B cell subsets in adult-onset Still's disease: potential candidates for disease pathogenesis and immunophenotyping

Abstract

Background: Adult-onset Still's disease (AOSD) is a systemic autoinflammatory disorder of unknown etiology. B cells are critical participants in different rheumatic diseases, and their roles in AOSD are rarely investigated. This study aimed to unveil the B cell subset features in AOSD and provide evidence for B cell-based diagnosis and targeted therapies of AOSD.

Methods: B cell subsets in the peripheral blood of AOSD patients and healthy controls (HCs) were detected by flow cytometry. Firstly, the frequencies of B cell subsets were compared. Then, the correlation analysis was performed to explore the correlation between B cell subsets and clinical manifestations in AOSD. Finally, unbiased hierarchical clustering was performed to divide AOSD patients into three groups with different B cell subset features, and the clinical characteristics of the three groups were compared.

Results: The frequencies of B cell subsets were altered in AOSD patients. Disease-promoting subsets (such as naïve B cells, double negative B cells (DN B cells), and plasmablasts) increased, and potential regulatory subsets (such as unswitched memory B cells (UM B cells) and CD24^hiCD27⁺ B cells (B10 cells)) decreased in the peripheral blood of AOSD patients. In addition, the altered B cell subsets in AOSD correlated with the clinical and immunological features, such as immune cells, coagulation features, and liver enzymes. Intriguingly, AOSD patients could be divided into three groups with distinct B cell immunophenotyping: group 1 (naïve B cells-dominant), group 2 (CD27⁺ memory B cells-dominant), and group 3 (precursors of autoantibody-producing plasma cells-dominant). Moreover, these three group patients demonstrated differential manifestations, including immune cells, liver or myocardial enzymes, coagulation features, and systemic score.

Conclusions: B cell subsets are significantly altered in AOSD patients, potentially contributing to the disease pathogenesis. These findings would inspire B cell-based diagnosis and targeted therapies for this refractory disease.

Keywords: Adult-onset Still’s disease; B cell subsets; Clinical correlation; Immunophenotyping.

Fig. 1

Gating strategy for flow cytometry analyses of the frequencies of B cell subsets. After lymphocyte gating according to FSC and SSC, CD3⁻CD19⁺ lymphocytes were selected and resolved into subsets by analyzing CD20, CD24, CD27, or IgD expression

Fig. 2

Altered frequencies of B cell subsets in AOSD PBMC. a The frequencies of B cell subsets in the peripheral blood of the healthy controls (n = 40) and AOSD patients (n = 27). b The relative frequencies of three CD27⁺ B cell subsets in the total CD27⁺ B cells. c The frequencies of B cell subsets in the peripheral blood of the healthy controls (n = 40), active AOSD patients (n = 18), and inactive AOSD patients (n = 9). d t-SNE analyses of B cell subsets in healthy controls’ and AOSD patients’ peripheral blood. *P < 0.05, **P < 0.01, ***P < 0.001, and ns, no significance (unpaired t test, naïve B cells, UM B cells, SM B cells, CD27⁺ B cells, DN B cells, and B10 cells in a and b; Mann–Whitney U test, B cells, and plasmablasts in a; One-way ANOVA followed by uncorrected Fisher’s LSD test, naïve B cells, UM B cells, SM B cells, CD27⁺ B cells, DN B cells and B10 cells in c; Kruskal–Wallis H test followed by uncorrected Dunn’s test, B cells and plasmablasts in c)

Fig. 3

Correlation analysis of B cell subsets with clinical manifestations of AOSD patients. Correlation matrix among B cell subsets and clinical manifestations. *P < 0.05, **P < 0.01, and ‘.’ marginal significance (Spearman’s rank correlation test)

Fig. 4

Unbiased cluster analysis of B cell subsets in AOSD. a Hierarchical cluster analysis divided patients with active AOSD (n = 18) into three subgroups. b Results of PCA based on B cell subsets in AOSD patients, first and second principal components were chosen to virtualize different B cell subsets. c Comp1 and Comp 2 values in individual patients with AOSD

Fig. 5

Differences of B cell subsets and clinical manifestations among the clustered groups. a Differences of B cell subsets among the clustered groups. Differences of clinical manifestations among the clusters, including liver and myocardial enzymes (b), other liver function indicators (c), coagulation parameters (d), immune cells (e), indexes and ratios (f), and mPss (g). *P < 0.05, **P < 0.01, ***P < 0.001, and ns, no significance (Kruskal–Wallis H test followed by uncorrected Dunn’s test)