Publications

1. Terminal deoxynucleotidyl transferase and CD84 identify human multi-potent lymphoid progenitors. Nature communications Kim, Y., Calderon, A. A., Favaro, P., Glass, D. R., Tsai, A. G., Ho, D., Borges, L., Greenleaf, W. J., Bendall, S. C. 2024; 15 (1): 5910

   Lymphoid specification in human hematopoietic progenitors is not fully understood. To better associate lymphoid identity with protein-level cell features, we conduct a highly multiplexed single-cell proteomic screen on human bone marrow progenitors. This screen identifies terminal deoxynucleotidyl transferase (TdT), a specialized DNA polymerase intrinsic to VDJ recombination, broadly expressed within CD34+ progenitors prior to B/T cell emergence. While these TdT+ cells coincide with granulocyte-monocyte progenitor (GMP) immunophenotype, their accessible chromatin regions show enrichment for lymphoid-associated transcription factor (TF) motifs. TdT expression on GMPs is inversely related to the SLAM family member CD84. Prospective isolation of CD84lo GMPs demonstrates robust lymphoid potentials ex vivo, while still retaining significant myeloid differentiation capacity, akin to LMPPs. This multi-omic study identifies human bone marrow lymphoid-primed progenitors, further defining the lympho-myeloid axis in human hematopoiesis.

   DOI 10.1038/s41467-024-49883-w

   PubMed 39003273

2. Unravelling human hematopoietic progenitor cell diversity through association with intrinsic regulatory factors. bioRxiv : the preprint server for biology Favaro, P., Glass, D. R., Borges, L., Baskar, R., Reynolds, W., Ho, D., Bruce, T., Tebaykin, D., Scanlon, V. M., Shestopalov, I., Bendall, S. C. 2023

   Hematopoietic stem and progenitor cell (HSPC) transplantation is an essential therapy for hematological conditions, but finer definitions of human HSPC subsets with associated function could enable better tuning of grafts and more routine, lower-risk application. To deeply phenotype HSPCs, following a screen of 328 antigens, we quantified 41 surface proteins and functional regulators on millions of CD34+ and CD34- cells, spanning four primary human hematopoietic tissues: bone marrow, mobilized peripheral blood, cord blood, and fetal liver. We propose more granular definitions of HSPC subsets and provide new, detailed differentiation trajectories of erythroid and myeloid lineages. These aspects of our revised human hematopoietic model were validated with corresponding epigenetic analysis and in vitro clonal differentiation assays. Overall, we demonstrate the utility of using molecular regulators as surrogates for cellular identity and functional potential, providing a framework for description, prospective isolation, and cross-tissue comparison of HSPCs in humans.

   DOI 10.1101/2023.08.30.555623

   PubMed 37693547

   PubMedCentralID PMC10491219

3. Spatial proteomics reveals human microglial states shaped by anatomy and neuropathology. Research square Mrdjen, D., Amouzgar, M., Cannon, B., Liu, C., Spence, A., McCaffrey, E., Bharadwaj, A., Tebaykin, D., Bukhari, S., Hartmann, F. J., Kagel, A., Vijayaragavan, K., Oliveria, J. P., Yakabi, K., Serrano, G. E., Corrada, M. M., Kawas, C. H., Camacho, C., Bosse, M., Tibshirani, R., Beach, T. G., Angelo, M., Montine, T., Bendall, S. C. 2023

   Microglia are implicated in aging, neurodegeneration, and Alzheimer's disease (AD). Traditional, low-plex, imaging methods fall short of capturing in situ cellular states and interactions in the human brain. We utilized Multiplexed Ion Beam Imaging (MIBI) and data-driven analysis to spatially map proteomic cellular states and niches in healthy human brain, identifying a spectrum of microglial profiles, called the microglial state continuum (MSC). The MSC ranged from senescent-like to active proteomic states that were skewed across large brain regions and compartmentalized locally according to their immediate microenvironment. While more active microglial states were proximal to amyloid plaques, globally, microglia significantly shifted towards a, presumably, dysfunctional low MSC in the AD hippocampus, as confirmed in an independent cohort (n=26). This provides an in situ single cell framework for mapping human microglial states along a continuous, shifting existence that is differentially enriched between healthy brain regions and disease, reinforcing differential microglial functions overall.

   DOI 10.21203/rs.3.rs-2987263/v1

   PubMed 37398389

   PubMedCentralID PMC10312937

4. Gestationally dependent immune organization at the maternal-fetal interface. Cell reports Moore, A. R., Vivanco Gonzalez, N., Plummer, K. A., Mitchel, O. R., Kaur, H., Rivera, M., Collica, B., Goldston, M., Filiz, F., Angelo, M., Palmer, T. D., Bendall, S. C. 2022; 41 (7): 111651

   The immune system and placenta have a dynamic relationship across gestation to accommodate fetal growth and development. High-resolution characterization of this maternal-fetal interface is necessary to better understand the immunology of pregnancy and its complications. We developed a single-cell framework to simultaneously immuno-phenotype circulating, endovascular, and tissue-resident cells at the maternal-fetal interface throughout gestation, discriminating maternal and fetal contributions. Our data reveal distinct immune profiles across the endovascular and tissue compartments with tractable dynamics throughout gestation that respond to a systemic immune challenge in a gestationally dependent manner. We uncover a significant role for the innate immune system where phagocytes and neutrophils drive temporal organization of the placenta through remarkably diverse populations, including PD-L1+ subsets having compartmental and early gestational bias. Our approach and accompanying datasets provide a resource for additional investigations into gestational immunology and evoke a more significant role for the innate immune system in establishing the microenvironment of early pregnancy.

   DOI 10.1016/j.celrep.2022.111651

   PubMed 36384130

5. 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

   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.

   DOI 10.1186/s40478-022-01465-x

   PubMed 36333818

6. Supervised dimensionality reduction for exploration of single-cell data by HSS-LDA. Patterns (New York, N.Y.) Amouzgar, M., Glass, D. R., Baskar, R., Averbukh, I., Kimmey, S. C., Tsai, A. G., Hartmann, F. J., Bendall, S. C. 2022; 3 (8): 100536

   Single-cell technologies generate large, high-dimensional datasets encompassing a diversity of omics. Dimensionality reduction captures the structure and heterogeneity of the original dataset, creating low-dimensional visualizations that contribute to the human understanding of data. Existing algorithms are typically unsupervised, using measured features to generate manifolds, disregarding known biological labels such as cell type or experimental time point. We repurpose the classification algorithm, linear discriminant analysis (LDA), for supervised dimensionality reduction of single-cell data. LDA identifies linear combinations of predictors that optimally separate a priori classes, enabling the study of specific aspects of cellular heterogeneity. We implement feature selection by hybrid subset selection (HSS) and demonstrate that this computationally efficient approach generates non-stochastic, interpretable axesamenable to diverse biological processes such as differentiation over time and cell cycle. We benchmark HSS-LDA against several popular dimensionality-reduction algorithms and illustrate its utility and versatility for the exploration of single-cell mass cytometry, transcriptomics, and chromatin accessibility data.

   DOI 10.1016/j.patter.2022.100536

   PubMed 36033591

7. Integrating transcription-factor abundance with chromatin accessibility in human erythroid lineage commitment. Cell reports methods Baskar, R., Chen, A. F., Favaro, P., Reynolds, W., Mueller, F., Borges, L., Jiang, S., Park, H. S., Kool, E. T., Greenleaf, W. J., Bendall, S. C. 2022; 2 (3)

   Master transcription factors (TFs) directly regulate present and future cell states by binding DNA regulatory elements and driving gene-expression programs. Their abundance influences epigenetic priming to different cell fates at the chromatin level, especially in the context of differentiation. In order to link TF protein abundance to changes in TF motif accessibility and open chromatin, we developed InTAC-seq, a method for simultaneous quantification of genome-wide chromatin accessibility and intracellular protein abundance in fixed cells. Our method produces high-quality data and is a cost-effective alternative to single-cell techniques. We showcase our method by purifying bone marrow (BM) progenitor cells based on GATA-1 protein levels and establish high GATA-1-expressing BM cells as both epigenetically and functionally similar to erythroid-committed progenitors.

   DOI 10.1016/j.crmeth.2022.100188

   PubMed 35463156

8. An Integrated Multi-omic Single-Cell Atlas of Human B Cell Identity. Immunity Glass, D. R., Tsai, A. G., Oliveria, J. P., Hartmann, F. J., Kimmey, S. C., Calderon, A. A., Borges, L., Glass, M. C., Wagar, L. E., Davis, M. M., Bendall, S. C. 2020; 53 (1): 217–32.e5

   B cells are capable of a wide range of effector functions including antibody secretion, antigen presentation, cytokine production, and generation of immunological memory. A consistent strategy for classifying human B cells by using surface molecules is essential to harness this functional diversity for clinical translation. We developed a highly multiplexed screen to quantify the co-expression of 351 surface molecules on millions of human B cells. We identified differentially expressed molecules and aligned their variance with isotype usage, VDJ sequence, metabolic profile, biosynthesis activity, and signaling response. Based on these analyses, we propose a classification scheme to segregate B cells from four lymphoid tissues into twelve unique subsets, including a CD45RB+CD27- early memory population, a class-switched CD39+ tonsil-resident population, and a CD19hiCD11c+ memory population that potently responds to immune activation. This classification framework and underlying datasets provide a resource for further investigations of human B cell identity and function.

   DOI 10.1016/j.immuni.2020.06.013

   PubMed 32668225

9. Multiplexed single-cell morphometry for hematopathology diagnostics. Nature medicine Tsai, A. G., Glass, D. R., Juntilla, M., Hartmann, F. J., Oak, J. S., Fernandez-Pol, S., Ohgami, R. S., Bendall, S. C. 2020; 26 (3): 408–17

   The diagnosis of lymphomas and leukemias requires hematopathologists to integrate microscopically visible cellular morphology with antibody-identified cell surface molecule expression. To merge these into one high-throughput, highly multiplexed, single-cell assay, we quantify cell morphological features by their underlying, antibody-measurable molecular components, which empowers mass cytometers to 'see' like pathologists. When applied to 71 diverse clinical samples, single-cell morphometric profiling reveals robust and distinct patterns of 'morphometric' markers for each major cell type. Individually, lamin B1 highlights acute leukemias, lamin A/C helps distinguish normal from neoplastic mature T cells, and VAMP-7 recapitulates light-cytometric side scatter. Combined with machine learning, morphometric markers form intuitive visualizations of normal and neoplastic cellular distribution and differentiation. When recalibrated for myelomonocytic blast enumeration, this approach is superior to flow cytometry and comparable to expert microscopy, bypassing years of specialized training. The contextualization of traditional surface markers on independent morphometric frameworks permits more sensitive and automated diagnosis of complex hematopoietic diseases.

   DOI 10.1038/s41591-020-0783-x

   PubMed 32161403

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

    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.

    DOI 10.1038/s41587-020-0651-8

    PubMed 32868913

11. MIBI-TOF: A multiplexed imaging platform relates cellular phenotypes and tissue structure. Science advances Keren, L., Bosse, M., Thompson, S., Risom, T., Vijayaragavan, K., McCaffrey, E., Marquez, D., Angoshtari, R., Greenwald, N. F., Fienberg, H., Wang, J., Kambham, N., Kirkwood, D., Nolan, G., Montine, T. J., Galli, S. J., West, R., Bendall, S. C., Angelo, M. 2019; 5 (10): eaax5851

    Understanding tissue structure and function requires tools that quantify the expression of multiple proteins while preserving spatial information. Here, we describe MIBI-TOF (multiplexed ion beam imaging by time of flight), an instrument that uses bright ion sources and orthogonal time-of-flight mass spectrometry to image metal-tagged antibodies at subcellular resolution in clinical tissue sections. We demonstrate quantitative, full periodic table coverage across a five-log dynamic range, imaging 36 labeled antibodies simultaneously with histochemical stains and endogenous elements. We image fields of view up to 800 mum * 800 mum at resolutions down to 260 nm with sensitivities approaching single-molecule detection. We leverage these properties to interrogate intrapatient heterogeneity in tumor organization in triple-negative breast cancer, revealing regional variability in tumor cell phenotypes in contrast to a structured immune response. Given its versatility and sample back-compatibility, MIBI-TOF is positioned to leverage existing annotated, archival tissue cohorts to explore emerging questions in cancer, immunology, and neurobiology.

    DOI 10.1126/sciadv.aax5851

    PubMed 31633026

12. Comprehensive Immune Monitoring of Clinical Trials to Advance Human Immunotherapy. Cell reports Hartmann, F. J., Babdor, J., Gherardini, P. F., Amir, E. D., Jones, K., Sahaf, B., Marquez, D. M., Krutzik, P., O'Donnell, E., Sigal, N., Maecker, H. T., Meyer, E., Spitzer, M. H., Bendall, S. C. 2019; 28 (3): 819

    The success of immunotherapy has led to a myriad of clinical trials accompanied by efforts to gain mechanistic insight and identify predictive signatures for personalization. However, many immune monitoring technologies face investigator bias, missing unanticipated cellular responses in limited clinical material. We present here a mass cytometry (CyTOF) workflow for standardized, systems-level biomarker discovery in immunotherapy trials. To broadly enumerate immune cell identity and activity, we established and extensively assessed a reference panel of 33 antibodies to cover major cell subsets, simultaneously quantifying activation and immune checkpoint molecules in a single assay. This assay enumerates ≥98% of peripheral immune cells with ≥4 positively identifying antigens. Robustness and reproducibility are demonstrated on multiple samples types, across two research centers and by orthogonal measurements. Using automated analysis, we identify stratifying immune signatures in bone marrow transplantation-associated graft-versus-host disease. Together, this validated workflow ensures comprehensive immunophenotypic analysis and data comparability and will accelerate biomarker discovery.

    DOI 10.1016/j.celrep.2019.06.049

    PubMed 31315057

13. Parallel analysis of tri-molecular biosynthesis with cell identity and function in single cells NATURE COMMUNICATIONS Kimmey, S. C., Borges, L., Baskar, R., Bendall, S. C. 2019; 10

    DOI 10.1038/s41467-019-09128-7

    Web of Science ID 000460930600017

14. Proliferation tracing with single-cell mass cytometry optimizes generation of stem cell memory-like T cells NATURE BIOTECHNOLOGY Good, Z., Borges, L., Gonzalez, N., Sahaf, B., Samusik, N., Tibshirani, R., Nolan, G. P., Bendall, S. C. 2019; 37 (3): 259-+

    DOI 10.1038/s41587-019-0033-2

    Web of Science ID 000460155900016

15. A Structured Tumor-Immune Microenvironment in Triple Negative Breast Cancer Revealed by Multiplexed Ion Beam Imaging CELL Keren, L., Bosse, M., Marquez, D., Angoshtari, R., Jain, S., Varma, S., Yang, S., Kurian, A., Van Valen, D., West, R., Bendall, S. C., Angelo, M. 2018; 174 (6): 1373-+

    DOI 10.1016/j.cell.2018.08.039

    Web of Science ID 000443841000009

16. Single-cell developmental classification of B cell precursor acute lymphoblastic leukemia at diagnosis reveals predictors of relapse. Nature medicine Good, Z., Sarno, J., Jager, A., Samusik, N., Aghaeepour, N., Simonds, E. F., White, L., Lacayo, N. J., Fantl, W. J., Fazio, G., Gaipa, G., Biondi, A., Tibshirani, R., Bendall, S. C., Nolan, G. P., Davis, K. L. 2018; 24 (4): 474–83

    Insight into the cancer cell populations that are responsible for relapsed disease is needed to improve outcomes. Here we report a single-cell-based study of B cell precursor acute lymphoblastic leukemia at diagnosis that reveals hidden developmentally dependent cell signaling states that are uniquely associated with relapse. By using mass cytometry we simultaneously quantified 35 proteins involved in B cell development in 60 primary diagnostic samples. Each leukemia cell was then matched to its nearest healthy B cell population by a developmental classifier that operated at the single-cell level. Machine learning identified six features of expanded leukemic populations that were sufficient to predict patient relapse at diagnosis. These features implicated the pro-BII subpopulation of B cells with activated mTOR signaling, and the pre-BI subpopulation of B cells with activated and unresponsive pre-B cell receptor signaling, to be associated with relapse. This model, termed 'developmentally dependent predictor of relapse' (DDPR), significantly improves currently established risk stratification methods. DDPR features exist at diagnosis and persist at relapse. By leveraging a data-driven approach, we demonstrate the predictive value of single-cell 'omics' for patient stratification in a translational setting and provide a framework for its application to human cancer.

    PubMed 29505032

17. Data-Driven Phenotypic Dissection of AML Reveals Progenitor-like Cells that Correlate with Prognosis CELL Levine, J. H., Simonds, E. F., Bendall, S. C., Davis, K. L., Amir, E. D., Tadmor, M. D., Litvin, O., Fienberg, H. G., Jager, A., Zunder, E. R., Finck, R., Gedman, A. L., Radtke, I., Downing, J. R., Pe'er, D., Nolan, G. P. 2015; 162 (1): 184-197

    Acute myeloid leukemia (AML) manifests as phenotypically and functionally diverse cells, often within the same patient. Intratumor phenotypic and functional heterogeneity have been linked primarily by physical sorting experiments, which assume that functionally distinct subpopulations can be prospectively isolated by surface phenotypes. This assumption has proven problematic, and we therefore developed a data-driven approach. Using mass cytometry, we profiled surface and intracellular signaling proteins simultaneously in millions of healthy and leukemic cells. We developed PhenoGraph, which algorithmically defines phenotypes in high-dimensional single-cell data. PhenoGraph revealed that the surface phenotypes of leukemic blasts do not necessarily reflect their intracellular state. Using hematopoietic progenitors, we defined a signaling-based measure of cellular phenotype, which led to isolation of a gene expression signature that was predictive of survival in independent cohorts. This study presents new methods for large-scale analysis of single-cell heterogeneity and demonstrates their utility, yielding insights into AML pathophysiology.

    DOI 10.1016/j.cell.2015.05.047

    Web of Science ID 000357542300019

    PubMed 26095251

    PubMedCentralID PMC4508757

18. Single-Cell Trajectory Detection Uncovers Progression and Regulatory Coordination in Human B Cell Development CELL Bendall, S. C., Davis, K. L., Amir, E. D., Tadmor, M. D., Simonds, E. F., Chen, T. J., Shenfeld, D. K., Nolan, G. P., Pe'er, D. 2014; 157 (3): 714-725

    Tissue regeneration is an orchestrated progression of cells from an immature state to a mature one, conventionally represented as distinctive cell subsets. A continuum of transitional cell states exists between these discrete stages. We combine the depth of single-cell mass cytometry and an algorithm developed to leverage this continuum by aligning single cells of a given lineage onto a unified trajectory that accurately predicts the developmental path de novo. Applied to human B cell lymphopoiesis, the algorithm (termed Wanderlust) constructed trajectories spanning from hematopoietic stem cells through to naive B cells. This trajectory revealed nascent fractions of B cell progenitors and aligned them with developmentally cued regulatory signaling including IL-7/STAT5 and cellular events such as immunoglobulin rearrangement, highlighting checkpoints across which regulatory signals are rewired paralleling changes in cellular state. This study provides a comprehensive analysis of human B lymphopoiesis, laying a foundation to apply this approach to other tissues and "corrupted" developmental processes including cancer.

    DOI 10.1016/j.cell.2014.04.005

    Web of Science ID 000335392100019

    PubMed 24766814

    PubMedCentralID PMC4045247

19. Multiplexed ion beam imaging of human breast tumors. Nature medicine Angelo, M., Bendall, S. C., Finck, R., Hale, M. B., Hitzman, C., Borowsky, A. D., Levenson, R. M., Lowe, J. B., Liu, S. D., Zhao, S., Natkunam, Y., Nolan, G. P. 2014; 20 (4): 436-442

    Immunohistochemistry (IHC) is a tool for visualizing protein expression that is employed as part of the diagnostic workup for the majority of solid tissue malignancies. Existing IHC methods use antibodies tagged with fluorophores or enzyme reporters that generate colored pigments. Because these reporters exhibit spectral and spatial overlap when used simultaneously, multiplexed IHC is not routinely used in clinical settings. We have developed a method that uses secondary ion mass spectrometry to image antibodies tagged with isotopically pure elemental metal reporters. Multiplexed ion beam imaging (MIBI) is capable of analyzing up to 100 targets simultaneously over a five-log dynamic range. Here, we used MIBI to analyze formalin-fixed, paraffin-embedded human breast tumor tissue sections stained with ten labels simultaneously. The resulting data suggest that MIBI can provide new insights into disease pathogenesis that will be valuable for basic research, drug discovery and clinical diagnostics.

    DOI 10.1038/nm.3488

    PubMed 24584119

20. Single-Cell Mass Cytometry of Differential Immune and Drug Responses Across a Human Hematopoietic Continuum SCIENCE Bendall, S. C., Simonds, E. F., Qiu, P., Amir, E. D., Krutzik, P. O., Finck, R., Bruggner, R. V., Melamed, R., Trejo, A., Ornatsky, O. I., Balderas, R. S., Plevritis, S. K., Sachs, K., Pe'er, D., Tanner, S. D., Nolan, G. P. 2011; 332 (6030): 687-696

    Flow cytometry is an essential tool for dissecting the functional complexity of hematopoiesis. We used single-cell "mass cytometry" to examine healthy human bone marrow, measuring 34 parameters simultaneously in single cells (binding of 31 antibodies, viability, DNA content, and relative cell size). The signaling behavior of cell subsets spanning a defined hematopoietic hierarchy was monitored with 18 simultaneous markers of functional signaling states perturbed by a set of ex vivo stimuli and inhibitors. The data set allowed for an algorithmically driven assembly of related cell types defined by surface antigen expression, providing a superimposable map of cell signaling responses in combination with drug inhibition. Visualized in this manner, the analysis revealed previously unappreciated instances of both precise signaling responses that were bounded within conventionally defined cell subsets and more continuous phosphorylation responses that crossed cell population boundaries in unexpected manners yet tracked closely with cellular phenotype. Collectively, such single-cell analyses provide system-wide views of immune signaling in healthy human hematopoiesis, against which drug action and disease can be compared for mechanistic studies and pharmacologic intervention.

    DOI 10.1126/science.1198704

    PubMed 21551058

21. Autophagy counters inflammation-driven glycolytic impairment in aging hematopoietic stem cells. Cell stem cell Dellorusso, P. V., Proven, M. A., Calero-Nieto, F. J., Wang, X., Mitchell, C. A., Hartmann, F., Amouzgar, M., Favaro, P., DeVilbiss, A., Swann, J. W., Ho, T. T., Zhao, Z., Bendall, S. C., Morrison, S., Göttgens, B., Passegué, E. 2024

    Autophagy is central to the benefits of longevity signaling programs and to hematopoietic stem cell (HSC) response to nutrient stress. With age, a subset of HSCs increases autophagy flux and preserves regenerative capacity, but the signals triggering autophagy and maintaining the functionality of autophagy-activated old HSCs (oHSCs) remain unknown. Here, we demonstrate that autophagy is an adaptive cytoprotective response to chronic inflammation in the aging murine bone marrow (BM) niche. We find that inflammation impairs glucose uptake and suppresses glycolysis in oHSCs through Socs3-mediated inhibition of AKT/FoxO-dependent signaling, with inflammation-mediated autophagy engagement preserving functional quiescence by enabling metabolic adaptation to glycolytic impairment. Moreover, we show that transient autophagy induction via a short-term fasting/refeeding paradigm normalizes glycolytic flux and significantly boosts oHSC regenerative potential. Our results identify inflammation-driven glucose hypometabolism as a key driver of HSC dysfunction with age and establish autophagy as a targetable node to reset oHSC regenerative capacity.

