signal, B cells are tolerogenic. Immunity 1995, 2:645-653. Shows that resting allogeneic B cells do not induce tolerance unless CD40 signalling is inhibited. The results differ from those reported in [64"'] for H-Y all•immunization where H-Y+ B cells alone were toleragenic, but note comment on [65].
Mechanisms of tolerance to self Jacques FAP Miller* and Antony Bastent Several mechanisms exist to prevent lymphocytes from reacting against self-antigens. As T cells develop in the thymus and express antigen-specific receptors, those with high-affinity to self-antigens existing within the thymus are deleted. Low-affinity self-reactive T cells and T cells with receptors against antigens not represented intrathymically will mature and join the peripheral T cell pool. They may either ignore self-antigens expressed by tissues unable to activate T cells through a lack of the appropriate costimulator signals, or they may, under certain conditions, be deleted or rendered anergic and unable to respond. Likewise, B cells that express surface Ig receptors with high binding affinity to membrane-bound self-antigens present in the bone marrow will be rescued by receptor editing or will be deleted, whereas those of lower affinity will migrate to the periphery in either an anergic or indifferent state depending on the degree of receptor engagement by antigen. Once there, their ultimate fate is determined by the availability of T cell help.
Addresses *The Walter and Eliza Hall Institute of Medical Research, Post Office, The Royal Melbourne Hospital, Victoria 3050, Australia; e-mail: [email protected] '~The Centenary Institute of Cancer Medicine and Cell Biology, Locked Bag Number 6, Newtown, NSW 2042, Australia
of the lymphocyte at the time it encounters antigen. This was clearly pointed out by Lederberg in 1959 [5], in his modification of the clonal selection theory: contact of immature lymphocytes with antigen should lead to deletion or functional inactivation, whereas contact with mature cells should result in immunity. Formal experimental proof for this concept was obtained only in the late eighties and early nineties with the demonstration of the physical deletion (i.e. negative selection) of clones of developing T and B cells specifically reactive to self-antigens presented intrathymically [6,7] or within the bone marrow, respectively [8,9]. Other key factors influencing responsiveness relate to how the antigen is presented and include the nature of the cells that are presenting epitopes, the affinity of epitope binding, the production by APCs of costimulatory molecules and the secretion of nonepitope chemical products. It thus comes as no surprise that the few mature T cells that are present in neonatal mice can be activated by professional APCs [10 °] and that, given the right conditions, priming for both T h l and Th2 T cells can be induced in neonatal mice [11°,12°]. In fact it has been known for more than 20 years that, even at the level of thymocytes, the mature T cells in neonatal (one day old) mice are strongly reactive to foreign antigens [131.
Current Opinion in Immunology 1996, 8:815-821
© Current Biology Ltd ISSN 0952-7915 Abbreviations APC antigen-presenting cell HEL hen egg lysozyme IL interleukin mOVA membrane-bound OVA OVA ovalbumin RIP rat insulin promoter TCR specific T cell receptor Tg transgenic
Tolerance resulting from the inoculation of foreign mate~ rial may not always mimic self-tolerance and under certain circumstances can be an epiphenomenon reflecting the induction of various immunoregulatory feedback loops. For this reason, and because recent reviews have dealt with mechanisms of tolerance in general (e.g. [14,15]), in the present article we shall concentrate mainly, although not exclusively, on issues pertaining to tolerance to self-components.
Self-tolerance in the T cell repertoire Intrathymic selection
Introduction Although Burner suggested that antigen encountered in early life selectively deletes specific clones, whereas it activates them in later life [1], it has been realized for close to four decades that there is nothing intrinsically unique about the prenatal or neonatal period as far as tolerance is concerned. In fact, Burnet himself and his colleagues [2,3] failed to induce tolerance in chicks or mice, even after in o v o or in utero exposure, presumably due to rapid antigen clearance. By contrast, T and B cell tolerance to the immunogenic form of a protein could easily be induced in adult mice by inoculating the protein in deaggregated form [4]. One key factor in determining the nature of the response, whether tolerance or immunity, therefore is not the developmental stage of the individual but rather the state of differentiation
Both positive and negative selection involve the recognition of self-peptides, in association with self-MHC molecules, presented to differentiating T cells by thymic APCs. How then do signals initiated through the same T C R lead to these two opposing processes? Models to explain this conundrum fall into the following two major groups: those determined by the strength of the interaction between the T C R and its ligands [15]; and those dependent on changes in the conformation or topology of the T C R after it has bound different ligands [16]. In a recent study [17°], the outcome of thymic selection was correlated with the agonist or antagonist activity of M H C - p e p t i d e complexes for peripheral T cell activation, the agonist complexes having high-affinity for the T C R and the antagonist having low-affinity. These results are in agreement with kinetic discrimination
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models in which the rate of ligand dissociation from the T C R determines the agonist or antagonist properties of the ligand [18]. How the coreceptors, CD4 and CD8, might influence the outcome of some of these interactions has not been determined, however. To what extent intrathymic T cell deletion during negative selection involves the costimulatory molecules CD28, CTLA-4, B-7.1 or B-7.2 [14] is not clear. It does not appear to involve the CD95 molecule [19]. By contrast, interactions between CD40 and gp-39 (CD40 ligand) [20] provide costimulatory signals for thymic deletion and CD30 is an important coreceptor that produces a death signal during intrathymic T cell development [21].
