CD19+CD24hiCD38hi B Cells Are Expanded in Juvenile Dermatomyositis and Exhibit a Pro-Inflammatory Phenotype After Activation Through Toll-Like Receptor 7 and Interferon-α

Abstract

Juvenile dermatomyositis (JDM) is a rare form of childhood autoimmune myositis that presents with proximal muscle weakness and skin rash. B cells are strongly implicated in the pathogenesis of the disease, but the underlying mechanisms are unknown. Therefore, the main objective of our study was to investigate mechanisms driving B cell lymphocytosis and define pathological features of B cells in JDM patients. Patients were recruited through the UK JDM Cohort and Biomarker study. Peripheral blood B cell subpopulations were immunophenotyped by flow cytometry. The results identified that immature transitional B cells were significantly expanded in active JDM, actively dividing, and correlated positively with disease activity. Protein and RNAseq analysis revealed high interferon alpha (IFNα) and TLR7-pathway signatures pre-treatment. Stimulation of B cells through TLR7/8 promoted both IL-10 and IL-6 production in controls but failed to induce IL-10 in JDM patient cells. Interrogation of the CD40-CD40L pathway (known to induce B cell IL-10 and IL-6) revealed similar expression of IL-10 and IL-6 in B cells cultured with CD40L from both JDM patients and controls. In conclusion, JDM patients with active disease have a significantly expanded immature transitional B cell population which correlated with the type I IFN signature. Activation through TLR7 and IFNα may drive the expansion of immature transitional B cells in JDM and skew the cells toward a pro-inflammatory phenotype.

Keywords: B cells; immature transitional B cells; interferon alpha; interleukin-10; juvenile dermatomyositis; toll-like receptor 7.

Figure 1

Immature transitional B cells are significantly expanded in juvenile dermatomyositis (JDM) patients before treatment. Peripheral blood mononuclear cells (PBMC) from patients and age-matched controls were analyzed by flow cytometry and B cell data compared across disease course and against clinical measures. (A) B cell (CD19⁺ cells within all PBMC) frequencies summarized according to time since treatment start in patients compared with controls. (B) Representative flow cytometry plots of B cell subsets, identified using expression of CD24 and CD38, shown for child controls (left plot), JDM pre-treatment (middle plot), and JDM (<6 months) on-treatment (right plot). Bar graphs of (C) frequency and (D) absolute numbers of immature transitional, mature, and memory B cells and plasmablasts (left–right) in patients and controls. For patients up to 6 months into treatment, (E) frequency and (F) absolute numbers of immature transitional B cells were correlated to Physicians Global Assessment. (G) Immature transitional frequency in JDM patients pre-treatment and after treatment with intravenous prednisolone or (H) cyclophosphamide. Pearson r values are shown for panel (E), Spearman r values for panel (F). For panels (A,C,D), lines represent mean values. For panels (G,H), bars represent mean ± SEM (*p < 0.05, **p < 0.01, and ***p < 0.001).

Figure 2

Immature transitional B cells are highly proliferative in juvenile dermatomyositis (JDM) patients before treatment. Peripheral blood mononuclear cell (PBMC) samples were stained ex vivo for B cell surface markers CD19, CD24, and CD38 and the intra-nuclear marker of proliferation, Ki-67. (A) Representative flow cytometry plots of Ki-67 expression in immature transitional (top row) and mature B cells (bottom row), in patients and controls. (B) The frequency of Ki-67⁺ B cells was summarized for total B cells, immature transitional B cells, and mature B cells (left–right) for patients split according to time from treatment start, and for child controls. (C) B cell subpopulations were sorted from patients on-treatment (n = 3) and child controls (n = 3) PBMC. DNA extracted from immature transitional (CD19⁺CD24^hiCD38^hiCD27⁻) and mature B cells (CD19⁺CD24^intCD38^intCD27⁻) were assessed for the levels of kappa-deleting recombination excision circles (KRECs). KREC copies per 10⁶ cells were calculated for immature transitional and mature B cells. Lines represent mean values for panel (B). For panel (C), bars represent mean ± SEM (*p < 0.05, **p < 0.01, and ***p < 0.001).

