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Results for “immunology”

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bioRxiv2019

Deep metagenomics examines the oral microbiome during dental caries, revealing novel taxa and co-occurrences with host molecules

Dental caries is the most common chronic infectious disease globally. The microbial communities associated with caries have mainly been examined using relatively low-resolution 16S rRNA gene amplicon sequencing and/or using downstream analyses that are unsound for the compositional nature of the data provided by sequencing. Additionally, the relationship between caries, oral microbiome composition, and host immunological markers has not been explored. In this study, the oral microbiome and a panel of 38 host markers was analyzed across the saliva from 23 children with dentin caries and 24 children with healthy dentition. Metagenomic sequencing, followed by investigation using tools designed to be robust for compositional data, illustrated that several Prevotella spp. were prevalent in caries, while Rothia spp. were associated with the health. The contributional diversity (extent to which multiple taxa contribute to each pathway) of functional pathways present in the oral microbiome was decreased in the caries group. This decrease was especially noticeable in several pathways known to impede caries pathogenesis, including arginine and branched-chain amino acid biosynthesis. 10 host immunological markers were found to be significantly elevated in the saliva of the caries group, and microbe-metabolite co-occurrence analysis provided an atlas of relationships contributing to the bi-directional influence between the oral microbiome and the host immune system. Finally, 527 metagenome-assembled genomes were obtained from the metagenomics data, representing 151 species. 23 taxa were novel genera/species and a further 20 taxa were novel species. This study thus serves as a model analysis pipeline that will tremendously expand our knowledge of the oral microbiome and its relationship to dental caries once applied to large populations.

microbiology↗

bioRxiv2019

Stochastic model of T Cell repolarization during target elimination (I)

Cytotoxic T lymphocytes (T) and natural killer (NK) cells are the main cytotoxic killer cells of the human body to eliminate pathogen-infected or tumorigenic cells (i.e. target cells). Once a NK or T cell has identified a target cell, they form a tight contact zone, the immunological synapse (IS). One then observes a re-polarization of the cell involving the rotation of the microtubule (MT) half-spindle and a movement of the microtubule organizing center (MTOC) to a position that is just underneath the plasma membrane at the center of the IS. Concomitantly a massive relocation of organelles attached to MTs is observed, including the Golgi apparatus, lytic granules and mitochondria. Since the mechanism of this relocation is still elusive we devise a theoretical model for the molecular motor driven motion of the MT half-spindle confined between plasma membrane and nucleus during T cell polarization. We analyze different scenarios currently discussed in the literature, the cortical sliding and the capture-shrinkage mechanisms, and compare quantitative predictions about the spatio-temporal evolution of MTOC position and spindle morphology with experimental observations. The model predicts the experimentally observed biphasic nature of the repositioning process due to an interplay between spindle geometry and motor forces and confirms the dominance of the capture-shrinkage over the cortical sliding mechanism when MTOC and IS are initially diametrically opposed. We also find that the two mechanisms act synergetically, thereby reducing the resources necessary for repositioning. Moreover, it turns out that the localization of dyneins in the pSMAC facilitates their interaction with the MTs. Our model also opens a way to infer details of the dynein distribution from the experimentally observed features of the MT half-spindle dynamics. In a subsequent publication we will address the issue of general initial configurations and situations in which the T cell established two immunological synapses.

biophysics↗

bioRxiv2019

Antibody Bank: the Synthetic Cell Repertoire for Immunotherapy

The immune surveillance hypothesis is regarded as the intellectual underpinning of cancer immunology. We find that engineered T cell library displaying CAR constructs, where the antibody repertoires took the place of the antigen-recognition region, has the capacity to recognize nonself-antigens and showed antigen dependent clonal expanding and persistence. This process coupled with long-lasting anti-tumour effect in a broad spectrum of epithelial tumours. Moreover, CAR-T library showed robust immunologic memory and maintenance of diversity to recognize mutated or evolved tumours in both immunodeficiency and immunoresponsive murine tumor models. Large CAR library was further developed in logic-gated NK-92 cells to develop an off-the shelf therapeutic library. Thus, the biological behaviour of synthetic cell library is highly accordance with the immune surveillance function, which proved the way to rebuilt functional MHC unrestricted cellular immunity in vitro using synthetic method.

