Human immunodeficiency virus (HIV) infection leads to a decline in CD4+ T cell numbers and is characterized by an impaired cellular immune function leading to development of AIDS. However, a functional defect in the cellular immune system is detectable early in HIV infection, at a time when numbers of CD4+ T cells are within the normal range. Also, in many pediatric AIDS cases, the number of CD4+ T cells in the peripheral blood remains within pediatric normal limits even when cellular function is severely decreased. The overall objective of this proposal is to elucidate which immature and mature thymocyte subsets express HIV, to study which cytokines are involved in augmenting HIV production and to study differences in function of HIV-infected versus non-infected mature thymocytes with the hope of gaining a better understanding of HIV pathogenesis in Pediatric HIV infection.
Our specific aims are intended to test our hypothesis, that HIV infection of the developing immune system results in an export to the periphery of mature thymocytes which are HIV- infected, but functionally deficient. We have shown that mature and immature thymocytes can be infected with clinical isolates of HIV-1 and that virus expression is enhanced by cytokines normally present in the thymus. Therefore an HIV-infected thymus may export a constant supply of HIV positive cells to the periphery as long as it is able to generate T cells. The following is an outline of our experimental strategy. First, we will investigate which subsets are infectable by HIV by performing in- vitro infection followed by isolation of phenotypically different thymocyte subsets by cell sorting. We will then analyze HIV infection of these subsets by PCR and use Elisa for viral core antigen (p24) to determine which subset is expressing virus. In addition, we will examine which cytokines influence the production of HIV by these thymocyte subsets. These experiments will determine whether all CD4+ cells are infectable including all CD4+CD8+ cells, whether infected CD4+ CD8+ thymocytes give rise to infected single CD8+ thymocytes and which cytokines are involved. We will also address the question if low concentrations of CD4 immunoadhesin (CD-IgG) can prevent infection of immature and mature thymocytes. Next, we will compare functions of HIV-infected and non- infected mature """"""""bright"""""""" CD3+ thymocytes since we expect that even though maturation in-vitro is taking place the HIV-infected thymocytes may be functionally deficient. Functional assays will include proliferation in response to anti-CD3, cytokine production, T cell help for B cells and cytotoxciity (only if CD4+CD8+ give rise to HIV-infected CD8+ thymocytes). Thereafter, we will determine if selected non-thymocyte populations can be infected with HIV. These studies will be done with cytopathic and non- cytopathic virus isolates. The results from these studies will contribute greatly to understanding the impact of HIV on T cell development in children.
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