Browse Articles

Impaired humoral immune response to hepatitis B vaccination in children with HIV and type 1 diabetes mellitus

A specific albumin receptor mediates antigen delivery and the induction of B cell responses in secondary lymphoid organs 2259364

Abstract Introduction The phenomenon of albumin-hitchhiking has been exploited in vaccination strategies. We hypothesized that albumin mediated protein delivery may have evolved to facilitate the presentation of conformational determinants directly to B cells to facilitate B cell activation. However, the receptor that potentially targets albumin to lymph nodes and facilitates the presentation of intact proteins to B cells has hitherto not been identified. Methods Surface binding, specificity and saturability studies were performed on human and murine immune cells. Membrane proteins were extracted from human Ramos B cell membranes and affinity chromatography was performed using an HSA (human serum albumin)-Sepharose matrix. Mass spectrometry was performed on eluted proteins. CRISPR/Cas9 mediated gene ablation was used to establish the identity of the HSA-receptor (HSA-R). HEK 293T cells were transfected with the cDNA for the HSA-R. In vitro culture of murine dendritic cells with HSA and HSA-bound proteins and immunization of a range of genetically altered recipient mice was performed to establish the in vivo function of the HSA-R. Results We purified the HSA-R using affinity chromatography and validated its identity using gene ablation and overexpression studies. CRISPR/Cas9 mediated gene ablation and overexpression in HEK 293T cells helped establish that the HSA-R is both necessary and sufficient to exhibit albumin on the surface of antigen presenting cells for at least 15 hours. In vivo experiments have established that this receptor on dendritic cells facilitates potent T dependent germinal center-based B cell responses. Conclusion A newly discovered poorly endocytic but specific albumin receptor on antigen presenting cells is likely crucial for facilitating antigen presentation to antigen-specific B cells and enhancing T-B cell collaboration against bacterial lipoproteins and other albumin-binding microbial proteins. Funding Source n/a Topic Categories Immune Response Regulation: Cellular Mechanisms (IRC)

Pleiotropic Cytokine Effects Drive Synchronized Proliferation and Emergent Chaos in Hematopoiesis 2309108

Abstract Introduction Hematopoiesis is an inherently dynamic process with variations shaping differential immune responsiveness. Our previous work demonstrated that homeostatic hematopoiesis operates as a chaotic system with large fluctuations in cell frequencies, enabling robust adaptability. However, the underlying biological mechanism remains unclear. We hypothesize that pleiotropic cytokine signaling drives variable cell kinetics that generate chaotic hematopoietic dynamics. Methods We performed pulse-labeling experiments using 5-iodo-2’- deoxyuridine (IdU) in 39 healthy C57BL/6 mice, followed by mass cytometry analysis of bone marrow, blood and spleen. We also treated isolated bone marrow cells with 30 individual cytokines for 24h and 48h, and quantified changes in proliferative subset (%Ki-67+) across leukocyte subsets by spectral flow cytometry. These measurements were integrated into a mathematical model simulating cytokine effect on hematopoietic dynamics. Results Proliferation rates of all cell types between left and right femurs within individual mice showed high similarity (slope = 0.98, R² = 0.97), confirming observed fluctuations reflect biological variation, rather than experimental artifact. All leukocyte types exhibited substantial proliferation variability (coefficient of variation ≈ 1). Strikingly, we observed globally synchronized cell proliferation. Individual cytokines simultaneously upregulated the %Ki-67+ subsets across multiple cell types, revealing pleiotropic proliferative effects. Even cell populations with strongly negative correlations in numbers (e.g., neutrophils and B cells) showed positively correlated proliferation rates. Mathematical modeling confirmed such cytokine-driven synchronized proliferation is sufficient to generate observed chaotic fluctuations in hematopoiesis. Conclusion Our findings reveal that pleiotropic cytokine signaling drives synchronized proliferation across cell types, which paradoxically generates chaotic population dynamics through nonlinear interactions. Funding Source This work was supported by the intramural research program of the NCI. Topic Categories Hematopoiesis and Immune System Development (HEM)

Comprehensive Biomarker Strategy to Monitor GNTI-122, an Engineered Regulatory T Cell Therapy in Clinical Development to Treat Type 1 Diabetes 2252996

