Cd45ro pe cy7

For the eight elderly donors, PBMCs were isolated from fresh blood samples using density gradient centrifugation with Ficoll (GE Healthcare). CD3⁺ T cells were enriched from PBMCs by immunomagnetic selection using CD3 MicroBeads (Miltenyi Biotec, Auburn, CA). Cells were stained in the dark for 15 min with the following anti-human Abs: CD45RO PE-Cy7 (BD Biosciences, San Jose, CA), CD3 Alexa Fluor 700 (BD Biosciences), CD62L-PE (BD Biosciences), CD45RA-allophycocyanin (BD Biosciences), CD8-Pacific Blue (BD Biosciences), CD4 allophycocyanin-Cy7 (BD Biosciences), and LIVE/DEAD Aqua fluorescent reactive dye (Invitrogen, Grand Island, NY).
CD8⁺ T cell subsets were isolated using the BD FACSAria cell-sorting system (BD Biosciences), including CD8⁺CD45RA⁻CD45RO⁺ (for CD8⁺ memory), CD8⁺CD45RA⁺CD45RO⁻CD62L^hi (CD8⁺ naive), and CD8⁺CD45RA⁺CD62L^lo/− (CD8⁺ T_EMRA). FlowJo (TreeStar, Ashland, OR) analysis was used to determine the proportions of the different subsets as a fraction of total CD8⁺ T cells.

Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll-Hypaque density gradient centrifugation. For surface staining, PBMCs were stained for 30 min in the dark at 4°C with the monoclonal antibodies PE-CF594 mouse anti-human CD4 (BD Biosciences, San Diego, CA, USA), PE anti-human CD25 (BioLegend, San Diego, CA, USA), FITC anti-human CD161 (BioLegend), 7AAD (BD Pharmingen, San Diego, CA, USA), and CD45RO PE-Cy7 (BD Biosciences) or with FITC Mouse IgG1 κ isotype control (BioLegend) and Mouse IgG1 κ isotype control PE (eBioscience, San Diego, CA, USA). For intracellular staining of cytokines, incubate PBMCs in RPMI 1640 medium (Gibco, Life Technologies, Shanghai, China) in 5% CO₂ at 37°C, then stimulate these cells for 5 h, PBMCs were stimulated for 5 h with 50 ng/mL phorbol 12-myristate 13-acetate (PMA; Sigma-Aldrich, Steinheim, Germany) and 1 μg/mL ionomycin (BD Pharmingen) in the presence of 10 μg/mL Brefeldin-A (BFA; BioLegend) and subsequently fixed and permeabilized by Foxp3/Transcription Factor Staining Buffer Set (eBioscience). Then the cells were stained for 30 min away from the light at 4°C with anti-human IL-17A APC (eBioscience) or Mouse IgG1 κ Isotype Control APC (eBioscience).

By use of an AriaII FACS sorter^™ (Becton Dickinson, BD, Franklin Lakes, NJ), pure CD28^POS cells (purity 98% [95–100%]) were isolated. PBMCs were stained with CD3 Brilliant Violet 510 (BioLegend, San Diego, CA), CD4 Pacific Blue (BD, Franklin Lakes, NJ), CD8 APC-Cy7 (BD Pharm, San Diego, CA), CD28 APC (BD), and the viability dye 7-AAD PerCP (BD). Pure memory subsets (≥95% pure) were isolated using CD3 Brilliant Violet 510 (BioLegend), CD45RO PE-Cy7 (BD) and CCR7 PE (BD): naïve (T_N cells: CCR7+CD45RO-), central-memory (T_CM cells: CCR7+, CD45RO+), effector-memory (T_EM cells: CCR7-, CD45RO+), and end-stage terminally-differentiated EMRA (T_EMRA cells: CCR7-CD45RO-) T-cells.

Isolated mononuclear cells or whole blood samples were stained with the following fluorochrome-conjugated monoclonal antibodies: CD4-PcP, CD8-APC-H7, CD31-PE, CD45RA-FITC, CD25-PE, CD45RO-PE-Cy7, CD45RO-FITC, CCR7-PcP-Cy5.5, TCRγδ-BV421, pSTAT5-PE-Cy7, Ki-67-PcP-Cy5.5, Tbet-PE, CXCR3-PE-Cy5, CD69-APC-Cy7, CCR4-PE-Cy7, CD5-APC, CRTH2-PE, GATA3-APC, CD5-APC (all BD), CD4-ECD, CD4-PC7, CD69-PC5, CD69-ECD, Beta Mark TCR V β kit (all Beckman Coulter, Woerden, The Netherlands), CD122-PE, CD132-PE, CCR6-PcP-Cy5.5, IL-2-AF700, IL-4-PE, IFN-γ-PcP-Cy5.5, FOXP3-AF647, Helios-AF488 (all Biolegend, Uithoorn, The Netherlands), CD4-ef450, CD25-APC, CD25-PE, CD45RA-PE, CD45RA-ef605, HLA-DR-APC-ef780, IL-17-AF488, CD27-AF700, CD28-PcP-cy5.5 (all eBioscience, Vienna, Austria). In case of whole blood staining, samples were lysed with BD FACS lysing solution. Samples were measured on a LSR-II (BD) or FC500 (Beckman Coulter) and analyzed with Kaluza Analysis Software (Beckman Coulter). Absolute numbers of CD4+ and CD8+ T cells were determined according to the BD MultiTest TruCount method, as described by the manufacturer. TruCount measurements were taken on a FACS Canto-II (BD) and analyzed with FACSCanto Clinical Software (BD).

Different immune cell subpopulations were immunophenotyped with flow cytometry from fresh PB samples with 6 panels of different cell-surface markers, including the following immune checkpoint receptors and cytotoxicity and migration markers: CD3-PerCP-Cy5.5 (BD, catalog 332771), CD4-PE-Cy7 (BD, catalog 560649), CD45-APC-H7 (BD, catalog 560178), CD8-BV510 (BD, 563919), CD56-BV421 (BD, 562751), CXCR1-FITC (BioLegend, catalog 341606), CD16-PE (BD, 561313), TCR γδ-APC (BD, catalog 555718), PD1-FITC (BD, catalog 557860), LAG3-PE (BD, catalog 125209), ICOS-PE-Cy7 (eBioscience, catalog 25-9948-42), CTLA-4–APC (BD, catalog 560938), HLA-DR-BB515 (BD, catalog 560938), CD27-PE (BD, catalog 555441), CD25-PE-Cy7 (BD, catalog 561405), CD11b-APC (BD, catalog 550019), NKG2C-AF488 (R&D Systems, catalog FAB138G), CD161-PE (BD, catalog 556081), NKG2D-PE-Cy7 (BD, catalog 562365), NKG2A-APC (R&D Systems, catalog FAB1059A), DNAM-BB515 (BD, catalog 565152), CD57-PE (BD, catalog 560844), NKp46-PE-Cy7 (BD, catalog 562101), NKp30-AF647 (BD, 558408), CXCR3-AF488 (BD, catalog 561730), CCR7-PE (R&D Systems, catalog FAB197P), CD45RO-PE-Cy7 (BD, catalog 560608), and CXCR4-APC (BD, catalog 560936). CD45⁺ lymphocytes were acquired with the BD FACS Verse, and the data were analyzed with FlowJo, version 10.4. The results are shown in Supplemental Table 1.