Fig 2: Following TLR stimulation, origin determines gene expression, chromatin profile and IL-6 gene accessibility.AMs were FACS-sorted based on congenic markers from the lungs of naïve and post-influenza Busulfan chimeras, and stimulated ex vivo with Pam3CSK4. PCA plot of (a) RNA-seq data and (b) ATAC-seq data from unstimulated and Pam3CSK4-stimulated CD45.2 (Ccr2-/-) resident and CD45.1 recruited AMs. c,d, Top 5 pathways (FDR q-val <0.01) from GSEA of Pam3CSK4 stimulated AMs from post-influenza lungs. NES = Normalized Enrichment Score. Representative GSEA plots are shown, with the top 10 highest ranked genes from each GSEA listed. e, ATAC-seq data as in (b) shown as heatmap of mean centred k-means clustering of rlog values generated by DESeq2 for differentially accessible intervals found to be significant (FDR ≤ 0.01) in at least one pairwise comparison. f, GREAT analysis of ATAC-seq-derived intervals from each cluster in (e). g,h, IGV tracks of ATAC-seq data for AMs, showing the IL-6 locus and upstream peaks. Significantly differentially accessible intervals in the comparison Flu_resident_Pam vs Flu_recruited_Pam are marked using bars. ImmGen chromatin tracks from lung macrophages and blood Ly6Chi monocytes are shown underneath (h). All tracks were group-autoscaled to enable comparison.

Fig 3: Zfp335 deficiency promotes thymocyte apoptosis in vivo and in vitro.(A, B) Thymi were harvested from 6- to 8-week-old WT and KO mice. The expression of BrdU in DN3 and DN4 thymocytes from WT and KO thymi was measured by flow cytometry (n = 3). (A) Representative FACS plots of BrdU expression in DN3 and DN4 thymocytes. (B) The percentages of BrdU+ thymocytes in DN3 and DN4 cells. (C, D) The binding of Annexin V in DN3 and DN4 thymocytes from WT and KO thymi was measured by flow cytometry (n = 3). (C) Representative FACS plots of Annexin V binding in DN3 and DN4 thymocytes. (D) The percentages of Annexin V+ DN3 and DN4 cells. (E, F) A mixed population of WT and KO DN3 thymocytes at a 1:4 ratio was cocultured with OP9-DL1 feeder cells in vitro in the presence of IL-7 (1 ng/ml) and Flt3L (5 ng/ml) for 4 days. The binding of Annexin V was measured by flow cytometry (n = 3). (E) Representative FACS plots of Annexin V+ DN3 and DN4 cells. (F) The percentage of Annexin V+ DN3 and DN4 cells. (G, H) Full chimeric mice were generated by transplanting a mixed population of WT (CD45.1+) and KO (CD45.2+) bone marrow progenitor cells at a 1:4 ratio into lethally irradiated WT recipient mice (CD45.1+CD45.2+) with 8.5 Gy. Five weeks after transplantation, thymi from recipient mice were harvested. The binding of Annexin V was measured by flow cytometry (n = 4). (G) Representative FACS plots of Annexin V+ DN3 and DN4 cells. (H) The percentage of Annexin V binding in DN3 and DN4 thymocytes. Results represent three independent experiments. **p < 0.01 and ****p < 0.0001. Figure 4—source data 1.Figure 4B. The percentages of BrdU+ thymocytes in DN3 and DN4 cells from WT and KO mice.Figure 4D. The percentages of Annexin V+ DN3 and DN4 cells from WT and KO mice. Figure 4F. The percentage of Annexin V+ DN3 and DN4 cells 4 days post coculture in vitro. Figure 4H. The percentage of Annexin V+ DN3 and DN4 cells in chimeric mice cotransferred with WT and KO bone marrow cells.

Fig 4: Analysis of skin-infiltrating T cells in IMQ-treated K14-I?B?–KO mice.All analyses were performed after 7 days of IMQ treatment. (A) Flow cytometry analysis of T cell subsets in the ears of IMQ-treated Ctrl and K14-KO mice. T cell subsets were detected as CD45+ and CD3+, aßTCR+, or ?dTCR+ cells. Single data points derive from 2 ears. Shown is the mean of 4–12 mice per group ± SEM. (B) Gene expression analysis of Il22 and Il17a in skin tissue of untreated and IMQ-treated control and K14-KO mice, normalized to the reference gene Actin. n = 4–14 ± SEM. (C) Determination of the percentage of IL-17A– and IL-22–producing aß and ?d T cells in IMQ-treated control and K14-KO mice. After fixation and permeabilization, cells were gated as in A, except for an additional gating on either IL-17A+ or IL-22+ cells. n = 3 ± SEM. (D) Gene expression analysis of Il1b and Il23a in untreated and IMQ-treated mice, similar as in B. (E) mRNA levels of Ccl2, Ccl17, and Ccl20 in untreated mice, similar as in B. (F) mRNA levels of Ccr2, Ccr4, and Ccr6 in the skin of untreated control and K14-KO mice, similar as in B. n = 5–14 ± SEM. P values were calculated using 2-tailed Student’s t test (*P < 0.05, **P < 0.01, and ***P < 0.001).

Fig 5: Recruited macrophages are distinct in their expression and chromatin profile, relative to resident macrophages at one month post-influenza.RNA-seq and ATAC-seq of AMs FACS-sorted from the lungs of naïve and post-influenza Busulfan chimeras. a, Heat map of genes differentially expressed (FC>1.5, 1-way ANOVA with Benjamini-Hochberg correction, p<0.05) between CD45.2 (Ccr2-/-) resident and CD45.1 recruited AMs. b, Principal component analysis (PCA) plot of RNA-seq data from resident and recruited macrophages from post-influenza and naive lungs of Ccr2-/- Busulfan chimeras. c,d, Gene set enrichment analysis (GSEA) of Flu resident (CD45.2) vs Flu recruited (CD45.1) macrophages using clusters from Lavin et al.30 representing (c) lung macrophages and (d) blood monocytes. e,f, IGV tracks from ATAC-seq data; depicting chromatin in unstimulated resident and recruited macrophages from naïve and post-influenza lungs of Ccr2-/- Busulfan chimeras. All tracks were group-autoscaled to enable comparison. g, PCA plot of ATAC-seq data from unstimulated resident and recruited macrophages from naïve and post-influenza lungs.