    DOI 10.1016/j.stem.2024.04.020

    PubMed 38754428

22. Author Correction: Advances and prospects for the Human BioMolecular Atlas Program (HuBMAP). Nature cell biology Jain, S., Pei, L., Spraggins, J. M., Angelo, M., Carson, J. P., Gehlenborg, N., Ginty, F., Goncalves, J. P., Hagood, J. S., Hickey, J. W., Kelleher, N. L., Laurent, L. C., Lin, S., Lin, Y., Liu, H., Naba, A., Nakayasu, E. S., Qian, W., Radtke, A., Robson, P., Stockwell, B. R., Van de Plas, R., Vlachos, I. S., Zhou, M., HuBMAP Consortium, Borner, K., Snyder, M. P., Ahn, K. J., Allen, J., Anderson, D. M., Anderton, C. R., Curcio, C., Angelin, A., Arvanitis, C., Atta, L., Awosika-Olumo, D., Bahmani, A., Bai, H., Balderrama, K., Balzano, L., Bandyopadhyay, G., Bandyopadhyay, S., Bar-Joseph, Z., Barnhart, K., Barwinska, D., Becich, M., Becker, L., Becker, W., Bedi, K., Bendall, S., Benninger, K., Betancur, D., Bettinger, K., Billings, S., Blood, P., Bolin, D., Border, S., Bosse, M., Bramer, L., Brewer, M., Brusko, M., Bueckle, A., Burke, K., Burnum-Johnson, K., Butcher, E., Butterworth, E., Cai, L., Calandrelli, R., Caldwell, M., Campbell-Thompson, M., Cao, D., Cao-Berg, I., Caprioli, R., Caraccio, C., Caron, A., Carroll, M., Chadwick, C., Chen, A., Chen, D., Chen, F., Chen, H., Chen, J., Chen, L., Chen, L., Chiacchia, K., Cho, S., Chou, P., Choy, L., Cisar, C., Clair, G., Clarke, L., Clouthier, K. A., Colley, M. E., Conlon, K., Conroy, J., Contrepois, K., Corbett, A., Corwin, A., Cotter, D., Courtois, E., Cruz, A., Csonka, C., Czupil, K., Daiya, V., Dale, K., Davanagere, S. A., Dayao, M., de Caestecker, M. P., Decker, A., Deems, S., Degnan, D., Desai, T., Deshpande, V., Deutsch, G., Devlin, M., Diep, D., Dodd, C., Donahue, S., Dong, W., Dos Santos Peixoto, R., Duffy, M., Dufresne, M., Duong, T. E., Dutra, J., Eadon, M. T., El-Achkar, T. M., Enninful, A., Eraslan, G., Eshelman, D., Espin-Perez, A., Esplin, E. D., Esselman, A., Falo, L. D., Falo, L., Fan, J., Fan, R., Farrow, M. A., Farzad, N., Favaro, P., Fermin, J., Filiz, F., Filus, S., Fisch, K., Fisher, E., Fisher, S., Flowers, K., Flynn, W. F., Fogo, A. B., Fu, D. A., Fulcher, J., Fung, A., Furst, D., Gallant, M., Gao, F., Gao, Y., Gaulton, K., Gaut, J. P., Gee, J., Ghag, R. R., Ghazanfar, S., Ghose, S., Gisch, D., Gold, I., Gondalia, A., Gorman, B., Greenleaf, W., Greenwald, N., Gregory, B., Guo, R., Gupta, R., Hakimian, H., Haltom, J., Halushka, M., Han, K. S., Hanson, C., Harbury, P., Hardi, J., Harlan, L., Harris, R. C., Hartman, A., Heidari, E., Helfer, J., Helminiak, D., Hemberg, M., Henning, N., Herr, B. W., Ho, J., Holden-Wiltse, J., Hong, S., Hong, Y., Honick, B., Hood, G., Hu, P., Hu, Q., Huang, M., Huyck, H., Imtiaz, T., Isberg, O. G., Itkin, M., Jackson, D., Jacobs, M., Jain, Y., Jewell, D., Jiang, L., Jiang, Z. G., Johnston, S., Joshi, P., Ju, Y., Judd, A., Kagel, A., Kahn, A., Kalavros, N., Kalhor, K., Karagkouni, D., Karathanos, T., Karunamurthy, A., Katari, S., Kates, H., Kaushal, M., Keener, N., Keller, M., Kenney, M., Kern, C., Kharchenko, P., Kim, J., Kingsford, C., Kirwan, J., Kiselev, V., Kishi, J., Kitata, R. B., Knoten, A., Kollar, C., Krishnamoorthy, P., Kruse, A. R., Da, K., Kundaje, A., Kutschera, E., Kwon, Y., Lake, B. B., Lancaster, S., Langlieb, J., Lardenoije, R., Laronda, M., Laskin, J., Lau, K., Lee, H., Lee, M., Lee, M., Strekalova, Y. L., Li, D., Li, J., Li, J., Li, X., Li, Z., Liao, Y., Liaw, T., Lin, P., Lin, Y., Lindsay, S., Liu, C., Liu, Y., Liu, Y., Lott, M., Lotz, M., Lowery, L., Lu, P., Lu, X., Lucarelli, N., Lun, X., Luo, Z., Ma, J., Macosko, E., Mahajan, M., Maier, L., Makowski, D., Malek, M., Manthey, D., Manz, T., Margulies, K., Marioni, J., Martindale, M., Mason, C., Mathews, C., Maye, P., McCallum, C., McDonough, E., McDonough, L., Mcdowell, H., Meads, M., Medina-Serpas, M., Ferreira, R. M., Messinger, J., Metis, K., Migas, L. G., Miller, B., Mimar, S., Minor, B., Misra, R., Missarova, A., Mistretta, C., Moens, R., Moerth, E., Moffitt, J., Molla, G., Monroe, M., Monte, E., Morgan, M., Muraro, D., Murphy, B. R., Murray, E., Musen, M. A., Naglah, A., Nasamran, C., Neelakantan, T., Nevins, S., Nguyen, H., Nguyen, N., Nguyen, T., Nguyen, T., Nigra, D., Nofal, M., Nolan, G., Nwanne, G., O'Connor, M., Okuda, K., Olmer, M., O'Neill, K., Otaluka, N., Pang, M., Parast, M., Pasa-Tolic, L., Paten, B., Patterson, N. H., Peng, T., Phillips, G., Pichavant, M., Piehowski, P., Pilner, H., Pingry, E., Pita-Juarez, Y., Plevritis, S., Ploumakis, A., Pouch, A., Pryhuber, G., Puerto, J., Qaurooni, D., Qin, L., Quardokus, E. M., Rajbhandari, P., Rakow-Penner, R., Ramasamy, R., Read, D., Record, E. G., Reeves, D., Ricarte, A., Rodriguez-Soto, A., Ropelewski, A., Rosario, J., Roselkis, M., Rowe, D., Roy, T. K., Ruffalo, M., Ruschman, N., Sabo, A., Sachdev, N., Saka, S., Salamon, D., Sarder, P., Sasaki, H., Satija, R., Saunders, D., Sawka, R., Schey, K., Schlehlein, H., Scholten, D., Schultz, S., Schwartz, L., Schwenk, M., Scibek, R., Segre, A., Serrata, M., Shands, W., Shen, X., Shendure, J., Shephard, H., Shi, L., Shi, T., Shin, D., Shirey, B., Sibilla, M., Silber, M., Silverstein, J., Simmel, D., Simmons, A., Singhal, D., Sivajothi, S., Smits, T., Soncin, F., Song, Q., Stanley, V., Stuart, T., Su, H., Su, P., Sun, X., Surrette, C., Swahn, H., Tan, K., Teichmann, S., Tejomay, A., Tellides, G., Thomas, K., Thomas, T., Thompson, M., Tian, H., Tideman, L., Trapnell, C., Tsai, A. G., Tsai, C., Tsai, L., Tsui, E., Tsui, T., Tung, J., Turner, M., Uranic, J., Vaishnav, E. D., Varra, S. R., Vaskivskyi, V., Velickovic, D., Velickovic, M., Verheyden, J., Waldrip, J., Wallace, D., Wan, X., Wang, A., Wang, F., Wang, M., Wang, S., Wang, X., Wasserfall, C., Wayne, L., Webber, J., Weber, G. M., Wei, B., Wei, J., Weimer, A., Welling, J., Wen, X., Wen, Z., Williams, M., Winfree, S., Winograd, N., Woodard, A., Wright, D., Wu, F., Wu, P., Wu, Q., Wu, X., Xing, Y., Xu, T., Yang, M., Yang, M., Yap, J., Ye, D. H., Yin, P., Yuan, Z., Yun, C. J., Zahraei, A., Zemaitis, K., Zhang, B., Zhang, C., Zhang, C., Zhang, C., Zhang, K., Zhang, S., Zhang, T., Zhang, Y., Zhao, B., Zhao, W., Zheng, J. W., Zhong, S., Zhu, B., Zhu, C., Zhu, D., Zhu, Q., Zhu, Y. 2024

    DOI 10.1038/s41556-024-01384-0

    PubMed 38429479

23. Human cerebrospinal fluid single exosomes in Parkinson's and Alzheimer's diseases. bioRxiv : the preprint server for biology Yakabi, K., Berson, E., Montine, K. S., Bendall, S. C., MacCoss, M. J., Poston, K. L., Montine, T. J. 2023

    Exosomes are proposed to be important in the pathogenesis of prevalent neurodegenerative diseases. We report the first application of solid-state technology to perform multiplex analysis of single exosomes in human cerebrospinal fluid (CSF) obtained from the lumbar sac of people diagnosed with Alzheimer's disease dementia (ADD, n=30) or Parkinson's disease dementia (PDD, n=30), as well as age-matched health controls (HCN, n=30). Single events were captured with mouse monoclonal antibodies to one of three different tetraspanins (CD9, CD63, or CD81) or with mouse (M) IgG control, and then probed with fluorescently labeled antibodies to prion protein (PrP) or CD47 to mark neuronal or presynaptic origin, as well as ADD- and PDD-related proteins: amyloid beta (Abeta), tau, alpha-synuclein, and Apolipoprotein (Apo) E. Data were collected only from captured events that were within the size range of 50 to 200 nm. Exosomes were present at approximately 100 billion per mL human CSF and were similarly abundant for CD9+ and CD81+ events, but CD63+ were only 22% to 25% of CD9+ (P<0.0001) or CD81+ (P<0.0001) events. Approximately 24% of CSF exosomes were PrP+, while only 2% were CD47+. The vast majority of exosomes were surface ApoE+, and the number of PrP-ApoE+ (P<0.001) and PrP+ApoE+ (P<0.01) exosomes were significantly reduced in ADD vs. HCN for CD9+ events only. Abeta, tau, and alpha-synuclein were not detected on the exosome surface or in permeabilized cargo. These data provide new insights into single exosome molecular features and highlight reduction in the CSF concentration of ApoE+ exosomes in patients with ADD.

    DOI 10.1101/2023.12.22.573124

    PubMed 38187636

24. Immune determinants of CAR-T cell expansion in solid tumor patients receiving GD2 CAR-T cell therapy. Cancer cell Kaczanowska, S., Murty, T., Alimadadi, A., Contreras, C. F., Duault, C., Subrahmanyam, P. B., Reynolds, W., Gutierrez, N. A., Baskar, R., Wu, C. J., Michor, F., Altreuter, J., Liu, Y., Jhaveri, A., Duong, V., Anbunathan, H., Ong, C., Zhang, H., Moravec, R., Yu, J., Biswas, R., Van Nostrand, S., Lindsay, J., Pichavant, M., Sotillo, E., Bernstein, D., Carbonell, A., Derdak, J., Klicka-Skeels, J., Segal, J. E., Dombi, E., Harmon, S. A., Turkbey, B., Sahaf, B., Bendall, S., Maecker, H., Highfill, S. L., Stroncek, D., Glod, J., Merchant, M., Hedrick, C. C., Mackall, C. L., Ramakrishna, S., Kaplan, R. N. 2023

    Chimeric antigen receptor T cells (CAR-Ts) have remarkable efficacy in liquid tumors, but limited responses in solid tumors. We conducted a Phase I trial (NCT02107963) of GD2 CAR-Ts (GD2-CAR.OX40.28.z.iC9), demonstrating feasibility and safety of administration in children and young adults with osteosarcoma and neuroblastoma. Since CAR-T efficacy requires adequate CAR-T expansion, patients were grouped into good or poor expanders across dose levels. Patient samples were evaluated by multi-dimensional proteomic, transcriptomic, and epigenetic analyses. T cell assessments identified naive T cells in pre-treatment apheresis associated with good expansion, and exhausted T cells in CAR-T products with poor expansion. Myeloid cell assessment identified CXCR3+ monocytes in pre-treatment apheresis associated with good expansion. Longitudinal analysis of post-treatment samples identified increased CXCR3- classical monocytes in all groups as CAR-T numbers waned. Together, our data uncover mediators of CAR-T biology and correlates of expansion that could be utilized to advance immunotherapies for solid tumor patients.

    DOI 10.1016/j.ccell.2023.11.011

    PubMed 38134936

25. Phenotyping EMT and MET cellular states in lung cancer patient liquid biopsies at a personalized level using mass cytometry. Scientific reports Karacosta, L. G., Pancirer, D., Preiss, J. S., Benson, J. A., Trope, W., Shrager, J. B., Sung, A. W., Neal, J. W., Bendall, S. C., Wakelee, H., Plevritis, S. K. 2023; 13 (1): 21781

    Malignant pleural effusions (MPEs) can be utilized as liquid biopsy for phenotyping malignant cells and for precision immunotherapy, yet MPEs are inadequately studied at the single-cell proteomic level. Here we leverage mass cytometry to interrogate immune and epithelial cellular profiles of primary tumors and pleural effusions (PEs) from early and late-stage non-small cell lung cancer (NSCLC) patients, with the goal of assessing epithelial-mesenchymal transition (EMT) and mesenchymal-epithelial transition (MET) states in patient specimens. By using the EMT-MET reference map PHENOSTAMP, we observe a variety of EMT states in cytokeratin positive (CK+) cells, and report for the first time MET-enriched CK+ cells in MPEs. We show that these states may be relevant to disease stage and therapy response. Furthermore, we found that the fraction of CD33+ myeloid cells in PEs was positively correlated to the fraction of CK+ cells. Longitudinal analysis of MPEs drawn 2 months apart from a patient undergoing therapy, revealed that CK+ cells acquired heterogeneous EMT features during treatment. We present this work as a feasibility study that justifies deeper characterization of EMT and MET states in malignant cells found in PEs as a promising clinical platform to better evaluate disease progression and treatment response at a personalized level.

    DOI 10.1038/s41598-023-46458-5

    PubMed 38065965

    PubMedCentralID 2689101

26. Immuno-metabolic dendritic cell vaccine signatures associate with overall survival in vaccinated melanoma patients. Nature communications Adamik, J., Munson, P. V., Maurer, D. M., Hartmann, F. J., Bendall, S. C., Argüello, R. J., Butterfield, L. H. 2023; 14 (1): 7211

    Efficacy of cancer vaccines remains low and mechanistic understanding of antigen presenting cell function in cancer may improve vaccine design and outcomes. Here, we analyze the transcriptomic and immune-metabolic profiles of Dendritic Cells (DCs) from 35 subjects enrolled in a trial of DC vaccines in late-stage melanoma (NCT01622933). Multiple platforms identify metabolism as an important biomarker of DC function and patient overall survival (OS). We demonstrate multiple immune and metabolic gene expression pathway alterations, a functional decrease in OCR/OXPHOS and increase in ECAR/glycolysis in patient vaccines. To dissect molecular mechanisms, we utilize single cell SCENITH functional profiling and show patient clinical outcomes (OS) correlate with DC metabolic profile, and that metabolism is linked to immune phenotype. With single cell metabolic regulome profiling, we show that MCT1 (monocarboxylate transporter-1), a lactate transporter, is increased in patient DCs, as is glucose uptake and lactate secretion. Importantly, pre-vaccination circulating myeloid cells in patients used as precursors for DC vaccine generation are significantly skewed metabolically as are several DC subsets. Together, we demonstrate that the metabolic profile of DC is tightly associated with the immunostimulatory potential of DC vaccines from cancer patients. We link phenotypic and functional metabolic changes to immune signatures that correspond to suppressed DC differentiation.

    DOI 10.1038/s41467-023-42881-4

    PubMed 37938561

    PubMedCentralID 5698905

27. Prior Knowledge Integration Improves Relapse Prediction and Identifies Relapse Associated Mechanisms in Childhood B Cell Acute Lymphoblastic Leukemia Koladiya, A., Jager, A., Culos, A., Merchant, M., Liu, Y., Stuani, L., Sarno, J., Domizi, P., Mullighan, C. G., Aghaeepour, N., Bendall, S., Davis, K. L. AMER SOC HEMATOLOGY. 2023

    DOI 10.1182/blood-2023-187264

    Web of Science ID 001159306706074

28. Loss-of-function mutations in Dnmt3a and Tet2 lead to accelerated atherosclerosis and concordant macrophage phenotypes. Nature cardiovascular research Rauch, P. J., Gopakumar, J., Silver, A. J., Nachun, D., Ahmad, H., McConkey, M., Nakao, T., Bosse, M., Rentz, T., Vivanco Gonzalez, N., Greenwald, N. F., McCaffrey, E. F., Khair, Z., Gopakumar, M., Rodrigues, K. B., Lin, A. E., Sinha, E., Fefer, M., Cohen, D. N., Vromman, A., Shvartz, E., Sukhova, G., Bendall, S., Angelo, M., Libby, P., Ebert, B. L., Jaiswal, S. 2023; 2 (9): 805-818

    Clonal hematopoiesis of indeterminate potential (CHIP) is defined by the presence of a cancer-associated somatic mutation in white blood cells in the absence of overt hematological malignancy. It arises most commonly from loss-of-function mutations in the epigenetic regulators DNMT3A and TET2. CHIP predisposes to both hematological malignancies and atherosclerotic cardiovascular disease in humans. Here we demonstrate that loss of Dnmt3a in myeloid cells increased murine atherosclerosis to a similar degree as previously seen with loss of Tet2. Loss of Dnmt3a enhanced inflammation in macrophages in vitro and generated a distinct adventitial macrophage population in vivo which merges a resident macrophage profile with an inflammatory cytokine signature. These changes surprisingly phenocopy the effect of loss of Tet2. Our results identify a common pathway promoting heightened innate immune cell activation with loss of either gene, providing a biological basis for the excess atherosclerotic disease burden in carriers of these two most prevalent CHIP mutations.

    DOI 10.1038/s44161-023-00326-7

    PubMed 39196062

    PubMedCentralID 8050831

29. Loss-of-function mutations in <i>Dnmt3a</i> and <i>Tet2</i> lead to accelerated atherosclerosis and concordant macrophage phenotypes NATURE CARDIOVASCULAR RESEARCH Rauch, P. J., Gopakumar, J., Silver, A. J., Nachun, D., Ahmad, H., Mcconkey, M., Nakao, T., Bosse, M., Rentz, T., Gonzalez, N., Greenwald, N. F., Mccaffrey, E. F., Khair, Z., Gopakumar, M., Rodrigues, K. B., Lin, A. E., Sinha, E., Fefer, M., Cohen, D. N., Vromman, A., Shvartz, E., Sukhova, G., Bendall, S., Angelo, M., Libby, P., Ebert, B. L., Jaiswal, S. 2023; 2 (9): 805-+

    DOI 10.1038/s44161-023-00326-7

    Web of Science ID 001127053400010

30. Cross-species comparative analysis of single presynapses. Scientific reports Berson, E., Gajera, C. R., Phongpreecha, T., Perna, A., Bukhari, S. A., Becker, M., Chang, A. L., De Francesco, D., Espinosa, C., Ravindra, N. G., Postupna, N., Latimer, C. S., Shively, C. A., Register, T. C., Craft, S., Montine, K. S., Fox, E. J., Keene, C. D., Bendall, S. C., Aghaeepour, N., Montine, T. J. 2023; 13 (1): 13849

    Comparing brain structure across species and regions enables key functional insights. Leveraging publicly available data from a novel mass cytometry-based method, synaptometry by time of flight (SynTOF), we applied an unsupervised machine learning approach to conduct a comparative study of presynapse molecular abundance across three species and three brain regions. We used neural networks and their attractive properties to model complex relationships among high dimensional data to develop a unified, unsupervised framework for comparing the profile of more than 4.5 million single presynapses among normal human, macaque, and mouse samples. An extensive validation showed the feasibility of performing cross-species comparison using SynTOF profiling. Integrative analysis of the abundance of 20 presynaptic proteins revealed near-complete separation between primates and mice involving synaptic pruning, cellular energy, lipid metabolism, and neurotransmission. In addition, our analysis revealed a strong overlap between the presynaptic composition of human and macaque in the cerebral cortex and neostriatum. Our unique approach illuminates species- and region-specific variation in presynapse molecular composition.

    DOI 10.1038/s41598-023-40683-8

    PubMed 37620363

    PubMedCentralID 3365257

31. High-dimensional profiling of pediatric immune responses to solid organ transplantation. Cell reports. Medicine Rao, M., Amouzgar, M., Harden, J. T., Lapasaran, M. G., Trickey, A., Armstrong, B., Odim, J., Debnam, T., Esquivel, C. O., Bendall, S. C., Martinez, O. M., Krams, S. M. 2023: 101147

    Solid organ transplant remains a life-saving therapy for children with end-stage heart, lung, liver, or kidney disease; however, ∼33% of allograft recipients experience acute rejection within the first year after transplant. Our ability to detect early rejection is hampered by an incomplete understanding of the immune changes associated with allograft health, particularly in the pediatric population. We performed detailed, multilineage, single-cell analysis of the peripheral blood immune composition in pediatric solid organ transplant recipients, with high-dimensional mass cytometry. Supervised and unsupervised analysis methods to study cell-type proportions indicate that the allograft type strongly influences the post-transplant immune profile. Further, when organ-specific differences are considered, graft health is associated with changes in the proportion of distinct T cell subpopulations. Together, these data form the basis for mechanistic studies into the pathobiology of rejection and allow for the development of new immunosuppressive agents with greater specificity.

    DOI 10.1016/j.xcrm.2023.101147

    PubMed 37552988

32. Expanded vacuum-stable gels for multiplexed high-resolution spatial histopathology. Nature communications Bai, Y., Zhu, B., Oliveria, J., Cannon, B. J., Feyaerts, D., Bosse, M., Vijayaragavan, K., Greenwald, N. F., Phillips, D., Schurch, C. M., Naik, S. M., Ganio, E. A., Gaudilliere, B., Rodig, S. J., Miller, M. B., Angelo, M., Bendall, S. C., Rovira-Clave, X., Nolan, G. P., Jiang, S. 2023; 14 (1): 4013

    Cellular organization and functions encompass multiple scales in vivo. Emerging high-plex imaging technologies are limited in resolving subcellular biomolecular features. Expansion Microscopy (ExM) and related techniques physically expand samples for enhanced spatial resolution, but are challenging to be combined with high-plex imaging technologies to enable integrative multiscaled tissue biology insights. Here, we introduce Expand and comPRESS hydrOgels (ExPRESSO), an ExM framework that allows high-plex protein staining, physical expansion, and removal of water, while retaining the lateral tissue expansion. We demonstrate ExPRESSO imaging of archival clinical tissue samples on Multiplexed Ion Beam Imaging and Imaging Mass Cytometry platforms, with detection capabilities of>40 markers. Application of ExPRESSO on archival human lymphoid and brain tissues resolved tissue architecture at the subcellular level, particularly that of the blood-brain barrier. ExPRESSO hence provides a platform for extending the analysis compatibility of hydrogel-expanded biospecimensto mass spectrometry, with minimal modifications to protocols and instrumentation.

    DOI 10.1038/s41467-023-39616-w

    PubMed 37419873

33. Synthesis, Characterization, and Applications of a Superior Dendrimer-Based Polymer for Multiplexed Ion Beam Imaging Time-of-Flight Analysis. Biomacromolecules Kumar, R., Liu, C. C., Bendall, S. C., Angelo, M. 2023

    High-dimensional single-cell mass spectrometric imaging techniques such as multiplexed ion beam imaging by time-of-flight mass spectrometry (MIBI-TOF), imaging mass cytometry (IMC), and flow cytometry-based CyTOF utilize antibodies conjugated to linear metal-chelating polymers. Here, we report on the synthesis and characterization of a dendrimer-based polymer and its utilization in tissue imaging using MIBI-TOF. We compared the staining performance in FFPE tissue of antibodies for lineage-specific immune proteins (CD20, CD3, CD45, FoxP3) that were conjugated with dendrimer or linear polymer. Staining of serial tissue sections with dendron-conjugated and linear-polymer-conjugated antibodies revealed comparable avidities of dendrons and linear polymers with log2 (ratio of mean positive pixel intensity of staining for linear polymers to dendrons) within the range ±0.25. Interestingly, dendron-conjugated antibodies were observed to have some advantages over linear polymer-conjugated antibodies. For example, tissue staining of a nuclear protein, FoxP3 with dendron-conjugated antibodies showed notably less background staining than that of linear-polymer-conjugated antibodies. Additionally, dendron-conjugated antibodies did not exhibit off-target cytosolic binding in neural tissue typically observed when using linear polymer conjugates. Taken together, this work provides a versatile framework for using third-generation dendron-conjugated antibodies with improved staining over conventional linear polymers.

    DOI 10.1021/acs.biomac.3c00174

    PubMed 37352475

34. Dasatinib overcomes glucocorticoid resistance in B-cell acute lymphoblastic leukemia. Nature communications Sarno, J., Domizi, P., Liu, Y., Merchant, M., Pedersen, C. B., Jedoui, D., Jager, A., Nolan, G. P., Gaipa, G., Bendall, S. C., Bava, F., Davis, K. L. 2023; 14 (1): 2935

    Resistance to glucocorticoids (GC) is associated with an increased risk of relapse in B-cell progenitor acute lymphoblastic leukemia (BCP-ALL). Performing transcriptomic and single-cell proteomic studies in healthy B-cell progenitors, we herein identify coordination between the glucocorticoid receptor pathway with B-cell developmental pathways. Healthy pro-B cells most highly express the glucocorticoid receptor, and this developmental expression is conserved in primary BCP-ALL cells from patients at diagnosis and relapse. In-vitro and in vivo glucocorticoid treatment of primary BCP-ALL cells demonstrate that the interplay between B-cell development and the glucocorticoid pathways is crucial for GC resistance in leukemic cells. Gene set enrichment analysis in BCP-ALL cell lines surviving GC treatment show enrichment of B cell receptor signaling pathways. In addition, primary BCP-ALL cells surviving GC treatment in vitro and in vivo demonstrate a late pre-B cell phenotype with activation of PI3K/mTOR and CREB signaling. Dasatinib, a multi-kinase inhibitor, most effectively targets this active signaling in GC-resistant cells, and when combined with glucocorticoids, results in increased cell death in vitro and decreased leukemic burden and prolonged survival in an in vivo xenograft model. Targeting the active signaling through the addition of dasatinib may represent a therapeutic approach to overcome GC resistance in BCP-ALL.

