Browse Articles

Dietary lipids are delivered to Tregs in chylomicrons and determine intestinal Treg survival 2310329

Abstract Introduction Regulatory T cells (Tregs) limit intestinal mucosal damage in inflammatory bowel disease (IBD), yet no clinical therapies currently exist to enhance Treg numbers or activity. Foundational studies have shown that fatty acids promote Treg differentiation, proliferation, and suppressive activity. Dietary fat, the largest source of fatty acids, is packaged exclusively into chylomicrons, which are secreted directly into the mesenteric lymph. We hypothesized that chylomicrons regulate intestinal Treg accumulation or function. Methods We cannulated mesenteric lymph from mice, from which we isolated chylomicrons for Treg culture. Analyses of Treg metabolism include radiolabeled tracers, quantitative beta-oxidation assays, and seahorse metabolic flux assays. The mouse models we used are: huAPOC3 transgenic mouse models, which overexpresses huAPOC3 under the human promoter; low density lipoprotein receptor (LDLr) knockout mice; APOC3 knockout mice; and littermate and/or appropriate C57BL6 WT controls. Colitis was modeled with both dextran sodium sulfate (DSS) and T cell transfer colitis in Rag-1 knockouts. All lymphocyte population counting and cytokine staining were conducted via flow cytometry. Results We found that Tregs are capable of endocytosing chylomicron lipids and that chylomicron-derived lipids impair Treg survival. Conversely, reducing chylomicron lipid uptake in vivo–via APOC3 overexpression or Treg-specific LDL receptor knockout–enhance Treg accumulation in the colon and protect mice from colitis. Mechanistically, we discovered that chylomicrons instigate Treg apoptosis, which can be reversed by blocking lipid uptake through excess APOC3. In vivo, these mechanisms can be exploited to protect mice from multiple models of colitis. Conclusion Our study shows that chylomicrons and their lipids are an underappreciated regulator of Tregs in the gut and suggests that postprandial chylomicron metabolism may be a novel target for regulating Treg accumulation in the intestine. Funding Source Rainin Foundation (Synergy Award) and the National Institutes of Health (R01DK118239, R03DK116011 ) Topic Categories Immune Response Regulation: Cellular Mechanisms (IRC)

Resident cDC2 subset drive CD4+ T-cell help-independent CD8+ T cell cross-priming for type 17-mediated fungal vaccine immunity 2255958

Abstract Introduction The global rise in invasive fungal infections, mainly in immunocompromised populations, underscores the urgent need for effective fungal vaccines. Accumulating evidence suggests that T cells are instrumental in providing fungal immunity. Dendritic cells (DCs) are essential sentinels that bridge innate with adaptive immunity, orchestrating T-cell activation and dictating vaccine immunity. Since CD4+ T cell help is known to license DC for CD8+ T cell cross-priming and our publications showing its dispensability for inducing sterilizing antifungal vaccine immunity, the nature of DC activation in CD4+ lymphopenia is unclear. Methods Using a murine model of fungal vaccination, we delineated the dynamics, kinetics, transcriptional profile, and cross-presentation of DC subsets in draining lymph nodes of immunocompromised hosts using flow cytometry and bulk RNA-Sequencing. Results Vaccination with experimental attenuated fungal strains elicited dominant cDC2 responses over cDC1 in draining lymph nodes, independent of CD4+ T cells. Transcriptomic profiling of sorted DC subsets revealed that cDC2 possessed an enriched inflammatory and chemokine-signaling signature, whereas cDC1 exhibited programs associated with growth and interleukin signaling. Notably, resident cDC2 expressed a regulatory costimulatory profile, whereas migratory cDC2 exhibited a pro-inflammatory gene signature. Functionally, resident cDC2 showed superior antigen processing, cross-presentation, and type 17-polarizing cytokine gene expression profile compared with migratory cDC2 or cDC1. Conclusion Collectively, these findings identify cDC2 as the principal cross-presenting subset driving antifungal vaccine immunity through type 17-biased CD8+ T cell programming. By defining the molecular and functional landscape of DC subset specialization, our study provides mechanistic insight for the rational, next-generation adjuvanted fungal vaccines optimized for immunocompromised hosts. Funding Source NIH-NIAID R01AI153522 (to SGN) Topic Categories Microbial, Parasitic, and Fungal Immunology (MPF)

