The success of targeted therapies for hematological malignancies has heralded their potential as both salvage treatment and early treatment lines, reducing the need for high-dose, intensive, and often toxic chemotherapeutic regimens. For young patients with classic Hodgkin lymphoma (cHL), immunotherapies provide the possibility to lessen long-term, treatment-related toxicities. However, suitable therapeutic targets are lacking. By integrating single-cell dissection of the tumor landscape and an in-depth, single-cell-based off-tumor antigen prediction, we identify CD86 as a promising therapeutic target in cHL. CD86 is highly expressed on Hodgkin and Reed-Sternberg cancer cells and cHL-specific tumor-associated macrophages. We reveal CD86-CTLA-4 as a key suppressive pathway in cHL, driving T-cell exhaustion. Cellular therapies targeting CD86 had extraordinary efficacy in vitro and in vivo and were safe in immunocompetent mouse models without compromising bacterial host defense in sepsis models. Our results prove the potential value of anti-CD86 immunotherapies for treating cHL.

Excessively restrictive inclusion and exclusion criteria in clinical trials are one of many barriers to clinical trial enrollment for patients with myelodysplastic syndromes/neoplasms (MDSs). Many organizations are developing efforts to increase clinical trial eligibility; yet, several recent publications focused on patients with MDS suggest that many patients with this disease may be excluded from clinical trials unnecessarily. Clinical trial eligibility should reflect the phase of the study and risks of the agent being studied. Phase 3 trials should be less restrictive than early-phase trials to represent the real-world population as closely as possible. We hypothesize that many clinical trials, particularly phase 3 trials, have unnecessarily restrictive eligibility criteria. This study aims to evaluate the most common eligibility criteria according to phase of trial and to determine whether criteria correspond with drug safety signals. We identified MDS clinical trials registered on ClinicalTrials.gov from 1 January 2000 to 1 September 2023 and analyzed the eligibility criteria of 191 therapeutic MDS trials. We found that categorical inclusion and exclusion criteria are remarkably similar in representation across trial phases. Additionally, only 13% of trials are concordant with drug safety signals, suggesting that the eligibility criteria are often arbitrary. On behalf of the icMDS (International Consortium for Myelodysplastic Syndromes), an association of international MDS experts, we provide a position statement on restrictive eligibility criteria for MDS clinical trials that should be avoided with the aim of removing barriers to clinical trial enrollment.

The half-life of recombinant VWF (rVWF) expressed in CHO cells (CHO-rVWF; Vonicog alfa, VonvendiÒ / VeyvondiÒ) is significantly longer than plasma-derived (pd) VWF concentrates. This finding is intriguing since CHO cells do not generate α2-6 sialylation, which constitutes the majority of human pdVWF sialylation. We hypothesized that glycan differences might regulate the longer half-life of CHO-rVWF. In lectin plate binding assays and liquid chromatography mass spectrometry analysis, we confirmed that CHO-rVWF lacked α2-6 linked sialylation. Conversely, however, α2-3 linked sialylation was significantly increased on CHO-rVWF, which also had reduced exposed β-Gal compared to pdVWF. Consistent with human data, CHO-rVWF clearance was significantly (p<0.001) reduced in VWF-/- mice compared to pdVWF. However, clearance of asialo-pdVWF and asialo-CHO-rVWF were identical. In keeping with the in vivo half-life prolongation, CHO-rVWF binding to murine macrophages (p=0.012) and HepG2 cells (p=0.001) was significantly decreased compared to pdVWF. Furthermore, CHO-rVWF binding to purified MGL and ASGPR receptors was also significantly reduced. In contrast to pdVWF, in vivo studies in MGL1-/- mice and Asgr1-/- mice demonstrated that neither MGL nor ASGPR play significant roles in regulating CHO-rVWF clearance. Together, our findings demonstrate that enhanced α2-3 linked sialylation on CHO-rVWF is responsible for its extended in vivo half-life.

