Clinical & Translational Immunology

PurposeSepsis-associated immunothrombosis significantly contributes to high mortality, yet the role of N-glycosylation in this process remains poorly understood. This study aimed to comprehensively profile the plasma N-glycosylation landscape in sepsis and elucidate how its specific reprogramming in the complement and coagulation cascades influences immunothrombotic balance and patient outcomes. MethodsWe performed in-depth 4D-DIA proteomic and N-glycomic analyses on plasma from 43 sepsis patients and 9 healthy controls. Differential expression, weighted gene co-expression network analysis (WGCNA), and protein-glycosylation correlation analyses were used to characterize molecular features. Clinical relevance was assessed via correlation and survival analyses. ResultsExtensive N-glycosylation reprogramming was observed in sepsis plasma,with marked enrichment in complement and coagulation pathways(KEGG p=7.76x10- {superscript 2}{superscript 1}).Pro-coagulant proteins(eg,vWF,fibrinogen)showed increased abundance together with enhanced site-specific glycosylation,potentially amplifying their activity.In contrast,key anticoagulant proteins(eg,SERPINC1)displayed unchanged glycosylation at critical sites despite abundance changes,which may impair function.Survival analysis revealed distinct prognostic values of glycoproteins and specific glycosylation sites.For instance,high vWF protein levels predicted mortality(HR=2.83),whereas elevated glycosylation at vWF N211 was associated with improved survival(HR=0.135),suggesting a negative regulatory role.These glycosylation markers correlated closely with disease severity and prognosis,representing potential early-warning biomarkers independent of current clinical coagulation indicators. ConclusionOur study demonstrates widespread reprogramming of the plasma proteome and N-glycome in sepsis.We propose that decoupling of protein function from abundance through N-glycosylation in the complement-coagulation network contributes to immunothrombotic imbalance.Specific N-glycosylation sites may serve as novel prognostic biomarkers,offering new perspectives for early risk stratification and glycosylation-targeted therapies in sepsis. Key PointsO_LISepsis plasma exhibits specific N-glycosylation reprogramming overwhelmingly focused on the complement and coagulation cascade. C_LIO_LIA dominant "glycosylation-dominated co-upregulation" mode in procoagulant factors, coupled with a "silent" glycosylation state in key anticoagulants, drives prothrombotic imbalance. C_LIO_LISite-specific N-glycosylation levels provide prognostic information distinct from, and often superior to, their carrier protein abundance, offering novel early-risk biomarkers. C_LI

Lymphatic vessels are formed during embryonic and postnatal development to facilitate interstitial fluid clearance and immune regulation after birth. Their organ-specific heterogeneity in organisation and function is preceded by heterogenous origins of lymphatic endothelial cells (LECs), the main building blocks of lymphatic vessels. In the dermis, a subset of LECs was reported to arise from blood capillaries, which themselves differentiate, in part, from paraxial mesoderm. However, it is not known whether additional cell lineages contribute to the dermal LEC population. Here, we have combined transcriptomic analyses with genetic lineage tracing and wholemount immunostaining to show that 60% of LECs in the embryonic day (E) 13.5 and E15.5 dermis are derived from a cell lineage that expresses Csf1r, a marker of myeloid cells and their progeny. Csf1r lineage LECs persist in adult dermal lymphatic vasculature and are indispensable for normal lymphatic development, because Prox1 deletion within the Csf1r lineage causes dermal oedema and blood-filled lymphatic vessels. As Csf1r lineage dermal LECs do not themselves express Csf1r and also do not arise from Csf1r-expressing differentiated myeloid cells, our findings imply the existence of a Csf1r-expressing non-LEC precursor population for the majority of dermal LECs and will prompt further work to identify this cell population.

