Background: Primary Sjögren’s syndrome (pSS) is an autoimmune disease primarily affecting exocrine glands, with a characteristic pathological feature of focal lymphocytic infiltration in the salivary glands. However, the mechanism underlying this infiltration remains unclear.

Objectives: This study aims to utilize spatial transcriptomics sequencing (stRNAseq) to elucidate the structure, function, and intercellular interactions of the small salivary glands in pSS patients within their spatial context, and to further corroborate findings through related in vitro experiments.

Methods: A total of 18 lip gland samples from 15 patients were collected (including 4 non-pSS controls, 5 pSS patients with no focal infiltration and mild pathology, and 8 pSS patients with focal infiltration and severe pathology; some patients underwent secondary biopsy). A 10x Visium spatial transcriptomics library was constructed. Fastq data were preprocessed using Spaceranger, and spatial transcriptomics were subjected to cell type deconvolution using Cell2location. Special was used to perform spatial joint clustering on all slices, while Scanpy and Metscape were employed to analyze differential genes and gene set enrichment. CellChat was used to analyze ligand-receptor interactions and their pathways within each region. Immunofluorescence and immunohistochemistry were employed to validate angiogenesis in the lip glands of pSS patients. Tube formation assays and inhibition experiments were conducted using conditioned medium prepared from primary human lip gland FIB cultures to confirm their pro-angiogenic activity.

Results: After data quality control, a total of 6,075 spots were obtained, with an average of 2,670 genes expressed per spot. By combining cell types and spatial locations, spatial joint partitioning of all slices was performed using SPECAL, resulting in 3 distinct spatial regions: domain1, domain2, and domain3. Corresponding to the pathological zones, domain1 primarily consisted of acinar regions, domain2 mainly comprised ductal regions, and domain3 was enriched in lymphocyte-rich areas. Gene Ontology (GO) functional enrichment analysis revealed that vascular development signals were enhanced in domain1 and domain2 of mild pSS samples, suggesting a process of angiogenesis in the salivary glands prior to the formation of the infiltration foci. The ligand-receptor pathways were most active in domain3, with high expression of various CCL and CXCL factors (CCL19, CCL2, CXCL11, CXCL9, CXCL10, CXCL12, CXCL14) in the CXCL10+ fibroblast (FIB) subpopulation. UMAP secondary dimensional reduction of FIBs identified 5 FIB subpopulations. Compared to the non-pSS group, the pSS patients’ lip glands showed high expression of CXCL10+FIBs across all FIB subpopulations, with this subpopulation predominantly distributed in domain3. GO functional enrichment analysis revealed that FIBs, especially CXCL10+FIBs, highly expressed VEGFA-VEGFR2 signaling pathway genes, suggesting a pro-angiogenic role. Immunofluorescence and histochemistry of human lip gland specimens confirmed that, compared to other groups, severe pSS patients exhibited higher vascular density in the salivary glands, particularly at the edges and peripheral areas of lymphocytic infiltration foci. Tube formation assays of human lip gland primary FIBs demonstrated their pro-angiogenic potential, which was inhibited by VEGFA blockers. Predicted potential ligand-receptor interaction analysis revealed that CXCL10+FIBs highly express IFNGR2, IL1R1, and IFNGR1, with the ligand IL1B and IFNG having the most pronounced effect on CXCL9+FIBs.

Conclusion: Our study suggests that in pSS, CXCL10+FIBs are highly expressed in domain3, regulated by IL-1β and IFN-γ, and produce multiple chemokines that promote lymphocyte aggregation. This subpopulation also promotes angiogenesis through the VEGF pathway, providing additional pathways for lymphocytes to reach the salivary glands and playing a crucial role in the formation of lymphocytic infiltration foci.

REFERENCES: NIL.

Acknowledgements: NIL.

Disclosure of Interests: None declared.

© The Authors 2025. This abstract is an open access article published in Annals of Rheumatic Diseases under the CC BY-NC-ND license ( http://creativecommons.org/licenses/by-nc-nd/4.0/ ). Neither EULAR nor the publisher make any representation as to the accuracy of the content. The authors are solely responsible for the content in their abstract including accuracy of the facts, statements, results, conclusion, citing resources etc.