Cd279 clone eh12.2h7

The following antibodies were used in experiments: InVivo anti-PD-1 (CD279, J43) (BioXCell), Elraglusib (9-ING-41) kindly provided by Actuate Therapeutics Ltd and OVA_257-264 peptide (Bachem Ag). Conjugated antibodies for flow cytometry: anti-CD8α (clone, 53–6.7), anti-CD3 (eBioscience) and CD279 (clone EH12.2H7) (BioLegend). Antibodies for IHC: anti-PD-1 (Cell Signaling Technology), and anti-CD8 (Abcam).

CAR T cells were stained with fluorescence-conjugated monoclonal antibodies against CD3 (clone SK7, Biolegend, Heidelberg, Germany), CD8 (clone RPA-T8, Biolegend), CD4 (clone SK3, Biolegend), CD45RO (clone UCHL1, BD Pharmingen, Heidelberg, Germany), CD197 (clone G043H7, Biolegend), CD279 (clone EH12.2H7, Biolegend), TIM-3 (clone F38.2E2 eBioscience, Frankfurt, Germany), LAG-3 (clone 3DS223H, eBioscience) and the CAR-specific anti-idiotype antibody ganglidiomab, provided by H. Lode, Greifswald, Germany, and fluorescence-labeled with the Mix-n-Stain Kit (Sigma-Aldrich, Taufkirchen, Germany) or alternatively with a fluorescein isothiocyanate (FITC)-labeled goat-anti-human Fcγ Ab recognizing the hinge domain (Jackson ImmunoResearch, Pennsylvania, USA, product no. 109-095-098) for the CD19CAR T cells. Samples were fixed with 1% paraformaldehyde (PFA) and acquired directly or not later than 24 h after staining. For each sample, 10 000 cells within the respective gates were analyzed with FACS Diva 9.0, using FACS Celesta flow cytometer (BD Biosciences, Heidelberg, Germany) and FlowJo version 10 (FlowJo, LLC, Ashland, OR, USA).