Neutrophils are the most abundant white blood cell, and they differentiate in homeostasis in the bone marrow from hematopoietic stem cells (HSCs) via multiple intermediate progenitor cells into mature cells that enter the circulation. Recent findings support a continuous model of differentiation in the bone marrow of heterogeneous HSCs and progenitor populations. Cell fate decisions at the levels of proliferation and differentiation are enforced through expression of lineage-determining transcription factors and their interactions, which are influenced by intrinsic (intracellular) and extrinsic (extracellular) mechanisms. Neutrophil homeostasis is subjected to positive-feedback loops, stemming from the gut microbiome, as well as negative-feedback loops resulting from the clearance of apoptotic neutrophils by mature macrophages. Finally, the cellular kinetics regarding the replenishing of the mature neutrophil pool is discussed in light of recent contradictory data.

COVID-19 is a primary respiratory illness that is frequently complicated by systemic involvement of the vasculature. Vascular involvement leads to an array of complications ranging from thrombosis to pulmonary edema secondary to loss of barrier function. This review will address the vasculopathy of COVID-19 with a focus on the role of the endothelium in orchestrating the systemic response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. The endothelial receptor systems and molecular pathways activated in the setting of COVID-19 and the consequences of these inflammatory and prothrombotic changes on endothelial cell function will be discussed. The sequelae of COVID-19 vascular involvement at the level of organ systems will also be addressed, with an emphasis on the pulmonary vasculature but with consideration of effects on other vascular beds. The dramatic changes in endothelial phenotypes associated with COVID-19 has enabled the identification of biomarkers that could help guide therapy and predict outcomes. Knowledge of vascular pathogenesis in COVID-19 has also informed therapeutic approaches that may control its systemic sequelae. Because our understanding of vascular response in COVID-19 continues to evolve, we will consider areas of controversy, such as the extent to which SARS-CoV-2 directly infects endothelium and the degree to which vascular responses to SARS-CoV-2 are unique or common to those of other viruses capable of causing severe respiratory disease. This conceptual framework describing how SARS-CoV-2 infection affects endothelial inflammation, prothrombotic transformation, and barrier dysfunction will provide a context for interpreting new information as it arises addressing the vascular complications of COVID-19.

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a potentially curative treatment of patients with nonmalignant or malignant blood disorders. Its success has been limited by graft-versus-host disease (GVHD). Current systemic nontargeted conditioning regimens mediate tissue injury and potentially incite and amplify GVHD, limiting the use of this potentially curative treatment beyond malignant disorders. Minimizing systemic nontargeted conditioning while achieving alloengraftment without global immune suppression is highly desirable. Antibody-drug-conjugates (ADCs) targeting hematopoietic cells can specifically deplete host stem and immune cells and enable alloengraftment. We report an anti-mouse CD45-targeted-ADC (CD45-ADC) that facilitates stable murine multilineage donor cell engraftment. Conditioning with CD45-ADC (3 mg/kg) was effective as a single agent in both congenic and minor-mismatch transplant models resulting in full donor chimerism comparable to lethal total body irradiation (TBI). In an MHC-disparate allo-HSCT model, pretransplant CD45-ADC (3 mg/kg) combined with low-dose TBI (150 cGy) and a short course of costimulatory blockade with anti-CD40 ligand antibody enabled 89% of recipients to achieve stable alloengraftment (mean value: 72%). When CD45-ADC was combined with pretransplant TBI (50 cGy) and posttransplant rapamycin, cyclophosphamide (Cytoxan), or a JAK inhibitor, 90% to 100% of recipients achieved stable chimerism (mean: 77%, 59%, 78%, respectively). At a higher dose (5 mg/kg), CD45-ADC as a single agent was sufficient for rapid, high-level multilineage chimerism sustained through the 22 weeks observation period. Therefore, CD45-ADC has the potential utility to confer the benefit of fully myeloablative conditioning but with substantially reduced toxicity when given as a single agent or at lower doses in conjunction with reduced-intensity conditioning.

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.

