Fig 2: Loss of Rorc in T cells does not Impact Depressive- or Anxiety- like Behaviors in Female Mice. No differences in (A) depressive-like (n = 12/group) or (B) anxiety-like (n = 12/group) behaviors between female Rorc KO and littermate controls at baseline. No differences in the (C) Kullback–Leibler divergence heatmap or (D) PCA plot of DeepLabCut analyzed baseline behaviors between groups (n = 12/group). (E) Depressive-like (n = 12/group) and (F) anxiety-like (n = 12/group) behaviors after 3 weeks of UCRS between female Rorc KO and littermate controls (Nestlet Shred: p = 0.0392, Elevated Plus Maze: p = 0.0453). (G) Kullback–Leibler divergence heatmap and (H) PCA plot of DeepLabCut analyzed behaviors after 3 weeks of UCRS between groups (n = 12/group). T tests, N = 1, female mice.

Fig 3: GPR183 and 7a,25-OHC Promote ILC3 Localization to CPs and ILFs(A) Distribution of Gpr183+/+ and Gpr183-deficient hematopoietic cells in the colon of mixed bone marrow chimeras. Bone marrow cells from Gpr183+/+ or Gpr183-/- mice (CD45.2+) were mixed 9:1 with B6 cells (CD45.1+) and injected into irradiated Rag1-/- recipients (CD45.1+) for the generation of bone marrow chimeras. Sections were stained for detection of Gpr183+/+ and Gpr183-/- cells (CD45.2, red) or B6 cells (CD45.1, green). Nuclei were visualized by DAPI staining (blue). Scale bars on the right (white) represent 100 µm.(B) Distribution of Gpr183+/+ and Gpr183-deficient ILCs in the colon of mixed bone marrow chimeras. Bone marrow cells from Gpr183+/+ or Gpr183-/- mice (CD90.2+) were mixed 9:1 with B6 cells (CD90.1+) and injected into irradiated B6 recipients (CD90.1+) for the generation of bone marrow chimeras. Colon sections were stained for detection of Gpr183+/+ and Gpr183-/- (CD90.2, red) or B6 (CD90.1, green) ILCs. Scale bars (red) represent 100 µm. The lower panel shows the number of clusters in Gpr183+/+-B6 and Gpr183-/--B6 chimeras consisting of CD90.2+ (red) or CD90.1+ (green) ILCs. Data are represented as means ± SEM. p value by two-way ANOVA.(C and D) Distribution of donor-derived ILC3s (ROR?t-GFP+, green) in the colon (C) and small intestine (D) of bone marrow chimeras. Bone marrow cells from Rag1-deficient Rorc(?t)GFP transgenic mice were injected into irradiated Ch25h+/+ or Ch25h-/- recipients for the generation of bone marrow chimeras. Sections were co-stained for detection of B cells (B220+, red). Scale bars (red) represent 100 µm. The lower panel in (C) shows the number of donor-derived ILC3s in the colon of Ch25h+/+ or Ch25h-/- hosts. Data are representative of or combined from two (B–D) or three (A) experiments. See also Figure S5.

Fig 4: ILC3-Expressed GPR183 Is Required for the Formation of Colonic Lymphoid Tissues(A) Distribution of GFP+ cells in the small intestine and colon of Gpr183GFP/+ mice. Tissue sections were co-stained with a-CD90.2 and a-B220 Abs. Scale bars (white) represent 100 µm.(B) Number of CPs and ILFs in the small intestine and colon of Rorc(?t)GFPGpr183+/+ and Rorc(?t)GFPGpr183-/- mice (n = 3–5). The upper panel shows representative images of a CP and an ILF from Rorc(?t)GFPGpr183+/+ mice. Scale bars (red) represent 100 µm.(C) Number of peripheral and mesenteric lymph node cells (n = 6–8), Peyer’s patches (n = 10), and colonic patches (n = 3) from Gpr183+/+ and Gpr183-/- mice.(D) Number of ROR?t+ clusters in the colon of bone marrow chimeras (n = 5–9). Bone marrow cells from Gpr183+/+ or Gpr183-/- mice were injected into either Gpr183+/+ or Gpr183-/- irradiated recipient mice.(E) Number of CPs in the colon of Rag1-deficient Gpr183+/+ and Gpr183-/- mice (n = 5).(F) Number of CPs, ILFs, Peyer’s patches, and colonic patches in Rorc-cre Gpr183flox/flox mice and Gpr183flox/flox or Gpr183flox/+ controls (n = 3–6).Data are represented as means ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 by Student’s t test or one-way ANOVA with Tukey’s post-test (D). Data are representative of or combined from two (F) or three (A–E) experiments. See also Figures S3 and S4.

Fig 5: LTi-like ILC3s Highly Express GPR183 and Migrate toward 7a,25-OHC(A) Gpr183 mRNA expression in the indicated cell populations from the spleen (n = 2–6). mRNA expression was normalized to Hprt.(B) GFP expression in lamina propria B cells and ILC subsets from the colon of Gpr183GFP/+ reporter mice (green histograms) and B6 control mice (gray histograms).(C) Left panel illustrates high GPR183-GFP expression in CD4+ LTi-like ILC3s from the colon. Right panel shows mean fluorescence intensity (MFI) of GPR183-GFP expression in the indicated cell populations from (B) (n = 6).(D–F) Transwell migration of splenic LTi-like ILC3s (Lin-CD90.2+CD127+NK1.1-) from Rag1-deficient Gpr183+/+ and Gpr183-/- mice (D), splenic B cells from B6 mice and ILC subsets from Rorc(?t)GFPRag1-/- mice (E), and colonic ILC subsets from Rag1-/- mice (F) to 7a,25-OHC (n = 2–3).Data are represented as means ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 by one-way ANOVA with Tukey’s post-test. Data are representative of or combined from two (D and E) or three (A–C and F) experiments. See also Figures S1 and S2.