Hematopoietic progenitors downstream of hematopoietic stem cells (HSC) are now recognized as the main drivers of day-to-day hematopoiesis. While embryonic and adult HSC fates have been studied in detail, less information exists on stages downstream from HSC, notably in the multipotent progenitor compartment. The early postnatal period represents an important growth phase of the animal and its immune system. Developing immune lineages must be generated in large numbers rapidly, and populate expanding organ niches. To shed light on this critical period, we focused our experiments on early postnatal Flt3+ hematopoietic progenitors, and combined genetic single progenitor barcoding using Polylox with Flt3-driven, inducible fate mapping. Key immune cell types, including T and B lymphocytes (lymphocytes), innate lymphocytes (ILC) 1-3, NK cells, and granulocytes and monocytes (myeloid) emerged from Flt3+ hematopoietic progenitors. Barcode analysis revealed that about 75% of Flt3+ hematopoietic progenitors had unipotent fates for lymphocytes, or ILC or myeloid cells, while the remaining fraction showed unprecedented fate combinations for these lineages. Focusing on ILC only, we uncovered clonal fate restriction towards ILC1, or ILC2, or ILC3 in tissues. These data indicate early tissue seeding by progenitors, and further differentiation towards discrete subsets in situ. In addition to these fate analyses, induction of fluorescent marker at this intermediate stage of hematopoiesis showed that Flt3+ progenitors generated a wave of progeny lasting for over one year. The washout of these cells over time provided kinetic data of cell turnover in major immune cell compartments (in the circulation and in tissues) in vivo. In conclusion, we tracked the fate of large numbers (in the order of hundreds) of Flt3+ progenitor clones in situ. These intermediate progenitors downstream of HSC displayed mostly lineage-restricted fates as well as strong fate complexity, thus serving as a source for early tissue seeding and durable immune lineage.

BackgroundIn lung transplantation, de novo immunodominant donor-specific anti-HLA antibodies recognizing HLA-DQ antigens (dn-iDSA-DQ) are predominant and can induce chronic lung allograft dysfunction (CLAD). We previously developed a method to measure the active concentration of dn-iDSA-DQ. We aimed to determine whether this new quantitative biomarker is associated with transplantation outcomes. MethodsThis retrospective multicentre cohort study included 90 lung transplant recipients (LTRs) developing dn-iDSA-DQ, evidenced through single antigen flow beads (SAFB) follow-up. We measured the active concentration of dn-iDSA-DQ at the time of their first detection (T0) for all LTRs, and within the 2 years after DSA detection, whenever possible. SAFB dn-iDSA-DQ characteristics and clinical data were retrieved up to 5 years after DSA detection. ResultsWe tested 184 sera with SPR (n=90 at T0, n=94 within the 2 years after DSA detection), among which 63 (34.4%) had a quantifiable concentration of the dn-iDSA-DQ ([&ge;]0.3 nM). The median SAFB mean fluorescence intensity (MFI) of the dn-iDSA-DQ with a concentration [&ge;]0.3 nM was higher (p<0.0001), yet the correlation between SAFB MFI and active concentration was low (r=0.758, p<0.0001). In multivariate analysis, a concentration of the dn-iDSA-DQ [&ge;]0.3 nM at T0 was independently associated with a lower 2-year CLAD-free survival (HR 2.06, p=0.02). A concentration of the dn-iDSA-DQ [&ge;]0.3 nM within the 2 years from DSA detection was associated with a lower graft survival in univariate analysis. ConclusionsActive concentration of dn-iDSA-DQ appears as a valuable biomarker to identify pathogenic DSA at their first detection because of its association with CLAD.

Abstract/SummaryImmune checkpoint blockade can produce long-lasting responses in patients with metastatic melanoma; notably, combined CTLA-4/PD-1 blockade has been associated with approximately 52% melanoma specific 10-year survival (1). Yet, nearly half of patients experience minimal clinical benefit, and intensified regimens come with substantial risk of severe immune-related toxicity. The precise determinants of immunotherapy response are incompletely defined, reflecting a complex interplay between tumor biology and host immunity. This is especially consequential for patients whose disease progresses on checkpoint blockade, for whom effective salvage options are limited. In a series of patients with NRAS-mutated melanoma refractory to checkpoint inhibitors, we found that intratumoral administration of talimogene laherparepvec (T-VEC) combined with MEK inhibitor binimetinib induced exceptional clinical responses by amplification of pre-existing T cell responses and induction of de novo tumor-reactive immunity.

