T regulatory cells maintain intestinal homeostasis by suppressing γδ T cells

Abstract

Immune tolerance against enteric commensal bacteria is important for preventing intestinal inflammation. Deletion of phosphoinositide-dependent protein kinase 1 (Pdk1) in T cells via Cd4-Cre induced chronic inflammation of the intestine despite the importance of PDK1 in T cell activation. Analysis of colonic intraepithelial lymphocytes of PDK1-deficient mice revealed markedly increased CD8α(+) T cell receptor (TCR)γδ(+) T cells, including an interleukin-17 (IL-17)-expressing population. TCRγδ(+) T cells were responsible for the inflammatory colitis as shown by the fact that deletion of Tcrd abolished spontaneous colitis in the PDK1-deficient mice. This dysregulation of intestinal TCRγδ(+) T cells was attributable to a reduction in the number and functional capacity of PDK1-deficient T regulatory (Treg) cells. Adoptive transfer of wild-type Treg cells abrogated the spontaneous activation and proliferation of intestinal TCRγδ(+) T cells observed in PDK1-deficient mice and prevented the development of colitis. Therefore, suppression of intestinal TCRγδ(+) T cells by Treg cells maintains enteric immune tolerance.

Figure 1

Pdk1 gene deletion by Cd4-Cre induces spontaneous colitis. (A) Photograph of the colon of a rectally prolapsed Pdk1^flox/flox; Cd4-Cre mouse (flox/flox) and its littermate control Pdk1^+/flox; Cd4-Cre mouse (+/flox) (original magnification 100×). (B) Incidence of rectal prolapse (n = 15 mice per each group). (C) ELISA analysis of serum IL-12p40 (samples pooled, n = 8 – 9 mice per each group). (D) ELISA analysis for IL-12p40 level of ileum and colon explant cultures. (E–H), Relative mRNA of IL-12p40 (E), IL-23p19 (F), IL-17A (G), TNF-α (H), IL-4 (I), IFN-γ (J), IL-12p35 (K), and TGF-β (L) normalized to β-actin in colon (n = 3 – 4 mice per each group). Error bars (C–L), s.d. of triplicate samples. *P<0.05, **P<0.01.

Figure 2

TCRγδ⁺ T cells are dramatically increased in PDK1 deficient mice. (A) Flow cytometry of IELs and lamina propria (LP) from colon. (B) Flow cytometry of IELs from colon. (C) Cytometry for analysis of CD69 expression on TCRγδ⁺ T cells in the colon IELs or in the colon LP. (D) Flow cytometry for analysis of IFN-γ and IL-17A-expressing TCRγδ⁺ T cells in the lymph node after stimulation with phorbol 12-myristate 13-acetate (PMA) and ionomycin. (E) Flow cytometry for analysis of IL-17A-expressing TCRγδ⁺ T cells in the colon IELs after stimulation with PMA and ionomycin. (F) Flow cytometry analysis for Vγ4-expressing TCRγδ⁺ T cells in the colon IELs. Numbers adjacent to outlined areas indicate percent cells in each (A–F).

Figure 3

TCRγδ⁺ T cells are responsible for the development of spontaneous colitis. (A) Incidence of rectal prolapse (n=7–10 mice per group). Photograph (B) and histological grade (C) of colon from each genotype (original magnification 100×).

Figure 4

PDK1 significantly affects the Treg cell development. (A) Flow cytometry analysis of Treg cells in the IELs from colon and gating on Thy1⁺ cells. (B) Flow cytometry analysis of Thy1⁺ cells in the IELs from colon. (C) Flow cytometry analysis of Treg cells from thymus and peripheral lymph nodes and gating on CD4⁺ cells. (D) The number of Treg cells in thymus and lymph node (n = 5 mice). Open bars, flox/flox; filled bars, +/flox. Numbers adjacent to outlined areas indicate percent cells in each (A–C).

Figure 5

PDK1 is necessary for the inhibitory function of Treg cells. (A–C), Relative mRNA amounts of IL-10 (A), TGF-β (B), and Ebi3 (C) in Treg cells after activation with anti-CD3 and anti-CD28. (D) ELISA analysis of IL-10 production by WT Treg cells after stimulation with either anti-CD3, anti-CD28, or anti-CD3 and anti-CD28. (E) ELISA analysis of IL-10 production by Treg cells after stimulation with anti-CD3 and anti-CD28 with or without IL-2. Open bars, flox/flox; filled bars, +/flox. (F) Flow cytometry analysis of intracellular CTLA-4, surface CTLA-4, CD103, GITR on Treg cells and of surface CTLA-4 and CD69 on Treg cells after activation with anti-CD3 and anti-CD28. All analyses were done after gating on CD4⁺CD25⁺Foxp3⁺. (G) In vitro inhibition of CD4⁺ T cell proliferation with Tregs cells. (H) Relative IDO mRNA expression in the pDC by Treg cells activated with anti-CD3 and anti-CD28. (I) Clinical score and weight changes of mice after transfer of CD4⁺CD25⁻CD45RB^high cells with or without Treg cells. (J) Photograph of colon and histological grade of the colitis 10 weeks after the transfer (original magnification 100×). Error bars (A–E and G–H), s.d. of triplicate samples. *P<0.05, **P<0.01.

Figure 6

Treg cells regulate proliferation of TCRγδ⁺ T cells. (A) Flow cytometry for analysis of homeostatic proliferation of CFSE-labeled IEL TCRγδ⁺ T cells in Rag1^−/− mice with or without Treg cells. (B) Flow cytometry of IELs after adoptive transfer of WT Treg cells(top panel) and flow cytometry for analysis of IL-17-expressing TCRγδ⁺ T cells in the IELs of Pdk1^flox/flox; Cd4-Cre mice (flox/flox) with or without adoptive transfer of WT Treg cells, after stimulation with PMA and ionomycin (bottom panel). (C) Treg cell-mediated in vitro inhibition of TCRγδ⁺ T cell proliferation activated with plate-bound anti-TCRγδ with or without anti-IL-10 neutralizing antibody. (D) Treg cell-mediated in vitro inhibition of CD4⁺ T cell proliferation activated by anti-CD3 and anti-CD28 with or without IL-10 neutralizing antibody. (E) Inhibition of TCRγδ⁺ T cell proliferation by IL-10 after activation with plate-bound anti-TCRγδ. (F) Flow cytometry for TCRγδ⁺ T cells in the IELs of Il-10^−/− or Il-10^+/+ mice. Error bars (C, D and E), s.d. of triplicate samples. *P<0.05, **P<0.01. Numbers adjacent to outlined areas indicate percent cells in each (A, B, and F).

Figure 7

Treg are essential for TCRγδ⁺ T cell regulation to prevent spontaneous colitis. (A) Photograph of colon and histological grade of the colitis 9 weeks after injection of WT Treg cells or PBS into Pdk1^flox/flox; Cd4-Cre mice (flox/flox) (n=7–12 mice per group) (original magnification 100×). (B) ELISA analysis of serum IL-12p40 3 weeks after injection of WT Treg cells or PBS into Pdk1^flox/flox; Cd4-Cre mice (flox/flox) (n=4–5 mice per group). (C) Relative weight changes of Pdk1^flox/flox; Cd4-Cre mice (flox/flox) after injection of WT Treg cells or PBS and of Pdk1^+/flox; Cd4-Cre mice (+/flox) (n=7–12 mice per group). (D–F) Relative mRNA amounts of IL-12p40 (D), IL-23p19 (E), and IL-17A (F) in colon (n=3–4 mice per each group). Error bars (D–F), s.d. of triplicate samples. *P<0.05, **P<0.01.