Generation and characterization of neo-T cells derived from healthy donors. (A) Reactivity of PBMCs after exposure to neoantigen mastermix (peptide library encompassing 14 selected AML neoantigens) or medium only (negative control), as measured by IFN-γ ELISpot. Data presented as SFC per 5 × 105 input cells; bars and error bars indicate mean and standard error of mean (SEM), respectively. (B) Schematic of ex vivo expansion protocol. (C) Fold expansion of neo-T cells achieved over 3 stimulations in culture, determined by cell counting based on trypan blue exclusion (n = 23). (D) Immunophenotyping of expanded cells as assessed by flow cytometry, for the expression of T-cell subset, memory, activation, and exhaustion markers (n = 23). (E) Specificity of expanded cells toward the neoantigen mastermix or medium only, as measured by IFN-γ ELISpot (n = 23). Specific cells are defined as ≥30 SFC per 2 × 105 cells (indicated by red dots).
