Fig. 6

Hp-TGM drives immune regulation in vitro and in vivo. a Mouse Foxp3⁺ Treg cells are suppressive in vitro. CD4⁺CD25⁺GFP⁺ cells, generated as Fig. 4a were isolated by FACS and co-cultured with CD4⁺CD25^–GFP^– responder cells from Foxp3-GFP mice together with, irradiated APCs and anti-CD3 for 5 days. Proliferation was assessed by thymidine incorporation, and percentage suppression calculated; data are means ± SEM from 3 replicates per concentration and represent two independent experiments. b Hp-TGM prolongs survival of fully allogeneic full-thickness BALB/c to C57BL/6 skin grafts. Kaplan–Meier curve of graft survival: allograft only (n = 6), allograft + HES or Hp-TGM following implantation of intraperitoneal minipumps, n = 6) and syngeneic controls, n = 3. Mantel-Cox comparison of allograft vs. allograft + Hp-TGM survival curves: p = 0.0136. c, d Histological analyses of graft sites 7 days following transplantation; c, representative images of tissues sections, scale bars = 100 µm; d, scoring of inflammation on 3 sections per graft performed in a blinded fashion: syngeneic control (n = 7), allograft + control protein (n = 14), allograft + HES (n = 13), allograft + TGM (n = 12); data shown combine two independent experiments, with mean ± SEM shown. HES vs untreated allogeneic control p = 0.0493; Hp-TGM v untreated control p = 0.0397, by unpaired t test. e, f Treg and Th17 CD4⁺ T cell populations 21 days after transplantation, in e draining lymph node Foxp3⁺ (syngeneic controls (n = 3), allograft + control protein (n = 6), allograft + TGM minipump (n = 6); two-tailed, unpaired t test: p = 0.0042) and f spleen RORγt⁺ (syngeneic controls (n = 3), allograft + control protein (n = 7), allograft + TGM minipump (n = 6); two-tailed, unpaired t test: p = 0.0112). Gating strategy is shown in Supplementary Fig. 6. g, h Fibrogenesis in human lung fibroblasts exposed to TGFβ1 or Hp-TGM, assayed by immunofluoresence for collagen deposition (g, see Supplementary Fig. 8A for quantitative summary) and smooth muscle actin (h, presented as quantitative summary, see Supplementary Fig. 8B for fluorescence micrographs). Human collagen I stained with AF488-conjugated scondary antibody (green) and counterstained with DAPI (blue). Scale bars = 200 µm. Data are means ± SEM from 1 of 3 replicate experiments, with 4 technical replicates per group