Granzyme b gb11

Cell surface markers were stained for 30 min in the dark at 4 °C. Intracellular cytokine staining was performed using the ebioscience Foxp3 staining kit (ThermoFisher Scientific, Waltham MA) per manufacturer’s protocol. The following mouse antibodies from BioLegend (San Diego CA) were used: CD4 (GK1.5); CD8 (53-6.7); Tbet (4B10); Gata3 (16E10A23); Foxp3 (MF-14); IFNγ (XMG1.2); IL-10 (JES5-16E3); IL17 (TC11-18H10.1); and TGFβ (TW7-16B4). RORγt (AFKJS-9) was ordered from eBioscience (ThermoFisher Scientific). To show T cell reactivity, splenocytes were isolated from tumor bearing mice and cultured with 4T1 cells at a ratio of 5:1 (splenocytes to tumor cells) in the presence anti-CD107A/CD107B (ThermoFisher Scientific) and Monensin for 4 h. After 4 h, cells were stained for surface and intracellular markers described above. Flow cytometry analysis of T cell markers on human PBMCs was performed using the following clones: CD8 (RPA-T8); CD4 (SK3); Tbet (4B10); Ki67 (Ki67) from BioLegend; RORγt and granzyme B (GB11) from ThermoFisher Scientific.

The LCMV glycoprotein peptides gp33-41 (gp33 peptide, KAVYNFATM) and gp61-80 (gp61, GLNGPDIYKGVYQFKSVEFD) were purchased from EMC microcolletions GmbH. Allophycocyanin (APC)- conjugated peptide/MHC class I tetrameric complexes were generated as previously described^{67 (link)}. The following anti-mouse monoclonal antibodies were used from Biolegend: CD4 (RM4-5 or GK1.5), PD-1 (29 F.1A12), CD44 (IM7), CD8 (53-6.7), IFN-γ (XMG1.2), Lag-3 (C9B7W), TIGIT (1G9), TNF (MP6-XT22). CD45.1 (A20) and CD90.1 (OX-7), CD49b (DX5), CD69 (H1.2F3), NKG2D (CX5), CD11b (M1/70), MHCII (M5/114.15.2), Ly6C (HK1.4), Ly6G (1A8), NK1.1 (PK136), F4/80 (BM8). TCF-1/TCF7 (C63D9) was purchased from Cell Signaling Technology. Tim-3 (215008) was purchased from R&D Systems. Granzyme B (GB11) was purchased from Thermo Fisher. Dilution factors for the antibodies used in this study can be found in Supplementary Table 1. The anti-TIGIT mAbs for blockade (clone 1B4) and stimulation (clone 1G9) were described previously^{31 (link)}. Control animals were treated with mouse IgG1 (BioXCell).