Journal of Clinical Investigation

Impaired cellular differentiation is a defining characteristic of myeloid malignancies and remains a major therapeutic challenge. The BRG1/Brahma-associated factor (BAF) chromatin remodeling complex, through the ATPases SMARCA4 and SMARCA2, maintains the stemness of leukemic blasts and thus represents a promising target for novel differentiation-based therapies. In a phase 1 study in advanced myeloid malignancies, the first-in-class dual SMARCA4/2 inhibitor FHD-286 combined with decitabine (DAC) was tolerated and produced an objective response rate of 12.8% (6/47) compared with no responses with FHD-286 monotherapy. To understand the basis of this activity, we integrated high-dimensional flow cytometry and single-cell genomic analyses of longitudinal bone marrow samples from responders and nonresponders. While FHD-286 monotherapy was predominantly associated with myeloid differentiation, responders to FHD-286+DAC combination therapy exhibited a range of myeloid and erythroid differentiation trajectories. FHD-286 potentiated the transcriptional impact of DAC, driving tumor clones to fully differentiate out of the immunophenotypically and transcriptionally defined blast compartment. Responders had a baseline transcriptional profile similar to that of CEBPA-mutant acute myeloid leukemia and showed further downregulation of CEBPA upon treatment. These findings reinforce tumor cell differentiation as a mechanism of response to pharmacologic SMARCA4/2 inhibition and support further evaluation of FHD-286+DAC in molecularly defined patient subsets.

Guillain-Barre syndrome (GBS) is an autoimmune disorder that causes weakness, sensory loss, autonomic dysfunction, and chronic neuropathic pain. The mediators responsible for driving early autoimmune injury in the most common GBS variant, acute inflammatory demyelinating polyradiculoneuropathy (AIDP), remain incompletely understood. We performed single-cell and bulk RNA sequencing on peripheral blood mononuclear cells collected from early untreated AIDP-variant GBS patients and healthy controls to comprehensively deduce leukocyte transcriptome alterations and predict disease- and pain-driving interactions between pathogenic leukocytes and peripheral nervous system cells. We found that classical, intermediate, and non-classical monocytes were expanded and upregulated genes associated with type I and II interferons, JAK/STAT signaling, and NLRP3 inflammasome engagement. CD8+ T cells were highly proliferative and likewise upregulated JAK/STAT signaling. CD4+FOXP3+ regulatory T cells upregulated PRDM1 and CD74 in a signature that may indicate functional exhaustion. A subpopulation of highly activated intermediate monocytes upregulated genes related to angiogenesis and oncostatin M. Differential expression-based cell-cell interaction analysis between GBS leukocytes, Schwann cells, and sensory neurons predicted engagement of ligand-receptor pairs with nerve integrity and pain functions, including epiregulin, interferon-beta, adrenomedullin, clusterin, IL-6, IL-15, and CCL4. Functional validation demonstrated that CCL4 sensitizes human sensory neurons in vitro. These results unearth molecular interactions by which specific leukocyte populations in AIDP-variant GBS may participate in peripheral nerve injury and drive neuropathic pain. Many of these targets may be amenable to therapeutic modulation using available approved and investigational drugs, potentially providing drug repurposing opportunities.

Purpose: Neurofibromatosis type 2-related schwannomatosis (NF2-SWN) is an autosomal dominant tumor syndrome with complete penetrance and variable expressivity. Underlying patterns of disease burden and severity are largely unexplained. Methods: We comprehensively phenotyped 168 NF2-SWN patients over a mean duration of 4.5 years. We used a custom sequencing panel of NF2 and schwannomatosis genes to identify germline (n=166) and somatic variants in tumors (n=37). An optimized composite severity (CSS) score based on clinical and radiological data was created to analyze the effect of genetic variants on phenotype. Results: We found significant variable expressivity not explainable by demographic variables. Germline variants included premature termination (42%), splice-site (18%), and large deletions (16%). The CSS successfully predicted worsening clinical function in patients. Unsupervised clustering of clinical data revealed distinct phenotypic clusters that corresponded to CSS. Mosaicism, however, was not associated with CSS or any other disease severity marker. CSS was significantly associated with germline variant location along the NF2 locus. Specifically, FERM-F1 and the -helical variants were associated with increased disease severity. Within tumors, germline variants with severe effects on merlin acquired milder somatic second-hits at the NF2 locus. Conclusion: We identified a second-hit modifier to the Mendelian first-hit: severe germline variants were associated with milder somatic variants, and vice versa. This phenomenon partly explains the variable expressivity in NF2-SWN.

