Blood Advances

Multiple myeloma remains a fatal, incurable disease. Most therapies are targeted to the cancer cell or T cell engagement. Little is known about the supporting myeloma microenvironment and its contribution to tumor fitness. Here, we expand upon the observation of human mast cells in the NSG-hIL6 myeloma patient derived xenograft mouse model to show mast cells decrease time to engraftment, promote increased myeloma engraftment and cause myeloma bone disease. We identify 10 mast cell secreted factors that together improve the survival of patient myeloma cells in vitro. Our results highlight the versatility of the NSG-hIL6 model to study microenvironmental interactions between human bone marrow cells and myeloma and confirm prior suggestions that clinical signs of disease, such as osteolytic lesions, may at least partially be related to non-malignant bone marrow microenvironmental cells, such as mast cells.

Therapy-driven genomic changes in multiple myeloma (MM) remain poorly defined. We analyzed whole-genome sequencing (WGS) data from relapsed/refractory MM (rrMM, N=386) and identified regional 1p31.1-p12 (hereafter 1pCEN, a region proximal to the centromere) loss-of-heterozygosity (LOH) as the only enriched aberration showing strong therapy-associated clonal selection (clonal timing rank fold-change = 3.7, P<2.2x10-16). This event showed enriched co-occurrence with 1qGain (OR = 2.3 (1.5-3.8), P=2x10-4) forming a recurrent "double-hit" in rrMM. To validate the clonal selection process, we examined three longitudinal cohorts (180 patients, 390 samples) and confirmed clonal expansion of 1pCEN and consistent prevalence of the 1pCEN+1q double-hit (20-24%). Survival analyses demonstrated significantly reduced progression-free survival in rrMM patients with this double-hit compared with those without. Comparison with a large newly diagnosed MM (ndMM) cohort confirmed previously-described 1p32 LOH is the prognostic locus at baseline, whereas 1pCEN is therapy-selected and largely independent of the 1p32 locus. Thus, 1pCEN+1q represents a recurrent double-hit event that clonally emerges in rrMM, conferring selective advantage under drug exposure and is distinct from the ndMM high-risk markers defined by current consensus guidelines. These findings nominate 1pCEN as a new genomic biomarker in rrMM and 1pCEN+1q may help patient stratification for therapeutic monitoring. Key PointsA therapy-driven common genomic double-hit (1p31.1-p12 LOH with 1q gain) clonally emerges in relapsed/refractory myeloma.

Sickle cell disease (SCD) is caused by a point mutation in the {beta}-globin gene that promotes hemoglobin polymerization, leading to chronic hemolytic anemia, vaso-occlusive episodes, and progressive organ damage. The most efficacious therapies focus on reactivating fetal hemoglobin (HbF) expression to mitigate the pathological effects of sickle hemoglobin (HbS) polymerization. However, the predominantly used HbF inducer, hydroxyurea (HU), exhibits substantial interpatient variability in efficacy, and curative approaches such as gene therapy remain inaccessible to the vast majority of patients. Although all SCD patients share the same causative HBB glu7val mutation, differences in genetic background significantly influence disease severity and therapeutic response. We describe a SCD-specific induced pluripotent stem cell (iPSC) platform as a renewable and scalable preclinical model to interrogate treatment responses across the genetically diverse SCD patient population. By generating patient-specific iPSC-derived erythroblasts (iEry) representing distinct SCD genetic backgrounds, we demonstrate that this system faithfully recapitulates the heterogeneous HbF induction observed clinically in response to HU. Moreover, this platform enables the identification and evaluation of alternative therapeutic agents for HU non-responders and provides sufficient resolution to dissect drug-specific effects on erythroid differentiation and cellular phenotypes. Together, these findings support the use of iPSC-derived erythroid models as a versatile tool to advance precision therapeutic strategies for SCD. KEY POINTS- SCD iPSC-derived erythroid cells (iEry) reflect the diversity in HU-mediated HbF induction seen in SCD patients - SCD iEry recapitulate patient-specific treatment responses and can be used to identify therapeutic alternatives for HU non-responders - iEry provide a versatile platform to study the impact of novel HbF inducers on erythroid cell characteristics and differentiation parameters

