Allelic exclusion ensures that most T and B lymphocytes express only one antigen receptor specificity - a central tenet of clonal selection theory. The discovery that some T and B cells in normal mice and humans express two different antigen receptor specificities demonstrated that allelic exclusion was imperfect. This led to concern that dual receptor lymphocytes pose a risk for autoimmunity - recognition of a foreign antigen through one receptor might activate a lymphocyte whose other antigen receptor is autoreactive. Indeed, dual receptor lymphocytes are enriched among autoreactive lymphocytes and can contribute to autoimmune disease pathogenesis in experimental settings. Dual T cell receptor (TCR) T cells are also enriched among alloreactive cells and contribute to graft-versus-host disease. On the other hand, dual receptor lymphocytes may broaden host protection against infection by increasing the number of antigen receptor specificities in the repertoire. The primary barrier to understanding the contribution of dual receptor lymphocytes in any of these immunological settings is simply that such cells are difficult to identify. For the case of B cells, it is now possible to identify dual immunoglobulin heavy chain-expressing cells (by exploiting natural allelic variants) and dual kappa light chain-expressing cells (via an engineered knockin of the human kappa constant region). Similar approaches to identify dual TCR T cells do not yet exist. Identification of dual T cell receptor T cells in mice is challenging due to the lack of allelic markers of the TCR? chain and because monoclonal antibodies exist for only ~16% of TCR V? segments. Estimating the number of dual TCR T cells in a population of lymphocytes has thus been accomplished by mathematical extrapolation rather than true quantification. The goal of the current project is to engineer a mouse that permits accurate identification, enumeration, and characterization of dual TCR?-expressing T cells in a normal polyclonal repertoire. Other investigators have successfully introduced epitope tags into the TCR? chain and TCR? chain in cell lines without disrupting the expression or function of the TCR. We propose here first to use a retroviral TCR expression system to identify an optimal site to insert an epitope tag in the mouse TCR? constant region. We will then engineer two gene targeted knockin mouse lines: one expressing a myc-tagged- and the other a FLAG-tagged-TCR? constant region. Crossing these mice will produce mice in which dual TCR?-expressing T cells can readily be identified based on their expression of both epitope tags. These mice will provide a powerful new approach to study dual TCR T cells in a wide range of immunological settings.
Most T and B lymphocytes express only one variety of antigen receptor. Dual antigen receptor T and B cells do exist, however, and have purported roles in defense against infection, autoimmunity, and organ transplant rejection. Reagents exist to identify dual receptor B cells in mice, but no similar reagents exist for dual receptor T cells. The objective of the proposed research is to engineer a mouse in which dual receptor T cells can be easily identified, thereby providing a powerful new way to study the role of dual receptor T cells in a wide range of immunologic settings.
