Requirement for T-cell apoptosis in the induction of peripheral ... - Nature

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Requirement for T-cell apoptosis in the induction of peripheral transplantation tolerance ANDREW D. WELLS2, XIAN CHANG LI1, YONGSHENG LI1, MATTHEW C. WALSH2, XIN XIAO ZHENG1, ZIHAO WU2, GABRIEL NUÑEZ3, AIMIN TANG2, MOHAMED SAYEGH5, WAYNE W. HANCOCK4, TERRY B. STROM1 & LAURENCE A. TURKA2


1

Harvard Medical School, Department of Medicine, Beth Israel Deaconess Medical Center, Boston, Massachusetts 02215, USA 2 Department of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA 3 Department of Pathology, University of Michigan, Ann Arbor, Michigan 48109, USA 4 LeukoSite Inc., & Department of Pathology, Harvard Medical School, Cambridge, Massachusetts 02142, USA 5 Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts 02115, USA A.D.W. and X.C.L. contributed equally to this paper. Correspondence should be addressed to L.A.T.; email: [email protected] or T.B.S.; email: [email protected]

The mechanisms of allograft tolerance have been classified as deletion, anergy, ignorance and suppression/regulation. Deletion has been implicated in central tolerance1, whereas peripheral tolerance has generally been ascribed to clonal anergy and/or active immunoregulatory states2. Here, we used two distinct systems to assess the requirement for T-cell deletion in peripheral tolerance induction. In mice transgenic for Bcl-xL, T cells were resistant to passive cell death through cytokine withdrawal, whereas T cells from interleukin-2-deficient mice did not undergo activation-induced cell death. Using either agents that block co-stimulatory pathways or the immunosuppressive drug rapamycin, which we have shown here blocks the proliferative component of interleukin-2 signaling but does not inhibit priming for activation-induced cell death, we found that mice with defective passive or active T-cell apoptotic pathways were resistant to induction of transplantation tolerance. Thus, deletion of activated T cells through activation-induced cell death or growth factor withdrawal seems necessary to achieve peripheral tolerance across major histocompatibility complex barriers. Peripheral transplantation tolerance can be induced by interference with T-cell growth factor production, use or signaling. This can be accomplished by blockade of the CD28 and/or the CD154 T-cell co-stimulatory pathways3–5, or by using rapamycin, which inhibits interleukin (IL)-2-mediated signal transduction6. In either event, the immediate effect of deprivation of IL-2 or its proliferative stimulus is the induction of T-cell anergy, which is reversible by exogenous IL-2 (ref. 7). The reversibility of anergy and the strength of the alloreactive response has generated questions regarding whether a mechanism that does not involve destruction of alloreactive clones can lead to durable transplantation tolerance. Apoptosis, an essential component of central (thymic) tolerance1, might also function in peripheral tolerance resulting from treatments that do not directly kill T cells. Two distinct forms of Tcell apoptosis have been identified8. ‘Passive’ cell death is mediated by growth factor deprivation of activated T cells9. In contrast, activation-induced cell death (AICD) occurs when previously primed T cells are repetitively activated by antigen8, and requires previous exposure to IL-2 (ref. 10). Here, we used models in which NATURE MEDICINE • VOLUME 5 • NUMBER 11 • NOVEMBER 1999