The CREBBP lysine acetyltransferase (KAT) is frequently mutated in follicular lymphoma (FL) and diffuse large B-cell lymphoma (DLBCL) and has been studied using gene knock-out (KO) in murine and human cells. However, the majority of CREBBP mutations encode amino acid substitutions within the catalytic KAT domain (CREBBP KAT-PM) that retain an inactive protein and have not been extensively characterized. Using CRISPR gene editing and extensive epigenomic characterization of lymphoma cell-lines, we found that CREBBP KAT-PM lead to unloading of CREBBP from chromatin, loss of enhancer acetylation and prevention of EP300 compensation. These enhancers were enriched for those that are dynamically loaded by CREBBP in the normal centroblast-to-centrocyte transition in the germinal center, including enhancers activated in response to CD40 signaling, leading to blunted molecular response to CD40 ligand in lymphoma cells. We provide evidence that CREBBP KAT-PM inhibits EP300 function by binding limiting quantities nuclear transcription factor, thereby preventing its compensatory activity. This effect can be experimentally overcome by expressing saturating quantities of transcription factor, or biologically attenuated by strong stimulation of CD40 signaling that increases nuclear transcription factor abundance. Importantly, epigenetic responses to CD40 signaling can be induced by enforcing CD4 T-cell engagement using a bispecific antibody, leading to CD40-dependent restoration of antigen presentation machinery in CREBBP KAT-PM cells and cell death. We therefore provide a mechanistic basis for enhancer deregulation by CREBBP KAT-PM and highlight enforced CD4 T-cell engagement as a potential approach for overcoming these effects.

Genetic screening for severe congenital immuno-hematological diseases offers potential for early intervention, particularly through preemptive allogeneic stem cell transplantation (HSCT). However, the clinical value of such screening depends on precise prognostic predictions based on genotype-phenotype correlations and/or functional confirmation. We investigated familial hemophagocytic lymphohistiocytosis type 2 (FHL2), caused by PRF1 variants. Specifically we evaluated the clinical significance of the frequent PRF1 A91V variant, if present in trans with a predicted loss-of-function (pLOF) PRF1 variant, defined as "disease mutation" listed in the Human Gene Mutation Database. We combined clinical and functional data from our HLH-network registry with UK Biobank data to evaluate disease penetrance and clinical outcomes. Among 52 A91V/pLOF individuals in the registry, 39 (72%) showed FHL2-related manifestations with mean onset at 20 years. Four patients had recurrent disease, 15 were transplanted, and 14 died. Among 14 A91V/pLOF individuals identified by family screening (mean 29 years), however, only one was symptomatic. Moreover, among 21 A91V/pLOF carriers identified in 200,000 UK Biobank participants, 12 with genotypes identical to symptomatic registry patients, none had developed HLH by age 73. Premature stop pLOF alleles appeared more penetrant than missense variants, but functional data including perforin expression or cytotoxicity failed to predict disease manifestation. Our combined registry and population-based approach reveals significant variability in disease penetrance and severity among PRF1 A91V/pLOF carriers, with no clear association between genotype, functional data and clinical outcomes. This complexity illustrates the challenges of genetic screening and highlights the need for careful clinical decision-making regarding preemptive HSCT in asymptomatic carriers.

We report our single center experience demonstrating that HLA Class I alloimmunization predicts longer time to platelet engraftment, increased bleeding complications and higher transfusion requirements in patients undergoing gene-modified hematopoietic stem cell transplant for transfusion-dependent beta thalassemia.

Donor blood saves lives, yet the potential impact of recurrent large-volume phlebotomy on donor health and hematopoietic stem cells (HSCs) remains largely unexplored. In our study, we conducted a comprehensive screening of 217 older male volunteer donors with a history of extensive blood donation (>100 life-time donations) to investigate the phenomenon of clonal hematopoiesis (CH). No significant difference in the overall incidence of CH was found in frequent donors (FD) compared to sporadic donors (<10 life-time donations, 212 donors). However, upon deeper analysis of mutations in DNMT3A, the most commonly affected gene in CH, we observed distinct mutational patterns between the FD and age/sex matched control donor (CD) cohorts. Functional analysis of FD enriched DNMT3A variants examined in CRISPR-edited human HSCs demonstrated their competitive outgrowth potential upon stimulation with erythropoietin (EPO), a hormone which increases in response to blood loss. In contrast, clones harboring leukemogenic DNMT3A R882 mutations increase upon stimulation with IFNy. Through concurrent mutational and immunophenotypic profiling of primary samples at single cell resolution, a myeloid bias of premalignant R882 mutant HSCs was found, while no significant lineage bias was observed in HSCs harboring EPO responsive DNMT3A variants. The latter exhibited preferential erythroid differentiation when persistent erythropoietic stress was applied to CRISPR-edited human HSC xenografts. Our data demonstrate a nuanced ongoing Darwinian evolution at the somatic stem cell level, with EPO identified as a novel environmental factor that favors HSCs carrying certain DNMT3A mutations.