Mechanisms preventing T cell autoimmunity
Although intrathymic negative selection of self-reactive T cells is generally considered to be the predominant mechanism for inducing self tolerance, there is ample evidence for the presence of autoreactive T cells in healthy individuals and animals (e.g. [22]). These cells may not have encountered their target autoantigen in the thymus or, if it was present intrathymically, they may have escaped negative selection because they expressed a T C R of too low an affinity for the self-peptide-MHC complex [17"]. Alternatively, the self-peptide may not have been able to produce a stable complex with M H C molecules in the thymus and thus could not be presented effectively [23]. To prevent autoimmunity, therefore, the autoaggressive potential of self-reactive T cell escapees must be held in check by a variety of mechanisms, some of which induce peripheral tolerance. These include indifference, deletion and immune regulation, as discussed here, and T C R and coreceptor downregulation and anergy, as reviewed elsewhere [24]. Because most naive T cells do not usually circulate through nonlymphoid tissues [25], unprimed autoreactive T cells should not inflict damage on target tissues [26]. This was clearly demonstrated in transgenic (Tg) mice expressing the class I molecule H-2K b in pancreatic islet 13 cells (rat insulin promoter [RIP]-Kb mice) and expressing in most of their CD8 ÷ T cells a Kb-specific T C R identifiable by a monoclonal clonotypic antibody. In the absence of intentional priming, the CD8 ÷ T cells ignored islet antigens. T h e mice were not tolerant of Kb as they rejected Kb-bearing skin grafts and most islets became infiltrated, resulting in specific destruction of 13 cells two weeks after such priming [27°]. Even if naive autoreactive T cells were to gain entry to a nonlymphoid tissue, as a result of local inflammation, they might still remain silent unless optimally stimulated by a strong immunogenic stimulus by professional APes, thus increasing their prect, rsor frequency. Indeed, one of the major factors that reverses indifference and leads to autoimmunity is an increase in the precursor frequency of autoreactive T cells [28",29"].
Induction of peripheral autoreactive T cell deletion
Antigen-induced proliferation of T cells is accompanied by the deletion of some of the activated cells; this is likely to represent a homeostatic mechanism that prevents unrestricted growth of these clones. Such a phenomenon, sometimes referred to as propriocidal regulation [30"], may also play a crucial role in preventing autoimmune reactions. Indeed, the failure to induce T cell apoptotic death is involved in the pathogenesis of autoimmunity in mice homozygous for the lpr gene and for the gld gene [31], and in mice in which the gene for CTLA-4 has been disrupted [32]. Highly relevant to the mechanism of preventing autoimmune responses against target nonlymphoid tissues cells is the question of which cell is responsible for inducing the deletion of activated T cells. Could any cell be involved or only professional APCs? T h e T cells of mice that are T g for the expression of the allogeneic M H C class I molecules H-2Kb, predominantly in the liver, and H-2Kb-specific TCR, in most of their CD8 ÷ T cells, showed signs of activation, then proliferated, caused damage to the hepatocytes, and eventually underwent apoptosis [33"]. In this and other T g models studied to date, it cannot be excluded that professional APCs were involved in activating T cells either because the APCs expressed the T g antigen aberrantly or acquired it in some way. This issue was resolved in another T g model in which ovalbumin (OVA)-specific T g CD8 ÷ T cells were injected into unirradiated T g RIP-mOVA mice, which express a membrane-bound form of OVA (mOVA) in pancreatic islet 13cells and in renal proximal tubular cells ([34°°]; C Kurts et al., unpublished data). In these studies, the nonlymphoid tissue-associated 'self' antigens, were presented, not by the tissue cells themselves, but by professional APCs in the context of MHC class I via a constitutive exogenous cross-over processing pathway. T h e CD8 + T cells were activated, proliferated (initially) and some of the progeny infiltrated the islets but had a curtailed lifespan. Their elimination may simply have reflected the operation of the normal homeostatic mechanism that regulates any CD8 ÷ T cell immune response, but as the tissue antigen persisted all the activated CD8 ÷ T cells may have eventually been doomed to die, particularly in the absence o f C D 4 ÷ T cell help. T h e results of these experiments provide a constitutive mechanism whereby T cells can be primed to self-antigens present in nonlymphoid tissues. T h e y therefore support the thesis that most naive T cells fail to migrate into nonlymphoid tissues [25] and argue against the recent suggestion that this is not true but that both naive and memory T cells migrate into both lymphoid and nonlymphoid organs [35]. T h e data also cast doubt on the physiological significance of the idea of naive T cells becoming anergized in vivo as a result of receiving only one signal from antigens expressed in tissues which lack the ability to generate a second signal such as costimulation.
Mechanisms of tolerance to self Miller and Basten
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Similarly they are difficult to reconcile with the 'danger hypothesis' [36] as the cells were activated by shed self-antigen in a perfectly normal unstressed situation [34 °°] and they did infiltrate the islets, the extent of the infiltration (and hence, damage) being dependent on the precursor frequency (C Kurts et al., unpublished data). Under physiological conditions, the frequency of a given autoreactive T cell clone is likely to be so low that after activation the cells would not gain entry to healthy tissues in sufficient numbers to cause disease, but would be subjected to the normal homeostatic regulation described above. Furthermore, bona fide self-antigens may not be expressed as abundantly on nonlymphoid tissue cells as the mOVA in this model. Nonetheless, these results provide a plausible mechanism whereby infected cells and perhaps even malignant cells, that lack the molecular machinery for direct immune induction, could activate CD8 ÷ T cells via APCs specialized to process shed antigen and shunt it to the class I pathway. How then would CD8 ÷ T cells distinguish between the virus-specified antigen on virus-infected nonlymphoid tissue cells which they must kill and the self-antigen on normal tissue cells which must be kept intact? T h e answer may lie in the fact that, in contrast to the situation with pathogens, the CD4 ÷ T cell repertoire is tolerant of shed self-antigens ([34°°]; C Kurts et al., unpublished data) and T cell help is not available to allow sufficient expansion of the activated CD8 + T cells.
