Fig 2: Loss of miR-15a/16-1 but not miR-497/195 promotes a CLL-like phenotype and impairs NK and early B-cell development. (A) Blood of control, miR-15a/16-1fl/fl Vav-Cre, and miR-497/195fl/fl Vav-Cre mice was taken monthly (10–17 months), and the percentage of CD5+ B cells out of total CD19+B220+ B cells was quantified by flow cytometry. Individual measurements are connected by black lines. (B) Statistical analysis for the percentage of CD5+ B cells in the spleen and spleen weight of the respective mouse genotypes at an age of 17 months. Each dot represents an individual mouse (controlVav-Cre, n = 3; miR-15a/16-1fl/fl Vav-Cre, n = 5; miR-497/195fl/fl Vav-Cre, n = 4). (C) NK cells defined by NK1.1 expression were classified as stage I to stage IV based on their expression of CD27 versus CD11b. (D) Percentages of NK stage I to stage IV cells in controlVav-Cre (n = 9), miR-15a/16-1fl/fl Vav-Cre (n = 6), and miR-497/195fl/fl Vav-Cre (n = 8) mice. (E) Representative FACS plot illustrating the gating of pro-B and pre-B (AA4.1+IgM-), and immature (AA4.1+IgM+) and mature B cells (AA4.1-) in the bone marrow. (F) Bar graphs show the percentage of pro- and pre-, immature, and mature B cells within the B220+CD19+ B-cell population (bone marrow) of controlVav-Cre, miR-15a/16-1fl/fl Vav-Cre, and miR-497/195fl/fl Vav-Cre mice (n = 6). Error bars depict the standard deviation of the mean. **P < 0.01; ***P < 0.001.

Fig 3: SNX25 in dermal macrophages regulates Ngf level.(a) A representative immunoblot showing NGF levels in the hind paw skin of Snx25fl/fl mice and Snx25Cx3cr1-cKO mice after two-week TAM treatment (schedule was shown in upper panel). Semi-quantitative analyses of the NGF levels are shown (n = 5, p = 0.059). (b) Flow cytometry strategy to sort dMacs, dMonos and dDCs from the back skin of mice using CD11b, CD24, Ly6C, CD64, MHCII, and lineage (CD3, CD19, NK1.1, TER119, Ly6G) marker expression. (c) Proportion of myeloid population (dMacs, dMonos, and dDCs) (% each cell / CD45+ CD11b+ Lin- × 100) in the back skin of WT or Snx25+/- mice (WT: n = 4; Snx25+/-: n = 6). dMacs, p = 0.577; dMonos, p = 0.289; dDCs, p = 0.898. (d) Expression patterns of Snx25 and Ngf in dMonos of WT and Snx25+/- mice (WT: n = 3; Snx25+/-: n = 3). Snx25, p = 0.009; Ngf, p = 0.328. (e) Expression patterns of Snx25 and Ngf in dMonos of Snx25fl/fl and Snx25Cx3cr1-cKO mice (Snx25fl/fl: n = 3; Snx25Cx3cr1-cKO: n = 3). Snx25, p = 0.748; Ngf, p = 0.988. (f) Expression patterns of Snx25 and Ngf in dDCs of WT and Snx25+/- mice (WT: n = 4; Snx25+/-: n = 4). Snx25, p = 0.001; Ngf, p = 0.256. (g) Expression patterns of Snx25 and Ngf in dDCs of Snx25fl/fl and Snx25Cx3cr1-cKO mice. (Snx25fl/fl: n = 3; Snx25Cx3cr1-cKO: n = 3). Snx25, p = 0.062; Ngf, p = 0.804. Results are represented as mean ± SEM. Statistical analyses were performed using the two-tailed Student’s t-test (c), Welch’s t-test (a, d, e, f, and g). *p < 0.05, **p < 0.01. n.s., not significant. Source data

Fig 4: BTB domain And CNC Homolog 2 (Bach2) expression at different natural killer (NK) cell developmental stages by analysis of Bach2Flag knock-in mouse.(A) Histogram plots of Bach2Flag expression (red) in common lymphoid progenitor (CLP), pre-NK progenitor (pre-NK), and refined NK progenitor (rNKp) as compared to cells from wild-type (WT) mice (gray fill). The geometric MFI (gMFI) number was indicated in the plot. Summary of gMFI was shown (n=5 for each group in three independent experiments). (B) Histogram plots of Bach2Flag expression (red) in NK cell (CD3-CD19-NK1.1+) from bone marrow (BM) and spleen as compared to cells from WT mice (gray fill). Numbers indicate gMFI and summary of gMFI is shown (n=7 or 9 for each group in three to four independent experiments). (C) CD3-CD122+ NK cells from BM and spleen were subdivided into iNK (DX5-NK1.1+) and mNK (DX5+NK1.1+) and analyzed for Bach2Flag expression (red for iNK, blue for mNK) as compared to cells from WT mice (gray fill). Numbers indicate gMFI and summary of gMFI is shown (n=7 for each group in three to four independent experiments). (D) Histogram plot of Bach2Flag expression in CD27+CD11b- (red), CD27+CD11b+ (blue), and CD27+CD11b+ (yellow) NK subsets as compared to cells from WT mice (gray fill). Note that WT plot represents expression of Bach2Flag from CD27-CD11b+ subsets. Numbers indicate gMFI and summary of gMFI is shown (n=6 for each group in three independent experiments). (E) Splenic NK cells were sorted into CD27+CD11b-, CD27+CD11b+ and CD27-CD11b+ subsets. Bach2Flag expression in the subsets was detected using Anti-FLAG M2-Peroxidase (HRP) antibody by western blot. Expression of Actin was used as an internal control. Two individual experiments have been done with one mouse each time. (F) Histogram plot of Bach2Flag expression in splenic NK cell (CD3-CD19-NK1.1+) from mice at 2 weeks’ (red), 5 weeks’ (blue), and 12 weeks’ (orange) age as compared to cells from WT mice (gray fill). Note that WT plot represents expression of Bach2Flag from 12-week age mice. Numbers indicate gMFI and summary of gMFI fold change is shown (n=6 or 11 for each group in three to four independent experiments). (G) Summary of gMFI data for Bach2Flag expression in NK cells from lymph node, spleen, lung, and liver (n=4 or 6 for each group in three independent experiments). NK cells from lymph node, spleen, and lung were gated as CD3-CD19-NK1.1+. NK cells from liver were gated as CD3-CD19-NK1.1+DX5+. Statistical significance was determined by two-way ANOVA (A, C, D, and G), one-way ANOVA, (F) or by Student’s t test (B). Error bars indicate SD. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. ns, not significant. See Figure 1—figure supplement 1 for gating strategies. Figure 1—source data 1.Bach2 expression in different subsets by western blot.Splenic NK cells were enriched from splenocytes of Bach2Flag mice. Enriched NK cells (CD3-NK1.1+) from Bach2Flag mice were further sorted into CD27+CD11b-, CD27+CD11b+, and CD27-CD11b+ subsets. Bach2 expression in the subsets was detected using Anti-FLAG M2-Peroxidase (HRP) antibody by western blot. Expression of Actin was used as an internal control. Splenocytes from wild-type (WT) mice were used as negative control. Splenocytes from Bach2Flag mice were used as positive control. Two individual experiments have been done with one mouse each time.

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.