Initiation of Antiretroviral Therapy Differentially Influences Genital and Systemic Immune Activation in HIV-Infected Women.

Antiretroviral therapy (ART) has significantly improved the quality of life of HIV-infected individuals: reducing plasma viremia, restoring CD4+ T cell numbers, and correcting imbalances in blood memory T cell subsets. While ART improves immune correlates at mucosal sites, including the lower female genital tract (FGT), ART initiation has been associated with reactivation of common FGT infections. We investigated the effect of ART on immune activation and inflammation in the genital tract. We measured blood and genital T cell activation, proliferation, and immunosenescence (CD38, HLADR, Ki67, CD127, and CD57), and cytokine levels in women on ART for ∼7 years (cross-sectional analysis) or initiating ART (immediately before and 1 month after). Effector memory T cells predominated in blood and FGT during chronic infection, irrespective of ART status. In women initiating ART, 1 month was insufficient for T cell reconstitution, or alterations in T cell subset distribution, despite both plasma and genital viral loads decreasing to undetectable levels in most participants. Initiating ART was accompanied by a decline in plasma IP-10 that correlated with decreased blood CD38 expression in blood (p = .0204) but not in the FGT. The reduction in plasma (but not genital) cytokine levels due to ART initiation was dependent on their concentrations before treatment. While T cell activation decreased significantly in blood (CD4: p = .032; CD8: p = .0137), activation levels remained similar in the genital tract despite 1 month of treatment. Overall, the decrease in cellular activation and inflammation seen in blood with ART initiation was not evident in the FGT.

CCR5 expression, haplotype and immune activation in protection from infection in HIV-exposed uninfected individuals in HIV-serodiscordant relationships.

Several host factors have been implicated in resistance to HIV infection in individuals who remain HIV-seronegative despite exposure. In a cohort of HIV-serodiscordant heterosexual couples, we investigated interactions between systemic inflammation and T-cell activation in resistance to HIV infection. Males and females in stable long-term relationships with either HIV-infected or uninfected partners were recruited, blood T-cell activation (CD38, HLA-DR, CCR5 and Ki67) and plasma cytokine concentrations were evaluated. The HIV-negative exposed individuals had significantly lower frequencies of CCR5+ CD4+ and CD8+ T cells than unexposed individuals. Mean fluorescence intensity of CCR5 expression on CD4+ T cells was significantly lower in HIV-negative exposed than unexposed individuals. Protective CCR5 haplotypes (HHA/HHF*2, HHF*2/HHF*2, HHC/HHF*2, HHA/HHA, HHA/HHC and HHA/HHD) tended to be over-represented in exposed compared with unexposed individuals (38% versus 28%, P = 0·58) whereas deleterious genotypes (HHC/HHD, HHC/HHE, HHD/HHE, HHD/HHD and HHE/HHE) were under-represented (26% versus 44%; P = 0·16). Plasma concentrations of interleukin-2 (P = 0·02), interferon-γ (P = 0·05) and granulocyte-macrophage colony-stimulating factor (P = 0·006) were lower in exposed compared with unexposed individuals. Activation marker expression and systemic cytokine concentrations were not influenced by gender. We conclude that the dominant signature of resistance to HIV infection in this cohort of exposed but uninfected individuals was lower T-cell CCR5 expression and plasma cytokine concentrations.

Female genital tract inflammation, HIV co-infection and persistent mucosal Human Papillomavirus (HPV) infections.

BACKGROUND: Persistent genital infections with high-risk HPV types increase risk of cervical disease and cancer. Since genital inflammation increases HIV acquisition risk and cancer progression, we evaluated whether HPV infection induces cytokine expression in the reproductive tract. METHODS: Genital cytokines concentrations were measured in 93 HIV-infected and 72 uninfected women. HPV typing was done by Roche Linear array. Persistence and clearance of HPV were evaluated using longitudinal data. RESULTS: Infection with HPV did not influence genital cytokine concentrations. In contrast, HIV-infected women had higher IL-1α, IL-6, IL-8, IP-10, MCP-1 and G-CSF concentrations compared to HIV-uninfected women, and HPV-infections that were more prevalent, persistent and multi-type. CONCLUSION: HPV did not influence inflammatory cytokine levels in the genital tract, although immune suppression may favor persistence.

Optimizing viable leukocyte sampling from the female genital tract for clinical trials: an international multi-site study.

