There is significant interest in immunotherapy for the treatment of high-risk smoldering multiple myeloma (SMM), but no available data on the immune status of this particular disease stage. Such information is important to understand the interplay between immunosurveillance and disease transformation, but also to define whether patients with high-risk SMM might benefit from immunotherapy. Here, we have characterized T lymphocytes (including CD4, CD8, T-cell receptor γδ, and regulatory T cells), natural killer (NK) cells, and dendritic cells from 31 high-risk SMM patients included in the treatment arm of the QUIREDEX trial, and with longitudinal peripheral blood samples at baseline and after 3 and 9 cycles of lenalidomide plus low-dose dexamethasone (LenDex). High-risk SMM patients showed at baseline decreased expression of activation-(CD25/CD28/CD54), type 1 T helper-(CD195/interferon-γ/tumor necrosis factor-α/interleukin-2), and proliferation-related markers (CD119/CD120b) as compared with age-matched healthy individuals. However, LenDex was able to restore the normal expression levels for those markers and induced a marked shift in T-lymphocyte and NK-cell phenotype. Accordingly, high-risk SMM patients treated with LenDex showed higher numbers of functionally active T lymphocytes. Together, our results indicate that high-risk SMM patients have an impaired immune system that could be reactivated by the immunomodulatory effects of lenalidomide, even when combined with low-dose dexamethasone, and support the value of therapeutic immunomodulation to delay the progression to multiple myeloma. The QUIREDEX trial was registered to www.clinicaltrials.gov as #NCT00480363.

Mutations in the calreticulin gene (CALR) represented by deletions and insertions in exon 9 inducing a -1/+2 frameshift are associated with a significant fraction of myeloproliferative neoplasms (MPNs). The mechanisms by which CALR mutants induce MPN are unknown. Here, we show by transcriptional, proliferation, biochemical, and primary cell assays that the pathogenic CALR mutants specifically activate the thrombopoietin receptor (TpoR/MPL). No activation is detected with a battery of type I and II cytokine receptors, except granulocyte colony-stimulating factor receptor, which supported only transient and weak activation. CALR mutants induce ligand-independent activation of JAK2/STAT/phosphatydylinositol-3'-kinase (PI3-K) and mitogen-activated protein (MAP) kinase pathways via TpoR, and autonomous growth in Ba/F3 cells. In these transformed cells, no synergy is observed between JAK2 and PI3-K inhibitors in inhibiting cytokine-independent proliferation, thus showing a major difference from JAK2V617F cells where such synergy is strong. TpoR activation was dependent on its extracellular domain and its N-glycosylation, especially at N117. The glycan binding site and the novel C-terminal tail of the mutant CALR proteins were required for TpoR activation. A soluble form of TpoR was able to prevent activation of full-length TpoR provided that it was N-glycosylated. By confocal microscopy and subcellular fractionation, CALR mutants exhibit different intracellular localization from that of wild-type CALR. Finally, knocking down either MPL/TpoR or JAK2 in megakaryocytic progenitors from patients carrying CALR mutations inhibited cytokine-independent megakaryocytic colony formation. Taken together, our study provides a novel signaling paradigm, whereby a mutated chaperone constitutively activates cytokine receptor signaling.

Evidence shows that factor VIII (FVIII) ectopically expressed in platelets (2bF8) is therapeutic in FVIII(null) mice even with anti-FVIII inhibitory antibodies (inhibitors). If current efforts to generate platelets in vitro succeed, genetically manipulated platelets containing FVIII may be used therapeutically in hemophilia A patients with inhibitors. One important concern is the immunogenicity of platelet-derived FVIII. To address this concern, we infused 2bF8 transgenic (2bF8(Tg)) platelets into naïve FVIII(null) mice weekly for 8 weeks. No anti-FVIII antibodies were detected in the infused animals during the study course. We then explored whether platelet-derived FVIII is immunogenic in FVIII(null) mice with inhibitors. The 2bF8(Tg) platelets were transfused into rhF8-primed FVIII(null) mice, resulting in no augmentation of anti-FVIII antibodies. To investigate whether preconditioning affects the immune response, animals were sublethally irradiated and subsequently transfused with 2bF8(Tg) platelets. No anti-FVIII antibodies were detected in the recipients after platelet infusions. Following further challenge with rhF8, the inhibitor titer in this group was significantly lower than in naïve FVIII(null) mice utilizing the same immunization protocol. Thus, our data demonstrate that infusion of platelets containing FVIII triggers neither primary nor memory anti-FVIII immune response in FVIII(null) mice and that sublethal irradiation plus 2bF8(Tg) platelet infusion suppresses anti-FVIII immune response in FVIII(null) mice.

