Figure 1. Workflow for isolation, identification, and characterization of TIL derived TCRs. (A) illustrates the diversity of TILs within the tumor microenvironment (TME), with different colors representing distinct T cell subsets. (B) details the tetramer staining process utilized to identify TILs with specific antigen recognition. Here, tetramers bind to the TCRs on TILs that specifically recognize the presented peptide antigen. (C) shows the gating strategy used to isolate antigen-specific T cells. Cells that are positive for both CD8 and the tetramer are identified as specific to the target antigen. (D) presents a schematic of the TCR vector construction process. TCR α and β chains are linked to form tandem expression vectors for subsequent transduction into recipient cells. The plasmids were sequenced to confirm the specific TCR sequences. (E) illustrates the TCR reporter cell, highlighting the integration of the specific TCR along with a reporter gene construct. The reporter genes are activated upon successful TCR-mediated cell activation following antigen recognition. (F) demonstrates flow cytometry analysis showing a shift in fluorescence intensity, indicating the expression of reporter genes triggered by antigen-specific TCR activation.