- Email: [email protected]
Do anergie T cells live or die?
304
42nd FORUM
IN IMMUNOLOGY
Do anergic T cells live or die? A. Ochi, K. Migita and K. Yuh Division of Neurobiology and Molecular Immunology, Samuel Lunenfeld Research Institute, and The Department of Immunology and Medical Genetics, University of Toronto, Mount Sinai Hospital, 600 University Avenue, Toronto, Ontario (Canada) MSG 1X5
Introduction Both death and functional unresponsiveness (anergy) of reactive T cells in the periphery have been documented in bacterial and viral superantigenchallenged mice (Rammensee et al., 1989 ; White et al., 1989; Kawabe and Ochi, 1990, 1991; Rellahan et al., 1990; Webb et al., 1990; MacDonald et al., 1991). Cell death follows an expansion of reactive T cells which takes place within 3 days after the in vivo injection with superantigens. Anergy is evident when spleen cells are assayed 4 days after bacterial superantigen priming. These two parameters, death and anergy, are currently recognized as the major mechanisms by which immunological tolerance develops in reactive T cells. Whereas both deletion and anergy occur at a clonal level and the recognition of class II major histocompatibility complex (MHC)superantigen complex by specific T-cell receptor (TCR) VP elements must be essential, the underlying cellular mechanisms for each are poorly understood. In this article, we will discuss the possible physiological relevance of clonal anergy and death of T cells in vivo.
(SEA) was comparable in both PBS-primed and 50 pg-SEB-primed mice. When spleen cells were assayed in vitro for their SEB-specific proliferative response 2 months after SEB priming, the response was higher than 10 days post-SEB-priming, but was still below that of control spleen cells. Thus, mice were apparently recovering from tolerance 2 months after SEB challenge. Four months later, SEB-primed spleen cells responded efficiently to SEB and the cell dose-dependent response curve was equivalent to that of control spleen cells. IL2 production of SEBstimulated spleen cells also recovered to the level of controls. The recovery of the proliferative response to SEB was further demonstrated when CD4+,VP8+ T cells of SEB-primed and control mice were purified and their response determined. The proliferation and IL2 production to different dosages of SEB were identical between recovered and normal cell samples but the percentage of VP8’,CD4+ or CD8+ T cells on day 10 was maintained even after 4 months and there was no increase in the number of CD4+,Vp8’ spleen T cells. The study indicates that unresponsiveness of CD4+ ,Vp8’ T cells is reversible and, after four months, cells regain a proliferative response comparable to that of control mice.
Anergic cells live Unresponsiveness of CD4+, VJ38’ T cells is reversible in vivo
A protein synthesis inhibitor, cycloheximide (CX), blocked anergy induction but not death of VpS’ T cells when injected with SEB
In order to investigate whether anergic T cells are terminally differentiated, we have induced staphylococcal enterotoxin B (SEB)-specific T-cell anergy in thymectomized mice and monitored the response to SEB at different times. As in normal mice, thymectomized mice became SEB-specifictolerant and the proportion of CD4+ ,Vp8’ T cells was reduced to 60 070of control in spleens when examined on day 10 post-SEB priming. Both proliferation and interleukin 2 (IL2) production by spleen cells were suppressed in SEB-primed mice to less than 30 070of PBS-primed control mice. The response to another superantigen staphylococcal enterotoxin A
Whereas the molecular change which induces the anergy of T cells is not yet clear, a report showed that de novo protein synthesis was essential for T-cell unresponsiveness in vitro (Quill and Schwartz, 1987). In order to understand the molecular mechanism of SEB-specific CD4+,VP8+ T-cell anergy in vivo, we have tested the effect of CX on tolerance induction. A sublethal dose of CX was injected i.p. with SEB into mice at different times. The tolerance to SEB stimulation was examined after 10 days. The results demonstrated that in vivo tolerance induction to SEB was dependent on de novo protein synthesis. The effective period was only within the first 12 h, and
CLONAL
DELETION
AND ANERGY:
after that period, the injection of CX did not prevent anergy. The duration of CX-induced inhibition of protein synthesis in vivo was determined by measuring the time required for resumption of protein synthesis in spleen after I-mg CX injection by pulsing mice with 35S-methionine. After 6 h, de nova protein synthesis equivalent to that of normal animals was detected. Probably owing to this short-lasting effect of CX in vivo, expansion and deletion of VPS’ T cells equivalent to those of normal mice were associated with all animals with CX, despite their lack of specific tolerance. Therefore, protein synthesis important for anergy induction seemed to take place within 12 h after SEB injection. The results also suggested that activation-induced cell death was able to take place in mice despite the prevention