Cheng-shan Guo

The effects of BAFF and BAFF-R-Fc fusion protein in immune thrombocytopenia

Elevated level of B-cell activating factor (BAFF) has been implicated in the pathogenesis of some autoimmune diseases. Blockade of receptor and ligand binding by decoy receptor has demonstrated a clinical benefit in both oncologic and immunologic diseases. In this report, we have detected plasma BAFF and BAFF mRNA expression in immune thrombocytopenia (ITP) patients by enzyme-linked immunosorbent assay (ELISA) and real-time quantitative reverse-transcription polymerase chain reaction (RT-PCR). The effects of recombinant human BAFF (rhBAFF) and BAFF-R-Fc fusion protein (BR3-Fc) on B cells, T cells, platelets, secretion of interferon gamma (IFNgamma), and interleukin-4 (IL-4) were measured by flow cytometry and ELISA. Patients with active disease had higher levels of plasma BAFF and BAFF mRNA than patients in remission and controls. In in vitro assays, rhBAFF promoted the survival of CD19(+) and CD8(+) cells, and increased the apoptosis of platelets and the secretion of IFN-gamma. BR3-Fc successfully corrected the effects of rhBAFF on lymphocytes, platelets, and cytokines. These findings suggest that BAFF may play a pathogenic role in ITP by promoting the survival of CD19(+) and CD8(+) cells, and increasing the apoptosis of platelets and the secretion of IFN-gamma. Blockade of BAFF by BR3-Fc might be a promising therapeutic approach for ITP.

De novo induction of platelet-specific CD4+CD25+ regulatory T cells from CD4+CD25- cells in patients with idiopathic thrombocytopenic purpura

CD4(+)CD25(+) regulatory T cells (Treg) play the critical role in maintenance of peripheral immune tolerance. However, the numbers of naturally occurring Treg (nTreg) that can be isolated from periphery are far too small to be clinically effective. The isolation and expansion of nTreg for treatment of autoimmune diseases encounter great difficulties. Whether autoantigen-specific Treg could be converted from CD4(+)CD25(-) T cells in patients with autoimmune diseases has not been reported. Here, we demonstrated that platelet glycoprotein (GP)-specific induced Treg (GP-iTreg) could be generated de novo from nonregulatory CD4(+)CD25(-)CD45RA(+) cells in patients with idiopathic thrombocytopenic purpura and induced both antigen-specific and linked suppression. GP-iTreg mediated regulatory effects via modulating the T cell-stimulatory capacity of dendritic cells. By investigating the gene expression profile of iTreg-modulated dendritic cells, we provided a genome-wide assessment of the changes induced by antigen-specific iTreg and identified that the Toll-like receptor, Notch and transforming growth factor-beta signaling pathways were related to the GP-specific tolerance, with the Toll-like receptor pathway being dominant. The findings in patients with idiopathic thrombocytopenic purpura will facilitate our understanding of the mechanisms of induction and maintenance of autoantigen-specific tolerance and highlight the considerable potential of antigen-specific iTreg for targeted immunotherapy in human auto-immune diseases.

High-dose dexamethasone shifts the balance of stimulatory and inhibitory Fcγ receptors on monocytes in patients with primary immune thrombocytopenia

The human Fcγ receptor (FcγR) system is composed of 2 opposing families, the activating FcγRs (FcγRI, FcγRIIa, and FcγRIII) and the inhibitory FcγR (FcγRIIb). The disturbed balance of the activating and inhibitory FcγRs has been implicated in the pathogenesis of many autoimmune diseases. In this study, the expression of FcγRs on monocytes was determined in 23 patients with primary immune thrombocytopenia (ITP) before and after high-dose dexamethasone (HD-DXM) treatment. The FcγRI expression was significantly higher in ITP patients and decreased after HD-DXM treatment. The ratio of FcγRIIa/IIb mRNA expression on monocytes was significantly higher in untreated patients than in healthy controls. After HD-DXM therapy, the ratio decreased and the increased expression of FcγRIIb mRNA and protein coincided with a remarkable decrease in the expression of FcγRIIa, FcγRI, and monocyte phagocytic capacity. There was no significant difference in FcγRIII expression on monocytes between patients and controls. In vitro cell-culture experiments showed that DXM could induce FcγRIIa and FcγRIIb expression in monocytes from ITP patients, with FcγRIIb at higher amplitudes. These findings suggested that the disturbed FcγR balance might play a role in the pathogenesis of ITP, and that HD-DXM therapy could shift monocyte FcγR balance toward the inhibitory FcγRIIb in patients with ITP.

