Dunja Mrdjen

All Publications

• Single-cell spatial proteomic imaging for human neuropathology. Acta neuropathologica communications Vijayaragavan, K., Cannon, B. J., Tebaykin, D., Bosse, M., Baranski, A., Oliveria, J. P., Bukhari, S. A., Mrdjen, D., Corces, M. R., McCaffrey, E. F., Greenwald, N. F., Sigal, Y., Marquez, D., Khair, Z., Bruce, T., Goldston, M., Bharadwaj, A., Montine, K. S., Angelo, R. M., Montine, T. J., Bendall, S. C. 2022; 10 (1): 158

  Abstract

  Neurodegenerative disorders are characterized by phenotypic changes and hallmark proteopathies. Quantifying these in archival human brain tissues remains indispensable for validating animal models and understanding disease mechanisms. We present a framework for nanometer-scale, spatial proteomics with multiplex ion beam imaging (MIBI) for capturing neuropathological features. MIBI facilitated simultaneous, quantitative imaging of 36 proteins on archival human hippocampus from individuals spanning cognitively normal to dementia. Customized analysis strategies identified cell types and proteopathies in the hippocampus across stages of Alzheimer's disease (AD) neuropathologic change. We show microglia-pathologic tau interactions in hippocampal CA1 subfield in AD dementia. Data driven, sample independent creation of spatial proteomic regions identified persistent neurons in pathologic tau neighborhoods expressing mitochondrial protein MFN2, regardless of cognitive status, suggesting a survival advantage. Our study revealed unique insights from multiplexed imaging and data-driven approaches for neuropathologic analysis and serves broadly as a methodology for spatial proteomic analysis of archival human neuropathology. TEASER: Multiplex Ion beam Imaging enables deep spatial phenotyping of human neuropathology-associated cellular and disease features.

  View details for DOI 10.1186/s40478-022-01465-x

  View details for PubMedID 36333818

• Multiplexed Ion Beam Imaging: Insights into Pathobiology. Annual review of pathology Liu, C. C., McCaffrey, E. F., Greenwald, N. F., Soon, E., Risom, T., Vijayaragavan, K., Oliveria, J., Mrdjen, D., Bosse, M., Tebaykin, D., Bendall, S. C., Angelo, M. 2021

  Abstract

  Next-generation tools for multiplexed imaging have driven a new wave of innovation in understanding how single-cell function and tissue structure are interrelated. In previous work, we developed multiplexed ion beam imaging by time of flight, a highly multiplexed platform that uses secondary ion mass spectrometry to image dozens of antibodies tagged with metal reporters. As instrument throughput has increased, the breadth and depth of imaging data have increased as well. To extract meaningful information from these data, we have developed tools for cell identification, cell classification, and spatial analysis. In this review, we discuss these tools and provide examples of their application in various contexts, including ductal carcinoma in situ, tuberculosis, and Alzheimer's disease. We hope the synergy between multiplexed imaging and automated image analysis will drive a new era in anatomic pathology and personalized medicine wherein quantitative spatial signatures are used routinely for more accurate diagnosis, prognosis, and therapeutic selection. Expected final online publication date for the Annual Review of Pathology: Mechanisms of Disease, Volume 17 is January 2022. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

  View details for DOI 10.1146/annurev-pathmechdis-030321-091459

  View details for PubMedID 34752710

• MAUI (MBI Analysis User Interface)-An image processing pipeline for Multiplexed Mass Based Imaging. PLoS computational biology Baranski, A., Milo, I., Greenbaum, S., Oliveria, J., Mrdjen, D., Angelo, M., Keren, L. 2021; 17 (4): e1008887

  Abstract

  Mass Based Imaging (MBI) technologies such as Multiplexed Ion Beam Imaging by time of flight (MIBI-TOF) and Imaging Mass Cytometry (IMC) allow for the simultaneous measurement of the expression levels of 40 or more proteins in biological tissue, providing insight into cellular phenotypes and organization in situ. Imaging artifacts, resulting from the sample, assay or instrumentation complicate downstream analyses and require correction by domain experts. Here, we present MBI Analysis User Interface (MAUI), a series of graphical user interfaces that facilitate this data pre-processing, including the removal of channel crosstalk, noise and antibody aggregates. Our software streamlines these steps and accelerates processing by enabling real-time and interactive parameter tuning across multiple images.

