Immunoprofiling of early, untreated rheumatoid arthritis using mass cytometry reveals an activated basophil subset inversely linked to ACPA status

Abstract

Background: Autoantibody production is a hallmark of rheumatoid arthritis (RA). Anti-citrullinated protein antibodies (ACPA) are highly disease-specific, and their presence is associated with more severe disease and poor prognosis compared to ACPA-negative patients. However, the immune cell composition associated with antibody-positive/negative disease is incompletely defined. Mass cytometry (MC) is a high-dimensional technique offering new possibilities in the determination of the immune cell composition in rheumatic diseases. Here, we set up a broad phenotyping panel to study the immune cell profile of early untreated RA to investigate if specific immune cell subsets are associated with ACPA+ versus ACPA− RA. Methods: Freshly obtained PBMCs of early, untreated RA patients (8 ACPA+ and 7 ACPA−) were analysed using a 36-marker MC panel, including markers related to various immune lineages. Data were processed using Cytosplore for dimensional reduction (HSNE) and clustering. Groups were compared using Cytofast. A second validation cohort of cryopreserved PBMCs obtained from early RA patients (27 ACPA+ and 20 ACPA−) was used to confirm MC data by flow cytometry (FC). FC data were processed and analysed using both an unsupervised analysis pipeline and through manual gating. Results: MC indicated no differences when comparing major immune lineages (i.e. monocytes, T and B cells), but highlighted two innate subsets: CD62L+ basophils (p = 0.33) and a subset of CD16− NK cells (p = 0.063). Although the NK cell subset did not replicate by FC, FC replication confirmed the difference in CD62L+ basophil frequency when comparing ACPA+ to ACPA− patients (mean 0.32% vs. 0.13%; p = 0.01). Conclusions: Although no differences in major lineages were found between early ACPA+ and ACPA− RA, this study identified the reduced presence of activated basophils in ACPA-negative disease as compared to ACPA-positive disease and thereby provides the first evidence for a connection between activated basophils and ACPA status.