Background/Purpose: Sjogren’s Disease(SjD) is a systemic autoimmune disease characterized by exocrine dysfunction. The pathogenesis is incompletely understood, but involves gene-environment interactions leading to infiltration of immune cells and autoimmunity. Immune-epithelial crosstalk and the mechanisms driving exocrine dysfunction remain ill-defined. High throughput transcriptional analyses have broadened our understanding the etiopathogenesis of SjD. However, ‘bulk’ approaches cannot uncouple disease-specific changes in cellularity and cell-state simultaneously and lack direct spatial context. We hypothesized that single cell(sc) and spatial transcriptomics(st) approaches can directly unravel the immunopathology of SjD in the salivary glands.

Methods: scRNAseq was performed on human minor salivary glands(MSG; SjD: n=12, HV: n=17) and paired peripheral blood mononuclear cells(PBMCs; SjD: n=8, HV: n=7) to assess cellular composition and transcriptional states. MSG(SjD: n=24; HV: n=24) were sectioned onto 10X Visium slides and sequenced. Cell2location was used to analyze altered cellularity and interactions from stRNAseq datasets. Utilization of signaling pathways in sc and st datasets was assessed across annotated pathways. MSG were also used for flow cytometry, HiPlex in situ hybridization, and immunohistochemistry as confirmation.

Results: scRNAseq of MSG, but not PBMCs, exhibited profound changes in cellularity and differentially expressed genes(DEG) in SjD(Figure 1). SjD MSG had more inflammatory cells(e.g., T cells, B cells, and antigen presenting cells[APC]; and loss of seromucous cells). In SjD, many cell types’ DEG sets showed increased expression of MHC class I, B2M and HLA-B, and interferon (IFN) stimulated genes(e.g., ISG15). These phenomena were dependent on autoantibody positivity, but not focus score. In acinar cells, secretory markers(MUC7, AQP5) were significantly decreased. In T cell clusters(CD8, CD4), functional annotation analysis revealed marked activation of T cells including: ‘positive regulation of the Type I IFN response’, ‘T cell receptor signaling’, and ‘response to IFNg signaling’. PMA/ionomycin stimulation of MSG lymphocytes showed that CD8⁺T-cells were significantly more cytotoxic than in HV(Figure 1). Profound cellularity-dependent architectural and transcriptional changes in the glands and disease-specific cell-cell interactions(e.g., T cells:APC, T cells:acinar cells) and altered cellular neighborhoods were identified using stRNAseq(Figure 2).

Conclusion: Multiomics single cell and spatially transcriptomic approaches disentangle the complex disease-dependent tissue disorganization, cellular compositional alterations, and transcriptional cellular states in the salivary glands of SjD patients. Our findings link loss of secretory cells and gland function, with the presence of cytotoxic CD8⁺T-cells. Further, these results emphasize the expression modules and spatial arrangement of immune cells, and their interactions, promoting tissue damage in the salivary glands. In summary, these data pinpoint pathogenic cell populations, and their interactions, that may be directly targetable for therapeutic intervention.

ACR Meeting Abstracts - https://acrabstracts.org/abstract/single-cell-and-spatial-transcriptomics-identifies-pathogenic-drivers-of-sjogrens-disease-in-humans/