IL-10 by the CD4 ÷ T cells and the suggestion was made that pathogenic T h l - t y p e T cells had been diverted to protective Th2-type T cells [46]. In these T g N O D mice, the M H C class II transgenes are thought to have acted both intrathymically and extrathymically. T h e most likely explanation is that intrathymic expression of non-NOD class II genes is essential for the positive selection of class II-restricted immunoregulatory T cells, while peripheral expression is required to bring about the interaction of these cells in a tricellular complex with N O D autoantigen-specific T cells and APCs, so as to deviate the response down a nonpathogenetic pathway. Significantly, for protection to occur in the I-E T g N O D mice, the I-E transgene had to be expressed both intrathymically and on B cells [47], which would support the notion that autoantigen displayed by B cells may be required to activate protective Th2-type immunoregulatory T cells.
Immunoregulation and dominant T cell tolerance
S e l f t o l e r a n c e in t h e B cell r e p e r t o i r e The decision between immunity and tolerance
T-cell-dependent immunoregulation may be considered as a fail safe mechanism to inhibit activated autoreactive T cells [37]. It has been demonstrated in vivo in several experimental models in sheep [38] rats [39] and in T g and non-Tg mice [40,41]. CD4 ÷ T cells and Th2-type cytokines have been implicated in mediating immunoregulatory effects in most models of dominant tolerance. Thus, for example, anti-IgD-peptide complexes protected rats from the development of experimental allergic encephalomyelitis induced by immunization with myelin basic protein in complete Freund's adjuvant, and this protection was associated with a Th2-type cytokine response [42"]. Protective Th2 cells, presumably induced by peptide-presenting B cells [43], might have inhibited autoaggressive cells by secreting cytokines such as IL-4, IL-10, or transforming growth factor-[3 or by recruiting protective CD8 ÷ T cells. Transforming growth factor-[3 has indeed been shown to diminish T cell activation and inflammatory responses [441. T h e T g introduction of non-nonobese diabetic (NOD) M H C class II genes (H-2A or H-2E) into N O D mice has prevented these mice from developing spontaneous diabetes (for review see [45]). Protection was not achieved by deletion or by permanent silencing of all autoreactive T cell clones. Purified CD4÷ T cells from N O D mice expressing a normal I-Ab d molecule prevented diabetogenic T cells from transferring disease to non-Tg recipients. This was associated with the production of IL-4 and
How do immunoregulatory CD4 ÷ T cells arise in the normal animal? One possibility is that they would be selected by self-antigens expressed intrathymically. If such cells had low-affinity for these antigens, they could sneak through the censorship mechanism and build up a peripheral repertoire of immunoregulatory T cells responsible for controlling autoreactive T ceils by the mechanism of immune deviation.
Recent work on self-tolerance in B cells, similar to that in T cells, has been focused on the cellular and molecular events responsible for determining whether interaction with antigen leads to persistent activation and survival or to transient activation and death by apoptosis. In this context it is becoming increasingly apparent that anergy in B cells (and possibly T cells) may represent an intermediate step in the activation pathway when the final decision between survival and deletion remains in the balance [481. Experiments conducted in T g mice including the well defined hen egg lysozyme (HEL)/anti-HEL T g model indicate that B cells are susceptible to tolerance induction at multiple stages during their development [49°°]. In practical terms, such a scenario makes good sense given the predilection of B cells to undergo hypermutation in response to antigen and T cell signals within peripheral lymphoid tissues: it also highlights the complexity of the events involved in the decision making process between immunity and tolerance. As a consequence of this complexity, even now the molecular basis for the long established sensitivity of immature B cells to tolerance induction remains elusive. To cite one example, the recent demonstration of a selective deficiency in thefvn (andfgr) members of the src family of tyrosine kinases in immature B cells [50 ° ] needs to be reconciled with the equally convincing finding that lyn -/- mice spontaneously develop
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Autoimmunity
an autoimmune syndrome characterized by circulating autoantibodies and immune complex glomerulonephritis [51°,52°]. On the other hand, the relative importance of the various factors responsible for the decision between immunity and tolerance in mature B cells is becoming clearer at least at the cellular level, with a major role being attributable to the degree of receptor engagement and the availability of T cell help. Role of T cell help in B cell tolerance
Irrespective of the precise mechanism involved, the selective susceptibility of immature self-reactive B cells to tolerance induction is thought to be an intrinsic property of the cells themselves and according to Monroe, may not be influenced by the availability of T cell help [53°]. On the other hand, mature self-reactive B cells that have escaped negative selection in the bone marrow are clearly capable of receiving T cell help which highlights the potential importance of the presence or absence of extrinsic second signals in determining the fate of B cells following antigen stimulation. This prediction receives support from previous experiments demonstrating the reversability of B cell tolerance by gp-39 in vitro and by strong antigenic stimuli in vivo [54,55], and from more recent studies of the effects of idiotype-specific [56] or antigen-specific T cells [57 -°] on B cell tolerance. In the case of the latter, Ig Tg B cells labelled with the inttacytoplasmic dye, 5,6-carboxy-succinimidyl-fluorescein-ester, were transferred into self-antigen-expressing Tg recipients where they homed within 18 hours to the outer T cell zone. When T cell help was provided through recognition of a peptide on the B cell surface, the B cells migrated to the outer T cell zone and then proliferated throughout the follicles with the formation of germinal centres, proliferative foci and the production of autoantibodies. By contrast, antigen-stimulated B cells, which could not interact with the transferred T cells, died in the outer T cell zone within three days; in other words, T cell help was crucial to the decision between activation and tolerance. A similar conclusion has been reached by Finkelman et al. [58-°] using the surrogate antigen, anti-IgD.