Impairment of normal hematopoiesis and leukemia progression are 2 well-linked processes during leukemia development and are controlled by the bone marrow (BM) niche. Extracellular matrix proteins, including laminin, are important BM niche components. However, their role in hematopoiesis regeneration and leukemia is unknown. Laminin α4 (Lama4), a major receptor-binding chain of several laminins, is altered in BM niches in mice with acute myeloid leukemia (AML). So far, the impact of Lama4 on leukemia progression remains unknown. We here report that Lama4 deletion in mice resulted in impaired hematopoiesis regeneration following irradiation-induced stress, which is accompanied by altered BM niche composition and inflammation. Importantly, in a transplantation-induced MLL-AF9 AML mouse model, we demonstrate accelerated AML progression and relapse in Lama4-/- mice. Upon AML exposure, Lama4-/- mesenchymal stem cells (MSCs) exhibited dramatic molecular alterations, including upregulation of inflammatory cytokines that favor AML growth. Lama4-/- MSCs displayed increased antioxidant activities and promoted AML stem cell proliferation and chemoresistance to cytarabine, which was accompanied by increased mitochondrial transfer from the MSCs to AML cells and reduced reactive oxygen species in AML cells in vitro. Similarly, we detected lower levels of reactive oxygen species in AML cells from Lama4-/- mice post-cytarabine treatment. Notably, LAMA4 inhibition or knockdown in human MSCs promoted human AML cell proliferation and chemoprotection. Together, our study for the first time demonstrates the critical role of Lama4 in impeding AML progression and chemoresistance. Targeting Lama4 signaling pathways may offer potential new therapeutic options for AML.

Fibrinogen plays a pathologic role in multiple diseases. It contributes to thrombosis and modifies inflammatory and immune responses, supported by studies in mice expressing fibrinogen variants with altered function or with a germline fibrinogen deficiency. However, therapeutic strategies to safely and effectively tailor plasma fibrinogen concentration are lacking. Here, we developed a strategy to tune fibrinogen expression by administering lipid nanoparticle (LNP)-encapsulated small interfering RNA (siRNA) targeting the fibrinogen α chain (siFga). Three distinct LNP-siFga reagents reduced both hepatic Fga messenger RNA and fibrinogen levels in platelets and plasma, with plasma levels decreased to 42%, 16%, and 4% of normal within 1 week of administration. Using the most potent siFga, circulating fibrinogen was controllably decreased to 32%, 14%, and 5% of baseline with 0.5, 1.0, and 2.0 mg/kg doses, respectively. Whole blood from mice treated with siFga formed clots with significantly decreased clot strength ex vivo, but siFga treatment did not compromise hemostasis following saphenous vein puncture or tail transection. In an endotoxemia model, siFga suppressed the acute phase response and decreased plasma fibrinogen, D-dimer, and proinflammatory cytokine levels. In a sterile peritonitis model, siFga restored normal macrophage migration in plasminogen-deficient mice. Finally, treatment of mice with siFga decreased the metastatic potential of tumor cells in a manner comparable to that observed in fibrinogen-deficient mice. The results indicate that siFga causes robust and controllable depletion of fibrinogen and provides the proof-of-concept that this strategy can modulate the pleiotropic effects of fibrinogen in relevant disease models.

