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Routine examination of entire histological slides at cellular resolution poses a significant if not insurmountable challenge to human observers. However, high-resolution data such as the cellular distribution of proteins in tissues, e.g., those obtained following immunochemical staining, are highly desirable. Our present study extends the applicability of the PathoFusion framework to the cellular level. We illustrate our approach using the detection of CD276 immunoreactive cells in glioblastoma as an example. Following automatic identification by means of PathoFusion’s bifocal convolutional neural network (BCNN) model, individual cells are automatically profiled and counted. Only discriminable cells selected through data filtering and thresholding were segmented for cell-level analysis. Subsequently, we converted the detection signals into the corresponding heatmaps visualizing the distribution of the detected cells in entire whole-slide images of adjacent H&E-stained sections using the Discrete Wavelet Transform (DWT). Our results demonstrate that PathoFusion is capable of autonomously detecting and counting individual immunochemically labelled cells with a high prediction performance of 0.992 AUC and 97.7% accuracy. The data can be used for whole-slide cross-modality analyses, e.g., relationships between immunochemical signals and anaplastic histological features. PathoFusion has the potential to be applied to additional problems that seek to correlate heterogeneous data streams and to serve as a clinically applicable, weakly supervised system for histological image analyses in (neuro)pathology.

Brain neoplasms represent a complex form of cancer that is one of the most challenging to classify and treat. Over 120 different tumor subtypes originate from various parts of the central nervous system, which makes identifying the composition of the tumor microenvironment (TME) vital for early assessment of progression, treatment, and prevention. We developed high-plex proteomic analysis tools to thoroughly characterize the TME of both human and mouse brain tissues using Imaging Mass Cytometry™ (IMC™). IMC offers unprecedented insight into the TME by uncovering the spatial distribution of 40-plus distinct molecular markers without autofluorescence, facilitating the research of brain neoplasms. Here, we demonstrate the application of high-plex human and mouse neuro-oncology IMC panels on normal and tumor formalin-fixed paraffin-embedded brain tissues. A basic neurophenotyping panel was developed and used to customize the Maxpar® Human and Maxpar OnDemand™ Mouse Immuno-Oncology IMC Panel Kits. Human and mouse neuro-oncology panels provide deep phenotyping and characterization of brain TME composition. These neuro panels consist of cross-reactive clones and enable flexible panel design for brain-specific research goals, such as brain tumor classification, and assessment of neuronal inflammation, degeneration, and development. We applied the neuro-oncology panels on tissue microarrays (TMAs) containing a variety of human brain tumors and mouse glioblastoma and neuroblastoma tissues. Normal brain tissues were used for comparative analysis as controls. The Hyperion™ Imaging System was utilized to digitize images from the tissues followed by quantitative analysis to assess the cellular composition of normal and cancerous brain TME. We successfully identified major cell populations that make up human and mouse brain matter, such as neurons, astrocytes, microglia, and oligodendrocytes. Various tumor cell phenotypes, resident and infiltrating cells, and resting and activated microglia were detected in multiple tumor subtypes. Subsequent single-cell analysis provided a comprehensive and quantitative assessment of the brain TME in our samples. We classified the distinct states of neurons and quantified myeloid and lymphoid immune cell infiltration across normal, astrocytoma, and glioblastoma tissues. Empowered by high-plex neuro-oncology panels, IMC can accelerate brain tumor research and provide insights into the spatial complexity of neuronal neoplasms. Citation Format: Nick Zabinyakov, Qanber Raza, Christina Loh. Neuro-oncology imaging mass cytometry panels enable spatial investigation of brain tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 676.

