Children with extended oligoarticular and polyarticular juvenile idiopathic arthritis have alterations in B and T follicular cell subsets in peripheral blood and a cytokine profile sustaining B cell activation

Abstract

Objectives: The main goal of this study was to characterise the frequency and phenotype of B, T follicular helper (Tfh) and T follicular regulatory (Tfr) cells in peripheral blood and the cytokine environment present in circulation in children with extended oligoarticular juvenile idiopathic arthritis (extended oligo JIA) and polyarticular JIA (poly JIA) when compared with healthy controls, children with persistent oligoarticular JIA (persistent oligo JIA) and adult JIA patients.

Methods: Blood samples were collected from 105 JIA patients (children and adults) and 50 age-matched healthy individuals. The frequency and phenotype of B, Tfh and Tfr cells were evaluated by flow cytometry. Serum levels of APRIL, BAFF, IL-1β, IL-2, IL-4, IL-6, IL-10, IL-17A, IL-21, IL-22, IFN-γ, PD-1, PD-L1, sCD40L, CXCL13 and TNF were measured by multiplex bead-based immunoassay and/or ELISA in all groups included.

Results: The frequency of B, Tfh and Tfr cells was similar between JIA patients and controls. Children with extended oligo JIA and poly JIA, but not persistent oligo JIA, had significantly lower frequencies of plasmablasts, regulatory T cells and higher levels of Th17-like Tfh cells in circulation when compared with controls. Furthermore, APRIL, BAFF, IL-6 and IL-17A serum levels were significantly higher in paediatric extended oligo JIA and poly JIA patients when compared with controls. These immunological alterations were not found in adult JIA patients in comparison to controls.

Conclusions: Our results suggest a potential role and/or activation profile of B and Th17-like Tfh cells in the pathogenesis of extended oligo JIA and poly JIA, but not persistent oligo JIA.

Keywords: B cells; Cytokines; Juvenile Idiopathic Arthritis; T-Lymphocyte subsets.

Figure 1

Children with extended oligoarticular and polyarticular JIA, but not persistent oligoarticular JIA, have lower levels of plasmablasts (CD19+IgD-CD27++CD38++) in circulation when compared with healthy controls. The frequency of total CD19+ B cells (A) and B cell subpopulations (B) was determined by flow cytometry in peripheral blood of children with extended oligoarticular and polyarticular JIA when compared with persistent oligoarticular JIA patients and healthy controls. Flow cytometry gating strategy for B cell subpopulations (defined in CD19+ B cells) based on IgD/CD27 and IgD/CD38 classification systems is shown in representative dot plots. Data are represented in box plots. Each dot represents an individual patient. Horizontal lines represent median values, quartiles and extremes (minimum and maximum). Differences were considered statistically significant for p<0.05. Non-parametric Mann-Whitney U test was used for comparisons between 2 independent groups. Kruskal-Wallis test with post hoc Dunn’s multiple comparisons was used to compare more than two groups. A correlation analysis between the frequency of total CD19+ B cells and B cell subpopulations and the clinical data (C) was performed using Spearman correlation test with Bonferroni correction to counteract multiple comparisons and differences were considered statistically significant for p<0.007 (*). Data are represented as heatmap distribution. CRP, C-reactive protein; eo+pJIA, extended oligoarticular and polyarticular JIA; ESR, erythrocyte sedimentation rate; HC, healthy controls; JADAS-27, juvenile arthritis disease activity score 27-joint reduced count; poJIA- persistent oligoarticular JIA.

Figure 2

Changes in CD19+ B cells phenotype are found in children with JIA when compared with healthy controls. Phenotypic analysis of total CD19+ B cells in peripheral blood from paediatric JIA patients. The expression levels of B cell activating factor receptor (BAFF-R), activation receptors (CD86, HLA-DR), chemokine receptor (CXCR5), B-cell differentiation (CD5), complement receptor type 2 (CD21), the low-affinity Fc-epsilon-receptor II (CD23), inhibitory receptor (Fc-gamma-RIIB), calcium signalling (CD38), costimulatory receptor (CD40), Fas-receptor (CD95) and toll-like receptor 9 (TLR9) were analysed (median fluorescence intensity, MFI) in total CD19+ B cells to characterise B cell phenotype in children with JIA when compared with healthy controls. Data are represented in box plots. Each dot represents an individual patient. Horizontal lines represent median values, quartiles and extremes (minimum and maximum). Differences were considered statistically significant for p<0.05. Non-parametric Mann-Whitney U test was used for comparisons between two independent groups. Kruskal-Wallis test with post hoc Dunn’s multiple comparison was used to compare more than two groups. eo+pJIA, extended oligoarticular and polyarticular JIA; FMO, fluorescence minus one; HC, healthy controls; JIA, juvenile idiopathic arthritis; poJIA, persistent oligoarticular JIA.