Selective Effects of Fenbendazole on Normal Human Dermal Fibroblasts 2310154

Abstract Introduction Fenbendazole, a benzimidazole anthelmintic, has recently gained attention for its reported anticancer activity. This study aimed to evaluate the selectivity of fenbendazole by examining its effects on normal human dermal fibroblasts (HDFa), building on prior cancer cell line findings and emphasizing normal-cell response as a critical component of therapeutic relevance. Methods HDFa fibroblasts were cultured under standard mammalian cell culture conditions and maintained through routine passaging and cell counting. Fenbendazole exposure was conducted within available laboratory constraints, and cell viability trends were assessed using Trypan Blue exclusion assays and serial cell counts over time. Results HDFa fibroblasts were successfully maintained and consistently quantified throughout the study period. Under the exposure conditions tested, fenbendazole treatment resulted in minimal loss of fibroblast viability relative to previously observed cytotoxic effects in cancer cell lines. Literature findings supported the observed variability in fenbendazole response across cell types and suggested that resistance mechanisms and cellular context may influence drug sensitivity. Conclusion This continuation study strengthens the selectivity analysis of fenbendazole by prioritizing normal-cell response profiling. Preliminary findings suggest that fenbendazole exhibits reduced cytotoxic effects on normal fibroblasts under comparable exposure conditions, supporting a potentially favorable selectivity profile. Future work will focus on increased replicate testing and incorporation of marker-based validation assays as resources permit. Funding Source n/a Topic Categories Tumor Immunology: Cellular Responses and Tumor Microevironment (TIME)

Reversible Control of Ferrimagnetic Skyrmion Generation and Motion via Hydrogen Migration Under Electric Field

ABSTRACT Skyrmions, chiral magnetic textures distinguished by their small dimensions and structural stability, are promising information carriers for advanced spintronic devices. Among various skyrmions, ferrimagnetic skyrmions exhibit a reduced skyrmion Hall angle, which makes them particularly suitable for practical applications. In this study, we demonstrate that ionic liquid gating can effectively tune the density, diameter, and magnetic field range of the skyrmion stability in ferrimagnetic Gd/Co‐based heterostructures by more than one order of magnitude at room temperature. Such significant changes are related to the electric‐field driven hydrogen ions migration: a positive gate voltage ( V G ) drives H + migration into Pt and Gd layers, reducing the perpendicular magnetic anisotropy and resultant skyrmion equilibrium and nucleation energy, whereas a negative V G produces the opposite effect. Furthermore, the H + accumulation at positive V G suppresses the current‐driven skyrmion motion owing to the decrease in the Dzyaloshinskii–Moriya interaction constant. The obtained results reveal that the electric‐field driven ion migration is a powerful tool for manipulating both the static and dynamic performance characteristics of ferrimagnetic skyrmions, which can advance the development of high‐efficiency skyrmion‐based spintronic devices with logic operations and neuromorphic computing functionalities.

Dimerization of natively folded molecules drives misfolding and aggregation in Cys-depleted variants of cataract-associated human lens γD-crystallin

Vitamin D levels and other factors associated with depressive symptoms and cognitive impairment in the oldest-old patients with diabetes

Quantitative Analysis of a Novel Systems-predicted Neutralizing Antibody Titer Assay 2305470

