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bioRxiv2024

Structural and immunological characterization of the H3 influenza hemagglutinin during antigenic drift

The quest for a universal influenza vaccine holds great promise for mitigating the global burden of influenza-related morbidity and mortality. However, challenges persist in identifying conserved epitopes capable of inducing protection. In this study, we explore the influence of glycan evolution on H3 hemagglutinin from 1968 to present day and its impacts on antigenicity and immunogenicity. We observe that the appearance of potential N-linked glycosylation sites in Sing/16 hemagglutinin head domain reduces the binding of broadly neutralizing antibodies and shifts the polyclonal immune response upon vaccination to target the stem. Furthermore, structural characterization of HK/68 and Sing/16 by cryo-electron microscopy shows that while HK/68 is resistant to enzymatic deglycosylation, removal of glycans destabilizes the hyperglycosylated head and membrane-proximal region in Sing/16. These insights expand our understanding of glycans beyond their role in protein folding and highlight the interplay among glycan integration and immune recognition to design a universal influenza vaccine.

immunology↗

bioRxiv2025

Generation of a T cell receptor, cytokine and cell repertoire synovial fluid atlas to define commonalities and dissimilarities between arthritic diseases through systems immunology approaches

Although different chronic arthritic diseases are defined by clinical factors like gender, psoriasis and auto-antibodies, the biology of inflamed joints while comparing the different diseases remains neglected. Here, after curating an inflamed joint derived T-cell receptor (TCR) database, our new TRIASSIC tool identified 66303 significantly convergent TCR clonotypes. Clustering TCR clonotypes showed that synovial fluid convergence clusters (SFCCs) characterized HLA-B27+ mediated diseases (spondyloarthritis, SpA, and enthesitis-related juvenile idiopathic arthritis, JIA-ERA), Lyme arthritis and oligoarticular JIA. Single-cell transcriptomics and bulk proteomics showed upregulated interferon type I and II and TNF- pathways in oJIA. Adult and juvenile psoriatic arthritis, (JIA-)PsA, was characterized by upregulated HSP expression in monocytes and TXNIP in T-cells. We discovered an abundance of CCL5 expressing CD8+ T-cells in SF from HLA-B27+ JIA-ERA and SpA patients. JIA-ERA patients showed upregulation of CD74 and LGALS1 in Th1 and Th17 cells and IGHV7-4.1 in B-cells. oJIA patients shared a TRBV28 RG-motif on CXCL13 producing helper T-cells. Rheumatoid arthritis and (JIA-)PsA patients carried EBV-reactive cytotoxic CD8+ T-cells. Annexin signalling was shown to be important in the intercellular communication for all arthritis groups. Collectively, our work showed that chronic arthritis is characterized by both disease-specific and broadly shared mechanisms.

immunology↗

bioRxiv2025

Immunological surveillance using gSG6-P1 biomarker reveals spatio-temporal dynamics of Anopheles exposure and gaps in malaria risk assessment

Malaria risk assessment often relies on the entomological inoculation rate (EIR), which quantifies infectious bites per person over time. However, this approach does not account for human behavioral variability, limiting its effectiveness in accurately reflecting malaria risk at the population level. Recent studies have attempted to estimate mosquito-bite risk through the measurement of antibody responses to Anopheles salivary peptides. However, this approach has yet to be applied to evaluate malaria transmission risks along the Thailand-Cambodia border. This study aims to employ an immune biomarker for Anopheles salivary peptide (anti-gSG6-P1) to identify key risk factors associated with exposure to Anopheles mosquitoes in Sisaket Province, a region bordering Thailand and Cambodia. Blood samples from the same set of 184 participants were collected via finger prick during each season: rainy (August 2022), cool-dry (December 2022), and hot-dry (April 2023). Anti-gSG6-P1 antibody levels were quantified using enzyme-linked immunosorbent assay (ELISA). Factor Analysis of Mixed Data (FAMD) revealed that seasonality exerts the strongest influence on antibody levels. This pattern was likely driven by human activities, particularly the frequency of rubber tapping activity in the rubber plantation area where Anopheles species, especially Anopheles dirus, are present. While this study successfully identifies seasonality and human factors as critical influences on antibody responses, it also highlights gaps in understanding the kinetics of anti-salivary peptide responses to Anopheles bites, particularly in species with low salivary peptides sequence similarity to that of Anopheles gambiae. These findings emphasize the need for the development of new serological tools tailored to malaria vectors in the Greater Mekong Subregion to enhance malaria risk assessment and improve vector control strategies.

