Biology subjects

Merkler, D.

Publications and source records attributed to Merkler, D..

18 recordsLinked to original sources

bioRxiv2025

Non-cytolytic re-engineering of a viral vaccine vector enables durable effector-memory T cell immunity by reinforcing type I IFN induction

Replication-deficient viral vector systems hold promise for CD8 T cell-based vaccination, but the molecular mechanisms accounting for platform-specific differences in immunogenicity remain ill- defined. When comparing prototypic single-cycle vaccine delivery platforms we found that lymphocytic choriomeningitis virus-based vectors (rLCMV), which are non-cytolytic, elicited more durable and effector-memory-differentiated CD8 T cell responses than vectors based on cytolytic vesicular stomatitis virus (rVSV). Hence we re-engineered rVSV to be non-cytolytic (rVSVMq). This vector induced more durable and effector-differentiated CD8 T cell memory than the parental rVSV and it afforded superior protection against Listeria challenge. Improved CD8 T cell responses of non-cytolytic rVSVMq were driven by a reinforced type I interferon (IFN-I) response and its direct sensing by vaccination-induced CD8 T cells. Many vector cargo-specific CD8 T cells in the splenic marginal zone of rVSVMq- or rLCMV-vaccinated mice were in contact with vector cargo-expressing cells that co- expressed type I interferon. In contrast, rVSV-vectored cargo-expressing contacts of specific CD8 T cells were largely IFN type I-negative. Thereby, vaccination with non-cytolytic viral vectors offered an opportunity for CD8 T cells to integrate peptide-MHC and IFN-I signals during priming. These mechanistic insights should help to refine vaccines aimed at eliciting durable and protective effector- memory CD8 T cell immunity.

immunology↗

bioRxiv2025

Chronic Infection Perturbs the Affinity Hierarchy of Antiviral B Cells

The germinal center (GC) subjects antigen-specific B cells to a Darwinian selection process. Whether and how persistent viral infection perturbs the intended affinity hierarchy remains ill-defined. Here we transferred monoclonal lymphocytic choriomeningitis virus-specific B cells into persistently infected hosts. High affinity B cells expanded vigorously, forming GCs and abundant antibody-secreting cells. When failing to gain the upper hand over the virus, the expanded B cell population contracted, ending in its quasi-complete disappearance, a process termed "attrition". In stark contrast, low-affinity B cells expanded and persisted irrespective of high viral loads. B cell attrition was associated with phenotypic and transcriptional alterations including a prominent Blimp-1 transcriptional signature in high-affinity GC B cells. Blimp-1-deficient B cells were resistant to attrition, suggesting a B cell-intrinsic process. Moreover, exogenously supplied antibody feedback prevented attrition, indicating the latter resulted from excessive stimulation. Our findings reveal that in chronic viral infection the incessant activation by overwhelming amounts of antigen perturbs B cell affinity hierarchies by preferentially dysregulating high-affinity B cells.

immunology↗

bioRxiv2024

Interleukin-33 from oligodendrocytes sustains effector differentiation of tissue-resident CD8+ T cells and is a druggable target in CNS autoimmune disease

In chronic inflammatory disorders of the central nervous system (CNS), tissue-resident self-reactive T cells perpetuate disease. The specific tissue factors governing the persistence and continuous differentiation of these cells remain undefined but could represent attractive therapeutic targets. In a model of chronic CNS autoimmunity, we find that oligodendrocyte-derived interleukin-33 (IL-33), an alarmin, is key for locally regulating the pathogenicity of self-reactive CD8+ T cells. The selective ablation of IL-33 from neo-self-antigen-expressing oligodendrocytes mitigates CNS disease. In this context, fewer self-reactive CD8+ T cells persist in the inflamed CNS, and the remaining cells are largely locked into a stem-like precursor program, failing to form TCF-1low effector progeny. Importantly, interventional IL-33 blockade by locally administered somatic gene therapy reduces T cell infiltrates and improves the disease course. Our study identifies oligodendrocyte-derived IL-33 as a druggable tissue factor regulating the differentiation and survival of self-reactive CD8+ T cell in the inflamed CNS. This finding introduces tissue factors as a novel category of immune targets for treating chronic CNS autoimmune diseases.

