In vivo equilibrium of proinflammatory IL-17+ and regulatory IL-10+ Foxp3+ RORgamma t+ T cells

Abstract

The nuclear hormone receptor retinoic acid receptor-related orphan receptor gamma t (RORgamma t) is required for the generation of T helper 17 cells expressing the proinflammatory cytokine interleukin (IL)-17. In vivo, however, less than half of RORgamma t(+) T cells express IL-17. We report here that RORgamma t(+) T alphabeta cells include Foxp3(+) cells that coexist with IL-17-producing RORgamma t(+) T alphabeta cells in all tissues examined. The Foxp3(+) RORgamma t(+) T alphabeta express IL-10 and CCL20, and function as regulatory T cells. Furthermore, the ratio of Foxp3(+) to IL-17-producing RORgamma t(+) T alphabeta cells remains remarkably constant in mice enduring infection and inflammation. This equilibrium is tuned in favor of IL-10 production by Foxp3 and CCL20, and in favor of IL-17 production by IL-6 and IL-23. In the lung and skin, the largest population of RORgamma t(+) T cells express the gammadelta T cell receptor and produce the highest levels of IL-17 independently of IL-6. Thus, potentially antagonistic proinflammatory IL-17-producing and regulatory Foxp3(+) RORgamma t(+) T cells coexist and are tightly controlled, suggesting that a perturbed equilibrium in RORgamma t(+) T cells might lead to decreased immunoreactivity or, in contrast, to pathological inflammation.

Figure 1.

Diversity and distribution of RORγt⁺ T cells in vivo. (A and B) Flow cytometry analysis of cells isolated from the organs of 8–12-wk-old Rorc(γt)-Gfp^TG mice. Plots are gated on CD3⁺ cells (A) or GFP⁺ CD3⁺ cells (B). Numbers indicate mean percent cells in quadrants ± SD obtained with at least three Rorc(γt)-Gfp^TG mice. LPLs, lamina propria lymphocytes isolated from small intestine; mLN, mesenteric LNs; BM, bone marrow. (C) Immunofluorescence histology of RORγt⁺ cells in the small intestine of Rorc(γt)-Gfp^TG mice. Most RORγt⁺ cells in villi are T cells, whereas RORγt⁺ cells in cryptopatches located between crypts are CD3⁻ LTi cells. Bar, 50 μm. (D) Expression of CD4 and CD8α by spleen GFP⁺TCR-β⁺ and lung or GFP⁺TCR-δ⁺ cells.

Figure 2.

RORγt⁺ T cells include Th17 and Foxp3⁺ T reg cells. (A and B) Flow cytometry analysis of cells isolated from Rorc(γt)-Gfp^TG mice. Cells were restimulated in vitro with PMA/ionomycin for 5 h and subjected to intracellular staining for GFP, IL-17, Foxp3, and/or IFN-γ. Plots are gated on TCR-β⁺ cells (A) and GFP⁺TCR-β⁺ or GFP⁺TCR-δ⁺ cells (B). Numbers indicate mean percent cells in quadrants ± SD obtained with at least three Rorc(γt)-Gfp^TG mice. (C) CD4⁺ T cells isolated from the spleen and mesenteric LNs of Rorc(γt)-Gfp^TG mice were sorted into two populations based on their expression of GFP and CD25. Sorted cells were subsequently restimulated in vitro and stained for IL-17 and Foxp3. Results are representative of three independent experiments. (D) Immunofluorescence histology staining for GFP, RORγt, Foxp3, and nuclei (DAPI) of a mesenteric LN isolated from a Rorc(γt)-Gfp^TG mouse. Arrowheads indicate cells expressing RORγt and/or Foxp3. Bar, 20 μm. (E) Proliferation assay of CD4⁺ T cells isolated from the spleen and mesenteric LNs of Rorc(γt)-Gfp^TG mice and sorted into two populations based on their expression of GFP and CD25. CFSE-labeled CD25⁻ responder T cells (2.5 × 10⁴) were cultured alone (left) or together with either GFP⁻CD25⁺ T cells or GFP⁺CD25⁺ T cells (2.5 × 10⁴ or 0.625 × 10⁴ cells). Cells were cultured in duplicates for 3 d in the presence of 10⁵ irradiated spleen cells and anti-CD3ε antibody. Numbers indicate frequency of proliferating cells (± SD) obtained from three independent experiments.

Figure 3.

RORγt⁺ T cells express genes involved in the Th17 or T reg cell pathway. (A) Cells isolated from the spleen and mesenteric LNs of Rorc(γt)-Gfp^TG mice were sorted into eight distinct populations based on their expression of GFP, CD3, TCR-β, TCR-δ, CD4, and CD25 (Fig. S1), and gene expression was assessed using real-time PCR. Ct values were normalized to the mean Ct of five housekeeping genes. Data are the mean of two or three independent experiments. (B) Foxp3⁺ RORγt⁺ T cells express IL-10. Cells isolated from LNs of Rorc(γt)-Gfp^TG mice were restimulated in vitro with PMA/ionomycin for 5 h and subjected to intracellular staining for GFP, IL-17, Foxp3, and IL-10 or an isotype control. Numbers indicate percent cells in quadrants. Results are representative of at least three individual experiments.

Figure 4.

