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•EBNA2 reduces ICOSL expression in B-cell lymphoma such as DLBCL through miR-24.•Silencing of miR-24 reconstitutes tumor immunogenicity and induces apoptosis in DLBCL.

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Superoxide dismutase 2 (SOD2) catalyzes the dismutation of superoxide to hydrogen peroxide in mitochondria, limiting mitochondrial damage. The SOD2 amino acid valine-to-alanine substitution at position 16 (V16A) in the mitochondrial leader sequence is a common genetic variant among patients with sickle cell disease (SCD). However, little is known about the cardiovascular consequences of SOD2V16A in SCD patients or its impact on endothelial cell function. Here, we show SOD2V16A associates with increased tricuspid regurgitant velocity (TRV), systolic blood pressure, right ventricle area at systole, and declined 6-minute walk distance in 410 SCD patients. Plasma lactate dehydrogenase, a marker of oxidative stress and hemolysis, significantly associated with higher TRV. To define the impact of SOD2V16A in the endothelium, we introduced the SOD2V16A variant into endothelial cells. SOD2V16A increases hydrogen peroxide and mitochondrial reactive oxygen species (ROS) production compared with controls. Unexpectedly, the increased ROS was not due to SOD2V16A mislocalization but was associated with mitochondrial complex IV and a concomitant decrease in basal respiration and complex IV activity. In sum, SOD2V16A is a novel clinical biomarker of cardiovascular dysfunction in SCD patients through its ability to decrease mitochondrial complex IV activity and amplify ROS production in the endothelium.

Clonal hematopoiesis of indeterminate potential (CHIP) is associated with increased risk of cancers and inflammation-related diseases. This phenomenon becomes common in persons aged ≥80 years, in whom the implications of CHIP are not well defined. We performed a mutational screening in 1794 persons aged ≥80 years and investigated the relationships between CHIP and associated pathologies. Mutations were observed in one-third of persons aged ≥80 years and were associated with reduced survival. Mutations in JAK2 and splicing genes, multiple mutations (DNMT3A, TET2, and ASXL1 with additional genetic lesions), and variant allele frequency ≥0.096 had positive predictive value for myeloid neoplasms. Combining mutation profiles with abnormalities in red blood cell indices improved the ability of myeloid neoplasm prediction. On this basis, we defined a predictive model that identifies 3 risk groups with different probabilities of developing myeloid neoplasms. Mutations in DNMT3A, TET2, ASXL1, or JAK2 were associated with coronary heart disease and rheumatoid arthritis. Cytopenia was common in persons aged ≥80 years, with the underlying cause remaining unexplained in 30% of cases. Among individuals with unexplained cytopenia, the presence of highly specific mutation patterns was associated with myelodysplastic-like phenotype and a probability of survival comparable to that of myeloid neoplasms. Accordingly, 7.5% of subjects aged ≥80 years with cytopenia had presumptive evidence of myeloid neoplasm. In summary, specific mutational patterns define different risk of developing myeloid neoplasms vs inflammatory-associated diseases in persons aged ≥80 years. In individuals with unexplained cytopenia, mutational status may identify those subjects with presumptive evidence of myeloid neoplasms.

Polyphosphate is a procoagulant inorganic polymer of linear-linked orthophosphate residues. Multiple investigations have established the importance of platelet polyphosphate in blood coagulation; however, the mechanistic details of polyphosphate homeostasis in mammalian species remain largely undefined. In this study, xenotropic and polytropic retrovirus receptor 1 (XPR1) regulated polyphosphate in platelets and was implicated in thrombosis in vivo. We used bioinformatic analyses of omics data to identify XPR1 as a major phosphate transporter in platelets. XPR1 messenger RNA and protein expression inversely correlated with intracellular polyphosphate content and release. Pharmacological interference with XPR1 activity increased polyphosphate stores, led to enhanced platelet-driven coagulation, and amplified thrombus formation under flow via the polyphosphate/factor XII pathway. Conditional gene deletion of Xpr1 in platelets resulted in polyphosphate accumulation, accelerated arterial thrombosis, and augmented activated platelet-driven pulmonary embolism without increasing bleeding in mice. These data identify platelet XPR1 as an integral regulator of platelet polyphosphate metabolism and reveal a fundamental role for phosphate homeostasis in thrombosis.

Systemic mastocytosis (SM) is a rare hematological neoplasm characterized by pleomorphic symptoms, related to mediators release from pathological mast cells. Tissue infiltration of liver, spleen, gastro-intestinal (GI) tract and osteolysis are present in aggressive disease. As KIT D816V mutation can be found in approximately 80% of patients, several additional somatic mutations of prognostic significance have been detected in a subset of patients.

Background:Unanchored MAIC (Matching-Adjusted Indirect Comparison) is an ITC (Indirect Treatment Comparison) method adjusting for cross-trial heterogeneity in patient demographic or disease that are believed to be either prognostic or treatment effect modifiers. In this analysis, two trials for adults with secondary acute myeloid leukemia (AML) were compared by an unanchored MAIC.

Introduction:Based on the efficacy of the fixed-duration venetoclax and rituximab combination (VenR) in the setting of relapsed/refractory (R/R) patients with chronic lymphocytic leukemia (CLL), we investigated the efficacy and safety of the front-line VenR regimen in young (≤65 years) and fit patients with CLL and an unfavorable biologic profile in the GIMEMA LLC1518 VERITAS trial. Here, we report the 36-month updated results of this phase II, single-arm, multicenter, front-line study for young patients with CLL carrying an unmutated IGHV profile and/or a TP53 disruption.