Ig receptor engagement Experiments involving the transfer of Ig Tg B cells into H E L T g recipients or non-Tg recipients given exogenous H E L indicated that B cells, irrespective of their specificity for self-antigen or foreign antigen or their stage of maturation, homed to the outer T cell zone of the follicles in search of T cell help, provided they received an antigenic signal above a critical threshold [57°°]. When expressed in terms of receptor occupanc'~; the threshold required to direct the B cells to the outer T cell zone and to cause significant downregulation of surface Ig was shown to lie between 30% and 75% of surface Ig molecules [57"°,59"], and in the case of soluble self-antigen resulted in anergy. On the other hand, a substantially lower level of receptor occupancy on the B cells was associated with a failure in migration to the outer T cell zone and
corresponded to a state of indifference. By inference, exposure to multivalent membrane-bound self-antigen that causes deletion must involve a receptor occupancy in excess of the figure responsible for anergy. These findings, if taken in conjunction with previous studies on longevity of tolerant B cells, point to a close relationship between Ig receptor occupancy by self-antigen, tolerance induction and lifespan. Thus, the lifespans of deleting, anergic and indifferent B cells were very short, intermediate and normal respectively [48]. Moreover, anergy in B cells may simply be a form of delayed deletion. One of the intriguing aspects of the transfer system, in which anti-HEL Ig T g B cells were injected into antigen-expressing Tg hosts, was the finding that naive B cells transferred into tolerant double Tg recipients expressing both Ig and H E L transgenes, as opposed to single H E L Tg recipients, failed to home to the outer T cell zone and moved into the follicles where they survived for at least seven days [57°',60]. When the effective concentration of antigen was measured in the serum of the two types (double T g and single Tg) of recipients it was found to be below the threshold required to cause significant downregulation of surface Ig or to direct the B cells to the outer T cell zone in the case of the double but not single Tg mice [57°°,59°]. In a related experiment, it was shown that anergic B cells in intact double Tg mice given zinc to increase the serum concentration of H E L and thereby the antigenic signal, could now home to the outer T cell zone. Moreover, Ig T g B cells pulsed with H E L in vitro also acquired the capacity to localize to the outer T cell zone on transfer into double Tg recipients (M Cook et al., unpublished data). In other words, the strength of the antigenic signal rather than either the functional state of the B cells (normal versus anergic [61°]) or the repertoire of specificities of the B cells in the follicle (polyclonal in single H E L Tg mice versus monoclonal in double Tg mice [60]) determines the positioning and ultimate fate of self-reactive B cells in this system. Presumably, the reason why B cells from double Tg mice are rendered tolerant, despite their failure to localize to the T cell zone, is that they are exposed to soluble self-antigen within the bone marrow at an immature stage in their development when the threshold of receptor occupancy and signalling required for tolerance induction is lower than in mature B cells. Role of B cells in T cell tolerance
B cells by virtue of their specific Ig receptors are highly efficient APCs for protein antigens and have been shown to cooperate effectively with CD4 + Th cells in antibody responses [62]. On the other hand, their role in the induction of peripheral T cell tolerance has been controversial. Among the reasons for this controversy is the inevitable presence of other APCs in addition to B cells in intact hosts and potential differences in their state of activation and that of cooperating T cells at the time of antigen encounter. Two strategies have
Mechanisms of tolerance to self Miller and Basten
been used in an attempt to resolve the problem. In one approach, gene-targeted B cell deficient ~ M T - / - mice were exposed to antigen in tolerogenic form. T h e results indicated that tolerance in the T cell compartment was as prolonged in mice lacking B cells as in B cell sufficient controls [63••,64°']. In other words, APCs other than B cells arc clearly capable of inducing class II-restricted T cell tolerance which is consistent with the great rarity of T cell autoimmunity in human subjects with X-linked agammaglobulinaemia. These findings, however, are not relevant to the reciprocal issue of the outcome of restricting antigen presentation to B cells. According to both Fuchs and Matzinger [65] and Eynon and Parker [66], the answer is tolerance not activation, although they and others have not reached a consensus on the activation state of the B cells involved. In order to re-examine this particular question, a different approach was used involving the transfer of anti-HEL [g T g B cells together with naive T C R T g T cells into severe combined immunodeficient recipients. When the conditions were such as to restrict antigen presentation exclusively to the B cells, the transferred T cells initially proliferated but were then rendered anergic on restimulation with antigen in vitro. By contrast, T cells underwent sustained activation if other APCs in addition to B cells were available to present antigen. Moreover, anergic B cells which express reduced levels of costimulatory molecules such as the CD80/86 complex induced less T cell proliferation and only partial tolerance (M Cook et al., unpublished data). These findings not only confirmed the toleragenic capacity of antigen-activated B cells for naive T cells, but also provided further evidence to suggest that the early stages of tolerance and immunity are remarkably similar. A similar conclusion concerning the toleragenicity of B cells was reached by Buhlmann et al. [67 ° ] who demonstrated allospecific T cell tolerance in recipients of resting allogeneic B cells in which CD40 signalling had been disrupted either genetically or by administration of anti-gp-39 antibody. Although these results confirm the potential therapeutic value of inhibiting CD40 signalling, their relevance to the role of B cells in T cell tolerance is less clear cut, because allogeneic B cells given on their own were not toleragenic. In addition to the explanation given here, namely the 'ease with which they acquire professional APC capacities upon transfer', it is possible that the B cell population may have been contaminated with very small numbers of other APCs [65].
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: • ,,,,
of special interest of outstanding interest Burnet FM: The Clonal Selection Theory of Acquired Immunity. Cambridge: Cambridge University Press; 1959.
