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bioRxiv2024

Characterization and Antiatherogenic Potential of P3R99 Monoclonal Antibody Against Sulfated Glycosaminoglycans: Physicochemical and Functional Insights

Atherosclerosis is initiated by the retention of ApoB-containing lipoproteins in the arterial wall, mediated by glycosaminoglycan chains of proteoglycans. At the Center for Molecular Immunology, we are developing the P3R99 monoclonal antibody (mAb) to target this process. This study characterizes new P3R99 mAb variants expressed in CHO-K1 and HEK-293 cell lines. We compared these variants with the parental mAb from NS0 cells using SDS-PAGE, size exclusion and cation exchange chromatography, dynamic light scattering, peptide mapping, far-UV circular dichroism, and PNGase F deglycosylation. All variants exhibited a molecular size of [~]150 kDa, [~]99% purity, and similar average particle sizes (12.5-13.7 nm). They displayed a high {beta}-sheet content (>40%) and basic amino acids on the surface, with minor differences in peptide maps compared to the parental mAb. Notable differences were found in the content of acidic and basic species and glycosylation profiles. NS0-derived P3R99 had lower G0F content (10.39%), higher G1F (38.29%) and G2F (30.44%) levels, with more terminal galactose (83.07%) and sialylation (15.33%). In contrast, CHO-K1 and HEK-293 variants showed similar glycosylation patterns. Despite these differences, the antigen and atherosclerotic lesion recognition properties of the mAb were unaffected in vitro. Biodistribution studies in Sprague Dawley rats (1 mg, IV, n=3) revealed preferential accumulation of the new P3R99 variants in aortas and reduced LDL arterial retention (1 mg, IP). Passive administration of the mAbs (2 mg every three days, three IV doses, n=6-7) in a Lipofundin 20%-induced atherosclerosis NZW rabbit model also demonstrated preferential accumulation in aortas and reduced atherosclerosis, with 60% of treated rabbits not developing lesions. These results suggest that the P3R99 mAb derived from CHO-K1 and HEK-293 cells retains its antiatherogenic properties despite structural differences from the NS0-derived mAb associated with the different expression systems.

immunology↗

bioRxiv2024

MHC class I / II restricted T cell epitopes from clinical isolate of Mycobacterium tuberculosis: A potential candidate for vaccine development for Tuberculosis

Tuberculosis is major challenge to the health care system with TB associated death rates increasing annually. Optimum management of TB (particularly latent or MDR cases) warrants use of immunological approaches like subunit or peptide-based vaccination for tailoring effector immunity in patients. Since MHC class I is a potent enhancer element of host immunity and effective in clearing large variety of intracellular pathogens or tumors. In this context, we explore whether MHC-I restricted peptides from clinical isolates of M. tuberculosis can be used as an adjuvant for augmenting host immune responses. In the present study, we have synthesized various peptides from clinical isolates of M. tuberculosis which were having high affinity for Class I MHC molecules as potential immune enhancer for T cell or iNKT cell populations. We have evaluated the immunogenic potential of various MHC class I restricted epitopes (Rv2588c, Rv1357, Rv0148, Rv2973, Rv2557 and Rv2445) which were derived from clinical isolates of M. tuberculosis on increased proliferation of T or iNKT cells, release of IFN gamma secreted by T cells as well as NO as indicative parameters of immuno-stimulation. As expected, FACS and ELISA data clearly revealed that these peptides were potentially immunogenic for PBMCs from both healthy as well as 10 HC PTB patients. Our data clearly demonstrated a significant immune response in the PBMC from w PTB patients over healthy individuals which mimicked booster response. Our cytokine and nitric oxide data further revealed the influence of these peptides on sensitizing innate immune response as well. SignificanceOur study demonstrates the significance of MHC class I restricted peptides from M. tuberculosis for inducing potential immunogenic responses in host that may qualify them as potent vaccine candidate. To the best of our knowledge this is the first immune monitoring protocol describing the impact of synthetic novel MHC class I restricted T-cell epitope (Rv2588c, Rv0148) on cell mediated and innate immune response in PBMC populations and suggests their potential as vaccine candidate

immunology↗

bioRxiv2024

Sca-1 expression depicts pro-inflammatory murine neutrophils under steady state and pathological conditions