Inherited thrombocytopenias (ITs) are a heterogeneous group of disorders characterized by low platelet count that may result in bleeding tendency. Despite progress being made in defining the genetic causes of ITs, nearly 50% of patients with familial thrombocytopenia are affected with forms of unknown origin. Here, through exome sequencing of 2 siblings with autosomal-recessive thrombocytopenia, we identified biallelic loss-of-function variants in PTPRJ. This gene encodes for a receptor-like PTP, PTPRJ (or CD148), which is expressed abundantly in platelets and megakaryocytes. Consistent with the predicted effects of the variants, both probands have an almost complete loss of PTPRJ at the messenger RNA and protein levels. To investigate the pathogenic role of PTPRJ deficiency in hematopoiesis in vivo, we carried out CRISPR/Cas9-mediated ablation of ptprja (the ortholog of human PTPRJ) in zebrafish, which induced a significantly decreased number of CD41+ thrombocytes in vivo. Moreover, megakaryocytes of our patients showed impaired maturation and profound defects in SDF1-driven migration and formation of proplatelets in vitro. Silencing of PTPRJ in a human megakaryocytic cell line reproduced the functional defects observed in patients’ megakaryocytes. The disorder caused by PTPRJ mutations presented as a nonsyndromic thrombocytopenia characterized by spontaneous bleeding, small-sized platelets, and impaired platelet responses to the GPVI agonists collagen and convulxin. These platelet functional defects could be attributed to reduced activation of Src family kinases. Taken together, our data identify a new form of IT and highlight a hitherto unknown fundamental role for PTPRJ in platelet biogenesis.

Inherited thrombocytopenias (ITs) are a heterogeneous group of disorders characterized by low platelet count that may result in bleeding tendency. Despite progress being made in defining the genetic causes of ITs, nearly 50% of patients with familial thrombocytopenia are affected with forms of unknown origin. Here, through exome sequencing of 2 siblings with autosomal-recessive thrombocytopenia, we identified biallelic loss-of-function variants in PTPRJ.This gene encodes for a receptor-like PTP, PTPRJ (or CD148), which is expressed abundantly in platelets and megakaryocytes. Consistent with the predicted effects of the variants, both probands have an almost complete loss of PTPRJ at the messenger RNA and protein levels. To investigate the pathogenic role of PTPRJ deficiency in hematopoiesis in vivo, we carried out CRISPR/Cas9-mediated ablation of ptprja(the ortholog of human PTPRJ) in zebrafish, which induced a significantly decreased number of CD41+thrombocytes in vivo. Moreover, megakaryocytes of our patients showed impaired maturation and profound defects in SDF1-driven migration and formation of proplatelets in vitro. Silencing of PTPRJin a human megakaryocytic cell line reproduced the functional defects observed in patients' megakaryocytes. The disorder caused by PTPRJmutations presented as a nonsyndromic thrombocytopenia characterized by spontaneous bleeding, small-sized platelets, and impaired platelet responses to the GPVI agonists collagen and convulxin. These platelet functional defects could be attributed to reduced activation of Src family kinases. Taken together, our data identify a new form of IT and highlight a hitherto unknown fundamental role for PTPRJ in platelet biogenesis.

The rarity of neoplastic cells in the biopsy imposes major technical hurdles that have so far limited genomic studies in classical Hodgkin lymphoma (cHL). By using a highly sensitive and robust deep next-generation sequencing approach for circulating tumor DNA (ctDNA), we aimed to identify the genetics of cHL in different clinical phases, as well as its modifications on treatment. The analysis was based on specimens collected from 80 newly diagnosed and 32 refractory patients with cHL, including longitudinal samples collected under ABVD (adriamycin, bleomycin, vinblastine, dacarbazine) chemotherapy and longitudinal samples from relapsing patients treated with chemotherapy and immunotherapy. ctDNA mirrored Hodgkin and Reed-Sternberg cell genetics, thus establishing ctDNA as an easily accessible source of tumor DNA for cHL genotyping. By identifying STAT6as the most frequently mutated gene in ∼40% of cases, we refined the current knowledge of cHL genetics. Longitudinal ctDNA profiling identified treatment-dependent patterns of clonal evolution in patients relapsing after chemotherapy and patients maintained in partial remission under immunotherapy. By measuring ctDNA changes during therapy, we propose ctDNA as a radiation-free tool to track residual disease that may integrate positron emission tomography imaging for the early identification of chemorefractory patients with cHL. Collectively, our results provide the proof of concept that ctDNA may serve as a novel precision medicine biomarker in cHL.

•AIHA in pregnancy/puerperium was severe, required therapy (steroids ± IVIG) in nearly all cases and blood transfusions in half of patients.•Adverse maternal-fetal outcomes occurred in ∼1 of 4 cases, with fetal complications being more frequent and severe than in healthy women.

Voso: Bristol Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Passamonti:Roche: Other: Support of parent study and funding of editorial support; Novartis: Speakers Bureau; BMS: Speakers Bureau. Santoro:Bristol-Myers Squibb, SERVIER, Gilead Sciences, Pfizer, Eisai, Bayer, MSD, Sanofi, ArQule: Consultancy; Takeda, Roche, Abbvie, Amgen, Celgene, AstraZeneca, ArQule, Lilly, Sandoz, Novartis, Bristol-Myers Squibb, Servier, Gilead Sciences, Pfizer, Eisai, Bayer, MSD: Speakers Bureau; Takeda, Roche, Abbvie, Amgen, Celgene, AstraZeneca, ArQule, Lilly, Sandoz, Novartis, Bristol-Myers Squibb, Servier, Gilead Sciences, Pfizer, Eisai, Bayer, MSD: Speakers Bureau; Arqule, Sanofi: Consultancy; Bristol-Myers Squibb, SERVIER, Gilead Sciences, Pfizer, Eisai, Bayer, MSD, Sanofi, ArQule: Consultancy, Speakers Bureau; Bristol-Myers Squibb, SERVIER, Gilead Sciences, Pfizer, Eisai, Bayer, MSD, Sanofi, ArQule: Consultancy; Bristol Myers Squibb, Servier, Gilead, Pfizer, Eisai, Bayer, MSD: Membership on an entity's Board of Directors or advisory committees. Santini:Menarini: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; Acceleron: Consultancy; Pfizer: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; BMS: Consultancy, Honoraria; Takeda: Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Honoraria; Johnson & Johnson: Honoraria. Sanz:Takeda Pharmaceutical Ltd.: Membership on an entity's Board of Directors or advisory committees; LaHoffman Roche Ltd.: Membership on an entity's Board of Directors or advisory committees; Abbvie Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Helsinn: Membership on an entity's Board of Directors or advisory committees. Diez-Campelo:Celgene BMS: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau.