ObjectivesCD4+ T cells play key roles in regulating immune responses during pregnancy, therefore we aimed to understand the CD4+ T cell surface proteome and transcriptome during pregnancy. MethodsCD4+ T cells were analysed in blood and decidua from term-pregnancies (>37 weeks), and non-pregnant blood. >350 surface proteins were screened via flow cytometry, and transcriptomes were analysed using single-cell RNA sequencing with >130 CITE-seq barcoded antibodies. ResultsSurface protein screening identified changes to ILT4/CD85d, CD9, IFN-{gamma} receptor {beta}-chain, CX3CR1 and CCR5 in the pregnant blood and decidual CD4+ T cells. CX3CR1 and CCR5 had the highest expression on the effector-memory T cell (TEM) subset in the blood, with expression consistent across subsets in decidua. CD126/IL-6R was lower in pregnant blood and decidual CD4+ T cells, while scRNAseq identified enrichment in the IL-6R signalling pathway in naive CD4+ T cells in pregnant blood. Both sIL-6R and IL-6 concentrations were increased in plasma during pregnancy, suggesting perturbations to the IL-6/IL-6R signalling axis. Meanwhile, decidual CD4+ T cells had increased expression of transcription factor RUNX3 in the CD69+ tissue-resident-like subset. ConclusionsOur findings demonstrate altered molecular expression in CD4+ T cells during pregnancy. This provides important mechanistic insight of their adaptation and regulation during placental development, which may drive placental dysfunction or pregnancy complications including preeclampsia, fetal growth restriction and stillbirth. These new data may inform future studies that focus on determining the significance of differentially- expressed immune features in pregnancy to identify potential targets for immune modulation to treat pregnancy complications and infections.

Rationale: Sepsis is a life-threatening syndrome causing significant morbidity and mortality especially in the aging population. Clonal hematopoiesis of indeterminate potential (CHIP) is an age-related condition of clonal expansion of hematopoietic stem cells harboring somatic mutations associated with increased incidence of chronic illness and all-cause mortality. Objective: Evaluate the association of pre-illness CHIP with mortality and morbidity in patients admitted to the ICU with sepsis. Methods: We performed a retrospective study using a de-identified electronic health record linked with a DNA biorepository. We identified adult patients with sepsis who had DNA collected prior to ICU admission. We tested the association between CHIP status, determined from whole-genome sequencing, and ICU mortality, organ support-free days, and long-term survival adjusting for age, sex, race and Sequential Organ Failure Assessment (SOFA) score on ICU admission. Measurements and Main Results: Pre-illness CHIP was associated with increased sepsis mortality (OR = 1.54, 95% CI 1.13 to 2.07, P = 0.005) and fewer days alive and free of organ support (-1.7 days, 95% CI -3.2 to -0.2, P = 0.028) after adjusting for age, sex, race, and SOFA score. In sepsis survivors, CHIP was also associated with increased long-term mortality after discharge (HR 1.40, 95% CI 1.01 to 1.93, P = 0.041). Conclusions: Pre-illness CHIP was independently associated with increased mortality and morbidity in critically-ill adults with sepsis. These findings suggest that CHIP is a risk factor for sepsis severity. Elucidating the mechanism underlying this association could uncover new therapeutic interventions for sepsis.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a glycoprotein cytokine with therapeutic potential in cancer and neutropenia treatment. While glycosylation of GM-CSF reduces immunogenicity and enhances serum bioavailability, it can also diminish receptor binding and bioactivity. Based on transcriptomic analysis of human T lymphocytes reported previously, GM-CSF-producing cells exhibit elevated expression of Alpha-1,6-Mannosylglycoprotein 6-Beta-N-Acetylglucosaminyltransferase (MGAT5), which encodes N-acetylglucosaminyltransferase V, an enzyme involved in N-glycan branching. Given this role of MGAT5 in glycosylation, we produced GM-CSF variants using glycoengineered Chinese hamster ovary cells to generate diverse glycoforms and assessed their bioactivity. Testing their activity on TF-1 cell proliferation, we found that decreases in GM-CSF N-glycan branching significantly suppressed its activity. These findings underscore the importance of glycosylation in modulating the efficacy and safety of GM-CSF-based therapeutics, suggesting that precise glycoengineering may be key to optimizing GM-CSF performance in clinical applications.

