IL-1β promotes the differentiation of polyfunctional human CCR6+CXCR3+ Th1/17 cells that are specific for pathogenic and commensal microbes

Abstract

In humans, Th1/17 cells, identified by coexpression of the chemokine receptors CCR6 and CXCR3, are proposed to be highly pathogenic in several autoimmune disorders due in part to their expression of the proinflammatory cytokines IL-17, IFN-γ, and GM-CSF. However, their developmental requirements, relationship with "classic" Th17 and Th1 cells and physiological role in normal immune responses are not well understood. In this study, we examined CCR6+ CXCR3+ Th1/17 cells from healthy individuals and found that ex vivo these cells produced the effector cytokines IL-17, IL-22, and IFN-γ in all possible combinations and were highly responsive to both IL-12 and IL-23. Moreover, although the Ag specificity of CCR6+ CXCR3+ Th1/17 cells showed substantial overlap with that of Th1 and Th17 cells, this population was enriched in cells recognizing certain extracellular bacteria and expressing the intestinal homing receptor integrin β7. Finally, we identified IL-1β as a key cytokine that renders Th17 cells sensitive to IL-12, and both cytokines together potently induced the differentiation of cells that produce IL-17, IFN-γ, and GM-CSF. Therefore, interfering with IL-1β and IL-12 signaling in Th17 cells during inflammation may be a promising therapeutic approach to reduce their differentiation into "pathogenic" CCR6+ CXCR3+ Th1/17 cells in patients with autoimmune diseases.

Figure 1. Phenotype, gene expression and cytokine production by human CCR6+CXCR3+ T cells

(A) Expression of CCR6, CXCR3, CCR4 and CCR10 by gated CD4+CD45RO+CD25-CD127+ Th cells from peripheral blood (left panels). Summary of several different donors with 6+3− Th17 cells, 6+3+ Th1/17 cells and 6−3+ Th1 cells (right panel). ***P<0.001 (ANOVA). Each symbol represents one donor; horizontal bars indicate mean. Data are from 12 donors. (B) Quantitative RT-PCR analysis of RORC and TBX21 gene expression by the indicated Th cell subsets (top panel). AU, arbitrary units. Data are mean ± SEM of 7 donors. Expression of RORγt and T-bet by sorted 6+3− Th17 cells, 6+3+ T cells and 6−3+ Th1 cells directly ex vivo (lower panel). Data are representative of 4 independent experiments. (C) Production of IL-17, IL-22 and IFN-γ by sorted Th cell subsets stimulated for 5h with PMA/ionomycin. (D) Boolean gating analysis showing each possible combination of IL-17, IL-22 and IFN-γ production representative of 6 different donors.

Figure 2. CCR6+CXCR3+ T cell respond to both IL-12 and IL-23

(A) Quantitative RT-PCR analysis of IL12RB2, IL23R and IL12RB1 gene expression by the indicated Th cell subsets. AU, arbitrary units. Data are mean ± SEM of 6 donors. (B) Phosphorylation of STAT4 and STAT3 by the indicated Th cell subsets in response to IL-12 and IL-23, respectively; untreated cells (filled histograms) and cytokine-treated cells (solid line). (C) Proportion of cells that phosphorylate STAT4 or STAT3 in response to IL-12 and IL-23. Data are mean ±SEM of 7 donors. ns, P>0.05; *P<0.05; ***P<0.001 (ANOVA). (D) Phosphorylation of STAT4 and STAT3 by the indicated Th cell subsets in response to IL-12 + IL-23; cells treated with IL-12 (upper panels) or IL-23 (lower panels) (filled histograms) and cells treated with both cytokines together (solid line). Plots are gated on pSTAT4+ (top) and pSTAT3+ (bottom) cells. Data are representative of 4 independent experiments. (E) Production of IL-17 and IFN-γ by 6+3+ T cells activated with CD3/CD28 beads and cultivated for 13 days with either medium, IL-12 or IL-23. Data are representative of 6 donors analyzed.

Figure 3. CCR6+CXCR3+ T cells recognize certain extracellular bacteria in addition to Th1- and Th17-associated antigens

(A and B) Antigen-specific proliferation of the indicated Th cell subsets stimulated with autologous monocytes pulsed with different antigens. Proliferation was measured after 16h pulse with [³H]thymidine on day 4 of the culture. Data are from 8 or 9 healthy donors.

Figure 4. Integrin α4β7 expression in Th17 cells and 6+3+ T cells discriminates between C. albicans and E. coli responses

(A) Expression of integrin β7 by the indicated Th cell subsets. (B) Proportion of cells that express integrin β7 among the different Th cell subsets. Data are from 10 donors. ***P<0.001 (ANOVA). Antigen-specific proliferation of 6+3− Th17 and 6+3+ Th1/17 cell subsets sorted based on integrin β7 expression and stimulated with autologous monocytes pulsed with S. aureus (C) or E. coli (D). Proliferation was measured after 16h pulse with [³H]thymidine on day 4 of the culture. Data are from 4 donors. *P<0.05; **P<0.01 (two-tailed paired t test).

Figure 5. TCR stimulation and IL-1β render Th17 cells responsive to IL-12 and favor the differentiation into IFN-γ⁺IL-17⁺ Th cells

(A) Quantitative RT-PCR analysis of IL1R1 gene expression by the indicated Th cell subsets. AU, arbitrary units. Data are mean ± SEM of 9 donors. (B) STAT4 phosphorylation and Ikbα degradation in response to IL-12 and IL-1β by the indicated Th cell subsets directly ex vivo. (C) STAT4 phosphorylation in response to IL-12 by 6+3− Th17 cells activated for 24h with CD3/CD28 beads in the presence or absence of IL-1β; untreated cells (filled histograms) and cytokine-treated cells (solid line). (D) STAT4 phosphorylation and T-bet expression by 6+3− Th17 cells activated for 24h with CD3/CD28 beads in the presence of absence of IL-1β and/or IL-12. (E) Production of IL-17 and IFN-γ by IL-1R1 – and + 6+3− Th17 cells expanded for 13 days with the indicated cytokines and stimulated for 5h with PMA/ionomycin. (F) Proportion of IL-1R1 – and + 6+3− Th17 cells producing IL-17, IFN-γ, GM-CSF or all three cytokines together after expansion as indicated above. Data are mean ±SEM of 4 donors. *P<0.05; **P<0.01; ***P<0.001 (ANOVA). (G) Production of IL-10 by expanded 6+3− Th17 and 6+3+ T cells. Data are mean ±SEM of 6 donors. ***P<0.001 (two-tailed paired t test).