Foreskin T-cell subsets differ substantially from blood with respect to HIV co-receptor expression, inflammatory profile, and memory status

Abstract

The foreskin is the main site of heterosexual human immunodeficiency virus (HIV) acquisition in uncircumcised men, but functional data regarding T-cell subsets present at this site are lacking. Foreskin tissue and blood were obtained from Ugandan men undergoing elective adult circumcision. Tissue was treated by mechanical and enzymatic digestion followed by T-cell subset identification and assessment of cytokine production using flow cytometry. Foreskin CD4(+) T cells were predominantly an effector memory phenotype, and compared with blood they displayed a higher frequency of CCR5 expression (42.0% vs. 9.9%) and interleukin-17 production. There was no difference in T-regulatory cell frequency, but interferon-γ and tumor necrosis factor-α production were increased in foreskin CD8(+) T cells. These novel techniques demonstrate that the foreskin represents a proinflammatory milieu that is enriched for HIV-susceptible T-cell subsets. Further characterization of foreskin T-cell subsets may help to define the correlates of HIV susceptibility in the foreskin.

Figure 1. CD4⁺ and CD8⁺ T cell subsets within the foreskin and peripheral blood

PBMC and foreskin cells from 46 men were stained with CD3-FITC, CD4-PE, and CD8-PerCP. Graphs show percentages of CD3⁺ cells within PBMC or foreskin cells that co-express (A) either CD4 or CD8, or (B) expressed neither CD4 nor CD8 (double negative, DN, T cells).

Figure 2. CCR5 expression on CD4⁺ T cells from the foreskin and peripheral blood

PBMC and foreskin cells from 46 men were stained with CD3-APC, CD4-PE, and CCR5-FITC. Plots in (A) were created by gating on CD3⁺/CD4⁺ events. The gate defining CCR5⁺ events was created based on PBMC staining for this marker and applied to foreskin plots. (B) Proportions of CD3⁺/CD4⁺ cells in PBMC and foreskin cells co-expressing CCR5.

Figure 3. Increased production of IL17a and IL22 by foreskin CD4⁺ T cells, with no increase in Treg frequency

PBMC and foreskin cells from 46 men were either left unstimulated (vehicle/Treg) or treated with PMA-ionomycin (stimulated). Representative plots of foreskin cells are shown (A, B). The gates in (A) defining IL17a⁺ and IL22⁺ events were created based on unstimulated PBMC staining for each patient, and then applied to stimulated PBMC and foreskin plots. The gate defining CD25⁺ events in (B) was created based on CD25-FMOs (fluorescence minus one = CD3, CD4 and FoxP3). (C) Proportions of Th17 cells in PBMC and foreskin samples (CD3⁺/CD4⁺/IL17a⁺). (D) IL22 production in stimulated PBMC and foreskin CD4 T cells. (E) Proportions of PBMC and foreskin CD3⁺/CD4⁺ cells that are Tregs. *p<0.0001

Figure 4. The foreskin contains primarily effector memory CD4 T cells (T_EM)

Memory phenotype of PBMC and foreskin mononuclear cells was assessed on a sub-group of three men by staining with CD3-FITC, CD4-PerCP, CCR7-PE and CD45RA-APC. Representative plots were created by gating on CD3⁺/CD4⁺ events. The gates defining CD45RA⁺ and CCR7⁺ events were created based on FMO (Fluorescence minus one) staining for each cell type.

Figure 5. Enhanced production of pro-inflammatory cytokines by foreskin CD8⁺ T cells

PBMC and foreskin cells from 46 men were either left unstimulated (vehicle) or treated with PMA-ionomycin. Cells were then stained with CD3-FITC, CD8-PerCP, TNFα-PE and IFNγ-APC. Plots in (A) were created by gating on CD3⁺/CD8⁺ events. The gates defining TNFα ⁺ and IFNγ⁺ events were created based on unstimulated PBMC staining for each patient, and then applied to stimulated PBMC and foreskin plots. (B) Proportions of CD8 T cells producing TNFα, (C) IFNγ, and (D) of bi-functional CD8 T cells (producing both TNFα and IFNγ).