Fig 2: Ischemia Increases Brain CD11c-eYFP+ Cells and Brain Flt3L(A) Brain eYFP+ cells increase 4 days post-ischemia in CD11c-eYFP mice. Flow cytometry values are percentages of viable cells. Mann-Whitney test, **p = 0.001, n = 5 controls, n = 10 ischemic mice.(B) Left image: coronal brain section of a CD11c-eYFP mouse (n = 6) showing eYFP+ cells (green) in the ischemic hemisphere (right); scale bar, 100 µm. Right image: higher magnification showing eYFP+ cells in parenchyma; blood vessels (Glut1+, red); nuclei (To-Pro3, blue); scale bar, 10 µm.(C) In control, eYFP+ cells are seen in ventricular region and rostral migratory pathway. After ischemia, eYFP+ cells increase in the parenchyma. Scale bar, 30 µm.(D) Mice received recombinant mouse Flt3L (10 µg/mouse) or vehicle subcutaneously (s.c.) for 7 days. Ischemia was induced 3 days after treatment onset, and tissue was studied by flow cytometry 4 days post-ischemia. Flt3L increased the percentage of CD11c+MHCII+ cells in spleen (Mann-Whitney test, **p = 0.001, n = 7 vehicle, n = 11 Flt3L) and ischemic brain (Mann-Whitney test, *p = 0.02, n = 7 vehicle, n = 9 Flt3L). Values are CD11c+MHCII+ cells expressed as percentages of CD45+ cells or CD45hiCD11bhi cells.(E) Brain Flt3L mRNA (Flt3lg) expression increases after ischemia (h, hours; d, days). One-way ANOVA and Dunnett’s multiple comparison test, ***p < 0.001 versus control, n = 3–6 mice per time point.(F) Flt3g mRNA in microglia, astroglia, and endothelial cells sorted from control and ischemic brains (day 4) (n = 3 or 4 samples per cell type and condition). Values are expressed as fold increase versus control microglia. Astrocytes show the highest Flt3lg mRNA expression versus microglia (***p = 0.0003) and endothelial cells (***p < 0.0001), and microglia show higher expression than endothelial cells (two-way ANOVA, p < 0.001, Sidak’s multiple-comparisons test, ***p = 0.004). Ischemia 'I' increases astrocyte Flt3lg mRNA vs. control 'C' (&p = 0.049).(G) Confocal microscope brain images of CD11c-eYFP mice (n = 3–6 mice per group). eYFP+ cells (green) are located near reactive GFAP+ astrocytes (red) at infarct periphery. Scale bar, 10 µm. Bars show mean ± SEM and symbols are values per mouse.See also Figures S1 and S2.

Fig 3: 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 4: Reduced BCR Levels on DZ GC B Cells(A and B) Mice were immunized with SRBCs and splenic IgDlow CD95+ GL7+ GC B cells were analyzed on day 7. (A) BCR levels on DZ populations (CXCR4high) were analyzed and compared to B220neg non-B cells. The gate indicates a BCRlow DZ population. (B) DZ and LZ cells are gated as shown and BCR levels overlaid with IgD+ CD95low follicular B cells.(C–G) HEL-specific SWHEL or MD4 B cells were adoptively co-transferred with OT-II CD4+ T cells and mice were immunized with HEL-OVA/adjuvant adjuvant. Splenic IgDlow CD95+ GL7+ CD45.1+ GC B cells were analyzed on day 7. (C) Surface and intracellular BCR (IgMa) levels were co-stained in fixed/permeabilized MD4 GC B cells revealing a subset of cells that is negative for both. (D and E) DZ cells with low BCR levels in MD4 transgenic (D) and SWHEL gene targeted GC B cell populations (E). (F and G) Single CXCR4high BCRhigh and CXCR4high BCRlow DZ SWHEL GC B cells (gated IgDlow, CD38low, GL7+, CD45.1+, or CD45.2+) were index sorted and cultured for 8 days on NB21 feeder cells. (F) The BCR and CXCR4 staining profiles (at the time of sort) of single GC B cells that grew and secreted detectable antibody after culture are shown. The plots for cells that did not grow, or that grew did not secrete detected antibody, are also shown and overlaid with the total GC B cell population for reference. Plots for additional mice are shown in Figure S1. (G) Summary of results. Numbers of cells in each group per experiment are indicated.(H) Surface BCR levels on Aicda+/+ and Aicda-/- IgDlow CD95+ GL7+ GC cells from mixed bone marrow chimeric mice 8 days after SRBC immunisation. Numbers in (C–E) and (H) represent mean ± SEM from 7 (C and D), 10 (E) and 11 (H) mice from 2–4 experiments. See also Figure S1.

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.