ObjectiveTo define cellular and molecular mechanisms distinguishing active systemic lupus erythematosus (SLE) from remission by profiling autoreactive antinuclear antigen- positive (ANA+) and non-autoreactive B cells subsets in three cohorts: active disease (SLE-A), long-term, drug free remission (SLE-R), and healthy controls (HC). MethodsPeripheral blood B cells were phenotyped by flow cytometry, including ANA reactivity. Single-cell RNA sequencing (scRNA-seq) was performed on sorted ANA+ and ANA- subsets. ResultsSeven transcriptomic B cell clusters were resolved: quiescent (Naive 1, Marginal Zone B cells [MZB], IgG Memory 1) and activated (Age-Associated B cells [ABCs], Naive 2, IgM Memory, IgG Memory 2). SLE-A showed expansion of activated clusters, MZB contraction, and a higher IgG:IgM B cell ratio. SLE-R exhibited an "immunological reset," distinct from healthy homeostasis, with reduced ABCs and IgG Memory 2, persistence of Naive 2, and partial restoration of MZB and Naive 1. Interferon- (IFNa) signaling was elevated across clusters in SLE-A (SLE-A > SLE-R > HC), whereas TNF signaling was enriched in activated clusters across cohorts, with minimal differences between SLE-R and SLE-A. IFNa and TNF scores were inversely correlated. B cells predominantly expressed TNFR2, suggesting immunomodulatory TNF effects in remission. ANA+ cells in HC and SLE-R showed enriched Fc{gamma}RIIb inhibitory and IL-4/STAT6 signaling, suggesting reinstated regulatory control. DiscussionCompared to SLE-A, SLE-R was characterized by partial reversion to HC homeostasis with residual activation. These findings delineate immunologic features of remission and suggest therapeutic opportunities, including modulation of TNFR2, Fc{gamma}RIIb, and IL-4 to help sustain remission. What is already known on this topicSome patients with SLE achieve complete clinical remission without treatment, referred as immune reset; the mechanisms that underlie this state have not been well characterized. Healthy individuals and patients with Systemic Lupus Erythematosus (SLE) normally harbor similar frequencies of autoreactive B cells; the checkpoints that regulate activation of these cells are not fully defined. What this study addsB cells, stratified by their reactivity to nuclear antigens (ANA), from active SLE (SLE-A), drug-free remission (SLE-R), and healthy controls (HC) were analyzed using single cell sequencing and flow cytometry. We identified B cells states associated with disease activity; SLE-R displayed a distinct profile that differed from SLE-A and HC. TNF signaling was present in activated B cell subsets in SLE-A and SLE-R. This persistence in SLE-R may reflect an immunomodulatory function of TNF on TNFR2, which is expressed on B cells. ANA+ cells in SLE-R and HC were enriched for inhibitory Fc{gamma}RIIb and IL-4/STAT6 programs. How this study might affect research, practice or policyThe signatures identified help define the "immunological reset" state in patients with SLE-R. We also identified pathways, such as type I IFN, TNFR2, Fc{gamma}RIIb, IL-4/STAT6 as potential targets for maintaining remission.

