Presence of CD8+ T cells in the ectocervical mucosa correlates with genital viral shedding in HIV-infected women despite a low prevalence of HIV RNA-expressing cells in the tissue

Abstract

The female genital tract is a portal of entry for sexual HIV transmission and a possible viral reservoir. In this study, the ectocervical CD8+ T cell distribution was explored in situ and was related to expression of CD3 and HLA-DR and presence of HIV RNA. For this purpose, ectocervical tissue samples and genital secretions were collected from HIV-seropositive (HIV+) Kenyan female sex workers (FSWs) (n = 20), HIV-seronegative (HIV-) FSWs (n = 17), and HIV(-) lower-risk women (n = 21). Cell markers were assessed by in situ staining and by quantitative PCR. HIV RNA expression in tissue was analyzed by in situ hybridization, and viral shedding was assessed by quantitative PCR. The HIV+ FSW group had a higher amount of total cells and CD8+, CD3+, and HLA-DR+ cells compared with the HIV(-)FSW group and HIV- lower-risk women. The majority of CD8+ cells were CD3+ T cells, and the numbers of CD8+ cells correlated significantly with plasma and cervical viral load. HIV RNA expression in situ was found in 4 of the 20 HIV+FSW women but did not correlate with cervical or plasma viral load. Thus, the HIV+ women displayed high numbers of CD8+, CD3+, and HLA-DR+ cells, as well as a limited number of HIV RNA+ cells, in their ectocervical mucosa; hence, this localization cannot be neglected as a potential viral reservoir. The elevated levels of CD8+ T cells may play a role in the immunopathogenesis of HIV in the female genital tract.

FIGURE 1

Enumeration and in situ staining of immune cells in ectocervical tissue. (A) Distribution and median of the percentage of total cells in the ectocervical epithelium and submucosa of tissue samples from the three study groups. A nonparametric, two-tailed Mann–Whitney U test was used to compare the HIV⁺FSW group versus the HIV⁻FSW group and the HIV⁺FSW group versus the HIV⁻LR group. (B) Immunofluorescent images of ectocervical tissue sections from an HIV⁺FSW subject. The image on the right for each pair is a magnified view of the region indicated in the box in the image to the left. The majority of CD8⁺ cells (red) were also CD3⁺ (green) and vice versa (left panels); the majority of CD3⁺ cells (red) were not CD4⁺ cells (green) (right panels). Scale bar, 100 μm. (C) Bright-field images of ectocervical tissue stained with hematoxylin (blue) for visualization of cell nuclei and stained for CD8⁺ cells (brown; upper panels) and CD3⁺ cells (brown; lower panels). Scale bar, 200 μm.

FIGURE 2

Enumeration of CD8⁺ and CD3⁺ cells in ectocervical tissue. Distribution and median of the percentage of positively stained (A) CD8⁺ cells in the total tissue analyzed (B) and in the ectocervical epithelial and submucosal tissue area analyzed separately. Percentage of positively stained (C) CD3⁺ cells in the total tissue analyzed and (D) in the ectocervical epithelial and sub-mucosal tissue area analyzed separately, as assessed by imaging analysis. (E) CD8⁺/CD3⁺ cell ratio in the total ectocervical epithelial area and submucosal area. (F) Correlation between the percentage of CD8⁺ cells and the percentage of the total cells within the ectocervical tissue area analyzed by imaging analysis. (G) Distribution and median relative quantification (RQ; UBC = 1) of CD8 mRNA expression, which was assessed by qPCR. (H) CD8/CD3 mRNA ratio. The Ct values for each target gene were normalized to UBC. Fold change of the target genes was calculated as 2^−dCt. A nonparametric, two-tailed Mann–Whitney U test was used to analyze the statistical significance between the study groups.

FIGURE 3

Detection and quantification of HIV RNA in ectocervical tissue and in plasma and cervical secretions. (A) Bright-field images of ectocervical tissue sections stained with H&E (in blue and red) to visualize cell nuclei and showing the presence of HIV RNA⁺ cells (a cluster of green silver grains illuminated under epipolarized light after radioautography), detected by in situ hybridization. Arrows are pointing at HIV RNA⁺ cells. Ectocervical tissue from an HIV⁺FSW (left panel), an HIV⁻FSW (middle panel), and a humanized mouse experimentally infected with HIV, which was used as a positive control (right panel). Scale bars, 25 μm (lower-magnification images); 50 μm (higher-magnification images). (B) Correlation between plasma and cervical viral load in the HIV⁺ FSW group, as determined by Spearman rank correlation coefficient test.

FIGURE 4

Comparisons between cellular markers versus plasma and cervical viral load. Correlation between plasma viral load (left panels) and cervical viral load (right panels) and the percentage of CD8⁺ cells (A) within the total ectocervical tissue area (B), the epithelial tissue area, and (C) the submucosal tissue area, as assessed by imaging analysis. Relative quantification (RQ; UBC = 1) of (D) CD8 mRNA and (E) CD69 mRNA levels, as assessed by qPCR. Spearman rank correlation coefficient test was used to assess correlations.

FIGURE 5

Enumeration of HLA-DR⁺ cells and HLA-DR mRNA expression in ectocervical tissue. Distribution and median of the percentage of positively stained HLA-DR⁺ cells in (A) the total tissue analyzed (B) and in the ectocervical epithelial and submucosal tissue area analyzed separately, as assessed by imaging analysis. (C) Relative quantification (RQ; UBC = 1) of HLA-DR mRNA expression, which was assessed by qPCR. A nonparametric, two-tailed Mann–Whitney U test was used to analyze statistical significance between the HIV⁺FSW group versus the HIV⁻FSW group and the HIV⁺FSW group versus the HIV⁻LR group. (D) Bright-field images of human ectocervical tissue stained with hematoxylin (blue) for visualization of cell nuclei and stained for HLA-DR (brown). Scale bars, 200 μm. (E) Correlation between the percentage of CD8⁺ cells and percentage of HLA-DR⁺ cells within the total ectocervical tissue area, as assessed by imaging analysis in the HIV⁺FSW group. (F) Correlation between the RQ of CD8 and HLA-DR mRNA levels, as assessed by qPCR, in the HIV⁺FSW group. Spearman rank correlation coefficient test was used to assess the correlation.