Deep-vein thrombosis (DVT) is regarded a chronic disease as it often recurs. DVT affects most frequently the lower limbs and hence DVT of the leg will be the focus of this article. Whereas algorithms were developed and validated for the diagnosis of a first DVT, no such well-defined strategies exist in the case of recurrence of DVT. Likewise, the scientific evidence regarding the treatment of recurrent DVT is sparse, in particular when it comes to deciding on the duration of anticoagulation. Two typical cases of recurrent DVT, one with an unprovoked DVT and one with DVT during anticoagulation, will be presented. Based on these two clinical scenarios, algorithms for the diagnosis and treatment of recurrent DVT will be put forward. The purpose of this article is to discuss strategies that can be applied in daily clinical practice by physicians who do not have access to means and measures available in specialized thrombosis centers.

Acute lymphoblastic leukemia (ALL) persisting or relapsing following bone marrow transplantation (BMT) has a dismal prognosis. Success with chimeric antigen receptor (CAR) T cells offers an opportunity to treat these patients with leukemia-redirected donor-derived T cells, which may be more functional than T cells derived from patients with leukemia but have the potential to mediate graft-versus-host disease (GVHD). We, together with others, have previously demonstrated tumor-specific T-cell dysfunction in the allogeneic environment. Here, we studied CAR T-cell function following BMT using an immunocompetent murine model of minor mismatched allogeneic transplantation followed by donor-derived CD19-CAR T cells. Allogeneic donor-derived CD19-CAR T cells eliminated residual ALL with equal potency to those administered after syngeneic BMT. Surprisingly, allogeneic CAR T cells mediated lethal acute GVHD with early mortality, which is atypical for this minor mismatch model. We demonstrated that both allogeneic and syngeneic CAR T cells show initial expansion as effector T cells, with a higher peak but rapid deletion of allogeneic CAR T cells. Interestingly, CAR-mediated acute GVHD was only seen in the presence of leukemia, suggesting CAR-target interactions induced GVHD. Indeed, serum interleukin (IL)-6 was elevated only in the presence of both leukemia and CAR T cells, and IL-6 neutralization ameliorated the severity of GVHD in a delayed donor lymphocyte infusion model. Finally, allogeneic CD4(+) CAR T cells were responsible for GVHD, which correlated with their ability to produce IL-6 upon CAR stimulation. Altogether, we demonstrate that donor-derived allogeneic CAR T cells are active but have the capacity to drive GVHD.

Acute myeloid leukemia (AML) is the most common type of acute leukemia in adults. AML is a heterogeneous malignancy characterized by distinct genetic abnormalities. Recent discoveries have highlighted an additional important role of dysregulated epigenetic mechanisms in the pathogenesis of the disease. In contrast to genetic changes, epigenetic modifications are frequently reversible, which provides opportunities for targeted treatment using specific inhibitors. In this review, we will provide an overview of the current state of epigenetics and epigenetic therapy in AML and will describe perspectives on how to identify promising new approaches for epigenetic targeted treatment.

Recent major advances in understanding the molecular basis of acute myeloid leukemia (AML) provide a double-edged sword. Although defining the topology and key features of the molecular landscape are fundamental to development of novel treatment approaches and provide opportunities for greater individualization of therapy, confirmation of the genetic complexity presents a huge challenge to successful translation into routine clinical practice. It is now clear that many genes are recurrently mutated in AML; moreover, individual leukemias harbor multiple mutations and are potentially composed of subclones with differing mutational composition, rendering each patient's AML genetically unique. In order to make sense of the overwhelming mutational data and capitalize on this clinically, it is important to identify (1) critical AML-defining molecular abnormalities that distinguish biological disease entities; (2) mutations, typically arising in subclones, that may influence prognosis but are unlikely to be ideal therapeutic targets; (3) mutations associated with preleukemic clones; and (4) mutations that have been robustly shown to confer independent prognostic information or are therapeutically relevant. The reward of identifying AML-defining molecular lesions present in all leukemic populations (including subclones) has been exemplified by acute promyelocytic leukemia, where successful targeting of the underlying PML-RARα oncoprotein has eliminated the need for chemotherapy for disease cure. Despite the molecular heterogeneity and recognizing that treatment options for other forms of AML are limited, this review will consider the scope for using novel molecular information to improve diagnosis, identify subsets of patients eligible for targeted therapies, refine outcome prediction, and track treatment response.

