Extended Data Fig. 8: CAR promotes IL-10 expression in mucosal Teff cells and regulates TR1 and TH17 cell development in the small intestine.
From: CAR directs T cell adaptation to bile acids in the small intestine
a, Equal numbers of CD45.1 wild-type (B6; blue) and CD45.2 CAR-deficient (B6.Nr1i3−/−; red) naive CD4+ T cells were transferred together into Rag1−/− mice. The resulting Teff cells were analysed using surface and intracellular flow cytometry after ex vivo stimulation with PMA and ionomycin at 2, 4, and 6 weeks from spleen, MLN, siLP, or cLP. Gating hierarchy is shown from a representative sample of MLN mononuclear cells at two weeks after T cell transfer. b, Intracellular IL-10 and IFNγ expression, determined by flow cytometry, in wild-type (B6, blue; left) and CAR-deficient (B6.Nr1i3−/−, red; right) non-TH17 Teff cells, gated as in a, from tissues of T cell-reconstituted B6.Rag1−/− mice over time. Numbers indicate percentages; representative of five mice per tissue and time point. c, d, Mean ± s.e.m. percentages (c) or numbers (d) (n = 5) of IL-10-expressing wild-type (B6, left) or CAR-deficient (B6.Nr1i3−/−, right) Teff cells, determined by ex vivo flow cytometry as in a, b from tissues of transferred B6.Rag1−/− mice over time. e, Specificity of IL-10 intracellular staining, as validated by analysis of IL-10 production by CD45.1 wild-type (B6; blue) or CD45.2 Il10−/− (red) Teff cells isolated from spleen or siLP of congenically co-transferred Rag1−/− mice. Representative of six mice analysed over two independent experiments. f, Expression of RORγt and IL-17A, determined by intracellular FACS analysis as in Extended Data Fig. 8a, in wild-type (B6) or CAR-deficient (B6.Nr1i3−/−) CD4+ Teff cells from tissues of reconstituted Rag1−/− mice two weeks after mixed T cell transfer. Numbers indicate percentages; representative of five mice per tissue and time point. g, Mean ± s.e.m. percentages of (n = 5) wild-type (B6; blue) or CAR-deficient (B6.Nr1i3−/−; red) RORγt+IL-17A− Teff cells, determined by intracellular flow cytometry as in a. *P = 0.029, paired two-tailed Student’s t-test. h, Expression of RORγt and IL-17A, determined by intracellular FACS analysis, in wild-type (B6) or Il10 −/− Teff cells from tissues of reconstituted Rag1−/− mice two weeks after mixed T cell transfer. Numbers indicate percentages; representative of five mice per tissue and time point. i, Mean ± s.e.m. percentages of (n = 7) wild-type (B6; blue) or Il10 −/− (red) RORγt+IL-17A− Teff cells, determined by intracellular flow cytometry as in c. *P = 0.033, **P = 0.0099, paired two-tailed Student’s t-test.
