Fig 2: Immuno-profiling revealed an overall immune-suppressive TME in Tmem30-/- (PSout) tumors(A–G) Flow cytometry quantitative analysis of TILs at the endpoint (day 16). Shown are no infiltrating differences in CD45+CD11b-CD3+CD4+ T cells (B), CD45+CD11b-CD3+CD8+ T cells (C), CD45+CD11b-CD3+CD4+CD25+ Foxp3+ Tregs (D), and CD45+CD11b+ Ly6G-Ly6C+ Mo-MDSC (F); but more infiltrating cells in CD45+CD11b+Ly6G-Ly6C-F4/80 TAMs (E) and CD45+CD11b+Ly6C-Ly6G+ PMN-MDSC (G).(H) Representative flow plots of MHC II expression in TAMs.(I) Flow cytometry analysis of MHC II+ and MHC II- TAM distribution (n = 5).(J and K) Flow cytometry quantitative analysis of MHC II+ and MHC II- TAMs (n = 5).(L) RNA-seq analysis of sorted TAMs in WT or PSout (Tmem30-/-) TMEs. Shown is the Z score of gene expression (n = 4 for PSout and n = 3 for WT).(M and N) Geometric mean fluorescence intensity (gMFI) of CD163 (M) and CD206 (N) shows higher expression on TAMs (n = 5).(O–Q) qPCR analysis of MHC II, CD163, and CD206 in MHC II+ and MHC II- TAMs from both MC38-WT and Tmem30a-/- TMEs (n = 4).(R and S) gMFI of PD-1 (R) and Tim-3 (S) shows lower expression on CD8+T cells (n = 5).(T) Flow cytometry analysis of PD-1 and TIM-3 CD8+ T cell distribution (n = 5).(U) Representative flow plots and analysis of tetramer+ in CD8+ T cells (n = 5). Data were representative of at least two independent experiments (except RNAseq). Data represent mean ± SEM and were analyzed with unpaired t test. Ns, no significant difference; *p < 0.05; **p < 0.01; ***p < 0.001. Tmem30a-/- clone #1 cells were used for these figures.

Fig 3: T and B GC Cells Express T-bet Following Influenza InfectionZsGreen_T-bet reporter mice were infected with influenza and dLNs analyzed.(A–C) Representative confocal micrograph at indicated days p.i. T-bet+ cells (yellow) in GC LZ (IgD-GL7+CD35+) and DZ (IgD-GL7+CD35+). Arrow indicates T-bet+GL7+ cell at d4 p.i. Dashed lines mark GC regions, bottom panels show T-bet+ cells without GC markers (A). Day 8 p.i. T-bet+ GC co-stained with CD4 (cyan) (B) and co-stained with B220 (cyan) (C).(D) Representative flow cytometry plots showing frequency of T-bet+ TFH (red, CD44+PSGL-1- Ly6c- PD-1+ CXCR5+) and T-bet+ TH1 (blue, CD44+ PSGL-1+Ly6c+) at indicated days p.i.(E and F) Frequency (E) and total numbers (F) of T-bet+ TFH and TH1 cells.(G) Flow cytometry plots showing frequency of T-bet+ GC B cells (red, B220+IgD-CD95+CD38-) and plasma cells (blue, B220lo CD138+).(H and I) Frequency (H) and total numbers (I) of T-bet+ GC B and plasma cells.In (E), (F), (H), and (I), data are pooled from three independent experiments. Data are mean ± SEM.

Fig 4: AR is important for bone marrow monocytic development. BM of WT and MARKO male mice was analyzed by flow cytometry for monocytic and macrophage cell populations. (A) The upper plot represents UMAP analysis of live singlet lineage negative (CD3, Ly-6G, B220, Ter119) BM cells from WT and MARKO male mice. The lower plot in (A) is colored according to sample group. (B) UMAP graphs indicating intensities of c-Kit, CD16/32, Ly-6C, CD115, CD11b and F4/80 expression in WT and MARKO BM. (C–G) Quantification of BM cell populations utilizing gating schemes in Supplementary Figures 1A, D, F. Plots depict percentage and numbers of singlet live (C) oligopotent GMP (oGMP), (D) monocytic progenitors (MoPs), (E) CD11b+Ly6C+CD115+F4/80- cells, (F) CD11b+Ly6C-CD115-F4/80- cells, and (G) CD11b+Ly6C-CD115-F4/80+ cells. Graphs show pooled data from two to four experiments with two to three mice per group. Black filled squares denote WT, and red empty squares indicate MARKO BM. Comparisons were done with non-parametric t-test. ns, not significant; *p = 0.05, **p = 0.01.

Fig 5: JAB0505 treatment causes a sustained increase in several immune cell populations following skeletal muscle injury of Acvr1tnR206H/+; Tie2-Cre FOP mice.Flow cytometry analysis was used to determine cell numbers of (A) total CD45+ hematopoietic cells, (B) myeloid cells, (C) lymphoid cells, (D) total macrophages, (E) Ly6C+ inflammatory monocytes/macrophages, (F) neutrophils, (G) mast cells, and (H) T cells in injured distal hind limb muscles of control (R26NG/+; Tie2-Cre) and FOP (Acvr1tnR206H/+; R26NG/+; Tie2-Cre) mice that were either untreated or administered 10 mg/kg JAB0505 i.p. (n = 3–4). Error bars represent ±SD. Significance was determined using 1-way ANOVA with Tukey’s multiple-comparison test within individual time points. Symbols representing significance were placed above FOP and FOP + JAB0505 bars to indicate a comparison to control (^), control + JAB0505 (#), and FOP (*). The numbers of symbols of each type denote levels of significance: (1) P = 0.05, (2) P = 0.01, (3) P = 0.001, and (4) P = 0.0001. No control vs. control + JAB0505 comparisons were significant.