SumrrlaryT cells play a major role in the development of insulin-dependent diabetes mellitus (IDDM) in nonobese diabetic (NOD) mice. Administration of interleukin 12 (IL-12), a key cytokine which guides the development of T helper type 1 (Thl) CD4 +~ T cells, induces rapid onset of IDDM in NOD, but not in BALB/c mice. Histologically, IL-12 administration induces massive infiltration of lymphoid cells, mostly T cells, in the pancreatic islets of NOD mice. CD4 + pancreasinfiltrating T cells, after activation by insolubilized anti T cell receptor antibody, secrete high levels of interferon 3/and low levels of IL-4. Therefore, IL-12 administration accelerates IDDM development in genetically susceptible NOD mice, and this correlates with increased Thl cytokine production by islet-infiltrating cells. These results hold implications for the pathogenesis, and possibly for the therapy of IDDM and of other Thl cell-mediated autoimmune diseases. Based on the repertoire of lymphokine production, mouse (1) and human (2) CD4 + T cells can be subdivided into two subsets, Thl and Th2, characterized by secretion of IFN-3' and IL-4, respectively. The generation of Thl and Th2 subsets is influenced by the cytokines present during the initial phase of the immune response, and a major role is played by 4). IL-12, a heterodimeric cytokine (5, 6) produced by activated monocytes and B cells (7), promotes Thl cell development. It also enhances proliferation and cytolytic activity of NK and T cells (8), and stimulates production of IFN-3, by these cell types (9). IFN-qr appears to play a role in the development of insulin-dependent diabetes mellitus (IDDM), as demonstrated by the prevention of disease by administration of anti-IFN-3, mAb to nonobese diabetic (NOD) mice (10, 11), or by IDDM induction in mice expressing genes encoding IFN-y under the control of the insulin promoter (12). It has been proposed that the pathological immune response of NOD mice to islet/~ cells is initiated by CD4 + T cells that recognize glutamic acid decarboxylase (13,14), and splenic CD4 + T cells from NOD mice secrete IFN-3' when stimulated with this enzyme (13). Conversely, the Th2-derived lymphokines IL-4 and IL-10 appear to inhibit progression to IDDM in NOD mice (15-17). These findings suggest that T cells that recognize pancreatic B cell antigens cause IDDM only if they develop into Thl cells.To test this assumption, we treated prediabetic NOD mice with IL-12, which has been shown to induce the development of 19) and in vivo (20). Results in the present paper demonstrate that administration of IL-12 to prediabetic NOD female mice induces rapid onset of IDDM. This is associated with enhanced production of Thl-type cytokines by islet-infiltrating lymphocytes, and with selective destruction of islet B cells. Materials and MethodsMice. 10-wk-old female BALB/c mice were obtained from Charles River (Calco, Italy). NOD/Lt mice from The Jackson Laboratory (Bar Harbor, ME) were bred and kept in conventional housing conditions in our animal facility. Mice we...

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Nonobese diabetic (NOD) mice develop spontaneous insulin-dependent diabetes mellitus (IDDM), and the pancreas-infiltrating T cells invariably show a Th1 phenotype. We demonstrated here that the interleukin (IL)-12 antagonist (p40)2 can deviate the default Th1 development of naive T cell receptor (TCR)-transgenic CD4+ cells to the Th2 pathway in vitro. Although (p40)2 does not modify the cytokine profile of polarized Th1 cells, it prevents further recruitment of CD4- cells into the Th1 subset. To study the involvement of Th1 and Th2 cells in the initiation and progression of IDDM, we targeted endogenous IL-12 by administration of (p40)2 in NOD mice. (p40)2 administration to NOD mice inhibits interferon-gamma but not IL-10 production in response to lipopolysaccharide (LPS) or to the putative autoantigen IA-2. Serum immunoglobulin isotypes determined after (p40)2 treatment indicate an increase in Th2 and a decrease in Th1 helper activity. Administration of (p40)2 from 3 weeks of age onwards, before the onset of insulitis, results in the deviation of pancreas-infiltrating CD4+ but not CD8+ cells to the Th2 phenotype as well as in the reduction of spontaneous and cyclophosphamide-accelerated IDDM. After treating NOD mice with (p40)2 from 9 weeks of age, when insulitis is well established, few Th2 and a reduced percentage of Th1 cells are found in the pancreas. This is associated with a slightly decreased incidence of spontaneous IDDM, but no protection from cyclophosphamide-accelerated IDDM. In conclusion, deviation of pancreas-infiltrating CD4+ cells to Th2 is associated with protection from IDDM. However, targeting IL-12 after the onset of insulitis, when the pancreas contains polarized Th1 cells, is not sufficient to induce an effective immune deviation able to significantly modify the course of disease.

