Circulating extracellular particles from severe COVID-19 patients show altered profiling and innate lymphoid cell-modulating ability.

Introduction: Extracellular vesicles (EVs) and particles (EPs) represent reliable biomarkers for disease detection. Their role in the inflammatory microenvironment of severe COVID-19 patients is not well determined. Here, we characterized the immunophenotype, the lipidomic cargo and the functional activity of circulating EPs from severe COVID-19 patients (Co-19-EPs) and healthy controls (HC-EPs) correlating the data with the clinical parameters including the partial pressure of oxygen to fraction of inspired oxygen ratio (PaO2/FiO2) and the sequential organ failure assessment (SOFA) score. Methods: Peripheral blood (PB) was collected from COVID-19 patients (n=10) and HC (n=10). EPs were purified from platelet-poor plasma by size exclusion chromatography (SEC) and ultrafiltration. Plasma cytokines and EPs were characterized by multiplex bead-based assay. Quantitative lipidomic profiling of EPs was performed by liquid chromatography/mass spectrometry combined with quadrupole time-of-flight (LC/MS Q-TOF). Innate lymphoid cells (ILC) were characterized by flow cytometry after co-cultures with HC-EPs or Co-19-EPs. Results: We observed that EPs from severe COVID-19 patients: 1) display an altered surface signature as assessed by multiplex protein analysis; 2) are characterized by distinct lipidomic profiling; 3) show correlations between lipidomic profiling and disease aggressiveness scores; 4) fail to dampen type 2 innate lymphoid cells (ILC2) cytokine secretion. As a consequence, ILC2 from severe COVID-19 patients show a more activated phenotype due to the presence of Co-19-EPs. Discussion: In summary, these data highlight that abnormal circulating EPs promote ILC2-driven inflammatory signals in severe COVID-19 patients and support further exploration to unravel the role of EPs (and EVs) in COVID-19 pathogenesis.

Process development and validation of expanded regulatory T cells for prospective applications: an example of manufacturing a personalized advanced therapy medicinal product.

BACKGROUND: A growing number of clinical trials have shown that regulatory T (Treg) cell transfer may have a favorable effect on the maintenance of self-tolerance and immune homeostasis in different conditions such as graft-versus-host disease (GvHD), solid organ transplantation, type 1 diabetes, and others. In this context, the availability of a robust manufacturing protocol that is able to produce a sufficient number of functional Treg cells represents a fundamental prerequisite for the success of a cell therapy clinical protocol. However, extended workflow guidelines for nonprofit manufacturers are currently lacking. Despite the fact that different successful manufacturing procedures and cell products with excellent safety profiles have been reported from early clinical trials, the selection and expansion protocols for Treg cells vary a lot. The objective of this study was to validate a Good Manufacturing Practice (GMP)-compliant protocol for the production of Treg cells that approaches the whole process with a risk-management methodology, from process design to completion of final product development. High emphasis was given to the description of the quality control (QC) methodologies used for the in-process and release tests (sterility, endotoxin test, mycoplasma, and immunophenotype). RESULTS: The GMP-compliant protocol defined in this work allows at least 4.11 × 109 Treg cells to be obtained with an average purity of 95.75 ± 4.38% and can be used in different clinical settings to exploit Treg cell immunomodulatory function. CONCLUSIONS: These results could be of great use for facilities implementing GMP-compliant cell therapy protocols of these cells for different conditions aimed at restoring the Treg cell number and function, which may slow the progression of certain diseases.

An Abnormal Host/Microbiomes Signature of Plasma-Derived Extracellular Vesicles Is Associated to Polycythemia Vera.

