Low-dose Decitabine Restores Immune Tolerance In Immune Thrombocytopenia Via Modulating Treg Cells And Inhibiting CTLs Toxicity

Part Ⅰ:Low-dose decitabine modulates T cell homeostasis and restores immune tolerance in immune thrombocytopeniaBackgroundImmune thrombocytopenia(ITP)is an acquired autoimmune disease characterized by increased platelet destruction and impaired platelet production.Loss of immune tolerance is pivotal to the pathogenesis of ITP.CD4+CD25+Foxp3+regulatory T(Treg)cells play an essential role in the maintenance of self-tolerance;their quantity and function are impaired in ITP patients,which contributes to the excessive proliferation and activation of auto-reactive effector T(Teff)cells.The imbalance of CD4+helper T(Th)cells is further evidenced in the development of ITP,along with the unrestricted activation of cytotoxic T lymphocytes(CTLs)and the production of antiplatelet autoantibodies.Restoring immune tolerance in patients with ITP by reversing the impairment of Treg cells and suppressing the expansion of Thl and Th17 cells was demonstrated to be an important mechanism for currently applied ITP-specific therapies.Decitabine is a hypomethylating agent that promotes cell differentiation at low dose and is used for the treatment of myelodysplastic syndrome(MDS)with a relatively high platelet response.Recently,decitabine was shown to induce the murine megakaryoblastic cell line to differentiate into megakaryocyte-like polyploidy CD41-expressing cells in vitro and increase platelet release in BALB/C mice in vivo.In our previous studies,low-dose decitabine significantly increased the number of mature polyploidy megakaryocytes and also showed long-term clinical efficacy,which could not be explained simply by its role in promoting platelet production in ITP patients.It has been reported that hypomethylating agents could decrease the production of interferon-γ(IFN-γ)and tumor necrosis factor-α(TNF-α)while increasing the number of immunosuppressive Treg cells.However,whether decitabine has the potential to restore immune tolerance in ITP is unknown.Our study aimed to investigate the therapeutic value and mechanisms of decitabine in treatment of ITP.Objective1.To investigate the effect of low-dose decitabine on the production and function of Treg cells in ITP in vivo and in vitro.2.To investigate whether decitabine rebalances CD4+T cell subsets through Treg cells.3.To analyze the therapeutic effect and underlying mechanism of low-dose decitabine by next-generation RNA sequencing and cytokine assay,to further verify the targets of decitabine.Methods1.Patients and controls:for in-vitro studies,40 ITP patients and 31 age-and sex-matched healthy volunteers were recruited.For in vivo studies,32 ITP patients were enrolled and received decitabine treatment.2.Active ITP murine model:splenocytes from CD61 knockout mice immunized with CD61+platelets were transferred into severe combined immunodeficient(SCID)mouse recipients to induce a murine model of ITP.Different doses of decitabine were administrated intravenously three times a week for three weeks.3.Isolation and culturing of peripheral blood mononuclear cells(PBMCs)and CD4+T cells:PBMCs were isolated from peripheral blood of ITP patients and healthy controls with Ficoll-Hypaque centrifugation.CD4+T cells were sorted by magnetic beads and MS columns.PBMCs or sorted CD4+T cells were cultured in RPMI-1640 medium supplemented with recombinant human IL-2,anti-human CD3 antibodies and incubated for 72 hours4.Flow cytometry:to analyze apoptosis,PBMCs were stained with FITC-annexin V and propidium iodide(PI).Treg,Th1,Th2,Th17,and Th22 cells were detected by Beckman Coulter.5.Immunosuppression assays of Treg cells:CFSE-labeled CD4+CD25-T cells(Teff cells)and CD4+CD25+CD127-T cells(Treg cells)were sorted and co-cultured for 6 days.Proliferation analysis were analyzed by Flow cytometry.6.Treg deletion and depletion in ITP murine models:for the deletion of Treg,CD4+CD25+T cells were deleted from splenocytes of CD61 knockout mice using magnetic beads before the transfer to SCID mice.For the depletion of CD25+T cells in vivo,ITP mice received intraperitoneal injection of anti-mouse CD25(IL-2Rα)antibody.7.Cytokine analysis:serum samples of ITP patients before and after decitabine treatment were tested using V-PLEX Human Proinflammatory Panel 1 Kits,V-PLEX Human 17A Kits and R&D Elisa Assay Kit.8.RNA sequencing:RNA of PBMCs from ITP patients receiving decitabine treatment were extracted and sequenced on an Illumina Hiseq platform.9.Western blot analysis:PBMCs from ITP patients were lysed in radio-immunoprecipitation