Regulatory T cells from patients with end-stage organ disease can be isolated, expanded and cryopreserved according good manufacturing practice improving their function.

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.

Mobilized Peripheral Blood versus Cord Blood: Insight into the Distinct Role of Proinflammatory Cytokines on Survival, Clonogenic Ability, and Migration of CD34+ Cells.

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 after in vitro exposure to combined crucial inflammatory factors such as interleukin- (IL-) 1ß, 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 the in vitro survival of CB-derived CD34+ cells by reducing apoptosis. Conversely, selected combinations of inflammatory cytokines (IL-1ß + TNF-α, IL-6 + TNF-α, and IL-1ß + TNF-α + TIMP-1) mainly enhanced the in vitro CXCR4-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 distinct in vitro functional activation of neonatal or adult normal HSPCs.

Molecular and functional characterization of CD133+ stem/progenitor cells infused in patients with end-stage liver disease reveals their interplay with stromal liver cells.

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.

Risk factors for infections in myelofibrosis: role of disease status and treatment. A multicenter study of 507 patients.

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 < 0.001). In conclusion, IPSS-category and splenomegaly, emerged as the main risk factors for infections in MF. RUX-treated patients experienced significantly more infection episodes; however, future prospective studies are needed to isolate the confounding contribution of other risk factors such as disease stage. Am. J. Hematol. 92:37-41, 2017. © 2016 Wiley Periodicals, Inc.

Have splenectomy rate and main outcomes of ITP changed after the introduction of new treatments? A monocentric study in the outpatient setting during 35 years.

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 < 0.001), mainly thanks to the optimization of front-line corticosteroids therapy and to the wider availability of second and third-line therapies.

Circulating Calreticulin Is Increased in Myelofibrosis: Correlation with Interleukin-6 Plasma Levels, Bone Marrow Fibrosis, and Splenomegaly.

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.

The choice of second-line therapy in steroid-resistant immune thrombocytopenia: role of platelet kinetics in a single-centre long-term study.

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.

Decreased expression of indoleamine 2,3-dioxygenase 1 in dendritic cells contributes to impaired regulatory T cell development in immune thrombocytopenia.

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.

Distinct profile of CD34+ cells and plasma-derived extracellular vesicles from triple-negative patients with Myelofibrosis reveals potential markers of aggressive disease.

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)-1ß, 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.

Mutations in JAK2 and Calreticulin genes are associated with specific alterations of the immune system in myelofibrosis.

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.

Crucial factors of the inflammatory microenvironment (IL-1ß/TNF-α/TIMP-1) promote the maintenance of the malignant hemopoietic clone of myelofibrosis: an in vitro study.

Along with molecular abnormalities (mutations in JAK2, Calreticulin (CALR) and MPL genes), chronic inflammation is the major hallmark of Myelofibrosis (MF). Here, we investigated the in vitro effects of crucial factors of the inflammatory microenvironment (Interleukin (IL)-1ß, Tumor Necrosis Factor (TNF)-α, Tissue Inhibitor of Metalloproteinases (TIMP)-1 and ATP) on the functional behaviour of MF-derived circulating CD34+ cells.We found that, regardless mutation status, IL-1ß or TNF-α increases the survival of MF-derived CD34+ cells. In addition, along with stimulation of cell cycle progression to the S-phase, IL-1ß or TNF-α ± TIMP-1 significantly stimulate(s) the in vitro clonogenic ability of CD34+ cells from JAK2V617 mutated patients. Whereas in the JAK2V617F mutated group, the addition of IL-1ß or TNF-α + TIMP-1 decreased the erythroid compartment of the CALR mutated patients. Megakaryocyte progenitors were stimulated by IL-1ß (JAK2V617F mutated patients only) and inhibited by TNF-α. IL-1ß + TNF-α + C-X-C motif chemokine 12 (CXCL12) ± TIMP-1 highly stimulates the in vitro migration of MF-derived CD34+ cells. Interestingly, after migration toward IL-1ß + TNF-α + CXCL12 ± TIMP-1, CD34+ cells from JAK2V617F mutated patients show increased clonogenic ability.Here we demonstrate that the interplay of these inflammatory factors promotes and selects the circulating MF-derived CD34+ cells with higher proliferative activity, clonogenic potential and migration ability. Targeting these micro-environmental interactions may be a clinically relevant approach.

Reinfusion of highly purified CD133+ bone marrow-derived stem/progenitor cells in patients with end-stage liver disease: A phase I clinical trial.

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 5×10(4)/kg up to 1×10(6)/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.

The CD47 pathway is deregulated in human immune thrombocytopenia.

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.