GPR15 mediates accumulation of TEM and Treg cells in the colon. (a) Gpr15–KO (CD45.2) or het (CD45.2) bone marrow cells were combined with Gpr15-het (CD45.1) cells and transferred into irradiated hosts (CD45.1 × CD45.2 F1) to generate experimental (Exp) or control (Ctrl) chimeras, respectively, as in . Ratios of Gpr15–KO to Gpr15–het derived cells (CD45.2/CD45.1) among Treg cells (CD4+, CD25hi, Foxp3+; top), Treg–excluded TEM cells (CD4+, Foxp3−, CD44hi, CD45RBlo; middle), and naive CD4+ T cells (CD4+, Foxp3−, CD44lo, CD45RBhi; bottom) in Exp mixed bone marrow chimeras (white bars); ratios of Gpr15- het to het cells (CD45.2/CD45.1) in control chimeras (black bars). Peripheral lymph nodes (PLN), mesenteric lymph node (MLN), Peyer’s patches (PP), small intestine (SI). n = 3, mean + SEM, **p <0.01 (2–way ANOVA, Bonferonni posttest). Representative of 3 experiments. (b) Splenocytes from DO11.10 Gpr15–het or DO11.10 Gpr15–KO were i.v. injected into Balb/c (Thy–1.1) mice. After 24 h, recipients were treated intra-rectally with ovalbumin (OVA) and cholera toxin (CT). Accumulation of OVA-specific CD4+ T cells in tissues was assessed 5 days later, as in . The percent antigen–specific (DO11+) of Gpr15-het or KO CD4+ TEM cells (CD44hi, CD45RBlo) 5 days after antigen exposure was normalized to the ratio in the spleen. Data from 9 mice from 3 independent experiments are shown. Mean ± SEM, *p <0.05 (2–way ANOVA, Bonferroni posttest). (c) Similar proliferation rate of antigen–reactive Gpr15–het and Gpr15–KO cells. CFSE–labeled splenocytes from Gpr15–het (Thy–1.1, Thy–1.2) and KO (Thy–1.2) DO11 mice were co–injected i.v. in equal proportion into congenic non–DO11 hosts (Thy–1.1) on day 0. Recipients were treated with OVA and CT per rectum on day 1, FTY720 or PBS i.p on day 3, and tissues were collected on day 5, as in . (d) Recipient MLN gated on donor DO11+ CD4+ T cells. n = 3; mean + SEM. Not significant (NS).