Fig 1.
Kinetics of activation of circulating CD8+ T-cells.
Kinetics of the expression of HLA-DR and CD38 (A), PD-1 (B), Annexin V (C), perforin and/or granzyme B (D), CD107a alone or together with granzyme B or perforin (E) and IFN-γ alone or together with granzyme B or CD107a (F) in CD8+ T-cells from seronegative individuals (n = 3) after stimulation with PMA-Ionomycin (at 50 and 500 ng/mL, respectively). In B, the expression of PD-1 in total and HLA-DR/CD38-expressing CD8+ T-cells is shown. *P = 0.04 vs 0 hours; &P = 0.04 vs 2 hours; #P = 0.04 vs 6 hours; +P = 0.04 vs 12 hours.
Fig 2.
Alteration in the cytotoxic program of CD8+ T-cells from HIV-infected patients.
Frequencies of granzyme B+ perforin+ (A), granzyme B+ IFN-γ+ (B) and CD107a+ (C) CD8+ T-cells in seronegative (n = 15) and HIV-infected (n = 30) individuals after polyclonal stimulation. D. Correlation between the frequency of granzyme B+ perforin+ and CD107a+ CD8+ T-cells in seronegative and HIV-infected individuals (n = 15 and n = 30, respectively). E. Levels of granzyme B in supernatant of cultured PBMC from seronegative and HIV-infected individuals (n = 7 and n = 15, respectively). The dashed line indicates the assay limit of detection. F. Correlation between the levels of granzyme B in supernatant of cultured PBMC and the frequency of CD107a+ CD8+ T-cells in seronegative and HIV-infected individuals after polyclonal stimulation (n = 7 and n = 15, respectively). In A-C and E, the P value of the Mann-Whitney test is shown. In D and F, the rho and P value of the Spearman test are shown.
Fig 3.
HLA-DR+ CD8+ T-cells exhibit an effector memory profile.
A. Expression of CCR7, CD28, CD45RA, CD45RO, CD95, CD57, and CTLA-4 in HLA-DR/CD38-expressing CD8+ T-cells from a representative seronegative and HIV-infected individual. The numbers indicate the percentage of positive cells for each marker. The summary of the results is shown in the right panels (n = 5 in both groups of individuals). B. Expression of HLA-DR and CD38 in CD107a+ CD8+ T-cells from a representative seronegative individual after 12 hours treatment with PMA-ionomycin. The summary of the results is shown in the lower panel (n = 15). *P≤0.01 vs all other subsets. C. Frequencies of HLA-DR/CD38-expressing CD8+ T-cells that are CD107a+ (upper panel), and granzyme B+ perforin+ (lower panel), in seronegative individuals (n = 15). The P value of the Wilcoxon test is shown. D. Expression of perforin and CD107a in CD57hi, CD57lo and CD57- cells among HLA-DR/CD38-expressing CD8+ T-cells from a representative seronegative individual after PMA-ionomycin stimulation, from a total of 5 individuals. NS: Not statistically significant.
Table 1.
Expression of CD8+ T-cell memory and functional markers in HLA-DR/CD38-expressing cells.
Fig 4.
Alteration in the cytotoxic program of HLA-DR/CD38-expressing CD8+ T-cells from HIV-infected patients.
Frequencies of HLA-DR/CD38-expressing CD8+ T-cells that are CD107a+ (A), granzyme B+ perforin+ (B) and granzyme B+ IFN-γ+ (C) in seronegative (n = 15) and HIV-infected (n = 30) individuals. The P value of the Mann-Whitney test is shown. NS: Not statistically significant. D. Cell ACCENSE t-SNE plots of HLA-DR, CD38, perforin, granzyme B, CD107a and IFN-γ expression in merged stimulated CD8+ T-cells from representative seronegative and HIV-infected individuals (n = 4 in each group; M = 60,000 cells). The circles indicate the cells positive for the indicated molecules. E-H. Correlation between the frequency of granzyme B+ perforin+ or CD107a+ cells and the frequency of their respective total HLA-DR+ CD38- (E and F) or HLA-DR- CD38+ (G and H) CD8+ T-cells from HIV-infected individuals (n = 30). The rho and P value of the Spearman test are shown.
Fig 5.
Cytotoxic program of HIV-specific CD8+ T-cells.
A-B. Expression of CD107a alone or together with perforin, granzyme B or IFN-γ, and granzyme B plus IFN-γ in CD8+ T-cells treated with medium or a pool of HIV Gag peptides (n = 30). The P value of the Wilcoxon test is shown. C. Levels of granzyme B in supernatant of cultured unstimulated or Gag peptides-stimulated PBMC from HIV-infected individuals (n = 15). The dashed line indicates the assay limit of detection. D. Expression of HLA-DR and CD38 in CD107a+ CD8+ T-cells from a representative HIV-infected individual, after stimulation with a pool of HIV Gag peptides. The summary of the results is shown in the lower panel (n = 30). E-F. Frequencies of HLA-DR/CD38-expressing CD8+ T-cells that are CD107a+ (E), and granzyme B+ IFN-γ+ (F) in HIV-infected individuals (n = 30), after stimulation with a pool of HIV Gag peptides. G. Correlation between the levels of granzyme B in supernatant of cultured PBMC and the frequency of CD107a+ CD8+ T-cells in HIV-infected individuals (n = 15) after stimulation with a pool of HIV Gag peptides. The rho and P value of the Spearman test are shown. In D, E and F, the P value of the Dunn’s post-hoc test is shown.
Fig 6.
Association between the treatment duration, the exhaustion/activation state and inflammation levels with the cytotoxic program of CD8+ T-cells from HIV-infected patients.
A. Heat map of the weight of the variables included in the Component 1 of the Principal Component Analysis performed, including the phenotypic and functional characterization of CD8+ T-cells, as well as other variables such as the length of the treatment, the CD4:CD8 ratio, and plasma levels of sCD14. B. Spatial distribution of seronegative (n = 15) and HIV-infected individuals, the latter classified in those with <24 months (n = 17) or ≥25 (n = 13) months of therapy, after Principal Component Analysis. C-D. Frequencies of CD107a+ (C) and granzyme B+ perforin+ (D) CD8+ T-cells in seronegative (n = 15) and HIV-infected individuals, the latter classified in those with <24 months or ≥25 months of therapy (n = 17 and n = 13, respectively). E. Correlation between the frequency of CD107a+ CD8+ T-cells after Gag peptides stimulation and the treatment time in months in HIV-infected patients (n = 30). F. Percentage of specific lysis after in vitro cytotoxicity assay in cells from HIV-infected patients with <24 or ≥25 months of therapy (n = 3 in each group, run in duplicate in each case). G. Correlation between the frequency of HLA-DR+ CD38+ CD8+ T-cells that are CD107a+ after PMA-ionomycin stimulation and that of HLA-DR+ CD38+ CD8+ T-cells that are PD-1+ in resting peripheral blood, in HIV-infected patients (n = 30). H. Correlation between the frequency of HLA-DR+ CD38+ CD8+ T-cells that are CD107a+ after Gag peptides stimulation, the frequency of HLA-DR+ CD38+ CD8+ T-cells that are CD107a+ after PMA-ionomycin stimulation and the levels of plasma sCD14, in HIV-infected patients (n = 30). In C and D, The P value of the Dunn’s post-hoc test is shown. In E, G and H, the rho and P value of the Spearman test are shown.