Intrathymic and extrathymic clonal deletion of T cells

Intrathymic and extrathymic clonal deletion of T cells Jonathan Sprent and Susan R Webb T h e Scripps Research Institute, La Jolla, USA Clonal elimination accounts for self-tolerance induction in the thymus and also affects mature T cells responding to exogenous antigens in the periphery. Recent evidence on the microenvironments, cell-cell interactions and signalling requirements for clonal deletion of immature and mature T cells is discussed. Current Opinion in Immunology 1995, 7:196-205

Introduction Clonal deletion in the thymus is the principal method for purging the immune system of autoreactive T cells. Clonal elimination also operates in the peripheral lymphoid tissues and affects mature T cells responding to foreign antigens. This review will cover the recent evidence on the factors controlling both intrathymic and extrathymic T-cell deletion.

Clonal deletion in the thymus The production of mature T cells in the thymus involves a combination of positive and negative selection directed towards an array of self peptides bound to class I or class II M H C molecules [1-3]. As discussed elsewhere in this issue (Fowlkes and Schweighoffer, pp 188-195), positive selection occurs in the cortex of the thymus and involves TCR-mediated contact of double-positive (DP) CD4+CD8 + thymocytes with MHC-peptide complexes displayed on epithehal cells. Most DP cells (>95%) have negligible binding avidity for the peptides presented by M H C molecules on epithelial cells and die in situ by neglect within a few days. A small proportion of DP cells do have significant avidity for the peptides on selfMHC molecules; these DP cells receive a gentle (low-level) fignal which rescues the cells from neglect and induces maturation into single-positive (SP) CD4+8 - and CD4-8 + cells. This protective signal is transduced via a ~ T C R molecules in consort with CD4 or CD8 co-receptors (which direct T C R contact with class II and class I M H C molecules, respectively). A third subset of DP cells has overt reactivity for self MHC-peptides, and these autoaggressive cells undergo clonal deletion (negative selection) via apoptosis. Signalling of early T cells via contact with self MHC-self peptide complexes can

thus have diametrically opposite consequences: survival versus death. This paradox raises a number of questions that are covered below.

Relationship of positive and negative selection Recent studies with mice that are deficient in M H C class I molecules ([~2-microglobulin negative mice) or in the transporter associated with antigen processing (TAP-l) have indicated that positive selection of CD8 + cells can be induced by M H C class I molecules bearing either moderate amounts of antagonist peptides (peptides with low affinity for TCR) or small quantities ofagonist peptides (peptides with high affinity for TCR) [4°°-6°',7°,8"]. Significantly, raising the concentration of peptides on M H C molecules above a certain threshold leads to negative selection rather than positive selection. Negative selection caused by higher doses of peptides is most conspicuous with agonist peptides but also applies to certain antagonist peptides. Collectively, these findings favor an affinity/avidity model of thymic selection in which weak signalling of T cells leads to positive selection and strong signalling causes negative selection [9,10"].

Sites of negative selection Full induction of negative selection requires intrathymic contact of T cells with bone marrow (BM)-derived antigen-presenting cells (APCs) such as dendritic cells (reviewed in [2]). The fact that these cells are largely confined to the medulla suggests that negative selection takes place mainly in the medulla rather than in the cortex. This notion is supported by the finding that, in the case of endogenous mouse mammary tumor virus (mtv) antigens, negative selection of mtv 8,9-reactive V[~5 T cells in I-E + mice leads to the appearance of dense ag-

Abbreviations APC--antigen-presenting cell; BM--bone marrow; CFA~complete Freunds adjuvant; CSA~cyclosporin A; DP---double positive; LCMV--lymphocytic choriomeningitis virus; MBP--myelin basic protein; mtv--mammary tumor virus; OVA--ovalbumin; SAg--superantigen; SEB--Staphylococcal enterotoxin B; SP--single positive; TCR--T-cell receptor; TxAz--thromboxane A2.