bioengineering↗

bioRxiv2019

ImmuCellAI: a unique method for comprehensive T-cell subsets abundance prediction and its application in cancer immunotherapy

The distribution and abundance of immune cells, particularly T-cell subsets, play pivotal roles in cancer immunology and therapy. There are many T-cell subsets with specific function, however current methods are limited in estimating them, thus, a method for predicting comprehensive T-cell subsets is urgently needed in cancer immunology research. Here we introduce Immune Cell Abundance Identifier (ImmuCellAI), a novel gene set signature-based method, for precisely estimating the abundance of 24 immune cell types including 18 T-cell subsets, from gene expression data. Performance evaluation on both our sequencing data with flow cytometry results and public expression data indicated that ImmuCellAI can estimate immune cells with superior accuracy than other methods especially on many T-cell subsets. Application of ImmuCellAI to immunotherapy datasets revealed that the abundance of dendritic cells (DC), cytotoxic T, and gamma delta T cells was significantly higher both in comparisons of on-treatment vs. pre-treatment and responders vs. non-responders. Meanwhile, we built an ImmuCellAI result-based model for predicting the immunotherapy response with high accuracy (AUC 0.80~0.91). These results demonstrated the powerful and unique function of ImmuCellAI in tumor immune infiltration estimation and immunotherapy response prediction. The ImmuCellAI online server is freely available at http://bioinfo.life.hust.edu.cn/web/ImmuCellAI/.

bioinformatics↗

bioRxiv2025

Insights into Aspergillus fumigatus morphogenesis and pathogenesis through the putative lipid transporter ArvA

Aspergillus fumigatus poses a significant threat to human well-being, in part due to the increasing emergence of strains resistant to frontline antifungal therapy. In this study, we observe that the gene, arvA, is required for A. fumigatus morphogenesis, antifungal drug susceptibility, and cell wall homeostasis. Intriguingly, our study reveals novel morphological and growth aberrations in the absence of arvA. Loss of arvA results in hyper-swollen conidia that give rise to stunted, polarity-deficient hyphae in numerous environmental conditions, indicating a pivotal role for arvA in A. fumigatus morphogenesis. Surprisingly, despite these severe in vitro morphological and cell wall defects, arvA was not required for morbidity and mortality in immunologically distinct murine models of invasive pulmonary aspergillosis (IPA). However, growth in natural calf lung surfactant was able to normalize{Delta} arvA growth with the wild-type strain suggesting lung surfactant may partially complement the severe in vitro morphological defects of arvA loss in vivo. Taken together our observations reveal arvA as a mediator of A. fumigatus antifungal drug susceptibility and highlight the complex and ill-defined pulmonary nutrient environments role in mediating A. fumigatus pathogenesis and disease progression. IMPORTANCEAspergillus fumigatus is a challenging fungal pathogen in the clinic in part due to increasing azole drug resistance. In this study, we observe that loss of the A. fumigatus gene arvA results in increased azole susceptibility and significant in vitro morphological changes highlighted by hyper-swollen conidia that yield stunted and polarity deficient hyphae. Importantly, despite these severe in vitro morphological and growth abnormalities,{Delta} arvA surprisingly retains full pathogenicity and virulence in two immunologically distinct murine models of invasive pulmonary aspergillosis. These results challenge our understanding of the in-host environment and how it mediates fungal morphogenesis and pathogenesis. These results, consequently, not only enhances our understanding of the role of arvA in A. fumigatus morphogenesis and drug susceptibility, but further emphasizes the importance of in vivo animal models in fully evaluating potential antifungal drug targets.

microbiology↗

bioRxiv2025

Using social network analysis and non-invasive antibody detection to explore pathogen exposure in wildlife communities

