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 α4β7 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

The bii4africa dataset of faunal and floral population intactness estimates across Africa’s major land uses

Sub-Saharan Africa is under-represented in global biodiversity datasets, particularly regarding the impact of land use on species’ population abundances. Drawing on recent advances in expert elicitation to ensure data consistency, 200 experts were convened using a modified-Delphi process to estimate ‘intactness scores’: the remaining proportion of an ‘intact’ reference population of a species group in a particular land use, on a scale from 0 (no remaining individuals) to 1 (same abundance as the reference) and, in rare cases, to 2 (populations that thrive in human-modified landscapes). The resulting bii4africa dataset contains intactness scores representing terrestrial vertebrates (tetrapods: ±5,400 amphibians, reptiles, birds, mammals) and vascular plants (±45,000 forbs, graminoids, trees, shrubs) in sub-Saharan Africa across the region’s major land uses (urban, cropland, rangeland, plantation, protected, etc.) and intensities (e.g., large-scale vs smallholder cropland). This dataset was co-produced as part of the Biodiversity Intactness Index for Africa Project. Additional uses include assessing ecosystem condition; rectifying geographic/ taxonomic biases in global biodiversity indicators and maps; and informing the Red List of Ecosystems

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 1β (IL-1β), 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 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

Optimizing Viable Leukocyte Sampling from the Female Genital Tract for Clinical Trials: An International Multi-Site Study

<div><p>Background</p><p>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.</p><p>Methods and Findings</p><p>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 α4β7 integrin, an HIV envelope-binding mucosal homing receptor.</p><p>Conclusions</p><p>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.</p></div

Inter-site variability and sample reproducibility from female genital tract immune cell sampling techniques.

<p>(<b>A</b>) Percentage contribution of each immune cell population to the total CD45⁺ population recovered from CVL, cytobrush, and biopsy samples collected at the Seattle, Chicago, and Nairobi study sites. (<b>B</b>) Numbers of CD45⁺ leukocytes recovered from cytobrush samples collected from all women recruited in Chicago. Filled circles and connecting lines indicate women participating in both Part 1 and Part 2 of the study (n = 12). Open circles indicate women sampled only in one part of the study. Boxes indicate median, interquartile range and total range. (<b>C</b>) Scatterplot of cell numbers from the twelve women sampled in both Part 1 (x axis) and Part 2 (y axis) of the study. (<b>D</b>) Scatterplot of cell numbers from replicate biopsies collected at the same clinic visit in seven participants at the Nairobi study site. Biopsies were taken from upper left and upper right quadrants of the ectocervix.</p

Median (IQR) numbers of leukocyte subpopulations in endocervical cytobrushes (CB) and cervicovaginal lavages (CVL).

<p>CD4, helper T lymphocytes; CD8, cytotoxic T lymphocytes; B, B lymphocytes; MΦ, macrophages; DC, dendritic cells.</p

Comparison of immune cell yield and distribution in endocervical cytobrush (CB) and ectocervical biopsy samples.

<p>(<b>A</b>) Representative gates for identification of CD45⁺ leukocytes from cytobrush and biopsy samples. (<b>B</b>) Numbers of CD45⁺ leukocytes, CD4⁺ T cells, CD8⁺ T cells, CD19⁺ B cells, CD14⁺ macrophages, and CD19^neg/HLA-DQ⁺ DC enumerated from cytobrush and biopsy samples. Cell numbers were log₁₀ transformed, and plotted per site (Chicago, red dots; Nairobi, blue dots; Seattle, black dots). Horizontal bars indicate median values for each site. (<b>C</b>) Percentage contribution of each cell subset, as well as of cells that do not fit one of the described populations (“unknown”), to the total CD45⁺ population from cytobrush and biopsy samples. Data are averaged across sites. P values are listed in the text, and median and IQR in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085675#pone-0085675-t002" target="_blank">Table 2</a>.</p