Isolation and TCR characterization of dextramer-reactive CD8 ⁺ T cells. (A) Gating strategy of activated CD8⁺ T cells (CD3⁺CD8⁺CD137⁺CD69⁺) pre-gated on viable (7AAD⁻) lymphocytes (SSC/FSC) after in vitro stimulation with PfCSP peptides or in unstimulated control culture. (B) Schematic overview of the in vitro stimulation and dextramer-based single-cell isolation strategy. PBMC samples collected 14 days after the third rCSP/ALFQ vaccine dose (III+14) were stimulated with seven CSP peptides and cultured for 10 days. CSP peptide-loaded MHC molecules were bound to dextramer backbone and used for flow cytometry staining of PBMC samples before and after in vitro stimulation. After the stimulation, dextramer⁺ cells were isolated by indexed flow cytometric single-cell sorting for paired TRA and TRB gene amplification and sequencing. (C) Frequency of dextramer⁺ CD8⁺ T cells before and after in vitro stimulation. (D)TRBV and TRAV gene segment usage among dextramer-binding CD8⁺ T cell clones. (E) Transgenic CD8⁺ Jurkat76 T cell lines were generated expressing TCRs of dextramer⁺ T cell clones and co-cultured with peptide-pulsed autologous B cells (pulsed with seven predicted CSP peptides) or unstimulated control (non-peptide–pulsed autologous B cells). Il-2 concentration in supernatants. A, C, and D represent data from one experiment. (E) One representative out of two independent experiments is shown.