Understanding the dynamics of infectious diseases in ecosystems shared with wildlife is a priority for preventing future threats to human, animal health and conservation. However, the collection of ecological and epidemiological data on wild populations has relied on invasive and costly methods which limited the capacity of investigation. Recent technological developments have changed this trend. In Hwange national park and its surrounding area in Zimbabwe, we combined camera trap-based ecological monitoring over a period of 12 months, covering 14 water holes with a fecal-based antibody survey in 16 large herbivore species. A survey involving 52,829 pictures as well as 629 faecal samples collected every 15 days, has shown Through the modelling of multispecies contact networks, the risk of infection exposure at species-level is predicted. Coupled with foot-and-mouth disease virus (FMDV) antibody information, the role of each species in the dynamics of infectious diseases is explored. This study highlights how community networks can provide valuable insights into the functional epidemiological role of wildlife populations. Rather than establishing transmission routes, our aim is to propose a scalable and non-invasive surveillance framework that identifies priority species and areas for epidemiological monitoring in complex ecological systems. Significance StatementMonitoring disease circulation in wildlife is often hindered by the difficulty of collecting epidemiological data. We propose a novel non-invasive approach that combines species interaction networks derived from camera-trap data with non-invasive antibody detection to explore the exposure patterns of large herbivore communities to foot-and-mouth disease virus (FMDV). By linking the position of species in contact networks with their immunological status, we demonstrate the potential of using ecological centrality as a proxy for identifying key hosts in transmission or indicator species for pathogen circulation. Beyond FMDV, this framework can be adapted to other pathogens for which non-invasive immunological assays are currently being developed, making it broadly relevant for wildlife disease surveillance in remote or protected areas.

ecology↗

bioRxiv2025

Immune-mediated Engagement of T Regulatory Cells with Tumor Cells Results in Trogocytosis and Tumor Cell Killing.

Despite advances in treatment, >50% of patients with advanced melanoma are unresponsive to current therapies. Using the B78-D14 melanoma model (GD2+/MHC-I-/MHC-II+), we can cure mice with a regimen that includes radiation therapy (RT) in combination with immunocytokine (IC; anti-GD2 monoclonal antibody linked to IL-2) while establishing immunological memory. We interrogated the role of T cells in the antitumor and memory responses following RT+IC. We show a requirement for CD4, but not CD8 T cells, to achieve both the initial and memory responses. Upon IC-induced cell-cell contact, subsets of CD4 T cells, including Foxp3 T regulatory cells, trogocytose GD2 from tumor cells, acquire cytotoxic granules, and kill tumor cells. These results were confirmed using human tumor cell lines. These findings reveal that CD4 T regulatory cells, upon immunologically-induced binding to tumor cells, can trogocytose tumor antigens and directly kill tumor cells, redefining their potential role in antitumor immunity.

cancer biology↗

bioRxiv2026

UnivAIRRse: A Unified Framework for Organizing and Comparing Adaptive Immune Receptor Repertoire Simulators

Adaptive immune receptor repertoire sequencing (AIRR-seq) enables large-scale profiling of B- and T-cell receptor diversity and has become a cornerstone of modern computational immunology. However, AIRR-seq provides only a partial and lossy molecular snapshot of immune dynamics, lacking explicit ground truth for clonal ancestry, lineage trajectories, antigen specificity, and longitudinal immune evolution. This limitation complicates benchmarking, method validation, and mechanistic interpretation of repertoire analysis pipelines. Here, we introduce UnivAIRRse, a unified hierarchical framework that organizes AIRR simulators within a shared conceptual coordinate system spanning five operational levels, from observed sequence data to the theoretical generative potential of the adaptive immune system. By explicitly distinguishing sequence-, clonal-, specificity-, repertoire-, and generative-level representations, UnivAIRRse enables systematic comparison of simulator assumptions, biological scope, abstraction level, and application focus. To our knowledge, this is the first review to formalize such a unified structure across biological, computational, and functional layers of AIRR simulation. Using this framework, we review how simulation supports benchmarking, strengthens computational inference, and enables multi-scale investigation of immune repertoire formation and evolution. We identify persistent limitations in existing simulators, including incomplete biological context, limited modularity, restricted interoperability, and overreliance on AIRR-seq as a molecular proxy for complex spatiotemporal immune processes. To operationalize this framework, we provide an interactive web-based AIRR Simulation Landscape Explorer (publicly available at https://www.imgt.org/AIRR-Simulator/) that enables dynamic filtering and comparison of simulators across biological scope, abstraction level, output fidelity, and application focus. Finally, we outline emerging directions toward digital-twin-ready immune simulation, emphasizing modular architectures, longitudinal multi-omic integration, uncertainty quantification, and dynamic model updating. By providing a coherent conceptual and operational coordinate system, UnivAIRRse establishes a foundation for reproducible, interpretable, and clinically actionable modeling of adaptive immune repertoires, bridging current simulation practices with the next generation of predictive and personalized immunological modeling. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=133 SRC="FIGDIR/small/706510v1_ufig1.gif" ALT="Figure 1"> View larger version (34K): org.highwire.dtl.DTLVardef@7a5a95org.highwire.dtl.DTLVardef@d127f1org.highwire.dtl.DTLVardef@19545c9org.highwire.dtl.DTLVardef@118cc74_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioinformatics↗