of anergy. This may indicate that anergy is not a conditional process for cell death. It is also probable that anergy and cell death are mechanistically differential. Current information does not clarify whether the prevention of T-cell anergy induction is because of protein synthesis inhibition in responding T cells or in other cellular components, like antigen-presenting cells (APC). However, SEB does not require processing by APC to stimulate T cells (Janeway et al., 1989) and the TCR P-chain spontaneously binds to high affinity complexed SEB and class II MHC. Therefore, the inhibition of anergy by CX is most probably because of the inhibition of protein synthesis essential for anergy in CD4+ ,Vp8’ T cells in vivo. DNA degradation occurs exclusively in T-cell blasts of SEB-primed spleen The results obtained in the current studies showed that the clonal deletion of reactive VP’ T cell took place only after the initial expansion of the same VP phenotype cells. The results suggested that PCD occurs in proliferating cells and its development depends on cellular activation signals. Similarly, results of studies on PCD in T-cell hybridomas linked this phenomenon with cell activation (Mercep et al., 1988; Ucker et al., 1989). These cells expressed increased amounts of IL2R. To test that deletion occurred in activated cells in our system, cell death was compared between density gradient separated blastic and non-blastic spleen cells obtained from SEBprimed mice 3 days after SEB injection. DNA fragmentation was observed exclusively in the blastic cell fraction, as is consistent with the contention that cells targeted for clonal deletion undergo activation and blastogenesis prior to their PCD in vivo. The T helper (Th) clones become unresponsive without proliferation in vitro .+The functional unresponsiveness of T lymphocytes has been reported by Schwartz using in vitro
FROM
MODELS
TO REALITY
305
cultured protein antigen-specific Th cell clones (Schwartz, 1990). In those, functional unresponsiveness was observed on Th clones which were in vitro antigen-stimulated with chemically fixed APC, antigen and purified class II MHC molecules inserted into planar membranes, or immobilized CD3 complex-specific antibodies (Quill and Schwartz, 1987 ; Jenkins and Schwartz, 1987 ; Jenkins et al., 1988, 1990). These experiments suggested that induction of protein antigen-specific unresponsiveness of Th cells was the result of occupancy of TCR in the absence of co-stimulatory signals (Mueller et al., 1989). The anergic Th cell clones expressed unchanged levels of TCR and IL2R, and were responsive to exogenous IL2. Antigen stimulation could not induce autocrine production of IL2 because of defective transcription of the IL2 gene in these tolerant cells. In these studies, the cells enlarged but other activation changes such as IL2R upregulation and thymidine incorporation were not observed (Quill and Schwartz, 1987; De Silva et al., 1991). Thus the data indicated that Th clones become anergic without cell division. Anergic cells die In contrast to the above mentioned results, which support the idea that anergic T cells are long-lived, there is some evidence that anergic T cells are dying in vivo. An observation that we believe supports the notion that a proportion of CD4+ ,Vp8’ cells continues to decline after SEB stimulation at a time when these cells are already tolerant. This occurs in the period when the CD4+ ,Vp8’ T-cell proportion is gradually decreasing after a major reduction between day 2 and 3 post-SEB injection. This type of cell death may be taking place on a small scale even before this period, but may be difficult to detect in this period of intense activity. It is an intriguing issue if these cells are slowly dying because of exhaustion from the strong activation. In any case, it seems to be the case that these cells lose responsiveness before they die. Discussion The current manuscript has tried to characterize the relationship of clonal anergy and cell death of T lymphocytes. Two lines of evidence presented here indicate that anergic T cells either die or live and recover responsiveness. This most probably indicates the heterogeneity of the mechanism by which functional unresponsiveness develops in T cells. This hypothesis is apparently supported by reported characteristics that distinguish T cells anergized in vivo by superantigen from anergic protein antigenspecific Th clones in vitro. The major difference be-
42nd FORUM
306
IN IMMUNOLOGY
tween the former and the latter exists in the responsiveness to exogenous IL2 ; anergic Th clones are still responsive to IL2, whereas anergic normal T cells lack responsiveness to exogenous IL2 (Rammensee et al., 1989 ; Rellahan et al., 1990 ; Blackman et al., 1991). This may reflect the difference in in vitro and in vivo experimental systems or, alternatively, the difference in trans-signalling pathways between protein antigens (for Th clones) and superantigens (for normal T cells) (O’Rourke et al., 1990; Liu et al., 1991). If T-cell anergy can be induced in different
1.
