Coagulation factor Va (fVa) is the cofactor component of the prothrombinase complex required for rapid generation of thrombin from prothrombin in the penultimate step of the coagulation cascade. In addition, fVa is a target for proteolytic inactivation by activated protein C (APC). Like other protein-protein interactions in the coagulation cascade, the fVa-APC interaction has long posed a challenge to structural biology and its molecular underpinnings remain unknown. A recent cryogenic electron microscopy (cryo-EM) structure of fVa has revealed the arrangement of its A1-A2-A3-C1-C2 domains and the environment of the sites of APC cleavage at R306 and R506. Here we report the cryo-EM structure of the fVa-APC complex at 3.15 Å resolution where the protease domain of APC engages R506 in the A2 domain of fVa mainly through electrostatic interactions between positively charged residues in the 30- and 70- loops of APC and an electronegative surface of fVa. The auxiliary Gla and EGF domains of APC are highly dynamic and point to solvent, without making contacts with fVa. Binding of APC displaces a large portion of the A2 domain of fVa and projects the 654VKCIPDDDEDSYEIFEP670 segment as a "latch", or exosite ligand, over the 70-loop of the enzyme. The latch induces a large conformational change of the autolysis loop of APC which in turn promotes docking of R506 into the primary specificity pocket. The cryo-EM structure of the fVa-APC complex validates the bulk of existing biochemical data and offers molecular context for a key regulatory interaction of the coagulation cascade.

Previous reports have highlighted that some low VWF patients with significant bleeding were diagnosed based upon an isolated but persistent reduction in plasma VWF activity levels in the 30-50 IU/dL range. These patients had plasma VWF:Ag levels > 50 IU/dL and thus had 'qualitative' rather than 'quantitative' low VWF. Although the clinical importance of functional VWF defects in type 2 VWD is well recognized, the translational implications of mild functional defects in patients with qualitative low VWF (low VWF-QL) have not been defined. To address this clinically important question, we combined low VWF datasets from the low VWF in Ireland cohort and the low VWF in Erasmus MC studies. Overall, we observed that low VWF-QL was common and accounted for approximately 50% of our combined low VWF cohort. Importantly, our findings demonstrate that many of these patients with mild isolated functional VWF defects in the 30-50 IU/dl range had significant bleeding phenotypes, even though their plasma VWF:Ag levels were within the normal range. In addition, we further show that low VWF-QL is a distinct clinic-pathological entity compared to type 2 VWD. Finally, our studies highlight that low VWF-QL is predominantly due to abnormalities in VWF biosynthesis within endothelial cells that are occurring largely independent of identifiable pathological VWF sequence variants. Cumulatively, these novel observations have important clinical implications for the diagnosis and management of patients with mild functional VWF defects.

Immune effector cell-associated hematotoxicity (ICAHT) was recently introduced as a distinct toxicity category of CAR-T therapy. While a grading system based solely on neutrophil counts was proposed (hereafter termed N-ICAHT), the prevalence and prognostic impact of thrombocytopenia remains poorly defined. In this multicenter observational study, we systematically examined patterns of thrombocytopenia in 744 patients treated with commercial CD19 CAR-T for B-cell Non-Hodgkin Lymphoma (B-NHL). We developed a grading system termed T-ICAHT with thresholds that closely aligned with N-ICAHT - based on depth, duration and timing of thrombocytopenia. In the core NHL dataset, 43% of patients developed any-grade early T-ICAHT (day 0-30), with 23% developing severe (G3+) manifestations. Late T-ICAHT (day 31-100) was observed in 42% (G3+: 13%). While T-ICAHT and N-ICAHT gradings showed some correlation, considerable discordance was noted. On multivariate analysis, bridging therapy, poor performance status, and high Hematotox scores were associated with increased risk of severe early T-ICAHT. Patients with higher T-ICAHT grades showed increased platelet and red blood cell transfusion burden (p<0.001) and more bleeding events (p=0.01). T-ICAHT grades were inversely associated with overall survival (OS), with landmarked 2-year estimates ranging from 67% (G0), to 48% (G1-2) and 35% (G3+). In multivariable Cox regression analysis, the independent prognostic capacity of T-ICAHT for OS was confirmed. Finally, we validated T-ICAHT's clinical and prognostic utility in 3 external cohorts spanning an additional 599 pediatric and adult patients (NHL, MM, B-ALL), confirming its broad applicability. These findings support integrating T-ICAHT into the ICAHT framework to standardize thrombocytopenia grading in CAR-T recipients.

