In Caucasians, maternal allo-immunization against human platelet antigen (HPA)-1a is the most common cause for fetal and neonatal alloimmune thrombocytopenia (FNAIT). Intracranial hemorrhage (ICH) is a serious complication. The lack of a consistent correlation between antibody concentration, severity of thrombocytopenia, and ICH makes it difficult to predict thrombocytopenia in subsequent pregnancies. HPA-1a is located on integrin β3, which forms heterodimers with different alpha chains. The αvβ3 heterodimer is present on endothelial cells (ECs), and a specific antibody interaction with fetal ECs may explain the sporadic nature of ICH. Analysis of human sera was consistent with the presence of anti-HPA-1a of the anti-αvβ3 type in ICH positive cases. We show evidence that these sera induce VEGFR2 fixation to the cell surface, lead to VEGFR2 phosphorylation, enhance ROS production, and interfere with endothelial sprouting. Analysis of blinded human FNAIT samples demonstrates that combined biochemical and functional testing correctly identifies 6/6 ICH positive and 4/4 ICH negative cases. In a murine model, female b3 knockout mice develop anti-platelet antibodies after immunization with wild-type platelets. After mating with b3 wildtype males, only pups born to mice with anti-αvβ3 develop ICH. Matching our findings obtained with human material, only murine sera containing anti-αvβ3 induce VEGFR2 fixation on the cell surface, lead to VEGFR2 phosphorylation, induce ROS production, and hamper endothelial loop formation. In conclusion, we find evidence that ICH in FNAIT is based on vascular impairment and substantiate previous reports that impaired angiogenesis could represent a mechanism of ICH in FNAIT.

Factor VIII Aurora (FVIII-R571S) is the first described naturally occurring enhanced-potency FVIII variant identified in a patient with recurrent thrombosis and early mortality. The patient's plasma exhibited increased procoagulant activity and reduced responsiveness to activated protein C (APC). To define the mechanism, we generated recombinant FVIII-R571S and performed in vitro and in vivo studies. Consistent with the clinical phenotype, FVIII-R571S demonstrated a 6-fold increase in one-stage assay activity, while chromogenic substrate assay activity was comparable to wild-type FVIII (FVIII-WT). This discrepancy was explained by biochemical studies showing that activated FVIII-R571S (FVIIIa-R571S) has 10-20-fold higher affinity for FIXa; notably, the chromogenic assay is insensitive to differences in FVIIIa-FIXa affinity. Additional analyses demonstrated that FVIII-R571S is inactivated by APC and protein S analogous to FVIII-WT, indicating that the variant is not intrinsically APC-resistant. However, the increased affinity of FVIIIa-R571S for FIXa confers FIXa-dependent reduced A2-domain dissociation and APC-mediated inactivation in purified and plasma-based studies. These enhanced biochemical properties translated in vivo to a more potent procoagulant phenotype in hemophilia A mice. In the tail clip assay, FVIII-R571S exhibited a 4-5-fold increase in potency compared to FVIII-WT. In a thrombosis model, FVIII-R571S promoted significantly increased platelet and fibrin accumulation relative to FVIII-WT at equivalent antigen levels. Collectively, these data demonstrate that the prothrombotic phenotype of FVIII-R571S is driven by increased FIXa affinity, which enhances FVIIIa-FIXa complex assembly and function. This same mechanism confers reduced A2 dissociation and functional APC resistance, providing a unifying explanation for the observed gain-of-function phenotype.

Anemia, the most prevalent hematologic disorder in older adults, imposes a significant burden of cardiovascular events, cognitive decline, and mortality. However, the mechanisms underlying aging-related anemia, especially epigenetic dysregulation in hematopoietic stem and progenitor cells (HSPCs), remain incompletely understood. Although the gut microbiota is critical for hematopoiesis, its specific contribution to aging-related erythropoiesis impairment remains unclear. Here, we reveal that aging markedly activates phenylalanine metabolism and elevates plasma phenylacetic acid (PAA) levels in both humans and mice. We identify Odoribacter splanchnicus (O.splanchnicus) as a key gut symbiont whose abundance is significantly increased in aged mice and which directly drives PAA production from phenylalanine via the oxoacid:ferredoxin oxidoreductase (OFOR) superfamily encoded by porA,nifJ, and iorA/iorB. Rifaximin treatment selectively reduces O.splanchnicus and plasma PAA, thereby alleviating aging-related anemia. Mechanistically, PAA promotes a novel post-translational modification (PTMs) termed histone lysine phenylacetylation (Kpa) through the acetyltransferases HBO1. Elevated histone Kpa increases chromatin accessibility at the GATA2 promoter, disrupts the GATA switch, and blocks erythroid differentiation of HSPCs. In vivo, supplementation with sodium phenylacetate (NaPA) exacerbates anemia in microbiota-depleted mice, whereas the HBO1 inhibitor WM-3835 restores erythropoiesis by reversing histone Kpa and normalizing the GATA switch. Furthermore, dietary phenylalanine restriction lowers circulating PAA and effectively ameliorates aging-related anemia in both naturally aged mice and O.splanchnicus-colonized mice. These findings provide the first evidence that gut microbiota-derived PAA plays a critical role in the development of aging-related erythropoiesis impairment and offer multiple translatable strategies for treating this condition.