Introduction

Chronic lymphocytic leukemia (CLL) is characterized by an accumulation of mature CD19+CD5+CD20dim B lymphocytes that typically express the B-cell activation marker CD23. In the present study, we cloned and expressed in T lymphocytes a novel chimeric antigen receptor (CAR) targeting the CD23 antigen (CD23.CAR). CD23.CAR+ T cells showed specific cytotoxic activity against CD23+ tumor cell lines (average lysis 42%) and primary CD23+ CLL cells (average lysis 58%). This effect was obtained without significant toxicity against normal B lymphocytes, in contrast to CARs targeting CD19 or CD20 antigens, which are also expressed physiologically by normal B lymphocytes. Moreover, CLL-derived CD23.CAR+ T cells released inflammatory cytokines (1445-fold more TNF-β, 20-fold more TNF-α, and 4-fold more IFN-γ). IL-2 was also produced (average release 2681 pg/mL) and sustained the antigen-dependent proliferation of CD23.CAR+ T cells. Redirected T cells were also effective in vivo in a CLL Rag2−/−γc−/− xenograft mouse model. Compared with mice treated with control T cells, the infusion of CD23.CAR+ T cells resulted in a significant delay in the growth of the MEC-1 CLL cell line. These data suggest that CD23.CAR+ T cells represent a selective immunotherapy for the elimination of CD23+ leukemic cells in patients with CLL.

Mechanisms governing stress-induced hematopoietic progenitor cell mobilization are not fully deciphered. We report that during granulocyte colony-stimulating factor–induced mobilization c-Met expression and signaling are up-regulated on immature bone marrow progenitors. Interestingly, stromal cell–derived factor 1/CXC chemokine receptor-4 signaling induced hepatocyte growth factor production and c-Met activation. We found that c-Met inhibition reduced mobilization of both immature progenitors and the more primitive Sca-1+/c-Kit+/Lin− cells and interfered with their enhanced chemotactic migration to stromal cell–derived factor 1. c-Met activation resulted in cellular accumulation of reactive oxygen species by mammalian target of rapamycin inhibition of Forkhead Box, subclass O3a. Blockage of mammalian target of rapamycin inhibition or reactive oxygen species signaling impaired c-Met–mediated mobilization. Our data show dynamic c-Met expression and function in the bone marrow and show that enhanced c-Met signaling is crucial to facilitate stress-induced mobilization of progenitor cells as part of host defense and repair mechanisms.

Using a combination high-throughput screening technology, multiple classes of drugs and targeted agents were identified that synergize with dexamethasone (Dex) in multiple myeloma (MM) cells. Performing combination screening with these enhancers, we discovered an unexpected synergistic interaction between adenosine receptor agonists and phosphodiesterase (PDE) inhibitors that displays substantial activity in a panel of MM and diffuse large B-cell lymphoma (DLBCL) cell lines and tumor cells from MM patients. We have used selective adenosine receptor agonists, antagonists, and PDE inhibitors as well as small interfering RNAs targeting specific molecular isoforms of these proteins to dissect the molecular mechanism of this synergy. The adenosine A2A receptor and PDE2, 3, 4, and 7 are important for activity. Drug combinations induce cyclic AMP (cAMP) accumulation and up-regulate PDE4B. We also observe rigorous mathematical synergy in 3-way combinations containing A2A agonists, PDE inhibitors, and Dex at multiple concentrations and ratios. Taken together, these data suggest that A2A agonist/PDE inhibitor combinations may be attractive as an adjunctive to clinical glucocorticoid containing regiments for patients with MM or DLBCL and confer benefit in both glucocorticoid-sensitive and -resistant populations.

Interaction of chronic lymphocytic leukemia (CLL) B cells with tissue microenvironment has been suggested to favor disease progression by promoting malignant B-cell growth. Previous work has shown expression in peripheral blood (PB) of CLL B cells of activation-induced cytidine deaminase (AID) among CLL patients with an unmutated (UM) profile of immunoglobulin genes and with ongoing class switch recombination (CSR) process. Because AID expression results from interaction with activated tissue microenvironment, we speculated whether the small subset with ongoing CSR is responsible for high levels of AID expression and could be derived from this particular microenvironment. In this work, we quantified AID expression and ongoing CSR in PB of 50 CLL patients and characterized the expression of different molecules related to microenvironment interaction. Our results show that among UM patients (1) high AID expression is restricted to the subpopulation of tumoral cells ongoing CSR; (2) this small subset expresses high levels of proliferation, antiapoptotic and progression markers (Ki-67, c-myc, Bcl-2, CD49d, and CCL3/4 chemokines). Overall, this work outlines the importance of a cellular subset in PB of UM CLL patients with a poor clinical outcome, high AID levels, and ongoing CSR, whose presence might be a hallmark of a recent contact with the microenvironment.

Hodgkin lymphoma relapsing after autologous transplantation (autoSCT) has a dismal outcome. Allogeneic transplantation (alloSCT) using reduced intensity conditioning (RIC) is a salvage option, but its effectiveness is still unclear. To evaluate the role of RIC alloSCT, we designed a retrospective study based on the commitment of attending physicians to perform a salvage alloSCT; thus, only Hodgkin lymphoma patients having human leukocyte antigen-typing immediately after the failed autoSCT were included. Of 185 patients, 122 found an identical sibling (55%), a matched unrelated (32%) or a haploidentical sibling (13%) donor; 63 patients did not find any donor. Clinical features of both groups did not differ. Two-year progression-free (PFS) and overall survival (OS) were better in the donor group (39.3% vs 14.2%, and 66% vs 42%, respectively, P < .001) with a median follow-up of 48 months. In multivariable analysis, having a donor was significant for better PFS and OS (P < .001). Patients allografted in complete remission showed a better PFS and OS. This is the largest study comparing RIC alloSCT versus conventional treatment after a failed autoSCT, indicating a survival benefit for patients having a donor.