immunology↗

bioRxiv2025

Immunological evaluation in African Green Monkeys of two nasal vaccine candidates to induce a broad immunity against coronaviruses

The COVID-19 pandemic has had a profound impact on the world. While the disease is currently under control, the emergence of new coronavirus variants with pandemic potential underscores the need for more universal coronavirus vaccines. This study evaluates the immunogenicity in non-human primates of two broad-spectrum nasal vaccine candidates called PanCoV. The vaccine preparations are based on highly conserved regions of SARS-CoV-2, specifically the nucleocapsid (N) and the S2 subunit from Spike protein, which include several relevant T-cell epitopes and have the potential to form virus-like particles. PanCoV1 candidate contains the N protein, while the PanCoV2 comprises a chimeric protein containing the C-terminal domain of N protein fused to a fragment of S2 subunit. Both vaccine candidates also include the receptor-binding domain (RBD) from Spike protein and the ODN-39M as CpG adjuvant. The results demonstrate that both nasal vaccine candidates can boost the anti-RBD immune response and induce anti-N immunity, this systemic response shows cross-reactivity with SARS-CoV, MERS-CoV, and H-CoV antigens. In addition, a neutralizing response against SARS-CoV-2 that persisted for almost three months after the last dose was induced. A mucosal-specific and cross-reactive IgA response was also observed, mainly in monkeys vaccinated with PanCoV2. Furthermore, the specific IFN{gamma}-secreting response observed in the vaccinated groups peripheral blood mononuclear cells highlights the ability of these candidates to stimulate cellular immunity with a Th1 bias. PanCoV vaccines emerge as promising candidates for use as a booster alternative, with the potential to amplify and broaden the scope of the immune response against coronaviruses.

immunology↗

bioRxiv2025

Tissue-resident neutrophils serve homeostatic and immunological functions in embryos

Development of neutrophils in the bone marrow and their crucial role in first-line defense are well understood in adults, but remarkably little is known about fetal neutrophils. Here, we analyzed the production, distribution, and functions of neutrophils during embryonic development in the mouse. We discovered that multiple non-hematopoietic steady-state organs harbor substantial numbers of immature and mature neutrophils, many of which are localized to tissue parenchyma outside the vessels. Using single-cell transcriptomic analyses, we revealed the presence of neutrophil progenitors and precursor cells in the blood and even in non-hematopoietic tissues in fetal and newborn mice. Embryonic tissue-resident neutrophils were transcriptionally different from embryonic blood-borne neutrophils and adult neutrophils. We demonstrated, through functional analyses, that embryonic neutrophils proliferated actively, had a high glycolytic capacity, and exhibited distinct diurnal rhythmicity. Embryonic neutrophils displayed lineage-specific innate immune effector functions and were responsive to maternal immunostimulation and immunosuppression. Using a genetic embryonic neutrophil depletion model, we discovered that neutrophils impact the piRNA pathway in the testis. Collectively, our data provides an atlas of the fetal neutrophil landscape and dissects their responses in steady-state. SummaryNon-hematopoietic steady-state tissues harbor extravascular immature and mature neutrophils during fetal development. Embryonic neutrophils are endowed with multiple effector mechanisms and also serve homeostatic roles during tissue development.

immunology↗

bioRxiv2025

TCR2HLA: calibrated inference of HLA genotypes from TCR repertoires enables identification of immunologically relevant metaclonotypes