Abstract Introduction Engineered regulatory T cell (EngTreg) therapy offers a novel approach to restore immune tolerance in type 1 diabetes (T1D). To support POLARIS, GentiBio’s first-in-human trial of GNTI-122, we developed a biomarker program to monitor drug kinetics, pharmacodynamics, and immunogenicity. Methods Kinetics of GNTI-122 will be quantified by droplet digital PCR (ddPCR) detecting engineered transgenes. An 18-color flow cytometry panel is designed to detect GNTI-122, its Treg profile, and impact on immune subsets. Mechanism of action of GNTI-122 will be assessed by reduction in β cell-specific effector T cell (Teffs) responses using two complementary assays: an Activation-Induced Marker (AIM) assay and a FluoroSpot assay. Immunogenicity will be evaluated by a cell-based anti-drug antibody (ADA) assay targeting the TCR of GNTI-122. Results Biomarker assays demonstrated robust sensitivity, specificity and precision. Validated assays show the ability to detect GNTI-122 at a level of sensitivity < 0.1% of total WBCs. AIM and FluoroSpot assays reproducibly detected β cell-specific Teffs. Our cell-based anti-TCR ADA assay detected anti-TCR antibodies at a sensitivity < 850 ng/ml. Conclusion This comprehensive biomarker strategy provides a rigorous translational framework to monitor cellular kinetics, pharmacodynamics, and immunogenicity, to elucidate biological mechanisms of GNTI-122 for GentiBio’s POLARIS Phase 1 safety trial of GNTI-122 in recently diagnosed T1D. Funding Source T1D Fund Topic Categories Immune Mechanisms of Human Disease (HUM)

Selective SIK2 Inhibitors Suppress Proinflammatory Responses while Upregulating Pro-Resolution Signals in Stimulated Myeloid Cells and Mice 2259968

Abstract Introduction Salt-Inducible Kinases (SIKs) amplify proinflammatory gene expression in myeloid cells via phosphorylation of transcriptional coregulators. Genetic loss-of-function (LOF) of SIKs in mice or pan-SIK inhibitors decrease proinflammatory cytokine production induced by toll-like receptor (TLR) agonists or IL-1. Among the three SIK isoforms, SIK2 shows the highest activity in myeloid cells. SIK2 LOF also enhances production of the anti-inflammatory cytokine IL-10, an effect not observed with SIK1 or SIK3 LOF. However, the lack of highly selective SIK2 inhibitors has prevented characterization of pharmacological SIK2 inhibition and its therapeutic potential. Methods Using structure-based drug design, we developed first-in-class highly SIK2-selective inhibitors with nanomolar cellular potency, favorable in vitro ADME and off-target profiles, and suitable PK properties to support in vivo evaluation. SIK2 inhibitors were assessed in vitro using differentiated mouse and human myeloid cells and in vivo in LPS challenged mice. Results In vitro, potent and selective SIK2 inhibitors reduced proinflammatory cytokines and chemokines in TLR-stimulated mouse and human inflammatory myeloid cells, while upregulating pro-resolution factors, IL-10, amphiregulin, and retaining the phagocytic activity of regulatory macrophages. Transcriptional analysis of SIK2 inhibitor-treated macrophages confirmed dual suppression of inflammatory pathways and concomitant induction of tissue repair pathways. In vivo, SIK2 inhibitors decreased multiple proinflammatory cytokines and increased IL-10 in LPS-challenged mice. This dual mechanism demonstrated by SIK2 inhibitors was not observed with JAK1 or SIK3-selective inhibitors, which only suppressed inflammatory cytokines. Conclusion Selective targeting of SIK2 in myeloid cells has the potential to suppress inflammation and promote tissue healing, offering a novel oral therapeutic approach for more complete disease control in autoimmune and inflammatory diseases. Funding Source n/a Topic Categories Therapeutic Approaches to Autoimmunity (THER)

Targeting the Transcription Factor Id2 Reveals Novel Regulatory Mechanisms in Chronic Graft-versus-Host Disease 2257336

