Nature Medicine: doi: /nm.3922
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1 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 < by Student s t test. Data represent two independent experiments. Nature Medicine: doi: /nm.3922
2 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.
3 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.
4 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.
5 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.
6 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 < by Student s t test. N.S. non-significant. Data represent two independent experiments.
7 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.
8 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 < by Student s t test. N.S. non-significant. Data represent two independent experiments.
9 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 < by Student s t test. Data represent at least two independent experiments.
10 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 < by Student s t test. Data represent two independent experiments.
11 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 < by Student s t test. Data represent two independent experiments.
12 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 < by Student s t test. Data represent two independent experiments.
13 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.
14 Supplementary Fig. 14 Inhibition of NF-κB signaling pathways by BAY 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 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.
15 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.
16 Original gel images for Figure 6a kda Long exposure kda 130 P <cytosol> kda kda Short exposure 55 Nature Medicine: doi: /nm.3922
17 Original gel images for Supplementary Fig. 4b and Supplementary Fig. 14a kda Supplementary Fig. 4b Stripped 55 Supplementary Fig. 14a Nature Medicine: doi: /nm.3922
Supplemental Materials
Supplemental Materials Programmed death one homolog maintains the pool size of regulatory T cells by promoting their differentiation and stability Qi Wang 1, Jianwei He 1, Dallas B. Flies 2, Liqun Luo
Supplemental Figure 1
Supplemental Figure 1 1a 1c PD-1 MFI fold change 6 5 4 3 2 1 IL-1α IL-2 IL-4 IL-6 IL-1 IL-12 IL-13 IL-15 IL-17 IL-18 IL-21 IL-23 IFN-α Mut Human PD-1 promoter SBE-D 5 -GTCTG- -1.2kb SBE-P -CAGAC- -1.kb
Supplementary Figure 1. Characterization of basophils after reconstitution of SCID mice
Supplementary figure legends Supplementary Figure 1. Characterization of after reconstitution of SCID mice with CD4 + CD62L + T cells. (A-C) SCID mice (n = 6 / group) were reconstituted with 2 x 1 6 CD4
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Supplemental Figure 1. Furin is efficiently deleted in CD4 + and CD8 + T cells. a, Western blot for furin and actin proteins in CD4cre-fur f/f and fur f/f Th1 cells. Wild-type and furin-deficient CD4 +
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doi:1.138/nature1554 a TNF-α + in CD4 + cells [%] 1 GF SPF 6 b IL-1 + in CD4 + cells [%] 5 4 3 2 1 Supplementary Figure 1. Effect of microbiota on cytokine profiles of T cells in GALT. Frequencies of TNF-α
Supplementary Fig. 1 p38 MAPK negatively regulates DC differentiation. (a) Western blot analysis of p38 isoform expression in BM cells, immature DCs
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A B16F1 s.c. Lung LN Distant lymph nodes Colon B B16F1 s.c. Supplementary Figure 1. Deletion of Smad3 prevents B16F1 melanoma invasion and metastasis in a mouse s.c. tumor model. Highly invasive growth
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SUPPLEMENTARY MATERIAL
SUPPLEMENTARY MATERIAL IL-1 signaling modulates activation of STAT transcription factors to antagonize retinoic acid signaling and control the T H 17 cell it reg cell balance Rajatava Basu 1,5, Sarah K.
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doi: 1.138/nature89 IFN- (ng ml ) 5 4 3 1 Splenocytes NS IFN- (ng ml ) 6 4 Lymph node cells NS Nfkbiz / Nfkbiz / Nfkbiz / Nfkbiz / IL- (ng ml ) 3 1 Splenocytes IL- (ng ml ) 1 8 6 4 *** ** Lymph node cells
Blocking antibodies and peptides. Rat anti-mouse PD-1 (29F.1A12, rat IgG2a, k), PD-
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Cell isolation. Spleen and lymph nodes (axillary, inguinal) were removed from mice
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D CD8 T cell number (x10 6 )
IFNγ Supplemental Figure 1. CD T cell number (x1 6 ) 18 15 1 9 6 3 CD CD T cells CD6L C CD5 CD T cells CD6L D CD8 T cell number (x1 6 ) 1 8 6 E CD CD8 T cells CD6L F Log(1)CFU/g Feces 1 8 6 p
Supplemental Figure Legends
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Supplementary Information:
Supplementary Information: Follicular regulatory T cells with Bcl6 expression suppress germinal center reactions by Yeonseok Chung, Shinya Tanaka, Fuliang Chu, Roza Nurieva, Gustavo J. Martinez, Seema
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Supplementary Figure 1.
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Supporting Information
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<10. IL-1β IL-6 TNF + _ TGF-β + IL-23
3 ns 25 ns 2 IL-17 (pg/ml) 15 1 ns ns 5 IL-1β IL-6 TNF
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Supplementary Figure 1. Efficiency of Mll4 deletion and its effect on T cell populations in the periphery. Nature Immunology: doi: /ni.
