This grant seeks to characterize autoimmune features of a post-infectious Lyme disease (LD) syndrome called antibiotic-refractory Lyme arthritis (LA), the only post-treatment LD syndrome for which a specific pathology has been defined. We have previously reported that excessive inflammation, immune dysregulation of the Teff/Treg cell ratio, up-regulation of certain microRNAs, and infection-induced autoimmunity are features of this untoward outcome. Moreover, the greatest genetic risk factor for refractory LA is certain HLA-DR alleles, and we have identified immunogenic HLA-DR-presented peptides directly from synovial tissue in these patients. In this way, we have shown that 4 autoantigens, endothelial cell growth factor (ECGF), MMP-10, apoB-100, and annexin A2, are targets of T and B cell responses in subsets of patients with each of the manifestations of LD; and nearly half of patients with antibiotic- refractory LA have autoantibody responses to 1 or more of these autoantigens. Based on RA- seq data, we report in this grant that the synovial lesion in refractory LA has a highly inflammatory expression signature, which includes up-regulation of genes associated with IFN- ?-responses, MHC class II antigen processing and presentation, cell-mediated cytotoxicity, and cell proliferation. We now propose that synovial fibroblast-like synoviocytes (FLS), the most common cell in the lesion, become unconventional antigen presenting cells (uAPC), and CD4+ T cells with cytotoxic potential may be directed against FLS. As detailed in Aim 1, we have identified two types of CD4+ SLAMF7+ T cells with cytotoxic potential in LA patients, and we will further determine their phenotype using single-cell RNA-seq. In Aim 2, we will identify a greater range of HLA-DR-peptides presented to CD4+ T cells by professional APCs or uAPC (FLS), and we will delineate molecular interactions between CD4+SLAMF7+T cells and FLS in cell cultures. In Aim 3, we present preliminary data that autoantibodies in refractory LA may participate in this disease process. In these patients, high levels of IgG4 autoantibodies to ECGF, MMP-10 and apoB-100 each correlate with marked fibrosis and obliterative microvasular lesions in synovial tissue. We will assess whether the binding characteristics and glycan composition of these autoantibodies shift from an anti-inflammatory to a pro-inflammatory phenotype in refractory patients. Finally, we will determine the utility of autoantibody determinations as part of a diagnostic platform for early identification of patients with maladaptive immune responses, which may allow earlier therapy to ameliorate or prevent this post-infectious syndrome.