Supplemental Information. Crosstalk between Regulatory T Cells. and Tumor-Associated Dendritic Cells. Negates Anti-tumor Immunity in Pancreatic Cancer

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1 Cell Reports, Volume 20 Supplemental Information Crosstalk between Regulatory T Cells and Tumor-Associated Dendritic Cells Negates Anti-tumor Immunity in Pancreatic Cancer Jung-Eun Jang, Cristina H. Hajdu, Caroline Liot, George Miller, Michael L. Dustin, and Dafna Bar-Sagi

2 Figure S1, related to Figure 1. (A) GFP-Kras G12D -PDECs were implanted (day 0) into the pancreta of synegeic WT mice. Pancreata were analyzed 2 weeks post-implantation. Flow cytometric quantification of CD4 + Foxp3 + Treg cells (left) and the expression of CTLA-4 (middle) and PD-1 (right) on Foxp3 + Treg cells in pancreata, tumor-draining pancreatic LNs (Pan LNs), and peripheral inguinal lymph nodes (ilns) (n= 4-5 mice). (B) Schematic of the experimental design. The arrows indicate either PBS (control) or DT (50 μg/kg) was injected i.p. on days 7, 9, 11, and 13 after implantation of GFP- Kras G12D -PDECs into pancreata of WT mice. Analyses were performed 14 days after orthotopic implantation. Orthotopic pancreatic tumors (left) and quantification of tumor volume (right) are shown (n= 3 mice). (C) Schematic of the experimental design. Either PBS or DT was injected i.p. once a week for 3 weeks starting 1 week after implantation of GFP-Kras G12D -PDECs into pancreata of Foxp3 DTR mice. Analyses were performed 5 weeks after orthotopic implantation. Quantification of orthotopic pancreatic tumor volume (right) (n= 5 mice). Data are represented as mean ± SEM. *p < 0.05, **p < 0.01.

3 Figure S2, related to Figure week-old KC mice were treated with 500 μg anti-cd25 neutralizing monoclonal antibody or isotype control i.p. every three days for 5 weeks. All analyses were performed at 5 weeks (n= 5-6 mice). (A) Representative images of H&E staining on pancreatic sections are shown. Scale bar, 1 mm. (B) The pancreatic area occupied by intact acinar structures, numbers of PanIN lesions, and the fraction of ductal structures exhibiting normal, acinar-to-ductal metaplasia (ADM), or graded PanIN I-III lesions are shown. (C) Flow cytometric plots (left) and quantification (right) of the percentage and number of CD4 + Foxp3 + Treg cells in the pancreas. Data are represented as mean ± SEM. *p < 0.05.

4 Figure S3, related to Figure 2. (A and B) Flow cytometric plots (left) and quantification (right) of expression of CD25, CD44 (A), and IFN- (B) in CD4 + Foxp3 - T cells (n= 4-5 mice). (C) Flow cytometric plots of negative and positive controls for Granzyme B and IFN- staining in Figure 2C and 2D and Figure S3B. Naïve T cells stimulated with anti-cd3 and anti-cd28 and unstimulated naïve T cells were used as positive and negative controls, respectively. (D) Flow cytometric quantification of expression of CD44 (left), GranzymeB (middle), and IFN- (right) in CD8 + cells in tumor-draining Pan LNs, and peripheral ilns are shown (n= 4-5 mice). (E) Schematic of the experimental design. The arrows indicate either PBS (control) or DT (50 μg/kg) was injected i.p. 7 and 9 days post-implantation, and 150 μg Rat IgG2b isotype or anti-cd4 (αcd4) antibody 8 and 11 days after implantation of GFP-Kras G12D -PDECs into pancreata of WT or Foxp3 DTR mice. Analysis was performed 15 days after orthotopic implantation. Orthotopic pancreatic tumors (left) and quantification of tumor volume (right) are shown (n= 4-5 mice). Data are represented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, NS (not significant) p > 0.05.

5 Figure S4, related to Figure 3. (A) The schematic illustrates the experimental system. The arrows indicate either 500 μg Rat IgG2a (isotype control) or anti-cd25 (αcd25) was injected i.p. every three days for 2 weeks starting 1 week after implantation of GFP- Kras G12D -PDECs into pancreata of CD8a-Cre;Rosa tdtomato ;CD11c-EYFP mice. Analyses were performed at 3 weeks post-implantation of GFP-Kras G12D -PDECs (n= 3-4 mice). (B) Percentage of CD4 + Foxp3 + Treg cells in the pancreas. (C) Average contact duration of CD11c + cells with CD8 + T cells. (D) Representative in vivo time-lapse images of contacts between CD8 + T cells and CD11c + cells in pancreata of CD8a-Cre;Rosa tdtomato ;CD11c-EYFP mice treated with isotype control (top) or anti-cd25 (bottom). Relative frequency of contact duration between CD8 + T cells and CD11c + cells is shown. Scale bar, 50 μm. Data are represented as mean ± SEM. *p < 0.05, ****p <

6 Figure S5, related to Figure 3. (A) 4-6 month-old KC mice were analyzed. (B) Either PBS (control) or DT (50 μg/kg) was injected i.p. on days 7, 9, 11, and 13 after implantation of GFP-Kras G12D -PDECs into pancreata of Foxp3 DTR mice. Analyses of orthotopic pancreatic tumors were performed 14 days post-implantation. Representative images from pancreatic sections of H&E staining and immunofluorescence staining of CD45 +, CD4 + T, CD8 + T, B220 +, and Foxp3 + cells are shown (n= 3-4 mice). Scale bar, 100 μm.

