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Background: Detailed molecular analyses of cells from rheumatoid arthritis (RA) synovium hold promise in identifying cellular phenotypes that drive tissue pathology and joint damage. The Accelerating Medicines Partnership (AMP) RA/SLE network aims to deconstruct autoimmune pathology by examining cells within target tissues through multiple high-dimensional assays.Robust standardized protocols need to be developed before cellular phenotypes at a single cell level can be effectively compared across patient samples. Methods: Multiple clinical sites collected cryopreserved synovial tissue fragments from arthroplasty and synovial biopsy in a 10%-DMSO solution. Mechanical and enzymatic dissociation parameters were optimized for viable cell extraction and surface protein preservation for cell sorting and mass cytometry, as well as for reproducibility in RNA sequencing (RNA-seq). Cryopreserved synovial samples were collectively analyzed at a central processing site by a custom-designed and validated 35-marker mass cytometry panel. In parallel, each sample was flow sorted into fibroblast, T cell, B cell, and macrophage suspensions for bulk population RNA-seq and plate-based single cell CEL-Seq2 RNA-seq. Results: Upon dissociation, cryopreserved synovial tissue fragments yielded a high frequency of viable cells, comparable to samples undergoing immediate processing. Optimization of synovial tissue dissociation across six clinical collection sites with ~30 arthroplasty and ~20 biopsy samples yielded a consensus digestion protocol using 100µg/mL of Liberase TL TM enzyme. This protocol yielded immune and stromal cell lineages with preserved surface markers and minimized variability across replicate RNA-seq transcriptomes. Mass cytometry analysis of cells from cryopreserved synovium distinguished: 1) diverse fibroblast phenotypes, 2) distinct populations of memory B cells and antibody-secreting cells, and 3) multiple CD4+ and 4 CD8+ T cell activation states. Bulk RNA sequencing of sorted cell populations demonstrated robust separation of synovial lymphocytes, fibroblasts, and macrophages. Single cell RNA-seq produced transcriptomes of over 1000 genes/cell, including transcripts encoding characteristic lineage markers identified. Conclusion: We have established a robust protocol to acquire viable cells from cryopreserved synovial tissue with intact transcriptomes and cell surface phenotypes. A centralized pipeline to generate multiple high-dimensional analyses of synovial tissue samples collected across a collaborative network was developed. Integrated analysis of such datasets from large patient cohorts may help define molecular heterogeneity within RA pathology and identify new therapeutic targets and biomarkers.

Background Tofacitinib is a novel oral Janus kinase (JAK) inhibitor for the treatment of rheumatoid arthritis (RA). The specific JAK-STAT (signal transducer and activator of transcription) pathways affected by tofacitinib and the downstream effects on gene expression in situ are not known. Objectives To understand how tofacitinib alters synovial biology in RA, we performed a prospective serial synovial biopsy study. The effects of JAK inhibition on synovial histopathology, gene expression, and signalling were determined and correlated with clinical response. Methods A randomised, double-blind, Phase 2a clinical trial (A3921073; NCT00976599) of seropositive RA patients (pts) with inadequate response to methotrexate compared tofacitinib 10 mg twice daily (BID) for 1 month (15 pts) with placebo (PBO; 14 pts). Synovial biopsies were performed on Days -7 and 28. Biopsies were analysed by immunohistochemistry (IHC), real-time polymerase chain reaction (PCR), and synovial tissue extracts by enzyme-linked immunosorbent assay. Clinical response was determined by disease activity score (DAS)28-4 (erythrocyte sedimentation rate) and EULAR criteria. Results Exposure to tofacitinib led to EULAR moderate to good responses (11/14 pts), while PBO was ineffective (1/14 pts) on Day 28. Tofacitinib treatment significantly reduced synovial mRNA expression of the matrix metalloproteases MMP1 and MMP3 (p<0.05), and chemokines CCL2, CXCL10, and CXCL13 (p<0.05). No overall changes were observed in synovial inflammation scores or the presence of any immune cell lineages at this time point using IHC e.g. CD20, CD3, CD68 (B cells, T cells and macrophages, respectively), as well as quantitative PCR to quantify cell specific genes e.g. CD19, CD3 (B cell, T cell, respectively). Clinical responses in the tofacitinib-treated group correlated with several synovial biomarkers e.g. mean pSTAT1 levels were significantly decreased (p<0.05) in tofacitinib responders (48%) but not in PBO non-responders (21%). In contrast, only a trend towards a modest decrease in pSTAT3 was observed with tofacitinib responders. Tofacitinib (bound plus unbound) peak and trough serum levels were approximately 290 nM and 30 nM, respectively; these drug exposures are within the half-maximal inhibitory concentration (IC50) range required to block STAT1 phosphorylation in T cells (54 nM), but below the IC50 for STAT3 phosphorylation (367 nM). Conclusions The data suggest that tofacitinib modulates synovial immune and inflammatory responses by suppressing JAK1-STAT1 signalling, and that these changes correlate with clinical response. The greater effect on STAT1 vs STAT3 is likely due to greater sensitivity of this pathway to tofacitinib inhibition and the serum concentrations achieved with tofacitinib 10 mg BID. The STAT1 pathway is critical for interleukin-6 and interferon (IFN) signalling, with the latter effect paralleling reductions in expression of IFN-regulated chemokines (e.g. CXCL10 and CCL2) in the blood and synovium of tofacitinib-treated pts. The data sug...