HIV-1-induced pathogenesis is manifested by depletion of the pool of peripheral T cells and (in children) by depletion of T cells within the thymic gland, the primary organ for T cell generation. Studies in children and in adults have demonstrated that following administration of HAART, some restoration of peripheral and intrathymic T cells could be achieved; thus indicating that there is a window of opportunity that allows reconstitution of the immune system even in the absence of full eradication of the virus. However, it is largely unknown, what cellular factors can contribute to the restoration of T cells in immune compromised individuals and how these factors are affected by HIV infection. Some of the vaccine candidates that are under development incorporate human cytokines, such as IL-2 and GM-CSF. In addition, recent studies demonstrated that IL-7 and IL-15 cytokines play an indispensable role in generating cytotoxic memory T cells and therefore may be included in the vaccination regiments in the close future. Although the benefits of these cytokines for enhancing immunogenicity have been shown in vitro and in vivo in animal models, it is not known how these and other cellular factors (cytokines, chemokines, growth factors) affect immune reconstitution following HIV infection and whether introduction of these cytokines can cause any adverse reactions in immunocompromised host. Our study is designed to identify the host cell genes that are important for immune reconstitution and how the expression of these genes is affected by HIV infection. By designing the in vivo models that mimic the scenario of HIV-induced T cell depletion, we will create a framework that will allow evaluation of the efficacy of restoration of T cells following administration of anti-HIV vaccines. In addition, it is possible that HIV-1 infection is inducing immunosuppressive cytokines in the thymic tissues thus affecting the intrinsic ability of the thymus to regenerate. This question is especially important since a combination of thymopoiesis-inducing cytokines with blocking of immunosuppressive cytokines may provide a possible therapeutic approach for restoration of full immune function in patients in addition to or as a replacement for HAART therapy. (1) Goals of the project: - To identify the host cell genes that are important for immune reconstitution. - To study how HIV infection affects the expression of these genes regulating immune reconstitution. - 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 death in both CD4 and CD8 cells. - To study in situ expression of chemokines and cytokines in normal and HIV-1-infected human thymic tissues. - To apply the knowledge gained towards development of anti-viral therapy that target both viral and cellular genes. (2) Experimental approach: - SCIDhu mice were reconstituted with fetal human thymus and liver tissues and were infected intrathymically with primary isolates of HIV. Frozen sections were subjected to immunohistochemistry with antibodies against HIV-1 p24, HIV-1 co-receptors, against CXCR4 ligand, SDF1 and by TUNEL assay for in situ detection of apoptosis. - FACS analysis of mouse thymuses and of human thymuses from SCIDhu mice was performed to evaluate the effects of dexamethasone and estradiol administration on various thymocyte subsets. - FACS analysis and real-time TREC-PCR analysis was used to evaluate the effects of thymus regeneration on repopulation of peripheral naive T cells. - RT-PCR analysis was used to determine the expression of IL-7 and of other cytokines and chemokines in regenerating thymic tissues both from mice and in SCIDhu mouse models. - DNA array analysis was initiated to search for new gene expressed during atrophy and at the regenerating stage. (3) Major Findings: During this year, the following assays and models have been established: in vivo model of estradiol-induced thymus atrophy, in vivo model of dexamethasone-induced thymus atrophy; model of transient atrophy in human thymus using SCID mice reconstituted with fetal human thymic and liver tissues, TREC-PCR analysis, analysis of gene expression by DNA arrays, IL-7 RT-PCR, immunohistochemistry analysis of HIV-infected of thymic tissues. The following goals of the study were achieved: - Immunohistochemistry analysis demonstrated increased levels of SDF-1 chemokine and increased levels of apoptosis in the medulla of HIV-infected human thymuses from SCIDhu mouse model. - All the essential assays to evaluate thymic atrophy/reconstitution and generation of naive cells in vivo were established - Transient thymic atrophy in mice was induced by a single injection with steroids or with estrogens in mice and was followed by full reconstitution of thymic cellularity. - Transient increase in the numbers of naive CD8+ T cells was observed following reconstitution of the thymus. - Thymus ablating treatment induced IL-7 expression in the stromal component of mouse thymus. - Importantly, IL-7 was also induced in human thymic tissues in SCIDhu mouse model at the initiation of thymic reconstitution following steroid-induced atrophy. - Data obtained with DNA array analysis identified high levels of expression of several genes in regenerating thymic tissues that have not been previously described.