Association of autoantibody specificity and response to intravenous immunoglobulin G therapy in immune thrombocytopenia: a multicenter cohort study

Immune thrombocytopenia (ITP) is a common autoimmune bleeding disorder, in which platelet glycoprotein (GP)IIb–IIIa and GPIb–IX are the two most frequently targeted autoantigens. Our previous studies in animal models of ITP demonstrated that intravenous immunoglobulin G (IVIG) could protect against anti‐GPIIb–IIIa autoantibody‐mediated thrombocytopenia but failed to ameliorate ITP induced by most anti‐GPIb–IX autoantibodies.

High-Dose Dexamethasone Inhibits BAFF Expression in Patients with Immune Thrombocytopenia

IntroductionB-cell activating factor belonging to the TNF family (BAFF) is elevated in several autoimmune diseases including immune thrombocytopenia (ITP). High-dose dexamethasone (HD-DXM) has shown its clinical efficacy in ITP patients. Materials and MethodsThe plasma BAFF concentration and BAFF mRNA were measured in ITP patients before and after oral administration of 40 mg/day DXM for four consecutive days by enzyme-linked immunosorbent assay (ELISA) and real-time quantitative PCR. Moreover, we evaluated the effects of DXM on BAFF expression and proliferation of lymphocytes by ELISA, real-time quantitative PCR and cell proliferation respectively in in vitro experiment.ResultsBoth plasma BAFF concentration and BAFF mRNA were significantly increased in active ITP patients at pretherapy when compared with controls (P < 0.001). After 4-day treatment with HD-DXM, the BAFF and BAFF mRNA were decreased, and lower than that for controls. In in vitro assays, we found DXM-inhibited BAFF, IFN-γ expression, and the proliferation of lymphocytes in a dose-dependent manner.ConclusionThese results suggest that BAFF expression is increased in ITP patients with active disease, and DXM is an effective inhibitor of BAFF production. As immunosuppressant, DXM may play its role in ITP treatment partly through regulating BAFF expression.

Modulation of immune response with cytotoxic T‐lymphocyte‐associated antigen 4 immunoglobulin‐induced anergic T cells in chronic idiopathic thrombocytopenic purpura2

Summary.  Background: Platelet glycoprotein (GP)‐reactive CD4+ T cells are essential for the stimulation and maintenance of antiplatelet autoantibody production in chronic idiopathic thrombocytopenic purpura (ITP). Blocking costimulatory signals could result in platelet‐specific T‐cell anergy.

A multicenter randomized open-label study of rituximab plus rhTPO vs rituximab in corticosteroid-resistant or relapsed ITP

This study aimed to compare the efficacy and safety of rituximab (RTX) plus recombinant human thrombopoietin (rhTPO) with RTX alone in patients with immune thrombocytopenia (ITP) who had failed to respond to corticosteroids or relapsed. Recruited patients were randomized at a ratio of 2:1 into 2 groups: the combination group (RTX + rhTPO, n = 77) and the monotherapy group (RTX, n = 38). Overall response was achieved in 79.2% of patients in the combination group vs 71.1% in the monotherapy group (P = .36), and the complete response (CR) rate was 45.4% in the combination group compared with 23.7% in the monotherapy group (P = .026). The combination group had significantly shorter time to response (TTR; median and range, 7 and 4-28 days) compared with the monotherapy group (28 and 4-90 days) (P < .01). There was no difference between these 2 groups in terms of the long-term response (P = .12). Our findings demonstrated that the combination of RTX and rhTPO significantly increased the CR rate and shortened TTR compared with RTX monotherapy in the treatment of corticosteroid-resistant or relapsed ITP but failed to show a beneficial effect on the long-lasting response. This study is registered at www.clinicaltrials.gov as #NCT01525836.