Although the bone marrow is the main site of blood cell production in adults, rare pools of hematopoietic stem and progenitor cells have been found in extramedullary organs. In mice, we have previously shown that the lung contains hematopoietic progenitor cells and is a site of platelet production. Here, in the adult human lung, we show that functional hematopoietic precursors reside in the extravascular spaces with a frequency similar to the bone marrow and are capable of proliferation and engraftment in mice. The gene signature of pulmonary and medullary CD34+ hematopoietic progenitors indicates greater baseline activation of immune-, megakaryocyte/platelet-, and erythroid-related pathways in lung progenitors. Spatial transcriptomics mapped blood progenitors in the lung to an alveolar interstitium niche with only a few cells identified in an intravascular location. In human blood samples collected for stem cell transplantation, CD34+ cells with a lung signature enriched the mobilized pool of hematopoietic stem cells. These results identify the lung as a pool for uniquely programmed blood stem and progenitor cells with the potential to support hematopoiesis in humans.

Multiple Myeloma is driven by clonal plasma cell (cPC)-intrinsic factors and changes in the tumorigenic microenvironment (TME). To investigate if residual polyclonal PCs (pPCs) are disrupted, single-cell (sc) RNAseq and sc B-cell receptor analysis were applied in a cohort of 46 samples with PC dyscrasias and 21 healthy donors (HDs). Out of n=234,789 PCs, 64,432 were genotypically identified as pPCs with frequencies decreasing over different disease stages, from 23.66% in monoclonal gammopathy of undetermined significance (MGUS) to 3.23% in MMs (p=0.00012). Both cPCs and pPCs had a comparable expression of typical lineage markers (i.e. CD38, CD138), while others were more variable (CD27, ITGB7). Only cPCs overexpressed oncogenes (e.g. CCND1/2, NSD2), but CCND3 was often expressed in pPCs. BCMA was expressed on both p- and cPCs, while GPRC5D was mostly upregulated in cPCs with implications for on-target, off-tumor activity of targeted immunotherapies. In comparison with HDs, pPCs from patients showed upregulated autophagy and disrupted interaction with TME. Importantly, interferon related pathways where significantly enriched in pPCs from patients vs HDs (p-adjusted < 0.05) showing an inflamed phenotype affecting genotypically normal PCs. Function of pPCs was consequently impacted and correlated with immunoparesis, driven by disrupted cellular interactions with TME. Leveraging our scRNAseq data, we derived a "healthy PC signature" that could be applied to bulk transcriptomics from the CoMMpass dataset and predicted significantly better PFS and OS (log rank p < 0.05 for both). Our findings show that genotypic, single-cell identification of pPCs in PC dyscrasias has relevant pathogenic and clinical implications.

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive malignancy characterized by an expansion of T-cell progenitors and DNA mutations that lead to an overactive NOTCH1 signaling in over 50% of T-ALL cases. Using synthetic models of human T-ALL, we report that NOTCH1 dimeric signaling was crucial for the leukemogenesis of human hematopoietic stem/progenitor cells (HSPCs) from cord blood. We also identified a Notch-dimerization-dependent gene signature, including HES4 transcription factor, which induced proliferative advantage in human HSPCs as well as in Notch-dimerization-dependent patient-derived xenografts (PDXs) of T-ALL. Interestingly, in human T-ALL cells, HES4 enforced the expression of D133p53 isoform with the concomitant block of pro-apoptotic p53 target genes and the induction of BCL2L1 gene expression and anti-apoptotic Bcl-xL protein. Additionally, through an integrated experimental approach including genetically modified cell lines, RNA/Chip-sequencing and single cell RNA-sequencing (scRNA-Seq) profiles of primary T-ALL samples, we revealed cell subsets with Notch-dimerization-dependent gene signature, which indirectly correlated with pro-apoptotic genes as well as directly associated with cell markers of poor clinical outcome in primary T-ALL samples. Taken together, these findings highlight the crucial role of NOTCH1 dimeric signaling in human T-cell leukemogenesis and T-ALL maintenance suggesting that a possible benefit can be obtained from a therapeutic strategy targeting NOTCH1-dimer signaling or its downstream effectors.