alloreactive T cells are resistant to either passive or active cell death to directly test the involvement of T-cell apoptosis in the induction of peripheral transplantation tolerance. To study the role of passive apoptosis, we used mice with transgenic expression of the long form of the Bcl-x gene (Bcl-xL) targeted to the T-cell lineage9. Bcl-xL protects lymphoid cell lines from apoptosis induced by cytokine insufficiency11. In normal mice, Bcl-xL was minimally expressed in resting splenic T cells, but was induced by T-cell receptor ligation, and its induction was augmented and prolonged by CD28 co-stimulation (Fig. 1a)(ref. 11). In Bcl-xL transgenic mice, Bcl-xL expression was detectable in unstimulated T cells and was constitutively expressed after activation with or without CD28 co-stimulation (Fig. 1a)(ref. 9). Transgenic Bcl-xL expression did not obviate the requirement for CD28 signaling in T-cell proliferation. However, this response was completely inhibited by cytotoxic T-lymphocyte A4 immunoglobulin (CTLA4Ig) (Fig. 1b). Bcl-xL transgenic T cells showed a smaller responder frequency and a slightly lower proliferative capacity than wild-type cells (Fig. 1b), accounting for the decreased number of mitotic events. This is consistent with delayed cell cycle entry/progression induced by overexpression of Bcl2 family proteins12. Constitutive expression of Bcl-xL in T cells enhanced their survival in vitro when unstimulated, or when activated without CD28 co-stimulation (Fig. 1c). In contrast, cell survival was similar between Bcl-xL transgenic and wild-type T cells stimulated in the presence of CD28 signals. Although supplemental IL-2 did not notably effect the survival of T cells activated along with CD28 signaling, IL-2 considerably decreased the survival of Bcl-xL transgenic T cells stimulated in the presence of CTLA4Ig (Fig. 1c). This indicates that although Bcl-xL expression alone protects resting T cells from apoptosis, activated T cells can still succumb to IL2-triggered AICD. When AICD is inhibited by blockade of IL-2 production with CTLA4Ig, maximum survival of activated, Bcl-xL transgenic T cells occurs. To determine whether activated Bcl-xL transgenic T cells also have increased long-term survival in vivo, we stimulated wild-type or Bcl-xL transgenic cells in vitro with antibody against CD3 plus either antibody against CD28 (‘priming’ conditions) or CTLA4Ig (‘tolerizing’ conditions). As anticipated, the combination of CD3 1303


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Fig. 1 Bcl-xL expression and survival in T cells of wild-type and Bcl-xL transgenic mice. a, CD4+ or CD8+ T-cells from Bcl-xL transgenic mice or C57BL/6 transgene-negative mice (Wild-type) were cultured in medium alone (Unstim.) or on plates coated with monoclonal antibodies against CD3 and CD28, or with monoclonal antibody against CD3 alone plus soluble CTLA4Ig. Cell extracts were assessed for Bcl-xL expression. b, Splenocytes from wild-type mice (open symbols) and Bcl-xL transgenic mice (filled symbols) were labeled with CFSE and stimulated with monoclonal antibody against CD3 (2.5–2000 ng/ml; horizontal axis) in the presence of 2 µg/ml antibody against CD28 (circles) or 15 µg/ml CTLA4Ig (squares). Cells were collected at 72 h, and T-cell proliferation was assessed by flow cytometry. c, Resistance of Bcl-xL transgenic T cells to apoptosis induction. Splenocytes from Bcl-xL-transgenic mice () or wild-type littermate mice () were cultured in media alone or stimulated with soluble antibody against CD3, in the presence of either 2 µg/ml agonistic antibody against CD28

(top) or 15 µg/ml CTLA4Ig (bottom). Right, 15 U/ml IL-2. Viability of T cells was assessed using TOPRO-3 exclusion. d, Spleen and lymph node cells from B6 Thy1.1+ wild-type mice () or B6 Thy1.2+ Bcl-xL transgenic mice () were cultured with antibody against CD3 in the presence of either monoclonal antibody against CD28 (left, primed) or CTLA4Ig (right, tolerized). Cells were transferred to congenic B6 (Thy1.1 × Thy1.2)F1 recipients, which were killed (horizontal axis, time). Top, The number of donor-derived CD8 cells in draining lymph nodes was assessed by flow cytometry. Bottom, A portion of the cells were restimulated and the production of IFN-γ was assessed by flow cytometry. e, Naive BALB/c mice adoptively transferred with lymph node cells from DO11.10 mice (WT) or Bcl-xL transgenic DO11.10 mice were then immunized with ovalbumin and treated with CTLA4Ig or control immunoglobulin on days 0 and 2; 6 d later, the recipients were killed and clonotypic (KJ-126+) T cells from draining lymph nodes were counted by flow cytometry.