Neutrophils play a crucial role in various pathophysiological conditions, yet targeting them for therapeutic intervention has been discouraged due to the associated risk of infections. Thus, identification of neutrophil subsets and their involvement in inflammatory conditions is warranted for targeted therapeutic strategies. This study, through screening of surface proteins on neutrophils isolated from different tissue microenvironments, identified a distinct neutrophil subset, CD11b+Ly6G+Sca1+ neutrophils, expressing Stem cell antigen-1 (Sca-1). Interestingly, these Sca1pos neutrophils were more abundant in the liver than BM, blood, and lungs. Further analysis revealed that Sca1pos neutrophils are mature and activated with enhanced effector functions, including superoxide generation, phagocytosis, degranulation, and NETosis. Tracing studies demonstrated ageing-independent characteristics of Sca1pos neutrophils. Remarkably, Sca1pos pro-inflammatory neutrophils promote T cell proliferation through ROS, while inhibition of Sca-1 restores T cell proliferation and ROS generation. Intriguingly, inflammatory as well as metabolic cues induce the transition of conventional neutrophils (Sca1neg) to Sca1pos neutrophils and differentiation of progenitors (granulocyte monocyte progenitors, GMPs) into Sca1pos neutrophils. Furthermore, in vivo models of acute inflammation, peritonitis, and chronic inflammatory condition, non-alcoholic steatohepatitis (NASH), exhibit an increase of Sca1pos neutrophils at inflammatory sites, while the pharmacological approach using NAC specifically mitigates the expansion of these pro-inflammatory neutrophils. Collectively, our findings unveil a novel subset of Sca1pos neutrophils with implications for inflammation. Significance StatementNeutrophilic inflammation remains the leading driver in infectious and inflammatory diseases. Targeting neutrophil populations remained un-recommended due to hampering the immunological functions of neutrophils. The heterogeneity of neutrophils provides the perspective to target altered neutrophil subsets, but subtle changes defining neutrophil subsets make it complex and ambiguous. Our study identified abundant expression of Sca1on distinct neutrophils under steady state and inflammation. Thus, we reported previously undefined Sca1pos pro-inflammatory neutrophil subsets and elucidated their regulation. This study further established their involvement in acute and chronic inflammatory conditions. This understanding may further pave the way toward targeting specific neutrophil subsets in pathologies characterized by neutrophilic inflammation. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=144 SRC="FIGDIR/small/613221v1_ufig1.gif" ALT="Figure 1"> View larger version (42K): org.highwire.dtl.DTLVardef@62f3eaorg.highwire.dtl.DTLVardef@7baed3org.highwire.dtl.DTLVardef@12da63org.highwire.dtl.DTLVardef@1bfcce9_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LICD11b+Ly6G+Sca1+ neutrophil subset identified with <1% presence in BM and >40% frequency in the liver. C_LIO_LIThese neutrophils are mature and activated, demonstrating enhanced effector functions. C_LIO_LISca1pos neutrophils promote T cell proliferation and display pro-inflammatory characteristics. C_LIO_LIConventional Sca1neg neutrophils transition into Sca1pos neutrophils in response to inflammatory signals, while progenitors undergo differentiation. C_LIO_LIBoth acute and chronic inflammatory models show the expansion of CD11b+Ly6G+Sca1+ neutrophils. C_LI

immunology↗

bioRxiv2024

Overcoming immune evasion from post-translational modification of a mutant KRAS epitope to achieve TCR-engineered T cell-mediated antitumor activity