Decreased expression of interleukin-27 in immune thrombocytopenia

Primary immune thrombocytopenia (ITP) is an immune‐mediated disorder in which disturbed cytokine profiles have been found. Interleukin‐27 (IL27) has been shown to bear both proinflammatory and anti‐inflammtory effects. In the present study, plasma levels of IL27, interferon gamma (IFNG), IL4, and IL17A were determined by enzyme‐linked immunosorbent assay in 23 active ITP patients, 20 patients in remission and 20 healthy controls. mRNA expression levels of IL27, EBI3, IL27 receptor (IL27RA), IL17A and RAR‐related orphan receptor C (RORC) were determined by real‐time quantitative polymerase chain reaction. Significantly lower levels of plasma IL27, IL4, mRNA expression of IL27, EBI3 and higher levels of plasma IFNG as well as mRNA expression of IL17A, RORC were observed in active ITP patients compared with healthy controls or patients in remission. No statistical difference was found in IL27RA mRNA expression or plasma IL17A levels among active ITP patients and controls. A negative correlation was found between the IL27 and RORC mRNA expression levels in active ITP patients. Our data demonstrated that active ITP patients had decreased plasma and mRNA expression levels of IL27, suggesting that it might be involved in the pathophysiological process of ITP.

Thalidomide corrects impaired mesenchymal stem cell function in inducing tolerogenic DCs in patients with immune thrombocytopenia

Thalidomide (THD) is an immunomodulatory agent used to treat immune-mediated diseases. Immune thrombocytopenia (ITP) is an autoimmune disorder in which impaired mesenchymal stem cells (MSCs) are potentially involved. We demonstrated that MSCs in ITP patients had reduced proliferative capacity and lost their immunosuppressive function, which could be corrected with THD treatment. According to the gene profile, the downregulation of caspase-8 and caspase-10, and upregulation of oct3/4 and tgf-β1, may be associated with THD modulation. Dendritic cells (DCs) played an important role in mediating the inhibitory activity of MSCs. To study the functional alteration of DCs elicited by MSCs, we sorted DCs after incubation with MSCs and performed T-lymphocyte reaction assays. The THD-modulated MSCs from ITP patients induced mature DCs to become tolerogenic DCs, whereas unmodulated MSCs had no effect. The induction of tolerogenicity in DCs by MSCs was dependent on the expression of TIEG1 in DCs. The study reveals the inability of MSCs from ITP patients to induce tolerogenic ability in DCs. THD could restore the regulatory effect of MSCs on DCs. These findings will help us understand the pathogenesis of ITP, and with appropriate safeguards, THD may benefit patients with ITP.

Decreased indoleamine 2,3-dioxygenase expression in dendritic cells and role of indoleamine 2,3-dioxygenase-expressing dendritic cells in immune thrombocytopenia

Indoleamine 2,3-dioxygenase (IDO) expression in dendritic cells (DCs) can induce or maintain peripheral immune tolerance. Impaired IDO-mediated tryptophan catabolism has been observed in autoimmune diseases. In order to investigate the effects of IDO-mediated tryptophan catabolism and IDO-expressing DCs in immune thrombocytopenia, the concentrations of kynurenine were detected by high-pressure liquid chromatography. The expressions of IDO were analyzed by flow cytometry and western blot analysis. The effects of IDO+ DCs stimulated with CTLA-4-Ig on T cells proliferation and activation, lymphocyte apoptosis, and Tregs were measured by flow cytometry. We found that the expression of IDO in DCs of immune thrombocytopenia (ITP) patients was significantly decreased. CTLA-4-Ig significantly increased the expression of functional IDO in DCs of ITP patients. IDO+ DCs stimulated with CTLA-4-Ig suppressed T cells proliferation and activation, promoted lymphocyte apoptosis, and increased the percentage of Tregs. These results suggest that decreased IDO expression in DCs may play a critical role in ITP. CTLA-4-Ig successfully corrected the disorder of IDO expression in ITP. IDO+ DCs stimulated with CTLA-4-Ig inhibited immune responses by an IDO-dependent mechanism. Increasing the expression and activity of IDO in DCs might be a promising therapeutic approach for ITP.