Mouse tumor models are widely utilized for cancer studies and preclinical drug development. An obstacle in predicting therapeutic drug efficacy is the ability to quantitatively evaluate the multi-parametric post-treatment response in the tumor microenvironment (TME). Identification of immuno-oncological processes that dictate tumor growth, metastasis, and immune response is essential for selecting promising drug candidates for further clinical evaluation. Imaging Mass Cytometry™ (IMC™) is a vital and proven high-plex imaging technology that enables deep characterization of the complexity and diversity of tumor tissue without disrupting spatial context. The Hyperion™ Imaging System utilizes IMC technology to simultaneously assess 40-plus individual structural and functional markers in tissues, providing unprecedented insight into the organization and function of the TME. We have previously demonstrated the application of IMC in combination with Maxpar® and Maxpar OnDemand™ antibodies to highlight cellular composition of normal mouse tissues. Here, we showcase the Maxpar OnDemand Mouse Immuno-Oncology IMC Panel Kit for application on a variety of mouse tumor tissues. We compiled the antibody panel to quantitatively assess IO-related processes and applied it to a tissue microarray (TMA) containing a large variety of mouse tumors. Antibodies in panel kits were selected from the Maxpar and Maxpar OnDemand catalogs. We digitized high-plex data from mouse tissues using the Hyperion Imaging System and generated images demonstrating the detailed layout of the TME. We further conducted single-cell analysis to identify specific populations of tumor and immune cells in the TME. The Mouse Immuno-Oncology IMC Panel Kit successfully identified pathophysiological processes such as immune cell infiltration and activation, signaling pathway activation, biomarkers of epithelial-to-mesenchymal transition (EMT), metabolic activity, growth, and the tissue architecture of the TME. Single-cell analysis of several highly relevant tumor types separated distinct cellular clusters representing tumor, immune, stromal, and vascular cells. Activation of cellular processes associated with signaling, growth, and metastasis were identified in tumor cells. In addition, cytotoxic and inflammatory activation in lymphoid and myeloid immune cell subtypes were detected. Application of IMC based multiparametric analysis successfully identified the spatial landscape of the TME at single-cell resolution. Quantitative analysis of tumor composition revealed critical insights regarding prognostic parameters such as metastatic and growth potential of tumors, and identification and activation of immune cell infiltrates. Overall, we demonstrate the power of IMC and provide evidence of its successful application in mouse tumor models. For Research Use Only. Not for use in diagnostic procedures. Citation Format: Qanber Raza, Liang Lim, Thomas D. Pfister, Nick Zabinyakov, Christina Loh. Identifying pathophysiological features of mouse tumors using imaging mass cytometry. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5640.

High-parameter flow cytometry is essential for human and mouse studies to discover novel immunological mechanisms of cancer, infections, and immunosenescence. It plays an increasingly important role in cancer research to ensure clinical therapeutic success but is limited by the large amount of cell samples needed for staining controls. CyTOF® technology has transformed flow cytometry by enabling 50-plus-marker analysis per tube of sample, with easy panel design and no need for single-stained or autofluorescence controls. Flow cytometry by CyTOF provides an efficient and unbiased approach to discovering novel subsets and unique functional states of immune cells, maximizing insights from precious samples. Immunosenescence perturbs lung cancer onset and development, yet the mechanisms remain largely unknown. To study the pulmonary immune populations in aged (75 weeks old) and young adult (6-8 weeks old) mice, we built a 50-parameter panel (2 for single live-cell identification, 6 for live-cell sample barcoding, and 42 for immune profiling). A core panel of 32 antibodies was selected from Standard BioTools™ catalogs to detect key cell lineage and functional surface markers. A complementary panel with 10 Maxpar® antibodies was added to further study functional cell states. Maxpar Pathsetter™ software was used to create an automated analytical model for high-dimensional analysis. The 50-parameter panel successfully identified over 30 lymphoid and myeloid cell subsets including but not limited to T cells, B cells, NK cells, alveolar macrophages (AMs), dendritic cells, and neutrophils. The panel enabled high-fidelity detection of over 15 functional markers mediating proliferation, activation, inhibition, migration, tissue residence, and cellular metabolism. Automated in-depth analysis by Maxpar Pathsetter efficiently identified many aging-associated alterations in cell frequencies and functional states such as the enrichment of PD-1+ T cells and CD27- γδ T cells that could potentially perturb anti-tumor immunity. Moreover, CyTOF technology is uniquely advanced in characterizing autofluorescent cells such as AMs. A pro-inflammatory state (higher expression of MHC-II, CD80, and PD-L1) was specifically defined for AMs in aged lungs. This study demonstrates comprehensive single-cell immune profiling of mouse tissues with the products and solutions provided by Standard BioTools. Both the 50-parameter panel and Maxpar Pathsetter analytical model can be customized for deep characterization of specific immune populations according to the requirements of various cancer studies in mice. By utilizing end-to-end solutions offered by Standard BioTools, flow cytometry by CyTOF can significantly facilitate the mechanistic studies of mouse models to expand the understanding of human cancers and accelerate therapeutic development. Citation Format: Wenxi Xu, Stephen Li, Alexandre Bouzekri, Lauren Tracey, Christina Loh. 50-parameter flow cytometry by CyTOF empowers comprehensive single-cell immune profiling of pulmonary immunosenescence in aged mice. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5142.

