Cells In Immune Thrombocytopenia Patients Research Articles

Nicotinamide enhances Treg differentiation by promoting Foxp3 acetylation in immune thrombocytopenia.

Imbalanced nicotinamide adenine dinucleotide (NAD+) homeostasis has been reported in multiple autoimmune diseases and supplementation with NAD+ precursors has consistently demonstrated positive therapeutic benefits for these conditions. Immune thrombocytopenia (ITP) is an acquired autoimmune disease, in which the decreased number and impaired function of regulatory T cells (Tregs) contribute to the main pathogenesis. Here we found NAD+ level was decreased in the plasma and CD4+ T cells of ITP patients. Supplementation with NAD+ precursor nicotinamide (NAM), but not nicotinamide mononucleotide (NMN), increased Treg frequency and ameliorated thrombocytopenia in an ITP murine model. Moreover, whilst both NAM and NMN restored cytosolic NAD+ level in the CD4+ T cells from ITP patients, only NAM promoted Treg differentiation. Mechanistically, Sirtuin1 (Sirt1), a major consumer of NAD+, was highly expressed in the CD4+ T cells of ITP patients, potentially contributing to the low level of NAD+. NAM, which could act as Sirt1 inhibitor, promoted Foxp3 acetylation and stability in induced Tregs derived from naïve CD4+ T cells of ITP patients. These findings suggest that NAM holds promise as a novel therapeutic strategy for restoring immune balance in ITP.

Analysis of Frequencies and Subsets of Peripheral Helper T Cells in Patients with Immune Thrombocytopenia

To investigate the frequencies and subset distribution of peripheral helper (Tph) T cells in patients with immune thrombocytopenia (ITP), and explore the pathogenesis of ITP. A total of 25 newly diagnosed ITP patients treated in The Second Affiliated Hospital of Dalian Medical University from January to December 2022 were selected, and 25 healthy volunteers (age- and sex-matched) were recruited as the control group. Flow cytometry was used to detect the subsets of CD4+ T cells and Tph cells. The frequency of effector memory (CCR7- CD45RO+CD4+) T cells in ITP patients was significantly higher than that in healthy controls(P < 0.05). The frequency of Tph cells in ITP patients was also significantly higher than that in healthy controls (P < 0.001), and most of the Tph cells in ITP patients were effector memory T cells. Furthermore, the expressions of T-cell costimulatory molecules in Tph cells, including ICOS and CD84, were similar to those in follicular helper T(Tfh) cells. CXCR3 - CCR6 -Tph (Tph2) subgroup was dominant in Tph cells, but the frequency of CXCR3+CCR6- Tph (Tph1) cells in ITP patients was much higher than that in healthy controls (P < 0.05). Tph cells, especially Tph1 cells, were abnormally expanded in ITP patients, which may be a potential etiology of ITP.

Identification and validation of key regulating circRNAs in Immune Thrombocytopenia by circRNAs sequencing

Dysfunction of T cells is a causative factor in Immune Thrombocytopenia (ITP), an autoimmune disorder. Circular RNAs (circRNAs), which have been associated with the pathophysiology of various immunological conditions, are of particular interest. Our study aimed to identify pivotal regulatory circRNAs within the peripheral T cells of ITP patients. We utilized circRNA sequencing to discern differences in circRNA expression between the ITP cohort and healthy controls. We identified 606 upregulated and 719 downregulated circRNAs. Subsequent Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were conducted to scrutinize the parent genes of these differentially expressed circRNAs, revealing their significant involvement in metabolic processes and T cell receptor signaling pathways. Following a rigorous selection process that included DEG analysis, KEGG, and GO pathways analysis, along with an assessment of the potential roles of their parent genes, five top differentially expressed circRNAs were subjected to further validation via quantitative Polymerase Chain Reaction (RT-qPCR). Specifically, in the peripheral T cells of ITP patients, hsa_circ_0008866 (TAOK1), hsa_circ_0006856 (MAP3K5), and hsa_circ_0007444 (RHOBTB3) emerged as key regulatory circRNAs. The potential miRNA targets of these circRNAs were predicted employing miRanda, RNAhybrid, and TargetScan algorithms.

The miR-641-STIM1 and SATB1 axes play important roles in the regulation of the Th17/Treg balance in ITP

Immune thrombocytopenia (ITP) is an autoimmune disease caused by T-cell dysfunction. Recently, several studies have shown that a disturbed Th17/Treg balance contributes to the development of ITP. MicroRNAs (miRNAs) are small noncoding RNA moleculesthat posttranscriptionally regulate gene expression. Emerging evidences have demonstrated that miRNAs play an important role in regulating the Th17/Treg balance. In the present study, we found that miR-641 was upregulated in ITP patients. In primary T cells, overexpression of miR-641 could cause downregulation of its target genes STIM1 and SATB1, thus inducing a Th17 (upregulated)/Treg (downregulated) imbalance. Inhibition of miR-641 by a miR-641 sponge in primary T cells of ITP patients or by antagomiR-641 in an ITP murine model could cause upregulation of STIM1 and SATB1, thus restoring Th17/Treg homeostasis. These results suggested that the miR-641-STIM/SATB1 axis plays an important role in regulating the Th17/Treg balance in ITP.