  View details for DOI 10.1371/journal.pcbi.1008887

  View details for PubMedID 33872301

• Mass-tag barcoding for multiplexed analysis of human synaptosomes and other anuclear events. Cytometry. Part A : the journal of the International Society for Analytical Cytology Gajera, C. R., Fernandez, R., Montine, K. S., Fox, E. J., Mrdjen, D., Postupna, N. O., Keene, C. D., Bendall, S. C., Montine, T. J. 2021

  Abstract

  Mass-tag cell barcoding has increased the throughput, multiplexing, and robustness of multiple cytometry approaches. Previously, we adapted mass cytometry for cells to analyze synaptosome preparations (mass synaptometry or SynTOF), extending mass cytometry to these smaller, anuclear particles. To improve throughput and individual event resolution, we report here the application of palladium-based barcoding in human synaptosomes. Up to 20 individual samples, each with a unique combinatorial barcode, were pooled for labeling with an antibody cocktail. Our synaptosome protocol used six palladium-based barcoding reagents, and in combination with sequential gating increased the identification of presynaptic events approximately fourfold. These same parameters also efficiently resolved two other anuclear particles: human red blood cells and platelets. The addition of palladium-based mass-tag barcoding to our approach improves mass cytometry of synaptic particles.

  View details for DOI 10.1002/cyto.a.24340

  View details for PubMedID 33818911

• Single-cell peripheral immunoprofiling of Alzheimer's and Parkinson's diseases. Science advances Phongpreecha, T., Fernandez, R., Mrdjen, D., Culos, A., Gajera, C. R., Wawro, A. M., Stanley, N., Gaudilliere, B., Poston, K. L., Aghaeepour, N., Montine, T. J. 2020; 6 (48)

  Abstract

  Peripheral blood mononuclear cells (PBMCs) may provide insight into the pathogenesis of Alzheimer's disease (AD) or Parkinson's disease (PD). We investigated PBMC samples from 132 well-characterized research participants using seven canonical immune stimulants, mass cytometric identification of 35 PBMC subsets, and single-cell quantification of 15 intracellular signaling markers, followed by machine learning model development to increase predictive power. From these, three main intracellular signaling pathways were identified specifically in PBMC subsets from people with AD versus controls: reduced activation of PLCgamma2 across many cell types and stimulations and selectively variable activation of STAT1 and STAT5, depending on stimulant and cell type. Our findings functionally buttress the now multiply-validated observation that a rare coding variant in PLCG2 is associated with a decreased risk of AD. Together, these data suggest enhanced PLCgamma2 activity as a potential new therapeutic target for AD with a readily accessible pharmacodynamic biomarker.

  View details for DOI 10.1126/sciadv.abd5575

  View details for PubMedID 33239300

• Single-cell metabolic profiling of human cytotoxic T cells. Nature biotechnology Hartmann, F. J., Mrdjen, D. n., McCaffrey, E. n., Glass, D. R., Greenwald, N. F., Bharadwaj, A. n., Khair, Z. n., Verberk, S. G., Baranski, A. n., Baskar, R. n., Graf, W. n., Van Valen, D. n., Van den Bossche, J. n., Angelo, M. n., Bendall, S. C. 2020

