Objective. Antinuclear antibody (ANA)-positive juvenile idiopathic arthritis (JIA) is characterized by synovial B cell hyperactivity, but the precise role of CD4+ T cells in promoting local B cell activation is unknown. This study was undertaken to determine the phenotype and function of synovial CD4+ T cells that promote aberrant B cell activation in JIA.Methods. Flow cytometry was performed to compare the phenotype and cytokine patterns of PD-1 high CD4+ T cells in the synovial fluid (SF) of patients with JIA and T follicular helper cells in the tonsils of control individuals. TCRVB next-generation sequencing was used to analyze T cell subsets for signs of clonal expansion. The functional impact of these T cell subsets on B cells was examined in cocultures in vitro.Results. Multidimensional flow cytometry revealed the expansion of interleukin-21 (IL-21) and interferon-γ (IFNγ)coexpressing PD-1 high CXCR5-HLA-DR+CD4+ T cells that accumulate in the joints of ANA-positive JIA patients. These T cells exhibited signs of clonal expansion with restricted T cell receptor clonotypes. The phenotype resembled peripheral T helper (Tph) cells with an extrafollicular chemokine receptor pattern and high T-bet and B lymphocyteinduced maturation protein 1 expression, but low B cell lymphoma 6 expression. SF Tph cells, by provision of IL-21 and IFNy, skewed B cell differentiation toward a CD21 low/-CD11c+ phenotype in vitro. Additionally, SF Tph cell frequencies correlated with the appearance of SF CD21 low/-CD11c+CD27-IgM-double-negative (DN) B cells in situ.Conclusion. Clonally expanded CD4+ Tph cells accumulate in the joints of ANA-positive JIA patients and, in particular, promote CD21 low/-CD11c+ DN B cell differentiation. The expansion of Tph cells and DN B cells might reflect the autoimmune response in the joints of ANA-positive JIA patients.

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Juvenile idiopathic arthritis (JIA) encompasses a heterogeneous group of diseases. The appearance of antinuclear antibodies (ANAs) in almost half of the patients suggests B cell dysregulation as a distinct pathomechanism in these patients. Additionally, ANAs were considered potential biomarkers encompassing a clinically homogenous subgroup of JIA patients. However, in ANA+ JIA patients, the site of dysregulated B cell activation as well as the B cell subsets involved in this process is still unknown. Hence, in this cross-sectional study, we aimed in an explorative approach at characterizing potential divergences in B cell differentiation in ANA+ JIA patients by assessing the distribution of peripheral blood (PB) and synovial fluid (SF) B cell subpopulations using flow cytometry. The frequency of transitional as well as switched-memory B cells was higher in PB of JIA patients than in healthy controls. There were no differences in the distribution of B cell subsets between ANA- and ANA+ patients in PB. However, the composition of SF B cells was different between ANA- and ANA+ patients with increased frequencies of CD21lo/−CD27−IgM− “double negative” (DN) B cells in the latter. DN B cells might be a characteristic subset expanding in the joints of ANA+ JIA patients and are potentially involved in the antinuclear immune response in these patients. The results of our explorative study might foster further research dissecting the pathogenesis of ANA+ JIA patients.

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