Anti cd27 pe clone m t271

biotinylated SARS-CoV-2 S protein and RBD were conjugated to streptavidin-bound fluorophores. Briefly, the biotinylated proteins were incubated for a minimum of 1 h at 4°C with the streptavidin-conjugates AF647 (Biolegend), BV421 (Biolegend) and BB515 (BD Biosciences) at a 1:2 protein to fluorochrome ratio. The fluorescent probes were incubated for at least 10 min with 10mM biotin (GeneCopoeia) to saturate the unconjugated streptavidin-fluorochrome complexes. Cryopreserved macaque PBMCs from 2 weeks after final immunization (week 12) were thawed and counted. 5x10⁶ cells were stained for 30 min at 4°C with the fluorescent probes, a viability marker (LiveDead-eF780, eBiosciences) and the following B cell-specific antibodies: anti-CD20-PE-CF594 (clone 2H7; BD Biosciences), anti-IgG-PE-Cy7 (clone G18-145;BD Biosciences), anti-CD27-PE (clone M-T271; BD Biosciences), and anti-IgM-BV605 (clone MHM-88; Biolegend). Cells were washed twice with FACS buffer and acquired on the FACS-ARIA-SORP 4 laser (BD Biosciences). Analysis was performed on FlowJo v.10.7.1.

Total T-cell activation and proliferation were assessed using fresh PBMCs and tissue cell suspensions. Cells were stained with the LIVE/DEAD Fixable Aqua Dead Cell Stain Kit (Thermo Fisher Scientific) and then surface stained for CD3, CD4, CD8, CD38, and HLA-DR. For differentiation phenotype analyses, cells were additionally stained with CD45RA, CCR7 and CD27. Then, the cells were fixed/permeabilized using a Cytofix/CytoPerm Kit (BD Biosciences) and stained for intracellular Ki-67. The following antibodies were used: anti-CD3–AF700 (clone SP34-2, BD Biosciences), anti-CD4–PerCP-Cy5.5 (clone L200, BD Biosciences), anti-CD8–APC-Cy7 (clone RPA-T8, BD Biosciences), anti-CD38–FITC (clone AT-1, StemCell Technologies), anti-HLA-DR–V450 (clone G46-6, BD Biosciences), anti-CD45RA–PE Cy7 (clone 5H9, BD Biosciences), anti-CCR7–PE-Dazzle594 (clone G043H7, Biolegend), anti-CD27–PE (clone M-T271, BD Biosciences), and anti-Ki-67– AF647 (clone B56, BD Biosciences). Data were acquired using an LSRII flow cytometer (BD Biosciences) and analyzed with FlowJo software version 10 (Tree Star Inc.). Cells were classified as follows: naive—CD45RA⁺CD27⁺CCR7⁺; central memory (CM)—CD45RA⁻CD27⁺CCR7⁺; transitional memory (TM)—CD45RA^-CD27⁺CCR7^-; effector memory (EM)—CD45RA⁻CD27⁻CCR7⁻; and effector—CD45RA⁺CD27^-CCR7⁻ (Fig. S2).

The following monoclonal antibodies (mAb) were used for phenotypic characterization and cell sorting: Anti-CD4 APC (clone RPA-T4), anti-CD27 PE (clone M-T271), anti-CD28 FITC (clone CD28.2), anti-CD28 PE (clone CD28.2), anti-IL-10 APC (clone JES3-19F1), anti-Granzyme B FITC (clone GB11), anti-CD147 FITC (clone HIM6), anti-CD39 APC (clone TU66), CTLA-4 PE (clone BNI3) (BD Biosciences), anti-FoxP3 PE (clone PCH101), anti-CD127 PE-Cy7 (clone eBioRDR5), anti-IL-35 PE (ebic6), anti-PD1 PE (clone eBioJ105) (eBiosciences), anti-TGFβ PE (clone TB21; Cedarlene). Growth medium was prepared using RPMI-1640 (Mediatech, Inc) supplemented with 2mM Glutamine (Gemini Bio-Products), 20mM HEPES (Mediatech, Inc), 1% penicillin/streptomycin (Gemini Bio-Products), and 10% inactivated human serum (Gemini Bio Products). Recombinant human IL2 (rhIL2) was obtained from R&D Systems, Inc. Staining medium was prepared using 2% fetal bovine serum (Gemini Bio Products) in PBS.