Supplemental Materials and Methods Mice Female C57BL/6 (B6, I-E null, H-2 b ), BALB/c (H-2 d ) + ), FVB/N (H-2 q, I-E null, CD45.1 + ), and B6D2F1 (H-2 b/d ) mice were purchased from the Animal Resources Centre. OT-II Tg (B6) and BALB/c CD45.1 + mice were bred and housed at the QIMR Berghofer Medical Research Institute (QIMR Berghofer). H2dlAb1-Ea (B6, MHC II-deficient; namedh2- Ab1 -/- ) mice were a gift from Mohammed Alsharifi (Australian National University, Canberra, Australia). CD11c-DTR-OVA-GFP (B6.CD11c-OVA), B6.luc + and FoxP3.LuciDTR-4 (B6.FoxP3-luc + ) mice were provided by Gunter Hammerling (German Cancer Research Center, Germany). B6 or BALB/c.FoxP3-DTR-GFP mice were provided by Alexander Rudenski (Memorial Sloan-Kettering Cancer Center, USA). Itgax(CD11c)-Cre-GFP (B6) mice were purchased from The Jackson Laboratory and kindly shared by Christian Engwerda (QIMR Berghofer, Brisbane, Australia). I-A b -loxp (B6) mice were provided by Pandelakis Koni (GRU Cancer Center, USA). B6.CD11c- Cre-GFP mice were bred with I-Ab-loxP mice to generate CD11c-Cre positive (I-A b - CD11c-Cre pos ) or negative (CD11c-Cre neg ) mice that were homozygous for the I-A b -loxp allele (Supplemental Figure 1). The mice were used between 8 to 14 weeks of age. All animal studies were performed in accordance with the QIMR Berghofer Animal Ethics Committee. Antibodies and FoxP3 intracellular staining CD3 (145-2C11), CD4 (RM4-5), CD8 (53-6.7), CD25 (7D4), MHC class II (I-A/I-E, M5/114.15.2), CD11c (N418), CD19 (1D3), F4/80 (BM8), CD90.2 (30-H12), CD45.1
(A20) and FoxP3 (150D) antibodies were purchased from Biolegend or BD Biosciences. Biotinylated antibodies to YAe and mouse IgG2b were purchased from ebioscience and BD Biosciences, respectively. FoxP3 intracellular staining was performed according to the manufacturer s protocols (ebioscience FoxP3 intracellular staining kit). Histology Tissues were sectioned and stained with haematoxylin and eosin (H&E) or Masson s trichrome. H&E-stained sections of skin were examined in a blinded fashion (by A.D.C.) using a previously published semi-quantitative scoring system for cgvhd. 1 Images were acquired using the Aperio Scanscope XT microscope and the Aperio ImageScope V10.2 software (original magnification x20). Ex vivo antigen presentation Lethally irradiated B6D2F1 recipients were transplanted with 5x10 6 TCD BM cells from B6.WT or B6.CD11c.OVA donor mice, with or without sort purified 0.5x10 6 CD3 + T cells from B6 donor mice. At day 28 post-bmt, sort-purified OT-II Tg T cells (2x10 6 cells, CD8 - CD90.2 + ) were labelled with CFSE (Sigma-Aldrich) and injected intravenously (i.v.). 2 CFSE dilution was analyzed 3 days later in the spleen. The index of proliferation was determined using ModFit LT 3.2 cell cycle analysis software (Verity Software House). 3 Using two GVHD models, donor cdc expression of MHC II associated hostderived alloantigen (B6 B6D2F1) or donor-derived self-antigen (B6D2F1 FVB/N) was assessed using the YAe mab which reacts with Ea peptide (derived uniquely from B6D2F1 in these models) bound to I-A b as previously described. 4 Lethally irradiated B6D2F1 or FVB/N mice were transplanted with 5 x 10 6 B6 or B6D2F1 TCD BM,
respectively, with or without 0.5 x 10 6 or 0.2 x 10 6 CD3 + T cells. Donor cdc reactivity with YAe antibody was examined on day 28. References 1. Hill GR, Olver SD, Kuns RD, et al. Stem cell mobilization with G-CSF induces type 17 differentiation and promotes scleroderma. Blood. Aug 5 2010;116(5):819-828. 2. Robb RJ, Kreijveld E, Kuns RD, et al. Type I-IFNs control GVHD and GVL responses after transplantation. Blood. Sep 22 2011;118(12):3399-3409. 3. Markey KA, Koyama M, Kuns RD, et al. Immune insufficiency during GVHD is due to defective antigen presentation within dendritic cell subsets. Blood. Jun 14 2012;119(24):5918-5930. 4. Koyama M, Cheong M, Markey KA, et al. Donor colonic CD103+ dendritic cells determine the severity of acute graft-versus-host disease. J Exp Med. Jul 27 2015;212(8):1303-1321.