Recent advances in acute myeloid leukemia (AML) biology and its genetic landscape should ultimately lead to more subset-specific AML therapies, ideally tailored to each patient's disease. Although a growing number of distinct AML subsets have been increasingly characterized, patient management has remained disappointingly uniform. If one excludes acute promyelocytic leukemia, current AML management still relies largely on intensive chemotherapy and allogeneic hematopoietic stem cell transplantation (HSCT), at least in younger patients who can tolerate such intensive treatments. Nevertheless, progress has been made, notably in terms of standard drug dose intensification and safer allogeneic HSCT procedures, allowing a larger proportion of patients to achieve durable remission. In addition, improved identification of patients at relatively low risk of relapse should limit their undue exposure to the risks of HSCT in first remission. The role of new effective agents, such as purine analogs or gemtuzumab ozogamicin, is still under investigation, whereas promising new targeted agents are under clinical development. In contrast, minimal advances have been made for patients unable to tolerate intensive treatment, mostly representing older patients. The availability of hypomethylating agents likely represents an encouraging first step for this latter population, and it is hoped will allow for more efficient combinations with novel agents.

Multiple new drugs are being developed to treat acute myeloid leukemia (AML), including novel formulations of traditional chemotherapy-antibody drug conjugates and agents that target specific mutant enzymes. Next-generation sequencing has allowed us to discover the genetic mutations that lead to the development and clinical progression of AML. Studies of clonal hierarchy suggest which mutations occur early and dominate. This has led to targeted therapy against mutant driver proteins as well as the development of drugs such as CPX-351 and SGN-CD33A whose mechanisms of action and efficacy may not be dependent on mutational complexity. In this brief review, we discuss drugs that may emerge as important for the treatment of AML in the next 10 years.

Postremission therapy in patients with acute myeloid leukemia (AML) may consist of continuing chemotherapy or transplantation using either autologous or allogeneic stem cells. Patients with favorable subtypes of AML generally receive chemotherapeutic consolidation, although recent studies have also suggested favorable outcome after hematopoietic stem cell transplantation (HSCT). Although allogeneic HSCT (alloHSCT) is considered the preferred type of postremission therapy in poor- and very-poor-risk AML, the place of alloHSCT in intermediate-risk AML is being debated, and autologous HSCT is considered a valuable alternative that may be preferred in patients without minimal residual disease after induction chemotherapy. Here, we review postremission transplantation strategies using either autologous or allogeneic stem cells. Recent developments in the field of alternative donors, including cord blood and haploidentical donors, are highlighted, and we discuss reduced-intensity alloHSCT in older AML recipients who represent the predominant category of patients with AML who have a high risk of relapse in first remission.

The sequential activation of distinct developmental gene networks governs the ultimate identity of a cell, but the mechanisms involved in initiating downstream programs are incompletely understood. The pre-B-cell receptor (pre-BCR) is an important checkpoint of B-cell development and is essential for a pre-B cell to traverse into an immature B cell. Here, we show that activation of myocyte enhancer factor 2 (Mef2) transcription factors (TFs) by the pre-BCR is necessary for initiating the subsequent genetic network. We demonstrate that B-cell development is blocked at the pre-B-cell stage in mice deficient for Mef2c and Mef2d TFs and that pre-BCR signaling enhances the transcriptional activity of Mef2c/d through phosphorylation by the Erk5 mitogen-activating kinase. This activation is instrumental in inducing Krüppel-like factor 2 and several immediate early genes of the AP1 and Egr family. Finally, we show that Mef2 proteins cooperate with the products of their target genes (Irf4 and Egr2) to induce secondary waves of transcriptional regulation. Our findings uncover a novel role for Mef2c/d in coordinating the transcriptional network that promotes early B-cell development.

In platelets, splicing and translation occur in the absence of a nucleus. However, the integrity and stability of mRNAs derived from megakaryocyte progenitor cells remain poorly quantified on a transcriptome-wide level. As circular RNAs (circRNAs) are resistant to degradation by exonucleases, their abundance relative to linear RNAs can be used as a surrogate marker for mRNA stability in the absence of transcription. Here we show that circRNAs are enriched in human platelets 17- to 188-fold relative to nucleated tissues and 14- to 26-fold relative to samples digested with RNAse R to selectively remove linear RNA. We compare RNAseq read depths inside and outside circRNAs to provide in silico evidence of transcript circularity, show that exons within circRNAs are enriched on average 12.7 times in platelets relative to nucleated tissues and identify 3162 genes significantly enriched for circRNAs, including some where all RNAseq reads appear to be derived from circular molecules. We also confirm that this is a feature of other anucleate cells through transcriptome sequencing of mature erythrocytes, demonstrate that circRNAs are not enriched in cultured megakaryocytes, and demonstrate that linear RNAs decay more rapidly than circRNAs in platelet preparations. Collectively, these results suggest that circulating platelets have lost >90% of their progenitor mRNAs and that translation in platelets occurs against the backdrop of a highly degraded transcriptome. Finally, we find that transcripts previously classified as products of reverse transcriptase template switching are both enriched in platelets and resistant to decay, countering the recent suggestion that up to 50% of rearranged RNAs are artifacts.