The concept of endothelial progenitor cells (EPCs) has attracted considerable interest in cardiovascular research, but despite a decade of research there are still no specific markers for EPCs and results from clinical trials remain controversial. Using liquid chromatography–tandem mass spectrometry, we analyzed the protein composition of microparticles (MPs) originating from the cell surface of EPC cultures. Our data revealed that the conventional methods for isolating mononuclear cells lead to a contamination with platelet proteins. Notably, platelets readily disintegrate into platelet MPs. These platelet MPs are taken up by the mononuclear cell population, which acquires “endothelial” characteristics (CD31, von Willebrand factor [VWF], lectin-binding), and angiogenic properties. In a large population-based study (n = 526), platelets emerged as a positive predictor for the number of colony-forming units and early outgrowth EPCs. Our study provides the first evidence that the cell type consistent with current definitions of an EPC phenotype may arise from an uptake of platelet MPs by mononuclear cells resulting in a gross misinterpretation of their cellular progeny. These findings demonstrate the advantage of using an unbiased proteomic approach to assess cellular phenotypes and advise caution in attributing the benefits in clinical trials using unselected bone marrow mononuclear cells (BMCs) to stem cell-mediated repair.

Patients with hematological malignancies (HM) undergoing hematopoietic stem cell transplantation (HSCT) have an increased vulnerability to SARS-Cov-2 (Sharma et al, Lancet Haematology 2020; Ljungman et al, Leukemia 2021), the reason why international guidelines strongly support the need for a protective vaccination for these subjects. The most relevant data currently available on the response to a complete anti-SARS-Cov-2 vaccination cycle in HM patients after HSCT refer to 314 patients reported in a Lithuanian national survey (Maneikis et al, Lancet Haematol 2021). In this study, the median titers of antibodies against SARS-Cov-2, determined 7-21 days after the second vaccination, were comparable to that of healthy controls (HC) in both autologous and allogeneic groups, with no patient found below the protective threshold of 50 arbitrary units (AU)/ml. Notably, the large majority of patients had received the transplant more than 1 year before vaccination.

The antitumor effect of adoptively transferred tumor-specific cytotoxic T lymphocytes (CTLs) is impaired by the limited capacity of these cells to expand within the tumor microenvironment. Administration of interleukin 2 (IL-2) has been used to overcome this limitation, but the systemic toxicity and the expansion of unwanted cells, including regulatory T cells, limit the clinical value of this strategy. To discover whether transgenic expression of lymphokines by the CTLs themselves might overcome these limitations, we evaluated the effects of transgenic expression of IL-2 and IL-15 in our model of Epstein Barr Virus–specific CTLs (EBV-CTLs). We found that transgenic expression of IL-2 or IL-15 increased the expansion of EBV-CTLs both in vitro and in vivo in a severe combined immunodeficiency disease (SCID) mouse model and enhanced antitumor activity. Although the proliferation of these cytokine genes transduced CTLs remained strictly antigen dependent, clinical application of this approach likely requires the inclusion of a suicide gene to deal with the potential development of T-cell mutants with autonomous growth. We found that the incorporation of an inducible caspase-9 suicide gene allowed efficient elimination of transgenic CTLs after exposure to a chemical inducer of dimerization, thereby increasing the safety and feasibility of the approach.

Burkitt lymphoma (BL) is a B-cell tumor whose characteristic gene aberration is the translocation t(8;14), which determines c-myc overexpression. Several genetic and epigenetic alterations other than c-myc overexpression have also been described in BL. It has been demonstrated that the RBL2/p130gene, a member of the retinoblastoma family (pRbs), is mutated in BL cell lines and primary tumors. The aim of this study was to investigate the biologic effect of RBL2/p130in BL cells and its possible role in lymphomagenesis. Therefore, we reintroduced a functional RBL2/p130in BL cell lines where this gene was mutated. Our results demonstrated that RBL2/p130-transfected cells regain growth control. This suggests that RBL2/p130may control the expression of several genes, which may be important for cell growth and viability. Gene-expression analysis revealed a modulation of several genes, including CGRRF1, RGS1, BTG1, TIA1, and PCDHA2, upon RBL2/p130reintroduction. We then monitored their expression in primary tumors of endemic BL as well, demonstrating that their expression resembled those of the BL cell lines. In conclusion, these data suggest that, as RBL2/p130modulates the expression of target genes, which are important for cell growth and viability, its inactivation may be relevant for the occurrence of BL.

Burkitt lymphoma (BL) is a B-cell tumor whose characteristic gene aberration is the translocation t(8;14), which determines c-myc overexpression. Several genetic and epigenetic alterations other than c-myc overexpression have also been described in BL. It has been demonstrated that the RBL2/p130 gene, a member of the retinoblastoma family (pRbs), is mutated in BL cell lines and primary tumors. The aim of this study was to investigate the biologic effect of RBL2/p130 in BL cells and its possible role in lymphomagenesis. Therefore, we reintroduced a functional RBL2/p130 in BL cell lines where this gene was mutated. Our results demonstrated that RBL2/p130-transfected cells regain growth control. This suggests that RBL2/p130 may control the expression of several genes, which may be important for cell growth and viability. Gene-expression analysis revealed a modulation of several genes, including CGRRF1, RGS1, BTG1, TIA1, and PCDHA2, upon RBL2/p130 reintroduction. We then monitored their expression in primary tumors of endemic BL as well, demonstrating that their expression resembled those of the BL cell lines. In conclusion, these data suggest that, as RBL2/p130 modulates the expression of target genes, which are important for cell growth and viability, its inactivation may be relevant for the occurrence of BL.