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Intrathymic and extrathymic clonal deletion of T cells Sprent and Webb gregates of apoptotic cells in the medulla but not in the cortex [11°°]. Because mtv antigens are expressed predominantly by BM-derived cells rather than epithehal cells [12], the finding that mtv-induced negative selection is confined to the medulla is not surprising. For non-mtv antigens, however, it is clear that negative selection can sometimes occur in the cortex, presumably through contact with antigen expressed on cortical epithelial cells (reviewed in [2,3]). This finding indicates that induction of negative selection is not a unique property of BM-derived APCs. Indeed, under in vitro conditions virtually all cell types, including epithelial cells, have the capacity to induce negative selection [2,13,14]; however, the key question is whether negative selection occurs in the cortex under normal physiological conditions. A prediction of the afffinity/avidity model is that any selfpeptide antigen that associates with M H C molecules on epithelial cells (or other cells) in the cortex will be capable oftolerizing T cells that have high affinity for the peptide. Generation of such agonist peptides by epithelial (and other) cells presumably occurs continuously through breakdown of various intracellular proteins [15°]. As most intracellular self proteins are unlikely to be tissue-specific [15°], the bulk of self tolerance could occur in the cortex through interaction with peptides on epithelial cells (or other cortical cells). Self tolerance induction to native blood-borne antigens is probably quite different. Certain soluble self antigens present in high concentrations in the blood have access to the cortex and presumably lead to negative selection in this site [16-18]. However, except via the transcapsular route [19], tl~e cortex is relatively impermeable to large protein molecules, and entry of these molecules into the thymus from the blood is likely to be largely restricted to the medulla. This fact is exemplified by studies on T-cell tolerance to C5, a common complement component present in serum at 50~gml-1 (10-7M) [20°,21"°]. This self protein, which is absent in certain mouse strains, is expressed strongly on thymic dendritic cells but rather weakly on cortical epithelial cells (thymic nurse cells) [20°]. Recent studies on a C5-specific class II restricted T C R transgenic line have shown that exposure to circulating C5 completely deletes CD4 + SP cells in the thymus but causes only marginal deletion of DP cells [21°']. In marked contrast, exposure to C5 in peptide form caused rapid elimination of DP cells. These important observations strongly suggest that, for native (non-peptide) antigens, self-tolerance induction to blood-borne antigens takes place predominantly in the medulla rather than in the cortex.

The depth of clonal deletion A corollary of the afffinity/aviditymodel is that the depth of self tolerance is a reflection of the concentration of antigen reaching the thymus: clonal deletion is complete with high concentrations of antigen, but only partial

with low concentrations and undetectable when antigen fails to enter the circulation (as is the case with tissue-specific antigens such as myelin basic protein). Immature T cells are extremely sensitive to tolerance induction (reviewed in [2,3]), and the concentration of self antigen required to induce full tolerance in the thymus is quite low, for example 10-8 M to 10-9 M for circulating liver F protein [20°,22]. With lower concentrations of antigen, tolerance is incomplete. This is apparent from the finding that T-cell tolerance to natural self antigens, such as insulin and thyroglobulin (10-10M), can be broken by injecting high concentrations of antigen suspended in adjuvant [23,24]. In such situations, one can envisage that tolerance does occur but is restricted to high-affinity cells: low-affinity cells escape tolerance and remain responsive to antigen, but only when antigen is present at unphysiologically high concentrations and supplemented with adjuvant. Results from recent studies with hen egg lysozyme specific TC1L transgenic mice expressing varying concentrations of hen egg lysozyme in the bloodstream are in accord with this line of reasoning [25]. Similarly, many forms of 'split-tolerance' to alloantigens (reviewed in [2,3]) and viral antigens (e.g. [26]) are probably a reflection of suboptimal exposure to antigen in the thymus. As discussed elsewhere [27], tolerance to self antigens is limited when, for one reason or another, the antigen fails to associate with M H C molecules. 'Cryptic' antigens fall into this category. An interesting example of the lack of tolerance to cryptic self antigens is provided by the finding that although human T cells display operational tolerance to autologous erythrocytes, they are able to give strong primary responses in vitro to synthetic 15-mer peptides corresponding to the sequence of erythrocyte Rhesus polypeptide [28°].

