ABSTRACT: Dermatomyositis (DM) is a severe idiopathic inflammatory myopathy, associated with significant morbidity and mortality. While the immunopathogenesis of DM is not fully understood, we have recently shown that interferon chemokine signatures track with changes in disease activity in patients with DM [1]. Another feature of active disease in DM is elevation of circulating T follicular helper (Tfh) cells [2, 3]. We recently studied circulating Tfh cells (CXCR3neg Tfh2/17 and CXCR3pos Tfh1) from DM patients and healthy controls using single cell RNA sequencing. We found that the Tfh2/17 cells had a type I interferon (IFN) signature, while the Tfh1 cells upregulated genes in the type II IFN pathway. The Tfh cells from DM patients had higher expression of factors that promote B-cell survival. Examining muscle biopsies from DM patients, we found extrafollicular Tfh and Tfh- like cells in very close proximity to B-cells in the muscle tissue when compared to other T cell types. These data suggest that Tfh and Tfh-like cells are pathogenic, promoting B cell activity in human DM muscle tissue. The lymphoid aggregates observed in DM muscle biopsies are a classical finding, but the immunological significance of these aggregates is not well understood. Recent studies have documented a number of Tfh-like cells in tissues that can provide B cell help in extra-follicular lymphoid aggregates, in addition to classical Tfh cells. We hypothesize that extrafollicular Tfh or Tfh-like cells are active in within lymphoid aggregates in DM muscle, providing B cell help and inducing local expansion of B cells. This may be stimulated by the type I and type II interferon pathways that are suggested by our preliminary data from circulating Tfh cells. Documenting this phenomenon in the lymphoid aggregates in JDM muscle tissue could suggest specific interventions directed at the T:B cell infiltrates that characterize active inflammation. Via the studies proposed, we will define the functional activity of circulating cells capable of providing B cell help in myositis, including Tfh and Tfh-like cells. It seems likely that extrafollicular T:B interactions are important in human myositis, and in Aim 1 we will be able to compare the effectiveness of Tfh vs. Tfh-like cells from human patients in providing B cell help. In Aim 2, we will comprehensively examine Tfh subsets in human muscle tissue using an advanced method that will allow for quantitation of the various Tfh subsets as well as full transcriptome assessment. The B cell receptor sequences will allow us to assess B cell clonality and local expansion, which would provide further evidence of B cell help in the tissue.