Polycythemia Vera (PV) is a myeloproliferative neoplasm with increased risk of thrombosis and progression to myelofibrosis. Chronic inflammation is commonly observed in myeloproliferative neoplasms including PV. The inflammatory network includes the extracellular vesicles (EVs), which play a role in cell-cell communication. Recent evidence points to circulating microbial components/microbes as potential players in hemopoiesis regulation. To address the role of EVs in PV, here we investigated phenotype and microbial DNA cargo of circulating EVs through multidimensional analysis. Peripheral blood and feces were collected from PV patients (n=38) and healthy donors (n=30). Circulating megakaryocyte (MK)- and platelet (PLT)-derived EVs were analyzed by flow cytometry. After microbial DNA extraction from feces and isolated EVs, the 16S rDNA V3-V4 region was sequenced. We found that the proportion of circulating MK-derived EVs was significantly decreased in PV patients as compared with the healthy donors. By contrast, the proportion of the PLT-derived EVs was increased. Interestingly, PV was also associated with a microbial DNA signature of the isolated EVs with higher diversity and distinct microbial composition than the healthy counterparts. Of note, increased proportion of isolated lipopolysaccharide-associated EVs has been demonstrated in PV patients. Conversely, the gut microbiome profile failed to identify a distinct layout between PV patients and healthy donors. In conclusion, PV is associated with circulating EVs harbouring abnormal phenotype and dysbiosis signature with a potential role in the (inflammatory) pathogenesis of the disease.

A Specific Host/Microbial Signature of Plasma-Derived Extracellular Vesicles Is Associated to Thrombosis and Marrow Fibrosis in Polycythemia Vera.

Polycythemia vera is a myeloproliferative neoplasm with increased risk of thrombosis and progression to myelofibrosis. However, no disease-specific risk factors have been identified so far. Circulating extracellular vesicles (EVs) are mostly of megakaryocyte (MK-EVs) and platelet (PLT-EVs) origin and, along with phosphatidylethanolamine (PE)-EVs, play a role in cancer and thrombosis. Interestingly, circulating microbial components/microbes have been recently indicated as potential modifiers of inflammation and coagulation. Here, we investigated phenotype and microbial DNA cargo of EVs after isolation from the plasma of 38 patients with polycythemia vera. Increased proportion of MK-EVs and reduced proportion of PLT-EVs identify patients with thrombosis history. Interestingly, EVs from patients with thrombosis history were depleted in Staphylococcus DNA but enriched in DNA from Actinobacteria members as well as Anaerococcus. In addition, patients with thrombosis history had also lower levels of lipopolysaccharide-associated EVs. In regard to fibrosis, along with increased proportion of PE-EVs, the EVs of patients with marrow fibrosis were enriched in DNA from Collinsella and Flavobacterium. Here, we identified a polycythemia-vera-specific host/microbial EV-based signature associated to thrombosis history and marrow fibrosis. These data may contribute to refining PV prognosis and to identifying novel druggable targets.

The Power of Extracellular Vesicles in Myeloproliferative Neoplasms: "Crafting" a Microenvironment That Matters.

Myeloproliferative Neoplasms (MPN) are acquired clonal disorders of the hematopoietic stem cells and include Essential Thrombocythemia, Polycythemia Vera and Myelofibrosis. MPN are characterized by mutations in three driver genes (JAK2, CALR and MPL) and by a state of chronic inflammation. Notably, MPN patients experience increased risk of thrombosis, disease progression, second neoplasia and evolution to acute leukemia. Extracellular vesicles (EVs) are a heterogeneous population of microparticles with a role in cell-cell communication. The EV-mediated cross-talk occurs via the trafficking of bioactive molecules such as nucleic acids, proteins, metabolites and lipids. Growing interest is focused on EVs and their potential impact on the regulation of blood cancers. Overall, EVs have been suggested to orchestrate the complex interplay between tumor cells and the microenvironment with a pivotal role in "education" and "crafting" of the microenvironment by regulating angiogenesis, coagulation, immune escape and drug resistance of tumors. This review is focused on the role of EVs in MPN. Specifically, we will provide an overview of recent findings on the involvement of EVs in MPN pathogenesis and discuss opportunities for their potential application as diagnostic and prognostic biomarkers.

The diagnostic role of Next Generation Sequencing in uncovering isolated splenomegaly: A case report.