Abstract Introduction Chronic graft-versus-host disease (cGVHD) is a severe complication of hematopoietic stem cell transplantation (HSCT) complication driven by immune dysregulation. A key feature is aberrant T follicular helper (Tfh) and germinal center B (GCB) cells, which promote pro-inflammatory cytokine release, pathogenic antibody production, and tissue fibrosis. Leveraging cGVHD patient T cell single-cell RNA sequencing data from the Kean Lab, we selected Id2, a transcription factor that inhibits Tfh differentiation while supporting antibody production, as a target for the treatment of cGvHD. Methods We used CRISPR/Cas9 to generate Id2-knockout (KO) naïve murine T cells and adoptively transferred into pre-conditioned B10.BR recipients of C57BL/6 bone marrow (BM) ± T cells to generate murine cGVHD model with bronchiolitis obliterans (BO) lung disease. Results Id2-KO T cells maintained ≥90% KO efficiency until study end (d49). Mice receiving Id2-KO vs control T cells had significantly improved pulmonary function (p < 0.0001), normalizing resistance, compliance, and elastance to BM-only (no disease) levels. Consistent with cGVHD amelioration, a 2-fold reduction in lung collagen deposition (p < 0.0001) and 4-fold decrease in serum allo-reactive antibody (p = 0.0286) were noted. On D49, despite similar GCB and Tfh ratios to control, Id2-KO T cells had enhanced Tfh differentiation: Bcl6+T cells increased from 4.9% to 10.8% (p = 0.0038) with 1.8-fold higher Bcl6 MFI(p = 0.0005). Id2-KO Tfh cells were functionally impaired with a 50% reduction in IL-4 production (p = 0.0078) and a 1.9-fold decrease in SLAMF1+ Tfh cells (from 63.3% to 33%, p < 0.0001), indicating disrupted Tfh-GC B cell interactions. Conclusion Id2 restrains Tfh differentiation while supporting their functional capacity to help GCB cells produce pathogenic antibody. Genetic Id2 loss uncouples Tfh differentiation from effector activity, positioning Id2 inhibition as a novel therapeutic strategy to selectively disrupt pathogenic Tfh-GCB crosstalk in cGVHD Funding Source NIH Topic Categories Transplantation Immunology (TRAN)

Immunometabolic Resistors of Aging in Long-Lived Golden Spiny Mice 2309529

Abstract Introduction Investigating long-lived wild rodents closely related to laboratory mice on the evolutionary scale presents an opportunity to uncover dormant pathways that combat inflammaging. Spiny mice (Acomys) are known for their exceptional regenerative capabilities, but their lifespan and resilience to aging remain unexplored. Methods We established cohorts of two Acomys species (A. russatus and A. dimidiatus), originating from a colony at Tel Aviv University Zoological Research Garden, and laboratory mice (Mus musculus) across their lifespans (0.5 to 4.5 years for Acomys and 0.5 to 2.5 years for Mus). We conducted behavioral tests and histological analyses of immune-metabolic organs. Bulk or single-nucleus RNA sequencing analysis was utilized in young and aged Mus and Acomys. Aged M. musculus were randomly assigned to receive either a vehicle or clusterin (50 µg/kg) for 2 weeks. Results We reveal that A. russatus, maintained in a non-pathogen-free environment, outlives M. musculus significantly both in the wild (>4 years) and in captivity (>5 years). We observe that A. russatus resists age-associated functional decline and exhibits a greater repair capacity with minimal thymic involution and splenic cellular senescence compared to its congener, A. dimidiatus, or M. musculus. Furthermore, aged A. russatus does not exhibit cellular and molecular immune aging hallmarks, such as the accumulation of age-associated B cells and exhausted T cells, nor inflammatory cytokine production. Notably, clusterin expression is significantly elevated in diverse immune cells in A. russatus, unlike in M. musculus. Interestingly, clusterin administration to aged mice improves healthspan, suppresses the accumulation of B cells and regulatory T cells, and reduces inflammatory cytokine production, mirroring the exceptional aging resilience observed in A. russatus. Conclusion A. russatus biology unveils therapeutically actionable targets that may enhance functional capacity and maintain immunometabolic homeostasis during aging. Funding Source National Research Foundation of Korea grant RS-2024-00412002 Yale University Waldemar Von Zedtwitz endowed chair Yale University School of Medicine’s intramural funding Israel Science Foundation grant 2129/20 Topic Categories Veterinary and Comparative Immunology (VET)

Evaluating memory B cell cross-reactivity between ancestral and future SARS-CoV-2 variants — evidence for original antigenic sin 2219641

