An IgD-Fc-Ig fusion protein restrains the activation of T and B cells by inhibiting IgD-IgDR-Lck signaling in rheumatoid arthritis

Abstract

Rheumatoid arthritis (RA) is a chronic systemic autoimmune disease characterized by synovitis and the destruction of small joints. Emerging evidence shows that immunoglobulin D (IgD) stimulation induces T-cell activation, which may contribute to diseases pathogenesis in RA. In this study, we investigated the downstream signaling pathways by which IgD activated T cells as well as the possible role of IgD in the T-B interaction. Peripheral blood mononuclear cells were isolated from peripheral blood of healthy controls and RA patients. We demonstrated that IgD activated T cells through IgD receptor (IgDR)-lymphocyte-specific protein tyrosine kinase (Lck)-zeta-associated protein 70 (ZAP70)/phosphatidylinositol 3-kinase (PI3K)/nuclear factor kappa-B (NF-κB) signaling pathways; IgD-induced CD4⁺ T cells promoted the proliferation of CD19⁺ B cells in RA patients. A novel fusion protein IgD-Fc-Ig (composed of human IgD-Fc domain and IgG₁ Fc domain, which specifically blocked the IgD-IgDR binding) inhibited the coexpression of IgDR and phosphorylated Lck (p-Lck) and the expression levels of p-Lck, p-ZAP70, p-PI3K on CD4⁺ T cells, and decreased NF-κB nuclear translocation in Jurkat cells. Meanwhile, IgD-Fc-Ig downregulated the expression levels of CD40L on CD4⁺ T cells as well as CD40, CD86 on CD19⁺ B cells in RA patients and healthy controls. It also decreased the expression levels of CD40L on CD4⁺ T cells and CD40 on CD19⁺ B cells from spleens of collagen-induced arthritis (CIA) mice and reduced IL-17A level in mouse serum. Moreover, administration of IgD-Fc-Ig (1.625-13 mg/kg, iv, twice a week for 4 weeks) in CIA mice dose-dependently decreased the protein expression levels of CD40, CD40L, and IgD in spleens. IgD-Fc-Ig restrains T-cell activation through inhibiting IgD-IgDR-Lck-ZAP70-PI3K-NF-κB signaling, thus inhibiting B-cell activation. Our data provide experimental evidences for application of IgD-Fc-Ig as a highly selective T cell-targeting treatment for RA.

Keywords: CD19+ B cells; CD4+ T cells; IgD-Fc-Ig; immunoglobulin D; immunoglobulin D receptor; rheumatoid arthritis.

Fig. 1. Effects of IgD-Fc-Ig (DG) on the IgDR-p-Lck interaction on CD4⁺ T cells in healthy controls induced by IgD.

To obtain CD4⁺ T cells from PBMCs of healthy controls, CD4⁺ T cells were incubated with IgD (3 μg/mL), IgD-Fc-Ig (10 μg/mL), and 0.5 μM Lck inhibitor (A770041) for 24 h. a Confocal microscopy of CD4⁺ T cells stained for IgDR and p-Lck: red, staining of CD4⁺ T cells with labeled IgDR protein; green, staining of CD4⁺ T cells with labeled p-Lck protein; blue, staining of nuclei with the DNA-binding dye DAPI. b Colocalization rate of IgDR and p-Lck proteins (%). c Pearson’s correlation of IgDR and p-Lck proteins. d Overlap coefficient of IgDR and p-Lck proteins. Data were analyzed by the “Colocalization Module” of a Leica SP8 TCS STED ×3 microscope system and are expressed as the mean ± SEM (n = 5). **P < 0.01 vs. control (T cells), ^##P < 0.01 vs. IgD (3 μg/mL) stimulation group. e Coimmunoprecipitation analysis of IgDR-p-Lck interactions on CD4⁺ T cells in healthy controls. Data were expressed as the mean ± SEM (n = 5). *P < 0.05 vs. control (T cells), ^#P < 0.05 vs. IgD (3 μg/mL) stimulation group.

Fig. 2. Effects of IgD-Fc-Ig (DG) on the protein expression of p-Lck, ZAP70, p-ZAP70, PI3K, and p-PI3K.

Western blot analysis (a) of p-Lck (b), ZAP70, p-ZAP70 (c), PI3K, and p-PI3K (d) expression on CD4⁺ T cells from PBMCs of healthy controls. CD4⁺ T cells were incubated with IgD (3, 10 μg/mL), DG (10 μg/mL), and 0.5 μM Lck inhibitor A770041 for 24 h and lysed. Data were expressed as the mean ± SEM (n = 3). **P < 0.01 vs. control, ^##P < 0.01 vs. IgD (3 μg/mL) stimulation group.

Fig. 3. Image Stream fluorescence imaging of NF-κB nuclear translocation in Jurkat cells after IgD induction and incubation with IgD-Fc-Ig (DG) and the Lck inhibitor A770041.

