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).