BackgroundCutaneous melanoma (CM) is an aggressive skin cancer with rising incidence, representing a growing public health concern. Despite the remarkable success of immune-checkpoint inhibitors (ICIs) in the management of advanced disease, mortality remains high due to therapy resistance. Identifying reliable prognostic and predictive biomarkers is therefore essential to improve patient stratification, optimize treatment selection, and minimize unnecessary toxicity. MethodsWe comprehensively profiled the circulating immune landscape of 54 treatment-naive CM patients by integrating flow cytometry immunophenotyping with clinicopathological data, and performed tumor gene expression analysis in a subset of 26 patients. ResultsElevated HLA-DR and CD69 expression on circulating CD4+ T cells, together with reduced circulating CD8+ T cell frequency, emerged as candidate prognostic biomarkers associated with improved survival. Prognostic models combining these immune variables with clinical covariates accurately stratified patients by overall survival (89.5% sensitivity, 72.7% specificity; AUC = 0.872, p < 0.0001) and progression/recurrence risk (75% sensitivity and 71.4% specificity; AUC = 0.763, p = 0.001). In a subset of 43 patients subsequently treated with ICIs, elevated baseline HLA-DR and CD69 expression on circulating CD4+ T cells was also associated with therapeutic benefit. A predictive model integrating these markers with clinical covariates achieved good discriminatory performance (65.2% sensitivity, 88.9% specificity; AUC = 0.775, p = 0.0027). Tumor gene expression profiling supported the role of IFN-{gamma}-related signatures, previously linked to ICI response, as complementary prognostic and predictive tools. ConclusionThese findings highlight systemic CD4+ T cell activation status as a promising, easily measurable biomarker in CM, laying the foundation for future strategies to refine patient stratification and guiding immunotherapy decisions.

There is growing evidence that neonates harbor innate-like CD8a+ T cell subsets that contribute to both protection and hyper-inflammatory states. It remains unclear, however, where these innate-like features are found among the many conventional and unconventional T cell populations that can upregulate the CD8 receptor. Further delineation of these unique populations and functions, with a focus on CD8ab co-expression, will enable studies that seek to understand the unique immune features in conventional T cell populations that are present during fetal and early postnatal life. We used cord blood from infants across the full viable gestational age range to examine phenotypic and transcriptional heterogeneity, with a particular focus on the naive T cell pool. We report a set of fetally-derived and innate-like naive CD8{beta}+ T cells ( FITs) that are marked by their KLRG1+CD161+ phenotype, unique transcriptomic features and which are sparsely detected in adult peripheral blood. Additionally, using T cell receptor repertoire profiling, we can distinguish FITs from well-described and semi-invariant unconventional T cell populations such as mucosa-associated invariant T cells. Our delineation of FITs unique features will enable future investigation into their ontogeny and tissue distribution, and ultimately their role in immune-related outcomes in preterm infants.

Children with acute lymphoblastic leukemia (ALL) are treated with multiagent chemotherapy that causes profound changes to the immune system. There are limited data on how disease and therapy impact antigen-specific immune memory, leading to inconsistent guidelines on best practices for revaccination of this population. Here, to inform vaccine guidance, we investigated whether immunity derived from routine childhood measles and varicella zoster virus (VZV) vaccines is maintained during and after therapy for childhood ALL. We report that antibodies against measles and VZV were significantly reduced in children with ALL (n=45) compared to healthy controls (n=13), particularly in older children in whom a longer time had passed since their most recent vaccine dose. However, the avidity of the measles and VZV-specific antibodies was indistinguishable between groups. Despite changes to the composition of the T cell compartment, both overall and antigen-specific T cell function were preserved in children with ALL. These data provide compelling evidence for revaccination of children following ALL treatment. Intact T cell responses suggest that post-treatment revaccination would be effective.

