Cd45ro apc cy7

Activation-induced marker (AIM) assays were performed as described by Reiss et al. [14 (link)]. The PBMC used in ELISpot assays were plated in round-bottom 96-well plates at a density of 1 × 10⁶ cells/well in AIM-V medium, and incubated with medium alone as an unstimulated control, or with the AID SARS-CoV-2 peptide library—containing peptides from the S, N, M, and O proteins—for 18 h. Cells were blocked with TruStain (BioLegend, Amsterdam, The Netherlands) for 30 min; stained with CD4-Pacific Blue, CD134-FITC, CD25-PE-Dazzle, CCR7-PerCP-Cy5.5, and CD45RO-APC-Cy7 (BioLegend, Amsterdam, The Netherlands) for 60 min at 4 °C; and washed and analyzed on a Cytoflex S cytometer using CytExpert software. Cells were gated on the forward scatter/side scatter live cell gate, and then on the CD3+CD4+ gate for quantification of CD25^hiCD134^hi SARS-CoV-2-specific T-cells and memory T-cell subsets based on expression of CCR7 and CD45RO. All measurements are shown as background subtracted values (CD25^hiCD134^hi stimulated cells minus CD25^hiCD134^hi unstimulated cells as % total CD4+ T-cells). AIM assays were performed using the same flow cytometer with the same settings and CD25+CD134+ gate. Batches of antibodies were the same for all cohorts, with the exception of anti-CCR7, for which reason the CCR7 gate was adjusted relative to the CCR7+ naïve T-cell population.

PBMCs thawed on day one were washed twice in PBS + 1% BSA (PBS/BSA) and resuspended in 100µL PBS/BSA containing previously titrated combinations or singles of fluorescently conjugated antibodies against human 1 µg/mL CD3 Pacific Blue (Clone UCHT1; BD Bioscience), 1 µg/mL CD4 FITC (Clone OKT4; BioLegend), 0.2 µg/mL CD28 PE (Clone CD28.2; BioLegend), 1.5 µg/mL CCR7 APC (Clone G043H7; BioLegend), 0.5 µg/mL CD45RO APC-Cy7 (Clone UCHL1; BioLegend), 0.5 µg/mL CD45RA PE-CY7 (Clone HI100; BioLegend), 0.1 µg/mL CD56 PE (Clone HCD56; BioLegend), 1.5 µg/mL CD19 APC-Cy7 (Clone HIB19; BioLegend), and 5µL CD16 FITC (Clone B73.1; BD Biosciences). Cells were analyzed separately for T cells (CD3, CD4, CD28, CD45RA, CD45RO, and CCR7), NK cells (CD3, CD56, and CD16), and B cells (CD3 and CD19). Cells were incubated with antibodies for 30 min on ice in the dark before washing once in PBS/BSA and fixed with 1% formalin (Sigma Aldrich) for 20 min at RT in the dark. Cells were then washed and resuspended in 300 µL PBS/BSA and immediately analyzed on a BD FACS Canto II equipped with 3 lasers at the Duke Cancer Institute Flow Cytometry Core. All analyses were completed after acquisition using FCS Express v6 (DeNovo Software, CA).