一种用于适应性免疫受体分析和抗原特异性克隆型筛选并应用于癌症的实用工作流程

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.