BackgroundIron metabolism disorder is highly prevalent before and after heart transplantation (HTx). The impact of pretransplant and posttransplant iron disorder on posttransplant outcomes is unclear. ObjectivePretransplant serum levels of key regulator proteins of iron metabolism (hepcidin, interleukin-6, erythroferrone) were tested for prediction of the composite outcome 1-year posttransplant all-cause mortality (ACM) or [&ge;]moderate acute cellular rejection (ACR). Furthermore, serum levels of these proteins were measured at 1-year posttransplant to explore their posttransplant course and association with ACR. ResultsIn a multicenter cohort including 276 consecutive HTx recipients, patients with or without outcome (n=118/158, respectively) did not differ for pretransplant demographics, mismatch of donor/recipient sex, mismatch of HLA epitopes, and hepcidin or interleukin-6 levels. However, pretransplant erythroferrone levels were higher (1.40 vs. 1.19 ng/mL; p=0.013) and hemoglobin levels were lower (124.5 vs. 127 g/L; p=0.004) among patients with the composite outcome. Pretransplant erythroferrone levels >2.25 ng/ml (4th-quartile) were significantly associated with the composite outcome in multivariable analysis (OR 2.17; 95% CI 1.19-3.94, p=0.011; reference: 1st-3rd quartiles). In adjusted predicted proportions analysis, the incidence of the composite outcome was higher in 4th-quartile patients when compared to 1-3rd -quartiles patients (58.0 vs. 37.7%; p=0.003). At 1-year posttransplant, 80.4% of patients with pretransplant erythroferrone levels >2.25 ng/ml remained high; 88.4% of patients with pretransplant erythroferrone levels [&le;]2.25 ng/ml had high levels posttransplant. In 1-year survivors with high erythroferrone levels and [&ge;]moderate ACR during the first postoperative year, the ratio of the opponent regulators of hepcidin gene expression, erythroferrone to interleukin-6, was higher when compared to those without ACR (1.18 vs. 0.41; p=0.016). Hepcidin levels were not different between these two subgroups indicating disproportionate ERFE increase. ConclusionHigh pretransplant erythroferrone levels predict the composite posttransplant outcome 1-year ACM and ACR. Disproportionately high posttransplant erythroferrone levels are related with [&ge;]moderate acute cellular rejection.

TRAF1 is a pro-survival signaling adaptor that contributes to NF-{kappa}B activation downstream of a subset of TNFR superfamily members. TRAF1 is overexpressed in many cancers of mature B cells, including chronic lymphocytic leukemia (CLL). Previous studies have established that TRAF1 S146 is a target of phosphorylation by the kinase PKN1 and that PKN1 is required to prevent cellular inhibitor of apoptosis protein (cIAP)-dependent degradation of TRAF1 in the CD40 signaling complex. The kinase inhibitor OSST167 inhibits PKN1 in the nm range and its addition to primary CLL cells was shown to induce dose-dependent loss of TRAF1 and concomitant increases in activated caspase 3 and cell death. These studies identified PKN1 as a target for therapy of CLL. To identify more potent and specific PKN1 inhibitors for therapy of B cell cancers it is important to measure a direct target of PKN1, such as phospho-TRAF1. To this end, here we use overexpression of an S146A mutant of human TRAF1 in 293 cells to validate a recently generated phospho-TRAF1 S146-specific antibody and to confirm that this phosphorylation is lost upon treatment with OTSSP167. Using Cas/Crispr knockout in RAJI cells we also show that both PKN1 and the closely related family member PKN2 can phosphorylate TRAF1 S146. We further show that TRAF1 S146 is constitutively phosphorylated in primary human CLL cells, including those with p53 mutations and that this phosphorylation is sensitive to inhibition with OTSSP167. These findings provide support the development of more potent PKN1/2 inhibitors for CLL.

Neuropsychiatric systemic lupus erythematosus (NPSLE) is a potentially severe complication of systemic lupus erythematosus (SLE), yet its pathogenesis remains largely elusive. By jointly probing the immune dynamics of subjects cerebrospinal fluid (CSF) and peripheral blood, we showed that both innate and adaptive immune responses jointly contribute to the pathogenesis of NPSLE. In particular, we found the remarkable enrichment of BAM-CCL3, a subtype of border-associated macrophages with strong recruitment capacity, implicating its potential role in central nervous system (CNS) inflammation. We also observed pronounced activation of memory B cells and CD4+ regulatory T cells in NPSLE CSF, along with the preferential blood-to-CSF migration and subsequent within-CSF clonal expansion of CD8+ effector memory T cells in NPSLE patients, suggesting a persistent CNS-localized adaptive immune dysregulation. Finally, we developed the single-cell CNS disease CSF-Blood Atlas (scCDCB), a comprehensive collection for CSF and peripheral blood of multiple CNS diseases, which is publicly available at (https://sccdcb.gao-lab.org) to serve as a reference for future research on CNS diseases.

