Expansion of CD25-Negative Forkhead Box P3-Positive T Cells during HIV and Mycobacterium tuberculosis Infection

Abstract

Tuberculosis (TB) and HIV alter the immune system, and coinfected (HIV-TB) individuals usually present deregulations of T-lymphocytic immune response. We previously observed an increased frequency of "unconventional" CD4⁺CD25^-FoxP3⁺ Treg (uTreg) population during HIV-TB disease. Therefore, we aimed to explore the phenotype and function of uTreg and conventional CD4⁺CD25⁺FoxP3⁺ Treg subsets (cTreg) in this context. We evaluated the expression of CD39, programmed cell death protein 1 (PD1), glucocorticoid-induced tumor necrosis factor receptor (GITR), and the effector/memory distribution by flow cytometry in cTreg and uTreg. Also, IL-10, TGF-β, IFN-γ production, and the suppressor capacity of uTregs were analyzed in cocultures with effector lymphocytes and compared with the effect of regulatory T cells (Tregs). We found diminished expression of CD39 and higher levels of PD1 on uTreg compared to cTreg in both HIV-TB and healthy donors (HD). In addition, uTreg and cTreg showed differences in maturation status in both HIV-TB and HD groups, due to the expansion of effector memory uTregs. Interestingly, both HIV-TB and HD showed a pronounced production of IFN-γ in uTreg population, though no significant differences were observed for IL-10 and TGF-β production between uTreg and cTreg. Moreover, IFN-γ⁺ cells were restricted to the CD39^- uTreg population. Finally, when the suppressor capacity was evaluated, both uTreg and cTreg inhibited polyclonal T cell-proliferation and IFN-γ production in a similar extent. These findings suggest that uTregs, which are expanded during HIV-TB coinfection, exert regulatory functions in a similar way to cTregs despite an altered surface expression of Treg characteristic markers and differences in cytokine production.

Keywords: cytokines; effector/memory Treg populations; human; infectious immunity bacteria; regulatory mechanisms.

Figure 1

Analysis of cTreg and uTreg populations across the spectrum of tuberculosis (TB) infection. PBMC from HIV-TB, HIV-LTB, HIV⁺, and healthy donors (HD) individuals were stained for cell surface expression of CD4, CD25, CD39, glucocorticoid-induced tumor necrosis factor receptor (GITR), programmed cell death protein 1 (PD1), CD45RA, CD27, and intracellular FoxP3 and analyzed by flow cytometry. Cells were gated based first on the basis of CD4 expression, then CD4⁺ cells were analyzed for the expression of CD25 and FoxP3 in order to define “conventional” and “unconventional” regulatory T cells (Tregs), and finally the expression of CD45RA/CD27 was determined in uTregs and cTregs. (A) Evaluation of the percentage of cTreg population in HIV-TB, HIV-LTB, HIV⁺, and HD. (B) Comparison of the percentage of uTreg between groups. (C) Mean fluorescence intensity (MFI) of FoxP3 was measured to compare the level of expression of this molecule in uTreg and cTreg of HIV-TB and HD. Wilcoxon test was used for statistical comparison between the two different subsets. (D) Correlation analysis between the absolute number of CD4⁺ T cells and the percentage of uTreg from HIV-infected patients (HIV-TB, HIV-LTB, and HIV). (E) The expression of CD27 and CD45RA on CD4⁺ T cells from HIV-TB, HIV-LTB, HIV⁺, and HD patients was analyzed by flow cytometry; (top) pie charts summarize the data and each slice corresponds to the mean proportion of uTreg cells for each phenotype. (Bottom) Possible phenotypes are shown on the x-axis whereas percentages of distinct T-cell subsets within uTreg cells are shown on the y-axis. (F) Representative dot plots of Treg identification, maturation status distribution and surface CD39, PD1, and GITR expression. For the comparison between the four groups, Kruskal–Wallis test was performed followed by Dunns posttest. Horizontal lines represent the median range and each point represents an individual subject; asterisks indicate a significant difference between groups; *p < 0.05; **p < 0.01; ***p < 0.001. Spearman test was performed to evaluate correlations between variables. Comparisons of phenotype distribution were performed using the partial permutation test as described in Ref. (14) and the Kruskal–Wallis test followed by Dunn’s multiple comparisons posttest.