2.
ways, it is probable that their fate is also heterogenous depending on the mechanism involved. Figure 1 shows a possible sequence of events of TCRstimulated resting T cells to develop anergy and death. From the top, resting T cells are activated and die without expressing anergy. This type of cell death may occur at early time points after SEB injection within 3 days (particularly between days 2 and 3). As shown in the second row, activated T cells may become functionally unresponsive before they die. This is probably what is taking place from day 4 to day 14 post-SEB injection when spleen cells become tolerant but the percent of CD4+,VP8+ T cells gradually decreases. The fifth pathway, which does not include the activation phase, may also be followed by these cells. The third and forth pathways are followed by anergic T cells which remain in the SEBtolerized animal after day 14. The authors prefer the fourth model because anergy seems to be independent of cell activation events. While we have proposed a number of ways that anergy or death of TCR-stimulated T cells can occur, it is mysterious why cells challenged with superantigen have such different fates. There is much work to be done to fully characterize these processes. Acknowledgements
3.
We wish to thank Drs. D. Spaner and J. Roder for critical reading of the manuscript. This work was supported by grants from the Arthritis Society of Canada and National Cancer Institute of Canada and from the Medical Research Council.
4.
5.
:. “.? 0.Y.
-
0
: Proliferated
0:.G 37 :
.,
blastic
T
References Blackman,M.A., Finkel, T.H., Kappler, J.W. & Marrack, P. (1991),Altered antigenreceptorsignallingin anergic T cellsfrom self-tolerant T-cell receptor P-chain transgenicmice. Proc. nat. Acud. Sci. (Wash.), 88,
T cells
6682-6686. : Proliferative unresponsiveness antigenic stimulation : Apoptic
Fig. 1. Hypothetical
to
cell death
pathways for anergy and death in stimulated T cells. In response to TCR-mediated stimulation, resting T cellsproliferate and becomeblast cells.The blast cellsdie by apoptosis (Pathway 1) or they become anergic before death (Pathway 2). Alternatively, these anergic T cells survive and regain responsiveness (Pathway 3). T cells may becomeanergic without activation and regain responsiveness (Pathway 4) or die (Pathway 5).
De Silva, D.R., Urdahl, K.B. & Jenkins, M.K. (1991), Clonal anergy is induced in vitro by T cell receptor
occupancy in the absence of proliferation. munol.; 147, 3261-3267.
J. Im-
Janeway, Jr. C.A., Yagi, J., Conrad, P.J., Katz, M.E., Jones, B., Vroegop, S. & Buxter, S. (1989), T cell responses to Mls and to bacterialproteinsthat mimic its behavior. Immunol. Rev., 107, 61-88. Jenkins,M.K. & Schwartz, R.H. (1987),Antigen presentation by chemically modified splenocytesinduces antigen-specificT cell unresponsiveness in vitro and in vivo. J. exp. Med., 165, 302-319. Jenkins, M.K., Ashwell, J.D. & Schwartz, R.H. (1988), Allogeneic non-T spleencellsrestorethe responsivenessof normal T cell clonesstimulatedwith antigen and chemically modified antigen-presentingcells. J. Immunol., 140, 3324-3330.