Obinutuzumab and lenalidomide (referred to as the GALEN combination) is an active immunomodulatory combination with a manageable safety profile in multiple types of lymphoma. We report efficacy and safety results for the phase 2 GALEN study in previously untreated patients with advanced follicular lymphoma (FL). Eligible patients aged ≥18 years had an Eastern Cooperative Oncology Group performance status ≤2 and high-tumor burden, grade 1 to 3a FL. Induction treatment was obinutuzumab (1000 mg IV, days 8, 15, and 22, cycle 1; day 1, cycles 2-6) plus lenalidomide (20 mg/d, days 1-21, cycle 1; days 2-22, cycles 2-6) for six 28-day cycles. Maintenance included obinutuzumab (1000 mg every 2 cycles) plus lenalidomide (10 mg, days 2-22) for ≤12 cycles (year 1) followed by obinutuzumab (1000 mg every 56 days) for 6 cycles (year 2). The primary end point was complete response rate (CRR) after induction per the 1999 International Working Group criteria. From October 2015 to February 2017, a total of 100 patients were enrolled. CRR after induction was 47%, and the overall response rate (ORR) was 92%. Post hoc analyses per the 2014 Lugano classification, including patients with missing bone marrow assessments, identified an additional 13 patients fulfilling CRR criteria, resulting in a complete metabolic response of 80% and an ORR of 94%. At a median follow-up of 3.7 years, 3-year progression-free survival and overall survival were 82% and 94%, respectively. The most common adverse event was neutropenia (48% any grade; 47% grade ≥3). Only 2% of patients presented with febrile neutropenia; others were mainly grade ≤2. No other specific grade ≥3 toxicity occurred at a frequency >3%. Overall, these results showed promising clinical efficacy for the chemotherapy-free GALEN backbone in previously untreated patients with high tumor burden FL. Except for neutropenia, the safety profile of the combination is remarkable. The study was registered at clinicaltrials.gov as #NCT01582776.

Immunomodulatory (IMiD) agents like lenalidomide and pomalidomide induce the recruitment of IKZF1 and other targets to the CRL4CRBN E3 ubiquitin ligase, resulting in their ubiquitination and degradation. These agents are highly active in B-cell lymphomas and a subset of myeloid diseases but have compromised effects in T-cell lymphomas (TCLs). Here, we show that 2 factors determine resistance to IMiDs among TCLs. First, limited CRBN expression reduces IMiD activity in TCLs but can be overcome by newer-generation degrader CC-92480. Using mass spectrometry, we show that CC-92480 selectively degrades IKZF1 and ZFP91 in TCL cells with greater potency than pomalidomide. As a result, CC-92480 is highly active against multiple TCL subtypes and showed greater efficacy than pomalidomide across 4 in vivo TCL models. Second, we demonstrate that ZFP91 functions as a bona fide transcription factor that coregulates cell survival with IKZF1 in IMiD-resistant TCLs. By activating keynote genes from WNT, NF-kB, and MAP kinase signaling, ZFP91 directly promotes resistance to IKZF1 loss. Moreover, lenalidomide-sensitive TCLs can acquire stable resistance via ZFP91 rewiring, which involves casein kinase 2-mediated c-Jun inactivation. Overall, these findings identify a critical transcription factor network within TCLs and provide clinical proof of concept for the novel therapy using next-generation degraders.

To enable effective oxygen transport, ∼200 billion red blood cells (RBCs) need to be produced every day in the bone marrow through the fine-tuned process of erythropoiesis. Erythropoiesis is regulated at multiple levels to ensure that defective RBC maturation or overproduction can be avoided. Here, we provide an overview of different layers of this control, ranging from cytokine signaling mechanisms that enable extrinsic regulation of RBC production to intrinsic transcriptional pathways necessary for effective erythropoiesis. Recent studies have also elucidated the importance of posttranscriptional regulation and highlighted additional gatekeeping mechanisms necessary for effective erythropoiesis. We additionally discuss the insights gained by studying human genetic variation affecting erythropoiesis and highlight the discovery of BCL11A as a regulator of hemoglobin switching through genetic studies. Finally, we provide an outlook of how our ability to measure multiple facets of this process at single-cell resolution, while accounting for the impact of human variation, will continue to refine our knowledge of erythropoiesis and how this process is perturbed in disease. As we learn more about this intricate and important process, additional opportunities to modulate erythropoiesis for therapeutic purposes will undoubtedly emerge.