Figure 2

Anti-TB treatment induces changes in uTreg but not in cTreg populations in HIV-TB patients. (A) Evaluation of uTreg (CD4⁺CD25^–FoxP3⁺), (B) cTreg (CD4⁺CD25⁺FoxP3⁺) frequencies by flow cytometry, (C) C-reactive protein (CRP) plasma concentrations, (D) CD4⁺ lymphocytes count, and (E) HIV RNA copies per milliliter in peripheral blood from HIV-TB individuals along anti-TB treatment (0, 3, and 6 months); values obtained from healthy donors (HD) were used as controls. Friedman test for paired samples was used for comparisons between consecutive visits for each population, cTreg and uTreg (**p < 0.01; *p < 0.05). For comparison between each visit and values in HD group, Mann–Whitney test was performed (^#p < 0.05 was consider significant). (F) Correlation analysis between the % of cTreg and the % of PD1⁺CD4⁺ T lymphocytes (left) and between the % of uTreg and the % of PD1⁺CD4 T cells (right) from HIV-TB individuals. Spearman rank test was used for the evaluation of the correlation. (G) Correlation analysis between the % of uTreg and the % of CD4⁺ T^CM lymphocytes (upper panel) and between the % of uTreg and the % of CD4⁺ T^EM cells (lower panel) from HIV-TB individuals. Spearman rank test was used to test correlation.

Figure 3

uTreg differ in CD39, programmed cell death protein 1 (PD1) and glucocorticoid-induced tumor necrosis factor receptor (GITR) expression levels with cTreg population. Comparison of the (A) CD39, (B) PD1, and (C) GITR expression between uTreg and cTreg from HIV-TB, HIV-LTB, HIV, and healthy donors (HD) individuals. Wilcoxon rank test was used for paired statistical analysis. For the comparison between the four groups, Kruskal–Wallis test was performed followed by Dunns posttest. *p < 0.05; **p < 0.01; ***p < 0.001; ^##p < 0.01; ^###p < 0.001.

Figure 4

Differing maturation status of uTreg and cTreg in HIV-TB and healthy donors (HD) individuals. Evaluation of effector/memory phenotype distribution between uTreg and cTreg in HIV-TB (upper panel) and healthy donors (HD) (lower panel) individuals. Pie charts summarize the data and each slice corresponds to the mean proportion of uTreg or cTreg cells for each phenotype. Each point represents a single individual. Comparisons between phenotype distributions were performed using the partial permutation test follow by Kruskal–Wallis test and the Dunn’s multiple comparisons posttest. Asterisks indicate significant difference between groups. *p < 0.05; **p < 0.01; ***p < 0.001.

Figure 5

A distinct effector/memory differentiation path occurs in Treg lymphocytes from coinfected patients. Correlation analysis between the % of uTreg^CM and the % of uTreg^EM and between the % of cTreg^CM and the % of cTreg^EM from (A,C) HIV-TB and (B,D) healthy donors (HD) individuals in order to infer recent effector/memory transitions as described previously (13). Spearman rank test was used for the evaluation of the correlation. p < 0.05 was considered significant.

Figure 6

uTregs produce a distinct pattern of cytokines and high levels of IFN-γ. PBMC from HIV-TB and healthy donors (HD) were stimulated with PMA/Io and analyzed by flow cytometry for the indicated cytokines. Comparison of latency-associated peptide (LAP) expression between uTreg and cTreg from (A) HIV-TB and (B) HD persons. Analysis of IL-10 production between uTreg and cTreg from (C) HIV-TB and (D) HD individuals. Evaluation of IFN-γ production in the uTreg and cTreg populations from (E) HIV-TB and (F) HD. (G) Correlation analysis between FoxP3 and IFN-γ mean fluorescence intensity (MFI) in Tregs. Wilcoxon test was used for statistical analysis. (H) Representative flow cytometry dot plots depicting IFN-γ production from uTregs (left) and cTregs (right). (I) Representative flow cytometry graphs showing IFN-γ production from CD39⁺ uTregs (left) and CD39⁻ uTregs (right). Each point represents an individual subject. Asterisks indicate significant differences between groups. *p < 0.05; ***p < 0.001. Spearman test was performed for correlation analysis.

Figure 7

Functional characterization of uTregs. FACS-isolated CD4⁺CD25⁻CD39⁻ effector T cells were stimulated with anti-CD3 plus allogeneic mitomycin-treated PBMCs and cocultured with CD4⁺CD25⁻CD39⁺ uTregs or CD4⁺CD25⁺CD39⁺ cTregs cells at the indicated Treg:Teff ratios. In addition, effector T cells were left unstimulated in order to determine basal proliferation. After 5 days, proliferation and IFN-γ secretion were evaluated. Proliferation levels of stimulated CD4⁺CD25⁻CD39⁻ effector T cells without any Treg population were defined as 100%. (A) Proliferation of effector T cells at different Teff/Treg ratios. (B) IFN-γ production in cell-free supernatants from cocultures was assessed by ELISA. Bars represent mean values ⁺ SEM from three individual experiments. *p < 0.05.