Richter transformation of Chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) into classic Hodgkin lymphoma (CHL-RT) is rare and remains incompletely understood. Two histologic subtypes are recognized: type 1 (CLL/SLL with scattered Hodgkin/Reed-Sternberg (HRS) cells) and type 2 (HRS cells within a polymorphous inflammatory background). In this multi institutional study of 77 patients with CHL-RT (27 type 1 and 50 type 2), we characterized immune evasion markers, PD-L1/PD-L2 copy number alterations, tumor microenvironment, and performed targeted next-generation sequencing on 37 CLL/SLL samples. HRS cells in CHL-RT displayed immune evasion phenotypes similar to de novo CHL, though PD-L1 expression was lower in type 1 cases. PD-L1/PD-L2 gain/polysomy were frequent (83.3%). CLL/SLL with CHL-RT harbored increased mutations in XPO1, FBXW7, BIRC3, TRAF3, and HLA-A versus reference CLL/SLL. Similar mutational profiles, demographics, and survival outcomes support a biological continuum between type 1 and type 2 CHL-RT, with distinct genetic features in CLL/SLL predisposing to CHL transformation.

Global mRNA translation is a defining functional property of hematopoietic stem cells (HSCs) and is increasingly recognized as a critical axis of dysregulation in myelodysplastic syndromes (MDS) and other clonal hematopoietic disorders. Yet the quantitative measurement of protein synthesis at single-cell resolution across phenotypically defined HSPC subpopulations, in parallel with apoptotic state, is technically challenging. Here we describe and validate a single-tube flow cytometry protocol that simultaneously quantifies global protein synthesis by O-propargyl-puromycin (OP-Puro) incorporation and intracellular cleaved Caspase-3 with cell immunophenotyping across the canonical CD34+ HSPC hierarchy in cryopreserved human cord blood (CB) CD34+ cells. The protocol enables quantitative assessment of key dynamic cell processes in defined subsets of primary hematopoietic cells on a standard flow cytometer. We apply this assay to a four-condition factor-omission analysis of the canonical SR1 + UM729 + dmPGE2 ex vivo expansion cocktail across three independent CB donors. The analysis assigns each compound a distinct functional profile: UM729 constrains protein synthesis and supports apoptotic priming across the hierarchy; SR1 maintains a pro-survival state without modulating translation; and dmPGE2 promotes HSC cycling and progressive exit from the primitive state, with minimal direct effect on the translation or apoptotic axes measured here. This analysis resolves three mechanistically distinct small-molecule signatures using a protocol directly transferable to clinical biobank specimens. We propose it as a functional-state analytic platform that may be useful for patient-derived CD34+ cells from MDS and other myeloid neoplasms in which translational dysregulation is a recognized pathological feature.

Antigen density critically influences CAR T-cell efficacy. We show that ibrutinib induces CD19 upregulation in B-cell lymphoma models, enhancing CAR T-cell cytotoxicity. Pharmacologic modulation of target antigen expression represents a promising strategy to overcome resistance.

Background: Multiple myeloma (MM) remains incurable despite therapeutic advances, reflecting limited understanding of the molecular mechanisms underlying disease initiation and progression. MM develops through asymptomatic precursor stages, monoclonal gammopathy of undetermined significance (MGUS) and smouldering multiple myeloma (SMM). This study aimed to investigate protein changes associated with disease progression and, through a further integrative approach, to highlight molecular changes of potential predictive and/or therapeutic value. Methods: We performed a comparative proteomic analysis of 94 bone marrow-derived CD138+-selected plasma cell samples (29 MGUS, 20 SMM, and 45 MM) using LC-MS/MS. Differential protein abundance was assessed using pairwise Mann-Whitney U tests between groups, with Benjamini-Hochberg correction. Pathway enrichment, protein-protein interaction, and co-expression network analyses were also conducted. Selected proteins were further evaluated using public transcriptomic datasets and experimentally validated in independent samples by flow cytometry and enzyme-linked immunosorbent assay (ELISA). Results: Following data processing, proteomic analysis identified 6,203 proteins. Pairwise comparisons revealed significant proteomic differences across disease stages, with 370 differentially abundant proteins exhibiting monotonic changes during disease progression. Pathway analysis showed that monotonically upregulated proteins were mainly associated with gene expression and cell proliferation, whereas downregulated proteins were linked to immune-related processes. Further co-expression network analysis, combined with criteria including detection frequency, biological relevance, and translational potential, highlighted a group of prioritised proteins. Representative examples include nucleolin (NCL) and U3 small nucleolar ribonucleoprotein IMP3 (IMP3), involved in nucleolar organisation, ribosome biogenesis and rRNA processing, as well as the immune-associated lactotransferrin (LTF) and serine protease cathepsin G (CTSG). Transcriptomic support and independent experimental validation by flow cytometry and ELISA confirmed the relevance of selected candidates. Conclusions: Taken together, our findings highlight coordinated changes in immune regulation, RNA processing and ribosome biogenesis during MM progression and identify candidate proteins and their networks, including the emerging pharmacologically tractable target NCL and the underexplored IMP3 of potential therapeutic relevance, opening new avenues for further investigation.

