Anti cd226

For multiparametric flow cytometry analysis, PBMC were stained and enriched with the matched MHC class I or class II tetramer. To exclude dead cells in the subsequent analysis, PBMC were stained with the LIVE/DEAD™ Fixable Near-IR dye (Thermo Fisher, Germany) according to the manufacturer’s protocol. PBMC were stained with appropriate fluorochrome-conjugated surface antibodies, including anti-CD3 (OKT3), anti-CD4 (RPA-T4) anti-CD45RO (UCHL1), anti-CCR7 (G043H7), anti-CD226 (DX11), anti-TIGIT (A15153G), anti-BTLA (MIH26), anti-LIGHT (115520), anti-Ceacam1 (ASL-32), anti-Tim-3 (F38-2E2), anti-PD-1 (EH12.2H7), anti-OX40 (ACT-35), anti-CD14 (63D3), anti-CD19 (HIB19) (from Biolegend, Koblenz or BD Biosciences, Heidelberg, Germany) for 20 min at RT in the dark. After surface staining, cells were washed once with PBS and were then resuspended in 0.5% paraformaldehyde.

NK cell activation assays were performed with PBMCs at a PBMC to target cell ratio of 5:1. PBMCs yielded 10% NK cells on average. Functional studies were performed in the presence and absence of blocking antibodies to NKG2D, CD226 or CD96 on NK cells and MICA, MICB, CD155, CD112 tumour cell surfaces respectively. Here, cells were incubated in 1ug/mL solutions of either anti-NKG2D (R&D clone# 149810), anti-MICA (R&D clone# 159227), anti-MICB (R&D clone# 236511), anti-CD226 (BioLegend clone# 11A8), anti-CD96 (BioLegend clone# 92.39), anti-CD155 (BioLegend clone# SKII.4) and anti-CD112 (BioLegend clone# TX.31) for 1 hour at room temperature and washed to remove excess soluble antibody. Recombinant MICA and MICB were used at a final concentration of 10μg/mL for 30 minutes with PBMCs. IgG controls were used for each experiment.
Recombinant TGFbeta was used to pre-treat PBMCs for 1 hour at room temperature prior to functional assay in relevant experiments.
Activated platelets were obtained by incubating washed platelets with 125μg/mL thrombin receptor-activated peptide (TRAP) for 30 minutes at room temperature under gentle rocking conditions. Activated platelets, and their releasate, were then used in functional assays as indicated within the text.