Presence of CD8+ T Cells in the Ectocervical Mucosa Correlateswith Genital Viral Shedding in HIV-Infected Women despite aLow Prevalence of HIV RNAâ•fiExpressing Cells in the Tissue

The female genital tract is a portal of entry for sexual HIV transmission and a possible viral reservoir. In this study, the ectocervical CD8+ T cell distribution was explored in situ and was related to expression of CD3 and HLA-DR and presence of HIV RNA. For this purpose, ectocervical tissue samples and genital secretions were collected from HIV-seropositive (HIV+) Kenyan female sex workers (FSWs) (n = 20), HIV-seronegative (HIV−) FSWs (n = 17), and HIV− lower-risk women (n = 21). Cell markers were assessed by in situ staining and by quantitative PCR. HIV RNA expression in tissue was analyzed by in situ hybridization, and viral shedding was assessed by quantitative PCR. The HIV+FSW group had a higher amount of total cells and CD8+, CD3+, and HLA-DR+ cells compared with the HIV−FSW group and HIV− lower-risk women. The majority of CD8+ cells were CD3+ T cells, and the numbers of CD8+ cells correlated significantly with plasma and cervical viral load. HIV RNA expression in situ was found in 4 of the 20 HIV+FSW women but did not correlate with cervical or plasma viral load. Thus, the HIV+ women displayed high numbers of CD8+, CD3+, and HLA-DR+ cells, as well as a limited number of HIV RNA+ cells, in their ectocervical mucosa; hence, this localization cannot be neglected as a Copyright © 2014 by The American Association of Immunologists, Inc. Address correspondence and reprint requests to Dr. Annelie Tjernlund, Unit of Infectious Diseases, Department of Medicine Solna, Center for Molecular Medicine, Karolinska Institutet, CMM L8:01, Karolinska University Hospital, S-17176 Stockholm, Sweden. [email protected]. Disclosures The authors have no financial conflicts of interest. NIH Public Access Author Manuscript J Immunol. Author manuscript; available in PMC 2014 July 15. Published in final edited form as: J Immunol. 2014 April 15; 192(8): 3947–3957. doi:10.4049/jimmunol.1302826. N IH -P A A uhor M anscript N IH -P A A uhor M anscript N IH -P A A uhor M anscript potential viral reservoir. The elevated levels of CD8+ T cells may play a role in the immunopathogenesis of HIV in the female genital tract. A broad range of immune cells in the epithelial and sub-mucosal layer of the female genital tract mucosa, as well as the epithelial cells themselves, have the capacity to respond rapidly to pathogen exposure. Because the distribution and function of various immune cells differ at the systemic and mucosal level, it is important to study the immune response in both of these compartments. The T cell responses, as well as influx of APCs and release of inflammatory cytokines at genital mucosal sites, have the capacity to affect viral replication and systemic spread of the infection. Cervical T cells are predominantly Ag experienced and highly differentiated, with effector memory T cells being the most predominant subset (1). However, although HIV-specific T cells are present in the cervix (2), they are suggested to be largely monofunctional and, thereby, may have limited functional antiviral capacity (3– 5). Unfortunately, exposure to seminal fluid and to various pathogens, including HIV, can cause mucosal inflammation and, thereby, also may increase the number of target cells for HIV and promote local viral replication (6–8). We recently documented a higher expression of immune activation markers, in situ, by intact ectocervical tissue samples from HIVinfected women. In the same study, it also was observed that, although the blood CD4+ T cell numbers were lower, the ectocervical CD4+ T cell numbers were comparable to those of healthy uninfected control women (9). Sexual HIV transmission is correlated with plasma viral RNA levels and likely occurs through mucosal contact with the virus in genital secretions (10). Furthermore, genital HIV RNA levels often correspond to plasma HIV RNA levels and can predict transmission risk in some cases (11–13). However, some studies (14–17) reported only a modest correlation between systemic and local virus levels, and this discrepancy may be explained, in part, by mucosal immune activation resulting in increased local viral replication without affecting other anatomical sites. Several conditions can increase the risk for genital viral shedding, including genital infections, general mucosal inflammation, vaginal douching, hormonal contraceptive use, and pregnancy (18–28). Genital viral shedding also may be intermittent and can be detected even though plasma viral load is low or undetectable (29, 30). The local T cell response against HIV and factors involved in genital viral shedding at the female genital tract have been investigated extensively, primarily by assessing cervicovaginal secretions (CVSs) and cytobrush-derived cervical cells (1, 4, 31, 32). Studies (5, 9, 33–36) at the single-cell level discriminating the epithelial and submucosal distribution of immune markers in intact cervical tissue of HIV-infected women have been limited in sample size and have been lacking information about corresponding plasma or cervical viral shedding. Using in situ techniques assessing snap-frozen human tissue samples, it is possible to visualize the exact distribution of local T cells and HIV RNA expression. Therefore, in the current study we investigated the in situ distribution and quantity of CD8-, CD3-, or HLA-DR–expressing cells, as well as the presence of HIV RNA+ cells. These results were correlated with plasma and cervical viral load. Gibbs et al. Page 2 J Immunol. Author manuscript; available in PMC 2014 July 15. N IH -P A A uhor M anscript N IH -P A A uhor M anscript N IH -P A A uhor M anscript Materials and Methods