Gpibα Caar-T Cells Induce Autoreactive B Cells Elimination to Treat Immune Thrombocytopenia

Background: Immune thrombocytopenia (ITP) is a bleeding disorder caused by autoantibodies against platelet glycoproteins (GPs). A key attempt to treat ITP is to eliminate the autoreactive B cells. Here, we proposed a new strategy for B cell-depletion therapy of ITP, chimeric autoantibody receptors T cells (CAAR-T), which could trap autoreactive B cells and perform specific killing. GPIbα is a significant autoantigen found in ITP patients and indicates a poor response to standard immunosuppressive treatments. We developed GPIbα-based CAARs in this study. Aim: To precisely eliminate the autoreactive B cells to treat ITP. Methods: We constructed various lengths of the native GPIbα ectodomain into the ligand-binding domain of a second-generation CAR structure and expressed the CAAR structure on the HEK293 cell surface to verify its conformational epitope. We immunized BALB/C mice with human platelet lysates to obtain anti-GPIbα hybridomas and performed a binding assay to confirm selected hybridoma antibodies' binding ability to GPIbα CAAR. We performed cytotoxicity assays to assess GPIbα CAAR-T-cell selective cytolysis against anti-GPIbα B cells in vitro and developed a xenograft model to assess the cytolytic efficiency and security of GPIbα CAAR-T cells in vivo. A plasma antibody binding assay was used to test GPIbα CAAR-T cells' ability in reaction with native GPIbα autoantibodies from ITP patients. We applied an enzyme-linked immunospot (ELISpot) assay to verify GPIbα CAAR-T cells' potential in eradicating autoreactive B cells of ITP patients. Results: We successfully expressed GPIbα CAAR structure on the cell surface and proved that the conformational epitope of GPIbα CAAR is consistent with that of the native human platelet GPIbα. The Schematic of GPIbα CAAR structure is presented in Figure 1A. By immunizing BALB/C mice with human platelet lysates, we obtained four anti-GPIbα hybridomas with different CAAR binding sites. Vitro cytotoxicity assays confirmed GPIbα CAAR-T-cell selective cytolysis of anti-GPIbα hybridoma cells. In a hybridoma xenograft model, we verified that the CAAR-T cells could persist and selectively lyse autoreactive B cells and subsequently reduce autoantibody titers without apparent off-target organ toxicity. Through the plasma antibody binding assay, we demonstrated that GPIbα CAAR could react with sera from ITP patients with anti-GPIbα antibodies (Figure 1B). ELISpot analysis showed that GPIbα CAAR-T cells successfully eliminated anti-GPIbα B cells from a patient with refractory ITP, and normal B cells were not destroyed compared to anti-CD19 CAR-T cells. Conclusion: Our study provides a proof of concept for CAAR-T cell therapy to eradicate autoimmune B cells while sparing healthy B cells, which would compensate for the defects of traditional B cell depletion therapy in ITP. GPIbα CAAR-T therapy would be a promising treatment for refractory and relapsed ITP patients.

Rapamycin Inhibits Cytotoxic T Lymphocytes-Mediated Platelet Destruction in Immune Thrombocytopenia

Primary immune thrombocytopenia (ITP) is an autoimmune disorder characterized with increased risk of bleeding. CD8+ cytotoxic T lymphocytes (CTLs) of ITP patients express high level of lethal molecules, leading to the lysis and apoptosis of their own platelets, which is an important mechanism of platelet destruction in ITP patients. Accumulating evidence have highlighted that the activation of mTOR signaling pathway promotes the differentiation of CD8+ T cells to CTLs, which leads to the up-regulation of cell-mediated immunity. Several studies have showed that rapamycin, a mTOR inhibitor, was effective in the treatment of relapsed/refractory ITP. However, the endogenous mechanism remains to be explored. The activation of mTOR signaling pathway in CD8+ T cells was determined by flow cytometry. We found that the phosphorylation level of p-mTOR, p-Akt, p-4E-BP1 and p-P70S6K in ITP patients were significantly higher than that in healthy controls. Meanwhile, we found that the levels of perforin and granzyme B in CD8+ T cells in ITP patients were significantly higher than that in healthy controls. In the in vitro experiments, CTLs were isolated from PBMCs of ITP patients or healthy controls and cultured with different concentrations of rapamycin (0-1 uM) for 3 days, and we chose an optimal concentration of 20 nM. There was no significant difference in the expression levels of mTOR, Akt, 4E-BP1, and P70S6K after rapamycin treatment, but the phosphorylation level of p-mTOR, p-Akt, p-4E-BP1, p-P70S6K was significantly inhibited by rapamycin treatment. Likewise, the ratios of p-mTOR/mTOR, p-AKT/AKT, p-4E-BP1/4E-BP1, p-P70S6K/P70S6K were also significantly down-regulated. Flow cytometry analysis found that rapamycin significantly inhibited the expression of perforin and granzyme B (Figure 1) in CD8+ T cells of ITP patients. We further established the ITP murine model by transferring splenocytes of C57BL/6 CD61 knockout mice, immunized against platelets from wild-type syngeneic C57BL/6 mice, into severe combined immune deficient (SCID) mice. Rapamycin was injected intraperitoneally daily at a final concentration 2.0 mg/kg. Our data showed that the rapamycin group had significantly higher platelet counts compared with control group. In summary, our findings suggest that the activation of mTOR signaling pathway-related molecules of CD8+ T cells in patients with ITP are abnormal. These data shed new light on the molecular mechanism of rapamycin action by inhibiting CTLs cytotoxicity. This novel mechanism of rapamycin underlies the potential therapeutic strategy for ITP. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