Germline variants in DDX41 are the most frequent genetic predisposition to adult hematologic malignancies. The most common variants are truncating, implicating loss of function in the pathogenesis. However, non-truncating variants account for 30-40% of cases, and their impact on essential DDX41 functions remains unknown. We utilized a genetic complementation assay to assess the functionality of 10 recurrent germline non-truncating variants of DDX41. All variants restored viability to Ddx41-deficient hematopoietic progenitor cells at exogenous expression levels. In contrast, the hotspot mutant p.R525H, which is somatically acquired at disease onset in >50% of patients, failed to restore viability. CRISPR-based modeling in cell lines and mice revealed heterogeneity: some variants were non-functional at endogenous expression levels whereas others maintained complete functionality, supporting normal cell proliferation and even lifelong hematopoiesis in a homozygous setting. Notably, co-expression of p.R525H with some variants caused impaired hematopoietic progenitor cell viability, indicating a dominant-negative effect of p.R525H. In contrast, other variants, all classified as variants of unknown significance, were unaffected by the presence of p.R525H. A screen of 100 disease-associated variants confirmed that many non-truncating germline variants are susceptible to p.R525H-mediated dominant-negative effects, whereas wild-type DDX41 is not. These findings indicate that DDX41 variant curation is complicated by variable effects on functionality and variant-specific interactions with somatically-acquired DDX41 mutations. The dominant-negative effect of p.R525H provides a mechanistic basis for the conclusion of recent patient cohort analyses that co-occurrence with a somatic hotspot mutation is a reliable indicator of DDX41-driven disease in carriers of non-truncating variants.

Background: Modern therapy for childhood and adolescent leukemia requires accurate risk classification of genomic subtype. Although short-read next-generation sequencing (NGS)- based approaches provide comprehensive clinical diagnostics in limited, highly resourced settings, they remain expensive, slow, and inaccessible to most children worldwide. Transformative approaches are needed to improve diagnostic classification for leukemia globally. Methods: We simultaneously continued to develop an analytical pipeline NASVar (Nanopore variant calling for adaptive sampling), and conducted a multicenter, type-two hybrid clinical validation study of an Oxford Nanopore Technologies (ONT) adaptive-sampling whole-genome sequencing (asWGS) assay across hospitals with varying diagnostic resources. In preparation for implementation, a global panel developed a leukemia-based standardized gene set and consensus laboratory-developed test (LDT) validation guidelines. Measures of assay effectiveness compared to both conventional and orthogonal NGS methods, where available, were simultaneously collected with data to measure the implementation outcomes of feasibility, fidelity, appropriateness, and cost. Results: All four centers successfully completed the LDT validation, with minimal adaptations required for regulatory compliance. A total of 457 specimens were sequenced (331 B-ALL, 83 AML, 43 T-ALL). For the 210 B-ALL cases with locally resolved genomic subtypes defined by DNA alterations, asWGS was 100% concordant (210/210). Cases locally defined as B-other were resolved via asWGS with disease-defining DNA alterations in 47% (49/105) of cases. An additional 41% (43/105) of locally defined B-other cases were classified by incorporation of DNA methylation, and all 16 B-ALL patient-derived xenograft controls were correct, for a total of 96% (318/331) of all B-ALL cases in the cohort resolved with single assay asWGS. For AML, 97% (56/58) of cases with locally resolved genomic subtypes were identified by automated asWGS analysis, while an additional two cases were identified after targeted manual review. At Indus Hospital in Pakistan, the B-ALL and AML diagnostic genomic subtype yield increased from 28% with local standard of care diagnostic testing, to 84% with asWGS. The cost of reagents and consumables in the United States, assuming pooled three-plexing, was $343/sample. Based on the combined hybrid validation results, all centers are independently preparing for clinical return of results. Conclusions: ONT asWGS was successfully validated as a clinical assay in four diverse hospital settings. As a single, multi-omic platform that delivers value across the continuum of high-resource to resource-limited contexts, the approach offers a disruptive solution to address the global equity gap in cancer diagnostics.