CLONAL
DELETION
AND
ANERGY:
Jenkins, M.K., Chen, C., Jung, G., Mueller, D. & Schwartz, R.H. (1990), Inhibition of antigen-specific proliferation of type 1 murine T cell clones after stimulation with immobilized anti-CD3 monoclonal antibody. J. Immunol., 144, 16-22. Kawabe, Y. & Ochi, A. (1990), Selective anergy of VP8 + ,CD4+ T cells in Stuphylococcus enferotoxin B-primed mice. J. exp. Med., 172, 10651070. Kawabe, Y. & Ochi, A. (1991), Programmed cell death and extrathymic reduction of Vj38+CD4+ T cells in mice tolerant to Stuphylococcus uurens enterotoxin B. Nuture (Lond.), 349, 245-248. Liu, H., Lampe, M.A., Iregui, M.V. &Cantor, H. (1991), Conventional antigen and superantigen may be coupled to distinct and cooperative T-cell activation pathways. Proc. nut. Acud. Sci. (Wash.), 88, 87058709. Mercep, M., Blueston, J.A., Noguchi, P.D. & Ashwell, J.D. (1988), Inhibition of transformed T cell growth in vitro by monoclonal antibodies directed against distinct activating molecules. J. Immunof., 140, 324-335. MacDonald, H.R., Baschieri, S. & Lees, R.K. (1991), Clonal expansion precedes anergy and death of VP8 + peripheral T cells responding to staphylococcal enterotoxin B in vivo. Europ. J. Immunol., 21, 1963-1966. Mueller, D.L., Jenkins, M.K. & Schwartz, R.H. (1989), Clonal expansion versus functional clonal inactivation : a costimulatory signalling pathway determines the outcome of T cell antigen receptor occupancy. Ann. Rev. Immunol.. 7, 445-480. O’Rourke, A.M., Mescher, M.F. & Webb, S.R. (1990)
Deletion Institute
MODELS
TO REALITY
for Cancer Research,
307
Activation of polyphosphoinositide hydrolysis in T cells by H-2 alloantigen but not Mls determinants. Science, 249, 171-174. Quill, H. & Schwartz, H.R. (1987), Stimulation of normal inducer T cell clones with antigen presented by purified Ia molecules in planar lipid membranes: specific induction of a long-lived state of proliferative nonresponsiveness. J. Immunof., 138, 3704-3712. Rammensee, H., Kroschewski, R. & Frangoulis, B. (1989). Clonal anergy induced in mature Vp6+ T lymphocytes immunized with Mls-la antigen. Nature (Lond.), 339, 541-544. Rellahan, B.L., Jones, L.A., Kruisbeek, A.M., Fry, A.M. & Matis, L.A. (1990), In vivo induction of anergy in peripheral V88+ T cells by Staphylococcal enterotoxin B. J. exp. Med., 172, 1091-1100. Schwartz, R.H. (1990), A cell culture model for T lymphocyte clonal anergy. Science, 248, 1349-1356. Ucker, D.S., Ashwell, J.D. & Nickas, G. (1989), Activation-driven T cell death. - I. Requirements for de novo transcription and translation and association with genome fragmentation. J. Immunol., 143, 3461-3469. Webb, S., Morris, C. & Sprent, J. (1990), Extrathymic tolerance of mature T cells: clonal elimination as a consequence of immunity. Cell, 63, 1249-1256. White, J., Herman, A., Pullen, A.M., Kubo, R., Kappler, J.W. & Marrack, P. (1989), The VP-specific superantigen Staphylococcal enterotoxin B : stimulation of mature T cells and clonal deletion in neonatal mice. Cell, 56, 27-35.
versus anergy : the superantigen H.R.