and CTLA4Ig resulted in a substantial decrease (67-88%) in the production of IL-2 or gamma interferon (IFN-γ) after re-stimulation, compared with that produced by CD3/CD28-activated T cells (data not shown). Next, we co-adoptively transferred in vitroprimed or -tolerized T cells, derived from Thy1.2+ Bcl-xL transgenic mice or Thy1.1+ wild-type mice, respectively, to non-transgenic (Thy1.1+/Thy1.2+)F1 congenic mice, and killed the recipients at serial time points to assess T-cell survival and function (Fig. 1d). Primed CD8+ T cells, both wild-type and Bcl-xL transgenic, had extended survival in vivo, although the Bcl-x transgene afforded a survival advantage at later times. In contrast, we detected very few tolerized wild-type cells by 10 days after adoptive transfer, whereas we were able to recover tolerized Bcl-xL transgenic T cells

in numbers similar to those of productively activated T cells. We obtained similar results with CD4+ T cells (data not shown). The survival of tolerized Bcl-xL transgenic cells in vivo was accompanied by their eventual functional recovery (Fig. 1d). By 30–60 days after adoptive transfer, cells derived from Bcl-xL transgenic mice were fully competent for IFN-γ (and IL-2) production whether they had been stimulated in priming or tolerizing condition. Even in priming conditions, a higher percentage of Bcl-xL transgenic T cells had demonstrable IFN-γ production than did wild-type cells. We also demonstrated preferential in vivo T-cell survival in a major histocompatibility complex (MHC) class II-restricted, antigen-specific system. We adoptively transferred DO11.10 T-cell receptor transgenic T cells (which respond to an ovalbumin-derived

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peptide fragment13) or Bcl-xL transgenic DO11.10 cells to congenic BALB/c mice, immunized the hosts with ovalbumin peptide in incomplete Freund’s adjuvant and treated them with control immunoglobulin or CTLA4Ig (ref. 14)(Fig. 1e). DO11.10/Bcl-xL double-transgenic T cells accumulated to a greater degree, and were more resistant to inhibition by CTLA4Ig, than were DO11.10 cells. Furthermore, the differential recovery of DO11.10 and DO11.10/Bcl-xL cells was most likely underestimated in this system, because the increased survival of Bcl-xL cells was counteracted by their decreased expansion (Fig. 1b). These data indicate that the initial responses of Bcl-xL transgenic T cells are inhibited by CD28 co-stimulatory blockade, but that overexpression of Bcl-xL in these conditions enables the survival of many cells that regain functional integrity. This indicates that tolerance induction with CTLA4Ig might be abrogated in Bcl-xL transgenic mice. We tested this in MHC-mismatched murine vascularized cardiac allografts. As shown before15, transfusion of donor cells in combination with either CTLA4Ig or monoclonal antibody against CD154 induced long-term allograft survival (more than 120 days) in all wild-type mice (Fig. 2a). In contrast, Bcl-xL transgenic recipient mice were resistant to tolerance induction by treatment with either CTLA4Ig or antibody against CD154. Although some Bcl-xL transgenic mice showed prolonged graft survival after treatment, all of these allografts evidenced severe chronic rejection and transplant arteriosclerosis (Fig. 2b). This indicates that induction of anergy by itself is not sufficient to prevent rejection in this model, and that inhibition of Bcl-xL induction in T cells with resultant apoptosis is a chief mechanism of action of co-stimulatory blockade. The Bcl-xL transgenic model emphasizes the importance of cytokine deprivation-induced apoptosis in tolerance induction by co-stimulatory blockade. However, Bcl-xL transgenic T cells remain susceptible to AICD, and the data in Fig. 1 demonstrate the importance of IL-2 in this process. To study the involvement of IL-2-dependent AICD in allograft tolerance, we used mice with targeted deletions of the interleukin-2 gene. IL-2-deficient mice develop age-dependent lymphoproliferative disease and autoimmunity due to the accumulation of activated, apoptosis-resistant T cells in the periphery. Furthermore, T cells from these mice are resistant to AICD after in vivo activation with ‘superantigen’16 or alloantigen (data not shown). IL-2 has at least two separable functions in Tcell responses: to serve as a growth factor, and to prime for AICD (ref. 8). These two functions of IL-2 can be dissociated using raNATURE MEDICINE • VOLUME 5 • NUMBER 11 • NOVEMBER 1999