Imaging Mass Cytometry™ (IMC™) is the leading platform for high-plex tissue imaging. IMC allows for detailed assessment of cell phenotype and function using 40-plus markers simultaneously at subcellular resolution on a single slide. A comprehensive IMC panel containing structural, functional, and immune markers enables us to reveal the complex heterogeneity of tumor tissues as well as the tumor microenvironment (TME). Driven by an increase in the number of antibody markers and the addition of mRNA markers, there is an increasing demand for larger panels. In addition, increasing the number of investigated target markers on a single tissue enriches spatial characterization that may facilitate a more accurate prediction of disease progression and preclinical outcome measures in clinical research projects using tumor biopsies or tissue microarrays (TMAs). Therefore, to increase the plexity of IMC panels, it is essential to expand the number of available metal channels. Here, we demonstrate the incorporation of conjugated antibodies with yttrium (89Y) and indium (115In), two low-mass metals, for IMC application. These metal tags have been previously tested as putative channels for IMC application. We performed IMC analysis of various tissue types stained with panels of conjugated antibodies including the novel 89Y- and 115In-conjugated antibodies. At least 3 different regions of interest (ROIs) were assessed for each of the investigated tissue types. We compared images for the 89Y- and 115In-conjugated antibodies with the images generated using Maxpar® catalog antibodies of the same clones, with a focus on marker specificity and background signal. Compared with the lanthanide-conjugated catalog antibodies, the 89Y- and 115In-conjugated antibodies showed equivalent specificity and staining quality. Our results open a new avenue to assign markers to 89Y and 115In, which enables a larger list of potential targets to be investigated in any IMC study. Expanding the number of markers to 40-plus in Imaging Mass Cytometry will improve the imaging results necessary to identify novel cell signatures (phenotype and interactions) in the TME. Citation Format: Thomas D. Pfister, Shaida Ouladan, Huihui Yao, Daniel Majonis, Christina Loh, Nick Zabinyakov. Increasing plexity of imaging mass cytometry for tumor tissue analysis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 677.

Mass cytometry, or cytometry by time-of-flight (the basis for Fluidigm

Mass cytometry, or cytometry by time-of-flight (the basis for Fluidigm® CyTOF® technology), is a system for single-cell detection using antibodies tagged with metal probes. Without the need for compensation, the highly parametric Helios™ mass cytometer has a detection range of 135 distinct mass channels (75-209 Da). Optimized for mass cytometry, the Maxpar® Direct™ Immune Profiling Assay™ is a dry, metal-tagged antibody cocktail for immunophenotyping 37 immune cell populations found in human peripheral blood in a single tube. The Maxpar Direct Assay utilizes 31 mass channels for marker detection and live/dead viability staining, with at least 14 additional marker channels available from the Fluidigm catalog for flexible custom panel design. Here, we describe a workflow combining the assay with additional surface and intracellular cytokine antibodies for peripheral blood mononuclear cell (PBMC) staining using lanthanide-, bismuth-, and cadmium-tagged antibodies.

High-parameter immune profiling is crucial in translational and clinical research to quantify changes in immune cell populations over time. CyTOF® mass cytometry is a high-plex, single-cell analysis platform that uses isotopically pure metal-labeled antibodies. The major advantage of CyTOF is its ability to resolve 40-plus markers in a single panel without compensation, making mass cytometry ideal for routine immunophenotyping. The autosampler module of CyTOF XT™ provides significant time savings by allowing automated sample acquisition. Tubes of pelleted stained samples are loaded into the Autosampler carousel, and the samples are resuspended with EQ™ Calibration Beads for acquisition. User input is only required during instrument startup, tuning, and batch setup. The added automation of CyTOF XT provides a streamlined workflow for suspension mass cytometry. Testing was performed to ensure that the data obtained on CyTOF XT was comparable to manual acquisition systems. The performance of CyTOF XT was tested in parallel with its predecessor, Helios™. Several workflows and applications for suspension mass cytometry including sample barcoding with the Cell-ID™ 20-Plex Pd Barcoding Kit, and surface, cytoplasmic, and nuclear staining and phosphostaining were evaluated on human PBMC. Manual gating analysis was performed to assess population frequencies and median intensities for each marker. Resolution index was calculated to assess how well positive and negative populations separated from each other. There was no significant difference between population frequencies analyzed between the two CyTOF systems. Moreover, samples acquired on CyTOF XT, on average, resulted in greater signal resolution between positive and negative populations compared to Helios. The Maxpar Direct Immune Profiling System was also compared on CyTOF XT and Helios using human whole blood and PBMC. The Maxpar® Direct™ Immune Profiling Assay™ and Maxpar Pathsetter™ software were developed as a sample-to-answer system for human immune profiling using CyTOF. The Maxpar Direct Immune Profiling Assay includes an optimized panel of 30 unique markers in a dry, single-tube format. Maxpar Pathsetter is an automated software used to report population statistics, stain assessments, and relevant data plots for the panel. The automated staining assessment in Maxpar Pathsetter was compared between files acquired on CyTOF XT and Helios. Comparable population frequencies were obtained between the two acquisition systems, and improved staining assessment was observed on CyTOF XT. Overall, these studies demonstrate that CyTOF XT generates better signal resolution as compared to Helios. The automated acquisition of CyTOF XT enables researchers to streamline immunophenotyping of human samples while accurately and reproducibly monitoring changes in immune cell subsets. For Research Use Only. Not for use in diagnostic procedures. Citation Format: Thiru Selvanantham, Stephen K.H. Li, Nick Zabinyakov, Alexandre Bouzekri, Raymond Jong, Michael Sullivan, Alexander Laboda, Daniel Majonis, Christina Loh. A streamlined and automated approach to high-content cytometric immunophenotyping with CyTOF XT [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3908.

