Rare PfCSP-specific CD8 ⁺ T cells target an HLA class II–restricted C-terminal epitope co-receptor independently. (A) Number of activated (CD69⁺CD137⁺) CD8⁺ T cells from seven vaccinated donors (F7+F9, F1–5) after CSP, N-CSP, or C-CSP peptide pool-mediated in vitro stimulation compared with unstimulated control cells from the same individuals. (B and C) Activated T cells were index single-cell sorted for paired TRA and TRB gene amplification and sequencing. Clonal composition of activated CD8⁺ T cells after CSP, N-CSP, or C-CSP peptide stimulation of samples from the indicated donors (F7, F9; F1, F3, F4). Expanded clones are shown in color, and non-expanded clones are combined in white. Shared clones across separate expansion cultures are connected by ribbon. (D) Transgenic CD8⁺ Jurkat76 T cell lines were generated expressing TCRs from CD8⁺ T cells with an activated phenotype after stimulation of PBMCs with N- or C-CSP peptide pools. All monoclonal T cell lines were 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). For each T cell line, the IL-2 concentrations in culture supernatants as measure for TCR-mediated activation were quantified by ELISA (individual dots). (E) IL-2 concentrations as quantified by ELISA in supernatants of C-CSP reactive T cell line co-cultured with autologous B cells pulsed with the CS.T3 peptide (CSP_367-381) (+) or left unstimulated (−). (F) CD4/CD8 phenotype of single-cell sorted activated T cells in the expansion culture. Clonally related cells (n = 6) among the PfCSP-reactive T cell clone are highlighted in red. (G) IL-2 concentrations in supernatants of TCR-transgenic CD4⁺ or CD8⁺ Jurkat76 T cells co-cultured with CSP_367–381 peptide-pulsed autologous B cells at the indicated peptide concentrations. (H) IL-2 concentrations in supernatants of TCR-transgenic CD4⁺ or CD8⁺ Jurkat76 T cells co-cultured with peptide-pulsed autologous B cells in the presence or absence of the indicated locus-specific HLA-blocking antibodies. (I) IL-2 concentrations in supernatants of TCR-transgenic CD8⁺ Jurkat76 T cells co-cultured with peptide-pulsed autologous B cells in the absence or presence of HLA-E or HLA-DP-specific HLA-blocking antibodies. A–C and F represent one experiment. (D, E, G, H, and I) One out of two independent experiments is shown.