Characterization of IDH2^R140Q neo-T cells with mutant-specific responses. Neo-T cells directed toward TIQ- and IQN-minimal epitopes were mutant-specific, and further profiled using donor 5 (left column) and donor 7 (right column) as representative examples, respectively. (A) FluoroSpot analysis to assess for IFN-γ, GrB, and TNF-α secretion from neo-T cells (FluoroSpot images, top; corresponding bar graphs, bottom). (B) Reactive neo-T cells specifically lysed autologous neopeptide-pulsed OKT3 blasts, as assessed in a 6-hour ⁵¹Cr-release cytotoxicity assay over a range of E:T ratios, from 40:1 to 2.5:1. (C) HLA-restriction studies using allogeneic HLA-mismatched OKT3 blasts transduced with individual class I HLA molecules from the donors as targets. Only neopeptide-pulsed OKT3b expressing B∗15:01 and B∗35:43 were lysed by the TIQ- and IQN-specific neo-T cells, respectively, indicating that these are the restricting HLA alleles (E:T ratio, 40:1). (D) Six-hour ⁵¹Cr-release assay showing specific lysis of AML cell lines Kg1A and MOLM13 modified to overexpress IDH2^R140Q and the restricting HLA alleles, and R140Q TF-1 that express the neoantigen at native levels (E:T, 60:1). (E) Specific lysis of primary AML blasts from 2 patients modified to express the relevant, or irrelevant HLA molecules (E:T, 60:1).