Colitis-associated tumors were induced as described previously (19) by administering DSS (MP Biomedicals) in the drinking water and injecting AOM (MilliporeSigma; 10 mg/kg in saline). Control mice received water and were injected with saline. Mice were euthanized 10 days after the last DSS treatment. After the third DSS cycle and at the end of the experiment, mice were anesthetized by i.p. injection of ketamine (90–120 mg/kg body weight; Vetoquinol) and xylazine (8 mg/kg body weight; Bayer), and a colonoscopy was performed to monitor tumor growth.

For injection models, MC38-GFP (donated by Lubor Borsig, Institute of Physiology, University of Zurich, Zurich, Switzerland) or B16-F10 tumor cells were suspended in culture medium, mixed 1:1 with Matrigel and 300,000 cells, and injected into the cecal wall or s.c. into the mouse flank. Mice were euthanized 2–3 weeks after injection. In the s.c. model, tumor development was measured every 3 days using a digital caliper. Tumor volume was calculated using the ellipsoid formula: 4/3 × 3.14 × length/2 × (width/2)2, where the shorter dimension was used as the width and depth. Mice were euthanized when the volume reached 1 cm3 or the length reached 2 cm.

CD8+ T cells depletion was performed using anti-CD8 (Lyt 3.2) antibody (Bio X Cell; clone 53-5.8) or IgG isotype control (Bio X Cell; clone HRPN). Antibodies were injected i.p. in a concentration of 200 mg/mouse on day –3 and 100 mg/mouse on day 0 and then once weekly thereafter. CD4+ T cells depletion was performed using an anti-CD4 antibody (Bio X Cell; clone GK 1.5) or an IgG isotype control (Bio X Cell; clone LTF-2). Antibodies were injected i.p. at a concentration of 200 mg/mouse on day –3, day 0, and every 3 days thereafter. PD-1 blockade was performed in the s.c. tumor model and achieved by i.p. injection of 200 mg/mouse anti–PD-1 (CD279) antibody (Bio X Cell; clone 29F.1A12) or IgG isotype control (Bio X Cell; clone 2a3) on days 9, 12, and 15 after injection of MC38 cells.

For adoptive transfer, 500,000 CD8+ T cells from Ptpn2fl/fl and Ptpn2fl/fl Cd4Cre+/– mice were transferred by i.p. injection into Rag2–/– mice on day 6 after the s.c. MC38 tumor injection.

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