a Percent graft survival

Fig. 2 Resistance of Bcl-xL transgenic mice to CTLA4Ig. a, Wild-type (WT) or Bcl-xL transgenic mice (B6 background) received vascularized cardiac allografts from BALB/c donors, and received BALB/c splenic mononuclear leukocytes on the day of transplantation (day 0). Mice received monoclonal antibody against CD154 on day 0, CTLA4Ig on day 2, or control hamster or human immunoglobulin on days 0 or 2, respectively. b, Wild-type or Bcl-xL transgenic mice treated with donor cells plus CTLA4Ig were killed on day 100, and their cardiac allografts were sectioned, stained for elastin and examined by light microscopy. Top, Despite an interstitial mononuclear cell infiltrate, hearts transplanted into wild-type mouse shows good preservation of myocardial architecture and normal vessels: large arrow, normal single-cell intimal layer in a representative intramyocardial artery; small arrow-heads, sparsely distributed apoptotic leukocytes. Bottom, Although this allograft in a Bcl-xL transgenic mouse also has a diffuse mononuclear cell infiltrate, it lacks cells undergoing apoptosis and shows many healed infarcts and severe transplant arteriosclerosis; arrow, substantially disrupted internal elastic lamina in a representative intramyocardial artery and the associated concentric increase in intimal cells (which clinically leads to cardiac hypoxia and gradual organ failure).

Day after transplant

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pamycin. Although IL-2-dependent T-cell proliferation was inhibited by rapamycin (Fig. 3a), rapamycin did not block IL-2 priming for AICD (Fig. 3b). As IL-2 priming for AICD was not affected by rapamycin, we used this model to assess the involvement of AICD in tolerance induction (Fig. 3c). IL-2-deficient mice readily rejected islet allografts, presumably because of the functional redundancy of T-cell growth factors. Treatment of wild-type mice with rapamycin induced long-term graft survival in most recipients. In contrast, all rapamycin-treated IL-2-deficient recipients rejected their grafts. This indicates that an IL-2-triggered apoptotic signal is required for tolerance induction in this model. These data show that intact T-cell apoptotic pathways are required for the induction of transplantation tolerance across MHC barriers with treatments that do not directly kill T cells. Consistent with this, peripheral deletion of host anti-donor alloreactive cells occurs with allogeneic bone marrow transplantation using CD28 and CD154 blockade17, and IL-2-deficient recipients resist immunosuppression with CTLA4Ig (ref. 18). However, we do not infer that clonal deletion is the sole mechanism of tolerance in these systems. Exogenous IL-2 can prevent or reverse tolerance induction by CTLA4Ig early after transplant19, a phenomenon most consistent with anergy. Furthermore, adoptive transfer of cells from mice tolerized with CTLA4Ig induces tolerance in naive recipients, indicating a involvement of active immunoregulation20. Anergy and active immunoregulation have also been demonstrated in transplant tolerance induced by nondepleting antibodies against CD4 and CD8 (ref. 21). The processes that may govern the emergence of regulatory cells in these systems are incompletely understood. In particular, it is not known if cells that survive AICD develop regulatory function by 1305


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Fig. 3 Rapamycin blocks IL-2-driven T-cell proliferation, but does not inhibit Fas-mediated cell death. a, Splenic mononuclear leukocytes from IL-2deficient mice were stimulated with 2 µg/ml monoclonal antibody against CD3 plus IL-2 for 3 d in the presence of rapamycin (doses, horizontal axis). Proliferation was measured as the incorporation of 3H-thymidine present for the last 18 h of culture. b, Splenic mononuclear leukocytes from wild-type mice were stimulated with 2 µg/ml monoclonal antibody against CD3 in

the presence or absence of 5 ng/ml rapamycin, with or without exogenous IL-2 for 3 d. Viable cells were isolated and treated with immunoglobulin (Cont Ig) or against Fas (Anti-fas). Apoptotic cell death was assessed by annexin V binding. c, Rapamycin fails to prolong islet allograft survival in IL-2deficient mice. Islet donors, DBA/2 mice (H-2d); recipients, 129 mice (H-2b), wild-type (WT) or IL-2-deficient (IL-2KO). Mice that received syngeneic islet grafts had 100% graft survival for more than 120 days (n = 3).

default, or if events occurring during AICD actively induce regulatory capabilities in cells that are refractory to apoptosis. We favor the hypothesis that depletion of alloreactive cells during the inductive phase of tolerance is required to reduce the clonal size of the allo-destructive population to a level that can be controlled through anergy and/or immunoregulation during a maintenance phase. In support of this, immune deviation Thelper type 1 to a T-helper type 2 phenotype can induce transplantation tolerance across minor histocompatibility barriers, where only a small alloreactive population exists, but immune deviation alone is unable to do so across MHC barriers, where the responding population is much larger22. Consistant with this, immune deviation is effective at ameliorating disease in many autoimmune models, and may be involved in tolerance to the H-Y minor transplantation antigen23. Reduction of the alloreactive pool may be achieved either by using depleting monoclonal antibodies, for which intact cell death pathways may not be required, or through protocols that block cytokine production and use, for which the induction of endogenous apoptotic mechanisms seems to be essential. This has implications for the design and implementation of clinical strategies to induce transplant tolerance.

