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Myeloid-derived suppressor cells (MDSCs) are heterogeneous immature cells and natural inhibitors of adaptive immunity. Metabolic fitness of MDSCs is fundamental for its suppressive activity toward effector T cells. Our previous studies showed that the number and inhibitory function of MDSCs were impaired in patients with immune thrombocytopenia (ITP) compared with healthy controls. In this study, we analyzed the effects of decitabine on MDSCs from patients with ITP, both in vitro and in vivo. We found that low-dose decitabine promoted the generation of MDSCs and enhanced their aerobic metabolism and immunosuppressive functions. Lower expression of liver kinase 1 (LKB1) was found in MDSCs from patients with ITP, which was corrected by decitabine therapy. LKB1 short hairpin RNA (shRNA) transfection effectively blocked the function of MDSCs and almost offset the enhanced effect of decitabine on impaired MDSCs. Subsequently, anti-CD61 immune-sensitized splenocytes were transferred into severe combined immunodeficient (SCID) mice to induce ITP in murine models. Passive transfer of decitabine-modulated MDSCs significantly raised platelet counts compared with that of phosphate buffered saline–modulated MDSCs. However, when LKB1 shRNA-transfected MDSCs were transferred into SCID mice, the therapeutic effect of decitabine in alleviating thrombocytopenia was quenched. In conclusion, our study suggests that the impaired aerobic metabolism of MDSCs is involved in the pathogenesis of ITP, and the modulatory effect of decitabine on MDSC metabolism contributes to the improvement of its immunosuppressive function. This provides a possible mechanism for sustained remission elicited by low-dose decitabine in patients with ITP.

Our previous clinical study showed that low-dose decitabine exhibited sustained responses in nearly half of patients with refractory immune thrombocytopenia (ITP). The long-term efficacy of decitabine in ITP is not likely due to its simple role in increasing platelet production. Whether decitabine has the potential to restore immune tolerance in ITP is unknown. In this study, we analyzed the effect of decitabine on T-cell subpopulations in ITP in vitro and in vivo. We found that low-dose decitabine promoted the generation and differentiation of regulatory T (Treg) cells and augmented their immunosuppressive function. Splenocytes from CD61 knockout mice immunized with CD61+ platelets were transferred into severe combined immunodeficient mouse recipients to induce a murine model of ITP. Low-dose decitabine alleviated thrombocytopenia and restored the balance between Treg and helper T (Th) cells in active ITP mice. Treg deletion and depletion offset the effect of decitabine in restoring CD4+ T-cell subpopulations in ITP mice. For patients who received low-dose decitabine, the quantity and function of Treg cells were substantially improved, whereas Th1 and Th17 cells were suppressed compared with the pretreatment levels. Next-generation RNA-sequencing and cytokine analysis showed that low-dose decitabine rebalanced T-cell homeostasis, decreased proinflammatory cytokines, and downregulated phosphorylated STAT3 in patients with ITP. STAT3 inhibition analysis suggested that low-dose decitabine might restore Treg cells by inhibiting STAT3 activation. In conclusion, our data indicate that the immunomodulatory effect of decitabine provides one possible mechanistic explanation for the sustained response achieved by low-dose decitabine in ITP.

Increased macrophage phagocytosis of antibody-coated platelets, as well as decreased numbers and/or impaired function of CD4+CD25+Foxp3+ regulatory T (Treg) cells, has been shown to participate in the pathogenesis of immune thrombocytopenia (ITP). Low-dose histone deacetylase inhibitors (HDACi’s) are anti-inflammatory and immunomodulatory agents that can enhance immunosuppression in graft-versus-host disease by increasing the number and function of Foxp3+ Treg cells, but it is unclear whether they have the potential to promote immune tolerance and platelet release in ITP. In this study, we performed in vitro and in vivo experiments and found that a low-dose HDACi (chidamide) alleviated thrombocytopenia in passive and active murine models of ITP. Further, low-dose HDACi’s attenuated macrophage phagocytosis of antibody-coated platelets, stimulated the production of natural Foxp3+ Treg cells, promoted the peripheral conversion of T cells into Treg cells, and restored Treg cell suppression in vivo and in vitro. Finally, we confirmed that low-dose HDACi’s could regulate CTLA4 expression in peripheral blood mononuclear cells through modulation of histone H3K27 acetylation. Low-dose HDACi treatment in ITP could be offset by blocking the effect of CTLA4. Therefore, we propose that low-dose chidamide administration has potential as a novel treatment for ITP in the clinic.

Background: Splenectomy is a therapy for many conditions the most common of which are trauma, Hodgkin lymphoma, thalassemia and hereditary or autoimmune hemolytic anemia, and immune thrombocytopenia (ITP). By 1960 splenectomy had been known to create a risk of over-whelming post splenectomy sepsis (OPSS) with a high mortality rate. More recently population-based studies from Denmark have suggested that there is also an increased risk of thromboembolic (TEE) complications. Thromboembolic events generally include venous thrombosis (deep vein thrombosis (DVT) and pulmonary embolism (PE)) and arterial thrombosis (stroke and myocardial infarction [MI]). ITP and hemolytic anemias have been shown to independently have increased risks of arterial and venous TEE which has complicated assessment of additive effects of splenectomy. While splenectomized patients appear to be at increased risk of arterial and venous thromboses, the incidence and which TEEs predominate remains unclear as does the timing of occurrence of the TEEs. Method: The Nurses' Health Study (NHS) was established in 1976 and enrolled 121,700 female nurses between 30-55 years old. The nurses completed baseline questionnaires and biennial follow up questionnaires. In 2004, NHS participants were asked whether they had undergone splenectomy. The primary outcomes of this study included DVT,PE, and MI, which were identified through self-report on biennial questionnaires. MIs were confirmed through medical record review. End of follow-up for this study went through 2016, date of death, or diagnosis of outcome, whichever came first. Loss to follow up was very low and causes of deaths were carefully tracked. Descriptive statistics compared the splenectomy vs non-splenectomy participants with respect to basic demographic factors and variables that may be related to the outcomes. Cox proportional hazards models were run to evaluate incidence rates for primary and secondary outcomes. We conducted age-adjusted, as well as multivariable adjusted models. Multivariate models were adjusted for age, body mass index, smoking history, diabetes mellitus, high blood pressure, and high cholesterol, thus taking into account known predictors of the specific TEEs. Multivariate models were updated every two years to account for time varying confounders. Results： In 2004, 323 participants reported having had a splenectomy, out of 96,000 completing the questionnaire. There was a strong, significant association between splenectomy and subsequent DVT (n=613) and PE (n=840). The multivariate adjusted HR was 3.73 (95%CI: 1.77-7.86) and 3.80 (95%CI: 2.04-7.10) respectively. When considered together as a composite outcome, the splenectomized participants had a 3.52-fold higher risk (95%CI: 2.12-5.85) of PE/DVT compared with non-splenectomy. There was no association between splenectomy and MI (n=1011; HR = 0.97, 95% CI: 0.31-3.01). Given the very limited number of stroke cases in the splenectomized population, we were underpowered/unable to evaluate the association of splenectomy and subsequent risk of stroke. Conclusion: This study found that splenectomized patients are at a 4-fold increased risk of PE and DVT as compared to not-splenectomized individuals. These findings were not dependent on any of the potentially confounding variable analyzed. We found no association between splenectomy and MI. There are limitations to these results. No information was available on family history and perioperative thrombo-prophylaxis. Also we do not know exactly when or why splenectomy was performed; there were at least 34 women who had splenectomy for ITP but in most cases the underlying disease was unknown. Since the nurses in 1976 were already between 30-55 years old, it is no longer possible to retrospectively obtain sufficient information. Therefore, the splenectomy question has been added to the current survey for NHS#2. We will pursue the relevant information in a follow up letter to respondents who reported having undergone splenectomy while seeing if the findings of NHS#2 confirm those of NHS#1. Figure 1 Figure 1. Disclosures Bussel: Principia/Sanofi: Consultancy, Membership on an entity's Board of Directors or advisory committees; UCB: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: DSMB; Momenta/Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Argenx: Consultancy, Membership on an entity's Board of Directors or advisory committees; Dova/Sobi: Consultancy, Membership on an entity's Board of Directors or advisory committees; CSL: Other: DSMB; RallyBio: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Rigel: Consultancy, Membership on an entity's Board of Directors or advisory committees; UptoDate: Honoraria.

The aim of this study was to determine the safety and efficacy of recombinant human thrombopoietin (rhTPO) for the management of immune thrombocytopenia (ITP) during pregnancy. Pregnant patients with ITP were enrolled in the study if they had a platelet count less than 30 × 109/L, were experiencing bleeding manifestations, had failed to respond to corticosteroids and/or intravenous immunoglobulin (IVIG), and had developed refractoriness to platelet transfusion. Thirty-one patients received rhTPO at an initial dose of 300 U/kg once daily for 14 days. Twenty-three patients responded (74.2%), including 10 complete responders (>100 × 109/L) and 13 responders (30-100 × 109/L). It appears that rhTPO ameliorated the bleeding symptoms remarkably, even in the nonresponders. rhTPO was well tolerated. Dizziness, fatigue, and pain at an injection site were reported in 1 patient each. No congenital disease or developmental delays were observed in the infants in a median follow-up of 53 (range, 39-68) weeks. In conclusion, rhTPO is a potentially safe and effective treatment choice for patients with ITP during pregnancy. Our work has paved the way for further study on the clinical application of rhTPO and other thrombopoietic agents for the management of ITP during pregnancy. This study is registered at www.clinicaltrials.gov as NCT02391272.

