Cloning and Expression of Antigen-Specific T-Cell Receptors

T cells, a component of the adaptive immune system, play an instrumental role in directing immune responses and direct cell killing in response to pathogens and cancers. T cells recognize and signal through the T cell receptor, a protein heterodimer on the surface of T cells. The T cell receptor is a highly variable structure formed via somatic recombination; the structure recognizes peptides presented on the surface of nucleated cells by major histocompatibility complex proteins in a specific receptor-restricted, peptide-restricted manner. This balance between T cell diversity and T cell specificity stands as a barrier to efficacious development of articificial T cell receptors capable of clearing disease. T cell receptors may be tailored to produce pathogen- or cancer-specific immune responses from autologous T cell populations. This necessitates a pipeline for amplification, cloning, and expression of antigen-specific T cell receptors. This study aims to utilize influenza-specific T cell receptor chains from healthy donor T cells to test a model for T cell receptor cloning and expression. This study utilizes Gateway recombination for high-throughput cloning into mammalian expression vectors. This study has successfully amplified and cloned T cell receptor chains from a population of influenza-specific T cells from donor cell transcripts into mammalian cell expression vectors. Additionally, CD8, a coreceptor for the T cell receptor complex, was successfully cloned and inserted into a vector for expression in mammalian cells. Sanger sequencing has confirmed sequences for influenza-specific T cell receptor chains and the CD8 chain. Future application of this project includes expression in mammalian non-T cells to test for efficacy of expression and, ultimately, expression in cytotoxic cells to create lymphocytes capable of antigen-specific recognition and cytolytic killing of cells of interest.