T cell receptors (TCRs) recognize peptides presented by polymorphic human leukocyte antigen (HLA) molecules, but HLA genotype data are often missing from TCR repertoire sequencing studies. To address this, we developed TCR2HLA, an open-source tool that infers HLA genotypes from TCR{beta} repertoires. Expanding on work linking public TRBV-CDR3 sequences to HLA genotypes, we incorporated "quasi-public" metaclonotypes - composed of rarer TCR{beta} sequences with shared amino acid features - enriched by HLA genotypes. Using four TCR{beta}seq datasets from 3,150 individuals, we applied TRBV gene partitioning and locality-sensitive hashing to identify [~]96,000 TCR{beta} features strongly associated with specific HLA alleles from 71M input TCRs. Binary HLA classifiers built with these features achieved high balanced accuracy (>0.9) across common HLA-A (9/12), B (9/12), C (6/13), DRB1 (11/11) alleles and prevalent DPA1/DPB1 (6/10), DQA1/DQB1 (8/17) heterodimers. We also introduced a high-sensitivity calibration to support predictions in samples with as few as 5,000 unique clonotypes. Calibrated predictions with confidence filtering improved reliability. Beyond genotype imputation, TCR2HLA enables the discovery of novel HLA- and exposure-associated TCRs, as shown by the identification of SARS-CoV-2 related TCRs in a large COVID-19 dataset lacking HLA data. TCR2HLA provides a scalable framework for bridging the gap between TCRseq data and HLA genotype for biomarker discovery.

immunology↗

bioRxiv2018

Effect of tributyltin chloride (TBT-Cl) exposure on expression of HSP90β1 in the river pufferfish (Takifugu obscurus): evidences for its immunologic function involving in exploring process

HSP90{beta}1 (known as GP96) is a vital endoplasmic reticulum depended chaperonin among the HSPs family. It plays important roles in regulating the growth, development, differentiation, and apoptosis of cells. Furthermore, it always processes and presents antigen of the tumor and keeps balance for the intracellular environment. In the present study, we explored the effect of tributyltin chloride (TBT-Cl) exposure on HSP90{beta}1 expression in river pufferfish, Takifugu obscurus. The full length of To-HSP90{beta}1 was gained with 2775 bp in length, an ORF obtained with 2412 bp encoding an 803 aa polypeptide. The phylogenetic tree was constructed and showed the close relationship to other fish species. The HSP90{beta}1 mRNA transcript was expressed in all tissues investigated. After the acute and chronic exposure of TBT-Cl, the mRNA level of To-HSP90{beta}1 significantly up-regulated in tissues of liver and gill. Moreover, the histochemistry study indicated the injury degree of TBT-Cl on liver and gill. Immunohistochemistry (IHC) staining results implied the cytoplasm reorganization after TBT-Cl stress and the function of immunoregulation for To-HSP90{beta}1. All the results indicated that HSP90{beta}1 may involve in the resistance to the invasion of TBT-Cl for keeping autoimmune homeostasis.

immunology↗

bioRxiv2018

Immunological characterization of a rat model of Duchenne’s disease and demonstration of improved muscle strength after anti-CD45RC antibody treatment

Duchenne muscular dystrophy (DMD) has as standard pharmacological therapy with corticoisteroids (CS) that decrease inflammation and immune responses present in patients and animal models. CS have however limited efficacy and important and numerous side effects. Therefore, there is a need for new anti-inflammatory and pro-tolerogenic treatments that could replace or decrease doses of CS. We first assessed the status of immune system of dystrophin-deficient rats (Dmdmdx) that closely reproduce the phenotype of DMD patients. Dmdmdx rats showed increased leukocyte infiltration in skeletal and cardiac muscles, containing mostly macrophages but also T cells, and increased expression of several cytokines. Anti-CD45RC Monoclonal antibody (Mab) treatment induced immune tolerance in models of organ transplantation and GVHD (Graft Versus Host Disease). We observed that muscles and blood of DMD patients contained T CD4+ and CD8+ expressing high levels of CD45RChigh cells. Treatment of young Dmdmdx rats with anti-CD45RC MAb corrected skeletal muscle strength associated to a depletion of effectors CD45RChigh T cells with no obvious side-effects. Prednisolone treatment of Dmdmdx rats similarly increased skeletal muscle strength and was also associated to a depletion of effectors CD45RChigh cells but resulted in severe weight loss.\n\nOverall, Dmdmdx rats display important immune inflammatory response and thus represent a useful model to analyze new anti-inflammatory and tolerogenic treatments for DMD. As an example, a new treatment with anti-CD45RC antibodies improved muscle strength in Dmdmdx rats as prednisolone did but without side effects. Anti-CD45RC therapy could complement other therapies in DMD patients.

immunology↗

bioRxiv2018

Meeting the challenges of high-dimensional data analysis in immunology

Recent advances in cytometry have radically altered the fate of single-cell proteomics by allowing a more accurate understanding of complex biological systems. Mass cytometry (CyTOF) provides simultaneous single-cell measurements that are crucial to understand cellular heterogeneity and identify novel cellular subsets. High-dimensional CyTOF data were traditionally analyzed by gating on bivariate dot plots, which are not only laborious given the quadratic increase of complexity with dimension but are also biased through manual gating. This review aims to discuss the impact of new analysis techniques for in-depths insights into the dynamics of immune regulation obtained from static snapshot data and to provide tools to immunologists to address the high dimensionality of their single-cell data.

immunology↗

bioRxiv2018

Immunological responses to the relapsing fever spirochete Borrelia turicatae in infected Rhesus macaques: implications for pathogenesis and diagnosis.

