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Background Myelofibrosis (MF) is a clonal disorder of hemopoietic stem/progenitor cells (HSPCs) with high prevalence in elderly patients and mutations in three driver genes (JAK2, MPL, or CALR). Around 10–15% of patients are triple-negative (TN) for the three driver mutations and display significantly worse survival. Circulating extracellular vesicles (EVs) play a role in intercellular signaling and are increased in inflammation and cancer. To identify a biomolecular signature of TN patients, we comparatively evaluated the circulating HSPCs and their functional interplay with the microenvironment focusing on EV analysis. Methods Peripheral blood was collected from MF patients (n = 29; JAK2V617F mutation, n = 23; TN, n = 6) and healthy donors (HD, n = 10). Immunomagnetically isolated CD34+ cells were characterized by gene expression profiling analysis (GEP), survival, migration, and clonogenic ability. EVs were purified from platelet-poor plasma by ultracentrifugation, quantified using the Nanosight technology and phenotypically characterized by flow cytometry together with microRNA expression. Migration and survival of CD34+ cells from patients were also analyzed after in vitro treatments with selected inflammatory factors, i.e. (Interleukin (IL)-1β, Tumor Necrosis Factor (TNF)-α, IL6) or after co-culture with EVs from MF patients/HD. Results The absolute numbers of circulating CD34+ cells were massively increased in TN patients. We found that TN CD34+ cells show in vitro defective functions and are unresponsive to the inflammatory microenvironment. Of note, the plasma levels of crucial inflammatory cytokines are mostly within the normal range in TN patients. Compared to JAK2V617F-mutated patients, the GEP of TN CD34+ cells revealed distinct signatures in key pathways such as survival, cell adhesion, and inflammation. Importantly, we observed the presence of mitochondrial components within plasma EVs and a distinct phenotype in TN-derived EVs compared to the JAK2V617F-mutated MF patients and HD counterparts. Notably, TN EVs promoted the survival of TN CD34+ cells. Along with a specific microRNA signature, the circulating EVs from TN patients are enriched with miR-361-5p. Conclusions Distinct EV-driven signals from the microenvironment are capable to promote the TN malignant hemopoiesis and their further investigation paves the way toward novel therapeutic approaches for rare MF.

Background Here, we isolated, expanded and functionally characterized regulatory T cells (Tregs) from patients with end stage kidney and liver disease, waiting for kidney/liver transplantation (KT/LT), with the aim to establish a suitable method to obtain large numbers of immunomodulatory cells for adoptive immunotherapy post-transplantation. Methods We first established a preclinical protocol for expansion/isolation of Tregs from peripheral blood of LT/KT patients. We then scaled up and optimized such protocol according to good manufacturing practice (GMP) to obtain high numbers of purified Tregs which were phenotypically and functionally characterized in vitro and in vivo in a xenogeneic acute graft-versus-host disease (aGVHD) mouse model. Specifically, immunodepressed mice (NOD-SCID-gamma KO mice) received human effector T cells with or without GMP-produced Tregs to prevent the onset of xenogeneic GVHD. Results Our small scale Treg isolation/expansion protocol generated functional Tregs. Interestingly, cryopreservation/thawing did not impair phenotype/function and DNA methylation pattern of FOXP3 gene of the expanded Tregs. Fully functional Tregs were also isolated/expanded from KT and LT patients according to GMP. In the mouse model, GMP Tregs from LT or KT patient proved to be safe and show a trend toward reduced lethality of acute GVHD. Conclusions These data demonstrate that expanded/thawed GMP-Tregs from patients with end-stage organ disease are fully functional in vitro. Moreover, their infusion is safe and results in a trend toward reduced lethality of acute GVHD in vivo, further supporting Tregs-based adoptive immunotherapy in solid organ transplantation. Electronic supplementary material The online version of this article (10.1186/s12967-019-2004-2) contains supplementary material, which is available to authorized users.

The role of circulating microvesicles (MVs) in Myelofibrosis (MF) and Essential Thrombocythemia (ET) is far to be defined. Here we found that 1) circulating megakaryocyte-MVs were reduced in MF and ET while platelet-MVs were increased; 2) the proportion of circulating megakaryocyte- and platelet-MVs was associated with disease severity in MF; 3) ruxolitinib normalized the profile of circulating megakaryocyte- and platelet-MVs in spleen responders MF patients only. Of note, a cut-off value of 19.95% of circulating megakaryocyte-MVs predicts ruxolitinib spleen response. In light of these findings, circulating megakaryocyte/platelet-MVs may have a tissue specific diagnostic and prognostic role in MF.

Although infectious complications represent a relevant cause of morbidity and mortality in patients with myelofibrosis (MF), little is known about their incidence, outcome and risk factors. We retrospectively evaluated a cohort of 507 MF patients, diagnosed between 1980 and 2014 in five Italian hematology centers, to define the epidemiology of infections and describe the impact of ruxolitinib (RUX) treatment. Overall, 112 patients (22%) experienced 160 infectious events (grade 3-4, 45%) for an incidence rate of 3.9% per patient-year. Infections were mainly bacterial (78%) and involving the respiratory tract (52% of cases). Also, viral (11%) and fungal infections (2%) were recorded. Overall, infections were fatal in 9% of the cases. Among baseline features, high/intermediate-2 IPSS category (HR 1.8, 95%CI:1.2-2.7; p=0.02) and spleen length ≥10 cm below left costal margin (HR 1.6, 95%CI:1.1-2.5; p=0.04) were associated with higher infectious risk in multivariate analysis. Overall, the rate of infections was higher in the cohort of 128 RUX-treated patients (44% vs. 20%, p

Introduction: Myelofibrosis (MF) is a myeloproliferative neoplasm (MPN) characterized by clonal hemopoiesis, inflammatory microenvironment and mutations in JAK2, MPL, or CALR genes. Around 10% of patients (pts) do not carry the 3 mutations (Triple negative (TN)). Microparticles (MPs; 0.1-1 μm) are small vesicles deriving from plasma membrane during homeostasis and cell activation of a wide variety of cells with a role in intercellular signalling. Circulating MPs are increased in inflammation and cancer including MPN. However, their role in the pathogenesis of MF is still elusive. Here we compared phenotype, function and microRNAs (miRs) expression of isolated MPs of JAK2V617F mutated and TN pts. Methods: Peripheral blood was collected from 10 MF pts (at diagnosis or out of cytotoxic treatment for at least 3 months) and 10 age/sex-matched healthy donors (HD). MF pts were JAK2V617F mutated (n=5) and TN n=5). MPs were purified from 2 mL of platelet poor plasma by ultracentrifugation and quantified using Nanosight technology. Isolated MPs were characterized by flow cytometry (CytoFLEX, Beckman Coulter) for the expression of platelets (PLT:CD61+CD62P+) and megakaryocytes (MK:CD61+CD62P-) markers. MPs were then analyzed for their ability to modulate the in vitro viability of immunomagnetically isolated CD34+ cells from MF pts and cord blood (CB). MiRs expression of isolated MPs (10e9) from 3 JAK2V617F mutated, 3 TN pts and 3 HD was investigated using TaqMan™ Array Human MicroRNA A Cards (ThermoFisher). Results: The mean number of plasma MPs isolated from MF pts was similar to HD (5*10e10/mL). However, flow cytometry analysis revealed that the mean percentage of PLT-MPs was significantly increased (p

Inflammation may play a role in cancer. However, the contribution of cytokine-mediated crosstalk between normal hemopoietic stem/progenitor cells (HSPCs) and their (inflammatory) microenvironment is largely elusive. Here we compared survival, phenotype, and function of neonatal (umbilical cord blood (CB)) and adult (normal G-CSF-mobilized peripheral blood (mPB)) CD34+cells afterin vitroexposure to combined crucial inflammatory factors such as interleukin- (IL-) 1β, IL-6, tumor necrosis factor- (TNF-)α, or tissue inhibitor of metalloproteinases-1 (TIMP-1). To mimic bone marrow (BM) niche, coculture experiments with normal BM stromal cells (BMSCs) were also performed. We found that combined inflammatory cytokines increased only thein vitrosurvival of CB-derived CD34+cells by reducing apoptosis. Conversely, selected combinations of inflammatory cytokines (IL-1β + TNF-α, IL-6 + TNF-α, and IL-1β + TNF-α + TIMP-1) mainly enhanced thein vitroCXCR4-driven migration of mPB-derived CD34+cells. TNF-α, alone or in combination, upregulated CD44 and CD13 expression in both sources. Finally, BMSCs alone increased survival/migration of CB- and mPB-derived CD34+cells at the same extent of the combined inflammatory cytokines; importantly, their copresence did not show additive/synergistic effect. Taken together, these data indicate that combined proinflammatory stimuli promote distinctin vitrofunctional activation of neonatal or adult normal HSPCs.

