Development of novel cryopreservation medium.

<p><b>A</b>, Viability of vaginal T cells (indicated “CD3”) and macrophages (indicated “CD14”) following cryopreservation with 1.5M glycerol, 1.5M propylene glycol (PG) or 10% dimethyl sulfoxide (DMSO). <b>B</b>, Effect of CPA concentration on cell viability after cryopreservation. <b>C</b>, Effect of trehalose concentration on cell viability after cryopreservation in the presence 0.84M DMSO, 1.5M PG, or 1.75M EG. <b>D</b>, Effect of CPA concentration on cell viability after cryopreservation in the presence of 4.8% trehalose. <b>E</b>, Effect of trehalose concentration on recovery after cryopreservation in the presence of 1.4M DMSO. <b>F</b>, Effect of mixtures of DMSO and EG on cell recovery after cryopreservation in the presence of 1.2% trehalose. Each line for a given color indicates a different tissue sample. The line with the circles indicates the mean across the different tissue samples. Measurements were made in duplicate for each tissue sample. For relative viability, 100% indicates the same viability as the fresh cells.</p

Overview of the three different cryopreservation procedures used.

<p>Overview of the three different cryopreservation procedures used.</p

Recovery and functionality of colorectal cells after cryopreservation.

<p><b>A</b>, Recovery of colorectal T cells (indicated “CD3”), macrophages (indicated “CD13”), and neutrophils (indicated “CD66b”), after cryopreservation with several cryopreservation media. GHRC I [Global HIV Vaccine Research Cryorepository] is 10% DMSO alone and GHRC II is 5% DMSO with 6% hydroxyethyl starch, both in RPMI with 12.5% bovine serum albumin. HANC is 10% DMSO in FBS. HANC was processed according to procedure C and the others were processed with procedure B (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0156293#pone.0156293.t001" target="_blank">Table 1</a>). <b>B</b>, Background-subtracted cytokine production from colorectal CD8 T cells fresh or after cryopreservation after stimulation with phorbol 12-myristate 13-acetate (PMA); staphylococcal enterotoxin B (SEB); or human cytomegalovirus, Epstein-Barr virus and influenza virus peptides (CEF). Gray symbols indicate the individual samples, with gray lines indicating pairing. Black symbols show the mean across all samples and black vertical lines show the 95% confidence interval of the mean. <b>C</b>, Polyfunctionality of colorectal CD8 cytokine responses to stimulation. Red bars indicate fresh samples and blue bars indicate samples tested after cryopreservation. Each bar corresponds to the percent of CD8 cells that expressed the indicated cytokines, averaged across the donors. Percentages are indicated explicitly for bars that exceed the axis limits of the graphs. In the legend, a dark square indicates presence of that cytokine and a light square its absence. White gaps divide the graphs and legends into groups expressing 0, 1, 2, 3, 4, or 5 cytokines.</p

Comparison of novel cryopreservation medium with published methods.

<p><b>A</b>, Recovery of vaginal T cells (indicated “CD3”) and macrophages (indicated “CD14”) with several cryopreservation media. GHRC I [Global HIV Vaccine Research Cryorepository] is 10% DMSO alone and GHRC II is 5% DMSO with 6% hydroxyethyl starch, both in RPMI with 12.5% bovine serum albumin. HANC is 10% DMSO in FBS. HANC was processed according to procedure C and the others were processed with procedure B (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0156293#pone.0156293.t001" target="_blank">Table 1</a>). <b>B</b>, Evaluation of which processing steps improve the recovery obtained relative to the standard HANC procedure, where positive values indicate improvements over HANC. <b>C</b>, Rescue of HANC procedure (“optimal HANC”) by use of optimal processing procedures for small sample sizes. <b>D</b>, Percentage of all vaginal cells that are CD45⁺ before and after freezing. Gray symbols indicate the average of duplicates, with gray lines indicating pairing. Black symbols show the mean across all samples and black vertical lines show the 95% confidence interval of the mean.</p

Cryopreservation of cells derived from endocervical cytobrushes.

<p><b>A</b>, Recovery of endocervical T cells (indicated “CD3”), macrophages (indicated “CD14”), and neutrophils (indicated “CD66b”), when cryopreserved after isolation from cytobrushes. <b>B</b>, Absolute number of live (top) and viability of total endocervical cells (bottom) after cryopreservation either as a cell suspension or still on the cytobrush. Gray symbols indicate the individual samples, with gray lines indicating pairing. Black symbols show the mean across all samples and black vertical lines show the 95% confidence interval of the mean.</p

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

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

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 ectocervical biopsies.

<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