assay(RIPA)buffer for western blot.10.STAT3 and AKT inhibition assay:either cryptotanshinone(STAT3 inhibitor)or ARQ 092(AKT inhibitor)were added to PBMCs culture media and were applied to ITP murine models.Results1.Low-dose decitabine promoted the differentiation of Treg cells in vitro and enhanced their immunosuppressive capacityIn the in-vitro study,low-dose decitabine(10 nM and 100 nM)significantly elevated the percentage of Treg cells in ITP patients without changing the percentage of CD4+T cells in PBMCs.Subsequently,to minimize the distraction of other cells,we sorted CD4+T cells co-cultured with decitabine and found that low-dose decitabine(100 nM)also significantly increased the number of Treg cells in ITP.We sorted CD4+CD25+CD127-Treg cells from ITP patients and healthy controls,and cultured with CFSE-labeled CD4+CD25-Teff cells in a 1:4 ratio to evaluate the suppressive activity of Treg cells in the presence of decitabine.The division index of Teff cells from ITP patients were significantly decreased after co-incubation with Treg cells and 100 nM decitabine compared with Treg cells in ITP.These results suggest that the immunosuppressive function of Treg cells was significantly enhanced by low-dose decitabine in ITP.2.Low-dose decitabine ameliorated thrombocytopenia in active ITP mice with increased Treg cells in the spleenWe further established an active ITP murine model to investigate whether our observations in vitro were reproducible in vivo.Our data showed a significant platelet increase at day 21 and 28 in decitabine group at the dose of 0.03 mg/kg compared with controls.Flow cytometry analysis revealed a significant increase in the percentage of Treg cells in CD4+T cells in the spleen of decitabine-treated ITP mice compared with controls.We also identified a decrease of Thl and Th17 cells in CD4+T cells,but no significant difference was observed in Th22 cells.3.Treg deletion and depletion offset the effect of decitabineTreg-deleted splenocytes transferred mice and anti-CD25 antibody-treated mice were significantly depleted of Treg cells in the spleens.Treg deletion and depletion therapy partly offset the effect of decitabine on increasing platelet counts.After Treg deletion and depletion,decitabine had no significant effect on splenic Th1,Th17 or Th22 cells.4.Low-dose decitabine restored the balance of T cell subsets in ITP patientsAfter decitabine treatment,there was a significant increase in the number of CD25+Foxp3+Treg cells in CD4+T cells.In contrast,decitabine significantly decreased the percentage of Thl and Th17 cells in CD4+T cell populations,yet the amounts of Th2 and Th22 cells remained unchanged.We also observed an increase in the suppressive function of Treg cells after decitabine treatment.5.Low-dose decitabine regulates cytokines related to T cell differentiation in ITP patientsSerum was isolated from peripheral blood of 32 ITP patients before and after decitabine treatment.Low-dose decitabine significantly reduced serum levels of TNF-α,IFN-y and IL-17a,as well as a panel of inflammatory cytokines and chemokines,such as IL-6,IL-8 and IL-12,which were associated with T cell differentiation.We also found asignificant increases in TGF-β and IL-2.However,there was no significant difference in serum levels of IL-1 β,IL-4,IL-10,or IL-13.6.RNA sequencing analysis correlated with decitabine efficacy in ITP patientsTo assess the impact of low-dose decitabine on ITP patients’ gene expression,we performed mRNA sequencing analysis in 4 responders and 3 non-responders.Compared with the pre-treatment point,responders shared a significant differential expression of 301 genes after decitabine treatmentBased on KEGG analysis,these genes were associated with cytokine-cytokine receptor interaction,hematopoietic cell lineage,IL-17 signaling pathway,Thl and Th2 cell differentiation,Th17 cell differentiation and Jak-STAT signaling pathway.7.Low-dose decitabine inhibited STAT3 in ITPWestern blot analysis of cell lysates from PBMCs of ITP patients after decitabine treatment revealed decreased STAT3 and AKT phosphorylation compared with pre-treatment levels.In in-vitro studies,the percentage of Treg cells in CD4+T cells from ITP patients and healthy controls in STAT3 inhibitor plus decitabine group had no difference from that in STAT3 inhibitor group.In murine studies,platelet counts in STAT3 inhibitor plus decitabine group were not significantly different from those in mice receiving STAT3 inhibitor.These results indicated that low-dose decitabine might restore Treg cells by inhibiting STAT3 in ITP.Conclusions1.Low-dose decitabine increased