T cell receptor (TCR)-T cell immunotherapy, in which T cells are engineered to express a TCR targeting a tumor epitope, is a form of adoptive cell therapy (ACT) that has exhibited promise against various tumor types. Mutants of oncoprotein KRAS, particularly at glycine-12 (G12), are frequent drivers of tumorigenicity, making them attractive targets for TCR-T cell therapy. However, class I-restricted TCRs specifically targeting G12-mutant KRAS epitopes in the context of tumors expressing HLA-A2, the most common human HLA-A allele, have remained elusive despite evidence an epitope encompassing such mutations can bind HLA-A2 and induce T cell responses. We report post-translational modifications (PTMs) on this epitope may allow tumor cells to evade immunologic pressure from TCR-T cells. A lysine side chain-methylated KRASG12V peptide, rather than the unmodified epitope, may be presented in HLA-A2 by tumor cells and impact TCR recognition. Using a novel computationally guided approach, we developed by mutagenesis TCRs that recognize this methylated peptide, enhancing tumor recognition and destruction. Additionally, we identified TCRs with similar functional activity in normal repertoires from primary T cells by stimulation with modified peptide, clonal expansion, and selection. Mechanistically, a gene knockout screen to identify mechanism(s) by which tumor cells methylate/demethylate this epitope unveiled SPT6 as a demethylating protein that could be targeted to improve effectiveness of these new TCRs. Our findings highlight the role of PTMs in immune evasion and suggest identifying and targeting such modifications should make effective ACTs available for a substantially greater range of tumors than the current therapeutic landscape. One-sentence summaryTumor cell methylation of KRASG12V epitope in HLA-A2 permits immune evasion, and new TCRs were generated to overcome this with engineered cell therapy.

immunology↗

bioRxiv2024

Dynamic responses of human lung innate and adaptive immune cells highlight the roles of genes at asthma risk loci

RationaleThe lung is a unique immunological niche with diverse immune cell types. The effects of stimulation through innate and adaptive immune receptors on human lung immune cells has largely been extrapolated from studies of blood immune cells. While multiple immune cell types and many genes have been implicated as contributing to asthma, the dynamics of these in human lung immune cells following activation will yield insights into asthma pathogenesis and lung immunity more broadly. Methods and MeasurementsHuman lung immune cells from 6 donors were isolated. Mixed leukocytes were treated separately with lipopolysaccharide (LPS), F(ab)2-anti-human-IgM/IgG + IL4 and anti-CD3/CD28 for 4 and 18 hours and underwent single cell RNA sequencing (scRNAseq). Lung immune cell types were annotated, and gene expression compared across conditions. Genes at prior asthma-associated genetic loci were characterized across cell types, treatments and timepoints. Expression of non-classical class II genes associated with asthma, HLA-DQA2 and HLA-DQB2, and their protein products was characterized with immunohistochemistry. Main ResultsWe characterized gene expression in 116,697 lung immune cells. Cell-, treatment-, and timepoint-specific effects on gene expression were detected in all lung immune cell populations. Correlation of gene expression between lung and blood lymphocyte populations decreased following stimulation. Among the genes that were differentially expressed, 97 receptor:ligand pairs had changes with treatments. 96.0% of genes at asthma risk loci demonstrated differential expression in at least one cell type and at least one treatment. B cells were the cell type with the highest expression of HLA-DQA2 and HLA-DQB2 which increased with anti-IgM/IgG treatment and the HLA-DQ{beta}2 protein was identified in lung B cells from a donor with asthma. ConclusionsHuman lung immune activation elicits a broad range of cellular responses that deviate from those of blood immune cells and are relevant to asthma. Lung B cells expressing HLA-DQA2 and HLA-DQB2 appear to be involved in a novel antigen presentation pathway that contributes to asthma risk.

immunology↗

bioRxiv2024

Simultaneous Profiling of the Blood and Gut T and B Cell Repertoires in Crohn's Disease and Symptomatic Controls Illustrates Tissue-specific Alterations in the Immune Repertoire of Crohn's Disease Patients

Crohns disease (CD) is a clinical subset of inflammatory bowel disease that is characterized by patchy transmural inflammation across the gastrointestinal tract. Although the exact etiology remains unknown, recent findings suggest that it is a complex multifactorial disease with contributions from the host genetics and environmental factors such as the microbiome. We have shown that the T cell repertoire of CD patients harbors a group of highly expanded T cells which hints toward an antigen-mediated pathology. To profile the immunological signature of CD at a high-resolution we simultaneously profiled the {beta} and {gamma}{delta} T cell repertoire in addition to the B cell repertoire of both the blood and the colonic mucosa for 27 treatment-naive CD patients and 27 age-matched symptomatic controls. Regardless of disease, we observed multiple physiological differences between the immune repertoire of blood and colonic mucosa. Additionally, by comparing the repertoire of CD patients relative to controls, we observed different alterations that were only detected in the blood or colonic mucosa. These include a depletion of mucosal-associated invariant T (MAIT) cells in the blood repertoire, an expansion of TRAV29/DV5-TRAJ5+ clonotypes and a significant depletion of multiple IGHV3-33-IGHJ4+and IGHV3-33-IGHJ6+ clonotypes in the blood and gut IGH repertoire of CD patients. In conclusion, our findings highlight the importance of studying the immune repertoire in a tissue-specific manner and the need to profile the T and B cell immune repertoire of gut tissues.