The global public health impact of relapsing fever (RF) spirochetosis is significant, as the pathogens exist on five of seven continents. The hallmark sign of infection is episodic fever and the greatest threat is to the unborn. With the goal of better understanding the specificity of B cell responses and the role of immune responses in pathogenicity, we infected Rhesus macaques with Borrelia turicatae (a new world RF spirochete species) by tick bite and monitored the immune responses generated in response to the pathogen. Specifically, we evaluated inflammatory mediator induction by the pathogen, host antibody responses to specific antigens, and peripheral lymphocyte population dynamics. Our results indicate that B. turicatae elicits from peripheral blood cells key inflammatory response mediators (IL-1{beta} and TNF-) which are associated with pre-term abortion. Moreover, a global decline in peripheral B cell populations was observed in all animals at 14 days post-infection. Serological responses were also evaluated to assess the antigenicity of three surface proteins, BipA, BrpA and Bta112. Interestingly, a distinction was observed between antibodies generated in non-human primates (NHPs) and mice. Our results provide support for the nonhuman primate model not only in studies of prenatal pathogenesis, but for diagnostic and vaccine antigen identification and testing.

immunology↗

bioRxiv2019

Analysis of immunological resistance to primary Mycobacterium tuberculosis infection in humans

BackgroundDespite recent advances in diagnosis and treatment, tuberculosis (TB) remains a major infectious disease killer in resource-poor settings. Strategies to prevent Mycobacterium tuberculosis (Mtb) infection are urgently required. By characterising natural protective immunity to Mtb infection we aimed to identify correlates of protection to guide vaccine development and other immune based therapies. MethodsTwo groups of Mtb-exposed contacts of TB patients were recruited in The Gambia and assessed for Mtb infection status using either tuberculin skin test (TST) reactivity at baseline and 3 months or QuantiFERON (QFT) reactivity at baseline and 6 months. For both groups, converters were defined as having a negative test at baseline and a positive one at follow-up, while those with a negative test at both time-points were defined as non-converters (Mtb resisters). Participants were analysed using RNA-sequencing and plasma Mtb proteome IgA and IgG arrays. ResultsSeveral genes were found to be differentially expressed at baseline between the groups prior to any signs of infection by current tests. Modular analysis revealed a distinct B cell gene signature in TST non-converters compared to converters (at q < 10-6, AUC > 0.7), which was only present in the most highly exposed group. Interestingly, when infection status was defined by QFT, enrichment of Type I IFN and antiviral gene signatures was observed. Plasma IgG and IgA antibody reactivity across the entire Mtb proteome showed the best differentiation in individuals with the highest exposure. An AUC of 1.0 (q<10-3) was observed for IgA reactivity to Rv0134 and an AUC of 0.98 for IgA reactivity to both Rv0629c and Rv2188c (all lower in TST non-converters). IgG reactivity to Rv3223c resulted in an AUC of 0.96 (q < 10-4) and was again lower in TST non-converters. The highest AUC for those with lower Mtb exposure were 0.84 (Rv2411c) for IgA and 0.83 (Rv2131c) for IgG. ConclusionsThese data provide insight into the early protective response to Mtb infection and possible avenues for novel therapeutic strategies to prevent Mtb infection.

immunology↗

bioRxiv2019

Systemic Lymphoid Architecture Response Assessment (SYLARAS): An approach to multi-organ, discovery-based immunophenotyping implicates a role for CD45R/B220+ CD8T cells in glioblastoma immunology