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Kappler JW, Roehm M, Marrack P: T cell tolerance by clonal elimination in the thymus. Cell 1987, 49:273-280.
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Kisielow P, Blethmann H, Staerz UD, Steinmetz M, Von Boehmer H: Tolerance in T-cell-receptor transgenic mice involves deletion of nonmature CD4+8 + thymocytes. Nature 1988, 333:742-746.
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Nemazee D, B0rki K: Clonal deletion of autoreactive B lymphocytes in bone marrow chimeras. Proc Natl Acad Sci USA 1989, 86:8039-8043.
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Hadley SB, Crosbie J, Brink R, Kantor AB, Basten A, Goodnow CC: Elimination from peripheral lymphoid tissues of selfreactive B lymphocytes recognizing membrane-boud antigens. Nature 1991, 353:765-769.
10. •
Ridge JP, Fuchs EJ, Matzinger P: Neonatal tolerance revisited: turning on newborn T cells with dendritic cells. Science 1996, 271:1723-1726. When 105 enriched dendritic cells from male C57BL/6 mice were injected into neonatal female C57BL/6 mice the newborn recipients were primed for an H-Y-specific cytotoxic T lymphocyte response, whereas when adult females were given 5 × 108 male spleen cells they did not generate efficient cytotoxic T lymphocytes. This confirms what was already known from previous work and was originally predicted by Lederberg [5] in his modification of the clonal selection theory, namely, that the neonatal period is different from adulthood as regards tolerance induction in quantitative but not in qualitative terms. 11. •
Sarzotti M, Robbins DS, Hoffman PM: Induction of protective CTL responses in newborn mice by a murine retrovirus. Science 1996, 271:1726-1729. A cytotoxic T lymphocyte response occurred in newborn mice inoculated with low doses of Cas-Br-M routine leukaemia virus, but when high doses of virus were injected, a nonprotective Th2-type response resulted. Thus, by reducing the dose of virus in proportion to the peripheral T cell number, neonatal mice were able to produce a protective CD8 + T cell response, again showing that toleragenicity is not related to the stage of maturity of the animal as a whole. 12. Forsthuber T, Yip HC, Lehmann PV: Induction of TH1 and TH2 • immunity in neonatal mice. Science 1996, 271:1728-1730. Newborn mice injected with protein in incomplete Freund's adjuvant produced a vigorous Th2 response when challenged with the protein in complete Freund's adjuvant. Interestingly, only spleen and not lymph node ceils, were responsible for this response presumably because T cell activation is associated with downregulation of L-selectin and this prevents re-entry into lymph nodes but allows accumulation in the spleen. 13.
Sprent J, Miller JFAP: Interaction of thymus lymphocytes with histoincompaUble cells. I. Quantitation of the proliferative response of thymus cells. Cell/mrnunol 1972, 3:361-384.
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Kruisbeek AM, Amsen D: Mechanisms underlying T cell tolerance. Curt Opin Immuno11996, 8:233-244.
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Sprent J, Webb SR: Intrathymic and extrathymic deletion of T cells. Curt Opin Immunol 1995, 7:196-205.
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Janeway CA Jr: Ligands for the T-cell receptor: hard times for avidity models. Immunol Today 1995, 16:223-225.
17. •
Alam SM, Travers PJ, Wun 9 JL, Nasholds W, Redpath S, Jameson SC, Gascoigne NRJ: T-cell receptor affinity and thymocyte positive selection. Nature 1996, 381:616-620. By using a soluble purified preparation of a well characterized T C R - M H C peptide complex and varying the peptide ligand, it was shown, using surface plasmon resonance, that the most stable TCR-MHC-peptide interactions (k°ff -0.02 s -1) occurred with agonist peptides that caused negative selection, whereas antagonistic peptides involved in positive selection produced less stable complexes (k°ff -0.039-0.146 s-I). Null peptides, those not involved in positive or negative selection, failed to interact detectably with the TCR. Only one TCR preparation was used and the possible contribution of coreceptor molecules, such as CD4 and CD8, was not investigated.
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Autoimmunity
18.
Rabinowitz JD, Beeson C, Lyons DS, Davis M, McConnell HM: Kinetic discrimination in T-cell activation. Proc Nat/Acad Sci USA 1996, 93:1401-1405.
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Cohen PL, Eisenberg RA: Ipr and g/d: single gene models of systemic autoimmunity and lymphoproliferative disease. Annu Rev Immunol 1991, 9:243-269.
19.
Singer GG, Abbas AK: The Fas antigen is involved in peripheral but not thymic deletion of T lymphocytes in T cell receptor transgenic mice. Immunity 1994, 1:365-371.
32.
20.
Foy TM, Page DM, Waldschmidt TJ, Sohoneveld A, Laman JD, Masters SR, Tygrett L, Ledbetter JA, Aruffo A, Claassen E et al.: An essential role for gp39, the ligand for CD40, in thymic selection. J Exp Med 1995, 182:1377-1388.
Tivol EA, Bofiello F, Schweitzer AN, Lynch WP, Bluestone JA, Sharpe AH: Loss of CTLA-4 leads to massive lymphoproliferation and fatal multiorgan tissue destruction, revealing a critical negative regulatory role of CTLA-4. Immunity 1995, 3:541-547.
21.
Amakawa R, Hakem A, Kundig TM, Matsuyama 1", Simard JJL, Timms E, Wakeham A, Mittreiecker H-W, Griesser H, Takimoto H et al.: Impaired negative selection of T cells in Hodgkin's disease antigen CD30-deficient mice. Cell 1996, 84:551-562.
22.