Measurable (or minimal) residual disease (MRD) predicts relapse in patients with acute myeloid leukemia (AML). However, the biological and spatial characteristics of the AML bone marrow (BM) microenvironment (BMME) in which MRD cells survive remain largely unexplored; in particular, little is known of the BMME in TP53 mutant (TP53mut) AML. Here, we applied sequential immunofluorescence to whole BM biopsy specimens obtained from patients with TP53 wild-type (TP53WT) AML and TP53mut AML at diagnosis and in morphological complete remission (CR) to generate a comprehensive spatial map of the hematopoietic and BMME components. We identified TP53mut leukemia cells based on high p53 expression and delineated their spatial organization relative to stromal and immune niches. Biopsy-based cell composition analysis revealed marked B-cell depletion and an increased abundance of regulatory T-cells (Tregs) in TP53mut BM at CR. Unlike TP53WT BM, TP53mut BM at CR exhibited persistent TP53mut erythroid and immature leukemia cell clusters, spatially segregated from T-cell clusters, in perisinusoidal niches, suggesting niche-level immune evasion. Spatial profiling further revealed that Tregs characterized by FOXP3 upregulation were enriched near TP53mut MRD cells, indicating a locally enhanced immunosuppressive activity. Single-cell RNA sequencing-based cell-cell communication analysis identified erythroid-T-cell interactions mediated by the GDF15-CD48 axis as a potential mechanism of T-cell suppression, suggesting that the erythroid differentiation of TP53mut AML cells enhances local immunosuppression. Collectively, our results show a spatially organized immunosuppressive BMME in TP53mut AML and highlight the potential of spatial proteomics to identify actionable MRD niches in leukemias. Key pointsO_LITP53 mutant erythroid and immature leukemia cells form spatial clusters segregated from T-cells in complete remission. C_LIO_LIAn erythroblast-centered immunosuppressive niche characterizes TP53 mutant leukemia cells. C_LI

Pediatric platelet disorders are commonly classified according to specific structural or functional abnormalities, yet it remains unclear how well these diagnoses capture overall hemostatic phenotype. Here, we combined quantitative single-cell platelet measurements with spatially resolved plasma clotting analysis to characterize pediatric patients with dense granule deficiency, platelet function defects, immune thrombocytopenia, and other inherited platelet disorders. Quantitative fluorescence microscopy revealed reduced dense granule abundance not only in dense granule deficiency but also in several patients from other diagnostic groups. Measurements of platelet adhesion, spreading, and calcium signaling identified substantial functional diversity, with individual patients exhibiting distinct combinations of abnormalities that were not predicted by diagnostic category. Unexpectedly, plasma clotting analysis frequently revealed hypercoagulable behavior, including accelerated fibrin clot growth and spontaneous fibrin formation, despite clinical diagnoses associated with platelet-related bleeding disorders. Hypercoagulable phenotypes occurred across multiple diagnostic groups and did not show a simple relationship with platelet functional abnormalities. Together, these findings reveal previously unrecognized complexity in pediatric platelet disorders and suggest that platelet and plasma pathways contribute independently to hemostatic variability. These findings argue that pediatric platelet disorders are best viewed as multidimensional functional phenotypes rather than isolated platelet defects and motivate broader integration of platelet and coagulation measurements in future studies.

Wiskott-Aldrich syndrome (WAS) and X-linked neutropenia (XLN) are caused by genetic variants in the WAS gene. How WAS variants lead to clinical disease remains unsolved in many cases. We expressed human WASp using a spider silk inspired solubility tag (NT*-tag) and inserted patients variants. Native mass spectrometry and pyrene actin assays showed that five variants (L270P, F271S, S272P, I290T, I294T) predicted to cause XLN led to open protein conformation and high actin polymerization rate in the absence of the WASp activator, Cdc42. One previously reported XLN variant (R268W), two loss-of-function WAS variants (A236G, D485N), and one variant of unknown significance (R431W) behaved similarly to wildtype WASp in terms of structural conformation and actin polymerization. Patient CD4+ T cells were used for analysis of WASp expression and phosphorylation, actin polymerization, anti-CD3 induced proliferation capacity, and upregulation of high affinity LFA-1, distinguishing loss-of-function and gain-of-function variants from benign WAS variants. This systematic approach reveals how WAS genetic variants cause severe human disease and stratify variants to guide clinical decision for definitive therapy. Key PointsO_LIGain-of-function WASp variant has extended protein conformation probed by native mass spectrometry and raised pyrene actin polymerization. C_LIO_LIFunctional analysis of patients CD4+ T cells classifies WASp variants as loss-of-function, reduced-function, gain-of-function, and benign. C_LI

