The PBMC cryovials were removed from storage and immediately thawed in a water bath at 37 °C. For analysis of the cell surface molecules, PBMC suspensions were prepared and incubated with the following antibodies: PreCP-Cyanine 5.5-anti-human CD3 (317336, Biolegend, San Diego, CA, USA), FITC-anti-human CD4 (357406, Biolegend, San Diego, CA, USA), APC-Cyanine 7-anti-human CD8 (344714, Biolegend, San Diego, CA, USA), PE-anti-human CD25 (302606, Biolegend, San Diego, CA, USA), PE-Cyanine 7-anti-human CD45RA (304126, Biolegend, San Diego, CA, USA), APC-anti-human CD127 (351316, Biolegend, San Diego, CA, USA), BV421TM-anti-human CCR7 (353208, Biolegend, San Diego, CA, USA) and BV785-anti-human HLA-DR (307624, Biolegend, San Diego, CA, USA). Flow cytometry data were acquired using a BD LSR Fortessa X-20 cell analyzer (BD Biosciences, San Jose, CA, USA) and analyzed with FlowJo software version 10.10.0 (Tree Star, OR, USA). The cells were sorted into CCR7+CD45RA+ (Tnaïve), CCR7−CD45RA+ (Teff), CCR7+CD45RA− (TCM), CCR7−CD45RA− (TEM), and CD4+CD25+CD127− (Treg) subpopulations, as shown in the gating strategy (Figure 1B). Since it is difficult to make comparisons based on absolute counts, the analysis of subpopulations was based on the percentage of each population. Spanning Tree Progression of Density Normalized Events (SPADE) analysis [15 (link)] was implemented using the R package.
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