immunology↗

bioRxiv2024

GDF3 promotes adipose tissue macrophage-mediated inflammation via altered chromatin accessibility during aging

Age-related susceptibility to sepsis and endotoxemia is poorly defined, although hyperactivation of the immune system and the expansion of the visceral adipose as an immunological reservoir are underlying features. Macrophages from older organisms exhibit substantial changes, including chronic NLRP3 inflammasome activation, genomic remodeling and a dysfunctional, amplified inflammatory response upon new exposure to pathogen. However, the mechanisms by which old macrophages maintain their inflammatory phenotype during endotoxemia remains elusive. We previously identified Gdf3, a TGF{beta} superfamily cytokine, as a top-regulated gene by age and the NLRP3 inflammasome in adipose tissue macrophages (ATMs). Here, we demonstrate that endotoxemia increases inflammatory (CD11c+) ATMs in a Gdf3- dependent manner in old mice. Lifelong systemic or myeloid-specific deletion of Gdf3 leads to reduced endotoxemia- induced inflammation, with decreased CD11c+ ATMs and inflammatory cytokines, and protection from hypothermia. Moreover, acute blockade of Gdf3 using JQ1, a BRD4 inhibitor, phenocopies old mice with lifelong Gdf3-deficiency. We show that GDF3 promotes the inflammatory phenotype in ATMs by phosphorylating SMAD2/3. Mechanistically, the differential chromatin landscape of ATMs from old mice with or without myeloid-driven Gdf3 indicates that GDF3- SMAD2/3 signaling axis shifts the chromatin accessibility of ATMs towards an inflammatory state during aging. Furthermore, pharmaceutical inhibition of SMAD3 with a specific inhibitor of SMAD3 (SIS3) mimics Gdf3 deletion. SIS3 reduces endotoxemia-mediated inflammation with fewer CD11c+ ATMs and less severe hypothermia in old, but not young mice, as well as reduced mortality. In human adipose tissue, age positively correlates with GDF3 level, while inflammation correlates with pSMAD2/3 level. Overall, these results highlight the importance of GDF3-SMAD2/3 axis in driving inflammation in older organisms and identify this signaling axis as a promising therapeutic target for mitigating endotoxemia-related inflammation in the aged.

immunology↗

bioRxiv2024

Perforin-2 is dispensable for host defense against Aspergillus fumigatus and Candida albicans

Myeloid phagocytes are essential for antifungal immunity against pulmonary Aspergillus fumigatus and systemic Candida albicans infections. However, the molecular mechanisms underlying fungal clearance by phagocytes remain incompletely understood. In this study, we investigated the role of perforin-2 (Mpeg1) in antifungal immunity. We found that Mpeg1-/- mice generated on a mixed C57BL/6J-DBA/2 background exhibited enhanced survival, reduced lung fungal burden, and greater neutrophil fungal killing activity compared to wild-type C57BL/6J (B6) mice, suggesting that perforin-2 may impair antifungal immune responses. However, when we compared Mpeg1-/- mice with co-housed Mpeg+/+ littermate controls, these differences were no longer observed, indicating that initial findings were likely influenced by differences in the murine genetic background or the microbiota composition. Furthermore, perforin-2 was dispensable for antifungal immunity during C. albicans bloodstream infection. These results suggest that perforin-2 is not essential for host defense against fungal infections in otherwise immune competent mice and highlight the importance of generating co-housed littermate controls to minimize murine genetic and microbiota-related factors in studies of host defense mechanisms. IMPORTANCEAspergillus fumigatus is the leading cause of invasive aspergillosis (IA), which is associated with significant mortality, particularly in immunocompromised patients such as those with acute leukemia or undergoing hematopoietic stem cell transplants, where death rates reach 40-50% despite standard care. Treatments for IA remain limited and resistance to antifungals is emerging, leading the World Health Organization to recently classify A. fumigatus as a critical priority fungal pathogen. A greater understanding of how the immune system clears A. fumigatus could lead to host-directed therapies that could complement our current armamentarium of antifungal drugs and improve patient outcomes. Our findings reveal that perforin-2 is not essential for antifungal immunity against A. fumigatus in otherwise immune-competent mice and underscore the necessity of using co-housed littermate controls to avoid confounding factors in immunological studies.