Platelet-activating antibodies, which recognize platelet factor 4 (PF4)/heparin complexes, induce spontaneous heparin-induced thrombocytopenia (HIT) syndrome or fondaparinux-associated HIT without exposure to unfractionated heparin (UFH) or low-molecular-weight heparin (LMWH). This condition mostly occurs after major orthopedic surgery, implying that surgery itself could trigger this immune response, although the mechanism is unclear. To investigate how surgery may do so, we performed a multicenter, prospective study of 2069 patients who underwent total knee arthroplasty (TKA) or hip arthroplasty. Approximately half of the patients received postoperative thromboprophylaxis with UFH, LMWH, or fondaparinux. The other half received only mechanical thromboprophylaxis, including dynamic (intermittent plantar or pneumatic compression device), static (graduated compression stockings [GCSs]), or both. We measured anti-PF4/heparin immunoglobulins G, A, and M before and 10 days after surgery using an immunoassay. Multivariate analysis revealed that dynamic mechanical thromboprophylaxis (DMT) was an independent risk factor for seroconversion (odds ratio [OR], 2.01; 95% confidence interval [CI], 1.34-3.02; P = .001), which was confirmed with propensity-score matching (OR, 1.99; 95% CI, 1.17-3.37; P = .018). For TKA, the seroconversion rates in patients treated with DMT but no anticoagulation and in patients treated with UFH or LMWH without DMT were similar, but significantly higher than in patients treated with only GCSs. The proportion of patients with ≥1.4 optical density units appeared to be higher among those treated with any anticoagulant plus DMT than among those not treated with DMT. Our study suggests that DMT increases risk of an anti-PF4/heparin immune response, even without heparin exposure. This trial was registered to www.umin.ac.jp/ctr as #UMIN000001366.

In times of physiological stress, platelet count can transiently rise. What initiates this reactive thrombocytosis is poorly understood. Intriguingly, we found that treating megakaryocytes (MKs) with the releasate from activated platelets increased proplatelet production by 47%. Platelets store inflammatory cytokines, including the chemokine ligand 5 (CCL5, RANTES); after TRAP activation, platelets release over 25 ng/mL CCL5. We hypothesized that CCL5 could regulate platelet production by binding to its receptor, CCR5, on MKs. Maraviroc (CCR5 antagonist) or CCL5 immunodepletion diminished 95% and 70% of the effect of platelet releasate, respectively, suggesting CCL5 derived from platelets is sufficient to drive increased platelet production through MK CCR5. MKs cultured with recombinant CCL5 increased proplatelet production by 50% and had significantly higher ploidy. Pretreating the MK cultures with maraviroc prior to exposure to CCL5 reversed the augmented proplatelet formation and ploidy, suggesting that CCL5 increases MK ploidy and proplatelet formation in a CCR5-dependent manner. Interrogation of the Akt signaling pathway suggested that CCL5/CCR5 may influence proplatelet production by suppressing apoptosis. In an in vivo murine acute colitis model, platelet count significantly correlated with inflammation whereas maraviroc treatment abolished this correlation. We propose that CCL5 signaling through CCR5 may increase platelet counts during physiological stress.

Extravascular fibrin deposition accompanies many human diseases and causes chronic inflammation and organ damage, unless removed in a timely manner. Here, we used intravital microscopy to investigate how fibrin is removed from extravascular space. Fibrin placed into the dermis of mice underwent cellular endocytosis and lysosomal targeting, revealing a novel intracellular pathway for extravascular fibrin degradation. A C-C chemokine receptor type 2 (CCR2)-positive macrophage subpopulation constituted the majority of fibrin-uptaking cells. Consequently, cellular fibrin uptake was diminished by elimination of CCR2-expressing cells. The CCR2-positive macrophage subtype was different from collagen-internalizing M2-like macrophages. Cellular fibrin uptake was strictly dependent on plasminogen and plasminogen activator. Surprisingly, however, fibrin endocytosis was unimpeded by the absence of the fibrin(ogen) receptors, αMβ2 and ICAM-1, the myeloid cell integrin-binding site on fibrin or the endocytic collagen receptor, the mannose receptor. The study identifies a novel fibrin endocytic pathway engaged in extravascular fibrin clearance and shows that interstitial fibrin and collagen are cleared by different subsets of macrophages employing distinct molecular pathways.

