Ex vivo Immune Cell Signaling Activity to Stratify Patients with Rheumatic Disease
Aberrant signal transduction in leukocytes is involved in the establishment and maintenance of chronic inflammation. Also, signaling events represent how cells sense and integrate their inflammatory environment. While the activity and abnormalities of these cascades are known to be cell subset-specific, we lack a systems-level understanding of which immune cells signal differently in particular chronic inflammatory conditions and through which pathways. In this study, we combined in-depth phenotyping of blood leukocytes with cell type-specific intracellular signal transducer phosphorylation analyses to define the cellular fingerprint of active Systemic Lupus Erythematosus (SLE), Rheumatoid Arthritis (RA), Spondyloarthritis (SpA) patients.
To this end, we applied a 40-parameter mass cytometry panel to leukocytes isolated from cryopreserved whole blood samples of patients suffering from SLE, RA or SpA, as well as age- and gender-matched healthy controls. Immune cell populations and their heterogeneity were defined based on the expression of 28 cell-surface antigens. 12 parameters were used to detect the ex vivo phosphorylation status of intracellular signal transducers. The data were analyzed by dimension reduction, FlowSOM clustering and supplementary manual gating.
Consistent with activation by type I interferon, the hallmark cytokine of SLE, this patient cohort showed increased phosphorylation of STAT1, and to a lesser extent of STAT3 and 5, across almost all innate and adaptive immune cell populations. In SpA patients, pNfkB signals were increased in NK, B and T cell populations, likely reflecting imprinting by TNF alpha, a key driver of disease pathology. Consistently, serum levels of these cytokines, as determined by Luminex analysis, were elevated in SLE and SpA patients. pSTAT1 and 3 signal intensities in T cell subsets were correlated with complement use in SLE and associated with autoantibody titer levels, but not correlated with SLE disease activity index SLEDAI. While phosphorylation of signal transducers was not significantly altered in RA and not associated with disease activity score DAS28, pNfkB and pSTAT5 levels in T cell subsets were correlated with swollen joint counts. Patients with especially high autoantibody titers and/or positivity for multiple autoantibodies were characterized by high ex vivo pSTAT signals, especially in myeloid cell clusters. In summary, phosphorylation readouts reflect stable imprints of inflammation and immunological activity in patients and could be suitable for the development of molecular markers for precision medicine.
Marie received both her Bachelor’s and Master’s degree in Biochemistry from Freie Universität in Berlin. Since 2017, she has been a graduate student at the department of immune monitoring and mass cytometry at the German Rheumatism Research Centre (DRFZ) in Berlin. Her research focuses on the identification of cellular biomarkers in chronic inflammatory diseases, with a special focus on signal transduction in peripheral blood immune cells.
Immune Monitoring & Mass Cytometry, Deutsches Rheuma-Forschungszentrum Berlin, a Leibniz-Institute