Despite decades of widespread vaccination, whooping cough caused by Bordetella pertussis (Bp) continues to circulate globally. To define correlates of protection and mechanisms underlying symptom development, we characterized systemic and mucosal immune responses in a controlled human infection model of Bp (NCT05136599). Healthy vaccinated adults were intranasally challenged with escalating Bp doses and classified as symptomatic, asymptomatic, or non-infected. Longitudinal sampling of blood and nasal mucosa allowed mapping of antibody titers, immune cell subset frequencies, cytokine concentrations, gene expression, and T cell activation and polarization. Non-infected participants exhibited significantly higher pre-challenge serum antigen-specific IgG titers. Post-challenge, only symptomatic participants developed robust antigen-specific IgG responses. Notably, infection-induced IgG responses displayed slower kinetics and a lower overall magnitude compared to the rapid day 7 peak observed following tetanus, diphtheria, and acellular pertussis (Tdap) booster vaccination. Furthermore, symptomatic infection was driven by pronounced nasal inflammation characterized by increased HLA-DR myeloid cells and upregulated mucosal NF-{kappa}B signaling on day 7. Systemic immune responses were comparatively modest post-challenge: plasma cytokine concentrations decreased independently of clinical outcome, peripheral blood mononuclear cell transcriptomes and antigen-specific T cell activation and polarization remained largely unchanged. These findings identify pre-existing serum antibodies as potential correlates of protection from Bp infection and suggest that symptom development is associated with localized mucosal inflammation dominated by a myeloid cell response. The predominance of nasal over systemic immune activation highlights the importance of mucosal immunity in controlling Bp and provides critical insights to guide the design of next-generation vaccines aimed at preventing both disease and transmission.

While recent therapeutic advances have extended the survival of patients with non-small cell lung cancer (NSCLC), overcoming metastatic progression in the CNS remains a significant challenge. Some patients with NSCLC may require concurrent management of CNS and extracranial metastases, while others develop isolated brain metastasis or leptomeningeal disease. These heterogenous clinical outcomes are difficult to predict and diagnose for early intervention with current surveillance modalities. Herein, we comprehensively analyzed gene mutations, copy number variations, and DNA methylation of NSCLC brain metastasis tissue collected at the time of craniotomy, combined with ctDNA sequencing of paired plasma and CSF liquid biopsies. We confirmed a high concordance between the molecular features of brain metastasis tissue with ctDNA from CSF which were largely distinct from ctDNA alterations in paired plasma samples. Plasma ctDNA tumor fraction and ctDNA hypermethylation were most significantly associated with extracranial metastasis and overall survival. Alternatively, we identified specific hypermethylated DNA loci in brain metastasis tissue and CSF ctDNA as significant correlates of brain metastasis progression and risk of leptomeningeal disease. Our findings support the utility of integrating ctDNA testing from CSF and plasma, while revealing distinct epigenetic features and biomarkers of brain metastasis or leptomeningeal disease.

