Cd45ro apc clone uchl1

CD4 subset analysis and sorting were performed as described previously^{12 (link)}. PBMC samples were stained by the following antibodies: CD3-Brilliant Violet 605 (clone OKT3, BioLegend), CD4-PerCP-Cy5.5 (clone OKT4, Biolegend), CCR7-PE-CF594 (clone 2-L1-A, BD Biosciences), CD27-PE (clone M-T271, BioLegend), CD45RO-APC (clone UCHL1, BioLegend). The cells were then stained by the Live-dead aqua (Invitrogen) prior to flow analysis. The stained cells were sorted on a BD FACSAria II cell sorter (BD Biosciences). Four CD4 subsets were defined as follows: naïve (CD45RO⁻, CCR7⁺, and CD27⁺), central memory (CD45RO⁺, CCR7⁺, and CD27⁺), transitional memory (CD45RO⁺, CCR7⁻, and CD27⁺) and effector memory (CD45RO⁺, CCR7⁻, and CD27⁻). The purity of each sorted CD4 subset was higher than 95%.

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).