Cd8 v450 clone rpa t8

Flow cytometry was performed on a BD FACSverse instrument. Antibodies used for mouse cell analysis: CD127-PE/V450 clone A7R34, CD25-FITC clone 7D4, CD44-PerCPCy5.5/FITC clone IM7, CD69-PECy7 clone HI.2F3, CD8-PerCPCy5.5 clone 53-6.7, CD8-PECy7 clone YTS156.7.7, PD1-PerCPCy5.5 clone 29F.1A12 Vβ13-PE/APC clone MR12-3/MR12-4 (Biolegend), CD4-APCCy7 clone RM4-5, CD62L-APC clone MEL-14, and KLRG1-APC/V450 clone 2F1 (BD). Antibodies used for human cell analysis: CD127-PECy7 clone A019D5, CD25-APCCy7 clone BC96, CD4-APCCy7 OKT4, CD45RO-APC clone UCHL1, CD62L-FITC clone DREG-56, CD8-PerCPCy5.5 clone SK1, TIM3-PE clone F38-2E2 (biolegend), CCR7-PECy7 clone CCR7, CD8-V450 clone RPA-T8, and PD1-FITC clone M1H4 (BD Biosciences). Cell viability assessed via Zombie Live/Dead Stain (biolegend). Data analyzed using Flowjo 10 software (Tree Star). For experiments with sorted cells, CD8⁺Vb13⁺ T cells were sorted from pmel-1 cultures using Dynabeads untouched mouse CD8⁺ T cell kit (Invitrogen) and a FACSAria cell sorting machine (BD Biosciences).

Cryopreserved stimulated whole blood samples were slowly thawed in a 37°C water bath, followed by fixation and permeabilization using Cytofix (BD Biosciences, 554722) and Cytoperm/Wash (BD Biosciences, 554723) according to the manufacturer’s instructions. Cells were stained with the following fluorochrome conjugated antibodies to assess polyfunctionality and memory: CD8 V450 (clone RPA-T8, Cat 560347), CD3 V500 (clone SP34-2, Cat 560770), CD4 FITC (clone RPA-T4, Cat 555346 and clone M-T477, Cat 556615), CD45RA-PE (clone HI100, Cat 555489) and IFN-γ APC (clone B27, Cat 554702) all from BD Biosciences as well as IL-2 PE (clone N7.48A, Beckman, Cat IM2718U) and TNF PCP-Cy5.5 (clone MAb11, eBioscience, Cat 45-7349-42). Acquisition of a minimum of 100,000 cells per sample was performed on a MACSQuant 10 analyzer (Miltenyi Biotec). Single-stained controls using mouse Ig/ κ compensation beads (BD Biosciences, 552843) were used to calculate compensation. Flow cytometry data was analyzed using FlowJo software v10.5.3 software (BD Life Sciences) [40 ]. Boolean combination gating was performed to assess polyfunctionality of T cells. The gating strategy for flow cytometry analysis of T cell-specific (CD4+ or CD8+) cytokine expression is presented in S1 Fig.

Peripheral blood samples (100 μL) were incubated with viability marker LIVE/DEAD Fixable Red Dead Cell Stain Kit (Invitrogen, Carlsbad, CA, USA) for 20 minutes at room temperature. After that, it was followed by incubation with surface antibodies to CD3/BV605 (clone SK7), CD4/V500 (clone RPA-T4), CD8/V450 (clone RPA-T8), CD19/PE-Cy7 (clone SJ25C1), CD14/PERCP (clone MφP9), or PD1/APC (clone MIH4) (BD Biosciences, CA, USA) for 30 minutes at room temperature. Next, the samples were fixed with 4% formalin for 15 minutes and the RBCs were lysed with FACS Lysing Reagent (BD Biosciences) for 15 minutes at room temperature. Subsequently, the cells were washed and resuspended in a phosphate buffer, and the acquisition was performed in the flow cytometer LSR Fortessa (BD Biosciences). Approximately 100,000 events per sample were acquired. FlowJo™ software was used to analyze the data obtained.