    DOI 10.1038/s41467-023-38456-y

    PubMed 37217509

35. Systems biology approaches to unravel lymphocyte subsets and function. Current opinion in immunology Kim, Y., Greenleaf, W. J., Bendall, S. C. 2023; 82: 102323

    Single-cell technologies have revealed the extensive heterogeneity and complexity of the immune system. Systems biology approaches in immunology have taken advantage of the high-parameter, high-throughput data and analyzed immune cell types in a 'bottom-up' data-driven method. This approach has discovered previously unrecognized cell types and functions. Especially for human immunology, in which experimental manipulations are challenging, systems approach has become a successful means to investigate physiologically relevant contexts. This review focuses on the recent findings in lymphocyte biology, from their development, differentiation into subsets, and heterogeneity in their functions, enabled by these systems approaches. Furthermore, we review examples of the application of findings from systems approach studies and discuss how now to leave the rich dataset in the curse of high dimensionality.

    DOI 10.1016/j.coi.2023.102323

    PubMed 37028221

36. NK-like CD8+ γδ T cells are expanded in persistent Mycobacterium tuberculosis infection. Science immunology Roy Chowdhury, R., Valainis, J. R., Dubey, M., von Boehmer, L., Sola, E., Wilhelmy, J., Guo, J., Kask, O., Ohanyan, M., Sun, M., Huang, H., Huang, X., Nguyen, P. K., Scriba, T. J., Davis, M. M., Bendall, S. C., Chien, Y. H. 2023; 8 (81): eade3525

    The response of gamma delta (γδ) T cells in the acute versus chronic phases of the same infection is unclear. How γδ T cells function in acute Mycobacterium tuberculosis (Mtb) infection is well characterized, but their response during persistent Mtb infection is not well understood, even though most infections with Mtb manifest as a chronic, clinically asymptomatic state. Here, we analyze peripheral blood γδ T cells from a South African adolescent cohort and show that a unique CD8+ γδ T cell subset with features of "memory inflation" expands in chronic Mtb infection. These cells are hyporesponsive to T cell receptor (TCR)-mediated signaling but, like NK cells, can mount robust CD16-mediated cytotoxic responses. These CD8+ γδ T cells comprise a highly focused TCR repertoire, with clonotypes that are Mycobacterium specific but not phosphoantigen reactive. Using multiparametric single-cell pseudo-time trajectory analysis, we identified the differentiation paths that these CD8+ γδ T cells follow to develop into effectors in this infection state. Last, we found that circulating CD8+ γδ T cells also expand in other chronic inflammatory conditions, including cardiovascular disease and cancer, suggesting that persistent antigenic exposure may drive similar γδ T cell effector programs and differentiation fates.

    DOI 10.1126/sciimmunol.ade3525

    PubMed 37000856

37. Magnitude and kinetics of the human immune cell response associated with severe dengue progression by single-cell proteomics. Science advances Robinson, M. L., Glass, D. R., Duran, V., Agudelo Rojas, O. L., Sanz, A. M., Consuegra, M., Sahoo, M. K., Hartmann, F. J., Bosse, M., Gelvez, R. M., Bueno, N., Pinsky, B. A., Montoya, J. G., Maecker, H., Estupiñan Cardenas, M. I., Villar Centeno, L. A., Garrido, E. M., Rosso, F., Bendall, S. C., Einav, S. 2023; 9 (12): eade7702

    Approximately 5 million dengue virus-infected patients progress to a potentially life-threatening severe dengue (SD) infection annually. To identify the immune features and temporal dynamics underlying SD progression, we performed deep immune profiling by mass cytometry of PBMCs collected longitudinally from SD progressors (SDp) and uncomplicated dengue (D) patients. While D is characterized by early activation of innate immune responses, in SDp there is rapid expansion and activation of IgG-secreting plasma cells and memory and regulatory T cells. Concurrently, SDp, particularly children, demonstrate increased proinflammatory NK cells, inadequate expansion of CD16+ monocytes, and high expression of the FcγR CD64 on myeloid cells, yet a signature of diminished antigen presentation. Syndrome-specific determinants include suppressed dendritic cell abundance in shock/hemorrhage versus enriched plasma cell expansion in organ impairment. This study reveals uncoordinated immune responses in SDp and provides insights into SD pathogenesis in humans with potential implications for prediction and treatment.

    DOI 10.1126/sciadv.ade7702

    PubMed 36961888

38. Spatial proteomics of tumor microenvironments reveal why location matters. Nature immunology Piyadasa, H., Angelo, M., Bendall, S. C. 2023

    DOI 10.1038/s41590-023-01471-8

    PubMed 36959293

    PubMedCentralID 5998822

39. High-dimensional profiling of pediatric immune responses to solid organ transplantation Rao, M., Amouzgar, M., Harden, J. T., Lapasaran, M. G., Trickey, A., Armstrong, B., Odim, J., Debnam, T., Esquivel, C. O., Bendall, S. C., Martinez, O. M., Krams, S. M. WILEY. 2023

    Web of Science ID 001002465500071

40. Polyunsaturated fatty acid-bound alpha-fetoprotein promotes immune suppression by altering human dendritic cell metabolism. Cancer research Munson, P. V., Adamik, J., Hartmann, F. J., Favaro, P. M., Ho, D., Bendall, S. C., Combes, A. J., Krummel, M. F., Zhang, K., Kelley, R. K., Butterfield, L. H. 2023

    Alpha-fetoprotein (AFP) is expressed by stem-like and poor outcome hepatocellular cancer tumors and is a clinical tumor biomarker. AFP has been demonstrated to inhibit dendritic cell differentiation and maturation and to block oxidative phosphorylation. To identify the critical metabolic pathways leading to human dendritic cell functional suppression, here we utilized two recently described single cell profiling methods, scMEP (single-cell metabolic profiling) and SCENITH (single-cell energetic metabolism by profiling translation inhibition). Glycolytic capacity and glucose dependence of dendritic cells was significantly increased by tumor-derived, but not normal cord blood-derived, AFP, leading to increased glucose uptake and lactate secretion. Key molecules in the electron transport chain in particular were regulated by tumor-derived AFP. These metabolic changes occurred at mRNA and protein levels, with negative impact on dendritic cell stimulatory capacity. Tumor-derived AFP bound significantly more polyunsaturated fatty acids than cord blood-derived AFP. Polyunsaturated fatty acids bound to AFP increased metabolic skewing and promoted dendritic cell functional suppression. Polyunsaturated fatty acids inhibited dendritic cell differentiation in vitro, and omega-6 polyunsaturated fatty acids conferred potent immunoregulation when bound to tumor-derived AFP. Together, these findings provide mechanistic insights into how AFP antagonizes the innate immune response to limit anti-tumor immunity.

    DOI 10.1158/0008-5472.CAN-22-3551

    PubMed 36847613

41. CELL TYPE-SPECIFIC TRANSCRIPTOMIC TRAJECTORIES UNDERLYING DISEASE PROGRESSION IN INCLUSION BODY MYOSITIS Wischnewski, S., Ikenaga, C., Kocharyan, A., Thaewel, T., Zulji, A., Rausch, H., Martin, C., Mrdjen, D., Brenner, D., Bunse, L., Tan, C., Thomas, L., Kutza, M., Trobisch, T., Preusse, C., Reed, N., Von Pein, H. D., Rosenbohm, A., Ludolph, A., Hoke, A., Platten, M., Bendall, S. C., Weishaupt, J. H., Sommer, C. J., Stenzel, W., Lloyd, T. E., Schirmer, L. CLINICAL & EXPER RHEUMATOLOGY. 2023: 506-507

    Web of Science ID 000944146200197

42. UNRAVELLING HUMAN HEMATOPOIETIC PROGENITOR CELL DIVERSITY THROUGH ASSOCIATION WITH INTRINSIC REGULATORY FACTORS Favaro, P., Glass, D., Borges, L., Baskar, R., Reynolds, W., Ho, D., Bruce, T., Tebaykin, D., Tsai, A., Shestopalov, I., Bendall, S. ELSEVIER SCIENCE INC. 2023: S79

    Web of Science ID 001057881700100

43. IKAROS MEDIATES ANTIGEN ESCAPE FOLLOWING CD19 CAR T CELL THERAPY IN R/R B-ALL Domizi, P., Sarno, J., Jager, A., Rotiroti, M., Baskar, R., Reynolds, W., Sahaf, B., Bendall, S., Mullighan, C., Leahy, A., Myers, R., Grupp, S., Majzner, R., Sotillo, E., Barrett, D., Davis, K. WILEY. 2022

    Web of Science ID 000859203900105

44. DEEP MYELOID CELL PROFILING PROVIDES NEW INSIGHTS INTO MODULATORS OF CAR T CELL EXPANSION IN PATIENTS WITH SOLID TUMOR MALIGNANCIES Kaczanowska, S., Ramakrishna, S., Murty, T., Contreras, C., Alimadadi, A., Gutierrez, N., Jhaveri, A., Liu, Y., Altreuter, J., Michor, F., Duault, C., Balasubrahmanyam, P., Reynolds, W., Baskar, R., Pichavant, M., Sahaf, B., Bendall, S., Maecker, H., Merchant, M., Mackall, C., Hedrick, C., Kaplan, R. BMJ PUBLISHING GROUP. 2022: A418

    DOI 10.1136/jitc-2022-SITC2022.0397

    Web of Science ID 000919423400393

45. DEFINING T CELL EXHAUSTION AND MEMORY CORRELATES OF GD2 CAR T CELL EXPANSION IN PEDIATRIC PATIENTS WITH SOLID TUMOR MALIGNANCIES Murty, T., Ramakrishna, S., Kaczanowska, S., Contreras, C., Duault, C., Balasubrahmanyam, P., Reynolds, W., Baskar, R., Jhaveri, A., Liu, Y., Altreuter, J., Michor, F., Pichavant, M., Sahaf, B., Bendall, S., Maecker, H., Merchant, M., Mackall, C., Kaplan, R. BMJ PUBLISHING GROUP. 2022: A381

    DOI 10.1136/jitc-2022-SITC2022.0362

    Web of Science ID 000919423400358

46. Post-infusion CAR T-Reg cells identify patients resistant to CD19-CAR therapy NATURE MEDICINE Good, Z., Spiegel, J. Y., Sahaf, B., Malipatlolla, M. B., Ehlinger, Z. J., Kurra, S., Desai, M. H., Reynolds, W. D., Lin, A., Vandris, P., Wu, F., Prabhu, S., Hamilton, M. P., Tamaresis, J. S., Hanson, P. J., Patel, S., Feldman, S. A., Frank, M. J., Baird, J. H., Muffly, L., Claire, G. K., Craig, J., Kong, K. A., Wagh, D., Coller, J., Bendall, S. C., Tibshirani, R. J., Plevritis, S. K., Miklos, D. B., Mackall, C. L. 2022

    Approximately 60% of patients with large B cell lymphoma treated with chimeric antigen receptor (CAR) T cell therapies targeting CD19 experience disease progression, and neurotoxicity remains a challenge. Biomarkers associated with resistance and toxicity are limited. In this study, single-cell proteomic profiling of circulating CAR T cells in 32 patients treated with CD19-CAR identified that CD4+Helios+ CAR T cells on day 7 after infusion are associated with progressive disease and less severe neurotoxicity. Deep profiling demonstrated that this population is non-clonal and manifests hallmark features of T regulatory (TReg) cells. Validation cohort analysis upheld the link between higher CAR TReg cells with clinical progression and less severe neurotoxicity. A model combining expansion of this subset with lactate dehydrogenase levels, as a surrogate for tumor burden, was superior for predicting durable clinical response compared to models relying on each feature alone. These data credential CAR TReg cell expansion as a novel biomarker of response and toxicity after CAR T cell therapy and raise the prospect that this subset may regulate CAR T cell responses in humans.

    DOI 10.1038/s41591-022-01960-7

    Web of Science ID 000852940800007

    PubMed 36097223

47. Distinct metabolic states guide maturation of inflammatory and tolerogenic dendritic cells. Nature communications Adamik, J., Munson, P. V., Hartmann, F. J., Combes, A. J., Pierre, P., Krummel, M. F., Bendall, S. C., Arguello, R. J., Butterfield, L. H. 2022; 13 (1): 5184

    Cellular metabolism underpins immune cell functionality, yet our understanding of metabolic influences in human dendritic cell biology and their ability to orchestrate immune responses is poorly developed. Here, we map single-cell metabolic states and immune profiles of inflammatory and tolerogenic monocytic dendritic cells using recently developed multiparametric approaches. Single-cell metabolic pathway activation scores reveal simultaneous engagement of multiple metabolic pathways in distinct monocytic dendritic cell differentiation stages. GM-CSF/IL4-induce rapid reprogramming of glycolytic monocytes and transient co-activation of mitochondrial pathways followed by TLR4-dependent maturation of dendritic cells. Skewing of the mTOR:AMPK phosphorylation balance and upregulation of OXPHOS, glycolytic and fatty acid oxidation metabolism underpin metabolic hyperactivity and an immunosuppressive phenotype of tolerogenic dendritic cells, which exhibit maturation-resistance and a de-differentiated immune phenotype marked by unique immunoregulatory receptor signatures. This single-cell dataset provides important insights into metabolic pathways impacting the immune profiles of human dendritic cells.

    DOI 10.1038/s41467-022-32849-1

    PubMed 36056019

48. The interaction of SWI/SNF with the ribosome regulates translation and confers sensitivity to translation pathway inhibitors in cancers with complex perturbations. Cancer research Ulicna, L., Kimmey, S. C., Weber, C. M., Allard, G. M., Wang, A., Bui, N. Q., Bendall, S. C., Crabtree, G. R., Bean, G. R., Van Rechem, C. 2022

    Subunits from the chromatin remodelers mammalian SWItch/Sucrose Non-Fermentable (mSWI/SNF) are mutated, deleted or amplified in more than 40% of cancers. Understanding their functions in normal cells and the consequences of cancerous alterations will provide insight into developing new targeted therapies. Here we examined whether mSWI/SNF mutations increase cellular sensitivity to specific drugs. Taking advantage of the DepMap studies, we demonstrate that cancer cells harboring mutations of specific mSWI/SNF subunits exhibit a genetic dependency on translation factors and are sensitive to translation pathway inhibitors. Furthermore, mSWI/SNF subunits were present in the cytoplasm and interacted with the translation initiation machinery, and short-term inhibition and depletion of specific subunits decreased global translation, implicating a direct role for these factors in translation. Depletion of specific mSWI/SNF subunits also increased sensitivity to mTOR-PI3K inhibitors. In patient-derived breast cancer samples, mSWI/SNF subunits expression in both the nucleus and the cytoplasm was substantially altered. In conclusion, an unexpected cytoplasmic role for mSWI/SNF complexes in translation suggests potential new therapeutic opportunities for patients afflicted by cancers demonstrating alterations in their subunits.

    DOI 10.1158/0008-5472.CAN-21-1360

    PubMed 35749589

49. An optimized protocol for phenotyping human granulocytes by mass cytometry. STAR protocols Vivanco Gonzalez, N., Oliveria, J., Tebaykin, D., Ivison, G. T., Mukai, K., Tsai, M. M., Borges, L., Nadeau, K. C., Galli, S. J., Tsai, A. G., Bendall, S. C. 2022; 3 (2): 101280

    Granulocytes encompass diverse roles, from fighting off pathogens to regulating inflammatory processes in allergies. These roles are represented by distinct cellular phenotypes that we captured with mass cytometry (CyTOF). Our protocol enables simultaneous evaluation of human basophils, eosinophils, and neutrophils under homeostasis and upon immune activation by anti-Immunoglobulin E (anti-IgE) or interleukin-3 (IL-3). Granulocyte integrity and detection of protein markers were optimized so that rare granulocyte populations could be deeply characterized by single cell mass cytometry. For complete details on the use and execution of this protocol, please refer to Vivanco Gonzalez etal. (2020).

    DOI 10.1016/j.xpro.2022.101280

    PubMed 35434655

50. Human IL-10-producing B cells have diverse states that are induced from multiple B cell subsets. Cell reports Glass, M. C., Glass, D. R., Oliveria, J. P., Mbiribindi, B., Esquivel, C. O., Krams, S. M., Bendall, S. C., Martinez, O. M. 2022; 39 (3): 110728

    Regulatory B cells (Bregs) suppress immune responses through the secretion of interleukin-10 (IL-10). This immunomodulatory capacity holds therapeutic potential, yet a definitional immunophenotype for enumeration and prospective isolation of B cells capable of IL-10 production remains elusive. Here, we simultaneously quantify cytokine production and immunophenotype in human peripheral B cells across a range of stimulatory conditions and time points using mass cytometry. Our analysis shows that multiple functional B cell subsets produce IL-10 and that no phenotype uniquely identifies IL-10+ B cells. Further, a significant portion of IL-10+ B cells co-express the pro-inflammatory cytokines IL-6 and tumor necrosis factor alpha (TNFα). Despite this heterogeneity, operationally tolerant liver transplant recipients have a unique enrichment of IL-10+, but not TNFα+ or IL-6+, B cells compared with transplant recipients receiving immunosuppression. Thus, human IL-10-producing B cells constitute an induced, transient state arising from a diversity of B cell subsets that may contribute to maintenance of immune homeostasis.

    DOI 10.1016/j.celrep.2022.110728

    PubMed 35443184

51. Reproducible, high-dimensional imaging in archival human tissue by multiplexed ion beam imaging by time-of-flight (MIBI-TOF). Laboratory investigation; a journal of technical methods and pathology Liu, C. C., Bosse, M., Kong, A., Kagel, A., Kinders, R., Hewitt, S. M., Varma, S., van de Rijn, M., Nowak, S. H., Bendall, S. C., Angelo, M. 2022

    Multiplexed ion beam imaging by time-of-flight (MIBI-TOF) is a form of mass spectrometry imaging that uses metal labeled antibodies and secondary ion mass spectrometry to image dozens of proteins simultaneously in the same tissue section. Working with the National Cancer Institute's (NCI) Cancer Immune Monitoring and Analysis Centers (CIMAC), we undertook a validation study, assessing concordance across a dozen serial sections of a tissue microarray of 21 samples that were independently processed and imaged by MIBI-TOF or single-plex immunohistochemistry (IHC) over 12 days. Pixel-level features were highly concordant across all 16 targets assessed in both staining intensity (R2=0.94±0.04) and frequency (R2=0.95±0.04). Comparison to digitized, single-plex IHC on adjacent serial sections revealed similar concordance (R2=0.85±0.08) as well. Lastly, automated segmentation and clustering of eight cell populations found that cell frequencies between serial sections yielded an average correlation of R2=0.94±0.05. Taken together, we demonstrate that MIBI-TOF, with well-vetted reagents and automated analysis, can generate consistent and quantitative annotations of clinically relevant cell states in archival human tissue, and more broadly, present a scalable framework for benchmarking multiplexed IHC approaches.

    DOI 10.1038/s41374-022-00778-8

    PubMed 35351966

52. Author Correction: The immunoregulatory landscape of human tuberculosis granulomas. Nature immunology McCaffrey, E. F., Donato, M., Keren, L., Chen, Z., Delmastro, A., Fitzpatrick, M. B., Gupta, S., Greenwald, N. F., Baranski, A., Graf, W., Kumar, R., Bosse, M., Fullaway, C. C., Ramdial, P. K., Forgo, E., Jojic, V., Van Valen, D., Mehra, S., Khader, S. A., Bendall, S. C., van de Rijn, M., Kalman, D., Kaushal, D., Hunter, R. L., Banaei, N., Steyn, A. J., Khatri, P., Angelo, M. 2022

    DOI 10.1038/s41590-022-01178-2

    PubMed 35277696

53. CytofIn enables integrated analysis of public mass cytometry datasets using generalized anchors. Nature communications Lo, Y., Keyes, T. J., Jager, A., Sarno, J., Domizi, P., Majeti, R., Sakamoto, K. M., Lacayo, N., Mullighan, C. G., Waters, J., Sahaf, B., Bendall, S. C., Davis, K. L. 2022; 13 (1): 934

    The increasing use of mass cytometry for analyzing clinical samples offers the possibility to perform comparative analyses across public datasets. However, challenges in batch normalization and data integration limit the comparison of datasets not intended to be analyzed together. Here, we present a data integration strategy, CytofIn, using generalized anchors to integrate mass cytometry datasets from the public domain. We show that low-variance controls, such as healthy samples and stable channels, are inherently homogeneous, robust against stimulation, and can serve as generalized anchors for batch correction. Single-cell quantification comparing mass cytometry data from 989 leukemia files pre- and post normalization with CytofIn demonstrates effective batch correction while recapitulating the gold-standard bead normalization. CytofIn integration of public cancer datasets enabled the comparison of immune features across histologies and treatments. We demonstrate the ability to integrate public datasets without necessitating identical control samples or bead standards for fast and robust analysis using CytofIn.

    DOI 10.1038/s41467-022-28484-5

    PubMed 35177627

54. Revealing new biology from multiplexed, metal-isotope-tagged, single-cell readouts. Trends in cell biology Baskar, R., Kimmey, S. C., Bendall, S. C. 2022

    Mass cytometry (MC) is a recent technology that pairs plasma-based ionization of cells in suspension with time-of-flight (TOF) mass spectrometry to sensitively quantify the single-cell abundance of metal-isotope-tagged affinity reagents to key proteins, RNA, and peptides. Given the ability to multiplex readouts (~50 per cell) and capture millions of cells per experiment, MC offers a robust way to assay rare, transitional cell states that are pertinent to human development and disease. Here, we review MC approaches that let us probe the dynamics of cellular regulation across multiple conditions and sample types in a single experiment. Additionally, we discuss current limitations and future extensions of MC as well as computational tools commonly used to extract biological insight from single-cell proteomic datasets.

    DOI 10.1016/j.tcb.2022.01.012

    PubMed 35181197

55. Natural Killer Cell Receptors and Ligands Are Associated With Markers of HIV-1 Persistence in Chronically Infected ART Suppressed Patients. Frontiers in cellular and infection microbiology Ivison, G. T., Vendrame, E., Martinez-Colon, G. J., Ranganath, T., Vergara, R., Zhao, N. Q., Martin, M. P., Bendall, S. C., Carrington, M., Cyktor, J. C., McMahon, D. K., Eron, J., Jones, R. B., Mellors, J. W., Bosch, R. J., Gandhi, R. T., Holmes, S., Blish, C. A., ACTG 5321 Team 2022; 12: 757846

    The latent HIV-1 reservoir represents a major barrier to achieving a long-term antiretroviral therapy (ART)-free remission or cure for HIV-1. Natural Killer (NK) cells are innate immune cells that play a critical role in controlling viral infections and have been shown to be involved in preventing HIV-1 infection and, in those who are infected, delaying time to progression to AIDS. However, their role in limiting HIV-1 persistence on long term ART is still uncharacterized. To identify associations between markers of HIV-1 persistence and the NK cell receptor-ligand repertoire, we used twin mass cytometry panels to characterize the peripheral blood NK receptor-ligand repertoire in individuals with long-term antiretroviral suppression enrolled in the AIDS Clinical Trial Group A5321 study. At the time of testing, participants had been on ART for a median of 7 years, with virological suppression <50 copies/mL since at most 48 weeks on ART. We found that the NK cell receptor and ligand repertoires did not change across three longitudinal samples over one year-a median of 25 weeks and 50 weeks after the initial sampling. To determine the features of the receptor-ligand repertoire that associate with markers of HIV-1 persistence, we performed a LASSO normalized regression. This analysis revealed that the NK cell ligands CD58, HLA-B, and CRACC, as well as the killer cell immunoglobulin-like receptors (KIRs) KIR2DL1, KIR2DL3, and KIR2DS4 were robustly predictive of markers of HIV-1 persistence, as measured by total HIV-1 cell-associated DNA, HIV-1 cell-associated RNA, and single copy HIV-RNA assays. To characterize the roles of cell populations defined by multiple markers, we augmented the LASSO analysis with FlowSOM clustering. This analysis found that a less mature NK cell phenotype (CD16+CD56dimCD57-LILRB1-NKG2C-) was associated with lower HIV-1 cell associated DNA. Finally, we found that surface expression of HLA-Bw6 measured by CyTOF was associated with lower HIV-1 persistence. Genetic analysis revealed that this was driven by lower HIV-1 persistence in HLA-Bw4/6 heterozygotes. These findings suggest that there may be a role for NK cells in controlling HIV-1 persistence in individuals on long-term ART, which must be corroborated by future studies.