Fetal hemoglobin (HbF) prevents the polymerization of sickle hemoglobin (HbS). HbF, measured usually as a percent of total hemoglobin (%HbF), is inversely associated with the severity of sickle cell disease (SCD) but fails to capture the distribution of HbF concentrations within red blood cells (RBCs). The relative proportion of HbF and HbS within a RBC is reflected by the HbF:HbS ratio whereas HbF/F-cell quantifies the absolute amount of HbF/RBC. While correlated, HbF:HbS ratio and HbF/F-cell are not interchangeable. In the context of mean corpuscular hemoglobin (MCH), HbF/F-cell approximates whether sufficient HbF is present to inhibit HbS polymerization. We examined the association of mean HbF/F-cell with sub-phenotypes of sickle cell disease in three independent cohorts. Both %HbF and HbF/F-cell were significantly associated with multiple clinical and laboratory features of SCD; however, HbF/F-cell demonstrated stronger associations with clinical severity measures across cohorts. Higher HbF/F-cell was associated with fewer clinical events, reduced hemolysis, and mortality. Changes in HbF/F-cell after hydroxyurea treatment were associated with ~11-13% reduction in acute events in patients with <1 pg increase and >60% reduction with a >5 pg increase in HbF/F-cell. For each pg increase in HbF/F-cell there was ~6% reduction in the rate of acute events. As a surrogate for the distribution of HbF concentrations among F-cells, HbF/F-cell adds physiologically relevant insights that could guide prognosis and treatment

Antibody-dependent cellular cytotoxicity (ADCC) is a major mechanism of action for many FDA-approved therapeutic antibodies that is driven by interactions between the antibody Fc and Fc{gamma} receptors (Fc{gamma}Rs) on immune effector cells. Murine models used for preclinical antibody evaluation currently have limited predictive value for clinical ADCC performance due to interspecies differences in Fc-Fc{gamma}R interactions. The molecular determinants governing Fc-Fc{gamma}R engagement in mice remain poorly defined, complicating the interpretation of murine ADCC data and its clinical relevance. To address this, we present the high-resolution crystal structure of the receptor that regulates Fc-mediated cytotoxicity in mice, mouse Fc{gamma}RIV, alone and in complex with mouse IgG2a Fc. This complex preserves key features of the human IgG1 Fc-human Fc{gamma}RIIIa interface which mediates ADCC in humans including salt bridges, hydrogen bonds, and a proline sandwich. However, subtle variations in receptor orientation, Fc-Fc{gamma}R electrostatics, and glycan positions reduce human IgG1 Fc- mouse Fc{gamma}RIV binding affinity, resulting in species-restricted Fc-Fc{gamma}R mediated immune responses. Modeling of human IgG1 Fc interactions with mouse Fc{gamma}RIV predicted steric clashes, suggesting opportunities to modulate the interaction. One structure-guided substitution variant of human IgG1, Fchumo, maintains comparable human Fc{gamma}RIIIa engagement with enhanced binding to and activation of mouse Fc{gamma}RIV, relative to human IgG1 Fc. This study provides proof-of-concept for engineering human Fc domains for cross-species Fc{gamma}R recognition and provides a strategic framework to improve the predictive power of in vivo preclinical models.