Reagents. Antibodies and fusion proteins used include antibody against CD3 (2C11; hybridoma obtained from J. Bluestone), antibody against CD28 (37.51; hybridoma obtained from J. Allison), antibody against Fas (Jo2; PharMingen, San Diego, California), CTLA4Ig (Bristol-Meyers-Squibb, Seattle, Washington). Growth factors used include IL-2, IL-4, IL-7 and IL-15 (PharMingen, San Diego, California). Rapamycin, for use in vitro and in vivo, was obtained from S. Sehgal (Wyeth Ayerst, Princeton, New Jersey). Ovalbumin peptide (323-339) was synthesized at the University of Pennsylvania protein core facility.

Methods

In vitro T-cell cultures. Single-cell lymphocyte suspensions were prepared from lymph nodes and spleen. Where indicated, suspensions were enriched for CD4+ or CD8+ T cells using magnetic beads coated with monoclonal antibody against the macrophage marker F4/80, MHC class II (M5/114), Mac-1 (M1/70.15), CD16 (2.4G2) and CD4 (GK1.5) or CD8 (2.43). In some experiments, lymphocytes were labeled with the fluorescent dye CFSE (carboxyfluorescein diacetate succinimidyl diester), as described24. T cells were stimulated with soluble or plate-bound antibody against CD3 alone, or in combination with antibody against CD28, CTLA4Ig, growth factors, rapamycin or control immunoglobulin. T-cell proliferation was assessed after 3 d by 3H-thymidine incorporation (1 µCi per well; 8-hour pulse), or at various times using CFSE fluorescence. Fas-mediated cell death was induced in primed T-cell populations by culture for 16 h on plate-bound monoclonal antibody against Fas. Cytokine production by primed T cells was stimulated in a 5-hour culture with plate-bound antibody against CD3 or CD28 in the presence of monensin14.

Mice. Mice with constitutive expression of the human Bcl-xL transgene in the T-cell lineage9 were back-crossed ten generations onto a C57/BL6 (H2b) background. Bcl-xL transgenic mice were back-crossed eight generations with BALB/c mice (H-2d) transgenic for the DO11.10 T-cell receptor13. The appropriate transgene-negative littermates were used as controls in all experiments.

In vivo T-cell studies.’Pooled’ spleen and lymph node cells from B6 Thy1.1+ wild-type mice or B6 Thy1.2+ Bcl-xL transgenic mice were cultured in vitro with 5 µg/ml antibody against CD3 in the presence of either 5 µg/ml agonistic monoclonal antibody against CD28 or 15 µg/ml CTLA4Ig for 3 d. The cells were collected and adoptively transferred to congenic B6 (Thy1.1 ×