Many diseases can induce splenomegaly, however, about 5% of splenomegalies are idiopathic. When there is no underlying treatable cause, and the splenomegaly significantly affects the quality of life, splenectomy is the best therapeutic choice. A 67-year-old woman had idiopathic and asymptomatic splenomegaly. The increase in splenomegaly resulted in hypersplenism with cytopenia and symptoms related to abdominal discomfort. The patient underwent splenectomy which led to clinical improvement. A histological examination showed the presence of hematopoietic tissue. Peripheral blood Next Generation Sequencing with the myeloid panel SOPHiA Genetics showed the following mutations: ASXL1, SRSF2, KRAS and TET2. Three out of these four mutations were also found in the splenic tissue. Next Generation Sequencing could be useful in the diagnosis of splenomegalies associated with myeloproliferative neoplasms otherwise defined as idiopathic, in order to address a therapeutic strategy.

The "Vesicular Intelligence" Strategy of Blood Cancers.

Blood cancers are a heterogeneous group of disorders including leukemia, multiple myeloma, and lymphoma. They may derive from the clonal evolution of the hemopoietic stem cell compartment or from the transformation of progenitors with immune potential. Extracellular vesicles (EVs) are membrane-bound nanovesicles which are released by cells into body fluids with a role in intercellular communication in physiology and pathology, including cancer. EV cargos are enriched in nucleic acids, proteins, and lipids, and these molecules can be delivered to target cells to influence their biological properties and modify surrounding or distant targets. In this review, we will describe the "smart strategy" on how blood cancer-derived EVs modulate tumor cell development and maintenance. Moreover, we will also depict the function of microenvironment-derived EVs in blood cancers and discuss how the interplay between tumor and microenvironment affects blood cancer cell growth and spreading, immune response, angiogenesis, thrombogenicity, and drug resistance. The potential of EVs as non-invasive biomarkers will be also discussed. Lastly, we discuss the clinical application viewpoint of EVs in blood cancers. Overall, blood cancers apply a 'vesicular intelligence' strategy to spread signals over their microenvironment, promoting the development and/or maintenance of the malignant clone.

Impact of comorbidities and body mass index on the outcome of polycythemia vera patients.

In 816 patients with 2016 World Health Organization-defined polycythemia vera (PV) enrolled in a multicenter retrospective study, we investigated the predictive value of Charlson comorbidity index (CCI) and body mass index (BMI) on thrombosis, progression to post-PV myelofibrosis (PPV-MF) and survival. Patients were subgrouped according to CCI = 0 (58.1%, no comorbidities) or CCI ≥ 1 (41.9%) and according to normal/underweight (BMI < 25, 54.5%) or overweight/obesity (BMI ≥ 25, 45.5%) at PV diagnosis. BMI was available for 529 patients. Patients with CCI ≥ 1 were older and more frequently presented cardiovascular risk factors compared to patients with CCI = 0 (p < 0.001), while overweight/obese patients were more frequently males (p < 0.001). Cumulative incidence of thromboses with death as competing risk was 13.3% at 10 years. Multivariable analysis with death as competing risk showed that previous thromboses (subdistribution hazard ratio [SHR]: 2.1, p = 0.01) and hypertension (SHR: 1.77, p = 0.04) were significantly associated with a higher thrombotic risk, while BMI ≥ 25 lost statistical significance (SHR: 1.69, p = 0.05) and CCI ≥ 1 was excluded after evaluation of goodness of fit. After a median follow-up of 6.1 years, progression to PPV-MF occurred in 44 patients, and 75 patients died. BMI ≥ 25 was associated with a lower probability of progression to PPV-MF (SHR: 0.38, CI95%: 0.15-0.94, p = 0.04) and better survival (hazard ratio [HR]: 0.42, CI95%: 0.18-0.97, p = 0.04). CCI ≥ 1 did not affect progression to PPV-MF (p = 0.44) or survival (p = 0.71).  The evaluation of CCI and BMI may improve the prognostic definition of PV. In patients with hypertension an accurate evaluation of thrombotic risk is warranted.

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.

Is there a gender effect in polycythemia vera?

In recent times, there has been a growing interest in understanding the impact of gender on disease biology and clinical outcomes in Philadelphia-negative chronic myeloproliferative neoplasms. Among those, polycythemia vera (PV) is characterized by increased thrombotic risk, systemic symptoms, and overall reduced survival. Here, we aim to summarize data on whether and to what extent female sex can affect PV biology and outcome. To this end, we will discuss the latest acquisitions in terms of pathogenesis, diagnosis, epidemiology, clinical presentation and symptoms burden, thrombotic risk and related treatment strategies, and prognosis in female patients affected by PV.