immunology↗

bioRxiv2024

Permanent lymphocyte subset elimination upon a single dose of AAV-delivered depletion antibody dissects immune control of chronic viral infection

To interrogate the role of specific immune cells in infection, cancer and autoimmunity, immunologists commonly use monoclonal depletion antibodies (depletion-mAbs) or genetically engineered mouse models (GEMMs). To generate a tool that combines specific advantages and avoids select drawbacks of the two methods we engineer adeno-associated viral vectors expressing depletion-mAbs (depletion-AAVs). Single-dose depletion-AAV administration permanently eliminates lymphocyte subsets in mice while avoiding accessory deficiencies of GEMMs such as marginal zone defects in B cell-deficient animals. Depletion-AAVs can be used irrespective of the animals genetic background, and multiple depletion-AAVs can readily be combined. Exploiting depletion-AAV technology, we show that B cells are required for unimpaired CD4 and CD8 T cell responses to chronic viral infection. Importantly, CD8 T cells fail to suppress viremia when B cells are depleted, and they only help resolving chronic infection if antibodies suppress viral loads. Our study positions depletion-AAVs as a versatile tool for immunological research.

immunology↗

bioRxiv2024

KIR3DL1 and Tox identify clonally expanded encephalitogenic neuron-specific CD8+ T cells in autoimmune encephalitis

Autoreactive CD8+ T cells are the principal suspects in autoimmune encephalitis (AIE) with antibodies targeting intracellular neuronal antigens So far, the search for neuron-autoreactive CD8+ T cells has been focused on a few autoantigens and did not yield convincing results. Here, we leveraged natural antigen presentation by hiPSC-derived neurons to look at the global autoreactive CD8+ T cell response, independently of pre-conceived hypothesis of the autoantigens involved in the disease. This unbiased approach allowed for the identification of rare polyclonal neuron-reactive CD8+ T cells in healthy donors, and contrastingly, expanded clonotypes in two patients with anti-Ri AIE. Detailed ex vivo phenotypic characterization of these clonotypes revealed a specific transcriptional program suggestive of a pathogenic potential. In particular, this subset can be identified by the expression of KIR3DL1 and TOX. Strikingly, we could also demonstrate that CD8+ T cells found in the brain of an anti-Ri AIE patient display a similar phenotype associated with cytotoxicity and encephalitogenic features.

immunology↗

bioRxiv2023

Targeting the TCA cycle can ameliorate widespread axonal energy deficiency in neuroinflammatory lesions

Inflammation in the central nervous system (CNS) can impair the function of neuronal mitochondria and contributes to axon degeneration in the common neuroinflammatory disease multiple sclerosis (MS). Here we combine cell type-specific mitochondrial proteomics with in vivo biosensor imaging to dissect how inflammation alters the molecular composition and functional capacity of neuronal mitochondria. We show that neuroinflammatory lesions in the mouse spinal cord cause widespread and persisting axonal ATP deficiency, which precedes mitochondrial oxidation and calcium overload. This axonal energy deficiency is associated with impaired electron transport chain function, but also an upstream imbalance of tricarboxylic acid (TCA) cycle enzymes, with several, including key rate-limiting, enzymes being depleted in neuronal mitochondria in experimental models and in MS lesions. Notably, viral overexpression of individual TCA enzymes can ameliorate the axonal energy deficits in neuroinflammatory lesions, suggesting that TCA cycle dysfunction in MS may be amendable to therapy.

neuroscience↗

bioRxiv2022

Persistent virus-specific and clonally expanded antibody secreting cells respond to induced self antigen in the CNS

