Mouse tumor models

B16F10 melanoma cells were kindly provided by Dr. Linda Sherman (TSRI, La Jolla, CA). MC38 cells were kindly provided by Dr. John Teijaro (TSRI, La Jolla, CA). Tumor cells were grown in complete DMEM (Invitrogen) supplemented with 10% heat-inactivated FCS. For tumor challenge studies, mice were shaved 24–48 h before tumor injection. For tumor cell injections, cells were detached using 0.05% trypsin-EDTA (Invitrogen), washed, and suspended in 1× Dulbecco’s PBS (DPBS). Cells were then injected intradermally with a 1-ml syringe and a 25-gauge needle in a total volume of 100 µl. To determine tumor latency, mice were injected with 10⁵ B16F10 melanoma cells and assessed daily for palpable tumors. To measure tumor growth rates, mice were injected with either 5 × 10⁵ B16F10 or 2 × 10⁵ MC38 cells. After tumor formation, tumor growth was measured using calipers every 2–3 d. Tumors were measured in two directions along the long (D) and short (d) axes in order to calculate tumor volume (0.5 × D × d²). Mouse survival was tracked until tumors grew to 1,200 mm³ in size. Mice that developed severe tumor ulceration or cachexia during experiments were euthanized. For in vivo antibody treatments, mice were treated with low-endotoxin, functional grade anti-JAML (clone HL4E10; Verdino et al., 2011; produced at the Scripps California Institute for Biomedical Research), anti–PD-1 (clone RMP1-14; Leinco), rat IgG2a isotype (clone 1-1; Leinco), or Armenian hamster IgG (clone PIP; Leinco) antibodies. 200 µg of each antibody was diluted in 1× DPBS and administered via i.p. injection in a total volume of 200 μl. Antibodies were administered on days 7, 10, and 13 after tumor challenge for tumor growth studies and on day 7 for analysis of TILs by flow cytometry. For CD8 T cell depletion studies, mice were treated with 200 µg anti-CD8 (clone YTS-169; Leinco) or rat IgG2a isotype (clone 1-1; Leinco) antibodies starting on day 4 after tumor challenge and then concurrently with Armenian hamster IgG or anti-JAML antibodies on days 7, 10, and 13 after tumor challenge.