Immune correlates of HIV exposure without infection in foreskins of men from Rakai, Uganda

Abstract

Human immunodeficiency virus (HIV) susceptibility is heterogenous, with some HIV-exposed but seronegative (HESN) individuals remaining uninfected despite repeated exposure. Previous studies in the cervix have shown that reduced HIV susceptibility may be mediated by immune alterations in the genital mucosa. However, immune correlates of HIV exposure without infection have not been investigated in the foreskin. We collected sub-preputial swabs and foreskin tissue from HESN (n=20) and unexposed control (n=57) men undergoing elective circumcision. Blinded investigators assayed swabs for HIV-neutralizing IgA, innate antimicrobial peptides, and cytokine levels. Functional T-cell subsets from foreskin tissue were assessed by flow cytometry. HESN foreskins had elevated α-defensins (3,027 vs. 1,795 pg ml(-1), P=0.011) and HIV-neutralizing IgA (50.0 vs. 13.5% of men, P=0.019). Foreskin tissue from HESN men contained a higher density of CD3 T cells (151.9 vs. 69.9 cells mm(-2), P=0.018), but a lower proportion of these was Th17 cells (6.12 vs. 8.04% of CD4 T cells, P=0.007), and fewer produced tumor necrosis factor α (TNFα) (34.3 vs. 41.8% of CD4 T cells, P=0.037; 36.9 vs. 45.7% of CD8 T cells, P=0.004). A decrease in the relative abundance of susceptible CD4 T cells and local TNFα production, in combination with HIV-neutralizing IgA and α-defensins, may represent a protective immune milieu at a site of HIV exposure.

Figures

Figure 1. Levels of soluble innate immune proteins in foreskin secretions

HESN (hatched bars) and unexposed control men (open bars). Sub-preputial swabs collected before surgery were assayed for levels of soluble innate immune proteins by ELISA. Median concentration (with range) of innate peptide in 1mL collection volume is displayed (HESN n=18; Controls n=37).

Figure 2. The ability of IgA isolated from the foreskin to neutralize HIV

IgA was purified from foreskin secretions and incubated with a primary clade C viral isolate, which was subsequently challenged with PBMCs. The ability of IgA-treated virus to infect PBMCs was measured through p24 production. Capacity to neutralization HIV was defined as 67% less p24 production compared to reference sample. The proportion of men in each group with HIV-neutralizing IgA is shown (HESN n=18; Controls n=37).

Figure 3. T cell subsets in the blood and foreskin

HESN (hatched bars) and unexposed control men (open bars). The proportion of CD3+ T cells expressing either CD4 or CD8 was measured on PBMCs and foreskin cells using flow cytometry (B); representative flow cytometry plots showing the gating strategy for foreskin cells are shown in A. Proportions of T cells subsets were normalized to the number of CD3 T cells per mm2 of foreskin tissue (obtained using IHC) to obtain absolute numbers of each cell type (C) (HESN n=20, Controls n=57).

Figure 4. CD4 T cell subsets in the blood and foreskin

HESN (hatched bars) and unexposed control men (open bars). CCR5 expression was measured on PBMCs and foreskin CD4 T cells using flow cytometry (representative foreskin plot in A). Th17 cells were identified by production of IL17a by CD4 T cells in response to stimulation with PMA and ionomycin (representative foreskin plot in D). Summary data of the proportion of CCR5+ CD4 T cells and Th17 cells in the blood and foreskin are shown in B and E. Absolute numbers of cells, obtained by IHC of CD3, are shown in C and F (HESN n=20, Controls n=57).

Figure 5. Inflammatory cytokine production by CD8 (A–E) and CD4 (F–J) T cells

HESN (hatched bars) and unexposed control men (open bars). T cells isolated from foreskin tissue were challenged with PMA and ionomycin and subsequent TNFα and IFNγ production by either CD8+ T cells or CD8- T cells (a proxy for CD4 T cells) was measured by flow cytometry (representative plots shown for CD8 (A) and CD4 T cells (F). Summary data of the proportion of CD8 T cells producing TNFα or IFNγ shown in B and D, respectively. Absolute numbers of each cell type, obtained through CD3 IHC, are shown in C and E. Similarly, proportions of CD4 T cells producing TNFα or IFNγ shown in G and I, and absolute numbers of each cell type are shown in H and J (HESN n=20, Controls n=57).

Figure 6. HIV-specific CD8 T cells responses in the foreskin

CD8 T cells isolated from foreskin tissue were challenged with a pool of cross-clade HIV peptides previously shown to be highly antigenic in an East African population. (A) TNFα and IFNγ production by CD8 T cells was measured by flow cytometry. (B) Frequency of response for HESN and unexposed control men. An HIV-specific response was defined as cytokine production 3 times greater than observed in unstimulated cells and a minimum of 0.3% (HESN n=20, Controls n=57).