Protein kinase Cδ (PKCδ) has been shown by pharmacologic approaches to negatively regulate collagen-induced platelet aggregation. Here we addressed the molecular and cellular mechanisms underlying this negative regulation. Using PKCδ–/–platelets, we show that the mechanism did not involve altered inside-out signaling to integrin αIIbβ3and did not affect early signaling events downstream of GPVI, because there was no difference in tyrosine phosphorylation of PLCγ2 between wild-type and PKCδ–/–platelets. There was also no increase in secretion of dense granule content, in contrast to studies using rottlerin where secretion was enhanced. Importantly, however, there was marked enhancement of filopodia generation in PKCδ–/–platelets upon adhesion to collagen compared with wild-type platelets. Filopodia play an essential role regulating adhesive events leading to platelet aggregation by increasing platelet-platelet contact. We show that the critical effector for PKCδ is vasodilator-stimulated phosphoprotein (VASP), a major regulator of actin cytoskeleton dynamics. PKCδ physically interacts with VASP constitutively and regulates its phosphorylation on Ser157. In VASP–/–platelets, the enhancement of filopodia generation, actin polymerization, and platelet aggregation by rottlerin is ablated. PKCδ is therefore a critical negative regulator of filopodia, and hence platelet aggregation, through a functional interaction with the actin organizer VASP.

Dendritic-cell (DC) migration to secondary lymphoid organs is crucial for the initiation of adaptive immune responses. Although LPS up-regulates CCR7 on DCs, a second signal is required to enable them to migrate toward the chemokine CCL19 (MIP-3β). We found that the nitric oxide (NO) donor NOR4 provides a signal allowing LPS-stimulated DCs to migrate toward CCL19. NO affects DC migration through both the initial activation of the cGMP/cGMP kinase (cGMP/cGK) pathway and a long-term effect that reduced cGK activity via negative feedback. Indeed, migration of DCs toward CCL19, unlike migration toward CXCL12 (SDF-1α), required inhibition of cGK. LPS increased both cGK expression and cGK activity as measured by phosphorylation of the key cGK target vasodilator-stimulated phosphoprotein (VASP). Because cGK phosphorylation of VASP can disrupt focal adhesions and inhibit cell migration, LPS-induced VASP phosphorylation may prevent DCs from migrating without a second signal. Long-term NOR4 treatment inhibited the increase in cGK-dependent VASP phosphorylation, releasing this brake so that DCs can migrate. NO has been implicated in the regulation of autoimmunity through its effect on T cells. Our results suggest that NO regulation of DC migration and cytokine production may contribute to the protective effects of NO in autoimmune disorders.

Bone marrow–derived mesenchymal stem cells (BM-MSCs) are stromal cells with the ability to proliferate and differentiate into many tissues. Although they represent powerful tools for several therapeutic settings, mechanisms regulating their migration to peripheral tissues are still unknown. Here, we report chemokine receptor expression on human BM-MSCs and their role in mediating migration to tissues. A minority of BM-MSCs (2% to 25%) expressed a restricted set of chemokine receptors (CXC receptor 4 [CXCR4], CX3C receptor 1 [CX3CR1], CXCR6, CC chemokine receptor 1 [CCR1], CCR7) and, accordingly, showed appreciable chemotactic migration in response to the chemokines CXC ligand 12 (CXCL12), CX3CL1, CXCL16, CC chemokine ligand 3 (CCL3), and CCL19. Using human pancreatic islets as an in vitro model of peripheral tissue, we showed that islet supernatants released factors able to attract BM-MSCs in vitro, and this attraction was principally mediated by CX3CL1 and CXCL12. Moreover, cells with features of BM-MSCs were detected within the pancreatic islets of mice injected with green fluorescent protein (GFP)–positive BM. A population of bona fide MSCs that also expressed CXCR4, CXCR6, CCR1, and CCR7 could be isolated from normal adult human pancreas. This study defines the chemokine receptor repertoire of human BM-MSCs that determines their migratory activity. Modulation of homing capacity may be instrumental for harnessing the therapeutic potential of BM-MSCs.

Semaphorins, a large family of membrane-bound and secreted proteins, signal through their transmembrane receptors, the plexins. Semaphorins and plexins share structural homologies with scatter factor receptors, a family of tyrosine kinase receptors for which Met is the prototype. Semaphorins have been studied primarily in the developing nervous system, where they act as repelling cues in axon guidance. However, they are widely expressed in several tissues, and their role in epithelial morphogenesis has been recently established. Not much is known about their role in angiogenesis, a key step during embryonic development and adulthood. Here we demonstrate that a semaphorin, Sema4D, is angiogenic in vitro and in vivo and that this effect is mediated by its high-affinity receptor, Plexin B1. Moreover, we prove that biologic effects elicited by Plexin B1 require coupling and activation of the Met tyrosine kinase. In sum, we identify a proangiogenic semaphorin and provide insight about the signaling machinery exploited by Plexin B1 to control angiogenesis.

The effects of estrogen on the immune system are still largely unknown. We have investigated the effect of 17β-estradiol (E2) on human monocyte-derived immature dendritic cells (iDCs). Short-term culture in E2 had no effect on iDC survival or the expression of cell surface markers. However, E2 treatment significantly increased the secretion of interleukin 6 (IL-6) in iDCs and also increased secretion of osteoprotegerin (OPG) by DCs. Furthermore, E2 significantly increased secretion of the inflammatory chemokines IL-8 and monocyte chemoattractant protein 1 (MCP-1) by iDCs, but not the production of the constitutive chemokines thymus and activation-regulated chemokine (TARC) and macrophage-derived chemokine (MDC). However, after E2 pretreatment the lipopolysaccharide (LPS)–induced production of MCP-1, TARC, and MDC by DCs was clearly enhanced. Moreover, mature DCs pretreated with E2 stimulated T cells better than control cells. Finally, we found that E2 provides an essential signal for migration of mature DCs toward CCL19/macrophage inflammatory protein 3β (MIP3β). In summary, E2 may affect DC regulation of T-cell and B-cell responses, as well as help to sustain inflammatory responses. This may explain, in part, the reason serum levels of estrogen correlate with the severity of certain autoimmune diseases.

