Hematopoietic stem and progenitor cells (HSPCs) balance self-renewal with on-demand differentiation of blood lineages. Modern bone marrow atlases specify static transcriptional states, but not HSPC dynamics, which are only partly understood, particularly in humans. Here, we introduce cHSPCTrack (clonal HSPC tracker), an in vitro framework for serially tracking clonal differentiation and fate acquisition from human HSPCs extracted from circulation. By following thousands of single-cell-derived clones stimulated by different cytokine combinations, cHSPCTrack reconstructs the balance between stochasticity and directed differentiation in human HSPCs. Single HSPCs stochastically yield multiple lineages under a uniform cytokine environment, with stimulatory inputs modulating differentiation probabilities by introducing biases toward preferred fates. In parallel, differentiation rates vary stochastically across and within clones. Clonal memory stabilizes the induction of a few genes per clone over otherwise archetypical differentiation trajectories. Within clones, multiple fates can interact with each other, as shown for clones mixing basophils, eosinophils, and mast cells with other fates. cHSPCTrack opens avenues for understanding hematopoietic clonal dynamics in health and disease.

Granulopoiesis is a tightly regulated process encompassing the production, maturation, and release of neutrophils in the bone marrow, ensuring their optimal physiological contribution to host defence. The mechanisms that maintain a balanced regulation of neutrophil effector functions during this process remain incompletely understood. Here, we identify bone marrow-resident erythroblasts as a key source of specialized pro-resolving mediators (SPMs) and show that they imprint neutrophil development and function. Terminally differentiating erythroblasts highly express the key SPM biosynthetic enzyme 12/15-lipoxygenase (Alox15) and accordingly generate several SPMs, including n-3 docosapentaenoic acid-derived Resolvin D5 (RvD5n-3 DPA). Conditional erythroblast-specific depletion of Alox15 decreased bone marrow SPM levels and caused altered neutrophil phenotypes, including augmented release of reactive oxygen species and neutrophil extracellular traps, as well as increased migration to chemotactic stimuli. This was accompanied by increased neutrophil sequestration in peripheral organs and concomitant neutropenia. Mice with erythroblast-specific Alox15 depletion displayed impaired bacterial clearance in experimental peritonitis and increased severity in DSS-colitis. Aberrant neutrophil phenotypes were rectified by the reconstitution of RvD5n-3 DPA and were largely recapitulated by the specific depletion of the SPM receptor Gpr101 in bone marrow macrophages, but not in neutrophil precursors, suggesting involvement of the macrophage niche in mediating the SPM effects on granulopoiesis. Our findings establish a central role for erythroblasts and their SPM production in instructing granulopoiesis for balanced functional neutrophil responses.

Azacitidine (Aza) plus venetoclax (Ven) is standard treatment for older/unfit patients with newly diagnosed (ND) acute myeloid leukemia (AML). The approved 28-day (D) Ven schedule is associated with prolonged cytopenias, causing frequent dose reductions and cycle delays. Retrospective studies show similar efficacy and reduced toxicity with abbreviated Ven dosing, but prospective data is lacking. We conducted OPTI-AML(NCT03013998), a prospective randomized phase 2 trial comparing 28D Ven (AV28) versus 14D (AV14) with Aza (75mg/m²x7D) for C1-2 in genomically agnostic ND-AML patients ≥60 years. The primary endpoint was complete remission (CR) rate achieved at any time with two cycles of therapy. Between 2023-2025, 169 patients received AV28 (n=83) or AV14 (n=86). CR across two cycles was 49.4% (AV28) versus 43% (AV14); difference of 6.4% [90%CI:-6.1% to 19.0%], not meeting non-inferiority criteria. Patients with NPM1/ IDH2 mutations had higher CR rates with AV28 (60.9% vs. 33.3%), while CR rates were equivalent (45%) for other subgroups. Composite CR rates were 80.7% (AV28) versus 68.6% (AV14) and MRD negativity was similar (77.6% vs. 76.5%). Although AV28 had more frequent treatment interruptions, count recovery after C2, grade ≥3 adverse events and early mortality were similar. In conclusion, the study did not demonstrate non-inferiority of AV14 compared with AV28 during C1-2 in an unselected ND-AML cohort. However, as the confidence interval for the difference covered 0, CR rate for AV28 was not significantly different than AV14. Certain subgroups may benefit from prolonged Ven exposure, but these findings require validation in larger studies, especially as triplet regimens evolve.