immunology↗

bioRxiv2024

Avian interferon regulatory factor (IRF) family reunion: IRF3 and IRF9 found

Interferon regulatory factors (IRFs) are a family of transcription factors in jawed vertebrates with important functions in immunity and many other key cellular processes. The genomes of most vertebrates encode ten IRF genes (IRF1 to IRF10). IRF3 and IRF9 have key roles in the interferon (IFN) induction and signaling. Most of our knowledge about the IFN pathways originates from the study of the mammalian IFN system, and the description of the corresponding avian components is not as complete. Both IRF3 and IRF9 were considered missing from the chicken genome and also from the genomes of all other avian species. Here we describe multiple avian IRF3 and IRF9 genes, all with difficult GC-rich sequence context which prevented their earlier characterization. IRF3 orthologs are more narrowly distributed and are present in the avian infraclass Palaeognathae residing in a syntenic genomic locus shared with other vertebrates. In contrast, IRF9 orthologs were found in most avian species with the notable exception of the order Galliformes. In about half of the avian orders analyzed, IRF9 was located in noncanonical chromosomal positions indicating past evolutionary translocations. Importantly, phylogenetic analysis confirmed the correct orthology of all newly described avian IRFs. We performed a series of experiments using duck (Anas platyrhynchos) IRF9, confirming its key role in the IFN signaling pathway. Knockout of IRF9 in duck embryonal fibroblasts decreases the induction of IFN-stimulated genes (ISGs). Full induction of ISGs in duck cells requires both intact IRF9 and canonical IFN-stimulated response element (ISRE). Lastly, intact IRF9 is needed for IFN-mediated protection of duck cells against vesicular stomatitis virus (VSV)-induced cytopathic effect. The identification of avian IRFs fills an important gap in our understanding of avian immunology and brings new questions related to the evolution of the IRF family.

immunology↗

bioRxiv2024

Relation of CMV and brain atrophy to trajectories of immunosenescence in diverse populations

Immunosenescence (ISC), the aging of the immune system, has largely been studied in populations of European descent. Here, circulating immune cell cytometric data from African-American, Hispanic, and non-Hispanic White participants were generated. Known and novel age effects were identified using either a meta-analysis approach or a parallel genetic approach. Most results are consistent across the three populations, but some cell populations display evidence of heterogeneity, such as a PD-L1+CD56+ NK cell subset. The study estimated "Immunological Age" (IA) during physiologic aging. While we found no relation of IA to Multiple Sclerosis, IA is associated with entorhinal cortex atrophy, a presymptomatic feature of Alzheimers disease, linking neurodegeneration and peripheral immunity. ISC trajectories were also inferred, highlighting age, CMV status, and genetic ancestry as key influences. Our assessment offers reference ISC trajectories for personalization of assessments of immune function over the life course in diverse populations.

immunology↗

bioRxiv2024

PD-L1+ Neutrophils mediate Susceptibility during Systemic Inflammatory Response in Non-Alcoholic Fatty Liver Disease

Metabolic Dysfunction-Associated Steatotic Liver Disease (MAFLD) is a pathological condition affecting many individuals worldwide. Patients with MAFLD are more susceptible to systemic inflammation, including endotoxemia, which accelerates the progressive liver damage. However, the immunological mechanisms that trigger the hyper-inflammatory responses in individuals with MAFLD remain unknown. In the present study, we reported that short-term HFCD (Choline Deficient High Fat Diet)-fed mice, which did not show significant signs of hepatic damage and inflammation in the first two weeks, are more susceptible to a non-severe sepsis-like systemic inflammation induced by LPS challenge. Mechanistically, endotoxemic mice show an excessive accumulation of NK-producing IFN-{gamma} cells in liver tissue, which trigger the recruitment and polarization of a distinct subset of neutrophils, characterized by high expression of PD-L1 and massive release of TNF-. Remarkably, genetic inhibition of IFN-{gamma} or pharmacological blockade of PD-L1 effectively modulated the excessive recruitment of these neutrophils to the liver and TNF- production, thereby preventing hepatic damage and reducing the severity of host mortality. Thus, these results support the design of novel effective strategies to control hyperinflammatory responses in septic HFCD patients and consequently improve their survival.

