Aim Immunoglobulin D （IgD） is a surface immunoglobulin that is expressed as either membrane IgD（mIgD） or secreted IgD （sIgD）. Researchers have shown that sIgD is often elevated in patients with autoimmune diseas...Aim Immunoglobulin D （IgD） is a surface immunoglobulin that is expressed as either membrane IgD（mIgD） or secreted IgD （sIgD）. Researchers have shown that sIgD is often elevated in patients with autoimmune diseases. The possible roles of sIgD on the function of peripheral blood mononuclear cells （PBMCs） in rheumatoid arthritis （RA） are still unclear and few studies have been performed. The objective of this study was to investigate the abnormal level of immunoglobulin D （IgD） and the effects of it by binding its receptor （IgDR） on peripheral blood mononuclear cells （PBMCs） in rheumatoid arthritis （RA）. Methods Blood samples were obtained from 54 RA patients and 42 healthy controls. The levels of sIgD, human soluble receptor activator of nuclear factor-KB lig- and （sRANKL）, anti-cyclic citrullinated peptide （anti-CCP）, C-reactive protein （CRP） were determined in ser- um samples by ELISA. Rheumatoid factor （RF） was detected by quantitative nephelometry. Erythrocytes sedimen- tation rate （ESR） was tested by Westergren method. IgDR and mIgD were detected by using flow cytometry. After PBMCs were cultured and treated with different concentrations of human IgD. PBMCs proliferation were measured by CCK-8, inflammatory cytokine production were assessed by inflammation antibody array, T-/B- cell subsets and IgDR expression were tested by flow cytometry. Results A significantly higher level of sIgD, mIgD and IgDR were detected in RA patients compared with healthy controls. The concentrations of sIgD were positively correlated with sRANKL, rheumatoid factor and C-reactive protein in RA patients. Strikingly, IgD could enhance the prolifer- ation of PBMCs and induce IL-lα, IL-1β, TNF-α, IL-6 and production from PBMCs. Moreover, the percentage of activated T cell subsets （ CD4 ＋ CD69 ＋ , CD4 ＋ CD154 ＋ ） and activated B cell subsets （ CD19 ＋ CD23 ＋ , CD19 ＋ CD21 ＋ , CD19 ＋ IgD ＋ and CD19- CD138 ＋ ） were increased by IgD. The percentage of unactivated T cell subset （CD4 ＋ CD62L ＋ ） and immature B cell subset （ CD19 ＋ IgM ＋ IgD- ） were decreased by IgD in PBMCs. Further- more, the expressions of IgDR on T and B cells were significantly increased by treatment with IgD. Conclusion IgD enhanced the activation of PBMCs through stimulation of IgDR, which may contribute to RA pathogenesis. IgD represents a potentially novel immunotherapeutic target for the management of RA.展开更多
OBJECTIVE To observe whether human CD4^+T cells could be activated by immuno-globulin D(IgD) via IgD receptor(IgDR)-Lck.METHODS Human CD4^+T cells were purified from peripheral blood mononuclear cells(PBMCs) with micr...OBJECTIVE To observe whether human CD4^+T cells could be activated by immuno-globulin D(IgD) via IgD receptor(IgDR)-Lck.METHODS Human CD4^+T cells were purified from peripheral blood mononuclear cells(PBMCs) with microbeads.The viability of T cells were detected by CCK-8.The binding affinity and expression of IgDR on T cells were detected by flow cytometry.The protein expression of IgDR,Lck and P-Lck were analyzed by western blot.RESULTS IgD could concentration-dependent bind to IgDR on CD4^+T cells.The expression of IgDR was increased in response to treatment with IgD in a time-dependent and concentration-dependent manner.Stimulating by IgD resulted in enhanced phosphorylation of Lck compared with that in the medium control sample.The expression of Lck was not changed.As inhibitor of PTK,Herbimycin A or A770041,which combined with IgD could significantly inhibit phosphorylation of Lck(Tyr^(394)).The proliferation promoting effect of IgD was blocked by Herbimycin A or A770041.IgD could stimulate CD4^+T cell activation and proliferation through upregulating activating tyrosine residue of Lck(Tyr^(394)) phosphorylation.CONCLUSION These results demonstrate that IgD exaggerates CD4^+T cell activities,which may be through promoting Lck phosphorylation.展开更多