Abstract Introduction Using systems serology, it is possible to create an assay that quantifies a human receptor’s binding levels to a virus receptor binding protein in the presence of neutralizing antibodies. For example, human influenza A virus predominantly binds to alpha 2’6-linked sialic acids, while avian influenza A prefers alpha 2’3-linked sialic acids. Modeling this binding in the presence of neutralizing antibodies using a systems serology approach would allow us to quantify neutralization breadth and capacity at a throughput and scale not previously known. Methods Using four parameter or Michaelis-Menton fitted models, we can quantify the complete binding and complete inhibition of binding for virus receptor binding proteins and human receptor pairs at the multiplex level. From this, percent inhibition can be quantified, and a systems-predicted neutralizing antibody (SNAb) titer can be calculated. This titer was compared against existing neutralization assays and readouts for validation. This was done using serum and lavage samples from vaccinated/boosted non-human primates. Results Neutralizing antibody levels, whether they were measured by microneutralization assay, hemagglutinin inhibition assay, or SNAb were tightly correlated, and showed dependence on route of vaccination. The SNAb assay could successfully multiplex hemagglutinin molecules to quantify the predicted neutralization of twelve analytes simultaneously. The SNAb assay was performance-validated and did not show an overcounting or undercounting of neutralizing antibody titers. Conclusion Neutralizing antibody levels can be quantified in an efficient way using a systems-based approach. This allows for the multiplexing of readouts in a high-throughput assay. The assay itself is built on four-parameter or Michaelis-Menton binding modeling, which does not bias the titer compared to other commonly used methods. Funding Source National Institutes of Health Grant CA260476, PO1AI165072, and U19AI135995, and National Institutes of Health Contract 75N93021C00029 Topic Categories Technological Innovations in Immunology (TECH)

Cardiac glycosides activate non-canonical STAT1 signaling to reprogram antigen-presenting cells 2310011

Abstract Introduction Effective activation of antigen-presenting cells (APCs) is essential for durable vaccine responses and successful cancer immunotherapy, yet pharmacologic strategies to achieve it remain limited. Methods Approximately 1000 compounds of Library of Pharmacologically Active Compounds (LOPAC) were screened to identify inducers of innate immune responses in activation of monocyte-derived dendritic cells (MDDCs). Results We identify cardiac glycosides (CGs) as a class of small-molecule immunopotentiators that trigger a non-canonical STAT1 pathway through the Na+/K+-ATPase-Src-p38 axis. In primary human dendritic cells, the CGs Bufalin and ouabain induce serine STAT1 phosphorylation (S727) independent of interferon signaling, driving a distinct proinflammatory and antigen-presentation program. Bufalin enhances CD8+ T-cell priming in response to mRNA vaccination and, in combination with an oncolytic Newcastle disease virus, promotes myeloid remodeling and enhances tumor regression in vivo. Conclusion These findings reveal a previously unrecognized mechanism linking Na+/K+-ATPase signaling to immune activation through non-canonical STAT1 pathway activation. Identification of non-toxic agonists of this pathway may lead to a next-generation of small-molecule adjuvants and immunotherapy co-agents. Funding Source •NIH/NCI R01 CA229818, • NIAID-DAIT-NIHAI201700100 Topic Categories Vaccines and Immunotherapy (VAC)

Vaccine-induced Immunometabolic Reprogramming in Neonates from The Gambia and Papua New Guinea 2260646

Abstract Introduction Neonatal immunization reprograms metabolism to support immune activation, yet early-life systemic responses across populations remain poorly defined. Understanding these shifts is key for precision newborn vaccine design. Methods Peripheral blood plasma was collected at birth (day of life [DoL] 0, pre-vaccination) and DoL7 from infants in The Gambia (GAM, n = 230; 426 samples) and Papua New Guinea (PNG, n = 50; 65 samples) within the Expanded Program on Immunization Consortium. Infants received BCG, HBV, BCG+HBV, or delayed control. Untargeted LC—MS/MS metabolomics was performed, and antibody titers were measured at DoL30 and DoL128. Results Generalized Additive Mixed Models (gamm4) of longitudinal WGCNA modules showed that within 48h after birth in GAM, vaccination induced distinct metabolic signatures in four modules: Mod1 (PC/Plasmalogens), Mod4 (Acylcarnitines), Mod7 (Met/Cys/SAM/Taurine), and Mod17 (PC), representing lipid, energy, and amino acid/sulfur metabolism. By 200h, vaccine-specific effects in Mod1, Mod7, and Mod17 subsided as metabolic trajectories converged between vaccinated and unvaccinated infants, indicating transient early shifts. In GAM, Lipid Fatty Acid Dicarboxylate metabolites differed between vaccine arms, being downregulated with HBV but not BCG. In PNG, vaccination led to broad reductions in plasma metabolites by DoL7, especially amino acids and lipids, whereas GAM infants showed no comparable reduction. Cohort-specific divergence was seen in tryptophan metabolism and lysophospholipids (LPLs), which decreased in PNG but not in GAM; anthranilate rose ∼15% with BCG in GAM (p = 0.037) but fell ∼40% in PNG (p = 0.0029). Conclusion Neonatal vaccination induces robust yet transient systemic metabolic shifts with both conserved and population-specific features. We are developing an Immuno-Metabolic Response Score (IMRS) using sparse regression and logistic modeling to identify metabolite signatures predictive of antibody responses, with ongoing validation in neonatal immune assays. Funding Source NIH NIAID U19AI118608 UO1 & ADP NIAID contracts↑ Boston Children’s Hospital Award to Precision Vaccines↑ Program (PVP) Topic Categories Vaccines and Immunotherapy (VAC)

