A practical workflow for adaptive immune receptor profiling and screening of antigen-specific clonotypes with applications in cancer

We developed a streamlined workflow linking adaptive immune receptor (AIR) profiling to antigen-specific functional screening for cancer-relevant T-cell receptor (TCR) and B-cell receptor (BCR) discovery. Bulk AIR sequencing of DNA and RNA from matched samples quantified clonal expansion while distinguishing transcriptionally activated tumor-infiltrating lymphocytes. Paired TCR chains were obtained using a 96-well plate-based, multiplex single-cell assay for TCR alpha beta chain-pairs together with 36 T-cell marker genes, enabling simultaneous identification of full-length receptor sequences and functional phenotype.Reconstructed paired TCRs were cloned into GFP-reporter Jurkat cells, which were then screened using antigen-binding dextramers and co-cultured with K562 APCs expressing tumor-associated peptides. Reporter activation provided a sensitive readout of antigen recognition and allowed ranking of tumor-specific clonotypes. In proof-of-principle studies, the single-cell assay identified the most abundant TCR-alpha beta clonotype in leukemic T cells (35 wells) and revealed co-expression of NKG7 and CCL5, markers associated with cytotoxic activation in cancer. Functional assays confirmed antigen-responsive signaling in the engineered Jurkat cells.This integrated workflow-from repertoire profiling to TCR-alpha beta chain-pair reconstruction and antigen validation-will enable rapid discovery of tumor-associated clonotypes, characterization of cancer-specific immune responses, and the development of receptor-based cellular immunotherapies.