These files contain intracellular cytokine staining flow cytometry data for CD4+ and CD8+ T cells after exposure of serial PBMC to SARS-CoV-2 spike peptides or control antigens. PBMC are from subjects recovered from SARS-CoV-2 infection that subsequently received mRNA vaccination. The data were analyzed and exported from FlowJo version 10 as individual gated events and related Boolean subsets for four functional markers: CD40L, IFN-g, IL-2, and TNF-a. Counts and frequencies are both included. Procedure for obtaining data: Cryopreserved peripheral blood mononuclear cells (PBMC) were thawed and rested overnight. PBMC (1 x 106 per well) were stimulated with a SARS-CoV-2 spike overlapping peptide pool (JPT, 1 μg/mL each peptide; 0.4% final DMSO concentration), 0.4% DMSO as a negative control, or PHA-P (Remel; 1.6 μg/mL final concentration) as a positive control, in the presence of anti-CD28 and anti-CD49d (BD Biosciences) antibodies at 37°C for 6 hours. Brefeldin A (Sigma) was added after 2 hours. Cells were stained with Live/Dead Near IR dye (Invitrogen), treated with FACS lyse (BD Biosciences) and frozen at -80oC. For staining, the cells were thawed, washed, and permeabilized with Permeabilizing solution 2 (BD Biosciences) then stained with fluorochrome labeled monoclonal antibodies to anti-human CD3 mAb (clone UCHT) conjugated with PE-Texas Red (ECD; Beckman Coulter), anti-human CD4 mAb (clone SK3) conjugated with BV510 (Biolegend), anti-human CD8 mAb (clone SK1) conjugated with PerCP-Cy5.5 (BD Biosciences), and the activation markers anti-human CD40L mAb (clone TRAP1) conjugated with BV421 (BD Biosciences), anti-human IFNγ mAb (clone 4S.B3) conjugated with PE (BD Biosciences), anti-human IL2 mAb (clone MQ1-17H12) conjugated with APC (BD Biosciences), and anti-human TNFα mAb (clone MAb11) conjugated with FITC (BD Biosciences). Events were recorded with BD Fortessa and analyzed with FlowJo (v10 for Mac; BD).