Background: IgG4-related disease (IgG4-RD) is an immune-mediated disease characterized by fibroinflammatory lesions affecting a broad spectrum of organs. Although IgG4-RD is thought to be driven by B-T-cell interactions and IgG4 ⁺ plasmablasts are known to play a central role in disease development, the specific B cell subsets underlying disease pathogenesis and the cellular pathways leading to the generation of IgG4 ⁺ plasmablasts remain incompletely defined

Objectives: Using mass cytometry, we aimed to identify rare peripheral blood B cell subsets that are associated with IgG4-RD and may contribute to the pathophysiology.

Methods: Cryopreserved PBMC from 25 IgG4-RD patients and 35 healthy controls (HC) were thawed, barcoded, pooled, and enriched for B cells by fluorescence-activated cell sorting. Samples were acquired on the mass cytometer in four runs following surface and intracellular staining using 50 antibodies. By applying FlowSOM, 60 B cell clusters were distinguishable and those significantly increased in patients were subsequently analyzed for their immunoglobulin (Ig) subclass expression. B cell-to-plasmablast differentiation trajectories were inferred from Spearman correlation analyses of cluster frequencies.

Results: Unsupervised hierarchical clustering of FlowSOM-derived B cell subsets identified three distinct groups of individuals, broadly segregating HC from patients with high or low disease activity, as measured by the IgG4 Responder Index. Differential abundance analysis of the B cell subset frequencies revealed that 8 out of 60 B cell subsets were significantly expanded in peripheral blood of IgG4-RD patients compared to HC. Among the most expanded were IgG1 and IgA1 dominated CXCR5 ^low CD62L ⁺ CD43 ⁺ Ki-67 ^high proliferating plasmablast precursors ( p <0.001), IgG1 and IgG4 expressing CXCR5 ⁺ CD62L ^high germinal-center-derived memory B cells ( p <0.001), IgA1 and IgA2 enriched CXCR5 ⁺ CD11c ⁺ β7integrin ^high CCR9 ⁺ CD43 ⁺ Ki-67 ^high proliferating mucosa-associated memory B cells ( p =0.02), and IgA1 enriched CXCR5 ^low IgD ^– CD27 ^– CD11c ^– Syk ^– extrafollicular DN3-like B cells ( p <0.001). IgG1 ⁺ , IgG2 ⁺ , IgG3 ⁺ , IgG4 ⁺ , IgA1 ⁺ , and IgA2 ⁺ plasmablasts were significantly increased in patients, and serum IgG4 significantly correlated with frequencies of IgG1 ⁺ , IgG2 ⁺ , IgG3 ⁺ , and IgG4 ⁺ plasmablasts (r= 0.55, 0.47, 0.44, and 0.46). Correlation analysis of B cell subset frequencies identified significant associations in patients, suggesting disease-relevant differentiation trajectories from proliferating plasmablast precursors, germinal-center-derived memory B cells, and proliferating mucosa-associated memory B cells towards IgG4 ⁺ plasmablasts (r=0.73, 0.55, and 0.54). Notably, the former two subsets also showed significant correlations with IgG1 ⁺ plasmablast frequencies (r=0.68 and 0.40), whereas the latter significantly correlated with IgA2 ⁺ plasmablasts (r=0.59).

Conclusions: Deep immunophenotyping uncovered several previously undescribed follicular, extrafollicular, and mucosa-associated blood B cell subsets implicated in IgG4-RD, with inferred trajectories largely converging on IgG4 ⁺ and IgG1 ⁺ plasmablasts, suggesting shared plasmablast differentiation endpoints across compartments. Validation in larger cohorts and with integrated BCR sequencing will further substantiate their involvement in disease pathogenesis and define their clonal relationships.

REFERENCES: NIL.

Acknowledgments: NIL.

Disclosure of Interests: None declared.