Myelofibrosis (MF) is a clonal neoplasia associated with chronic inflammation due to aberrant cytokine production. Mutations in Janus Kinase-2 (JAK2), calreticulin (CALR) and myeloproliferative leukemia protein (MPL) genes have been recently associated to MF and they all activate the JAK/STAT signaling pathway. Since this pathway is essential in shaping the immune response, we investigated the role of circulating immune subsets and cytokines in 38 patients (20 carrying JAK2(V617F),13 exon-9 CALR mutation and 5 triple negative). In comparison to healthy donors, patients presented a reduced amount of circulating dendritic cells (DCs) associated with a defective ability of monocytes in differentiating into DCs. In addition, we found a reduction in circulating T-helper (Th)1 and Th17 and hypo-functional innate lymphoid cells (ILC). Results analyzed according to the mutational status showed that patients carrying JAK2(V617F) mutation had a reduction in Th17, myeloid-DCs and effector Tregs as well as increased ILC1 and cytokine producing Tregs. The CALR mutated patients revealed high ILC3 levels, reduced Th1 and their monocytes had a reduced capacity to mature in vitro into fully committed DCs. Their Tregs were also less effective in inhibiting the proliferation of autologous effector T-cells due to an increased proliferative status induced by CALR mutation. Triple negative patients presented a reduced amount of total circulating CD3, effectors Tregs and Th1 with increased ILC1.

Background aims Growing evidence supports the therapeutic potential of bone marrow (BM)-derived stem/progenitor cells for end-stage liver disease (ESLD). We recently demonstrated that CD133+ stem/progenitor cell (SPC) reinfusion in patients with ESLD is feasible and safe and improve, albeit transiently, liver function. However, the mechanism(s) through which BM-derived SPCs may improve liver function are not fully elucidated. Methods Here, we characterized the circulating SPCs compartment of patients with ESLD undergoing CD133+ cell therapy. Next, we set up an in vitro model mimicking SPCs/liver microenvironment interaction by culturing granulocyte colony-stimulating factor (G-CSF)-mobilized CD133+and LX-2 hepatic stellate cells. Results We found that patients with ESLD show normal basal levels of circulating hematopoietic and endothelial progenitors with impaired clonogenic ability. After G-CSF treatment, patients with ESLD were capable to mobilize significant numbers of functional multipotent SPCs, and interestingly, this was associated with increased levels of selected cytokines potentially facilitating SPC function. Co-culture experiments showed, at the molecular and functional levels, the bi-directional cross-talk between CD133+ SPCs and human hepatic stellate cells LX-2. Human hepatic stellate cells LX-2 showed reduced activation and fibrotic potential. In turn, hepatic stellate cells enhanced the proliferation and survival of CD133+ SPCs as well as their endothelial and hematopoietic function while promoting an anti-inflammatory profile. Discussion We demonstrated that the interaction between CD133+ SPCs from patients with ESLD and hepatic stellate cells induces significant functional changes in both cellular types that may be instrumental for the improvement of liver function in cirrhotic patients undergoing cell therapy.

Although infectious complications represent a relevant cause of morbidity and mortality in patients with myelofibrosis (MF), little is known about their incidence, outcome and risk factors. We retrospectively evaluated a cohort of 507 MF patients, diagnosed between 1980 and 2014 in five Italian hematology centers, to define the epidemiology of infections and describe the impact of ruxolitinib (RUX) treatment. Overall, 112 patients (22%) experienced 160 infectious events (grade 3-4, 45%) for an incidence rate of 3.9% per patient-year. Infections were mainly bacterial (78%) and involving the respiratory tract (52% of cases). Also, viral (11%) and fungal infections (2%) were recorded. Overall, infections were fatal in 9% of the cases. Among baseline features, high/intermediate-2 IPSS category (HR 1.8, 95%CI:1.2-2.7; P = 0.02) and spleen length ≥10 cm below left costal margin (HR 1.6, 95%CI:1.1-2.5; P = 0.04) were associated with higher infectious risk in multivariate analysis. Overall, the rate of infections was higher in the cohort of 128 RUX-treated patients (44% vs. 20%, P

Myelofibrosis (MF) is a clonal neoplasia associated with chronic inflammation due to aberrant cytokine production. Mutations in Janus Kinase-2 (JAK2), calreticulin (CALR) and myeloproliferative leukemia protein (MPL) genes have been recently associated to MF and they all activate the JAK/STAT signaling pathway. Since this pathway is essential in shaping the immune response, we investigated the role of circulating immune subsets and cytokines in 38 patients (20 carrying JAK2(V617F),13 exon-9 CALR mutation and 5 triple negative). In comparison to healthy donors, patients presented a reduced amount of circulating dendritic cells (DCs) associated with a defective ability of monocytes in differentiating into DCs. In addition, we found a reduction in circulating T-helper (Th)1 and Th17 and hypo-functional innate lymphoid cells (ILC). Results analyzed according to the mutational status showed that patients carrying JAK2(V617F) mutation had a reduction in Th17, myeloid-DCs and effector Tregs as well as increased ILC1 and cytokine producing Tregs. The CALR mutated patients revealed high ILC3 levels, reduced Th1 and their monocytes had a reduced capacity to mature in vitro into fully committed DCs. Their Tregs were also less effective in inhibiting the proliferation of autologous effector T-cells due to an increased proliferative status induced by CALR mutation. Triple negative patients presented a reduced amount of total circulating CD3, effectors Tregs and Th1 with increased ILC1. Overall, we have demonstrated that in MF different mutations lead to phenotypic and functional alterations in different immune subsets that may have a potential role in disease progression and susceptibility to infections.

Splenectomy is a time-honoured well established approach for patients with steroid-resistant immune thrombocytopenia (ITP). However, due to the more recent availability of therapeutic options alternative to splenectomy, such as rituximab and agonists of the thrombopoietin-receptor, the choice of second-line therapy is challenging. Platelet kinetics has been widely used to predict response to splenectomy. We describe the outcome of 70 chronic ITP patients who performed a platelet kinetic study after failure of front-line corticosteroids and subsequently underwent open splenectomy. After a median follow-up from surgery of 20 years, 62 (88.5%) patients responded to splenectomy and 9 patients (13%) relapsed. Achieving a complete response (CR) significantly predicted a higher probability long-term stable response. The pattern of platelet sequestration was predominantly splenic in 52 patients (74%), predominantly hepatic in 12 patients (17%), and diffuse in 6 (9%). Patients with nonsplenic (diffuse and hepatic) sequestration showed significantly lower overall responses compared to patients with splenic captation (P = 0.002). A nonsplenic sequestration significantly correlated with lower CR rate and, among CR patients, predicted an increased risk of relapse. Also, the probability of stable responses in nonsplenic uptake patients was substantially lower than in patients with splenic uptake (85% vs. 50%, P = 0.0083). Platelet life span and platelet turnover did not correlate with response and relapse rate. Overall, splenic sequestration was able to predict not only a better quality, but also a higher durability of the responses. However, it should be enphasized that the response rate and duration of response even in patients with nonsplenic uptake were similar or even superior to those reported in patients treated with rituximab as first option. Am. J. Hematol. 89:1047–1050, 2014. © 2014 Wiley Periodicals, Inc.

Growing evidence supports the therapeutic potential of bone marrow (BM)-derived stem/progenitor cells for end-stage liver disease (ESLD). We recently demonstrated that CD133Here, we characterized the circulating SPCs compartment of patients with ESLD undergoing CD133We found that patients with ESLD show normal basal levels of circulating hematopoietic and endothelial progenitors with impaired clonogenic ability. After G-CSF treatment, patients with ESLD were capable to mobilize significant numbers of functional multipotent SPCs, and interestingly, this was associated with increased levels of selected cytokines potentially facilitating SPC function. Co-culture experiments showed, at the molecular and functional levels, the bi-directional cross-talk between CD133We demonstrated that the interaction between CD133

Myelofibrosis (MF) is a clonal neoplasia of the hemopoietic stem/progenitor cells associated with genetic mutations in the Janus kinase 2 (JAK2), myeloproliferative leukemia virus oncogene (MPL), and calreticulin (CALR) genes. MF is also characterized by a state of chronic inflammation. Calreticulin (CRT), as a multifunctional protein, is involved in a spectrum of cellular processes including inflammation, autoimmunity, and cancer initiation/progression. Based on this background, we hypothesised that in MF circulating CRT might reflect the inflammatory process. In the present study we show that circulating CRT is increased in MF patients compared to healthy controls. Also, in MF, CRT levels highly correlate with bone marrow fibrosis, splenomegaly, and Interleukin-6 (IL-6) plasma levels. In turn, higher IL-6 levels also correlated with disease severity in terms of increased spleen size, bone marrow fibrosis, number of circulating CD34+cells, and lower hemoglobin values. These results demonstrate that the circulating CRT takes part in the inflammatory network of MF and correlates with aggressiveness of the disease.

