Cd8 bv650

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.

Mononuclear cells were isolated from mice spleens by Ficoll-Hypaque density gradient centrifugation and the count and viability of the lymphocytes were determined using a Guava EasyCyte Flow Cytometer and Viacount Kit (both Merck, USA). For this, 2 × 10⁶ cells/ml were placed in RPMI containing 10% FBS, 2 mM glutamine, 100 U/ml penicillin, and 100 μg/ml of streptomycin, and with/without 10 μg/ml of recombinant gB. After incubation for 20 h at 37°C, the cells were used for phenotypic analysis of lymphocytes. Then, the cells were washed three times with PBS and viability staining was performed with FVS780 dye (Becton Dickinson, USA) according to the manufacturer’s protocol. Subsequently, the cells were washed and placed in vials containing PBS supplemented with 2% FBS and 0.09% sodium azide (staining buffer). Cell surface marker staining was then performed on the combination with the following conjugated antibodies: CD3-FITC, CD4-PE, CD8-BV650, and CD69-BV421 (all from Becton Dickinson). After incubation in the dark for 30 min on ice, the cells were washed three times with staining buffer. Subsequently, at least 10.000 CD3⁺ events were analyzed by flow cytometric acquisition using the Guava EasyCyte 16ST Flow Cytometer (Merck). Data were analyzed using InCyte software (Merck).