Cutting edge: Generation of colitogenic Th17 CD4 T cells is enhanced by IL-17+ γδ T cells

Abstract

Th 17 cells have been implicated in the pathogenesis of colitis; however, a cellular mechanism by which colitogenic Th17 immunity arises in vivo remains unclear. In this study, we report that a subset of IL-17(+) γδ T cells plays a crucial role in enhancing in vivo Th17 differentiation and T cell-mediated colitis. TCRβ(-/-) mice were highly susceptible to T cell-mediated colitis, whereas TCRβδ(-/-) mice were resistant to the disease. Importantly, cotransfer of IL-17(+) but not of IL-17(-) γδ T cells with CD4 T cells was sufficient to enhance Th17 differentiation and induce full-blown colitis in TCRβδ(-/-) recipients. Collectively, our results provide a novel function of IL-17(+) γδ T cell subsets in supporting in vivo Th17 differentiation and possibly in fostering the development of intestinal inflammation.

Figure 1. TCRβ-/- recipients of naïve CD4 T cells develop exacerbated colitis while TCRβδ-/- recipients are fully resistant to the colitis, and adoptive transfer of γδ T cells restores colitis in TCRβδ-/- recipients

(A) FACS purified CD4⁺CD25^-CD44^low Thy1.1 naïve T cells (2.5 × 10⁵) were transferred into TCRβ-/- or TCRβδ-/- mice. In some experiments, FACS sorted γδ T cells (2.5 × 10⁵) were cotransferred with CD4 T cells into TCRβδ-/- mice. Body weight was weekly monitored and presented as a percentage of the initial weight at day 0. ** p<0.01; *** p <0.001. Data shown are from 5-8 individually tested mice. (B) Colitis score of each group. (C) Representative H&E staining of colon 5 weeks post transfer. 20× original magnification. (D) Total donor cell recovery of IL-17+ cells were calculated. Each symbol represents individually tested mouse. *** p<0.001.

Figure 2. Increased IL-17+ γδ T cells after CD4 T cell transfer

TCRβ-/- mice were transferred with naïve CD4 T cells as described above. Spleen and LN cells were harvested every week post transfer and ex vivo stimulated and γδ T cell cytokine expression was examined. The results shown represent the mean ± SD of 4-5 individually tested mice. * p<0.05; ** p<0.01.

Figure 3. CCR6+ γδ T cells support Th17 differentiation and colitogenicity of CD4 T cells in vivo

CD4⁺CD25^-CD44^low Ly5.1 naïve T cells (2.5 × 10⁵) were transferred into TCRβδ-/- recipients with sorted CCR6- γδ T cells or CCR6+ γδ T cells (2.5 × 10⁵). (A) Weight was weekly monitored. * p<0.05; *** p<0.001 (compared with CD4 alone group). (B) Representative H&E staining and immunohistochemistry for CD3 staining of colon 5 weeks post transfer of CD4 alone, CD4/CCR6- γδ T cell co-transfer, and CD4/CCR6+ γδ T cell co-transfer. 20X original magnification. (C) Colitis score of each group. (D and E) mLN and LP cells were isolated 5 weeks post transfer. The percentage of IL-17 producing Ly5.1 CD4 T cells from TCRβδ-/- recipients of CD4 alone, CD4/CCR6- γδ T cell co-transfer, and CD4/CCR6+ γδ T cell cotransfer is shown. Each symbol represents individually tested mouse. * p<0.05; ** p<0.01.

Figure 4. Cytokine profiles of γδ T cells are stable in vivo

Peripheral LN γδ T cells from Ly5.1 mice were sorted based on CCR6 expression and separately transferred into Rag2-/- mice (2.5×10⁵). Spleen cells were harvested 14 days post transfer and ex vivo stimulated for cytokine expression. Shown are IFNγ and IL-17 expression of Ly5.1 gated γδ T cells. The results shown represent the mean ± SD from two independent experiments.