  Abstract

  Cellular metabolism regulates immune cell activation, differentiation and effector functions, but current metabolic approaches lack single-cell resolution and simultaneous characterization of cellular phenotype. In this study, we developed an approach to characterize the metabolic regulome of single cells together with their phenotypic identity. The method, termed single-cell metabolic regulome profiling (scMEP), quantifies proteins that regulate metabolic pathway activity using high-dimensional antibody-based technologies. We employed mass cytometry (cytometry by time of flight, CyTOF) to benchmark scMEP against bulk metabolic assays by reconstructing the metabolic remodeling of in vitro-activated naive and memory CD8+ T cells. We applied the approach to clinical samples and identified tissue-restricted, metabolically repressed cytotoxic T cells in human colorectal carcinoma. Combining our method with multiplexed ion beam imaging by time of flight (MIBI-TOF), we uncovered the spatial organization of metabolic programs in human tissues, which indicated exclusion of metabolically repressed immune cells from the tumor-immune boundary. Overall, our approach enables robust approximation of metabolic and functional states in individual cells.

  View details for DOI 10.1038/s41587-020-0651-8

  View details for PubMedID 32868913

• GM-CSF and CXCR4 define a T helper cell signature in multiple sclerosis. Nature medicine Galli, E., Hartmann, F. J., Schreiner, B., Ingelfinger, F., Arvaniti, E., Diebold, M., Mrdjen, D., van der Meer, F., Krieg, C., Nimer, F. A., Sanderson, N., Stadelmann, C., Khademi, M., Piehl, F., Claassen, M., Derfuss, T., Olsson, T., Becher, B. 2019

  Abstract

  Cytokine dysregulation is a central driver of chronic inflammatory diseases such as multiple sclerosis (MS). Here, we sought to determine the characteristic cellular and cytokine polarization profile in patients with relapsing-remitting multiple sclerosis (RRMS) by high-dimensional single-cell mass cytometry (CyTOF). Using a combination of neural network-based representation learning algorithms, we identified an expanded T helper cell subset in patients with MS, characterized by the expression of granulocyte-macrophage colony-stimulating factor and the C-X-C chemokine receptor type 4. This cellular signature, which includes expression of very late antigen 4 in peripheral blood, was also enriched in the central nervous system of patients with relapsing-remitting multiple sclerosis. In independent validation cohorts, we confirmed that this cell population is increased in patients with MS compared with other inflammatory and non-inflammatory conditions. Lastly, we also found the population to be reduced under effective disease-modifying therapy, suggesting that the identified T cell profile represents a specific therapeutic target in MS.

  View details for DOI 10.1038/s41591-019-0521-4

  View details for PubMedID 31332391

• Pre-conception maternal helminth infection transfers via nursing long-lasting cellular immunity against helminths to offspring SCIENCE ADVANCES Darby, M. G., Chetty, A., Mrjden, D., Rolot, M., Smith, K., Mackowiak, C., Sedda, D., Nyangahu, D., Jaspan, H., Toellner, K., Waisman, A., Quesniaux, V., Ryffel, B., Cunningham, A. F., Dewals, B. G., Brombacher, F., Horsnell, W. C. 2019; 5 (5): eaav3058

  Abstract

  Maternal immune transfer is the most significant source of protection from early-life infection, but whether maternal transfer of immunity by nursing permanently alters offspring immunity is poorly understood. Here, we identify maternal immune imprinting of offspring nursed by mothers who had a pre-conception helminth infection. Nursing of pups by helminth-exposed mothers transferred protective cellular immunity to these offspring against helminth infection. Enhanced control of infection was not dependent on maternal antibody. Protection associated with systemic development of protective type 2 immunity in T helper 2 (TH2) impaired IL-4Rα-/- offspring. This maternally acquired immunity was maintained into maturity and required transfer (via nursing) to the offspring of maternally derived TH2-competent CD4 T cells. Our data therefore reveal that maternal exposure to a globally prevalent source of infection before pregnancy provides long-term nursing-acquired immune benefits to offspring mediated by maternally derived pathogen-experienced lymphocytes.

