Ox40 clone ber act35

Cryopreserved PBMCs were thawed and 10⁶ live cells were plated out in 96 well U-bottom plates. Spike protein peptide pool (GenScript, New Jersey, USA), or Chimpanzee Adenovirus Y25 hexon protein peptide pool (JPT Peptide Technologies, Berlin, Germany) was added to each well at 1 μg/mL and cells incubated for 20–24 h at 37°C. For surface staining of AIM markers, cells were incubated in 1 μg/mL human Fc block (BD Biosciences) and fixable viability solution 780 (BD Biosciences) in PBS for 15 min and washed in PBS. An antibody cocktail containing antibodies against CD3 (clone UCHT1, BD Biosciences), CD4 (clone M-T477, BD Biosciences), CD8 (G42-8, BD Biosciences), CXCR5 (J252D4, BD Biosciences), CD38 (HIT2, BD Biosciences), PD-1 (EHI2.1, BD Biosciences), CD69 (clone FN50, BD Biosciences), CD137 (clone 4B4-1, Biolegend), OX40 (clone Ber-ACT35, Biolegend), CCR7 (clone 2-L1-A, BD Biosciences), and CD45RA (clone HI100, BD Biosciences)were added directly to cells and incubated for a further 30 min at 4°C. Following surface staining, cells were washed twice in PBS. All samples were acquired on a BD FACS Symphony and analyzed using FlowJO software v18 (FlowJo, Ashland, USA). The gating strategy for AIM⁺ T cells is shown in (Methods S3). Data were imported into R v4.2 and visualized with the ggplot2 v3.3.6 package.