Fixed-duration venetoclax-rituximab (VenR) in patients with relapsed/refractory chronic lymphocytic leukemia (CLL) in the phase 3 MURANO trial (NCT02005471) resulted in superior progression-free survival (PFS) and overall survival (OS) vs bendamustine-rituximab (BR). We report the final analyses of MURANO (median 7 years follow-up). Patients were randomized to VenR (venetoclax 400 mg daily for 2 years plus monthly rituximab for 6 months; n = 194) or BR (6 months; n = 195). In a substudy, patients with progressive disease (PD) received VenR as retreatment or crossover from BR. At the final data cut (3 August 2022), median PFS with VenR was 54.7 vs 17.0 months with BR. Seven-year PFS with VenR was 23.0%. Seven-year OS was 69.6% and 51.0%, respectively. Among VenR-treated patients with undetectable (u) minimal residual disease (MRD) and no PD at end of treatment (EOT) (n = 83), median PFS from EOT was 52.5 vs 18.0 months in patients with MRD at EOT (n = 35; P < 0.0001). Fourteen patients had enduring uMRD. Three distinct mutations in BCL2 in four patients were identified. In the substudy, 25 patients were retreated with VenR and nine patients crossed over to VenR; median PFS was 23 and 27 months, and best overall response rate was 72% and 89%, respectively. At the end of combination treatment, following retreatment or crossover, eight and six patients achieved uMRD, respectively. No new safety findings were observed. Overall, these final MURANO analyses support consideration of fixed-duration VenR therapy for patients with relapsed/refractory CLL.

T-cell-recruiting bispecific antibodies (BsAbs) are in clinical development for relapsed/refractory acute myeloid leukemia (AML). Despite promising results, early clinical trials have failed to demonstrate durable responses. We investigated whether activation of the innate immune system through stimulator of interferon (IFN) genes (STING) can enhance target cell killing by a BsAb targeting CD33 (CD33 bispecific T-cell engager molecule; AMG 330). Indeed, we show that cytotoxicity against AML mediated by AMG 330 can be greatly enhanced when combined with the STING agonist 2',3'-cyclic guanosine monophosphate-adenosine monophosphate (cGAMP) or diamidobenzimidazole (diABZI). We used in vitro cytotoxicity assays, immunoblotting, transcriptomic analyses, and extensive CRISPR-Cas9 knockout experiments to investigate the enhancing effect of a STING agonist on the cytotoxicity of AMG 330 against AML. Importantly, we validated our findings with primary AML cells and in a xenograft AML model. Mechanistically, in addition to direct cytotoxic effects of STING activation on AML cells, activated T cells render AML cells more susceptible to STING activation through their effector cytokines, IFN-γ and tumor necrosis factor, resulting in enhanced type I IFN production and induction of IFN-stimulated genes. This feeds back to the T cells, leading to a further increase in effector cytokines and an overall cytotoxic T-cell phenotype, contributing to the beneficial effect of cGAMP/diABZI in enhancing AMG 330-mediated lysis. We established a key role for IFN-γ in AMG 330-mediated cytotoxicity against AML cells and in rendering AML cells responsive to STING agonism. Here, we propose to improve the efficacy of CD33-targeting BsAbs by combining them with a STING agonist.

Cardiovascular disease has been recognized as the main cause of death in adults with sickle cell disease (SCD). Although the exact mechanism linking SCD to cardiomyopathy remains elusive, a possible role of subclinical acute transient myocardial ischemia during acute sickle cell-related vaso-occlusive crises (VOCs) has been suggested. We approached SCD cardiomyopathy by integrated omics using humanized SS mice exposed to hypoxia/reoxygenation (H/R; 10 hours hypoxia followed by 3 hours reoxygenation) stress, mimicking acute VOCs. In sickle cell (SS) mice exposed to H/R, a neutrophil-driven cardiac hypertrophic response is initiated by cardiac proinflammatory pathways, intersecting proteins and micro RNA involved in profibrotic signaling. This response may be facilitated by local unresolved inflammation. We then examined the effect of 17(R)-resolvin D1 (17R-RvD1), a member of the specialized proresolving lipid mediator superfamily, administration on H/R-activated profibrotic and proangiogenic pathways. In SS mice, we found that 17R-RvD1 (1) modulates miRNAome; (2) prevents the activation of NF-κB p65; (3) protects against the H/R-induced activation of both platelet derived growth factor receptor and transforming growth factor (TGF)-β1/Smad2-3 canonical pathways; (4) reduces the expression of hypoxia-inducible factor-dependent proangiogenic signaling; and (5) decreases the H/R-induced proapoptotic cell signature. The protective role of 17R-RvD1 against H/R-induced maladaptive heart remodeling was supported by the reduction of galectin-3, procollagen C-proteinase enhancer-1, and endothelin-1 expression and perivascular fibrosis in SS mice at 3 days after H/R stress compared with vehicle-treated SS animals. Collectively, our data support the novel role of unresolved inflammation in pathologic heart remodeling in SCD mice in response to H/R stress. Our study provides new evidence for protective effects of 17R-RvD1 against SCD-related cardiovascular disease.

