Fig 2: J- and N-HSPC Subpopulations Display Similar Lineage Output Post TransplantHSPC subpopulations (LT-HSC, ST-HSC, MPP2, MPP3, and MPP4) from J or N mice (CD45.2) were transplanted into sublethally irradiated recipients (CD45.1/CD45.2). Recipient PB was examined for CD45.2+ cells and myeloid (GR1+CD11b+) and lymphoid (B cells as B220+ and T cells as CD4+CD8+) cells by flow cytometry every 3–4 days for 33–100 days. Percentage of myeloid, B, and T cells among the CD45.2+ cells produced by HSPC subpopulations post transplant. Data from two independent transplants are presented as mean ± SEM. Donor mice, n = 3 pooled per transplant. Recipient mice, n = 5 per transplant. *p < 0.05, significantly different to recipients of J-HSPCs.

Fig 3: J-HSPCs Display a Short-Term Repopulating Defect(A) N- or J-LSK cells (CD45.2) were mixed 1:1 with competitor LSK cells (CD45.1) and injected into lethally irradiated recipient mice (CD45.1/CD45.2). CD45.2+ chimerism of the PB was examined by flow cytometry every 4 weeks for 20 weeks post transplant.(B) Normalized CD45.2+ chimerism in total recipient PB after transplant.(C) Normalized CD45.2+ chimerism among the myeloid (GR1+CD11B+) and lymphoid (B cells as B220+ and T cells as CD4+CD8+) lineages in the recipient PB post transplant.(D) Normalized CD45.2+ chimerism among the BMC at 4, 8, and 12 weeks post transplant. Data presented are normalized to the CD45.2+ chimerism observed in recipients of J-LSK cells.Data from three independent transplants are presented as mean ± SEM. Donor mice, n = 3 pooled per transplant. Recipient mice, n = 5 per transplant. *p < 0.05, **p < 0.005, ***p < 0.001, significantly different from recipients of J-LSK cells.

Fig 4: TGFß endothelial-specific induction of inflammatory response.(A, B) Bulk RNA-seq analysis of HUVEC (EC), HASMC (SMC), CD4+ effector memory T cells (T) and macrophages (M) gene expression before and after stimulation with TGFß1 (A) and inflammatory gene expression (B). Each lane is an average of two biologically independent samples. (C) ChIP-seq analysis of canonical TGFß signaling in EC and SMCs. Note increased SMAD2/3 binding to regulatory elements of CCL2, CLDN5 and SERPINE1 in ECs but not SMCs. Pol II S2 and Pol II S5 ChIP in combination with bulk RNA-seq indicate increased expression of CCL2 and SERPINE1 and decreased expression of CLDN5 after TGFß1 stimulation. N=1 per experimental condition. (D) Relative CLDN5 expression in primary ECs isolated from control (Cdh5-CreERT2; Tgfbr1fl/fl-Tgfbr2fl/fl-mT/mG) and TGFßRiEC mice treated with corn oil. Bars: mean ± SEM. 0 hr P=0.004, 6 hr P=0.001. Two-tailed Student’s t tests (N=3 animals per group for 0 hr, N=6 animals per group for 6 hr). (E) i.v. Evans blue dye administration following a subcutaneous TNFa injection into mouse ear. Scale bar: 2 mm; mean ± SEM. 0 min P=0.56, 15 min P=0.000066, Two-tailed Student’s t tests (N=6 animals per group for 0 min, N=7 animals per group for 15 min). (F,G) Mouse ear sections were stained with anti-ICAM, VCAM-1, CD45 and Ly6G antibodies 6 hrs and 3 days following a s.c. TNFa injection. Note a strong reduction in expression of both adhesion molecules (F) and reduced presence of inflammatory cells (G) in TGFßRiEC mice. Scale bar: 16 µm. N=7 animals per group. Bars: mean ± SEM. ICAM1+ area 6 hrs P=0.001, ICAM1+ area 3 days P=0.0002, VCAM1+ area 6 hrs P=0.0001, VCAM1+ area 3 days P=0.00001, Ly6G+ cells 6 hrs P=0.0499, Ly6G+ cells a 3 days P=0.0004, CD45+ cells 6 hrs P=0.051, CD45+ cells a 3 days P=0.00004. Two-tailed Student’s t tests.

Fig 5: scRNA-seq analysis reveals adrenal gland leukocyte diversity and monocyte contribution to the macrophage pool(A) scRNA-seq analysis of adrenal gland CD45+ cells from 7-week-old male and female wild-type mice.(B) Proportion of each cluster identified in scRNA-seq analysis.(C) Heatmap showing normalized expression levels of cluster-specific genes.(D) Violin plots showing Cx3cr1, Timd4, and Lyve1 expression by cells from clusters 1–6.(E) Flow-cytometry plot showing Ly6C and CCR2GFP expression among adrenal gland CD45+CD11b+CD64+ cells in male and female CCR2GFP/+ mice. Data are representative of three independent experiments.(F) Flow-cytometry plot showing Ly6C and TdTomato expression among adrenal gland CD45+CD11b+CD64+ cells from male and female CCR2creERT2 x R26TdTomato mice 48 h after TAM gavage. Data are representative of two independent experiments.(G) Quantification of TdTomato+ macrophages in 16- to 20-week-old male and female heterozygous (CCR2+/-, ? n = 6 and ? n = 6) or double knockin (CCR2-/-, ? n = 4 and ? n = 3) CCR2creERT2 x R26TdTomato mice 48 h after TAM gavage. Data are pooled from two independent experiments.(H) Histograms representing R26TdTomato expression in AGMs from 10-week-old female CCR2creERT2/+ x R26TdTomato mice 2, 7, and 14 days after TAM gavage. Data are representative of one (days 7 and 14) or two (day 2) experiments.(I) Proportions of TdTomato+ macrophages from 10-week-old female CCR2creERT2/+ x R26TdTomato mice 2 (n = 8), 7 (n = 4), and 14 (n = 3) days after TAM gavage. Data from one (days 7 and 14) or two (day 2) experiments.(J) Proportions of TdTomato+ macrophages from 8 (? n = 2, ? n = 6) or 16 (? n = 5, ? n = 5)-week-old male and female Ms4a3cre/+ x R26TdTomato mice. Data are from one experiment.(K) Quantification of AGMs in male and female CCR2+/- (? n = 18, ? n = 21) and CCR2-/- (? n = 10, ? n = 10) mice. Data are pooled from four independent experiments.(L) Proportions of Ki67+ AGMs in male and female CCR2+/- (? n = 11, ? n = 11) and CCR2-/- (? n = 6, ? n = 5) mice. Data are pooled from two independent experiments.Statistical analysis was performed using two-way ANOVA with Bonferroni’s post-test.See also Figure S2.