Supplemental data Supplemental Figure 1: The restricted Cre deletion is specific to the cdcs compartment. (A) Gating strategy to analyze cdcs (CD11c + MHC II + ), T cells (CD3 + ), B cells (CD19 + ) and macrophages (F4/80 + ) in the splenocytes from CD11c-Cre neg or I-Ab- CD11c-Cre pos naïve mice. (B) Frequency of CD11c +, CD3 +, CD19 + and F4/80 + cells from the spleen of CD11c-Cre neg or I-Ab-CD11c-Cre pos naïve mice. (C) Expression of CD11c-Cre GFP + gated within CD11c + MHC II +, CD3 +, CD19 + and F4/80 + cells. (D) Expression of MHC class II gated within CD11c + cells (1 experiment).
BALB/c B6 TCD BM BM + T A B6 B6D2F1 TCD BM BM + T Masson B TCD BM liver B6 B6D2F1 lung salivary gland H&E Masson BM + T H&E Masson Supplemental Figure 2: Histopathology of GVHD target organs in BALB/c B6 and B6 B6D2F1 cgvhd models. (A) Representative images of Masson s trichrome staining of skin sections from lethally irradiated C57BL/6 mice transplanted with 10x106 TCD BM cells and no T cells (TCD BM) or with 3x106 CD3+ T cells (BM + T) from BALB/c donors (day 35) (left panel) or from lethally irradiated B6D2F1 mice transplanted with 5x106 TCD BM cells and no T cells (TCD BM) or with 0.5x106 CD3+ T
cells (BM + T) from C57BL/6 donors (day 28) (right panel) (n=3-4, 1 experiment). (B) Representative images of H&E and Masson s trichrome staining of liver, lung and salivary gland sections of transplanted mice from B6 B6D2F1 cgvhd model described in panel A (n=4, 1 experiment). (Original magnification x20, scale bar 200 µm).
TCD BM skin liver lung salivary gland BM + T Supplemental Figure 3: Histopathology of GVHD target organs in B6D2F1 FVB/N model. Representative images of H&E staining of skin, liver, lung and salivary gland sections of lethally irradiated FVB/N mice transplanted with 5x10 6 TCD BM cells and no T cells (TCD BM) or with 0.2x10 6 CD3 + T cells (BM + T) from B6D2F1 (day 28) (n=4, 1 experiment). (Original magnification x20, scale bar 200 µm).
Supplemental Figure 4: Donor Treg prevent the generation of scleroderma (BALB/c FoxP3-DTR-GFP donor to B6 recipients). (A) Lethally irradiated C57BL/6 recipients were transplanted with 10x10 6 TCD BM cells with 3x10 6 CD3 + T cells from BALB/c.FoxP3-DTR-GFP donor mice. (B) After transplantation, the mice were injected i.p. twice a week with saline or DT from day 7. (C) Representative dot plots of CD4 + FoxP3 + cells gated on CD3 + CD4 + T cells in spleen after saline or DT treatment at day 39. (D) The frequency and absolute number of CD4 + FoxP3 GFP + cells gated within
CD3 + CD4 + T cells in spleen were analyzed at day 39 post-bmt (n=6-12, 2 experiments). (E) Clinical scores of the recipients. The mice injected with DT were sick and sacrifice at day 39. (F) Representative images of H&E staining of skin histology collected at day 39 post-bmt. GVHD histopathology was quantified (n=6-7, 1 experiment). Statistically significant differences were calculated using 2-tailed Mann- Whitney U tests. Data represent the mean ± SEM.
A B C D Spleen Spleen mlns mlns 100 * 0.8 **** NS 100 0.15 NS % YAe + in donor DC 80 60 40 20 donor DC (x 10 6 ) 0.6 0.4 0.2 % YAe + in donor DC 80 60 40 20 ** donor DC (x 10 6 ) 0.10 0.05 0 Donor Ag Host Ag GVHD - - + + + + - - - + - + 0.0 Donor Ag Host Ag GVHD - - + + + + - - - + - + 0 Donor Ag Host Ag GVHD - - + + + + - - - + - + 0.00 Donor Ag Host Ag GVHD - - + + + + - - - + - + TCDBM BM+T TCDBM BM+T B6 B6D2F1 (Host Ag) B6D2F1 FVB (Donor Ag) Supplemental Figure 5: Endogenous Ag presentation by donor splenic DC is impaired during chronic GVHD. Lethally irradiated B6D2F1 or FVB/N recipients were transplanted with 5 10 6 TCD BM cells from B6 or B6D2F1, respectively, with or without supplementation with CD3 + T cells from the same donor. Splenic and mln donor cdc reactivity with YAe antibody (A,C), and absolute DC numbers (B,D) was assessed by flow cytometry on day 28 after BMT. Mann-Whitney test (A, C, D) or T-test with Welch s correction (B) was performed for each analysis. *p<0.05; **p<0.01; ****p<0.0001, NS (not significant); p>0.05.