Second primary malignancy in myelofibrosis patients treated with ruxolitinib.

Ruxolitinib (RUX), the first JAK1/JAK2 inhibitor approved for myelofibrosis (MF) therapy, has recently been associated with the occurrence of second primary malignancies (SPMs), mainly lymphomas and non-melanoma skin cancers (NMSCs). We analyzed the incidence, risk factors and outcome of SPMs in 700 MF patients treated with RUX in a real-world context. Median follow-up from starting RUX was 2·9 years. Overall, 80 (11·4%) patients developed 87 SPMs after RUX start. NMSCs were the most common SPMs (50·6% of the cases). Multivariate analysis demonstrated that male sex [hazard ratio (HR): 2·37, 95% confidence interval (95%CI): 1·22-4·60, P = 0·01] and thrombocytosis> 400 × 109 /l at RUX start (HR:1·98, 95%CI: 1·10-4·60, P = 0·02) were associated with increased risk for SPMs. Risk factors for NMSC alone were male sex (HR: 3·14, 95%CI: 1·24-7·92, P = 0·02) and duration of hydroxycarbamide and RUX therapy > 5 years (HR: 3·20, 95%CI: 1·17-8·75, P = 0·02 and HR: 2·93, 95%CI: 1·39-6·17, P = 0·005 respectively). In SPMs excluding NMSCs, male sex (HR: 2·41, 95%CI: 1·11-5·25, P = 0·03), platelet > 400 × 109 /l (HR: 3·30, 95%CI: 1·67-6·50, P = 0·001) and previous arterial thromboses (HR: 3·47, 95%CI: 1·48-8·14, P = 0·004) were shown to be associated with higher risk of SPMs. While it is reassuring that no aggressive lymphoma was documented, active skin surveillance is recommended in all patients and particularly after prolonged hydroxycaramide therapy; oncological screening should be triggered by thrombocytosis and arterial thrombosis, particularly in males.

The role of circulating monocytes and JAK inhibition in the infectious-driven inflammatory response of myelofibrosis.

Myelofibrosis (MF) is characterized by chronic inflammation and hyper-activation of the JAK-STAT pathway. Infections are one of the main causes of morbidity/mortality. Therapy with Ruxolitinib (RUX), a JAK1/2 inhibitor, may further increase the infectious risk. Monocytes are critical players in inflammation/immunity through cytokine production and release of bioactive extracellular vesicles. However, the functional behavior of MF monocytes, particularly during RUX therapy, is still unclear. In this study, we found that monocytes from JAK2V617F-mutated MF patients show an altered expression of chemokine (CCR2, CXCR3, CCR5) and cytokine (TNF-α-R, IL10-R, IL1ß-R, IL6-R) receptors. Furthermore, their ability to produce and secrete free and extracellular vesicles-linked cytokines (IL1ß, TNF-α, IL6, IL10) under lipopolysaccharides (LPS) stimulation is severely impaired. Interestingly, monocytes from RUX-treated patients show normal level of chemokine, IL10, IL1ß, and IL6 receptors together with a restored ability to produce intracellular and to secrete extracellular vesicles-linked cytokines after LPS stimulation. Conversely, RUX therapy does not normalize TNF-R1/2 receptors expression and the LPS-driven secretion of free pro/anti-inflammatory cytokines. Accordingly, upon LPS stimulation, in vitro RUX treatment of monocytes from MF patients increases their secretion of extracellular vesicles-linked cytokines but inhibits the secretion of free pro/anti-inflammatory cytokines. In conclusion, we demonstrated that in MF the infection-driven response of circulating monocytes is defective. Importantly, RUX promotes their infection-driven cytokine production suggesting that infections following RUX therapy may not be due to monocyte failure. These findings contribute to better interpreting the immune vulnerability of MF and to envisaging strategies to improve the infection-driven immune response.