Abstract Introduction A hallmark of adaptive immunity is the generation of immunological memory and ability to mount a robust recall response following encounter with the same antigen. In the context of mutable pathogens, the specificity of memory B cells (Bmem) generated following the initial exposure can constrain the response elicited by an antigenic drift variant — a phenomenon referred to as “original antigenic sin”. Methods To investigate whether human donors with verified SARS-CoV-2 infection in early 2022, likely with Omicron (BA.1), generated variant-specific Bmem responses or whether WH1-reactive Bmem were preferentially boosted, we established a novel ImmunoSpot assay to assess cross-reactivity at single-cell resolution. Results Bmem capable of recognizing BA.1 RBD were already present in convalescent donors collected early in the COVID-19 pandemic, and such secretory footprints were equally labelled with WH1 and BA.1 RBD probes. In contrast, assessment of Bmem following BA.1 infection revealed an increased frequency of WH1/BA.1 dual-reactivity and few, if any, BA.1 strain-specific footprints providing evidence for preferential boosting of Bmem recognizing shared epitopes. Moreover, we observed increased heterogeneity in the size of the secretory footprints revealed by the RBD probes, indicating cross-reactive Bmem maintained an increased affinity for the WH1 strain. Conclusion Collectively, multi-color inverted assays provide a suitable methodology for assessment of Bmem cross-reactivity in larger donor cohorts. Funding Source N/A Topic Categories Viral Immunology (VIR)

Chemical Probes Targeting CTLA-4: Building the Foundation for Non-Antibody Immune Modulattors 2310508

Abstract Introduction CTLA-4 is a critical inhibitory receptor that maintains immune tolerance by restricting CD28 co-stimulation. Clinical biologics have demonstrated the therapeutic value of modulating this pathway in autoimmune disease. However, antibody-based therapies have limitations that constrain mechanistic studies and limit precision in tuning immune responses. This work aims to develop small-molecule chemical probes that target CTLA-4 or its ligands, enabling a non-antibody approach to modulating immune regulation Methods Recombinant human CTLA-4, CD80, and CD86 extracellular domains were expressed and purified for biophysical characterization. Candidate small molecules predicted to engage CTLA-4 or the CTLA-4/CD80 interface were evaluated using biolayer interferometry (BLI) to assess preliminary interaction patterns. An allogeneic human T cell—dendritic cell co-culture and a multicolor flow cytometry panel were established to measure early activation markers and CTLA-4 expression dynamics. Results BLI studies identified initial interaction signatures with CTLA-4 and the CTLA-4/CD80 complex, demonstrating successful biochemical engagement. The T cell—dendritic cell assay produced consistent activation and checkpoint-expression readouts, establishing a functional system in which these compounds can be evaluated. Together, these early data support continued development of small-molecule probes to explore CTLA-4—mediated immune regulation. Conclusion This work establishes a chemical-immunology platform for identifying and assessing small-molecule modulators of CTLA-4. By enabling non-antibody engagement of this checkpoint pathway, these tools will support future studies aimed at refining CTLA-4—mediated immune regulation and informing therapeutic approaches for autoimmune disease. Funding Source n/a Topic Categories Therapeutic Approaches to Autoimmunity (THER)

Rapid Water‐Soluble Sacrificial Scaffolds for Unconstrained 3D Microfabrication via Two‐Photon Lithography

ABSTRACT Two‐photon lithography (TPL) enables high‐resolution fabrication of complex microarchitectures but remains fundamentally constrained in realizing truly arbitrary three‐dimensional geometries due to the lack of effective microscale support‐removal strategies. Here, we report a universal sacrificial‐support‐assisted TPL approach enabled by a newly developed water‐soluble (W‐S) photoresist. The W‐S photoresist integrates high printing resolution with rapid dissolution, allowing sacrificial scaffolds to be completely removed in mild aqueous media within 10 min. To the best of our knowledge, this represents one of the fastest dissolution performances achieved under such gentle conditions among reported dissolvable photoresists for TPL. This combination of structural stability under dry conditions and rapid aqueous removability decouples fabrication fidelity from post‐processing constraints, thereby enabling unconstrained 3D microfabrication. As representative demonstrations, long single‐clamped cantilevers and micro‐biconvex lenses were fabricated with high geometric fidelity and surface smoothness. After selective removal of the soluble supports, the microstructures retained precise morphology and exhibited excellent optical performance. Overall, this work introduces a rapidly dissolvable photoresist and establishes a versatile materials‐enabled fabrication strategy for constructing previously inaccessible 3D microarchitectures, opening new opportunities in micro‐optics, photonics, and bio‐integrated systems.