Premature birth and cesarean section are major perinatal factors influencing immune development and are associated with increased morbidity and inflammatory diseases. However, their impact on neonatal immunity remains incompletely defined. To determine how gestational age and mode of delivery shape early immune programming, we analyzed CD4 T cells, central regulators of adaptive responses, from preterm neonates and full-term neonates born by cesarean section or natural birth. We performed transcriptomic profiling (mRNA-seq) and functional assessment of T cell activation, proliferation, and cytokine production following stimulation. The mode of delivery exerted a dominant effect on the CD4 T cell transcriptome and function. CD4 T cells from full-term neonates delivered by natural birth exhibited an immune activation signature, produced higher levels of multiple cytokines, but showed reduced proliferative capacity. In contrast, prematurity induced modest changes in basal gene expression relative to full- term cesarean section neonates. CD4+ T cells from preterm neonates displayed enhanced proliferation and increased secretion of inflammatory cytokines IL-13, TNF, IL-6, and IL- 17F upon stimulation, indicating heightened responsiveness. Collectively, our findings show that CD4 T cells from preterm neonates exhibit augmented inflammatory capacity, which becomes more regulated at term. The mode of delivery further refines this developmental trajectory: cesarean section is associated with a restrained functional profile, whereas natural birth is associated with an immune activation signature and increased responsiveness. These results provide evidence that neonatal CD4 T cell trajectories are established during fetal life and subsequently modulated at birth, underscoring the layered influence of perinatal factors on immune development.

Chimeric antigen receptor T-cell (CAR-T) therapy targeting CD19 has transformed outcomes for children with relapsed or refractory (R/R) B-cell acute lymphoblastic leukemia (B-ALL), yet the influence of molecular subtype on outcomes remains unclear. We evaluated the impact of cytogenetic and molecular signatures on complete response (CR), overall survival (OS), and leukemia-free survival (LFS) after CD19 CAR-T therapy in eighty-six pediatric patients with R/R B-ALL treated with tisagenlecleucel. CR was assessed 30 days after infusion. Cytogenetic data were available for 84 patients and molecular profiling for 62. Survival analyses included 72 patients who received CD19 CAR-T as the sole cellular therapy. Seventy-seven patients achieved CR (89.5%). Pre-infusion bone marrow blasts of [&ge;]20% were associated with lower CR rates (53.8% vs 95.9%, p<0.0001) and significantly reduced OS and LFS (both p<0.0001). Among molecular markers, RAS mutations correlated with inferior OS (p=0.0222) and LFS (0.0402). In multivariate analysis, bone marrow blasts >20% and RAS mutations independently predicted inferior OS. Post CAR-T, CD19 negative relapses showed almost twice higher prevalence of RAS mutations (66% vs 37.5%). These findings highlight RAS mutations as a key molecular predictor of outcome after CD19 CAR-T therapy and suggest emergence of unique risk stratification for patients receiving CD19-targeting therapy.

ABSTRACT/SUMMARYVascular remodeling within the developing fetus and placenta is essential for supporting the growth and function of emerging tissues and organs. Pericytes (PCs) play a central role in stabilizing and maturing microvascular networks by extending along endothelial cells (ECs) and reinforcing vessel integrity. In the placenta, as in other organs, PC-EC communication is mediated in part by platelet-derived growth factor-BB (PDGF-BB) signaling, which governs PC differentiation, proliferation, migration, and survival, ultimately enabling their recruitment and retention along capillaries. In this study, we identified progressive PC investment along feto-placental capillaries in both murine and human tissues across gestation, supported by morphological and molecular evidence. Placental PCs displayed phenotypic heterogeneity comparable to that observed in the brain and heart, suggesting conserved diversity across organ systems. In addition to characterizing PC dynamics, we examined the expression of recently identified soluble PDGF Receptor-{beta} (sPDGFR{beta}) isoforms. These variants were detected at the protein and transcript levels in mouse and human placentas, as well as in a murine trophoblast-embryonic stem cell (TESC) differentiation model that recapitulates aspects of early placental vascular development. Within this model, sPDGFR{beta} expression was independent of ADAM10 activity and exogenous growth factors during early vessel formation but was markedly upregulated during hypoxia. To assess how elevated sPDGFR{beta} might influence PDGF-BB signaling, we exposed TESCl-derived vascular networks to excess PDGF-BB with or without a sPDGFR{beta} mimetic. PDGF-BB alone reduced full-length PDGFR{beta} levels while increasing receptor phosphorylation, consistent with known ligand-induced regulatory mechanisms. Inclusion of the sPDGFR{beta} mimetic shifted these responses toward baseline, suggesting a potential modulatory or feedback role for soluble receptor variants. Together, these findings demonstrate that PCs are progressively recruited to placental capillaries and exhibit diverse phenotypes during development, and that soluble PDGFR{beta} isoforms may modulate PDGF-BB signaling in a manner sensitive to oxygen tension. Understanding these mechanisms provides insight into the regulation of placental vascular maturation and may inform strategies to improve human health by targeting disorders rooted in impaired placental development.