Bone Marrow Mesenchymal Stem Cells Support Acute Myeloid Leukemia Bioenergetics and Enhance Antioxidant Defense and Escape from Chemotherapy.

Like normal hematopoietic stem cells, leukemic stem cells depend on their bone marrow (BM) microenvironment for survival, but the underlying mechanisms remain largely unknown. We have studied the contribution of nestin+ BM mesenchymal stem cells (BMSCs) to MLL-AF9-driven acute myeloid leukemia (AML) development and chemoresistance in vivo. Unlike bulk stroma, nestin+ BMSC numbers are not reduced in AML, but their function changes to support AML cells, at the expense of non-mutated hematopoietic stem cells (HSCs). Nestin+ cell depletion delays leukemogenesis in primary AML mice and selectively decreases AML, but not normal, cells in chimeric mice. Nestin+ BMSCs support survival and chemotherapy relapse of AML through increased oxidative phosphorylation, tricarboxylic acid (TCA) cycle activity, and glutathione (GSH)-mediated antioxidant defense. Therefore, AML cells co-opt energy sources and antioxidant defense mechanisms from BMSCs to survive chemotherapy.

Disease-Specific Derangement of Circulating Endocannabinoids and N-Acylethanolamines in Myeloproliferative Neoplasms.

Growing evidence highlights the endocannabinoid (EC) system involvement in cancer progression. Lipid mediators of this system are secreted by hematopoietic cells, including the ECs 2-arachidonoyl-glycerol (2AG) and arachidonoyl-ethanolamide (AEA), the 2AG metabolite 1AG, and members of N-acylethanolamine (NAE) family-palmitoyl-ethanolamide (PEA) and oleoyl-ethanolamide (OEA). However, the relevance of the EC system in myeloproliferative neoplasms (MPN) was never investigated. We explored the EC plasma profile in 55 MPN patients, including myelofibrosis (MF; n = 41), polycythemia vera (PV; n = 9), and essential thrombocythemia (ET; n = 5) subclasses and in 10 healthy controls (HC). AEA, PEA, OEA, 2AG, and 1AG plasma levels were measured by LC-MS/MS. Overall considered, MPN patients displayed similar EC and NAE levels compared to HC. Nonetheless, AEA levels in MPN were directly associated with the platelet count. MF patients showed higher levels of the sum of 2AG and 1AG compared to ET and PV patients, higher OEA/AEA ratios compared to HC and ET patients, and higher OEA/PEA ratios compared to HC. Furthermore, the sum of 2AG and 1AG positively correlated with JAK2V617F variant allele frequency and splenomegaly in MF and was elevated in high-risk PV patients compared to in low-risk PV patients. In conclusion, our work revealed specific alterations of ECs and NAE plasma profile in MPN subclasses and potentially relevant associations with disease severity.

Life after ruxolitinib: Reasons for discontinuation, impact of disease phase, and outcomes in 218 patients with myelofibrosis.

BACKGROUND: After discontinuing ruxolitinib, the outcome of patients with myelofibrosis reportedly has been poor. The authors investigated whether disease characteristics before the receipt of ruxolitinib may predict drug discontinuation in patients with myelofibrosis and whether reasons for drug discontinuation, disease phase at discontinuation, and salvage therapies may influence the outcome. METHODS: A centralized electronic clinical database was created in 20 European hematology centers, including clinical and laboratory data for 524 patients who received ruxolitinib for myelofibrosis. RESULTS: At 3 years, 40.8% of patients had stopped ruxolitinib. Baseline predictors of drug discontinuation were: intermediate-2-risk/high-risk category (Dynamic International Prognostic Score System), a platelet count <100 ×109 per liter, transfusion dependency, and unfavorable karyotype. At last contact, 268 patients (51.1%) had discontinued therapy, and the median drug exposure was 17.5 months. Fifty patients (18.7%) died while taking ruxolitinib. The reasons for discontinuation in the remaining 218 patients were the lack (22.9%) or loss (11.9%) of a spleen response, ruxolitinib-related adverse events (27.5%), progression to blast phase (23.4%), ruxolitinib-unrelated adverse events (9.2%), and allogeneic transplantation during response (5.1%). The median survival after ruxolitinib was 13.2 months and was significantly better in the 167 patients who discontinued ruxolitinib in chronic phase (27.5 vs 3.9 months for those who discontinued in blast phase; P < .001). No survival differences were observed among patients who discontinued ruxolitinib in chronic phase because of lack of response, loss of response, or ruxolitinib-related adverse events. The use of investigational agents and/or ruxolitinib rechallenge were associated with improved outcome. CONCLUSIONS: The survival of patients with myelofibrosis after discontinuation of ruxolitinib is poor, particularly for those who discontinue in blast phase. Salvage therapies can improve outcome, emphasizing the need for novel therapies.