the production of Treg cells and enhanced their immunosuppressive function in ITP2.Thrombocytopenia was ameliorated by low-dose decitabine with an increase of splenic Treg cells and reduction of Thl and Th17 cells in ITP murine models.3.Low-dose decitabine modulated T cell homeostasis along with the down-regulation of STAT3 in ITP.Part Ⅱ:Low-dose decitabine inhibits cytotoxic T lymphocytes-mediated platelet destruction via modulating PD-1 methylation in immune thrombocytopeniaBackgroundPrimary immune thrombocytopenia(ITP)is an acquired autoimmune disease characterized by immune-mediated platelet destruction and impaired platelet production.In addition to platelet autoantibodies,abnormal T cell immunity,especially CD8+cytotoxic T lymphocytes(CTLs)play a vital role in the pathogenesis of ITP.CTLs have been demonstrated to cause direct platelet lysis in patients with active ITP.Programmed cell death protein 1(PD-1),a member of the CD28/CTLA-4 family,is inducibly expressed on peripheral T cells,B cells,natural killer T cells,monocytes and some dendritic cells upon their activation.In combination with T cell receptor(TCR)signal,PD-1/PD-L1 signaling pathway is critical in turning off autoreactive T cells and reducing CTLs cytotoxicity to target cells.Aberrant PD-1/PD-L1 signaling results in the breakout of peripheral tolerance and is an important contributor to autoimmune diseases,such as rheumatoid arthritis(RA),systemic lupus erythematosus(SLE),and encephalomyelitis.Previous studies have shown that PD-1 and PD-L1 expression on peripheral blood mononuclear cells(PBMCs)and CD4+T cells were lower in ITP patients.In vitro studies have proved that PD-L1-Fc recombinant fusion protein(PD-L1-Fc,linking the extracellular domains of PD-1)increased T cell apoptosis and inhibited the activation and proliferation of T cells in ITP,suggesting the important role of PD-1 pathway in the pathogenesis of ITP.Decitabine,a hypomethylating agent(HMA),has been used in the treatment of myelodysplastic syndrome(MDS).Recently,low-dose decitabine was shown to promote megakaryocyte maturation in vitro and platelet release in vivo,which was closely related to the demethylating effect of decitabine.Our previous studies showed that low-dose decitabine significantly increased the number of mature polyploidy megakaryocytes and had a clinical long-term efficacy that could not be annotated by the effect of decitabine on promoting platelet release from these megakaryocytes.It has been found that hypomethylating agents resulted in the demethylation of the PD-1 CpG island and increased PD-1 expression in MDS.We hypothesized that PD 1/PD-L1 signaling pathway plays an important role in maintaining immune tolerance in ITP by inhibiting cytotoxic T lymphocytes-mediated platelet destruction.Low-dose decitabine may regulate PD-1 methylation thus have therapeutic potential in ITP.Objective1.to investigate the role of PD-1/PD-L1 pathway in maintaining immune tolerance in ITP by inhibiting cytotoxic T lymphocytes-mediated platelet destruction.2.to investigate the regulatory effect of low-dose decitabine on PD-1 methylation and expression,and to clarify the therapeutic effect of decitabine on inhibiting cytotoxic T lymphocytes-mediated platelet destruction in ITPMethods1.Patients and controls:sixty-five patients with active ITP and Forty-three geographically matched healthy individuals were consented in the study.2.Active ITP murine model:splenocytes from CD61 knockout mice immunized with CD61+platelets were transferred into severe combined immunodeficient(SCID)mouse recipients to induce a murine model of ITP.3.Isolation and culturing of CD8+T cells:PBMCs were isolated from peripheral venous blood of ITP patients and healthy controls with Ficoll-Hypaque centrifugation.CD8+T cells were sorted from PBMCs by magnetic beads and MS columns.CD8+T cells were cultured in RPMI 1640 medium supplemented with recombinant human IL-2,anti-human CD3 antibodies and anti-human CD28 antibodies for 72 hours.Recombinant fusion protein PD-L1-Fc,decitabine,anti-human PD-1 antibody or PBS were added to the incubation media.4.Quantitative real-time PCR:the mRNA expression of PD-1 and endogenous control GAPDH was quantified by real-time PCR.5.Flow cytometry:to determine the expression of PD-1 and PD-L1,all suspensions were subsequently stained by anti-human or anti-mouse conjugated-antibodies.Cells were subsequently analyzed on a Gallios Flow cytometer.6.CTLs-induced platelet apoptosis:CD8+T cells and platelets were adjusted to 105/mL and 106/mL.After co-culturing for 4 hours,the cells were harvested to analyze the platelet