Elevated expression of the activating Fcγ receptor (FcγR) I and FcγRIIa together with decreased expression of the inhibitory FcγRIIb are involved in the pathogenesis of primary immune thrombocytopenia (ITP). Thrombopoietin receptor agonists (TPO-RAs) have been used clinically for the management of ITP; however, little is known about the effect of TPO-RAs on FcγR modulation in ITP. In this prospective study, we measured the alteration in monocyte FcγR expression from 21 corticosteroid-resistant/relapsed patients with chronic ITP receiving eltrombopag therapy. Results showed that the mRNA and protein levels of FcγRIIb were significantly elevated after 6-week eltrombopag treatment. Concurrently, FcγRI and IIa levels decreased remarkably, whereas FcγRIII expression did not change. In vitro phagocytosis assays indicated that a shift in the balance of FcγR toward inhibitory FcγRIIb on monocytes was accompanied with a considerable decrease in monocyte/macrophage phagocytic capacity. The response to eltrombopag therapy in patients with ITP was associated with FcγR phenotype and functional changes of monocytes/macrophages. Moreover, the plasma transforming growth factor-β1 (TGF-β1) concentrations increased significantly in eltrombopag responders. Modulation of monocyte FcγR balance by TPO-RAs was also found in a murine model of ITP established by transferring splenocytes from immunized CD61 knockout mice into CD61(+) severe combined immunodeficient mice. Romiplostim administration in ITP mice significantly upregulated inhibitory FcγRII expression and downregulated activating FcγRI expression. These findings showed that recovery of platelet counts after TPO-RA treatment in ITP is associated with the restoration of FcγR balance toward the inhibitory FcγRIIb on monocytes, and suggested that thrombopoietic agents have a profound effect on immune modulation in ITP. This study is registered at ClinicalTrials.gov as #NCT01864512.

Myeloid-derived suppressor cells (MDSCs) are heterogeneous immature cells and natural inhibitors of adaptive immunity. In this study, the MDSC population was evaluated in adult patients with primary immune thrombocytopenia (ITP), where cell-mediated immune mechanisms are involved in platelet destruction. Our data demonstrated that both the numbers and suppressive functions of MDSCs were impaired in the peripheral blood and spleens of patients with ITP compared with healthy control patients. High-dose dexamethasone (HD-DXM) treatment rescued MDSC numbers in patients with ITP. And DXM modulation promoted the suppressive function of MDSCs induced in vitro. Moreover, the expression of interleukin 10 and transforming growth factor β was significantly upregulated in DXM-modulated MDSCs compared with the unmodulated cultures. DXM-modulated MDSCs inhibited autologous CD4(+)T-cell proliferation and significantly attenuated cytotoxic T lymphocyte-mediated platelet lysis, further indicating enhanced control over T-cell responses. Elevated expression of the transcription factor Ets1 was identified in DXM-modulated MDSCs. Transfection of Ets-1 small interfering RNA efficiently blocked regulatory effects of MDSCs, which almost offset the augmentation of MDSC function by DXM. Meanwhile, splenocytes from CD61 knockout mice immunized with CD61(+)platelets were transferred into severe combined immunodeficient (SCID) mouse recipients (C57/B6 background) to induce a murine model of severe ITP. We passively transferred the DXM-modulated MDSCs induced from bone marrow of wild-type C57/B6 mice into the SCID mouse recipients, which significantly increased platelet counts in vivo compared with those receiving splenocyte engraftment alone. These findings suggested that impaired MDSCs are involved in the pathogenesis of ITP, and that HD-DXM corrected MDSC functions via a mechanism underlying glucocorticoid action and Ets1.

This study compared the efficacy and safety of high-dose dexamethasone (HD-DXM) and conventional prednisone (PDN) on the largest cohort to date as first-line strategies for newly diagnosed adult primary immune thrombocytopenia (ITP). Patients enrolled were randomized to receive DXM 40 mg/d for 4 days (n = 95, nonresponders received an additional 4-day course of DXM) or prednisone 1.0 mg/kg daily for 4 weeks and then tapered (n = 97). One or 2 courses of HD-DXM resulted in a higher incidence of overall initial response (82.1% vs 67.4%, P = .044) and complete response (50.5% vs 26.8%, P = .001) compared with prednisone. Time to response was shorter in the HD-DXM arm (P < .001), and a baseline bleeding score ≥8 was associated with a decreased likelihood of initial response. Sustained response was achieved by 40.0% of patients in the HD-DXM arm and 41.2% in the PDN arm (P = .884). Initial complete response was a positive indicator of sustained response, whereas presence of antiplatelet autoantibodies was a negative indicator. HD-DXM was generally tolerated better. We concluded that HD-DXM could be a preferred corticosteroid strategy for first-line management of adult primary ITP. This study is registered at www.clinicaltrials.gov as #NCT01356511.

Introduction: Primary immune thrombocytopenia (ITP) is an acquired hemorrhagic disorder caused by immune-mediated platelet destruction and insufficient platelet production. As the first-line treatment of ITP, corticosteroids enable the recovery of platelet count in 60% to 80% of cases. However, a large proportion of patients would relapse during steroid tapering or withdrawal. Recently, sporadic studies reported successful treatment of ITP with oseltamivir which decreased platelet destruction through inhibition of platelet desialylation, a possible mechanism leading to Fc-independent platelet phagocytosis by hepatocytes. We hypothesize that corticosteroids and oseltamivir might complement each other in terms of mechanisms and induce a better response in ITP. Therefore, a prospective randomized controlled trial was conducted to compare the efficacy and safety of high-dose dexamethasone (HD-DXM) plus oseltamivir with HD-DXM monotherapy in adult ITP. Here we report the interim analysis of this study. Methods: From February 2016 to May 2018, 80 newly diagnosed treatment-naïve ITP patients from 5 tertiary medical centers in China were screened in the trial (NCT01965626). Dexamethasone was administered orally at 40 mg daily to both arms for 4 days (days 1 - 4). If platelet counts remained < 30 × 109/L or there were bleeding symptoms by day 10, another course of HD-DXM was given (days 11 -14). For the combination arm, oral oseltamivir was given concomitantly at a dosage of 75 mg twice daily for the first 10 days (days 1 - 10). The primary endpoints were initial response and sustained response (SR). Platelet counts should be confirmed on two separate occasions at least 7 days apart when defining responses (CR: platelet count ≥ 100×109/L and absence of bleeding; R: platelet count ≥ 30 × 109/L but < 100 × 109/L and at least a doubling of baseline and absence of bleeding). Initial responses were assessed by day 14. Responders underwent follow-up for at least 12 months or until relapse. Response lasting for at least 6 consecutive months without additional ITP-specific intervention was regarded as SR. Secondary endpoints included time to response (TTR), bleeding scores, duration of response and adverse events (AEs). Platelet transfusion was permitted in patients with severe bleeding symptoms and recorded. Requirements for any additional ITP-specific intervention were considered as treatment failure. Results: Fifty-nine patients were enrolled, of whom 28 were randomly to receive HD-DXM plus oseltamivir and 31 to HD-DXM monotherapy. Demographics and baseline characteristics were balanced between the 2 arms (Table 1). Results showed that HD-DXM plus oseltamivir achieved a higher incidence of initial response than HD-DXM alone at day 14 (82.1% vs. 58.1%, P = 0.045). At the end of 6 months, SR rate in the HD-DXM plus oseltamivir arm was higher than that in the HD-DXM monotherapy arm even though it did not reach statistical significance (42.8% vs. 25.8%, P = 0.167). Relapse-free survival in the HD-DXM plus oseltamivir arm was significantly improved compared with the HD-DXM monotherapy arm during follow-up period (HR 1.96, Log Rank P =0.043), as estimated by the Kaplan-Meier analysis. There was no statistical difference in median TTR between the two arms (P > 0.05). Bleeding was well controlled in both arms, with comparable post-treatment bleeding events (7.1% vs.19.3%, P> 0.05) and lower bleeding scores in the HD-DXM plus oseltamivir arm (0 vs. 0, P = 0.036). Incidence of AEs was similar between the 2 arms (P > 0.05). A slight increase in gastrointestinal reactions was observed in the HD-DXM plus oseltamivir arm but it did not reach statistical significance. Most of the AEs were mild to moderate (grade 1 to 2) and usually resolved spontaneously after medication was completed. No previously undescribed AEs was observed. Conclusions: The combination of HD-DXM with oseltamivir significantly improved the initial response in newly diagnosed ITP patients. However, due to the limitation of sample size, the conclusion of SR superiority can hardly be drawn at the moment. Disclosures No relevant conflicts of interest to declare. OffLabel Disclosure: Oseltamivir, a sialidase inhibitor which is extensively used in the treatment of or prophylaxis against influenza virus A and B. It has been reported that the treatment of oseltamivir could concomitantly increase platelet counts, which was independent whether influenza was diagnosed. Along those lines, several studies showed successful treatment of ITP patients with oseltamivir. We hypothesize that HD-DXM and oseltamivir might complement each other in terms of the mechanisms and achieve better response rates in ITP patients.

Thrombopoietin (TPO), a hematopoietic growth factor produced predominantly by the liver, is essential for thrombopoiesis. Prevailing theory posits that circulating TPO levels are maintained through its clearance by platelets and megakaryocytes via surface c-Mpl receptor internalization. Interestingly, we found a two- to threefold decrease in circulating TPO in GPIbα-/- mice compared with wild-type (WT) controls, which was consistent in GPIbα-deficient human Bernard-Soulier syndrome (BSS) patients. We showed that lower TPO levels in GPIbα-deficient conditions were not due to increased TPO clearance by GPIbα-/- platelets but rather to decreased hepatic TPO mRNA transcription and production. We found that WT, but not GPIbα-/-, platelet transfusions rescued hepatic TPO mRNA and circulating TPO levels in GPIbα-/- mice. In vitro hepatocyte cocultures with platelets or GPIbα-coupled beads further confirm the disruption of platelet-mediated hepatic TPO generation in the absence of GPIbα. Treatment of GPIbα-/- platelets with neuraminidase caused significant desialylation; however, strikingly, desialylated GPIbα-/- platelets could not rescue impaired hepatic TPO production in vivo or in vitro, suggesting that GPIbα, independent of platelet desialylation, is a prerequisite for hepatic TPO generation. Additionally, impaired hepatic TPO production was recapitulated in interleukin-4/GPIbα-transgenic mice, as well as with antibodies targeting the extracellular portion of GPIbα, demonstrating that the N terminus of GPIbα is required for platelet-mediated hepatic TPO generation. These findings reveal a novel nonredundant regulatory role for platelets in hepatic TPO homeostasis, which improves our understanding of constitutive TPO regulation and has important implications in diseases related to GPIbα, such as BSS and auto- and alloimmune-mediated thrombocytopenias.

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.