    DOI 10.3389/fcimb.2022.757846

    PubMed 35223535

56. Variation of Immune Cell Responses in Humans Reveals Sex-Specific Coordinated Signaling Across Cell Types. Frontiers in immunology Fragiadakis, G. K., Bjornson-Hooper, Z. B., Madhireddy, D., Sachs, K., Chen, H., McIlwain, D. R., Spitzer, M. H., Bendall, S. C., Nolan, G. P. 2022; 13: 867016

    Assessing the health and competence of the immune system is central to evaluating vaccination responses, autoimmune conditions, cancer prognosis, and treatment. With an increasing number of studies examining immune dysregulation, there is a growing need for a curated reference of variation in immune parameters in healthy individuals. We used mass cytometry (CyTOF) to profile blood from 86 humans in response to 15 ex vivo immune stimuli. We present reference ranges for cell-specific immune markers and highlight differences that appear across sex and age. We identified modules of immune features that suggest there exists an underlying structure to the immune system based on signaling pathway responses across cell types. We observed increased MAPK signaling in inflammatory pathways in innate immune cells and greater overall coordination of immune cell responses in females. In contrast, males exhibited stronger pSTAT1 and pTBK1 responses. These reference data are publicly available as a resource for immune profiling studies.

    DOI 10.3389/fimmu.2022.867016

    PubMed 35419006

57. The immunoregulatory landscape of human tuberculosis granulomas. Nature immunology McCaffrey, E. F., Donato, M., Keren, L., Chen, Z., Delmastro, A., Fitzpatrick, M. B., Gupta, S., Greenwald, N. F., Baranski, A., Graf, W., Kumar, R., Bosse, M., Fullaway, C. C., Ramdial, P. K., Forgó, E., Jojic, V., Van Valen, D., Mehra, S., Khader, S. A., Bendall, S. C., van de Rijn, M., Kalman, D., Kaushal, D., Hunter, R. L., Banaei, N., Steyn, A. J., Khatri, P., Angelo, M. 2022

    Tuberculosis (TB) in humans is characterized by formation of immune-rich granulomas in infected tissues, the architecture and composition of which are thought to affect disease outcome. However, our understanding of the spatial relationships that control human granulomas is limited. Here, we used multiplexed ion beam imaging by time of flight (MIBI-TOF) to image 37 proteins in tissues from patients with active TB. We constructed a comprehensive atlas that maps 19 cell subsets across 8 spatial microenvironments. This atlas shows an IFN-γ-depleted microenvironment enriched for TGF-β, regulatory T cells and IDO1+ PD-L1+ myeloid cells. In a further transcriptomic meta-analysis of peripheral blood from patients with TB, immunoregulatory trends mirror those identified by granuloma imaging. Notably, PD-L1 expression is associated with progression to active TB and treatment response. These data indicate that in TB granulomas, there are local spatially coordinated immunoregulatory programs with systemic manifestations that define active TB.

    DOI 10.1038/s41590-021-01121-x

    PubMed 35058616

58. Transition to invasive breast cancer is associated with progressive changes in the structure and composition of tumor stroma. Cell Risom, T., Glass, D. R., Averbukh, I., Liu, C. C., Baranski, A., Kagel, A., McCaffrey, E. F., Greenwald, N. F., Rivero-Gutiérrez, B., Strand, S. H., Varma, S., Kong, A., Keren, L., Srivastava, S., Zhu, C., Khair, Z., Veis, D. J., Deschryver, K., Vennam, S., Maley, C., Hwang, E. S., Marks, J. R., Bendall, S. C., Colditz, G. A., West, R. B., Angelo, M. 2022; 185 (2): 299-310.e18

    Ductal carcinoma in situ (DCIS) is a pre-invasive lesion that is thought to be a precursor to invasive breast cancer (IBC). To understand the changes in the tumor microenvironment (TME) accompanying transition to IBC, we used multiplexed ion beam imaging by time of flight (MIBI-TOF) and a 37-plex antibody staining panel to interrogate 79 clinically annotated surgical resections using machine learning tools for cell segmentation, pixel-based clustering, and object morphometrics. Comparison of normal breast with patient-matched DCIS and IBC revealed coordinated transitions between four TME states that were delineated based on the location and function of myoepithelium, fibroblasts, and immune cells. Surprisingly, myoepithelial disruption was more advanced in DCIS patients that did not develop IBC, suggesting this process could be protective against recurrence. Taken together, this HTAN Breast PreCancer Atlas study offers insight into drivers of IBC relapse and emphasizes the importance of the TME in regulating these processes.

    DOI 10.1016/j.cell.2021.12.023

    PubMed 35063072

59. Mass Synaptometry: Applying Mass Cytometry to Single Synapse Analysis. Methods in molecular biology (Clifton, N.J.) Gajera, C. R., Fernandez, R., Postupna, N., Montine, K. S., Keene, C. D., Bendall, S. C., Montine, T. J. 1800; 2417: 69-88

    Synaptic degeneration is one of the earliest and phenotypically most significant features associated with numerous neurodegenerative conditions, including Alzheimer's and Parkinson's diseases. Synaptic changes are also known to be important in neurocognitive disorders such as schizophrenia and autism spectrum disorders. Several labs, including ours, have demonstrated that conventional (fluorescence-based) flow cytometry of individual synaptosomes is a robust and reproducible method. However, the repertoire of probes needed to assess comprehensively the type of synapse, pathologic proteins (including protein products of risk genes discovered in GWAS), and markers of stress and injury far exceeds what is achievable with conventional flow cytometry. We recently developed a method that applies CyTOF (Cytometry by Time-Of-Flight mass spectrometry) to high-dimensional analysis of individual human synaptosomes, overcoming many of the multiplexing limitations of conventional flow cytometry. We call this new method Mass Synaptometry. Here we describe the preparation of synaptosomes from human and mouse brain, the generation and quality control of the "SynTOF" (Synapse by Time-Of-Flight mass spectrometry) antibody panel, the staining protocol, and CyTOF parameter setup for acquisition, post-acquisition processing, and analysis.

    DOI 10.1007/978-1-0716-1916-2_6

    PubMed 35099792

60. Macrophages are metabolically heterogeneous within the tumor microenvironment. Cell reports Geeraerts, X., Fernandez-Garcia, J., Hartmann, F. J., de Goede, K. E., Martens, L., Elkrim, Y., Debraekeleer, A., Stijlemans, B., Vandekeere, A., Rinaldi, G., De Rycke, R., Planque, M., Broekaert, D., Meinster, E., Clappaert, E., Bardet, P., Murgaski, A., Gysemans, C., Nana, F. A., Saeys, Y., Bendall, S. C., Laoui, D., Van den Bossche, J., Fendt, S., Van Ginderachter, J. A. 1800; 37 (13): 110171

    Macrophages are often prominently present in the tumor microenvironment, where distinct macrophage populations can differentially affect tumor progression. Although metabolism influences macrophage function, studies on the metabolic characteristics of exvivo tumor-associated macrophage (TAM) subsets are rather limited. Using transcriptomic and metabolic analyses, we now reveal that pro-inflammatory major histocompatibility complex (MHC)-IIhi TAMs display a hampered tricarboxylic acid (TCA) cycle, while reparative MHC-IIlo TAMs show higher oxidative and glycolytic metabolism. Although both TAM subsets rapidly exchange lactate in high-lactate conditions, only MHC-IIlo TAMs use lactate as an additional carbon source. Accordingly, lactate supports the oxidative metabolism in MHC-IIlo TAMs, while it decreases the metabolic activity of MHC-IIhi TAMs. Lactate subtly affects the transcriptome of MHC-IIlo TAMs, increases L-arginine metabolism, and enhances the Tcell suppressive capacity of these TAMs. Overall, our data uncover the metabolic intricacies of distinct TAM subsets and identify lactate as a carbon source and metabolic and functional regulator of TAMs.

    DOI 10.1016/j.celrep.2021.110171

    PubMed 34965415

61. Single-synapse analyses of Alzheimer's disease implicate pathologic tau, DJ1, CD47, and ApoE. Science advances Phongpreecha, T., Gajera, C. R., Liu, C. C., Vijayaragavan, K., Chang, A. L., Becker, M., Fallahzadeh, R., Fernandez, R., Postupna, N., Sherfield, E., Tebaykin, D., Latimer, C., Shively, C. A., Register, T. C., Craft, S., Montine, K. S., Fox, E. J., Poston, K. L., Keene, C. D., Angelo, M., Bendall, S. C., Aghaeepour, N., Montine, T. J. 1800; 7 (51): eabk0473

    [Figure: see text].

    DOI 10.1126/sciadv.abk0473

    PubMed 34910503

62. Chromatin Content Capture Reveals Acute Leukaemia Oncogenic Vulnerability Point in Human B Cell Development Baskar, R., Favaro, P., Reynolds, W. D., Domizi, P., Tsai, A. G., Davis, K. L., Bendall, S. AMER SOC HEMATOLOGY. 2021

    DOI 10.1182/blood-2021-153114

    Web of Science ID 000736398802212

63. Ikaros Mediates Antigen Escape Following CD19 CAR T Cell Therapy in r/r B-ALL Domizi, P., Sarno, J., Jager, A., Baskar, R., Reynolds, W. D., Sahaf, B., Bendall, S., Mullighan, C. G., Leahy, A., Myers, R. M., Grupp, S. A., Sotillo, E., Barrett, D., Davis, K. L. AMER SOC HEMATOLOGY. 2021

    DOI 10.1182/blood-2021-151002

    Web of Science ID 000736398802154

64. Inhibition of Pre-BCR Signaling Mediates a Metabolic Switch in B-Cell Progenitor Acute Lymphoblastic Leukemia Liu, Y., Stuani, L., Jedoui, D., Merchant, M., Jager, A., Sarno, J., Mullighan, C. G., Hartmann, F. J., Bendall, S., Davis, K. L. AMER SOC HEMATOLOGY. 2021

    DOI 10.1182/blood-2021-145956

    Web of Science ID 000736398802156

65. Whole-cell segmentation of tissue images with human-level performance using large-scale data annotation and deep learning. Nature biotechnology Greenwald, N. F., Miller, G., Moen, E., Kong, A., Kagel, A., Dougherty, T., Fullaway, C. C., McIntosh, B. J., Leow, K. X., Schwartz, M. S., Pavelchek, C., Cui, S., Camplisson, I., Bar-Tal, O., Singh, J., Fong, M., Chaudhry, G., Abraham, Z., Moseley, J., Warshawsky, S., Soon, E., Greenbaum, S., Risom, T., Hollmann, T., Bendall, S. C., Keren, L., Graf, W., Angelo, M., Van Valen, D. 2021

    A principal challenge in the analysis of tissue imaging data is cell segmentation-the task of identifying the precise boundary of every cell in an image. To address this problem we constructed TissueNet, a dataset for training segmentation models that contains more than 1million manually labeled cells, an order of magnitude more than all previously published segmentation training datasets. We used TissueNet to train Mesmer, a deep-learning-enabled segmentation algorithm. We demonstrated that Mesmer is more accurate than previous methods, generalizes to the full diversity of tissue types and imaging platforms in TissueNet, and achieves human-level performance. Mesmer enabled the automated extraction of key cellular features, such as subcellular localization of protein signal, which was challenging with previous approaches. We then adapted Mesmer to harness cell lineage information in highly multiplexed datasets and used this enhanced version to quantify cell morphology changes during human gestation. All code, data and models are released as a community resource.

    DOI 10.1038/s41587-021-01094-0

    PubMed 34795433

66. 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

    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.

    DOI 10.1146/annurev-pathmechdis-030321-091459

    PubMed 34752710

67. Multiplexed imaging reveals an IFN-gamma-driven inflammatory state in nivolumab-associated gastritis CELL REPORTS MEDICINE Ferrian, S., Liu, C. C., McCaffrey, E. F., Kumar, R., Nowicki, T. S., Dawson, D. W., Baranski, A., Glaspy, J. A., Ribas, A., Bendall, S. C., Angelo, M. 2021; 2 (10)

    DOI 10.1016/j.xcrm.2021.100419

    Web of Science ID 000709853700005

68. Multiplexed imaging reveals an IFN-γ-driven inflammatory state in nivolumab-associated gastritis. Cell reports. Medicine Ferrian, S., Liu, C. C., McCaffrey, E. F., Kumar, R., Nowicki, T. S., Dawson, D. W., Baranski, A., Glaspy, J. A., Ribas, A., Bendall, S. C., Angelo, M. 2021; 2 (10): 100419

    Immune checkpoint blockade using PD-1 inhibition is an effective approach for treating a wide variety of cancer subtypes. While lower gastrointestinal (GI) side effects are more common, upper gastrointestinal adverse events are rarely reported. Here, we present a case of nivolumab-associated autoimmune gastritis. To elucidate the immunology underlying this condition, we leverage multiplexed ion beam imaging by time-of-flight (MIBI-TOF) to identify the presence and proportion of infiltrating immune cells from a single section of biopsy specimen. Using MIBI-TOF, we analyze formalin-fixed, paraffin-embedded human gastric tissue with 28 labels simultaneously. Our analyses reveal a gastritis characterized by severe mucosal injury, interferon gamma (IFN-γ)-producing gastric epithelial cells, and mixed inflammation that includes CD8 and CD4 T cell infiltrates with reduced expression of granzyme B and FOXP3, respectively. Here, we provide a comprehensive multiplexed histopathological mapping of gastric tissue, which identifies IFN-γ-producing epithelial cells as possible contributors to the nivolumab-associated gastritis.

    DOI 10.1016/j.xcrm.2021.100419

    PubMed 34755133

    PubMedCentralID PMC8561237

69. Multi-Center Immune Profiling Mass Cytometry Assay Harmonization. Clinical cancer research : an official journal of the American Association for Cancer Research Sahaf, B., Pichavant, M., Lee, B. H., Duault, C., Thrash, E. M., Davila, M., Fernandez, N., Millerchip, K., Bentebibel, S., Haymaker, C., Sigal, N., Del Valle, D. M., Ranasinghe, S., Fayle, S., Sanchez-Espiridion, B., Zhang, J., Bernatchez, C., Wu, C. J., Wistuba, I. I., Kim-Schulze, S., Gnjatic, S., Bendall, S. C., Song, M., Thurin, M., Lee, J. J., Maecker, H. T., Rahman, A. 2021

    PURPOSE: The Cancer Immune Monitoring and Analysis Centers - Cancer Immunologic Data Commons (CIMAC-CIDC) Network is supported by the National Cancer Institute to identify biomarkers of response to cancer immunotherapies across clinical trials using state-of-the-art assays. A primary platform for CIMAC-CIDC studies is cytometry by time-of-flight (CyTOF), performed at all CIMAC laboratories. To ensure the ability to generate comparable CyTOF data across labs, a multistep cross-site harmonization effort was undertaken.EXPERIMENTAL DESIGN: We first harmonized standard operating procedures (SOPs) across the CIMAC sites. Because of a new acquisition protocol comparing original narrow - or new wide bore injector introduced by the vendor (Fluidigm), we also tested this protocol across sites before finalizing the harmonized SOP. We then performed cross-site assay harmonization experiments using 5 shared cryopreserved and one lyophilized internal control PBMCs with a shared lyophilized antibody cocktail consisting of 14 isotype-tagged antibodies previously validated, plus additional liquid antibodies. These reagents and samples were distributed to the CIMAC sites and the data were centrally analyzed by manual gating and automated methods (Astrolabe).RESULTS: Average coefficients of variation (CVs) across sites for each cell population were reported and compared to a previous multisite CyTOF study. We reached an inter-site CV of under 20% for most cell subsets, similar to a previously published study.CONCLUSIONS: These results establish the ability to reproduce CyTOF data across sites in multi-center clinical trials, and also highlight the importance of quality control procedures, such as the use of spike-in control samples, for tracking variability in this assay.

    DOI 10.1158/1078-0432.CCR-21-2052

    PubMed 34266889

70. Multiplexed Ion Beam Imaging Readout of Single-Cell Immunoblotting. Analytical chemistry Lomeli, G., Bosse, M., Bendall, S. C., Angelo, M., Herr, A. E. 2021

    Improvements in single-cell protein analysis are required to study the cell-to-cell variation inherent to diseases, including cancer. Single-cell immunoblotting (scIB) offers proteoform detection specificity, but often relies on fluorescence-based readout and is therefore limited in multiplexing capability. Among rising multiplexed imaging methods is multiplexed ion beam imaging by time-of-flight (MIBI-TOF), a mass spectrometry imaging technology. MIBI-TOF employs metal-tagged antibodies that do not suffer from spectral overlap to the same degree as fluorophore-tagged antibodies. We report for the first-time MIBI-TOF of single-cell immunoblotting (scIB-MIBI-TOF). The scIB assay subjects single-cell lysate to protein immunoblotting on a microscale device consisting of a 50- to 75-mum thick hydrated polyacrylamide (PA) gel matrix for protein immobilization prior to in-gel immunoprobing. We confirm antibody-protein binding in the PA gel with indirect fluorescence readout of metal-tagged antibodies. Since MIBI-TOF is a layer-by-layer imaging technique, and our protein target is immobilized within a 3D PA gel layer, we characterize the protein distribution throughout the PA gel depth by fluorescence confocal microscopy and confirm that the highest signal-to-noise ratio is achieved by imaging the entirety of the PA gel depth. Accordingly, we report the required MIBI-TOF ion dose strength needed to image varying PA gel depths. Lastly, by imaging 42% of PA gel depth with MIBI-TOF, we detect two isoelectrically separated TurboGFP (tGFP) proteoforms from individual glioblastoma cells, demonstrating that highly multiplexed mass spectrometry-based readout is compatible with scIB.

    DOI 10.1021/acs.analchem.1c01050

    PubMed 34106685

71. 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

    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.

    DOI 10.1002/cyto.a.24340

    PubMed 33818911

72. Network for biomarker immunoprofiling for cancer immunotherapy: Cancer Immune Monitoring and Analysis Centers and Cancer Immunologic Data Commons (CIMAC-CIDC). Clinical cancer research : an official journal of the American Association for Cancer Research Chen, H. X., Song, M., Maecker, H. T., Gnjatic, S., Patton, D., Lee, J. J., Adam, S. J., Moravec, R., Liu, X. S., Cerami, E., Lindsay, J., Hodi, F. S., Wu, C., Wistuba, I. I., Al-Atrash, G., Bernatchez, C., Bendall, S. C., Hewitt, S. M., Sharon, E., Streicher, H., Enos, R. A., Bowman, M. D., Tatard-Leitman, V. M., Sanchez-Espiridion, B., Ranasinghe, S., Pichavant, M., Del Valle, D. M., Yu, J., Janssens, S., Peterson-Klaus, J., Rowe, C., Bongers, G., Jenq, R. R., Chang, C., Abrams, J. S., Mooney, M., Doroshow, J. H., Harris, L. N., Thurin, M. 2021

    Immunoprofiling to identify biomarkers and integration with clinical trials outcome are critical to improve immunotherapy approaches for cancer patients. However, the translational potential of individual studies is often limited by small sample size of trials and the complexity of immuno-oncology biomarkers. Variability in assays further limits comparison and interpretation of data across studies and laboratories. To enable a systematic approach to biomarker identification and correlation with clinical outcome across trials, the Cancer Immune Monitoring and Analysis Centers and Cancer Immunologic Data Commons (CIMAC-CIDC) Network was established through support of the Cancer MoonshotSM Initiative of the National Cancer Institute and the Partnership for Accelerating Cancer Therapies (PACT) with industry partners via the Foundation for the National Institutes of Health. The CIMAC-CIDC Network is composed of four academic centers (CIMACs) with multidisciplinary expertise in the field of cancer immunotherapy that provide validated and harmonized assays for immune profiling. A data coordinating center (CIDC) provides the computational expertise and resources for biomarker data storage and analysis platforms for correlation with clinical data. This overview highlights strategies for assay harmonization to enable cross-trial and cross-site data analysis and describes key elements for establishing a network to enhance immuno-oncology biomarker development. These include an operational infrastructure; validation and harmonization of core immunoprofiling assays; platforms for data ingestion and integration; and access to specimens from clinical trials. Published in the same volume are reports of harmonization for core analyses: whole exome sequencing, RNA sequencing, cytometry by time of flight, and immunohistochemistry/immunofluorescence.

    DOI 10.1158/1078-0432.CCR-20-3241

    PubMed 33419780

73. Immune-stimulating antibody conjugates elicit robust myeloid activation and durable antitumor immunity. Nature cancer Ackerman, S. E., Pearson, C. I., Gregorio, J. D., Gonzalez, J. C., Kenkel, J. A., Hartmann, F. J., Luo, A., Ho, P. Y., LeBlanc, H., Blum, L. K., Kimmey, S. C., Luo, A., Nguyen, M. L., Paik, J. C., Sheu, L. Y., Ackerman, B., Lee, A., Li, H., Melrose, J., Laura, R. P., Ramani, V. C., Henning, K. A., Jackson, D. Y., Safina, B. S., Yonehiro, G., Devens, B. H., Carmi, Y., Chapin, S. J., Bendall, S. C., Kowanetz, M., Dornan, D., Engleman, E. G., Alonso, M. N. 2021; 2 (1): 18-33

    Innate pattern recognition receptor agonists, including Toll-like receptors (TLRs), alter the tumor microenvironment and prime adaptive antitumor immunity. However, TLR agonists present toxicities associated with widespread immune activation after systemic administration. To design a TLR-based therapeutic suitable for systemic delivery and capable of safely eliciting tumor-targeted responses, we developed immune-stimulating antibody conjugates (ISACs) comprising a TLR7/8 dual agonist conjugated to tumor-targeting antibodies. Systemically administered human epidermal growth factor receptor 2 (HER2)-targeted ISACs were well tolerated and triggered a localized immune response in the tumor microenvironment that resulted in tumor clearance and immunological memory. Mechanistically, ISACs required tumor antigen recognition, Fcγ-receptor-dependent phagocytosis and TLR-mediated activation to drive tumor killing by myeloid cells and subsequent T-cell-mediated antitumor immunity. ISAC-mediated immunological memory was not limited to the HER2 ISAC target antigen since ISAC-treated mice were protected from rechallenge with the HER2- parental tumor. These results provide a strong rationale for the clinical development of ISACs.

    DOI 10.1038/s43018-020-00136-x

    PubMed 35121890

74. Immune-stimulating antibody conjugates elicit robust myeloid activation and durable antitumor immunity NATURE CANCER Ackerman, S. E., Pearson, C. I., Gregorio, J. D., Gonzalez, J. C., Kenkel, J. A., Hartmann, F. J., Luo, A., Ho, P. Y., LeBlanc, H., Blum, L. K., Kimmey, S. C., Luo, A., Nguyen, M. L., Paik, J. C., Sheu, L. Y., Ackerman, B., Lee, A., Li, H., Melrose, J., Laura, R. P., Ramani, V. C., Henning, K. A., Jackson, D. Y., Safina, B. S., Yonehiro, G., Devens, B. H., Carmi, Y., Chapin, S. J., Bendall, S. C., Kowanetz, M., Dornan, D., Engleman, E. G., Alonso, M. N. 2021; 2 (1): 18-+

    DOI 10.1038/s43018-020-00136-x

    Web of Science ID 000610051100003

75. Mass Cytometry Phenotyping of Human Granulocytes Reveals Novel Basophil Functional Heterogeneity. iScience Vivanco Gonzalez, N., Oliveria, J., Tebaykin, D., Ivison, G. T., Mukai, K., Tsai, M. M., Borges, L., Nadeau, K. C., Galli, S. J., Tsai, A. G., Bendall, S. C. 2020; 23 (11): 101724

    Basophils, the rarest granulocyte, play critical roles in parasite- and allergen-induced inflammation. We applied mass cytometry (CyTOF) to simultaneously asses 44 proteins to phenotype and functionally characterize neutrophils, eosinophils, and basophils from 19 healthy donors. There was minimal heterogeneity seen in eosinophils and neutrophils, but data-driven analyses revealed four unique subpopulations within phenotypically basophilic granulocytes (PBG; CD45+HLA-DR-CD123+). Through CyTOF and fluorescence-activated cell sorting (FACS), we classified these four PBG subpopulations as (I) CD16lowFcepsilonRIhighCD244high (88.5± 1.2%), (II) CD16highFcepsilonRIhighCD244high (9.1± 0.4%), (III) CD16lowFcepsilonRIlowCD244low (2.3± 1.3), and (IV) CD16highFcepsilonRIlowCD244low (0.4± 0.1%). Prospective isolation confirmed basophilic-morphology of PBG I-III, but neutrophilic-morphology of PBG IV. Functional interrogation via IgE-crosslinking or IL-3 stimulation demonstrated that PBG I-II had significant increases in CD203c expression, whereas PBG III-IV remained unchanged compared with media-alone conditions. Thus, PBG III-IV could serve roles in non-IgE-mediated immunity. Our findings offer new perspectives in human basophil heterogeneity and the varying functional potential of these new subsets in health and disease.

