Polyclonal expansion of cervical cytobrush-derived T cells to investigate HIV-specific responses in the female genital tract

Abstract

Human immunodeficiency virus (HIV) -specific T-cell responses are detectable in the female genital tract of HIV-infected women but little is known about their frequency or the factors that influence their detection. We investigated the feasibility of polyclonal in vitro expansion of cervical cytobrush-derived T cells to investigate HIV-specific responses in the female genital tract in HIV-infected women. Cytobrush-derived cervical cells were isolated from 22 HIV-infected women and expanded with anti-CD3 and recombinant interleukin-2. Cervical T-cell lines were investigated for Gag-specific responses by interferon-gamma ELISPOT and compared with those detected in matched blood samples. Cervical T-cell lines were established from 16/22 (72.7%) participants. Although the absolute number of CD3(+/-) cells recovered after expansion was positively associated with the number of cells isolated ex vivo (P = 0.01; R = 0.62), we observed a significant negative correlation between fold expansion and ex vivo cell number (P = 0.004; R = -0.68). We show that both the magnitude (P = 0.002; R = 0.7) and specific Gag regions targeted by cervical T-cell lines (P < 0.0001; R = 0.5) correlated significantly with those detected in blood. With one exception, cervical interferon-gamma T-cell responses to Gag were detected only in HIV-infected women with blood Gag-specific response > 1000 spot-forming units/10(6) cells. We conclude that cervical Gag-specific T-cell responses in expanded lines are most easily detectable in women who have corresponding high-magnitude Gag-specific T-cell responses in blood.

Figure 1

Impact of ex vivo yield on expansion of cervical cytobrush-derived cervical mononuclear cells (CMC). (a) Comparison between day 0 and day 28 CMC yields in donors whose cervical CD3 cells expanded. (b) Comparison of CD4 : CD8 ratio ex vivo (day 0) and day 28 in expanders. CMC were cultured in vitro with anti-CD3 monoclonal antibody in the presence of recombinant human interleukin-2 (rhIL-2). The CMC counts and viability were performed by Trypan blue counting. CD4 : CD8 ratios on cervical cell lines were analysed before and after polyclonal expansion using allophycocyanin conjugated CD3 and fluorescein isothiocyanate-conjugated CD8 and CD4 staining on a FACSCalibur flow cytometer and analysis was performed using flowjo software. Wilcoxon Rank Test was applied to compare yield and CD4 : CD8 ratios at day 0 and day 28. P-values < 0·05 were considered significant. Each data point represents an individual donor.

Figure 2

Comparison of cervical and blood human immunodeficiency virus (HIV) Gag-specific interferon-γ (IFN-γ) responses in women with chronic HIV infection. Blood (top panel) and cervical (bottom panel) IFN-γ responses to HIV-1 subtype C Gag peptide pools 1–5. Net spot forming units (SFU)/10⁶ was calculated by subtracting background IFN-γ frequency from Gag peptide pool-specific IFN-γ frequencies in 16 women with chronic HIV infection. Each stacked bar represents the cumulative IFN-γ frequency for all five Gag peptide pools and the subjects have been ordered from highest to lowest blood T-cell magnitude to Gag. The contribution of CD4⁺ and CD8⁺ T-cell subsets to overall IFN-γ responses to HIV Gag peptides was determined by intracellular cytokine staining and fluorescence-activated cell sorting. The T-cell subset contributing to Gag-specific responses per donor is shown above each stacked bar for blood responses. CD8 indicates CD8-only response, CD4 indicates CD4 responses only and CD8/CD4 indicates that responses in both subsets were detected. Asterisks indicate that responses were not evaluated in these individuals. ‘Fine mapping done’ denotes the four women for whom this analysis was carried out.

Figure 3

Correlation between human immunodeficiency virus (HIV) Gag-specific interferon-γ (IFN-γ) response magnitudes detected at the cervix and in blood of HIV-infected subjects. (a) Correlation between cumulative net IFN-γ responses to Gag pools (sum of five pools) per chronically HIV-infected individual in each compartment (n = 16 women for whom matched enzyme-linked immunosorbent spot-forming cell assay was conducted on cervical lines and peripheral blood mononuclear cells). (b) Correlation between individual Gag pools (1–5) at the cervix and in blood in HIV-infected women. Each data point represents an individual’s IFN-γ response at the cervix and in blood. Spearman Rank Test was applied to test correlations and P-values < 0·05 were considered significant. Spearman R-value is shown on each plot.

Figure 4

Correlation between ex vivo and expanded human immunodeficiency virus (HIV) Gag-specific interferon-γ (IFN-γ) enzyme-linked immunosorbent spot-forming cell assay responses in blood from chronically HIV-infected subjects. (a) Peripheral blood mononuclear cells (PBMCs) from four HIV-infected women were assessed for HIV-specific IFN-γ production ex vivo (top panel) and following in vitro expansion with anti-CD3 monoclonal antibody for 28 days (bottom panel). (b) Correlation between IFN-γ responses detected in PBMCs ex vivo and after 28 days expansion. HIV-1 subtype C Gag overlapping peptides were divided into five pools and responses in these donors to each Gag pool were assessed. Net IFN-γ response to Gag was calculated by subtracting background IFN-γ production in each individual from Gag-specific responses. Each dot (b) represents an individual donor’s matched IFN-γ response to each of the five Gag pools in blood ex vivo and following expansion (day 28). Spearman Rank Test was used to test the correlation and a P-value <0·05 were considered significant.