bioRxiv2026

A druggable redox switch on SHP1 controls macrophage inflammation

Immunological proteins are major disease targets, yet most remain undrugged. Post-translational redox modification of cysteine residues has emerged as an important mode of immune cell regulation, particularly in macrophage cytokine responses. Here, we develop a strategy for systematic discovery and small-molecule functionalization of redox-regulated cysteines on immunological proteins. Using deep redox proteomics, we annotate 788 in vivo redox-regulated cysteines across diverse immune-relevant protein domains. We demonstrate how these sites enable cysteine-directed pharmacology through discovery of a novel cysteine activation site on the immune regulator SHP1. Targeting Cys102, we develop a highly selective covalent agonist, SCA, which binds the N-SH2 domain to relieve autoinhibition and activate SHP1. In mouse and human macrophages, SCA selectively engages SHP1 Cys102, antagonizing IRAK signaling and LPS-induced pro-inflammatory cytokine production. Together, this work identifies a druggable cysteine redox switch controlling macrophage cytokine responses and provides a compendium of redox-regulated sites for therapeutic development.

biochemistry↗

bioRxiv2026

SR2P: an efficient stacking method to predict protein abundance from gene expression in spatial transcriptomics data

Spatial transcriptomics data are largely available with RNA expression alone, limiting the detection of cell states defined by surface protein abundance. The lack of multi-omics spatial data limits the ability to identify immune cells and their signaling in the tumor microenvironment, as most solid tumors are immunologically poor and exhibit protein-RNA abundance discordance in critical immune cell surface markers. Although emerging technologies enable spatial multi-omics profiling, technical and cost constraints remain a hurdle. We introduce SR2P, a stacking-based machine-learning framework for predicting spatial protein abundance from RNA expression. SR2P integrates 11 complementary predictive models and consistently outperforms existing methods across multiple spatial multi-omics benchmark. We showcased an application of SR2P recovered macrophage-enriched regions and identified potential immune markers associated with therapeutic response from head-and-neck squamous cell carcinoma patients. SR2P enables protein-abundance inference from RNA-only spatial data, extending the analytical capabilities of current spatial platforms for studies of tumor immunology.

bioinformatics↗

bioRxiv2026

ENSO-driven climate variability reconfigures the altitudinal frontier of dengue risk in the Andes

