Apoptosis of Mature T Lymphocytes G. BIAS17b A. FACCHINETTI; M. PANOZZO: AND P. PERTILEC ahtitUte of Experimental Pathology University of Ancona Ancona, Italy cInstitute of Oncology Ist Biotechnology Section University of Padua Padua, Italy Superantigens, produced by microbial agents and encoded by murine tumor viruses, activate T cells bearing receptors encoded by specific Vp gene segments, but appear to exert opposite in vitro and in vivo effects on the T-cell repertoire. Superantigens in vitro induce specific clonal expansion of mature T cells,lJ whereas in vivo exposure of mice to Mlsa or Staphylococcus aureus enterotoxin B (SEB) produces a reduction in MISa-reactiveVp6 T cells” and extrathymic deletion of Vp8 T cells$ respectively. Our investigations into the reasons for these contrasting findings indicate that additional signals directed to the TCR induce the clonal elimination of SEBactivated cells both in vivo and in vitro. Three days following SEB administration, BALB/c mice were injected with the anti-Vp8 TCR (F23.1 mAb); the next day their peripheral T-cell repertoire was analyzed cytofluorimetrically. In comparison to controls (untreated, SEB-treated, or anti-Vp8 treated mice), a very strong reduction (40-70%) in CD4+/VP8+ and CD8+/Vp8+, but not in Vp6 cell percentages was detected in the lymph nodes of experimental mice. A similar contraction in Vp8+ cell percentages was also found when lymph node cells, activated in vitro for 3 days with SEB, were subsequently exposed for an additional 20 hours to insolubilized anti-Vp8 mAb in Petri dish cultures. VpS T-cell deletion in these cultures appeared due to death by apoptosis. In fact, agarose gel electrophoresis of the DNA extracted from these SEB + anti-Vp8 mAb triggered cells disclosed intense fragmentation characteristic of apoptosis (FIGla). Apoptosis, the major mechanism involved in intrathymic T-cell negative selection, was recently described also in mature Vp8+ cells during extrathymic deletion caused by SEB inje~tion.~ We therefore evaluated apoptosis in mature T cells using the Southern blot technique, which improves detection of electrophoresed DNA fragments.6 This analysis disclosed that apoptosis became likewise operative in vitro following T-cell proliferation in response to SEB. Interestingly enough, studies on both fresh nylon wool-enriched lymph node T cells and liquid nitrogen-chilled lymph nodes clearly indicated that intense DNA fragmentation also occurs naturally in mature T cells in vivo (FIG.lb). These findings indicate that clonal elimination may be selectively induced either in vivo or in vitro through apoptosis in SEB-activated cells, by means of specific signals directed to the TCR or CD4 co-receptor (G. Biasi et al., in preparation); these results recall those obtained in other studies in which mature T-cell death was achieved by TCR mAb ligation following CD4+ cell-coating with an anti-CD4 bAddress for correspondence: G. Biasi, Institute of Oncology, via Gattamelata 64, 35128 Padua, Italy. 481

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a FIGURE 1. DNA fragmentation in mature T cells. (a) Nylon wool-purified and in birr0 SEB-activated lymph node cells (lanes I and 2, respectively) from BALB/c mice were plated on anti-Vp8TCR-pre-coated Petri dishes. After 20 hours of incubation, fragmented DNA was extracted from gradient-isolated viable cells, separated on agarose gel, and visualized by ethydium bromide staining. (b) DNA fragmentation pattern in a nylon wool-enriched lymph node T-cell suspension 2 x lo6 (lane I) and in a 30-mg frozen lymph node specimen (lane 2). These DNA fragments were separated by agarose gel electrophoresis and visualized using a radiolabeled probe following Southern blot transfer.6

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mAb.'~~ We also detected conspicuous DNA fragmentation in SEB-activated lymph node cultures in which Vp8 T cells increased in response to the ligand. Therefore, as far as the apoptotic event in SEB-activated cells is concerned, no differences between in vitro and in vivo5 findings exist. The unexpectedly high degree of DNA fragmentation in the lymphoid organs might result from repeated exposure of the living organism to superantigens produced by various microbial invaders. If this is the case, and in view of our finding that additional TCR signals induced specific T-cell deletion, we advance the idea that environmental factors would then play an important role in the homeostatic control of the T-cell repertoire by means of continuous shaping due to specific TCR signals from exogenous or self antigens. REFERENCES 1.

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KAWABE, Y. & A. OCHI.1990. Selective anergy of Vp8+, CD4+ T cells in Staphylococcus enterotoxin B-primed mice. J. Exp. Med. 172: 1065-1070. JANEWAY, C. A., JR., J. YAGI,P. J. CONRAD,M. E. KATZ, B. JONES, S. VROEGOP& S. BUXSER.1989. T-cell responses to MIS and to bacterial proteins that mimic its behavior. Immunol. Rev. 107: 61-88. WEBB,S., C. MORRIS& J. SPRENT.1990. Extrathymic tolerance of mature T cells: Clonal elimination as a consequence of immunity. Cell 63: 1249-1256. RELLAHAN, B. L., L. A. JONES,A. M. KRUISBEEK, A. M. FRY& L. A. MATIS.1990. In vivo induction of anergy in peripheral Vp8+ T cells by staphylococcal enterotoxin B. J. Exp. Med. 172: 1091-1100. KAWABE,Y. & A. OCHI. 1991. Programmed cell death and extrathymic reduction of V@+CD4+ T cells in mice tolerant to Staphylococcus aureus enterotoxin B. Nature 349 245-248. M.MAZZOCCHI, R. KINGSTON, D. COLLAVO & G . BIASI. FACCHINEITI, A., L. TESSAROLLO, 1991. An improved method for the detection of DNA fragmentation. J. Immunol. Meth. 136 125-131. NEWELL,M. K., L. J. HAUGHN, C. R. MAROUN& M. H. JULIUS.1990. Death of mature T cells by separate ligation of CD4 and the T-cell receptor for antigen. Nature 347: 286289. BIASI, G., A. FACCHINETTI, M. PANOZZO, P. ZANOVELLO, L. CHIECO-BIANCHI & D. COLMVO.1991. Moloney murine leukemia virus tolerance in anti-CD4 mAb-treated adult mice. J. Immunol. 147: 22862289.