Accurately profiling systemic immune responses to cancer initiation and progression is necessary for understanding tumor surveillance and, ultimately, improving therapy. Here, we describe the SYLARAS software tool (SYstemic Lymphoid Architecture Response ASsessment) and a data set collected with SYLARAS that describes the frequencies of immune cells in primary and secondary lymphoid organs and in the tumor microenvironment of mice engrafted with a standard syngeneic glioblastoma (GBM) model. The data resource involves profiles of 5 lymphoid tissues in 48 mice and shows that GBM causes wide-spread changes in the local and systemic immune architecture. We perform in-depth analysis of one significant tumor-induced change: depletion of a specialized subset of CD45R/B220+ CD8+ T cells from the circulation and their accumulation in the tumor mass. Immunoprofiling of tissue microarrays demonstrates the presence of similar cells in human GBM.

immunology↗

bioRxiv2019

Quantitative Immunology for Physicists

The adaptive immune system is a dynamical, self-organized multiscale system that protects vertebrates from both pathogens and internal irregularities, such as tumours. For these reason it fascinates physicists, yet the multitude of different cells, molecules and sub-systems is often also petrifying. Despite this complexity, as experiments on different scales of the adaptive immune system become more quantitative, many physicists have made both theoretical and experimental contributions that help predict the behaviour of ensembles of cells and molecules that participate in an immune response. Here we review some recent contributions with an emphasis on quantitative questions and methodologies. We also provide a more general methods section that presents some of the wide array of theoretical tools used in the field.

immunology↗

bioRxiv2026

Investigating the impact of obesity as an immunological instigator on young-onset arthritis using perturbation-based simulation

PurposeYoung-onset inflammatory arthritis (YOA; disease onset at age <25) incidence has risen since 1990, coinciding with the childhood obesity epidemic. Traditional epidemiology cannot easily quantify obesitys contribution because early-life exposures can precede clinical onset by years and may be poorly measured at diagnosis. We developed a perturbation-based simulation to estimate young-age inflammatory arthritis burden by calibrating to age-stratified RA estimates from GBD (GBD does not report JIA) under varying obesity scenarios - an approach that allows counterfactual testing, difficult to achieve in observational studies. MethodsWe built a Monte Carlo model generating arthritis prevalence estimates for 1,000,000 individuals. The model incorporated published odds ratios: BMI per SD, smoking, HLA-DR shared epitope allele dose (0/1/2), and interactions. We systematically perturbed average BMI in the <25 stratum (weighted towards adolescents and young adults) from 25-29 kg/m2 as a stress-test while holding other factors constant, then compared predicted prevalence against Global Burden of Disease data. Each scenario ran 2,500 iterations to propagate parameter uncertainty. ResultsOur model predicted the current YOA prevalence of 0.07% (observed: 0.06%, 95% CI: 0.05%-0.07%). Under perturbation analysis, each unit increase in average BMI yielded an additional 0.005% (95% CI: 0.0025%-0.0075%) in YOA cases - small but meaningful given the rarity of young-onset inflammatory arthritis. The relationship was locally linear. Significantly, the model saw that returning average BMI toward 1990 levels (<26) predicted around a 30% drop in BMI-attributable diagnoses and a 3% decrease in YOA prevalence. ConclusionsPerturbation modeling identifies childhood obesity as a potentially modifiable driver of young-onset RA, accounting for upwards of 5% of prevalence increases since 1990. This approach uniquely enables testing of prevention scenarios: our model predicts that lowering average BMI by one to two units over the next decade could prevent 3-5% of YOA cases in <25 under the modeled scenarios. These estimates provide a quantitative basis for incorporating arthritis prevention in childhood and adolescent obesity intervention cost-effectiveness analyses.

immunology↗

bioRxiv2026

Evaluating 6- and 18-hour stimulation durations for natural killer cell degranulation (CD107a assay) to optimize workflow efficiency in a clinical immunology laboratory