Figure 3

Children with extended oligoarticular and polyarticular JIA, but not persistent oligoarticular JIA have lower levels of regulatory T cells (CD4+CD25+FoxP3+) in circulation when compared with healthy controls. The frequencies of total CD3+ T cells (A), CD4+ and CD8+ T cells (B), CD4+ T conventional (Tconv, CD4+CD25-FoxP3−), regulatory T cells (Tregs, CD4+CD25+FoxP3+) and Tregs/Tconv ratio (C), T follicular helper (Tfh, CD4+CD25-FoxP3-CD45RO+CXCR5+), T follicular regulatory (Tfr, CD4+CD25+FoxP3+CXCR5+) cells and Tfr/Tfh ratio (D), were evaluated by flow cytometry in peripheral blood of children with extended oligoarticular JIA and polyarticular JIA when compared with persistent oligoarticular JIA patients and healthy controls. Data are represented in box plots. Each dot represents an individual patient. Horizontal lines represent median values, quartiles and extremes (minimum and maximum). Differences were considered statistically significant for p<0.05. Non-parametric Mann-Whitney U test was used for comparisons between two independent groups. Kruskal-Wallis test with post hoc Dunn’s multiple comparisons was used to compare more than two groups. eo+pJIA, extended oligoarticular and polyarticular JIA; HC, healthy controls; JIA, juvenile idiopathic arthritis; poJIA, persistent oligoarticular JIA.

Figure 4

Children with extended oligoarticular and polyarticular JIA, but not persistent oligoarticular JIA have an imbalance in T follicular cell subsets in circulation when compared with healthy controls. The frequencies CD4+ Tfh cell subsets (Tfh1, CXCR3+CCR6−; Tfh2, CXCR3-CCR6−; Tfh17, CXCR3-CCR6+) (A), PD1+ Tfh cells (B), PD1+ICOS+ Tfh cells (C) and Tph (CXCR5-PD-1++) cells (D) were evaluated by flow cytometry in peripheral blood of children with extended oligoarticular JIA and polyarticular JIA when compared with persistent oligoarticular JIA patients and healthy controls. Data are represented in box plots. Each dot represents an individual patient. Horizontal lines represent median values, quartiles and extremes (minimum and maximum). Differences were considered statistically significant for p<0.05. Non-parametric Mann-Whitney U test was used for comparisons between two independent groups. Kruskal-Wallis test with post hoc Dunn’s multiple comparisons was used to compare more than two groups. A correlation analysis between the frequency of total T and T cell subpopulations and clinical data (E) was performed using Spearman correlation test with Bonferroni correction to counteract multiple comparisons and differences were considered statistically significant for p<0.007 (*). Data are represented as heatmap distribution. CRP, C-reactive protein; eo+pJIA, extended oligoarticular and polyarticular JIA; ESR, erythrocyte sedimentation rate; HC, healthy controls; JADAS-27, juvenile arthritis disease activity score 27-joint reduced count; JIA, juvenile idiopathic arthritis; poJIA, persistent oligoarticular JIA.

Figure 5

Changes in Tfh cells phenotype are found in children with JIA when compared with healthy controls. A phenotypic analysis of circulating Tfh (CD4+CD25-FoxP3−CD45RO+CXCR5+) cells was performed by flow cytometry in peripheral blood from paediatric JIA patients. The expression levels of T-cell costimulatory receptor CD28, CD40 ligand (CD40-L), programmed cell death protein 1 (PD-1), inducible T-cell costimulator (ICOS), cytotoxic T lymphocyte antigen-4 (CTLA-4) and activation markers (CD69, HLA-DR) were analysed (median fluorescence intensity, MFI) in Tfh (CD4+CD25-FoxP3-CD45RO+CXCR5+) cells to characterise Tfh cell phenotype in children with JIA when compared with healthy controls. Data are represented in box plots. Each dot represents an individual patient. Horizontal lines represent median values, quartiles and extremes (minimum and maximum). Differences were considered statistically significant for p<0.05. Non-parametric Mann-Whitney U test was used for comparisons between two independent groups. Kruskal-Wallis test with post hoc Dunn’s multiple comparisons was used to compare more than two groups. eo+pJIA, extended oligoarticular and polyarticular JIA; FMO, fluorescence minus one; HC, healthy controls; JIA, juvenile idiopathic arthritis; poJIA, persistent oligoarticular JIA.

Figure 6

Children with extended oligoarticular and polyarticular JIA, but not persistent oligoarticular JIA patients, have a cytokine pattern in circulation sustaining B cell activation. Serum levels of A proliferation-inducing ligand (APRIL), B cell activating factor (BAFF), interleukin (IL)-1β, IL-2, IL-4, IL-6, IL-10, IL-17A, IL-21, IL-22, interferon gamma (IFN-γ), programmed cell death-protein 1 (PD-1), programmed death-ligand 1 (PD-L1), soluble CD40 ligand (sCD40L), chemokine (C-X-C motif) ligand 13 (CXCL13) and tumour necrosis factor (TNF) were measured by multiplex bead-based immunoassay and/ or ELISA in children with extended oligoarticular and polyarticular JIA when compared with persistent oligoarticular JIA patients and healthy controls (A) Data are represented in box plots. Each dot represents an individual patient. Horizontal lines represent median values, quartiles and extremes (minimum and maximum). Differences were considered statistically significant for p<0.05. Non-parametric Mann-Whitney U test was used for comparisons between two independent groups. Kruskal-Wallis test with post hoc Dunn’s multiple comparisons was used to compare more than two groups. A correlation analysis between cytokine serum levels and clinical data (B) was performed using Spearman correlation test with Bonferroni correction to counteract multiple comparisons and differences were considered statistically significant for p<0.007 (*). Data are represented as heatmap distribution. CRP, C-reactive protein; eo+pJIA, extended oligoarticular and polyarticular JIA; ESR, erythrocyte sedimentation rate; HC, healthy controls; JADAS-27, juvenile arthritis disease activity score 27-joint reduced count; JIA, juvenile idiopathic arthritis; poJIA, persistent oligoarticular JIA.