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Charline Miot, Rahul Arya, Thomas Burn, Edward M Behrens, Craig Bassing, Elucidating roles of the Rag1 N-terminus and RAG DSBs in shaping the cellular response to TCRa recombination, The Journal of Immunology, Volume 204, Issue 1_Supplement, May 2020, Page 80.8, https://doi.org/10.4049/jimmunol.204.Supp.80.8
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Abstract
The RAG1/RAG2 (RAG) endonuclease generates lymphocyte antigen receptor gene diversity via V(D)J recombination. The large numbers of V, D, and J segments and inherent imprecision in repair of RAG DNA double strand breaks (DSBs) together establish a vast diversity of antigen receptor specificities, including self-reactive receptors. Mechanisms have evolved to negatively select self-reactive cells and inhibit autoimmunity. In humans, deletion of the RAG1 N-terminus causes Omenn Syndrome, a fatal immunodeficiency with ab T cell-based autoimmunity. We discovered impaired negative selection in a spontaneous homozygous Rag1 mutant mouse with loss of the Rag1 N-terminus. This Rag1 region, which is not required for V(D)J recombination, has intrinsic ubiquitin ligase activity and interacts with another ubiquitin ligase and a kinase. We hypothesize the RAG1 N-terminus signals from RAG DSBs induced during TCRa recombination to shape the thymocyte proteome and enhance negative selection. To test our hypothesis, we have made mice whose thymocytes lack RAG DSBs or harbor RAG DSBs induced by wild-type Rag1 or mutant Rag1 lacking the N-terminus or intrinsic ubiquitin ligase activity. Our preliminary data suggest that RAG DSBs and distinct RAG1 N-terminus activities up-regulate expression of Zap70, an intracellular protein that transmits TCR signals, and CD80, a transmembrane protein that co-stimulates T cells engaging antigen. These data are consistent with a model wherein the RAG1 N-terminus facilitates negative selection through stimulating TCR signaling in thymocytes that bind self-antigens presented by thymocytes, dendritic cells, or thymic epithelial cells.