Management of elderly patients with immune thrombocytopenia: Real-world evidence from 451 patients older than 60 years.

INTRODUCTION: Primary Immune thrombocytopenia (ITP) in the elderly is a major clinical challenge which is increasingly frequent due to global ageing population. MATERIALS AND METHODS: To describe baseline ITP features, management, and outcome, a centralized electronic database was established, including data of 451 patients aged ≥60 years that were treated from 2000 onwards and were observed for ≥1 year (total observation of 2704 patient-years). RESULTS: At ITP diagnosis, median age was 71.1 years (age ≥ 75: 42.8%); 237 (53.9%) patients presented with haemorrhages (grade ≥ 3: 7.5%). First-line therapy included prednisone (82.9%), dexamethasone (14.6%), thrombopoietin-receptor agonists (TRAs, 1.3%), and oral immunosuppressive agents (1.1%). Prednisone starting dose ≥1 mg/kg/d (p = .01) and dexamethasone 40 mg/d (p < .001) were mainly reserved to patients aged 60-74, who were more treated with rituximab (RTX, p = .02) and splenectomy (p = .03) second-line. Overall response rates to first and second-line therapies were 83.8% and 84.5%, respectively, regardless of age and treatment type/dose. A total of 178 haemorrhages in 101 patients (grade ≥ 3: n. 52, 29.2%; intracranial in 6 patients), 49 thromboses in 43 patients (grade ≥ 3: n. 26, 53.1%) and 115 infections in 94 patients (grade ≥ 3: n. 23, 20%) were observed during follow-up. Incidence rates of complications per 100 patient-years were: 4.5 (haemorrhages, grade ≥ 3: 1.7), 1.7 (thromboses, grade ≥ 3: 0.9), and 3.9 (infections, grade ≥ 3: 0.7). TRAs use were associated with reduced risk of bleeding and infections, while cardiovascular risk factors (particularly, diabetes) significantly predicted thromboses and infections. CONCLUSIONS: Age-adapted treatment strategies are required in elderly and very elderly patients.

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.

Impact of comorbidities and body mass index in patients with myelofibrosis treated with ruxolitinib.

Comorbidities defined by the Charlson comorbidity index (CCI) and body mass index (BMI) are significantly associated with outcome in patients who receive continuous treatment with tyrosine kinase inhibitors. We evaluated the impact of CCI and BMI on responses, drug-related toxicities, and outcome in a cohort of 402 patients with myelofibrosis (MF) treated with ruxolitinib in 23 European Hematology Centers. Comorbidities were evaluable in all 402 patients. A higher (≥ 3) CCI did not correlate with a lower spleen reduction at any time (p = 0.68) or symptoms' response (p = 0.11), but influenced the onset of anemia during the first 3 months of treatment and later (p = 0.02 and p = 0.03, respectively) in patients without anemia baseline. BMI was evaluable in 380 patients and did not correlate with differences in spleen and symptoms response (p = 0.57 and p = 0.49, respectively). A higher CCI and a lower BMI correlated also with a reduced overall survival (p < 0.001 and p = 0.02, respectively). The achievement of a spleen response at 6 months could counterbalance the negative impact of comorbidities, while patients who were underweight when starting ruxolitinib and did not achieve a spleen response at 6 months were projected to the worse outcome. In MF patients treated with ruxolitinib, BMI and comorbidities did not influence the achievement of spleen/symptom responses, but they contributed to the early identification of patients who deserve a strict monitoring during treatment.