Genetic variants of TYR and MC1R in biosynthesis of melanin and skin pigmentation are correlated with JDM and cSLE with photosensitive rash 2260556

Abstract Introduction Juvenile dermatomyositis (JDM) and Systemic Lupus Erythematosus (SLE) are systemic autoimmune diseases characterized by production of autoantibodies against cellular and nuclear antigens and complement-mediated tissue injuries. Common clinical presentations of JDM and cSLE include photosensitive rashes on the skin, although the patterns of rash are different. We seek to determine the genetic features of these two diseases. Methods Data from whole exomes sequencing of 51 patients with JDM and 43 patients with cSLE revealed two nonsynonymous variants, S192Y and R402Q in the tyrosinase gene (TYR), which encodes a key enzyme in melanin biosynthesis and skin pigmentation. We developed restriction fragment length polymorphisms (RFLP) of PCR amplified genomic DNA fragments using Hpy188I digest or HpyAV digest to identify the S192Y and R402Q variations, respectively. Results The S192Y variant has significantly higher frequency in JDM than controls (41% vs 25%, p = 0.0003), while R402Q has higher frequency in cSLE than controls (32.6% vs 17.6%, p = 0.0006). Intriguingly, another protein engaged in the melanin synthesis pathway, melanocortin-1 receptor MC1R, has non-synonymous variant for R160W. FauI RFLP was developed to detect this R160W polymorphism in MC1R. Close to 21.6% JDM patients have a homozygous or heterozygous variant of R160W in MC1R (p = 0.001). Conclusion Overall, 88% of patients with JDM or cSLE have a variant in S192Y or R402Q of TYR, or R160W of MC1R. Further experiments are in progress to manipulate the expression and functional activities of TYR and MC1R. Funding Source Cure JM Topic Categories Basic Autoimmunity (BA)

Persistent uterine inflammation following murine chorioamnionitis predisposes to poor outcomes in subsequent pregnancy 2260652

Abstract Introduction Chorioamnionitis, an infection and/or inflammation of the placenta and fetal membranes, is the leading cause of preterm birth, and an independent risk factor for a variety of neonatal morbidities. Women with a history of chorioamnionitis are at increased risk for complications (including implantation failure, recurrent chorioamnionitis, and adverse neonatal outcomes) in subsequent pregnancies; however, the mechanisms underlying these subsequent poor outcomes remain unclear. Pathological in-utero inflammation is a primary driver of the morbidities seen with chorioamnionitis. Thus, we hypothesized that chorioamnionitis-associated inflammation persists postpartum in the uterus, thereby predisposing to adverse outcomes in subsequent pregnancies. Methods To test this, we induced chorioamnionitis in pregnant syngeneic C57BL/6 mice at embryonic day 14.5 via intravaginal inoculation with 100 CFU of E. coli K1 (or sterile PBS as a control). Four weeks postpartum, mice were either rebred or euthanized for uterine immune profiling via flow cytometry. Vaginal and uterine swabs were collected to assess for persistent E. coli K1 infection. Results Chorioamnionitis-exposed dams (CED) had poor outcomes in their second pregnancy, including dystocia, ∼50% perinatal mortality, and growth-restricted pups. Uterine tissues from CED showed increased frequencies and total numbers of T cells, activated (CD44hi) CD4+ T cells, and Ly6Chi pro-inflammatory macrophages, as well as dendritic cells with significantly high MHC II expression. Uterine and vaginal E. coli K1 colonization persisted in only 12% of CED. Conclusion These findings suggest that chorioamnionitis drives chronic uterine inflammation postpartum. This inflammation appears to be associated with chronic T cell activation, thereby providing a potential immune-mediated mechanism for poor outcomes in subsequent pregnancies. Funding Source R01 AI 141673 Topic Categories Mucosal and Regional Immunology (MUC)

