GPR15 mediates accumulation of T_EM and T_reg 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 T_reg cells (CD4⁺, CD25^hi, Foxp3⁺; top), T_reg–excluded T_EM cells (CD4⁺, Foxp3⁻, CD44^hi, CD45RB^lo; middle), and naive CD4⁺ T cells (CD4⁺, Foxp3⁻, CD44^lo, CD45RB^hi; 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⁺ T_EM cells (CD44^hi, CD45RB^lo) 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).

GPR15–deficient naive T cells fail to induce colitis in the CD45RB^hi CD4⁺ T cell transfer model. (a) Weight loss of Rag2^−/− mice that received either WT naive CD4⁺ T cells, Gpr15–KO naive CD4⁺ T cells, WT naive plus WT T_reg cells, WT naive plus KO T_reg cells, or KO naive plus WT T_reg cells. n = 5, mean + SEM, *p <0.05, **p <0.01 between WT naive (disease control) and Gpr15 KO naive (2-way ANOVA, Bonferroni posttest). Representative of 3 experiments. (b) Clinical score for histological inflammation in colon. *p <0.05, **p < 0.01, ***p <0.001 (1–way ANOVA, Tukey’s posttest). (c) H&E staining of distal colon section: WT naive (top left), Gpr15–KO naive (top right), WT naive + WT T_reg (middle left), WT naive + Gpr15–KO T_reg (middle right) and Gpr15–KO naive and WT T_reg (bottom). Scale bar denotes 10 μm.

GPR15 deficiency inhibits effector T cell accumulation in the colon, but not T_H1 or T_H17 differentiation. Naive CD45RB^hi CD4⁺ T cells sorted from Gpr15–KO (CD45.2) or het (CD45.1, CD45.2) mice were co–injected into Rag2^−/− recipients in equal proportion, as in . (a) Ratios Gpr15–het to Gpr15–KO GFP⁺ or GFP⁻ CD4⁺ T cells in recipient tissues after 2 weeks, normalized to those in the spleen. n = 5, mean + SEM, *p <0.001. (2–way ANOVA, Bonferroni posttest). (b) Similar percentage of Gpr15–het- and KO-derived IFN-γ and IL–17⁺ CD4⁺ T cells in recipient colon after 2 weeks (left). Ratio of Gpr15– het- to KO-derived IFN–γ⁺ or IL–17⁺ CD4⁺ T cells, normalized to that of the spleen (right). Representative of 2 experiments. (c) BrdU incorporation 2 weeks after cell transfer as in (a); baseline (grey shade). n = 3; mean + SEM.

Human proinflammatory CD4⁺ T cells express GPR15 in the colon. (a) Expression of GPR15 on cytokine–producing CD45RO⁺CD4⁺ T cells from ulcerative colitis (UC) or non–UC colons (top). In UC colons, GPR15 is expressed on IL–5⁺ and IL–13⁺ CD45RO⁺CD4⁺ T cells (bottom). n = 5 UC specimens, 11 Non–UC or normal samples, and 2 Crohn’s disease (CD) colons, which showed similar cytokine expression among GPR15⁺ cells as normal samples. Mean + SEM; *p <0.05, **p < 0.01, ***p <0.001 (2–way ANOVA, Bonferroni posttest). Statistics not determined for CD samples. (b) Lower expression of GPR15 on T_reg cells (CD127^lo, Foxp3⁺; red histogram) as compared to non–T memory CD4⁺ T_reg, cells (CD127^hi, Foxp3⁻; blue).

Distinct patterns of GPR15 expression by in vitro–polarized human and mouse T cells. Naive CD4⁺ T cells enriched from human PBMC (a) or mouse Gpr15–het spleen (b) were polarized as described in the online methods. GPR15 is expressed on human but not mouse in vitro–polarized T_H2 cells. Human cells are gated on CD45RO⁺ CD4⁺ T cells, and mouse cells are gated on CD44^hi CD4⁺ T cells. Combined results of 3 mouse and 4 human experiments; mean + SEM.

Differential binding of GATA–3 to human and mouse Gpr15 enhancer sequences. (a) ChIP–Seq data showing binding of GATA–3 at the human GPR15 locus in T_H2 cells. The number of sequencing reads from ChIP–enriched DNA are plotted per million background–subtracted total reads and aligned with the Hg18 build of the human genome. Association with H3K4me3 and H3K27ac in human T_H1 and T_H2 cells are shown below. The position of the upstream enhancer is marked with a dashed box. The positions of qPCR primers used in (c) are marked by arrows. (b) As (a), except for Gata–3 and H3K4me3 at the mouse Gpr15 locus (Mm9 build). (c) Enrichment of DNA corresponding to the human and mouse upstream and downstream GATA–3 binding sites marked in (a) and (b), relative to input DNA and a control non–bound region (also marked in (a) and (b)), measured by ChIP–qPCR. Mean and SD from triplicate wells from one ChIP per species. *p < 0.05, unpaired Student’s t–test.