    DOI 10.1016/j.isci.2020.101724

    PubMed 33205028

76. COVALENT ATTACHMENT OF A TLR7/8 AGONIST TO TUMOR-TARGETING ANTIBODIES DRIVES POTENT ANTI-TUMOR EFFICACY BY SYNERGISTICALLY ACTIVATING FCGR- AND TLR- SIGNALING AND ENABLES SAFE SYSTEMIC ADMINISTRATION Ackerman, S., Hartmann, F., Pearson, C., Gonzalez, J., Ho, P., Kimmey, S., Luo, A., Ackerman, B., Lee, A., Laura, R., Paik, J., Henning, K., Jackson, D., Chapin, S., Devens, B., Dornan, D., Bendall, S., Engleman, E., Alonso, M. BMJ PUBLISHING GROUP. 2020: A360

    DOI 10.1136/jitc-2020-SITC2020.0603

    Web of Science ID 000616665301125

77. Single cell proteomics to capture human dysplasia and dysfunction Bendall, S. AMER ASSOC CANCER RESEARCH. 2020

    Web of Science ID 000587913100019

78. Mapping the tumor and microenvironmental evolution underlying DCIS progression through multiplexed ion beam imaging. Risom, T., Rivero, B., Liu, C., Baranski, A., Strand, S., Greenwald, N., McCaffrey, E., Varma, S., Keren, L., Srivastava, S., Zhu, C., Vennam, S., Hwang, S., Colditz, G., Bendall, S., West, R., Angelo, M. AMER ASSOC CANCER RESEARCH. 2020

    Web of Science ID 000587913100120

79. PLEIOTROPIC EFFECTS OF IL-7 IN PROSTATE CANCER PATIENTS RECEIVING SIPULEUCEL-T VACCINATION Duault, C., Ramchurren, N., Pachynski, R., Fong, L., Bendall, S., Pichavant, M., Sahaf, B., Morishima, C., D'Amico, L., Cheever, M., Fling, S., Maecker, H. BMJ PUBLISHING GROUP. 2020: A160–A161

    DOI 10.1136/jitc-2020-SITC2020.0263

    Web of Science ID 000616665300255

80. Integration of mechanistic immunological knowledge into a machine learning pipeline improves predictions NATURE MACHINE INTELLIGENCE Culos, A., Tsai, A. S., Stanley, N., Becker, M., Ghaemi, M. S., McIlwain, D. R., Fallahzadeh, R., Tanada, A., Nassar, H., Espinosa, C., Xenochristou, M., Ganio, E., Peterson, L., Han, X., Stelzer, I. A., Ando, K., Gaudilliere, D., Phongpreecha, T., Maric, I., Chang, A. L., Shaw, G. M., Stevenson, D. K., Bendall, S., Davis, K. L., Fantl, W., Nolan, G. P., Hastie, T., Tibshirani, R., Angst, M. S., Gaudilliere, B., Aghaeepour, N. 2020

    DOI 10.1038/s42256-020-00232-8

    Web of Science ID 000579336000001

81. Integration of mechanistic immunological knowledge into a machine learning pipeline improves predictions. Nature machine intelligence Culos, A., Tsai, A. S., Stanley, N., Becker, M., Ghaemi, M. S., McIlwain, D. R., Fallahzadeh, R., Tanada, A., Nassar, H., Espinosa, C., Xenochristou, M., Ganio, E., Peterson, L., Han, X., Stelzer, I. A., Ando, K., Gaudilliere, D., Phongpreecha, T., Marić, I., Chang, A. L., Shaw, G. M., Stevenson, D. K., Bendall, S., Davis, K. L., Fantl, W., Nolan, G. P., Hastie, T., Tibshirani, R., Angst, M. S., Gaudilliere, B., Aghaeepour, N. 2020; 2 (10): 619-628

    The dense network of interconnected cellular signalling responses that are quantifiable in peripheral immune cells provides a wealth of actionable immunological insights. Although high-throughput single-cell profiling techniques, including polychromatic flow and mass cytometry, have matured to a point that enables detailed immune profiling of patients in numerous clinical settings, the limited cohort size and high dimensionality of data increase the possibility of false-positive discoveries and model overfitting. We introduce a generalizable machine learning platform, the immunological Elastic-Net (iEN), which incorporates immunological knowledge directly into the predictive models. Importantly, the algorithm maintains the exploratory nature of the high-dimensional dataset, allowing for the inclusion of immune features with strong predictive capabilities even if not consistent with prior knowledge. In three independent studies our method demonstrates improved predictions for clinically relevant outcomes from mass cytometry data generated from whole blood, as well as a large simulated dataset. The iEN is available under an open-source licence.

    DOI 10.1038/s42256-020-00232-8

    PubMed 33294774

    PubMedCentralID PMC7720904

82. A HIGHLY MULTIPLEXED SINGLE CELL PROTEOMIC SCREEN REVEALS THE PHENOTYPIC AND FUNCTIONAL LANDSCAPE OF THE HUMAN LYMPHO-MYELOID DIFFERENTIATION AXIS Kim, Y., Caleron, A., Glass, D., Tsai, A., Favaro, P., Baskar, R., Hartmann, F., Greenleaf, W., Bendall, S. ELSEVIER SCIENCE INC. 2020: S33

    Web of Science ID 000655609700035

83. Validation of a model of pediatric leukemia based on pluripotent stem cells using mass cytometry Domingo-Reines, J., Kimmey, S., Vijayaragavan, K., Bosse, M., Bendall, S., Davis, K., Ramos-Mejia, V. AMER ASSOC CANCER RESEARCH. 2020: 95

    Web of Science ID 000551367400138

84. CIMAC-CIDC CyTOF harmonization. Rahman, A., Sahaf, B., Davila, M., Fernandez, N., McWilliams, E., Millerchip, K., Bentebibel, S. E., Haymaker, C. L., Sigal, N., Duault, C., Thrash, E., Del Valle, D., Espiridion, B., Pichavant, M., Bernatchez, C., Wistuba, I., Gnjatic, S., Bendall, S., Maecker, H., CIMAC-CIDC Network LIPPINCOTT WILLIAMS & WILKINS. 2020

    Web of Science ID 000560368305212

85. Diamonds in the doublets. Nature biotechnology Bendall, S. C. 2020

    DOI 10.1038/s41587-020-0511-6

    PubMed 32317762

86. In-depth characterization of immune cells in preeclampsia using Multiplexed Ion Beam Imaging by Time-of-Flight (MIBI-TOF) Greenbaum, S., Bosse, M., Baranski, A., Khair, Z., Bruce, T., Tebaykin, D., Oliveria, J., Bendall, S. C., Winn, V. D., Angelo, M. MOSBY-ELSEVIER. 2020: S156–S157

    DOI 10.1016/j.ajog.2019.11.241

    Web of Science ID 000504997300225

87. High-Parameter Immune Profiling with CyTOF. Methods in molecular biology (Clifton, N.J.) Sahaf, B., Rahman, A., Maecker, H. T., Bendall, S. C. 2020; 2055: 351–68

    Mass cytometry, or CyTOF, is a useful technology for high-parameter single-cell phenotyping, especially from suspension cells such as blood or PBMC. It is particularly appealing to monitor the systemic immune changes that could accompany cancer immunotherapy. Here we present a reference panel for identification of all major immune cell populations, with flexibility for addition of trial-specific markers. We also describe best-practice measures for minimizing and tracking batch variability. These include: sample barcoding, use of spiked-in reference cells, and lyophilization of the antibody cocktail. Our protocol assumes the use of cryopreserved PBMC, both for convenience of batching samples and for maximum comparability across patients and time points. Finally, we show an option for automated analysis using the Astrolabe platform (Astrolabe Diagnostics, Inc.).

    DOI 10.1007/978-1-4939-9773-2_16

    PubMed 31502160

88. Single-cell mass cytometry reveals cross-talk between inflammation-dampening and inflammation-amplifying cells in osteoarthritic cartilage Science Advances Grandi, F. ., Baskar, R., Smeriglio, P., Murkherjee, S., Indelli, P., F. Amanatullah, D., Goodman, S., Chu, C., Bendall , S., Bhutani, N. 2020; 6 (11)

    DOI 10.1126/sciadv.aay5352

89. Immune monitoring usingmass cytometry and related high-dimensional imaging approaches. Nature reviews. Rheumatology Hartmann, F. J., Bendall, S. C. 2019

    The cellular complexity and functional diversity of the human immune system necessitate the use of high-dimensional single-cell tools to uncover its role in multifaceted diseases such as rheumatic diseases, as well as other autoimmune and inflammatory disorders. Proteomic technologies that use elemental (heavy metal) reporter ions, such as mass cytometry (also known as CyTOF) and analogous high-dimensional imaging approaches (including multiplexed ion beam imaging (MIBI) and imaging mass cytometry (IMC)), have been developed from their low-dimensional counterparts, flow cytometry and immunohistochemistry, to meet this need. A growing number of studies have been published that use these technologies to identify functional biomarkers and therapeutic targets in rheumatic diseases, but the full potential of their application to rheumatic disease research has yet to be fulfilled. This Review introduces the underlying technologies for high-dimensional immune monitoring and discusses aspects necessary for their successful implementation, including study design principles, analytical tools and future developments for the field of rheumatology.

    DOI 10.1038/s41584-019-0338-z

    PubMed 31892734

90. TRAIL-induced variation of cell signaling states provides nonheritable resistance to apoptosis. Life science alliance Baskar, R., Fienberg, H. G., Khair, Z., Favaro, P., Kimmey, S., Green, D. R., Nolan, G. P., Plevritis, S., Bendall, S. C. 2019; 2 (6)

    TNFalpha-related apoptosis-inducing ligand (TRAIL), specifically initiates programmed cell death, but often fails to eradicate all cells, making it an ineffective therapy for cancer. This fractional killing is linked to cellular variation that bulk assays cannot capture. Here, we quantify the diversity in cellular signaling responses to TRAIL, linking it to apoptotic frequency across numerous cell systems with single-cell mass cytometry (CyTOF). Although all cells respond to TRAIL, a variable fraction persists without apoptotic progression. This cell-specific behavior is nonheritable where both the TRAIL-induced signaling responses and frequency of apoptotic resistance remain unaffected by prior exposure. The diversity of signaling states upon exposure is correlated to TRAIL resistance. Concomitantly, constricting the variation in signaling response with kinase inhibitors proportionally decreases TRAIL resistance. Simultaneously, TRAIL-induced de novo translation in resistant cells, when blocked by cycloheximide, abrogated all TRAIL resistance. This work highlights how cell signaling diversity, and subsequent translation response, relates to nonheritable fractional escape from TRAIL-induced apoptosis. This refined view of TRAIL resistance provides new avenues to study death ligands in general.

    DOI 10.26508/lsa.201900554

    PubMed 31704709

91. Multiplexed Imaging for the simultaneous detection of nucleic acids and proteins to dissect the tissue immune landscape and microenvironment of viral diseases Jiang, S., Clave, X., Chan, C., Zhu, B., Bai, Y., Bosse, M., McIlwain, D., Bendall, S., Angelo, M., Estes, J., Nolan, G. BMC. 2019

    Web of Science ID 000496473200465

92. IN-DEPTH CHARACTERIZATION OF GESTATIONAL IMMUNE DYNAMICS USING MASS CYTOMETRY Moore, A., Vivanco-Gonzalez, N., Plummer, K., Mitchel, O., Kaur, H., Rivera, M., Bendall, S., Palmer, T. W B SAUNDERS CO LTD. 2019: E87–E88

    Web of Science ID 000483998500276

93. Glucose Metabolism Drives Histone Acetylation Landscape Transitions that Dictate Muscle Stem Cell Function. Cell reports Yucel, N., Wang, Y. X., Mai, T., Porpiglia, E., Lund, P. J., Markov, G., Garcia, B. A., Bendall, S. C., Angelo, M., Blau, H. M. 2019; 27 (13): 3939

    The impact of glucose metabolism on muscle regeneration remains unresolved. We identify glucose metabolism as a crucial driver of histone acetylation and myogenic cell fate. We use single-cell mass cytometry (CyTOF) and flow cytometry to characterize the histone acetylation and metabolic states of quiescent, activated, and differentiating muscle stem cells (MuSCs). We find glucose is dispensable for mitochondrial respiration in proliferating MuSCs, so that glucose becomes available for maintaining high histone acetylation via acetyl-CoA. Conversely, quiescent and differentiating MuSCs increase glucose utilization for respiration and have consequently reduced acetylation. Pyruvate dehydrogenase (PDH) activity serves as a rheostat for histone acetylation and must be controlled for muscle regeneration. Increased PDH activity in proliferation increases histone acetylation and chromatin accessibility at genes that must be silenced for differentiation to proceed, and thus promotes self-renewal. These results highlight metabolism as a determinant of MuSC histone acetylation, fate, and function during muscle regeneration.

    DOI 10.1016/j.celrep.2019.05.092

    PubMed 31242425

94. Parallel analysis of tri-molecular biosynthesis with cell identity and function in single cells. Nature communications Kimmey, S. C., Borges, L., Baskar, R., Bendall, S. C. 2019; 10 (1): 1185

    Cellular products derived from the activity of DNA, RNA, and protein synthesis collectively control cell identity and function. Yet there is little information on how these three biosynthesis activities are coordinated during transient and sparse cellular processes, such as activation and differentiation. Here, we describe Simultaneous Overview of tri-Molecule Biosynthesis (SOM3B), a molecular labeling and simultaneous detection strategy to quantify DNA, RNA, and protein synthesis in individual cells. Comprehensive interrogation of biosynthesis activities during transient cell states, such as progression through cell cycle or cellular differentiation, is achieved by partnering SOM3B with parallel quantification of select biomolecules with conjugated antibody reagents. Here, we investigate differential de novo DNA, RNA, and protein synthesis dynamics in transformed human cell lines, primary activated human immune cells, and across the healthy human hematopoietic continuum, all at a single-cell resolution.

    PubMed 30862852

95. An Immune Atlas of Mid to Late Mouse Gestation. Moore, A. R., Vivanco-Gonzalez, N., Plummer, K., Kaur, H., Mitchel, O., Rivera, M., Bendall, S. C., Palmer, T. D. SAGE PUBLICATIONS INC. 2019: 345A–346A

    Web of Science ID 000459610400837

96. A topological view of human CD34(+) cell state trajectories from integrated single-cell output and proteomic data BLOOD Knapp, D. F., Hammond, C. A., Wang, F., Aghaeepour, N., Miller, P. H., Beer, P. A., Pellacani, D., VanInsberghe, M., Hansen, C., Bendall, S. C., Nolan, G. P., Eaves, C. J. 2019; 133 (9): 927–39

    DOI 10.1182/blood-2018-10-878025

    Web of Science ID 000461501400010

97. Proliferation tracing with single-cell mass cytometry optimizes generation of stem cell memory-like T cells. Nature biotechnology Good, Z., Borges, L., Vivanco Gonzalez, N., Sahaf, B., Samusik, N., Tibshirani, R., Nolan, G. P., Bendall, S. C. 2019

    Selective differentiation of naive T cells into multipotent T cells is of great interest clinically for the generation of cell-based cancer immunotherapies. Cellular differentiation depends crucially on division state and time. Here we adapt a dye dilution assay for tracking cell proliferative history through mass cytometry and uncouple division, time and regulatory protein expression in single naive human T cells during their activation and expansion in a complex ex vivo milieu. Using 23 markers, we defined groups of proteins controlled predominantly by division state or time and found that undivided cells account for the majority of phenotypic diversity. We next built a map of cell state changes during naive T-cell expansion. By examining cell signaling on this map, we rationally selected ibrutinib, a BTK and ITK inhibitor, and administered it before T cell activation to direct differentiation toward a T stem cell memory (TSCM)-like phenotype. This method for tracing cell fate across division states and time can be broadly applied for directing cellular differentiation.

    PubMed 30742126

98. A topological view of human CD34+ cell state trajectories from integrated single-cell output and proteomic data. Blood Knapp, D. J., Hammond, C. A., Wang, F., Aghaeepour, N., Miller, P. H., Beer, P. A., Pellacani, D., VanInsberghe, M., Hansen, C., Bendall, S. C., Nolan, G. P., Eaves, C. J. 2019

    Recent advances in single-cell molecular analytical methods and clonal growth assays are enabling more refined models of human hematopoietic lineage restriction processes to be conceptualized. Here, we report the results of integrating single-cell proteome measurements with clonally-determined lymphoid, neutrophilic/monocytic, and/or erythroid progeny outputs from over 1,000 index-sorted CD34+ human cord blood cells in short-term cultures with and without stromal cells. Surface phenotypes of functionally examined cells were individually mapped onto a molecular landscape of the entire CD34+ compartment constructed from single-cell mass cytometric measurements of 14 cell surface markers, 20 signaling/cell cycle proteins and 6 transcription factors in approximately 300,000 cells. This analysis demonstrated that conventionally defined subsets of CD34+ CB cells are quite heterogeneous in their functional properties, transcription factor content, and signaling activities. Importantly, this molecular heterogeneity was reduced, but not eliminated in phenotypes that we showed display highly restricted lineage outputs. Integration of the complete proteomic and functional datasets obtained revealed a continuous probabilistic topology of change that includes a multiplicity of lineage restriction trajectories. Each of these reflects progressive but variable changes in the levels of specific signaling intermediates and transcription factors, but shared features of decreasing quiescence. Taken together, our results suggest a model in which increasingly narrowed hematopoietic output capabilities in neonatal CD34+ cord blood cells are determined by a history of external stimulation in combination with innately programmed cell state changes.

    PubMed 30622121

99. Scalable Conjugation and Characterization of Immunoglobulins with Stable Mass Isotope Reporters for Single-Cell Mass Cytometry Analysis. Methods in molecular biology (Clifton, N.J.) Hartmann, F. J., Simonds, E. F., Vivanco, N., Bruce, T., Borges, L., Nolan, G. P., Spitzer, M. H., Bendall, S. C. 2019; 1989: 55–81

    The advent of mass cytometry (CyTOF®) has permitted simultaneous detection of more than 40 antibody parameters at the single-cell level, although a limited number of metal-labeled antibodies are commercially available. Here we present optimized and scalable protocols for conjugation of lanthanide as well as bismuth ions to immunoglobulin (Ig) using a maleimide-functionalized chelating polymer and for characterization of the conjugate. The maleimide functional group is reactive with cysteine sulfhydryl groups generated through partial reduction of the Ig Fc region. Incubation of Ig with polymer pre-loaded with lanthanide ions produces metal-labeled Ig without disrupting antigen specificity. Antibody recovery rates can be determined by UV spectrophotometry and frequently exceeds 60%. Each custom-conjugated antibody is validated using positive and negative cellular control populations and is titrated for optimal staining at concentrations ranging from 0.1 to 10 μg/mL. The preparation of metal-labeled antibodies can be completed in 4.5 h, and titration requires an additional 3-5 h.

    PubMed 31077099

100. Mapping lung cancer epithelial-mesenchymal transition states and trajectories with single-cell resolution. Nature communications Karacosta, L. G., Anchang, B., Ignatiadis, N., Kimmey, S. C., Benson, J. A., Shrager, J. B., Tibshirani, R., Bendall, S. C., Plevritis, S. K. 2019; 10 (1): 5587

     Elucidating the spectrum of epithelial-mesenchymal transition (EMT) and mesenchymal-epithelial transition (MET) states in clinical samples promises insights on cancer progression and drug resistance. Using mass cytometry time-course analysis, we resolve lung cancer EMT states through TGFβ-treatment and identify, through TGFβ-withdrawal, a distinct MET state. We demonstrate significant differences between EMT and MET trajectories using a computational tool (TRACER) for reconstructing trajectories between cell states. In addition, we construct a lung cancer reference map of EMT and MET states referred to as the EMT-MET PHENOtypic STAte MaP (PHENOSTAMP). Using a neural net algorithm, we project clinical samples onto the EMT-MET PHENOSTAMP to characterize their phenotypic profile with single-cell resolution in terms of our in vitro EMT-MET analysis. In summary, we provide a framework to phenotypically characterize clinical samples in the context of in vitro EMT-MET findings which could help assess clinical relevance of EMT in cancer in future studies.

     DOI 10.1038/s41467-019-13441-6

     PubMed 31811131

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

     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.

     DOI 10.1007/s00401-019-02054-4

     PubMed 31392412

102. Comprehensive characterization of human decidual immune cells involvement in spiral artery remodelling Greenbaum, S., Rizzuto, G., Bosse, M., Keren, L., Kowk, S., van de Rijn, M., Bendall, S., Angelo, M. MOSBY-ELSEVIER. 2019: S27–S28

     DOI 10.1016/j.ajog.2018.11.038

     Web of Science ID 000454249400035

103. Scalable Conjugation and Characterization of Immunoglobulins with Stable Mass Isotope Reporters for Single-Cell Mass Cytometry Analysis MASS CYTOMETRY: METHODS AND PROTOCOLS Hartmann, F. J., Simonds, E. F., Vivanco, N., Bruce, T., Borges, L., Nolan, G. P., Spitzer, M. H., Bendall, S. C., McGuire, H. M., Ashhurst, T. M. 2019; 1989: 55–81

     DOI 10.1007/978-1-4939-9454-0_5

     Web of Science ID 000489270700006

104. Serial transplantation reveals a critical role for endoglin in hematopoietic stem cell quiescence. Blood Borges, L., Oliveira, V. K., Baik, J., Bendall, S., Perlingeiro, R. C. 2018

     TGF-beta is well-known for its important function in hematopoietic stem cell (HSC) quiescence. However the molecular mechanism underlining this function remains obscure. Endoglin (Eng), a type III receptor for the TGF-beta superfamily, has been shown to selectively mark the long-term HSC, however its necessity in adult HSC is unknown due to embryonic lethality. Using conditional deletion of Eng combined with serial transplantation, here we show that this TGF-beta receptor is critical to maintain the HSC pool. Transplantation of Eng-deleted whole bone marrow or purified HSCs into lethally irradiated mice results in a profound engraftment defect in tertiary and quaternary recipients. Cell cycle analysis of primary grafts revealed decreased frequency of HSCs in G0, suggesting that lack of Eng impairs re-entry of HSCs to quiescence. Using CyTOF to evaluate activity of signaling pathways in individual HSCs, we find that endoglin is required within the LSK-SLAM fraction for both canonical and non-canonical TGF-beta signaling, as indicated by decreased phosphorylation of both SMAD2/3 and the p38 MAPK-activated protein kinase 2 (MAPKAPK2), respectively.

     PubMed 30593445

105. Mass synaptometry: High-dimensional multi parametric assay for single synapses. Journal of neuroscience methods Gajera, C. R., Fernandez, R., Postupna, N., Montine, K. S., Fox, E. J., Tebaykin, D., Angelo, M., Bendall, S. C., Keene, C. D., Montine, T. J. 2018

     BACKGROUND: Synaptic alterations, especially presynaptic changes, are cardinal features of neurodegenerative diseases and strongly correlate with cognitive decline.NEW METHOD: We report "Mass Synaptometry" for the high-dimensional analysis of individual human synaptosomes, enriched nerve terminals from brain. This method was adapted from cytometry by time-of-flight mass spectrometry (CyTOF), which is commonly used for single-cell analysis of immune and blood cells.RESULT: Here we overcome challenges for single synapse analysis by optimizing synaptosome preparations, generating a 'SynTOF panel,' recalibrating acquisition settings, and applying computational analyses. Through the analysis of 390,000 individual synaptosomes, we also provide proof-of principle validation by characterizing changes in synaptic diversity in Lewy Body Disease (LBD), Alzheimer's disease and normal brain.COMPARISON WITH EXISTING METHOD(S): Current imaging methods to study synapses in humans are capable of analyzing a limited number of synapses, and conventional flow cytometric techniques are typically restricted to fewer than 6 parameters. Our method allows for the simultaneous detection of 34 parameters from tens of thousands of individual synapses.CONCLUSION: We applied Mass Synaptometry to analyze 34 parameters simultaneously on more than 390,000 synaptosomes from 13 human brain samples. This new approach revealed regional and disease-specific changes in synaptic phenotypes, including validation of this method with the expected changes in the molecular composition of striatal dopaminergic synapses in Lewy body disease and Alzheimer's disease. Mass synaptometry enables highly parallel molecular profiling of individual synaptic terminals.

     PubMed 30465796

106. Metal-isotope-tagged monoclonal antibodies for high-dimensional mass cytometry. Nature protocols Han, G., Spitzer, M. H., Bendall, S. C., Fantl, W. J., Nolan, G. P. 2018

     Advances in single-cell mass cytometry have increasingly improved highly multidimensional characterization of immune cell heterogeneity. The immunoassay multiplexing capacity relies on monoclonal antibodies labeled with stable heavy-metal isotopes. To date, a variety of rare-earth elements and noble and post-transition metal isotopes have been used in mass cytometry; nevertheless, the methods used for antibody conjugation differ because of the individual metal coordination chemistries and distinct stabilities of various metal cations. Herein, we provide three optimized protocols for conjugating monoclonal IgG antibodies with 48 high-purity heavy-metal isotopes: (i) 38 isotopes of lanthanides, 2 isotopes of indium, and 1 isotope of yttrium; (ii) 6 isotopes of palladium; and (iii) 1 isotope of bismuth. Bifunctional chelating agents containing coordinative ligands of monomeric DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) or polymeric pentetic acid (DTPA) were used to stably sequester isotopic cations in aqueous solutions and were subsequently coupled to IgG antibodies using site-specific biorthogonal reactions. Furthermore, quantification methods based on antibody inherent absorption at 280 nm and on extrinsic absorption at 562 nm after staining with bicinchoninic acid (BCA) are reported to determine metal-isotope-tagged antibodies. In addition, a freeze-drying procedure to prepare palladium isotopic mass tags is described. To demonstrate the utility, experiments using six palladium-tagged CD45 antibodies for barcoding assays of live immune cells in cytometry by time-of-flight (CyTOF) are described. Conjugation of pure isotopes of lanthanides, indium, or yttrium takes ~3.5 h. Conjugation of bismuth takes ~4 h. Preparation of palladium mass tags takes ~8 h. Conjugation of pure isotopes of palladium takes ~2.5 h. Antibody titration takes ~4 h.