Immune thrombocytopenia (ITP) is a prevalent autoimmune disease characterized by autoantibody-induced platelet clearance. Some ITP patients are refractory to standard immunosuppressive treatments such as intravenous immunoglobulin (IVIg). These patients often have autoantibodies that target the ligand-binding domain (LBD) of glycoprotein Ibα (GPIbα), a major subunit of the platelet mechanoreceptor complex GPIb-IX. However, the molecular mechanism of this Fc-independent platelet clearance is not clear. Here, we report that many anti-LBD monoclonal antibodies such as 6B4, but not AK2, activated GPIb-IX in a shear-dependent manner and induced IVIg-resistant platelet clearance in mice. Single-molecule optical tweezer measurements of antibodies pulling on full-length GPIb-IX demonstrated that the unbinding force needed to dissociate 6B4 from the LBD far exceeds the force required to unfold the juxtamembrane mechanosensory domain (MSD) in GPIbα, unlike the AK2-LBD unbinding force. Binding of 6B4, not AK2, induced shear-dependent unfolding of the MSD on the platelet, as evidenced by increased exposure of a linear sequence therein. Imaging flow cytometry and aggregometry measurements of platelets and LBD-coated platelet-mimetic beads revealed that 6B4 can sustain crosslinking of platelets under shear, whereas 6B4 Fab and AK2 cannot. These results suggest a novel mechanism by which anti-LBD antibodies can exert a pulling force on GPIb-IX via platelet crosslinking, activating GPIb-IX by unfolding its MSD and inducing Fc-independent platelet clearance.

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.

The roles of Heterogeneous nuclear ribonucleoproteins(hnRNPs) in regulating tumor development and progression, either as oncogenes or as tumor suppressors, were well documented. HnRNP C is one of the members of hnRNPs,and differential expression of hnRNP C has been found in series of tumor cells. However, the role of hnRNP C in leukemia has not been reported to date. Here, we report the first novel gene fusion event between HNRNPC and retinoic acid receptor gamma (RARG) in acute myeloid leukemia mimicking acute promyelocytic leukemia. This translocation produced the HNRNPC-RARG fusion gene and its reciprocal, RARG-HNRNPC. A 43-year-old man was referred to our hospital with fever and a sore throat.Laboratory investigations revealed the following patient characteristics: (1) white blood cell count 12 × 109/L (blasts 1% and abnormal promyelocytes 86%). (2) Morphologic analysis of the bone marrow aspirate showed 86.5% microgranular atypical promyelocytes (Figure 1a, 1b). (3) Analysis from flow cytometry showed that the blasts were positive for CD33, CD13, CD45, and cMPO and negative for CD14, CD34, CD16, CD56, HLA-DR, B- or T-cell markers. Thus, the patient started all-trans retinoic acid (ATRA) treatment immediately. Afterwards, chromosomal analysis revealed 47 metaphases, and most of them were involved in t(14;17). Fluorescence in situ hybridization and RT-PCR assays did not identify the PML/RARA, NPM-RARA, PLZF-RARArearrangement. ATRA therapy lasted for 3 weeks, but no response was observed. Next, the patient received 2 cycles of induction chemotherapy until a complete response. Afterwards, he received 6 cycles of chemotherapy. Unfortunately, the leukemia relapsed 1 year later, and all treatments (including ATRA and arsenious acid) failed to produce any effects. The patient died from sepsis. To identify molecular alterations, transcriptome sequencing analysis was performed. A 213-bp RARG-HNRNPC fusion product was specifically amplified from the patient's cDNA, as predicted (Figure 1c). Sanger sequencing showed that RARG exon 9 was fused in-frame to HNRNPC exon 3(Figure 1d). The RARG 5'-region encoding the ligand-binding domain was fused to the HNRNPC3'-region, where a cluster of phosphorylation sites is located(Figure 1e). We also found a reciprocal chimeric transcript. The amplicon size of HNRNPC-RARG fusion was 186-bp (Figure 2a). Sanger sequencing demonstrated that HNRNPC exon 3 was fused in-frame to RARG exon 5 (Figures 2b). The HNRNPC 5'-region encodes an RNA recognition motif (RRM), and the segment from RARG encodes a DNA binding domain (DBD, Figure 2c). HnRNP C ubiquitously expressed RNA-binding protein (RBP) which are believed to influence pre-mRNA metabolism such as splicing, polyadenylation, stability, transport, andtranslation mediated by internal ribosome entry site. HnRNP C also plays an essential role in cell progression and the regulation of several DNA repair proteins. Retinoic acid receptors (RARs) are transcription factors that belong to the nuclear hormone receptor family.RARA, RARB, and RARG are three RARs subtypes which share highly similar sequences and functions. A study showed RARG seems to act as a major regulator maintaining the balance between HSC self-renewal and differentiation. Acute myeloid leukemias mimicking acute promyelocytic leukemia, or acute promyelocytic-like leukemias (APLL), share the same morphology and immunocytochemistry features with typical acute promyelocytic leukemia (APL) except the RARA rearrangements, and little is known about the molecular mechanisms of APLL. The sequences and function of the RARG and RARA are highly alike, and therefore can logically explain the similarity of biological characteristics between the two entities. Three other fusion genes harboring RARG ( including NUP98-RARG , PML-RARG and CPSF6-RARG) have been found in APLL. Unfortunately they showed resistance to treatment with ATRA or ATRA plus arsenic. Moreover, poor prognosis was observed likewise. All the above confirm that RARG rearrangements are not random but recurrent genetic abnormalities. In conclusion, we present a novel HNRNPC-RARG fusion gene and its reciprocal in APLL, and suggest that at least a portion of APLLs have RARG gene rearrangements. We propose that RARG-rearranged APLL may be a novel candidate subtype of acute myelocytic leukemia, or even of APL. Disclosures No relevant conflicts of interest to declare.

Backgrounds:Primary immune thrombocytopenia (ITP) is an acquired autoimmune disease characterized by reduced platelet count and an increased risk of bleeding. The imbalance of Treg/Th17 cells has been demonstrated in ITP, but the mechanism of Th17/Treg cells imbalance is still not clear. In this study, we aimed to investigate whether the expression of helper T (Th) or Treg cell-related microRNAs, such as miR-183-96-182 cluster, miR-17-5p, miR-99a, miR-146-5p, miR-155-5p, miR-181-5p, and miR-326, regulates the ratio of Th17/Treg in CD4+ T cells and could be used to evaluate the clinical implications of ITP patients. Methods: Peripheral blood was obtained from 54 patients with active ITP and 34 healthy controls. Peripheral blood mononuclear cells (PBMCs) were isolated using Ficoll density-gradient centrifugation and the CD4+ cells were separated by immuno-magnetic microbeads selection. Amplification technique of RT-PCR using stem-loop primers was applied to detect the relative expression of microRNAs (miR-17-5p, miR-99a, miR-96-5p, miR-146a-5p, miR-155-5p, miR-181a-5p, miR-182-5p, miR-183-5, miR-326) and U6 was normalized as control for miRNA quantification. The frequencies of Th17 and Treg cells in peripheral blood were analyzed by flow cytometry. The mRNA expression levels of Il-6, Il-10, Il-17, Rorγ-t and Foxp-3 in CD4+ cells were determined by RT-PCR. Platelet autoantibodies specific for GPIIb/IIIaor GPIb/IX were measured using MAIPA method. CD4+ cells were transfected with miRNAs (miR-99a, miR-182-5p, miR-183-5), mimics or inhibitors, which were used to detect the function of miRNAs. Cytokines in culture medium were determined by ELISA. Results: Our results showed that the relative expression of miR-182-5p and miR-183-5p in CD4+ cells was significantly increased in active ITP patients, compared to healthy controls (miR-182-5p, median 9.2678 vs 5.2723, p < 0.05, Fig. 1a; miR-183-5p, median 5.4435 vs 2.009, p < 0.05, Fig. 1b). In addition, the relative expression of miR-99a in ITP patients was lower than that of healthy controls (median 3.4214 vs 7.9648, p < 0.05; Fig. 1c). Moreover, the frequency of Treg cells decreased significantly in ITP patients compared to those in controls (1.89±1.59% vs 4.12±1.42%, p < 0.05; Fig. 2a), and the percentage of Treg cells was positively correlated with the relative expression of miR-99a in ITP patients(r=0.461, p< 0.05; Fig. 2c) and health controls(r=0.729, p< 0.05; Fig. 2d). Though the percentage of Th17 cells increased in ITP patients compared to the health controls (3.51±2.13%vs 1.85±0.63%, p < 0.05; Fig. 2b), there was no correlation between the percentage of Th17 and the relative expression of microRNAs in ITP patients or health controls. Besides, there was no correlation between the expression of mRNAs (Il-10, Il-17, Rorγ-t and Foxp-3) and microRNAs (miR-99a, miRNA-182-5p or miR-183-5p). No significant correlation was found between the microRNAs expression and platelets counts or different autoantibody subsets in ITP patients. The relative expression of other microRNAs (miR-17-5p, miR-96-5p, miR-146a-5p, miR-155-5p, miR-181-5p, miR-326) revealed no difference in CD4+ cells between ITP patients and health controls. Furthermore, the down-regulated expression of miR-183-5p with inhibitors promoted to the differentiation of Th17 cells(Fig. 3a), while up-regulated expression of miR-99a with mimics contributed to Treg cells in CD4+ cells from ITP patients (Fig. 3b). Meanwhile, the IL-17A in culture medium decreased in inhibitor group of miR-183-5p or miR-183-5p. However, miR-182-5p inhibitor had no effect on the differentiation of Th17 cells. Conclusions: Our results show the abnormal expression of microRNAs (miR-99a, miRNA-182-5p and miR-183-5p) in CD4+ cells and the miR-99a was closely correlated with the Treg cells. The aberrant expression of microRNAs may contribute to the imbalance of Th17/Treg cells in the development of ITP patients and potentially constitute a novel therapeutic target. Disclosures No relevant conflicts of interest to declare.

