Background/Purpose: Current data suggests that immune events in the gut may impact on joint inflammation in ankylosing spondylitis (AS) but what directs cells in the gut-joint axis is undefined. For this reason, we examined the expression of trafficking molecules on immune cells using Cytometry by Time-of-flight (CyTOF), and the expression of differentially regulated genes using bulk RNA-sequencing (RNA-seq). Our objectives are to utilize proteomic and transcriptomic analysis to 1) assess differential expression patterns of trafficking molecules between patients and controls, 2) generate joint-specific cellular signatures, and 3) obtain genetic profiles of noteworthy cell subpopulations.

Methods: Male subjects under 40 years of age fulfilling the mNY criteria were recruited. The following cells were surface stained using a 36-marker antibody panel: (i) Peripheral blood mononuclear cells (PBMC) from AS patients, and healthy controls; (ii) Synovial fluid mononuclear cells (SFMC) from AS and rheumatoid arthritis (RA) patients. After acquiring on CyTOF2, data were analysed using SPADE, viSNE and FlowJo programs for data visualization and statistical analysis. Additionally, bulk RNA-seq was performed on CD8+ T cell subpopulations from the synovial fluid. Pathway analysis of differentially expressed genes was conducted using the ClueGo application from the Cytoscape program.

Results: Mature CD8+ T cells were increased in frequency in AS SFMC, with significant changes in their phenotype: β7+, CD103+, CD29+ and CD49a+ integrin expression was increased in CD8+CD45RO+ cells in AS SFMC vs paired AS PBMC (mean 7.51% vs 0.87%, p=0.0035). A similar comparison in RA SFMC vs paired RA PBMC revealed less dramatic changes (mean 3.11% vs 0.33%, p=0.0056). RNA-seq data analysis of CD103+CD49a+ cells in AS SFMC revealed elevated GZMA, GZMB, PRF1 and IL-10 cytokines, in addition to a cytotoxicity regulation profile. Signaling molecules, such as TNFAIP3 and TIAF1, and transcription factors, such as RUNX1 and IRF4, were elevated as well.

Conclusion: We identified a novel integrin-expressing mature CD8+ T cell subset (CD49a+CD103+β7+CD29+) that appears to be more prevalent in AS SF than RA SF. These cells possess a dual proinflammatory and regulatory profile, suggesting these roles may be microenvironment-dependent. Further experiments are ongoing to provide evidence of gut-joint trafficking capabilities using murine models. Examinations of patient gut and synovial tissue biopsies, as well as murine gut and joint tissue are essential to determine the arthritogenic potential of these cells. A global analysis encompassing transcriptional and proteomic changes is mandated to provide crucial insights into the inflammatory relationship between the gut and joint, which in turn would be important to design innovative immunotherapy for AS.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/proteomic-and-transcriptomic-profiling-of-cells-in-ankylosing-spondylitis-patients-identifies-a-novel-synovial-resident-cd8-t-cell/