Clone l106

Detection of AIM was performed as described previously (8 (link)). Briefly, cells were stained with the following antibodies in their respective dilutions: anti–CD3-PerCP (1:25, clone SK7, BD), anti–CD4-V50 (1:50, clone L200; BD), anti–CD8–fluorescein isothiocyanate (1:25, clone DK25; Dako), anti–CD45RA-phycoerythrin (PE)–Cy7 (1:50, clone L48; BD), anti–CCR7-BV711, anti–CD69-allophycocyanin (APC)-H7 (1:50, clone FN50; BD), anti–CD137-PE (1:50, clone 4B4-1; Miltenyi), and anti–OX40-BV605 (1:25, clone L106; BD). LIVE/DEAD Fixable Aqua Dead Cell staining was included (1:100, AmCyan; Invitrogen) and acquired on a FACSLyric (BD Biosciences). After setting a time gate, LIVE CD3+ T-cells were gated, singlets were selected, and T-cells were subtyped into CD3+CD4+ and CD3+CD8+ cells. Within the CD4+ and CD8+ T-cells, T_naive were defined as CD45RA+CCR7+, T_CM as CD45RA-CCR7+, T_EM as CD45RA-CCR7-, and T_EMRA as CD45RA+CCR7-. S-specific T-cells were detected by co-expression of AIM on CD4+ (OX40 and CD137) or CD8+ (CD69 and CD137) T-cells in the combination of memory subsets (Fig. 3A). The DMSO-stimulated sample was used to set the cutoff gate for activation markers. On average, 500,000 cells were acquired per sample.

After ex vivo stimulation of PBMCs, cells were stained at 4°C for 15 min for phenotypical lymphocyte markers and AIM expression. Surface staining was performed with the following antibodies in their respective dilutions: anti–CD3-PerCP (1:25, clone SK7, BD), anti–CD4-V50 (1:50, clone L200; BD), anti–CD8–fluorescein isothiocyanate (1:25, clone DK25; Dako), anti–CD45RA-phycoerythrin (PE)–Cy7 (1:50, clone L48; BD), anti–CCR7-BV711, anti–CD69-allophycocyanin (APC)-H7 (1:50, clone FN50; BD), anti–CD137-PE (1:50, clone 4B4-1; Miltenyi), and anti–OX40-BV605 (1:25, clone L106; BD). LIVE/DEAD Fixable Aqua Dead Cell staining was included (1:100, AmCyan; Invitrogen). Cells were first gated for LIVE CD3⁺ T cells and then subdivided into CD3⁺CD4⁺ T helper cells and CD3⁺CD8⁺ T-cytotoxic cells (Fig. 4A). SARS-CoV-2–specific T cells were identified by gating the CD69⁺CD137⁺ cells (within CD4⁺ or CD8⁺ subsets). For N = 11 of 20 donors assessed, SARS-CoV-2 specificity in the CD4⁺ subset was confirmed by gating CD137⁺OX40⁺ cells. The DMSO-stimulated sample was used to set the cutoff gate for activation markers. On average, 500,000 cells were acquired per sample. Low-frequency samples (<10,000 cells in CD4 gate and <5000 cells in CD8 gate) were excluded from analysis.