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Thy1.2)F1 recipients (3 × 107 per recipient). The recipient mice were killed and the number of donor-derived cells in draining lymph nodes was assessed by flow cytometry. A portion of the cells were also re-stimulated in vitro for 5 h, and the production of IFN-γ and IL-2 was assessed by flow cytometry. Naive BALB/c mice were adoptively transferred, by intravenous injection, with lymph node cells from DO11.10 mice or Bcl-xL transgenic DO11.10 mice. Recipients were then immunized subcutaneously at the base of the tail with ovalbumin peptide, amino acids 323-339, in incomplete Freund’s adjuvant and treated with 200 µg CTLA4Ig or control immunoglobulin on day 0 and day 2 of immunization as described14. Then, 6 d later, the recipients were killed and clonotypic (KJ-126+) T cells from the draining lymph nodes (inguinal and paraaortic) were counted by flow cytometry. Transplantation. For the vascularized cardiac allograft model, wild-type or transgenic C57/BL6 mice (H-2b; 8-10 weeks old) received vascularized cardiac allografts from BALB/c donors (H-2d). All mice received 5 × 106 BALB/c splenocytes by intravenous infusion on the day of transplantation. Mice also received 200 µg monoclonal antibody against CD154 on the day of transplantation, 200 µg CTLA4Ig on day 2 after transplantation, or 200 µg control hamster immunoglobulin or human immunoglobulin on days 0 or 2, respectively. Graft survival was monitored daily by palpation, and rejection (cessation of palpable contractions) was verified by direct visualization. Allografts were sectioned and stained for elastin, and myocardial, intimal and vascular architecture, as well as mononuclear cell infiltration, were assessed by light microscopy. For the pancreatic islet allograft model, strain 129 mice (H-2b) were rendered diabetic by a single intraperitoneal injection of 225 mg/kg streptozotocin (Sigma), and hyperglycemia was verified by measurements of glucose in blood obtained from the tail vein. Islets from DBA/2 donors (H-2d) were transplanted as described22, and allograft function was monitored as serial blood glucose measurements. Where indicated, mice received rapamycin: 0.2 mg/kg per day intraperitoneally daily for three days, followed by 0.2 mg/kg every other day for a total treatment period of 14 days. Primary graft function was defined as blood glucose less than 200 µg/dl on day 3 after transplantation, and graft rejection was defined as an increase in blood glucose exceeding 300 mg/dl after a period of primary graft function. Immunoblot analysis of Bcl-xL expression. Spleen and lymph node cells were isolated from Bcl-xL transgenic mice or transgene negative littermate control mice (Wild-type;, C57BL/6). Purified CD4+ or CD8+ T-cell subsets were cultured 48 h in medium alone, or on tissue culture plates coated with monoclonal antibodies against CD3 (5 µg/ml) and CD28 (5 µg/ml), or with monoclonal antibody against CD3 alone plus soluble CTLA4Ig (15 µg/ml). Cells were collected at 48 h and cell extracts (1 × 106 cell equivalents per sample) were separated by SDS-PAGE, and then transferred to nitrocellulose membranes. Blots were probed with a polyclonal rabbit antiserum specific for Bcl-x (Transduction Laboratories, Lexington, Kentucky), and immunoreactive proteins were visualized using peroxidase-conjugated goat antibodies against rabbit followed by the addition of chemiluminescent peroxidase substrate (Boehringer). Thymocytes from a Bcl-xL transgenic mouse and unfractionated splenic mononuclear leukocytes from a wildtype mouse served as positive and negative controls, respectively. The blots were ‘stripped’ and re-probed with a polyclonal rabbit antiserum specific for actin (Sigma) to confirm equivalent loading in each lane. Flow cytometric analysis of T-cell proliferation, survival and cytokine production. Specific T-cell subsets were identified using fluorochrome-conjugated monoclonal antibodies against CD4, CD8, Thy1.1 and Thy1.2 (PharMingen, San Diego, California). D011.10 T-cell receptor transgenic T cells were identified using KJ-126 (hybridoma obtained from M. Jenkins) clonotypic monoclonal antibody, followed by fluorochrome-conjugated streptavidin (PharMingen, San Diego, California). Cell division within distinct live T-cell subsets was monitored on the basis of CFSE fluorescence,


and was quantified as described24. T-cell apoptosis was assessed by vital dye exclusion (TOPRO-3; Molecular Probes, Eugene, Oregon) or by annexin V binding. Cytokine production by individual primed T cells was detected after fixation, permeabilization, and staining with fluorochrome-conjugated monoclonal antibody against IFN-γ or IL-2 (PharMingen, San Diego, California)14.

Acknowledgments This work was supported by the National Kidney Foundation of America (X.C.L.), the Juvenile Diabetes Foundation (X.X.Z., T.B.S., M.H.S. and L.A.T.), the American Heart Association (L.A.T.) and the National Institutes of Health (AI-34665, AI-37691, AI-37798, AI-41521, AI-42298 and CA09140).

