Extended Data Figure 6. TCR reconstruction and expression in T cells for reactivity screening.

T cell receptors were selected upon TCR sequencing of tetramer positive CD4⁺ T cells sorted from 3 patients (Pt 2, 3, 6). These TCRs represented clonotypes that were observed in more than one cell (either as replicates at a single timepoint or at more than one timepoint) or as singletons (observed as a single replicate at a single timepoint). The full-length TCRA and TCRB chains were cloned into a lentiviral vector and expressed in donor T cells through lentiviral transduction (Methods). Transduced T cells were co-cultured with patient-derived B cell lines immortalized with EBV virus and pulsed with different doses of mutated or wild-type neoantigen peptides. TCR reactivity was measured through detection of CD137 surface expression on CD4⁺ TCR transduced lymphocytes by flow cytometry. The values in the graphs report the percentage of CD137 positive cells with subtraction of background, measured upon coculture of T cells with patient-derived B cells pulsed with DMSO (negative control). Bulk data is showing IFN-γ secretion by neoantigen-reactive T-cell lines against mutated and wild-type peptides at several doses (figure from previous publication)¹, tested in triplicate wells/condition (error bars, s.e.m.).