Impact of Mucosal Inflammation on Cervical Human Immunodeficiency Virus (HIV-1)-Specific CD8 T-Cell Responses in the Female Genital Tract during Chronic HIV Infection▿

The female genital tract is the major route of heterosexual human immunodeficiency virus (HIV) acquisition and transmission. Here, we investigated whether HIV-specific CD8 T-cell-mediated immune responses could be detected in the genital mucosa of chronically HIV-infected women and whether these were associated with either local mucosal HIV shedding or local immune factors. We found that CD8+ T-cell gamma interferon responses to Gag were detectable at the cervix of HIV-infected women but that the magnitude of genital responses did not correlate with those similarly detected in blood. This indicates that ex vivo HIV responses in one compartment may not be predictive of those in the other. We found that increased genital tumor necrosis factor alpha (TNF-α) and interleukin-10 (IL-10) levels correlated significantly with levels of Gag-specific CD8+ T cells at the cervix. Women who were detectably shedding virus in the genital tract had significantly increased cervical levels of TNF-α, IL-1β, IL-6, and IL-8 compared to women who were not detectably shedding virus. We were, however, unable to detect any association between the magnitude of cervical HIV-specific responses and mucosal HIV shedding. Our results support the hypothesis that proinflammatory cytokines in the female genital tract may promote HIV replication and shedding. In addition, we further show that inflammatory cytokines are associated with increased levels of HIV-specific CD8 effector cells at the genital mucosa but that these were not able to control genital HIV shedding

Stability and transport of cervical cytobrushes for isolation of mononuclear cells from the female genital tract

Cervical cytobrushing, biopsy, or lavages have previously been used to collect mononuclear cells from the female genital tract. Compared with blood, obtaining cells from the female genital tract is more invasive and generally yields few cells for subsequent immune studies. Because of the value of including mucosal sampling in HIV vaccine trials, standardisation of methods for collection, processing, and analysis of immunity from cells derived from the female genital tract is important. The aim of this study was to assess the effect of transport conditions on the viability, recovery and antigenic responsiveness of cervical T cells. This was investigated in cervical cytobrush specimens collected from 215 chronically HIV-infected women. Cytobrushes were either processed immediately, after cryopreservation, or after 24 h at 37 °C, 4 °C or room temperature. CD3+ T cell numbers were quantified using Guava automated cell counting. Viability was assessed using Trypan and Annexin/PI staining. Intracellular cytokine staining was used to evaluate IFN-γ responses to PMA, PHA and CEF peptides in cytobrush-derived T cells ex vivo and after delayed processing. In vitro polyclonal expansion of thawed cervical lymphocytes was conducted for 14 days in the presence of anti-CD3 and IL-2. We found that CD3+ T cell recovery and viability was similar in cytobrushes processed immediately or after 24 h irrespective of the conditions at which they were maintained. Fifty percent of the CD3+ T cells could be recovered after cryopreservation of cytobrushes and these could be polyclonally expanded in half of the cryopreserved samples. IFN-γ production following mitogenic stimulation was similar in ex vivo and delayed processing cytobrushes. Maintaining cytobrushes at 37 °C prior to processing significantly improved the detection of CEF-specific T cell responses compared to ex vivo. We conclude that cervical cytobrush-derived T cells are robust and can preserve their viability, phenotype and function over 24 h of mock transport