Figure 3: Uni-Vect Expression Cassettes for Characterization of T-cell Receptors.

(A) Schematic representation of Uni-Vect-Jurkat reporter cell lines J^pASP90 and J^pASP89. (B) Transduction of TCR constructs into J^pASP90 reporter cell lines. Titrated concentrations of peptide (LLHGFSFYL) antigen in the presence of antigen presenting cells led to upregulation of NFAT-inducible eGFP expression as a surrogate for activation via TCR signaling. Blue bars represent percentage of eGFP positive cells, red line represents MFI of eGFP at each concentration. (n=3). (C) Flow cytometry analysis of 6 neoantigen-specific/HLA-A*02:01 restricted TCRs engineered into the J^pASP90 reporter cell line. (D) NFAT reporter EC₅₀ curves for neoantigen-specific TCRs expressed in J^pASP90 reporter cell line upon activation with mutated (blue) and wild-type (red) peptides. (E) J^pASP90 reporter cell line expressing the various TCRs were cultured for 16 h with melanoma cell line DM6 expressing tandem mini-gene constructs encoding mutated or wild-type antigen and analyzed for inducible eGFP expression. All data are representative of two or more experiments. All data with error bars are presented as mean ± SD. See also Figure S4 and Tables S1 and S2.