Diffuse large B-cell lymphoma (DLBCL), the most common lymphoma subtype, is localized in 25% to 30% of patients. Prognosis in patients with limited-stage DLBCL (LS-DLBCL) is excellent with 10-year overall survival of at least 70% to 80%. Improved insights into the disease biology, the availability of positron-emission tomography (PET) scans, and recent dedicated clinical trials within this unique population have led to evolving treatment paradigms. However, no standard definition of LS-DLBCL exists, and although generally defined as Ann Arbor stages I to II disease with largest mass size <10 cm in diameter, variations across studies cause challenges in interpretation. Similar to advanced-stage disease, rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisolone (R-CHOP) immunochemotherapy forms the basis of treatment, with combined modality therapy including 3 cycles of systemic treatment and involved-site radiation therapy being a predominant historical standard. Yet the well-described continuous risk of relapse beyond 5 years and established late complications of radiotherapy have challenged previous strategies. More rigorous baseline staging and response assessment with PET may improve decision making. Recent clinical studies have focused on minimizing toxicities while maximizing disease outcomes using strategies such as abbreviated immunochemotherapy alone and PET-adapted radiotherapy delivery. This comprehensive review provides an update of recent literature with recommendations for integration into clinical practice for LS-DLBCL patients.

Inadequate molecular and clinical stratification of the patients with high-risk diffuse large B-cell lymphoma (DLBCL) is a clinical challenge hampering the establishment of personalized therapeutic options. We studied the translational significance of liquid biopsy in a uniformly treated trial cohort. Pretreatment circulating tumor DNA (ctDNA) revealed hidden clinical and biological heterogeneity, and high ctDNA burden determined increased risk of relapse and death independently of conventional risk factors. Genomic dissection of pretreatment ctDNA revealed translationally relevant phenotypic, molecular, and prognostic information that extended beyond diagnostic tissue biopsies. During therapy, chemorefractory lymphomas exhibited diverging ctDNA kinetics, whereas end-of-therapy negativity for minimal residual disease (MRD) characterized cured patients and resolved clinical enigmas, including false residual PET positivity. Furthermore, we discovered fragmentation disparities in the cell-free DNA that characterize lymphoma-derived ctDNA and, as a proof-of-concept for their clinical application, used machine learning to show that end-of-therapy fragmentation patterns predict outcome. Altogether, we have discovered novel molecular determinants in the liquid biopsy that can noninvasively guide treatment decisions.

The erythroid marrow and circulating red blood cells (RBCs) are the key components of the human erythron. Abnormalities of the erythron that are responsible for anemia can be separated into 3 major categories: erythroid hypoproliferation, ineffective erythropoiesis, and peripheral hemolysis. Ineffective erythropoiesis is characterized by erythropoietin-driven expansion of early-stage erythroid precursors, associated with apoptosis of late-stage precursors. This mechanism is primarily responsible for anemia in inherited disorders like β-thalassemia, inherited sideroblastic anemias, and congenital dyserythropoietic anemias, as well as in acquired conditions like some subtypes of myelodysplastic syndrome (MDS). The inherited anemias that are due to ineffective erythropoiesis are also defined as iron-loading anemias because of the associated parenchymal iron loading caused by the release of erythroid factors that suppress hepcidin production. Novel treatments specifically targeting ineffective erythropoiesis are being developed. Iron restriction through enhancement of hepcidin activity or inhibition of ferroportin function has been shown to reduce ineffective erythropoiesis in murine models of β-thalassemia. Luspatercept is a transforming growth factor-β ligand trap that inhibits SMAD2/3 signaling. Based on preclinical and clinical studies, this compound is now approved for the treatment of anemia in adult patients with β-thalassemia who require regular RBC transfusions. Luspatercept is also approved for the treatment of transfusion-dependent anemia in patients with MDS with ring sideroblasts, most of whom carry a somatic SF3B1 mutation. While the long-term effectiveness and safety of luspatercept need to be evaluated in β-thalassemia and MDS, defining the molecular mechanisms of ineffective erythropoiesis in different disorders might allow the discovery of new effective compounds.