Despite the high burden of dengue in Latin America, high elevations have historically protected megacities such as Mexico City, Quito, and Bogota. Climate change is expected to shift elevational suitability frontiers, yet the mechanisms governing disease expansion into highland regions and the timescales over which it occurs remain poorly characterized. Using spatiotemporal decomposition techniques, we identified a mechanistic cascade linking (1) the El Nino-Southern Oscillation (ENSO) to local Colombian climate, accounting for [~]85% of interannual temperature variability and [~]53% of rainfall variability, and (2) local climate to dengue dynamics, accounting for [~]66-76% of variation in incidence and [~]44-55% of its altitudinal range. Multiple lines of evidence, including spatial ENSO signatures across Andean ridges, temporal predictive performance, and vectorial-capacity modeling, identified the local temperature expression of the ENSO teleconnection as the dominant driver of transmission. During warm ENSO phases, dengue incidence increases exponentially and transmission expands into higher elevations. This shifts the altitudinal distribution of dengue transmission upward, exposing immunologically naive populations in highland areas that were previously considered unsuitable. Mechanistically, this altitudinal shift arises because a coherent warming across elevational strata disproportionately increases transmission in highland regions, where small temperature increments produce the largest increase in vectorial capacity. These findings challenge the view that disease expansion is primarily a gradual response to long-term warming and highlight the importance of climate variability for disease projection, attribution, and public health preparedness. Significance statementWhile increasing mean temperatures are a known threat to the changing landscape of infectious diseases, this study reveals that interannual climate variability can be a more immediate driver of disease burden and expansion. We show that the El Nino phenomenon produces local temperature anomalies in Colombia that exponentially increase dengue cases and extend dengues reach into high-altitude areas, potentially exposing millions of immunologically naive people. This finding is highly relevant to the elevated dengue-free megacities in Latin America, such as Mexico City, Quito, and Bogota. By shifting the focus from multi-decadal trends to interannual climate variability, this paper provides a methodological blueprint for more accurate predictions of how infectious diseases will behave as our climate becomes increasingly unstable.

ecology↗

bioRxiv2026

Therapeutic scheduling of WEE1 inhibition preserves T cell function and promotes immune control of HPV⁺ tumors

Human papillomavirus-associated oropharyngeal squamous cell carcinoma (HPV OPC) is driven by viral E6 and E7 oncoproteins, which disrupt G1 checkpoint control and impose selective dependency on WEE1-mediated G2/M regulation. While this vulnerability confers sensitivity to WEE1 inhibition, its immunologic consequences remain poorly defined, and the challenge of eliciting antitumor immunity without compromising immune fitness has limited clinical translation. Here, we show that WEE1 inhibition elicits durable antitumor immunity in immunocompetent models of HPV OPC. Using murine and human preclinical systems, we demonstrate that the WEE1 inhibitor azenosertib (ZN-c3) mediates tumor control through both cell-autonomous cytotoxicity and immune-dependent mechanisms requiring T cells and conventional dendritic cells. Mechanistically, HPV tumor cells are deficient in STING signaling and fail to mount canonical type I interferon responses. Instead, tumor cell-intrinsic cGAS drives immune activation through STING-competent host cells within the tumor microenvironment, revealing a non-cell-autonomous relay that circumvents viral immune evasion. Intermittent WEE1 inhibition preserves T cell fitness while maintaining antitumor efficacy, and mice achieving complete responses develop immunologic memory capable of rejecting tumor rechallenge. These findings establish intermittent WEE1 inhibition as an immune-permissive therapeutic strategy that enables antigen-specific T cell responses in HPV-driven malignancies and provides a mechanistic rationale for combination with immunotherapy.

cancer biology↗

bioRxiv2026

SARM1 is required for macrophage immunophenotype switching that is essential for nerve repair

SARM1 is a key executor of Wallerian degeneration in axons. Global knockout of sarm1 in mice delays degeneration for several weeks. Recently, we reported that Schwann cell reprogramming, inflammation, and axon regeneration are also delayed in these animals. Several studies have also indicated that SARM1 has essential regulatory functions in macrophages (M{phi}). However, the role of SARM1 in M{phi} in the context of peripheral nerve injury remains unknown. Here, we report that loss of sarm1 impairs splenic M{phi} from adopting immunological stimuli driven immunophenotypes in culture. Through a combination of cell culture, Western blotting, gene expression analysis, in vivo injection of M{phi} into sciatic nerves, and generation of cell specific sarm1 conditional knockout mouse lines, we found that SARM1 is required for proper immunophenotypes in M{phi}. Loss of sarm1 in macrophages increases neurite length of sensory neurons in culture but delays regeneration in a model of peripheral nerve injury. We identified dysregulation of several inflammatory and anti-inflammatory immunological stimuli pathways and altered regulation of both iNOS and Arginase-1 in Sarm1-/- M{phi}. In culture, Sarm1-/- M{phi} display difficulty phagocytosing and clearing myelin debris and this was recapitulated in vivo with a M{phi} specific sarm1 knockout line. Generation of M{phi} and neuronal sarm1 conditional knockout mice further indicated that SARM1 is required in both cell types for an efficient response to peripheral nerve injury. This study provides the first evidence that SARM1 signaling in M{phi} is required for injury induced inflammation, degeneration, and axon regeneration.