Sulfur‐Mediated Charge Delocalization Enables High‐Voltage p‐Type Polymer Cathodes for Ultralow‐Temperature Potassium Dual‐Ion Full Cells

ABSTRACT High‐voltage organic cathodes based on nitrogen‐rich aromatic systems have been widely explored for sustainable potassium dual‐ion batteries, yet their practical application remains hindered by insufficient redox potential, poor structural stability, and sluggish kinetics under extreme conditions. Here, we establish sulfur‐mediated charge delocalization as a design strategy to overcome these fundamental limitations. By strategically incorporating electron‐delocalizing sulfur atoms into the phenazine framework, we achieve extended π‐conjugation and efficient delocalization of charges and electrons, thereby enhancing electronic conductivity and elevating the redox potential. Specifically, we develop a sulfur‐substituted p‐type conjugated polymer, poly[10‐methyl‐3‐(phenazin‐5(10H)‐yl)‐10H‐phenothiazine] (P(PhTz)), as a model system to realize this concept. P(PhTz) exhibits an average discharge potential of 3.70 V (vs. K + /K), a specific capacity of 176 mAh g −1 , and rate capability up to 20C, outperforming its nitrogen‐only analogue poly[5‐phenyl‐5,10‐dihydrophenazine] (PPZ). When paired with a hard carbon anode, the full cell delivers a high specific capacity of 162 mAh g −1 at a 3.55 V average discharge voltage, with stable cycling over 1300 cycles. Crucially, the full cell operates efficiently at −40°C, retaining 87% capacity retention after 850 cycles and achieving an energy density of 434 Wh kg −1 , which represents the highest reported energy density for potassium‐ion full cells under low‐temperature conditions.

Structural and mechanistic insights into the catalytic versatility of lysyl hydroxylases in collagen post-translational modifications

Integrated 3D modeling of train-induced ground vibrations considering track irregularities and wave impeding barrier performance under different subgrade stiffness conditions

Role of B cell activation through Toll-like receptors in Systemic Lupus Erythematosus 2259300

Abstract Introduction Systemic Lupus Erythematosus (SLE) is an autoimmune disease and a leading cause of death in young women. Its high clinical heterogeneity makes therapy development challenging. SLE is marked by autoantibodies targeting DNA and associated proteins, patients often display interferon and plasmablast transcriptomic signatures. One notable B cell subset in SLE is the double negative 2 (DN2; CD27⁻ IgD⁻ CD11c+ CD21⁻) population, expanded in patients with active disease and are poised to differentiate into autoreactive antibody-secreting cells, contributing to disease progression. Methods We used novel activation and culture protocols based on TLR7/9 stimulation and CD40L co-stimulation of naïve B cells (nBCs) from healthy donors and SLE patients, and the generation of a human tonsil organoid model aimed to mimic the heterogeneity observed in late B cell developmental stages in SLE, including likely pathogenic DN2 B cells, plasmablasts (PBs) and plasma cells (PCs), as well as the B cell receptor (BCR) repertoire encoded by VDJ recombination commonly associated with autoreactive B cells in SLE. Results Data showed that TLR7 and CD40L co-stimulation of peripheral nBCs enhanced B cell survival and differentiation into late developmental stages, such as PBs and PCs after 7 and 14 days of stimulation. DN2 expansion was also showed as previously reported. Nevertheless, differentiation of nBCs was controlled when stimulated through TLR9 or the mixed activation cocktail containing ligands for both TLR7 and TLR9 and co-stimulation. Single Cell RNA sequencing (sc-RNAseq) data of VDJ rearrangements after TLR stimulation showed the presence of at least one BCR rearrangement, denominated VH4-34, previously reported as autoreactive. Similar behavior was observed on tonsil organoids consisting of nBCs from tonsil samples of healthy individuals. Conclusion B cell differentiation and fate is controlled by the kind of TLR used to activate these cells, enhancing autoreactivity when TLR7 is stimulated. Funding Source LRA-EMPOWERING LUPUS RESEARCH POSTDOCTORAL AWARD Topic Categories Basic Autoimmunity (BA)