In the last years, rituximab (RTX) and agonists of the thrombopoietin receptor (TPO-R) eltrombopag and romiplostim have provided new treatment options in persistent and chronic immune thrombocytopenia (ITP). Here, we analyzed the changes in therapeutic choices over time and their impact on clinical outcomes in a cohort of 557 ITP outpatients followed at the "L. and A. Seràgnoli" Institute of Hematology, Bologna, Italy, from 1980 to 2015. Overall 397 patients (71%) required front-line corticosteroids, mainly prednisone. Over the decades, splenectomy was delayed from second to third-line, but was steadily used in around 15-25% of patients refractory or relapsing after first-line treatment. Consensually, RTX and TPO-R agonists emerged as second and third-line therapy of choice, respectively. Splenectomy was associated with the best response rates and the lower incidences of relapse, while the relapse rate after RTX was comparable to that observed with corticosteroids and other immunosuppressive agents. The introduction of TPO-R agonists gave an alternative to the administration of immunosuppressive drugs and probably contributed to moderate the incidence of infectious complications that remained stable over the decades, despite an increasing use of RTX from the 2000s onwards. Overall responses were similar over time, with over 97% achieving a response in all time-periods. However, the cumulative risk of bleeding significantly decreased [14.3% (1980-89) vs. 7% (1990-99) vs. 5.6% (2000-09) vs. 0.2% (2010-15)] (P

OBJECTIVE: A novel mechanism of platelet destruction involving the CD47/ signal regulatory protein-α (SIRPα) system has recently been suggested in a mouse model of immune thrombocytopenia (ITP). The CD47 molecule serves as ligand for SIRPα receptor and as receptor for thrombospondin acting as antagonistic to phagocyte activity and a regulator of apoptosis, respectively. In this study, we evaluated if the CD47/SIRPα axis may be involved in the apoptosis and clearance of platelets in human ITP. MATERIALS AND METHODS: Using flow cytometry, we characterized whether expression of CD47 on fresh and in vitro-aged platelets- and of SIRPα receptor on CD14-derived dendritic cells (DCs), macrophages, circulating DCs, and monocytes is reduced is ITP; whether the in vitro platelet phagocytic capacity of CD14-derived DCs and macrophages is differentially modulated in the presence or absence of antibodies against CD47 and SIRPα in ITP; and whether platelets are more susceptible to the CD47-induced death signal in ITP. RESULTS: We demonstrated that low platelet count in ITP is not due to increased phagocytosis associated with decreased expression of CD47 on the platelet surface and, despite reduced SIRPα expression, blockage of SIRPα on immature CD14-derived DCs or CD47 on platelets by specific antibodies failed to modify platelet uptake/phagocytosis of DCs. In contrast, targeting platelet CD47 with specific antibody significantly increases platelet phagocytosis of CD14-derived macrophages, and platelets are not healthy because they show increased apoptosis and are resistant to CD47-induced death signal. CONCLUSIONS: Our results demonstrate that the CD47 pathway in ITP patients abnormally modulates platelet homeostasis.

Background Myelofibrosis (MF) is a clonal hemopoietic stem/progenitor cells (HSPCs) disorder with mutations in 3 driver genes (JAK2, MPL, or CALR) and inflammatory microenvironment. Triple negative (TN) patients (pts) do not carry the 3 driver mutations and display a significantly worse survival-rate which can be partially due to a greater mutation complexity. Circulating microvesicles (MVs; 0.1-1 μm), most of megakaryocytes (MKMVs) and platelets (PMVs) origin, play a role in intercellular signalling and are increased in inflammation and cancer including MF. We hypothesized that in TN pts the HSPCs and/or the inflammatory microenvironment show a more aggressive and malignant phenotype. Aims To identify a biomolecular signature of TN pts by comparatively evaluating the circulating HSPCs behaviour and their functional interplay with the inflammatory microenvironment. Methods EDTA-anticoagulated peripheral blood (PB) was collected from 16 MF pts (WHO-2016 criteria-JAK2(V617F) mutated (n=10) and TN (n=6)) and 20 age/sex-matched healthy donors (HD). Driver mutation status was obtained by PCR/NGS. Plasma MVs were isolated by ultracentrifugation (45.000 rpm for 2 hours), counted by Nanosight technology and phenotypically characterized for PMVs (CD61+CD62P+) and MKMVs (CD61+CD62P-) by flow cytometry (size 0.5-0.9 µm). in vitro survival (24 hours), CXCL12-driven migration (12 hours) and clonogenic ability of immunomagnetically isolated CD34+ cells from pts PB or cord blood (CB; n=10) was performed in the presence/absence of the isolated MVs or IL-1β, TNF-α and IL-6 (alone/ in combination). Incorporation of CFSE-labelled MVs into CD34+ cells was analyzed by confocal microscopy. MicroRNA (miRs) profile analysis of the isolated MVs and Gene expression profile (GEP) analysis of CD34+ cells from 3 JAK2(V617F) mutated, 3 TN pts and 3 HD/CB was performed. Plasma inflammatory cytokines levels were evaluated by ELISA. Results We firstly compared the TN and JAK2(V617F) CD34+ cells. TN CD34+ cells showed decreased in vitro survival and clonogenic ability but comparable migration capacity (Fig. 1 A, B, C). Consistently, comparing the GEP, the expression of selected anti-apoptotic (TSPYL5, GFI-1 and FCMR) and pro-apototic (TNFSF10, TP53INP1) genes was significantly down- and up-regulated, respectively, in TN CD34+ cells. We then investigated whether signals from the microenvironment may affect the in vitro behaviour of the TN CD34+ cells. IL-1β, IL-6 and TNF-α plasma levels were significantly increased (p Conclusions Our results demonstrate that TN CD34+ cells show in vitro defective function and are unresponsive to the inflammatory microenvironment. Interestingly, only the autologous circulating MVs promoted the malignant clone, suggesting that MVs, as signals from microenvironment, may have a pathogenetic role in TN MF and may be the target of novel therapeutic approaches. Figure 1. Figure 1. Disclosures Fabbri: Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST) IRCCS: Employment. Cavo:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; AbbVie: Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees; Adaptive Biotechnologies: Honoraria, Membership on an entity's Board of Directors or advisory committees; GlaxoSmithKline: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Palandri:Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.

Along with their immunogenic role, dendritic cells (DCs) are also critical in maintaining tolerance to self-antigens by inducing regulatory T cells (Tregs) via the expression of the immunomodulatory enzyme indoleamine 2,3-dioxygenase 1 (IDO1). In turn, Tregs modulate the maturation and/or function of DCs. In immune thrombocytopenia (ITP), the interaction between DCs and Tregs has never been investigated although decreased number/function of Tregs as well as altered DCs have been described. Here, we ask whether, in ITP: (1) IDO1 expression/activity is decreased in mature DCs; (2) IDO1-mediated Treg generation is impaired; and (3) DC maturation is abnormally modulated by Tregs. We found that in ITP, DCs show reduced capability of upregulating the expression/activity of IDO1. This finding results in the reduced ability of mature DCs of converting T cells into Tregs. In turn, Tregs are characterized by decreased interleukin-10 production and show lower ability of inhibiting DC maturation. In conclusion, these data point out the role of IDO1 in the impaired regulatory T cell development of ITP patients and suggest that the cross-talk between Tregs and DCs is hampered and plays a pathogenetic role.

Background Bone marrow stem/progenitor cells seem to be effective in liver regeneration after tissue injury. Aim To evaluate the feasibility and safety of the mobilization and reinfusion of CD133+ stem/progenitor cells in patients with end-stage liver disease. Methods Autologous CD133+ stem/progenitor cells, mobilized with granulocyte-colony stimulating factor, were collected by leukapheresis and reinfused at increasing doses through the hepatic artery starting from 5 × 104/kg up to 1 × 106/kg. Results 16 subjects with Model for End-stage Liver Disease (MELD) score between 17 and 25 were enrolled, 14 mobilized an adequate number of CD133+ stem/progenitor cells and 12 were reinfused. No severe adverse events related to the procedure were reported. MELD score significantly worsened during mobilization in Child Turcotte Pugh-C patients. A significant improvement of liver function was observed 2 months after reinfusion (MELD 19.5 vs 16; P = 0.045). Overall, 5 patients underwent liver transplantation within 12 months from reinfusion and 2 died because of progressive liver failure. Conclusions CD133+ stem/progenitor cells reinfusion in patients with end-stage liver disease is feasible and safe. A worsening of liver function was observed during mobilization in Child Turcotte Pugh-C patients. The temporary improvement of MELD score after reinfusion suggests that stem cells therapy may be a “bridge to transplant” approach for these patients.

n/a

Immune thrombocytopenia (ITP) is an autoimmune disorder in which, for still unknown reasons, platelet surface proteins become antigenic and stimulate the immune system to produce autoantibodies and self-reactive cytotoxic T lymphocytes. These findings result in immune-induced platelet destruction

• In the last years several authors studied a regulatory T cells subset lacking CD25 (CD4 + CD25 − Foxp3 + cells) in systemic autoimmune disorders.