Ludwig
FROM
paradigm
MacDonald
Ch. des Boveresses
Introduction It is now well accepted that tolerance to self antigens may be achieved by distinct mechanisms, including clonal deletion and functional unresponsiveness (anergy). The recent availability of T-cell receptor (TCR) transgenic mice as well as superantigens reactive with specific TCR VP domains have provided novel tools to investigate the mechanism of T-cell tolerance induction in vivo. In keeping with the aims of this Forum, the present contribution will be restricted to a comparison of the respective roles of deletion and anergy in the establishment and
155, 1066 Epalinges
(Switzerland)
maintenance of central (thymic) and peripheral T-cell tolerance, with particular emphasis on superantigens. Central (thymic) tolerance During their development in the thymus, T cells with high-affinity receptors for self antigens are clonally deleted. This phenomenon has been extensively studied using superantigen models, particularly those involving Mls(MMTV)-encoded endogenous superantigens. Our own experiments (reviewed in MacDonald et al., 1989) suggest that clonal deletion
42nd FORUM
IN IMMUNOLOGY
Do anergic T cells live or die? A. Ochi, K. Migita and K. Yuh Division of Neurobiology and Molecular Immunology, Samuel Lunenfeld Research Institute, and The Department of Immunology and Medical Genetics, University of Toronto, Mount Sinai Hospital, 600 University Avenue, Toronto, Ontario (Canada) MSG 1X5
Introduction Both death and functional unresponsiveness (anergy) of reactive T cells in the periphery have been documented in bacterial and viral superantigenchallenged mice (Rammensee et al., 1989 ; White et al., 1989; Kawabe and Ochi, 1990, 1991; Rellahan et al., 1990; Webb et al., 1990; MacDonald et al., 1991). Cell death follows an expansion of reactive T cells which takes place within 3 days after the in vivo injection with superantigens. Anergy is evident when spleen cells are assayed 4 days after bacterial superantigen priming. These two parameters, death and anergy, are currently recognized as the major mechanisms by which immunological tolerance develops in reactive T cells. Whereas both deletion and anergy occur at a clonal level and the recognition of class II major histocompatibility complex (MHC)superantigen complex by specific T-cell receptor (TCR) VP elements must be essential, the underlying cellular mechanisms for each are poorly understood. In this article, we will discuss the possible physiological relevance of clonal anergy and death of T cells in vivo.
(SEA) was comparable in both PBS-primed and 50 pg-SEB-primed mice. When spleen cells were assayed in vitro for their SEB-specific proliferative response 2 months after SEB priming, the response was higher than 10 days post-SEB-priming, but was still below that of control spleen cells. Thus, mice were apparently recovering from tolerance 2 months after SEB challenge. Four months later, SEB-primed spleen cells responded efficiently to SEB and the cell dose-dependent response curve was equivalent to that of control spleen cells. IL2 production of SEBstimulated spleen cells also recovered to the level of controls. The recovery of the proliferative response to SEB was further demonstrated when CD4+,VP8+ T cells of SEB-primed and control mice were purified and their response determined. The proliferation and IL2 production to different dosages of SEB were identical between recovered and normal cell samples but the percentage of VP8’,CD4+ or CD8+ T cells on day 10 was maintained even after 4 months and there was no increase in the number of CD4+,Vp8’ spleen T cells. The study indicates that unresponsiveness of CD4+ ,Vp8’ T cells is reversible and, after four months, cells regain a proliferative response comparable to that of control mice.
