CP-25 Attenuates the Activation of CD4+ T Cells Stimulated with Immunoglobulin D in Human

Abstract

Researchers have shown that the level of immunoglobulin D (IgD) is often elevated in patients with autoimmune diseases. The possible roles of IgD on the function of human T cell activation are still unclear. Paeoniflorin-6′-O-benzene sulfonate (code: CP-25), the chemistry structural modifications of paeoniflorin, was a novel drug of anti-inflammation and immunomodulation. The aims of this study were to determine if human CD4+ T cells could be activated by IgD via the IgD receptor (IgDR)-Lck pathway and whether the novel compound CP-25 could affect the activation of T cells by regulating Lck. Human CD4+ T cells were purified from peripheral blood mononuclear cells using microbeads. T cell viability and proliferation were detected by Cell Counting Kit-8 and CFSE Cell Proliferation Kit. Cytokines secreted by T cells were assessed with the Quantibody Human Inflammation Array. The binding affinity and expression of IgDR on T cells were detected by flow cytometry, and protein expression of IgDR, Lck, and P-Lck were analyzed by western blot. IgD was shown to bind to IgDR on CD4+ T cells in a concentration-dependent manner and stimulate the activation and proliferation of these cells by enhancing phosphorylation of the activating tyrosine residue of Lck (Tyr394). CP-25 inhibited the IgD-stimulated activation and proliferation of CD4+ T cells, as well as the production of inflammatory cytokines; it was thus suggested that this process might be related to the downregulation of Lck (Tyr394) phosphorylation. These results demonstrate that IgD amplifies the activation of CD4+ T cells, which could be mediated by Lck phosphorylation. Further, CP-25, via its ability to modulate Lck, is a novel potential therapeutic agent for the treatment of human autoimmune diseases.

Regulation of T Cell Activities in Rheumatoid Arthritis by the Novel Fusion Protein IgD-Fc-Ig

“…Activated T cells, together with high expression of CD69 and CD154 (CD40L), play vital roles in RA-associated inflammation by producing inflammatory cytokines, which induce synovium inflammation and immune responses (33,34). We previously demonstrated that IgD increases the percentage of CD69 + and CD154 + cells, which might contribute to the elevated IgDR expression and Lck tyrosine phosphorylation (19). The specific blocking of the activated T cells induced by IgD suggests that IgD-Fc-Ig may prevent T cell over-proliferation and activation through selectively targeting IgDR.…”

“…Save for the control group, cells were stimulated with 3 µg/mL of IgD or anti-CD3/CD28 (0.4 µg/mL) in combination with different concentrations of IgD-Fc-Ig fusion protein (1, 3, and 10 µg/mL) for 48 h at 37 • C. A Lck inhibitor A770041 group was used as a positive control, while the IgG 1 -Fc protein treatment group was used as a negative control. After treatment, a Cell Counting Kit-8 (CCK-8) was used to evaluate cell proliferation using stimulation indices according to published protocols (17,19).…”

“…Our previous studies have shown that serum levels of sIgD and IgDR expression on T/B cells in RA patients were higher than in healthy controls (17,18). IgD may also be involved in enhancing the proliferation of peripheral blood mononuclear cells (PBMCs) in RA patients, inducing T cell activation and increasing IgDR expression (19). The IgD-induced IgDR expression levels in T cells was found to be higher than in B cells both in RA patients and healthy controls (17), which shifted our focus to IgD function in T cells, specifically in RA.…”

Immunoglobulin D (IgD) and IgD receptor expression in diffuse large B-cell lymphoma

“…Binding between IgD and IgDR was clearly concentration-dependent. We have estimated the IgD binding affinity by performing a fluorescence-based receptor binding assay, with high-affinity (KD value was 0.216 nmol/L) [17]. We also identified IgDbinding IgDR as a ∼70 kDa protein complex that was detected on rheumatoid arthritis (RA)-fibroblast-like synoviocytes (FLSs) by using flow cytometry (FCM), confocal laser scanning microscopy and western blot [18].…”

“…Previous reports also suggested significantly high levels of sIgD, mIgD, and IgDR in RA patients. In human, treatment with IgD could enhance the activation of peripheral blood mononuclear cells (PBMCs) [15], and amplify the activation of CD4 + T cells, which could be mediated by Lck phosphorylation [17]. In Daudi cell line, which is the human Burkitt lymphoma B-cell line, we have investigated that human IgD antibody could induce cell proliferation potentially by activating the putative IgDR to initiate tyrosine phosphorylation signaling cascade that accelerated G1/S transition [19].…”