Introduction: Myelofibrosis (MF), a clonal disorder of the hemopoietic stem/progenitor cell, is characterized by distinctive abnormalities in megakaryocyte (MK) development. A proliferation of dysplastic MK with disturbance of nuclear/cytoplasmic maturation is observed. In addition to driver mutations in Janus Kinase 2 (JAK2) and Calreticulin (CALR) genes, chronic inflammation has emerged as a key-player in MF pathogenesis. Here, we analyzed the potential contribution of crucial factors of the inflammatory microenvironment (Interleukin (IL)-1β, Tumor Necrosis Factor (TNF)-α and Tissue Inhibitor of Metalloproteinases (TIMP)-1 to MF dysmegakaryocytopoiesis. Methods: We studied 10 patients with MF at diagnosis while 6 patients had received previous treatment for MF. In all cases, therapies had been discontinued for at least two months before peripheral blood samples collection. To characterize the mutational status, JAK2V617F and CALR exon 9 mutations were assessed by quantitative PCR-based allelic discrimination assay and Next Generation Sequencing approach, respectively. To quantify MK progenitors, circulating CD34+CD41+ cells of MF patients (10 JAK2V617F and 6 CALR mutated) and controls (cord blood (CB); 8 cases) were identified by flow cytometry analysis. To evaluate the pro-inflammatory profile, IL-1β, TNF-α and TIMP-1 were measured in plasma of patients (10 JAK2V617F and 6 CALR mutated) and healthy subjects (10 cases) by ELISA. To determine whether and to what extent crucial factors of the inflammatory microenvironment may affect the in vitro growth of MK progenitors, Colony Forming Units-Megakaryocyte (CFU-MK) growth of circulating CD34+ cells from MF patients (6 JAK2V617F and 6 CALR mutated) and CB (6 cases) was assessed with a collagen-based medium in the presence or absence of TIMP-1, TNF-α and IL-1β (alone or in combination). Pure and mixed CFU-MK were quantified on the basis of CD41 immunostaining. Results: We found that, irrespective of mutational status, IL-1β, TNF-α and TIMP-1 plasma levels were significantly increased in MF patients. The frequency of circulating MF-derived CD34+CD41+ cells was significantly higher than the CB counterparts. However, a significantly higher proportion of cells was associated with CALR compared to JAK2V617F mutated patients. The growth of pure CFU-MK from untreated cells of the JAK2V617F mutated patients was significantly decreased as compared to that of CALR mutated patients and the CB counterparts. Factors alone did not stimulate the CFU-MK growth from CB CD34+ cells. Conversely, IL-1β had stimulatory activity on pure MK colony formation of JAK2V617F, but not CALR, mutated patients and the growth of MK progenitors from MF patients was significantly inhibited by TNF-α, irrespective of mutational status. TIMP-1 was ineffective. Factors in combination did not significantly modify the growth of pure CFU-MK from patients/CB as compared with factors alone. The growth of mixed MK colonies from untreated cells was absent (JAK2V617F mutated patients) or very low (CALR mutated patients and CB). Only TNF-α (alone or in combination (IL-1β+TNF-α and IL-1β+TNF-α+TIMP-1)) increases the growth of mixed MK colonies of patients harboring CALR mutation and CB. Interestingly, factors alone or in combination failed to stimulate the growth of mixed MK colonies from JAK2V617F mutated patients. Conclusions: Taken together these results demonstrate that in MF signals provided by the inflammatory microenvironment promote a mutation-associated functional dysregulation of MK progenitors and suggest a mechanism by which chronic inflammation may play a role in the development/maintenance of the abnormal megakaryocytopoiesis. Disclosures Martinelli: BMS: Speakers Bureau; MSD: Consultancy; Pfizer: Consultancy, Speakers Bureau; Novartis: Speakers Bureau; Roche: Consultancy, Speakers Bureau; Ariad: Consultancy, Speakers Bureau; Amgen: Consultancy, Speakers Bureau; Genentech: Consultancy; Celgene: Consultancy, Speakers Bureau. Cavo:Janssen-Cilag: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Celgene: Consultancy, Honoraria; Millennium: Consultancy, Honoraria.

Myelofibrosis (MF) is a clonal disorder of the hemopoietic stem/progenitor cells (HSPCs). Together with molecular abnormalities (mutations in JAK2, Calreticulin (CALR) and MPL genes), chronic inflammation is the major hallmark of MF pathogenesis. It is argued that the up-regulated production of pro-inflammatory cytokines selects for the malignant clone. However, the key players linking inflammation and cancer in MF are still poorly known. Here, we investigated the in vitro effects of crucial factors of the inflammatory microenvironment (Interleukin (IL)-1β, Tumor Necrosis Factor (TNF)-α, Tissue Inhibitor of Metalloproteinases (TIMP)-1 and ATP) on the functional behaviour of MF-derived HSPCs. Plasma TIMP-1, TNF-α and IL-1β were measured by ELISA assay. Circulating CD34+ cells from MF patients (JAK2V617F (10 cases) and CALR (10 cases) mutations) or cord blood (CB; 8 samples) were functionally characterized after incubation with or without ATP (1000 μM), IL-1β (10 ng/mL), TNF-α (10 ng/mL) or TIMP-1 (100 ng/mL) (alone or in combination). Cells were then analyzed for survival/apoptosis (Annexin-V/Propidium Iodide staining), cell cycle and clonogenic capacity. Migration was assessed towards a C-X-C motif chemokine 12 (CXCL12) gradient in the presence or absence of the pro-inflammatory factors. MF-derived CD34+ cells were co-cultured with normal mesenchymal stromal cells (MSCs) in the presence of the pro-inflammatory factors and then evaluated for survival. Regardless mutation status, the plasma levels of IL-1β, TNF-α and TIMP-1 are increased in MF patients and the presence of pro-inflammatory cytokines confers a survival advantage of MF-derived CD34+/CD34+CD38- cells. In addition, MF-derived CD34+ cells promoted cell-cycle progression to the S-phase. Whereas in the JAK2V617F mutated group, the addition of IL-1β or TNF-α ± TIMP-1 impaired the erythroid clonogenic output of the CALR mutated patients. Of note, IL-1β + TNF-α ± TIMP-1 highly promoted the in vitro migration of MF-derived CD34+ cells in the presence of CXCL12. Intriguingly, MF-derived CD34+ cells revealed increased clonogenic ability after migration toward CXCL12 and IL-1β + TNF-α ± TIMP-1, suggesting a pivotal role of inflammation in the selection of the malignant clone. Finally, co-cultures of normal MSCs with MF-derived CD34+ cells sustained the survival effects of IL-1β+TNF-α±TIMP-1. Overall, our data indicate that the behavior of the MF-derived HSPCs can be influenced by regulatory signals provided by the microenvironment through the cooperation between various pro-inflammatory factors. The interplay of these pro-inflammatory factors promotes and selects the circulating MF-derived HSPCs with higher proliferative activity, clonogenic potential and migration ability. Targeting these micro-environmental interactions may be a clinically relevant approach. Citation Format: Daria Sollazzo, Dorian Forte, Nicola Polverelli, Marco Romano, Margherita Perricone, Sofia Fatica, Francesco Lapi, Emanuela Ottaviani, Martinelli Giovanni, Nicola Vianelli, Michele Cavo, Maria Pantaleo, Francesca Palandri, Lucia Catani. Crucial factors of the inflammatory microenvironment (IL-1β/TNF-α/TIMP-1) promote the selection of highly malignant hemopoietic clone of myelofibrosis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4077.

Previous studies have shown that bone marrow (BM) cells contribute to liver regeneration after tissue injury. The main objective of the present study was to evaluate the feasibility and the safety of the purification and intrahepatic reinfusion of increasing numbers of autologous BM-derived G-CSF-mobilized CD133+ stem cells (SCs) in patients with end-stage liver disease (ESLD). For this purpose, G-CSF at 7.5 µg/Kg/b.i.d. is administered subcutaneously (sc) from day 1 until the completion of peripheral blood stem cells (PBSC) collection. Collection of PBSC begin on day + 5 only if the concentration of CD133+ cells is > 8/µL. CliniMacs device is used for the positive selection of CD133+ SCs (under GMP conditions) from PB of mobilized standard-volume leukapheresis. At least 4 weeks after SC mobilization, collection and cryopreservation, highly purified autologous G-CSF-mobilized CD133+ cells are re-infused through the hepatic artery by transfemoral or transbrachial arteriography. CD133+ cells are administered to patients starting from 5x104/Kg patient’s body weight and increased every 3 patients up to 1x106/kg. G-CSF at 5µg/Kg/day is administered sc for 3 days after the reinfusion of SCs for their expansion and to induce a selective proliferative advantage of reinfused cells in vivo. Biological assays (phenotype of circulating SCs, clonogenic assays, serum cytokines) were done during the mobilization and re-infusion phases together with the phenotypic characterization of the isolated CD133+ SCs. The clinical trial is ongoing. Up to date, 9 patients have been successfully mobilized with G-CSF and highly purified autologous CD133+ SCs have been re-infused in 7 cases. Based on preliminary data, we suggest the feasibility and safety of intrahepatic reinfusion of highly purified CD133+ stem cells in patients with ESLD. Biological studies show that: 1) circulating hematopoietic and endothelial progenitors are increased after G-CSF treatment; 2) highly purified CD133+ cells express hematopoietic and endothelial markers; 3) serum concentration of HGF, SDF-1, VEGF and MMP9 and clonogenic capability of hematopoietic progenitors are increased during the mobilization and re-infusion phases; 4) clonogenic potential of endothelial progenitors shows variable expression.

No abstract is available for this article.