neuroscience↗

bioRxiv2026

Temporal shifts in polygenic traits track major epidemics in Western Eurasia

Infectious diseases are recognized as one of the strongest selective forces, exerting a profound influence on the genetic makeup of human populations over time. Recently, large-scale genome-wide association studies (GWAS) on immunological traits have underscored the notion that genetic predisposition to infectious diseases likely stems from the contribution of several thousand loci across the human genome. To model the polygenic inheritance of these traits, multiple variants can be combined into polygenic risk scores (PRS), which estimate an individuals genetic predisposition for a trait. By combining present-day GWAS statistics from large European biobanks with genomic data from more than 3,500 ancient individuals from Western Eurasia, we characterize temporal changes in four highly heritable infectious disease-related traits over a span of 10,000 years. In doing so, we account for variation in these traits across time, space, and genetic ancestries, and demonstrate that the observed patterns cannot be explained by genetic drift alone. Our findings suggest that major disease outbreaks in human history are associated with shifts in polygenic traits in human populations. Specifically, three events - the Justinian Plague, Antonine Plague, and early medieval measles outbreaks - coincide with significant shifts in the polygenic profiles of these traits. Using a Gene Ontology enrichment approach, we show that these shifts involve multiple systemic biological processes, with a consistent emphasis on metabolic pathways modulating immunological responses both directly and indirectly.

genomics↗

bioRxiv2026

R8-Stabilized Multi-Epitope mRNA Vaccine Triggers Potent Intratumoral T Cell Infiltration and Suppresses Breast Cancer Progression.

BackgroundBreast cancer remains a significant therapeutic challenge due to the heterogeneity of tumor antigens and the presence of "immunologically cold" tumor microenvironments (TME) that resist conventional immunotherapy. mRNA vaccines offer a versatile platform for multi-epitope targeting, but their clinical utility is often limited by inherent instability and poor cellular internalization. ObjectiveTo design, characterize, and evaluate an R8-stabilized multi-epitope mRNA vaccine targeting HER2, MUC1, and Survivin for the treatment of aggressive breast cancer. MethodsA multi-epitope mRNA construct (R8-CTL1- 7-HTL1- 2) was designed and synthesized via in vitro transcription (IVT). The mRNA was complexed with an octa-arginine (R8) domain at an N/P ratio of 4:1 to form stable nanoparticles. Characterization included Minimum Free Energy (MFE) modeling and Dynamic Light Scattering (DLS). In vitro uptake and antigen expression were quantified in breast cancer cell lines. In vivo efficacy was assessed in female BALB/c mice (n=6) challenged with 4T1 cells, focusing on tumor growth inhibition, CD8+ T cell cytotoxicity, and intratumoral T cell infiltration (counts/mm2) over a 28-day period. ResultsThe mRNA construct exhibited high structural stability (MFE = -450 kcal/mol) and formed uniform nanoparticles (mean diameter [~]92 nm). R8-complexation significantly enhanced cellular uptake to 88%, resulting in robust relative expression of HER2 and MUC1. In vivo results demonstrated potent systemic immunity with a marked increase in CD8+ T cell cytotoxicity (p<0.05). Most notably, vaccinated mice showed a 65% increase in intratumoral T cell recruitment (from 1.4 to 2.3 counts/mm2), correlating with significant tumor growth suppression compared to the control group by Day 28. ConclusionThe R8-stabilized mRNA platform effectively overcomes the delivery barriers and "warms up" the immunosuppressive tumor microenvironment. By inducing high-density T cell infiltration and systemic cytotoxicity, this multi-epitope approach provides a promising therapeutic strategy for converting "cold" breast tumors into immunologically active, treatable targets.

cancer biology↗

bioRxiv2026

Nephrotoxicity of Immune Checkpoint Inhibitors in Mice with a Human Immune System