The role of IL-6, IL-12Rβ1, and CCR6 in the generation of RORγt⁺ T cell subsets. (A and B) Cells were isolated from the spleen of Rorc(γt)-Gfp^TG (WT) or Il6^−/−× Rorc(γt)-Gfp^TG (IL-6°) mice, restimulated in vitro with PMA/ionomycin for 5 h and subjected to intracellular staining for GFP, IL-17, Foxp3, and IL-10. Plots are gated on GFP⁺TCR-β⁺ cells (A) or GFP⁺TCR-δ⁺ cells (B). Numbers indicate percent cells in quadrants and are representative of two independent experiments. (C) Cells were isolated from the spleen, mesenteric LN, and lung of Rorc(γt)-Gfp^TG (WT), Il12rb1^−/−× Rorc(γt)-Gfp^TG (IL12Rβ1°), Ccr6^−/− × Rorc(γt)-Gfp^TG (CCR6°), or Il6^−/−× Rorc(γt)-Gfp^TG mice (IL-6°) and processed as in A and B. Histograms report percent GFP⁺TCR-β⁺ cell subsets in total T cells. Each bar also shows the percentages of IL-17⁺ and Foxp3⁺ cells within the GFP⁺TCR-β⁺ cell population. (D) Histograms report the ratio of the frequencies of IL-17–producing GFP⁺TCR-β⁺ cells to Foxp3⁺ GFP⁺TCR-β⁺ cells as indicated in the figure. Three to five mice were analyzed per group. *, P < 0.05 as compared with WT.

Figure 5.

Regulation of RORγt⁺ T cells by Foxp3. (A and B) Bone marrow cells isolated from CD45.2⁺Rorc(γt)-Gfp^TG (WT) or Foxp3^sf × Rorc(γt)-Gfp^TG (Scurfy) mice were mixed with bone marrow cells isolated from CD45.1⁺ C57BL/6 mice and injected into irradiated lymphopenic mice. 6 wk after transfer, cells were isolated from the spleen, LN, and lung and restimulated in vitro with PMA/ionomycin for 5 h and subjected to intracellular staining for GFP, IL-17, Foxp3, and IL-10. (A) Spleen cells are gated on GFP⁺TCR-β⁺ cells. Numbers indicate percent cells in quadrants. Reconstitutions with CD45.1⁺ and CD45.2⁺ cells were comparable in all chimeras. Results are representative of two independent experiments. (B) Histograms report percent GFP⁺TCR-β⁺ cell subsets in total CD45.2⁺ T cells (see Fig. 4 C). *, P < 0.05 as compared with WT. (C) Immunoprecipitation of RORγt and Foxp3. Foxp3 was expressed in HEK293T cells alone or together with N-terminally FLAG-tagged RORγt. Total cell lysates or immunoprecipitates (IP) using anti-FLAG antibody were blotted and probed with antibodies to Foxp3, FLAG, or β-actin. Results are representative of two independent experiments.

Figure 6.

Equilibrium in RORγt⁺ T cell subsets during pathology. (A) Rorc(γt)-Gfp^TG mice were treated with DSS in the drinking water for 6 d, followed by water for 10 d. This protocol was repeated for a total of three cycles. After the last cycle, cells isolated from the colon were restimulated in vitro with PMA/ionomycin for 5 h and subjected to intracellular staining for GFP, IL-17, and Foxp3. Histograms (from left to right) report percent GFP⁺TCR-β⁺ cell subsets in total T cells (see Fig. 4 C), total numbers of RORγt⁺ Tαβ cells present in the organ, and the ratio of IL-17–producing to Foxp3⁺ cells within RORγt⁺ Tαβ cells (see Fig. 4 D). Right panels show immunofluorescence histology of a colon from a healthy or a treated Rorc(γt)-Gfp^TG mouse. Bar, 100 μm. (B) Rorc(γt)-Gfp^TG mice were infected intranasally with 100 PFUs of influenza A virus for 7 d. Cells were then isolated from the lung and processed as in A. Right panels show immunofluorescence histology of a lung from healthy or an infected Rorc(γt)-Gfp^TG mouse. Bar, 50 μm. (C) Cells were isolated from the mesenteric LNs of a 4-mo-old Rorc(γt)-Gfp^TG × Apc^min/+ mouse and processed as in A. Right panels show immunofluorescence histology of a mesenteric LN from a normal or a tumor-bearing mouse. Bar, 100 μm. Data shown are representative of at least three independent experiments. Three to four mice were analyzed per group. *, P < 0.05 as compared with control (mock-treated or WT mice).

Figure 7.

Generation and differentiation of RORγt⁺ Tαβ cells in vitro. MACS-sorted naive (CD62L⁺) CD4⁺ T cells from the spleens of Rorc(γt)-Gfp^TG mice were stimulated in duplicates with anti-CD3ε and anti-CD28 in the presence of blocking anti–IFN-γ and anti–IL-4 antibodies and the indicated cytokines or RA. After different periods of time, cells were restimulated with PMA/ionomycin for 5 h and analyzed by flow cytometry for the expression of GFP, Foxp3, IL-17, and IL-10. All plots are gated on TCR-β⁺ cells, except plots for IL-10 that are gated on GFP⁺TCR-β⁺ cells. Numbers indicate percent cells in quadrants. Data are representative of three independent experiments.