Introduction. Myelofibrosis (MF), an acquired clonal disorder of the hematopoietic stem/progenitor cell (HSPC) with a dysregulation in JAK/STAT signalling (mutations in JAK2, MPL and Calreticulin (CALR) genes), is characterized by a state of chronic inflammation. It is argued that the up-regulated production of proinflammatory cytokines by both HSPCs and the surrounding stromal cells generates a microenvironment that selects for the malignant clone. Only recently, it has been hypothesized that the sustained inflammatory microenvironment of MF can alter crucial biological processes, leading to genomic instability and cancer progression. Here we tested the in vitro functional effects of pivotal players of the inflammatory microenvironment (the extracellular ATP nucleotide and selected cytokines, such as Interleukin (IL)-1β, Tumor Necrosis Factor (TNF)-α or the Tissue Inhibitor of Metalloproteinases-1 (TIMP-1)) on the HSPCs from MF patients. Methods: Circulating CD34+/CD34+ CD38- cells from MF patients (JAK2V617F (17 cases) and CALR (9 cases) mutations) or cord blood (CB; 8 samples) were phenotypically and functionally characterized after in vitro incubation with or without ATP (1000 μM), IL-1β (10 ng/mL), TNF-α (10 ng/mL) or TIMP-1 (100 ng/mL) (alone or in combination). Cells were then analyzed for survival/apoptosis (Annexin-V/Propidium Iodide staining), phenotype (evaluation of CD63 (TIMP-1 receptor), CXCR4 and CD38 expression), cell cycle and clonogenic capacity. Migration was assessed first towards a CXCL12 gradient in the presence or absence of the pro-inflammatory factors. In parallel experiments, CD34+ cells from MF patients were co-cultured with normal mesenchymal stromal cells (MSCs) in the presence or absence of the pro-inflammatory cytokines and then evaluated for their ability to migrate towards a CXCL12 gradient. Plasma TIMP-1, TNF-α, IL-1β and CXCL12 were measured by ELISA assay. Results: The plasma levels of TIMP-1, TNF-α, IL-1β, CXCL12 and the number of circulating CD34+, CD34+ CD38-, CD34+ CD63+, CD34+ CD184+ cells were increased in MF patients. According to mutational status, the CD34+ CD63+ cells were higher in the CALR+ patients. The survival of MF CD34+ cells was strongly stimulated by in vitro incubation with TNF-α or IL-1β as compared with the CB-derived CD34+ cells or untreated cells. By multiple cytokine combinations, IL-1β/TIMP-1, IL-1β /ATP or IL-1β /TNF-α treatments significantly promote the survival of MF CD34+ cells as compared with the normal counterparts or the untreated cells. Various combinations with IL-1β were also effective in stimulating survival of CD34+CD38- cells. IL-1β/TIMP-1 and IL-1β/TNF-α/TIMP-1, but not factors alone, significantly increased the CFU-C growth of MF patients as compared with the CB-derived counterparts and the untreated cells. Moreover, comparing CALR+ vs JAK2V617F+ patients, the colony formation of JAK2V617F+ patients was mainly promoted by the IL-1β/TNF-α treatment. Along with clonogenic capacity stimulation, exposure of CD34+ cells from MF patients to IL-1β/TNF-α/TIMP-1 significantly increases the S-phase cells, suggesting that these pro-inflammatory factors stimulated cell-cycle progression in dormant CD34+ MF cells. Migration of CD34+ cells from MF was significantly increased in CXCL12 treated cells. In addition, exposure of MF CD34+ cells to IL-1β/TNF-α, IL-1β/TIMP-1 or IL-1β/TNF-α/TIMP-1 significantly promotes cell migration in comparison with the CB-derived counterparts or SDF-1 alone. MF migrated cells in the presence of IL-1β/TNF-α significantly upregulate CD63 expression. Intriguingly, colony formation of MF migrated CD34+ cells in the presence of IL-1β/TNF-α or IL-1β/TNF-α/TIMP-1 was potently increased. Finally, co-culture systems with normal MSCs in the presence of pro-inflammatory factors revealed that MF CD34+ cells display increased migration ability toward CXCL12 gradient. Conclusions: Altogether our findings suggest that in MF the inflammatory niche plays a key role in the maintenance of the malignant clone. Thus, the interplay between the pro-inflammatory cytokines promote and select the HSPCs with higher proliferative activity, clonogenic potential and migration capability. Targeting these microenvironmental interactions may be a clinically relevant approach. D.F. and D.S. equally contributed Disclosures Martinelli: Pfizer: Consultancy; Ariad: Consultancy; Novartis: Consultancy, Speakers Bureau; MSD: Consultancy; AMGEN: Consultancy; BMS: Consultancy, Speakers Bureau; ROCHE: Consultancy.

Introduction: Myelofibrosis (MF) is a clonal disorder associated mainly with JAK2V617F and MPL mutations. Recently, a new mutation in the gene encoding calreticulin (CALR) was discovered in the majority of JAK2/MPL negative patients. MF is burdened by a high rate of potentially life-threatening infections. The issue of recurrent and opportunistic infections is increased after the introduction in clinical practice of JAK inhibitors with immunosuppressive activity. However, the role of crucial immune cell subsets is still poorly characterized. Here, we investigated the phenotype/function of selected immune cells in MF. Specifically, we focused on circulating regulatory (Tregs) and IL-17-producing T cells (Th17 cells), monocytes and dendritic cells (DCs). Monocyte-derived DCs were also characterized. Methods: We characterized circulating Th17 cells, Tregs, monocytes and DCs of 17 untreated MF patients and 8 healthy controls (HC) by flow cytometry. Th17 cells were identified as CD4+ CD161+ CD196+ cells while Tregs were enumerated as CD4+ CD25high CD127low T cells. We also tested the in vitro suppressive activity of circulating CD4+ CD25+ Tregs with a mixed leukocyte reaction assay. Two subpopulations of circulating DCs, myeloid CD11c+ and plasmacytoid CD123+cells, were enumerated as well. In addition, after immunomagnetic selection, we tested both phenotype of circulating monocytes and their capacity to differentiate into CD14-derived immature and mature DCs, using a specific cytokines cocktail. JAK2V617F and MPL mutations were detected with RT-PCR while the presence of CALR mutations were tested with Exon 9 Next Generation Sequencing assay. Results: JAK2V617F (11 cases), MPL (3 cases), and CARL (3 cases) mutations were detected. We found that circulating CD4+CD25highCD127low Tregs were reduced in MF patients as compared with healthy controls (p=0.043), although their suppressive ability was maintained. We also found a lower number of circulating Th17 cells (p=0.0026) in MF patients. This finding was particularly evident in JAK2V617F+(p=0.008) and CARL+(p=0.03) patients. Despite their number was in the normal range, circulating monocytes from MF patients showed reduced expression of the CD86 co-stimulatory molecule. Moreover, as compared with the normal counterparts, immature monocytes-derived DCs from patients maintained low CD14 expression without upregulating the CD80 co-stimulatory molecule expression (p=0.0063). Interestingly, at variance with plasmacytoid DCs, a reduced number of circulating myeloid DCs was observed in MF patients as compared with that of HC (p=0.01). Conclusions: Here we demonstrated that specific crucial subsets of immune cells show quantitative and/or qualitative abnormalities in MF patients. These findings may be useful to better understand the increased susceptibility of these patients to infections, since Th17 cells play a role in bacterial and fungal infections while myeloid DCs regulate Th1 activity. Of note, DCs inhibition might result in increased propensity to infections and compromised immune response to cancer.In addition, since monocytes are DC precursors, alterations in their differentiation pathway may contribute to develop defective immune responses. Disclosures Martinelli: NOVARTIS: Consultancy, Speakers Bureau; BMS: Consultancy, Speakers Bureau; PFIZER: Consultancy; ARIAD: Consultancy.

Abstract 696 Regulatory T cells (Tregs) are a subset of T cells involved in the maintenance of peripheral self-tolerance. Specifically, Tregs modulate the maturation and/or function of dendritic cells (DCs). In turn, along with their immunogenic role, DCs are also critical in maintaining tolerance to self-antigens by inducing Tregs via the expression of the immunomodulatory enzyme indoleamine 2,3-dioxygenase 1 (IDO1). IDO1-expressing DCs may play a role in preventing the initiation of autoimmune disorders. Immune Thrombocytopenia (ITP) is an autoimmune disorder in which platelet surface proteins become antigenic and stimulate the immune system. However, in ITP, the interaction between DCs and Tregs has never been investigated, although decreased numbers of Tregs as well as altered DCs have been described. Therefore, in the present study, we investigated whether, in ITP: 1) IDO1 expression/activity is decreased in monocyte-derived DCs (Mo-DCs); 2) the mechanism of Tregs generation is impaired; 3) Mo-DCs maturation is abnormally modulated by Tregs. We studied 54 patients with active ITP. Nineteen patients were newly diagnosed and 35 patients had persistent (25 cases) or chronic (10 cases) ITP. At the time of samples collection, all patients with persistent or chronic ITP were out of any treatment for at least two months and none of the patients received previous treatment with rituximab or were splenectomized. We demonstrated that in mature Mo-DCs from ITP patients the absolute number of IDO1 transcripts was significantly reduced as compared with healthy donors (973.000±771.000 vs 2.409.000±1.592.000 (IDO1 copy number/ABL1 copy number)*10.000; p In summary, this study demonstrates that in ITP low IDO1 expression/activity in DCs results in the reduced ability of generating Tregs. In turn, circulating Tregs with impaired IL-10 production show decreased capacity of inhibiting DCs maturation. Taken together these data suggest that in ITP the cross-talk between Tregs and DCs is hampered and plays a pathogenetic role. As a consequence, DCs are less “tolerogenic” and Tregs are defective in their regulatory function. Disclosures: No relevant conflicts of interest to declare.