CD8+ TEMRA Clones Cause Platelet Lysis in Immune Thrombocytopenia

Background Immune thrombocytopenia (ITP) is traditionally thought of as an antibody-mediated disease. However, there are a number of features to suggest alternative mechanisms of platelet destruction. In this study we use a multi-dimensional approach to explore the role of terminally differentiated effector memory CD8+ T cells (TEMRA). Methods We characterised 83 adults with chronic ITP and 30 age-matched controls using immunophenotyping, next generation sequencing of T cell receptor (TCR), single cell (sc) RNA sequencing and functional assays. Next generation DNA sequencing of TCRβ was carried out using the Illumina MiSeq platform. Analysis of the raw TCR sequences was performed using MiXCR. Single cell immune profiling (Gene expression and TCR) was done using Chromium Single Cell V(D)J and 5' Library kits from 10x Genomics. The constructed library was sequenced on the HiSeq platform (Illumina). Seurat v.2.3.4 was used for gene expression analysis. Interactions between CD8+ T cells and platelets were measured by co-culturing CD8+ T cells with autologous platelets overnight and by perfusing them on the autologous platelet covered VenaFluoro8+ microchip channel. Results We found an expansion of CD8+ CD45RA+ CD62L- TEMRA cells expressing IFN gamma, TNF alpha and granzyme B, with no evidence of physiological exhaustion (failure to upregulate TIM-3 or PD-1 expression) in ITP patients which correlated with disease activity. DNA TCR sequencing revealed that expansion of TEMRA cells in ITP was associated with reduced T cell repertoire diversity (p ≤ 0.0001) and lower Simpson's diversity index (p ≤ 0.05). Patients with ITP had a higher number of T cell clones occupying more than 5% of the T cell repertoire when compared to age matched controls (p ≤ 0.05). Expanded clones were not shared across ITP patients and displayed no known antigen specificities when compared to available databases of TCR sequences, providing evidence for the lack of virally driven clonal expansion. Clonally expanded CD8+ T cells persisted over many years in ITP patients and characterised disease severity. Longitudinal samples from 9 patients showed expanded clones and reduced T cell repertoire diversity during active disease (platelet count of less than 30 x 109/L) compared to stable disease (platelet count greater or equal to 30 x 109/L) (p ≤ 0.01; r = 0.56 - Figure 1 shows an example patient). To better characterise CD8+ T cells, we performed paired scRNA and scTCR sequencing on isolated whole CD8+ T cells from patients with active and stable disease. Paired sequencing enables transcriptomic characterisation of T cell clones. ScRNA sequencing revealed four distinct clusters in CD8+ T cells: naïve, TEMRA, central memory (CM) and natural killer T cells (NKT). By mapping scTCR CD8+ sequencing to single cell gene expression, we found that the largest expanded clones showed an aggregative distribution, indicating transcriptional homogeneity and were almost exclusively comprised of TEMRA cells (Figure 2). Consistent with immunophenotyping and DNA TCR sequencing, expanded TEMRA cell clones were expanded in active disease compared to stable disease. To assess the impact of these CD8+ T cells on platelets in ITP, PBMCs from patients with ITP and HC were passed across a platelet layer at flow rates consistent with venous blood flow. CD8+ T cells in ITP patients interacted with platelets 4x more frequently than HCs. Isolated CD8+ T cells were cultured over night with autologous platelets to further explore these interactions: CD8+ T cell:platelet aggregates were identified more frequently in the co-cultures from patients with ITP (p ≤ 0.05) and were inhibited by blocking MHC Class I (p ≤ 0.01). CD8+ T cells within the aggregates had increased CD107a expression compared to non-aggregated CD8+ T cells (p ≤ 0.01), indicating cytolytic activity and release of Granzyme and Perforin. Correspondingly, platelets in the aggregates showed increased CD62P expression, a marker of activation and impending death (p ≤ 0.001). Conclusions Here we demonstrate clonal expansion of disease-associated TEMRA CD8+ T cells in ITP. These cells bind to platelets and cause TCR-mediated platelet activation and death providing an antibody-independent mechanism of platelet destruction. This could serve as biomarker to direct treatment in patients with refractory ITP and targeting specific T-cell clones could be a novel therapeutic option in refractory ITP. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

TCR CDR3 Sequencing as a Clue to Elucidate the Landscape of Dysimmunity in Patients with Primary Immune Thrombocytopenia.

Background: Primary immune thrombocytopenia (ITP) is an autoimmune disorder. The existence of autoreactive T cells has long been proposed in ITP. Yet the identification of autoreactive T cells has not been achieved, which is an important step to elucidate the pathogenesis of ITP. Methods: ITP patients’ peripheral blood was collected prior to the treatment and one month after initiating dexamethasone treatment per related therapeutic guideline. Serum cytokines were profiled to examine T cell subtypes imbalance using a protein chip. TCR Vβ analysis in CD8+T cells of ITP patients, and TCR CDR3 DNA sequencing of CD4+T and CD8+T cells were performed to determine the autoreactive T cells’ clones. Results: Cytokine profiling revealed imbalanced distribution of T cells subtypes, which was confirmed by CD4+T and CD8+T cells’ oligoclonal expansion of TCR Vβ analysis and TCR CDR3 DNA sequencing. VDJ segments were found to be more frequently presented in ITP patients, when compared with health controls. There was an individualized CD4+T cell or CD8+T cell CDR3 sequence in each ITP patient. Conclusions: The present study revealed that T cell clones expanded in ITP patients’ peripheral blood, and each clone had an individualized TCR CDR3 sequence. The expanded T cell clones preferred to use some specific VDJ segment. Further studies are warranted to get access to individualized treatment such as Car-T in patients with ITP.