The autoimmune/lymphoproliferative syndrome (ALPS) displays defective function of Fas, autoimmunities, lymphadenopathy/splenomegaly, and expansion of CD4/CD8 double-negative (DN) T cells. Dianzani autoimmune/lymphoproliferative disease (DALD) is an ALPS variant lacking DN cells. Both forms have been ascribed to inherited mutations hitting the Fas system but other factors may be involved. A pilot cDNA array analysis on a DALD patient detected overexpression of the cytokine osteopontin (OPN). This observation was confirmed by enzyme-linked immunosorbent assay (ELISA) detection of higher OPN serum levels in DALD patients (n = 25) than in controls (n = 50). Analysis of the OPNcDNA identified 4 polymorphisms forming 3 haplotypes (A, B, and C). Their overall distribution and genotypic combinations were different in patients (N = 26) and controls (N = 158) (P< .01). Subjects carrying haplotype B and/or C had an 8-fold higher risk of developing DALD than haplotype A homozygotes. Several data suggest that these haplotypes influence OPN levels: (1) in DALD families, high levels cosegregated with haplotype B or C; (2) in healthy controls, haplotype B or C carriers displayed higher levels than haplotype A homozygotes; and (3) in AB and AC heterozygotes, mRNA for haplotype B or C was more abundant than that for haplotype A. In vitro, exogenous OPN decreased activation-induced T-cell death, which suggests that high OPN levels are involved in the apoptosis defect.

Growth and survival of chronic B-cell tumors are favored by the malignant cell's capacity to respond to selected microenvironmental stimuli provided by nontumoral bystander cells. To investigate which mechanisms operate in these crosstalks and whether they are malignancy-related or reproduce the mechanisms used by normal B cells we have studied the expression and functional role of semaphorin CD100 (now called Sema4D) in chronic lymphocytic leukemia (CLL) cells and normal CD5+ B cells. We demonstrate here that (1) leukemic and normal CD5+ B lymphocytes uniformly express CD100; (2) the CD100 high-affinity receptor Plexin-B1 is expressed by bone marrow stromal cells, follicular dendritic cells, and activated T lymphocytes, and is thus available to CD100+ lymphocytes in different specific microenvironments; and (3) upon interaction between CD100 and Plexin-B1 both CLL and normal CD5+ B cells increase their proliferative activity and extend their life span. These findings establish that Plexin-B1 is an easily accessible receptor for CD100 within the immune system. The encounter of CD100+ leukemic cells with Plexin-B1 may promote the proliferation and survival of malignant cells. The crosstalk operated by the CD100/Plexin-B1 interaction is not malignancy related but reproduces a mechanism used by normal CD5+ B cells.

Growth and survival of chronic B-cell tumors are favored by the malignant cell's capacity to respond to selected microenvironmental stimuli provided by nontumoral bystander cells. To investigate which mechanisms operate in these crosstalks and whether they are malignancy-related or reproduce the mechanisms used by normal B cells we have studied the expression and functional role of semaphorin CD100 (now called Sema4D) in chronic lymphocytic leukemia (CLL) cells and normal CD5+B cells. We demonstrate here that (1) leukemic and normal CD5+B lymphocytes uniformly express CD100; (2) the CD100 high-affinity receptor Plexin-B1 is expressed by bone marrow stromal cells, follicular dendritic cells, and activated T lymphocytes, and is thus available to CD100+lymphocytes in different specific microenvironments; and (3) upon interaction between CD100 and Plexin-B1 both CLL and normal CD5+B cells increase their proliferative activity and extend their life span. These findings establish that Plexin-B1 is an easily accessible receptor for CD100 within the immune system. The encounter of CD100+leukemic cells with Plexin-B1 may promote the proliferation and survival of malignant cells. The crosstalk operated by the CD100/Plexin-B1 interaction is not malignancy related but reproduces a mechanism used by normal CD5+B cells.

Enhancing the pharmacologic activity of all-transretinoic acid (ATRA) is potentially useful in the management of acute promyelocytic leukemia (APL) and other types of myeloid leukemia. In this report, we identify a novel class of experimental agents selectively potentiating the cytodifferentiating activity of ATRA and synthetic retinoic acid receptor α agonists in APL and other myeloid leukemia cell lines. These agents have a bis-indolic structure (BISINDS), and ST1346 is the prototypical compound of the series. Gene-profiling experiments and determination of the level of expression of myeloid-associated markers indicate that ST1346 stimulates many aspects of the granulocytic maturation process set in motion by ATRA. Stimulation of the cytodifferentiating activity of ATRA by ST1346 enhances the efficacy of the retinoid in vivo, as demonstrated in the APL model of the severe combined immunodeficiency (SCID) mouse receiving transplants of NB4 cells. Although the molecular mechanisms underlying the ATRA-potentiating action of ST1346 and congeners have not been completely clarified, bis-indols are not ligands and do not exert any direct effect on the ATRA-dependent transactivation of nuclear receptors. However, ST1346 inhibits the down-regulation of cyclic adenosine monophosphate (cAMP)–dependent CREB transcriptional complexes and enhances the level of expression of signal transducers and activators of transcription-1 (STAT1), 2 putative molecular determinants of the differentiation process activated by ATRA in APL cells. More importantly, ST1346 relieves the down-regulation of Jun N-terminal kinases (JNK) afforded by ATRA. In addition, a specific JNK inhibitor blocks the enhancing effect of ST1346 on ATRA-induced maturation of NB4 cells. This demonstrates an important role for the mitogen-activated protein kinase in the molecular mechanisms underlying the pharmacologic activity of the bis-indol.