Study of Mycoplasma pneumoniae CARDS Toxin in a lung fibroblast transfection model 2310308

Abstract Introduction Mycoplasma pneumoniae is an atypical respiratory pathogen that causes bronchiolitis and bacterial pneumonia. During infection, M. pneumoniae generates a three domain protein called Community-Acquired Respiratory Distress Syndrome (CARDS) toxin. Intracellular trafficking and processing activate its ADP-ribosylation and vacuolization properties. While ADP-ribosylation activity originates from the first domain, it is unclear where vacuolization activity originates. Lung fibroblasts play a crucial role in maintaining lung tissue integrity throughout life by recruiting immune cells during infection, regulating inflammation, and shaping lung tissue repair. Chronic M. pneumoniae infection is associated with airway remodeling and fibrosis, however the effects of CARDS toxin on fibroblast function are unclear. We hypothesize that CARDS toxin vacuolization disrupts lung fibroblast ability to regulate inflammation and initiate lung repair. Methods An ADP-ribosylation-deficient mutant of CARDS toxin was cloned for mammalian and bacterial overexpression and validated by sequencing. Mammalian vectors were transfected into IMR-90 lung fibroblasts to visualize CARDS toxin localization and fibroblast function and viability. Results Transfected fibroblasts showed similar pathology as recombinant CARDS toxin-treated fibroblasts, as well as high counts of cell death and vacuolization. CARDS-GFP signal was localized around the nucleus, confirming previous immunofluorescence observations. Comparable vacuolization was also seen between transfected and recombinant CARDS toxin-treated fibroblasts. Conclusion IMR-90 fibroblast transfection serves as an effective model for studying effects of M. pneumoniae CARDS toxin. Visualization of GFP-CARDS toxin movement in real time also provides critical insight into CARDS toxin trafficking and fibroblast responses. Funding Source n/a Topic Categories Microbial, Parasitic, and Fungal Immunology (MPF)

Immune-Stromal Co-culture System to Interrogate Cytokine Responses in Rheumatoid Arthritis 2328589

Abstract Introduction A critical component of rheumatoid arthritis (RA) pathogenesis is the complex communication between immune and stromal cells. While this has been historically challenging to model in vitro, we set out to build a robust transwell culture system using human PBMCs and RA fibroblast-like-synoviocytes (FLS). Utilizing a disease relevant inflammatory stimulus (anti-CD3/CD28), this platform enables us to profile multiple cytokine responses and identify downstream pathway activation with translational relevance. Methods PBMCs and immortalized RA FLS were cultured for 48 h following the addition of anti-CD3/CD28 to the PBMC compartment. Readouts include multiplex cytokine profiling and single cell RNA-sequencing of both RA FLS and PBMCs. The assay was validated by inhibiting clinically relevant mechanisms (IL-6, TNFa, JAK/STAT) and comparing against clinical datasets. Results This co-culture system exhibited dynamic immune—stromal crosstalk, resulting in emergent properties that are more physiologically relevant than single pathway activation. Inhibition of clinically validated pathways (IL-6, TNFa, JAK/STAT) revealed robust transcriptomic effects unique to the individual pathways. These results were aligned with gene expression changes observed in clinical trial responder datasets. Single cell RNA sequencing allowed us to project transcriptomic profiles of individual cell types in vitro onto an internally compiled human RA Single Cell Atlas. This analysis revealed shared pathways and gene modules between our in vitro assay and human disease tissue, highlighting the translatability of this co-culture system. Conclusion This transwell co culture provides a scalable, robust platform for RA research. It supports parallel evaluation of cytokine targets and identification of orthogonal downstream mechanisms in both immune and stromal compartments. Future studies will further elucidate translatable pathways relevant to RA and novel therapeutic targets to explore. Funding Source n/a Topic Categories Technological Innovations in Immunology (TECH)

Modulating Local Structure of Amorphous Oxyhalide to Achieve High‐Rate and Ultra‐Stable All‐Solid‐State Lithium Battery