Expression and Significance of PD-1 and ICOS in Patients with Primary Immune Thrombocytopenia

To investigate the expression of programmed death receptor-1 (PD-1) and inducible costimulator (ICOS) on the surface of CD8+ T cells in peripheral blood of patients with primary immune thrombocytopenia (ITP), and explore the roles of PD-1 and ICOS in the occurrence and development of ITP. A total of 28 ITP patients treated in Tianjin Medical University General Hospital from September to December 2020 were selected, including 13 patients with newly diagnosed ITP, 15 patients with chronic ITP, and 22 healthy volunteers were recruited as control group. Flow cytometry was used to detect the expression levels of PD-1 and ICOS, and evaluate their correlation with clinical indicators. The percentage of CD8 + T cells in ITP patients of chronic group was higher than that of the newly diagnosed group and the control group (P<0.05). The expression level of PD-1 on CD8+ T cells in ITP patients of newly diagnosed group and chronic group were significantly lower than that of the control group (P<0.05), while the expression level of ICOS were significantly higher (P<0.05). In ITP patients, PD-1 was negatively correlated with platelet count (r=-0.4942, P<0.01), but positively with ICOS (r=0.4342). PD-1 and ICOS were both negatively correlated with lymphocyte count (rPD-1=-0.4374; rICOS=-0.4492). In ITP patients, the unbalanced expression of PD-1 and ICOS may interfere with the immune homeostasis of the body, which can be used as a therapeutic target for ITP patients.

CD4+CD39 T CELLS IN THE PERIPHERAL BLOOD AND SPLEEN OF PATIENTS WITH IMMUNE THROMBOCYTOPENIA

Primary immune thrombocytopenia (ITP) is characterized with decreased platelet count and increased risk of bleeding. The mechanism of thrombocytopenia in ITP is incompletely understood but thought to involve autoantibodies which are produced by the B cells and are stimulated by helper T cells. Regulatory T cells (Treg) have been seen to be significant in ITP pathogenesis. Recent studies have reported reduction of circulating Treg cells in ITP patients but similar levels with controls have also been observed. The ectoenzyme CD39 is highly expressed on the surface of Treg cells and can suggest its immunosuppressive function.In this study we aimed to analyze CD4+CD39+ T lymphocytes both in the blood and spleen of patients with ITP who did not respond to the first line treatment and underwent splenectomy as a second line therapy. Non-ITP patients undergoing splenectomy were involved in the control group. Our data demonstrates significant diminution of in splenic but not circulating CD4+CD39+ T cells in ITP patients compared to controls. Of note, the comparison of spleen and peripheral CD4+CD39+T lymphocytes indicates that the frequency of CD39+ Treg cells is more stable in spleen compared to blood in ITP patients. Our data suggests the potential of CD39 as an important biomarker for ITP and underlines the clinical and scientific value of spleen immune analyzes in this pathology.

Orelabrutinib, a Selective Bruton's Tyrosine Kinase (BTK) Inhibitor in the Treatment of Primary Immune Thrombocytopenia (ITP)

Background:Primary immune thrombocytopenia (ITP) is the most common autoimmune hemorrhagic disorder characterized by decreased platelet count. increased risk of bleeding, and poor quality of life. Only about 70% patients response to first-line treatments, some patients are still refractory or relapsed after combined therapies, therefore it is necessary to explore new therapeutic targets. Bruton's tytosine kinase (BTK) is a non-receptor tyrosine kinase of Tec family, which is widely expressed in hematopoietic cells including B cells, monocytes/macrophages, and others. BTK participates in a variety of signaling pathways of innate and adaptive immunity, and plays an important role in cell survival and maturation. Platelet destruction mediated by anti-platelet glycoprotein antibodies is considered to be the main cause of ITP. B cells differentiate into plasma cells and produce autoantibodies due to the intolerance to autoantigens, which are important effectors in the pathogenesis of ITP. We speculated that inhibition of BTK may reduce platelet destruction by inhibiting B cell activation and autoantibody production. Orelabrutinib is a new generation of BTK inhibitor which has been used in hematological malignancies, this is the first study to explore the mechanisms of BTK inhibitor in the treatment of ITP.Methods:The concentrations of orelabrutinib were set at 1 nM, 10 nM, 100 nM and 1 μM in the in vitro study. Peripheral blood mononuclear cells (PBMCs) were isolated from active ITP patients and healthy controls and cultured for 72 hours, the apoptosis rate of PBMCs in each group was measured by Annexin V/PI double staining. CD19 + B cells of ITP patients were sorted by magnetic beads and stimulated with anti-human IgM to evaluate the activation of B-cell receptor (BCR) pathway and differentiation of plasma cells, respectively. Further, we transfused the splenocytes of immunized CD61-KO mice (C57BL/6) into the severe combined immunodeficient (SCID) mice to establish the active ITP murine models. Orelabrutinib was administered intragastrically at 10mg/kg, once a day. The control group was treated with 0.5% CMC at the same volume and frequency. Platelet count was measured weekly, the peripheral blood was collected and the B cell subsets in spleen were detected by flow cytometry at days 28 after splenocyte transfusion.Results:The proportion of early apoptotic cells (Annexin V +PI -) in PBMCs from both ITP patients and healthy controls was increased by orelabrutinib at 1μM,but there was no statistical difference. Orelabrutinib significantly inhibited the expression of CD69 in a dose-dependent manner at the concentrations of 10nM, 100nM and 1μM. Another early activation marker of BCR signaling pathway, CD86, was also found to be inhibited by orelabrutinib at 100nM and 1μM. The number of CD138 + plasma cells was decreased after treated with orelabrutinib at 10nM, 100nM and 1μM without dose-dependent manner. In the murine models, mice administered with orelabrutinib had significantly higher platelet count than the control mice at days 7, 14, 28 after splenocyte transfusion. The frequency of total B cells in peripheral blood leukocytes, the proportion of GL-7 + germinal center B cells and plasma cells in splenocytes were all determined to be lower in mice treated with orelabrutinib than the control group, though did not reach the statistical significance.Conculsion:Orelabrutinib could effectively suppress the activation and differentiation of B cells in vitro and in vivo, thus alleviate the thrombocytopenia in active ITP murine models. It could be the new treatment strategy for refractory/relapsed ITP patients. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

