Anti ctla4 pe cy7

Immunophenotyping was undertaken following APC-conjugated HLA class I tetramer staining of PBMCs at 37°C for 15 min. Details of the tetramers used can be found in Supplementary Table 5. Tetramers were conjugated to APC and a true tetramer response was verified through the lack of background staining by gating all CD3+ T cells, against CD8+ T cells and using a tetramer negative control. Surface staining with the following antibodies was then performed: live/dead blue dye (Invitrogen), anti-CD8 Amcyan (BD Biosciences), anti-CD3 APC-Cy7 (Biolegend), anti-PD-1 PercpCy5.5 (BD Biosciences), anti-CTLA4 PE-Cy7, anti-CD244 FITC, and anti-CD160 PE (Biolegend) before washing and flow cytometric analysis. Memory subset analysis utilized the same panel but included anti-CCR7 FITC (R&D systems) and anti-CD45RA AF700 (Biolegend) and omitted anti-CTLA4, anti-CD244, and anti-CD160. Example flow plots can be found in (Figure S1).

For fluorescence-activated cell sorting (FACS) analysis, cells were labelled with saturating amounts of antibodies in FACS buffer (PBS containing 0.1% BSA and 0.02% NaN₃) to stain cell-surface antigens for 15 min on ice, followed by a fixation/permeabilization step (Fix/Perm buffer, eBioscience) for 30 min at room temperature and intracellular staining for 45 min at room temperature in permeabilization buffer (eBioscience). Stained cells were analysed on an LSR II flow cytometer (BD Biosciences).
For the staining of PBMC, the following Abs were used: Anti-CD3-PE-Cy7, anti-CD4-PerCP, anti-CD4-Pacific Blue, anti-CD4-FITC, anti-CD4-Alexa Fluor 700, anti-CD8-PE, anti-CD25-APC, mIgG₁-APC, anti-CD45RA-PE, anti-CD45RA-PErCP-Cy5.5, anti-CD127-PE, anti-Foxp3-APC anti-Foxp3-Pacific Blue, anti-CCR7-Alexa Fluor 488, anti-Ki-67-Alexa Fluor 700, anti-CTLA-4-PE, anti-CTLA-4-PE-Cy7 (all Biolegend), anti-CD25-PE, anti-CD8-FITC, PE-Cy5-streptavidin, anti-CD86-biotin (all BD Bioscience) and viability dye (Thermo Fischer).
For di-4-ANEPPDHQ (ANE) staining, PBMC were incubated with 4 mM of ANE (Invitrogen) together with anti-CD4 APC-Cy7, anti-CD45RA BV510 and anti-CD25 APC (all from Biolegend) in RPMI medium for 30 min at 37°C and were immediately analysed by FACS.

Peripheral blood mononuclear cells were isolated from peripheral blood using Lymphoprep density gradient (STEMCELL Technologies, Vancouver, Canada). Aliquots of 200,000 cells were frozen in media that contained 90% fetal bovine serum (HyClone, Logan, UT) and 10% dimethyl sulfoxide (Sigma-Aldrich, St. Louis, MO) and stored at -70°C for <1 week to preserve viability and run as a single batch to avoid intrarun variability. Cells were blocked with 2% BSA solution for 5 min. at RT. A cocktail containing the following antibodies and reagents was added: Brilliant Stain Buffer (BD Biosciences, San Jose, CA), True-Stain Monocyte Blocker, anti-CD19-PE-Dazzle594, anti-CTLA-4 PE-Cy7, anti-CD3-APC, anti-CD16-Alexa Fluor 700 (all BioLegend, San Diego, CA), and anti-SARS-CoV-2 Spike S1 Subunit-Alexa Fluor 405 (R&D Systems, Minneapolis, MN). The following antibodies were then added: anti-CD8 BUV496, anti-CD4-BUV661, anti-CD45-BUV805, anti-PD-1-BB700 (all BD Biosciences, San Jose, CA), anti-CD56-BV711 (BioLegend, San Diego, CA), and anti-CD14 BV786 (BioLegend, San Diego, CA). Cells were stained for 30 min. at RT, and then washed twice with 2%BSA. Cells were fixed for 1 hour at RT in 1 mL incellMAX (IncellDx, San Carlos, CA), and then incubated with anti-FoxP3-PE antibody (BD Biosciences, San Jose, CA) for 30 min. Cells were washed twice with 2%BSA and then acquired on a 5-laser CytoFLEX LX.