Chimeric antigen receptor (CAR) T-cells have represented a groundbreaking advance in the control of hematological cancers. However, their efficacy in controlling solid tumors has been rather limited, highlighting the importance of new cell-based therapies strategies to curb the progression of solid cancers. Here, we generated functional macrophages from human umbilical cord blood derived CD34+ hematopoietic stem cells (HSCs) engineered to express CARs. Approximately 50% of the CAR-MacCD34 population expressed anti-HER2 CARs and maintained high viability throughout differentiation. Mass spectrometry (MS) and multiparametric flow cytometry analysis revealed upregulation of proteins associated with phagocytosis, matrix remodeling, and degradation, indicating enhanced tumor infiltration potential. In vitro, CAR-MacCD34 exhibited a significantly higher capacity to phagocytose HER2-positive tumor cells compared to untransduced MacCD34 cells. Additionally, CAR-MacCD34 cells that phagocytosed cancer cells showed increased nuclear translocation of NF-kB, suggesting CAR-mediated intracellular signaling. To assess functionality in a more physiologically relevant context, we used tumor spheroids embedded in a dense 3D collagen matrix. Confocal microscopy and live imaging revealed that CAR-MacCD34 cells exhibited superior infiltration of dense tumor spheroids compared to untransduced MacCD34 cells. Notably, we observed multiple instances of tumor cell phagocytosis by CAR-MacCD34 cells in this 3D model. In addition, we employed in vivo zebrafish larvae models of HER2-positive tumors. We noted that CAR-MacCD34 cells persisted for over 8 days post-injection and demonstrated significantly greater efficacy in controlling tumor growth compared to untransduced MacCD34 cells. Our findings introduce a novel CAR-macrophage therapeutic approach with promising clinical potential, leveraging a renewable and accessible cellular source. Optimizing CAR-MacCD34 functionality in combination with existing therapies may lead to durable and effective anti-tumor responses for patients with solid tumors.

Immunoglobulin E (IgE) drives allergic disease, yet what restrains the persistence of IgE production remains poorly understood. Mouse studies suggest that BCR-induced apoptosis limits the survival of IgE-producing plasma cells (PCs). Whether this mechanism applies to human IgE PCs is unclear. Using a human IgE class-switching system, we show that BCR crosslinking preferentially kills IgE PCs compared to IgG1+ PCs. However, this selective sensitivity is not explained by surface BCR levels or proximal BCR signaling as suggested in mice. Instead, elevated PTEN expression in IgE PCs constrains PI3K/Akt pro-survival signaling and lowers the apoptotic threshold by upregulating BIM, while JNK signaling sustains PTEN expression and amplifies their apoptotic sensitivity. CRISPR/Cas9 targeting of PTEN or BIM, or JNK inhibition protects IgE PCs from BCR-mediated killing. Therapeutic anti-IgE antibodies, including omalizumab and extracellular membrane-proximal domain (EMPD)-targeting antibodies, exploit this sensitivity to selectively eliminate IgE PCs and suppress IgE production, providing a mechanistic rationale for depleting IgE PCs in allergic disease. SummaryRamadani et al. identify a JNK/PTEN/BIM signaling axis that intrinsically limits human IgE plasma cell survival and drives their preferential sensitivity to BCR-induced apoptosis. This mechanism is distinct from that established in mice and has direct implications for anti-IgE therapeutic strategies.

Defining the genetic and cellular programs that allow solid tumours to evade immune control requires preclinical models that preserve the complexity of the human tumour immune microenvironment. Most available systems capture only part of this biology. Organoid cultures and ex vivo tumour fragments can retain patient-derived tumour architecture and associated immune cells, but immune populations are typically maintained only for short periods. These models also cannot capture antitumour immune responses in the physiological setting of a living organism. Patient-derived xenografts propagated in humanized mice offer a potential path to overcome these limitations by combining patient-derived tumour tissue with a reconstituted human immune system. However, few studies have systematically tested whether these models reproduce the diverse immune cell phenotypes present in the parental tumours from which they are derived. This has limited their use for studying tumour-intrinsic mechanisms that shape immune composition and promote immune evasion. To address this gap, we profiled tumour-infiltrating, splenic, and bone marrow immune cells from ovarian, head and neck, and renal PDX models propagated in CD34+ hematopoietic stem cell (HSC)-derived huNOG-EXL mice expressing human IL-3 and GM-CSF. By comparing tumours grown across distinct HSC donor backgrounds with their matched primary tumour samples, we found that tumour-intrinsic factors are a dominant determinant of immune composition in humanized PDX tumours. Across models, these immune infiltrates generally resembled those of the corresponding parental tumours. These findings support humanized PDX models as a platform for functionally interrogating tumour-intrinsic drivers of immune composition and immune evasion in solid tumours.

