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: Bach2 deficiency increases natural killer (NK) cells with terminally differentiated phenotype.Representative flow cytometry plots show NK cells (CD3-CD19-NK1.1+NKp46+) separated into maturation stages by CD27 and CD11b expression. The percentage of CD27+CD11b-, CD27+CD11b+, CD27-CD11b+ NK cells among all NK cells (left, representative flow plots; right, bar graph summary) from bone marrow (BM) (A), spleen (C), lymph node (E), liver, (G) and lung (I) in Bach2cKO mice and control mice were plotted (n=5–8 for each group from three independent experiments). Note that NK cells from liver were gated on CD3-CD19-DX5+NK1.1+NKp46+. Summary of the total number of NK cells from BM (B), spleen (D), lymph node (F), liver (H), and lung (J) in Bach2cKO mice and control mice were plotted (n=5–8 for each group from three independent experiments). Statistical significance was determined by two-way ANOVA (A, C, E, G, and I) or by Student’s t test (B, D, F, H, and J). Error bars indicate SD. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. ns, not significant.

Fig 4: RNA-seq analysis reveals BTB domain And CNC Homolog 2 (Bach2) deficiency in natural killer (NK) cells promotes the terminal maturation of NK cells with elevated effector function.(A) Heatmap of differentially expressed genes in NK cells compared between control and Bach2cKO mice in RNA-seq analysis (FDR < 0.01 and log2 fold change >1.5). Each column represents total splenic CD3-CD19-NK1.1+NKp46+ cells from an individual mouse. Three biological replicates per group from two individual sorting experiments are shown. The data were analyzed with the Database for Annotation, Visualization and Integrated Discovery (DAVID) Gene Ontology (GO) analysis for the biological process using the genes differentially expressed from NK cells between control and Bach2cKO mice. (B) Volcano plot shows differential gene expression between control and Bach2cKO splenic NK cells. Highlighted are genes discussed in the text. (C and D) RNA expression of indicated genes in Bach2cKO mice or control mice determined by quantitative PCR (qPCR). Data are shown with four mice per group from two independent experiments. (E) Summary of the percentage of NK cells expressing the indicated genes in Bach2cKO mice or control mice (n=7 or 8 for each group in three independent experiments). Representative histogram plots show the expression of indicated protein in NK cells from Bach2cKO mice (red) as compared to NK cells from control mice (gray fill). (F) Summary of the geometric MFI (gMFI) of indicated gene in total NK cells in Bach2cKO mice or control mice (n=5–8 for each group in three independent experiments). Representative histogram plots show the expression of indicated proteins in NK cells in Bach2cKO mice (red) as compared to NK cells from control mice (gray fill). (G and H) Gene set enrichment analysis (GSEA) illustrating enrichment of effector-like (G) and stem-like (H) gene signatures in Bach2cKO and control splenic NK cells. Statistical significance was determined by Student’s t test. Error bars indicate SD. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. ns, not significant.

Fig 5: 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