immunology↗

bioRxiv2024

Early spatiotemporal evolution of the immune response elicited by adenovirus serotype 26 vector vaccination in mice

As the first responder to immunological challenges, the innate immune system shapes and regulates the ensuing adaptive immune response. Many clinical studies evaluating the role of innate immunity in initiating vaccine-elicited adaptive immune responses have largely been confined to blood due to inherent difficulty in acquiring tissue samples. However, the absence of vaccine-site and draining lymph node information limits understanding of early events induced by vaccination that could potentially shape vaccine-elicited immunity. We therefore utilized a mouse model to investigate the spatiotemporal evolution of the immune response within the first 24 hours following intramuscular adenovirus serotype 26 (Ad26) vector vaccination in tissues. We show that the Ad26 vaccine-elicited innate immune response commences by one hour and rapidly evolves in tissues and blood within the first 24 hours as reflected by the detection of cytokines, chemokines, cellular responses, and transcriptomic pathways. Furthermore, serum levels of IL-6, MIG, MIP-1, and MIP-1{beta} at 6 hours post-vaccination correlated with the frequency of vaccine-elicited memory CD8+ T cell responses evaluated at 60 days post-vaccination in blood and tissues. Taken together, our data suggests that the immune response to Ad26 vector vaccination commences quickly in tissues by one hour and that events by as early as 6 hours post-vaccination can shape vaccine-elicited CD8+ T cell responses at later memory time points. IMPORTANCEPrior studies have largely concentrated on innate immune activation in peripheral blood following vaccination. In this study, we report the detailed spatial and temporal innate immune activation in tissues following Ad26 vaccination in mice. We observed rapid innate activation rapidly not only in peripheral blood but also in draining lymph nodes and at the site of inoculation. Our findings provide a more detailed picture of host response to vaccination than previously reported.

immunology↗

bioRxiv2024

Systemic effects of oral tolerance improve the healing of several and concomitant wounds in different body parts

Throughout our lives, we are continually subjected to different situations that can result in several and concomitant wounds to different parts of our body. The healing of these wounds is essential to maintaining health. Inflammation is an important step in wound healing, but in cases of intense or prolonged inflammation, pathological scarring or non-closure of the wound may occur. Assuming that leukocytes participate in wound healing and that it is possible to intervene systemically with inflammation, we investigated a way to promote better repair of multiple wounds that may occur at the same time. Oral tolerance is an immunological phenomenon that result from protein intake and that have systemic effects on inflammation. Previous works have shown that parenteral injection of tolerated proteins reduces the inflammatory infiltrate and improves skin wound healing. Herein we tested whether the injection of tolerated proteins improves the healing of several wounds in different body parts, such as on the skin of the back and in the external ear (the auricle). To induce oral tolerance to ovalbumin (OVA), eight weeks old C57BL/6 mice drank egg white diluted 1:5 in water for 3 consecutive days. Control mice drank water. Seven days after oral treatment, mice were submitted to excisional injuries in the skin of the back (6 mm) and in the ears (4 mm). Minutes before the injuries, the mice received an intraperitoneal injection of OVA + Al(OH)3. Seven and 40 days after injuries, tissue samples were collected and processed for histological analysis of the wounds. The results show that the injection of OVA in animals that drank OVA reduced the inflammatory infiltrate in all lesions. Besides, injection of OVA in animals that drank OVA promoted better organization of the extracellular matrix, with thicker and intertwined collagen fibers in the neodermis, resulting in smaller scars in the skin. Furthermore, the healing area of the ears of OVA-tolerant animals showed chondrocyte aggregates and less obvious fibrous scar tissue compared with control animals. In conclusion, systemic effects of oral tolerance positively influenced the healing of several lesions in different body parts.

