Apc anti human cd127

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.

Surface staining (phenotyping) was performed using fresh peripheral blood collected in the lavender tube with EDTA, BD Vacutainer. A 150-μL sample of fresh blood was used for each test tube (Th1, Th17, Treg,). Blood was incubated with a specific antibody mix for each population, for 30 min in the dark.
Th1: FITC anti-human CCR5 (BD), PE anti-human CXCR3 (Biolegend), APC anti-human CD4 (BD);
Th17: FITC anti-human CCR6 (Biolegend), PE anti-human CD161 (BD), APC anti-human CD14 (BD);
Treg: FITC anti-human CD4 (BD Pharmigen), PE anti-human CD25 (BD Pharmigen), APC anti-human CD127 (Biolegend).
Subsequently, FACS lysing (BD) was added to remove red blood cells (15 min, in darkness at room temperature). Finally, cells were washed with cold FACS Flow (BD) then resuspended in 1 mL of FACS Flow (BD). Ten thousand gated CD4+ cells were acquired by Cell-Quest four colors Facs Calibur cytometer (BD). The analysis of the results was carried out using the FlowJo software v.8.4.

T/CAR-T cells were centrifuged at 300×g for 5 minutes and resuspended in 100 μL PBS with 2% FBS. The T/CAR-T cells were stained at 4°C for 30 minutes with the following surface marker-specific antibodies: APC/Cy7-anti-CD25 (BD Pharmingen, San Diego, CA, USA), APC-anti-CD69 (BioLegend, San Diego, CA, USA), PE-anti-CD4 (BD Pharmingen), PE/Cy7-anti-CD8 (BioLegend), PE-anti-CD45RO (BD Pharmingen), APC-anti-CD62L (BD Pharmingen), PE-anti-PD-1 (BioLegend), PE/Cy7-anti-CTLA-4 (BioLegend), APC-anti-TIM-3 (BioLegend), and APC anti-human CD127 (BioLegend). The cells were then washed twice and resuspended in 400 μL PBS with 2% FBS for flow cytometry.