Increased Cervical CD4+CCR5+ T Cells Among Kenyan Sex Working Women Using Depot Medroxyprogesterone Acetate

Abstract

Depot medroxyprogesterone acetate (DMPA) is the most common hormonal contraceptive used by women in sub-Saharan Africa, however, it has been epidemiologically associated with HIV infections. To assess whether DMPA has an effect on the number and activation of HIV target cells, this study assessed the levels and phenotype of blood- and mucosal-derived HIV target cells among women using DMPA. Thirty-five HIV uninfected women from the Pumwani Sex Worker cohort from Nairobi, Kenya were enrolled in the study (15 using DMPA and 20 not using hormonal contraception). Blood (plasma and peripheral blood mononuclear cells) and cervicovaginal (lavage, cervical cells, and ectocervical biopsies) samples were collected. Cellular phenotype and activation status were determined by flow cytometry, cytokine levels were assessed by bead array and image analysis assessed cell number and phenotype in situ. In blood, the proportion of HIV target cells and activated T cells was lower in DMPA users versus those not using hormonal contraceptives. However, analysis of cervical mononuclear cells showed that DMPA users had elevated levels of activated T cells (CD4⁺CD69⁺) and expressed lower levels of the HIV co-receptor CCR5 on a per cell basis, while tissue samples showed that in the ectocervix, DMPA users had a higher proportion of CD4⁺CCR5⁺ T cells. This study demonstrates that DMPA users had higher levels of activated T cells and HIV target cells in the genital tract. The increased pool of mucosal HIV target cells provides new biological information about the potential impact of DMPA on HIV susceptibility.

Keywords: HIV target cells; cervical biopsy; contraception; depot medroxyprogesterone acetate (DMPA); ectocervix; immune activation.

FIG. 1.

The impact of DMPA in blood samples. (A) PBMC expression of cellular markers of activation and HIV co-receptor assessed by flow cytometry; (B) Expression of proinflammatory cytokines and chemokines derived from plasma. p-Values are from the multivariate analyses; graphs are presented with median and interquartile range; “▴” indicate participants sexually transmitted infections positive. ▪, DMPA-users (square); •, no HC users (circle). DMPA, depot medroxyprogesterone acetate; HC, hormonal contraception; MFI, median fluorescence intensity; PBMC, peripheral blood mononuclear cells.

FIG. 2.

The impact of DMPA on mucosal milieu. (A) The impact of DMPA on mucosal CMCs. Expression of cellular markers of activation and HIV co-receptors on CMCs as assessed by flow cytometry. (B) The impact of DMPA on MIP-3α in CVL. Expression of the proinflammatory chemokine MIP-3α in the CVL. p -Values are from the multivariate analyses; graphs are presented as median and interquartile range. CMCs, cervical mononuclear cells; CVL, cervico-vaginal lavage.

FIG. 3.

The transformation zone is highly populated with HIV target cells. Immunofluorescence images of cervical tissue sections from a DMPA user stained for CD4⁺ (red) and CCR5⁺ (green). DAPI (blue) was used as a counterstain for visualization of cell nuclei. The white line indicates the basal membrane, which separates the ectocervical epithelium from the underlying submucosal compartment, and the yellow line indicates the single columnar epithelium present in the endocervix. The green staining shown in the apical layer of the ectocervix and the endocervix is autofluorescent mucus. The scale bar represents 100 μm.

FIG. 4.

Visualization and enumeration of HIV target cells in the ectocervical epithelium of DMPA- and no HC users. (A) Representative immunofluorescence images of ectocervical tissue sections from a DMPA user (upper row) and a no HC user (lower row), stained for CD4⁺ (red) together with CCR5⁺ (green, left column), and CD4⁺ (red) together with Langerin⁺ (green, middle column); double-positive cells are shown in yellow. DAPI (blue) was used as a counterstain for visualization of cell nuclei. The images in the right column are magnified view of the regions indicated in the respective box in the images to the left. The scale bars represent 100 μm. (B) A scatter plot of percentage positively stained CD4⁺CCR5⁺ cell area out of total CD4⁺ stained tissue area. Each square/circle represents a different subject; DMPA-users (square) and no HC users (circle). As compared with the other analyses (PBMC, CMC, and CVL), two additional control subjects (no HC) were included and they are therefore clearly marked. One woman was infected with Neisseria gonorrhoeae (green circle) and one with Chlamydia trachomatis (red circle). These women were infected after enrollment and were therefore not excluded from entering the study. Horizontal lines represent median ± interquartile range. p values are from multivariate analyses.

FIG. 5.

Proportion of CD4⁺CCR5⁺ T cells present in the blood, CMC, and biopsy (ectocervical tissue).