Cellular genes as targets for anti-HIV Therapies

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Summary: (1) Goals of project: - To identify cellular genes required for optimal HIV-1 infection, or involved in HIV-1 pathogenesis. - To study the mechanism by which intrathymic HIV-1 infection affects the development of different T cell subsets. - To understand how HIV-1 infection leads to high levels of programmed cell cell in both CD4 and CD8 cells. - To apply the knowledge gained towards development of anti-viral therapy which target both viral and cellular genes. - To study in situ expression of chemokines and cytokines in normal and HIV-1-infected human thymic tissues. (2) Experimental approach: - Scid/Hu mice were infeected intrathymically with primary isolate of HIV. Frozen sections were stained by immunohistochemistry with antibodies against HIV-1 p24, HIV-1 coreceptors, and against the CXCR4 ligand SDF1. - Rabbit Igs specific for the CXCR4 and CCR5 extracellular domains were produced and tested for their ability to stain primary human cells and to block viral fusion and infection. - Study coreceptor expression and function on human thymocyte subpoulations and correlate coreceptor expression with susceptibility to infection with different hiv strains. - Develop imuunohistochemisry staining of thymic tissues to determine the sites and cellular origin of various chemokines and cytokines that may affect HIV infections and potential thymic regeneration in infected children on HAART. (3) Major Findings: - Studies with human thymocyte subpopulations from infants, revealed very high expression of the CXCR4 coreceptor on immature thymocytes, including CD34+ intrathymic precursors. Thymocytes responded to the chemokine SDF1 by chemotaxis and Ca++ mobilization. They fused readily with T-tropic HIV envelopes and the fusion was blocked by our rabbit anti-CXCR4 IgG. In addition, we have evidence that another chemokine receptor (I-309R) is expressed in human thymocytes and provides co-recptor function for infection with T-tropic and M-trpic strains. These data explain the exquisite sensitivity of neonatal thymuses to infection with T-tropic HIV strains that often result in sever thymic depletion. - IHC staining demonstrated that SDF-1 is highly expressed in normal thymus in the stromal cells surrounding Hassels' bodies in the medulla. SDF1/CXCR4 interactions play a pivotal role in thymopoiesis, i.e., removal of apoptotic thymocytes by thymic dendritic cells. In HIV-1 infected thymuses, the Hassels' bodies were significantly enlarged and constituted > 50% of the medulla. The staining with anti-SDF1 mAb was eventually reduced. These changes may represent the pathologic process leading to eventual destruction of the thymic architecture. Manuscript submitted - New findings suggest that, in addition to the chemokine receptors CXCR4 and CCR5, infection of human thymocytes can occur via the CCR8 chemokine receptor. Such infection can be blocked by the CCR8 ligand I309. This is the first evidence for in vivo usage of chemokine receptor other than CCR5 and CXCR4. Manuscript published. - Preliminary data suggest that IL7 is produced by epithelial cells in the thymus. Apoptosis inducing regiments (i.e., in vivo treatment with corticosteroids) upregulate IL7 and re-start thymopoiesis.