BackgroundNatural killer (NK) cell degranulation is a key immune defense mechanism where exposure to tumor or virus-infected cells triggers the fusion of cytoplasmic granules containing apoptotic proteins, perforin, and granzyme with the cell membrane. This process transiently expresses CD107a on the NK cell surface, and measuring CD107a is a standard method to assess NK cell activity. MethodsWe compared two stimulation protocols differing only in duration (6-hour vs. 18-hour) using K562 target cells to induce NK cell degranulation. Isolated PBMCs without stimulation served as controls to assess spontaneous degranulation. Anti-CD107a-PE antibody was present throughout stimulation in both test and control samples. After stimulation, cells were stained with anti-CD45, anti-CD3, and anti-CD56 and analyzed by flow cytometry. ResultsFor 6 of 7 healthy controls, results from both methods fell within 2 standard deviations. Notably, longer (18-hour) stimulation resulted in lower CD107a expression than the 6-hour assay. Interlaboratory comparisons of two samples showed no significant difference (p>0.05). In a suspected hemophagocytic lymphohistiocytosis (HLH) case, two labs reported similarly reduced CD107a expression (9% and 7%). Inter-day variability was observed in a donor across both time points. The 6-hour assay showed higher sensitivity and specificity than the 18-hour assay. A resting period before ex vivo PBMC assays was found necessary. ConclusionStimulation periods beyond 6 hours are unsuitable for clinical NK degranulation assays. Screening for HLH should include multiple stimulants to improve assay reliability.

immunology↗

bioRxiv2026

Immunological imprinting shapes the cross-reactive antibody responses to the KP.2 and LP.8.1 vaccine doses

The emergence of the SARS-CoV-2 Omicron BA.2.86 subvariant, a lineage derived from the BA.2 strain, led to the 2024-2025 COVID-19 vaccine update to include KP.2 or related JN.1-lineage spike antigens. We evaluated the magnitude, breadth, and durability of humoral immune responses following a single KP.2 vaccine dose in a longitudinal cohort of 21 individuals up to six months. KP.2 vaccination increased spike-specific binding and neutralizing antibodies against the ancestral WA1 strain, alongside the BA.5, XBB.1.5, and KP.2 variants. Power law modeling estimated half-lives for WA1- and KP.2-specific IgG responses at 770 and 248 days, respectively. Additionally, the KP.2 dose increased IgG1 and IgG4 subclasses more than IgG2 and IgG3 responses to both spike proteins. Serum depletion experiments using WA1 or KP.2 proteins demonstrated most vaccine-elicited antibodies were cross-reactive. Consequently, KP.2 vaccine-induced antibodies retained broad neutralizing activity against recently circulating Omicron subvariants (BA.2.86, KP.3.1.1, XEC, LP.8.1, LF.7, XFG.3.12, PQ.1, BA.3.2.1, and RE.2). Using a live virus neutralization assay, XFG.3.12 showed the greatest reduction in neutralizing titers relative to KP.2 (4.2-fold). In a small subset, an LP.8.1 vaccine dose increased neutralizing activity against the matched variant while maintaining WA1 and KP.2 cross-reactivity, but only modestly increased antibodies to divergent variants BA.3.2.1 and RE.2. Ultimately, these data indicate the KP.2 mRNA vaccine generates durable, cross-reactive responses against current Omicron subvariants. However, ongoing spike evolution impacts neutralization of emerging lineages, highlighting the need for continued viral monitoring and timely vaccine updates. IMPORTANCESARS-CoV-2 continues to evolve, raising ongoing concerns about how well updated vaccines protect against emerging variants. This study evaluates antibody responses after KP.2 spike mRNA vaccine dose and shows that a single dose induces durable and broadly cross-reactive immunity against both earlier strains and recently circulating Omicron subvariants. Despite this breadth, reduced neutralizing activity against certain emerging variants indicates that ongoing antigenic changes can impact vaccine induced antibody effectiveness. These findings provide insight into how current vaccines perform over time and highlight the need to track viral evolution and update vaccine antigens to maintain broad protection against severe disease, hospitalization, and death.

immunology↗

bioRxiv2026

Using the Collaborative Cross to Dissect Immunological Mechanisms Governing Latent Infection, Granuloma Formation, and Neuroinvasive Disease Following Repeated Very Low-Dose Inhalation Exposure to Cryptococcus

