Clinical & Translational Immunology

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.

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.

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.

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.

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.

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.

Patients with myasthenia gravis (MG) may produce autoantibodies neutralizing type I interferons (AAN-I-IFN), which have been shown to underlie severe viral diseases, including critical COVID-19 pneumonia, in patients without MG. We studied an international cohort of 85 unvaccinated SARS-CoV-2-infected MG patients with no antiviral treatment. Hypoxemic pneumonia occurred in 48 of these patients, including 22 (45.8%) with AAN-I-IFN, which neutralized both IFN-2 and IFN-{omega} in 14 (29.2%) patients. Six (16.2%) of the remaining 37 patients had AAN-I-IFN, which neutralized both IFN-2 and IFN-{omega} in three patients. The risk of hypoxemic pneumonia was greater in MG patients with AAN-I-IFN neutralizing 10 ng/mL of both IFN-2 and IFN-{omega} (odds ratio and 95% confidence interval (OR [95% CI]): 12.7 [2.1-78.9], p=0. 0010) or IFN-2 at any dose (4.7 [1.5-15.0], p=0.0054) than in those without such autoantibodies. The risk of AAN-I-IFN production was much higher in MG patients than in the general population (28.9 [10.8-77.7], p=4.9x10-27). Fourteen patients had thymoma, which increased the risk of AAN-I-IFN (64% versus 27%, (OR [95% CI]: 5.6 [1.6-19.4], p=0.0050) and hypoxemic pneumonia (9.2 [1.9-44.2]; p=0.0019). Thymoma is, thus, associated with a higher risk of producing AAN-I-IFN, and these autoantibodies are associated with a higher risk of developing life-threatening COVID-19 pneumonia in patients with MG.

Sarcoidosis is a heterogenous disease of unknown etiology characterized by non-caseating granulomas. Disease prevalence and presentation vary significantly by ancestry and ranges from acute, self-resolving disease to severe, chronic disease. Following previous reports suggesting B cells in the development and pathogenesis of sarcoidosis, we present here results of single-cell RNA sequencing, supporting B cell involvement in sarcoidosis through altered immediate early response, rewiring of MAPK signaling, and ancestry-specific preferential expansion of B cell receptors. Peripheral blood mononuclear cells were obtained from individuals of African or European Ancestry (AA and EA, respectively) including 48 healthy controls, 59 sarcoidosis patients, and 28 systemic lupus erythematosus (SLE) patients. SLE samples were used as a disease control. Differential expression analysis highlighted many differentially expressed genes (DEGs) with almost 5x more in the AA sarcoidosis versus AA control group compared to the EA sarcoidosis versus EA control group. B cells had the most DEGs of all cell types and expression patterns were similar between ancestries, however, sarcoidosis had an opposite transcription pattern than SLE, demonstrating an alternative immune response to acute activation than that seen in a prototypical autoinflammatory disease. This trend was maintained when examining specialized B cell subsets, with the most pronounced effect in the AA sarcoidosis versus AA control comparison. Our results strongly support further investigation of the role of humoral immune response in sarcoidosis and the potential to highlight patient groups likely to benefit from existing B cell therapies.

PURPOSE: Bispecific antibodies (BsAbs) represent a major advancement in the management of relapsed/refractory multiple myeloma (RRMM), offering high response rates even in heavily pretreated patients. However, their use presents operational, safety, and supportive care complexities that require coordinated care teams, and evolving infrastructure. This manuscript summarizes best practice recommendations for adverse event (AE) management, outpatient operational models, referral pathways, and emerging strategies to optimize long-term tolerability. METHODS: Medlive, A PlatformQ Health Brand, conducted qualitative interviews of academic and community-based clinicians. Discussions focused on BsAb implementation, patient selection and counseling, and AE management. Experts provided recommendations on team-based protocols, transitions of care, and inpatient versus outpatient considerations. RESULTS: Ten hematologists/oncologists (academic n=4; community n=6) described practice patterns, barriers, and perspectives on BsAb use. BsAbs were consistently regarded as highly effective across multiple lines of therapy, particularly for patients without alternatives. Cytokine release syndrome (CRS) was the most common acute toxicity, generally low grade and managed effectively with early tocilizumab, including prophylactic use in outpatient settings. Immune effector cell-associated neurotoxicity syndrome (ICANS) was rare, mild, and best mitigated through early recognition and caregiver support. Infections, largely from BCMA-associated hypogammaglobulinemia, frequently interrupted therapy, necessitating antiviral prophylaxis, pneumocystis jirovecii pneumonia (PJP) prophylaxis, and intravenous immunoglobulin (IVIG). Outpatient step-up dosing is expanding, supported by prophylactic strategies and academic-community collaboration. Timely referral was emphasized to preserving eligibility. Major outpatient challenges included sequencing, infrastructure readiness, and standardized caregiver and staff education. CONCLUSION: Effective community implementation of BsAbs requires multidisciplinary coordination, standardized AE protocols, infection prevention, and infrastructure to support monitoring, referrals, and equitable access. These measures are critical to ensure safe, sustainable integration of bispecific therapies and to optimize patient outcomes.