Malaria is caused by Plasmodium parasites, and its clinical symptoms are a result of parasite invasion of red blood cells and the subsequent cycles of replication and proliferation. In human populations, Plasmodium vivax is responsible for the most widely distributed recurring malaria infections whereas Plasmodium falciparum inflicts the most mortality and morbidity. One well-characterized family of adhesins involved in red blood cell invasion is the reticulocyte-binding-like protein homolog family, known as the RBL superfamily which includes the PfRh family in P. falciparum and PvRBP family in P. vivax. Here we report a collection of nanobodies against three members of this adhesin family, PfRh5, PfRh4 and PvRBP2b. Nanobodies against these Plasmodium adhesins bind with high affinity across several epitopes and can block receptor engagement and inhibit parasite invasion of red blood cells. Using computational design, we generated stabilized PfRh4 variants that encompass the conserved scaffold present in the PfRh and PvRBP families of adhesins and show that several variants with improved expression retained binding to mouse monoclonal antibodies, nanobodies and Complement Receptor 1, the human receptor for PfRh4. We also observed that most of the inhibitory nanobodies against the three antigens recognized the conserved structural scaffold that define this family of adhesins. These results demonstrate the potential of nanobodies to block malaria parasite invasion into red blood cells.

Objective: To evaluate whether glucagon-like peptide-1 receptor agonists (GLP-1 RAs) are associated with improvements in pain severity, disability, quality of life, and physical function in adults with obesity and chronic low back pain (cLBP), and to explore potential mechanisms. Design: Prospective, single-arm cohort study. Subjects: Thirty-five adults (median age 41 years; 86% women) with obesity (median BMI 39.9 kg/m2) and cLBP initiating GLP-1 RAs (tirzepatide, n=24; semaglutide, n=11). Methods: Participants completed questionnaires at baseline, 3, 6, 9, and 12 months. The primary outcome was Brief Pain Inventory-Short Form (BPI-SF) pain severity. Secondary outcomes included body mass index (BMI), BPI-SF pain interference, Numerical Rating Scale (NRS) back pain, Oswestry Disability Index (ODI), and Short Form-12 (SF-12). At baseline and 6 months, a subset (n=24) underwent quantitative sensory testing, physical performance testing, and blood draws for inflammatory biomarkers (C-reactive protein, TNF-, IL-6, IL-10), adipokines (leptin, adiponectin), and hemoglobin A1c. Results: Over 12 months, BMI decreased by 12.5% (median 39.9 to 34.9 kg/m2, 95% CI [-6.6, -4.2]). BPI-SF pain severity improved (median 4.8 to 2.0, 95% CI [-2.1, -0.8]), as did pain interference, ODI, NRS back pain, and SF-12 physical component scores. Hemoglobin A1c, leptin, and C-reactive protein decreased. Adiponectin increased and physical performance improved, but neither reached significance. Experimental pain sensitivity was unchanged. Conclusions: GLP-1 RAs were associated with clinically meaningful improvements in pain, disability, and quality of life. These findings suggest GLP-1 RAs may be a promising nonsurgical therapy for cLBP; randomized controlled trials are needed to establish causality and mechanisms.

WHIM (warts, hypogammaglobulinemia, infections, and myelokathexis) syndrome is a primary immunodeficiency caused by gain-of-function in CXCR4 chemokine receptor (CXCR4GOF) in response to its chemokine ligand CXCL12. The patients suffering from this syndrome display lymphopenia and neutropenia, and most of them show exacerbated susceptibility to human papillomavirus pathogenesis. In a mouse model harboring a WHIM-associated CXCR4 mutation and expressing HPV16 oncoproteins in keratinocytes, we previously reported reduced circulating plasmacytoid dendritic cells (pDCs), mirroring patients blood, and impaired dendritic cell (DC) trafficking from the skin to lymphoid organs, with the few migrating DCs displaying an overactivated phenotype. Given the promising results of CXCR4-targeted therapies in WHIM patients, we investigated whether and how the orally available CXCR4-specific antagonist, X4-136, affects DC localization, activation, and trafficking at the subset level, as well as skin immune landscape. CXCR4GOF inhibition corrected defects in circulating myeloid cells and pDCs, as well as in lymph node-resident DCs. Furthermore, it rescued skin DC migration to lymph nodes in WHIM mice, in a context- and subset-dependent manner, by promoting their activation and relocation within the dermis. Taken together, these findings indicate that inhibiting CXCR4GOF may restore skin immunity in WHIM syndrome by rescuing DC counts and functions. Key pointsO_LICXC R4 gain-of-function inhibition promotes subset-selective dermal dendritic cell migration to lymph nodes in a WHIM syndrome mouse model. C_LIO_LIInhibiting CXCR4 corrects migratory WHIM dendritic cell hyperactivation with subset-specific effects tied to the inflammatory context. C_LI