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. 2025 Jan 1;15(1):300–323. doi: 10.7150/thno.104594

Figure 6.

Figure 6

TCR screening using display libraries and pMHC tetramer-based assays. A) TCR libraries displayed on yeast, phages, and mammalian cells enable the high-throughput screening of antigen specificity. Yeast display incorporates TCRs anchored to the cell wall via Aga2p, facilitating the screening against labeled pMHC complexes. Phage display vectors present TCRs on capsid proteins, allowing for rapid affinity maturation through successive biopanning rounds. CHO cells expressing TCR-pMHC constructs provide a mammalian context for functional analyses. The resulting interactions are analyzed via biochemical screening, flow cytometry, and other downstream assays to identify TCRs with desired antigen specificities. B) pMHC tetramer-based screening utilizes DNA barcode-labeled pMHC tetramers linked to fluorochromes, facilitating simultaneous analysis of multiple TCR-pMHC interactions through flow cytometry. The barcodes allow for precise identification and quantification of TCR engagement, while TCR sequencing and droplet digital PCR provide detailed insights into the TCR repertoire. Mass spectrometry complements these analyses by identifying specific peptide-MHC interactions, enhancing the understanding of TCR specificity and affinity. The images in the figures were created using BioRender (https://www.biorender.com/).