Immunometabolic Defects in Intestinal CD8+ T Cells Disrupt Epithelial Integrity in treated people with HIV 2309248

Abstract Introduction The intestinal mucosa requires tightly regulated immune—epithelial metabolic crosstalk to maintain barrier integrity. In people with HIV (PWH) receiving suppressive antiretroviral therapy (ART), persistent gut epithelial damage and inflammation remain unresolved. The mechanisms by which tissue-resident immune cells contribute to epithelial injury in this setting are poorly understood. We hypothesized that immunometabolic dysfunction in intestinal CD8+ T cells drive pathological epithelial remodeling in treated HIV. Methods We analyzed patient-matched colonic biopsies from PWH on ART and HIV-negative controls using transcriptional profiling, metabolic assays, imaging, and functional perturbations. Autologous human intestinal organoid—CD8+ T-cell co-culture systems were used to define immune—epithelial interactions. Pharmacologic modulation of lipid metabolism and PPARγ signaling was performed to assess causality. Results Intestinal CD8+ T cells from PWH exhibited profound lipid depletion and heightened activation compared to peripheral counterparts, independent of HIV antigen specificity. This phenotype was driven by transcriptional repression of the PPARγ lipid-metabolic program. Lipid-deficient CD8+ T cells scavenged plasma membrane fragments from adjacent epithelial cells, inducing epithelial apoptosis and barrier disruption. Restoration of epithelial integrity occurred only with PPARγ agonist treatment, not with nonspecific lipid supplementation, demonstrating a cell-intrinsic requirement for PPARγ-regulated lipid metabolism. Conclusion These findings uncover a previously unrecognized nutrient-signaling axis in which lipid stress in intestinal CD8+ T cells drive epithelial damage in treated HIV. Targeting tissue-resident immunometabolism–specifically the PPARγ—lipid axis–represents a promising therapeutic strategy to restore mucosal barrier homeostasis in HIV and other chronic inflammatory diseases. Funding Source K99 Topic Categories Immune Mechanisms of Human Disease (HUM)

ImmgenT Regulatory T Cell Atlas 2252825

Abstract Introduction CD4+Foxp3+ regulatory T cells (Tregs) are essential mediators of immune tolerance, memory formation, tissue repair, and responses to harmless antigens. Their broad functional repertoire is reflected in extensive phenotypic and spatial diversity. To systematically characterize this complexity, the ImmGenT consortium established a comprehensive single-cell atlas of mouse Tregs across diverse tissues and immune perturbations. Methods We analyzed 658 samples spanning lymphoid and non-lymphoid tissues under steady-state and immune perturbations. Through multi-omic integration, we defined a consistent and reproducible Treg framework across tissues and conditions. In parallel, we developed a 7-marker flow cytometry panel resolving major clusters, enabling broad applications. Results This ontology reframes the resting—effector paradigm as a continuum. Cl.1/rTregs are not restricted to lymphoid organs but extend into non-lymphoid tissues (NLTs), and both compartments are represented by a mixture of the same populations. Classic cl.2/KLRG1+ and cl.8/RORγt+ populations are enriched at barrier sites and within tumors, where they constitute up to ∼30% of TILs, yet remain detectable in secondary lymphoid organs (SLOs). We further identify a previously unappreciated CD49b+CD29+ cluster, rare in barrier tissues but enriched in liver, uterus, and placenta. Under perturbation, the distribution of Tregs entirely reshuffles rather than generating new subsets; for instance, cl.2/KLRG1+ and cl.8/RORγt+ Tregs dominate infected lungs where they are scarce at steady state. Finally, we established a Reference-Based Integration algorithm that enables accurate mapping of Tregs from external datasets, demonstrating broad applicability across studies. Conclusion This atlas provides a unified framework and practical tools to dissect Treg diversity, adaptation, and function across tissues and immune perturbations, supporting community-wide integration and exploration. Funding Source 5R24AI072073-14, Cancer Research Institute. Topic Categories Computational and Systems Immunology (COMP)

G6PD deficiency decreases B cell production of IgM to oxidation specific neoepitopes and increases atherosclerosis 2260194