  View details for DOI 10.1126/sciadv.aav3058

  View details for Web of Science ID 000470125000038

  View details for PubMedID 31236458

  View details for PubMedCentralID PMC6587632

• Conventional DCs sample and present myelin antigens in the healthy CNS and allow parenchymal T cell entry to initiate neuroinflammation SCIENCE IMMUNOLOGY Mundt, S., Mrdjen, D., Utz, S. G., Greter, M., Schreiner, B., Becher, B. 2019; 4 (31)

  Abstract

  The central nervous system (CNS) is under close surveillance by immune cells, which mediate tissue homeostasis, protection, and repair. Conversely, in neuroinflammation, dysregulated leukocyte invasion into the CNS leads to immunopathology and neurological disability. To invade the brain parenchyma, autoimmune encephalitogenic T helper (TH) cells must encounter their cognate antigens (Ags) presented via local Ag-presenting cells (APCs). The precise identity of the APC that samples, processes, and presents CNS-derived Ags to autoaggressive T cells is unknown. Here, we used a combination of high-dimensional single-cell mapping and conditional MHC class II ablation across all CNS APCs to systematically interrogate each population for its ability to reactivate encephalitogenic TH cells in vivo. We found a population of conventional dendritic cells, but not border-associated macrophages or microglia, to be essential for licensing T cells to initiate neuroinflammation.

  View details for DOI 10.1126/sciimmunol.aau8380

  View details for Web of Science ID 000457561900005

  View details for PubMedID 30679199

• The basis of cellular and regional vulnerability in Alzheimer's disease. Acta neuropathologica Mrdjen, D. n., Fox, E. J., Bukhari, S. A., Montine, K. S., Bendall, S. C., Montine, T. J. 2019

  Abstract

  Alzheimer's disease (AD) differentially and specifically affects brain regions and neuronal cell types in a predictable pattern. Damage to the brain appears to spread and worsens with time, taking over more regions and activating multiple stressors that can converge to promote vulnerability of certain cell types. At the same time, other cell types and brain regions remain intact in the face of this onslaught of neuropathology. Although neuropathologic descriptions of AD have been extensively expanded and mapped over the last several decades, our understanding of the mechanisms underlying how certain regions and cell populations are specifically vulnerable or resistant has lagged behind. In this review, we detail what is known about the selectivity of local initiation of AD pathology in the hippocampus, its proposed spread via synaptic connections, and the diversity of clinical phenotypes and brain atrophy patterns that may arise from different fibrillar strains of pathologic proteins or genetic predispositions. We summarize accumulated and emerging knowledge of the cellular and molecular basis for neuroanatomic selectivity, consider potential disease-relevant differences between vulnerable and resistant neuronal cell types and isolate molecular markers to identify them.

  View details for DOI 10.1007/s00401-019-02054-4

  View details for PubMedID 31392412

• Elevated IgG Responses in Infants Are Associated With Reduced Prevalence of Mycobacterium tuberculosis Infection FRONTIERS IN IMMUNOLOGY Logan, E., Luabeya, A., Mulenga, H., Mrdjen, D., Ontong, C., Cunningham, A. F., Tameris, M., McShane, H., Scriba, T. J., Horsnell, W. C., Hatherill, M. 2018; 9: 1529

  Abstract

  It is unclear whether antibodies can prevent Mycobacterium tuberculosis (Mtb) infection. In this study, we examined the relationship between total plasma IgG levels, IgG elicited by childhood vaccines and soil-transmitted helminths, and Mtb infection prevalence, defined by positive QuantiFERON (QFT) test.We studied 100 Mtb uninfected infants, aged 4-6 months. Ten infants (10%) converted to positive QFT test (QFT+) within 2 years of follow-up for Mtb infection. Antibody responses in plasma samples acquired at baseline and tuberculosis investigation were analyzed by enzyme-linked immunosorbent assay and ImmunoCAP® assay.QFT- infants displayed a significant increase in total IgG titers when re-tested, compared to IgG titers at baseline, which was not observed in QFT+ infants. Bacille Calmette-Guérin (BCG) vaccine-specific IgG2 and live-attenuated measles vaccine-specific IgG were raised in QFT- infants, and infants who acquired an Mtb infection did not appear to launch a BCG-specific IgG2 response. IgG titers against the endemic helminth Ascaris lumbricoides increased from baseline to QFT re-testing in all infants.These data show raised IgG associates with a QFT-status. Importantly, this effect was also associated with a trend showing raised IgG titers to BCG and measles vaccine. Our data suggest a possible protective association between raised antibody titers and acquisition of Mtb infection, potentially mediated by exposure to antigens both related and unrelated to Mtb.