apoptosis.Platelets were stained with JC-1 using mitochondrial membrane potential assay kit.7.DNA methylation analysis:methylation of PD-1 CpG island was evaluated using a bisulfite-sequencing PCR(BSP).8.Western blots analysis:CD8+T cells cultured with 100 nM decitabine or PBS were lysed were lysed in radio-immunoprecipitation assay(RIPA)buffer for western blot.Results1.Decreased PD-1 and PD-L1 expression on CD8+T cells from ITP patientsThe mRNA level of PD-1 in PBMCs from ITP patients had no difference with that of healthy controls.The expression of PD-1 on CD8+T cells and CD4+T cells in peripheral blood of ITP patients was significantly lower than that in healthy controls.ITP patients expressed significantly lower PD-L1 on CD8+T cells and CD4+T cells compared with healthy controls.The expression level of PD-L1 on platelets from ITP patients had no difference with that of healthy controls.2.PD-Ll-Fc inhibited CTLs-mediated platelet apoptosis in ITP in vitroTo investigate the effect of PD-L1-Fc on CD8+T cells in ITP,CTLs-mediated platelet apoptosis was analyzed to determine the cytotoxicity of CD8+T cells after 72h incubation.Compared with controls,PD-L1-Fc significantly decreased CTLs-mediated platelet apoptosis in ITP in vitro.PD-L1-Fc itself had no effect on platelet apoptosis.3.Increased methylation of PD-1 in CD8+T cells from ITP patientsWe found that the percentage of methylated residues in PD-1 promoter was significantly higher in ITP patients than that in controls,suggesting a significantly higher PD-1 methylation level in ITP patients.4.Low-dose decitabine restored PD-1 expression on CD8+T cells and ameliorated thrombocytopenia in ITPLow-dose decitabine significantly increased PD-1 expression on CD8+T cells from ITP patients in vitro.To assess whether our in vitro observations were reproducible in vivo,we adopted an active ITP murine model.On day 21 and 28,significantly higher platelet counts were observed in the decitabine-treated group compared to control group.We quantified the expression of PD-1 in the active murine ITP models and ITP patients receiving decitabine administration.Compared with controls,PD-1 was increased on splenic CD8+T cells in decitabine-treated ITP mice.Furthermore,a significant increase in platelet counts and in PD-1 expression on CD8+T cells in ITP patients was observed after decitabine treatment.5.Low-dose decitabine decreased PD-1 promoter methylation in ITPBSP was performed to detect PD-1 gene methylation.Decitabine-Treated cells showed significantly lower methylated CpG residues compared with controls.6.Low-dose decitabine inhibited CTLs-mediated platelet apoptosis in ITPA significant reduction in CTLs induced apoptosis of platelets co-cultured with low-dose decitabine was observed in vitro.Further,we investigated CTLs cytotoxicity of ITP patients receiving decitabine treatment and found that CTLs induced platelet apoptosis was significantly inhibited after 12-week decitabine administration.7.Low-dose decitabine inhibited the phosphorylation of PI3K and AKTWestern blot analysis of cell lysates from CD8+T cells cultured with 100nM decitabine revealed that there was no significant change in the expression levels of PI3K and AKT However,the phosphorylation levels of PI3K and AKT were significantly downregulated by low-dose decitabine.8.Blocking PD-1 can partly counteract the therapeutic effect of low-dose decitabine in inhibiting CTLs-mediated platelet apoptosis in ITPTo determine whether the mechanism of decitabine was dependent on PD-1 pathway or not,we performed assays for PD-1 blockade test in vitro.No difference in CTLs induced platelet apoptosis among anti-human PD-1 antibody(anti-hPD-1)group,100nM decitabine group plus anti-hPD-1 group,or control group were found.To further evaluate the effect of PD-1 blockade in vivo,we applied anti-mouse PD-1 antibody(Anti-mPD-1)in ITP murine model.Platelet counts in decitabine-treated ITP mice plus anti-mPD-1 were not different from those in anti-mPD-1 or control mice,but decreased significantly compared with decitabine-treated ITP mice on day21 and on day 28.Conclusions1.The expression of PD-1 and PD-L1 on CD8+T cells was decreased.Enhancing PD-1/PD-L1 signaling by PD-L1-Fc could reduce CTLs cytotoxicity.PD-1 promoter methylation in CD8+T cells was increased in ITP patients compared with healthy controls.2.Low-dose decitabine decreased PD-1 promoter methylation,increased PD-1 expression,and inhibited CTLs-mediated platelet destruction in ITP.3.Low-dose decitabine decreased PD-1 promoter methylation,increased PD-1 expression,and inhibited CTLs-mediated platelet destruction in ITP.