Immune thrombocytopenia (ITP) is a common autoimmune bleeding disorder in which loss of immune tolerance leads to platelet destruction. Human leukocyte antigen-G (HLA-G) is a non-classical major histocompatibility complex class I antigen including four membrane-bound (mHLA-G, HLA-G1 to -G4) and three soluble (sHLA-G, HLA-G5 to -G7) isoforms due to alternative splicing of the HLA-G primary transcript. HLA-G exerts immunosuppressive functions by interacting with its inhibitory receptors, immunoglobulin-like transcripts (ILTs), expressing differentially on natural killer cells, T cells, and antigen presenting cells. Upon binding to ILTs, HLA-G suppressed lymphocyte proliferation, promoted apoptosis of cytotoxic lymphocytes and natural killer cells, inhibited antibody secretion from activated B cells, and induced expansion of suppressive T cells, tolerogenic dendritic cells and myeloid-derived suppressor cells. HLA-G supports immune escape, and high level of HLA-G is associated with unfavorable outcomes in cancer patients. Meanwhile, HLA-G-induced tolerance can benefit human allotransplantation and autoimmune diseases, such as rheumatoid arthritis and systemic lupus erythematosus, but the role of HLA-G in the treatment of ITP is still unclear. Here we observed significantly less soluble HLA-G in plasma from ITP patients positive for anti-glycoprotein (GP) IIb/IIIa and/or anti-GPIb/IX autoantibodies compared with autoantibody-negative patients or healthy controls. However, no significant difference was found in the sHLA-G levels between anti-glycoprotein (GP) IIbIIIa and anti-GPIb-IX autoantibodies groups. Interestingly, sHLA-G is positively correlated with platelet counts in ITP patients both with and without autoantibodies, suggesting that HLA-G can also protect platelets from destruction through other mechanisms. To elucidate this, we then analyzed both expression of membrane HLA-G and ILTs on PBMCs. Our data found less membrane-bound HLA-G and ILTs on CD4+T cells and CD14+cells in ITP patients. Moreover, recombinant human HLA-G (rhHLA-G) upregulated expression of ILT2 on CD4+and ILT4 on CD14+cells. The upregulation of ILT by rhHLA-G indicates that sHLA-G might also benefit ITP patients negative for anti-platelet autoantibodies. To determine whether rhHLA-G affects T cell differentiation, we tested 17 cytokines by ELISA to investigate the immune environment with or without rhHLA-G, and found that rhHLA-G upregulated Interleukin (IL)-4 and IL-10, and downregulated tumor necrosis factor-α, IL-12, and IL-17 secreted by ITP patient peripheral blood mononuclear cells (PBMCs), indicating a promotion of helper T(Th)2 and inhibition of Th1 and Th17 differentiation. However, our results indicated that rhHLA-G did not expand the CD4+CD25+Foxp3+regulatory T cells(Tregs). In addition, rhHLA-G exerted their immunosuppressive effects by upregulating IL-1β, IL-2, Granulocyte colony-stimulating factor (G-CSF), and granulocyte-macrophage colony stimulating factor (GM-CSF). By reprogramming the cytokine profile in ITP patients, HLA-G established an optimal environment for the impaired cell populations, such as dendritic cells (DCs), to regain their tolerogenic function. Our results showed that rhHLA-G inhibited monocyte-derived DCs maturation by downregulating CD80 and CD86 expression. To further assess whether the rhHLA-G modulated-DCs were functionally tolerogenic, PBMCs were then cocultured with rhHLA-G modulated-DCs. Our results showed that rhHLA-G-modulated DCs from ITP patients suppressed CD4+T cell proliferation by 38.8% compared to unmodulated cells. Besides, PBMCs exposed to rhHLA-G were less cytotoxic toward plateletsfrom ITP patients or healthy subjects. This study provides a potential strategy to protect exogenous platelets from destruction in ITP patients. In conclusion, our data demonstrated that impaired expression of HLA-G and/or ILT is involved in the pathogenesis of ITP, and recombinant human HLA-G can restore immune tolerance in ITP patients via upregulation of ILTs, which indicates that HLA-G can be a diagnostic marker and also a therapeutic option for ITP treatment. Disclosures No relevant conflicts of interest to declare.

Introduction Immune thrombocytopenia (ITP) is an autoimmune disease in which autoreactive T and B cells are activated by platelet autoantigens resulting in immune-mediated platelet destruction and/or suppression of platelet production. The binding of programmed death 1 (PD-1) to its ligands PD-L1 and PD-L2 on antigen-presenting cells turns off autoreactive T cells and induces peripheral tolerance. Aberrant PD-1/PD-L signalling could result in a breakdown of peripheral tolerance and lead to autoimmune diseases. Methods Thirty-four patients with primary active ITP who were diagnosed and/or followed up and 26 healthy controls were enrolled in this study. Platelet counts in all ITP patients were less than 30×109 /L at sampling. They had not been treated with any immunosuppressive agents for at least one week prior to sampling for this study. To determine the role of the PD-1/PD-L signalling pathway in ITP, we detected PD-1 expression on T cells and PD-L expression on dendritic cells (DCs) in both ITP patients with active disease and healthy controls by flow cytometry. To investigate the effects of PD-L1-Fc fusion protein (PD-L1-Fc) on T cells, PBMCs from ITP patients and healthy controls with autologous platelets were cultured with soluble anti-CD3 monoclonal antibody (mAb) and anti-CD28 mAb in the presence (PD-L1-Fc+ group) or absence (PD-L1-Fc- group) of the PD-L1-Fc (0.5 μg/mL) at 37 ˚C with 5% CO2 for 4 days. The cells were harvested and stained for flow cytometry to detect the apoptosis, activation and proliferation of T cells. IL-2 and IFN-γ levels in the co-culture supernatant were assayed by ELISA. Results Enhanced PD-1 expression on T cells and decreased PD-L1 expression on mDCs in ITP patients We found that PD-1 mean fluorescence intensities (MFIs) increased in CD4+ cells (P < 0.01) and in CD8+ cells (P < 0.01) from ITP patients compared with those from healthy controls. However, PD-L1 expression on monocyte-derived DCs was lower in patients with active ITP than in healthy controls (P < 0.01, Figure 1). PD-L1-Fc promoted T cell apoptosis Annexin V-FITC and propidium iodide (PI) were used to detect T cell apoptosis. The percentage of apoptotic cells and dead cells were analysed to determine T cell apoptosis levels. In ITP patients, the percentage of apoptotic cells and dead cells were higher in PD-L1-Fc+ group than in the PD-L1-Fc- group (P < 0.01 for both CD4+ and CD8+ T cells). However, we found no significant difference in apoptosis between PD-L1-Fc- and PD-L1-Fc+ groups in healthy controls (Figure 2). PD-L1-Fc inhibited T cell activation and proliferation CD25 MFIs were analysed to determine the activation level of cocultured T lymphocytes. Compared with healthy controls, CD25 expression on CD4+ and CD8+ T cells was significantly increased in ITP patients (P < 0.05 for both CD4+ and CD8+ T cells). These results suggest that ITP patients had more activated CD4+ and CD8+ T cells than in healthy controls. PD-L1-Fc significantly inhibited T cell activation in ITP patients (P < 0.01, n=34) but not in healthy controls (P = 0.0834 in CD4+ T cells, P = 0.6834 in CD8+ T cells, n=26, Figure 3). To analyse proliferation, the frequency of divided cells was calculated according to the loss of CFSE fluorescence intensity. PD-L1-Fc inhibited proliferation of T cells from ITP patients (P < 0.01 in both CD4+ T and CD8+ T cells, n=34, Figure 4). We found no significant difference in proliferation in T cells from healthy controls (CD4+ T cells P = 0.9758, CD8+ T cells P = 0.5658, n=26). PD-L1-Fc inhibited IL-2 and IFN-γ production IL-2 secretion was higher in ITP than in healthy controls in the absence of PD-L1-Fc (P = 0.0308, Figure 5). PD-L1-Fc significantly inhibited IL-2 and IFN-γ production in ITP patients. IL-2 and IFN-γ levels were lower in the PD-L1-Fc+ group than in the PD-L1-Fc- group in ITP (P < 0.01 for both IL-2 and IFN-γ, n=34). However, we did not detect a significant difference in secretion of IL-2 (P = 0.2016, n=26) or IFN-γ (P = 0.2989, n=26) in healthy controls. Conclusions In summary, our study suggests that the aberrant PD-1/PD-L negative costimulatory pathway may play a role in ITP. Enhancing PD-1/PD-L signalling might be a promising therapeutic approach for ITP by promoting T cell apoptosis, inhibiting T cell activation and proliferation, and reducing secretion of inflammatory factors. Disclosures No relevant conflicts of interest to declare.

Introduction: Adult primary immune thrombocytopenia (ITP) is an autoimmune-mediated haemorrhagic disorder characterized by excessive platelet destruction and decreased platelet production. ITP patients have a Th1 dominant profile, which was involved in the pathogenesis of ITP. Interleukin-16 (IL-16) can directly affect cellular or humoral immunity by mediating the cellular cross-talk among T cells, B cells and dendritic cells. Several studies have focused on IL-16 as an immunomodulatory cytokine that takes part in Th1 polarization in autoimmune diseases, but the roles of IL-16 in ITP remain unknown. Treatment with high-dose dexamethasone (HD-DXM) as a single-agent for four days has been widely recognized as the first-line therapy for ITP patients in need of clinical management. The aim of this study is to investigate the roles of IL-16 and effect of HD-DXM on IL-16 in ITP. Methods: We investigated the differences in IL-16 expression in the bone marrow and peripheral blood between ITP patients and healthy controls by ELISA. The mRNA expression of pro-IL-16, caspase-3 and T-bet in active ITP patients and healthy controls were quantified by real-time PCR. Furthermore, we detected changes in IL-16 concentration and gene expression before and after the 4-day HD-DXM therapy. Bone marrow samples were obtained from 23 patients and 10 healthy bone marrow donors, and peripheral blood samples were obtained from 64 patients (among them, 21 received single-agent HD-DXM therapy, the peripheral blood samples were obtained before HD-DXM therapy and 28 days after HD-DXM administration) and 38 healthy blood donors. Adult primary ITP patients with active disease were enrolled in this study between May 2015 and January 2018 at the Department of Haematology, Qilu Hospital, Shandong University, Jinan, China. Results: The concentration of IL-16 in the bone marrow supernatants and plasma of active ITP patients was significantly higher compared with that of the healthy controls (P < 0.05) (Fig. 1A, B). Gene expression of pro-IL-16 and caspase-3 in bone marrow mononuclear cells (BMMCs) in ITP patients elevated compared with healthy controls (P < 0.05) (Fig. 2A). Besides, gene expression of pro-IL-16, caspase-3 and T-bet in peripheral blood mononuclear cells (PBMCs) in ITP patients elevated (P < 0.05) (Fig. 2B). Among the 21 patients which received single-agent HD-DXM therapy, 18 responded effectively to the HD-DXM therapy according to the standard definition. The post-treatment plasma (n=21) IL-16 level decreased significantly compared with that of pre-treatment level (P < 0.05), and the level of IL-16 in the plasma of ITP patients before and after HD-DXM treatment was significantly higher than that of healthy controls (P < 0.05) (Fig. 1C). There was no correlation between IL-16 levels in the bone marrow supernatant or plasma and platelet count (data not shown). In addition, pro-IL-16, caspase-3, T-bet mRNA expression was significantly decreased after treatment with HD-DMX (P < 0.05) (Fig. 2C), although the corresponding values in patients were not statistically higher than those of healthy controls. The correlation between the plasma IL-16 concentration and pro-IL-16, caspase-3 and T-bet mRNA levels was also analysed in ITP patients (n = 21). The results demonstrated that the mRNA levels of the three detected factors were positively correlated with plasma IL-16 concentration (r2 = 0.5846, P < 0.05, r2 = 0.3980, P < 0.05 and r2 = 0.3145, P Conclusions: ITP is an autoimmune disorder; complex interactions among antigen-presenting cells, T cells and B cells are pivotal to its pathogenesis. Our data suggest that IL-16 plays an important role in the pathogenesis of ITP by polarization of Th1. Furthermore, by modulating the abnormal IL-16 level associated with the Th1 imbalance via treatment with pulsed HD-DXM provided us with new insights into the immune regulatory mechanisms for the treatment of ITP. In our future study, we will use an in vitro study system to test and verify if the anti-IL-16 antibody can be used to treat adult ITP. Disclosures No relevant conflicts of interest to declare.

