Th22 Cells Are a Major Contributor to the Mycobacterial CD4+ T Cell Response and Are Depleted During HIV Infection

Abstract

HIV-1 infection substantially increases the risk of developing tuberculosis (TB). Mechanisms such as defects in the Th1 response to Mycobacterium tuberculosis in HIV-infected persons have been widely reported. However, Th1-independent mechanisms also contribute to protection against TB. To identify a broader spectrum of defects in TB immunity during HIV infection, we examined IL-17A and IL-22 production in response to mycobacterial Ags in peripheral blood of persons with latent TB infection and HIV coinfection. Upon stimulating with mycobacterial Ags, we observed a distinct CD4⁺ Th lineage producing IL-22 in the absence of IL-17A and IFN-γ. Mycobacteria-specific Th22 cells were present at high frequencies in blood and contributed up to 50% to the CD4⁺ T cell response to mycobacteria, comparable in magnitude to the IFN-γ Th1 response (median 0.91% and 0.55%, respectively). Phenotypic characterization of Th22 cells revealed that their memory differentiation was similar to M. tuberculosis-specific Th1 cells (i.e., predominantly early differentiated CD45RO⁺CD27⁺ phenotype). Moreover, CCR6 and CXCR3 expression profiles of Th22 cells were similar to Th17 cells, whereas their CCR4 and CCR10 expression patterns displayed an intermediate phenotype between Th1 and Th17 cells. Strikingly, mycobacterial IL-22 responses were 3-fold lower in HIV-infected persons compared with uninfected persons, and the magnitude of responses correlated inversely with HIV viral load. These data provide important insights into mycobacteria-specific Th subsets in humans and suggest a potential role for IL-22 in protection against TB during HIV infection. Further studies are needed to fully elucidate the role of IL-22 in protective TB immunity.

Figure 1:. CD4+ T cell cytokine responses to mycobacterial antigens in latent TB infection.

(A) Representative flow cytometry plots of the production of IFN-γ, IL-22 and IL-17A from CD4+ T cells after stimulation with M. bovis BCG, M.tb PPD and ESAT-6/CFP-10 peptides, in one study participant. UNS corresponds to the unstimulated control. The frequency of cytokine-producing cells is shown as a percentage of the total CD4+ T cell population, after gating on live, CD3+ lymphocytes. (B) Individual IFN-γ (blue), IL-22 (red) or IL-17A (green) responses to BCG, PPD or ESAT-6/CFP-10 in individuals with latent TB infection (LTBI; n=25). The frequency of cytokine-producing cells is shown as a percentage of the total CD4+ T cell population, after gating on live, CD3+ lymphocytes. (C) The relationship between the frequency of CD4+ T cells producing IFN-γ and IL-22 in response to BCG (n=24). (D) Populations of CD4+ T cells producing different combinations of IFN-γ, IL-22 and IL-17A in response to BCG. The pie charts indicate the proportion of cytokine combinations that makes up the BCG response. Each slice of the pie represents a specific subset of cells, defined by a combination of cytokines shown by the color at the bottom of the graphs. Data are shown as box and whisker (interquartile range) plots and horizontal bars represent the median. Each dot represents one individual. Statistical comparisons were performed using Friedman’s test with Dunn’s multiple comparison, and a non-parametric Spearman rank test for the correlation. **p≤0.01, ***p≤0.001, ****p≤0.0001.

Figure 2:. Cytokine secretion in M.tb-stimulated whole blood.

The concentration of soluble IFN-γ (white), IL-22 (black) and IL-17A (gray) was measured by ELISA (pg/ml) in plasma from whole blood stimulated with M.tb lysate in individuals with LTBI (n=9). Plasma was harvested at 12 and 24 hours after stimulation. Horizontal bars represent the median. Statistical comparisons were performed using a Wilcoxon test. *p≤0.05, **p≤0.01.

Figure 3:. Detection of the CD4+ T cell IL-22 responses in PBMC.