ABSTRACT Halides‐based all‐solid‐state lithium batteries (ASSLBs) attract great attention because of their wide electrochemical window and fine processibility. However, low ionic conductivity of halides and solid‐solid interface incompatibility result in inferior rate capability and poor cycling stability. Herein, we modulate local structure (short‐range‐structure disorder degree, coordination diversity, and so forth) via a synergistic anion–cation strategy to achieve amorphous solid electrolyte 1.6Li 2 O‐TaCl 5 ‐0.3MgF 2 (LTOC‐M) with a high ionic conductivity (11.15 mS cm −1 ) and favorable interfacial compatibility. F incorporation at Cl/O sites strengthens Ta─F bonding and Li–F interactions, enhancing long‐term cycling stability, while Mg incorporation modulates the local cationic environment, increases coordination diversity, and facilitates Li + transport within the amorphous matrix. ASSLBs with LiNi 0.8 Co 0.1 Mn 0.1 O 2 or LiCoO 2 demonstrate superhigh rate capability and long‐term cycling stability (LiNi 0.8 Co 0.1 Mn 0.1 O 2 :92.29%@4000cycles@5C; LiCoO 2 :80.85%@5000cycles@10C). When paired with Li‐rich Li 1.2 Mn 0.54 Ni 0.13 Co 0.13 O 2 , the cell delivers a high initial capacity of 270.38 mAh g −1 with a cycling stability (92.75%@120cycles@0.2C). Moreover, Li‐In| Li 6 PS 5 Cl‐LTOC‐M|LiCoO 2 delivers a high discharge capacity of 128.80 mAh g −1 at −20°C and demonstrates a cycling stability (96.62%@550cycles@0.2C), and the battery functions even at −75°C for over 400 h. The proposed strategy effectively enhances high‐rate performance, long‐term cycling stability and low‐temperature performance of halides‐based ASSLBs, accelerating their practical application.

Rapid reduction in global chromatin loop size after acute STAG2 reconstitution in human cancer cells

Feasibility of energy expenditure estimation during outdoor walking using multimodal in-ear sensors: a preliminary validation study against indirect calorimetry

Abstract A growing number of portable devices has become available for daily physical activity and energy expenditure (EE) monitoring. Although several studies have examined EE estimation using heart rate (HR) and accelerometry (ACC), no study to date has explored EE prediction based on HR and ACC data measured simultaneously in the ear canal by a single commercial wearable device. Therefore, the aim of this study was to assess the feasibility of estimating EE using the cosinuss $$^\circ$$ in-ear sensors $$^\circ$$ One and $$^\circ$$ Two. Twenty-four healthy adults (12 female, 12 male, age 26.91 ± 2.91 years, body mass 65.43 ± 8.01 kg) completed two test sessions: a laboratory treadmill test (Modified Bruce Protocol) and an outdoor walking test consisting of three phases (uphill at comfortable pace, downhill at comfortable pace, and uphill as fast as possible). EE was measured by the portable indirect calorimetry device MetaMax 3B (criterion measure), while HR and ACC were recorded continuously by the in-ear sensors $$^\circ$$ One and $$^\circ$$ Two. Linear mixed-effects models were developed from laboratory data and applied to outdoor walking data from the same participants (participant-calibrated prediction). Prediction accuracy was assessed using root mean square error (RMSE), mean absolute percentage error (MAPE), and Bland-Altman analysis. Both in-ear sensors showed preliminary potential for EE prediction during outdoor walking. The integration of HR, ACC, and biological sex produced the best-fitting calibration models, with marginal $$R^2$$ values of 0.79 and 0.80 for $$^\circ$$ One and $$^\circ$$ Two respectively. In the outdoor walking test, no significant systematic bias was detected across conditions ( $$p> 0.05$$ ). However, prediction accuracy was variable across conditions and subgroups, with MAPE ranging from 22% to 37% for $$^\circ$$ One and 22% to 38% for $$^\circ$$ Two (following exclusion of one participant with implausible reference data in the Down phase). Pearson correlation coefficients were stronger for female participants, while male subgroup correlations were weak or negative in several conditions, limiting conclusions about individual-level prediction accuracy for this subgroup. The cosinuss $$^\circ$$ in-ear sensors demonstrated preliminary feasibility for group-level EE estimation during outdoor walking, with accuracy broadly comparable to several commercial wrist-worn devices. Given the variable prediction accuracy, particularly for male participants and during downhill walking, further validation with larger and more diverse samples is needed before clinical or sports application.

