Molecular and spatial analysis unveils the functional basis of tertiary lymphoid structures in Sjogren's syndrome[scRNA-seq]

The key role of tertiary lymphoid structures in autoimmune and non-autoimmune conditions has been recently appreciated. While many of the molecular mechanisms involved in tertiary lymphoid structure formation have been identified, their cellular sources and temporal and spatial relationship remain unknown. Using single-cell RNA-sequencing, spatial transcriptomics and proteomics of minor salivary glands of patients with Sjogren's disease and Sicca Syndrome, ex-vivo and in vivo functional studies, we construct a cellular and spatial map of key components involved in the formation and function of tertiary lymphoid structures. We confirm the presence of a fibroblast cell state and identify an undescribed pericyte/mural cell state with potential immunological functions. The identification of novel cellular properties associated with these structures and the molecular and functional interactions identified by this analysis provide key therapeutic cues for tertiary lymphoid structures associated conditions in autoimmunity and cancer. Overall design: Labial minor salivary gland samples were obtained from patients recruited in the Optimising Assessment in Sjögren's Syndrome (OASIS) cohort which recruits new patients attending the multidisciplinary Sjögren's clinic at the Queen Elizabeth Hospital Birmingham, UK for assessment. Sjögren's syndrome patients had a physician diagnosis of primary Sjögren's syndrome and fulfilled the 2016 ACR/EULAR classification criteria. Participants with non-Sjögren's sicca syndrome had signs and/or symptoms of dryness but did not have a physician diagnosis of SS or fulfill 2016 classification criteria. Salivary gland biopsy samples were divided in two: one for the scRNAseq study and the second for histological analysis to confirm diagnosis. Histological diagnosis is reported as presence of focal lymphocytic sialadenitis (FLS, suggestive of Primary Sjögren's Syndrome, PSS) or non-specific chronic sialadenitis (NSCS), in the case of non-Sjögren's sicca syndrome. All OASIS participants provided written informed consent and the study was approved by the Wales Research Ethics Committee 7 (WREC 7) formerly Dyfed Powys REC; 13/WA/0392. Minor salivary gland biopsies were taken surgically from the lip and frozen in 1mL of CryoStor® CS10 (StemCell Technologies) at -80°C. For preparation of single-cell suspension, firstly the frozen tissue sample in Cryotube were quickly thawed in water bath at 37°C and washed twice in pre-warmed 5%FBS RPMI media. The salivary gland biopsies were then enzymatically digested as previously described. Dead cells were removed using the EasySepTM Dead Cell Removal (Annexin V) kit from the digested samples following manufacturer's instructions before proceeding for the scRNA sequencing using the 10x platform. Library preparation and sequencing was completed by Oxford Genomics Centre. Cell counts in each sample were quantified using the Bio-Rad TC20. 10,000 cells per sample were used to generate libraries with the 10x Genomics v3 Single Cell 3' kit before sequencing to a minimum depth of 50,000 reads per cell using the NovaSeq 6000.