7 Figure S6, related to Figure 5 and 6. (A) Histograms displaying surface expression of MHC class II, CD40, C80 and CD86 on CD11c hi cells from the pancreas, pan LNs, and LNs in 5-8 month-old KC mice (n= 5-6 mice). Relative surface expression levels were measured by geo mean fluorescence intensity (MFI) minus isotype MFI. (B) Comparison of relative surface expression levels of MHC class II, CD40, C80 and CD86 on CD11c + cells from the pancreas and pan LNs in WT and KC mice are shown (n= 5-6 mice). (C) Relative surface expression levels of CD40, CD80 and CD86 on CD11c + cells 5 weeks after orthotopic pancreatic grafts of Foxp3 DTR mice treated with either PBS (red) or DT (green) compared to respective isotype controls (gray) (n= 4-5 mice). Data are represented as mean ± SEM. *p < 0.05, **p < 0.01.

8 Figure S7, related to Figure 7. (A) Schematic of the experimental design (left). Either 500 μg hamster IgG isotype control or anti-cd11c (clone N418) mab was injected i.p. in WT mice. Analysis was performed 3 days post-injection (n= 3 mice). Mononuclear cells isolated from the spleen and pancreas were stained using APC-conjugated anti-cd45 and biotin-conjugated anti-cd11c (clone HL3) followed by streptavidin-conjugated PE-cy5, and then analyzed by flow cytometry. Flow cytometric plot (middle) and quantification (right) of expression of CD11c + cells after gating on CD45 + population in the spleen and pancreas. (B and C) GFP-Kras G12D -PDEC were implanted into the pancreata of syngeneic WT or Foxp3 DTR mice. PBS or DT (50 μg/kg) was injected 7 and 9 days post-implantation and 500 μg hamster IgG isotype or anti-cd11c (N418) antibody 7 and 10 days post-implantation. Immune cells in orthotopic pancreatic grafts were analyzed by flow cytometry 14 days after implantation (n= 5-7 mice). Representative flow cytometric plots of CD44 and IFN- in CD4 + Foxp3 - cells (B) and CD44, granzyme B, and IFN- in CD8 + T cells (C).

9 SUPPLEMENTAL EXPERIMENTAL PROCEDURES Generation of bone marrow chimeras For bone marrow chimeras, recipients were irradiated with two 6.5Gy doses of γ irradiation from a cesium source at an interval of 3 hrs. CT or KCT bone marrow chimeras were generated by transplantation of 5 x 10 6 bone marrow cells isolated from CD11c-EYFP mice into lethally irradiated CT or KCT mice. Chimeras were analyzed 3 months after transplantation. Orthotopic tumor model For orthotopic implantation, a small (~10 mm) left abdominal side incision of mouse was made. Kras G12D -PDECs (1 x10 6 cells/mouse) or KPC cells (1 x10 5 cells/mouse) were suspended in Matrigel (Becton Dickinson) diluted 1:1 with PBS in a total volume of 50 μl and injected into the tail region of the pancreas using an insulin syringe. A successful injection was verified by the appearance of a fluid bubble without intraperitoneal leakage. The abdominal wall was closed with absorbable Vicryl Rapid sutures (Ethicon, VR834) and the skin was closed with Perma-hand silk sutures (Ethicon, K871). Mice were sacrificed at the indicated time points, and tumors were dissected away from surrounding pancreatic tissue. The length and width of the ovoid tumors was measured and the graft processed for flow cytometry analysis. Intravital two-photon laser-scanning microscopy (TPLSM) of pancreas The stage and mouse were maintained at 37 C by flowing heated air over the system (BioTherm Micro S37; Biogenics) and the pancreata and spleen were kept moist with saline throughout the experiment. To visualize vasculature, mice were injected i.v. with 25 μg Evan Blue (Sigma)10 min before imaging. Images were acquired with a Zeiss LSM 710 inverted microscope (Zeiss 25X 0.8 NA oil immersion objective) with a MaiTai Ti:Sapphire laser (Spectra-Physics) tuned to nm to excite all fluorescent probes used. Emitted fluorescence was detected through 420/40, 465/30, 520/30, 575/70, and 660/50 nm band-pass filters and nondescanned detectors to generate second harmonic signals (collagen fibers) and 4-color images. ZEN 2009 software (Zeiss) was used for operating the microscope, image acquisition and data handling. All the images were acquired at least μm below the tumor surface. For time lapse 10 to 15 images separated by 3 μm in the z axis were acquired with 30 second intervals between 3D stacks for min. All images and cell movement analysis were performed using Imaris v software (Bitplane). Individual cells were identified and tracked automatically using Imaris, annotated for region and pruned manually. Cell speed and displacement were calculated from tracks. Contact duration was quantified manually by assessing cell-cell juxtaposition in individual z-planes. Movies were then annotated and edited for presentation using ImageJ (NIH). Immunofluorescence μm-thick cryostat sections of pancreas were fixed with cold acetone for 10 min at -20 C and blocked/permeabilized with 0.2% Triton X-100 in PBS with 5% BSA and 10% FCS for 10 min at room temperature. Endogenous biotin and avidin were blocked with an avidin and biotin blocking kit (Vector laboratories) according to the manufacturer s instructions. Sections were stained using the following antibodies diluted in PBS containing 5% BSA and 10% goat serum overnight at 4 C: biotinylated anti-foxp3 (clone FJK-16s), APC-conjugated anti-cd11c (clone N418), PE-conjugated CD45 (RA3-6B2), APC conjugated CD4 (clone GK1.5), CD8a (clone ) from ebioscience, and unconjugated anti-ido (clone mido-48), biotinylated anti-cd45 (clone 30-F11) from Biolegend for mouse samples, unconjugated anti-human IDO (clone VINC3IDO, ebioscience), and Alexa Fluor 647 antihuman CD11c (clone 3.9, Biolegend) for human samples. Alexa Fluor 555-conjugated Streptavidin, goat anti-rabbit or anti-mouse (Invitrogen) diluted in PBS with 5% BSA were incubated for 30 min at room temperature and sections were mounted using mounting medium with DAPI (Vector Laboratories). Confocal images were acquired on Zeiss LSM 710 microscope equipped with a 25x or 40x oil lens. Immunohistochemistry and histology Immunohistochemistry was performed on sections from formalin-fixed paraffin embedded tissues. Deparaffinized sections (5 μm) were rehydrated and quenched in 1% hydrogen peroxide/methanol for 15 min, and antigen retrieval was performed in 10 mm sodium citrate and 0.05% Tween-20 (ph 6.0) for 15 min in a microwave oven. Blocking was performed in 10% serum, 1% BSA, and 0.5% Tween-20 for 1 hr at room temperature, followed by incubation with the primary antibodies diluted in 2% BSA overnight at 4 C. The following primary antibodies were used: rabbit anti-gfp (Cell Signaling) and rabbit anti-cleaved caspase-3 (Cell Signaling). After incubating with secondary biotinylated antibodies and ABC solution (both from Vector Laboratories), sections were developed with 3,3 - diaminobenzidine (DAB substrate kit, Vector Laboratories). After counterstaining with Harris hematoxylin (Sigma-