Abstract Introduction Glucose-6-phosphate dehydrogenase (G6PD) deficiency (G6PDdef) is the most common enzymopathy worldwide, resulting in impaired redox homeostasis and oxidative stress. Until now, no studies have primarily focused on the role of G6PDdef in lymphocytes. Given the importance of redox homeostasis in regulating B cells, this study sought to investigate how G6PDdef induced oxidative stress regulates B cell function in atherosclerosis. Methods To address this, we engineered novel humanized mice harboring the most common variant of G6PDdef in the US, the African variant (hG6PDA-) or the nondeficient human G6PD allele (hG6PDND) and induced oxidative stress through hyperlipidemia. Results To our knowledge, this is the first study to demonstrate that G6PDdef leads to a loss of G6PD enzymatic activity in B cells. Moreover, our study supports that hG6PDA- has functional effects on the humoral immune response as hyperlipidemic hG6PDA- mice had significantly reduced levels of IgM antibodies to oxidation specific neoepitopes (IgMOSE) compared to hG6PDND controls, along with increased atherosclerosis. Importantly, B cell G6PD enzymatic activity levels directly correlated with the number of IgMOSE antibody secreting cells. To determine if differences in IgMOSE in hG6PDA- mice were intrinsic to the B cell, we utilized a G6PD specific inhibitor (G6PDi-1) in-vitro on cultured peritoneal B cells. We found that in-vitro G6PD inhibition of B cells resulted in increased intracellular reactive oxygen species accumulation and reduced LPS-induced production of IgMOSE. In parallel, human B cells were treated with G6PDi-1 and we found that G6PD inhibition resulted in a significant reduction in the CPG-induced production of IgMOSE, indicating a conserved mechanism of G6PDdef impairing B cell function in both murine and human models. Conclusion Together, these data provide evidence that G6PD may act as a metabolic regulator of antibody production by B cells and demonstrate that hG6PDA- may promote atherosclerosis. Funding Source n/a Topic Categories Immune Response Regulation: Molecular Mechanisms (IRM)

Antigen-specific depletion of gluten-reactive T cells for the potential treatment of celiac disease 2257650

Abstract Introduction Celiac disease (CeD) is a gluten-triggered chronic inflammatory disease of the small intestine that affects roughly 1.4% of the global population. As a strict gluten-free diet is the only treatment option, the development of novel treatment strategies is necessary to alleviate physical, psychological, and financial burdens associated with CeD. Roughly 95% of CeD patients express the MHCII variant HLA-DQ2.5. In susceptible individuals, pathogenesis is mediated by gluten-specific CD4+ T cell populations, which are activated upon presentation of gluten peptides by HLA-DQ molecules. This homogeneity presents the opportunity to design HLA-DQ2.5-based therapies to target pathogenic T cell populations for the elimination of celiac disease. Methods As a potential treatment modality, we have engineered human T cells expressing novel ‘chimeric, antigen-presenting’ (CAP) constructs consisting of 1) a gluten peptide-linked HLA-DQ2.5 extracellular domain 2) intracellular CD28/CD3z signaling domains routinely utilized in CAR T cell applications, and 3) an engineered “di-sulfide trap” to stabilize peptide:HLA interactions and CAP surface expression. We hypothesize that upon engagement with gluten-reactive T cells, CAP T cells will specifically eliminate gluten-reactive targets, but leave the remainder of the T cell repertoire untouched. To test this hypothesis, we cultured CAP T cells (effectors) with target cells engineered to express patient-derived gluten-reactive T cell receptors. Results Strikingly, CAP T cells eliminated >98% of target cells within 48h. This effect was specific, as non-specific targets cultured with CAP T cells remained untouched. Further, CAP T cells were efficient killers: even at an 8:1 target:effector ratio, there was almost complete ablation of targets within 72h. Conclusion This work provides a proof-of-concept for CAP T cells, and demonstrates their potential as a novel, T cell-based immunotherapeutic strategy for the treatment of Celiac disease. Funding Source The Colton Consortium for Autoimmunity at the University of Pennsylvania Topic Categories Therapeutic Approaches to Autoimmunity (THER)

Transcriptomic Analysis Identifies Tfrchi Acinar Population As Possible Driver of Terminal Exhaustion via CEACAM1-TIM-3 axis 2309656