A growing body of pre-clinical research has demonstrated the therapeutic potential of chronic, continuous hypoxia (11% FIO2) for treating both rare and common forms of neurodegeneration (1). However, the chronic delivery of hypoxic gas poses both practical challenges and long-term safety concerns. We previously introduced a small molecule, ''hypoxia-in-a-pill'' regimen that combines the hemoglobin affinity enhancer (GBT440) -- which limits oxygen delivery to tissues -- with a HIF-2 inhibitor (PT2399) to prevent compensatory erythropoiesis that can be detrimental. While this regimen extended the lifespan of the Ndufs4 KO mouse model of Leigh syndrome, its efficacy still did not match that of chronic 11% FIO2. Here we report an optimized combination that now utilizes GBT601, a second-generation hemoglobin affinity enhancer with longer half-life and greater hemoglobin occupancy, again with PT2399. Here we report that the GBT601/PT2399 combination achieved therapeutic hypoxia and demonstrated strong efficacy comparable to continuous breathing of 11% FIO2 by halting neurodegeneration and even reversing neurological symptoms in three different mouse models: Leigh syndrome, Friedreich's ataxia, and Parkinson's disease. The dual targeting regimen led to a striking extension in median lifespan in the Leigh syndrome model, from a median of ~62 day to 158 days, when initiated after onset of advanced disease. Importantly, body weight was stable with the combination and it did not induce any signs of pulmonary hypertension, likely due to attenuation of HIF-2. Our findings motivate additional pre-clinical and even clinical studies to evaluate the safety and efficacy of the GBT601/PT2399 combination.

Genetic mitochondrial diseases (GMDs) are major challenges to human health accounting for a significant fraction of heritable neurologic diseases, myopathies, and inborn errors of metabolism. Leigh syndrome (LS) is the most common clinical presentation of GMD in pediatric patients. LS is a severe and complex disease for which effective clinical therapies are currently lacking. Preclinical therapies identified in the Ndufs4(-/-) mouse model of LS include immune-targeting interventions and chronic mild hypoxia (11% oxygen). Immune-targeting interventions include rapamycin and high-dose pexidartinib, the latter appearing to fully suppress disease. The mechanisms underlying the benefits of hypoxia remain unclear, and the relationship between hypoxia and immune interventions have not been assessed. Here, we report the immune profile of brainstem of the Ndufs4(-/-) mouse model prior to and after disease onset and the impact of pexidartinib treatment. We provide evidence that macrophages/monocytes drive pathology, consistent with recent genetic studies. We additionally find that pre-disease onset animals lack signs of inflammation, and that the elimination of leukocytes fully suppresses the molecular signature of disease. Finally, using distinct post-developmental periods of treatment, we find pexidartinib and rapamycin provide benefits which persist long beyond treatment cessation, while cessation of hypoxia results in rapid disease onset and an acceleration of disease progression. These findings are consistent with hypoxia acting upstream of immune cell activation and have major implications for the therapeutic translation of both hypoxia and immune targeting interventions. Our findings establish hypoxia-cessation as a novel method for synchronizing inflammatory disease onset in the Ndufs4(-/-) model which will be useful in future mechanistic studies.

Clonal hematopoiesis (CH) is an age-associated condiion defined by over-representation of hematopoietic stem cells (HSCs) and their progeny carrying somatic mutations or variants that confer a selective advantage. CH is associated with increased risk of hematologic malignancies (1), cardiovascular disease and inflammatory bone loss (2, 3). Chronic inflammation is increasingly recognized as a central mediator of CH-mutant hematopoietic stem and progenitor cell (HSPC) expansion underlying CH (4). DNA methyltransferase 3a ( Dnmt3a )-mutant cells produce higher levels of tumor necrosis factor-α (TNFα) and interleukin-6 (IL-6) (5), and blocking these pathways reduces the competitive advantage of Dnmt3a -mutant HSPCs (4, 6). The upstream mediators initiating inflammatory signaling in CH are unknown. Strong candidates are S100A8 and S100A9, members of the S100 calcium-binding protein family that regulate inflammatory signaling in the hematopoietic system. These proteins form a heterodimer complex and activate innate immune signaling through receptors including Toll-like receptor 4 (TLR4) and the receptor for advanced glycation end products (RAGE) (7). S100A8/A9 signaling promotes production of pro-inflammatory cytokines and inflammasome activation leading to poor prognosis in myelodysplastic syndrome and myeloproliferative neoplasms (8, 9). Across multiple myeloid malignancies, neutrophils are the primary bone marrow (BM) source of this alarmin (8, 10, 11) and pharmacologic inhibition of S100A9 with tasquinimod reduces disease severity without disrupting normal hematopoiesis (10, 12). Given the role of S100A8/A9 in establishing an inflammatory milieu, here we investigated the role of S100A8/A9 in Dnmt3a -mutant hematopoiesis. We identify neutrophils as a major source of elevated S100A8/A9 in the BM of Dnmt3a -mutant mice and this increase correlates with production of the inflammatory cytokines TNFα and IL-6. We show that tasquinimod reduces TNFα and IL-6 levels and selectively reduces the Dnmt3a -mutant HSPC compartment.