Red blood cell (RBC) alloimmunization is a clinically significant complication in transfused patients whose immunological determinants remain incompletely understood. Type I interferon (IFN-I) signaling drives RBC alloimmunization in murine models, and systemic lupus erythematosus (SLE) is characterized by constitutive IFN-I hyperactivation alongside elevated alloimmunization rates. We analyzed three publicly available SLE RNA-seq cohorts (GSE72509, GSE112087, GSE122459; whole blood and PBMC; total n = 150 SLE) in a pre-specified discovery-replication-validation design. A 14-gene IFN-I signature score was computed per sample; differential expression, gene set enrichment analysis, and Spearman correlation were performed independently per cohort. IFN-I scores were significantly elevated in SLE versus healthy controls in all three cohorts (p < 0.01 each). IFN-high SLE patients showed 665 differentially expressed genes, with enrichment of alloimmunization-associated and plasmablast differentiation gene sets confirmed by GSEA. The alloimmunization signature score correlated significantly with IFN-I score across all three independent cohorts ({rho} = +0.77, +0.51, +0.60; all FDR q < 0.05); Tfh differentiation showed no association in any cohort. To our knowledge, this represents the first human transcriptomic evidence that IFN-I pathway activity in SLE is coupled to alloimmunization-associated immune programs in vivo. These findings identify IFN-I score as a candidate biomarker of alloimmunization susceptibility in SLE and provide translational rationale for prospective studies incorporating transfusion outcome data.

The intestinal microbiome influences immune recovery and long-term outcomes after allogeneic hematopoietic stem cell transplantation (allo-SCT). While reduced bacterial diversity and depletion of immunomodulatory microbial metabolites during peri-engraftment have been linked to acute graft-versus-host disease (aGvHD) and mortality, it remains unclear whether microbiome recovery after engraftment and immune reconstitution is better reflected by bacterial diversity or by microbial metabolic output. We aimed to define microbiome recovery in the late post-transplant period and test whether a metabolite-based biomarker improves the prediction of clinical outcomes, including overall survival (OS) and chronic (c) GvHD. In this two-center longitudinal observational study, serial stool samples were collected from pre-transplant baseline to day +100 after allo-SCT in a discovery cohort (n = 20, Technical University Munich University Hospital (TUM)) and an independent validation cohort (n = 100, University Hospital Regensburg (UKR)). Gut microbiome composition was assessed by 16S rRNA gene amplicon sequencing, with metagenomic profiling in selected patients, and stool metabolites were quantified using targeted mass spectrometry. Patients were classified as RECOVERY or NO RECOVERY based on changes in bacterial richness between baseline and the post-transplant period. To capture microbial metabolic output, the previously established Immune-Modulatory Metabolite Risk Index (IMM-RI), comprising butyric, propionic, and isovaleric acids, desaminotyrosine and indole-3-carboxaldehyde, was adapted to the late post-transplant period (IMM-RI post-TX). Bacterial alpha diversity frequently improved by day +100; however, this did not consistently indicate restoration of baseline community structure and was not paralleled by recovery of stool metabolite profiles. Accordingly, RECOVERY status showed a limited association with survival or transplant-related mortality (TRM). In contrast, IMM-RI post-TX low-risk identified patients with preserved butyrate-associated biosynthetic capacity and was significantly associated with improved OS in both cohorts (UKR: HR 0.2052, 95% CI 0.07703 - 0.5466, p < 0.0001). In the validation cohort, IMM-RI post-TX low-risk was significantly associated with reduced relapse-related mortality. Interestingly, stool butyric-, propionic and valeric acid concentrations were increased in cGvHD of the skin, indicating context-dependent metabolite effects. These findings suggest that metabolite profiling outperforms bacterial diversity for predicting outcomes after allo-SCT and support microbial metabolites as promising biomarkers for risk stratification and actionable candidates for precision microbiome interventions after allo-SCT.

Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) and long COVID are complex chronic conditions that often follow infectious triggers with overlapping clinical features but poorly defined pathophysiological relationships. This study aimed to identify disease-specific immune signatures through multiparameter immunophenotyping of monocytes, dendritic cells, and T-cell subsets. A total of 207 participants were included (ME/CFS: n = 103; long COVID: n = 63; healthy controls: n = 41). Peripheral blood mononuclear cells were analyzed using multiparameter flow cytometry. Statistical analyses included non-parametric testing, age-adjusted ANCOVA, correlation network analysis, and principal component analysis (PCA). Long COVID was characterized by increased M2-like monocyte polarization, elevated CD80 expression across monocyte subsets, expansion of dendritic cells, and reduced expression of activation markers, indicating persistent immune activation with features of immune exhaustion. In contrast, ME/CFS exhibited reduced costimulatory molecule expression, impaired CCR7-mediated immune cell trafficking, and less coordinated activation patterns, consistent with a state of immune suppression. Correlation network analysis revealed more extensive and integrated immune interactions in long COVID, while PCA identified distinct immunophenotypic components and enabled moderate discrimination between the two conditions. These findings demonstrate that ME/CFS and long COVID are characterized by distinct immune profiles, supporting the concept of divergent immunopathological mechanisms. The identified signatures may contribute to biomarker development and guide targeted therapeutic approaches.

African swine fever virus (ASFV) causes a lethal haemorrhagic fever in pigs and spread of this disease threatens many pig species (Suidae) globally. By contrast, ASFV infections in the natural evolved hosts, the warthog and bushpig, are subclinical. The macrophage (M{varphi}) is the primary target of ASFV and species-dependent responses in M{varphi}s are presumed to influence disease susceptibility. In an attempt to model these differences in vitro, we generated transgene-regulated induced pluripotent stem cells (iPSCs) from domestic pig, wild boar, red river hog and warthog, and confirmed that their corresponding iPSC-derived M{varphi}s (iPSCdMs) supported infection and replication of ASFV. In contrast to the other species, however, warthog iPSCdMs did not induce interferon upon infection by either virulent or attenuated ASFV. iPSCdMs may therefore represent an experimental system to understand how ASFV infection of M{varphi}s contributes to disease and aid development of strategies to combat this economically and environmentally devastating pathogen.

Casein Kinase 2 (CK2) is a constitutively active kinase regulating proliferation and immune signaling and is frequently dysregulated in cancer, including acute myeloid leukemia (AML), making it a therapeutic target. CK2 comprises two catalytic subunits, CK2 or CK2, with two regulatory {beta} subunits. The role of CK2, the predominant catalytic subunit and principal mediator of CK2 kinase activity in hematopoietic cells, in steady-state hematopoiesis remains undefined. To define how CK2 shapes hematopoietic cells, we used bone marrow and spleen tissue samples of wild type control and conditional knock out (KO) of CK2 (Csnk2a1) in the hematopoietic compartment of transgenic mice. Using single-cell RNA sequencing, we profiled the transcriptomic changes associated with CK2 loss. Although HSC abundance was comparable between the control and CK2-deficient samples, HSCs experienced the largest transcriptional response to CK2 loss among all cell types. CK2-deficient HSCs displayed transcriptional remodeling for inflammatory and immune-associated programs, interferon signaling, and antigen presentation. Expression of inflammatory genes such as S100a8 and S100a9, changed in opposite directions in bone marrow and spleen HSCs, demonstrating the transcriptional consequences of CK2 loss shaped by tissue context. Using a network-based approach, we identified immune-associated transcription factors Nfkb1, Rfx5, Hes1, and AP-1 family members as regulatory hubs driving these inflammatory transcriptional states in CK2-deficient HSCs. Cell-cell communication profiling revealed multiple gains and losses in ligand-receptor communication between the HSCs and their immune microenvironment in KO. Our findings identify CK2 as a regulator of immune transcriptional programs in HSCs and suggest that disruption of CK2 signaling influences stem cell behavior and immune activation in contexts relevant to hematologic malignancies and CK2-targeted cancer therapies. Statement of significanceThis study reveals that inhibiting the protein CK2 forces blood stem cells into a stressed, immune-primed state. These tissue-specific findings highlight potential side effects for cancer therapies targeting this essential regulatory kinase.