  View details for DOI 10.3389/fimmu.2018.01529

  View details for Web of Science ID 000436996900002

  View details for PubMedID 30013573

  View details for PubMedCentralID PMC6036805

• High-Dimensional Single-Cell Mapping of Central Nervous System Immune Cells Reveals Distinct Myeloid Subsets in Health, Aging, and Disease. Immunity Mrdjen, D., Pavlovic, A., Hartmann, F. J., Schreiner, B., Utz, S. G., Leung, B. P., Lelios, I., Heppner, F. L., Kipnis, J., Merkler, D., Greter, M., Becher, B. 2018; 48 (3): 599

  View details for DOI 10.1016/j.immuni.2018.02.014

  View details for PubMedID 29562204

• High Dimensional Cytometry of Central Nervous System Leukocytes During Neuroinflammation. Methods in molecular biology (Clifton, N.J.) Mrdjen, D., Hartmann, F. J., Becher, B. 2017; 1559: 321-332

  Abstract

  Autoimmune diseases like multiple sclerosis (MS) develop from the activation and complex interactions of a wide network of immune cells, which penetrate the central nervous system (CNS) and cause tissue damage and neurological deficits. Experimental autoimmune encephalomyelitis (EAE) is a model used to study various aspects of MS, including the infiltration of autoaggressive T cells and pathogenic, inflammatory myeloid cells into the CNS. Various signature landscapes of immune cell infiltrates have proven useful in shedding light on the causes of specific EAE symptoms in transgenic mice. However, single cell analysis of these infiltrates has thus far been limited in conventional fluorescent flow cytometry methods by 14-16 parameter staining panels. With the advent of mass cytometry and metal-tagged antibodies, a staining panel of 35-45 parameters is now possible. With the aid of dimensionality reducing and clustering algorithms to visualize and analyze this high dimensional data, this allows for a more comprehensive picture of the different cell populations in an inflamed CNS, at a single cell resolution level. Here, we describe the induction of active EAE in C56BL/6 mice and, in particular, the staining of microglia and CNS invading immune cells for mass cytometry with subsequent data visualization and analysis.

  View details for DOI 10.1007/978-1-4939-6786-5_22

  View details for PubMedID 28063054

• High-dimensional single-cell analysis reveals the immune signature of narcolepsy. journal of experimental medicine Hartmann, F. J., Bernard-Valnet, R., Quériault, C., Mrdjen, D., Weber, L. M., Galli, E., Krieg, C., Robinson, M. D., Nguyen, X., Dauvilliers, Y., Liblau, R. S., Becher, B. 2016

  Abstract

  Narcolepsy type 1 is a devastating neurological sleep disorder resulting from the destruction of orexin-producing neurons in the central nervous system (CNS). Despite its striking association with the HLA-DQB1*06:02 allele, the autoimmune etiology of narcolepsy has remained largely hypothetical. Here, we compared peripheral mononucleated cells from narcolepsy patients with HLA-DQB1*06:02-matched healthy controls using high-dimensional mass cytometry in combination with algorithm-guided data analysis. Narcolepsy patients displayed multifaceted immune activation in CD4(+) and CD8(+) T cells dominated by elevated levels of B cell-supporting cytokines. Additionally, T cells from narcolepsy patients showed increased production of the proinflammatory cytokines IL-2 and TNF. Although it remains to be established whether these changes are primary to an autoimmune process in narcolepsy or secondary to orexin deficiency, these findings are indicative of inflammatory processes in the pathogenesis of this enigmatic disease.