GNB1 encephalopathy (GNB1E) is a rare neurodevelopmental disorder caused by mutations in GNB1 gene encoding the G protein subunit G{beta}1. Mechanisms linking these variants to neurological dysfunction remain unclear. We investigated the prevalent p.Ile80Thr (I80T) variant using combined clinical, cellular, and in vivo approaches. Longitudinal evaluation of a GNB1E patient revealed developmental delay, progressive peripheral spasticity, and epilepsy with Spike-Wave Activation in Sleep. Heterozygous knock-in Gnb1I80T/+ mice exhibited disease-relevant phenotypes, including impaired early development, mild adult motor and cognitive deficits and epileptiform cortical spike-and-wave discharges. Transcriptomic analysis identified 323 genes concordantly dysregulated in mouse cortex and cortical human neuronal cultures from patient-derived induced pluripotent cells. This gene set was enriched for ion-channel function, epilepsy-associated genes, and Gs/adenylyl cyclase signaling pathway. Our integrated analysis establishes the first cross-species model for GNB1E, suggests common neurological mechanisms and molecular pathways linked to GNB1E, and provides a framework for mechanistic and therapeutic studies. TeaserConserved human/mouse neurological and transcriptomic signatures in GNB1 encephalopathy.

Background & Aims: Hepatocellular carcinoma (HCC) remains a leading cause of cancer mortality, with most patients presenting at advanced stages requiring systemic therapy. Despite promising outcomes with immune checkpoint inhibitors (ICI), responses remain variable due to an immunosuppressive tumor microenvironment. Y90 radioembolization offers potential immune priming, but only a subset of patients benefit. Here, we apply spatial multi-omics to delineate baseline and treatment-induced immune features and identify predictive signatures of progressive disease (PD) for early detection of patients unlikely to benefit from therapy. Approach & Results: Paired baseline (Day 0) and on-treatment (Day 35) biopsies were obtained from 33 patients, following Y90 radioembolization (Day 14) and nivolumab. Multiplex immunohistochemistry (mIHC) was used for cell-cell interaction analysis. A subset was further profiled using Visium (n=13) for tissue category-specific analysis and NanoString GeoMx DSP (n=12) for cell type-resolved transcriptomic and pathway analyses. Global spatial transcriptomics analysis revealed minimal baseline immune activity in PD, indicating an intrinsically immune-deficient TME. Despite treatment-induced activation, PD exhibited reduced CD8+ T cell abundance and limited reinvigoration of exhausted subsets, and persistent LAG-3-associated exhaustion. DSP showed downregulation of antigen presentation and T cell activation pathways. Macrophage profiling revealed enrichment of CD38+ phenotypes, contrasting CXCL9-CXCR3-associated responses in responders. Furthermore, a 72-gene PD signature was identified and validated in TCGA, associating with poorer survival. Conclusions: Integrated spatial multi-omics reveals that PD in HCC is associated with an immune-deficient TME, characterized by LAG-3-associated CD8+ exhaustion and immunosuppressive macrophages. A 72-gene signature enables early identification and supports alternative therapeutic strategies.

