Flow Cytometry
A multicolor flow cytometry protocol was used to analyze the expression of immune checkpoint molecules (PD-1, TIGIT, and Tim-3) and activating receptors (DNAM-1, NKG2D, CD16) on study group PBMCs separated into the following populations: CD3-CD56dimand CD3-CD56bright NK cells, and CD56-CD3+ T cells. We used the following antibodies to stain 5X105 PBMCs: anti-CD3-ε (FITC, clone UCHT1), anti-CD56 (PE-Cy7, clone 5.1H11), anti-CD16 (Percp-Cy5.5, clone 3G8), anti-TIGIT (APC, clone A15153G), anti-Tim-3 (BV510, clone F38-2E2), and anti-PD-1 (BV421, clone EH12.2H7) (all antibodies purchased from BioLegend, San Diego, California, USA). Data acquisition was performed using the BD FACSCANTO II flow cytometer (Becton Dickinson, Franklin Lakes, New Jersey, USA). First, for acquisition of each sample we derived an initial dot plot (FSC-A vs FSC-H) for singlets. This selection generated a dot plot with the combination of FSC-A vs SSC-A, after which we acquired 250,000 events from the lymphocyte gate. The results were analyzed with Kaluza software V2.1 (Beckman Coulter, Brea, California, USA). Isotype controls were used to adjust background fluorescence. To establish the parameters for gating we employed Fluorescence Minus One (FMOs) staining controls. CD3negCD56dim and CD3negCD56bright NK cells, and CD56negCD3+ T cells data were visualized in t-Distributed Stochastic Neighbor Embedding (t-SNE) density plots generated in FCS express 7 Plus (De Novo Software). Normalized protein expression levels for CD3, CD56, CD16, PD-1, TIGIT, Tim-3, DNAM-1 and NKG2D in t-SNE fields were represented in red for high expression, and in blue for low expression (hot-to-cold heat map).