Cell-interaction molecules required for negative selection Another corollary of the affinity/avidity model is that interaction between complementary sets of adhesion/signalling molecules will augment T-cell-APC interaction and thereby accentuate negative selection. In the case of CD4 and CD8 co-receptors, these molecules are known to play a critical role in positive selection and probably also in negative selection [1,8°,29°]. Interestingly, for CD8 molecules, the CD8 ~-chain is highly important for both positive and negative selection [30,31]. This is surprising because association of the protein tyrosine kinase p56 lck (which plays an important role in T C R / C D 3 signalling) with CD8 is limited to the CD8 ~t-chain [32,33]. O f the spectrum of other surface molecules expressed by early T ceils, there is currently no direct evidence that any of these molecules play a mandatory role in negative selection [29°]. Nevertheless, under defined conditions, CD28-B7 (CD80) interaction [34] and other 'second signals' from APCs [35,36] can augment negative selection. It is also notable that thymic selection is

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Lymphocytedevelopment severely impaired in mice overexpressing the costimulatory molecule heat soluble antigen HSA [37]. These findings raise the question of the types of signals required for negative selection.

Signals for negative selection For negative selection, the prevailing view is that, unlike mature T cells, DP immature thymocytes are hypersensitive to strong signalling: instead of stimulating DP cells, strong signals via the TCtk initiate DNA fragmentation and cause rapid death via apoptosis. Before apoptosis, TCK-stimulated DP cells show downregulation of CD4 and CD8 [35,38,39"] and RAG-1 [40"], and upregulation of CD5 [34], CD69 ([39",40°]; see also [41"]) and IL-2R~ [42"]. Under in vitro conditions, subjecting DP thymocytes to strong T C R / C D 3 signalling initiates a cascade of events which include activation of protein tyrosine kinases such as p561ck, hydrolysis of phosphatidylinositol, sustained elevation of cytoplasmic-free Ca2+, and activation of protein kinase C followed by induction of various transcription factors [2,43-45,46"]; these factors then somehow induce DNA degradation (see below). In line with this scheme, TCR-mediated apoptosis of DP thymocytes can be blocked by inhibitors of protein tyrosine kinases [44,45,46",47], phosphatidylinositol [46"] or protein kinase C [46"] and by Ca 2+ chelators [45,46"]. Whether Ca2+-mediated signalling is essential for negative selection, however, is unclear because cyclosporin A (CSA), an inhibitor of calcineurin, has variable effects on negative selection. Under in vivo conditions CSA causes partial inhibition of negative selection to mtv antigens and the HY antigen [48]. In vitro, however, CSA either has no effect on negative selection [39",49] or causes a paradoxical increase in selection [39"]. These findings suggest that under defined conditions, negative selection can take place in the absence of Ca2+-mediated signals. In support of this possibility deletion of DP thymocytes in vitro can occur in the presence of KN-62, an inhibitor of Ca2+/calmodulin-dependent protein kinase II [46"]. The notion that negative selection may proceed in the absence of Ca2+-mediated signalling is interesting because a pathway involving a Ca2÷-independent protein kinase C (PKCe) has been invoked to explain apoptosis of thymocytes induced by glucocorticoids [50"] and also by epipodophyllotoxins [51]. DP thymocytes are exquisitely sensitive to these compounds, and glucocorticoids in the thymus [52 °] could play a key role in destroying the DP cells that fail positive selection [9,52°,53]. This raises the question of whether glucocorticoids can potentiate TCR-mediated negative selection of DP cells. In favor of this idea, apoptosis of DP cells induced by high concentrations o f a n t i - T C R antibody is augmented by glucocorticoids and blocked by the glucocorticoid inhibitor, RU486 [53,54"]. Interestingly, however, glucocorticoids can also have the reverse effect and protect against apoptosis when the dose of anti-TCK is lowered below a certain threshold [52",53]. The impli-

