Title: Glucocorticoid-induced tumor necrosis factor receptor-related protein co-stimulation facilitates tumor regression by inducing IL-9-producing helper T cells Authors: Il-Kyu Kim, Byung-Seok Kim, Choong-Hyun Koh, Jae-Won Seok, Jun-Seok Park, Kwang-Soo Shin, Eun-Ah Bae, Ga-Eun Lee, Hyewon Jeon, Jaebeom Cho, Yujin Jung, Daehee Han, Byoung S. Kwon, Ho-Young Lee, Yeonseok Chung, and Chang-Yuil Kang Supplementary Fig. 1 T H 9 cells are superior to T H 2 cells in rejecting tumors. (a) Flow cytometry of in vitro-generated T H 2 and T H 9 cells from naïve OT-II mice. (b) Tumor size in C57BL/6 mice with s.c. inoculation of B16F10-OVA cells and i.v. transfer of in vitrogenerated T H 2 or T H 9 cells on the same day (n = 5 per group). * P < 0.05, *** P < 0.001 by Student s t test. Data represent two independent experiments. Nature Medicine: doi:10.1038/nm.3922
Supplementary Fig. 2 DTA-1 treatment considerably upregulates IFN-γ production by CD4 + T cells at a later phase of tumor progression. Representative plots of flow cytometry showing IFN-γ + CD4 + T cells in the spleens, TdLNs and tumors of CT26 tumor-bearing mice at the indicated days after tumor inoculation. Numbers in each quadrant indicate each percent. Data represent two independent experiments.
Supplementary Fig. 3 IL-9-dependent regulation of chemical-induced colorectal cancer and spontaneous lung tumor formation following DTA-1 treatment. (a) The averages of colon tumor sizes in all mice in each group are depicted. A dot means an individual mouse. Bars indicate mean values (n = 9 to 10 per group). (b) In vivo imaging of K-Ras transgenic (Tg) mice (day 87) treated with or without DTA-1 and IL-9-neutralizing antibody 24 h after i.v. injection of the probe for matrix metalloproteinase (top). Whole lung images (middle) and H&E staining of lung sections (bottom) of K- Ras Tg mice. Scale bar 250 μm, (c) Tumor multiplicity in the lungs of K-Ras Tg mice within the indicated range of tumor sizes (n = 7 per group). * P < 0.05, ** P < 0.01 by Student s t test.
Supplementary Fig. 4 IL-9 receptor expression on tumor and immune cells. (a) Expression of the Il9r transcript in tumor cell lines and immune cells freshly isolated or stimulated with anti-cd3 and anti-cd28 for T cells and with LPS (0.5 µg ml -1 ) for dendritic cells. (b) Immunoblot analysis of the expression of IL-9 receptor in cells prepared as in a. Data represent two independent experiments.
Supplementary Fig. 5 GITR stimulation enhances IL-21 expression in CD4 + T cells in vitro and in tumor-bearing mice. (a) Expression of the Il21 transcript in CD4 + T cells stimulated with either isotype control antibody or DTA-1 in the presence or absence of T H 9 condition (triplicate). (b) IL-21 production by activated total, CD4 + T cells and non-cd4 cells from TdLNs of CT26-tumor bearing mice 10 d after tumor inoculation (n = 3 per group). * P < 0.05, ** P < 0.01 by Student s t test. Data represent two independent experiments.
Supplementary Fig. 6 GITR ligation by factors other than IL-9 promotes the induction of tumor-specific CD8 + CTLs. The number of tumor-specific AH1 tetramer + CD8 + T cells in the TdLNs of CT26 tumor-bearing mice was analyzed ex vivo by flow cytometry two weeks after tumor inoculation. A dot means an individual mouse. Bars indicate mean values (n = 6 per group). *** P < 0.001 by Student s t test. N.S. non-significant. Data represent two independent experiments.
Supplementary Fig. 7 IL-9 does not directly affect the cytotoxicity of CD8 + CTLs. Freshly isolated or pre-activated OVA-specific CD8 + T cells from OT-I mice were cocultured with CFSE-labeled B16F10-OVA tumor cells in the presence or absence of recombinant IL-9 (20 ng ml -1 ). After 36 h, 7-AAD expression on CFSE + tumor cells was analyzed by flow cytometry (triplicate). Data represent two independent experiments.
Supplementary Fig. 8 Mast cells might not be involved in DTA-1-induced tumor rejection. (a) Expression of the transcripts of mast cell-related enzymes in the TdLNs of mice received control IgG or anti-c-kit antibody (ACK2). (b) The number of mast cells per area of the tumor section was defined by toluidine blue staining. Scale bar 100 μm. (c,d) Tumor growth in CT26-bearing mice with i.p. administration of DTA-1 or ACK2 (n = 5 to 6 per group). Total rat IgG was used as a control. *** P < 0.001 by Student s t test. N.S. non-significant. Data represent two independent experiments.
