Anti cd3 petr

Fresh PBMCs (10⁶) were stained with Aqua fluorescent live/dead (Fixable Dead Cells Stain Kit, Invitrogen) to exclude dead cells. To analyze the frequency of different Th cell subsets, PBMCs were stained with anti-CD3-PETR (clone S4.1, Invitrogen), anti-CD4-PerCP (SK3, Becton Dickinson, USA), anti-CD45RO-PECy7 (UCHL1, BD), anti-CXCR3-PE (1C6/CXCR3, BD) and anti-CCR6-BV421 (11A9, BD). To analyze the purity of CD4-depleted PBMCs, cells were stained with anti-CD3-PETR (clone S4.1, Invitrogen) and anti-CD4-PerCP (SK3, BD). After staining, cells were acquired (100,000 total events) using FACSAria™ Fusion (BD, USA). Results were analyzed using FlowJo software (version 10, FlowJo LLC, BD, USA). Among the lymphocytes, 99% cells were found to be alive as determined by live/dead staining. Different subsets of live T cells were analyzed following the gating strategy shown in Supplementary Figure 1.

The expression of immune exhaustion (PD-1) and senescence (CD57) markers was assessed as described previously (26 (link)). Briefly, after revival and resting overnight, PBMCs were resuspended in 1 ml PBS and then stained with violet amine reactive dye (Invitrogen, Carlsbad, CA, USA) for 30 min at room temperature for differentiation of dead and live cells. The samples having more than 90% viability were considered for further analysis. The cells were washed again and incubated with a cocktail of antibodies [anti-Vα24 PE, anti-Vβ11 FITC (Beckman Coulter, Marseilles, France), anti-CD57 APC (Biolegend, USA), anti-CD3 PETR (Invitrogen, Carlsbad, CA, USA), anti-CD3 APC, and anti-PD1 PerCpCy5.5 (BD Biosciences)] for 30 min at room temperature. After washing, the cells were fixed in 3% paraformaldehyde, acquired on FACSAria-I (BD Biosciences, USA) and analyzed using FACSAria-I (BD Biosciences, USA) and analyzed using FACSDiva software version 6.1.3 (BD Biosciences, USA).