Fig 2: Neutralisation of TGF-ß1 decreases lymphedema and inflammation. (A) Left panel: Representative cross-sectional H&E stain of a mouse tail treated with isotype control or TGF-ß1 neutralising antibody. Brackets show subcutaneous fibroadipose tissues; note decreased subcutaneous thickness in TGF-ß1 neutralising antibody-treated mice. Right panel: Quantification of skin thickness in control or TGF-ß1 neutralising antibody-treated mice. Each circle represents an average of three HPF views per animal (N = 4). (B) Left panel: Representative IF localisation of TGF-ß1 (top) and pSmad3 (bottom) in isotype and TGF-ß1 neutralising antibody-treated mice. Right panel: Quantification of TGF-ß1 area and Smad3+ cells. Each circle represents an average of three HPF views per animal (N = 5). (C) mRNA expression of TGF-ß1 isoforms and downstream signaling pathways. Relative change to isotype control-treated mice is shown. Each circle represents an individual animal (N = 5). Genes shown in the green- and red-shaded zones represent upregulated (green) and downregulated (red) molecules. *p < .05, **p < .01. (D) mRNA expression of inflammatory mediators. Relative change to isotype control-treated mice is shown. Each circle represents an individual animal (N = 5). Genes shown in the green- and red-shaded zones represent upregulated (green) and downregulated (red) molecules. *p < .05, **p < .01. (E) Quantification of flow cytometry for leukocytes (CD45+), CD45+/CD4+ cells, Th1 (CD45+/CD4+/CXCR3+/CCR5+) cells, Th2 (CD45+/CD4+/CCR4+/CCR8+) cells, macrophages (CD45+/CD11b+/F480+) and neutrophils (CD45+/CD11b+/Ly-6G+). Each circle represents an average of two flow cytometry runs for each animal (N = 5). (F) Quantification of CD4 T cells (CD45+/CD4+), macrophages (CD45+/CD11b+/F480+) and DCs (CD45+/CD11c+/CD86+) in draining lymph nodes and foot pad injection site after injection of LE lysate. Each circle represents an individual animal (N = 5). TGF-ß1, transforming growth factor-beta 1; H&E haematoxylin and eosin; HPF, high-power field; IF, immunofluorescence; DCs, dendritic cells; LE, lymphedema

Fig 3: Topical PFD decreases the pathology of lymphedema. (A) Change in tail volume over time in mice treated with vehicle or PFD. Each circle is an average of duplicate measurements from each mouse (N = 9 animals per group). Results are presented as mean ± SEM (*p < .05, **p < .01). Statistical comparisons are between groups at the same time points. (B) Left panel: Representative cross-sectional H&E stain of vehicle and PFD-treated mouse tails. Brackets show subcutaneous fibroadipose tissue deposition. Right panel: Quantification of skin thickness in vehicle and PFD-treated mouse tails. Each circle represents an average of three HPF views from 5 animals. Bar = 500 µm. (C) Quantification of type I collagen deposition in vehicle and PFD-treated mouse tails. Each circle represents an average of three HPF views from 4 animals. (D) Quantification of TGF- ß1 protein in tissues collected from vehicle and PFD-treated mice. Each circle represents an average of duplicate ELISAs per animal (N = 9). (E) Quantification of the number of pSmad3+ cells in vehicle and PFD-treated mouse tails. Each circle represents an average of cell counts in three HPF views from 7 animals. (F) mRNA expression of TGF-ß1 isoforms and downstream signaling pathways. Relative change to vehicle control-treated mice is shown. Each circle represents an individual animal (N = 6). Genes shown in the green- and red-shaded zones represent upregulated (green) and downregulated (red) molecules. *p < .05, **p < .01, ***p < .001. (G) mRNA expression of fibrosis and ECM genes. Relative change to vehicle control-treated mice is shown. Each circle represents an individual animal (N = 6). Genes shown in the green- and red-shaded zones represent upregulated (green) and downregulated (red) molecules. *p < .05, **p < .01, ***p < .001. (H) mRNA expression of inflammatory cytokines. Relative change to vehicle control-treated mice is shown. Each circle represents an individual animal (N = 6). Genes shown in the green- and red-shaded zones represent upregulated (green) and downregulated (red) molecules. *p < .05, **p < .01, ***p < .001. (I) Quantification of flow cytometry for leukocytes (CD45+), macrophages (CD45+/CD11b+/F480+), CD45+/CD4+ cells, Th1 (CD45+/CD4+/CXCR3+/CCR5+) cells and Th2 (CD45+/CD4+/CCR4+/CCR8+) cells. Each circle represents an average of two flow cytometry runs per animal (N = 4). (J) Left panel: Representative IF localisation of CD4 (red) and LYVE-1 (green) in vehicle and PFD-treated mouse tails. Right panel: Quantification of perilymphatic CD4+ cells. Each circle represents an average of three HPF views from 9 animals. PFD, pirfenidone; SEM, standard error of the mean; H&E haematoxylin and eosin; HPF, high-power field; TGF-ß1, transforming growth factor-beta 1; ELISA, enzyme-linked immunosorbent assay; ECM, extracellular matrix molecules; IF, immunofluorescence

