Gating and isolation strategy of activated CD4 ⁺ and CD8 ⁺ T cells and TCR analysis. (A) Gating strategy of activated CD4⁺ T cells (CD3⁺CD4⁺OX40⁺CD25⁺) pre-gated on viable (7AAD⁻) lymphocytes (SSC/FSC) after in vitro stimulation with PfCSP peptides or in unstimulated control culture. (B) Left: Number of activated (CD25⁺OX40⁺) CD4⁺ T cells after in vitro PfCSP peptide pool stimulation compared with unstimulated control cells from the same individuals. Each symbol represents a volunteer. Right: Clonal composition of single activated CD4⁺ T cells after N-CSP or C-CSP peptide pool stimulation or unstimulated samples from the indicated donors. Individual expanded clones are shown in color, and all non-expanded clones are shown in white. (C) Gating strategy of TCR-transgenic Jurkat76 cell lines based on mCherry (mCherry⁺) and TCR (TCR⁺) expression quantified by flow cytometric analysis. (D) Gating strategy for flow cytometric analysis of CD8⁺ T effector memory (T_EM) cells or terminally differentiated T_EM re-expressing CD45RA (T_EMRA) cells in PBMC samples from FMP013/ALFQ vaccinees. (E) Frequency of activated (PD1⁺CD137⁺ICOS⁺CXCR5⁺) T_EM or T_EMRA cells as frequency of CD3⁺CD8⁺CCR7⁻ T cells for five donors (F1–5). Bar plots of individual time points before first (I–7), after second (II+28), and after third (III+14) vaccine dose show mean and standard deviation, with individual dots representing individual donors. (F) Activated CD8⁺ T_EM+EMRA cells were indexed single-cell sorted, and TR genes were amplified and sequenced for subsequent repertoire analysis. Clonal composition of the TCR gene repertoire in individual donors across multiple blood collection time points. Expanded clones are shown in color, while unique TCR sequences are combined in the white compartment. Same color within each donor represents the same clone, while color sharing across different donors does not. (G) Number of TCRs with TCR beta chain match in the Immune Epitope Database identified by TCRmatch tool (Chronister et al., 2021). Identified TCRs are stratified by donor (F1–F5) and origin of the target epitope. SARS: severe acute respiratory syndrome. (H) Transgenic CD8⁺ Jurkat76 T cell lines expressing TCRs from T cell clones without TCR sequence features of common virus-specific TCRs or presence before vaccination were generated. IL-2 concentrations in supernatants of 67 TCR-transgenic CD8⁺ Jurkat76 T cells co-cultured with autologous B cells pulsed with N-CSP or C-CSP peptide pools or with non-peptide–pulsed autologous B cells (unstimulated control). Data in A–G are from one experiment. (H) Data represent one out of two independent experiments.