     PubMed 30258176

107. A Universal Live Cell Barcoding-Platform for Multiplexed Human Single Cell Analysis. Scientific reports Hartmann, F. J., Simonds, E. F., Bendall, S. C. 2018; 8 (1): 10770

     Single-cell barcoding enables the combined processing and acquisition of multiple individual samples as one. This maximizes assay efficiency and eliminates technical variability in both sample preparation and analysis. Remaining challenges are the barcoding of live, unprocessed cells to increase downstream assay performance combined with the flexibility of the approach towards a broad range of cell types. To that end, we developed a novel antibody-based platform that allows the robust barcoding of live human cells for mass cytometry (CyTOF). By targeting both the MHC class I complex (beta-2-microglobulin) and a broadly expressed sodium-potassium ATPase-subunit (CD298) with platinum-conjugated antibodies, human immune cells, stem cells as well as tumor cells could be multiplexed in the same single-cell assay. In addition, we present a novel palladium-based covalent viability reagent compatible with this barcoding strategy. Altogether, this platform enables mass cytometry-based, live-cell barcoding across a multitude of human sample types and provides a scheme for multiplexed barcoding of human single-cell assays in general.

     PubMed 30018331

108. Individualized drug combination based on single-cell drug perturbations Anchang, B., Davis, K., Fienberg, H., Bendall, S., Karacosta, L., Nolan, G., Plevritis, S. K. AMER ASSOC CANCER RESEARCH. 2018

     DOI 10.1158/1538-7445.AM2018-2275

     Web of Science ID 000468818905139

109. Identifying dynamic EMT states and constructing a proteomic EMT landscape of lung cancer using single cell multidimensional analysis Karacosta, L. G., Anchang, B., Kimmey, S., van de Rijn, M., Shrager, J. B., Bendall, S. C., Plevritis, S. K. AMER ASSOC CANCER RESEARCH. 2018

     DOI 10.1158/1538-7445.AM2018-4997

     Web of Science ID 000468819504024

110. Publisher Correction: High-resolution myogenic lineage mapping by single-cell mass cytometry. Nature cell biology Porpiglia, E., Samusik, N., Van Ho, A. T., Cosgrove, B. D., Mai, T., Davis, K. L., Jager, A., Nolan, G. P., Bendall, S. C., Fantl, W. J., Blau, H. M. 2018

     In the version of this Article originally published, the name of author Andrew Tri Van Ho was coded wrongly, resulting in it being incorrect when exported to citation databases. This has been corrected, though no visible changes will be apparent.

     PubMed 29507406

111. Ibrutinib-Mediated Inhibition of cGVHD Pathogenic Pre-Germinal Center B-Cells and Follicular Helper Cells While Preserving Immune Memory and Thi T-Cells Sahaf, B., Tebaykin, D., Hopper, M., Cheung, P., Bittencourt, F., Cutler, C., Arora, M., Waller, E. K., Jagasia, M., Pusic, I., Flowers, M. E., Logan, A. C., Jaglowski, S., Lih, J., Solman, I., Lal, I. D., Styles, L., Hill, J. S., James, D. F., Bendall, S., Dubovsky, J., Miklos, D. ELSEVIER SCIENCE INC. 2018: S20–S21

     Web of Science ID 000425476000006

112. DRUG-NEM: Optimizing drug combinations using single-cell perturbation response to account for intratumoral heterogeneity. Proceedings of the National Academy of Sciences of the United States of America Anchang, B., Davis, K. L., Fienberg, H. G., Williamson, B. D., Bendall, S. C., Karacosta, L. G., Tibshirani, R., Nolan, G. P., Plevritis, S. K. 2018; 115 (18): E4294–E4303

     An individual malignant tumor is composed of a heterogeneous collection of single cells with distinct molecular and phenotypic features, a phenomenon termed intratumoral heterogeneity. Intratumoral heterogeneity poses challenges for cancer treatment, motivating the need for combination therapies. Single-cell technologies are now available to guide effective drug combinations by accounting for intratumoral heterogeneity through the analysis of the signaling perturbations of an individual tumor sample screened by a drug panel. In particular, Mass Cytometry Time-of-Flight (CyTOF) is a high-throughput single-cell technology that enables the simultaneous measurements of multiple ([Formula: see text]40) intracellular and surface markers at the level of single cells for hundreds of thousands of cells in a sample. We developed a computational framework, entitled Drug Nested Effects Models (DRUG-NEM), to analyze CyTOF single-drug perturbation data for the purpose of individualizing drug combinations. DRUG-NEM optimizes drug combinations by choosing the minimum number of drugs that produce the maximal desired intracellular effects based on nested effects modeling. We demonstrate the performance of DRUG-NEM using single-cell drug perturbation data from tumor cell lines and primary leukemia samples.

     PubMed 29654148

113. GateFinder: Projection-based Gating Strategy Optimization for Flow and Mass Cytometry. Bioinformatics (Oxford, England) Aghaeepour, N., Simonds, E. F., Knapp, D. J., Bruggner, R., Sachs, K., Culos, A., Gherardini, P. F., Samusik, N., Fragiadakis, G., Bendall, S., Gaudilliere, B., Angst, M. S., Eaves, C. J., Weiss, W. A., Fantl, W., Nolan, G. 2018

     High-parameter single-cell technologies can reveal novel cell populations of interest, but studying or validating these populations using lower-parameter methods remains challenging.Here we present GateFinder, an algorithm that enriches high-dimensional cell types with simple, stepwise polygon gates requiring only two markers at a time. A series of case studies of complex cell types illustrates how simplified enrichment strategies can enable more efficient assays, reveal novel biomarkers, and clarify underlying biology.The GateFinder algorithm is implemented as a free and open-source package for BioConductor: https://nalab.stanford.edu/gatefinder.gnolan@stanford.edu or naghaeep@stanford.edu.Supplementary data are available at Bioinformatics online.

     PubMed 29850785

114. High-resolution myogenic lineage mapping by single-cell mass cytometry NATURE CELL BIOLOGY Porpiglia, E., Samusik, N., Van Ho, A. T., Cosgrove, B. D., Mai, T., Davis, K. L., Jager, A., Nolan, G. P., Bendall, S. C., Fantl, W. J., Blau, H. M. 2017; 19 (5): 558-?

     Muscle regeneration is a dynamic process during which cell state and identity change over time. A major roadblock has been a lack of tools to resolve a myogenic progression in vivo. Here we capitalize on a transformative technology, single-cell mass cytometry (CyTOF), to identify in vivo skeletal muscle stem cell and previously unrecognized progenitor populations that precede differentiation. We discovered two cell surface markers, CD9 and CD104, whose combined expression enabled in vivo identification and prospective isolation of stem and progenitor cells. Data analysis using the X-shift algorithm paired with single-cell force-directed layout visualization defined a molecular signature of the activated stem cell state (CD44(+)/CD98(+)/MyoD(+)) and delineated a myogenic trajectory during recovery from acute muscle injury. Our studies uncover the dynamics of skeletal muscle regeneration in vivo and pave the way for the elucidation of the regulatory networks that underlie cell-state transitions in muscle diseases and ageing.

     DOI 10.1038/ncb3507

     Web of Science ID 000400376100019

     PubMed 28414312

115. Assessing basophil activation by using flow cytometry and mass cytometry in blood stored 24 hours before analysis JOURNAL OF ALLERGY AND CLINICAL IMMUNOLOGY Mukai, K., Gaudenzio, N., Gupta, S., Vivanco, N., Bendall, S. C., Maecker, H. T., Chinthrajah, R. S., Tsai, M., Nadeau, K. C., Galli, S. J. 2017; 139 (3): 889-?

     Basophil activation tests (BATs) have promise for research and for clinical monitoring of patients with allergies. However, BAT protocols vary in blood anticoagulant used and temperature and time of storage before testing, complicating comparisons of results from various studies.We attempted to establish a BAT protocol that would permit analysis of blood within 24 hours of obtaining the sample.Blood from 46 healthy donors and 120 patients with peanut allergy was collected into EDTA or heparin tubes, and samples were stored at 4°C or room temperature for 4 or 24 hours before performing BATs.Stimulation with anti-IgE or IL-3 resulted in strong upregulation of basophil CD203c in samples collected in EDTA or heparin, stored at 4°C, and analyzed 24 hours after sample collection. However, a CD63(hi) population of basophils was not observed in any conditions in EDTA-treated samples unless exogenous calcium/magnesium was added at the time of anti-IgE stimulation. By contrast, blood samples collected in heparin tubes were adequate for quantification of upregulation of basophil CD203c and identification of a population of CD63(hi) basophils, irrespective of whether the specimens were analyzed by means of conventional flow cytometry or cytometry by time-of-flight mass spectrometry, and such tests could be performed after blood was stored for 24 hours at 4°C.BATs to measure upregulation of basophil CD203c and induction of a CD63(hi) basophil population can be conducted with blood obtained in heparin tubes and stored at 4°C for 24 hours.

     DOI 10.1016/j.jaci.2016.04.060

     Web of Science ID 000397295800022

     PubMedCentralID PMC5237629

116. Assessing basophil activation by flow cytometry and mass cytometry in blood stored 24 hours before analysis Mukai, K., Gaudenzio, N., Gupta, S., Vivanco, N., Bendall, S. C., Maecker, H. T., Chinthrajah, R., Tsai, M., Nadeau, K. C., Galli, S. J. MOSBY-ELSEVIER. 2017: AB124

     DOI 10.1016/j.jaci.2016.12.402

     Web of Science ID 000401699800293

117. Systemic Immunity Is Required for Effective Cancer Immunotherapy. Cell Spitzer, M. H., Carmi, Y., Reticker-Flynn, N. E., Kwek, S. S., Madhireddy, D., Martins, M. M., Gherardini, P. F., Prestwood, T. R., Chabon, J., Bendall, S. C., Fong, L., Nolan, G. P., Engleman, E. G. 2017; 168 (3): 487-502 e15

     Immune responses involve coordination across cell types and tissues. However, studies in cancer immunotherapy have focused heavily on local immune responses in the tumor microenvironment. To investigate immune activity more broadly, we performed an organism-wide study in genetically engineered cancer models using mass cytometry. We analyzed immune responses in several tissues after immunotherapy by developing intuitive models for visualizing single-cell data with statistical inference. Immune activation was evident in the tumor and systemically shortly after effective therapy was administered. However, during tumor rejection, only peripheral immune cells sustained their proliferation. This systemic response was coordinated across tissues and required for tumor eradication in several immunotherapy models. An emergent population of peripheral CD4 T cells conferred protection against new tumors and was significantly expanded in patients responding to immunotherapy. These studies demonstrate the critical impact of systemic immune responses that drive tumor rejection.

     DOI 10.1016/j.cell.2016.12.022

     PubMed 28111070

118. Distinct signaling programs control human hematopoietic stem cell survival and proliferation. Blood Knapp, D. J., Hammond, C. A., Aghaeepour, N., Miller, P. H., Pellacani, D., Beer, P. A., Sachs, K., Qiao, W., Wang, W., Humphries, R. K., Sauvageau, G., Zandstra, P. W., Bendall, S. C., Nolan, G. P., Hansen, C., Eaves, C. J. 2017; 129 (3): 307-318

     Several growth factors (GFs) that together promote quiescent human hematopoietic stem cell (HSC) expansion ex vivo have been identified; however, the molecular mechanisms by which these GFs regulate the survival, proliferation. and differentiation of human HSCs remain poorly understood. We now describe experiments in which we used mass cytometry to simultaneously measure multiple surface markers, transcription factors, active signaling intermediates, viability, and cell-cycle indicators in single CD34(+) cord blood cells before and up to 2 hours after their stimulation with stem cell factor, Fms-like tyrosine kinase 3 ligand, interleukin-3, interleukin-6, and granulocyte colony-stimulating factor (5 GFs) either alone or combined. Cells with a CD34(+)CD38(-)CD45RA(-)CD90(+)CD49f(+) (CD49f(+)) phenotype (∼10% HSCs with >6-month repopulating activity in immunodeficient mice) displayed rapid increases in activated STAT1/3/5, extracellular signal-regulated kinase 1/2, AKT, CREB, and S6 by 1 or more of these GFs, and β-catenin only when the 5 GFs were combined. Certain minority subsets within the CD49f(+) compartment were poorly GF-responsive and, among the more GF-responsive subsets of CD49f(+) cells, different signaling intermediates correlated with the levels of the myeloid- and lymphoid-associated transcription factors measured. Phenotypically similar, but CD90(-)CD49f(-) cells (MPPs) contained lower baseline levels of multiple signaling intermediates than the CD90(+)CD49f(+) cells, but showed similar response amplitudes to the same GFs. Importantly, we found activation or inhibition of AKT and β-catenin directly altered immediate CD49f(+) cell survival and proliferation. These findings identify rapid signaling events that 5 GFs elicit directly in the most primitive human hematopoietic cell types to promote their survival and proliferation.

     DOI 10.1182/blood-2016-09-740654

     PubMed 27827829

119. APPLYING SINGLE-CELL MASS CYTOMETRY TO INVESTIGATE THE IMMUNE SYSTEM OF HIGHLY SENSITIZED PATIENTS WHO UNDERGO INTRAVENOUS IMMUNOGLOBULIN DESENSITIZATION TREATMENT. Wang, L., Bendall, S., Chang, C., Chen, G., Tyan, D. B. ELSEVIER SCIENCE INC. 2017: 37–38

     DOI 10.1016/j.humimm.2017.06.046

     Web of Science ID 000411658700041

120. Mutant IDH1 Downregulates ATM and Alters DNA Repair and Sensitivity to DNA Damage Independent of TET2. Cancer cell Inoue, S., Li, W. Y., Tseng, A., Beerman, I., Elia, A. J., Bendall, S. C., Lemonnier, F., Kron, K. J., Cescon, D. W., Hao, Z., Lind, E. F., Takayama, N., Planello, A. C., Shen, S. Y., Shih, A. H., Larsen, D. M., Li, Q., Snow, B. E., Wakeham, A., Haight, J., Gorrini, C., Bassi, C., Thu, K. L., Murakami, K., Elford, A. R., Ueda, T., Straley, K., Yen, K. E., Melino, G., Cimmino, L., Aifantis, I., Levine, R. L., De Carvalho, D. D., Lupien, M., Rossi, D. J., Nolan, G. P., Cairns, R. A., Mak, T. W. 2016; 30 (2): 337-348

     Mutations in the isocitrate dehydrogenase-1 gene (IDH1) are common drivers of acute myeloid leukemia (AML) but their mechanism is not fully understood. It is thought that IDH1 mutants act by inhibiting TET2 to alter DNA methylation, but there are significant unexplained clinical differences between IDH1- and TET2-mutant diseases. We have discovered that mice expressing endogenous mutant IDH1 have reduced numbers of hematopoietic stem cells (HSCs), in contrast to Tet2 knockout (TET2-KO) mice. Mutant IDH1 downregulates the DNA damage (DD) sensor ATM by altering histone methylation, leading to impaired DNA repair, increased sensitivity to DD, and reduced HSC self-renewal, independent of TET2. ATM expression is also decreased in human IDH1-mutated AML. These findings may have implications for treatment of IDH-mutant leukemia.

     DOI 10.1016/j.ccell.2016.05.018

     PubMed 27424808

     PubMedCentralID PMC5022794

121. Assessing basophil activation by using flow cytometry and mass cytometry in blood stored 24 hours before analysis. journal of allergy and clinical immunology Mukai, K., Gaudenzio, N., Gupta, S., Vivanco, N., Bendall, S. C., Maecker, H. T., Chinthrajah, R. S., Tsai, M., Nadeau, K. C., Galli, S. J. 2016

     Basophil activation tests (BATs) have promise for research and for clinical monitoring of patients with allergies. However, BAT protocols vary in blood anticoagulant used and temperature and time of storage before testing, complicating comparisons of results from various studies.We attempted to establish a BAT protocol that would permit analysis of blood within 24 hours of obtaining the sample.Blood from 46 healthy donors and 120 patients with peanut allergy was collected into EDTA or heparin tubes, and samples were stored at 4°C or room temperature for 4 or 24 hours before performing BATs.Stimulation with anti-IgE or IL-3 resulted in strong upregulation of basophil CD203c in samples collected in EDTA or heparin, stored at 4°C, and analyzed 24 hours after sample collection. However, a CD63(hi) population of basophils was not observed in any conditions in EDTA-treated samples unless exogenous calcium/magnesium was added at the time of anti-IgE stimulation. By contrast, blood samples collected in heparin tubes were adequate for quantification of upregulation of basophil CD203c and identification of a population of CD63(hi) basophils, irrespective of whether the specimens were analyzed by means of conventional flow cytometry or cytometry by time-of-flight mass spectrometry, and such tests could be performed after blood was stored for 24 hours at 4°C.BATs to measure upregulation of basophil CD203c and induction of a CD63(hi) basophil population can be conducted with blood obtained in heparin tubes and stored at 4°C for 24 hours.

     DOI 10.1016/j.jaci.2016.04.060

     PubMed 27527263

     PubMedCentralID PMC5237629

122. Visualization and cellular hierarchy inference of single-cell data using SPADE. Nature protocols Anchang, B., Hart, T. D., Bendall, S. C., Qiu, P., Bjornson, Z., Linderman, M., Nolan, G. P., Plevritis, S. K. 2016; 11 (7): 1264-1279

     High-throughput single-cell technologies provide an unprecedented view into cellular heterogeneity, yet they pose new challenges in data analysis and interpretation. In this protocol, we describe the use of Spanning-tree Progression Analysis of Density-normalized Events (SPADE), a density-based algorithm for visualizing single-cell data and enabling cellular hierarchy inference among subpopulations of similar cells. It was initially developed for flow and mass cytometry single-cell data. We describe SPADE's implementation and application using an open-source R package that runs on Mac OS X, Linux and Windows systems. A typical SPADE analysis on a 2.27-GHz processor laptop takes ∼5 min. We demonstrate the applicability of SPADE to single-cell RNA-seq data. We compare SPADE with recently developed single-cell visualization approaches based on the t-distribution stochastic neighborhood embedding (t-SNE) algorithm. We contrast the implementation and outputs of these methods for normal and malignant hematopoietic cells analyzed by mass cytometry and provide recommendations for appropriate use. Finally, we provide an integrative strategy that combines the strengths of t-SNE and SPADE to infer cellular hierarchy from high-dimensional single-cell data.

     DOI 10.1038/nprot.2016.066

     PubMed 27310265

123. Wishbone identifies bifurcating developmental trajectories from single-cell data NATURE BIOTECHNOLOGY Setty, M., Tadmor, M. D., Reich-Zeliger, S., Ange, O., Salame, T. M., Kathail, P., Choi, K., Bendall, S., Friedman, N., Pe'er, D. 2016; 34 (6): 637-645

     Recent single-cell analysis technologies offer an unprecedented opportunity to elucidate developmental pathways. Here we present Wishbone, an algorithm for positioning single cells along bifurcating developmental trajectories with high resolution. Wishbone uses multi-dimensional single-cell data, such as mass cytometry or RNA-Seq data, as input and orders cells according to their developmental progression, and it pinpoints bifurcation points by labeling each cell as pre-bifurcation or as one of two post-bifurcation cell fates. Using 30-channel mass cytometry data, we show that Wishbone accurately recovers the known stages of T-cell development in the mouse thymus, including the bifurcation point. We also apply the algorithm to mouse myeloid differentiation and demonstrate its generalization to additional lineages. A comparison of Wishbone to diffusion maps, SCUBA and Monocle shows that it outperforms these methods both in the accuracy of ordering cells and in the correct identification of branch points.

     DOI 10.1038/nbt.3569

     Web of Science ID 000377846400031

     PubMed 27136076

     PubMedCentralID PMC4900897

124. SESSION INTRODUCTION. Pacific Symposium on Biocomputing. Pacific Symposium on Biocomputing Samusik, N., Aghaeepour, N., Bendall, S. 2016; 22: 557-563

     Recent technological developments allow gathering single-cell measurements across different domains (genomic, transcriptomics, proteomics, imaging etc). Sophisticated computational algorithms are required in order to harness the power of single-cell data. This session is dedicated to computational methods for single-cell analysis in various biological domains, modelling of population heterogeneity, as well as translational applications of single cell data.

     PubMed 27897006

125. Single-cell systems-level analysis of human Toll-like receptor activation defines a chemokine signature in patients with systemic lupus erythematosus JOURNAL OF ALLERGY AND CLINICAL IMMUNOLOGY O'Gorman, W. E., Hsieh, E. W., Savig, E. S., Gherardini, P. F., Hernandez, J. D., Hansmann, L., Balboni, I. M., Utz, P. J., Bendall, S. C., Fantl, W. J., Lewis, D. B., Nolan, G. P., Davis, M. M. 2015; 136 (5): 1326-1336

     Activation of Toll-like receptors (TLRs) induces inflammatory responses involved in immunity to pathogens and autoimmune pathogenesis, such as in patients with systemic lupus erythematosus (SLE). Although TLRs are differentially expressed across the immune system, a comprehensive analysis of how multiple immune cell subsets respond in a system-wide manner has not been described.We sought to characterize TLR activation across multiple immune cell subsets and subjects, with the goal of establishing a reference framework against which to compare pathologic processes.Peripheral whole-blood samples were stimulated with TLR ligands and analyzed by means of mass cytometry simultaneously for surface marker expression, activation states of intracellular signaling proteins, and cytokine production. We developed a novel data visualization tool to provide an integrated view of TLR signaling networks with single-cell resolution. We studied 17 healthy volunteer donors and 8 patients with newly diagnosed and untreated SLE.Our data revealed the diversity of TLR-induced responses within cell types, with TLR ligand specificity. Subsets of natural killer cells and T cells selectively induced nuclear factor κ light chain enhancer of activated B cells in response to TLR2 ligands. CD14(hi) monocytes exhibited the most polyfunctional cytokine expression patterns, with more than 80 distinct cytokine combinations. Monocytic TLR-induced cytokine patterns were shared among a group of healthy donors, with minimal intraindividual and interindividual variability. Furthermore, autoimmune disease altered baseline cytokine production; newly diagnosed untreated SLE patients shared a distinct monocytic chemokine signature, despite clinical heterogeneity.Mass cytometry defined a systems-level reference framework for human TLR activation, which can be applied to study perturbations in patients with inflammatory diseases, such as SLE.

     DOI 10.1016/j.jaci.2015.04.008

     Web of Science ID 000364787200023

     PubMed 26037552

126. Synthetically Modified Viral Capsids as Versatile Carriers for Use in Antibody-Based Cell Targeting. Bioconjugate chemistry ElSohly, A. M., Netirojjanakul, C., Aanei, I. L., Jager, A., Bendall, S. C., Farkas, M. E., Nolan, G. P., Francis, M. B. 2015; 26 (8): 1590-1596

     The present study describes an efficient and reliable method for the preparation of MS2 viral capsids that are synthetically modified with antibodies using a rapid oxidative coupling strategy. The overall protocol delivers conjugates in high yields and recoveries, requires a minimal excess of antibody to achieve modification of more than 95% of capsids, and can be completed in a short period of time. Antibody-capsid conjugates targeting extracellular receptors on human breast cancer cell lines were prepared and characterized. Notably, conjugation to the capsid did not significantly perturb the binding of the antibodies, as indicated by binding affinities similar to those obtained for the parent antibodies. An array of conjugates was synthesized with various reporters on the interior surface of the capsids to be used in cell studies, including fluorescence-based flow cytometry, confocal microscopy, and mass cytometry. The results of these studies lay the foundation for further exploration of these constructs in the context of clinically relevant applications, including drug delivery and in vivo diagnostics.

     DOI 10.1021/acs.bioconjchem.5b00226

     PubMed 26076186

127. Synthetically Modified Viral Capsids as Versatile Carriers for Use in Antibody-Based Cell Targeting BIOCONJUGATE CHEMISTRY ElSohly, A. M., Netirojjanakul, C., Aanei, I. L., Jager, A., Bendall, S. C., Farkas, M. E., Nolan, G. P., Francis, M. B. 2015; 26 (8): 1590-1596

     The present study describes an efficient and reliable method for the preparation of MS2 viral capsids that are synthetically modified with antibodies using a rapid oxidative coupling strategy. The overall protocol delivers conjugates in high yields and recoveries, requires a minimal excess of antibody to achieve modification of more than 95% of capsids, and can be completed in a short period of time. Antibody-capsid conjugates targeting extracellular receptors on human breast cancer cell lines were prepared and characterized. Notably, conjugation to the capsid did not significantly perturb the binding of the antibodies, as indicated by binding affinities similar to those obtained for the parent antibodies. An array of conjugates was synthesized with various reporters on the interior surface of the capsids to be used in cell studies, including fluorescence-based flow cytometry, confocal microscopy, and mass cytometry. The results of these studies lay the foundation for further exploration of these constructs in the context of clinically relevant applications, including drug delivery and in vivo diagnostics.