Cerebral malaria is a severe neurological complication of Plasmodium falciparum infection. Damage of the blood-brain barrier (BBB) and endothelial dysfunction are established drivers of the disease pathology, however, whether astrocytes, a major constituent of the BBB, influence the disease outcome remains unclear. Using the murine model of experimental cerebral malaria (ECM), we show that astrocytes decisively regulate the outcome of ECM and the deubiquitinating enzyme OTUD7B in astrocytes fosters the disease. Mice lacking astrocytic OTUD7B showed reduced brain pathology and were protected from ECM compared with wildtype littermate controls. Transcriptomic profiling of ex vivo-isolated astrocytes revealed reduced proinflammatory chemokines and cytokines in the absence of OTUD7B. Plasmodium infection-associated microvesicles triggered a pro-inflammatory response in astrocytes, which was dependent on OTUD7B. Mechanistically, OTUD7B cleaved K48-linked ubiquitin chains from TRAF3 and TRAF6 upon stimulation with microvesicles or activation of TLR3/TLR9 by plasmodial nucleic acids. The OTUD7B-dependent TRAF3 and TRAF6 stabilization led to sustained NF-{kappa}B and p38 MAP kinase signaling and CXCL10 expression. Therapeutic silencing of CNS Otud7b or Cxcl10 expression after disease onset protected mice from ECM, identifying the cerebral OTUD7B-Cxcl10 axis as an attractive therapeutic target.

Background: Prolonged SARS-CoV-2 infection in immunocompromised individuals may accelerate virus evolution within the host, raising concerns about the virus evading immunity, developing resistance, and forming novel variants of concern. However, the determinants and public health implications of within-host viral evolution in this population remain incompletely understood. Methods: We performed longitudinal analyses of SARS-CoV-2 genomes from 91 patients with COVID-19 who were classified as being severely or moderately immunocompromised. Using serial measurements of viral RNA loads and infectious titers, we modeled the shedding dynamics of the virus and stratified the infected cases by upper respiratory virus shedding duration to assess associations with within-host evolutionary dynamics. Results: Shedding modeling identified two profiles of shedding duration: intermediate and long. The long shedding profile (shedding lasting >21 days) was found in 14.8% of moderately immunocompromised cases and 72.1% of severely immunocompromised cases. Frequent single-nucleotide variants accumulated specifically in severely immunocompromised individuals with the long shedding phenotype, correlating positively with shedding duration. By contrast, mutations remained limited in moderately immunocompromised individuals with the long shedding phenotype and in severely immunocompromised individuals with the intermediate shedding phenotype. We identified mutations in the spike receptor-binding domain associated with monoclonal antibody resistance; however, we found no fitness-enhancing mutations for inter-host transmission, and antiviral drug resistance mutations were rare. Instead, mutations were introduced frequently and randomly across the entire viral genome. Conclusions: Prolonged upper respiratory virus shedding exceeding 21 days combined with severe immunocompromise is a risk factor of the accumulation of within-host SARS-CoV-2 mutations. Although no variants of concern emerged, the introduction of genome-wide random mutations suggests that the risk for novel variant generation cannot be excluded. These findings highlight the need for intensive antiviral strategies to limit shedding duration to less than 21 days in severely immunocompromised patients, and for immunological investigations to elucidate the host factors underlying residual shedding control in those who achieve clearance within this threshold.