Aim To determine the effect of VIR576, a dimeric 20-mer peptide potently inhibits HIV-1 entry, on antigen-specific T cell and non-antigen-specific T cell activation. Methods In vitro T-cell proliferation assays were e...Aim To determine the effect of VIR576, a dimeric 20-mer peptide potently inhibits HIV-1 entry, on antigen-specific T cell and non-antigen-specific T cell activation. Methods In vitro T-cell proliferation assays were estalished to investigate the potential effect of FP16, VIR576 on the proliferation of A2b cells in response to MOG35-55. A fluorescence-based binding assay using Rhodamine （Rho）-conjugated VIR576 was estalished to e- valuate the potential interaction between VIR576 and TCR-TMD. Fluorescence confocal microscopy was used to to study whether VIR576 could colocalize with CD4 molecule in the CD4 ＋ T cell membrane to interact with TCR. Re- suits The effects of VIR576 on the proliferation of MOG-specific A2b T cells in response to the stimulation of MOG 35 -55 peptide wasevaluated. VIR576 itself could directly inhibit antigen-specific T-cell activation. Further studies confirmed that VIR576 also inhibited the proliferation of splenocytes and primary CD4 ＋ CD25- T cells iso- lated from the spleens of DOll. 10 OVA Tg mice in response to OVA stimulation in vitro. However, VIR576 had no effect on the proliferation of normal mouse splenocytes and T lymphocytes stimulated with Con A or anti-CD3 anti- body. The FRET assay confirmed that VIR576 effectively binds to the core peptide （CP） , corresponding to the N- terminal 9-residue region of TCR-TMD. Confocal microscopy revealed that VIR576 colocalizes with CD4 on the ac- tivated CD4 ＋ T-cell membrane, particularly within the activation cluster including re-assembled CD4 and TCR mol- ecules. Conclusion These results suggest that VIR576 is effective in suppressing antigen-specific T-cell activa- tion, but it has no effect on non-specific T-cell proliferation, and VIR576 has the ability to down-regulate antigen- specific T-cell activation by interaction with TCR transmembrane domain.展开更多
文摘Aim Immunoglobulin D （IgD） is a surface immunoglobulin that is expressed as either membrane IgD（mIgD） or secreted IgD （sIgD）. Researchers have shown that sIgD is often elevated in patients with autoimmune diseases. The possible roles of sIgD on the function of peripheral blood mononuclear cells （PBMCs） in rheumatoid arthritis （RA） are still unclear and few studies have been performed. The objective of this study was to investigate the abnormal level of immunoglobulin D （IgD） and the effects of it by binding its receptor （IgDR） on peripheral blood mononuclear cells （PBMCs） in rheumatoid arthritis （RA）. Methods Blood samples were obtained from 54 RA patients and 42 healthy controls. The levels of sIgD, human soluble receptor activator of nuclear factor-KB lig- and （sRANKL）, anti-cyclic citrullinated peptide （anti-CCP）, C-reactive protein （CRP） were determined in ser- um samples by ELISA. Rheumatoid factor （RF） was detected by quantitative nephelometry. Erythrocytes sedimen- tation rate （ESR） was tested by Westergren method. IgDR and mIgD were detected by using flow cytometry. After PBMCs were cultured and treated with different concentrations of human IgD. PBMCs proliferation were measured by CCK-8, inflammatory cytokine production were assessed by inflammation antibody array, T-/B- cell subsets and IgDR expression were tested by flow cytometry. Results A significantly higher level of sIgD, mIgD and IgDR were detected in RA patients compared with healthy controls. The concentrations of sIgD were positively correlated with sRANKL, rheumatoid factor and C-reactive protein in RA patients. Strikingly, IgD could enhance the prolifer- ation of PBMCs and induce IL-lα, IL-1β, TNF-α, IL-6 and production from PBMCs. Moreover, the percentage of activated T cell subsets （ CD4 ＋ CD69 ＋ , CD4 ＋ CD154 ＋ ） and activated B cell subsets （ CD19 ＋ CD23 ＋ , CD19 ＋ CD21 ＋ , CD19 ＋ IgD ＋ and CD19- CD138 ＋ ） were increased by IgD. The percentage of unactivated T cell subset （CD4 ＋ CD62L ＋ ） and immature B cell subset （ CD19 ＋ IgM ＋ IgD- ） were decreased by IgD in PBMCs. Further- more, the expressions of IgDR on T and B cells were significantly increased by treatment with IgD. Conclusion IgD enhanced the activation of PBMCs through stimulation of IgDR, which may contribute to RA pathogenesis. IgD represents a potentially novel immunotherapeutic target for the management of RA.