RECEIVED 20 JULY; ACCEPTED 20 SEPTEMBER 1999 1. Sykes, M. & Sachs, D. H. Bone marrow transplantation as a means of inducing tolerance. Semin. Immunol. 2, 401–417 (1990). 2. Schwartz, R. H. Acquisition of immunologic self-tolerance. Cell 57, 1073–1081 (1989). 3. Lenschow, D. et al. A. Long-term survival of xenogeneic pancreatic islet grafts induced by CTLA4Ig. Science 257, 789–792 (1992). 4. Parker, D.C. et al. Survival of mouse pancreatic islet allografts in recipients treated with allogeneic small lymphocytes and antibody to CD40 ligand. Proc. Natl. Acad. Sci. USA 92, 9560–9564 (1995). 5. Larsen, C. P. et al. Long-term acceptance of skin and cardiac allografts after blocking CD40 and CD28 pathways. Nature 381, 434–438 (1996). 6. Price, D. J., Grove, J. R., Calvo, V., Avruch, J. & Bierer, B. E. Rapamycin-induced inhibition of the 70-kilodalton S6 protein kinase. Science 257, 973–977 (1992). 7. Jenkins, M. K. & Schwartz, R. H. Antigen-presentation by chemically modified splenocytes induces antigen-specific T cell unresponsiveness in vitro and in vivo. J. Exp. Med. 165, 302–319 (1987). 8. Van Parijs, L. & Abbas, A. K. Homeostasis and self-tolerance in the immune system: turning lymphocytes off. Science 280, 243–248 (1998). 9. Grillot, D. A., Merino, R. & Nunez, G. Bcl-XL displays restricted distribution during T cell development and inhibits multiple forms of apoptosis but not clonal deletion in transgenic mice. J. Exp. Med. 182, 1973–1983 (1995). 10. Lenardo, M. J. Interleukin-2 programs mouse alpha beta T lymphocytes for apoptosis. Nature 353, 858–861 (1991). 11. Boise, L.H. et al. CD28 Co-stimulation can promote T cell survival by enhancing the expression of Bcl-xL. Immunity 3, 87–98 (1995). 12. O’Reilly, L. A., Huang, D. C. & Strasser, A. The cell death inhibitor Bcl-2 and its homologues influence control of cell cycle entry. EMBO J. 15, 6979–6990 (1996). 13. Murphy, K. M., Heimberger, A. B. & Loh, D. Y. Induction by antigen of intrathymic apoptosis of CD4+CD8+TCRlo thymocytes in vivo. Science 1990, 1720–1723 (1990). 14. Gudmundsdottir, H., Wells, A. & Turka, L. Dynamics and requirements of T cell clonal expansion in vivo at the single-cell level: Effector function is linked to proliferative capacity. J. Immunol. 162, 5212–5223 (1999). 15. Hancock, W.W. et al. Co-stimulatory function and expression of CD40-ligand, CD80, and CD86 in vascularized murine cardiac allograft rejection. Proc. Natl. Acad. Sci. USA 93, 13967–13972 (1996). 16. Kneitz, B., Herrmann, T., Yonehara, S. & Schimpl, A. Normal clonal expansion but impaired Fas-mediated cell death and anergy induction in interleukin-2-deficient mice. Eur. J. Immunol. 25, 2572–2577 (1995). 17. Wekerle, T. et al. Extrathymic T cell deletion and allogeneic stem cell engraftment induced with co-stimulatory blockade is followed by central T cell tolerance. J. Exp. Med. 187, 2037–2044 (1998). 18. Dai, Z., Konieczny, B.T., Baddoura, F.K. & Lakkis, F.G. Impaired alloantigen-mediated T cell apoptosis and failure to induce long-term allograft survival in IL-2-deficient mice. J. Immunol. 161, 1659–1663 (1998). 19. Sayegh, M.H. et al. CD28-B7 blockade after alloantigenic challenge in vivo inhibits Th1 cytokines but spares Th2. J. Exp. Med. 181, 1869–1874 (1995). 20. Tran, H.M. et al. J. Distinct mechanisms for the induction and maintenance of allograft tolerance with CTLA4-Fc treatment. J. Immunol. 159, 2232–2239 (1997). 21. Waldmann, H. & Cobbold, S. How do monoclonal antibodies induce tolerance? A role for infectious tolerance? Annu. Rev. Immunol. 16, 619–644 (1998). 22. Li, X.C., Zand, M.S., Li, Y., Zheng, X.X. & Strom, T.B. On histocompatibility barriers, Th1 to Th2 immune deviation, and the nature of the allograft responses. J. Immunol. 161, 2241–2247 (1998). 23. Tanchot, C. et al. Modifications of CD8+ T cell function during in vivo memory or tolerance induction. Immunity 8, 581–590 (1998). 24. Wells, A., Gudmundsdottir, H. & Turka, L. Following the fate of individual T cells throughout activation and clonal expansion: signals from T cell receptor and CD28 differentially regulate the induction and duration of a proliferative response. J. Clin. Invest. 100, 3173–3183 (1997).

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