Dengue virus (DENV) is a major burden to global public health, affecting hundreds of millions annually. Children represent the major proportion of global dengue cases, ranging from asymptomatic or subclinical presentation to dengue fever (DF) and severe dengue hemorrhagic fever or shock syndrome (DHF/DSS). The factors that distinguish this range of disease severity are still poorly understood. To identify biomarkers of severity, we analyzed the plasma proteome of acute DENV infected children including both subclinical and hospitalized cases. Proteins associated with the acute-phase response, innate immune and lysosomal activation, and components of the coagulation cascade showed marked differences between hospitalized and subclinical cases during early infection. Longitudinal profiling demonstrated that endothelial dysfunction emerges early, with PTX3 showing the strongest and most rapid upregulation in hospitalized patients, supporting its potential role as a marker of imminent vascular involvement. When comparing severe (DHF/DSS) and classical DF hospitalized cases, CLEC11A displayed the highest fold change at hospital admittance. We used machine-learning analysis to predict disease severity at the acute phase of infection, distinguishing subclinical from hospitalized cases and patients that develop classical dengue fever or severe disease based on the identified complement regulators and inflammatory markers. The panel of identified plasma proteins shed light on the mechanisms of dengue related disease progression and may provide a handle to predict disease severity based on blood markers present during the acute phase of infection.

BackgroundPreterm birth is one of the most significant etiologies for neonatal morbidity and mortality. Preterm delivery is classified as iatrogenic preterm delivery and spontaneous preterm delivery. The role of placental pathology is studied. Materials and methodsWe have previously collected placental pathology data with maternal pregnancy and neonatal birth data, and we investigated the role of placental pathology in preterm delivery. Preterm delivery was categorized as late preterm (34-36 weeks), moderate preterm (32 to 33 weeks), and extreme preterm (less than 32 weeks). Neonatal, maternal, placental gross and histologic features, and laboratory parameters were compared across groups using chi-square tests for categorical variables and Kruskal-Wallis tests for continuous variables using various programs in R-package. ResultsTotally 3723 singleton placentas including 3307 term (88.8%) and 416 preterm placentas (11.2%) were examined with maternal pregnancy data and neonatal birth data. There were 614 placentas from patients with preeclampsia/pregnancy induced hypertension (PRE/PIH) (16.5%). Preterm delivery showed significantly lower fetal birth weight, placental weight, and fetal-placental ratio (all p<0.01). Maternal Black race was more prevalent in preterm groups (up to 50.8% in extreme preterm vs. 33.2% in term, p<0.01). Preterm delivery was statistically associated with PRE/PIH and maternal vascular malperfusion (MVM), maternal and fetal inflammatory response (MIR and FIR), and increased pre-delivery white blood count (WBC). Extreme preterm deliveries were markedly associated with intrauterine fetal death (27.5%, p<0.01) and MIR/FIR (56.7%, p<0.01). After excluding PRE/PIH patients, preterm delivery was statistically associated with MIR/FIR and increased WBC. ConclusionsDistinct clinicopathologic profiles exist across preterm subcategories, with MVM predominating in late/moderate preterm and severe pathologic features (including fetal demise and acute inflammation) in extreme preterm. These findings highlight heterogeneous etiologies of preterm delivery.