BackgroundDecades of cryptococcal research have relied on a small number of hypervirulent laboratory strains, principally H99 and its derivatives, which cause rapid and lethal central nervous system infection and bypass the latent pulmonary phase that defines the natural history of human disease. As a result, the immune mechanisms that govern durable fungal containment in the lung, the reasons these mechanisms fail in genetically susceptible hosts, and whether the pulmonary immune response can engage the brain before any fungal cell reaches the central nervous system remain poorly understood. We address these gaps using a physiologically relevant repeated low-dose inhalation model in members of the Collaborative Cross and the BXD mouse panels. MethodsA repeated low-dose intranasal exposure model, consisting of four doses of 50 C. neoformans UgCl223 cells over 18 days, was compared with a single primary challenge in C57BL/6J mice. Six Collaborative Cross founder strains (A/J, C57BL/6J, 129S1/SvImJ, NOD/ShiLtJ, NZO/H1LtJ, CAST/EiJ) and one BXD founder (DBA/2J) were assessed for fungal burden, granuloma architecture, and CD4 T cell polarisation. The neuroimmune axis was interrogated by exposing BV2 microglia to serum from infected mice with confirmed lung-restricted infection or disseminated disease. ResultsRepeated low-dose exposure, in contrast to single-dose primary challenge, was linked with enhanced fungal containment, elevated frequency of CD44+ CD4 T cells, expansion of regulatory T cells, and organised granuloma formation. Across seven genetically diverse strains, host genetic background determined disease outcome, spanning four orders of magnitude in lung fungal burden, from near-sterilising control in NZO mice to overt central nervous system dissemination with brain lesions in CAST mice. Granuloma architecture varied markedly across backgrounds in a pattern that mirrors human granuloma heterogeneity. The Th17 and regulatory T cell axis, rather than Th1 or Th2 polarisation, distinguished progressors from controllers. Critically, serum from mice with confirmed lung-restricted infection induced morphological microglial activation in a strain-dependent pattern associated with the disease spectrum, providing initial evidence supporting the existence of a lung-brain immune axis in cryptococcal disease. ConclusionsThese data demonstrate that the immune dynamics of latent cryptococcal infection are fundamentally distinct from those characterised in primary high-dose models. Host genetic background influences not only pulmonary containment of Cryptococcus but may also impact the magnitude of distal brain immune responses, potentially through mechanisms that extend beyond direct fungal dissemination to the central nervous system. These findings suggest that early pulmonary immune events may shape subsequent neuroimmune outcomes and identify a previously underappreciated lung-brain axis in cryptococcosis that warrants in vivo validation.

immunology↗

bioRxiv2016

Tumor evolution of glioma intrinsic gene expression subtype associates with immunological changes in the microenvironment

SummaryWe leveraged IDH wild type glioblastomas and derivative neurospheres to define tumor-intrinsic transcription phenotypes. Transcriptomic multiplicity correlated with increased intratumoral heterogeneity and tumor microenvironment presence. In silico cell sorting demonstrated that M2 macrophages/microglia are the most frequent type of immune cells in the glioma microenvironment, followed by CD4 T lymphocytes and neutrophils. Hypermutation associated with CD8+ T cell enrichment. Longitudinal transcriptome analysis of 124 pairs of primary and recurrent gliomas showed expression subtype is retained in 53% of cases with no proneural to mesenchymal transition being apparent. Inference of the tumor microenvironment through gene signatures revealed a decrease in invading monocytes but a subtype dependent increase in M2 macrophages/microglia cells after disease recurrence. All expression datasets are accessible through http://recur.bioinfo.cnio.es/.\n\nSignificanceIDH wild type glioblastoma expression phenotypes have been related to tumor characteristics including genomic abnormalities and treatment response. We explored the intratumoral transcriptomic landscape, including a definition of tumor-intrinsic gene expression subtypes and how they relate to the different cellular components of the tumor immune environment. Comparison of matching primary and recurrent gliomas provided insights into the treatment-induced phenotypic tumor evolution. Proneural to mesenchymal transitions have long been suspected but were not apparent, while intratumoral heterogeneity was a predictor of subtype transition upon recurrence. Characterizing the evolving glioblastoma transcriptome en tumor microenvironment aids in designing more effective immunotherapy trials. Our study provides a comprehensive transcriptional and cellular landscape of IDH wild type GBM during treatment modulated tumor evolution.\n\nHighlightsO_LINext generation GBM-intrinsic transcriptional subtypes: proneural, classical, mesenchymal\nC_LIO_LIM2 macrophages, CD4+ T-lymphocytes and neutrophils dominate glioblastoma microenvironment\nC_LIO_LISensitivity to radiotherapy may associate with M2 macrophage presence\nC_LIO_LICD8+ T cells are enriched in hypermutated GBMs at diagnosis and recurrence\nC_LI

Preprint↗