Figure 8. DP CD4⁺ Th TILs recognize tumor-specific neoantigens.

(A) Schematic representation of the methodology used to identify neoantigen-reactive CD4⁺ Th cells. (B) In vitro–expanded CD4⁺ and CD8⁺ T cell subsets (DN, SP, and DP) from patient 1 were cocultured with autologous B cells pulsed with DMSO or the indicated peptide pools. T cell reactivity was measured by IFN-γ ELISPOT assay. (C) Reactivity of DP CD4⁺ and DP CD8⁺ TILs to B cells pulsed with individual 25 mer peptides from pool 1. Flow cytometric analysis of OX40 or 4-1BB upregulation on CD4⁺ or CD8⁺ T cells, respectively is shown. The mutations recognized are highlighted in red. (D) DP CD4⁺ Th TILs were cocultured with autologous B cells pulsed with decreasing concentrations of WT or mutated (Mut) PRKCI^D378N 25 mer peptides. Reactivity was measured by flow cytometric analysis of OX40 upregulation on CD4 cells. (E) DP CD8⁺ TILs were cocultured with autologous B cells pulsed with decreasing concentrations of WT or mutated 8 mer MVK^N190S peptides. Reactivity was measured by flow cytometric analysis of 4-1BB upregulation on CD8⁺ T cells. (F) In vitro–expanded CD4⁺ T cell subsets (DN, SP, and DP) from patient 2 were cocultured with autologous B cells pulsed with DMSO or the indicated peptide pools. T cell reactivity was measured by IFN-γ ELISPOT assay. (G) Reactivity of DP CD4⁺ Th TILs to B cells pulsed with individual 25 mer peptides from peptide pools 2 and 5. Flow cytometric analysis of OX40 upregulation on CD4⁺ cells is shown. The mutations recognized are highlighted in red. (H) DP CD4⁺ Th TILs were cocultured with autologous B cells pulsed with decreasing concentrations of WT or mutated CEP162^E1003K or FBXL3^W410L 25 mer peptides. Reactivity was measured by flow cytometric analysis of OX40 upregulation on CD4⁺ cells.