B cells contribute to the pathogenesis of both cellular- and humoral-mediated central nervous system (CNS) inflammatory diseases through a variety of mechanisms. In such conditions, B cells may enter the CNS parenchyma and contribute to local tissue destruction. It remains unexplored, however, how infection and autoimmunity drive transcriptional phenotypes, repertoire features, and antibody functionality. Here, we profiled B cells from the CNS of murine models of intracranial (i.c.) viral infections and autoimmunity. We identified a population of clonally expanded, antibody secreting cells (ASCs) that had undergone class-switch recombination and extensive somatic hypermutation following i.c. infection with attenuated lymphocytic choriomeningitis virus (rLCMV). Recombinant expression and characterisation of these antibodies revealed specificity to viral antigens (LCMV glycoprotein GP), correlating with ASC persistence in the brain weeks after resolved infection. Furthermore, these virus-specific ASCs upregulated proliferation and expansion programs in response to the conditional and transient induction of the LCMV GP as a neo-self antigen by astrocytes. This class-switched, clonally expanded, and mutated population persisted and was even more pronounced when peripheral B cells were depleted prior to autoantigen induction in the CNS. In contrast, the most expanded B cell clones in mice with persistent expression of LCMV GP in the CNS did not exhibit neo-self antigen specificity, potentially a consequence of local tolerance induction. Finally, a comparable population of clonally expanded, class-switched, proliferating ASCs was detected in the cerebrospinal fluid of multiple sclerosis patients. Taken together, our findings support the existence of B cells that populate the CNS and are capable of responding to locally encountered autoantigens. Graphical abstract O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY

immunology↗

bioRxiv2022

Single-cell immune repertoire sequencing of B and T cells in murine models of infection and autoimmunity

Adaptive immune repertoires are composed by the ensemble of B and T cell receptors (BCR, TCR) within an individual and reflect both past and current immune responses. Recent advances in single-cell sequencing enable recovery of the complete adaptive immune receptor sequences in addition to transcriptional information. Such high-dimensional datasets enable the molecular quantification of clonal selection of B and T cells across a wide variety of conditions such as infection and disease. Due to costs, time required for the analysis and current practices of academic publishing, small-scale sequencing studies are often not made publicly available, despite having informative potential to elucidate immunological principles and guide future-studies. Here, we performed single-cell sequencing of B and T cells to profile clonal selection across murine models of viral infection and autoimmune disease. Specifically, we recovered transcriptome and immune repertoire information for polyclonal T follicular helper cells following acute and chronic viral infection, CD8+ T cells with binding specificity restricted to two distinct peptides of lymphocytic choriomeningitis virus, and B and T cells isolated from the nervous system in the context of experimental autoimmune encephalomyelitis. We could relate repertoire features such as clonal expansion, germline gene usage, and clonal convergence to cell phenotypes spanning activation, memory, naive, antibody secretion, T cell inflation, and regulation. Together, this dataset provides a resource for experimental and computational immunologists that can be integrated with future single-cell immune repertoire and transcriptome sequencing datasets.

immunology↗

bioRxiv2021

Brain endothelial antigen presentation detains CD8+ T cells at the blood-brain barrier leading to its breakdown

Blood-brain barrier (BBB) breakdown and immune cell infiltration into the central nervous system (CNS) are early hallmarks of multiple sclerosis (MS). High numbers of CD8+ T cells are found in MS lesions and antigen (Ag)-presentation at the BBB was proposed to promote CD8+ T-cell entry into the CNS. Employing live cell imaging and primary mouse brain microvascular endothelial cells (pMBMECs) as in vitro model of the BBB and a mouse model of CNS autoimmunity, we here show that pMBMECs process and present antigens leading to effector CD8+ T-cell differentiation. Under physiological flow, endothelial Ag-presentation prohibited CD8+ T-cell crawling and diapedesis leading to pMBMEC apoptosis. Reduced motility of Ag-specific CD8+ T cells was also observed in CNS microvessels in neuroinflammation in vivo. Luminal MHC class I Ag-presentation at the BBB thus prohibits CD8+ T-cell entry into the CNS and rather triggers CD8+ T cell mediated focal BBB breakdown.

