Suml'l'lary A significant proportion of previously activated human T cells undergo apoptosis when triggered through the CD3/T cell receptor complex, a process termed activation-induced cell death (AICD). Ligation of Fas on activated T cells by either Fas antibodies or recombinant human Fas-ligand (Fas-L) also results in cytolysis. We demonstrate that these two pathways of apoptosis are causally related. Stimulation of previously activated T ceils resulted in the expression of Fas-L mILNA and lysis of Fas-positive target cells. Fas-L antagonists inhibited AICD of T cell clones and staphylococcus enterotoxin B (SEB)-specific T cell lines. The data indicate AICD in previously stimulated T ceils is mediated by Fas/Fas-L interactions. Mature peripheral T cells generally undergo activation . and proliferation when stimulated through the CD3/ TCR complex. Under certain circumstances, however, thymocytes, T cell hybridomas, and both CD4 + and CD8 + T cell clones (TCC) 1 undergo cell death when stimulated through the TCR with CD3 antibody in the absence of APC (1-6). This process is rapid and exhibits classic characteristics of apoptosis such as membrane blebbing, chromatin condensation, and the formation of DNA fragments of ",,200 bp. Deletion of T cells by apoptosis appears to be important not only in regulating autoreactive T cells in the thymus, but also in regulating the peripheral T cell pool (7,8). Little is known, however, about the mechanism that mediates the lytic process that has been termed activation-induced cell death (AICD).Fas/APO-1 (CD95) is a protein expressed on the surface of a variety of transformed cell lines and chronically stimulated T cells that can mediate apoptosis after ligation with a Fas-specific antibody (9-12). Under appropriate conditions Fas also transduces a stimulatory signal to certain B cell lines (13) and to .freshly isolated human peripheral blood T cells and thymocytes (14). To investigate a possible relationship between CD3-stimulated AICD and Fas-mediated T cell apoptosis, we have used a mAb directed against human Fas (Fas M3). Immobilized Fas M3 mAb is able to lyse Fas-expressing tumor cell lines in a manner analogous to Fas-ligand (Fas-L) or the prototypic Fas mAb, CH-11, whereas soluble Fas M3 blocks Fas-mediated killing (15).1 Abbreviations used in this paper: AICD, activation-induced cell death; Fas-L, Fas-ligand; SEB, staphylococcal enterotoxin B; TCC, T cell done, Materials and MethodsT Cell Lines and Clones. The alloreactive TCC used in this study were generated by establishing MLC in bulk culture for 7 d followed by limit dilution cloning in 96-well round-bottomed plates in the presence of 10 s irradiated allogeneic PBMC and 10 ng/mt of Ib2. TCC were maintained by stimulation with irradiated PBMC and soluble CD3 antibody (10 ng/ml) approximately every 2 wk and maintenance in IL-2 (10 ng/ml) between stimulations. Shortterm staphylococcal enterotoxin B (SEB)-specific T cell lines were established by stimulation of PBMC (106) with 5 /~g/ml SEB (Sigma Chemical Co., St. Loui...

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OBJECTIVETo investigate if recurrent autoimmunity explained hyperglycemia and C-peptide loss in three immunosuppressed simultaneous pancreas-kidney (SPK) transplant recipients.RESEARCH DESIGN AND METHODSWe monitored autoantibodies and autoreactive T-cells (using tetramers) and performed biopsy. The function of autoreactive T-cells was studied with in vitro and in vivo assays.RESULTSAutoantibodies were present pretransplant and persisted on follow-up in one patient. They appeared years after transplantation but before the development of hyperglycemia in the remaining patients. Pancreas transplant biopsies were taken within ∼1 year from hyperglycemia recurrence and revealed β-cell loss and insulitis. We studied autoreactive T-cells from the time of biopsy and repeatedly demonstrated their presence on further follow-up, together with autoantibodies. Treatment with T-cell–directed therapies (thymoglobulin and daclizumab, all patients), alone or with the addition of B-cell–directed therapy (rituximab, two patients), nonspecifically depleted T-cells and was associated with C-peptide secretion for >1 year. Autoreactive T-cells with the same autoantigen specificity and conserved T-cell receptor later reappeared with further C-peptide loss over the next 2 years. Purified autoreactive CD4 T-cells from two patients were cotransplanted with HLA-mismatched human islets into immunodeficient mice. Grafts showed β-cell loss in mice receiving autoreactive T-cells but not control T-cells.CONCLUSIONSWe demonstrate the cardinal features of recurrent autoimmunity in three such patients, including the reappearance of CD4 T-cells capable of mediating β-cell destruction. Markers of autoimmunity can help diagnose this underappreciated cause of graft loss. Immune monitoring during therapy showed that autoimmunity was not resolved by the immunosuppressive agents used.

The autoimmune process that destroys the insulin-producing pancreatic β cells in type 1 diabetes (T1D) is targeted at insulin and its precursor, proinsulin. T cells that recognize the proximal A-chain of human insulin were identified recently in the pancreatic lymph nodes of subjects who had T1D. To investigate the specificity of proinsulin-specific T cells in T1D, we isolated human CD4+ T cell clones to proinsulin from the blood of a donor who had T1D. The clones recognized a naturally processed, HLA DR4–restricted epitope within the first 13 amino acids of the A-chain (A1–13) of human insulin. T cell recognition was dependent on the formation of a vicinal disulfide bond between adjacent cysteine residues at A6 and A7, which did not alter binding of the peptide to HLA DR4. CD4+ T cell clones that recognized this epitope were isolated from an HLA DR4+ child with autoantibodies to insulin, and therefore, at risk for T1D, but not from two healthy HLA DR4+ donors. We define for the first time a novel posttranslational modification that is required for T cell recognition of the insulin A-chain in T1D.