Combined manual and automated immunophenotypisation identified disease-specific peripheral blood immune subpopulations in rheumatoid arthritis, ankylosing spondylitis and psoriatic arthritis

Objectives: Rheumatoid arthritis (RA), ankylosing spondylitis (AS) and psoriatic arthritis (PsA) are associated with abnormal immune cell functions. We combined manual and automated profiling in subpopulations of T-cells, B-cells and monocytes, in parallel to functional testing and clinical correlation. ----- Methods: Using flow cytometry, we analysed the expression of CCR4, CCR6 and CXCR5 on helper and cyotoxic T-cells, CD32B and CD86 on naïve and memory B-cells, and CCR1, CCR2, CCR4 and CXCR4 on monocytes in chronic high-disease activity patients to identify peripheral blood subpopulations. Cell activation, proliferative capability and osteoclastogenic effects were tested in vitro. Comparison with synovial compartment, clinical data and anti-TNF treatment were added to peripheral blood analysis. ----- Results: PsA had lower double-negative T-cell frequency, while RA had lower double-positive T-cell frequency and expanded Th1-like and cytotoxic T-cell subsets. CD32B expression was increased on naïve and memory B-cells in AS and associated with disease activity. CCR6+ and CXCR5+ cytotoxic T-cells and CD32B+ naïve and memory B-cells were highly enriched within the synovial compartment. T-cells and B-cells from AS exhibited enhanced activation and proliferation in vitro, whereas T-cell conditioned medium from RA produced an increased osteoclastogenic effect. CCR1 and CXCR4 were upregulated on osteoclastogenic monocyte subsets of RA, AS and PsA patients. Bioinformatic Citrus analysis identified additional T-cell, B-cell and monocyte clusters specifically associated with each disease. ----- Conclusions: By combining manual and automated data analysis, our study revealed several disease-specific immune cell subpopulations, particularly cytotoxic T-cell subsets in RA and memory B-cell subsets in AS, which may serve as an indicator of active disease or possible therapeutic target