Fig 2: GPR183 Expressed on Innate Immune Cells Promotes Colitis(A) Colonic Ch25h, Cyp7b1, and Hsd3b7 mRNA expression in Rag1-/- mice injected with 100 µg CD40 Ab (n = 6–7). d, day.(B) Transwell migration of GPR183-transduced B cell line (M12-GPR183-GFP) to colon homogenates from CD40-Ab-treated Rag1-/- mice (n = 4). Chemotaxis of GFP- M12 cells was used as a negative control.(C) Correlation of CH25H, CYP7B1, and HSD3B7 with CXCL8 mRNA expression in the colon of healthy controls (black dots; n = 8) and patients with ulcerative colitis (red dots; n = 6).(D) Immunofluorescence microscopy of proximal colon from Rag1-deficient Gpr183GFP/+ mice 7 days after CD40 Ab injection. Sections were co-stained for detection of nuclei (DAPI) and myeloid cells (CD11c) or ILCs (CD90.2). Inflammatory foci are shown. Scale bars (red) represent 100 µm.(E) H&E staining of proximal colon from Rag1-deficient Gpr183+/+ and Gpr183-/- mice 7 days after CD40 Ab treatment. Inflammatory foci at the tip of colonic folds are indicated. Scale bars (blue) represent 500 µm.(F) Number of inflammatory foci and colitis score in CD40-Ab-treated Gpr183+/+Rag1-/- and Gpr183-/-Rag1-/- mice (n = 7–15). PBS-treated Gpr183+/+Rag1-/- mice were used as controls.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 experiments.

Fig 3: Assessment of the role of Foxp3 feedforward loop in maintaining Foxp3 transcription and Treg cell fitness(A) Schematic of Foxp3gfp and Foxp3LSL knockin mice.(B) Experimental procedures for Thy1.1 induction and stability assays in vitro.(C) Stability of Foxp3 expression in iTreg cells and Thy1.1 expression in wannabe iTreg cells pretreated with or without ASC. Data show means + SEMs of triplicates and represent 2 experiments.(D) Quantification of nascent Thy1.1 transcripts in wannabe iTreg cells upon acute JQ1 treatment by EU pulse labeling and RT-qPCR. Data represent 2 experiments. Unpaired, two-tailed t test.(E) Experimental procedures for assessing the stability of Thy1.1 expression in wannabe iTreg cells in vivo.(F and G) Thy1.1 expression in recovered live CD45.1-CD4+TCRß+ cells. n = 5 each group. LN, lymph node; MLN, mesenteric lymph node. Data represent 2 experiments. Mann-Whitney test.(H–K) Assessment of the stability of Foxp3 or Thy1.1 transcription in nTreg or wannabe Treg cells in vivo. The percentages of GFP+ or Thy1.1+ cells among live CD45.1-CD4+TCRß+ cells (H, I) and CD25 (J) and CTLA-4 (K) MFIs were calculated in live GFP+ or live Thy1.1+ cells. n = 6 each group. Data represent 2 experiments. Mann-Whitney test.See also Figure S6.

Fig 4: VRC01-class BGC cell kinetics in response to MD39-GT3.1(A) Schematic of the experiment. Day 21 corresponds to either day 21 or day 22, depending on the replicate. Control: N = 3, n = 5; HYCAP1: N = 1, n = 5; HYCAP3: N = 3, n = 5.(B) Representative flow plot showing BGC VRC01gHL gating on day 10, from a HYCAP3 T cell recipient mouse. Cells were gated as in Figure 1C.(C) Representative flow plot showing the AIM gating strategy from a HYCAP1 T cell recipient mouse on day 10. Cells are gated on CD4+ T cells. Frequencies shown in the AIM+ (CD40L+CD69+) gates shows the percentage of AIM+ cells among total CD4 T cells. Control: N = 2, n = 5; HYCAP1: N = 1, n = 5; HYCAP3: N = 2, n = 5.(D) Frequency of total BGC cells.(E) Percentage of VRC01gHL cells among BGC cells; gated as CD45.1+ or CD45.1-CD45.2+, depending on the congenic marker of transferred VRC01gHL cells.(F) Number of BGC VRC01gHL cells in the spleen, back-calculated using the number of total splenocytes.(G) Total MD39-specific CD4 T cell responses, gated as in (C).(H) MD39-specific TFH responses, gated as in (C).(I) Percentage of HYCAP (CD90.1+TVß12+) cells among total MD39-specific TFH cells.(J) Area under the curve (AUC) analysis of total serum response to the trimer with and without RM19R competition.Pooled data from 3 experiments are shown in (D–F). Pooled data from 2 experiments are shown in (G–I). Mean and standard deviation are shown for graphs on linear scales. Geometric mean and geometric standard deviation are shown for data graphed in log scale. Two-tailed Student’s t test: ns, p > 0.05; *p <0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Fig 5: 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.