Figure 4.

DoubletSeeker excels in identifying the cognate antigens for public F5‐TCR and neoantigen‐specific TCR. A) Schematic diagram of DoubletSeeker for screening TCR cognate epitopes from an SCT library. CTV‐labelled TCR‐expressing Jurkat cells co‐incubated with CMFDA‐labelled SCT library‐expressing K562 cells at a 1:1 ratio. Subsequently, FACS‐sorted (CTV⁺CMFDA⁺) cell doublets were then subjected to NGS to identify the enriched epitopes based on read counts. B,C) Schematic and representative flow cytometry plots for the identification of doublets in K562 cells expressing an A2‐restricted SCT library containing 12055 epitopes (B) and a neoepitope SCT library containing 3251 unique neoepitopes C) co‐incubated with F5‐TCR‐Jurkat (B) or Neo‐TCR‐Jurkat (C) cells (TCR‐expressing cells: SCT‐expressing cells = 1:1), respectively. Identification of enriched epitopes after one round of selection by NGS is shown on the right. The rank average is determined by calculating the ranking of each peptide based on its abundance among all peptides, from three independent experiments. The SCT library‐K562 cells were labeled with CMFDA, and the TCR‐Jurkat cells were labeled with CTV. Data are represented as the means ± SEM. n = 3.