Seronegative MSM at high risk of HIV-1 acquisition show an immune quiescent profile with a normal immune response against common antigens

Abstract

Human immunodeficiency virus (HIV) infection still represents a major public health problem worldwide, and its vaccine remains elusive. The study of HIV-exposed seronegative individuals (HESN) brings important information about the natural resistance to HIV, allows a better understanding of the infection, and opens doors for new preventive and therapeutic strategies. Among HESN groups, there are some men who have sex with men (MSM) with high-risk sexual behaviors, who represent an adequate cohort for HESN study because of their major HIV exposure without infection. This study aimed to compare the immunological profile of Colombian seronegative MSM with different risk sexual behaviors. This study included 60 MSM at high-risk (n = 16) and low-risk (n = 44) of HIV-1 acquisition. No sex worker nor homozygous delta 32 mutation subjects were included. All participants were negative for anti-HIV-1/2 antibodies and HIV-1 proviral DNA. A higher frequency of sexual partners in the last 3 months before the study participation (median, 30 vs. 2), lifetime sexual partners (median, 1,708 vs. 26), and unprotected anal intercourse (median 12.5 vs. 2) was determined in high-risk MSM than low-risk MSM. High-risk MSM also showed a quiescent profile of T cells and natural killer (NK) cells, with a significantly lower percentage of CD4+CD38+, CD4+HLADR-CD38+, CD4+Ki67+ T cells, and NKG2D+ NK cells (CD3-CD16+CD56+), a significantly higher percentage of CD4+HLADR-CD38-, and a tendency to show a higher percentage of CD8+HLADR+CD38- T cells than the low-risk group. Likewise, they showed higher mRNA levels of Serpin A1 from PBMCs. The results suggest that this MSM cohort could be HESN individuals and their resistance would be explained by a quiescent profile of T cells and NK cells and an increased Serpin A1 expression. Further study on MSM at high risk of exposure to HIV-1 is necessary to better understand the natural resistance to HIV.

Fig 1. CD4+ and CD8+ T cells basal activation profile in both MSM groups.

Comparison of the percentage of (A) CD4+ and (B) CD8+ T cells expressing HLA-DR, Ki-67, CD38, and CD69 activation markers between the groups. High-risk group (n = 16), low-risk group (n = 44). All data are reported after background correction. The comparison was performed with the Mann–Whitney U-test or Student T-test according to the data distribution.

Fig 2. T cell responses against a pool of HIV-1 Gag peptides.

(A) IFN-γ expression by unstimulated CD4+ and CD8+ T cells, or after SEB and HIV-1 Gag peptides stimulus. (B) Comparison of the polyfunctional profiles of Gag-specific responses in CD4+ T cells from both MSM groups. The slices of the pies correspond to the proportions of Gag-specific CD4+ T cells expressing 1 (grey), 2 (light blue), 3 (dark blue), or 4 (red) functions, until n simultaneous parameters (n + 1 dimensions) are calculated using the Boolean gating; the results are presented as mean. In the permutation analysis conducted in the SPICE platform, only data higher than 0.1% were included (after background subtraction). (C) INDEX of polyfunctionality (pINDEX) of CD4+ and CD8+ T cells from both MSM groups based on the proportions of cells producing intracellular combinations of Granzyme B, MIP1-β, TNF-α, and IFN-γ; the pINDEX was calculated with Funky Cells software [31]; the comparison was performed with the Mann–Whitney U-test. For all the panels: high-risk group n = 16, low-risk group n = 44.

Fig 3. NK cells subset distribution, NKG2D expression, and polyfunctional index.

(A) Comparison between both MSM groups of NK cells subsets (CD56bright, CD56dim CD16+, CD56dim CD16−) from fresh peripheral blood. The comparison was realized using the Mann–Whitney U-test. Low-risk n = 15, high-risk n = 12. (B) Comparison of percentage of total NK cells (CD3−CD56+CD16− and CD3−CD56+CD16+ PBMCs) expressing Granzyme B, Perforin, IFN-γ, or NKG2D between both MSM groups. The comparison was realized using the Mann–Whitney U-test. Data are presented after background subtraction. Low-risk n = 43, high-risk n = 14. (C) Comparison of the polyfunctional profiles of total NK cells (CD3−CD56+CD16− and CD3−CD56+CD16+ PBMCs) from both MSM groups. The slices of the pies correspond to the proportions of total NK cells expressing 1 (grey), 2 (light blue), 3 (dark blue), or 4 (red) functions, until n simultaneous parameters (n + 1 dimensions) are calculated using the Boolean gating; the results are presented as mean. The permutation analysis conducted in the SPICE platform only included data higher than 0.1% (after background subtraction). (D) INDEX of polyfunctionality (pINDEX) of total NK cells from both MSM groups based on the proportions of cells expressing Granzyme B, Perforin, IFN-γ, and NKG2D; the pINDEX was calculated with Funky Cells software; the comparison was performed using the Mann–Whitney U-test. Low-risk n = 43, high-risk n = 14.

Fig 4. Symptoms, macroscopic, and microscopic findings in anal mucosa tissue of both MSM groups.

Comparison of symptoms, macroscopic, and microscopic findings in the anal mucosa tissue of both MSM groups. Data represent the percentages of people presenting each condition. No statistical differences were found between both groups through the Chi-square test. Low-risk (n = 29), high-risk (n = 9). ASCUS: atypical squamous cells of uncertain significance; LISIL: low-grade squamous intraepithelial lesion; HSIL: high-grade squamous intraepithelial lesion.