Delayed type hypersensitivity (DTH) is an antigen-specific mononuclear leukocyte response to an antigen to which the host has previously been sensitized. In this reaction, lymphocytes enter the tissues by passing through the tightly apposed intercellular junctions of the endothelial cells (EC) lining postcapillary venules. There is presently no good in vitro model of DTH. I have developed a quantitative assay of transendothelial migration (TEM) across EC monolayers. We will use this assay to identify the conditions and CAMs necessary for TEM by lymphocytes and establish an in vitro model of DTH. We will use this system to study cell mediated immunity in tuberculosis (TB) and AIDS. We will first test the hypothesis that activation of T cells is necessary for the initial emigration across resting EC. We will activate T cells with alloantigen or by crosslinking CD3 and T cell receptor and determine whether these cells now undergo efficient TEM in our assay. We will next test the hypothesis that in DTH, EC activated by the proper cytokines will be induced to express CAMs that promote TEM of resting, non antigen- specific memory T cells, accounting for the fact that >99% of the T cells in a DTH lesion are not specific for the inciting antigen. We will next identify the CAMs employed by both T cell and EC under both these migration conditions using specific monoclonal antibodies (mAb) to block TEM. My colleagues in this Program have demonstrated that intradermal instillation of interleukin 2 (IL-2) leads to an antigen-independent influx of mononuclear cells otherwise identical to DTH. We will study the cellular basis of this phenomenon by implanting IL-2 responsive cells under the EC monolayer and testing their ability to stimulate TEM when pulsed with IL-2. We will use this system to investigate the reason that AIDS patients with low (<200) CD4 counts no longer respond to IL-2 treatment. Finally, in a model of DTH, we will place PPD-responsive T cells or T cell clones from TB patients proliferating in response to PPD, under the EC and determine whether the cytokines generated in this antigen-specific response are sufficient to stimulate TEM of mononuclear cells in a manner resembling DTH chronologically and histologically. T cells from TB patients and PPD+ volunteers will be tested for TEM. Migrated and nonmigrated cells will be recovered and tested for proliferation in response to PPD to determine whether migrating cells are enriched for memory T cells. Hypotheses made about AIDS T cells in the IL-2 system (above) will be tested in this more physiologic model of DTH.
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