IntroductionImmune checkpoint inhibitors (ICIs) enhance antitumor responses by blocking inhibitory receptors, including PD-1 and CTLA-4. Overactivation can trigger systemic toxicity akin to autoimmune diseases, including kidney manifestations. We sought to 1) profile immune signaling and 2) interrogate potential mechanisms of ICI-related kidney injury in a Human Immune System (HIS) tumor-bearing mouse model treated with nivolumab and ipilimumab. MethodsImmunodeficient BRGS (BALB/c-Rag2nullIl2r{gamma}nullSirpNOD) neonates were engrafted with human CD34+ cells to generate HIS-BRGS mice. Human MDA-MB-231 tumor cells were implanted subcutaneously; once tumors reached [~]150 mm3, mice received weekly intraperitoneal vehicle (PBS) or ICI (nivolumab 20 mg/kg + ipilimumab 10 mg/kg) for 4 weeks (Veh BRGS n=4; ICI BRGS n=6; Veh HIS-BRGS n=7; ICI HIS-BRGS n=7). Kidneys were evaluated by histopathology (H&E, TEM), flow cytometry for human immune phenotypes, multiplex ELISA (80 human proteins; 10 injury biomarkers), bulk RNA sequencing, and targeted qPCR. Pearson correlations identified predictors of histopathological injury. ResultsRenal vasculitis and interstitial nephritis were observed only in ICI-treated HIS-BRGS mice. These kidneys showed a shift toward CD4+ T-cell enrichment with an increased TNF- production capacity compared to CD8+ counterparts. Toxicity was accompanied by increased renal concentrations of human cytokines, chemokines, and soluble receptors. ICI treatment significantly elevated serine proteases (Granzyme A/B) and NGF-{beta}, while decreasing IL-4. Interstitial nephritis correlated with renal PD-1 and MIF. Renal vasculitis correlated with kidney PD-1, CCL1, MIF, Granzyme A, IL-15, and BAFF. Traditional injury biomarkers (KIM-1, NGAL) remained unchanged; however, a trending decrease in EGF was observed. ConclusionsOur study suggests that shifts in human T-cell populations and specific immune proteins could serve as promising biomarkers and mechanistic targets for ICI nephrotoxicity. The tumor-bearing HIS-BRGS mouse model reproducibly recapitulates the histopathological and immunological features of human ICI-induced nephrotoxicity and represents a validated preclinical platform for testing novel therapeutic interventions to preserve kidney function during cancer immunotherapy. Translational StatementImmune checkpoint inhibitor (ICI)-associated nephrotoxicity occurs in up to 25% of treated patients, yet the immunological mechanisms driving renal injury remain poorly characterized due to the scarcity of human biopsy material and the absence of robust preclinical models that recapitulate human immune responses. This study demonstrates that tumor-bearing humanized immune system (HIS) mice treated with combined nivolumab and ipilimumab reproducibly develop renal vasculitis and interstitial nephritis mediated by a human CD4+ T cell-dominant infiltrate, mirroring the clinicopathological features reported in patients with ICI-associated acute kidney injury. By integrating histopathology, flow cytometry, multiplex proteomics, and transcriptomics, we identify a coordinated immune network, including IL-15, CCL1, MIF, GZMA, and BAFF, that correlates with the severity of renal pathology and represents tractable mechanistic targets and candidate biomarkers. These findings provide a validated preclinical platform for dissecting irAE mechanisms and testing novel therapeutic strategies to preserve kidney function during cancer immunotherapy.

pharmacology and toxicology↗

bioRxiv2026

Beyond the Billion: Dose-Response Immunophenotyping and Machine Learning Classification of Live versus Heat-Inactivated Gram-Positive Probiotic Strains in Human Peripheral Blood Mononuclear Cells