immunology↗

bioRxiv2021

Clonally expanded virus-specific CD8 T cells acquire diverse transcriptional phenotypes during acute, chronic, and latent infections

CD8+ T cells play a crucial role in the control and resolution of viral infections and can adopt a wide range of phenotypes and effector functions depending on the inflammatory context and the duration and extent of antigen exposure. Similarly, viral infections can exert diverse selective pressures on populations of clonally related T cells. Technical limitations have nevertheless made it challenging to investigate the relationship between clonal selection and transcriptional phenotypes of virus-specific T cells. We therefore performed single-cell T cell receptor (TCR) repertoire and transcriptome sequencing of virus-specific CD8 T cells in murine models of acute, chronic and latent infection. We observed clear infection-specific populations corresponding to memory, effector, exhausted, and inflationary phenotypes. We further uncovered a mouse-specific and polyclonal T cell response, despite all T cells sharing specificity to a single viral epitope, which was accompanied by stereotypic TCR germline gene usage in all three infection types. Persistent antigen exposure during chronic and latent viral infections resulted in a higher proportion of clonally expanded T cells relative to acute infection. We furthermore observed a relationship between transcriptional heterogeneity and clonal expansion for all three infections, with highly expanded clones having distinct transcriptional phenotypes relative to lowly expanded clones. Finally, we developed and utilized a bioinformatic pipeline integrating pseudotime and clonality, termed Clonotyme, to further support a model in which expanded virus-specific CD8+ T cells adopt heterogenic, yet preferentially, effector-like phenotypes. Together our work relates clonal selection to gene expression in the context of viral infection and further provides a dataset and accompanying software for the immunological community.

immunology↗

bioRxiv2021

Comparative multi-tissue profiling reveals extensive tissue-specificity in transcriptome reprogramming upon cold exposure

Cold exposure is an extensively used intervention for enhancing thermogenic and mitochondrial activity in adipose tissues. As such, it has been suggested as a potential lifestyle intervention for body weight maintenance. The metabolic consequences of cold acclimation are not limited to the adipose tissues, however the impact on rest of the tissues in context of their gene expression profile remains unclear. Here we provide a systematic characterization of cold exposure-mediated effects in a comparative multi-tissue RNA sequencing approach using wide range of organs including spleen, bone marrow, spinal cord, brain, hypothalamus, ileum, liver, subcutaneous-, visceral- and brown adipose tissues. Our findings highlight that transcriptional responses to cold exposure exhibit high degree of tissue-specificity both at the gene level and at GO enrichment gene sets, which is not directed by the basal gene expression pattern exhibited by the various organs. Our study places the cold adaptation of individual tissues in a whole-organism framework and provides an integrative transcriptional analysis necessary for understanding the cold exposure-mediated biological reprograming.

physiology↗

bioRxiv2021

Single-cell sequencing reveals clonally expanded plasma cells during chronic viral infection produce virus-specific and cross-reactive antibodies

