The T cell receptor (TCR) requirements in the pathogenesis of insulin-dependent diabetes were examined with transgenic NOD mice bearing nondisease-related TCR α and β chains. In both TCRβ and TCRαβ transgenic NOD mice the β chain transgene was expressed by > 98% of peripheral T cells. The α chain transgene was also highly expressed. Insulitis developed in both sets of transgenic animals with most of the lymphocytes in the lesion expressing the transgenic β chain and with depletion of the endogenous TCR V
β
genes. Nonetheless, NOD animals transgenic for TCRβ and TCRαβ developed diabetes similar to controls. Thus, skewing the TCR repertoire did not diminish autoimmune susceptibility in NOD mice.
We report the isolation of a panel of CD4+ T helper type 1 autoreactive T cell clones from the spleen of unprimed nonobese diabetic mice, a murine model of human insulin-dependent diabetes mellitus. The T cell clones express a diverse repertoire of T cell receptors, three of which recognize beta islet cell autoantigen(s). The islet cell-reactive T cell clones inhibit adoptive transfer of insulin-dependent diabetes mellitus and intraislet lymphocytic infiltration. The protective capacity of the T cell clones correlates with their ability to produce a novel immunoregulatory activity that potently inhibits in vitro allogeneic mixed lymphocyte reaction. The partially purified activity significantly inhibited the adoptive transfer of diabetes. Our work provides evidence in support of the existence of T helper type 1, CD4+ T cells reactive to beta islet cell autoantigens that have acquired a protective instead of a diabetogenic effector function. These T cells mediate their protective action in part by production of an immunoregulatory activity capable of down-regulating immune responses, and they are likely to represent a population of regulatory T cells that normally plays a role in maintaining peripheral tolerance.
The T cell receptor (TcR) for antigen, on the majority of T cells, is a disulfide-linked heterodimer composed of the alpha and beta chains, noncovalently associated with the CD3 complex of polypeptides (gamma, delta, epsilon and zeta). In this report, two murine thymoma cell lines are described which synthesized incomplete TcR/CD3 complexes and expressed low levels of CD3 on their surface in the absence of the TcR chains. The partial TcR/CD3 complexes were composed primarily of the inherently metabolically stable CD3 gamma and epsilon subunits. These results were in contrast to previous studies, which suggested that synthesis of all of the component chains of the TcR/CD3 complex is required for the successful transport of any of the chains to the cell surface. The efficiency of transport of the partial TcR/CD3 complexes from the endoplasmic reticulum (ER) to medial Golgi in the two thymomas was similar to complete complexes. However, the transport of the incomplete receptors was impaired at some point between the medial Golgi and the plasma membrane. Taken together with previous studies, these results suggested that T cells have mechanisms to retain partial TcR/CD3 complexes intracellularly both in the ER and in an undefined post-ER compartment. However, the transport of low levels of partial TcR/CD3 complexes to the cell surface in some T cell lines implied that the retention mechanisms may not always be completely efficient. Cross-linking of the surface, partial TcR/CD3 complexes with anti-CD3 epsilon antibodies did not stimulate interleukin 2 (IL 2) production. It is possible, however, that the partial TcR/CD3 complexes have some function which is unrelated to the stimulation of IL 2 production.
The cell surface expression of the T cell receptor (TcR)/CD3 complex and, consequently, the functional competence of the cell is partly dependent on CD3 zeta. In its absence, a pentameric complex (TcR alpha/beta/CD3 gamma delta epsilon) is formed which is inefficiently transported to the cell surface. Reconstitution of CD3 zeta by transfection, in turn, restores the cell surface expression and function of the complex. Through the use of transfection experiments, we here provide direct evidence that the association of CD3 zeta 2 with the TcR/CD3 complex is dependent on the presence of both the TcR alpha and beta polypeptide chains. Despite wild-type levels of the CD3 zeta protein in a TcR alpha-negative mutant human T cell line, a complex was formed intracellularly which lacked CD3 zeta 2 and consisted of beta gamma delta epsilon and beta 2 gamma delta epsilon. Upon transfection of the mutant with a TcR alpha cDNA, a TcR/CD3 complex which contained CD3 zeta 2 was observed intracellularly. In contrast to the partial subcomplex on the cell surface of the untransfected cell line, the TcR/CD3 complex on the transfectant was functional as demonstrated by its ability to mobilize intracellular calcium after stimulation with a mitogenic CD3 epsilon-specific monoclonal antibody. Transient transfection studies performed in COS cell fibroblasts indicated that CD3 zeta 2 was not interacting with the TcR alpha protein alone, implying that a conformation provided by either the TcR alpha/beta heterodimer or the TcR alpha/beta/CD3 gamma delta epsilon complex was necessary for the association of CD3 zeta 2. Transfection studies performed in a TcR alpha/beta-negative murine T-T hybridoma confirmed the requirement of both the TcR alpha and beta proteins in CD3 zeta 2 binding. We conclude that the TcR alpha and beta chains harbor polypeptide sequences essential for the association of CD3 zeta 2 with the TcR/CD3 complex.
We previously reported the generation and characterization of a panel of CD4(+) autoreactive T cell clones that suppress development of autoimmune diabetes in non-obese diabetic (NOD) mice. We showed that the protective capacity of the T cell clones correlated with secretion of an activity that potently inhibits allogeneic mixed lymphocyte reaction (allo-MLR). In this report, we describe the biological characteristics of the allo-MLR inhibitory activity (MLR-IA, short for mixed lymphocyte reaction inhibitory activity) secreted by the protective T cell clone, NOD-5. MLR-IA has little effect on initiation of proliferation in an allo-MLR, but it potently inhibits the maintenance and amplification of the proliferative response. MLR-IA is also capable of inhibiting concanavalin A (Con A) stimulated splenic responder T cell proliferation. MLR-IA is reversible in vitro and is not restricted by MHC class I or II proteins. MLR-IA does not affect IL-2 receptor expression of responding T cells and has no effect on IL-2-dependent proliferation of CTLL-20 T cells. Partially purified MLR-IA inhibits IL-2 production in a primary allo-MLR, and decreases IFN-gamma production during secondary allo-MLR and Con A activation, whereas it enhances IL-4 production in both primary and secondary Con A activation. MLR-IA is not neutralized by combination of antibodies specific for transforming growth factor-beta, IL-10, tumor necrosis factor-alpha/beta or IFN-gamma, suggestive of a novel activity. MLR-IA is ammonium sulfate precipitable, sensitive to protease digestion and is destroyed by boiling, indicating that a protein moiety is part of its active structure. Our work suggests that a potentially novel immunoregulatory activity, capable of inhibiting T lymphocyte proliferation and IFN-gamma production, and stimulating IL-4 production, may regulate development of autoimmune diabetes in NOD mice.
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