immunology↗

bioRxiv2024

Distinct maternofetal immune signatures delineate preterm birth onset following urinary tract infection

Preterm birth is the leading cause of infant mortality resulting in over one million neonatal deaths annually. Maternal urinary tract infection (UTI) during pregnancy increases risk for preterm birth; however, biological processes mediating UTI-associated preterm birth are not well-described. We established a murine maternal UTI model in which challenge with uropathogenic E. coli resulted in preterm birth in about half of dams. Dams experiencing preterm birth displayed excessive bladder inflammation and altered uteroplacental T cell polarization compared to non-laboring infected dams, with no differences in bacterial burdens. Additional factors associated with preterm birth included higher proportions of male fetuses and lower maternal serum IL-10. Furthermore, exogenous maternal IL-10 treatment absolved UTI-associated preterm birth but contributed to fetal growth restriction in this model. Using urine samples from a cohort of human pregnancies with or without UTI, we correlated urinary cytokines with birth outcomes and urine culture status. These analyses yielded a non-invasive, highly predictive three-model system for evaluating preterm birth risk implicating cytokines IL-10, IL-15, IL-1{beta}, and IL-1RA. Our unique bimodal murine model coupled with patient samples provides a platform to investigate immunological and microbial factors governing UTI-associated preterm birth, revealing novel therapeutic opportunities to predict or prevent preterm birth.

immunology↗

bioRxiv2024

Focused learning by antibody language models using preferential masking of non-templated regions

Existing antibody language models (LMs) are pre-trained using a masked language modeling (MLM) objective with uniform masking probabilities. While these models excel at predicting germline residues, they often struggle with mutated and non-templated residues, which are crucial for antigen-binding specificity and concentrate in the complementarity-determining regions (CDRs). Here, we demonstrate that preferential masking of the non-templated CDR3 is a compute-efficient strategy to enhance model performance. We pre-trained two antibody LMs (AbLMs) using either uniform or preferential masking and observed that the latter improves residue prediction accuracy in the highly variable CDR3. Preferential masking also improves antibody classification by native chain pairing and binding specificity, suggesting improved CDR3 understanding and indicating that non-random, learnable patterns help govern antibody chain pairing. We further show that specificity classification is largely informed by residues in the CDRs, demonstrating that AbLMs learn meaningful patterns that align with immunological understanding.

immunology↗

bioRxiv2024

Dissecting the Heterogeneity and Tumor-Associated Dynamics of Human Liver Group I ILC via scRNA Sequencing Data

Single-cell transcriptome analysis has made outstanding contributions to the identification of new cell lineages and the study of cancer immune microenvironment. Yet, the characterization of human liver ILC1s and their dynamic changes in the tumor microenvironment have not been thoroughly studied at this detailed level. Here, we performed an integrated analysis of mouse and human liver immune cells to identify human liver ILC1 based on identified mouse liver ILC1 and to verify its functional similarity. Additionally, our findings highlighted the different expression patterns of the EOMES gene in human versus mouse group I ILCs, suggesting its reduced regulatory significance in human liver NK cells and ILC1 compared to murine models. A unique subset of CD127hi intermediate ILCs (intILCs) was identified, exhibiting traits of both human liver NK cells and ILC1. Single-cell sequencing data analysis and TCGA were utilized to characterize the distinct alterations in the genes and functions of NK cells, ILC1, and intILCs in human hepatocellular carcinoma. It was found that the dynamic changes of ILC1 and intILCs, along with some of their subpopulations, may be key factors in tumor progression. This study provided new insights into the identification of ILC1 in human liver and the immunologic changes and mechanism of group I ILCs in the tumor microenvironment, and these findings may be applicable to improving the diagnosis and treatment of hepatocellular carcinoma. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=193 SRC="FIGDIR/small/619932v1_ufig1.gif" ALT="Figure 1"> View larger version (52K): org.highwire.dtl.DTLVardef@1976bbeorg.highwire.dtl.DTLVardef@228ce4org.highwire.dtl.DTLVardef@4fcdd4org.highwire.dtl.DTLVardef@1522eff_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2024