This first clinical evaluation of BCMA CAR-T cell therapy in four patients with refractory primary ITP demonstrated manageable safety, rapid and sustained platelet recovery, and durable drug-free complete remission lasting up to 15 months, supporting further evaluation in larger studies.

The clinical significance of one or two measurable residual disease (MRD) assessments is established in multiple myeloma (MM). However, how to stratify patients according to ≥3 MRD assessments remains unknown. The traits of MRD resistance and if treatment of persistent MRD vs relapse could improve outcomes also remains unknown. MRD dynamics were computed using next-generation flow cytometry and Connector in 539 newly-diagnosed MM patients with ≥3 assessments in the GEM2012MENOS65/GEM2014MAIN and GEM2017FIT trials. Molecular and immune profiling were performed in matched diagnostic and MRD samples. The survival impact of treating persistent MRD vs relapse was investigated with anti-BCMA CAR T cells in MIcγ1huCRBN mice. Computed MRD dynamics based on 3,610 MRD assessments identified five subgroups with different survival. Patients with late-sustained MRD response had excellent outcomes, similar to those with early-sustained MRD response. Patients with volatile results and those with primarily and resurgent MRD resistance had dismal survival. MRD dynamics outperformed transplant-eligibility and the R-ISS. These results were validated in 249 MM patients treated in routine practice. Multiomics characterization of MRD dynamics in patients and mouse models of MRD resistance revealed genomic evolution, transcriptional adaption and a pro-inflammatory tumor-immune microenvironment. Increasing clonality and exhaustion of endogenous T cells throughout disease progression urged investigating if MRD interception with anti-BCMA CAR-T cells could improve outcomes. Infusion at MRD resistance prolonged mouse survival compared to identical treatment at relapse. Altogether, MRD dynamics is the strongest predictor of progression and may help tailoring treatment to prevent additional tumor and immune alterations prior to relapse.

Factor V (FV) links procoagulant amplification to anticoagulant feedback, but how FV limits tissue factor-initiated coagulation are not fully defined. We hypothesized that procofactor FV downregulates factor X (FX) activation by tissue factor:factor VIIa (TF:FVIIa), independently of tissue factor pathway inhibitor α (TFPIα), and that this mechanism is especially important in hemophilia. Thrombin generation was measured in FV/FVIII‑immunodepleted plasma and synthetic plasma while titrating FV, with TFPIα removed, blocked, or re-added. TF:FVIIa activation of FX was measured on phosphatidylserine‑containing or phosphatidylserine‑free liposomes with antibodies against the FV light chain and C2 domain. FV and TFPIα levels modulated thrombin generation in plasma from people with hemophilia A. In TF‑initiated coagulation lacking TFPIα, thrombin generation peaked at 2 nM FV and decreased as FV increased; at 20 nM FV (normal concentration), peak thrombin and thrombin generation rate were reduced by up to 50-80%, with larger effects at low FVIII. In purified TF:FVIIa assays, FV reduced FX activation by ~80% at physiologic concentration and inhibited FX activation on TF-expressing fibroblasts. Increasing PS content enhanced FX activation and increased the FV-sensitive component, while blocking the FV light chain or C2 domain partially relieved inhibition. In hemophilia A plasma, higher FV was associated with longer lag time and time-to-peak, and lower peak thrombin independent of TFPIα. Thus, FV is an endogenous anticoagulant that inhibits TF-initiated coagulation by limiting FX activation by TF:FVIIa through a membrane-dependent mechanism. This mechanism refines models of coagulation initiation and may help explain how FV variation contributes to bleeding and thrombosis.