Abstract 2770 Recent studies suggest a bi-directional interaction between regulatory T cells (Tregs) and dendritic cells (DCs). Despite their role as inducers of immunity, DCs may also be critical in maintaining tolerance to self-antigens by inducing Tregs. Specifically, Indoleamine 2,3-dioxygenase 1 (IDO 1) expressing DCs expand Tregs. In turn, Tregs, which express CTLA4, engage its receptors B7-1(CD80)/B7-2(CD86), thus inducing the expression of IDO1 enzyme in DCs. IDO1 expression leads to tryptophan depletion and immunosuppressive kynurenine enrichment in the tissue. Both factors drive the de novo generation of Tregs from CD4+ T cells. In addition, Tregs may modulate the maturation and/or function of DCs, which are required for the activation of effector T cells. Tregs out compete with naïve T cells in aggregating around DCs. After forming aggregates, Tregs specifically downregulate the expression of CD80 and CD86 on DCs. The result of the interaction between Tregs and DCs is the impaired capacity of the antigen presenting cells to establish long lasting interactions with effector T cells. Therefore, given the crucial role of DCs and Tregs in initiating, regulating, maintaining or repressing immune responses, investigations on their interaction is of great importance for better elucidating the pathogenesis of ITP. Of note, in ITP this interaction and its pathogenetic role have never been investigated in depth. In this paper, we enumerated and functionally characterized Treg subsets, exploring whether in ITP abnormal Tregs interactions with DCs and/or effector T cells might play a pathogenetic role. Specifically, we studied whether, in ITP patients: 1) DCs maturation is differentially modulated by Tregs from ITP patients as compared with healthy donors; 2) the mechanism of Tregs generation is altered. Forty adult ITP patients, newly diagnosed (14 cases) or with persistent (20 cases) or chronic ITP (9 cases) were studied after informed consent. At the time of the study, patients with persistent or chronic ITP were out off therapy by at least two months. None of the patients were splenectomized. The median platelet count at the time of the study was 53×109/L (range 8–99). Allogeneic Mixed Leukocyte Reaction (MLR) was performed to test the suppressive activity of Tregs. To analyze the in vitro ability of highly purified CD4+CD25+ T cells to inhibit DCs maturation, normal immature CD14-derived DCs were cultured alone and with allogeneic CD4+CD25+ T cells from healthy subjects or ITP patients in the presence of Lipopolysaccaryde. CD80 and CD86 expression of CD14-derived DCs was then tested at flow cytometry. To evaluate the in vitro conversion of non Tregs into Tregs, CD4+CD25- T cells were cultured alone and with autologous mature CD14-derived DCs from ITP patients and healthy subjects. The percentages of CD4+CD25highFoxP3+ T cells were quantitated at flow cytometry. mRNA IDO1 expression of immature and mature CD14-derived DCs was then evaluated by Real-time RT-PCR. To determine IDO1 enzyme activity kynurenine levels were measured in the supernantants of mature CD14-derived DCs. We found that in ITP Tregs show lower ability to suppress T cell proliferation and to inhibit CD14-derived DCs maturation because they do not affect the expression of the costimulatory molecules CD80 and CD86. We show that the absolute number of Tregs was significantly decreased in ITP patients in comparison to healthy subjects (CD4+CD25highFoxp3+ T cells (5.5±4.3 vs 11.6±6.9 cells/microL; p below 0.01) and CD4+CD25highCD127low/negative T cells (50.9±27.3 vs 80.5±37.7 cells/microL; p below 0.02)). In addition, in ITP patients we document that the low number of circulating Tregs may be due to the reduced ability of mature CD14-derived DCs to convert non-Treg cells (CD4+CD25-) into Tregs (CD4+CD25highFoxP3+ Tregs). This is related to reduced expression and activity of the IDO1 enzyme in mature CD14-derived DCs from ITP patients, since DCs expressing IDO1 favour Treg generation. In conclusion, taken together our data demonstrate that in ITP the cross-talk between Tregs and DCs is impaired and plays a pathogenetic role. As a consequence, we found the generation of more immunogenic DCs and defective Tregs. Disclosures: No relevant conflicts of interest to declare.

Abstract 3776 The CD47 antigen is a transmembrane glycoprotein ubiquitously expressed on hematopoietic and non-hematopoietic cells. It serves as a receptor for Thrombospondin (TSP) and a ligand for signal regulatory protein-alpha (SIRP-alpha) receptor, acting, respectively, as a regulator of apoptosis and as antagonistic to phagocyte activity. Ligation of CD47 with antibodies, its natural physiological ligand TSP or the specific CD47-binding peptide 41NK induces apoptosis in nucleated blood cells. This apoptosis is characterized by mitochondrial damage and the exposure of phosphatydilserine on the outerleaflet of the plasma membrane. Interaction of SIRP-alpha with CD47 is important also for the regulation of phagocytosis. SIRP-alpha is an immunoglobulin superfamily member and is predominantly expressed in neurons, dendritic cells (DCs) and monocytes/macrophages. Phagocytes engulf foreign cells but not “self” in part because “self” cells express CD47 as a ligand for SIRP-alpha, which inhibits phagocytosis. Thus CD47 functions as a “don't eat me” signal. Based on studies in mice, a novel mechanism of platelet destruction involving the CD47/SIRP-alpha system has been recently suggested in Immune Thrombocytopenia (ITP). Specifically, it has been demonstrated that: 1) platelet homeostasis is regulated by platelet expression of CD47 under normal conditions and in immune thrombocytopenia in a mouse model; 2) interaction between platelet CD47 and macrophage SIRP-alpha is important in regulating normal platelet turnover and FcgammaR-mediated clearance of IgG-sensitized platelets; 3) CD47-deficient platelets have a shortened half-life in the circulation of CD47 wild-type mice and are also more sensitive to Fcgamma receptor-mediated clearance, both in vivo and in vitro. However, the role of CD47 pathway in the pathogenesis of human ITP has not yet been studied. Therefore, the main purpose of the present study was to evaluate whether alterations of this system (platelets/phagocytes) might play a pathogenetic role in human ITP. In particular, we investigated whether in ITP: i) platelets are more susceptible to CD47-induced cell death; ii) expression of CD47 on fresh and in vitro aged platelets is reduced; iii) the platelet phagocytic capacity of CD14-derived DCs and macrophages is differentially modulated in the presence or absence of antibodies against CD47 and SIRP-alpha. Phenotypical and functional analysis of the expression of CD47 on platelets and SIRP-αlpha on CD14-derived/circulating DCs and on CD14-derived macrophages was performed in 32 ITP patients. Patients were newly diagnosed (14 cases) or with persistent (15 cases) or chronic (3 cases) ITP. At the time of the study, patients with persistent or chronic ITP were off therapy by at least two months. None of the patients were splenectomized. The median platelet count at the time of the study was 49×109/L (range 14–98). We found that in healthy subjects CD47 expression increased in in vitro aged platelets and ligation of CD47 with anti-CD47 antibody induced a dose-dependent increase of platelet apoptosis. Immature and mature CD14-derived DCs and circulating myeloid DCs were strongly positive for SIRP-α. Conversely, we demonstrated that in ITP: 1) CD47 expression was unchanged in freshly isolated and in vitro aged platelets; 2) increased platelet apoptosis was not due to the activation of the CD47-induced cell death pathway, which instead was shown to be blocked; 3) the blockage of SIRP-αlpha on immature CD14-derived DCs or CD47 on platelets by specific antibodies failed to modify platelet uptake/phagocytosis of DCs; in contrast, targeting platelet CD47 with specific antibody significantly increases platelet phagocytosis of CD14-derived macrophages. In conclusion, our data demonstrate that in ITP the increased platelet clearance is not due to reduced CD47 expression on platelets. However, platelets from ITP patients are not healthy because 1) apoptosis is increased; 2) platelet apoptosis is independent from CD47 death signal; 3) CD47 expression is not modified by in vitro ageing/apoptosis. In addition, we show that the CD47 pathway plays a role in platelet phagocytosis of macrophages, but not in DCs. We conclude that in ITP patients platelet homeostasis is differentially modulated by the CD47 pathway. Disclosures: No relevant conflicts of interest to declare.

Abstract 1309 Poster Board I-331 Introduction The CD47 antigen is a transmembrane glycoprotein ubiquitously expressed on hematopoietic and non-hematopoietic cells. It serves as a ligand for SIRP-alpha (signal regulatory protein-alpha) receptor and as a receptor for Thrombospondin, acting, respectively, as antagonistic to phagocyte activity and as regulator of apoptosis. Based on mouse studies, a novel mechanism of platelet destruction involving the CD47/SIRP-alpha system has been recently proposed in immune thrombocytopenia. This mechanism suggests that platelet homeostasis is regulated by platelet expression of CD47 and that interaction between platelet CD47 and macrophage SIRP-alpha receptor is important in regulating platelet macrophage phagocytosis. However, the role of this system in platelet uptake/phagocytosis by dendritic cells (DCs) has never been investigated in immune thrombocytopenia (ITP) in humans. Therefore, our purpose was to evaluate whether alterations of the CD47/SIRP-alpha system may have a role in the pathogenesis of ITP in humans. Patients and methods Twenty five ITP patients were studied. We phenotypically characterized apoptosis (Annexin-V FITC staining) and the expression of CD47 on platelets and SIRP-alpha on CD14-derived and circulating DCs by flow cytometry. To determine whether platelet apoptosis was due to activation of CD47-cell death pathway, in parallel experiments, we assessed the in vitro sensitivity of platelets to antibody CD47 ligation. In addition, to investigate the role of CD47/SIRP-alpha system on platelet phagocytic capacity of CD14-derived DCs, immature DCs were coincubated with PKH26-labelled platelets in the presence or absence of antibodies against CD47 and SIRP-alpha. The percentage of ingested platelets was then evaluated by flow cytometry. Results We demonstrate that in ITP: 1) CD47 expression is not altered in freshly isolated platelets; 2) after in vitro aging, platelet apoptosis is increased as compared with the normal counterparts; 3) CD47 expression is unchanged in apoptotic platelets; by contrast, it increases in normal platelets; 4) the increased platelet apoptosis is not due to the activation of the CD47-induced cell death pathway; 5) despite low level expression of SIRP-alpha in CD14-derived DCs and in circulating DCs, the CD47/ SIRP-alpha system does not play a central role for in vitro platelet phagocytosis of DCs, since blockage of SIRP-alpha on DCs or CD47 on platelets by specific antibodies failed to modify phagocytosis. Conclusions In conclusion, we demonstrate that in ITP platelet CD47 expression does not play a role in the pathogenesis of the disease. We also show that, in ITP patients, higher platelet apoptosis is not due to different CD47-induced cell death susceptibility. Whether this is due to the fact that CD47 expressed on apoptotic platelets from ITP patients may have a peculiar conformation, avoiding the delivery of cell death signal, remains open question. Furthermore, the platelet uptake/phagocytosis by DCs is not significantly influenced by the CD47/ SIRP-alpha system. Supported in part by BolognaAIL (Italian association against Leukemia, Bologna section) Disclosures No relevant conflicts of interest to declare.