Quantification of nuclear translocation using the similarity algorithm. a The region High Sim drawn on the dark field/DAPI similarity plot represents the region for positive image correlation for the similarity algorithm. This region is applied to the NF-κB/DAPI similarity plot. The percentages of cells that fall within the High Sim region are displayed on the right of each histogram. b Control unstimulated Jurkat cells (b1), Jurkat cells treated with 3 μg/mL IgD for 4 h (b2), and IgD-induced Jurkat cells incubated with 10 μg/mL DG (b3) and 0.5 μM Lck inhibitor A770041 (b4) for 4 h were probed for NF-κB expression and DAPI as described in the “Materials and Methods” and run on the Image Stream. The multispectral imaging system acquires up to six images per cell in three different imaging modes: bright field (morphology) (1) and fluorescence: Ch07-DAPI (purple) (2), Ch03-NF-κB (yellow-PE) (3), and NF-κB/DAPI composite images (4) for five representative (of 10,000 images) cells and are shown for each treatment group. Pixel intensities from the NF-κB images are plotted against the corresponding pixel intensities from the DAPI or dark-field images. c NF-κB nuclear translocation in Jurkat cells. The G-means represents the geometric means of the similarity of the nuclear/cytoplasmic NF-κB intensity ratio to DAPI intensity. Jurkat cells were either left unstimulated or stimulated with IgD 3 μg/mL. IgD-induced Jurkat cells were incubated with 10 μg/mL DG and 0.5 μM Lck inhibitor A770041. The results show the mean ± SEM of five independent experiments in which the similarity ratio was calculated from 10,000 cells in each independent experiment. **P < 0.01 vs. control, ^##P < 0.01 vs. IgD (3 μg/mL) stimulation group.

Fig. 4. Effects of IgD on CD4⁺ T-cell and CD19⁺ B-cell activities in healthy controls.

Obtained CD4⁺ T cells and CD19⁺ B cells from PBMCs of healthy controls. CD4⁺ T cells were incubated with IgD (10 μg/mL) cocultured with CD19⁺ B cells in vitro for 24 and 48 h, and CD19⁺ B cells were incubated with IgD (10 μg/mL) in vitro for 24 and 48 h. a, b After coculture, CD19⁺ B cells were obtained, and CCK-8 analysis of the effect of IgD-induced CD4⁺ T cells on the proliferation of CD19⁺ B cells was performed. Western blot analysis of CD40L expression on CD4⁺ T cells (c) and CD40 expression on CD19⁺ B cells (d) from PBMCs of healthy controls. CD4⁺ T cells were incubated with IgD (3, 10 μg/mL), cocultured with CD19⁺ B cells in vitro for 48 h, and lysed. Flow cytometry analysis of CD40L expression on CD4⁺ T cells (e) and CD40 expression on CD19⁺ B cells after coculture (f). Data were expressed as the mean ± SEM (n = 5). *P < 0.05, **P < 0.01 vs. control; ^##P < 0.01, ^###P < 0.001 vs. T cells + B cells.

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Fig. 6. Effects of IgD-Fc-Ig (DG) on CD40, CD40L, and IgD expression in the spleens of CIA mice in vivo.

CIA mice were treated with IgD-Fc-Ig, IgG₁-Fc, rhTNFR:Fc, and anti-mouse IgD antibodies by intravenous administration via the tail. Immunohistochemical analysis of CD40 (a), CD40L (b), and IgD (c) expression in the spleens of CIA mice. Data are expressed as the mean ± SEM (for each group, five mice). **P < 0.01 vs. normal, ^#P < 0.05 and ^##P < 0.01 vs. model.

Fig. 7. Effects of IgD-Fc-Ig (DG) on CD4⁺ T-cell and CD19⁺ B-cell actions in CIA mice in vitro.

To obtain CD4⁺ T cells and CD19⁺ B cells from the spleens of CIA mice, CD4⁺ T cells were incubated with IgD (3 μg/mL), DG (3, 10 μg/mL), 0.5 μM Lck inhibitor A770041, and IgG₁-Fc (10 μg/mL) in coculture with CD19⁺ B cells in vitro for 48 h. Western blot analysis of CD40L (a) and CD40 (b) expression on CD4⁺ T cells and CD19⁺ B cells from the spleens of CIA mice. After coculture, CD4⁺ T cells and CD19⁺ B cells were obtained and lysed. c ELISA analysis of IL-17A expression in CD4⁺ T-cell supernatant of CIA mice. Data are expressed as the mean ± SEM (both normal group and CIA group 3–5 mice). **P < 0.01 vs. control, ^#P < 0.05 and ^##P < 0.01 vs. IgD (3 μg/mL) stimulation group.

Fig. 8. Hypothetical schematic diagram of the IgD-Fc-Ig mechanism. IgD induces the abnormal activation of T cells by affecting IgDR-Lck-ZAP70-PI3K-NF-κB signaling in T cells, which can upregulate the expression of CD40L on CD4⁺ T cells.

The cocultivation of IgD-activated T cells can upregulate the expression of the costimulatory molecules CD40–CD40L and then promote the activation of B cells. IgD-Fc-Ig downregulated CD86 expression and IL-17A levels and decreased CD40 and CD40L expression. IgD-Fc-Ig restrains the activation of T cells by inhibiting IgD-IgDR-Lck-ZAP70-PI3K-NF-κB signaling, thus inhibiting the activation of B cells.