Chronic granulomatous disease (CGD) is an inborn error of immunity caused by defects in any one of the 6 subunits (gp91phox, p47phox, p22phox, p67phox, p40phox or chaperone EROS) forming the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase complex 2 (NOX2) and resulting in defective phagocyte-derived reactive oxygen species (ROS) production. Almost 50% of patients with CGD have inflammatory bowel disease (IBD) associated with dysbiosis and age of IBD onset may vary according to CGD genotype. While we previously demonstrated that the intestinal microbiota determines colitis susceptibility in CGD mice, underlying mechanisms remain unknown. We hypothesized that NOX2 defects are associated with distinct intestinal microbiome signatures and immune responses, which impact colitis severity. Chemical colitis susceptibility was evaluated in 2 strains of CGD mice (gp91phox-/- and p47phox-/-) with distinct microbiota, from 2 different animal facilities, while also evaluating the impact of microbiota standardization and colitogenic microbiota transfer on mucosal immune responses at the intestinal barrier. While p47phox-/- and gp91phox-/-mice harbouring colitogenic microbiota had increased colitis severity, the intestinal epithelial cells from p47phox-/- mice produced more ROS which was associated with increased NOX isoform gene expression. In contrast, gp91phox-/- mice had decreased mucin production and a mucosal immune response profile suggestive of increased inflammasome activation at the intestinal barrier compared to control and p47phox-/- mice. Our findings suggest that the microbiota impacts colitis susceptibility in a CGD genotype-specific manner, thereby potentially explaining differences in the timing of IBD onset in patients with different CGD genotypes while identifying potential novel and personalized therapeutic targets.

Patients with acute myeloid leukemia (AML) ineligible for intensive chemotherapy (IC) have limited treatment options. The phase 3 ENHANCE-3 study aimed to determine whether magrolimab (magrolimab arm) was superior to placebo (control arm) when either was combined with venetoclax and azacitidine. Adults with previously untreated AML who were ineligible for IC were randomized to receive magrolimab (1 mg/kg on days 1 and 4, 15 mg/kg on day 8, 30 mg/kg on days 11 and 15, then weekly for 5 weeks, then every 2 weeks) or placebo, venetoclax (100 mg on day 1, 200 mg on day 2, and 400 mg daily thereafter), and azacitidine (75 mg/m2 days 1-7) in 28-day cycles. The primary endpoint was overall survival (OS); key secondary endpoints included complete remission (CR) rate and safety. After randomization of 378 patients, the trial was stopped at a prespecified interim analysis due to futility. At final analysis, with median follow-up of 7.6 months (magrolimab arm) vs 7.4 months (control arm), median OS was 10.7 vs 14.1 months (HR, 1.178 [95% CI, 0.848-1.637]). The CR rate within 6 cycles was 41.3% vs 46.0%. Addition of magrolimab to venetoclax and azacitidine resulted in more fatal adverse events (19.0% vs 11.4%), primarily driven by grade 5 infections (11.1% vs 6.5%) and respiratory events (2.6% vs 0%). There were similar incidences of any-grade infections, febrile neutropenia, and neutropenia between arms. These results highlight the difficulty in improving outcomes for patients with AML ineligible for IC. This trial was registered at www.clinicaltrials.gov as #NCT05079230.

The antenatal role of the hepcidin-regulating protease Tmprss6 has never been elucidated because knockout dams are infertile. Using an in vivo knockdown approach, we confirm Tmprsss6 is critical for hepcidin suppression in pregnancy, and Tmprss6 inhibition drives deleterious fetal outcomes.