Fig 4: Adoptively transferred CD4+ T cells differentiate into a mixed Th1/Th2 phenotype. a Schematic diagram of adoptive transfer following PLND versus sham surgery. Mice killed 4 weeks after surgery. b, c Representative FACS plots (b) and quantification (c) of single, live CD45+CD4+ cells (n = 4 for PLND, n = 5 for sham). d, e Representative FACS plots (d) and quantification (e) of single, live CD4+CCR5+CXCR3+ Th1 cells (n = 4 for PLND, n = 5 for sham). f, g Representative FACS plots (f) and quantification (g) of single, live CD4+CCR4+CCR8+ Th2 cells (n = 4 for PLND, n = 5 for sham). h Representative immunofluorescent images co-localizing CD4 and IFN-? with inset images demonstrating CD4 alone (inset upper) and IFN-? alone (inset lower); scale bar, 10 µm. Isotype control refers to use of control for IFN-? in mice without CD4-GFP expression. i Quantification of CD4+IFN-?+ cells per 0.25 mm2 (n = 5 per group; 4–5 hpf per mouse). j Representative immunofluorescent images co-localizing CD4 and IL-4 with inset images demonstrating CD4 alone (inset upper) and IL-4 alone (inset lower); scale bar, 10 µm. Isotype control refers to use of control for IL-4 in mice without CD4-GFP expression. k Quantification of CD4+IL-4+ cells per 0.25 mm2 (n = 5 per group; 4–5 hpf per mouse). Data representative of a minimum of two independent experiments with similar results; statistical analyses of one experiment shown. Mean ± s.d.; *P < 0.05, **P < 0.01, and ***P < 0.001 by unpaired student’s t-test. FACS, fluorescence-activated cell sorting; hpf, high-powered field; PLND, popliteal lymph node dissection; Th1, T helper type 1; Th2, T helper type 2

Fig 5: Regulatory T cells (Tregs) represents the majority of CD4+ tumor infiltrating Puro+ cells.(A) Schematic diagram for the experiment. (B) Representative plots (left) and statistical analysis (mean ± SEM) of gated CD44+ CD4+ tumor-infiltrating lymphocytes (TILs) analyzed for puromycin incorporation. Quantitation shows an enrichment for the expression of CD44. Percentages of positive cells in each gate are shown. Data from three experiments pooled together (n = 6 mice per experiment) **p < 0.01. (C) Statistical analysis (mean ± SEM) for the indicated markers within Puro+ and Puro- CD4+ TILs shows that Puro+ translating CD4+ TILs are enriched for CTLA4, CD39, CD27, ICOS, CCR5, and CD103 expression. Data from two experiments pooled together (n = 5 mice per experiment) *p < 0.05; **p < 0.01; ****p < 0.0001. (D) Representative plots (left) and statistical analysis (mean ± SEM) of gated CD25+ Foxp3+ CD4+ lymphocytes show that the number of Puro+ Tregs is higher in tumors versus spleen. Percentages of positive cells in each gate are shown. Data from two experiments pooled together (n = 4–5 mice per experiment) ***p < 0.001. (E) Immunohistochemical analysis of puromycin in Foxp3+ TILs shows that clusters of highly translating Foxp3+ cells are found. Scale bar, 20 µm. (F) Representative plots (left) and statistical analysis (mean ± SEM) for pS6(S235/236) within Puro+ and Puro- Tregs showing that, in vivo, most of translating Tregs have the mTORC1-S6K pathway active. Percentages of positive cells in each gate are shown. Data from one experiment representative of two (n = 7) ***p < 0.001. Figure 5—source data 1.Source data for Figure 5B, C, D, and F.