Temporal dynamics of inflammatory and transcriptome changes in the myocardium in a murine model of cardiac arrest

BackgroundIt is increasingly recognized that inflammatory changes play a significant role in myocardial injury following cardiac arrest. However, the pathophysiological mechanisms responsible for functional changes in the myocardium following the event remain incompletely understood. Here, we characterize the transcriptomic and immunological changes in a mouse model of cardiac arrest to explore the roles of epinephrine and ischemia/reperfusion on myocardial inflammation. Methods and ResultsMale and female mice (C57BL/6J) were divided into three groups: (1) Naive (anesthesia only), (2) Sham (anesthesia + saline injection + epinephrine), and (3) Arrest (8-minute cardiac arrest followed by resuscitation (anesthesia + potassium chloride + epinephrine). We monitored the animals over 0.5, 1, 3, and 7 days with serial echocardiography. Hearts were harvested for transcriptomic analysis and inflammatory changes as assessed by flow cytometry and immunohistochemistry. Plasma samples were collected for cytokine signaling analysis by multiplex ELISA. Our data indicate that the cardiac arrest group have increased infiltrating neutrophils in the myocardium and elevated plasma levels of pro-inflammatory cytokines, peaking at 0.5 days after the initial insult and subsequently resolving. In contrast, Sham mice displayed less pronounced inflammatory changes, which peaked at 1 day after the procedure. Notably, these inflammatory alterations coincided with cardiac stunning, as demonstrated by echocardiography. ConclusionsIn our murine model of cardiac arrest, epinephrine exposure and ischemia/reperfusion induce significant inflammatory changes in the myocardium with increased infiltrating neutrophils and elevated circulating cytokines which subsequently resolve in a time-dependent manner. These changes correlate with improving cardiac function by echocardiography.

immunology↗

bioRxiv2024

Predicting Immunogenic CD4+ T Cell Epitopes in Bacteria Using Antigen and Peptide Features

BackgroundT cell epitope prediction methods have been broadly utilized to facilitate epitope discovery in infectious agents and help design reagents, diagnostics, and vaccines. Current prediction methods are mainly focused on peptide presentation by MHC molecules, which is a necessary but not sufficient requirement for an epitope. For complex pathogens such as bacteria, it would be desirable to make such predictions more specific to limit the number of candidates that have to be experimentally tested. ObjectiveTo develop a machine learning-based prediction model that integrates both peptide-level and antigen-level features to improve the specificity of CD4+ T cell epitope predictions for bacteria. MethodsWe used a dataset of 20,216 peptides from Mycobacterium tuberculosis (Mtb), tested for T cell recognition in Mtb-infected participants, that led to the discovery of n = 144 peptide epitopes. For each peptide, we calculated six peptide-level features (e.g. MHC class II binding predictions and conservation scores) and six antigen-level features (e.g. including RNA expression levels and subcellular localization scores). Three machine learning algorithms--Random Forest, Gradient Boosting, and XGBoost--were trained using stratified, 5-fold cross-validation and combined into an ensemble model. Experimental validation was performed on Streptococcus pneumoniae peptides, using ex vivo IFN{gamma} assays to confirm the predictive performance. ResultsThe ensemble model achieved an ROC-AUC of 0.91 in predicting immunogenic peptides in the Mycobacterium tuberculosis (Mtb) dataset. Gene expression and conservation were identified as the most impactful features, followed by MHC class II binding predictions. When validated on an independent Bordetella pertussis dataset, the model demonstrated accurate predictive capability, especially for peptides with broad recognition in the participant cohort (ROC-AUC up to 0.82). Prospectively applying the model to Streptococcus pneumoniae, we synthesized peptides predicted by our ensemble model to be immunogenic or non-immunogenic. Ex vivo testing with PBMCs from healthy participants showed that peptides predicted to be immunogenic elicited significantly higher IFN{gamma} responses than non-immunogenic peptides, validating the model. ConclusionsOur machine learning approach, integrating both peptide and antigen features, effectively predicts immunogenic CD4+ T cell epitopes across different bacterial pathogens. This method enhances epitope selection efficiency, aiding vaccine development and immunological research by reducing the need for extensive experimental screening.

immunology↗