The major goal of this project is to further our understanding of the role of regulatory T cells in preventing autoimmunity. Organ-specific autoimmune disease develops in certain strains of mice following thymectomy on day 3 of life (d3TX). This disease process is mediated by CD4+ T cells and can be transferred to immunodeficient mice by CD4+ T cells. CD4+ T cells from euthymic animals can inhibit the development of disease when transferred into the d3Tx animals before 14 days of age. It is therefore likely that regulatory T cells whose development is prevented by thymectomy are responsible for preventing autoimmunity throughout the life span of the animal. We have focused our efforts on the characterization of the autoimmune effector cells responsible for the pathogenesis of the disease and on the suppressor/regulatory cells which control the activity of disease inducing effector cells. It has been demonstrated recently that the regulatory CD4+ T cell that prevent disease co-express CD25. We have further characterized the population of CD4+CD25+ immunoregulatory cells and demonstrated that they can suppress not only the induction of disease post-thymectomy, but can also suppress disease induced by cloned autoantigen specific effector cells. Furthermore, the CD4+CD25+ cells appear to be members of unique lineage of regulatory cells, as the induction of CD25 expression on a mono- specific population of T cells derived from TCR-transgenic SCID mice did not result in suppression of post-thymectomy autoimmunity. In addition, the TCR transgenic SCID mice were highly susceptible to autoimmune disease induced by cloned lines of autoantigen-specific effectors, while normal mice were relatively resistant. The capacity of the cloned line to transfer disease to nu/nu recipients could be inhibited by normal spleen cell populations containing CD4+CD25+ and by purified CD4+ CD25+ T cells. One of the major problems in the analysis of the pathogenesis of any model of spontaneous organ specific autoimmunity is the identification of the initiating antigen. We have defined the target antigen responsible for the pathogenesis of autoimmune gastritis in d3Tx BALB/c mouse as the proton pump, H/K ATPase, of the gastric parietal cell. Freshly explanted gastric LN cells from d3Tx mice react significantly to the H/K ATPase a-chain, but only marginally to the b-chain. Two H/K ATPAse reactive T cell lines were derived from the gastric LN of d3Tx mice. Both were CD4+, TCR a/b+ and recognized two distinct peptides derived from the a-chain in association with I-Ad. One cell line secreted Th1 and the other Th2 cytokines, but both were equally potent inducers of gastritis with distinct profiles of cellular infilration in nu/nu recipent animals. Neither of the cell lines induced disease in normal BALB/C animals and transfer of disease to nu/nu recipients was blocked by cotransfer of normal BALB/c spleen cells containing CD25+ cells. Our demonstration that the pathogenic activity in vivo of both activated Th1/Th2 lines can be abrogated by cotransfer of CD4+CD25+ cells, strongly suggests that the regulatory T cell population may have a therapeutic role in other models of established autoimmunity. Although these studies offer strong evidence for the existence of a population of CD4+CD25+immmunoregulatory T cells in vivo, the activity of these suppressor populations has been measured in systems that require weeks to months of assessment of disease activity. It has therefore proven difficult to determine their mechanism of action, their antigen specificity, or their cellular targets. To analyze the mechanism of action of the CD4+CD25+ cells, we have established an in vitro model system that mimics the function of these cells in vivo. Purified CD4+CD25+ cells failed to proliferate following stimulation with IL-2 alone of through the TCR. When co-cultured with CD4+CD25- cells, the CD4+ CD25+ cells markedly suppressed proliferation by specifically inhibiting the production of IL-2. The inhibition was not cytokine mediated, was dependent on cell contact between the regulatory cells and the responders, and required activation of the suppressors via the TCR. Inhibition could be overcome by the addition to the cultures of IL-2 or anti-CD28, suggesting that the CD4+CD25+ cells may function by blocking the delivery of a costimulatory signal. Induction of CD25 expression on CD25- negative T cells in vitro or in vivo did not result in the generation of suppressor activity. Collectively, these studies support the concept that CD4+CD25+ T cells in normal mice may represent a distinct lineage of professional suppressor cells.
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