Fig 2: Deletion of Srsf1 in Treg cells results in peripheral immune cell activationSpleen cells were isolated from 3- to 4-week-old WT and Treg Srsf1-KO mice and analyzed by flow cytometry.(A) Plots show Thy1.2+TCRβ+ T cells gated on live cells from spleen. Graph on the right shows the percentage of T cells (n = 5 each).(B) Plots show CD62L and CD44 staining gated on live CD4 T cells in spleen and PLN from WT and KO mice.(C) Graphs show the percentage of naive (CD44lo CD62Lhi) and effector/effector memory (CD44hi CD62Llo) cells among CD4 T cells in spleen and PLN of WT and Treg Srsf1-KO (n = 5 each).(D) Spleen cells were stimulated with phorbol myristic acid (PMA) and ionomycin with monensin for 4 h. Plots show IFN-γ- and IL-4-producing CD4 T cells.(E) Graphs show the percentage of IFN-γ- and IL-4-producing CD4 T cells (IFN-γ: n = 7, IL-4: n = 6 each).(F) Plots show GL7+Fas+ germinal center (GC) B cells gated on live B cells in spleen from WT and KO mice. Graph shows the percentage of GC B cells in spleen (n = 5 WT, n = 6 KO).*p < 0.05 and ***p < 0.0005, unpaired t test (A, C, E, and F); mean ± SEM.

Fig 3: Significant strain heterogeneity of respiratory T and B lymphocytes, NK cells, γδ T cells and Linnegleukocytes. Dissection of respiratory lymphocyte subsets suggests elevated CD3 T cell and CD4 T helper cell numbers in BALB/c mice (A, B). CD8+ T cell frequencies are elevated in C57BL/6 (BL6) mice when compared to either C3H or DBA mice (C). Putative T regulatory cell (CD4+CD25+ T cells) numbers are significantly elevated in 129SV mice compared to BL6 or C3H strains (D). Please note that the CD4+CD25+ T cells are not CD25high. A more detailed gating strategy of Treg subsets including isotpye controls is shown in Figure 8. B lymphocyte numbers are significantly elevated in BL6 mice (E). Natural killer (NK) cells and γδ T cell numbers are elevated in C3H and DBA strains (F, G). BALB/c and 129SV mice are rich in Lin-neg CD90+ leukocytes (H) whereas C3H mice exhibit high numbers of Lin-neg CD90neg leukocytes (I). Results are representative for n≥ 8 (A, B, C, F, G) or n ≥ 5 (D, E) independent animals per strain (mean ± SEM; *p<0.05; **p<0.01; ***p<0.001).

Fig 4: HDACi reduce the activation of SASP‐related signaling pathways in old mdx FAPsYC: young mdx control; YT: young mdx in vivo treated with TSA for 15 days; OC: old mdx control; OT: old mdx in vivo treated with TSA for 15 days. Acetylation tracks for H3K9/14ac and H3K27ac ChIP‐seq (on the left) and corresponding FPKM values by RNA‐seq (on the right) for representative genes in YC, YT, OC, and OT FAPs.Graphs showing RNA levels for the representative genes shown in (A) measured by qPCR.Graphs showing the H3K9/14 acetylation levels for the representative genes shown in (A) measured by ChIP‐qPCR.Representative images of the phospho‐NFkB staining in FAPs (pNFkB‐p65 in red/CD90 in green/Laminin in grey/DAPI in blue) on tibialis anterior muscle transversal section of YC, YT, OC, and OT mice; Scale bar = 25 μm.Box plot showing the percentage of pNFkB‐p65‐positive FAPs in the staining shown in (D).Representative images of the phospho‐p38 staining in FAPs (pP38 in red/CD90 in green/Laminin in grey/DAPI in blue) on tibialis anterior muscle transversal sections of YC, YT, OC, and OT mice. Scale bar = 25 μm.Box plot showing the percentage of pP38‐positive FAPs in the staining shown in (F).Representative images of the phospho‐SMAD2/3 staining in FAPs (pSMAD2/3 in red/CD90 in green/Laminin in grey/DAPI in blue) on tibialis anterior muscle transversal sections of YC, YT, OC and OT mice. Scale bar = 25 μm.Box plot showing the percentage of pSMAD2/3‐positive FAPs in the staining shown in (H).Data information: In (B, C) data are presented as average ± SEM, in (E, G, I) the central band of the boxplot shows the median, the box extends from the 25th to 75th percentiles, whiskers are down to the minimum and up to the maximum value (n = 3, biological replicates); (*) indicates statistical analysis by Student’s t‐test in the comparison against YC, * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001; (†) indicates statistical analysis by Student’s t‐test in the comparison against OC, † P ≤ 0.05, †† P ≤ 0.01, ††† P ≤ 0.001, †††† P ≤ 0.0001; (#) indicates statistical analysis by one‐way ANOVA, # P ≤ 0.05, ## P ≤ 0.01, ### P ≤ 0.001 #### P ≤ 0.0001.

Fig 5: Environmental Cues Drive Distinct DC2 Differentiation Pathways within the Spleen(A) Derivation of splenic CD11b+ DC subsets from bone marrow (BM) progenitors. Shown are splenic cDC2s 7 days post transfer. Data representative of MDP and CDP recipients (n = 3) or pre-DC recipients (n = 6) from 3 independent experiments.(B) Analysis of YFP in BM progenitors from RorcCreRosa26lsl-YFP mice (top) or RFP expression in cells from Tbx21RFP-cre mice (bottom).(C) t-SNE embedding of diffusion map of T-bet+, Tbet– cDC2, and Siglec-H+ pre-DC clusters identified in Figure 1E.(D) Palantir branch probabilities from Siglec-H+ pre-DC to T-bet+ cDC2 (top) or Tbet– cDC2 (bottom) terminal states. Gene expression trends in pseudo-time along corresponding trajectories.(E) Expression of genes identified as varying significantly along the trajectory from the pre-DC cluster to T-bet+ or Tbet– cDC2.(F) Expression of Siglec-H in Lin–CD90–Ly6C–CD64–CD11c+MHCII+ cells. Far right: overlay of Siglec-H+ DCs against all MHCII+CD11c+ DCs.(G) Representative plot showing percentage of YFP+ cells in splenic cDC1 and cDC2 populations in RorcCreRosa26lsl-YFP mice.(H) Heatmap showing expression of genes differentially expressed between bulk T-bet+ and Tbet– cDC2s across T-bet+, Tbet–, and Ror?t fate-mapped (fm) cDC2s.(I) Frequency of T-bet+ cDC2s within the spleen, MLN, large intestine lamina propria (LI), and small intestine lamina propria (SI) at indicated time point post birth. n = 3 per time point, error bars represent mean ± SEM. For the day 7 analysis SI and LI tissues were pooled from 2 mice per sample.(J) Generation of T-bet+ cDC2s in mice treated with a broad-spectrum antibiotic cocktail (AVKM) or H2O (control). Each symbol represents one mouse. Error bars represent mean ± SEM.See also Figure S4 and Table S5.