cation therefore is that glucocorticoid signals synergize with strong TCR-mediated signals to augment negative selection, but somehow antagonize weaker signals and thereby promote positive selection [52",53]. Such modulation of T C K signalling also applies to retinoic acid [55,56]. In the case of other soluble mediators, DP thymocytes express high levels of receptors for thromboxane A2 (TxA2) and are hypersensitive to this compound [57]. Recent evidence suggests that TxA 2 contributes to negative selection in vivo, perhaps through release ofTxA 2 by thymic dendritic cells [58]. O f the multiplicity of other molecules on DP cells, several cell-surface molecules have known costimulatory activity and could provide synergistic signals for TCR-mediated apoptosis. These T-cell molecules include CD28 [34], heat-stable antigen [37], Fas/Apo-1 [59-61,62"], CD45 [63] and Thyl [47,64]. Although several intracellular mediators such as p53, nur77, c-Myc and interleukin-lB converting enzyme (ICE) have been proposed as the final effectors of apoptosis, the downstream events involved in activating and regulating these effector molecules are still poorly understood [43,65]. Currently, there is much interest in the capacity of repressor molecules such as Bcl-2 and Bcl-XL to inhibit apoptosis (reviewed in [65]). Levels of Bcl-2 are very low on TCKIo DP cells but are upregulated on T C R hi DP cells, in other words, at an early stage of positive selection [66-71]. As constitutive upregulation of Bcl-2 in transgenic mice is highly effective in blocking the action of glucocorticoids and irradiation (reviewed in [65]), one might expect Bcl-2 to impair negative selection. Surprisingly, however, negative selection in the thymus of Bcl-2 trausgenic mice is only slightly less effective than in normal mice [65,70,72]. This finding may signify that multiple fail-safe mechanisms have evolved to ensure the efficiency of negative selection.

Clonal elimination of mature T cells in the periphery Akhough exposure of mature T cells to antigen generally evokes a vigorous immune response and the generation of long-lived memory cells, it is now clear that many of the effector T cells generated in the immune response subsequently disappear. Under in vivo conditions, the elimination of mature T cells a~er contact with specific antigen has been demonstrated for a variety of antigens, including both superantigens (SAgs) (reviewed in [62,73,74]) and conventional antigens [62,75-77"]. Three general patterns of T-cell deletion in response to SAgs have emerged. The first pattern is evident within 12-20 hours of in vivo injection of bacterial toxins, such as Staphylococcal enterotoxin B (SEB), and appears to involve apoptosis of a variable proportion of the T cells bearing the relevant Vi3's specific for the injected SAg [78,79]. This pattern of acute, rapid

Intrathymic and extrathymic clonal deletion of T cells Sprent and Webb

cell death seems to occur without cell division. Subsequently, the remaining SAg-reactive T cells mount a marked proliferative response which peaks between days 3-5 depending on the particular SAg. Thereafter, a substantial proportion of the responding cells are eliminated, leading (in some situations) to antigen-specific tolerance (see [73,74]). This second pattern of subacute deletion appears to be intimately connected with the preceding proliferative response as strong proliferative responses lead to more extensive deletion that weaker responses. These two patterns of acute and subacute deletion occur following a single injection of SAg. A third pattern of T-cell deletion, which is slow and chronic, is seen following repeated injections of low doses of bacterial SAg [80]. This pattern also occurs in mice expressing poorly-stimulatory endogenous SAg [81°]. The chronic deletion seen in these situations may not require extensive proliferation [80], although there can be a marked increase in the expression of activation markers (VLA-4 and CD44) on the antigen-specific T cells prior to their elimination [81"]. Slow chronic deletion may reflect lowavidity interactions between T cells and APCs, and may require repeated contact with antigen. Whether these three patterns of deletion also apply to conventional antigens is less clear. To date, the majority of studies on T-cell responses to infectious virus or peptide antigens have shown a subacute pattern of deletion following a proliferative response [62",75,76,77°]. The failure to see other patterns of deletion may reflect that these experiments involved TC1K transgenic mice, a situation where the responding T cells were monoclonal.

Factors that influence deletion of mature T cells For both SAgs and conventional antigens, the extent of T-cell elinfination is variable and highly dependent on the particular T C R - a n t i g e n - M H C studied. Several factors have been identified that influence the degree of deletion.