Venetoclax (Ven), when combined with intensive chemotherapy, shows promise for untreated acute myeloid leukemia (AML), but its integration with the 7+3 regimen remains underexplored. In a phase 1b study, we assessed the safety and efficacy of Ven with daunorubicin and cytarabine in patients with newly diagnosed AML. A total of 34 patients (median age, 59 years; 62% non-White) received Ven at escalating durations (8, 11, or 14 days). Adverse events included febrile neutropenia (100%), sepsis (29%), and enterocolitis (23.5%), but there were no induction deaths. The median recovery times for neutrophils (>1.0 × 103/μL) and platelets (>100 × 103/μL) were less than 30 days. Composite complete remission was achieved in 85.3% of patients, and 86.2% were negative for measurable residual disease (MRD). Responses spanned all European Leukemia Net 2022 risk categories. With a median follow-up of 9.6 (2-20) months, the median duration of response, event-free survival, and overall survival were not reached. Ven (400 mg), when combined with 7+3 chemotherapy, was safe and effective in achieving MRD-negative remissions across all durations. Ven dose optimization is being explored in the expansion phase of this trial. Future multicenter studies should confirm our findings. This trial was registered at clinicaltrials.gov as #NCT05342584.

Although described more than a decade ago, the mechanism by which the JAK2 46/1 haplotype increases the risk of developing JAK2-mutated myeloproliferative neoplasms (MPNs) remains unexplained. Inflammation and immunity are linked to MPN development and thus could be relevant to the mechanism by which 46/1 mediates its effect. Here, we show that programmed death-1 receptor ligand (PD-L1) expression is elevated in 46/1 haplotype, both in healthy carriers and in CD34+ cells from patients with MPN. Using circular chromosome conformation capture, we observed that PD-L1 and the neighboring PD-L2 loci physically interact with JAK2 in a manner that differs between 46/1 and nonrisk haplotypes. CRISPR/Cas9 genome editing identified a region within JAK2 intron 2 that influences both JAK2 and PD-L1 expression. We suggest that increased PD-L1 expression may be relevant to the mechanism by which 46/1 leads to an increased inherited risk of developing MPN.

Desmopressin (1-desamino-8-d-arginine vasopressin [DDAVP]) can be used to prevent or stop bleeding. However, large interindividual variability is observed in DDAVP response and determinants are largely unknown. In this systematic review and meta-analysis, we aimed to identify the response to DDAVP and the factors that determine DDAVP response in patients. We included studies with patients with any bleeding disorder receiving DDAVP. First and second screening round and risk of bias assessment were performed by independent reviewers. The main outcome was proportion of patients with complete (factor level >50 U/dL) or partial (30-50 U/dL) response to DDAVP. Determinants of response including disease type, age, sex, von Willebrand factor (VWF) and factor VIII (FVIII) mutations, and baseline factor levels were investigated. In total, 591 articles were found and 103 were included. Of these, 71 articles (1772 patients) were suitable for the study's definition of response. Meta-analysis showed a pooled response proportion of 0.71 (0.64; 0.78) and a significant difference in response between disease subtypes. For hemophilia A, baseline FVIII activity (FVIII:C) was a borderline significant determinant of response. In patients with von Willebrand disease (VWD) type 1, VWF antigen (VWF:Ag), VWF activity, and FVIII:C were significant determinants. A large variation in response was observed for specific mutations in VWF and FVIII. Response to DDAVP varied between disease subtypes and was largely determined by the baseline levels of FVIII:C for hemophilia A and VWF:Ag for VWD. Our findings highlight the significant differences in response and emphasize the need for a standardized response definition and further research into response mechanisms.

