Figure 3.

Activated memory CD4⁺ T cells specific for SARS-CoV-2 peptides. (A) Representative FlowJo analysis of activated (CD25⁺CD137⁺OX40⁺) memory T CD4⁺ T cells (in red) at four time points. Memory CD4⁺ T cells here encompass all cell subsets based on surface expression of CD45RA and CCR7 except the naive subset (CD45RA⁺CCR7⁺) (B) Frequency of activated memory CD4⁺ T cells specific for SARS-CoV-2 before and after the first and second vaccine doses. (C) Frequency of activated memory CD4⁺ T cells specific for SARS-CoV-2 Spike and MP-R peptides. (D) FlowJo analysis strategy example showing the subsets of memory CD4⁺ T cells: central memory (TCM) (CCR7⁺CD45RA^-) in blue; effector memory (TEM) (CCR7^-CD45RA^-) in green; and terminally differentiated effector memory cells (TEMRA) (CCR7^-CD45RA⁺) in red. (E) Distribution of the activated memory CD4⁺ T cell subsets among total SARS-CoV-2-specific CD4⁺ T cells. The percentage of cells co-expressing activation and other memory cell markers was determined by using a Boolean gate on the total live CD4⁺ T cells. In the scatter dot plot graphs: symbol colors represent age-groups (black: 18-59, grey: ≥60) and symbol shapes represent different volunteer sub-groups (unfilled circle: placebos (n = 8), filled circle: vaccinees (n = 52), filled triangles: SARS-CoV-2 infected hospitalized individuals (n = 8), 19 days median of symptoms onset (IQR 17-22)). 0d: vaccinees baseline, day of first vaccine dose (n = 52). 14d: two-weeks after the first dose, day of the second vaccine dose (n = 52). 28-42d: from 28 42 days after the first vaccine dose (n = 49). 90-180d: from 90 to 180 days after the first vaccine dose (n = 51). Three individuals missed the 28-42d visit, one individual was excluded at the latest visit due to the detection of SARS-CoV-2 infection and one was excluded due to lack of baseline PBMC sample. Statistical comparisons using the Kruskall-Wallis with post hoc Dunn’s test against baseline values were used.