In the hematopoietic compartment, the CD13/APN metalloprotease is one of the earliest markers of cells committed to the myeloid lineage where it is expressed exclusively on the surface of myeloid progenitors and their differentiated progeny. CD13/APN is also found in nonhematopoietic tissues, and its novel expression on the endothelial cells of angiogenic, but not normal, vasculature was recently described. Treatment of animals with CD13/APN inhibitors significantly impaired retinal neovascularization, chorioallantoic membrane angiogenesis, and xenograft tumor growth, indicating that CD13/APN plays an important functional role in vasculogenesis and identifying it as a critical regulator of angiogenesis. To investigate the mechanisms of CD13/APN induction in tumor vasculature, the regulation ofCD13/APN by factors contributing to angiogenic progression was studied. In this report, it is shown that endogenous CD13/APN levels in primary cells and cell lines are up-regulated in response to hypoxia, angiogenic growth factors, and signals regulating capillary tube formation during angiogenesis. Transcription of reporter plasmids containing CD13/APNproximal promoter sequences is significantly increased in response to the same angiogenic signals that regulate the expression of the endogenous gene and in human tumor xenografts, indicating that this fragment contains elements essential for the angiogenic induction ofCD13/APN expression. Finally, functional antagonists of CD13/APN interfere with tube formation but not proliferation of primary vascular endothelial cells, suggesting that CD13/APN functions in the control of endothelial cell morphogenesis. These studies clearly establish the CD13/APN metalloprotease as an important regulator of endothelial morphogenesis during angiogenesis.

Intralipid, a fat emulsion widely used in parenteral nutrition, can produce marked functional changes of the mononuclear phagocyte system. We investigated the effect of Intralipid administration on the generation of procoagulant activity by rabbit mononuclear phagocytes. Two groups of ten rabbits given either a single infusion of Intralipid 10% or a similar volume of sterile saline were studied before and after infusion. Procoagulant activity was measured on isolated blood mononuclear cells after incubation with and without endotoxin, using a one-stage clotting assay. Cells from animals infused with Intralipid produced significantly more procoagulant activity than controls (P less than .01). Results were similar when freshly collected whole blood was incubated with and without endotoxin, and procoagulant activity was measured on subsequently isolated mononuclear cells (P less than .01). In addition, when rabbits were given a single injection of endotoxin, blood and spleen mononuclear cells harvested 50 to 60 minutes after the injection from animals pretreated with Intralipid expressed five to seven times more procoagulant activity than did cells from animals pretreated with saline. In all instances, procoagulant activity was identified as tissue factor. These findings suggest that Intralipid may cause functional changes in mononuclear phagocytes, resulting in increased production of tissue factor on incubation in short-term culture in vitro and in response to endotoxin in vivo.

The t(12;21) (p 13; q22) results in the fusion of the TEL gene located on chromosome 12 with the AML1 gene located on the derivative chromosome 21. Because this translocation is difficult to detect using standard cytogenetic techniques, 27 previously karyotyped B-lineage acute lymphoblastic leukemia (ALL) cell lines were evaluated for the presence of the TEL-AML1 fusion using the reverse transcriptase- polymerase chain reaction (RT-PCR), fluorescence in situ hybridization (FISH), and cDNA sequencing. Six cell lines expressed the TEL-AML1 chimeric transcript by RT-PCR and the t(12;21) was confirmed by FISH analysis with probes for TEL, AML1, and chromosome 12. While only one of the 6 cell lines with the t(12;21) lost the der(12)t(12;21)-encoded AML1-TEL fusion transcript, 4 cell lines lacked expression of the nontranslocated allele of TEL and 5 cell lines lacked expression of CDKN2. Moreover, in 2 patients (1 with the TEL-AML1 transcript and 1 without), TEL expression was lost with disease progression; le, TEL was expressed in the initial cell lines (established at diagnosis or first relapse) whereas TEL was not expressed in the cell lines established from these patients in late-stage disease. These data show the coexistence of multiple genetic defects in childhood B-lineage ALL Cell lines with t(12;21) will facilitate the study of TEL-AML1 and AML1-TEL fusion proteins as well as TEL and CDKN2 gene inactivation in leukemia transformation and progression.

Employing the myeloblastic leukemia M1 cell line, which does not express endogenous p53, and genetically engineered variants, it was recently shown that activation of p53, using a p53 temperature-sensitive mutant transgene (p53ts), resulted in rapid apoptosis that was delayed by high level ectopic expression of bcl-2. In this report, advantage has been taken of these M1 variants to investigate the relationship between p53-mediated G1 arrest and apoptosis. Flow cytometric cell cycle analysis has provided evidence that activation of wild-type (wt) p53 function in M1 cells resulted in the induction of G1 growth arrest; this was clearly seen in the M1p53/bcl-2 cells because of the delay in apoptosis that unmasked p53-induced G1 growth arrest. This finding was further corroborated at the molecular level by analysis of the expression and function of key cell cycle regulatory genes in M1p53 versus M1p53/bcl-2 cells after the activation of wt p53 function; events that take place at early times during the p53-induced G1 arrest occur in both the M1p53 and the M1p53/bcl-2 cells, whereas later events occur only in the M1p53/bcl-2 cells, which undergo delayed apoptosis, thereby allowing the cells to complete G1 arrest. Finally, it was observed that a spectrum of p53 target genes implicated in p53-induced growth suppression and apoptosis were similarly regulated, either induced (gadd45, waf1, mdm2, and bax) or suppressed (c-myc and bcl-2), after activation of wt p53 function in M1p53 and M1p53/bcl-2 cells. Taken together, these findings show that wt p53 can simultaneously induce the genetic programs of both G1 growth arrest and apoptosis within the same cell type, in which the genetic program of cell death can proceed in either G1-arrested (M1p53/bcl-2) or cycling (M1p53) cells. These findings increase our understanding of the functions of p53 as a tumor suppressor and how alterations in these functions could contribute to malignancy.