基金supported by National Natural Science Foundation of China(81330081,81673444,81603121)BSKY(XJ201629 and XJ201630) from Anhui Medical University
文摘OBJECTIVE To observe whether human CD4^+T cells could be activated by immuno-globulin D(IgD) via IgD receptor(IgDR)-Lck.METHODS Human CD4^+T cells were purified from peripheral blood mononuclear cells(PBMCs) with microbeads.The viability of T cells were detected by CCK-8.The binding affinity and expression of IgDR on T cells were detected by flow cytometry.The protein expression of IgDR,Lck and P-Lck were analyzed by western blot.RESULTS IgD could concentration-dependent bind to IgDR on CD4^+T cells.The expression of IgDR was increased in response to treatment with IgD in a time-dependent and concentration-dependent manner.Stimulating by IgD resulted in enhanced phosphorylation of Lck compared with that in the medium control sample.The expression of Lck was not changed.As inhibitor of PTK,Herbimycin A or A770041,which combined with IgD could significantly inhibit phosphorylation of Lck(Tyr^(394)).The proliferation promoting effect of IgD was blocked by Herbimycin A or A770041.IgD could stimulate CD4^+T cell activation and proliferation through upregulating activating tyrosine residue of Lck(Tyr^(394)) phosphorylation.CONCLUSION These results demonstrate that IgD exaggerates CD4^+T cell activities,which may be through promoting Lck phosphorylation.
文摘Aim To determine the effect of VIR576, a dimeric 20-mer peptide potently inhibits HIV-1 entry, on antigen-specific T cell and non-antigen-specific T cell activation. Methods In vitro T-cell proliferation assays were estalished to investigate the potential effect of FP16, VIR576 on the proliferation of A2b cells in response to MOG35-55. A fluorescence-based binding assay using Rhodamine （Rho）-conjugated VIR576 was estalished to e- valuate the potential interaction between VIR576 and TCR-TMD. Fluorescence confocal microscopy was used to to study whether VIR576 could colocalize with CD4 molecule in the CD4 ＋ T cell membrane to interact with TCR. Re- suits The effects of VIR576 on the proliferation of MOG-specific A2b T cells in response to the stimulation of MOG 35 -55 peptide wasevaluated. VIR576 itself could directly inhibit antigen-specific T-cell activation. Further studies confirmed that VIR576 also inhibited the proliferation of splenocytes and primary CD4 ＋ CD25- T cells iso- lated from the spleens of DOll. 10 OVA Tg mice in response to OVA stimulation in vitro. However, VIR576 had no effect on the proliferation of normal mouse splenocytes and T lymphocytes stimulated with Con A or anti-CD3 anti- body. The FRET assay confirmed that VIR576 effectively binds to the core peptide （CP） , corresponding to the N- terminal 9-residue region of TCR-TMD. Confocal microscopy revealed that VIR576 colocalizes with CD4 on the ac- tivated CD4 ＋ T-cell membrane, particularly within the activation cluster including re-assembled CD4 and TCR mol- ecules. Conclusion These results suggest that VIR576 is effective in suppressing antigen-specific T-cell activa- tion, but it has no effect on non-specific T-cell proliferation, and VIR576 has the ability to down-regulate antigen- specific T-cell activation by interaction with TCR transmembrane domain.
