Anti cd49a ha31 8

BRPKp110 tumor cells (4×10⁵) were injected SC into C57BL/6 mice. Mice were treated IP with anti-PD-1 (RMP1), anti-LAG-3 (C9B7W), and anti-TIM-3 (RMT2–23) or matching IgG isotype controls (HRPN and 2A3)(all from BioXCell, 250μg/mouse per antibody) 48 hours prior to tumor harvest at day 19. In other experiments, mice were treated IV with 200μg/mouse anti-CD49a (Ha31/8, BD Biosciences) 24 hours prior to tumor harvest at day 21.

Fluorescent-labeled antibodies used were anti-NK1.1 (clone PK136), anti-NKp46 (29A1.4), anti-CD3 (145–2C11), anti-CD19 (eBio1D3), anti-CD45.1 (A20), anti-CD45.2(104), anti-Eomes (Dan11mag), anti-TNFα (MP6-XT22), and anti-IFNγ (XMG1.2), all from Thermo Fisher Scientific. Fluorescent-labeled anti-CD49a (Ha31/8) was purchased form BD Biosciences. Biotinylated anti-Ly49H (3D10) was produced in-house. MG-132(Cell Signaling 2094S), Actinomycin D (Sigma A9415), Cycloheximide (Sigma C7698), CGP-57380 (Sigma C0993), ISRIB (Sigma SML0843), Pyr-41 (Sigma N2915), BMS-345541 (Sigma B9935), Tpl2 kinase inhibitor (Santa Cruz sc-204351), PD90859 (Sigma P215) were used at the indicated concentrations. Where indicated, NK cells were enriched to a purity >90% NK1.1+CD3-CD19- cells, verified by flow cytometry for each experiment, from RAG1^−/− splenocytes using CD49b microbeads with LS columns (Miltenyi Biotec) or from C57BL/6 mice using EasySep Mouse NK cell isolation kit (StemCell Technologies) according to manufacturer’s instructions. m157-Tg or WT murine embryonic fibroblasts (MEFs) were isolated from day 11.5–13.5 embryos.