Germline-targeting vaccine elicits HIV broadly neutralizing antibodies in nonhuman primates 2303354

Abstract Introduction HIV vaccine development has been exceptionally challenging, for myriad reasons. Nevertheless, protective HIV broadly neutralizing antibodies (bnAbs) exist and have been proposed as templates for vaccine development. Germline-targeting is a conceptually radical approach to vaccine design, aiming to prime rare bnAb-precursor B cells possessing pre-determined human genetic sequence motifs, and then guide B cell affinity maturation to potent bnAb evolution with heterologous booster vaccines. While the approach has shown promise, it faces many novel immunological challenges, commonly thought to be insurmountable in aggregate. To date, germline-targeting has not succeeded in generating bnAbs in nontransgenic animals. Methods Here, we report a germline-targeting vaccine tested in outbred nonhuman primates that successfully generated bnAb-class memory B cells and sera capable of neutralizing diverse HIV clinical isolates. Results Neutralizing Ab clones were generated in ≥ 50% of animals, achieving a remarkable aggregate 62% neutralization breadth compared to the reference bnAb. 200 different bnAb-class clonal families were primed. The vaccine generated large and diverse bnAb-class clonal lineages, including multiple lineages containing > 1,000 memory cells. Furthermore, serum bnAb activity developed in 44% of animals; in the most striking instance serum bnAb breadth reached 84% that of BG18. Conclusion These results demonstrate that germline-targeting vaccines can reproducibly elicit prespecified classes of bnAbs through the priming and maturation of rare precursor B cells under endogenous conditions. Simultaneously, these results also demonstrate fundamental immunological findings, including demonstration that exceptionally rare B cells can be primed and recruited into germinal centers, competitive within germinal centers, affinity matured for extended periods, differentiated into memory B cells, boosted repeatedly with new immunogens, and differentiated into large numbers of bnAb plasma cells. Funding Source NIH NIAID CHAVD UM1 AI144462 Topic Categories Vaccines and Immunotherapy (VAC)

Impaired B Cell and cTfh Immune Pathways Underlie Influenza Vaccine Hyporesponsiveness in Older, Frail, and Multimorbid Adults 2309992

Abstract Introduction Older adults face the highest rates of influenza-related hospitalization and death due to immunosenescence and diminished vaccine responsiveness. Despite the use of high-dose and adjuvanted flu vaccines, substantial variability persists. The biological features distinguishing robust (“Hyper”) from poor (“Hypo”) vaccine responders (HyperR and HypoR) remain unclear. By integrating B cell and circulating T follicular helper cell (cTfh) activation, and RNA-seq transcriptional analysis, we identified coordinated signatures underlying response heterogeneity between HyperR and HypoR cohorts. Methods Samples from 21 frail multimorbid nursing home residents receiving flu vaccine were analyzed across visits (D0, D28). Hemagglutinin inhibition assays (HAI) quantified ab response to define HypoR and HyperR cohorts. Differential expression and pathway analyses of RNAseq data were performed after mitogenic stimulation of purified cTfh and B-cells. Results Transcriptomic profiling showed enrichment of antigen presentation, germinal-center differentiation, and metabolic activation pathways in HyperR. A Custom Nakaya B-cell pathway showed markedly higher expression in HyperR and reduced expression in HypoR, with AICDA (somatic hypermutation enzyme) as the most highly differentially expressed gene. Additional B-cell genes associated with survival and differentiation (BCL2L10, TBX21, CD27) were increased in HyperR. cTfh cells from HyperR showed increased expression of helper and trafficking genes (IL21, CXCR5, CXCL13, CRTAM). In contrast, HypoR exhibited attenuated pathway activity, increased TIGIT (checkpoint receptor) expression, and weakened concordance between cellular and antibody responses. Conclusion HyperR to flu vaccination mount coordinated cellular and molecular responses, whereas HypoR show reduced activation and fragmented B-cell proliferation and plasmablast maturation signaling. Our findings highlight putative mechanisms to inform improvements in influenza protection in frail adults. Funding Source 1R01AI129709-01A1 Topic Categories Vaccines and Immunotherapy (VAC)

