Pd 1 brilliant violet 605

Cryopreserved PBMC were thawed and rested at 37 °C overnight in R10 (RPMI 1640 medium supplemented with 10% fetal bovine serum; Penicillin/streptomycin; 2 nM L-glutamine; 1 nM sodium pyruvate and 10 mM HEPES). PBMC were cultured in the presence of LRAs at 37 °C. Drug was removed by washing PBMC twice with R10. R10 + 0.5% DMSO was used as the vehicle control. The positive control was 3 μg/mL PHA + 60 units/mL IL-2. Supernatants were harvested for cytokine analysis (see below). PBMC were stained with Zombie NIR viability dye, then CD3-PE-Dazzle 594; CD4-Alexa fluor 488; CD8-Brilliant Violet 510; PerCP-conjugated CD14, 16, 19, and 56 (dump channel); CD25-PE; CD38-PE-Cy7; CD45RO-Brilliant Violet 650; CD69-APC; HLA-DR-Alexa-fluor 700; MHC Class I-Pacific blue (clone W6/32); and PD-1-Brilliant Violet 605 (all Biolegend). Cells were acquired on an LSRII flow cytometer (BD Biosciences) and analyzed using FlowJo version 10 (Tree Star). Gates for positive events were positioned using fluorescence minus one (FMO) controls (Supplementary Fig. S1). Due to the previously reported downmodulation of CD4 by the PKCms Bryostatin-1 and Prostratin4 (link)59 (link), in assays where these compounds were used CD3+ CD8- lymphocytes were considered CD4+ T cells.