Amyotrophic lateral sclerosis is characterized by the progressive dismantling of the motor unit. While dying back hypothesis suggests that peripheral neuromuscular dysfunction precedes motor neuron loss, the molecular mechanisms limiting endogenous compensatory responses remain poorly understood. We longitudinally examined neuromuscular decline and GDF5-SMAD1/5/8 signaling in SOD1G93A mice. Our findings revealed a translational checkpoint linked to the lncRNA Myoparr that suppresses GDF5 production at symptom onset. To overcome this deficit, we delivered AAV9-GDF5 at the symptomatic stage. GDF5 supplementation restored SMAD signaling balance, shifting the motor unit from a pro-atrophic TGF-{beta}-SMAD2/3 toward a pro-myogenic SMAD1/5 profile. Treatment preserved muscle mass, reduced mitochondrial reactive oxygen species, and maintained neuromuscular junction integrity, including peri-synaptic glial support. GDF5 also promoted molecular recovery of spinal MNs by enhancing homeostatic marker expression. Together, these findings identify GDF5 as a multimodal stabilizer of the motor unit and highlight its potential as therapeutic target in combinatorial strategies aimed at coupling motor unit stabilization with central neuroprotective interventions.

Background: Myotonic dystrophy type 1 (DM1) is caused by a CTG repeat expansion in the DMPK gene and represents the most common adult-onset myopathy. Current molecular diagnostics rely on labor-intensive assays that limit accessibility and scalability. Haplotype-based approaches offer a promising alternative for detecting pathogenic expansions indirectly. Methods: We performed genome-wide genotyping in 226 genetically confirmed DM1 patients from the Saguenay-Lac-Saint-Jean founder population and reconstructed haplotypes surrounding the DMPK pathogenic repeat expansion. Based on these haplotypes, we performed a phylogenetic analysis that was further integrated with genealogical reconstruction from the BALSAC database to investigate the origin and transmission of DM1 haplotypes. To evaluate epidemiological utility, we implemented gene dropping simulations within the SLSJ extended genealogies (>80,000 starting individuals) to estimate DM1 incidence at birth. Results: A DM1-associated haplotype was identified in all patients (226/226), consistent with a single major ancestral origin in the SLSJ population. This complete concordance supports the robustness of haplotype-based approaches to infer carrier status without direct repeat sizing. Integrating phylogenetic analysis and genealogical data identified a single couple as the most likely entry point of DM1 in Quebec. Simulation-based estimates of incidence at birth exceeded observed prevalence, suggesting underdiagnosis in the region. Marked geographic heterogeneity in the SLSJ is also observed. Conclusions: Our results demonstrate that haplotype-based approaches can provide a reliable, cost-effective alternative to conventional pathogenic DM1 repeat carriers identification and familial screening strategies.

In TLR7-driven macrophage activation syndrome (MAS), inflammatory hemophagocytes (iHPCs) differentiate from Ly6CHI monocytes, phagocytose red blood cells and promote disease, including anemia and thrombocytopenia. We demonstrate here that IRF5 is required for iHPC differentiation and MAS in TLR7-overexpressing (TLR7.1) mice. Both constitutive and myeloid-specific Irf5 deletion reduced iHPCs and improved anemia, thrombocytopenia and survival. Furthermore, therapeutic inhibition of IRF5 ameliorated MAS features and reduced splenic and circulating iHPCs. While cell-intrinsic IRF5 expression was required for iHPC differentiation, it was not required for TLR7.1 Ly6CHI monocyte differentiation and monocyte transcriptional programs. We further show that the transcriptome and chromatin landscape changed dramatically as iHPCs differentiated from TLR7.1 Ly6CHI monocytes. Many transcriptional programs gained in iHPCs were enriched in genes associated with IRF5-binding accessible chromatin regions, including those associated with NF-kB signaling, cytokine and chemokine production, and complement activation. Our data suggest that IRF5 collaborates with other transcription factor families, including NF-kB, ETS and AP1 members, to regulate iHPC gene programs. Together, our findings demonstrate that expression of IRF5 in myeloid cells is critical for MAS, for iHPC differentiation, and acts broadly across iHPC-specific gene programs in TLR7-driven inflammation.