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Supplementary. presence of the. (c) mrna expression. Error. in naive or
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Supplemental Table I Male / Mean ± SEM n Mean ± SEM n Body weight, g 29.2±0.4 17 29.7±0.5 17 Total cholesterol, mg/dl 534.0±30.8 17 561.6±26.1 17 HDL-cholesterol, mg/dl 9.6±0.8 17 10.1±0.7 17 Triglycerides,
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SUPPLEMENT Supplementary Figure 1: (A) (B)
SUPPLEMENT Supplementary Figure 1: CD4 + naïve effector T cells (CD4 effector) were labeled with CFSE, stimulated with α-cd2/cd3/cd28 coated beads (at 2 beads/cell) and cultured alone or cocultured with
Pearson r = P (one-tailed) = n = 9
8F4-Specific Lysis, % 1 UPN1 UPN3 8 UPN7 6 Pearson r =.69 UPN2 UPN5 P (one-tailed) =.192 4 UPN8 n = 9 2 UPN9 UPN4 UPN6 5 1 15 2 25 8 8F4, % Max MFI Supplementary Figure S1. AML samples UPN1-UPN9 show variable
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doi: 1.138/nature775 4 O.D. (595-655) 3 1 -ζ no antibody isotype ctrl Plated Soluble 1F6 397 7H11 Supplementary Figure 1 Soluble and plated anti- Abs induce -! signalling. B3Z cells stably expressing!
Supplementary Figures
MiR-29 controls innate and adaptive immune responses against intracellular bacterial infection by targeting IFN-γ Feng Ma 1,2,5, Sheng Xu 1,5, Xingguang Liu 1, Qian Zhang 1, Xiongfei Xu 1, Mofang Liu 3,
Excerpt from MACS&more Vol 17 1/2016. Kenji Miki¹, Koji Nagaoka¹, Hermann Bohnenkamp², Takayuki Yoshimoto³, Ryuji Maekawa¹*, and Takashi Kamigaki⁴
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Supplementary Figure 1 Protease allergens induce IgE and IgG1 production. (a-c)
1 Supplementary Figure 1 Protease allergens induce IgE and IgG1 production. (a-c) Serum IgG1 (a), IgM (b) and IgG2 (c) concentrations in response to papain immediately before primary immunization (day
pro-b large pre-b small pre-b CCCP (µm) Rag1 -/- ;33.C9HCki
a TMRM FI (Median) b TMRM FI (Median) c 20 15 10 5 0 8 6 4 2 0 pro-b large pre-b small pre-b 0 10 20 30 40 50 60 70 80 90 100 TMRM (nm) pro-b large pre-b small pre-b 0 1 2 4 8 16 32 64 128 256 CCCP (mm)
well for 2 h at rt. Each dot represents an individual mouse and bar is the mean ±
Supplementary data: Control DC Blimp-1 ko DC 8 6 4 2-2 IL-1β p=.5 medium 8 6 4 2 IL-2 Medium p=.16 8 6 4 2 IL-6 medium p=.3 5 4 3 2 1-1 medium IL-1 n.s. 25 2 15 1 5 IL-12(p7) p=.15 5 IFNγ p=.65 4 3 2 1
Supplementary Figures
Supplementary Figures Supplementary Fig. 1. Galectin-3 is present within tumors. (A) mrna expression levels of Lgals3 (galectin-3) and Lgals8 (galectin-8) in the four classes of cell lines as determined
% of live splenocytes. STAT5 deletion. (open shapes) % ROSA + % floxed
Supp. Figure 1. a 14 1 1 8 6 spleen cells (x1 6 ) 16 % of live splenocytes 5 4 3 1 % of live splenocytes 8 6 4 b 1 1 c % of CD11c + splenocytes (closed shapes) 8 6 4 8 6 4 % ROSA + (open shapes) % floxed
ILC1 and ILC3 isolation and culture Following cell sorting, we confirmed that the recovered cells belonged to the ILC1, ILC2 and
Supplementary Methods and isolation and culture Following cell sorting, we confirmed that the recovered cells belonged to the, ILC2 and subsets. For this purpose we performed intracellular flow cytometry
Supplementary information
Supplementary information Intrahepatic myeloid cell-aggregates enable local CD8 + T cell expansion and successful immunotherapy against chronic viral liver infection Li- Rung Huang, Dirk Wohlleber, Florian
Supplementary Figures
Supplementary Figures Supplementary Figure 1 Characterization of stable expression of GlucB and sshbira in the CT26 cell line (a) Live cell imaging of stable CT26 cells expressing green fluorescent protein
S1a S1b S1c. S1d. S1f S1g S1h SUPPLEMENTARY FIGURE 1. - si sc Il17rd Il17ra bp. rig/s IL-17RD (ng) -100 IL-17RD
SUPPLEMENTARY FIGURE 1 0 20 50 80 100 IL-17RD (ng) S1a S1b S1c IL-17RD β-actin kda S1d - si sc Il17rd Il17ra rig/s15-574 - 458-361 bp S1f S1g S1h S1i S1j Supplementary Figure 1. Knockdown of IL-17RD enhances
of whole cell cultures in U-bottomed wells of a 96-well plate are shown. 2
Supplementary online material Supplementary figure legends Supplementary Figure 1 Exposure to T reg cells causes loss of T resp cells in co-cultures. T resp cells were stimulated with CD3+CD28 alone or
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Complete but curtailed T-cell response to very-low-affinity antigen Dietmar Zehn, Sarah Y. Lee & Michael J. Bevan Supp. Fig. 1: TCR chain usage among endogenous K b /Ova reactive T cells. C57BL/6 mice
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