Introduction: Primary immunodeficiency diseases (PID) are caused by gene defects that impair function of the innate or adaptive immune systems. An increasing number of patients have been identified with a causative monogenic defect. More than 300 different genetic defects have been found [J Clin Immunol (2018) 38:96-128]. The NFKB1 gene is strongly associated with PID. Heterozygous variants of NFKB1 cause a progressive defect in formation of immunoglobulin-producing B cells [Cell (2017) 168:37-57]. Here, we introduce a new NFKB1 mutation. Methods: A patient with a 20-year history of diarrhea was recently hospitalized due to three months of interrupted fever. Chest computed tomography (CT) showed bilateral pneumonia, splenomegaly, and retro-peritoneal lymphadenopathy. We highly suspect that he has primary immunodeficiency and collected blood samples from all family members to identify the gene mutation. Family history. The patient's father (I.2) died early and his mother (I.1) died of cerebral infarction a few months ago. The patient has two brothers and two sisters. One brother (II.1) died of tuberculosis, the other (II.4) is healthy. One sister (II.3) died of stomach cancer, the other (II.2) has a history of left breast cancer and has received chemotherapy three times. His son (III.1) is clinically asymptomatic. His wife (II.6) is healthy (Figure 1). Blood samples. We evaluated complement components and quantified immunoglobulin levels of the family members, and determined the B cell ratio of the patient. Next, we performed whole exon sequencing by next-generation sequencing. We also predicted the protein structure of the mutant gene. Results: The patient has severely decreased levels of serum IgG, IgA, and IgM. Unexpectedly, his son has moderately reduced IgG levels, while others' immunoglobulin is normal. The patient's lymphocyte subgroups revealed a high ratio of CD3+, CD3+/CD8+ lymphocytes and low ratio of CD19+, CD56+CD16+ lymphocytes, which suggests a decreased proportion of B lymphocytes (Table 1). Next-generation sequencing revealed all known gene mutations of this family. Using Phenolyzer software (a tool that uses prior information to implicate genes involved in diseases), we selected three candidate genes: RAG1, C2, and NFKB1. The patient (II.5), his sister (II.2), and his son (III.1) all have a heterozygous mutation of RAG1. Thus, we ruled out RAG1, as it does not conform to Mendel's laws in this family. C2 was also excluded due to the low haploinsufficiency score (0.178). Interestingly, the patient (II.5) and his son (III.1) both have a heterozygous mutation of NFKB1, while others do not. NFKB1 shows a high haploinsufficiency score (0.945), suggesting that the single functional copy of the gene may not produce enough protein. Thus, we hypothesize that NFKB1 is the disease-causing gene in this family. Further investigation revealed a heterozygous NFKB1 frame shift mutation (c.2053delG: p.G685fs) in the patient and his son. Other family members possess wild-type NFKB1. The novel frameshift mutation influences three transcriptomes with a similar coding sequence to the NFKB1 gene. Sanger sequencing verified the results. The NFKB1 gene consists of four regions: Rel homology, glycine-rich, ankyrin repeats, and DEATH domain. Our prediction of the protein structure suggests that the frameshift mutation occurred in the ankyrin repeats region. Studies have shown that large deletions in the ankyrin repeats region may cause deficiency in class-switched memory B cell generation. This mutation results in a loss of 283 amino acids and addition of 40 new amino acids. Prediction of the tertiary structure illustrated that the coding protein is terminated early. The mutation results in loss of some helixes and formation of a new helix at the C-terminal. This is a novel mutation of NFKB1 that has not previously been reported in PID, and which forms a new protein structure (Figure 2). Conclusions: Our findings broaden the scope of phenotypes caused by mutations in NFKB1. We suspect that this heterozygous mutation of NFKB1 may lead to fewer immunoglobulins produced.Onset was delayed for this patient, at the age of 20. His son is 25 years old now, with moderately reduced levels of IgG but without symptoms. We suspect that he may be ill in the future and recommended that he seek genetic counseling when he is ready to have a child. Disclosures No relevant conflicts of interest to declare.

In this issue of Blood , Guo et al used an anti-CD20 monoclonal antibody (mAb) in a murine model of immune thrombocytopenia (ITP) to show for the first time that B-cell depletion therapy inhibits splenic CD8 + T-cell proliferation and rescues T-cell–mediated ITP. 1

Tissue inhibitor of metalloproteinase-3 (TIMP-3) is one of a family of proteins inhibiting matrix metalloproteinases, which has also been identified as a mediator for checking inflammation. Meanwhile, it is well known that inflammation causes the activation of the immune response. However, it is not clear whether TIMP-3 plays a role in the immune system. In the present study, we demonstrated a novel function of TIMP-3 in Th1/Th2 polarization through its influence on the antigen-presenting cells. First, TIMP-3 was found strikingly up-regulated by IL-4 during the differentiation of human dendritic cells via the p38MAPK pathway. Second, the expression of costimulatory molecule-CD86 was repressed by TIMP-3. Besides, the induction of IL-12 in matured dendritic cells was significantly inhibited in a PI3K-dependent manner. Furthermore, dendritic cells matured in the presence of TIMP-3 could stimulate allogeneic naive T helper (Th) cells to display a prominent Th2 polarization. Importantly, in an autoimmune disorder–primary immune thrombocytopenia, TIMP-3 showed a statistically positive correlation with IL-4 and platelet count, but a negative correlation with IFN-γ in patient blood samples. Collectively, these in vitro and in vivo data clearly suggested a novel role of TIMP-3 in Th1/Th2 balance in humans.

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-β 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.

Cytotoxic T-lymphocyte (CTL)-mediated platelet destruction and aberrant cytokine profiles play important roles in the pathogenesis of primary immune thrombocytopenia (ITP). Interleukin-27 (IL-27) has pleiotropic immunomodulatory effects. However, the effect of IL-27 on CTL activity in ITP has not been reported. In the present study, platelets from ITP patients were cultured with autologous CTLs in the presence of IL-27. We found that IL-27 could inhibit CTL-mediated platelet destruction. In these IL-27–treated CTLs, granzyme B and T-bet expression decreased significantly, whereas granzyme A, perforin, and eomesodermin were not affected. To further investigate the role of granzyme B in CTL-mediated platelet destruction, granzyme B inhibitor was added and platelet apoptosis was significantly inhibited. These results suggest that IL-27 negatively regulates CTL cytotoxicity toward platelets in ITP by decreasing granzyme B expression, which is associated with reduced T-bet expression. IL-27 may have a therapeutic role in treating ITP patients.

Chronic autoimmune thrombocytopenic purpura (AITP) is characterized by platelet-specific autoantibody production that is influenced by enhanced antiplatelet T-helper cell reactivity. Costimulatory signals are absolutely required for T-cell activation and play key roles in the decision between tolerance and immunity. In this study we cultured T cells isolated from patients with chronic AITP to investigate the effects of the B7-blocking agent cytologic T-lymphocyte-associated antigen 4-immunoglobulin (CTLA4-Ig), and cyclosporin A (CsA), alone or in combination, on induction of platelet-specific T-cell anergy. The data showed that in most cases CTLA4-Ig and/or CsA could induce tolerance toward platelet antigens based on anergy. It could be overcome by stimulation with unrelated antigens, demonstrating its platelet specificity. The anergy is associated with lack of interleukin 2 (IL-2) and withheld by exogenous IL-2, emphasizing the pivotal role of IL-2 suppression in the induction of platelet-specific anergy. We also prospectively evaluated the efficacy of CsA therapy in patients with refractory AITP and observed that the response to CsA treatment in vivo was associated with the inhibiting sensitivity of platelet-reactive T cells to CsA in vitro. This suggests that the sensitivity of T cells to CsA in vitro could serve as a reliable parameter in predicting the efficacy of CsA for patients with refractory AITP. CTLA4-Ig may become a promising new therapeutic agent for the treatment of chronic AITP, and the combination of CTLA4-Ig and CsA would be a more powerful strategy for the management of refractory AITP.

