Cd4 cre

Six-to eight-week-old C57BL/6, Cd11c^cre, Zbtb46^cre, Cd4^cre, E8i^cre, Lysm^cre, Cx3cr1^cre and Foxp3-ERT2^cre mice were purchased from the Jackson Laboratory. Havcr2^fl/fl mice were generated as described in supplementary materials. TIM-3 conditional knockout mice were generated by crossing to the above cre lines. For knockin/knockout alleles (Lysm^cre) the appropriate controls were used; that is, Havcr2^fl/+ × Lysm^cre+/−, for all other cre lines fl/fl mice were used as controls. For experiments with Foxp3-ERT2^cre mice, mice were orally gavaged with 8 mg tamoxifen 3 days before tumour implantation and every 3 days thereafter for the duration of the experiments. Havcr2^+/+Foxp3-ERT2^cre were used as an additional wild-type control but results were comparable to Havcr2^fl/fl and were therefore not included. Deletion efficiency was determined by flow cytometry (not shown). Animal experiments were done in accordance with the guidelines of the institutional Animal Care and Use Committee (IACUC) at Brigham and Women’s Hospital and Harvard Medical School. All animals were euthanized before reaching humane endpoint, with tumour growth no greater than 2 cm in any one direction or of a total of 400 mm² overall. Mice of both sexes were used throughout the study; sex-matched and age-matched (8–12 weeks) controls were used in individual experiments.

All mice were housed in specific-pathogen free facilities at the University of Minnesota and experiments were performed in accordance with protocols approved by the Institutional Animal Care and Use Committee. Gitr ^−/− mice were previously described^{19 (link)} and provided by Drs. Carlo Riccardi and Ethan Shevach. Ox40 ^−/−, Foxp3-GFP, Foxp3-RFP, Rag2 ^−/−, and Cd4^Cre mice were purchased from the Jackson Laboratory (Bar Harbor, ME, stock numbers 012839, 006772, 008374, 008449, and 013234 respectively). CD45.1 (B6.SJL) mice were purchased from the NCI (Bethesda, MD). Nur77-GFP BAC reporter mice^{24 (link)}, Tak1 ^fl/fl and Bcl2-Tg mice were previously described^{44 (link), 45 (link)}. Cd28^−/− and Cd28^AYAA knock-in mice were previously described^{46 (link)}. All mice used were on the C57Bl/6 background, except for Cd28^−/− and Cd28^AYAA mice and their wild type littermates, which were on a BALB/c background. Mice were randomly selected for experiments from our mouse facility in age-matched cohorts. The investigators were not blinded to genotype during data acquisition.

Zranb1-targeted mice(Zranb1^{tm1a(EUCOMM)Hmgu}) were generated at Knockout Mouse Project (KOMP) by targeting exon 3 of Zranb1 gene using a FRT-LoxP vector (Supplementary Fig. 1a). Zranb1-floxed mice (in C57BL/6 × 129/Sv mixed background) were generated by crossing the Zranb1-targeted mice with FLP deleter mice (Rosa26-FLPe; Jackson Laboratory). The Zranb1-floxed mice were further crossed with CMV-Cre, Cd4-Cre, Cd11c-Cre, and Lyz2-Cre mice (all from Jackson Laboratory, C57BL/6 background) to generate Zranb1 germline KO, T-cKO (Zranb1^f/fCd4-Cre), DC-cKO (Zranb1^f/fCd11c-Cre), and M-cKO (Trabid^f/fLyz2-Cre) mice, respectively. Heterozygous mice were bred to generate littermate controls and KO (or conditional KO) mice for experiments. Outcomes of animal experiments were collected blindly and recorded based on ear-tag numbers of the experimental mice. Genotyping was performed as indicated in Supplementary Fig. 1. Mice were maintained in specific pathogen-free facility, and all animal experiments were conducted in accordance with protocols approved by the Institutional Animal Care and Use Committee of the University of Texas MD Anderson Cancer Center.