We report two different disorders of the beta-globin gene cluster segregating in a Belgian family: a novel deletion that results in (G) gamma + ((A) gamma delta beta)(0)-thalassemia (thal) and a heterocellular hereditary persistence of foetal hemoglobin of the Swiss type linked to a delta(0)-thal gene (delta (0)-HPFH). Heterozygosity for the heterocellular HPFH brings about a moderate (3.4% to 8.24%) increase of hemoglobin (Hb) F having a G gamma/A gamma ratio of 4:1, whereas carriers of the G gamma + ((A) gamma delta beta)(0)-thal deletion show in their peripheral blood a considerably higher (15%) percentage of Hb F. Both defects interact in the compound heterozygotes for G gamma + ((A) gamma delta beta)(0)-thal and delta(0)-HPFH producing a further increase (up to 24%) of fetal Hb consisting entirely of G gamma chains. Molecular characterization of the (G) gamma + ((A) gamma delta beta)(0)-thal by means of Southern analysis showed that the deletion spans about 50 kb, removing the 3' end of the A gamma- gene, the psi beta-, delta-, and beta-genes. A number of possible mechanisms leading to the overproduction of Hb F in HPFH and (G) gamma + ((A) gamma delta beta)(0)-thal will be discussed.

The antigenic profile and the proliferative pathways in leukemic cells from the patient TRT with T-prolymphocytic leukemia (T-PLL) were analyzed using monoclonal antibodies (MoAbs) and cytokines. T-PLL cells expressed the phenotype CD1- CD3+ CD4+ CD8+. Incubation with the differentiating agent phorbol-12-myristate-13-acetate markedly increased the percentage of cells with the CD4- CD8+ phenotype, suggesting that leukemic cells were already committed towards a differentiated element with the CD4- CD8+ phenotype. T-PLL cells were induced to proliferate by anti-CD2 MoAb 9–1 + 9.6 and by anti-CD3 MoAb OKT3. The two pathways exhibited normal functional interactions and were susceptible to modulation by anti-HLA class I MoAbs. These results indicate that regulation of cell proliferation was preserved to a significant extent in the T-PLL cells analyzed. At variance with normal resting T cells that require previous activation to proliferate when incubated with interleukin-1 (IL-1) or interleukin-2 (IL-2), T-PLL cells proliferated vigorously when incubated with either interleukin. Furthermore, T-PLL cells proliferated when incubated with immune interferon (IFN-gamma). The latter finding parallels the enhancement by IFN-gamma of the proliferative response of lectin-activated murine T lymphocytes. These results suggest that T-PLL cells, which express a high constitutive level of c-myc mRNA, may be in an activated state. The antigenic phenotype and the characteristics of the proliferative pathways of T-PLL cells from the patient TRT are compatible with the possibility that they may be derived from an intermediate thymocyte.

The antigenic profile and the proliferative pathways in leukemic cells from the patient TRT with T-prolymphocytic leukemia (T-PLL) were analyzed using monoclonal antibodies (MoAbs) and cytokines. T-PLL cells expressed the phenotype CD1-CD3+CD4+CD8\ Incubation with the differentiating agent phorbol-12-myristate-13-acetate markedly increased the percentage of cells with the CD4 CD8+phenotype, suggesting that leukemic cells were already committed towards a differentiated element with the CD4 CD8+phenotype. T-PLL cells were induced to proliferate by anti-CD2 MoAb 9-1+9.6 and by anti-CD3 MoAb OKT3. The two pathways exhibited normal functional interactions and were susceptible to modulation by anti-HLA class I MoAbs. These results indicate that regulation of cell proliferation was preserved to a significant extent in the T-PLL cells analyzed. At variance with normal resting T cells that require previous activation to proliferate when incubated with interleukin-1 (IL-1) or interleukin-2 (IL-2), T-PLL cells proliferated vigorously when incubated with either interleukin. Furthermore, T-PLL cells proliferated when incubated with immune interferon (IFN- γ). The latter finding parallels the enhancement by IFN- γof the proliferative response of lectin-activated murine T lymphocytes. These results suggest that T-PLL cells, which express a high constitutive level of c-mycmRNA, may be in an activated state. The antigenic phenotype and the characteristics of the proliferative pathways of T-PLL cells from the patient TRT are compatible with the possibility that they may be derived from an intermediate thymocyte. © 1989 by Grune & Stratton. Inc.

Clonal hematopoiesis of indeterminate potential (CHIP) is associated with increased risk of cancers and inflammation-related diseases. This phenomenon becomes very common in oldest-old individuals, in whom the implications of CHIP are not well defined. We performed a mutational screening in 1794 oldest-old individuals enrolled in two population-based studies and investigate the relationships between CHIP and associated pathologies. Clonal mutations were observed in one third of oldest-old individuals and were associated with reduced survival. Mutations in JAK2and splicing genes, multiple mutations (DNMT3A, TET2, ASXL1with additional genetic lesions) and variant allele frequency ≥0.096 had positive predictive value for myeloid neoplasms. Combining mutation profiles with abnormalities in red blood cell indices improved the ability of myeloid neoplasm prediction. On this basis, we defined a predictive model that identifies 3 risk groups with different probabilities of developing myeloid neoplasms. Mutations in DNMT3A, TET2, ASXL1or JAK2(most occurring as single lesion) were associated with coronary heart disease and rheumatoid arthritis. Cytopenia was a common finding in oldest-old population, the underlying cause remaining unexplained in 30% of cases. Among individuals with unexplained cytopenia, the presence of highly-specific mutation patterns was associated with myelodysplastic-like phenotype and a probability of survival comparable to that of myeloid neoplasms. Accordingly, 7.5% of oldest-old subjects with cytopenia had presumptive evidence of myeloid neoplasm. In conclusion, specific mutational patterns define different risk of developing myeloid neoplasms vs.inflammatory-associated diseases in oldest-old population. In individuals with unexplained cytopenia, mutational status may identify those subjects with presumptive evidence of myeloid neoplasms.