Research Article Functional TCR Retrieval from Single Antigen-Specific Human T Cells

The determination of the epitope specificity of disease-associated T-cell responses is relevant for the development of biomarkers and targeted immunotherapies against cancer, autoimmune, and infectious diseases. The lack of known T-cell epitopes and corresponding T-cell receptors (TCR) for novel antigens hinders the efficient development andmonitoring of new therapies. We developed an integrated approach for the systematic retrieval and functional characterization of TCRs from single antigen-reactive T cells that includes the identification of epitope specificity. This is accomplished through the rapid cloning of full-length TCR-a and TCR-b chains directly from single antigen-specific CD8þ or CD4þ T lymphocytes. The functional validation of cloned TCRs is conducted using in vitro–transcribed RNA transfer for expression of TCRs in T cells and HLA molecules in antigen-presenting cells. Thismethod avoids thework and bias associatedwith repetitive cycles of in vitro T-cell stimulation, and enables fast characterization of antigen-specific T-cell responses. We applied this strategy to viral and tumor-associated antigens (TAA), resulting in the retrieval of 56 unique functional antigen-specific TCRs from human CD8þ and CD4þ T cells (13 specific for CMV-pp65, 16 specific for the well-known TAA NY-ESO-1, and 27 for the novel TAATPTE), which are directed against 39 different epitopes. The proof-of-concept studies with TAAs NY-ESO-1 and TPTE revealed multiple novel TCR specificities. Our approach enables the rational development of immunotherapy strategies by providing antigen-specific TCRs and immunogenic epitopes. Cancer Immunol Res; 2(12); 1–15. 2014 AACR