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Insulin is a major target of the autoimmune response associated with destruction of pancreatic β cells in type 1 diabetes. A peptide that spans the junction of the insulin B chain and the connecting (C) peptide in proinsulin has been reported to stimulate T cells from humans at risk for type 1 diabetes and autoimmune diabetes–prone NOD mice. Here we show that proinsulin B24–C36 peptide binds to I-Ag7, the MHC class II molecule of the NOD mouse, and, after intranasal administration, induces regulatory CD4+ T cells that, in the absence of CD8+ T cells, block the adoptive transfer of diabetes. Curiously, however, intranasal B24–C36 did not inhibit development of spontaneous diabetes in treated mice. We then determined that B24–C36, and its core sequence B25–C34, bind to Kd, the NOD mouse MHC class I molecule, and elicit CD8+ CTLs. When the CD8+ T lymphocyte epitope was truncated at the C34 valine anchor residue for binding to Kd, the residual CD4+ T cell epitope, B24–C32/33, significantly inhibited diabetes development after a single intranasal dose. This study identifies a novel CTL epitope in proinsulin and demonstrates that the therapeutic potential of a “tolerogenic” autoantigen peptide can be compromised by the presence of an integral CTL epitope

The cytotoxic effects of anticancer immune cells are mediated by perforin/granzyme-B, Fas ligand and tumour necrosis factor-related apoptosis-inducing ligand (TRAIL), and therefore depend on intact apoptotic responses in target tumour cells. As killing by all three of these mechanisms is blocked by the frequently overexpressed antiapoptotic oncoprotein Bcl-2, we hypothesised that coexposure to a Bcl-2 inhibitor might enhance anticancer immune responses. We evaluated this in U937 lymphoma cells, and A02 melanoma cells, which both show strong Bcl-2 expression. Va24 þ Vb11 þ natural killer T (NKT) cells expanded from peripheral blood of normal donors (n ¼ 3) were coincubated with PKH26-labelled U937 cells, and cytotoxicity was determined by flow cytometry after annexin-V-FITC and 7-AAD staining. In all cases, addition of the HA14-1 small-molecule Bcl-2 inhibitor to the cocultures significantly increased apoptosis in the target U937 cells. Using a similar assay, killing of A02 cells by the cytotoxic T-lymphocyte clone 1H3 was shown to be amplified by coexposure to the potent small-molecule Bcl-2 inhibitor ABT-737. Experiments with immune effectors preincubated with concanamycin-A suggested that sensitisation to perforin/granzyme-B may underlie enhanced target-cell killing observed in the presence of Bcl-2 inhibitors. We conclude that immune destruction of malignant cells can be amplified by molecular interventions that overcome Bcl-2-mediated resistance to apoptosis.

Active boosting of the antitumour immune response of patients with solid malignancies has been tested in a large number of trials. Isolated complete clinical responses have been reported, however, they have not been replicated in subsequent studies. We recently reported objective clinical responses to a dendritic cell/irradiated autologous tumour cell 'vaccine' in patients with distant metastatic (stage IV) melanoma. Here we describe our experience in a second cohort of patients with stage IV melanoma, using this dendritic cell-based immunotherapy in a cryopreserved format. Of 46 patients enrolled into the study, three had complete remission of all detectable disease, and a further three had partial clinical responses. These data confirm that dendritic cell-based immunotherapy has potential as a therapy in a limited number of patients with stage IV melanoma. To our knowledge, this is the first demonstration that cryopreserved dendritic cells can elicit complete clinical responses in patients with advanced cancer. Our observations support randomized controlled trials to validate the findings.