University of Birmingham Birmingham, United Kingdom
SABA NAYAR1, Jason D. turner1, Saba Asam1, Charlotte G. Smith1, Serena Colfrancesco2, Ana Teodósio1, Joe Flint1, David H Gardner1, Professor Simon Bowman3, Adam Croft1, Andrew Filer1, Benjamin A. Fisher1, Christopher Buckley4 and Francesca Barone5, 1University of Birmingham, Birmingham, United Kingdom, 2University of Rome, Rome, Italy, 3University Hospitals Birmingham, Birmingham, United Kingdom, 4University of Oxford, Oxford, United Kingdom, 5University of Birmingham, Needham, MA
Background/Purpose: Sjögren's syndrome (SjS) shares a series of symptoms with non-SjS Sicca syndrome, a salivary gland disease characterised by glandular dysfunction and dryness. However, unlike SjS, Sicca is not defined by tertiary lymphoid structure (TLS) formation in the salivary glands. TLS germinal centres provide a local hub for the maturation and proliferation of auto-reactive B-cells and expansion of malignant B-cell clones. TLS that form within salivary glands (SGs) of SjS patients are associated with poor disease outcome, autoantibody production and lymphoma development. In contrast, some Sicca cases may be associated with poorly defined T cell infiltration in the absence of clear T/B cell aggregation without TLS formation. The cellular and biological processes that drive organ permissiveness for TLS establishment in SjS as opposed to Sicca are largely unclear, but could act as an exemplar for TLS formation at other sites.
Methods: Single cell atlases of SGs were generated from SjS and sicca (n=eight per group) patients using the 10x scRNAseq Genomics platform. SjS patients fulfilled 2016 ACR/EULAR classification criteria. Sicca patients were anti-Ro and biopsy negative for FLS, but displayed scattered T cell infiltration in some cases. Data analysis was performed using R and the harmony, Seurat and Cellchat packages. Multiplex IHC, RNAscope and quantitative-PCR were used to validate scRNAseq findings.
Results: Cell clustering of SjS and Sicca SGs unveiled key differences in both immune cells and stromal cell compartment between the two disease groups. Sicca samples had significant differences in the immune-cell compartment, in particular, sicca SGs lacked activated ICOS+IL17+IFNG+TNF+ CD4 T helper and presented major differences in the IL7R+CD8+ T and Treg population. Small numbers of poorly differentiated populations of B cells and plasma cells were detected in Sicca, as compared to SjS SGs. Both disease groups had populations of CD8+GranzymeK+CD8 T cells.
Sicca SGs lacked crucial alterations in their stromal cell populations including an absence of CXCL9+BAFF+CCL19+CCL21+CD40+ immunofibroblasts that are classically associated with SjS TLS. Whilst CD40 expression was broadly expressed across SjS cell clusters, limited expression of both CD40 and CD40L was observed in Sicca SG, supporting the clinical observations on the importance of the CD40/CD40L pathway in SjS pathology. Cellular interactome and in vitro analysis revealed complex interplay between LTBR, TNF, IFNγ and CD40 signals on both immune and stromal cell populations for TLS establishment. No major differences were observed in the presence of myeloid and pericyte populations between the two disease groups. Analysis of differentially expressed genes highlighted variances in activation and function of epithelial and endothelial cells. Interestingly, sicca presented a significant inflammatory signature on endothelial cells.
Conclusion: Our work is the first, systematic map and atlas of micro-anatomical and functional differences between SjS and Sicca providing critical insight in pathogenic players and therapeutic targets in these two different conditions. Acknowledgment Disclosures: S. NAYAR, None; J. turner, None; S. Asam, None; C. Smith, None; S. Colfrancesco, None; A. Teodósio, None; J. Flint, None; D. Gardner, None; P. Bowman, Novartis, AstraZeneca, AbbVie/Abbott, Galapagos; A. Croft, None; A. Filer, None; B. Fisher, Servier, Galapagos, Janssen, Novartis, Bristol-Myers Squibb(BMS); C. Buckley, None; F. Barone, Candel Therapeutics.
Acknowledgment 