The class II major histocompatibility complex molecule I-Ag7 is strongly linked to the development of spontaneous insulin-dependent diabetes mellitus (IDDM) in non obese diabetic mice and to the induction of experimental allergic encephalomyelitis in Biozzi AB/H mice. Structurally, it resembles the HLA-DQ molecules associated with human IDDM, in having a non-Asp residue at position 57 in its β chain. To identify the requirements for peptide binding to I-Ag7 and thereby potentially pathogenic T cell epitopes, we analyzed a known I-Ag7-restricted T cell epitope, hen egg white lysozyme (HEL) amino acids 9–27. NH2- and COOH-terminal truncations demonstrated that the minimal epitope for activation of the T cell hybridoma 2D12.1 was M12-R21 and the minimum sequence for direct binding to purified I-Ag7 M12-Y20/ K13-R21. Alanine (A) scanning revealed two primary anchors for binding at relative positions (p) 6 (L) and 9 (Y) in the HEL epitope. The critical role of both anchors was demonstrated by incorporating L and Y in poly(A) backbones at the same relative positions as in the HEL epitope. Well-tolerated, weakly tolerated, and nontolerated residues were identified by analyzing the binding of peptides containing multiple substitutions at individual positions. Optimally, p6 was a large, hydrophobic residue (L, I, V, M), whereas p9 was aromatic and hydrophobic (Y or F) or positively charged (K, R). Specific residues were not tolerated at these and some other positions. A motif for binding to I-Ag7 deduced from analysis of the model HEL epitope was present in 27/30 (90%) of peptides reported to be I-Ag7–restricted T cell epitopes or eluted from I-Ag7. Scanning a set of overlapping peptides encompassing human proinsulin revealed the motif in 6/6 good binders (sensitivity = 100%) and 4/13 weak or non-binders (specificity = 70%). This motif should facilitate identification of autoantigenic epitopes relevant to the pathogenesis and immunotherapy of IDDM.

SummsryHow peptide-major histocompatibility complex (MHC) class II complexes are naturally generated is still unknown, but accumulating evidence suggests that unfolding proteins or long peptides can become bound to class II molecules at the dominant determinant before proteolytic cleavage. We have compared the immunogenicity of hen egg-white lysozyme (HEL) in nonobese diabetic (NOD), (NOD x BALB/c)F1, and E~ transgenic NOD mice. We find that a response to the subdominant AN~ determinant disappears upon introduction of an E a molecule, and is restored when scission of HEL separates this determinant from its adjoining, competitively dominant, Ed-restricted determinant. This suggests that the E d molecule binds and protects its dominant determinant on a long peptide while captured neighboring determinants are lost during proteolysis. These results provide dear evidence for "determinant capture" as a mechanism of determinant selection during antigen processing and a possible explanation for MHC-protective effects in insulin-dependent diabetes mellitus.