The typical pattern ofsubacute deletion affecting T cells responding to a single injection of mtv-expressing APCs was found to be influenced by the M H C haplotype of both the APCs and the responding T cells, as well as by the particular SAg studied [82°]. Because of the relationship between the strength of the proliferative response and the extent of subsequent deletion in these studies, we suggested [82 °] that high-avidity T-cell-APC interactions promote deletion. Avidity in this context would reflect not only the affinity of the T C R for the particular antigen-MHC complex but also the hgand density and the triggering-enhancing contributions from accessory molecule interactions. Consistent with this view, several other studies have demonstrated the importance of antigen dose in determining the extent of deletion. In experiments assessing the acute (12-20 hours) pattern of deletion of SEB-reactive T cells, Miethke et al. [83 °] found that the antigen dose played a key role in determining the out-

come of SEB injection: both moderate and high doses of SEB induced anergy, whereas high doses were most effective in generating T cells with an unusual ct~TC1K1o CD3 + phenotype which appeared characteristic of cells programmed for apoptosis. Likewise, for the deletion of T cells following an in vivo proliferative response to lymphocytic choriomeningitis virus (LCMV), the extent of functional deletion was strongly dependent on the dose of virus [76]. At low doses of LCMV, some effector cells survived elimination, leading to clearance of the virus. By contrast, at high doses of LCMV the effector cells disappeared after a brief proliferative response, and the virus persisted. In this system, it was found that only certain LCMV isolates were effective at mediating T-cell deletion. High doses of other isolates induced a somewhat weaker response associated with viral clearance and persistence of effector cells. Although it is unclear why some isolates were more effective than others, these studies highlight the importance of antigen immunogenicity in influencing the efficiency of deletion. Interestingly, the elimination of mature T cells in this system applied equally well to naive and memory (primed) T cells [84°]. For soluble antigens, the way in which the antigen is administered (e.g. as a complex with adjuvant and/or the route of administration) appears to have a marked influence on the extent of T-cell elimination. In comparing the fate of adoptively transferred ovalbumin (OVA)-specific TC1K transgenic T cells following injection of OVA peptide by various routes with and without adjuvant, Kearney et al. [77"] found that all forms of antigen administration induced a measurable proliferative response followed by the disappearance of a substantial proportion of the responding T cells. However, the extent of elimination and the responsiveness of the surviving cells varied considerably. When antigen was injected subcutaneously in complete Freunds adjuvant (CFA), the T cells surviving elimination retained strong reactivity to OVA peptide and behaved like memory cells. By contrast, fewer T cells survived after injecting the peptide intravenously without adjuvant and these cells responded poorly when restimulated in vitro. The authors suggested that T-cell printing in the presence of a strong adjuvant (CFA) induces an inflammatory environment in which APCs show heightened expression of costimulatory molecules, favoring T-cell survival and/or the development of memory cells. In addition, the slow, continual release of peptide induced by CFA may be conducive to long-term survival of the responding cells (M Jenkins, personal communication). By contrast, printing in a non-inflammatory environment (peptide without CFA) limits expression of costimulatory molecules on APCs and thus favors tolerance. This scenario is in line with the requirements for inducing functional tolerance to protein antigens [85]. The issue of whether antigen-induced deletion of mature T cells requires repeated contact with antigen is difficult to address directly. In the case o f T cells responding to endogenous SAg, one injection of antigen-bearing APCs is sufficient to induce maximal deletion. Given the