A Landscape of Transcriptome Sequencing in Childhood Immune Thrombocytopenia(ITP)

Background: Immune thrombocytopenia(ITP) is a complex autoimmune disease characterized by an isolated thrombocytopenia. It has long been known that B cells produce autoantibodies that are directed against various platelet membrane receptors, eventually resulting in platelet destruction by the reticuloendothelial system. T cell has recently been shown to play a critical role in ITP pathogenesis. In addition to platelet destruction, platelet production can be decreased by an immune-mediated megakaryocyte maturation abnormality and abnormal apoptosis. Despite all this, the pathogenesis of childhood ITP has been proven complex, better knowledge of the childhood ITP pathogenesis continues to be a major objective for the near future. And to our knowledge, there is no specialized gene expression study by means of RNA sequencing (RNA-seq) to characterize the whole transcriptome change of childhood ITP. Thus, we performed RNA-Seq of two childhood ITP patients to identify differentially expressed genes and key biological processes involved in childhood ITP.Methods: Mononuclear cells were separated from the bone marrow samples of two childhood ITP patients. Then we used bioinformatics methods to identify statistically differentially expressed genes and performed functional enrichment analysis to characterize the important functions or signal pathways. To investigate the change of whole transcriptome between ITP and control group and identify potential new functions associated with ITP pathogenesis, we performed Gene Set Enrichment Analysis (GSEA) through multiple functional databases including GO, KEGG and Reactome to determine statistically significant functional gene sets.Results: We obtained more than 16 million paired-end reads in two ITP patients. Each sample could well map to reference genome with average 85.31% rate of unique mapping reads. By comparing the differences of transcriptome between ITP and control group, we identified 2197 significantly differentially expressed genes, including 1396 up-regulated genes and 801 down-regulated genes (Figure A). Among these genes, we found 12 genes were not only known disease genes of ITP but also significantly differentially expressed between ITP and control group, such as FOXP3, HES1 and ITGA2B. These differentially expressed genes were involved in GO functions associated with immune, blood coagulation and platelet development, for example, platelet aggregation, platelet degranulation and immune system development (Figure B). Moreover, these genes were also significantly involved in KEGG pathways, such as oxidative phosphorylation and cell cycle pathway.To analyze the function impact of B cells and T cells in ITP patients, we performed GSEA for gene expression profile. The results showed that genes were significantly enriched with B cell and T cell-mediated immunity and the core enrichment genes were clearly up-regulation in ITP patients. In addition, the differentially expressed genes between ITP and control group were enriched in functions related with cytokines (Figure C). By comparing the differential expression genes with known genes encoding cytokines, 9 cytokine genes were significantly differentially expressed between ITP patients and controls, such as CXCL12, BMP3 and IL-24. Further more, we found that the gene expression profile was positively enriched with genes involved in megakaryocyte development and platelet production (Figure D). 5 megakaryopoiesis-associated genes showed significantly abnormal expression, including GATA1, MYB, CXCL12, ANGPT2 and COL1A1. Last but not least, several potential new pathways were also discovered, such as ECM receptor interaction, cell cycle, oxidative phosphorylation, focal adhesion and leukocyte transendothelial migration.Conclusions: Our results characterized the whole transcriptome change of childhood ITP patients and elucidated the genes and pathways consistently aberrant in ITP. These abnormal expressed genes, cytokines and pathways may play important roles in the pathogenesis of childhood ITP patients, and may serve as potential targets of diagnosis or treatment. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Low-Dose Decitabine Restores Immune Tolerance in ITP By Modulating Regulatory T Cells

