Prevention of sexual acquisition of HIV in women requires a substantial increase in our knowledge about HIV-target cell availability and regulation in the female reproductive tract (FRT). In this study, we analyzed the phenotype and susceptibility to HIV-infection of CD4+ T cell in the endometrium (EM), endocervix (END) and ectocervix (ECT) of the FRT. We found that Th17 cells represent a major subset in FRT tissues analyzed, and that Th17 cells were the main CD4+ T cell population expressing CCR5 and CD90. In pre-menopausal women, CD4+ T cells and Th17 cells in particular, were significantly lower in EM relative to END and ECT. Th17 cells were elevated in EM from post-menopausal women relative to pre-menopausal tissues, but not changed in END and ECT. Susceptibility of CD4+ T cells to HIV infection measured as intracellular p24 was lowest in the EM and highest in ECT. Additionally, we found that Th17 cells co-expressing CCR5 and CD90 were the most susceptible to HIV-infection. Our results provide valuable information for designing preventive strategies directed at targeting highly susceptible target cells in the FRT.

The magnitude of the HIV epidemic in women requires urgent efforts to find effective preventive methods. Even though sex hormones have been described to influence HIV infection in epidemiological studies and regulate different immune responses that may affect HIV infection, the direct role that female sex hormones play in altering the susceptibility of target cells to HIV-infection is largely unknown. Here we evaluated the direct effect of 17-β-estradiol (E2) and ethinyl estradiol (EE) in HIV-infection of CD4+ T-cells and macrophages. Purified CD4+ T-cells and monocyte-derived macrophages were generated in vitro from peripheral blood and infected with R5 and X4 viruses. Treatment of CD4+ T-cells and macrophages with E2 prior to viral challenge reduced their susceptibility to HIV infection in a dose-dependent manner. Addition of E2 2 h after viral challenge however did not result in reduced infection. In contrast, EE reduced infection in macrophages to a lesser extent than E2 and had no effect on CD4+ T-cell infection. Reduction of HIV-infection induced by E2 in CD4+ T-cells was not due to CCR5 down-regulation, but was an entry-mediated mechanism since infection with VSV-G pseudotyped HIV was not modified by E2. In macrophages, despite the lack of an effect of E2 on CCR5 expression, E2–treatment reduced viral entry 2 h after challenge and increased MIP-1β secretion. These results demonstrate the direct effect of E2 on susceptibility of HIV-target cells to infection and indicate that inhibition of target cell infection involves cell-entry related mechanisms.

IL-15 is known to be critical in the homeostasis of Ag-specific memory CD8+ T cells following acute viral infection. However, little is known about the homeostatic requirements of memory CD8+ T cells during a latent viral infection. We have used the murine gammaherpesvirus-68 (MHV-68) model system to investigate whether IL-15 is necessary for the maintenance of memory CD8+ T cells during a latent viral infection. IL-15 is not essential either for the initial control of MHV-68 infection or for the maintenance of MHV-68-specific memory CD8+ T cells. Even at 140 days postinfection, the proportion of CD8+ T cells recognizing the MHV-68 epitopes were the same as in control mice. The maintenance of these memory CD8+ T cells was attributable to their ability to turn over in vivo, probably in response to the presence of low levels of Ag. IL-15−/− mice had a significantly higher turnover rate within the virus-specific memory CD8+ T cell population, which was the result of increased levels of viral gene expression rather than an increase in viral load. These cells did not accumulate in the spleens of the IL-15−/− mice due to an increased sensitivity to apoptosis as a result of decreased Bcl-2 levels. Intriguingly, memory CD8+ T cells from latently infected mice failed to undergo homeostatic proliferation in a naive secondary host. These data highlight fundamental differences between memory CD8+ T cells engaged in active immune surveillance of latent viral infections vs memory CD8+ T cells found after acute viral infections.

Gammaherpesviruses can persist in the host in the face of an aggressive immune response. T cells recognize Ags expressed in both the productive and latent phases of the virus life cycle, however little is known about their relative roles in the long-term control of the infection. In this study we used the murine gammaherpesvirus 68 model system to investigate the relative properties of CD8 T cells recognizing lytic and latent viral Ags. We report that the CD8 T cell response to lytic phase epitopes is maximal in the lungs of infected mice at ∼10 days postinfection, and is of progressively lesser magnitude in the mediastinal lymph nodes and spleen. In contrast, the CD8 T cell response to the latent M2 protein is maximal at ∼19 days postinfection and is most prominent in the spleen, then progressively less in the mediastinal lymph node and the lung. Latent and lytic Ag-specific CD8 T cells had markedly different cell surface phenotypes during chronic infection, with latent Ag-specific cells being predominantly CD62Lhigh or CD43 (1B11)high. Lytic Ag-specific T cells had significantly lower expression of these markers. Importantly, latent but not lytic Ag-specific T cells could kill target cells rapidly in vivo during the chronic infection. These two different sets of CD8 T cells also responded differentially to IL-7, a cytokine involved in T cell homeostasis and the maintenance of T cell memory. These data have important implications for our understanding of immunological control during chronic gammaherpesvirus infections.

The use of topical and oral adenosine derivatives in HIV prevention that need to be maintained in tissues and cells at effective levels to prevent transmission prompted us to ask whether estradiol could influence the regulation of catabolic nucleotidase enzymes in epithelial cells and fibroblasts from the upper and lower female reproductive tract (FRT) as these might affect cellular TFV-DP levels. Epithelial cells and fibroblasts were isolated from endometrium (EM), endocervix (CX) and ectocervix (ECX) tissues from hysterectomy patients, grown to confluence and treated with or without estradiol prior to RNA isolation. The expression of nucleotidase (NT) genes was measurable by RT-PCR in epithelial cells and fibroblasts from all FRT tissues. To determine if sex hormones have the potential to regulate NT, we evaluated NT gene expression and NT biological activity in FRT cells following hormone treatment. Estradiol increased expression of Cytosolic 5′-nucleotidase after 2 or 4 h in endometrial epithelial cells but not epithelial cells or fibroblasts from other sites. In studies using a modified 5′-Nucleotidase biological assay for nucleotidases, estradiol increased NT activity in epithelial cells and fibroblasts from the EM, CX and ECX at 24 and 48 h. In related studies, HUVEC primary cells and a HUVEC cell line were unresponsive to estradiol in terms of nucleotidase expression or biological activity. Our findings of an increase in nucleotidase expression and biological activity induced by estradiol do not directly assess changes in microbicide metabolism. However, they do suggest that when estradiol levels are elevated during the menstrual cycle, FRT epithelial cells and fibroblasts from the EM, CX and ECX have the potential to influence microbicide levels that could enhance protection of HIV-target cells (CD4+T cells, macrophages and dendritic cells) throughout the FRT.