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relatively short in vivo half-life of the injected APCs, the responding T cells would appear to be programmed for elimination or survival very early in the response, although multiple T-cell-APC interactions are possible during this limited time frame. Similarly, in the OVAspecific TCR transgenic system discussed above, one intravenous injection of peptide was sufficient to markedly reduce the proportion of functional transgenic T cells in an adoptive transfer system. By contrast, when myelin basic protein (MBP)-reactive TCR transgenic T cells were adoptively transferred to naive recipients, multiple injections of MBP (or peptide) were required for effective deletion [86-l. This discrepancy might reflect the differences in the in vivo half-life of the antigens concerned. Alternatively, the MBP data may be representative of the more chronic type of deletion mentioned earlier for SAgs and might reflect lower-avidity interactions, which only lead to deletion following repeated contact with antigen. In fact, there is increasing evidence to suggest that tolerance can be a stepwise process. This is exemplified by studies on Kb-specific TCR transgenic cells in mice that express very low levels of Kb in the liver [87*]. The T cells from these mice were functionally tolerant to Kb in vivo, tolerance being associated with reduced TCR expression, but were responsive to Kb under in vitro conditions. When the level of Kb in these mice was upregulated, however, the ‘tolerant’ T cells further downregulated their TCR expression and became unresponsive in vitro; in addition, some of the cells may have undergone elimination. In another study using this TCR transgenic line [88*], differentiation of the Kb-reactive T cells in a Kbml thymus (Kbml is a weaker antigen for the T cells than Kb) generated a population of CD810 TCRhi cells, which were tolerant in vivo but not in vitro . These cells were apparently eliminated following subsequent extrathymic contact with Kb, such as when Kb was expressed exclusively in the liver. These two studies suggest that, at least under some circumstances, T cells that avoid deletion following their initial encounter with antigen, for example by downregulating their TCR or co-receptor molecules, remain susceptible to additional tolerizing (deleting) signals upon subsequent exposure either to a higher dose of antigen [87*] or to a stronger antigen [88*].

of blast cells to non-lymphoid tissues, such as the intestine, where substantial numbers of Vg6+ cells accumulate in the lamina propria and Peyer’s patches. Indeed, the marked disappearance of cells from the lymphoid tissues could be largely attributed to homing of effector cells to the liver ([89]; S Webb, unpublished data), and to non-lymphoid sites of potential antigen entry, notably the intestine (S Webb, unpublished data). In this respect, it has proven difficult to demonstrate apoptosis in spleen, lymph nodes or thoracic duct lymph blast cells tested directly ex vivo (without in vitro culture) or by in sirtr staining of lymphoid tissues for apoptotic cells (S Webb, unpublished data). These negative findings do not rule out local death of blast cells, however, as apoptotic cells are known to be cleared rapidly by macrophages. Ingestion of apoptotic lymphocytes by macrophages can occur before DNA fragmentation ofthe lymphocyte and was recently shown to involve a vitronectin receptor system similar to that used for recognition of apoptotic neutrophils [90*], although a phosphatidyl serine receptor has also been implicated [91].

Mechanisms involved in the elimination of mature T cells

The capacity of the Bcl-2 Eunily of molecules to act as death-inhibitory proteins for mature T cells is now established [97-991, and constitutive expression of cl2 substantially impairs the antigen-induced elimination of mature T cells in vivo [97]. It is of interest that the activation of mature T cells causes downregulation of Bcl-2 in parallel with upregulation of Fas [98,100*,101], a cell-surface molecule that transduces death signals [99]. Thus, Bcl-2 expression is most prominent in CD45RBhi (naive) T cells, which express low levels of Fas antigen; CD45RO+ (activated/memory) T cells, by contrast, are strongly positive for Fas but express only low levels of Bcl-2 [lOO”]. This reciprocal pattern of Bcl-2 and Fas

In most of the above systems, contact with antigen generally causes a subacute pattern of deletion following an initial phase of T-cell proliferation. In the case of responses to mtv-7 encoded SAgs, the disappearance of V86f blast cells from the spleen and lymph nodes is paralleled by the appearance of the cells in the circulation (i.e. the blood and thoracic duct lymph) and also in the intestine and the liver (S Webb, unpublished data). The V86+ blast cells in lymph express high levels of a4fi7, other a4 integrins, lymphocyte function-associated antigen (LFA)-1, CD44 and low levels of L-selectin. This phenotype is consistent with the migration

