Anti cd3 alexa fluor 700 clone sp34.2

For Cohort A, cryopreserved PBMCs were thawed at 37°C and washed once in R10 media prior to staining. As samples permitted, 1 to 2 million PBMCs were stained with 2 μl anti-CD3 Alexa Fluor 700 (clone SP34-2; BD Biosciences, San Jose, CA, USA) and 1 μl anti-CD8 Pacific Blue (clone RPA-T8; BD Biosciences) in 150 μl R10 for 30 minutes at room temperature. Cells were then washed with fluorescence-activated cell sorting (FACS) buffer, and fixed with 1% paraformaldehyde. Cells were fixed for 30 minutes at 4°C, and washed once with FACS buffer. Cells were permeabilized by adding 75 μl medium B (Life Technologies, Grand Island, NY, USA), and simultaneously stained for granzyme B with 1 μl anti-GZMB allophycocyanin (clone GB12; Life Technologies) for 30 minutes at room temperature. Cells were washed twice with FACS buffer, fixed in 1% paraformaldehyde, and placed at 4°C until being run on a BD-LSRII (BD Biosciences). Analysis was performed using FlowJo software (version 9.7.1, Tree Star, Ashland, OR, USA). This methodology was then used to assess GZMB expression in Cohort B, with the exception of staining freshly processed cells, and not cryopreserved cells.