Wekerle H, Bradl M, Linington C, Kaab G, Kojima K: The shaping of the brain-specific T lymphocyte repertoire in the thymus. Immunol Rev 1996, 149:231-243.
23.
Liu GY, Fairchild PJ, Smith RM, Prowle JR, Kioussis D, Wraith DC: Low avidity recognition of self-antigen by T cells permit escape from central tolerance. Immunity 1995, 3:407-415.
24.
Ferber I, Schonrich G, Schenkel J, Mellor AL, Hammerling G J, Arnold B: Levels of peripheral T cell tolerance induced by different doses of tolerogen. Science 1994, 263:675-676.
25.
Mackay CR, Marston WL, Dudler L: Naive and memory T cells show distinct pathways of lymphocyte recirculation. J Exp Med 1990, 171:801-817.
26.
Miller JFAP, Heath WR: Self ignorance in the peripheral T cell pool. Immuno/Rev 1993, 133:131-150.
27. •
Heath WR, Karamalis F, Donoghue J, Miller JFAP: Autoimmunity caused by ignorant CD8 + T cells is transient and depends on avidity. J Immuno/1995, 155:2339-2349. Transgenic mice expressing H-2K b (K b) in pancreatic islet 13 cells were tolerant of Kb-bearing skin due to intrathymic expression of a few molecules of transgenic K b. To prevent thymus expression, RIP-K b mice were thymectomized, irradiated with 90OR, protected with bone marrow from Des-TCR transgenic mice (which expressed in most of their CD8 + T cells a Kb-specific TCR identifiable by a clonotypic antibody) and grafted with an irradiated (IO00R) thymus from nontransgenic syngeneic newborn donors. RIP-K b mice manipulated in this way had circulating Kb-specific CD8 + T cells with a high density of surface clonotype + TCR, but only two out of twenty-two mice showed very few CD8 + T cells in some of the 20 islets scored. In the absence of intentional priming, therefore, these T cells ignored islet antigens. The mice were not tolerant of Kb as they rejected Kb-bearing skin grafts and most islets were infiltrated two weeks after such priming. Analysis revealed that ~ cells were specifically destroyed in this response. Many manipulated mice rapidly lost weight and died. Hence, high density T cells, presumably those with high avidity for K b, were capable of inducing lethal diabetes after priming and in the absence of constitutive local IL-2 production. Von Herrath MG, Guerder S, Lewicki H, Flavell RA, Oldstone MAB: Coexpression of B7-1 and viral ("selF') transgenes in pancreatic [3 cells can break peripheral ignorance and lead to spontaneous autoimmune diabetes. Immunity 1995, 3:727-738. Tg mice expressing either B7-1 alone (RIP-B7-1) or only the glycoprotein (RIP-GP) or nucleoprotein (RIP-NP) of the lymphocytic choriomeningitis virus, in pancreatic islet ~ cells did not develop insulin-dependent diabetes mellitus. The RIP-NP, but not the RIP-GP mice, aberrantly expressed the transgene in the thymus. RIP-B7-1 x RIP-NP bigenic mice deleted most of their autoreactive CD8 + T cells intrathymically and failed to develop insulindependent diabetes mellitus, but did so within 14 days of lymphocytic choriomeningitis virus infection. On the other hand, RIP-B7-1 xRIP-GP bigenic mice developed insulin-dependent diabetes mellitus in the absence of viral infection. These results suggest a correlation between the development and frequency of insulin-dependent diabetes mellitus and the precursor frequency of autoreactive T cells.
33. •
Bertolino P, Heath WR, Hardy CL, Morahan G, Miller JFAP: Peripheral deletion of autoreactive CD8 + T cells in transgenic mice expressing H-2K b in the liver. Eur J Immuno11995, 25:1932-1942. B10.BR Tg mice expressing K b in hepatocytes under the control of the metallothionein promoter (MET-K b mice) were manipulated as the RIP-K b mice in [27"] to prevent thymus expression of the K b transgene. The livers were heavily infiltrated with lymphocytes by four weeks after thymus grafting and to a lesser extent by 20 weeks, many of the CD8 + T cells showing apoptosis. As the liver has great regenerative ability, continuous antigenic stimulation must have confronted the infiltrating T cells a,~d this was probably the reason for their deletion. It was thought unlikely that the immunogenic stimulus originated from radioresistant and persisting professional APCs and Kupffer cells which might have aberrantly expressed the K b transgene. 34. •-
Kurts C, Heath WR, Carbone F, Allison J, Miller JFAP, Kosaka H: Constitutive class I-restricted exogenous presentation of self antigens in vivo. J Exp Med 1996, in press. Two sets of Tg mice were used: mice of the RIP-mOVA line which expressed mOVA in the pancreatic islet [3 cells and the renal proximal tubular cells and mice of the OT-I line which produced OVA-specific CD8 + T cells. OT-I cells injected intravenously into nonirradiated RIP-mOVA mice (shown to be tolerant to OVA in the CD4 + T cell population), selectively homed to the draining lymph nodes of the kidneys and pancreas, expressed activation markers there and some entered the cell-cycle. Unilateral nephrectomy 7-14 days prior to OT-1 cell inoculation allowed the OT-I cells to home only to the regional lymph node of the remaining kidney and of the pancreas, but when nephrectomy was performed four hours before injecting the T cells, homing to the regional nodes on both sides occurred. The use of bone marrow chimeras (in which marrow ceils from bin1 or bin8 - the cells of which cannot present the OVA peptide to OT-I cells - or from syngeneic donors was given to protect irradiated C57BL/6 RIP-mOVA mice) proved that the OT-I cells were activated by OVA presented by short lived APCs of bone marrow origin in the draining lymph nodes of the OVA-expressing tissues and not in the tissues themselves. 35.
