Follicular helper T cells as drivers of epitope spreading

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Project Summary: Autoantibodies mediate tissue damage, organ failure, and clinical decline in a range of autoimmune diseases. The reactivities of these autoantibodies may change over time leading to evolving autoimmune sequalae, termed epitope spreading. Epitope spreading correlates with disease severity and can be used for both clinical diagnosis and prognosis, but the driving forces of autoantibody responses and epitope spreading remain unclear. Characterization of these mechanisms might provide therapeutic insight to the prevention and treatment of autoantibody-mediated diseases. We have developed a model of epitope spreading in mice, in which mixed bone marrow chimeric mice develop autoantibodies to a diverse set of self-antigens. To characterize the cellular mechanisms contributing to this clonal evolution, I performed single cell RNA sequencing (scRNA-seq) of follicular T cells. Preliminary analyses have found that follicular helper T (Tfh) cells and follicular regulatory T (Tfr) cells from autoimmune chimeras expressed increased levels of several long non-coding RNAs (lncRNAs). In parallel, I have performed TCR sequencing of follicular T cells to provide paired clonotypic information, revealing T cell clones enriched in autoimmune chimeras as well as differential gene expression within individual clonotypes. These initial findings suggest that follicular T cells are transcriptionally and clonally distinct in B cell-driven autoimmune disease. I hypothesize that lncRNA expression in autoreactive Tfh cells promotes loss of peripheral tolerance, serving as a necessary and sufficient driver of epitope spreading in autoreactive germinal centers. This proposal seeks to determine whether lncRNA expression alters Tfh cell function (Aim 1), whether Tfh cells are autoreactive in B cell-driven autoimmune disease (Aim 2), and whether these Tfh cells are responsible for epitope spreading (Aim 3). Aim 1 will test if lncRNAs can alter Tfh functionality by retrovirally transducing primary T cells and characterizing their function in vitro and in vivo. Aim 2 will determine the reactivity of autoimmune-associated Tfh clonotypes by expressing these TCRs in vitro and screening their reactivity using MHC-TCR chimeric receptors. Aim 3 will determine the necessity and sufficiency of Tfh cells to promote epitope spreading by generating mixed bone marrow chimeric mice that lack Tfh cells, and adoptively transferring Tfh cells into these mice and measuring autoantibody production. The proposed project aims to provide insight into the cellular and molecular mechanisms of epitope spreading, while potentially revealing new therapeutic strategies for both autoantibody- mediated disease and other diseases of germinal center dysfunction.