Introduction: Approximately 30% of patients with immune thrombocytopenia (ITP) fail to respond to the first- and/or second-line treatments (corticosteroids, intravenous immunoglobulin, rituximab, splenectomy, and thrombopoietin-receptor agonists). For those patients, the management is challenging. Decitabine (DAC), a demethylating agent with a dual mechanism of action: the demethylating effect leading to cell differentiation at low dose and cytotoxic activity leading to cell death at high concentration, has been used in the management of myelodysplastic syndrome (MDS) with a considerable platelet response during the past decade. Recent studies proved that low-dose DAC was sufficient to show a therapeutic effect with no obvious cytotoxicity. Our previous and other studies have demonstrated that low-dose DAC could promote megakaryocyte maturation and platelet production. These findings suggest a possible therapeutic role of low-dose DAC in the management of ITP. We hereby present the preliminary results of a prospective, multicenter, open-labeled study evaluating the efficacy and safety of low-dose DAC for ITP patients. Methods: ITP patients, who failed to respond to corticosteroids, intravenous immunoglobulin, rituximab, and/or thrombopoietin-receptor agonists from 9 centers, were enrolled in the study. The study protocol was approved by the ethics committee on medical research of each participating site. All patients provided written informed consent in accordance with the Declaration of Helsinki. DAC was given intravenously at 3.5mg/m2 for 3 days/cycle for 3 cycles with a 4-week interval between cycles. The primary end points were complete response (CR), response (R), overall response (OR). All the criteria were consistent with the standardization of terminology, definitions and outcome criteria in immune thrombocytopenia proposed by the international working group (RodeghieroF, et al. Blood, 2009, 113:2386-2393). Secondary end points were bleeding scores, time to response (TTR), duration of response and adverse events. Adverse events were evaluated according to Common Terminology Criteria for Adverse Events, version3.0. This clinical trial was registered at http://clinicaltrials.gov as NCT 01568333. Results: A total of 20 ITP patients were recruited. The clinical characteristics were shown in Table 1. At the end of the 12th week of the initial treatment, CR was achieved in 1 patient (5%) and R was achieved in 9 patients (45%). The OR rate was 50%. During the follow-up period, 1 patient initially stabilized at R and subsequently improved to CR at the 20th week. Therefore, CR, R and OR rates were 10% (2/20), 40% (8/20) and 50% (10/20), respectively. In patients who achieved CR and R, the median (range) TTR was 22 days (8-38 days). The median (range) follow-up time was 24 weeks (13-40 weeks). The platelet counts of patients who achieved CR and R were shown in Figure 1. The follow-up of our study is in progress. Adverse events were observed in 2 patients, one had nausea and the other was mild fever. No adverse events exceeded grade 1. Conclusion: Although the sample size is small, with a relatively short follow-up period limited by now, our study suggests that low-dose DAC is effective and safe in the management of ITP patients. Disclosures No relevant conflicts of interest to declare.

Immune thrombocytopenia (ITP) is an acquired autoimmune disease characterized by transient or persistent decrease of platelet count. ITP is the most common cause of thrombocytopenia in early pregnancy. Patients with severe thrombocytopenia (platelet count < 20 ×109/L) are at risk of spontaneous bleeding, postpartum hemorrhage and placental abruption. The aim of this study is to determine the efficacy and the safety of recombinant human thrombopoietin (rhTPO) in the management of ITP in pregnancy. This study is registered at www.clinicaltrials.gov as NCT02391272. Patients at eight centers in China were enrolled. Enrollment criteria were pregnant women aged between 18 and 50, who failed to respond to first-line treatments of ITP and/or were refractory to platelet transfusion. Patients' platelet counts were below 30×109/L with bleeding manifestations. Gestational age of the patients was over 12 weeks. Informed consent was obtained from each patient in accordance with the Declaration of Helsinki. Thirty-one patients were enrolled into the study. The median age of the pregnant ITP patients was 26 years (range 19 - 39 years) and 90.6% (29/31) were primigravidae. The median gestational age was 24 weeks (12 - 38 weeks). The median baseline platelet count was 10×109/L (range 1 - 29×109/L). 74.2% (23/31) of these patients were diagnosed as ITP before pregnancy and 25.8 % (8/31) during pregnancy. All eligible participants received rhTPO at an initial dose of 300U/kg once daily subcutaneously for 14 days, 74.2% (23/31) of these patients responded to the initial 14-day rhTPO therapy, including 10 CR and 13 R. Eight patients were NR though their platelet counts rose mildly. The median platelet count of responder was 100×109/L (range from 30 to 250×109/L) at day 14. Then the responders received sequential maintenance therapy through the end of week 12 after delivery. To reduce the risk of thrombocytosis during maintenance, dose was tapered to 300U/kg every other day when platelet counts exceeded 50 ×109/L and treatment stopped when platelet counts above 100×109/L. Only one responder had a transient loss of response due to influenza. After dose adjustment of rhTPO from 300U/kg every other day to 300U/kg every day, the platelet count exceeded 50×109/L in the next visit. The platelet counts gradually dropped after withdrawal of rhTPO. The relapse free survival (platelet count at least 30×109/L) at week 4 and week 12 after withdrawal of rhTPO was 69.6%(16/23) and 21.7%(5/23), respectively (Figure 1). Safety and adverse events were evaluated in all 31 participants. rhTPO was well tolerated. Only mild previously reported adverse events were observed, including one case of dizziness, one of fatigue and one of pain at injection site. There were no new reported adverse events during the observation period and no adverse event-related study withdrawals. In all the 31 newborns, the median age of gestation was 39 weeks (range 36-40, 3 cases had age of gestation< 37 weeks); median birth weight was 3.1 kg (range 2.3-4.2kg, 2 had birth weight This study demonstrates that rhTPO is a potentially effective, safe and fast-onset treatment for ITP in pregnancy refractory to fist-line therapy and platelet transfusion. Our study paved the way for the clinical application of rhTPO and other thrombopoietic agents in the management of ITP in pregnancy. Disclosures No relevant conflicts of interest to declare.

INTRODUCTION Primary immune thrombocytopenia (ITP) is an acquired autoimmune disease characterized by a reduced platelet count and an increased risk of bleeding. Although immense research has improved our understanding of ITP and led to impressive therapeutics, the pathogenesis is still not completely clear and a large subset of patients are refractory to first- and second-line therapies.In this study, we investigated the involvement of a cluster of inflammation-related genes, including CD24, FCRL3, CD226, IL2, IRF5, ITGAM, NLRP3, CARD8, PTPN22, SH2B3, STAT4, TNFAIP3, and TRAF1 genes. METHODS In this case-control study, 261 ITP inpatients (92 males and 169 females) with a mean age of 39.59 ± 17.52 years were recruited from Qilu Hospital, Shandong University between January 2007 to April 2016. In addition, 154 healthy participants (60 males and 94 females) with a mean age of 44.82 ± 13.63 years were enrolled as a control group. Inflammation-related single nucleotide polymorphism (SNP) genotyping was performed on the Sequenom MassARRAY iPLEX platform. SNPs included CD24 rs8734/rs52812045, FCRL3 rs11264799, rs7528684, rs945635, rs3761959, CD226 rs763361, IL2 rs6822844, IRF5 rs2004640, rs2280714, rs10954213, ITGAM rs1143679, NLRP3 rs35829419, rs4353135, rs10754558, CARD8 rs2043211, PTPN22 rs33996649, rs1310182, SH2B3 rs3184504, STAT4 rs7574865, rs10181656, TNFAIP3 rs6920220, rs10499194, rs2230926, rs5029939, TRAF1 rs10818488. SPSS (Ver 22.0, Chicago, IL) was used to perform statistical analysis. The results of genotyping were analyzed with t or ¦Ö2 tests. A Fisher's exact test was performed when expected frequencies were less than 5. Univariate and multivariate analyses were then performed by non-conditional logistic regression adjusting for age and gender. P values < 0.05 were considered significant. The two-tailed significance level was set at 0.05. RESULTS Genotype frequencies of these inflammation-related SNPs in healthy controls were consistent with the Hardy-Weinberg equilibrium (p > 0.05). Genotype distributions in ITP patients and controls are detailed in Table 1. Statistical analysis revealed a significant difference in TNFAIP3 rs10499194 and CD24 rs52812045 between patients and controls (Table 1). Genotype frequencies of other SNPs were not significantly different (p > 0.05). However, when allelic frequencies were compared, significant differences were not found in any genotyped SNPs (p > 0.05). ITP is classified by duration into newly diagnosed (less than 3-month duration), persistent (3- to 12-month duration) and chronic (1-year duration). According to ordinal logistic regression, the genotype frequency of IRF5 rs2004640 was significantly different between the 3 ITP phases. Compared with heterozygous carriers, homozygous carriers of major allele were found in higher frequency in newly diagnosed patients. The OR for GG vs. GT was 0.472 (95%CI = 0.289-0.770, p = 0.003). Patients with clinically relevant bleeding who require mandated treatment or additional therapeutic intervention are defined as having severe ITP. The inflammation-related SNPs were not significantly different between severe and non-severe patients (p > 0.05). Interestingly, genotype frequency distribution of CARD8 rs2043211 was significantly higher in refractory patients than in non-refractory patients (OR = 4.191, 95%CI = 1.026-14.567, p = 0.040), while allelic frequencies were not significantly different. We also compared corticosteroid-sensitive and corticosteroid-resistant ITP patients. We found that the genotype frequency of NLRP3 rs4353135 was significantly different between the two groups, and heterozygous genotype was more frequent in corticosteroid-sensitive patients (OR = 0.413, 95%CI = 0.203-0.841, p = 0.015). No significant difference was observed in other polymorphisms. CONCLUSIONS Our study identifies SNPs that differ based on the presence of ITP (TNFAIP3 rs10499194 and CD24 rs52812045), the phase of ITP (IRF5 rs2004640), refractoriness (CARD8 rs2043211), and corticosteroid sensitivity (NLRP3 rs4353135) in Chinese Han population. These important findings may lead to more tailored treatment strategies for ITP patients. Further study will dissect how these SNPs exert their roles to affect the pathogenesis of ITP. Disclosures No relevant conflicts of interest to declare.

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.

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 γ (IFNγ), 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-γ. 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-γ. Blockade of BAFF by BR3-Fc might be a promising therapeutic approach for ITP.

