(A–D) mCherry-expressing retrovirus encoding the anti-Rpl18^mut TCRs A09, I20, and I02 were used to infect 4G4R cells over a range of MOIs, and TCRβ expression Rpl18^mut -dextramer binding were quantitated. (A) Representative flow. (B) Plot of percentage of mCherry⁺ versus TCRβ MFI of mCherry⁺ cells; each transduction was done in triplicate. (C and D) At a matched transduction (see red box in B), dex^Rpl18 binding over a range of concentrations was determined. (E) To test functionality, Jurkat reporter cells were transduced with TCRs A09, I20, C05, B22, H03, F05, H17, and J12 and puromycin-selected. (F and G) These lines were cultured with different concentrations of Rpl18^mut peptide-pulsed B6WT3 cells (in triplicate) and the percentages that were CD69⁺ were determined. (F) Representative flow for TCRs A09, I20 and I02. (G) Peptide concentration/response plots for all TCRs. (H and I) Retrovirus encoding anti-ADPGK TCRs A10 and TCR30 and control anti-Rpl18 TCR A09 were used in dilution to infect 4G4R cells (in triplicate). Shown are TCRβ expression of mCherry⁺ cells (H) and dex^ADPGK staining of mCherry⁺ cells at about a 10% transduction (red box in H and I). (J) TCR A10-expressing Jurkat cells were cocultured with mutant or WT ADPGK peptide-pulsed B6WT3 cells (in triplicate) and the percentages of CD69⁺ cells were determined. (K) 4G4R cells were retrovirally transduced with anti-Zc3H7b TCRs over a range of MOIs in triplicate, and TCRβ MFIs were determined (of TCRβ⁺ cells). (L) Jurkat cells transduced with anti-Zc3H7b TCRs were reacted with B6WT3 cells pulsed with different concentrations of Zc3H7b-mutant peptide, and the percentages of CD69⁺ cells were determined. (M) Anti-PSMD2^mut G13 TCR-transduced Jurkat cells were cocultured with B6WT3 cells transduced with PSMD2^mut-encoding TMG 3.9 and 3.23 or versions reverted (REV) to express the WT PSMD2-derived peptide (PSMD2^WT). (N) Anti-PSMD2^mut G13 and A09 anti-Rpl18^mut TCR-expressing Jurkat cells were reacted against MC38-Leiden cells and MC38-Kerafast cells, which lack the targeted Rpl18 and PSMD2 mutant peptides.