SummaryInsulin-dependent diabetes meUitus (IDDM) is an autoimmune disease that results in the destruction of the pancreatic islet 3 cells. Glutamic acid decarboxylase (GAD) has been recently indicated as a key autoantigen in the induction of IDDM in nonobese diabetic mice. In human diabetes, the mechanism by which the 3 cells are destroyed is still unknown. Here we report the first evidence for the presence of GAD-specific cytotoxic T cells in asymptomatic and recent diabetic patients. GAD65 peptides displaying the human histocompatibility leukocyte antigen (HLA)-A*0201 binding motif have been synthesized. One of these peptides, GAD114-123, binds to HLA-A*0201 molecules in an HLA assembly assay. Peripheral blood mononudear cells from individuals with preclinical IDDM, recent-onset IDDM, and from healthy controls were stimulated in vitro with the selected peptide in the presence of autologous antigen-presenting cells. In three cases (one preclinical IDDM and two recent-onset IDDM), we detected specific killing of autologous antigen-presenting cells when incubated with GAD114-123 peptide or when infected with a recombinant vaccinia virus expressing GAD65. These patients were the only three carrying the HLA-A*0201 allele among the subjects studied. Our finding suggests that GAD-specific cytotoxic T lymphocytes may play a critical role in the initial events of IDDM.I nsulin-dependent diabetes mellitus (IDDM) is caused by 9 pathological T cell responses that lead to the destruction of 3 cells in the pancreatic islets. Recent studies suggest that MHC class II-restricted CD4 + T cells specific for peptides derived from glutamic acid decarboxylase (GAD65) play a crucial role in the initial phase of IDDM in nonobese diabetic (NOD) mice (1, 2). GAD-specific CD4 + T cells have also been observed in recent-onset IDDM patients and in relatives of IDDM patients at risk to develop diabetes (3). In addition to CD4 +, CD8 + T cells have also been shown to be involved in the pathogenesis of the disease. Induction of diabetes in adoptive transfer experiments requires both CD4 + and CD8 + T cells (4, 5). In addition, CD8 + T lymphocytes were the predominant cells in the inflamed islets of an acutely diabetic patient (6).In this study we demonstrate that MHC class I HLA-A*0201-restricted CD8 + CTLs specific for a GAD peptide (GAD114-123) are present in the peripheral blood of subjects with recent-onset IDDM and at high risk to develop IDDM. This peptide is generated by natural processing of human GAD65. GAD-specific CTLs were not found in healthy individuals expressing HLA-A*0201. These findings argue for a critical role of GAD-specific CTLs in the initial events of IDDM. Materials and MethodsPeptide Synthesis. Nine peptides conforming to the 3,'0201 motif were identified and synthesized by the solid-phase peptide methodology using a synthesizer (Fmoc/tBu chemistry) (431A; Applied Biosystems, Inc., Foster City, CA). Crude peptides were purified by reverse-phase HPLC. The identities of the purified peptides were confirmed by amino ac...

Insulin, glutamate decarboxylase (GAD) and the protein tyrosine phosphatase-like molecule IA-2 are major targets of humoral autoimmunity in insulin-dependent diabetes mellitus (IDDM). These autoantibodies are heterogeneous with respect to age and patient human leukocyte antigen (HLA) phenotype. We have previously demonstrated that GAD and IA-2 antibodies potentially identify different subsets of IDDM patients. The aim of this study was to determine whether GAD and IA-2 autoantibodies were associated with different HLA DR phenotypes. We studied 160 patients with IDDM onset before age 16 years. At disease onset serum was tested for GAD and IA-2 antibodies by immunoprecipitation by in vitro-translated 35S-methionine labelled recombinant proteins. IA-2 antibodies were significantly associated with HLA DR4: 67 (86%) of 78 patients with HLA DR4 vs 31 (38%) of 82 non-DR4 patients had IA-2 antibodies (Pc < 0.0001) and IA-2 antibody levels were higher in patients with HLA DR4 (Pc < 0.0001). In contrast, GAD antibodies were more prevalent (Pc < 0.05) and antibody levels highest (Pc < 0.01) in patients with HLA DR3 phenotypes. These data provide further evidence that, in IDDM, production and titre of major autoantibody specificities are associated with HLA class II alleles.

The infusion of donor lymphocytes after allogeneic bone marrow transplantation is a promising therapeutic tool for achieving a graft versus leukemia (GvL) effect in case of leukemic relapse (1-7), and for the treatment of other complications related to the severe immunosuppressive status of transplanted patients, such as Epstein Barr virus-induced lymphoproliferative disorders (EBV-BLPD) (8) or reactivation of CMV infection (9). Although the delay in the administration of T lymphocytes is expected to reduce the risk of severe GvHD, this risk is still present at higher doses of donor T-cells. The transfer of a suicide gene into donor lymphocytes could allow the in vivo selective elimination of cells responsible for severe GvHD. Additionally, under appropriate conditions, it may allow in vivo modulation of donor anti-tumor responses, and to separate GvL from GvHD. Finally, crucial questions concerning survival and function of donor lymphocytes could be answered by their gene marking. Previous studies documented that T lymphocytes are suitable targets for gene transfer through retroviral vectors (10, 11). This protocol has been designed to evaluate in the contest of allogeneic BMT: 1--the safety of increasing doses of donor lymphocytes transduced with a suicide retroviral vector; 2--the efficacy in terms of survival and immunologic potential of donor lymphocytes after in vitro activation, gene transduction, and immunoselection; 3--the possibility of in vivo down regulation of GvHD by the administration of ganciclovir to patients treated by tk-transduced donor lymphocytes.(ABSTRACT TRUNCATED AT 250 WORDS)

Little is known about the molecular characteristics of alloantigens recognized by alloreactive T cells mediating hematologic stem cell graft rejection. In particular, it has never been shown that such alloantigens can be encoded by HLA-DP␤ alleles. Indeed, matching for HLA-DP antigens is generally not considered to be of functional importance for the outcome of allogeneic bone marrow or peripheral blood stem cell transplantation. In this study, a case of peripheral blood stem

Antibody-mediated rejection is a complex and ongoing phenomenon with different phenotypic features. C4d positive predicts worse prognosis. C4d positive [corrected] and DSA can be used as early mortality predictors in patients without signs of graft dysfunction.