Anergic cells live Unresponsiveness of CD4+, VJ38’ T cells is reversible in vivo
A protein synthesis inhibitor, cycloheximide (CX), blocked anergy induction but not death of VpS’ T cells when injected with SEB
In order to investigate whether anergic T cells are terminally differentiated, we have induced staphylococcal enterotoxin B (SEB)-specific T-cell anergy in thymectomized mice and monitored the response to SEB at different times. As in normal mice, thymectomized mice became SEB-specifictolerant and the proportion of CD4+ ,Vp8’ T cells was reduced to 60 070of control in spleens when examined on day 10 post-SEB priming. Both proliferation and interleukin 2 (IL2) production by spleen cells were suppressed in SEB-primed mice to less than 30 070of PBS-primed control mice. The response to another superantigen staphylococcal enterotoxin A
Whereas the molecular change which induces the anergy of T cells is not yet clear, a report showed that de novo protein synthesis was essential for T-cell unresponsiveness in vitro (Quill and Schwartz, 1987). In order to understand the molecular mechanism of SEB-specific CD4+,VP8+ T-cell anergy in vivo, we have tested the effect of CX on tolerance induction. A sublethal dose of CX was injected i.p. with SEB into mice at different times. The tolerance to SEB stimulation was examined after 10 days. The results demonstrated that in vivo tolerance induction to SEB was dependent on de novo protein synthesis. The effective period was only within the first 12 h, and
CLONAL
DELETION
AND ANERGY:
after that period, the injection of CX did not prevent anergy. The duration of CX-induced inhibition of protein synthesis in vivo was determined by measuring the time required for resumption of protein synthesis in spleen after I-mg CX injection by pulsing mice with 35S-methionine. After 6 h, de nova protein synthesis equivalent to that of normal animals was detected. Probably owing to this short-lasting effect of CX in vivo, expansion and deletion of VPS’ T cells equivalent to those of normal mice were associated with all animals with CX, despite their lack of specific tolerance. Therefore, protein synthesis important for anergy induction seemed to take place within 12 h after SEB injection. The results also suggested that activation-induced cell death was able to take place in mice despite the prevention of anergy. This may indicate that anergy is not a conditional process for cell death. It is also probable that anergy and cell death are mechanistically differential. Current information does not clarify whether the prevention of T-cell anergy induction is because of protein synthesis inhibition in responding T cells or in other cellular components, like antigen-presenting cells (APC). However, SEB does not require processing by APC to stimulate T cells (Janeway et al., 1989) and the TCR P-chain spontaneously binds to high affinity complexed SEB and class II MHC. Therefore, the inhibition of anergy by CX is most probably because of the inhibition of protein synthesis essential for anergy in CD4+ ,Vp8’ T cells in vivo. DNA degradation occurs exclusively in T-cell blasts of SEB-primed spleen The results obtained in the current studies showed that the clonal deletion of reactive VP’ T cell took place only after the initial expansion of the same VP phenotype cells. The results suggested that PCD occurs in proliferating cells and its development depends on cellular activation signals. Similarly, results of studies on PCD in T-cell hybridomas linked this phenomenon with cell activation (Mercep et al., 1988; Ucker et al., 1989). These cells expressed increased amounts of IL2R. To test that deletion occurred in activated cells in our system, cell death was compared between density gradient separated blastic and non-blastic spleen cells obtained from SEBprimed mice 3 days after SEB injection. DNA fragmentation was observed exclusively in the blastic cell fraction, as is consistent with the contention that cells targeted for clonal deletion undergo activation and blastogenesis prior to their PCD in vivo. The T helper (Th) clones become unresponsive without proliferation in vitro .+The functional unresponsiveness of T lymphocytes has been reported by Schwartz using in vitro
FROM
MODELS
TO REALITY
305
