Figure 5. Increased expression of cytokines related to mucosal immunity in HIV-specific CD4⁺ T cells of elite controllers compared to chronic progressors.

(a,b). Representative flow cytometry plots depicting detection of IL22 and IL17F in CD69⁺CD40L⁺ CD4⁺ T cells in one EC after stimulation with a Gag peptide pool by delayed ICS for cytokine protein and RNA flow FISH for cytokine mRNA, respectively. Numbers represent frequencies of mRNA⁺ HIV-specific CD4⁺ T cells among (blue) CD69⁺CD40L⁺ and (grey) total CD4⁺ T cells (n= 8 CPs and 8 ECs). (c,d) Statistical comparison from (a) of frequencies of cytokine mRNA⁺ antigen-specific CD4⁺ T cells between CPs and EC after (c) a Gag peptide pool (n= 8 CPs and 8 ECs) or (d) SEB stimulation (n=6 CPs and 8 ECs) (two-tailed Mann-Whitney test). (e) Statistical comparison of IL-22 and IL-17F protein levels detected by Luminex beads array in the supernatant of CD8-depleted PBMCs 48h after stimulation with a Gag peptide pool (n= 8 CPs and 8 ECs). (c,d,e) Bars represent median with +/−interquartile range. *p<0.05, **p<0.01 by two-tailed Mann-Whitney test. (f) Representative flow cytometry plots of co-expression patterns of IL22 mRNA and IL17F mRNA in HIV-specific CD4⁺ T cells in CP and EC people (n=6 CPs and ^ ECs). (g) Statistical analysis from (f) by SPICE (*p<0.05 by permutation test, 10000 permutations). Slices represent median frequency of IL22/IL17F mRNA⁺ subpopulations of HIV-specific CD4⁺ T cells (n= 6 CPs and 6 ECs). (h,i) Comparison of the frequencies of CCR6 and CXCR3 expression on IL22 mRNA⁺ and IL17F mRNA⁺ HIV-specific CD4⁺ T cells compared to total HIV-specific CD4⁺ T cells in CPs (n=6) and ECs (n=6) by two-tailed Mann-Whitney test and by two-tailed Wilcoxon matched-pairs signed-ranked test. Only p˂0.05 are displayed for clarity. Bars represent median with interquartile range. (j,k) Correlation of (j) IL22 and (k) IL-17F mRNA levels assessed by qRT-PCR on sorted HIV Gag-specific CD4⁺ T cells and CD4⁺ T cell activation measured by HLA-DR and CD38 co-expression (two-tailed Spearman test; n= 10 CPs, 6 VCs, 12 ECs). (l,m) Statistical comparisons of microbial translocation in plasma in cohorts of CP, EC, and uninfected control donors (UD) (l) quantitation of bacterial RNA reads in plasma (transcripts per million, TPM); (m) bacterial RNA species diversity (Shannon entropy score) (Kruskal Wallis test; n= 10 CPs, 8 ECs and 6 UDs). Bars represent median +/−interquartile range. (n,o) Correlation between bacterial RNA species diversity in plasma and frequencies of (n) HIV-specific IL22⁺ CD4⁺ T cells and (o) HIV-specific IL17⁺ CD4⁺ T cells (two-tailed Spearman test; n=10 CPs and 8 ECs). (p,q) Correlation between abundance of Proteobacteria translocation and frequencies of (p) HIV-specific IL22⁺ CD4⁺ T cells and (q) HIV-specific IL17⁺ CD4⁺ T cells (two-tailed Spearman test; n=10 CPs and 8 ECs).