CD8+ T cells exert antiretroviral activity through multiple mechanisms including cytotoxicity, inhibition of HIV- 1 transcription, and blockade of HIV-1 entry into cells. Nevertheless, HIV-1 replication continues inexorably and is concentrated in lymphoid tissues. The reasons why CD8+ T cells are unable to fully suppress HIV-1 replication are unknown. In the previous grant cycle, we assessed HIV-1-specific CD8+ T cell responses in inguinal lymph nodes and found no evidence of a deficiency to explain ongoing virus replication. The major goals of this proposal are to expand these studies to gut-associated lymphoid tissue (GALT), which harbors the majority of the body's lymphocytes, and to evaluate several hypotheses that explain how HIV-1- producing cells may evade CD8+ T cell antiretroviral mechanisms.
In specific aim 1, we will assess the relationship between PBMC and lymphoid tissue HIV-1-specific CD8+ T cell responses using assays for interferon (IFN)-gamma production, tetramer staining, cytolysis, and apoptosis in peripheral blood, lymph node and sigmoid colon biopsies. IFN-gamma responses in subjects who donate both lymph node and GALT specimens will be compared to determine whether there are differences between lymphoid compartments.
In specific aim 2, the hypothesis that germinal centers are an immune privileged site will be tested using immunohistochemical and immunofluorescent staining techniques to quantify and compare virus-producing cells, CD8+ cell antiretroviral effector proteins, and HIV-1-specific CD8+ cells in follicular and extrafollicular regions of lymphoid tissue sections.
In specific aim 3, the hypotheses that HIV-1-producing cells evade CD8+ cell elimination by downregulation of MHC class I (MHC-I), through Fas-Fas ligand (FasL) killing, or through transforming growth factor (TGF)-beta1-induced immune suppression will be evaluated using immunofluorescent techniques to detect these cells in lymphoid tissue sections. The ability of infected CD4+ T cells to lyse CTL cell lines by Fas-FasL killing in vitro will be tested as well. Lastly, the relationship between CD8+ T cell antiretroviral mechanisms and plasma HIV-1 RNA concentration will be assessed in a mixed-effects model taking into consideration selected virologic and host factors known to be associated with disease progression (specific aim 4). These studies could provide important insight into the immunopathogenesis of HIV-1 infection and suggest alternative targets for immune therapies.