The fate of T cells homing to non-lymphoid tissues is not clear; however, these cells disappear within a few days of their appearance, implying that the majority ultimately undergo some form of cell death, be it in the lymphoid tissues or at extralymphoid sites. With regard to how mature T cells die, there is now considerable evidence that, like thymocytes, normal mature T cells are susceptible to apoptosis (reviewed in [92,93]), though there may be differences in the particular pathways used for apoptosis and/or the regulation of these pathways for mature versus immature T cells. Whereas apoptosis of thymocytes is generally blocked by inhibitors of protein and RNA synthesis (reviewed in [91]), death of mature T cells can be resistant to, or increased by, these inhibitors [94-961. Similarly, the capacity of protein kinase C inhibitors to prevent apoptosis of thymocytes does not appear to apply to mature T cells [95]. These findings suggest that, under certain conditions, death of mature T cells may reflect a release mechanism [91] whereby death-inhibitory proteins sensitive to protein kinase C inhibitors and perhaps protein/RNA synthesis inhibitors maintain viability in mature T cells after activation of a death-inducing pathway

Intrathymic and extrathymic clonal deletion of T cells Sprent and W e b b

expression, as well as the delayed sensitivity of Fas-expressing T cells to Fas-mediated death [99,102°], fits in well with the finding that activation-induced death of mature T cells applies preferentially to previously activated T cells [103-105]. Although Fas appears to have little effect on negative selection in the thymus [106], the importance of Fas-mediated apoptosis in mature T-cell death is supported by the finding that mature T cells from Fas-defective lpr and Fas-ligand defective gld mice are resistant to activationinduced cell death, both in vitro [62•,1077-109], and in vivo [62°,110"]. In particular, it was fourld that the subacute elimination of mature T cells in vivo following exposure to conventional antigens (cytochrome C) [62"] and SAgs (SEB) [110 °] was clearly less effective in Fas-defective animals. Nonetheless, at least in the case of SAg, the resistance to elimination was much less apparent when high concentrations of antigen were used. Thus, when lpr/lpr mice were injected with high doses of SEB, the elimination of T cells was delayed but eventually reached the same level seen in control + / + mice. The implication, therefore, is that with high-avidity interactions, the onset of T-cell elimination/apoptosis is relatively Fas-independent and must involve other mechanisms. Glucocorticoids could play a significant role in T-cell elimination as mature T cells become highly sensitive to cortisone-induced death following activation [111], especially after in vivo exposure to SAgs [112]. Indeed, the early acute (<24 hours) deletion of SEB-reactive VI38+ cells appears to be steroid-dependent, as the steroid inhibitor RU486 markedly blocked the early disappearance of these cells [79]. However, P,.U486 did not appear to block the subacute elimination of VI38+ cells following the proliferative response, implying that this latter pattern of deletion involves glucocorticoid independent pathways. Fas-independent elimination of T cells could also reflect withdrawal from growth promoting (or apoptosis protecting) cytokines such as IL-2 [91,100",113,114]. This possibility is challenged, however, by the finding that high levels of IL-2 (provided by an IL-2 producing recombinant vaccinia virus) did not have any effect on the subacute form of SEB-induced elimination of T cells [115]. Other workers have suggested that IL-2 can augment apoptosis, especially when T cells are exposed to IL-2 before contact with antigen [116]. The mechanisms involved in this type of death are not clear, although it was suggested that IL-2 may promote cell cycle progression, rendering the cells vulnerable to antigen-induced death [117]. It is also possible that other cytokines such as IL-1 and IFN-y play a role in regulating T-cell elimination [114,118], though direct evidence on this question in vivo is lacking.

Conclusion

The factors controlling the elimination of T cells in the thymus and in the extrathymic environment are becoming increasingly well understood. Nevertheless, much remains to be learned about the complex series of receptor-ligand interactions occurring at the cell surface and about how these interactions trigger various intracellular signalling pathways to activate and regulate the effectors of apoptosis. These topics are now coming under close scrutiny and rapid progress is to be expected.

Acknowledgements

This work was supported by grants CA38355, CA25803, CA41993 and A121487 from the United States Public Health Service. Publication no. 9105-1MM from The Scripps Research Institute.

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J Sprent and SR Webb, Department of Immunology, ]MM4, The Scripps Research Institute, 10666 North Torrey Pines Road, La Jolla, CA 92037, USA. J Sprent E-mail: [email protected] S Webb E-mail: [email protected]

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