Background/Purpose: Pathogenic immune cell types and functions have not been identified yet in rheumatoid arthritis (RA). This is explained by the impact of disease heterogeneity on study power and by the limited number of immune markers tested so far. Our lack of understanding of RA pathophysiology results in practising trial and error medicine with the prescription of biologic drugs: 30% of patients fail to respond to the first drug prescribed. Using the world-wide largest prospective cohort of RA patients undergoing treatment with biologics, we aim to identify immunological signatures of RA endotypes using a T cell mass cytometry (CyTOF) panel to define treatment response groups at baseline.

Methods: Peripheral blood mononuclear cells (PBMCs) are isolated from patients enrolled in the Biologics in Rheumatoid Arthritis Genetics and Genomics Study Syndicate (BRAGGSS) prior to treatment with a biologic drug. One hundred RA patients with a DAS28 > 5.1 are enrolled prospectively each year in addition to healthy controls. Treatment response is assessed at 3, 6 and 12 months. PBMCs are stimulated for 4 hours using anti-CD3/anti-CD28 beads and stained with a 37-channel CyTOF panel which includes intracellular cytokines, transcription factors and co-stimulatory molecules to allow a detailed characterization of the function of T-cells (including Th1, Th2, Th17 and Treg). Data will be analysed using in house developed unbiased advanced computational strategies and clustering algorithms to define cellular clusters agnostically (Raychaudhuri’s lab). Our in house analytical pipeline (MASC: “Mixed model association of single cells”) will be compared with conventional biaxial gating and commercially available packages like CITRUS.

Results: Preliminary data on 10 healthy controls (HC) and 10 RA patients were available for analysis. Traditional biaxial gating showed large differences in the proportions of both Th1 cells (1-9 % in RA and 5-15 % in HC) and Th17 cells (0-9 %) within and between RA and HC. Depending on IFNγ and IL-17A expression in CD4⁺ T cells, individuals could be classified into 4 immunophenotypes: ‘Th1’, ‘Th17’, ‘double positive’, and ‘double negative’. CITRUS identified 3 clusters of cells which were significantly different in abundance between the HC and RA groups. As an example, one cluster was CD4⁺CD38⁺, had characteristics of regulatory T cells and was less abundant in RA. Preliminary analysis of the drug response data showed a strong increase in Th1 cells in responders seen only after in vitro stimulation, together with an increase of CD40L⁺ CD4⁺ and CD40L⁺ CD8⁺ T-cells.

Conclusion: These preliminary analyses show the potential of our study design to capture RA heterogeneity at the single cell level. Importantly, sample size needs to be increased and analytical algorithms further developed, which is achievable between now and November 2017. The systematic quantification of intracellular cytokines in targeted cell types is likely to identify cell functions involved in RA pathogenesis and treatment response.

« Back to 2017 ACR/ARHP Annual Meeting

ACR Meeting Abstracts - https://acrabstracts.org/abstract/deep-immunophenotyping-of-t-lymphocytes-with-a-37-channel-mass-cytometry-cytof-panel-for-the-identification-of-pathological-cell-functions-and-the-prediction-of-response-to-biologic-drugs-in-rheumat/