181. Tissue immunophenotyping of three refractory cases of EGPA using single cell RNA sequencing
Creators: name: Coates1, Matthew
name: Jayne1, David
Publisher: name: Zenodo
Year: 2022
Subjects: Vasculitis
ANCA
Abstracts
Giant Cell Arthritis
MPA
GPA
EGPA
Takayasu
IgA vasculitis

description: <strong>Background: </strong>Eosinophilic granulomatosis with polyangiitis (EGPA) is a multisystem inflammatory disorder currently classified as an Antineutrophil Cytoplasmic Antibody (ANCA) associated vasculitis. Core features of the condition include eosinophilia and respiratory tract involvement, often manifesting as early and treatment resistant involvement of both the upper and lower respiratory tract. However, the underlying pathophysiology of EGPA remains poorly understood, with a particular lack of studies exploring the tissue immune response in the affected organs of subjects with the disease. Clinical phenotype varies within the condition, with an absence of ANCA and biopsy evidence of vasculitis in the majority of patients. Variations in clinical phenotype and genotype between ANCA positive and negative cohorts suggesting that the term could encompass at least two distinct conditions with similar clinical manifestations<sup>1,2</sup>. We aim to characterise the tissue immune response in MPO positive and ANCA negative EGPA using single cell transcriptomics. We show pilot data from a novel postnasal space biopsy technique, which we have used to sample respiratory tract secondary lymphoid tissue from EGPA subjects and controls, at a disease relevant site. <strong>Methods: </strong>Subjects with active (BVAS&gt;0) EGPA, disease controls with active GPA (Granulomatosis with polyangiitis) or Chronic rhinosinusitis, and healthy controls were recruited from Cambridge University Hospitals. EGPA was diagnosed based on MIRRA study criteria<sup>3</sup>. All subjects underwent single timepoint paired endoscopic postnasal space lymphoid tissue biopsy and peripheral blood samples. Biopsies were performed using topical local anaesthesia in the outpatient department. Tissue was minced, enzymatically digested (DNAse/Liberase) and filtered to create a single cell suspension. Blood underwent density centrifugation (Histopaque 1077) for red cell removal. Single cell transcriptome libraries were created from the resulting fresh single cell suspensions using the 10x genomics platform. Transcriptomes were aligned with Cellranger software and processed in R and Python. <strong>Results: </strong>Three EGPA subjects (one MPO ANCA positive, two ANCA negative), one Chronic rhinosinusitis subject, seven PR3 ANCA positive GPA, and six healthy controls were included in this pilot analysis. After quality control and preprocessing, 126822 cells were obtained, across 51 distinct celltypes. Celltypes included epithelial, stromal and immune cell populations, including germinal centre related celltypes. Eotaxins are chemokines with eosinophil chemotactic activity. Eotaxin expression profiles varied between ANCA positive and ANCA negative subjects and controls. Whilst Eotaxin 1(CCL11) and Eotaxin 2 (CCL24) predominated in the two ANCA negative subjects, Eotaxin 3 (CCL26) predominated in the MPO ANCA positive subject. Eotaxin expression in EGPA subjects was increased compared with healthy controls despite concurrent corticosteroid therapy. The source of Eotaxins also varied. Eotaxin 1 expression localised to a distinct cluster of Fibroblasts expressing Prostaglandin D2 Synthase, complement genes (C1S, C3), and the chemokines CXCL14 and RARRES2. Eotaxin 2 expression localised to a population of nasal macrophages expressing a tissue residency signature, and Eotaxin 3 was localised to the basal Epithelial cell cluster. <strong>Conclusions: </strong>Postnasal space biopsy can be used to safety and effectively obtain adequate tissue samples in vasculitis for single cell transciptomics. This pilot data suggests that different Eotaxin profiles may exist in ANCA positive and ANCA negative EGPA, which persist during corticosteroid therapy. <strong>Disclosures: </strong>None <strong>Figure 1: </strong>Uniform manifold projection (UMAP) of single cell RNA sequencing data in EGPA subjects and controls. A: Broad celltype named clusters. B: UMAP of celltype split by sample type. C: UMAP of Eotaxin expression in EGPA subjects and controls.