Intracellular adhesion molecule-1 (ICAM-1) is a transmembrane glycoprotein expressed on the cell surface of numerous cell types such as endothelial and epithelial cells, vascular smooth muscle cells, and certain leukocyte subsets. With respect to the latter, ICAM-1 has been detected on neutrophils in several clinical and experimental settings, but little is known about the regulation of expression or function of neutrophil ICAM-1. In this study, we report on the de novo induction of ICAM-1 on the cell surface of murine neutrophils by lipopolysaccharide (LPS), tumor necrosis factor, and zymosan particles in vitro. The induction of neutrophil ICAM-1 was associated with enhanced phagocytosis of zymosan particles and reactive oxygen species (ROS) generation. Conversely, neutrophils from ICAM-1-deficient mice were defective in these effector functions. Mechanistically, ICAM-1-mediated intracellular signaling appeared to support neutrophil ROS generation and phagocytosis. In vivo, LPS-induced inflammation in the mouse cremaster muscle and peritoneal cavity led to ICAM-1 expression on intravascular and locally transmigrated neutrophils. The use of chimeric mice deficient in ICAM-1 on myeloid cells demonstrated that neutrophil ICAM-1 was not required for local neutrophil transmigration, but supported optimal intravascular and extravascular phagocytosis of zymosan particles. Collectively, the present results shed light on regulation of expression and function of ICAM-1 on neutrophils and identify it as an additional regulator of neutrophil effector responses in host defense.

The effect of sympathetic stimulation on thrombopoiesis is not well understood. Here, we demonstrate that both continual noise and exhaustive exercise elevate peripheral platelet levels in normal and splenectomized mice, but not in dopamine β-hydroxylase-deficient (Dbh(-/-)) mice that lack norepinephrine (NE) and epinephrine (EPI). Further investigation demonstrates that sympathetic stimulation via NE or EPI injection markedly promotes platelet recovery in mice with thrombocytopenia induced by 6.0 Gy of total-body irradiation and in mice that received bone marrow transplants after 10.0 Gy of lethal irradiation. Unfavorably, sympathetic stress-stimulated thrombopoiesis may also contribute to the pathogenesis of atherosclerosis by increasing both the amount and activity of platelets in apolipoprotein E-deficient (ApoE(-/-)) mice. In vitro studies reveal that both NE and EPI promote megakaryocyte adhesion, migration, and proplatelet formation (PPF) in addition to the expansion of CD34(+) cells, thereby facilitating platelet production. It is found that α2-adrenoceptor-mediated extracellular signal-regulated kinase 1/2 (ERK1/2) activation is involved in NE- and EPI-induced megakaryocyte adhesion and migration, and PPF is regulated by ERK1/2 activation-mediated RhoA GTPase signaling. Our data deeply characterize the role of sympathetic stimulation in the regulation of thrombopoiesis and reevaluate its physiopathological implications.

Reduced intensity conditioning (RIC) allogeneic hematopoietic cell transplantation (HCT) with alemtuzumab, fludarabine, and melphalan is an effective approach for patients with nonmalignant disorders. Mixed chimerism and graft-versus-host-disease (GVHD) remain limitations on success. We hypothesized that higher levels of alemtuzumab at day 0 would result in a low risk of acute GVHD, a higher risk of mixed chimerism, and delayed early lymphocyte recovery and that alemtuzumab level thresholds for increased risks of these outcomes would be definable. We collected data from 105 patients to examine the influence of peritransplant alemtuzumab levels on acute GVHD, mixed chimerism, and lymphocyte recovery. The cumulative incidences of initial grades I-IV, II-IV, and III-IV acute GVHD in patients with alemtuzumab levels ≤0.15 vs ≥0.16 μg/mL were 68% vs 18% (P < .0001), 47% vs 13% (P = .0002), and 32% vs 8%, respectively (P = .005). The cumulative incidence of mixed chimerism in patients with an alemtuzumab level ≤0.15 μg/mL was 21%, vs 42% with levels of 0.16 to 4.35 μg/mL, and 100% with levels >4.35 μg/mL (P = .003). Patients with alemtuzumab levels ≤0.15 or 0.16 to 0.56 μg/mL had higher lymphocyte counts at day +30 and higher T-cell counts at day +100 compared with patients with levels ≥0.57 μg/mL (all P < .05). We conclude that peritransplant alemtuzumab levels impact acute GVHD, mixed chimerism, and lymphocyte recovery following RIC HCT with alemtuzumab, fludarabine, and melphalan. Precision dosing trials are warranted. We recommend a day 0 therapeutic range of 0.2 to 0.4 μg/mL.