Fig 2: Myeloid-specific MafB-deficient mice exhibit drastic changes in the proportion and phenotype of tissue RTMs.(A) Representative contour plots of MHC-II and F4/80 expression in peritoneal CD45+Lin−Ly6C−CD11b+CD115+ cells from Mafbfl/fl and Lyz2CreMafbfl/fl mice (left) and percentages of SPM and LPM, as gated on the contour plots (right) (n = 7–9). All subsequent panels compare Mafbfl/fl and Lyz2CreMafbfl/fl mice.(B) Absolute numbers of SPM and LPM, as in A.(C) Histograms (top) and bar graphs (bottom) of MHC-II and RELMα expression in SPM, as in A.(D) Histograms (top) and bar graphs (bottom) of F4/80, MerTK, Tim4, and Vsig4 expression in LPM, as in A.(E) Representative contour plots of F4/80 and CD11b expression in liver CD45+Lin−Ly6C− cells (left) and percentages of F4/80−CD11blo cells, CD11bhi Mo-Macs, and KCs, as gated on the contour plots (right) (n = 8).(F) Absolute numbers of CD11bhi Mo-Macs and KCs, as in E.(G) Histograms (top) and bar graphs (bottom) of MerTK, Clec4F, Tim4, Vsig4, FR-β, and CD163 expression in KCs, as in E.(H) Representative contour plots of CD206 and MHC-II expression in brain CD45+Lin−Ly6C−F4/80+CD64+ cells (left) and percentages of MG and BAM, as gated on the contour plots (right) (n = 6–8).(I) Absolute numbers of MG and BAM, as in H.(J) Histograms (top) and bar graphs (bottom) of P2RY12 and FCRLS expression in MG, as in H.(K) Representative contour plots of CD206 and MHC-II expression in BAM (left) and percentages of MHC-II− and MHC-II+ BAM, as gated on the contour plot (right) (n = 6–8).(L) Absolute numbers of MHC-II− and MHC-II+ BAM, as in K.(M) Representative contour plots of CD206 and CD11b expression in spleen CD45+Lin−Ly6C−F4/80+CD64+ cells (left) and percentages of RPM and CD11b+ mac, as gated on the contour plots (right) (n = 6–7).(N) Absolute numbers of RPM and CD11b+ macs, as in M.(O) Histograms (top) and bar graphs (bottom) of CD163 expression in RPM, as in M.Data are mean ± SEM and are pooled from two independent experiments. P values were calculated using a two-tailed Student’s t test. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. ns, not significant.See also Figures S4 and S5.

Fig 3: MafB binding sites and MafB-dependent regulation of mac identity are highly conserved across species.(A) CUT&RUN tracks depicting MafB binding at the FIRE enhancer within the second intron of the Csf1r gene in BMDMs from Mafbfl/fl and Lyz2CreMafbfl/fl mice (top), and phastCons and phyloP conservation scoring tracks around the FIRE enhancer (bottom), visualized with IGV. Horizontal red bars, MafB binding sites in BMDMs; horizontal blue bar, FIRE enhancer.(B) Genomic distribution of cREs in mice.(C) Overlap between MafB binding sites in BMDMs and cREs.(D and E) Distribution of mean phastCons (D) and phyloP (E) scores around PU.1 and MafB binding sites (−500 to +500 bp from peak center).(F and G) Histograms (left) and heatmaps (right) (F) and scatterplot (G) of normalized tag counts at MafB peaks in MDMs, as quantified by CUT&RUN.(H) Pie chart showing the genomic distribution of MafB binding sites in MDMs.(I and J) Venn diagrams showing the overlap between MafB binding sites (I) and target genes(J) in human MDMs and mouse BMDMs.(K) CUT&RUN tracks depicting MafB binding around representative mac signature genes in MDMs, visualized with IGV. Horizontal purple bars, MafB binding sites in MDMs; horizontal red bars, conserved MafB binding sites in human MDMs and mouse BMDMs, as in I.(L) Overview of cross-species single-cell RNA-seq datasets from mouse (TMS), human (TS), mouse lemur (TM), pig (PCA), clawed frog (XCL), and zebrafish (ZCL), used to calculate correlations of Mafb expression with mac and conserved MafB target gene signature scores, as in Figure S7E–J.(M and N) Heatmaps of Pearson correlations between Mafb expression and either mac (M) or conserved MafB target gene (N) signature scores across species, as in L.P values were calculated using a pairwise Wilcoxon signed-rank test with Benjamini–Hochberg correction (D and E) or a Wilcoxon signed rank exact test (M and N). **, P < 0.01; ****P < 0.0001.See also Figure S7 and Table S4

Fig 4: Elevated MafB expression and activity define mature RTMs in mice(A) Heatmap showing the top 50 genes upregulated in macs compared with monocytes (Mo) and DCs, inferred from microarray data on the Gene Expression Commons platform.(B) UMAP of myeloid cell transcriptional identity from the Tabula Muris Senis (TMS) dataset.(C) Feature plot of Mafb expression, as in B.(D) Violin plot of Mafb expression across myeloid cell clusters, as in C (height, expression; width, cell abundance).(E) Feature plot of MafB activity inferred with decoupleR, as in B.(F) Violin plot of MafB activity across myeloid cell clusters, as in E.(G) Bar graphs of MafB expression in blood Mo (blue) and RTM (red) populations, measured byflow cytometry (n = 4).(H) Bar graphs of MafB expression in DC and RTM populations from indicated organs, measured by flow cytometry (n = 4).(I) Scatter plot of Ly6C and MHC-II expression in colon CD45+Lin−F4/80+CD64+ cells colored by Wanderlust pseudotime (merged from 4 mice).(J) MafB expression along pseudotime, as in I.(K) Scatter plot of Ly6C and F4/80 expression in CD45+Lin−CD11b+CD115+ peritoneal cells colored by Wanderlust pseudotime (merged from 4 mice).(L) MafB expression along pseudotime, as in K.Data are mean ± SEM (G and H). P values were calculated using one-way ANOVA with Tukey’s post hoc test (G), two-tailed Student’s t test (H, spleen, kidney, and colon), or one-way ANOVA with Dunnett’s post hoc test (H and other organs). *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. cDC, conventional DC; C LPM, colonic lamina propria macs; cMoP, common Mo progenitors;DM, dermal macs; GMP, granulocyte–monocyte progenitors; LC, Langerhans cells; LCM, liver capsular macs; LN, lymph node; MFI, mean fluorescence intensity; MLN, mesenteric lymph nodes; Neu, neutrophils; pDC, plasmacytoid DC; pMo, patrolling Mo; RM, resident mac; SI LPM, small intestinal lamina propria macs; SI MM, small intestinal muscularis macs; SLN, skin-draining lymph nodes.See also Figure S1.