<p>Supplementary Table S1. Primers used in this study. Supplementary Table S2. A/B Coverage of TCR-α (A) and TCR-β (B) variable genes with clonotype-specific primers. Supplementary Table S3. Characteristics of cloned TCRs. Supplementary Figure S4. Flow cytometric sorting of pp65-specific CD8+ T cells from CMV-seropositive donor ID3 after one week of expansion. Supplementary Table S5. Synthetic peptides used in this study. Supplementary Figure S6. Functionality of TCRCD8-NY#5 in CD4+ and CD8+ T cells. Supplementary Figure S7. Functionality of NY-ESO-1-specific CD4-TCRs in CD8+ T cells. Supplementary Table S8. HLA haplotypes from healthy donors and NSCLC patients. Supplementary Methods. Detailed materials and methods.</p>
ARTICLE ABSTRACT
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 and monitoring 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-α and TCR-β chains directly from single antigen-specific CD8+ or CD4+ 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. This method avoids the work and bias associated with 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 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 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); 1230–44. ©2014 AACR.