     DOI 10.1021/acs.bioconjchem.5b00226

     Web of Science ID 000359962900024

128. An interactive reference framework for modeling a dynamic immune system SCIENCE Spitzer, M. H., Gherardini, P. F., Fragiadakis, G. K., Bhattacharya, N., Yuan, R. T., Hotson, A. N., Finck, R., Carmi, Y., Zunder, E. R., Fantl, W. J., Bendall, S. C., Engleman, E. G., Nolan, G. P. 2015; 349 (6244): 155-?

     Immune cells function in an interacting hierarchy that coordinates the activities of various cell types according to genetic and environmental contexts. We developed graphical approaches to construct an extensible immune reference map from mass cytometry data of cells from different organs, incorporating landmark cell populations as flags on the map to compare cells from distinct samples. The maps recapitulated canonical cellular phenotypes and revealed reproducible, tissue-specific deviations. The approach revealed influences of genetic variation and circadian rhythms on immune system structure, enabled direct comparisons of murine and human blood cell phenotypes, and even enabled archival fluorescence-based flow cytometry data to be mapped onto the reference framework. This foundational reference map provides a working definition of systemic immune organization to which new data can be integrated to reveal deviations driven by genetics, environment, or pathology.

     DOI 10.1126/science.1259425

     Web of Science ID 000357664300034

     PubMedCentralID PMC4537647

129. IMMUNOLOGY. An interactive reference framework for modeling a dynamic immune system. Science Spitzer, M. H., Gherardini, P. F., Fragiadakis, G. K., Bhattacharya, N., Yuan, R. T., Hotson, A. N., Finck, R., Carmi, Y., Zunder, E. R., Fantl, W. J., Bendall, S. C., Engleman, E. G., Nolan, G. P. 2015; 349 (6244)

     Immune cells function in an interacting hierarchy that coordinates the activities of various cell types according to genetic and environmental contexts. We developed graphical approaches to construct an extensible immune reference map from mass cytometry data of cells from different organs, incorporating landmark cell populations as flags on the map to compare cells from distinct samples. The maps recapitulated canonical cellular phenotypes and revealed reproducible, tissue-specific deviations. The approach revealed influences of genetic variation and circadian rhythms on immune system structure, enabled direct comparisons of murine and human blood cell phenotypes, and even enabled archival fluorescence-based flow cytometry data to be mapped onto the reference framework. This foundational reference map provides a working definition of systemic immune organization to which new data can be integrated to reveal deviations driven by genetics, environment, or pathology.

     DOI 10.1126/science.1259425

     PubMed 26160952

130. Conditional density-based analysis of T cell signaling in single-cell data SCIENCE Krishnaswamy, S., Spitzer, M. H., Mingueneau, M., Bendall, S. C., Litvin, O., Stone, E., Pe'er, D., Nolan, G. P. 2014; 346 (6213): 1079-?

     Cellular circuits sense the environment, process signals, and compute decisions using networks of interacting proteins. To model such a system, the abundance of each activated protein species can be described as a stochastic function of the abundance of other proteins. High-dimensional single-cell technologies, such as mass cytometry, offer an opportunity to characterize signaling circuit-wide. However, the challenge of developing and applying computational approaches to interpret such complex data remains. Here, we developed computational methods, based on established statistical concepts, to characterize signaling network relationships by quantifying the strengths of network edges and deriving signaling response functions. In comparing signaling between naïve and antigen-exposed CD4(+) T lymphocytes, we find that although these two cell subtypes had similarly wired networks, naïve cells transmitted more information along a key signaling cascade than did antigen-exposed cells. We validated our characterization on mice lacking the extracellular-regulated mitogen-activated protein kinase (MAPK) ERK2, which showed stronger influence of pERK on pS6 (phosphorylated-ribosomal protein S6), in naïve cells as compared with antigen-exposed cells, as predicted. We demonstrate that by using cell-to-cell variation inherent in single-cell data, we can derive response functions underlying molecular circuits and drive the understanding of how cells process signals.

     DOI 10.1126/science/1250689

     Web of Science ID 000345763400033

131. NRASG12V oncogene facilitates self-renewal in a murine model of acute myelogenous leukemia. Blood Sachs, Z., LaRue, R. S., Nguyen, H. T., Sachs, K., Noble, K. E., Mohd Hassan, N. A., Diaz-Flores, E., Rathe, S. K., Sarver, A. L., Bendall, S. C., Ha, N. A., Diers, M. D., Nolan, G. P., Shannon, K. M., Largaespada, D. A. 2014; 124 (22): 3274-3283

     Mutant RAS oncoproteins activate signaling molecules that drive oncogenesis in multiple human tumors including acute myelogenous leukemia (AML). However, the specific functions of these pathways in AML are unclear, thwarting the rational application of targeted therapeutics. To elucidate the downstream functions of activated NRAS in AML, we used a murine model that harbors Mll-AF9 and a tetracycline-repressible, activated NRAS (NRAS(G12V)). Using computational approaches to explore our gene-expression data sets, we found that NRAS(G12V) enforced the leukemia self-renewal gene-expression signature and was required to maintain an MLL-AF9- and Myb-dependent leukemia self-renewal gene-expression program. NRAS(G12V) was required for leukemia self-renewal independent of its effects on growth and survival. Analysis of the gene-expression patterns of leukemic subpopulations revealed that the NRAS(G12V)-mediated leukemia self-renewal signature is preferentially expressed in the leukemia stem cell-enriched subpopulation. In a multiplexed analysis of RAS-dependent signaling, Mac-1(Low) cells, which harbor leukemia stem cells, were preferentially sensitive to NRAS(G12V) withdrawal. NRAS(G12V) maintained leukemia self-renewal through mTOR and MEK pathway activation, implicating these pathways as potential targets for cancer stem cell-specific therapies. Together, these experimental results define a RAS oncogene-driven function that is critical for leukemia maintenance and represents a novel mechanism of oncogene addiction.

     DOI 10.1182/blood-2013-08-521708

     PubMed 25316678

132. NRAS9(G12V) oncogene facilitates self-renewal in a murine model of acute myelogenous leukemia BLOOD Sachs, Z., LaRue, R. S., Nguyen, H. T., Sachs, K., Noble, K. E., Hassan, N. A., Diaz-Flores, E., Rathe, S. K., Sarver, A. L., Bendall, S. C., Ha, N. A., Diers, M. D., Nolan, G. P., Shannon, K. M., Largaespada, D. A. 2014; 124 (22): 3274-3283

     Mutant RAS oncoproteins activate signaling molecules that drive oncogenesis in multiple human tumors including acute myelogenous leukemia (AML). However, the specific functions of these pathways in AML are unclear, thwarting the rational application of targeted therapeutics. To elucidate the downstream functions of activated NRAS in AML, we used a murine model that harbors Mll-AF9 and a tetracycline-repressible, activated NRAS (NRAS(G12V)). Using computational approaches to explore our gene-expression data sets, we found that NRAS(G12V) enforced the leukemia self-renewal gene-expression signature and was required to maintain an MLL-AF9- and Myb-dependent leukemia self-renewal gene-expression program. NRAS(G12V) was required for leukemia self-renewal independent of its effects on growth and survival. Analysis of the gene-expression patterns of leukemic subpopulations revealed that the NRAS(G12V)-mediated leukemia self-renewal signature is preferentially expressed in the leukemia stem cell-enriched subpopulation. In a multiplexed analysis of RAS-dependent signaling, Mac-1(Low) cells, which harbor leukemia stem cells, were preferentially sensitive to NRAS(G12V) withdrawal. NRAS(G12V) maintained leukemia self-renewal through mTOR and MEK pathway activation, implicating these pathways as potential targets for cancer stem cell-specific therapies. Together, these experimental results define a RAS oncogene-driven function that is critical for leukemia maintenance and represents a novel mechanism of oncogene addiction.

     DOI 10.1182/blood-2013-08-521708

     Web of Science ID 000347463100016

     PubMedCentralID PMC4239336

133. Single-cell mass cytometry of TCR signaling: Amplification of small initial differences results in low ERK activation in NOD mice PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Mingueneau, M., Krishnaswamy, S., Spitzer, M. H., Bendall, S. C., Stone, E. L., Hedrick, S. M., Pe'er, D., Mathis, D., Nolan, G. P., Benoist, C. 2014; 111 (46): 16466-16471

     Signaling from the T-cell receptor (TCR) conditions T-cell differentiation and activation, requiring exquisite sensitivity and discrimination. Using mass cytometry, a high-dimensional technique that can probe multiple signaling nodes at the single-cell level, we interrogate TCR signaling dynamics in control C57BL/6 and autoimmunity-prone nonobese diabetic (NOD) mice, which show ineffective ERK activation after TCR triggering. By quantitating signals at multiple steps along the signaling cascade and parsing the phosphorylation level of each node as a function of its predecessors, we show that a small impairment in initial pCD3ζ activation resonates farther down the signaling cascade and results in larger defects in activation of the ERK1/2-S6 and IκBα modules. This nonlinear property of TCR signaling networks, which magnifies small initial differences during signal propagation, also applies in cells from B6 mice activated at different levels of intensity. Impairment in pCD3ζ and pSLP76 is not a feedback consequence of a primary deficiency in ERK activation because no proximal signaling defect was observed in Erk2 KO T cells. These defects, which were manifest at all stages of T-cell differentiation from early thymic pre-T cells to memory T cells, may condition the imbalanced immunoregulation and tolerance in NOD T cells. More generally, this amplification of small initial differences in signal intensity may explain how T cells discriminate between closely related ligands and adopt strongly delineated cell fates.

     DOI 10.1073/pnas.1419337111

     Web of Science ID 000345153300062

     PubMedCentralID PMC4246343

134. Single-cell mass cytometry of TCR signaling: amplification of small initial differences results in low ERK activation in NOD mice. Proceedings of the National Academy of Sciences of the United States of America Mingueneau, M., Krishnaswamy, S., Spitzer, M. H., Bendall, S. C., Stone, E. L., Hedrick, S. M., Pe'er, D., Mathis, D., Nolan, G. P., Benoist, C. 2014; 111 (46): 16466-16471

     Signaling from the T-cell receptor (TCR) conditions T-cell differentiation and activation, requiring exquisite sensitivity and discrimination. Using mass cytometry, a high-dimensional technique that can probe multiple signaling nodes at the single-cell level, we interrogate TCR signaling dynamics in control C57BL/6 and autoimmunity-prone nonobese diabetic (NOD) mice, which show ineffective ERK activation after TCR triggering. By quantitating signals at multiple steps along the signaling cascade and parsing the phosphorylation level of each node as a function of its predecessors, we show that a small impairment in initial pCD3ζ activation resonates farther down the signaling cascade and results in larger defects in activation of the ERK1/2-S6 and IκBα modules. This nonlinear property of TCR signaling networks, which magnifies small initial differences during signal propagation, also applies in cells from B6 mice activated at different levels of intensity. Impairment in pCD3ζ and pSLP76 is not a feedback consequence of a primary deficiency in ERK activation because no proximal signaling defect was observed in Erk2 KO T cells. These defects, which were manifest at all stages of T-cell differentiation from early thymic pre-T cells to memory T cells, may condition the imbalanced immunoregulation and tolerance in NOD T cells. More generally, this amplification of small initial differences in signal intensity may explain how T cells discriminate between closely related ligands and adopt strongly delineated cell fates.

     DOI 10.1073/pnas.1419337111

     PubMed 25362052

135. The Split Virus Influenza Vaccine rapidly activates immune cells through Fc gamma receptors VACCINE O'Gorman, W. E., Huang, H., Wei, Y., Davis, K. L., Leipold, M. D., Bendall, S. C., Kidd, B. A., Dekker, C. L., Maecker, H. T., Chien, Y., Davis, M. M. 2014; 32 (45): 5989-5997

     Seasonal influenza vaccination is one of the most common medical procedures and yet the extent to which it activates the immune system beyond inducing antibody production is not well understood. In the United States, the most prevalent formulations of the vaccine consist of degraded or "split" viral particles distributed without any adjuvants. Based on previous reports we sought to determine whether the split influenza vaccine activates innate immune receptors-specifically Toll-like receptors. High-dimensional proteomic profiling of human whole-blood using Cytometry by Time-of-Flight (CyTOF) was used to compare signaling pathway activation and cytokine production between the split influenza vaccine and a prototypical TLR response ex vivo. This analysis revealed that the split vaccine rapidly and potently activates multiple immune cell types but yields a proteomic signature quite distinct from TLR activation. Importantly, vaccine induced activity was dependent upon the presence of human sera indicating that a serum factor was necessary for vaccine-dependent immune activation. We found this serum factor to be human antibodies specific for influenza proteins and therefore immediate immune activation by the split vaccine is immune-complex dependent. These studies demonstrate that influenza virus "splitting" inactivates any potential adjuvants endogenous to influenza, such as RNA, but in previously exposed individuals can elicit a potent immune response by facilitating the rapid formation of immune complexes.

     DOI 10.1016/j.vaccine.2014.07.115

     Web of Science ID 000343629900016

     PubMedCentralID PMC4191649

136. The Split Virus Influenza Vaccine rapidly activates immune cells through Fc? receptors. Vaccine O'Gorman, W. E., Huang, H., Wei, Y., Davis, K. L., Leipold, M. D., Bendall, S. C., Kidd, B. A., Dekker, C. L., Maecker, H. T., Chien, Y., Davis, M. M. 2014; 32 (45): 5989-5997

     Seasonal influenza vaccination is one of the most common medical procedures and yet the extent to which it activates the immune system beyond inducing antibody production is not well understood. In the United States, the most prevalent formulations of the vaccine consist of degraded or "split" viral particles distributed without any adjuvants. Based on previous reports we sought to determine whether the split influenza vaccine activates innate immune receptors-specifically Toll-like receptors. High-dimensional proteomic profiling of human whole-blood using Cytometry by Time-of-Flight (CyTOF) was used to compare signaling pathway activation and cytokine production between the split influenza vaccine and a prototypical TLR response ex vivo. This analysis revealed that the split vaccine rapidly and potently activates multiple immune cell types but yields a proteomic signature quite distinct from TLR activation. Importantly, vaccine induced activity was dependent upon the presence of human sera indicating that a serum factor was necessary for vaccine-dependent immune activation. We found this serum factor to be human antibodies specific for influenza proteins and therefore immediate immune activation by the split vaccine is immune-complex dependent. These studies demonstrate that influenza virus "splitting" inactivates any potential adjuvants endogenous to influenza, such as RNA, but in previously exposed individuals can elicit a potent immune response by facilitating the rapid formation of immune complexes.

     DOI 10.1016/j.vaccine.2014.07.115

     PubMed 25203448

137. Clinical recovery from surgery correlates with single-cell immune signatures SCIENCE TRANSLATIONAL MEDICINE Gaudilliere, B., Fragiadakis, G. K., Bruggner, R. V., Nicolau, M., Finck, R., Tingle, M., Silva, J., Ganio, E. A., Yeh, C. G., Maloney, W. J., Huddleston, J. I., Goodman, S. B., Davis, M. M., Bendall, S. C., Fantl, W. J., Angst, M. S., Nolan, G. P. 2014; 6 (255)

     Delayed recovery from surgery causes personal suffering and substantial societal and economic costs. Whether immune mechanisms determine recovery after surgical trauma remains ill-defined. Single-cell mass cytometry was applied to serial whole-blood samples from 32 patients undergoing hip replacement to comprehensively characterize the phenotypic and functional immune response to surgical trauma. The simultaneous analysis of 14,000 phosphorylation events in precisely phenotyped immune cell subsets revealed uniform signaling responses among patients, demarcating a surgical immune signature. When regressed against clinical parameters of surgical recovery, including functional impairment and pain, strong correlations were found with STAT3 (signal transducer and activator of transcription), CREB (adenosine 3',5'-monophosphate response element-binding protein), and NF-κB (nuclear factor κB) signaling responses in subsets of CD14(+) monocytes (R = 0.7 to 0.8, false discovery rate <0.01). These sentinel results demonstrate the capacity of mass cytometry to survey the human immune system in a relevant clinical context. The mechanistically derived immune correlates point to diagnostic signatures, and potential therapeutic targets, that could postoperatively improve patient recovery.

     DOI 10.1126/scitranslmed.3009701

     Web of Science ID 000343316800006

138. Clinical recovery from surgery correlates with single-cell immune signatures. Science translational medicine Gaudillière, B., Fragiadakis, G. K., Bruggner, R. V., Nicolau, M., Finck, R., Tingle, M., Silva, J., Ganio, E. A., Yeh, C. G., Maloney, W. J., Huddleston, J. I., Goodman, S. B., Davis, M. M., Bendall, S. C., Fantl, W. J., Angst, M. S., Nolan, G. P. 2014; 6 (255): 255ra131-?

     Delayed recovery from surgery causes personal suffering and substantial societal and economic costs. Whether immune mechanisms determine recovery after surgical trauma remains ill-defined. Single-cell mass cytometry was applied to serial whole-blood samples from 32 patients undergoing hip replacement to comprehensively characterize the phenotypic and functional immune response to surgical trauma. The simultaneous analysis of 14,000 phosphorylation events in precisely phenotyped immune cell subsets revealed uniform signaling responses among patients, demarcating a surgical immune signature. When regressed against clinical parameters of surgical recovery, including functional impairment and pain, strong correlations were found with STAT3 (signal transducer and activator of transcription), CREB (adenosine 3',5'-monophosphate response element-binding protein), and NF-κB (nuclear factor κB) signaling responses in subsets of CD14(+) monocytes (R = 0.7 to 0.8, false discovery rate <0.01). These sentinel results demonstrate the capacity of mass cytometry to survey the human immune system in a relevant clinical context. The mechanistically derived immune correlates point to diagnostic signatures, and potential therapeutic targets, that could postoperatively improve patient recovery.

     DOI 10.1126/scitranslmed.3009701

     PubMed 25253674

139. Single-cell mass cytometry analysis of human tonsil T cell remodeling by varicella zoster virus. Cell reports Sen, N., Mukherjee, G., Sen, A., Bendall, S. C., Sung, P., Nolan, G. P., Arvin, A. M. 2014; 8 (2): 633-645

     Although pathogens must infect differentiated host cells that exhibit substantial diversity, documenting the consequences of infection against this heterogeneity is challenging. Single-cell mass cytometry permits deep profiling based on combinatorial expression of surface and intracellular proteins. We used this method to investigate varicella-zoster virus (VZV) infection of tonsil T cells, which mediate viral transport to skin. Our results indicate that VZV induces a continuum of changes regardless of basal phenotypic and functional T cell characteristics. Contrary to the premise that VZV selectively infects T cells with skin trafficking profiles, VZV infection altered T cell surface proteins to enhance or induce these properties. Zap70 and Akt signaling pathways that trigger such surface changes were activated in VZV-infected naive and memory cells by a T cell receptor (TCR)-independent process. Single-cell mass cytometry is likely to be broadly relevant for demonstrating how intracellular pathogens modulate differentiated cells to support pathogenesis in the natural host.

     DOI 10.1016/j.celrep.2014.06.024

     PubMed 25043183

     PubMedCentralID PMC4127309

140. Single-Cell Mass Cytometry Analysis of Human Tonsil T Cell Remodeling by Varicella Zoster Virus CELL REPORTS Sen, N., Mukherjee, G., Sen, A., Bendall, S. C., Sung, P., Nolan, G. P., Arvin, A. M. 2014; 8 (2): 632-644

     Although pathogens must infect differentiated host cells that exhibit substantial diversity, documenting the consequences of infection against this heterogeneity is challenging. Single-cell mass cytometry permits deep profiling based on combinatorial expression of surface and intracellular proteins. We used this method to investigate varicella-zoster virus (VZV) infection of tonsil T cells, which mediate viral transport to skin. Our results indicate that VZV induces a continuum of changes regardless of basal phenotypic and functional T cell characteristics. Contrary to the premise that VZV selectively infects T cells with skin trafficking profiles, VZV infection altered T cell surface proteins to enhance or induce these properties. Zap70 and Akt signaling pathways that trigger such surface changes were activated in VZV-infected naive and memory cells by a T cell receptor (TCR)-independent process. Single-cell mass cytometry is likely to be broadly relevant for demonstrating how intracellular pathogens modulate differentiated cells to support pathogenesis in the natural host.

     DOI 10.1016/j.celrep.2014.06.024

     Web of Science ID 000341569800030

     PubMedCentralID PMC4127309

141. The split-virus influenza vaccine activates Fc gamma receptors instead of Toll-like receptors O'Gorman, W., Huang, H., Wei, Y., Davis, K., Leipold, M., Bendall, S., Kidd, B., Dekker, C., Maecker, H., Chien, Y., Davis, M. AMER ASSOC IMMUNOLOGISTS. 2014

     Web of Science ID 000209765002128

142. Antigen-Dependent Integration of Opposing Proximal TCR-Signaling Cascades Determines the Functional Fate of T Lymphocytes JOURNAL OF IMMUNOLOGY Wolchinsky, R., Hod-Marco, M., Oved, K., Shen-Orr, S. S., Bendall, S. C., Nolan, G. P., Reiter, Y. 2014; 192 (5): 2109-2119

     T cell anergy is a key tolerance mechanism to mitigate unwanted T cell activation against self by rendering lymphocytes functionally inactive following Ag encounter. Ag plays an important role in anergy induction where high supraoptimal doses lead to the unresponsive phenotype. How T cells "measure" Ag dose and how this determines functional output to a given antigenic dose remain unclear. Using multiparametric phospho-flow and mass cytometry, we measured the intracellular phosphorylation-dependent signaling events at a single-cell resolution and studied the phosphorylation levels of key proximal human TCR activation- and inhibition-signaling molecules. We show that the intracellular balance and signal integration between these opposing signaling cascades serve as the molecular switch gauging Ag dose. An Ag density of 100 peptide-MHC complexes/cell was found to be the transition point between dominant activation and inhibition cascades, whereas higher Ag doses induced an anergic functional state. Finally, the neutralization of key inhibitory molecules reversed T cell unresponsiveness and enabled maximal T cell functions, even in the presence of very high Ag doses. This mechanism permits T cells to make integrated "measurements" of Ag dose that determine subsequent functional outcomes.

     DOI 10.4049/jimmunol.1301142

     Web of Science ID 000332701400015

     PubMed 24489091

143. viSNE enables visualization of high dimensional single-cell data and reveals phenotypic heterogeneity of leukemia. Nature biotechnology Amir, E. D., Davis, K. L., Tadmor, M. D., Simonds, E. F., Levine, J. H., Bendall, S. C., Shenfeld, D. K., Krishnaswamy, S., Nolan, G. P., Pe'er, D. 2013; 31 (6): 545-552

     New high-dimensional, single-cell technologies offer unprecedented resolution in the analysis of heterogeneous tissues. However, because these technologies can measure dozens of parameters simultaneously in individual cells, data interpretation can be challenging. Here we present viSNE, a tool that allows one to map high-dimensional cytometry data onto two dimensions, yet conserve the high-dimensional structure of the data. viSNE plots individual cells in a visual similar to a scatter plot, while using all pairwise distances in high dimension to determine each cell's location in the plot. We integrated mass cytometry with viSNE to map healthy and cancerous bone marrow samples. Healthy bone marrow automatically maps into a consistent shape, whereas leukemia samples map into malformed shapes that are distinct from healthy bone marrow and from each other. We also use viSNE and mass cytometry to compare leukemia diagnosis and relapse samples, and to identify a rare leukemia population reminiscent of minimal residual disease. viSNE can be applied to any multi-dimensional single-cell technology.

     DOI 10.1038/nbt.2594

     PubMed 23685480

144. The transcriptional landscape of aß T cell differentiation. Nature immunology Mingueneau, M., Kreslavsky, T., Gray, D., Heng, T., Cruse, R., Ericson, J., Bendall, S., Spitzer, M. H., Nolan, G. P., Kobayashi, K., Von Boehmer, H., Mathis, D., Benoist, C., Best, A. J., Knell, J., Goldrath, A., Jojic, V., Koller, D., Shay, T., Regev, A., Cohen, N., Brennan, P., Brenner, M., Kim, F., Rao, T. N., Wagers, A., Heng, T., Ericson, J., Rothamel, K., Ortiz-Lopez, A., Mathis, D., Benoist, C., Bezman, N. A., Sun, J. C., Min-Oo, G., Kim, C. C., Lanier, L. L., Miller, J., Brown, B., Merad, M., Gautier, E. L., Jakubzick, C., Randolph, G. J., Monach, P., Blair, D. A., Dustin, M. L., Shinton, S. A., Hardy, R. R., Laidlaw, D., Collins, J., Gazit, R., Rossi, D. J., Malhotra, N., Sylvia, K., Kang, J., Kreslavsky, T., Fletcher, A., Elpek, K., Bellemare-Pelletier, A., Malhotra, D., Turley, S. 2013; 14 (6): 619-632

     The differentiation of αβT cells from thymic precursors is a complex process essential for adaptive immunity. Here we exploited the breadth of expression data sets from the Immunological Genome Project to analyze how the differentiation of thymic precursors gives rise to mature T cell transcriptomes. We found that early T cell commitment was driven by unexpectedly gradual changes. In contrast, transit through the CD4(+)CD8(+) stage involved a global shutdown of housekeeping genes that is rare among cells of the immune system and correlated tightly with expression of the transcription factor c-Myc. Selection driven by major histocompatibility complex (MHC) molecules promoted a large-scale transcriptional reactivation. We identified distinct signatures that marked cells destined for positive selection versus apoptotic deletion. Differences in the expression of unexpectedly few genes accompanied commitment to the CD4(+) or CD8(+) lineage, a similarity that carried through to peripheral T cells and their activation, demonstrated by mass cytometry phosphoproteomics. The transcripts newly identified as encoding candidate mediators of key transitions help define the 'known unknowns' of thymocyte differentiation.

