Vaccination-induced polyclonal CD4 ⁺ T cells targeting the PfCSP N and C terminus. (A) Schematic overview of the sample collection, in vitro stimulation, and single-cell isolation strategy. PBMC samples collected 28 days after two FMP013/ALFQ vaccine doses (II+28) were left untreated or stimulated with peptide pools covering the complete FMP013 aa sequence (CSP), the FMP013 N terminus, junction, and repeat domain (N-CSP), or the FMP013 C terminus (C-CSP). Indexed flow cytometric single-cell sorting was performed 10 days later. (B and D) Number of activated (CD25⁺OX40⁺) CD4⁺ T cells after in vitro peptide pool stimulation compared with unstimulated control cells from the same individuals. Each symbol represents a volunteer. (C and E) Clonal composition and TRAV and TRBV gene segment usage of single activated CD4⁺ T cells after CSP (C) or N-CSP and C-CSP (E) peptide pool stimulation of samples from the indicated donors. Individual expanded clones are shown in color, and all non-expanded clones are shown in white. (F)TRAV and TRBV gene usage after in vitro stimulation, TRAV and TRBV gene segments (>7%) enriched in activated cells isolated from peptide-stimulated cultures are labelled, and nonoverlapping segments highlighted in color. B–F represent data from one experiment.