The investigator hypothesize that for stem cell gene therapy to be effective for the generation of newly differentiated T cells in AIDS that a functional thymic environment must be present. In our studies as well as from others, thymic function is impaired in AIDS. In order to study the mechanism(s) underlying HIV impairment of thymopoiesis, our group has developed a novel in vitro model of CD34+ stem cells and cultured thymic epithelia fragment cocultures to evaluate the effect of HIV on thymopoiesis. In preliminary studies, HIV impaired production of CD4+ thymocytes as well as earlier immature cells. HIV could disrupt thymopoiesis by a variety of mechanisms, including infection of thymic epithelia, stem cells and/or thymocytes and disruption of the thymic matrix microenvironment. The specific goals of this proposal will be to elucidate the mechanisms by which HIV disrupts thymopoiesis. First, to determine whether HIV infection of CD34+ stem cells alters thymopoiesis, stem cells will be HIV-infected with pseudotyped HIV (env deficient; provided by Dr. Akkina) to achieve high efficiency infection prior to coculture in the thymic organ culture. Secondly, the cultured thymic epithelia will be HIV-infected prior to the coculture with normal stem cells. By examining thymocyte maturation in this in vitro model of HIV infection of the stem cell-thymic epithelia paradigm, it can be determined whether decreased T cell maturation in AIDS is due to HIV- induced thymic dysfunction and/or to stem cell impairment. In addition, anti-retroviral drugs, such as zidovudine, will be added after the model has been infected with HIV to analyze whether thymopoiesis can be restored. Thirdly, the thymopoietic potential of CD34+ stem cells transduced with combinations of anti-CCR-5 ribozymes and other anti-HIV genes (provided by Dr. Rossi) will be examined in the thymic organ culture model to determine whether transduction alters lineage commitment and confers protection of thymopoiesis in the HIV-infected thymus. Fourth, since HIV infection of the thymus has been reported to decrease thymic hormones, such as thymosin-alpha, replenishment of thymosin will be performed to determine whether thymopoiesis of the HIV-infected thymus can be restored. Thymosin-alpha in preliminary studies was observed to enhance thymopoiesis by appearance of mature CD4+ T cells. These studies, which are complimentary to the human-SCID model proposed by Dr. Akkina, will provide insight into the mechanism(s) underlying HIV-induced thymic dysfunction.