     DOI 10.1038/ni.2590

     PubMed 23644507

145. Normalization of mass cytometry data with bead standards. Cytometry. Part A : the journal of the International Society for Analytical Cytology Finck, R., Simonds, E. F., Jager, A., Krishnaswamy, S., Sachs, K., Fantl, W., Pe'er, D., Nolan, G. P., Bendall, S. C. 2013; 83 (5): 483-494

     Mass cytometry uses atomic mass spectrometry combined with isotopically pure reporter elements to currently measure as many as 40 parameters per single cell. As with any quantitative technology, there is a fundamental need for quality assurance and normalization protocols. In the case of mass cytometry, the signal variation over time due to changes in instrument performance combined with intervals between scheduled maintenance must be accounted for and then normalized. Here, samples were mixed with polystyrene beads embedded with metal lanthanides, allowing monitoring of mass cytometry instrument performance over multiple days of data acquisition. The protocol described here includes simultaneous measurements of beads and cells on the mass cytometer, subsequent extraction of the bead-based signature, and the application of an algorithm enabling correction of both short- and long-term signal fluctuations. The variation in the intensity of the beads that remains after normalization may also be used to determine data quality. Application of the algorithm to a one-month longitudinal analysis of a human peripheral blood sample reduced the range of median signal fluctuation from 4.9-fold to 1.3-fold.

     DOI 10.1002/cyto.a.22271

     PubMed 23512433

146. Single Cell Mass Cytometry of Dysregulated Signaling Networks in Myeloproliferative Neoplasms and Secondary Acute Myeloid Leukemia 54th Annual Meeting and Exposition of the American-Society-of-Hematology (ASH) Fisher, D. A., Simonds, E. F., Behbehani, G. K., Nolan, G. P., Bendall, S. C., Oh, S. T. AMER SOC HEMATOLOGY. 2012

     Web of Science ID 000313838900254

147. MASS CYTOMETRY TO COMPREHENSIVELY STUDY SINGLE CELL SIGNALING IN BIOLOGY AND DISEASE 12th Euroconference on Clinical Cell Analysis / 8th European Clinical Cytometry Course Bodenmiller, B., Zunder, E., Finck, R., Chen, T., Savig, E., Bruggner, R., Simonds, E., Bendall, S., Sachs, K., Krutzik, P., Nolan, G. WILEY-BLACKWELL. 2012: 376–77

     Web of Science ID 000310386300014

148. Multiplexed mass cytometry profiling of cellular states perturbed by small-molecule regulators NATURE BIOTECHNOLOGY Bodenmiller, B., Zunder, E. R., Finck, R., Chen, T. J., Savig, E. S., Bruggner, R. V., Simonds, E. F., Bendall, S. C., Sachs, K., Krutzik, P. O., Nolan, G. P. 2012; 30 (9): 858-U89

     Mass cytometry facilitates high-dimensional, quantitative analysis of the effects of bioactive molecules on human samples at single-cell resolution, but instruments process only one sample at a time. Here we describe mass-tag cellular barcoding (MCB), which increases mass cytometry throughput by using n metal ion tags to multiplex up to 2n samples. We used seven tags to multiplex an entire 96-well plate, and applied MCB to characterize human peripheral blood mononuclear cell (PBMC) signaling dynamics and cell-to-cell communication, signaling variability between PBMCs from eight human donors, and the effects of 27 inhibitors on this system. For each inhibitor, we measured 14 phosphorylation sites in 14 PBMC types at 96 conditions, resulting in 18,816 quantified phosphorylation levels from each multiplexed sample. This high-dimensional, systems-level inquiry allowed analysis across cell-type and signaling space, reclassified inhibitors and revealed off-target effects. High-content, high-throughput screening with MCB should be useful for drug discovery, preclinical testing and mechanistic investigation of human disease.

     DOI 10.1038/nbt.2317

     Web of Science ID 000308705700020

     PubMed 22902532

     PubMedCentralID PMC3627543

149. Single-cell mass cytometry adapted to measurements of the cell cycle. Cytometry. Part A : the journal of the International Society for Analytical Cytology Behbehani, G. K., Bendall, S. C., Clutter, M. R., Fantl, W. J., Nolan, G. P. 2012; 81 (7): 552-566

     Mass cytometry is a recently introduced technology that utilizes transition element isotope-tagged antibodies for protein detection on a single-cell basis. By circumventing the limitations of emission spectral overlap associated with fluorochromes utilized in traditional flow cytometry, mass cytometry currently allows measurement of up to 40 parameters per cell. Recently, a comprehensive mass cytometry analysis was described for the hematopoietic differentiation program in human bone marrow from a healthy donor. The current study describes approaches to delineate cell cycle stages utilizing 5-iodo-2-deoxyuridine (IdU) to mark cells in S phase, simultaneously with antibodies against cyclin B1, cyclin A, and phosphorylated histone H3 (S28) that characterize the other cell cycle phases. Protocols were developed in which an antibody against phosphorylated retinoblastoma protein (Rb) at serines 807 and 811 was used to separate cells in G0 and G1 phases of the cell cycle. This mass cytometry method yielded cell cycle distributions of both normal and cancer cell populations that were equivalent to those obtained by traditional fluorescence cytometry techniques. We applied this to map the cell cycle phases of cells spanning the hematopoietic hierarchy in healthy human bone marrow as a prelude to later studies with cancers and other disorders of this lineage.

     DOI 10.1002/cyto.a.22075

     PubMed 22693166

     PubMedCentralID PMC3667754

150. Single-cell mass cytometry adapted to measurements of the cell cycle CYTOMETRY PART A Behbehani, G. K., Bendall, S. C., Clutter, M. R., Fantl, W. J., Nolan, G. P. 2012; 81A (7): 552-566

     Mass cytometry is a recently introduced technology that utilizes transition element isotope-tagged antibodies for protein detection on a single-cell basis. By circumventing the limitations of emission spectral overlap associated with fluorochromes utilized in traditional flow cytometry, mass cytometry currently allows measurement of up to 40 parameters per cell. Recently, a comprehensive mass cytometry analysis was described for the hematopoietic differentiation program in human bone marrow from a healthy donor. The current study describes approaches to delineate cell cycle stages utilizing 5-iodo-2-deoxyuridine (IdU) to mark cells in S phase, simultaneously with antibodies against cyclin B1, cyclin A, and phosphorylated histone H3 (S28) that characterize the other cell cycle phases. Protocols were developed in which an antibody against phosphorylated retinoblastoma protein (Rb) at serines 807 and 811 was used to separate cells in G0 and G1 phases of the cell cycle. This mass cytometry method yielded cell cycle distributions of both normal and cancer cell populations that were equivalent to those obtained by traditional fluorescence cytometry techniques. We applied this to map the cell cycle phases of cells spanning the hematopoietic hierarchy in healthy human bone marrow as a prelude to later studies with cancers and other disorders of this lineage.

     DOI 10.1002/cyto.a.22075

     Web of Science ID 000305558700006

     PubMedCentralID PMC3667754

151. From single cells to deep phenotypes in cancer NATURE BIOTECHNOLOGY Bendall, S. C., Nolan, G. P. 2012; 30 (7): 639-647

     In recent years, major advances in single-cell measurement systems have included the introduction of high-throughput versions of traditional flow cytometry that are now capable of measuring intracellular network activity, the emergence of isotope labels that can enable the tracking of a greater variety of cell markers and the development of super-resolution microscopy techniques that allow measurement of RNA expression in single living cells. These technologies will facilitate our capacity to catalog and bring order to the inherent diversity present in cancer cell populations. Alongside these developments, new computational approaches that mine deep data sets are facilitating the visualization of the shape of the data and enabling the extraction of meaningful outputs. These applications have the potential to reveal new insights into cancer biology at the intersections of stem cell function, tumor-initiating cells and multilineage tumor development. In the clinic, they may also prove important not only in the development of new diagnostic modalities but also in understanding how the emergence of tumor cell clones harboring different sets of mutations predispose patients to relapse or disease progression.

     DOI 10.1038/nbt.2283

     Web of Science ID 000306293400023

     PubMed 22781693

152. A deep profiler's guide to cytometry TRENDS IN IMMUNOLOGY Bendall, S. C., Nolan, G. P., Roederer, M., Chattopadhyay, P. K. 2012; 33 (7): 323-332

     In recent years, advances in technology have provided us with tools to quantify the expression of multiple genes in individual cells. The ability to measure simultaneously multiple genes in the same cell is necessary to resolve the great diversity of cell subsets, as well as to define their function in the host. Fluorescence-based flow cytometry is the benchmark for this; with it, we can quantify 18 proteins per cell, at >10 000 cells/s. Mass cytometry is a new technology that promises to extend these capabilities significantly. Immunophenotyping by mass spectrometry provides the ability to measure >36 proteins at a rate of 1000 cells/s. We review these cytometric technologies, capable of high-content, high-throughput single-cell assays.

     DOI 10.1016/j.it.2012.02.010

     Web of Science ID 000306639500001

     PubMed 22476049

     PubMedCentralID PMC3383392

153. Cytometry by Time-of-Flight Shows Combinatorial Cytokine Expression and Virus-Specific Cell Niches within a Continuum of CD8(+) T Cell Phenotypes IMMUNITY Newell, E. W., Sigal, N., Bendall, S. C., Nolan, G. P., Davis, M. M. 2012; 36 (1): 142-152

     Cytotoxic CD8(+) T lymphocytes directly kill infected or aberrant cells and secrete proinflammatory cytokines. By using metal-labeled probes and mass spectrometric analysis (cytometry by time-of-flight, or CyTOF) of human CD8(+) T cells, we analyzed the expression of many more proteins than previously possible with fluorescent labels, including surface markers, cytokines, and antigen specificity with modified peptide-MHC tetramers. With 3-dimensional principal component analysis (3D-PCA) to display phenotypic diversity, we observed a relatively uniform pattern of variation in all subjects tested, highlighting the interrelatedness of previously described subsets and the continuous nature of CD8(+) T cell differentiation. These data also showed much greater complexity in the CD8(+) T cell compartment than previously appreciated, including a nearly combinatorial pattern of cytokine expression, with distinct niches occupied by virus-specific cells. This large degree of functional diversity even between cells with the same specificity gives CD8(+) T cells a remarkable degree of flexibility in responding to pathogens.

     DOI 10.1016/j.immuni.2012.01.002

     Web of Science ID 000299766000017

     PubMed 22265676

154. Signaling and Immunophenotypic Diversity in Pediatric Acute Myeloid Leukemia As Defined by 31-Parameter Single-Cell Mass Cytometry 53rd Annual Meeting and Exposition of the American-Society-of-Hematology (ASH) Simonds, E. F., Bendall, S. C., Davis, K. L., Fienberg, H. G., Finck, R. L., Amir, E. D., Radtke, I., Downing, J. R., Pe'er, D., Nolan, G. P. AMER SOC HEMATOLOGY. 2011: 1100–1101

     Web of Science ID 000299597103431

155. Extracting a cellular hierarchy from high-dimensional cytometry data with SPADE NATURE BIOTECHNOLOGY Qiu, P., Simonds, E. F., Bendall, S. C., Gibbs, K. D., Bruggner, R. V., Linderman, M. D., Sachs, K., Nolan, G. P., Plevritis, S. K. 2011; 29 (10): 886-U181

     The ability to analyze multiple single-cell parameters is critical for understanding cellular heterogeneity. Despite recent advances in measurement technology, methods for analyzing high-dimensional single-cell data are often subjective, labor intensive and require prior knowledge of the biological system. To objectively uncover cellular heterogeneity from single-cell measurements, we present a versatile computational approach, spanning-tree progression analysis of density-normalized events (SPADE). We applied SPADE to flow cytometry data of mouse bone marrow and to mass cytometry data of human bone marrow. In both cases, SPADE organized cells in a hierarchy of related phenotypes that partially recapitulated well-described patterns of hematopoiesis. We demonstrate that SPADE is robust to measurement noise and to the choice of cellular markers. SPADE facilitates the analysis of cellular heterogeneity, the identification of cell types and comparison of functional markers in response to perturbations.

     DOI 10.1038/nbt.1991

     PubMed 21964415

156. Clonal tracking of hESCs reveals differential contribution to functional assays NATURE METHODS Stewart, M. H., Bendall, S. C., Levadoux-Martin, M., Bhatia, M. 2010; 7 (11): 917-U75

     Human embryonic stem cells (hESCs) have unique self-renewal and differentiation properties, which are experimentally measured using functional assays. hESC cultures are known to be heterogeneous, but whether subsets of cells contribute differently to functional assays has yet to be examined. Here, using clonal tracking by retroviral integration, we analyzed in situ the propensity of individual hESCs to contribute to different functional assays. We observed different clonal distributions in teratomas versus in vitro differentiation assays. Some hESC subsets apparently contributed substantially to lineage-specific embryoid body differentiation and lacked clonogenic capacity, although they had self-renewal ability. In contrast, other subsets of self-renewing hESCs with clonogenic ability contributed to teratoma formation but were less frequently observed after in vitro differentiation. Our study suggests that assays used to measure pluripotency may detect distinct subsets of hESCs. These findings have direct implications for hESC-based therapies that may be optimized based on such functional assays.

     DOI 10.1038/nmeth.1519

     Web of Science ID 000283586600014

     PubMed 20953174

157. A HUPO test sample study reveals common problems in mass spectrometry-based proteomics NATURE METHODS Bell, A. W., Deutsch, E. W., Au, C. E., Kearney, R. E., Beavis, R., Sechi, S., Nilsson, T., Bergeron, J. J. 2009; 6 (6): 423-U40

     We performed a test sample study to try to identify errors leading to irreproducibility, including incompleteness of peptide sampling, in liquid chromatography-mass spectrometry-based proteomics. We distributed an equimolar test sample, comprising 20 highly purified recombinant human proteins, to 27 laboratories. Each protein contained one or more unique tryptic peptides of 1,250 Da to test for ion selection and sampling in the mass spectrometer. Of the 27 labs, members of only 7 labs initially reported all 20 proteins correctly, and members of only 1 lab reported all tryptic peptides of 1,250 Da. Centralized analysis of the raw data, however, revealed that all 20 proteins and most of the 1,250 Da peptides had been detected in all 27 labs. Our centralized analysis determined missed identifications (false negatives), environmental contamination, database matching and curation of protein identifications as sources of problems. Improved search engines and databases are needed for mass spectrometry-based proteomics.

     DOI 10.1038/NMETH.1333

     Web of Science ID 000266493300012

     PubMed 19448641

158. An Enhanced Mass Spectrometry Approach Reveals Human Embryonic Stem Cell Growth Factors in Culture MOLECULAR & CELLULAR PROTEOMICS Bendall, S. C., Hughes, C., Campbell, J. L., Stewart, M. H., Pittock, P., Liu, S., Bonneil, E., Thibault, P., Bhatia, M., Lajoie, G. A. 2009; 8 (3): 421-432

     The derivation and long-term maintenance of human embryonic stem cells (hESCs) has been established in culture formats that are both dependent and independent of support (feeder) cells. However, the factors responsible for preserving the viability of hESCs in a nascent state remain unknown. We describe a mass spectrometry-based method for probing the secretome of the hESC culture microenvironment to identify potential regulating protein factors that are in low abundance. Individual samples were analyzed several times, using successive mass (m/z) and retention time-directed exclusion, without sampling the same peptide ion twice. This iterative exclusion -mass spectrometry (IE-MS) approach more than doubled protein and peptide metrics in comparison to a simple repeat analysis method on the same instrument, even after extensive sample pre-fractionation. Furthermore, implementation of the IE-MS approach was shown to enhance the performance of an older quadrupole time of flight (Q-ToF) MS. The resulting number of identified peptides approached that of a parallel repeat analysis on a newer LTQ-Orbitrap MS. The combination of the results of both instruments proved to be superior to that achieved by a single instrument in the identification of additional proteins. Using the IE-MS strategy, combined with complementary gel- and solution-based fractionation methods, the hESC culture microenvironment was extensively probed. Over 10 to 12 times more extracellular proteins were observed compared with previously published surveys. The detection of previously undetectable growth factors, present at concentrations ranging from 10(-9) to 10(-11) g/ml, highlights the depth of our profiling. The IE-MS approach provides a simple and reliable technique that greatly enhances instrument performance by increasing the effective depth of MS-based proteomic profiling. This approach should be widely applicable to any LC-MS/MS instrument platform or biological system.

     DOI 10.1074/mcp.M800190-MCP200

     Web of Science ID 000264240000003

     PubMed 18936058

159. Prevention of amino acid conversion in SILAC experiments with embryonic stem cells MOLECULAR & CELLULAR PROTEOMICS Bendall, S. C., Hughes, C., Stewart, M. H., Doble, B., Bhatia, M., Lajoie, G. A. 2008; 7 (9): 1587-1597

     Recent studies using stable isotope labeling with amino acids in culture (SILAC) in quantitative proteomics have made mention of the problematic conversion of isotope-coded arginine to proline in cells. The resulting converted proline peptide divides the heavy peptide ion signal causing inaccuracy when compared with the light peptide ion signal. This is of particular concern as it can effect up to half of all peptides in a proteomic experiment. Strategies to both compensate for and limit the inadvertent conversion have been demonstrated, but none have been shown to prevent it. Additionally, these methods combined with SILAC labeling in general have proven problematic in their large scale application to sensitive cell types including embryonic stem cells (ESCs) from the mouse and human. Here, we show that by providing as little as 200 mg/liter L-proline in SILAC media, the conversion of arginine to proline can be rendered completely undetectable. At the same time, there was no compromise in labeling with isotope-coded arginine, indicating there is no observable back conversion from the proline supplement. As a result, when supplemented with proline, correct interpretation of "light" and "heavy" peptide ratios could be achieved even in the worst cases of conversion. By extending these principles to ESC culture protocols and reagents we were able to routinely SILAC label both mouse and human ESCs in the absence of feeder cells and without compromising the pluripotent phenotype. This study provides the simplest protocol to prevent proline artifacts in SILAC labeling experiments with arginine. Moreover, it presents a robust, feeder cell-free, protocol for performing SILAC experiments on ESCs from both the mouse and the human.

     DOI 10.1074/mcp.M800113-MCP200

     Web of Science ID 000259154800001

     PubMed 18487603

160. Deconstructing human embryonic stem cell cultures: niche regulation of self-renewal and pluripotency JOURNAL OF MOLECULAR MEDICINE-JMM Stewart, M. H., Bendall, S. C., Bhatia, M. 2008; 86 (8): 875-886

     The factors and signaling pathways controlling pluripotent human cell properties, both embryonic and induced, have not been fully investigated. Failure to account for functional heterogeneity within human embryonic stem cell (hESC) cultures has led to inconclusive results in previous work examining extrinsic influences governing hESC fate (self renewal vs. differentiation vs. death). Here, we attempt to reconcile these inconsistencies with recent reports demonstrating that an autologously produced in vitro niche regulates hESCs. Moreover, we focus on the reciprocal paracrine signals within the in vitro hESC niche allowing for the maintenance and/or expansion of the hESC colony-initiating cell (CIC). Based on this, it is clear that separation of hESC-CICs, apart from their differentiated derivatives, will be essential in future studies involving their molecular regulation. Understanding how extrinsic factors control hESC self-renewal and differentiation will allow us to culture and differentiate these pluripotent cells with higher efficiency. This knowledge will be essential for clinical applications using human pluripotent cells in regenerative medicine.

     DOI 10.1007/s00109-008-0356-9

     Web of Science ID 000257945800003

     PubMed 18521556

161. Human embryonic stem cells: lessons from stem cell niches in vivo REGENERATIVE MEDICINE Bendall, S. C., Stewart, M. H., Bhatia, M. 2008; 3 (3): 365-376

     In vivo the stem cell niche is an essential component in controlling and maintaining the stem cells' ability to survive and respond to injury. Human embryonic stem cells (hESCs) appear to be an exception to this rule as they can be removed from their blastocytic microenvironment and maintained indefinitely in vitro. However, recent observations reveal the existence of an autonomously derived in vitro hESC niche. This provides a previously unappreciated mechanism to control hESC expansion and differentiation. Recognizing this, it may now be possible to take aspects of in vivo stem cell niches, namely extracellular matrices, paracrine signals and accessory cell types, and exploit them in order to gain fidelity in directed hESC differentiation. In doing so, routine customization of hESC lines and their application in regenerative therapies may be further enhanced using unique hESC niche-based approaches.

     DOI 10.2217/17460751.3.3.365

     Web of Science ID 000257995200020

     PubMed 18462059

162. IGF and FGF cooperatively establish the regulatory stem cell niche of pluripotent human cells in vitro NATURE Bendall, S. C., Stewart, M. H., Menendez, P., George, D., Vijayaragavan, K., Werbowetski-Ogilvie, T., Ramos-Mejia, V., Rouleau, A., Yang, J., Bosse, M., Lajoie, G., Bhatia, M. 2007; 448 (7157): 1015-U3

     Distinctive properties of stem cells are not autonomously achieved, and recent evidence points to a level of external control from the microenvironment. Here, we demonstrate that self-renewal and pluripotent properties of human embryonic stem (ES) cells depend on a dynamic interplay between human ES cells and autologously derived human ES cell fibroblast-like cells (hdFs). Human ES cells and hdFs are uniquely defined by insulin-like growth factor (IGF)- and fibroblast growth factor (FGF)-dependence. IGF 1 receptor (IGF1R) expression was exclusive to the human ES cells, whereas FGF receptor 1 (FGFR1) expression was restricted to surrounding hdFs. Blocking the IGF-II/IGF1R pathway reduced survival and clonogenicity of human ES cells, whereas inhibition of the FGF pathway indirectly caused differentiation. IGF-II is expressed by hdFs in response to FGF, and alone was sufficient in maintaining human ES cell cultures. Our study demonstrates a direct role of the IGF-II/IGF1R axis on human ES cell physiology and establishes that hdFs produced by human ES cells themselves define the stem cell niche of pluripotent human stem cells.

     DOI 10.1038/nature06027

     Web of Science ID 000249097600030

     PubMed 17625568

163. Proteomic analysis of pluripotent stem cells. Current protocols in stem cell biology Bendall, S. C., Booy, A. T., Lajoie, G. 2007; Chapter 1: Unit 1B 1-?

     Mass spectrometry (MS)-based proteomics has become one of the most powerful tools for identifying expressed proteins, providing quick insights into molecular and cellular biology. Traditionally, proteins isolated by either one- or two-dimensional gel electrophoresis are digested with a site specific protease. The resulting peptides are subject to one of two forms of analysis: (1) matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS, where a "mass fingerprint" of all the peptides in a sample is generated, or (2) electrospray ionization tandem MS (ESI-MS/MS), where a mass fragmentation spectra is generated for each peptide in a sample. The resulting mass information is then compared to that of a theoretical database created with available genomic sequence information. This unit provides protocols for this type of assessment in embryonic stem cells (ESCs).

     DOI 10.1002/9780470151808.sc01b01s2

     PubMed 18785159

164. Clonal isolation of hESCs reveals heterogeneity within the pluripotent stem cell compartment NATURE METHODS Stewart, M. H., Bosse, M., Chadwick, K., Menendez, P., Bendall, S. C., Bhatia, M. 2006; 3 (10): 807-815

     Human embryonic stem cell (hESC) lines are known to be morphologically and phenotypically heterogeneous. The functional nature and relationship of cells residing within hESC cultures, however, has not been evaluated because isolation of single hESCs is limited to drug or manual selection. Here we provide a quantitative method using flow cytometry to isolate and clonally expand hESCs based on undifferentiated markers, alone or in combination with a fluorescent reporter. This method allowed for isolation of stage-specific embryonic antigen-3-positive (SSEA-3+) and SSEA-3- cells from hESC cultures. Although both SSEA-3+ and SSEA-3- cells could initiate pluripotent hESC cultures, we show that they possess distinct cell-cycle properties, clonogenic capacity and expression of ESC transcription factors. Our study provides formal evidence for heterogeneity among self-renewing pluripotent hESCs, illustrating that this isolation technique will be instrumental in further dissecting the biology of hESC lines.

     DOI 10.1038/nmeth939

     Web of Science ID 000240942600015

     PubMed 16990813

165. Complement targeting of nonhuman sialic acid does not mediate cell death of human embryonic stem cells NATURE MEDICINE Cerdan, C., Bendall, S. C., Wang, L., Stewart, M., Werbowetski, T., Bhatia, M. 2006; 12 (10): 1113-1114

     DOI 10.1038/nm1006-1113

     Web of Science ID 000241102200009

     PubMed 17024197

166. Multi-target drug combinations from single drug responses measured at the level of single cells using Mixture Nested Effects Models (MNEMs) applied to cancer. Special Conference on Computational and Systems Biology of Cancer Anchang, B., Fienberg, H., et al 2015

     DOI 10.1158/1538-7445.COMPSYSBIO-B1-39

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