Fig 2: Immunophenotyping of 1D3-CD19CAR-GFP (blue, circle), FMC63-CD19CAR-GFP (red, square), or control (GFP only, green, triangle) transduced T cells in peripheral blood of treated miceValues shown are the percentage of total CD3+, CD90.2+ T cells represented by each subtype (effector = CD44high, CD62Llow CD127low; effector memory = CD44high, CD62Llow CD127high; central memory = CD44high, CD62Lhigh CD127high; intermediate phenotype = CD44high CD62Lhigh CD127low; naive = CD44low, CD62Lhigh CD127high; regulatory = CD4+FOXP3+). N equals eight mice per group and error bars represent the SD from the mean value reported for each group. (A) At week 5, the 1D3-CD19CAR-GFP T cells were predominantly CD8+ effector memory T cells (5.1% ± 0.9% of total CD3+ T cells), effector T cells (1.7% ± 0.2% of total CD3+ T cells), and CD4+ intermediate T cells (1.3% ± 0.2% of total CD3+ T cells). At week 5, the majority of FMC63-CD19CAR-GFP T cells were CD8+ effector memory T cells (2.0% ± 0.2% of total CD3+ T cells) and effector T cells (0.7% ± 0.1% of total CD3+ T cells). (B) At week 8, the majority of 1D3-CD19CAR-GFP T cells were effector memory T cells (0.7% ± 0.6% of total CD3+ T cells) and CD8+ effector T cells (1.3% ± 0.4% of total CD3+ T cells). The majority of FMC63-CD19CAR-GFP T cells at week 8 were CD8+ effector memory T cells (2.60% ± 0.538% of total CD3+ T cells) and effector T cells (1.1% ± 0.6% of total CD3+ T cells). GFP-expressing CD4+ FOXP3+ regulator T cells were present at both time points but comprised a small percentage (<1%) of total T cells. Flow gating strategy outlined in Figure S5 and data in Table S5.

Fig 3: The abundance of immune cell subsets in treated mice over time, as a percentage of total PBMCsC57BL/6 wild-type mice were treated with a single intravenous injection of lentivirus (3.6–4.0 × 106 IU in 200 µL of PBS), to deliver either the 1D3-CD19CAR-GFP CAR, FMC63-CD19CAR, or control (GFP only). N equals eight mice per group and error bars represent the standard deviation (SD) from the mean value reported for each group. T cells, but not other immune cell subsets tested, show a substantial transduced cell population. (A) Representative flowgrams showing increasing GFP-positive T cells in PBMCs of treated mice, over time. The x axis of the flowgrams is CD3 positivity and GFP positivity on the y axis. (B–E). (B) Total T cells versus 1D3-, FMC63-CD19CAR, and GFP-transduced T cells (CD3+, CD90.2+ T cells). (C) Total B cells versus 1D3-, FMC63-CD19CAR, and GFP-transduced B cells (CD20+, CD45R/B220+ B cells). (D) Total macrophage versus 1D3-, FMC63-CD19CAR, and GFP-transduced macrophage (CD11b+ macrophage and other non-T cells). (E) Total NK/NK-T cells versus 1D3-, FMC63-CD19CAR, and GFP-transduced NK/NK-T cells (CD335+ NK/NKT cells). Flow gating strategy outlined in Figure S4 and data in Table S3 and S4.

Fig 4: 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.

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.