Introduction Oral prednisone (PDN) is conventional standard initial management for adult immune thrombocytopenia (ITP). In recent years single arm studies propose that high-dose dexamethasone (HD-DXM) improves response rate and remission duration and reduces adverse effects, though a randomized trial demonstrated no superiority of HD-DXM. It still lacks evidence-based conclusion on which one is better in efficacy and safety. We conducted a prospective randomized multicenter clinical trial to compare the efficacy and safety of HD-DXM and PDN as first-line strategy for adult newly diagnosed ITP. Methods We enrolled adult newly diagnosed ITP patients aging between 18 and 80 years, peripheral PLT count

Background:Immune thrombocytopenia (ITP) is a common bleeding disorder caused primarily by autoantibodies against platelet GPIIbIIIa (70-80%) and/or GPIb-complex (20-40%). Current theory suggests antibody-mediated platelet destruction occurs in the spleen, via macrophages through Fc-FcγR interactions. However, evidence from us and others demonstrated that anti-GPIbα, but not anti-GPIIbIIIa, can induce thrombocytopenia via an Fc-independent pathway, which is resistant to intravenous IgG (IVIG) therapy in murine ITP models (Blood 2006) and subsequent IVIG studies in human ITP patients, including our recent large patient cohort study (JTH 2014). This suggests that binding of anti-GPIbα antibodies may induce platelet clearance through a presently unidentified mechanism different than that of anti-GPIIbIIIa. Methods: We developed unique mouse anti-mouse monoclonal antibodies (mAbs) in GPIIIa-/- or GPIba-/- mice, which also recognize GPIbα and GPIIbIIIa of different species including human. Flow cytometry, immunofluorescence, and western blotting were used to evaluate whether these mAbs induced platelet activation, neuraminidase-1 translocation and desialylation of the heavily glycosylated GPIbα in the presence of sialidase inhibitor N-acetyl-2,3-dehydro-2-deoxy neuraminic acid (DANA). These experiments were repeated with human platelets and human ITP patient plasma. We further investigated the effects of anti-GPIbα antibodies on platelet activation, desialylation and clearance in vivo; BALB/c mice were injected with anti-GPIbα or anti-GPIIbIIIa mAbs and following, platelet activation and desialylation were measured by flow cytometry. Hepatocytic Ashwell-Morell receptor (AMR) mediated anti-GPIbα platelet clearance in the liver was examined using immunohistochemistry or blocking the AMR with asialofetuin in both wild-type and macrophage depleted mice. Therapeutic administration of DANA in a murine ITP model assessed the significance of Fc-independent anti-GPIbα mediated platelet clearance in ITP. Results and Discussion: We found that anti-GPIbα, but not anti-GPIIbIIIa antibodies, induced significant P-selectin expression, JON/A binding, neuraminidase-1 translocation and desialylation in murine platelets. Interestingly, certain human platelets were activated (P-selectin expression) and desialylated in the presence of both anti-GPIbα and anti-GPIIbIIIa mAbs or ITP patient plasma. However, we demonstrate that the anti-GPIIbIIIa antibody mediated platelet effects are dependent on the FcγRIIa present exclusively on human platelets as FcγRII blocker IV.3 completely attenuated the response. In contrast, IV.3 had little effect on anti-GPIbα mediated platelet activation or desialylation. Anti-GPIbα Fab fragments and platelet signal pathway inhibitors demonstrate that anti-GPIbα mediated platelet activation and desialylation are consequences of GPIbα cross linking and are reinforced by a positive feedback loop. In vivo, we found significant increases in P-selectin and desialylation in anti-GPIbα injected mice, independent of IgG subclass. A significant role for the hepatic AMR in the clearance of deglycosylated platelets was observed; particularly in macrophage depleted mice whereby, although anti-GPIIbIIIa mediated platelet clearance was completely attenuated, anti-GPIbα mediated platelets clearance still occurred, but was completely rescued with asialofetuin. Immunohistochemistry revealed significant co-localization of anti-GPIbα opsonized platelets with AMR. These suggest the AMR is the dominant Fc-independent anti-GPIbα mediated platelet clearance pathway in the absence of macrophages. Remarkably, sialidase inhibitor DANA ameliorated anti-GPIbα mediated thrombocytopenia in mice. Thus, we demonstrate for the first time that anti-GPIbα antibodies induce platelet activation leading to GPIbα desialyation and platelet clearance via a novel Fc-independent pathway: the hepatic AMR. Our data also suggested that some anti-GPIIbIIIa autoantibodies in human patients may also induce platelet activation and desialylation via the platelet FcR signaling pathway. These findings may lead to novel therapeutic regimens including designating desialylation as a potential diagnostic biomarker and therapeutic target in the treatment of both anti-GPIIbIIIa and anti-GPIbα mediated and/or refractory ITP. Disclosures No relevant conflicts of interest to declare.

Introduction: ITP is an autoimmune hemorrhagic disorder characterized by increased platelet destruction and insufficient platelet production. The classical mechanisms of platelet destruction include premature platelet clearance mediated by antiplatelet autoantibodies in the reticuloendothelial system and cytotoxic T lymphocyte (CTL)-mediated platelet lysis. Platelet desialylation, the process of removing sialic acid residues from glycoproteins, which results in the exposure of beta-galactose on platelet surface, can initiate platelet recognition by asialoglycoprotein receptors (ASGPRs)-expressing liver macrophages and hepatocytes and then lead to platelet clearance in the liver. The status and role of platelet desialylation in ITP have not been studied yet. In this study, we detected the level of platelet desialylation and analyzed its association with antiplatelet autoantibody- and CTL- mediated platelet destruction in ITP. Methods: Ethylenediaminetetraacetic acid anti-coagulated whole blood was obtained from 74 active ITP patients and 34 matched controls. Platelets, CD8+ T lymphocytes and plasma were isolated. The levels of platelet desialylation in ITP and controls were evaluated by the binding level of FITC conjugated Ricinus communis agglutinin I (RCA-I) on platelets by flow cytometry. In addition, the modified antibody-specific immobilization of platelet antigens (MAIPA) and assay of CTL-mediated cytotoxicity towards platelets were performed to evaluate antiplatelet autoantibody- and CTL-mediated platelet destruction in ITP patients. Results: Our results showed that the binding level of RCA-I on platelets was significantly elevated in ITP patients compared with controls (Figure 1). Among these 74 ITP patients, 10 were single anti-GPIIb/IIIa positive, 13 were single anti-GPIb positive, 16 were positive for both, and 35 were negative for autoantibodies. There was neither significant difference in the binding level of RCA-I on platelets between patients with anti-GPIIb/IIIa and those without anti-GPIIb/IIIa antibodies, nor between patients with anti-GPIb and those without anti-GPIb antibodies (data not shown). Moreover, we conducted the assay of CTL-mediated cytotoxicity towards platelets in 40 ITP patients and 13 controls. Positively induced platelet apoptosis (greater than mean plus 2 SD for the controls) was detected in 22 of 40 ITP patients, which was regarded as group A. The other 18 patients was referred to as group B. Interestingly, the binding level of RCA-I was significantly higher in group A than that in group B (Figure 2). These findings indicated that the level of platelet desialylation was markedly elevated in ITP patients than controls, and significantly higher in ITP patients with CTL-mediated platelet destruction. We supposed that neuraminidase-1, which could translocate to platelet surface after platelet lesion and cause platelet desialylation, was elevated from an internal platelet store after CTL-mediated platelet destruction, and then caused increased beta-galactose exposure and led to further platelet clearance in the liver. The specific mechanism still needs further study. Conclusion: In conclusion, our results suggest that platelet desialylation may be involved in the pathogenesis of ITP and closely associated with CTL-mediated platelet destruction. Inhibiting platelet desialylation may be a novel therapeutic strategy for ITP patients with CTL-mediated platelet destruction. Figure 1 The level of platelet desialylation in ITP patients and controls. A. The representative flow cytometry results. B. The binding level of RCA-I was higher in ITP patients than controls (***P < 0.001). Figure 1. The level of platelet desialylation in ITP patients and controls. A. The representative flow cytometry results. B. The binding level of RCA-I was higher in ITP patients than controls (***P < 0.001). Figure 2 Figure 2. The level of platelet desialylation was higher in ITP patients with CTL-mediated platelet destruction. A. Induced platelet apoptosis by CTLs in controls, group A and group B. B. The representative scattergrams of platelet apoptosis. Dots in the lower right corner represented apoptotic platelets. C. The binding level of RCA-I was significantly higher in group A or B than controls (***P < 0.001; *P = 0.042). Moreover, it was higher in group A than group B (*P = 0.015). Disclosures No relevant conflicts of interest to declare.

Thrombopoietin (TPO) and TPO receptor agonists have substantially broadened the therapeutic options for patients with immune thrombocytopenia (ITP). Platelet count response of ITP is usually maintained during the medication; however, once it is stopped, platelet counts commonly drop to pretreatment levels. We report three corticosteroid-resistant ITP patients achieving sustained complete remission with one short-term application of recombinant human thrombopoietin (rhTPO). Thirty-two adult ITP patients (18 females and 14 males; age range 18-72 years, median 45 years) were enrolled between December, 2011 and July, 2013 at the Department of Hematology, Qilu Hospital, Shandong University. Patients were diagnosed according to the recently published criteria (Rodeghiero F et al, Blood 2009). All patients had a baseline platelet count (PC) of < 30 x 109/L and bleeding symptoms, and were resistant to prior corticosteroid therapy or splenectomy. Recombinant human thrombopoietin (rhTPO, a truncated and non-glycosylated TPO developed by 3SBIO Pharmaceutical Co., LTD, Shenyang, China, approved by China State Food and Drug Administration) was given subcutaneously at a daily dose of 1.0 µg/kg for 14 days. Withdrawal of rhTPO could be performed while platelet count rose to above 100 x 109/L in less than 14 days. Responses were required to be independent of supportive medications. The criteria for response were defined as follows: (1) complete response (CR): platelet count > 100 x 109/L; (2) response (R): platelet count > 30 x 109/L and at least 2-fold increase of the baseline platelet count and absence of bleeding; (3) no response (NR): platelet count < 30 x 109/L or less than 2-fold increase of the baseline platelet count or bleeding. Of the 32 ITP patients treated with rhTPO, the complete response (CR), response (R), overall response (OR) and no response (NR) rates were 37.50% (12/32), 28.12% (9/32), 65.62% (21/32) and 34.38% (11/32), respectively. Among the 32 patients, three (9.38%) acquired sustained complete response (i.e., platelet count > 100 x 109/L and no clinical symptoms for at least six months after the medication). Of these three patients, the time to peak response was 28, 20 and 28 days, and the peak values of platelet counts were 215 x 109/L, 245 x 109/L and 296 x 109/L, respectively. These three patients’ information was shown in Table 1. Their platelet count responses sustained for 56, 40 and 28 weeks without any ITP-specific treatments after the cessation of rhTPO (Figure 1). In addition, only mild adverse events (WHO grades 1-2) were observed, including fever, fatigue and insomnia.Table 1Characteristics of the three ITP patients who achieved a durable complete response associated with the use of rhTPO.PatientAge(years)SexBaseline Platelet Count (x109/L)rhTPO Daily Dose(µg/kg)rhTPO Duration(days)Time to Peak Response(days)Peak Value of Platelet Count(x109/L)Complete Remission off rhTPO(Weeks)172Female21122821556234Female51102024540354Male81142829628Figure 1Serial platelet counts of three ITP patients who achieved a definite rhTPO-induced sustained remission.Figure 1. Serial platelet counts of three ITP patients who achieved a definite rhTPO-induced sustained remission. In conclusion, some ITP patients can achieve sustained complete remission after a short-course of rhTPO. However, the mechanism behind this is unclear. Disclosures: No relevant conflicts of interest to declare.