Primary tumor biopsy in retinoblastoma carries an unacceptable risk of extraocular dissemination. As a result, children treated with eye-sparing approaches currently lack access to tumor-derived genomic information at diagnosis, limiting accurate risk stratification, preventing subtype-guided therapy, and obscuring insight into tumor evolution during conservative treatment. Aqueous humor (AH) liquid biopsy has emerged as a promising window into circulating tumor DNA (ctDNA) from eyes managed conservatively, yet its ability to comprehensively capture the genomic and epigenomic landscape of retinoblastoma and to deliver clinically actionable molecular stratification has not been rigorously evaluated. We analyzed 18 matched AH-tumor pairs using genome-wide methylation profiling, copy-number analysis, and targeted sequencing. AH samples consistently contained high ctDNA fractions (median 0.65), enabling robust detection of single-nucleotide variants, canonical copy-number alterations, and methylation signatures defining established retinoblastoma subtypes. Importantly, promoter methylation patterns associated with RB1 inactivation and optic nerve invasion were confidently detected in AH, highlighting that liquid biopsy enables functional interrogation of disease-relevant genes and pathways. To enable biopsy-independent molecular classification, we developed a methylation-based machine learning classifier trained on combined AH and tumor datasets (n=114). The classifier demonstrated exceptional performance, with AUCs of 0.96-1.00 in cross-validation and 0.97-1.00 in independent validation across 63 additional retinoblastoma cases. Together, these findings position AH liquid biopsy as powerful, minimally invasive platform for comprehensive molecular profiling in retinoblastoma. This work establishes the first clinically viable non-invasive molecular stratification tool for the disease, enabling pretreatment risk assessment and paving the way for next-generation precision diagnostics in eye-preserving care.

Molecular characterization of human monogenic inborn errors of T cell immunity provides both biological insights and medical progress. We report rare biallelic deleterious variants in GTF3A, encoding transcription factor IIIA (TFIIIA), a zinc-finger protein required for transcription and chaperoning of 5S ribosomal RNA (rRNA). These variants were identified in ten patients from eight unrelated families and eight countries presenting with either T-B+NK+ severe combined immunodeficiency (SCID) or combined immune deficiency (CID), characterized by T cell lymphopenia and variable antibody deficiency. The GTF3A variants disrupt TFIIIA function through distinct mechanisms, including defective DNA binding, aberrant nuclear localization, and reduced protein stability compromising TFIIIA-mediated transcription and chaperoning of 5S rRNA. Using artificial thymic organoids derived from TFIIIA-deficient CD34+ progenitors, an early developmental arrest at the T cell commitment stage was documented in vitro. Zebrafish deficient for gtf3aa recapitulated the impaired thymocyte development in vivo. Together, these findings establish TFIIIA deficiency as a novel cause of (S)CID, expanding the genetic and mechanistic landscape of inborn errors of T cell immunity and uncovering an essential role for TFIIIA in human adaptive immunity.

Immune-related adverse events (irAEs), particularly colitis, are major limitations of immune checkpoint inhibitor (ICI) therapy, but their mechanisms remain poorly understood. Here we show that endogenous autoantibodies (AAbs) can promote ICI-associated colitis through Fc{gamma} receptor-dependent pathways. IgG from melanoma patients treated with pembrolizumab, nivolumab, or ipilimumab, with or without severe colitis, was transferred into wild-type or humanized Fc{gamma}R (hFc{gamma}R) mice receiving comparable ICI therapy. Wild-type mice did not develop changes in the colon. In contrast, hFc{gamma}R mice given IgG from patients with colitis developed colon inflammation marked by a significant increase in submucosal lymphocyte infiltration, goblet cell loss, and circulating cytokines, including IL-1{beta}, IL-17a, and IL-22. Single-cell RNA sequencing identified an IgG-regulated inflammatory network involving IFN{gamma}-producing ILC1, Th1 and cytotoxic T cells, IL-1{beta}+ M1 macrophages, plasma B cells/plasmablasts, and IL-22-producing ILC3-LTi cells. Patient serum autoantibody profiling further identified CCR5 and CXCR4 receptors as candidate immune-related targets associated with ICC susceptibility. Immune-related adverse events (irAEs), particularly colitis, are major limitations of immune checkpoint inhibitor (ICI) therapy, but their mechanisms remain poorly understood. Here we show that endogenous autoantibodies (AAbs) can promote ICI-associated colitis through Fc gamma receptor (FcgR)-dependent pathways. IgG from melanoma patients treated with pembrolizumab, nivolumab, or ipilimumab, with or without severe colitis, was transferred into wild-type or humanized FcgR (hFcgR) mice receiving comparable ICI therapy. Wild-type mice did not develop changes in the colon. In contrast, hFcgR mice given IgG from patients with colitis developed colon inflammation marked by a significant increase in submucosal lymphocyte infiltration, goblet cell loss, and circulating cytokines, including IL-6, IL-17, and IL-22. Single-cell RNA sequencing identified an IgG-regulated inflammatory network involving IFNg-producing ILC1, Th1 and cytotoxic T cells, IL-1betta-M1 macrophages, plasma B cells/plasmablasts, and IL-22-producing ILC3-LTi cells. Patient serum autoantibody profiling further identified CCR5 and CXCR4 receptors as candidate immune-related targets associated with ICC susceptibility.