Westermann J, Pabst R: How organ-specific is the migration of 'naive' and 'memory' T cells? Immunol Today 1996, 17:278-282.
36.
Matzinger P: Tolerance, danger and the extended family. Annu Rev/mmunol 1994, 12:991-1045.
37.
Charlton B, Lafferty K: The T h l / T h 2 balance in autoimmunity. Curr Opin /mmuno11995, 7:793-798.
36.
McCullagh P: The significance of immune suppression in normal self tolerance./mmunol Rev 1996, 149:127-153.
39.
Saoudi A, Seddon B, Heath V, Fowell D, Mason D: The physiological role of regulatory T cells in the prevention of autoimmunity: the function of the thymus in the generation of the regulatory T cell subset. Immunol Rev 1996, 149:195-216.
40.
Liblau RS, Singer SNM, McDevitt HO: Thl and Th2 CD4 + T cells in the pathogenesis of organ-specific autoimmune diseases. /mmuno/Today 1995, 16:34-38.
41.
Kumar V, Sercarz EE: The involvement of T cell receptor peptide-specific regulatory CD4 + T cells in recovery from antigen-induced autoimmune disease. J Exp Med 1993, 178:909-916.
28. •
F6rster I, Hirose R, Arbeit JM, Clausen BE, Hanahan D: Limited capacity for tolerization of CD4 + T cells specific for a pancreatic 13cell neoantigen. Immunity 1995, 2:573-585. As in the previous study [28°], there is a correlation between the number of circulating autoreactive transgenic T cells specific for a known 13 cell transgenic antigen and heavy intraislet infiltration. 29. •
30. •
Lenardo MJ, Boehme SA, Chen L, Combadiere B, Fisher G, Freedman M, McFarland H, Pelfrey C, Zheng L: Autocrine feedback death and the regulation of mature T lymphocyte antigen responses. Intern Rev Immunol 1995, 13:115-134. A very thoughtful review relating to antigen-induced T cell death as an important regulatory mechanism in the peripheral immune system.
42. •-
Saoudi A, Simmonds S, Huitinga I, Mason D: Prevention of experimental allergic encephalomyelitis in rats by targeting autoantigen to B cells: evidence that the protective mechanism depends on changes in the cytokine response and migratory properties of the autoantigen-specific T cells. J Exp Med 1995, 182:335-344. Intravenous injection of anti-lgD-myelin basic protein conjugated was administered to Lewis rats one or two weeks before immunization with myelin basic protein in adjuvant and prevented the development of encephalomyelitis but not the production of myelin basic protein-specific antibodies. Lymph node cells from pretreated rats proliferated m vitro in response to myelin basic protein as vigorously as cells from nonpretreated controls, but produced less interferon-y and more RNA for IL-4 and IL-13. They were inferior in their ability to transfer disease after in vitro activation. Pretreated rats also showed greatly reduced levels of leukocyte infiltration in the central nervous system. Protection afforded by pretreatment thus appears to be mediated by T cells with a Th2-type cytokine response and these cells seem to have an intrinsic inability to ir'iltrate the noninflamed central nervous system.
Mechanisms of tolerance to self Miller and Basten
43.
Mason D: The role of B cells in the programming of T cells for IL-4 synthesis. J Exp Med 1996, 183:717-719.
44.
KehrlJH, Wakefield LM, Roberts AB, Jakolew S, Alvarez-Mon M, Derynck R, Spore MB, Fauci AS: Production of transforming growth factor b by human T lymphocytes and its potential role in the regulation of T cell growth. J Exp Mad 1986, 163:1037-1050.
45.
Slattery RM, Miller JFAP: Influence of T lymphocytes and major histocompaUbility complex class II genes on diabetes susceptibility in the NOD mouse. Curt Topics Microbiol Immunol 1 9 9 5 , 206:51-66.
46.
Singer SM, Tisch R, Yang XD, McDevitt HO: An Abd transgene prevents diabetes in nonobese diabetic mice by inducing regulatory T cells. Proc Nat/Acad Sci USA 1993, 90:9566-9570.
4"7.
B~hme J, Schuhbaur B, Kanagawa O, Benoist C, Mathis D: MHC-linked protection from diabetes dissociated from clonal deletion of T cells. Science 1987, 249:293-295.
48.
FulcherDA, Basten A: Reduced life span of anergic selfreactive B cells in a double transgenic model. J Exp Med 1994, 179:125-134.
49. o•
Goodnow CC, Cyster JG, Hartley SB, Bell SE, Cooke MP, Healy JJ, Akkaraju S, Rathmell JC, Pogue SL, Shokat KP: Self-tolerance checkpoints in B lymphocyte development. Advlmmuno11995, 59:279-369. A comprehensive overview of recent work on the cellular and molecular mechanisms underlying self-tolerance in the B cell repertoire. It makes the important point that tolerance can be imposed at multiple stages of the development of the B cell lineage and draws attention where appropriate to the 'clinical' consequences of a breakdown in self-tolerance at the various checkpoints. 50. •
Wechsler RJ, Monroe JG: Immature B lymphocytes are deficient in expression of the src-family kinases p59fYn and p55fgrl. J Immuno/1995, 154:1919-1929. See annotation [53"]. 51. Hibbs ML, Tarlinton DM, Armes J, Grail D, Hodgson G, Maglitto R, • Stacker SS, Dunn AR: Multiple defects in the immune system of lyn-deficient mice culminating in autoimmune disease. Ca// 1995, 83:301-311. See annotation [52"]. 52. •
NishizumiH, Taniuchi I, Yamanashi Y, Kitamura D, Ilic D, Mori S, Watanabe T, Yamamoto T: Impaired proliferation of peripheral B cells and indication of eutoimmune disease in lyn-deficient mice. Ce/11995, 3:549-560. This paper and [51 ",52"] shows that disruption of the src family protein-tyrosine kinase lyn leads to a profound disturbance in B cell function manifest by reduced B cell numbers, hyper-gammaglobulinaemiaand a lupus-like autoimmune syndrome. Interestingly, deliberate or spontaneous disruption of genes encoding other tyrosine kinases and phosphatases [e.g. PTP1C] involved in Ig receptor signalling also leads to immunodeficiency and autoimmunity, highlighting the complexity of the molecular events underlying B cell regulation. 53. Monroe JG: Tolerance sensitivity of immature-stage B cells. J • Immuno11996, 156:2657-2660. This paper and [50"] discuss the issue of the relative importance of the stage of maturation in induction of B cell tolerance and suggest that a selective deficiency in fyn and fgr kinases may define the immature (transitional) stage in B cell development, but see [52"]. 54.