Immunomodulatory drug (IMiD) resistance is a key clinical challenge in myeloma treatment. Previous data suggests almost one third of myeloma patients acquire mutations in the key IMiD effector cereblon by the time they are pomalidomide refractory. Some events, including stop codons/frameshift mutations and copy loss, having clearly explicable effects on cereblon function. Missense mutations have also been reported throughout the length of cereblon but their functional impact has not been systematically studied. This study modelled selected missense mutations and examined their effect on cereblon function also analysing whether any mutations deleterious to IMiD action could be overcome using the novel cereblon binding agents (CELMoDs). Three patterns of response to missense mutations were apparent, mutations that led to complete loss of CRBN function for all agents, those that had no effect on CRBN function and those with agent-dependent effect on CRBN function. The latter group of 4 mutations were profiled in more detail with confirmatory experiments demonstrating an ability of the more potent CELMoDs to lead to neosubstrate degradation and cell death even though IMiDs were not active. Dynamic modelling based on a newly generated crystal structure of the DDB1/CRBN/lenalidomide complex, with greater resolution than those published to date, helped to understand the impact of these mutations. These results have important implications for the interpretation of CRBN sequencing results from patients for future therapy decisions, particularly differentiating those who may, despite relapsing on IMiDs with CRBN mutations, have the potential to still benefit from the use of CELMoD agents.

Leukemic stem cells (LSCs) fuel acute myeloid leukemia (AML) growth and relapse, but therapies tailored towards eradicating LSCs without harming normal hematopoietic stem cells (HSCs) are lacking. FLT3 is considered an important therapeutic target due to frequent mutation in AML and association with relapse. However, there has been limited clinical success with FLT3 drug targeting, suggesting either that FLT3 is not a vulnerability in LSC, or that more potent inhibition is required, a scenario where HSC toxicity could become limiting. We tested these possibilities by ablating FLT3 using CRISPR/Cas9-mediated FLT3 knock-out (FLT3-KO) in human LSCs and HSCs followed by functional xenograft assays. FLT3-KO in LSCs from FLT3-ITD mutated, but not FLT3-wild type (WT) AMLs, resulted in short-term leukemic grafts of FLT3-KO edited cells that disappeared by 12 weeks. By contrast, FLT3-KO in HSCs from fetal liver, cord blood and adult bone marrow did not impair multilineage hematopoiesis in primary and secondary xenografts. Our study establishes FLT3 as an ideal therapeutic target where ITD+ LSC are eradicated upon FLT3 deletion, while HSCs are spared. These findings support the development of more potent FLT3-targeting drugs or gene-editing approaches for LSC eradication to improve clinical outcomes.

Platelets are crucial players in hemostasis and thrombosis but also contribute to immune regulation and host defense, using different receptors, signaling pathways, and effector functions, respectively. Whether distinct subsets of platelets specialize in these diverse tasks is insufficiently understood. Here, we used a pulse-labeling method in Mus musculus models for tracking in vivo platelet aging and its functional implications. Using in vitro and in vivo assays, we reveal that young, reticulated platelets show heightened responses in the setting of clot formation, with corresponding, increased responses to agonists, adhesion, and retractile function. Unexpectedly, aged platelets lose their hemostatic proficiency but are more prone to react to inflammatory challenge: compared with reticulated platelets, this cohort was more likely to form platelet-leukocyte aggregates and showed increased adhesion to neutrophils in vitro, as well as enhanced bactericidal function. In vivo, this was reflected in increased pulmonary recruitment of aged platelets in an acute lung injury model. Proteomic analyses confirmed the upregulation of immune pathways in this cohort, including enhanced procoagulant function. In mouse models of prolonged platelet half-life, this resulted in increased pulmonary leukocyte infiltration and inflammation upon acute lung injury. Similarly, human platelet concentrates decreased their hemostatic function and elevated their putative immunomodulatory potential in vitro over time, and in a mouse model of platelet transfusion, aged platelet concentrates resulted in augmented inflammation. In summary, we show that platelets exhibit age-dependent phenotypic shifts, allowing them to fulfill their diverse tasks in the vasculature. Because functional alterations of aging platelets extend to platelet concentrates, this may hold important implications for transfusion medicine.