Cd127 bv711

The following antibodies were used: cluster of differentiation (CD)4 BV605, CD8 BV650, CCR7 PE-CF594 (Becton Dickinson), CD45RA PECy7, CD28 BV421, CD127 BV711, PD-1 PerCP-Cy5.5, CD95 APC, CD62L BV785 (BioLegend, Dedham, MA), CD3 AF700, CD45RO FITC, CD27 APC-ef780 (eBioscience, San Diego, CA). The dead cell exclusion stain (Live/Dead Aqua) was purchased from Invitrogen (Carlsbad, CA). To detect NY-ESO-1^c259TCR-expressing cells, purified anti-phycoerythrin-conjugated dextramer reagents specific for the HLA-A*02:01 SLLMWITQC complex (Immudex) were used at the manufacturer’s recommended concentrations.

The procedure of flow cytometric analysis of CMV- and IAV-specific CD8⁺ T cells prior to and post in vitro stimulation was described [40 (link)]. Briefly, freshly PMBCs were stained with fluorescently labeled dextramers or tetramers specific to CMV-pp65 (NLVPMVATV) and IAV-M1 (GILGFVFTL) (Immudex, Copenhagen, Denmark and NIAID tetramer core) first at room temperature for 20 min; followed by staining of cell surface markers including antibody against CD3, CD8, CD62L, CD45RA, CD95, CD27, CD28 CD70, CD127, and CD69 at 4°C for 30 min. Cells were washed again with FACS buffer (Hanks solution with 0.3% Sodium Azide), and then fixed immediately with 3% formaldehyde and 1% FBS in FACS buffer. Fixed cells were further stained with intracellular markers (perforin and granzyme B) at 4°C for 30 min. Stained cells were collected by BD_Symphony and were analyzed using FlowJo version 7.6.5 software.
Antibodies (CD3-BV570, CD62L-FITC, CD62L-PE-Cy7, CD45RA-PE-Cy7, CD45RA-APC, CD95-PE-Cy5, CD27-PE, CD28-BV785, CD28-PerCP-Cy5.5, CD70-PE, CD127--BV711, CD69-BV650, and granzyme B) were purchased from Biolegend, and antibodies against CD8-BUV496, CD27-BUV395, and perforin-PE-CF594 were purchased from BD.