Somatic mutations in murine antigen-experienced T cells

The body is a mosaic of cellular clones carrying somatic mutations, a pool of genetic variation with the potential to generate phenotypic variation in otherwise homogeneous cell populations. T cells acquire spontaneous somatic mutations during intense proliferative bursts that characterize their development and response to immune challenges. Somatic mutations in turn have the potential to alter the function of T cells and contribute to autoimmune diseases. We hypothesized that T cell somatic mutations found in autoimmune disease contexts can also be present under homeostatic conditions, where they do not cause disease but alter physiologic immune responses such as response to an infectious pathogen. In this case, the pool of latent somatic mutations present under homeostatic conditions could increase heterogeneity of the immune response. As a first step in testing this hypothesis using mouse models, we performed a genetic screen for somatic mutations in clonally expanded T cells in a T cell-mediated autoimmune disease context. Nonobese diabetic (NOD) mice are a model for autoimmune diabetes in which T cells are necessary and sufficient for pancreatic islet inflammation. The pancreatic lymph nodes (pLNs) of pre-diabetic NOD mice are highly enriched in antigen-experienced T cells specific for pancreatic islet self-antigens. We screened pLN T cells from pre-diabetic NOD mice by performing targeted sequencing to 500X mean coverage with a sequencing panel (Mouse-IMPACT) that covers the exonic regions of 578 cancer-associated genes, including lymphoma-associated genes with roles in T cell function. We reasoned that since NOD mouse pLN contains self-reactive T cells chronically stimulated by self-antigen, pLNs might be enriched in T cells with somatic driver mutations that impart a proliferative advantage to activated T cells reacting to antigen in an inflamed environment. We isolated by fluorescence-activated cell sorting (FACS) antigen-experienced (CD44hi CD62Llo) CD4 and CD8 T cells from the pLN of three NOD mice and sequenced the cell subsets with the Mouse-IMPACT panel, using matched tail tissue from the same mouse as germline control.