  View details for PubMedID 27821550

  View details for PubMedCentralID PMC5110028

• The end of gating? An introduction to automated analysis of high dimensional cytometry data EUROPEAN JOURNAL OF IMMUNOLOGY Mair, F., Hartmann, F. J., Mrdjen, D., Tosevski, V., Krieg, C., Becher, B. 2016; 46 (1): 34-43

  Abstract

  Ever since its invention half a century ago, flow cytometry has been a major tool for single-cell analysis, fueling advances in our understanding of a variety of complex cellular systems, in particular the immune system. The last decade has witnessed significant technical improvements in available cytometry platforms, such that more than 20 parameters can be analyzed on a single-cell level by fluorescence-based flow cytometry. The advent of mass cytometry has pushed this limit up to, currently, 50 parameters. However, traditional analysis approaches for the resulting high-dimensional datasets, such as gating on bivariate dot plots, have proven to be inefficient. Although a variety of novel computational analysis approaches to interpret these datasets are already available, they have not yet made it into the mainstream and remain largely unknown to many immunologists. Therefore, this review aims at providing a practical overview of novel analysis techniques for high-dimensional cytometry data including SPADE, t-SNE, Wanderlust, Citrus, and PhenoGraph, and how these applications can be used advantageously not only for the most complex datasets, but also for standard 14-parameter cytometry datasets.

  View details for DOI 10.1002/eji.201545774

  View details for Web of Science ID 000368234800005

  View details for PubMedID 26548301

• Natural and Vaccine-Mediated Immunity to Salmonella Typhimurium is Impaired by the Helminth Nippostrongylus brasiliensis PLOS NEGLECTED TROPICAL DISEASES Bobat, S., Darby, M., Mrdjen, D., Cook, C., Logan, E., Auret, J., Jones, E., Schnoeller, C., Flores-Langarica, A., Ross, E. A., Vira, A., Lopez-Macias, C., Henderson, I. R., Alexander, J., Brombacher, F., Horsnell, W. G., Cunningham, A. F. 2014; 8 (12)

  Abstract

  The impact of exposure to multiple pathogens concurrently or consecutively on immune function is unclear. Here, immune responses induced by combinations of the bacterium Salmonella Typhimurium (STm) and the helminth Nippostrongylus brasiliensis (Nb), which causes a murine hookworm infection and an experimental porin protein vaccine against STm, were examined.Mice infected with both STm and Nb induced similar numbers of Th1 and Th2 lymphocytes compared with singly infected mice, as determined by flow cytometry, although lower levels of secreted Th2, but not Th1 cytokines were detected by ELISA after re-stimulation of splenocytes. Furthermore, the density of FoxP3+ T cells in the T zone of co-infected mice was lower compared to mice that only received Nb, but was greater than those that received STm. This reflected the intermediate levels of IL-10 detected from splenocytes. Co-infection compromised clearance of both pathogens, with worms still detectable in mice weeks after they were cleared in the control group. Despite altered control of bacterial and helminth colonization in co-infected mice, robust extrafollicular Th1 and Th2-reflecting immunoglobulin-switching profiles were detected, with IgG2a, IgG1 and IgE plasma cells all detected in parallel. Whilst extrafollicular antibody responses were maintained in the first weeks after co-infection, the GC response was less than that in mice infected with Nb only. Nb infection resulted in some abrogation of the longer-term development of anti-STm IgG responses. This suggested that prior Nb infection may modulate the induction of protective antibody responses to vaccination. To assess this we immunized mice with porins, which confer protection in an antibody-dependent manner, before challenging with STm. Mice that had resolved a Nb infection prior to immunization induced less anti-porin IgG and had compromised protection against infection.These findings demonstrate that co-infection can radically alter the development of protective immunity during natural infection and in response to immunization.

  View details for DOI 10.1371/journal.pntd.0003341

  View details for Web of Science ID 000346701000024

  View details for PubMedID 25474738

  View details for PubMedCentralID PMC4256288