Bone marrow niche cells have been reported to fine-tune hematopoietic stem cell (HSC) stemness via direct interaction or secreted components. Nevertheless, how niche cells control HSC activities remains largely unknown. We previously showed that angiopoietin-like protein 2 (ANGPTL2) can support the ex vivo expansion of HSCs by binding to human leukocyte immunoglobulin-like receptor B2. However, how ANGPTL2 from specific niche cell types regulates HSC activities under physiological conditions is still not clear. Herein, we generated an Angptl2-flox/flox transgenic mouse line and conditionally deleted Angptl2 expression in several niche cells, including Cdh5+ or Tie2+ endothelial cells, Prx1+ mesenchymal stem cells, and Pf4+ megakaryocytes, to evaluate its role in the regulation of HSC fate. Interestingly, we demonstrated that only endothelial cell-derived ANGPTL2 and not ANGPTL2 from other niche cell types plays important roles in supporting repopulation capacity, quiescent status, and niche localization. Mechanistically, ANGPTL2 enhances peroxisome-proliferator-activated receptor D (PPARD) expression to transactivate G0s2 to sustain the perinuclear localization of nucleolin to prevent HSCs from entering the cell cycle. These findings reveal that endothelial cell-derived ANGPTL2 serves as a critical niche component to maintain HSC stemness, which may benefit the understanding of stem cell biology in bone marrow niches and the development of a unique strategy for the ex vivo expansion of HSCs.

Patients with B-lineage acute lymphoblastic leukemia (ALL) are at high-risk for relapse after allogeneic hematopoietic cell transplantation (HCT). We conducted a single-center phase 2 study evaluating the feasibility of 4 cycles of blinatumomab administered every 3 months during the first year after HCT in an effort to mitigate relapse in high-risk ALL patients. Twenty-one of 23 enrolled patients received at least 1 cycle of blinatumomab and were included in the analysis. The median time from HCT to the first cycle of blinatumomab was 78 days (range, 44 to 105). Twelve patients (57%) completed all 4 treatment cycles. Neutropenia was the only grade 4 adverse event (19%). Rates of cytokine release (5% G1) and neurotoxicity (5% G2) were minimal. The cumulative incidence of acute graft-versus-host disease (GVHD) grades 2 to 4 and 3 to 4 were 33% and 5%, respectively; 2 cases of mild (10%) and 1 case of moderate (5%) chronic GVHD were noted. With a median follow-up of 14.3 months, the 1-year overall survival (OS), progression-free survival (PFS), and nonrelapse mortality (NRM) rates were 85%, 71%, and 0%, respectively. In a matched analysis with a contemporary cohort of 57 patients, we found no significant difference between groups regarding blinatumomab's efficacy. Correlative studies of baseline and posttreatment samples identified patients with specific T-cell profiles as "responders" or "nonresponders" to therapy. Responders had higher proportions of effector memory CD8 T-cell subsets. Nonresponders were T-cell deficient and expressed more inhibitory checkpoint molecules, including T-cell immunoglobulin and mucin domain 3 (TIM3). We found that blinatumomab postallogeneic HCT is feasible, and its benefit is dependent on the immune milieu at time of treatment. This paper is posted on ClinicalTrials.gov, study ID: NCT02807883.

Genetic variants within the fibrinogen Aα chain encoding the αC-region commonly result in hypodysfibrinogenemia in patients. However, the (patho)physiological consequences and underlying mechanisms of such mutations remain undefined. Here, we generated Fga270 mice carrying a premature termination codon within the Fga gene at residue 271. The Fga270 mutation was compatible with Mendelian inheritance for offspring of heterozygous crosses. Adult Fga270/270 mice were hypofibrinogenemic with ∼10% plasma fibrinogen levels relative to FgaWT/WT mice, linked to 90% reduction in hepatic Fga messenger RNA (mRNA) because of nonsense-mediated decay of the mutant mRNA. Fga270/270 mice had preserved hemostatic potential in vitro and in vivo in models of tail bleeding and laser-induced saphenous vein injury, whereas Fga-/- mice had continuous bleeding. Platelets from FgaWT/WT and Fga270/270 mice displayed comparable initial aggregation following adenosine 5'-diphosphate stimulation, but Fga270/270 platelets quickly disaggregated. Despite ∼10% plasma fibrinogen, the fibrinogen level in Fga270/270 platelets was ∼30% of FgaWT/WT platelets with a compensatory increase in fibronectin. Notably, Fga270/270 mice showed complete protection from thrombosis in the inferior vena cava stasis model. In a model of Staphylococcus aureus peritonitis, Fga270/270 mice supported local, fibrinogen-mediated bacterial clearance and host survival comparable to FgaWT/WT, unlike Fga-/- mice. Decreasing the normal fibrinogen levels to ∼10% with small interfering RNA in mice also provided significant protection from venous thrombosis without compromising hemostatic potential and antimicrobial function. These findings both reveal novel molecular mechanisms underpinning fibrinogen αC-region truncation mutations and highlight the concept that selective fibrinogen reduction may be efficacious for limiting thrombosis while preserving hemostatic and immune protective functions.