cultured protein antigen-specific Th cell clones (Schwartz, 1990). In those, functional unresponsiveness was observed on Th clones which were in vitro antigen-stimulated with chemically fixed APC, antigen and purified class II MHC molecules inserted into planar membranes, or immobilized CD3 complex-specific antibodies (Quill and Schwartz, 1987 ; Jenkins and Schwartz, 1987 ; Jenkins et al., 1988, 1990). These experiments suggested that induction of protein antigen-specific unresponsiveness of Th cells was the result of occupancy of TCR in the absence of co-stimulatory signals (Mueller et al., 1989). The anergic Th cell clones expressed unchanged levels of TCR and IL2R, and were responsive to exogenous IL2. Antigen stimulation could not induce autocrine production of IL2 because of defective transcription of the IL2 gene in these tolerant cells. In these studies, the cells enlarged but other activation changes such as IL2R upregulation and thymidine incorporation were not observed (Quill and Schwartz, 1987; De Silva et al., 1991). Thus the data indicated that Th clones become anergic without cell division. Anergic cells die In contrast to the above mentioned results, which support the idea that anergic T cells are long-lived, there is some evidence that anergic T cells are dying in vivo. An observation that we believe supports the notion that a proportion of CD4+ ,Vp8’ cells continues to decline after SEB stimulation at a time when these cells are already tolerant. This occurs in the period when the CD4+ ,Vp8’ T-cell proportion is gradually decreasing after a major reduction between day 2 and 3 post-SEB injection. This type of cell death may be taking place on a small scale even before this period, but may be difficult to detect in this period of intense activity. It is an intriguing issue if these cells are slowly dying because of exhaustion from the strong activation. In any case, it seems to be the case that these cells lose responsiveness before they die. Discussion The current manuscript has tried to characterize the relationship of clonal anergy and cell death of T lymphocytes. Two lines of evidence presented here indicate that anergic T cells either die or live and recover responsiveness. This most probably indicates the heterogeneity of the mechanism by which functional unresponsiveness develops in T cells. This hypothesis is apparently supported by reported characteristics that distinguish T cells anergized in vivo by superantigen from anergic protein antigenspecific Th clones in vitro. The major difference be-
42nd FORUM
306
IN IMMUNOLOGY
tween the former and the latter exists in the responsiveness to exogenous IL2 ; anergic Th clones are still responsive to IL2, whereas anergic normal T cells lack responsiveness to exogenous IL2 (Rammensee et al., 1989 ; Rellahan et al., 1990 ; Blackman et al., 1991). This may reflect the difference in in vitro and in vivo experimental systems or, alternatively, the difference in trans-signalling pathways between protein antigens (for Th clones) and superantigens (for normal T cells) (O’Rourke et al., 1990; Liu et al., 1991). If T-cell anergy can be induced in different
1.
2.
ways, it is probable that their fate is also heterogenous depending on the mechanism involved. Figure 1 shows a possible sequence of events of TCRstimulated resting T cells to develop anergy and death. From the top, resting T cells are activated and die without expressing anergy. This type of cell death may occur at early time points after SEB injection within 3 days (particularly between days 2 and 3). As shown in the second row, activated T cells may become functionally unresponsive before they die. This is probably what is taking place from day 4 to day 14 post-SEB injection when spleen cells become tolerant but the percent of CD4+,VP8+ T cells gradually decreases. The fifth pathway, which does not include the activation phase, may also be followed by these cells. The third and forth pathways are followed by anergic T cells which remain in the SEBtolerized animal after day 14. The authors prefer the fourth model because anergy seems to be independent of cell activation events. While we have proposed a number of ways that anergy or death of TCR-stimulated T cells can occur, it is mysterious why cells challenged with superantigen have such different fates. There is much work to be done to fully characterize these processes. Acknowledgements
3.
We wish to thank Drs. D. Spaner and J. Roder for critical reading of the manuscript. This work was supported by grants from the Arthritis Society of Canada and National Cancer Institute of Canada and from the Medical Research Council.
4.
5.
:. “.? 0.Y.
-
0
: Proliferated
0:.G 37 :
.,
blastic
T
References Blackman,M.A., Finkel, T.H., Kappler, J.W. & Marrack, P. (1991),Altered antigenreceptorsignallingin anergic T cellsfrom self-tolerant T-cell receptor P-chain transgenicmice. Proc. nat. Acud. Sci. (Wash.), 88,
T cells
6682-6686. : Proliferative unresponsiveness antigenic stimulation : Apoptic
Fig. 1. Hypothetical
to
cell death
pathways for anergy and death in stimulated T cells. In response to TCR-mediated stimulation, resting T cellsproliferate and becomeblast cells.The blast cellsdie by apoptosis (Pathway 1) or they become anergic before death (Pathway 2). Alternatively, these anergic T cells survive and regain responsiveness (Pathway 3). T cells may becomeanergic without activation and regain responsiveness (Pathway 4) or die (Pathway 5).