Lineage switch (LS), defined as the immunophenotypic transformation of acute leukemia, has emerged as a mechanism of relapse following antigen-targeted immunotherapy which is associated with dismal outcomes. Through an international collaborative effort, we identified cases of LS following a host of antigen-targeted therapies (e.g., CD19, CD22, CD38 and CD7), described how LS was diagnosed, reviewed treatment approaches, and analyzed overall outcomes for this form of post-immunotherapy relapse. Collectively, 75 cases of LS were evaluated, including 53 (70.7%) cases of B-ALL to AML, 17 (22.7%) cases of B-ALL to mixed phenotypic acute leukemia (MPAL)/acute leukemias of ambiguous lineage (ALAL), and 5 (6.7%) cases of rare LS presentation (i.e., T-cell ALL to AML). An additional 10 cases with incomplete changes in immunophenotype, referred to as "lineage drift" were also described. With a primary focus on the 70 cases of LS from B-ALL to AML or MPAL/ALAL, LS emerged at a median of 1.5 months (range, 0-36.5 months) post-immunotherapy, with 81.4% presenting with LS within the first 6 months from the most proximal immunotherapy. While the majority involved KMT2A rearrangements (n=45, 64.3%), other rare cytogenetic and/or molecular alterations were uniquely observed. Treatment outcomes were generally poor with < 40% remission rates. The median overall survival following LS diagnosis was 4.8 months. Outcomes were similarly poor for those with rare immunophenotypes of LS or "lineage drift." This global initiative robustly categorizes lineage changes post-immunotherapy and, through enhanced understanding, establishes a foundation for improving outcomes of LS.

Inflammation is increasingly recognized as a critical factor in acute myeloid leukemia (AML) pathogenesis. We performed blood-based proteomic profiling of 251 inflammatory proteins in 543 patients with newly diagnosed AML. Using a machine learning model, we derived an 8-protein prognostic score termed the leukemia inflammatory risk score (LIRS). Individual proteins were evaluated in multivariable Cox models, and model performance was assessed by cumulative concordance index. Findings were validated in internal and external cohorts across 2 institutions. Blood-based LIRS significantly outperformed the European LeukemiaNet 2022 risk model and was independently prognostic of overall survival after accounting for known clinical and molecular prognostic factors. Oncostatin M receptor was uniquely identified as the strongest independent predictor of survival, early mortality, and induction chemotherapy response, and further validated in an independent assay. These blood-based biomarkers could have significant clinical implications for risk stratification and prognostication in patients with newly diagnosed AML.

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.

Acute myeloid leukemia (AML) that evolves from myeloproliferative neoplasm (MPN) is known as post-MPN AML. Current treatments don't significantly extend survival beyond 12 months. BCL-xL has been found to be overexpressed in leucocytes from MPN patients, making it a potential therapeutic target. We investigated the role of BCL-xL in post-MPN AML and tested the efficacy of DT2216, a platelet-sparing BCL-xL proteolysis-targeting chimera (PROTAC), in preclinical models of post-MPN AML. We found that BCL2L1, the gene encoding BCL-xL, is expressed at higher levels in post-MPN AML patients compared to those with de novo AML. Single-cell multi-omics analysis revealed that leukemia cells harboring both MPN-driver and TP53 mutations exhibited higher BCL2L1 expression, elevated scores for leukemia stem cell, megakaryocyte development, and erythroid progenitor than wild-type cells. BH3 profiling confirmed a strong dependence on BCL-xL in post-MPN AML cells. DT2216 alone, or in combination with standard AML/MPN therapies, effectively degraded BCL-xL, reduced the apoptotic threshold, and induced apoptosis in post-MPN AML cells. DT2216 effectively eliminated viable cells in JAK2-mutant AML cell lines, induced pluripotent stem cell-derived hematopoietic progenitor cells (iPSC-HPCs), primary samples, and reduced tumor burden in cell line-derived xenograft model in vivo by degrading BCL-xL. DT2216, either as a single agent or in combination with azacytidine, effectively inhibited the clonogenic potential of CD34+ leukemia cells from post-MPN AML patients. In summary, our data indicate that the survival of post-MPN AML is BCL-xL dependent, and DT2216 may offer therapeutic advantage in this high-risk leukemia subset with limited treatment options.