Immune thrombocytopenia (ITP) is an aquired autoimmune disease triggered by increased platelet destruction and impaired platelet production. Loss of immune tolerance is considered to be the pivotal mechanism of ITP. Decitabine, a hypomethylating agent promoting cell differentiation at low dose, has been applied in the treatment of myelodysplastic syndrome (MDS) with a considerable platelet response. Our previous and current studies demonstrated that low-dose decitabine could significantly increase the number of mature polyploidy megakaryocytes and had a long-term efficacy that could not be explained by its role on promoting platelet production in ITP patients. Recent reports have demonstrated that decitabine could decrease the production of interferon-γ (IFN-γ) and tumor necrosis factor-a (TNF-a), and increase immune suppressive regulatory T cells. Thus, low-dose decitabine might correct the loss of immune tolerance to induce a more durable response in patients with ITP.CD4+CD25+Foxp3+ regulatory T cells (Tregs), which play an important role in maintenance of peripheral tolerance, were reported to be decreased in number and function in ITP (Stasi R et. al. Blood 2008). In the in vitro study, peripheral blood mononuclear cells (PBMCs) were isolated and cultured with 100nM decitabine or PBS for 72h. We performed flow cytometry to determine the percentage of Tregs. It was shown that the percentage of Tregs in PBMCs and CD4+ T cells were significantly elevated after decitabine treatment. We also sorted the CD4+CD25+CD127- Tregs from ITP patients and co-cultured them with CFSE-labeled CD4+CD25-T cells to evaluate the suppressive activity of Tregs. The results indicated that the inhibitory function of Tregs after decitbine treatments was significantly elevated compared with controls.As compared to healthy individuals, patients with ITP also had increased number of TH1, TH17, TH22, Bregs and CD16+ monocytes. We subsequently investigated the effects of low-dose decitabine on those cells in patients with active ITP. Three cycles of decitabine were administered intravenously at a dose of at 3.5 mg/m2 for 3 days per cycle with a 4-week interval between cycles. After decitabine treatment, there was a significant increase in the relative number of CD25+Foxp3+ Tregs in the CD4+ T cell population. We also observed an increase in Tregs function after the decitabine treatment. In contrast, decitabine could significantly decrease the percentage of CD4+IFN-γ+TH1 in the CD4+T cell population and the percentage of CD16+monoyctes. However, we have not observed the effect of decitabine on the CD4+IFN-γ-CD17+TH17, CD4+IFN-γ-CD22+TH22, and CD19+CD24+CD38+Bregs by now.We further established the murine ITP model by transferring the splenocytes of C57BL/6 CD61 knockout mice, which were immunized against platelets from wild-type syngeneic C57BL/6 mice into severe combined immunodeficient (SCID) mice. We treated the ITP mice model with a low-dose range of decitabine (0.01, 0.03 or 0.10 mg/kg) intravenously three times a week. Our data showed a significant effect on PLT increase and the expansion of the Tregs population and the suppression of TH1 cells and CD16+ monocytes .In addition, programmed death-1 (PD-1) is a co-receptor that is expressed predominantly by T cells as well as B cells, monocytes and was found to be lower on the PBMCs and in serum of ITP patients (Zhong J et. al. Hematology. 2016). And research has reported that PD-1 signaling pathway is important in the pathogenesis of autoimmune diseases by inhibition of self-reactive T cells and increasing the development and functions of Tregs. Furthermore, it was reported that the expression of PD-1 is enhanced by decitabine in MDS (Yang H et. al. Leukemia 2014). In our current studies, decreased PD-1 expression on CD4+T cells of ITP patients was observed and decitabine could elevate the PD-1 expression on CD4+T cells after the decitabine treatment in ITP patients. Therefore, decitabine may correct the loss of immune tolerance by upregulating the Expression of PD-1 and be a promising therapeutic strategy for the management in ITP patients. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Defective Early B Cell Tolerance in Primary Immune Thrombocytopenia

AIMs:Primary immune thrombocytopenia (ITP) is a common bleeding disorder characterized by low platelet counts, which could cause serious bleeding. One hallmark of ITP is the production of platelet-specific autoantibodies, resulting in accelerated platelet destruction and impaired platelet production. The anti-platelet antibodies normally react with components of platelet glycoproteins (GPs), which are rich in negatively charged amino acids. However, it is not clear how these autoantibodies are generated in ITP patients. The aim of this study is to investigate the status of B cell central tolerance in ITP.Methods:To gain an insight into the early B cell tolerance in ITP, we investigated the B cell receptor (BCR) specificity and immunoglobulin genes of naïve B cells in ITP patients. Naïve B cells were sorted and distributed into 96-well PCR plates at one single cell per well by single cell flow sorting. Paired immunoglobulin heave (IgH) and light (IgL) chain genes were amplified by reverse transcription PCR from single naïve B cells. The correct PCR products were subcloned into corresponding vectors and sequenced. Matched IgH and IgL genes from the same B cell were co-transfected into HEK293 cells to express recombinant antibodies (Fig. 1). The reactivity of these antibodies against platelet antigens was determined by ELISA, immunofluorescence assays, and flow cytometry. Ig gene sequence features were analyzed by programs including IMGT, IgBLAST, and VHRFA-1.Results:So far, 228 recombinant antibodies were expressed derived from naïve B cells in 4 ITP patients and 4 healthy controls. The absolute numbers of somatic mutations (nucleotide exchanges compared with germ line) of VH and VL between naïve B cells and peripheral memory B cells revealed significant difference (5.1 and 4.4 v.s. 19.0 and 14.8, p < 0.05 in both comparisons), indicating that the sorted cells were actually naive B cells (Fig. 2). ELISA assay showed that 17.0% (21/124) of the ITP-derived naïve B cells were platelet-reactive, in contrast to the 2.8% (3/104) in healthy donor-derived naïve B cells (Fig. 3), suggesting a defective B cell tolerance in the early developing stage. The anti-platelet reactivity was also confirmed by immunofluorescence assays and flow cytometry. Sequence analysis revealed that positively charged amino acids were highly enriched in the complementarity determining region 3 (CDR3) of the anti-platelet antibodies, which possibly generates the potential to interact with the negatively charged glycoproteins. The IgL genes derived from ITP patients preferentially used upstream JΚ segments (25.7% v.s. 49.1 %, p < 0.01) compared with healthy controls, indicating an insufficient IgL receptor editing in ITP patients (Fig. 4). In addition, the frequencies of VH replacement products in ITP were elevated in IgH genes from ITP patients (8.6% v.s. 4.4%, p < 0.05) compared to healthy controls (Fig. 5).Conclusion:In summary, this study provides evidence of defective early B cell tolerance and accumulation of anti-platelet naïve B cells in patients with ITP. This defect might be a result of insufficient receptor editing in IgL as well as excessive VH replacement in IgH. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Bone marrow mesenchymal stem cell-derived exosomes induce the Th17/Treg imbalance in immune thrombocytopenia through miR-146a-5p/IRAK1 axis.