Within the vasculature, pannexin 1 (PANX1) channels in smooth muscle cells (SMCs) regulate -adrenergic constriction and blood pressure. PANX1 channel activity is regulated by phosphorylation at Y198, S205 and Y308 residues, but the physiological significance of these modifications is unknown. Here, we utilize newly developed PANX1 Y198F, S205A and Y308F phospho-dead mutant mice to test physiological changes related to hemodynamics. Radiotelemetry-measured blood pressure was decreased in Y198F, increased in Y308F, but unchanged in S205A mice at baseline. Clonidine-sensitive sympathetic-driven hypertension was observed in all mouse lines except Y198F. Pressure myography of third-order mesenteric arteries revealed -adrenergic contractile responses were decreased in Y198F, slightly enhanced in Y308F, but unchanged in S205A, with responses in Y198F vessels mimicking controls treated with PANX1 inhibitors. To understand signaling changes driving these phenotypes, we performed mesenteric artery bulk RNA sequencing, but found a minimal number of differentially expressed genes between phospho-dead mutants and controls. Similarly, co-immunoprecipitation-mass spectrometry of wildtype or phospho-dead mutant-expressing vascular SMCs revealed few interacting proteins distinct to each PANX1 variant. However, PANX1 channel activity assessments in HEK293T cells expressing the 1D-adrenergic receptor as well as each phospho-dead mutant PANX1 showed that phenylephrine-induced ATP release from Y198F channels was significantly decreased compared to wildtype, but current was unaffected. Conversely, basal and phenylephrine-induced S205A and Y308F currents were reduced, but ATP release resembled controls. Taken together, these findings indicate that distinct PANX1 phosphorylation determines PANX1 metabolite release versus current conducting properties and in turn, regulates physiological outcomes in the vasculature. One Sentence SummaryPANX1 Y198 phosphorylation-mediated ATP release is a major driver of -adrenergic vasoconstriction in vascular smooth muscle cells.