Abstract 3511 Poster Board III-448 CD4+CD25+ regulatory T cells (Tregs) are critical in maintaining self-tolerance and preventing organ-specific autoimmune diseases. However, the role of Tregs in the pathogenesis of immune thrombocytopenia (ITP), an immune disorder in which increased platelet clearance is caused by antiplatelet autoantibodies, has not yet been clarified. The purpose of the present study was to investigate whether the interaction between dendritic cells (DCs) and regulatory T cells (Tregs) may play a pathogenetic role in ITP. Forty patients with active disease and 35 healthy subjects were enrolled into the study. We firstly characterized the number (by flow cytometry) and the suppressive activity (by modified allogeneic mixed leukocyte reaction) of Tregs in ITP. We found that the absolute number of Tregs was significantly decreased in ITP patients in comparison to healthy subjects (CD4+CD25highFoxp3+ T cells (5.5±4.3 vs 11.6±6.9 cells/microL; p below 0.01) and CD4+CD25highCD127low-negative T cells (50.9±27.3 vs 80.5±37.7 cells/microL; p below 0.02)). We documented also that in ITP suppressive activity of Tregs was defective as compared to healthy subjects. In parallel experiments we studied the in vitro conversion of CD4+CD25- T cells, either from ITP patients or healthy subjects, into CD4+CD25+Foxp3+ Tregs by co-coltures with mature autologous/allogeneic DCs. The flow cytometry analysis showed that in ITP the low number of circulating Tregs may be partly due to the reduced ability of DCs to convert non-Treg cells into Tregs. We then explored the in vitro capability of CD4+CD25+ Tregs, either from ITP patients or healthy subjects, to inhibit allogeneic DCs maturation by flow cytometry evaluation of the expression of the costimulatory molecules CD80 and CD86. We demonstrated that in ITP patients CD4+CD25+ Tregs show lower ability to inhibit DCs maturation because they do not affect the expression of CD80 and CD86 molecules. This finding may be related to the lower level of Interleukin-10 and Interleukin-6 in the cocoltures. Taken together, these findings document that the interaction between DCs and Tregs is altered in ITP and suggest that this dysfunction may play a pathogenetic role. Supported in part by BolognaAIL (Italian association against Leukemia, Bologna section) Disclosures: No relevant conflicts of interest to declare.

Introduction: Myelofibrosis (MF) is characterized by clonal hemopoiesis, inflammatory microenvironment and mutations in JAK2, MPL, or CALR genes. Around 10% of patients (pts) do not carry the 3 mutations (Triple negative, TN). Microparticles (MPs; 0.1 - 1 µm) are small vesicles deriving from the cell plasma membrane with a role in intercellular signalling. Circulating MPs are increased in inflammation and cancer including MF. MicroRNAs (miRs) contribute to MF pathogenesis. However, the miRs profile of MPs has never been investigated in MF. Here we studied miR expression of circulating MPs from JAK2(V617F) mutated and TN pts. Methods: Peripheral blood was collected from 6 MF pts (at diagnosis or out of cytotoxic treatment for at least 3 months) and 3 age/sex-matched healthy donors (HD). MF pts were JAK2(V617F) mutated (n=3) and TN (n=3). MPs were purified from platelet poor plasma by ultracentrifugation and quantified using the Nanosight technology. MiR expression of isolated pts/HD-MPs (10e9) was investigated using miRNeasy Mini Kit and TaqMan™ Array Human MicroRNA A Cards (ThermoFisher). RT-PCR validation assays are under way. Results: As compared with the HD counterparts, many miRs were significantly upregulated in MPs from both JAK2(V617F) mutated and TN pts. Among them miR-21, known to be the most commonly upregulated miR in haematological tumours, is an anti-apoptotic factor with oncogenic potential and able to inhibit megakaryocyto-erithropoiesis. MiR-155, 222, 24 were upregulated in TN-MPs only. MiR-155 is upregulated in response to inflammation and, targeting p53 pathway, inhibits apoptosis. Its overexpression stimulates granulo-monocytopoiesis while impairing megakaryocytopoiesis. MiR-221/222, markers of poor prognosis in aggressive tumours and both increased in TN-MPs, show anti-apoptotic effect acting downstream of the oncogenic RAS-RAF-MEK pathway. MiR-423-5p, a pro-apoptic miR, shows high expression in JAK2(V617F)/TN MPs but is almost absent in the HD counterparts. MiR-34a-5p, shown to be associated with MF, is upregulated in pts-MPs; however, its expression is lower in TN-MPs. Interestingly, many miRs regulating inflammation (miR-21, 146a, 223, 19a) and proliferation (miR-199a-3p, 21, 99b) were overexpressed in MPs-pts. Finally, miR-155, 146a, 19a and 194 overexpression affects the JAK-STAT pathway by blocking SOCS3, a negative regulator of the JAK-STAT signalling, and promoting cell survival. Comparing TN vs JAK2(V617F) MPs, the expression of 6 miRs (miR-122, 27a, 744, 584c, 365, 483-5p) was increased, whereas 2 onco-suppressors miRs (let-7b and miR-361) were less expressed. Of note, 6 out of the above mentioned 8 miRs have SRSF1 gene as a common target which is both a protooncogene and a splicing regulator. Conclusions: Circulating TN-MPs show distinct miR signature as compared with the JAK2(V617F) mutated counterparts. This study has the potential to identify disease-related biomarker(s).

Abstract 1185 Background. The potential role of bone marrow (BM)-derived stem cells (SCs) in patients with end-stage liver disease has been addressed by our group in four studies. Main objectives were: 1) to assess stem/progenitor cell mobilization in 24 patients receiving orthotopic liver transplantation (OLT); 2) to evaluate whether G-CSF can be safely administered to patients with liver cirrhosis in order to expand and mobilize BM-derived SCs; 3) to investigate the effects of transplantation of human G-CSF-mobilized CD34+ and CD133+ SCs in mice with chronic liver injury and fibrosis; 4) to evaluate the feasibility and the safety of the purification and intrahepatic reinfusion of increasing numbers of autologous BM-derived G-CSF-mobilized CD133+ SCs in patients with end-stage liver disease. Methods. 1) Flow cytometry analysis, clonogenic assays and RT-PCR have been performed after OLT; 2) 18 patients with advanced liver disease were consecutively treated with increasing doses of G-CSF starting from 2 μg/kg/daily; 3) C57BL/6N mice received CCl4 by inhalation for thirteen weeks and were treated with Cyclosporin-A. Transplantation was performed by injection (tail vein) of 106 CD34+ or CD133+ SCs of three cirrhotic patients. After four weeks from transplantation all mice were sacrificed; 4) G-CSF at 7.5μg/Kg/b.i.d. is administered subcutaneosly (sc) from day 1 until the completion of peripheral blood stem cells (PBSC) collection. Collection of PBSC will begin on day + 4 only if the concentration of CD133+ cells is 38/mL. PB mononuclear cells obtained from mobilized standard-volume leukapheresis will be incubated with Macs colloidal superparamagnetic CD133 microbeads. CliniMacs device is used for the positive selection of CD133+ SCs under GMP conditions. At least 4 weeks after SC mobilization, collection and cryopreservation, highly purified autologous G-CSF-mobilized CD133+ cells are re-infused through the hepatic artery by transfemoral or transbranchial arteriography. CD133+ cells are administered to patients starting from 5×104/Kg patient's body weight and increased every 3 patients. The maximum infused cell dose will be 1×106/kg. G-CSF at 5μg/Kg/day is administered sc for 3 days after the reinfusion of SCs for their expansion and to induce a selective proliferative advantage of reinfused cells in vivo. Results and Discussion. 1) We demonstrated that both early subsets of the hematopoietic SC compartment (CD34+/CD90+ cells) and more mature committed progenitors (CFU-C) were mobilized into PB after OLT. We also demonstrated the release from the BM of liver-committed HSCs co-expressing epithelial markers after OLT; 2) We show that the administration of G-CSF to patients with liver cirrhosis is safe and feasible and allows the mobilization and collection of BM-derived SCs at the dose of 15 mg/kg/day. 3) We demonstrated that mice transplanted with either CD133+ or CD34+ human cells appear to have less fibrotic septa than mice without SC transplantation, suggesting the potential therapeutic role of human SCs on the recovery of liver fibrosis. 4) Up to date, three patients with end stage liver disease have been successfully mobilized with G-CSF and highly purified autologous CD133+ SCs have been re-infused. The number of collected CD133+ SCs is 0,7, 0,2 and 0.35×106/Kg, respectively. The number of the re-infused highly purified CD133+ SCs is 4.7, 5.0 and 5.4×104/Kg, respectively. No adverse events have been recorded during mobilization or intrahepatic SCs re-infusion. Updated results on current patients and future patients will be presented at the Meeting. Disclosures: No relevant conflicts of interest to declare.

