The determination of epitope-specificity of disease-associated T cell responses is of relevance 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 (TCRs) for novel antigens hinders efficient development and monitoring of new therapies. We developed an integrated approach for systematic retrieval and functional characterization of TCRs from single antigen-reactive T cells that includes identification of epitope specificity. This is accomplished through rapid cloning of full-length TCR-α and -β chains directly from single antigen-specific CD8+ or CD4+ T lymphocytes. Functional validation of cloned TCRs is subsequently conducted using IVT RNA transfer for versatile expression of TCRs in T cells and HLA molecules in antigen-presenting cells. This method avoids the work and bias associated with repetitive cycles of in vitro T cell stimulation and enables extremely 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 CD8+ and CD4+ T cells (13 specific for CMV-pp65, 16 specific for the well-known TAA NY-ESO-1 and 27 for the novel TAA TPTE), which are directed against 39 different epitopes. The proof of concept studies with tumor antigens 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.
- Received June 5, 2014.
- Revision received August 20, 2014.
- Accepted September 3, 2014.
- Copyright © 2014, American Association for Cancer Research.