Figure 1. Enumeration of SARS-CoV-2 spike-specific CD4+ Th1, CD8+ CTL and CD4+ CD25high Treg and their memory phenotype in 22 healthy vaccinated subjects. (A) PBMC were separated from 22 vaccinated subjects and stimulated in vitro with the SARS-CoV-2 spike peptides pool. Twenty-four hours after stimulation cells were collected and stained with specific monoclonal antibodies to determine their activation state (AIM assay). First gating on CD3+ T cells (anti-CD3-AF700, clone OKT3, mouse IgG2aκ, BioLegend), CD4+ Th1 were studied with anti-CD4-BV605 (clone RPA-T4, mouse IgG1κ, BD Biosciences), anti-4-1BB-allophycocyanin (clone 4B4-1, mouse IgG1κ, BioLegend), and anti-OX40-PE/Cy7 (clone Ber-ACT35, mouse IgG1κ, BioLegend). The percentage of AIM + CD4+ Th1 cells from un-stimulated control and SARS-CoV-2 CD4 S-all-stimulated cells were compared by the Wilcoxon matched-pairs signed rank test. CD8+ CTL were studied with anti-CD8-PE/CF594 (clone RPA-T8, mouse IgG1κ, BD Biosciences), anti-4-1BB-allophycocyanin (clone 4B4-1, mouse IgG1κ, BioLegend), and anti-CD69-PE (clone FN50, mouse IgG1κ, BD Biosciences). The percentage of AIM + CD8+ CTL from unstimulated control and CD4+ Th1 SARS-CoV-2 spike stimulated cells were compared by the Wilcoxon matched-pairs signed rank test. Regulatory T cells (Treg) were defined as CD4+ CD25high (anti-CD4-BV605, clone RPA-T4, mouse IgG1κ, BD Biosciences, and anti-CD25-BV421, clone M-A251, mouse IgG1κ, BD Biosciences). The percentage of CD4+ CD25high Treg from unstimulated control and SARS-CoV-2 spike-stimulated cells were compared by the Wilcoxon matched- pairs signed rank test. CCR6 expression on CD4 + Th1, CD8 + CTL and Treg was determined by staining with anti-CCR6-PerCp/Cy5.5 (clone 11A9, mouse IgG1κ, BD Biosciences). CCR6 expression was found in a large percentage of SARS-CoV-2-spike-specific CD4+ Th1 cells (median: 34.35%) and CD4+ CD25high Treg (median: 27.50%) in most subjects, unless CD8+ CTL where CCR6 was found in a much lower percentage (median: 11.66%). (B) T cell Memory phenotypes. Anti-CD45RA-APC-H7 (clone HI100, mouse IgG2bκ, BD Biosciences), and anti-CCR7- FITC (clone G043H7, mouse IgG2aκ, BioLegend) were used to determine memory populations on gated AIM + CD4+ Th1 and CD8+ CTL and Treg. Each dot shows the percentage of memory populations: terminally differentiated effector T cells (CD45RA + CCR7- TEMRA), median 2.66% on CD4+ Th1; median 44.75% on CD8+ CTL; and median 1.77% on Treg), effector memory T cells (CD45RA- CCR7- TEM), median 62.1% on CD4+ Th1, median 27.75% on CD8+ CTL, median 58.45% on Treg, and central memory T cells (CD45RA- CCR7+ TCM, median) 29.15% on CD4+ Th1, median 4.41% on CD8+ CTL, and median 22.70% on Treg).