PD-1 regulates CD4 T cell differentiation and trafficking in response to hyperlipidemia in mice and humans 2260972

Abstract Introduction Programmed cell death protein 1 (PD-1) is a well-studied regulator of T cell activation. It is also highly expressed on T follicular helper cells, and global deletion of PD-1 or its ligands, PD-L1 and PD-L2, has been shown to diminish antigen-specific B cell responses and formation of memory B cells. Given that PD-1 blockade immunotherapy has recently been associated with accelerated progression of atherosclerosis, we have developed a murine model with CD4-specific inducible loss of PD-1 to further investigate how PD-1 regulates the CD4 T cell, and consequently, the B cell response, both at baseline and in response to hyperlipidemia. We have also probed two cohorts of patients with coronary artery disease (CAD) to investigate the prevalence and phenotype of PD-1-expressing CD4 T cells among PBMCs. Methods To study loss-of-function in mice, we have generated CD4CreERT2Pdcd1fl/fl mice, with tamoxifen-inducible CD4-specific knockout of PD-1. In our human studies, we assembled a matched cohort of patients with low and high severity CAD, determined by coronary angiography, and performed CITE-Seq on PBMCs collected at the time of imaging. Results During hyperlipidemia, CD4-specific loss of PD-1 results in increased CD4 T cell activation and differentiation into Th subtypes, with corresponding decreases in the frequency of naïve CD4 T cells. We also observe increases in CXCR3 and CCR5 in CD4 T cells, both associated with trafficking to atherosclerotic lesions. In CAD patients, PD-1+ CD4s express these same markers. In the B cells, we observe major changes to B cell surface markers without PD-1 signaling, suggesting disruptions in T-dependent B cell activation. Conclusion Altogether, these data suggest that PD-1 is not only an important regulator of T cell activation, but also a regulator of T cell trafficking during atherosclerosis. Furthermore, the PD-1 signaling axis between CD4s and B cells may maintain B cell homeostasis. Funding Source Leducq Foundation, NIH T32AI007496-29, NIH R01 AI17211 Topic Categories Immune Response Regulation: Cellular Mechanisms (IRC)

A unified network systems approach uncovers a core novel program underlying T follicular helper cell differentiation 2260705

Abstract Introduction The complexity of T follicular helper (Tfh) cell populations and inconsistent findings across experimental systems have fueled ongoing debate regarding Tfh differentiation. Without a unified systems approach, it remains unclear whether these inconsistencies stem from technical artifacts or true biological differences between human and murine systems. Methods We adopted a novel multi-scale network approach that incorporates both regulatory and physical interactions. This systems approach elucidates circuits underlying Tfh differentiation by integrating data from ‘omic profiles. We then interrogate the circuits across data types, disease contexts and species to evaluate them and validate them in-vivo. Results We capture multi-level regulation of immune system organization and recapitulate known drivers. While components have been individually reported, the novel aspect lies in the discovery and connectivity of a core signature using an unbiased approach. We found a core Tfh set which is conserved across humans and mice addressing a fundamental open question in the field, as different systems (in-vitro, ex-vivo and in-vivo) have led to discovery of varying components. Further, we shed light on the scientific debate and demonstrate how IL-12 specifically blocks the differentiation of Tfh precursors into GC Tfh cells, in both humans and mice. Our in-vivo validation also demonstrated that at present, in-vitro systems fail to represent ex-vivo GC-Tfh, and combining high quality human cell analysis with murine experiments remains crucial for dissecting molecular mechanisms that are difficult to model in-vitro. Conclusion The gene sets identified in this analysis converge on a core regulatory network that is conserved across species, tissues, disease states, and experimental systems, an outcome only possible if the underlying biology is truly consistent. Notably, the necessity of IL-12 for Tfh differentiation has important implications for vaccine strategies that utilize IL-12 based adjuvants. Funding Source NIAID DP2AI164325, NIAID R01AI170108, NIAID U01AI179514, NHGRI U01HG012041, NIAID U19AI110495. Topic Categories Computational and Systems Immunology (COMP)