Extended Data Figure 3: Characterization of selected TCRs obtained by single-cell cloning.
From: Personalized RNA mutanome vaccines mobilize poly-specific therapeutic immunity against cancer
a, Specificity of NARFL(E62K)-specific TCRs cloned from CD8+ T cells of patient P01. CD8+ T cells transfected with four TCRs directed against a mutation in the NARFL gene were tested by ELISpot for recognition of HLA-A*3101-transfected K562 cells pulsed with individual 15mer OLPs representing the mutant or the wild-type sequence. Control, irrelevant OLPs. b–g, Cloning and characterization of TCRs directed against mutations in PPFIA4 and HPN proteins of P02. b, e, Activation-induced IFNγ-secretion-based single-cell sorting from in vitro stimulation cultures of neo-epitope-specific T cells after co-culture with autologous DCs transfected with RNA (b) or pulsed with OLPs (e) encoding the respective neo-epitope. Control, RNA or OLPs encoding an irrelevant neo-epitope. c, d, f, g, Determination of HLA-restriction and specificity of the cloned TCRs by ELISpot with healthy donor-derived T cells co-transfected with RNAs encoding the identified TCR-α/β chains and peptide-pulsed K562 cells expressing single HLA molecules of the patient. Controls, OLPs encoding an irrelevant neo-epitope (c), HIV-gag (f), target cells without OLPs (d, g), Staphylococcal-enterotoxin-B (SEB) (g).
