Induction Of Platelet-Specific T Cell Tolerance In Chronic Idiopathic Thrombocytopenic Purpura

Chronic idiopathic thrombocytopenic purpura (ITP) is an immune-mediated disorder in which platelets are prematurely destroyed in the reticuloendothelial system by platelet autoantibodies. Most adult patients have a prolonged history of mucocutaneous bleeding. A number of features suggest this destruction is immune-mediated and that it may involve in not only the destruction of the platelet, but also inhibition of platelet release by the megakaryocyte. Platelets from 50%～60% of ITP patients are coated with IgG antibodies that recognize one or more platelet surface glycoproteins. Approximately 75% of the antigenic determinants lie on the glycoprotein (GP) complex GPIIb/IIIa and Ib/IX. The antibody-coated platelets are destructed either by phagocytic cells or by autoantibody-induced activation of complement. However, to date no drug explored has shown an antigen-specific immunological mechanism where only the platelet-antigen-autoreactive T or B cells are targeted.Although it is now evident that autoantibody production by B-cells requires autoantigen-specific T-cell help, there is also increasing evidence that a variety of subset abnormalities within the T-cell population play an important role in the pathogenesis of this disorder. There is evidence that autoreactive CD4~+ T cells to GPIIb-IIIa exist in ITP patients. These T-cells are considered pathogenic because they help B-cells produce antibodies that bind to normal platelet surfaces. Therefore inactivation of GP-reactive CD4~+ T cells may be a selection of therapy for suppressing antiplatelet antibody production in ITP.Part I Modulation of immune response with CTLA4-Ig-induced anergic T cells in chronic idiopathic thrombocytopenic purpuraObjective: Lack of co-stimulation leads to a state of functional unresponsiveness that has been termed 'anergy'. When co-stimulatory signals are present, T cells proliferate and proceed to make a full-fledged immune response. By contrast, when co-stimulatory signals are absent, T cells become anergic-unresponsive to secondary stimulation, even if this includes both TCR and co-stimulatory signals.The goal in treating autoimmune and transplant patients is to re-establish specific tolerance to self-antigen, without causing generalized immunosuppression. Many of the strategies attempted have been shown to work at least in part through co-stimulatory blockade and the resulting development of anergy. In the present study we sought to explore the mechanisms whereby anergic T cells suppress autoreactive CD4~+ T cells in patients with ITP.Methods:* Preparation of peripheral blood mononuclear cells* GPIIb/IIIa- and GPIb/IX-reactive T cell lines* Generation of DCs and characterization of T-cell stimulating capacity of DCs from ITP patients* Induction of angeric T cells* Anergic T cells suppression assays* Cytokines assays* Transwell experiments* Statistical analysisResults: Our studies demonstrated that the anergized GP-specific T cells have profound effects on both GP-specific T cell proliferation and antibody production. The GP-specific anergic T cells exerted their suppressive effects in a cell-contact-dependent but IL-10- or TGF-β-independent manner. The GP-specific anergic T cells were not constitutively suppressive but required specific antigen stimulation to make DCs tolerogenic. The anergic T cell-modulated DCs could induce the autoreactive T cells tolerant, which was not restricted to T cells of the same specificity. The GP-specific anergic T cells could modulate the DCs to shift into a pathway of tolerogenic phenotype and function.Conclusion: Our studies demonstrate the efficacy of CTLA4-Ig in suppressing the pathologic autoimmune responses in ITP. These findings provide new insights into the underlying mechanisms.Part II CD4+CD25+ regulatory T cells induced from CD4+CD25- cells display platelet-glycoprotein specific suppressor activity in chronic idiopathicthrombocytopenic purpuraObjective: CD4+CD25+ regulatory T cells (Treg) play a critical role in maintenance of peripheral immune tolerance. And ITP is associated with decreased numbers and function of CD4+CD25+ naturally occurring regulatory T cells (nTreg). Therefore, we sought to induce platelet GP-specific CD4+CD25+ iTreg (GP-iTreg) from CD4+CD25- T cells in patients with chronic ITP, explore the molecular mechanisms by which the iTreg exert their suppressive effects and form a base for developing protocols to induce and expand antigen-specific Treg ex vivo to control and reverse the course of autoimmune diseases.Methods:* Purification of GP-reactive T cells* Generation of DCs from ITP patients* Induction of GP-specific regulatory T cells* T cell suppression assays* Measurement of stimulatory capacity of GP-iTreg-modulated DCs* Cytokine assays * Transwell experiments* Microarray analysis: RNA isolation; Labeling and hybridizations: Microarray data analysis.* Quantitative RT-PCR* Statistical analysisResulsts: We demonstrated the de novo generation of GP-specific CD4+CD25+ induced Treg (GP-iTreg) from CD4+CD25- cells in ITP. These GP-iTreg could render autologous GP-reactive T cells anergic. They were not constitutively suppressive but required specific antigen stimulation. Their functional suppression was not restricted to T cells of the same specificity, but also T cells specific for a different antigen, leading to both antigen-specific and linked suppression. Following exposure to GP-iTreg, dendritic cells (DCs) were rendered tolerogenic, suggesting that GP-iTreg mediate regulatory effects via modulating the T-cell-stimulatory capacity of DCs. By investigating the gene expression profile of iTreg-modulated DCs, we identified that the TLR, Notch, IFN-gamma and TGF-beta signaling pathways were related to the GP-specific tolerance induced by iTreg, with the TLR pathway being dominant.Conclusion: These findings facilitate our insights into the tolerizing mechanism of iTreg and highlight the considerable potential of GP-iTreg for targeted immunotherapy in patients with chronic ITP.