Supplementary Fig. 9 Effects of GITR co-stimulation on the differentiation of T H subsets. (a) Expression of IFN-γ, IL-17A and Foxp3 in naïve CD4 + T cells stimulated for 3 d under T H 1-, T H 17- and it reg -polarizing conditions in the presence of isotype control antibody or DTA-1. (b,c) IL-9 production by naïve CD4 + T cells stimulated for 3 d under the indicated T H conditions plus either isotype control antibody or DTA-1, measured by flow cytometry (b) and by ELISA (c) in accumulated supernatants of the culture (triplicate). * P < 0.05, ** P < 0.01, *** P < 0.001 by Student s t test. Data represent at least two independent experiments.
Supplementary Fig. 10 Effects of GITR co-stimulation on the integrity and function of T reg cells. (a) Left, sorted CD4 + Foxp3 + natural T reg (nt reg ) cells (purity>99%) from Foxp3 GFP reporter mice were activated for 4 d with anti-cd3 plus anti-cd28 and either isotype control antibody or DTA-1, with or without TGF-β or IL-4, and were analyzed for IL-9 and Foxp3 (GFP) expression by flow cytometry. Right, the frequencies of IL-9 + cells in nt reg cell cultures are depicted (triplicate). (b) IL-9 production by nt reg cells stimulated as in a. (c) Suppressive function of T reg cells isolated from naïve Foxp3 GFP mice or mice bearing B16F10-OVA tumors with in vivo treatment of control IgG or DTA-1 (n = 3 per group). ** P < 0.01, *** P < 0.001 by Student s t test. Data represent two independent experiments.
Supplementary Fig. 11 Autocrine IL-4 is dispensable for GITR-induced T H 9 differentiation. (a) Il4 mrna expression in naïve CD4 + T cells stimulated for 48 h with anti-cd3 plus anti-cd28 and isotype control antibody or DTA-1, with or without TGF-β or IL-4. (b) IL-4 in accumulated supernatants of CD4 + T cells cultured as in a for 3 d. (c) The frequencies of IL-9 + cells in CD4 + T cells activated as in a plus TGF-β and different concentrations of IL-4 (0-100 ng/ml). (d,e) IL-9 production in naïve CD4 + T cells from WT and Il4 / mice stimulated for 3 d as in a, measured by flow cytometry (d) and by ELISA (e). n.s. nonsignificant, * P < 0.05, ** P < 0.01, *** P < 0.001 by Student s t test. Data represent two independent experiments.
Supplementary Fig. 12 IL-4R signaling is not a prerequisite for GITR-induced T H 9 differentiation but is required for optimal IL-9 production. (a,b) IL-9 production by naïve CD4 + T cells from wild-type littermates and Il4ra / mice stimulated for 3 d with anti-cd3 plus anti-cd28 and either isotype control antibody or DTA-1, with or without TGF-β or IL-4, measured by flow cytometry (a) and by ELISA (b) in the accumulated supernatant of cultures (triplicate). N.D. Not done. * P < 0.05, ** P < 0.01, *** P < 0.001 by Student s t test. Data represent two independent experiments.
Supplementary Fig. 13 Kinetic analyses of the T H 9-related gene expression following DTA-1 treatment. Expression of the indicated genes in naïve CD4 + T cells activated with anti-cd3 and anti-cd28 in the presence of isotype control antibody or DTA-1, with or without T H 9-polarizing condition. Data represent two independent experiments.
Supplementary Fig. 14 Inhibition of NF-κB signaling pathways by BAY 11-82 dose not alter cell survival and proliferation. (a) Blot images of the NF-κB signaling subunits in the nuclear of naïve CD4 + T cells pre-activated with anti-cd3 and anti-cd28 for 24 h, treated with the vehicle (Veh.) or indicated concentrations of BAY 11-82 for 30 min and stimulated with either isotype control antibody or DTA-1 for 6 h. (b) FSC vs. SSC and CFSE dilution in CFSE-labeled naïve CD4 + T cells cultured as in Figure 6b. Data represent two independent experiments.
Supplementary Fig. 15 GITR co-stimulation upregulates TRAF6 expression in mouse and human CD4 + T cells. Expression of TRAF family transcripts in mouse (a) and human (b) naïve CD4 + T cells stimulated for 48 h with isotype control or GITR agonistic antibody in the presence of irradiated T cell-depleted total splenocytes plus anti-mcd3 and mitomycin C-treated allogenic human DCs plus anti-hcd3, respectively (triplicate). * P < 0.05, ** P < 0.01 by Student s t test. Data represent two independent experiments.
Original gel images for Figure 6a kda 130 95 130 95 55 55 43 Long exposure kda 130 P100 95 <cytosol> kda 130 95 kda 130 95 55 Short exposure 55 Nature Medicine: doi:10.1038/nm.3922
Original gel images for Supplementary Fig. 4b and Supplementary Fig. 14a kda 130 95 55 43 55 Supplementary Fig. 4b Stripped 55 Supplementary Fig. 14a Nature Medicine: doi:10.1038/nm.3922