56.
Eris JM, Basten A, Brink R, Doherty K, Kehry MR, Hodgkin PD: Anergic self-reactive B cells present antigen and respond normally to CD40 dependent signals but ere defective in antigen receptor mediated functions. Proc Nat/Acad Sci USA 1994, 91:4392-4396. Cooke MP, Heath AW, Shokat KM, Zeng Y, Finkelman FD, Linsley PS, Howard M, Goodnow CC: Immunoglobulin signal trensduction guides the specificity of B celI-T cell interactions end is blocked in tolerant self-reactive B cells. J Exp Mad 1994, 179:425-438.
56.
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Caulfield MJ, Stank• D: T-cell dependent response to immune complexes abrogates B-cell unresponsiveness to pneumococcal cell wall polysaccharide. Immunology 1995, 86:331-335.
57. o•
Fulcher DA, Lyons AB, Korn SL, Cook MC, Kileda C, Parish C, Fazekasde St.Groth B, Basten A: The fate of self-reactive B cells depends primarily on the degree of antigen receptor engagement and availability of T cell help. J Exp Med 1996, 183:2313-2328. Utilises the well defined HEL/anti-HEL transgenic model to analyze the relative importance of various extrinsic factors in induction of self-tolerance in the B cell repertoire. By labelling B cells with a novel intracytoplasmic dye, 5,6-carboxy-succinimidyl-fiuorescein-ester, it was possible to track them in tolerant hosts. The results indicated that the absence of T cell help and the strength of the antigenic signal play a major role in determining whether or not self-reactive B cells become tolerant. 58. o•
FinkelmanFD, Holmes JM, Dukhanina OI, Morris SC: Crosslinking of membrane immunoglobulin D, in the absence of T cell help, kills mature B cells in vivo. J Exp Mad 1995, 181:515-525. Utilises this group's neat in vivo system of T cell and complement depletion to study the role of T cell help in determining the fate of antigen stimulated B cells. The results are in agreement with those described in [57"'] for specific antigens. 59. •
Fulcher DA, Basten A: B-cell activation versus tolerance-the central role of immunoglobulin receptor engagement and T-cell help. Int Rev Immuno11996, in press. Provides a quantitative estimate of the relationship between the receptor occupancy, B cell homing to the T cell zone and lifespan in the HEL/anti-HEL transgenic model mentioned in [49"'] and [57"']. 60.
Cyster JG, Hartley SB, Goodnow CC: Competition for follicular niches excludes self-reactive cells from the recirculeting B-cell repertoire. Nature 1994, 371:389-395.
61. •
Cyster JG, Goodnow CC: Antigen-induced exclusion from follicles and anergy ere separate end complementary processes that influence peripheral B-cell fate. Immunity 1995, 3:691-701. This paper suggests that the functional state of self-reactive B cells and the repertoire of B cells within follicles (polyclonal versus monoclonal) are important but discrete factors involved in determining the outcome of interactions between B cells and antigen. This conclusion differs from that drawn in [57"] and [59"]. 62.
Hodgkin PD, Basten A: B-cell activation, tolerance end antigenpresenting function. Curt Opin/mmuno/1995, 7:121-129.
63. •.
Phillips JA, Romball CG, Hobbs MV, Ernst DN, Shultz L, Weigle WO: CD4 + T cell activation and tolerance induction in B-cell knockout mice. J Exp Med 1996, 183:1339-1344. See annotation [64"']. 64. ••
Vella AT, Scherer MT, Shultz L, Kappler JW, Marrack P: B cells are not essential for peripheral T cell tolerance. Proc Nat/Acad Sci USA 1996, 93:951-955. This paper and [63"'] utilised B-cell knockout mice [I~MT-/-] to demonstrate that T cell tolerance to soluble antigen, peptide and superantigen could be induced by APCs other than B cells. It would be interesting to know whether the thymus played any role in this form of B cell independent tolerance. 65.
FuchsEJ, Matzinger P: B cells turn off virgin but not memory T cells. Science 1992, 2 5 8 : 1 1 5 6 - 1 1 5 9 .
66.
EynonEE, Parker DC: Small B-cells as antigen-presenting cells in the induction of tolerance to soluble protein antigens. J Exp Med 1992, 175:131-138.
67. •
BuhlmannJE, Foy TM, Aruffo A, Crassi KM, Ledbetter JA, Green WR, Xu JC, Shultz LD, Roopesian D, Flavell RA et al.: In the absence of a CD40 signal, B cells are tolerogenic. Immunity 1995, 2:645-653. Shows that resting allogeneic B cells do not induce tolerance unless CD40 signalling is inhibited. The results differ from those reported in [64"'] for H-Y all•immunization where H-Y+ B cells alone were toleragenic, but note comment on [65].