Fig 2: Compromised transition of wound macrophages to fibroblast-like cells in diabetic mice. a qRT-PCR analysis of miR-21 expression in d5 wound tissue db/db and control db/+ mice, n = 5, Student’s t test p < 0.05. b Immunostaining of F4/80 (macrophage, red) and FSP1 (fibroblast, green), immunostaining of d5 wound tissue from db/+ and db/db mice. n = 5, p < 0.05. FSP1 intensity is expressed in arbitrary units, n = 5, Student’s t test p < 0.05, scale bar = 10 µm. c Multicolor flow cytometry of CD11b+ of d7 wound macrophages from db/+ and db/db mice F4/80 (macrophage, x-axis) and CD90 (fibroblast marker, y-axis) of db/+ and db/db mice, n = 5, Student’s t test p < 0.05. d Linear stiffness of the repaired skin in db/+ and db/db mice on d20 PW measured using mechanical tester, n = 6, Student’s t test p < 0.05. e, f Collagen expression as visualized using e Masson’s trichrome staining (collagen, blue) and f picrosirius staining (collagen, yellow) of healed wounds in db/+ and db/db mice, n = 5, Student’s t test p < 0.05, scale bar = 20 µm. g FSP1 (green) and F4/80(red) immunostained d5 wounds from db/db mice delivered with miR-21 via nanoparticles. FSP1 intensity presented in arbitrary units, n = 5, Student’s t test p < 0.05 compared to db/db mice delivered with control mimic, scale bar = 10 µm

Fig 3: CD8+ T Cells with Increased L-Arginine Levels Display Improved Anti-tumor Activity In Vivo(A) Survival of activated mouse CD8+ OT-I T cells (4 days) after IL-2 withdrawal. Data points represent the difference between the percentage of living T cells from cultures performed in L-Arg medium or Ctrl medium; n = 11.(B) CD90.1+ CD45.1/2+ and CD90.1+ CD45.1+ naive CD8+ OT-I T cells were activated for 4 days in Ctrl medium or L-Arg medium, respectively. Equal numbers of the congenically marked activated OT-I cells were co-transferred into Cd3e–/– mouse and the number of living T cells was measured in pooled spleen and lymph nodes at the indicated time points; n = 3 at each time point.(C) Naive CD8+ OT-I T cells were activated with CD3 + CD28 antibodies in L-Arg medium or Ctrl medium. Five days after activation, the percentage of Tcm-like cells (CD44hi, CD62L+) was measured by flow cytometry; n = 15.(D) Naive OT-I CD8+ T cells were activated in L-Arg medium or Ctrl medium and IFN-? was quantified in culture supernatants after 5 days; n = 15.(E) Same as in (D) but T cells were re-activated on day 5 day with PMA/Ionomycin; n = 15.(F and G) B16.OVA melanoma cells were injected into C57BL/6 mice and tumors were allowed to grow for 10 days. Naive OT-I CD8+ T cells were activated in vitro in L-Arg medium or Ctrl medium and injected into tumor bearing mice. Tumor burden (F) and survival (G) were assessed over time. Data are representative of three independent experiments, each performed with seven to nine mice per group.(H) B16.OVA melanoma cells were injected into C57BL/6 mice and tumors were allowed to grow for 6 days. At day 6, naive CD8+ OT-I T cells were transferred into tumor bearing mice and at day 7 mice were immunized with OVA peptide. Starting one day before the T cell transfer, PBS or L-arginine (1.5 mg/g body weight) was orally administered daily; n = 19 from three independent experiments. Bars represent the SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 (Student’s t test). In (G), *p < 0.05 as determined by log-rank test comparison between curves.Error bars represent SEM throughout.

Fig 4: Majority of FSP1+ fibroblast-like cells in wound granulation tissue are of myeloid origin. a LysMCreRosamT/mG mice express cell membrane-localized td Tomato (red) fluorescence while cells of myeloid origin express GFP (green) fluorescence. b FSP1 (blue) immunostaining of LysMCreRosamT/mG mice d5 wound. Colocalization was performed using Olympus Fluoview® software. Estimated (65 ± 5%) FSP1+ cells (blue) were GFP+ demonstrating their myeloid origin, n = 5, scale bar = 10 µm. c Macrophage (F4/80, red) and fibroblast (FSP1, green) immunostaining at wound-edge tissue (d5) from C57BL/6 mice, n = 5, scale bar = 10 µm. d Laser capture micro-dissected (LCM) GFP+ cells from full thickness excisional wounds on d3 (early inflammation phase) or d7 (inflammation resolution phase) post-wounding (PW), scale bar = 10 µm. qRT-PCR of LCM-captured GFP+ cells showed increased expression of FSP1 and Col1A1 at d7, n = 5, Student’s t test p < 0.05. e, f Multicolor flow cytometry of CD11b+ of wound macrophages on d3 and d7 PW with the F4/80 (macrophage, x-axis) and CD90 (fibroblast marker, y-axis) displayed increase (68 ± 2%) in F4/80+CD90+ cells on d7 PW, n = 4, Student’s t test p < 0.05. g Chronic wound fluid derived from human subjects contain CD68+ FSP1+ cells, scale bar = 10 µm. h Fibrocyte marker CD34 (blue), immune-staining of LysMCreRosamT/mG mice d5 wound tissue. Estimated (15 ± 2%) of CD34+ cells (blue) were GFP+, n = 5, scale bar = 10 µm. Data presented as ±SD

Fig 5: Alloreactive CD4+ T cells are robustly activated in the transplanted mLNs under aGvHD inflammatory conditions. (A) Experimental strategy: mLNs from B6.WT mice were transplanted into B6.WT recipient; 5-6 weeks post-surgery; MHC major mismatch aGvHD was induced by lethal irradiation (9 Gy) and allogenically transplanting with 5x106 TCD BM cells and 5x106 CD4+ T cells from FVB/N mice. (B) Mean fluorescence intensity (MFI) of CD44 and CD25 on donor T cells d+3 of allo-HCT. (C) Proliferative capacity of donor CD4+ T cells were analyzed by expression of Ki67 d+3 of allo-HCT. (D) Experimental strategy: mLNs of B6.MHCII? mouse were surgically removed and transplanted with donor mLNs from B6.CD11c.DOG mice that had been depleted of CD11c+ cells by i.p. administration with diphtheria toxin 20 ng/gram body weight day -5, -3, and -1 day before surgery. 10 weeks post-surgery MHCII? (CD11c.DOG mLNs), untreated B6.WT and MHCII? mice were myeloablatively irradiated with 9 Gy and i.v. transplanted with 5x106 T cell-depleted (TCD) BM and 5x106 CD4+ T cells from MHCII? and FVB mice respectively. (E) Analysis for T cell subsets - CD44 and CD62L (pre-gated on CD4+CD90.1+) on day +13 of allo-HCT from mLNs. Each data point represents one mouse, data pooled from two experiments; unpaired non-parametric Mann-Whitney test, (Mean± SD); **p < 0.01, ***p < 0.001 and ****p < 0.0001.