De Silva, D.R., Urdahl, K.B. & Jenkins, M.K. (1991), Clonal anergy is induced in vitro by T cell receptor
occupancy in the absence of proliferation. munol.; 147, 3261-3267.
J. Im-
Janeway, Jr. C.A., Yagi, J., Conrad, P.J., Katz, M.E., Jones, B., Vroegop, S. & Buxter, S. (1989), T cell responses to Mls and to bacterialproteinsthat mimic its behavior. Immunol. Rev., 107, 61-88. Jenkins,M.K. & Schwartz, R.H. (1987),Antigen presentation by chemically modified splenocytesinduces antigen-specificT cell unresponsiveness in vitro and in vivo. J. exp. Med., 165, 302-319. Jenkins, M.K., Ashwell, J.D. & Schwartz, R.H. (1988), Allogeneic non-T spleencellsrestorethe responsivenessof normal T cell clonesstimulatedwith antigen and chemically modified antigen-presentingcells. J. Immunol., 140, 3324-3330.
CLONAL
DELETION
AND
ANERGY:
Jenkins, M.K., Chen, C., Jung, G., Mueller, D. & Schwartz, R.H. (1990), Inhibition of antigen-specific proliferation of type 1 murine T cell clones after stimulation with immobilized anti-CD3 monoclonal antibody. J. Immunol., 144, 16-22. Kawabe, Y. & Ochi, A. (1990), Selective anergy of VP8 + ,CD4+ T cells in Stuphylococcus enferotoxin B-primed mice. J. exp. Med., 172, 10651070. Kawabe, Y. & Ochi, A. (1991), Programmed cell death and extrathymic reduction of Vj38+CD4+ T cells in mice tolerant to Stuphylococcus uurens enterotoxin B. Nuture (Lond.), 349, 245-248. Liu, H., Lampe, M.A., Iregui, M.V. &Cantor, H. (1991), Conventional antigen and superantigen may be coupled to distinct and cooperative T-cell activation pathways. Proc. nut. Acud. Sci. (Wash.), 88, 87058709. Mercep, M., Blueston, J.A., Noguchi, P.D. & Ashwell, J.D. (1988), Inhibition of transformed T cell growth in vitro by monoclonal antibodies directed against distinct activating molecules. J. Immunof., 140, 324-335. MacDonald, H.R., Baschieri, S. & Lees, R.K. (1991), Clonal expansion precedes anergy and death of VP8 + peripheral T cells responding to staphylococcal enterotoxin B in vivo. Europ. J. Immunol., 21, 1963-1966. Mueller, D.L., Jenkins, M.K. & Schwartz, R.H. (1989), Clonal expansion versus functional clonal inactivation : a costimulatory signalling pathway determines the outcome of T cell antigen receptor occupancy. Ann. Rev. Immunol.. 7, 445-480. O’Rourke, A.M., Mescher, M.F. & Webb, S.R. (1990)
Deletion Institute
MODELS
TO REALITY
for Cancer Research,
307
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versus anergy : the superantigen H.R.
Ludwig
FROM
paradigm
MacDonald
Ch. des Boveresses
Introduction It is now well accepted that tolerance to self antigens may be achieved by distinct mechanisms, including clonal deletion and functional unresponsiveness (anergy). The recent availability of T-cell receptor (TCR) transgenic mice as well as superantigens reactive with specific TCR VP domains have provided novel tools to investigate the mechanism of T-cell tolerance induction in vivo. In keeping with the aims of this Forum, the present contribution will be restricted to a comparison of the respective roles of deletion and anergy in the establishment and
155, 1066 Epalinges
(Switzerland)
maintenance of central (thymic) and peripheral T-cell tolerance, with particular emphasis on superantigens. Central (thymic) tolerance During their development in the thymus, T cells with high-affinity receptors for self antigens are clonally deleted. This phenomenon has been extensively studied using superantigen models, particularly those involving Mls(MMTV)-encoded endogenous superantigens. Our own experiments (reviewed in MacDonald et al., 1989) suggest that clonal deletion