Identification of antigen-specific functional CD8+ T cells using an optofluidic system independent of epitope information

Most studies detect and isolate antigen-specific CD8⁺ T cells solely by measuring T cell receptor (TCR) binding affinity to peptide-major histocompatibility complexes (p-MHCs) without considering T cell function. Here, we present a function-based strategy to screen antigen-specific CD8⁺ T cells using the Lightning optofluidic platform, named MicroFAST (microfluidic function-based screening of antigen-specific single T lymphocyte) fitting for limited sample volume. We validated this method by using it to isolate human cytomegalovirus (HCMV)-specific CD8⁺ T cells targeting a known HLA-A∗02:01-restricted epitope. We finally applied this method to identify several SARS-CoV-2-specific TCRs, which were characterized by recognizing HLA-B∗35:01-restricted TPS epitope (TPSGTWLTY), demonstrating the utility of our system. TCR binding affinity did not associate with its functionality. Collectively, we developed a workflow of isolating antigen-specific functional CD8⁺ T cells with or without a prior knowledge of peptide-MHC specificities, which will facilitate the development of engineered TCR-T therapies and T cell-based vaccines.