Fig. 6. Ahr promotes gut homing of Tregs by regulating GPR15.

(A) Migration index of donor cells (iTregs) from Ahr^+/+ (CD45.1/CD45.1) and Ahr^−/− (CD45.2/CD45.2) mice in different organs of recipients 10 h post transfer. The migration index was calculated using the following formula: migration index = (ratio of CD45.1⁻/CD45.1⁺ Tregs in post-transfer)/(ratio of CD45.1⁻/CD45.1⁺ Tregs in input). Data are representative of two independent experiments and shown as mean ± SEM (n = 3 mice per group). One-way ANOVA with Dunnett’s multiple comparisons test was used to compare large intestine or small intestine to the other organs. (B) GPR15 transduction rescued the colon homing deficiency of Tregs from Ahr^−/− mice. Migration index of MIT-GPR15- and MIG-EV- transduced iTregs from Ahr^−/− mice in different organs 10 h post transfer. The migration index was calculated using the following formula: migration index = (ratio of Ahr^−/− transduced with MIT-GPR15/ Ahr^−/− transduced with MIG-EV Tregs in post-transfer)/(ratio of Ahr^−/− transduced with MIT-GPR15/ Ahr^−/− transduced with MIG-EV Tregs in input). Data are representative of two independent experiments and shown as mean ± SEM (n = 4 mice per group). One-way ANOVA with Dunnett’s multiple comparisons test was used to compare large intestine or small intestine to the other organs. (C) Migration index of MIT-GPR15-transduced iTregs from Ahr^−/− (CD45.2/CD45.2) and MIT-EV-transduced iTregs from Ahr^+/+ (CD45.1/CD45.1) mice in different organs 10 h post transfer. The migration index was calculated using the following formula: migration index = (ratio of Ahr^−/− transduced with MIT-GPR15/ Ahr^−/− transduced with MIT-EV Tregs in post-transfer)/(ratio of Ahr^−/− transduced with MIT-GPR15/ Ahr^+/+ transduced with MIT-EV Tregs in input). Data are representative of two independent experiments and shown as mean ± SEM (n = 4 mice per group). One-way ANOVA with Dunnett’s multiple comparisons test was used to compare large intestine or small intestine to the other organs. (D–H) Adoptive transfer of Ahr-deficient iTregs transduced with MIT-GPR15 promoted their homing to the large intestine and suppressed gut inflammation in the α-CD40 colitis model. (D) Absolute Treg cell numbers in the colon of recipient Rag1^–/– mice transferred with MIT-GPR15- or MIT-EV- transduced iTregs from Ahr^f/+ Foxp3^Yfp-Cre or Ahr^f/_ Foxp3^Yfp-Cre mice (n = 3–4 mice per group). (E and F) Hematoxylin/Eosin (H&E) histology sections of representative colon (10×; scar bar: 150 μm) (E) and clinical histology score (F). (G and H) Realtime RT-PCR of proinflammatory cytokine expression (i.e., Tnf and Il1b). Data are representative of two independent experiments and shown as mean ± SEM (n = 3–4 mice per group). Also see fig. S5.