While Vegfc-Vegfr3 signaling is the primary driver of lymphangiogenesis, the role of G protein-coupled receptors (GPCRs), the most successful class of druggable targets in the human genome, remains far less understood. A previous study has implicated Apelin signaling and its receptor Apelin receptor (Aplnr), a class A GPCR, in lymphatic development, yet the underlying cellular and molecular mechanisms remain unclear. Here, we show that Apelin signaling is indispensable for Vegfc-Vegfr3-dependent lymphatic sprouting and promotes lymphatic endothelial cell (LEC) migration without affecting LEC specification. Loss of Apelin signaling resulted in defective sprouting of ECs from the posterior cardinal vein (PCV) and subsequent failure of lymphatic vessel formation. Conversely, Apelin overexpression induces ectopic endothelial extensions from the PCV, an effect that is suppressed by reducing Vegfr3 signaling. Mechanistically, we show that Vegfc signaling through ERK activation regulates Aplnr expression. We propose that the specific upregulation of Aplnrb in LECs renders them migration-competent, establishing Apelin signaling as a critical and non-redundant regulator of lymphatic sprouting. Overall, our results reveal a tightly coordinated signaling axis between growth factor and GPCR pathways that governs lymphatic endothelial behavior.

Unbiased classification of equine dendritic cells (DC) is necessary to address various research questions such as the role of DC subsets in immune-mediated diseases of horses. We applied single-cell RNA sequencing (scRNA-seq) on DC enriched from the blood of two horses. All main DC subsets were detected by key gene expression, including conventional DC type 1 (cDC1; XCR1) and type 2 (cDC2; FCER1A, CD1E) as well as plasmacytoid DC (pDC; TCF4). In addition, we detected a small cluster of hematopoietic progenitors, as well as transitional DC (tDC; FCER1A, TCF4) and putative DC type 3 (DC3; FLT3, CD163). Our data confirms the previously reported phenotype of equine pDC (Flt3+MHC-IIlowCADM1lowCD172aint), cDC1 (Flt3+MHC-IIhighCADM1highCD172alow-int) and cDC2 (Flt3+MHC-IIhighCADM1intCD172ahigh), while also highlighting considerable CD14 expression for cDC2. Two subclusters of equine cDC2 were found to be enriched in FCER1A or CX3CR1 transcripts (cDC2.1 and cDC2.2, respectively), with suggested enhanced extravasation and T-cell stimulatory capacities of the latter. Conservation of DC subsets across species (horse, pig, human, mouse) was illustrated by enrichment analyses with subset-specific gene signatures and by cross-species data integration with publicly available scRNA-seq datasets. Our atlas of equine blood DC is a valuable resource for comparative analyses, and it forms the foundation for understanding the involvement of distinct DC subsets in infections and immune-mediated pathologies.

BackgroundLong COVID is characterised by persistent systemic inflammation and endothelial dysfunction, with increasing evidence implicating thromboinflammatory mechanisms. Platelet-monocyte aggregates (PMA) represent a sensitive marker of platelet activation and immune-vascular interactions, but their role in Long COVID remains incompletely defined. MethodsThis study quantified circulating PMA in 20 Long COVID patients and 20 healthy controls using a two-colour imaging flow cytometry assay targeting CD14 (a monocyte receptor for pathogen-associated molecular patterns, PAMPs) and CD62P (P-selectin). PMA were expressed as a percentage of total monocytes, and platelet attachment patterns were classified into single versus multiple platelet binding. Statistical analyses included Shapiro-Wilk normality testing, unpaired t-tests, Mann-Whitney U tests or two-way ANOVA as appropriate, and linear regression for correlation analysis. ResultsCirculating PMA were significantly elevated in Long COVID patients compared with controls (29.19 [20.02-37.26] vs 4.59 [2.67-7.16], p < 0.0001). Long COVID samples showed a reduced proportion of monocytes with single platelet attachment and a corresponding increase in multiple platelet binding (p < 0.0001). In controls, %PMA increased with age (p < 0.01), whereas no age association was observed in Long COVID, indicating an elevated baseline independent of age. ConclusionsLong COVID is associated with markedly increased platelet-monocyte aggregation and altered platelet attachment dynamics, consistent with sustained thromboinflammatory activity. PMA represent a sensitive cellular marker of platelet-driven immune activation and may have utility as an accessible biomarker for stratifying thromboinflammatory burden in Long COVID.