Pd 1 clone j105

Cryopreserved patient pre and post samples PBMCs were thawed togetherand stained with dead cell exclusion dye and Fluorochrome conjugated anti-human antibodies CD3, CD4 and CD8 (all from BD Pharmingen) and CD56 (BioLegend Inc.), CD25 (clone 4E3, MiltenyiBiotec GmbH), CD45RO (BD Horizon) as well as PD-1 (clone J105; eBioscience Inc). For some samples, cells were fixed and permeabilized. After permeabilization of cells, fluorochrome conjugated antibodies against human Granzyme B, (BD Biosciences) and Ki-67(eBiosciences), were used to stain and detect the respective intracellular molecules. For detection of cytokine production, cells were rested overnight after thawing and then stimulated with PMA (Phorbol 12-myristate 13-acetate) and Ionomycin, both at 500ng/ml in the presence of protein transport inhibitor BD Golgi Stop™ (0.7ul/ml). After 5 hours of stimulation, the cells were stained with the dead cell exclusion dye, fixed, permeabilized and stained with fluorochrome-conjugated antibodies against human CD3, CD4, CD8, IFN-γ (all from BD Biosciences).. All live cell stains were acquired on BD-LSR Fortessa™ and the data was analyzed using Flowjo v9.7.5 software (Tree Star Inc). Intracellular staining samples were acquired on BD™LSR II and the data analysis was done using Flowjo software.

Cells were resuspended in FACS staining buffer (PBS + 3% fetal bovine serum) using the following antibodies: CD3 (clone OKT3, eBiosciences), PD-1 (clone J105, eBiosciences), TIM3 (clone F38–2E2, BioLegend), Lag3 (clone 3DS223H, Invitrogen). CARs were detected using a PE or APC-conjugated CD22, CD19 or CD33 protein. All changes in overall tumor or T cell counts reflect changes in absolute cell counts, which were determined using CountBright absolute counting beads (ThermoFisher). Cell viability was established using Live/Dead Aqua fixable staining kit (ThermoFisher), and data were acquired on an LSRII Fortessa Cytometer (BD). All data analysis was performed using FlowJo 9.0 or 10 software (FlowJo, LLC). See Supplementary Figure 6 for gating strategy.

Cells were resuspended in FACS staining buffer (PBS + 3% fetal bovine serum) using the following antibodies: CD3 (clone OKT3, eBiosciences), CD4 (clone OKT4, eBiosciences), CD8 (clone SK1, BD), PD-1 (clone J105, eBiosciences), TIM3 (clones F38–2E2, BioLegend), Tbet (clone 4B10, eBiosciences). CD19 CAR was detected using an anti-idiotype antibody provided by Novartis Pharmaceuticals. All changes in overall tumor or T cell counts reflect changes in absolute cell counts, which were determined using CountBright absolute counting beads (ThermoFisher). Cell viability was established using Live/Dead Aqua fixable staining kit (ThermoFisher), and data were acquired on an LSRII Fortessa Cytometer (BD). All data analysis was performed using FlowJo 9.0 software (FlowJo, LLC).