Bone marrow mesenchymal stem cells (BMSCs) are associated with immune thrombocytopenia (ITP), the underlying mechanism has not been fully elucidated. Here, we attempted to investigate whether BMSCs can regulate Th17/Treg imbalance in ITP through the exosome pathway. We first assessed the proportions of Th17 cells and Tregs in ITP patients, showing that ITP patients exhibited an evident imbalance of Th17/Treg. BMSCs-exosomes' treatment significantly reduced Th17/Treg ratio in the CD4+ T cells of ITP patients. Moreover, miR-146a-5p was highly expressed in BMSCs-exosomes. The expression of miR-146a-5p was obviously increased in CD4+ T cells following the treatment of BMSCs-exosomes. BMSCs-exosomal miR-146a-5p silencing promoted the proportions of Th17 cells and repressed the proportions of Tregs in CD4+ T cells. In addition, miR-146a-5p directly interacted with IL-1R-associated kinase-1 (IRAK), and repressed IRAK1 expression. IRAK1 overexpression promoted Th17/Treg ratio in CD4+ T cells, which was abolished by BMSCs-exosomal miR-146a-5p. In conclusion, these findings demonstrate that BMSC-derived exosomal miR-146a-5p regulates Th17/Treg imbalance in ITP by repressing IRAK1 expression. Thus, this work suggests that BMSCs-exosomal miR-146a-5p may be a potential therapeutic target for ITP.

Regulation of Th1/Th2 and Th17/Treg by pDC/mDC imbalance in primary immune thrombocytopenia.

This study investigates the regulatory effect of plasmacytoid dendritic cells (pDC)/myeloid dendritic cells (mDC) imbalance on balance of Th1/Th2 and Th17/Treg in primary immune thrombocytopenia (ITP). A total of 30 untreated ITP patients and 20 healthy controls were recruited. Compared with healthy control, the pDC proportion of ITP patients was significantly reduced (P = 0.004), while the mDC proportion was not significantly changed (P = 0.681), resulting in a decrease in the pDC/mDC ratio (P = 0.001). Additionally, compared with controls, serum levels of interleukin (IL)-6, IL-12, and IL-23 were increased in ITP patients (P < 0.001), and mRNA levels of IL-12p40, IL-12p35, and IL-23p19 were also increased (P =0.014, P = 0.043, P < 0.001). Compared with the healthy control, the proportion of Th1 and Th17 cells in ITP patients increased (P = 0.001, P = 0.031). Serum levels of interferon gamma (IFN-γ) and IL-17 in ITP patients also increased (P = 0.025, P = 0.005). Furthermore, T-bet and RORγt mRNA levels were increased in peripheral blood of ITP patients (P = 0.018, P < 0.001). Correspondingly, the proportion of Th2 and Treg cells decreased (P = 0.007, P < 0.001), along with a decrease in serum IL-4 and transforming growth factor beta (TGF-β) (P = 0.028, P = 0.042), and an increase in GATA-3 mRNA (P < 0.001). However, there was no significant difference in Foxp3 mRNA levels (P = 0.587). Pearson correlation analysis showed that the proportion of total dendritic cells (DCs) was positively correlated with IL-12 (r = 0.526, P = 0.003) and IL-23 (r = 0.501, P = 0.005) in ITP patients. Th1/Th2 ratio, IFN-γ, and IL-12 levels were negatively correlated with platelet counts (r = -0.494, P = 0.009; r = -0.415, P = 0.028; r = -0.492, P = 0.032). However, IL-23 was positively correlated with IL-17 (r = 0.489, P = 0.006) and negatively correlated with platelet count (r = -0.564, P = 0.001). The ratio of IL-6 and Th17 cells was negatively correlated with platelet count (r = -0.443, P = 0.014; r = -0.471, P = 0.011). The imbalance of pDC/mDC and the increase of IL-6, IL-12, and IL-23 lead to the increased differentiation of CD4+ T cells into Th1 and Th17 cells, which might be the important mechanisms underlying the imbalance of Th1/Th2 and Th17/Treg in ITP patients.