Introduction: Myelofibrosis (MF) is an acquired clonal disorder of the hematopoietic stem/progenitor cell (HSPC). Mutations in JAK2, Calreticulin (CALR) and MPL genes are associated with MF. MF is characterized by a state of chronic inflammation and it is argued that the up-regulated production of proinflammatory cytokines by both HSPCs and the surrounding stromal cells generates a microenvironment that selects for the malignant clone. Interestingly, recent findings showed that HSPCs actively sense pro-inflammatory factors. However, the key players linking inflammation and cancer in MF as well as the underlying pathogenetic mechanism are still to be defined. Extracellular nucleotides (ATP) and selected cytokines, such as Tumor Necrosis Factor (TNF)-alpha or the Tissue Inhibitor of Metalloproteinases-1 (TIMP-1), are crucial factors of the inflammatory microenvironment. Here we investigated the in vitro effects of ATP, TNF-alpha or TIMP-1 on phenotype/function of HSPCs from MF patients. Methods: Circulating CD34+ HSPCs from MF patients with JAK2V617F (7 cases), CALR (3 cases) and MPL (3 cases) mutations or from cord blood (5 samples) have been phenotypically and functionally characterized at baseline or after in vitro incubation with or without increasing doses of ATP, TNF-alpha or TIMP-1 (alone or in combination) at different time points. The expression of selected hemopoietic/endothelial markers (CD45,CD133, CD41, CD105, KDR, CD184,CD44) has been analyzed on CD34+ cells by flow cytometry. The expression of the TIMP-1 receptor CD63 was also assessed. Immunomagnetically isolated CD34+ cells were stained with Annexin-V/Propidium Iodide and survival/apoptosis were analyzed by flow cytometry. Migration of purified CD34+ cells were also assayed towards a CXCL12 gradient (150ng/mL) in transwell chambers. Clonogenic assay (BFU-E and CFU-GM in vitro growth) of CD34+cells was also tested. Results: We found that the survival of MF CD34+ cells was strongly stimulated by TNF-alpha (10 ng/mL; 2,9 fold increase) as compared with that of cord blood-derived CD34+ cells (1,7 fold increase). TIMP-1 (100ng/mL) treatment also promotes the survival of MF CD34+ cells. Accordingly, MF patients show increased number and percentages of circulating CD63+CD34+ cells. However, at variance with cord blood-derived CD34+ cells, MF CD34+ cells were more resistant to the citotoxic activity of high dose ATP (1000 nM). Interestingly, when we tested ATP, TNF-alpha and TIMP-1 in combination, the survival of CD34+ cells from MF patients was mainly promoted by the TNF-alpha/TIMP-1 treatment. At variance, ATP addition reduced the TNF-alpha or TIMP-1 pro-survival activity. We also found that TNF-alpha (1,6 fold increase) and TIMP-1 (1,4 fold increase), tested alone, slightly increased the BFU-E formation as compared with that of cord blood- derived CD34+ cells (2,2 and 2,0 fold increase, respectively), without affecting the number of CFU-GM progenitors. By contrast, the number of BFU-E and CFU-GM was significantly reduced when CD34+ cells were incubated in the presence of ATP+TNF-alpha (4,5 and 2,1 fold reduction as compared with untreated cells, respectively), whereas TIMP-1 addition slightly reduced the inhibitory activity of ATP+TNF-alpha (4,0 and 1,8 fold reduction, respectively). Of note, survival and proliferation of MF CD34+ cells were independent from the occurrence of JAK2, MPL or CALR mutations. In addition, despite CXCR4 (CD184) expression of CD34+ cells was significantly increased as compared with the cord blood-derived counterparts, when we analyzed the in vitro migration of CD34+ cells toward a CXCL12 gradient, we found the migration rate of MF CD34+ cells to be highly heterogeneous, ranging from 0,9 to 2,9 fold increase as compared with the corresponding untreated samples. Interestingly, CD34+ cells from 2 out of 3 CALR+patients showed the highest migration capacity. Conclusions: Here we demonstrated that crucial factors of the inflammatory microenvironment abnormally modulate the HSPC functions of MF and may contribute to the maintenance of the malignant clone. Specifically, TNF-alpha together with TIMP-1 promote the survival of the CD34+ HSPCs of MF, while ATP/TNF-alpha combination reduces their proliferation. Interestingly, the inflammatory factors tested in combination differentially modulate the proliferation of the MF CD34+ cells as compared with the factors alone. Disclosures Martinelli: NOVARTIS: Consultancy, Speakers Bureau; BMS: Consultancy, Speakers Bureau; PFIZER: Consultancy; ARIAD: Consultancy.

Introduction: Myelofibrosis (MF) is characterized by clonal hemopoiesis, inflammatory microenvironment and mutations in JAK2, MPL, or CALR genes. Around 10% of patients (pts) does not carry the 3 driver mutations (Triple negative (TN)) displaying, along with lower haemoglobin level and platelet number, significantly worse survival-rates. The malignant hemopoietic stem/progenitor cells compartment of TN MF pts has never been characterized. Here, we compared the in vitro effects of crucial factors of the inflammatory microenvironment on the functional behaviour of TN circulating CD34+ cells vs the JAK2(V617F) mutated counterparts. Methods: Peripheral blood was collected from 9 MF pts (at diagnosis or out of cytotoxic treatment for at least 3 months) and 10 age/sex-matched healthy donors (HD). MF pts were JAK2(V617F) (n=5) mutated and TN (n=4). Circulating CD34+ cells from pts were enumerated by flow cytometry. Immunomagnetically isolated CD34+ cells were in vitro treated with selected inflammatory factors (Interleukin (IL)-1β, Tumor Necrosis Factor (TNF)-α, Interleukin-6) for 12/24 hours and migration/survival ability was investigated. The plasma concentration of these cytokines was evaluated by ELISA. Gene expression profiling analysis (GEP) was performed in CD34+ cells from 3 TN vs 3 JAK2(V617F) mutated pts (Human Transcriptome array 2.0, Affymetrix) and gene set enrichment analysis was conducted by GSEA. Results: GEP analysis showed a total of 165 genes differentially expressed (121 downmodulated and 44 upregulated in CD34+ cells from TN- vs JAK2(V617F) mutated pts). Specifically, we found that the expression of selected anti-apoptotic (TSPYL5, GFI-1 and FCMR) and pro-apototic (TNFSF10, TP53INP1) genes was significantly down- and up-regulated, respectively, in TN-CD34+ cells. Consistent with the GEP data, the in vitro survival of untreated TN-CD34+ cells was significantly decreased as compared with the JAK2(V617F) mutated counterparts. GEP analysis identified also a network of genes involved in cell adhesion, proliferation and inflammation which were mainly down-regulated in TN-CD34+ cells. Specifically, GSEA highlighted an enrichment of cell adhesion, migration and chemotaxis signatures in TN-CD34+ cells. Accordingly, we found that the absolute number of circulating CD34+ and CD34+ CD184+ cells was significantly increased in TN pts as compared with the JAK2(V617F) mutated counterparts (p

Background: Microparticles (MPs) are small vesicles (0.1-1 micron) deriving from plasma membrane budding during homeostasis and cell activation. MPs express antigens and contain constituents from cell of origin and are increased in conditions that are characterized by high cell turnover or death, particularly inflammatory, autoimmune and neoplastic diseases. Myelofibrosis (MF) is a clonal neoplasia of the hematopoietic stem/progenitor cells characterized by distinctive abnormalities in megakaryocyte (MKC) development and platelet (PLT) activation. Mutations in 3 genes (JAK2, CALR, MPL) and chronic inflammation are the main pathogenetic drivers of MF. Ruxolitinib (RUX), a JAK1/2 inhibitor, suppresses both clonal myeloproliferation and release of proinflammatory cytokines, reducing splenomegaly and constitutional symptoms in around 50% of patients (pts). We hypothesized that MPs, as mediators of inflammation, could be overexpressed in MF and possibly predict responses to RUX. Aims: This study aims to: 1) enumerate circulating MK and PLT-derived MPs of MF pts; 2) evaluate the effect of RUX on MPs production by PLT and MK; 3) investigate whether circulating MK and PLT- MPs may be a biomarker of response to RUX. Methods: EDTA-anticoagulated peripheral blood from healthy donors (HD, n=10) and JAK2V617F positive MF pts (n=12) at intermediate-2/high IPSS risk was collected at baseline and 3 and 6 months after RUX therapy and immediately centrifuged. PLT (CD61+CD62P+) and MK (CD61+CD62P-)-derived MPs were analysed in PLT poor plasma samples by flow cytometry (CytoFLEX, Flow Cytometer-Beckman Coulter). The instrument was calibrated with MEGAMIX Beads (Beckman Coulter) with various diameters to cover the MPs (0.5 and 0.9 μm). Results: At 3 and 6 months, 5 out of 12 pts achieved a spleen response (R) according to 2013-IWG-MRT criteria. At baseline, the mean percentage of MK-derived MPs was significantly decreased (29±6 vs 72±5; p