Cd107a h4a3

Multiparameter analysis of PBMC and blister NK cell phenotype was performed on an ARIA II (BD Biosciences). PBMCs and blister cells were stained with different combinations of antibodies specific to human CD3(UCHT1;Biolegend), CD8(HIT8a;Biolegend), CD16(3G8;Biolegend), CD45(HI30;Biolegend), CD56(MEM-188;Biolegend), CD62L(DREG-56;Biolegend), CD69(FN50;Biolegend), CD107a(H4A3;Biolegend), NKG2D(1D11;Biolegend), CXCR6(K041E5;Biolegend), T-bet(04–46; BD), and EOMES(WD1928;ThermoFisher). All surface staining was performed for 30 min on ice after prior incubation of cells with Human Fc block and Murine Fc block (BD) for 10 minutes on ice. Following surface staining, cells were fixed and permeabilized using Foxp3 Transcription Factor Fixation/Permeabilization Concentrate and Diluent Kit (eBiosciences, Thermo Fisher), as per the manufacturers protocol, and stained for the expression of the transcription factors T-bet and Eomes. Fluorescence minus one control stains were performed using PBMC to verify the staining specificity and as a guide for setting markers to delineate positive and negative populations. Gating was set on the live lymphocyte population using forward- and side-scatter profiles to include lymphocytic blasts, followed by single cell gating using forward- and side-scatter heights and widths, before identification of hematopoietic cells using human CD45 staining.