The enteric nervous system (ENS) is a complex peripheral neural network that coordinates gastrointestinal motility through highly organized synaptic communication. Although tissue-resident muscularis macrophages (MMs) closely associate with enteric neurons, whether they regulate enteric synaptic organization remains unknown. In the central nervous system (CNS), microglia sculpt neural circuits through complement-dependent synaptic remodeling, raising the possibility that analogous neuroimmune mechanisms operate in the gut. Here, we identify a previously unrecognized role for C1qa{square} MMs in regulating enteric synaptic homeostasis and gastrointestinal motility. Using macrophage-specific constitutive and inducible C1qa deletion models, single-cell RNA sequencing, enteric synaptosome proteomics, physiology, and advanced imaging, we demonstrate that loss of MMs-derived C1qa increases enteric synaptic density without altering neuronal numbers. C1qa deficiency induced broad transcriptional changes in enteric neurons and macrophages, including altered synapse-associated, lysosomal, and endocytic programs. Proteomic analysis revealed that enteric synapses share a conserved molecular architecture with brain synapses while exhibiting distinct gastrointestinal-specific complement-associated synaptic networks enriched for structural and receptor-localization pathways. Functionally, macrophage-specific C1qa deletion altered excitatory and inhibitory enteric neurotransmission, enhanced cholinergic signaling, reduced nitrergic responses, and accelerated gastrointestinal transit, while smooth muscle responsiveness remained preserved. C1qa{square} MMs displayed transcriptional and functional features consistent with a phagocytic synapse-remodeling phenotype, including enrichment of complement, lysosomal, and engulfment pathways. Loss of C1qa impaired macrophage phagocytic activity both in vitro and in vivo and was associated with synapse accumulation and altered macrophage morphology. Importantly, inducible deletion of C1qa in adulthood recapitulated the synaptic and motility phenotypes, demonstrating that C1qa{square} MMs continuously regulate enteric synaptic organization beyond development. Together, these findings identify a complement-dependent neuroimmune mechanism that regulates enteric circuit organization and gut motility, establishing MMs as active modulators of adult ENS synaptic homeostasis. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=117 SRC="FIGDIR/small/729640v1_ufig1.gif" ALT="Figure 1"> View larger version (52K): org.highwire.dtl.DTLVardef@16aadd8org.highwire.dtl.DTLVardef@bb74f3org.highwire.dtl.DTLVardef@fb040corg.highwire.dtl.DTLVardef@1c6e4a5_HPS_FORMAT_FIGEXP M_FIG C_FIG

Sex-specific differences in cardiovascular disease outcomes remain incompletely understood at the molecular level. Here, we identified the platelet complement receptor C5aR1 as a critical mediator of sex-specific revascularization following hindlimb ischemia through an estradiol-regulated mechanism. Ischemic tissue exhibited robust complement activation with C3b and C5a accumulation that correlated strongly with deposition of the anti-angiogenic factor CXCL4 (PF4). Mechanistically, C5a stimulation of platelets triggered CXCL4 secretion, and platelet-specific deletion of C5aR1 (using PF4-Cre-C5aR1fl/fl mice) significantly improved revascularization in male mice associated with decreased CXCL4 deposition, while sex-specific differences were not observed in cre-negative animals. Male mice exhibited substantially higher platelet C5aR1 expression and enhanced C5a-induced CXCL4 secretion compared to females, resulting in greater CXCL4 accumulation in the ischemic tissue. Importantly, estradiol stimulation of megakaryocytes suppressed C5aR1 expression during pro-platelet formation, uncovering a hormone-dependent regulatory mechanism. This estradiol-C5aR1-CXCL4 axis provides a molecular explanation for sex-specific differences in ischemic revascularization known from patient studies, as sex-specific deposition of the anti-angiogenic platelet-derived factor CXCL4 was C5aR1-dependent. These findings establish a novel and unexpected mechanistic link between sex hormones, a complement-platelet crosstalk and the angiogenic response to ischemia with potential clinical implications for sex-tailored therapeutic strategies.

Immune effector cell-associated neurotoxicity syndrome (ICANS) is a major complication after CAR T cell therapy, but its underlying mechanisms remain poorly understood. We performed longitudinal immune profiling of paired whole blood and serum samples from patients with relapsed or refractory diffuse large B cell lymphoma (DLBCL) treated with CD19 CAR T cells. At peak neurotoxicity, high-dimensional mass cytometry and serum proteomics identified the expansion of CD163 monocytes and immature CD10lowCD101low neutrophils correlated with elevated serum ST2 and IL-2RA concentrations. Integrative immune module analysis identified these features among the strongest predictors of ICANS severity. Independent single-cell transcriptomic profiling validated the emergence of immunoregulatory CD163 monocytes and identified CD177 as a biomarker of ICANS-associated immature neutrophils. Together, these findings reveal a coordinated myeloid inflammatory network associated with ICANS and nominate candidate biomarkers and therapeutic targets for improving the safety of CAR T cell therapy. Significance: We demonstrate that immunoregulatory CD163+ monocytes and immature, activated CD177hiCD10lowCD101low neutrophils emerge in patients with moderate to severe ICANS at peak toxicity following CD19 CAR T cell therapy. These findings identify an uncharacterized myeloid network potentially contributing towards ICANS pathogenesis.