BACKGROUND: Functional analysis of mononuclear leukocytes in the female genital mucosa is essential for understanding the immunologic effects of HIV vaccines and microbicides at the site of HIV exposure. However, the best female genital tract sampling technique is unclear. METHODS AND FINDINGS: We enrolled women from four sites in Africa and the US to compare three genital leukocyte sampling methods: cervicovaginal lavages (CVL), endocervical cytobrushes, and ectocervical biopsies. Absolute yields of mononuclear leukocyte subpopulations were determined by flow cytometric bead-based cell counting. Of the non-invasive sampling types, two combined sequential cytobrushes yielded significantly more viable mononuclear leukocytes than a CVL (p<0.0001). In a subsequent comparison, two cytobrushes yielded as many leukocytes (∼ 10,000) as one biopsy, with macrophages/monocytes being more prominent in cytobrushes and T lymphocytes in biopsies. Sample yields were consistent between sites. In a subgroup analysis, we observed significant reproducibility between replicate same-day biopsies (r = 0.89, p = 0.0123). Visible red blood cells in cytobrushes increased leukocyte yields more than three-fold (p = 0.0078), but did not change their subpopulation profile, indicating that these leukocytes were still largely derived from the mucosa and not peripheral blood. We also confirmed that many CD4(+) T cells in the female genital tract express the α4ß7 integrin, an HIV envelope-binding mucosal homing receptor. CONCLUSIONS: CVL sampling recovered the lowest number of viable mononuclear leukocytes. Two cervical cytobrushes yielded comparable total numbers of viable leukocytes to one biopsy, but cytobrushes and biopsies were biased toward macrophages and T lymphocytes, respectively. Our study also established the feasibility of obtaining consistent flow cytometric analyses of isolated genital cells from four study sites in the US and Africa. These data represent an important step towards implementing mucosal cell sampling in international clinical trials of HIV prevention.

Persistence of genital tract T cell responses in HIV-infected women on highly active antiretroviral therapy.

Initiation of highly active antiretroviral therapy (HAART) for HIV-infected individuals is associated with control of viremia, improved CD4 counts, and declining systemic HIV-specific immune responses. While HAART effectively reduces plasma viremia, it remains unclear how effectively antiretroviral drugs reach mucosal surfaces, such as those of the genital tract. The aim of this study was to determine the effect of HAART on genital tract CD4 T cell reconstitution, HIV shedding, and HIV-specific T cell responses. Cervical cytobrush and blood specimens were obtained from 35 HIV-infected, HAART-naïve women and 27 women on HAART in order to investigate HIV Gag-specific T cell responses by intracellular gamma interferon (IFN-γ) staining. Interleukin 1ß (IL-1ß), IL-6, and IL-8 concentrations were measured by enzyme-linked immunosorbent assays (ELISA). We show that for HIV-infected women, HAART is associated with significantly improved CD4 T cell counts both in blood and at the cervix. While HAART effectively suppressed both blood and cervical viremia, HIV-specific CD8 T cell responses in blood were lost, while those at the cervix were preserved.

Impact of human immunodeficiency virus 1 infection and inflammation on the composition and yield of cervical mononuclear cells in the female genital tract.

Cervical cytobrush sampling is a relatively non-invasive method for obtaining mucosal cells from the female genital tract. To define mucosal immune cells sampled by cervical cytobrushing and to validate this approach for local immunity studies, we investigated the impact of human immunodeficiency virus (HIV) status and inflammation on the yield and composition of cervical cytobrush specimens. Cervical cytobrush samples were obtained from 89 chronically HIV-infected and 46 HIV-negative women. The HIV-infected women had significantly higher yields of CD3(+), CD45(+), CD19(+), CD14(+), Langerin(+) and CD24(+) cells than the uninfected women. While cytobrush-derived T cells from uninfected women were predominantly CD4(+) (4.2 CD4 : 1 CD8), CD8(+) T cells were predominant in HIV-infected women (0.6 CD4 : 1 CD8). The majority of CD4(+) and CD8(+) T cells from HIV-infected and uninfected women were of the effector memory (CD45RA(-) CCR7(-) CD27(-)) phenotype. HIV-infected women had significantly elevated levels of interleukin (IL)-1beta, IL-6 and IL-8 in cervical supernatants compared with uninfected women. We observed a significant positive correlation between T-cell counts and IL-1beta, tumour necrosis factor (TNF)-alpha and IL-12 concentrations. Neutrophil counts correlated significantly with cervical concentrations of IL-1beta, TNF-alpha, IL-8, IL-6 and IL-10. Antigen-presenting cell numbers correlated significantly with TNF-alpha and IL-12 concentrations. HIV-infected women on antiretroviral therapy had similar levels of cervical lymphocyte infiltration and inflammation to women naïve to therapy. In conclusion, we suggest that inflammation at the cervix and HIV infection are likely to be key determinants in the absolute number of mucosal immune cells recovered by cervical cytobrushing.

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-alpha) 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-alpha, IL-1beta, 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.