Background Though great progresses had been achieved in the management of primary immune thrombocytopenia (ITP), still 25%-30% of the patients show no response to the conventional therapy. Both thrombopoietin (TPO) and rituximab (RTX) are recommended as the second-line treatments. TPO has shown potent activity in ITP, but the therapeutic efficacy is dependent on continual administration. Rituximab is an anti-CD20 chimeric monoclonal antibody which could induce prolonged remission in ITP. However, longer time to response was also reported. The combination of rhTPO and rituximab could complement each other in both mechanism of action and time window, exert powerful effect, which may be a choice for the glucocorticosteroids-resistant/relapsed patients. Method Patients with platelet counts ≤ 30 x109/L, or ≤ 50 x109/L with bleeding symptoms from 12 centers were enrolled in the study. Subjects enrolled in the study were assigned randomly as 1: 2 to RTX group and rhTPO plus RTX group. Eligibility criteria were set according to the recently published guideline (Rodeghiero F, et al, Blood 2009). Approval for the study was obtained from the Ethics Committee of the School of Medicine and Qilu Hospital. Informed consent in accordance with the Declaration of Helsinki was given to each patient. Rituximab (MabThera; Roche) was given with a fixed dose of 100mg weekly for four weeks. And in the combination group, rhTPO (Tebiao, 3SBio, China) was added in the regimen with a dose of 300U/kg/day in the first 14 days, and a modified frequency of rhTPO was given according to the platelet counts after two weeks, but no longer than four weeks. Primary outcomes include response (R), complete response (CR), no response (NR) and relapse. All the criteria were consent with the definition and outcome criteria. (Rodeghiero F, et al. Blood, 2009.) Secondary outcomes are listed below. Time to response (TTR) was considered as the duration from baseline to response. Time to relapse was also measured with Kaplan-Meier analysis. Besides, safety was reflected as adverse events graded according to the Common Toxicity Criteria. This clinical trial was registered at http://clinicaltrials.gov as NCT 01525836. Result A total of 114 patients were recruited: monotherapy group (n = 35) and combination group (n = 79). All patients failed to response to glucocorticosteroids or relapsed after corticosteroids treatment. Response rate was reached in 71.4% (25/35) of patients in monotherapy group vs. 78.5% (62/79) in the RTX and rhTPO combination group, while complete responses were achieved as 10/35 (28.6%) in RTX monotherapy group and 34/79 (43.0%) in rhTPO plus RTX group respectively. Median time to response was 28 days (range 4-90 days) in RTX group, while in RhTPO plus RTX group, TTR was 7 days (range 4-28 days) (P = 0.002). There was significantly longer time to relapse in the combination group (shown in Fig. 1). Adverse effects were observed in RTX group in 5 patients, most of whom were affected with inflammation. One patient was reported as viral myocarditis, and there were also complaints about chill, rash and hyperpyrexia. And incidence of adverse events was slightly increased in rhTPO plus RTX group. There were 14 adverse effects reported in the combination group, such as fatigue, pulmonary inflammatory, but all were below Grade 2. Conclusion Our findings suggest that combination of RTX and RTX in ITP yields shorter time to response compared with monotherapy of RTX, and moreover, combination extended time to relapse. Thus, combination therapy may represent an effective treatment option for glucocorticosteroids-resistant or relapsed patients. The results were compared with Gehan-Breslow-Wilcoxon Test. (P = 0.0140) Disclosures: No relevant conflicts of interest to declare.

Abstract 2223 Background: Both thrombopoietin (TPO) receptor agonist and rituximab were recommended as second-line options for adult primary immune thrombocytopenia (ITP) according to the 2010 International Consensus Guideline. The mechanism of rituximab therapy mainly refers to inhibition of accelerated platelet destruction mediated by autoantibody, while the latter, TPO receptor agonist, is reported to be effective in the salvage of impaired platelet production in patients. Despite the considerable response rate (about 50–70%) and sustained response (about 20–40%), the median time to response (TTR) was about 5.5 weeks for patients undergoing rituximab therapy, indicating high bleeding risk in the early stage of the treatment (Arnold, et al. Ann Intern Med. 2007;146:25–33.). On the other hand, novel TPO receptor agonists could initiate fast but unenduring responses. We hereby report a novel therapy which combines low-dose rituximab with short-term application of a TPO receptor agonist, the recombinant human thrombopoietin (rh-TPO), based on the preliminary results of an ongoing multi-centered, non-randomized clinical study in corticosteroid-resistant adult ITP patients. Patients: Twenty-one adult ITP patients were enrolled since December, 2009. All the subjects had a baseline platelet count (PC) of Methods: Rituximab was given intravenously at a dose of 100 mg weekly for 4 consecutive weeks (Day 1, 8, 15, 22). Rh-TPO (TPIAO™, a product of Sunshine Pharmaceutical Co Ltd, China, approved by China State Food and Drug Administration) was given subcutaneously at a dose of 1.0 μg/kg daily for 14 days (Day 1–14). PC was monitored every three or four days until day 22, followed by tests every week. Responses were required to be independent of measures and supportive platelet transfusion. The criteria for response were defined as follows: (1) complete response (CR): platelet count >100×109/L and; (2) response (R): platelet count>30×109/L and at least 2-fold increase the baseline count and absence of bleeding; (3) No response (NR): platelet counts Results: All the patients received the therapy, but 4 were lost during follow-up. The efficacy of treatment is outlined in Table 1. Compared with rituximab alone, the combination therapy resulted in a rapid response (TTR 9 vs. 21–44 days) with similar response rate (CR 57% vs. 30–60%, OR 76% vs. 50–70%). Moreover, the rh-TPO seemed to not only shorten the TTR but also significantly increase the sustained response (69% vs. 20–40%), indicating a synergistic effect with rituximab. In addition, there was no toxicity overlap observed. Adverse events recorded were mild (WHO grades 1–2), including fever (2), fatigue (2), myalgia (2), secondary infection (2), chills (1),and insomnia (1). No serious adverse events occurred during the course of treatment and the period of follow-up. Conclusions: The efficacy of this novel combination therapy are very satisfactory, considering the higher overall and complete response, shorter TTR as well as higher sustained response, in comparison with administrating rituximab alone. However, the combination should be used with caution and needs further and larger scale observation, especially to monitor for serious adverse effects such as thrombosis, bone marrow fibrosis and infection. Disclosures: No relevant conflicts of interest to declare.

There is growing evidence that tolerogenic dendritic cells (DCs) play an important role in maintaining peripheral tolerance through the induction of anergic or regulatory T cells. However, in humans, little is known about the ability of tolerogenic DCs to induce tolerance to autoantigens in autoimmune patients. Idiopathic thrombocytopenic purpura (ITP) is an immune-mediated disease in which platelets are destroyed by antiplatelet autoantibodies. Here, we explored in vitro the ability of four subsets of tolerogenic DCs (i.e., immature DCs (imDCs), IL-10-modulated DCs (IL-10-DCs), vasoactive intestinal peptide-modulated DCs (VIP-DCs) or plasmacytoid DCs (pDCs)) derived from patients with ITP, to induce an anergic state or regulatory T cells in autologous platelet glycoprotein (GP)-specific T cells. GPIIb/IIIa-reactive T cells were preincubated with GPIIb/IIIa-loaded imDCs, IL- 10-DCs, pDCs or VIP-DCs, and then rechallenged with autologous mature DCs (mDCs) in the presence of GPIIb/IIIa. Only when T cells were cultured with GPIIb/IIIa-loaded VIP-DCs in primary incubation, inhibited proliferation of GPIIb/IIIa-reactive T cells could be observed at rechallenge with GPIIb/IIIa-loaded mDCs. The anergic state of VIPDC- primed GPIIb/IIIa-reactive T cells could be reversed when rechallenged with GPIIb/ IIIa-loaded mDCs in the presence of a high concentration of exogenous IL-2. Meanwhile, GPIIb/IIIa-reactive T cells were also cultured with VIP-DCs loaded with tetanus toxoid (TT). In contrast to T cells pretreated with GPIIb/IIIa-loaded VIP-DCs, GPIIb/IIIareactive T cells pretreated with TT-loaded VIP-DCs proliferated when rechallenged with GPIIb/IIIa-loaded mDCs, which demonstrated that the induced anergy of autoreactive T cells is antigen specific. Additionally, functional analysis showed that VIP-DC-modulated T cells could not suppress the proliferation of newly induced GPIIb/IIIa-reactive T cells when cocultured with GPIIb/IIIa-loaded mDCs. These results indicated that VIP-DCs could induce autoreactive T cells anergic but not functionally suppressive. Moreover, we found that coculture of VIP-DCs with autologous PBMCs resulted in reduced production of anti-GPIIb/IIIa antibodies, suggesting that GPIIb/IIIa-reactive T cells lost their helper function for inducing autoantibody production by B cells. In contrast, reduced antibody production could not be found when autologous PBMCs were cocultured with imDCs, IL-10-DCs or pDCs. In conclusion, our studies revealed the therapeutic potential of VIPDCs, compared with imDCs, IL-10-DCs or pDCs, to induce autoreactive T-cell anergy to GP antigens, which would in turn facilitate the reestablishment of autoantigen-specific tolerance in patients with ITP.