Plasma cells and their secreted antibodies play a central role in the long-term protection against chronic viral infection. However, due to experimental limitations, a comprehensive description of linked genotypic, phenotypic, and antibody repertoire features of plasma cells (gene expression, clonal frequency, virus specificity, and affinity) has been challenging to obtain. To address this, we performed single-cell transcriptome and antibody repertoire sequencing of the murine bone marrow plasma cell population following chronic lymphocytic choriomeningitis virus infection. Our single-cell sequencing approach recovered full-length and paired heavy and light chain sequence information for thousands of plasma cells and enabled us to perform recombinant antibody expression and specificity screening. Antibody repertoire analysis revealed that, relative to protein immunization, chronic infection led to increased levels of clonal expansion, class-switching, and somatic variants. Furthermore, antibodies from the highly expanded and class-switched (IgG) plasma cells were found to be specific for multiple viral antigens and a subset of clones exhibited cross-reactivity to non-viral- and auto-antigens. Integrating single-cell transcriptome data with antibody specificity suggested that plasma cell transcriptional phenotype was correlated to viral antigen specificity. Our findings demonstrate that chronic viral infection can induce and sustain plasma cell clonal expansion, combined with significant somatic hypermutation, and can generate cross-reactive antibodies. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=69 SRC="FIGDIR/small/428852v1_ufig1.gif" ALT="Figure 1"> View larger version (22K): org.highwire.dtl.DTLVardef@9d0852org.highwire.dtl.DTLVardef@18d46fcorg.highwire.dtl.DTLVardef@2f7f80org.highwire.dtl.DTLVardef@5be3fd_HPS_FORMAT_FIGEXP M_FIG Graphical abstract.Single-cell sequencing reveals clonally expanded plasma cells during chronic viral infection produce virus-specific and cross-reactive antibodies. C_FIG

immunology↗

bioRxiv2020

Cold Exposure Protects from Neuroinflammation Through Immunologic Reprogramming

Autoimmunity is energetically costly, but the impact of a metabolically active state on immunity and immune-mediated diseases is unclear. Ly6Chi monocytes are key effectors in CNS autoimmunity with elusive role in priming naive autoreactive T cells. Here we provide unbiased analysis of the immune changes in various compartments during cold exposure, and show that this energetically costly stimulus markedly ameliorates active experimental autoimmune encephalomyelitis (EAE). Cold exposure decreases MHCII on monocytes at steady-state and in various inflammatory mouse models, and suppresses T cell priming and pathogenicity through the modulation of monocytes. Genetic, or antibody-mediated monocyte depletion, or adoptive transfer of Th1- or Th17-polarized cells for EAE abolish the cold-induced effects on T cells or EAE, respectively. These findings provide a mechanistic link between environmental temperature and neuroinflammation, and suggest competition between cold-induced metabolic adaptations and autoimmunity as energetic trade-off beneficial for the immune-mediated diseases.

immunology↗

bioRxiv2020

Single-cell immune repertoire and transcriptome sequencing reveals that clonally expanded and transcriptionally distinct lymphocytes populate the aged central nervous system in mice

Neuroinflammation plays a crucial role during ageing and various neurological conditions, including Alzheimers disease, multiple sclerosis and infection. Technical limitations, however, have prevented an integrative analysis of how lymphocyte immune receptor repertoires and their accompanying transcriptional states change with age in the central nervous system (CNS). Here, we leveraged single-cell sequencing to simultaneously profile B cell receptor (BCR) and T cell receptor (TCR) repertoires and accompanying gene expression profiles in young and old mouse brains. We observed the presence of clonally expanded B and T cells in the central nervous system (CNS) of aged mice. Furthermore, many of these B cells were of the IgM and IgD isotype and had low levels of somatic hypermutation. Integrating gene expression information additionally revealed distinct transcriptional profiles of these clonally expanded lymphocytes. Our findings implicate that clonally related T and B cells in the CNS of elderly mice may contribute to neuroinflammation accompanying homeostatic ageing.

immunology↗

bioRxiv2019

Localized calcium accumulations prime synapses for phagocyte removal in cortical neuroinflammation

Cortical pathology contributes to chronic cognitive impairment of patients suffering from the neuroinflammatory disease multiple sclerosis (MS). How such gray matter inflammation affects neuronal structure and function is not well understood. Here we use functional and structural in vivo imaging in a mouse model of cortical MS to demonstrate that bouts of cortical inflammation disrupt cortical circuit activity coincident with a widespread but transient loss of dendritic spines. Spines destined for removal show a local calcium accumulation and are subsequently removed by invading macrophages and activated microglia. Targeting phagocyte activation with a new antagonist of the colony-stimulating factor 1 receptor prevents cortical synapse loss. Overall, our study identifies synapse loss as a key pathological feature of inflammatory gray matter lesions that is amenable to immunomodulatory therapy.\n\nHIGHLIGHTSO_LIWidespread, but transient loss of synapses in inflammatory lesions and beyond\nC_LIO_LIReversible impairment of neuronal firing and circuit function in the inflamed cortex\nC_LIO_LICalcium dyshomeostasis of single spines precedes swift synapse loss\nC_LIO_LIPhagocyte-mediated spine pruning as targetable mechanism of synapse loss\nC_LI

neuroscience↗

bioRxiv2019

Salivary gland macrophages assist tissue-resident CD8+ T cell immune surveillance

