Human granzyme b

For quantification of granzyme B and IFN-γ in cell culture supernatants a Cytometric Bead Array Assay was used (Human Granzyme B and Human IFN- γ Flex Set from BD Biosciences). Measurements were performed according to the manufacturer’s instructions. The lower limit of detection was 4 pg/ml for Granzyme B and 0.8 pg/ml for IFN- γ.

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.