(A) Schematic of mixed stimulation assay with representative flow cytometry data. PBMC were stained with a fluorescent dye, Oregon Green (Or Green), and added to autologous whole blood, and subsequently stimulated with M.tb lysate. Representative flow cytometry plots show the production of IFN-γ and IL-22 from CD4+ T cells in fresh PBMC (PBMC+ M.tb) alone, whole blood (WB + M.tb) alone, and the mixed stimulation assay (WB in mix, PBMC in mix), after stimulation with M.tb lysate in one participant. (B) Comparison of the frequency of CD4+ T cells producing IL-22 and IFN-γ in each experimental condition in the same individuals (n=8). Horizontal bars represent the median. Statistical comparisons were performed using Friedman’s test with Dunn’s multiple comparison. **p≤0.01, ***p≤0.001.

Figure 4:. Memory profiles of CD4+ T cells producing IFN-γ, IL-22 or IL-17A in response to BCG.

(A) Representative flow cytometry plots of total CD4+ memory subset distribution in one individual based on CD45RO and CD27 staining. Naïve: CD45RO-CD27+, early differentiated (ED: CD45RO+CD27+), late differentiated (LD: CD45RO+CD27-) and terminally differentiated (TD: CD45RO-CD27-). The overlays indicate the antigen specific CD4+ T cells producing IFN-γ (blue), IL-22 (red) or IL-17A (green). The frequencies of each subset are indicated. (B) The memory distribution of cells producing IFN-γ (blue), IL-22 (red) or IL-17A (green) in response to BCG (n=20, 25 and 7, respectively). Only individuals with a positive cytokine response and more than 30 cytokine events were included in the phenotyping. Each dot represents one individual. Data are shown as box and whisker (interquartile range) plots and horizontal bars represent the median. Statistical comparisons were performed using Kruskal-Wallis and Dunn’s multiple comparison test.

Figure 5:. Chemokine receptor expression of CD4+ T cells producing IFN-γ, IL-22 or IL-17A in response to M.tb whole cell lysate.

(A) Representative flow cytometry plots of the expression of CCR6, CCR4, CXCR3 and CCR10 on total CD4+ T cells in one individual. The overlays indicate the antigen specific CD4+ T cells producing IFN-γ (blue), IL-22 (red) or IL-17A (green). The frequencies of each subset are indicated. (B) The chemokine receptor distribution of cells producing IFN-γ (blue), IL-22 (red) or IL-17A (green) in response to M.tb lysate (n=19, 19 and 11, respectively). Only individuals with a positive cytokine response and more than 30 cytokine events were included in the phenotyping. Each dot represents one individual. Data are shown as box and whisker (interquartile range) plots and horizontal bars represent the median. Statistical comparisons were performed using Kruskal-Wallis and Dunn’s multiple comparison test. *p≤0.05, **p≤0.01, ***p≤0.001, ****p≤0.0001

Figure 6:. CD4+ T cell responses to BCG in HIV-infected and uninfected individuals and their relationship with CD4 count and HIV viral load.

(A) The individual IFN-γ, IL-22 or IL-17A responses in HIV uninfected or infected individuals in response to BCG (n=24 in each group). (B) The cytokine frequency adjusted for CD4 count in HIV-infected and HIV-uninfected individuals in response to BCG. (C) The median fluorescent intensity (MFI) of IFN-γ, (n=24 and n=15 for HIV-uninfected and infected, respectively), IL-22 (n=23 and n=20 for HIV-uninfected and infected, respectively) and IL-17A (n=22 and n=8 for HIV-uninfected and infected, respectively) in response to BCG. For each cytokine, MFI was only graphed for individuals with positive cytokine responses. HIV-uninfected participants are shown with open circles and HIV-uninfected individuals with closed circles. Each dot represents one individual. Horizontal bars represent the median. (D) The association between IFN-γ (white), IL-22 (black) or IL-17A (gray) responses to BCG and CD4 count or (E) viral load (n=24). The dotted line indicates linear regression for statistically significant correlations. Statistical comparisons were performed using a non-parametric Mann Whitney test, and a non-parametric Spearman rank test for the correlations. *p≤0.05, **p≤0.01, ***p≤0.001, ****p≤0.0001.