Ellezam, Benjamin

ABSTRACT Autoantibody internalization has been implicated in autoimmune disease pathogenesis, yet its mechanisms, and generality across different diseases, cell types, and affected tissues remain poorly defined. Using bulk RNA sequencing, we identified reproducible, autoantibody-specific transcriptomic signatures consistent with autoantigen dysfunction in muscle biopsies from patients with anti-Mi2 dermatomyositis and anti-PM/Scl scleromyositis across independent cohorts. Electroporation of purified patient IgG into primary cultures of healthy cells was sufficient to induce the corresponding transcriptomic programs in vitro . Direct immunofluorescence demonstrated immunoglobulin internalization into subcellular compartments matching the localization of the autoantigen in different affected tissues. Spatial transcriptomic analyses revealed that antibody-secreting cells translocated cytoplasmic material (i.e., immunoglobulin RNA) into adjacent affected cells expressing autoantibody-specific transcripts. The disease-specific transcripts were present not only in muscle fibers, but also in other cells, including macrophages, endothelial cells, and fibroblasts. Autoantibody-induced transcriptomic programs were associated with cell damage and autoantibody-specific reactive inflammatory programs, including activation of type I interferon and TGF-ß1 signaling in anti-Mi2 dermatomyositis and activation of type II interferon in anti-PM/Scl scleromyositis. Antibody internalization was also observed in different tissues from patients with other autoimmune diseases, including anti-U1RNP mixed connective tissue disease, anti-Ku overlap syndrome, and anti-Scl70 systemic sclerosis. Together, these findings establish autoantibody internalization as a shared pathogenic mechanism across diverse autoimmune diseases, providing a unifying framework for conditions driven by autoantibodies against intracellular antigens.

Abstract Posterior Fossa Group A (PFA) ependymomas are pediatric brain tumors with extremely poor survival outcomes. As protein-coding mutations in PFA are exceedingly rare, the underlying etiology of these tumors remains elusive. Elevated CpG island methylation and depletion of H3K27me3 have been described in PFA, leading to the hypothesis that PFA may be driven by a dysregulated epigenetic state. In this study, we sought to determine how three-dimensional (3D) genome features (such as DNA loops, domains, and compartments) differ between pediatric brain tumors. We performed Hi-C sequencing on a collection of 64 patient specimens and patient-derived primary cultures that collectively span multiple subgroups of ependymoma, medulloblastoma, high-grade glioma, and non-neoplastic brain. For certain samples, we further performed RNA-seq, histone modification ChIP-seq, or whole-genome bisulfite sequencing to allow multiomic data integration. Overall, the 3D genome organization of PFA samples appeared distinct from other tumor types. We identified and defined TULIPs: a subset of type B compartments, separated by genomic distances greater than 10 Mbp, that exhibit a striking fivefold increase in reciprocal interaction strength. These TULIPs recurred at the same genomic positions across the vast majority of PFA samples with minimal representation among other tumor or non-tumor samples. TULIPs displayed enrichment for heterochromatic features such as H3K9me3 and late replication timing and were depleted of euchromatic features such as H3K27ac and protein-coding genes. By using immuno-fluorescence for H3K9me3 and oligo-FISH to label TULIP regions, we demonstrated that TULIP regions are more compact in PFA than other tumors. Finally, by applying inhibitors of H3K9 lysine methylation to PFA cultures we showed that TULIPs become more diffuse and cell viability is reduced. Altogether, this work defines TULIPs as highly recurrent epigenetic features of PFA tumors.