Figure 3

Juvenile dermatomyositis (JDM) B cells have a prominent interferon alpha (IFNα) and TLR7-pathway signature. (A) Gene set enrichment analysis plot showing the hallmark IFNα response gene-set in pre- vs on-treatment patient B cells. (B) Patient sera from pre- and on-treatment JDM patients were analyzed for chemokines known to be downstream of the IFNα pathway. Serum concentrations of CXCL10, CXCL11, MCP-1, and MCP-2 (left–right) measured by Luminex multiplex array are summarized according to time since treatment start. Patient sera and peripheral blood mononuclear cells were collected on the same day. (C) Immature transitional B cell frequency and serum levels of CXCL10, CXCL11, MCP-1, and MCP-2 (left–right) were correlated in JDM patients (pre-/on-treatment). (D) Volcano plot highlighting differentially expressed genes downstream of the IFNα pathway in pre- vs on-treatment patients. (E) Normalized counts (reads per kilobase of transcript per million mapped reads—RPKM) for pre-treatment, on-treatment patients and child controls are shown for TLR7 and interferon-regulatory factor 7 (IRF7). For panel (E), N = 10 for pre-treatment, 9 for on-treatment, and 4 for child controls. For panel (B), lines represent mean values. For panel (C), Pearson r values are shown. For panel (E), data are represented as box and whisker plots with means. The whiskers represent minimum and maximum data points (*p < 0.05, **p < 0.01, and ***p < 0.001).

Figure 4

Juvenile dermatomyositis (JDM) B cells fail to induce IL-10 after TLR7 stimulation. B cells purified from active on-treatment patients [patients with flares had high disease activity (Physicians Global Assessment > 3.5) and were >6 months into treatment], and age-matched child control peripheral blood mononuclear cells, were stimulated with interferon alpha (IFNα) (1,000 IU/ml), 1 µg/ml R848 (a TLR7/8 agonist) or both, for 48 h. Culture supernatants taken from total B cells at 48 h were analyzed for (A) IL-10 and (B) IL-6 concentrations for patients and controls. Representative flow cytometry plots showing percentages of (C) IL-10⁺ and (D) IL-6⁺ B cells after 48 h stimulation with R848 (top row) and R848⁺ IFNα (bottom row). FMO plots are also shown for each cytokine. The frequencies of (E) IL-10⁺ and (F) IL-6⁺ B cells for child controls and active JDM patients are summarized. A two-way analysis of variance with Sidak multiple comparisons test was used for panels (A,B,E,F). N = 3 for both patients and controls. For panel (A,B,E,F), bars represent mean ± SEM (*p < 0.05).

Figure 5

B cells from juvenile dermatomyositis (JDM) patients are able to express IL-10 upon CD40 stimulation. Peripheral blood mononuclear cells (PBMC) were stimulated with CD40L-expressing Chinese Hamster Ovary cells for 72 h, and B cell IL-10 expression was analyzed by flow cytometry, following addition of PMA, Ionomycin, and Brefeldin A for the last 4 h of culture. (A) Representative flow cytometry plots of B cell IL-10 expression are shown for JDM pre-treatment, on-treatment, and child controls. (B) The frequency of IL-10-expressing B cells after 72 h culture with CD40L stimulation is summarized for each of the groups. PBMC were stimulated for 4 h with PMA/Ionomycin (C) Representative flow cytometry plots of total B cell IL-6 staining are shown (left–right: FMO, child control, and JDM pre-treatment). (D) IL-6 expression by B cells are summarized for JDM subgroups and child controls. For panels (B,D), lines represent mean values (*p < 0.05 and **p < 0.01).