Expression of CD19+B Cells and Involvement of Serum Breg in Pathogenesis of Immune Thrombocytopenia

To study the expression of CD19+B cells in immune thrombocytopenia patients, and to analyze the involvement of serum Breg in the pathogenesis of ITP. A total of 68 ITP patients admitted in department of hematology in our hospital from August 2015 to August 2018 were selected and enrolled in ITP group. 30 healthy individuals who were admitted to physical examination center at the same time were selected and enrolled in control group. The expression of peripheral blood lymphocyte subsets CD19+ and peripheral blood Breg cells were measured by flow cytometry. The difference between the ITP group and the control group was compared. The IL-10 mRNA and TGF-β1 mRNA levels were detected by RT-PCR. Correlations between the ratio of peripheral blood Breg cells and the expression of IL-10 mRNA and TGF-β1 mRNA in patients with immune thrombocytopenia, were analyzed. Expression of CD19+ in ITP group was significantly higher than that in control group. The difference between the two groups was statistically significant. Expression level of serum Breg cells in ITP group was lower than that in control group. IL-10 mRNA level in ITP group was significantly lower than that in control group. IL-10 mRNA level after treatment was significantly higher than that before treatment. In addition, TGF-β1 mRNA level in newly diagnosed ITP group was significantly higher than that in control group. After treatment, the TGF-β1 mRNA level was higher than that before treatment. The ratio of peripheral blood Breg cells positively correlated with IL-10 mRNA expression (r=0.968, P＜0.05), however, there was no correlation between peripheral blood Breg cell ratio and TGF-β1 mRNA expression (r=0.502, P＞0.05). Expression of CD19+ cells in ITP patients is increased. Breg cells may participate in the genesis of immune thrombocytopenia. The abnormal expression of Breg cells relates with the pathogenesis of immune thrombocytopenia.

Aberrant expression of microRNA in CD4+ cells contributes to Th17/Treg imbalance in primary immune thrombocytopenia

IntroductionImbalance of T helper 17 (Th17) cells and regulatory T (Treg) cells occurs in primary immune thrombocytopenia (ITP), but the mechanism remains unclear. We investigated whether expression of microRNAs (miRNAs) related to helper T or Treg cells regulate the Th17/Treg ratio in CD4+ T cells. Materials and methodsPeripheral blood was obtained from 52 active ITP patients and 56 healthy controls. We detected miRNA expression using RT-PCR with stem-loop primers and U6 as control.Th17 and Treg percentages were analyzed by flow cytometry. CD4+ cells were transfected with miRNA (miR-99a, miR-182-5p, miR-183-5p) mimics or inhibitors to investigate their function. ResultsmiR-99a expression in CD4+ cells in ITP patients was lower than in controls, while expression of miR-182-5p and miR-183-5p were higher in ITP patients. Moreover, Treg percentage correlated positively with miR-99a expression in ITP patients. We found no significant correlation between Th17 percentage and miR-182-5p or miR-183-5p expression. miR-183-5p expression correlated negatively with platelet count, while we found no significant difference between platelet count and miR-99a or miR-182-5p. miR-183-5p expression in CD4+ T cells from severe patients was significantly higher than in those from non-severe patients. Furthermore, down-regulating miR-183-5p expression repressed Th17 differentiation, while up-regulating miR-99a increased Tregs detected in CD4+ cells from ITP patients. In addition, up-regulated miR-99a repressed mTOR and p-mTOR expression. ConclusionsmiR-99a, miR-182-5p, and miR-183-5p expression levels in CD4+ cells were abnormal in ITP patients. Aberrant expression of miRNAs may contribute to the Th17/Treg imbalance in ITP patients and may represent a novel therapeutic target.

CB2 Receptor Stimulation and Dexamethasone Restore the Anti-Inflammatory and Immune-Regulatory Properties of Mesenchymal Stromal Cells of Children with Immune Thrombocytopenia.

Immune thrombocytopenia (ITP) is an autoimmune disorder characterized by antibody-mediated platelet destruction, with a complex and unclear pathogenesis. The impaired immunosuppressive capacity of mesenchymal stromal cells in ITP patients (ITP-MSCs) might play a role in the development of the disease. Correcting the MSC defects could represent an alternative therapeutic approach for ITP. High-dose dexamethasone (HD-Dexa) is the mainstay of the ITP therapeutic regimen, although it has several side effects. We previously demonstrated a role for cannabinoid receptor 2 (CB2) as a mediator of anti-inflammatory and immunoregulatory properties of human MSCs. We analyzed the effects of CB2 stimulation, with the selective agonist JWH-133, and of Dexa alone and in combination on ITP-MSC survival and immunosuppressive capacity. We provided new insights into the pathogenesis of ITP, suggesting CB2 receptor involvement in the impairment of ITP-MSC function and confirming MSCs as responsive cellular targets of Dexa. Moreover, we demonstrated that CB2 stimulation and Dexa attenuate apoptosis, via Bcl2 signaling, and restore the immune-modulatory properties of MSCs derived from ITP patients. These data suggest the possibility of using Dexa in combination with JWH-133 in ITP, reducing its dose and side effects but maintaining its therapeutic benefits.

Level of Regulatory B Cells in Patients with Immune Thrombocytopenia and Its Clinical Significance

To investigate the role of regulatory B cells (Breg) in pathogenesis of immune thrombocytopenia(ITP) and its clinical significance. A total of 40 ITP patients and 20 normal controls were enrolled in this study. The content of Breg, Th1, Th2, Th17 and Treg cells were detected by flow cytometry (FCM). The expression level of IL-10,TGF-β, CD40 and CD40L was detected by AimPlex Flow High Throughput Screening Technology. The of Breg cells in ITP patients was significantly lower than that in normal controls (P<0.05)，the expression levels of IL-10,TGF-β and CD40L in ITP patients were also significantly lower than those in normal controls (P<0.05). The contents of Th1 cells in ITP patients were significantly higher than that in normal controls (P<0.05), whereas the contents of Th2, Th17 and Treg cells in ITP patients were significantly lower than those in normal controls (P<0.05). The Breg cells may play an important role in the pathogenesis of ITP.