Neutrophils are acute responders to bacterial and fungal infection; however, their roles in disease extend far beyond their critical roles in countering pathogens. In this review, we seek to provide some broader context based on recent literature that highlights the contributions of the neutrophil lineage to cancer and chronic inflammation. Their importance for immunomodulation is underscored by their capacity to generate cytokines and chemokines, reactive oxygen species, granule secretory proteins, and use regulated inflammatory cell death pathways for the release of neutrophil extracellular traps. The potential to alter the balance between circumscribed inflammation and collateral tissue damage is most notable when considering the diverse contents and functions of secretory vesicles, and tertiary, specific, and azurophilic granules. We still have much to learn about the generation of granules in neutrophil progenitors, the factors controlling their release from mature neutrophils, and the cellular consequences for misfolding, misplacement, and premature release of granule contents from neutrophils. This review discusses recent advances in our understanding of neutrophil biology, roles in disease, and opportunities for depletion, inhibition, or supplementation and restoration of function.

Sickle cell disease attenuates nitric oxide signaling, limiting soluble guanylyl cyclase (sGC) stimulation needed for hemo-vascular function. Cytochrome b5 reductase 3 (CYB5R3) regulates sGC activity and expression. We show CYB5R3 T117S associates with reduced efficacy of the sGC stimulator riociguat. (NCT02633397).

The impact of chimeric antigen receptor (CAR)-T cell expansion and persistence on clinical outcomes and treatment-related morbidity in patients with relapsed/refractory multiple myeloma (RRMM) remains incompletely defined, in part due to limited availability of standardized CAR-T cell quantification assays. We evaluated CAR-T cell kinetics and their association with efficacy and toxicity in RRMM patients treated with idecabtagene vicleucel (ide-cel) or ciltacabtagene autoleucel (cilta-cel). Using a uniform flow cytometry-based platform (N=90; cilta-cel, n=54; ide-cel, n=36), we observed significantly greater CAR-T cell expansion with cilta-cel than with ide-cel (median 106 vs 49 cells/uL). Peak CAR-T cell expansion was associated with clinical response in the ide-cel cohort but not with cilta-cel, where rapid and excessive expansion was instead associated with an increased risk of delayed neurotoxicities (DNTs), a complication with potential long-term functional consequences (median peak 1,009 vs 96 cells/uL). To identify clinically accessible biomarkers of CAR-T cell expansion, we analyzed absolute lymphocyte count (ALC) as a surrogate biomarker in a larger multicenter cohort (N=532; cilta-cel, n=256; ide-cel, n=276). Higher peak ALC was significantly associated with the development of DNTs, particularly Parkinsonism after cilta-cel. A peak ALC ³3000/uL - or ³2500/uL following a daily twofold increase - predicted elevated DNT risk (sensitivity 81%, specificity 59%). Together, these findings delineate distinct expansion-toxicity relationships in cilta-cel and ide-cel therapy, establish ALC as a practical, uniformly available surrogate for CAR-T cell expansion, and define quantitative thresholds that may enable early recognition of patients at risk for DNT, informing preemptive strategies to mitigate morbidity following cilta-cel.

Patients with acute leukemias harboring translocations involving gene lysine methyltransferase 2A (KMT2A) have a poor prognosis due to chemotherapy resistance with rapid relapse following standard treatments. The resulting KMT2A fusion proteins dysregulate gene expression, leading to an upregulation of leukemogenic transcription factors such as HOXA9 and MEIS1, which drives leukemic transformation. Although Menin inhibitors are proving to be promising new therapeutics for patients with KMT2A-rearranged (KMT2Ar) acute leukemia, resistance mechanisms have already been described and new therapeutic approaches for this patient subgroup must be identified. Here, a genome-wide CRISPR/Cas9 screen in a KMT2Ar B-cell acute lymphoblastic leukemia (ALL) cell line identified the deubiquitinase USP22 as a novel regulator of MEIS1 protein stability. USP22 is a member of the Spt-Ada-Gcn5 acetyltransferase (SAGA) multiprotein complex, which has crucial functions in shaping the chromatin landscape and modulating transcription. Genetic depletion of USP22 impaired cellular growth and proliferation in KMT2Ar acute leukemia models. Chromatin immunoprecipitation revealed cooperative binding between USP22 and MEIS1 at critical oncogenic target genes suggesting that USP22 safeguards leukemogenic transcription by protecting MEIS1 from proteasomal degradation. Genetic or chemical inhibition of USP22 led to polyubiquitination of MEIS1 resulting in proteasomal degradation and downregulation of the expression of target genes. Our study identifies USP22 as a novel regulator of MEIS1 protein stability, that could potentially be exploited as a therapeutic target in the future in KMT2Ar leukemias.

Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is an orphan, aggressive hematologic malignancy characterized by high CD123 expression. Tagraxofusp (TAG), a CD123-directed toxin, is the only approved therapy. Prior studies demonstrated BPDCN dependence on BCL2, sensitivity to venetoclax (VEN), and reversal of TAG resistance with azacitidine (AZA). We conducted a phase 2 study evaluating combination therapy with TAG, AZA, and VEN in patients with BPDCN (NCT03113643). Patients with previously untreated (1L) or relapsed/refractory (R/R) BPDCN received 28-day cycles of AZA (75 mg/m² days 1-7), VEN (400 mg days 1-21), and TAG (12 µg/kg days 4-6). Eligibility followed TAG guidelines to mitigate risk of capillary leak syndrome (CLS). Twenty-seven patients were enrolled (16 1L, 11 R/R), with median age of 70 years (range 21-81). Composite complete remission (CR/CRi/CRc) rates were 88% in 1L and 64% in R/R cohorts. Median duration of response was not reached in 1L and was 7.2 months in R/R patients. CLS occurred in 15% of patients; most were grade 2. In the 1L cohort, median overall and progression-free survival were not reached; the 2-year overall survival was 65% and 2-year progression-free survival was 53%. Median overall survival in R/R patients was 8.4 months. A high proportion of patients proceeded to allogeneic stem cell transplantation in remission (63% 1L, including 10 of 11 patients age 75 or younger; and 55% R/R). TAG-AZA-VEN is highly active in both untreated and relapsed BPDCN with a predictable and manageable safety profile, supporting its use as a new therapeutic option.

BCMA and GPRC5D-directed therapies have shown high efficacy in the treatment of relapsed or refractory myeloma with multiple new immune therapies now approved. Preclinical studies have shown that expression of BCMA and GPRC5D are heterogenous in malignant plasma cells. We conducted a phase I dose escalation trial of the BCMA CAR T cell therapy MCARH125 alone or in combination with the GPRC5D CAR T cell therapy MCARH109 in patients with heavily pre-treated relapsed or refractory multiple myeloma. Patients were treated across three dose levels and the primary objective of establishing safety of the concurrent infusion. The trial is registered in ClinTrials.gov, NCT05431608 and has now completed accrual. We treated 15 patients across 3 dose levels; this included 6 patients who received MCARH125 alone and 9 patients treated with concurrent infusion of MCARH125 and MCARH109. 1 patient each with MCARH 125 alone and the co-infusion of MCARH125 and MCARH109 developed immune effector cell associated hemophagocytic syndrome (IEC-HS). There were no instances of grade 3 or higher cytokine release syndrome (CRS) or immune effector cell associated neurologic syndrome (ICANS). The overall response for co-infusion was 78% with a median PFS of 18.2 months. Correlative analysis suggests that antigen loss maybe an important mechanism of resistance even with dual-antigen targeting and we show for the first time that expansion of one CAR population can limit expansion of the second population, as dual-CAR treated patients had significantly less BCMA-CAR expansion than BCMA-CAR alone treated patients, despite equivalent BCMA CAR T cell doses at infusion.

Multiple myeloma progresses from precursor states to active disease, and studying tumor microenvironment (TME) evolution across these stages is key to understanding immune dysregulation and therapeutic resistance. Here, we integrated paired single-cell RNA, T-, and B-cell receptor sequencing data from bone marrow samples of 235 patients spanning the disease spectrum. This dataset revealed dynamic changes in the abundance and functional states of diverse immune populations, including T-, natural killer, B-, and myeloid cells. Using non-negative factorization of cell-subset composition, we identified five reproducible TME subtypes, or "ecotypes," defined by coordinated cellular architectures. These ecotypes captured structured variation beyond disease stage and were associated with distinct cell-cell communication networks, cytokine signaling landscapes, transcription factor programs, and shared gene modules, reflecting coordinated immune adaptation to microenvironmental constraints. By linking tumor features with immune changes and ecotype distributions, we identified context-dependent associations influenced by both disease biology and treatment effects on the TME. We found that an ecotype enriched for bone marrow-resident populations and limited immune infiltration was associated with tumor expansion and inferior clinical outcomes, whereas ecotypes reflecting T-cell functional states showed distinct associations with immunotherapy response and survival. Our study provides a comprehensive single-cell immune atlas of multiple myeloma and its precursors, offering insights into TME organization and progression, and may guide development of stage-specific or ecotype-targeted immunotherapies.

Decentralized clinical trials (DCTs) are clinical studies where part or all trial-related activities occur remotely, reducing or eliminating the need for patients to physically visit a research site. By applying a more patient-centered design, DCTs have the potential to enhance patient access to clinical trials, improve enrollment and retention, and increase convenience, while simultaneously decreasing costs. This perspective explores the growing importance of DCTs, including the opportunity to facilitate engagement of patients with rare diseases and their broader implications for improving access to care. It also examines the challenges of implementing a DCT, comprising logistical complexities, reliance on technology, and regulatory considerations. Drawing upon our direct experience designing and executing a DCT for patients with Clonal Cytopenia of Undetermined Significance, we discuss our study design, share key pitfalls encountered and lessons learned regarding technology integration, site coordination, and patient engagement. Based on these experiences, we offer recommendations for optimizing DCT design, addressing challenges, and guiding future trials to fully realize the potential of decentralized models, ultimately fostering more equitable and patient-centric clinical research.