Background/Purpose: Ankylosing spondylitis (AS) has a strong connection with gut inflammation: 10% of AS patients have inflammatory bowel disease (IBD) and 60% have subclinical ileal inflammation. Further, there is strong overlap between genetic susceptibility to IBD and to AS. This suggests that immune events in the gut may impact on joint inflammation but what directs cells in the gut-joint axis is undefined. For this reason, we are examining trafficking molecule expression on immune cells. Identification of trafficking molecules at a single-cell level has been possible through flow cytometry, but spectral overlap limits the number of parameters that can be tested simultaneously. Advances in single-cell mass cytometry have led to platforms such as Cytometry by Time-of-flight (CyTOF) that utilize stable lanthanide metal-conjugated antibodies bound to cells. This eliminates spectral overlap, thereby allowing for analysis of >30 parameters/cell. Our objectives are 1) to assess differential expression patterns of trafficking molecules between AS patients and controls, and 2) to generate tissue-specific cellular signatures.

Methods: Male subjects under 40 years of age fulfilling the mNY criteria were recruited from a longitudinal AS cohort. The following cells were surface stained using a 36-marker antibody panel: (i) Peripheral blood mononuclear cells (PBMC) from 30 AS patients, 20 healthy controls and 18 rheumatoid arthritis (RA) patients; (ii) Synovial fluid mononuclear cells (SFMC) from 10 AS patients and 4 RA patients. After acquiring on CyTOF2, data were subjected to SPADE and viSNE analysis for data visualization and Citrus and FlowJo for statistical analysis.

Results: Few differences were detected in PBMC trafficking marker expression between patients and controls. In AS SFMC mature CD4+ T cells, CXCR4 was reduced but CCR2, CCR5 and CD49a median expression was elevated (FDR<0.05). Furthermore, CCR2, CCR5 and CD49a were co-expressed in CD4+CD45RO+ cells. Mature CD8+ T cells were increased in frequency in AS SFMC, with significant changes in their phenotype: β7+, CD103+, CD18+, CD29+ and CD49a+ integrin expression was increased in CD8+CD45RO+ cells in AS SFMC vs paired AS PBMC (mean 9.05% vs 0.97%, p=0.0061), whereas similar comparison of RA SFMC vs RA PBMC showed less significant differences (mean 4.5% vs 0.63%, p=0.0578).

Conclusion: Analysis using mass cytometry revealed disease- and tissue-specific alterations in trafficking marker expression on AS patient T cells. We identified a novel integrin manifesting CD8+ mature T cell subset (CD49a+CD103+β7+CD29+CD18+) with some specificity for AS. Further experiments to determine similar expression profile on gut tissue biopsies from AS patients, as well as murine gut and joint tissues from experimental AS, are in progress to examine the arthritogenic potential of these cells. CyTOF represents an innovative new technology which can provide new insights into the immune mechanisms linking inflammation in the AS gut and the joint. These insights could lay the groundwork for innovative immunotherapy for AS in the future.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/discovery-of-a-novel-cd8-t-cell-population-in-ankylosing-spondylitis-implicates-gut-joint-trafficking-in-the-disease/