Abstract Introduction Ocular graft-versus-host disease severity correlates with inflammation and fibrotic damage in the lacrimal gland (LG). Long-living stem-like progenitor exhausted TCF1+ PD-1+ T cells (Tpex) have been reported to maintain and replenish the reservoir of exhausted-like effector T cells (Texef). In chronic settings, the cumulative cytotoxicity of CD8+ Texef mediates tissue damage. A comprehensive characterization of the phenotypic and transcriptomic profiles of these pathogenic exhausted populations has not yet been reported. Methods To this end, we used an established minor MHC-mismatched murine model of sclerodermatous cGVHD (B10.D2 (H-2d) into BALB/c (H-2d), allogeneic (allo) cGVHD) using syngeneic (syn) controls (Balb/c into Balb/c) assessed at 14-, 21-, and 42-days post-transplant. Here, we longitudinally investigated transcriptional changes in exocrine tissue immune population. Results Microscopy identified increased lymphocyte infiltration in the lacrimal gland and conjunctiva. Single cell sequencing was performed on lacrimal gland. Datasets were processed using a robust integrative approach based on scVI deep learning architecture. We identified four distinct exhausted T cell subpopulations expanded in allo LG. Exhaustion, cytotoxic and residency scores were highest for these four subsets. Proportional distribution analysis revealed a substantial early increase in Tcf7+, Sell+, Il7r+, Slamf6+, Pdcd1+ CD8+ Tpex cells in the allo group. Importantly, allo lacrimal glands were marked by the expansion of Tcf7− CD8+ Texef cells that were not present in the syn group. Cell-cell communication analyses inferred an interaction between Tfrchi acinar cells and exhausted T cells through Ceacam1-Havcr2, signaling shown to drive terminal exhaustion of Texef. Conclusion Overall, scRNAseq results evince a major shift in T-cell fate towards a pathogenic exhausted-like phenotype possibly stemming from an early Tcf7+ expressing precursor exhausted population in cGVHD-affected LG. Funding Source Intramural program of the NIDCR, NIH Topic Categories Transplantation Immunology (TRAN)

Dissecting the Role of Type I Interferon as Regulator of Innate Antifungal Immunity 2227148

Abstract Introduction Aspergillus fumigatus (Af) is the main cause of invasive pulmonary aspergillosis (IA), responsible for over 90% of cases, and has been identified by the World Health Organization (WHO) as the only mold among its list of critical fungal pathogens.Myeloid cells, CCR2+ inflammatory monocytes (CCR2+Mo), and neutrophils are necessary to control pulmonary Af infection.CCR2+Mo gives rise to monocyte-derived dendritic cells (mo-DCs) and alveolar macrophages (AMs), key effector populations within the CD11c+ cell compartment.Our lab found that mice with defective expression of the type I interferon receptor (IFNAR1) had increased mortality and fungal burden.Furthermore, mice with a deficiency in STAT1, a key transcription factor activated by IFNs, on CCR2+Mo and CD11c+cells had increased mortality, reduced ROS formation, and diminished control of fungal burden.Therefore, we hypothesize that type I IFNs regulate antifungal immunity through CCR2+Mo and CD11c+cells, enhancing host defense against Af infections. Methods Using CCR2creIFNAR1fl/fl and CD11ccreIFNAR1fl/fl mice, we evaluated the immune cell recruitment in bronchoalveolar lavage fluid and lung tissue.Antifungal responses were examined by ROS production and visualization of conidia-killing using the Af-FLARE strain.Furthermore, monocytes were sorted and analyzed for bioenergetic function using the Seahorse Analyzer and fungal burden by colony-forming units. Results Mice deficient in type I IFNs in CCR2+Mo and CD11c+cells succumbed to IA with impaired ROS production and increased fungal burden.Af viability was significantly increased in CCR2creIFNAR1fl/fl mice, indicating decreased fungal clearance.The seahorse analysis demonstrated decreased oxygen consumption rate, spare respiratory capacity, and extracellular acidification rate in monocytes from CCR2creIFNAR1fl/fl. Conclusion These findings can reveal new immunological mechanisms to enhance host defense against Af and could also help improve outcomes in patients at risk for aspergillosis. Funding Source n/a Topic Categories Microbial, Parasitic, and Fungal Immunology (MPF)