Probiotic research is constrained by three pervasive yet insufficiently challenged assumptions: the requirement for a minimum of one billion colony-forming units for efficacy, the necessity for gut colonization, and the inherent superiority of live over heat-treated preparations. This study addresses these gaps through a fully factorial experimental design evaluating ten Gram-positive probiotic strains in both viable (Active Fluorescent Units, AFU) and heat-treated (Total Fluorescent Units, TFU) forms across three flow cytometry-verified concentrations (105, 106, 107 cells/well per ISO 19344:2015) in primary human peripheral blood mononuclear cells (PBMC) from a single healthy male Caucasian donor (58 years), with simultaneous quantification of 17 cytokines by BioPlex suspension array. Viable preparations induced profoundly greater absolute cytokine responses than heat-treated preparations across 16 of 17 analytes, heat-treated preparations demonstrated stronger dose-response correlations (mean within-strain Spearman {rho} up to 1.00) for 13 of 17 cytokines, a finding we attribute to the uncontrolled proliferation of live bacteria during 24-hour co-culture compressing the effective concentration range. Six of ten viable strains exhibited monotonically increasing profiles; two strains displayed non-monotonic bell-shaped kinetics with peak activity at 106 AFU/well and significant attenuation at 107, directly falsifying the assumption that dose escalation uniformly increases immunological activity. MCP-1 was the sole cytokine showing no significant difference between viability states at the observation level (p = 0.61, fold-change 1.0), providing an internal methodological control. In this single-donor model, unsupervised hierarchical clustering identified three immunological phenotype clusters, requiring multi-donor validation before these groupings can be treated as generalizable biological phenotypes, with Random Forest classification achieving 83.3% internal partition-recovery consistency (clusters derived from the same data; not an estimate of generalization to novel strains) versus 33.3% chance; In this single-donor experiment, IL-12p70, IL-13, and IFN-{gamma}, not IL-6 or IL-1{beta}, were the primary discriminators of strain identity; generalizability of this ranking requires multi-donor validation. Heat-treated preparations achieved [≥]70% functional equivalence relative to viable preparations at 107 TFU/well for the majority of responsive strains (Functional Equivalence Dose, FED70), while one strain showed strongly attenuated activity in heat-treated form across all concentrations, a finding subject to the caveat that no positive control stimulus was included to formally verify PBMC functional competence on the experimental day. These findings establish a methodological framework integrating flow cytometric standardization, multiplex immunophenotyping, and machine learning for evidence-based dose characterization, postbiotic functional equivalence assessment, and data-driven strain classification in probiotic research (all p-values are descriptive within a single-donor experimental context).

microbiology↗

bioRxiv2026

Preclinical antiviral study of a liver-targeted TLR1/2 agonist in an immune-competent mouse model of HBV infection

Chronic hepatitis B cure requires the inactivation and/or elimination of covalently closed circular DNA (cccDNA), together with silencing of integrated viral genomes and restoration of HBV-specific immune responses. The TLR1/2 agonist Pam3CSK4 has previously been identified as a potent direct anti-HBV agent in vitro. In the present study, we engineered a liver-targeting polymeric nanoparticle formulation of Pam3CSK4 to enhance its in vivo immunostimulatory and antiviral activity. We evaluated the antiviral efficacy of this novel nanoformulation carrying the TLR1/2 agonist (NP-Pam3CSK4) in monotherapy and started to investigate its mechanism of action through immunological correlates in an immune-competent AAV-HBV mouse model. AAV-HBV-infected mice received intravenous administrations of NP-Pam3CSK4 at doses of 5 or 20 g twice per treatment cycle over four cycles, followed by a 2-week follow-up period. Soluble Pam3CSK4 was administered at substantially higher doses (100 g). Serial blood samples were regularly collected to monitor virological and host immune parameters. At study completion, liver tissues were harvested for intrahepatic quantification of viral and immunological markers using immunoassays, quantitative PCR, and histological analyses. The most pronounced antiviral effects were observed in mice treated with NP-Pam3CSK4 formulations, which achieved greater viral suppression than free Pam3CSK4 despite markedly lower administered doses. Histological examination of liver biopsies from treated animals revealed prominent immune cell infiltration, including macrophages, monocytes, and T cells, organized in dense cluster-like structures. These findings support the induction of coordinated innate and adaptive immune responses contributing to HBV control and clearance. Collectively, our results demonstrate that nanoparticle-based delivery of TLR1/2 agonist represents a promising therapeutic strategy for chronic HBV infection and may improve the likelihood of achieving functional cure. Further mechanistic and translational studies (combination) are warranted to support clinical development.

microbiology↗