Platelet α-granules regulate hemostasis and myriad other physiological processes, but their biogenesis is unclear. Mutations in only 3 proteins are known to cause α-granule defects and bleeding disorders in humans. Two such proteins, VPS16B and VPS33B, form a complex mediating transport of newly synthesized α-granule proteins through megakaryocyte (MK) endosomal compartments. It is unclear how the VPS16B/VPS33B complex accomplishes this function. Here we report VPS16B/VPS33B associates physically with Syntaxin 12 (Stx12), a SNARE protein that mediates vesicle fusion at endosomes. Importantly, Stx12-deficient MKs display reduced α-granule numbers and overall levels of α-granule proteins, thus revealing Stx12 as a new component of the α-granule biogenesis machinery. VPS16B/VPS33B also binds CCDC22, a component of the CCC complex working at endosome exit sites. CCDC22 competes with Stx12 for binding to VPS16B/VPS33B, suggesting a possible hand-off mechanism. Moreover, the major CCC form expressed in MKs contains COMMD3, one of 10 COMMD proteins. Deficiency of COMMD3/CCDC22 causes reduced α-granule numbers and overall levels of α-granule proteins, establishing the COMMD3/CCC complex as a new factor in α-granule biogenesis. Furthermore, P-selectin traffics through the cell surface in a COMMD3-dependent manner and depletion of COMMD3 results in lysosomal degradation of P-selectin and PF4. Stx12 and COMMD3/CCC deficiency cause less severe phenotypes than VPS16B/VPS33B deficiency, suggesting Stx12 and COMMD3/CCC assist but are less important than VPS16B/VPS33B in α-granule biogenesis. Mechanistically, our results suggest VPS16B/VPS33B coordinates the endosomal entry and exit of α-granule proteins by linking the fusogenic machinery with a ubiquitous endosomal retrieval complex that is repurposed in MKs to make α-granules.

Cutaneous T-cell lymphomas (CTCLs) are rare malignancies involving primarily the skin. Responses to treatment are usually short-lived in advanced CTCL. The determinants of long-term CTCL control are unclear. Mogamulizumab, an anti-human CCR4 antibody that acts by antibody-dependent cell cytotoxicity against CCR4+ CTCL tumor cells and peripheral memory blood regulatory T cells, has been associated with long-lasting remissions and immune adverse events. Here, we reported skin rashes in 32% of 44 patients with CTCL treated with mogamulizumab, associated with significantly higher overall survival (hazard ratio, 0.16; 0.04-0.73; P = .01). Rash occurred in patients with Sézary syndrome and was associated with longer time to progression. These rashes were characterized by a CD163+ granulomatous and/or CD8+ lichenoid skin infiltrate. High-throughput sequencing analysis of T-cell receptor β genes in skin and blood flow cytometry confirmed the depletion of CTCL tumor cells, as well as the recruitment of new reactive T-cell clones in skin at the time of skin rash. CXCL9 and CXCL11, two macrophage-derived chemokines that recruit CXCR3+ T cells to skin, were overexpressed in skin rashes. A higher frequency of TIGIT+ and PD1+ exhausted reactive blood T cells was observed at baseline in patients with rash, and this frequency decreased with mogamulizumab treatment. These data are consistent with mogamulizumab-induced long-term immune CTCL control by activation of the macrophage and T-cell responses in patients with rash.