IL-12 administration to nonobese diabetic (NOD) mice induces IFN-γ-secreting type 1 T cells and high circulating IFN-γ levels and accelerates insulin-dependent diabetes mellitus (IDDM). Here we show that IL-12-induced IFN-γ production is dispensable for diabetes acceleration, because exogenous IL-12 could enhance IDDM development in IFN-γ-deficient as well as in IFN-γ-sufficient NOD mice. Both in IFN-γ+/− and IFN-γ−/− NOD mice, IL-12 administration generates a massive and destructive insulitis characterized by T cells, macrophages, and CD11c+ dendritic cells, and increases the number of pancreatic CD4+ cells secreting IL-2 and TNF-α. Surprisingly, IL-12-induced IFN-γ hinders pancreatic B cell infiltration and inhibits the capacity of APCs to activate T cells. Although pancreatic CD4+ T cells from IL-12-treated IFN-γ−/− mice fail to up-regulate the P-selectin ligand, suggesting that their entry into the pancreas may be impaired, T cell expansion is favored in these mice compared with IL-12-treated IFN-γ+/− mice because IL-12 administration in the absence of IFN-γ leads to enhanced cell proliferation and reduced T cell apoptosis. NO, an effector molecule in β cell destruction, is produced ex vivo in high quantity by pancreas-infiltrating cells through a mechanism involving IL-12-induced IFN-γ. Conversely, in IL-12-treated IFN-γ-deficient mice, other pathways of β cell death appear to be increased, as indicated by the up-regulated expression of Fas ligand on Th1 cells in the absence of IFN-γ. These data demonstrate that IFN-γ has a dual role, pathogenic and protective, in IDDM development, and its deletion allows IL-12 to establish alternative pathways leading to diabetes acceleration.

Human TNF-α transgenic (hTNFtg) mice develop erosive arthritis closely resembling rheumatoid arthritis (RA). To investigate mechanisms leading to pathological autoimmune reactions in RA, we examined hTNFtg animals for the presence of RA-associated autoantibodies including Abs to citrullinated epitopes (anti-cyclic citrullinated peptide), heterogeneous nuclear ribonucleoprotein (hnRNP)-A2 (anti-RA33), and heat shock proteins (hsp) (anti-hsp). Although IgM anti-hsp Abs were detected in 40% of hTNFtg and control mice, IgG anti-hsp Abs were rarely seen, and anti-cyclic citrullinated peptide Abs were not seen at all. In contrast, >50% of hTNFtg mice showed IgG anti-RA33 autoantibodies, which became detectable shortly after the onset of arthritis. These Abs were predominantly directed to a short epitope, which was identical with an epitope previously described in MRL/lpr mice. Incidence of anti-RA33 was significantly decreased in mice treated with the osteoclast inhibitor osteoprotegerin and also in c-fos-deficient mice lacking osteoclasts. Pronounced expression of hnRNP-A2 and a smaller splice variant was seen in joints of hTNFtg mice, whereas expression was low in control animals. Although the closely related hnRNP-A1 was also overexpressed, autoantibodies to this protein were infrequently detected. Because expression of hnRNP-A2 in thymus, spleen, brain, and lung was similar in hTNFtg and control mice, aberrant expression appeared to be restricted to the inflamed joint. Finally, immunization of hTNFtg mice with recombinant hnRNP-A2 or a peptide harboring the major B cell epitope aggravated arthritis. These findings suggest that overproduction of TNF-α leads to aberrant expression of hnRNP-A2 in the rheumatoid joint and subsequently to autoimmune reactions, which may enhance the inflammatory and destructive process.

Mice with a null mutation in the terminal deoxynucleotidyl transferase (TdT) gene harbor immunoglobulin and T cell receptor repertoires essentially devoid of N-region diversity. Consequently, the CDR3 loops important for antigen recognition are shorter and considerably less diverse than those of wild-type controls. We find surprisingly normal immune responses in TdT0 mice, as regards both efficiency and specificity. This provokes a reconsideration of the assumption that N-region diversity is required for an effective T and B cell repertoire.