HFE-related hemochromatosis (HH) induces systemic iron overload. Although extensive studies indicate a pivotal role for iron homeostasis in αβ-T-cell immunity, its effect on γδ-T cells is unknown. Here we show a reversal of the Vδ2+/Vδ2- ratio in the γδ-T-cell compartment as a feature of hemochromatosis, which is associated with a Vδ2+ population that cannot be enriched by ZOL-stimulation, despite evidence of TCR-ligand formation and strong proliferative behavior. Already in vivo, ROS production and exhaustion marker expression are significantly increased on Vδ2+-T-cells in hemochromatosis compared with healthy individuals. Ex vivo, hemochromatosis-donor-derived Vδ2+-cells are hyporesponsive to TCR stimulation in terms of ROS production, but significantly increase their paramount expression of exhaustion markers further. Fas-Fas Ligand coexpression indicates their high susceptibility to activation-induced cell death. Consistent therewith FeSO4 alone induces Vδ2+ subset-specific proliferation in healthy PBMCs comparable to stimulation by ZOL, and blocking experiments identify FeSO4-induced proliferation as BTN3A1/TCR-mediated. Pyrophosphates are key for Vδ2+-TCR-ligand formation. Iron, by suppressing pyrophosphatase ALP promotes their stability. In light of this our data suggest that the transcriptional repression of pyrophosphatases - as under the conditions of iron overload in hemochromatosis in vivo - leads to the constitutive availability of stress-signaling Vδ2+-TCR ligand thus permanent TCR-triggering in Vδ2+-T-cells even under homeostatic conditions, ultimately leading to their subset-specific activation-induced cell death. A similar phenotype was observed in patients with iron overload due to inborn hemoglobinopathies, suggesting an inverted Vδ2+/Vδ2- ratio in the γδ-T-cell compartment as a hallmark of iron overload.

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.

With the incorporation of effective therapies for myelofibrosis (MF), accurately predicting outcomes after allogeneic hematopoietic cell transplantation (allo-HCT) is crucial for determining the optimal timing for this procedure. Using data from 5,183 MF patients who underwent first allo-HCT between 2005 and 2020 at EBMT centers, we examined different machine learning (ML) models to predict overall survival (OS) after transplant. The cohort was divided into a training set (75%) and a test set (25%) for model validation. A Random Survival Forests (RSF) model was developed based on 10 variables: patient age, comorbidity index, performance status, blood blasts, hemoglobin, leukocytes, platelets, donor type, conditioning intensity, and graft-versus-host disease prophylaxis. Its performance was compared with a four-level Cox regression-based score and other ML-based models derived from the same dataset, and with the CIBMTR score. The RSF outperformed all comparators, achieving better concordance indices across both primary and post-essential thrombocythemia/polycythemia vera MF subgroups. The robustness and generalizability of the RSF model was confirmed by Akaike's Information Criterion and time-dependent Receiver Operating Characteristic (ROC) Area Under the Curve (AUC) metrics in both sets. While all models were prognostic for non-relapse mortality, the RSF provided better curve separation, effectively identifying a high-risk group comprising 25% of patients. In conclusion, ML enhances risk stratification in MF patients undergoing allo-HCT, paving the way for personalized medicine. A web application (https://gemfin.click/ebmt) based on the RSF model offers a practical tool to identify patients at high risk for poor transplantation outcomes, supporting informed treatment decisions and advancing individualized care.

We comprehensively profiled the landscape of immunoglobulin (IG) and T-cell receptor (TR) rearrangements at diagnosis in 1212 acute lymphoblastic leukemia (ALL) patients (573 children, 639 adults) diagnosed in Germany between 2017 and 2022.Our findings indicate that immunogenetic maturity in ALL patients is age-dependent, with younger patients exhibiting more mature profiles. In fact, 68.7% of pediatric B-ALL and 85.7% T-ALL patients carried IGK, or complete TRB and/or TRD rearrangements, respectively; compared to 39.0% and 67.3% in adults (B-ALL: p<2.2e-16, T-ALL: p=6.7e-03). Additionally, children carried more IG/TR markers compared to adults (mean 6/patient versus 4/patient, respectively; p=2.5e-38). Only 0.5% of pediatric patients lacked markers, contrasted with 6.7% of adults.IGH clonal evolution was most pronounced among pro-B ALL cases (60.9%), with the V-to-DJ mechanism driving pro-B evolution (78.6%), while V-replacement dominated other immunophenotypes. Additionally, we observed that the presence of expanded accompanying T-cell clones of unknown significance in B-ALL patients increased with age.This next-generation sequencing (NGS)-based study offers an unprecedented characterization of IG/TR rearrangement patterns across ALL subtypes and age groups. It highlights the higher immunogenetic maturity in children, which may be explained by the infection-driven abnormal activity of the IG/TR recombination machinery in pediatric ALL.