Tissue macrophages and tissue resident memory CD8+ T cells (TRM) play important roles for pathogen sensing and rapid protection of barrier tissues. To date, it is incompletely understood how these two cell types cooperate for efficient organ surveillance during homeostasis. Here, we used intravital imaging to show that TRM dynamically crawled along tissue macrophages in murine submandibular salivary glands (SMG) during the memory phase following a viral infection. Ex vivo confined SMG TRM integrated an unexpectedly wide range of migration modes: in addition to chemokine-and adhesion receptor-driven motility, SMG TRM displayed a remarkable capacity of autonomous motility in the absence of chemoattractants and adhesive ligands. This unique intrinsic SMG TRM motility was transmitted by friction and adaptation to microenvironmental topography through protrusion insertion into permissive gaps. Analysis of extracellular space in SMG using super-resolution shadow imaging showed discontinuous attachment of tissue macrophages to neighboring epithelial cells, offering paths of least resistance for patrolling TRM. Upon tissue macrophage depletion, intraepithelial SMG TRM showed decreased motility and reduced epithelial crossing events, and failed to cluster in response to local inflammatory chemokine stimuli. In sum, our data uncover a continuum of SMG TRM migration modes and identify a new accessory function of tissue macrophages to facilitate TRM patrolling of the complex exocrine gland architecture.\n\nOne sentence summaryCombined in vitro and in vivo imaging of salivary gland-resident tissue memory CD8+ T cells (TRM) uncovers their unique migratory behavior and describes a novel accessory function of tissue macrophages to assist TRM surveillance.

immunology↗

bioRxiv2018

In vivo function of the lipid raft protein Flotillin 1 during CD8+ T cell-mediated host surveillance

Flotillin-1 (Flot1) is a highly conserved, ubiquitously expressed lipid raft-associated scaffolding protein. Migration of Flot1-deficient neutrophils is impaired due to a decrease in myosin II-mediated contractility. Flot1 also accumulates in the uropod of polarized T cells, suggesting an analogous role in T cell migration. Here, we analyzed morphology and migration of naive and memory WT and Flot1-/- CD8+ T cells in lymphoid and non-lymphoid tissues with intravital two-photon microscopy, as well as their clonal expansion during antiviral immune responses. Flot1-/- CD8+ T cells displayed minor alterations in cell shape and motility parameters in vivo but showed comparable homing to lymphoid organs and infiltration into non-lymphoid tissues. Taken together, Flot1 plays a detectable but unexpectedly minor role for CD8+ T cell behavior under physiological conditions.

immunology↗

bioRxiv2018

Profound functional and molecular diversity of mitochondria revealed by cell type-specific profiling in vivo

Mitochondria vary in morphology and function in different tissues, however little is known about their molecular diversity among cell types. To investigate mitochondrial diversity in vivo, we developed an efficient protocol to isolate cell type-specific mitochondria based on a new MitoTag mouse. We profiled the mitochondrial proteome of three major neural cell types in cerebellum and identified a substantial number of differential mitochondrial markers for these cell types in mice and humans. Based on predictions from these proteomes, we demonstrate that astrocytic mitochondria metabolize long-chain fatty acids more efficiently than neurons. Moreover, we identified Rmdn3 as a major determinant of ER-mitochondria proximity in Purkinje cells. Our novel approach enables exploring mitochondrial diversity on the functional and molecular level in many in vivo contexts.

cell biology↗