10 Aldrich), slides were subjected to an alcohol dehydration series and mounted with Permount (Fisher). Slides were examined on a Nikon Eclipse 80i microscope. For histology analysis, excised tumors were fixed in 10% neutral buffered formalin and processed for routine hematoxylin and eosin staining. The fraction of preserved acinar area and the faction and number of ducts containing all grades of PanIN lesions was evaluated as previously described (Daley et al., 2016). Foxp3 and intracellular cytokine staining for flow cytometry For the detection of transcription factors, cells were fixed and stained using the Foxp3-staining kit (ebioscience) according to the manufacturer s instructions. For intracellular cytokine staining, cells were stimulated in the presence of phorbol 12-myristate 13-acetate (PMA) (50 ng/ ml, Sigma), Ionomycin (500 ng/ml, Sigma) with Monensin (BD GolgiStop) in RPMI-10 media (RPMI-1640 containing 10% FBS, 1% nonessential amino acid, 50 μm β-mercaptoethanol, 1% Sodium pyruvate, 1% HEPES, 100 u/ml penicillin and 100 μg/ml streptomycin sulfate) at 37 C for 4 hours. The cells were fixed and permeabilized with a Cytofix/Cytoperm Kit (BD biosciences) according to the manufacture s protocol. Antibodies. Antibodies were obtained as follows: CD45 (clone 30-F11), CD3e (clone 17A2), CD4 (clone GK1.5 & RM4-5), CD8a (clone 5H10-1), CD19 (clone ebio1d3), CD11c (clone N418 & clone HL3), MHC class II (clone M5/ ), CD11b (clone M1/70), F4/80 (clone BM8), CD135 (Flt3) (clone A2F10), CD44 (clone IM7), Granzyme B (clone NGZB), Perforin (clone ebioomak-d), Foxp3 (clone FJK-16s), IDO (clone mido-48), CTLA-4 (clone UC10-4B9) from ebioscience, CD25 (cone PC61), IFN-γ (clone XMG1.2), CD86 (clone GL-1), CD40 (clone 3/23), CD80 (16-10A1) from Biolegend, CD8a (clone ) from BD Pharmingen. Secondary antibodies, isotype controls and fluorophore-conjugated streptavidin were purchased from Biolegend and ebioscience. Reference Daley, D., Zambirinis, C. P., Seifert, L., Akkad, N., Mohan, N., Werba, G., Barilla, R., Torres-Hernandez, A., Hundeyin, M., Mani, V. R., et al. (2016). gammadelta T Cells Support Pancreatic Oncogenesis by Restraining alphabeta T Cell Activation. Cell 166, e1415.