Multiple myeloma (MM) initiation is dictated by genomic events. However, its progression from asymptomatic stages to an aggressive disease that ultimately fails to respond to treatments is also dependent on changes of the tumor microenvironment (TME). Clonal hematopoiesis of indeterminate potential (CHIP) is a prevalent clonal condition of the hematopoietic stem cell whose presence is causally linked to a more inflamed microenvironment. Here, we show in 106 patients with MM that CHIP is frequently co-existing with MM at diagnosis, associates with a more advanced R-ISS stage, higher age and shows a non-significant trend towards lower median hemoglobin. In our cohort the two conditions do not share a clonal origin. Single cell RNA-sequencing in 16 MM patients highlights significant TME changes when CHIP is present: decreased naïve T cells, a pro-inflammatory TME, decreased antigen-presenting function by dendritic cells and expression of exhaustion markers in CD8 cells. Inferred interactions between cell types in CHIP-positive TME suggested that especially monocytes, T cells and clonal plasma cells may have a prominent role in mediating inflammation, immune evasion and pro-survival signals in favor of MM cells. Altogether, our data show that, in the presence of CHIP, the TME of MM at diagnosis is significantly disrupted in line with what usually seen in more advanced disease, with potential translational implications. Our data highlight the relevance of this association and prompt for further studies on the modifier role of CHIP in the MM TME.

The half-life of recombinant human von Willebrand factor (rVWF) expressed in CHO cells (CHO-rVWF; Vonicog alfa; and Vonvendi/Veyvondi) is significantly longer than that of plasma-derived VWF (pdVWF). This finding is intriguing because 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-galactose (β-Gal) compared to pdVWF. Consistent with human data, CHO-rVWF clearance was significantly (P < .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 = .012) and HepG2 cells (P = .001) was significantly decreased compared to pdVWF. Furthermore, CHO-rVWF binding to purified macrophage-galactose-type lectin (MGL) receptor and asialoglycoprotein receptor (ASGPR) was also significantly reduced. In contrast to pdVWF, in vivo studies in MGL1-/- mice and Asgr1-/- mice demonstrated that neither MGL nor ASGPR plays 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 half-life.

Rilzabrutinib is a covalent, reversible Bruton tyrosine kinase inhibitor targeting multiple immune thrombocytopenia (ITP)-related mechanisms. The phase 3 LUNA3 study in previously treated adults with persistent/chronic ITP evaluated oral rilzabrutinib 400 mg twice daily (n = 133) vs placebo (n = 69) for 24 weeks. At baseline overall, median age was 47 years, 63% female, 7.7 year median ITP duration, and 28% prior splenectomy. Overall (N = 202), 85 (64%) rilzabrutinib and 22 (32%) placebo patients achieved platelet response (≥50 × 109/L or 30 × 109/L to <50 × 109/L and doubled from baseline) during the first 12 weeks and were eligible to continue. The primary end point, durable platelet response (platelet count ≥50 × 109/L for ≥two-thirds of ≥8 of the last 12 of 24 weeks without rescue therapy), was observed in 31 (23%) rilzabrutinib vs 0 placebo patients (P < .0001). All secondary efficacy end points were significantly superior for rilzabrutinib (P < .05). Median time to first platelet response was 15 days in rilzabrutinib responders. Rilzabrutinib significantly reduced rescue therapy use by 52% (P = .0007) and improved week 25 bleeding scores (P = .0006). Improved physical fatigue was sustained from week 13 (P = .01) through 25 (P = .0003). Treatment-related adverse events were mainly grade 1/2. One rilzabrutinib patient with multiple risk factors had serious treatment-related grade 3 peripheral embolism (lower left leg), and another died from unrelated pneumonia. Rilzabrutinib in patients who failed multiple previous ITP therapies showed rapid and durable platelet response, reduced rescue medication and bleeding, improved physical fatigue, and favorable safety. Trial registration: www.clinicaltrials.gov (#NCT04562766) and www.clinicaltrialsregister.eu (#2020-002063-60).

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 immunohematological diseases offers potential for early intervention, particularly through preemptive allogeneic hematopoietic 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 hemophagocytic lymphohistiocytosis (HLH)-network registry with UK Biobank data to evaluate disease penetrance and clinical outcomes. Among 52 individuals with A91V/pLOF genotype in the registry, 39 (72%) showed FHL2-related manifestations with mean onset at 20 years. Four patients had recurrent disease, 15 received transplantation, and 14 died. Among 14 individuals with A91V/pLOF genotype identified by family screening (mean age, 29 years), however, only 1 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 years. 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 β thalassemia.