Imaging Mass Cytometry™ (IMC™) is a vital tool to deeply characterize the complexity and diversity of any tissue without disrupting spatial context. The Hyperion™ Imaging System utilizes IMC, based on CyTOF® technology, to simultaneously assess up to 40 individual structural and functional markers in tissues, providing unprecedented insight into the organization and function of tissue microenvironment. We have previously demonstrated the application of IMC in combination with Maxpar® panel kits to highlight cellular composition of human tissues. Here, we showcase the recently released Maxpar OnDemand Antibodies for IMC application on mouse tissue. We introduced 11 additional biomarkers to our existing mouse antibody catalog, providing the basis for the use of high-multiplex imaging in preclinical investigations. To demonstrate the IMC workflow on mouse tissue, we analyzed a normal mouse tissue microarray using IMC spatial proteomic analysis. Tissues were stained with a 20-marker panel designed to highlight tissue architecture and major immune lineage markers combined with our IMC Cell Segmentation Kit*. The IMC Cell Segmentation Kit facilitates identification of cellular borders using plasma membrane markers that lead to improved nucleus and plasma membrane demarcation. We generated a detailed spatial map of the heterogeneous tissue architecture and successfully identified immune, epithelial, and stromal cell populations in various mouse tissues. Additionally, we classified the activation state of immune cell populations, adhesion state of epithelial cells, and molecular composition of the extracellular matrix.Overall, this work demonstrates the capability of IMC to identify subcellular localization of cellular and structural markers in the mouse tissue microenvironment. Information gained from IMC studies will enable in-depth high-throughput phenotypic characterization of the tissue microenvironment in various mouse models of development and disease, and thus accelerate preclinical discoveries. *The IMC Cell Segmentation Kit is part of the Innovative Solutions menu of custom-made reagents and workflows developed and tested by Fluidigm scientists to give faster access to new cutting-edge solutions for high-multiplex single-cell analysis. Innovative Solutions are not part of the Maxpar catalog. For Research Use Only. Not for use in diagnostic procedures. Citation Format: Qanber Raza, Michael Cohen, Smriti Kala, Liang Lim, Geneve Awong, Andrew Quong, Christina Loh. Imaging mass cytometry identifies structural and cellular composition of the mouse tissue microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2035.

CyTOF® mass cytometry is a single-cell analysis platform that uses isotope-tagged antibodies to resolve 50-plus markers in a single tube without signal compensation, making CyTOF ideal for routine immunophenotyping. CyTOF XT™, the latest CyTOF system, features automated sample acquisition. Stained samples were acquired in parallel using the automated CyTOF XT system and manually, using the Helios™ system, to assess performance of the automated system. Multiple suspension mass cytometry staining workflows were evaluated. Population frequencies and resolution indices for markers were assessed by manual gating. There was no significant difference between population frequencies analyzed between the two CyTOF systems. On average, samples acquired on CyTOF XT resulted in greater resolution between positive and negative populations compared to Helios. The Maxpar® Direct™ Immune Profiling System, which comprises the Maxpar® Direct™ Immune Profiling Assay™ and Maxpar Pathsetter™ software, was also compared on the CyTOF XT and Helios systems. The Maxpar Direct Immune Profiling Assay includes a 30-marker panel in a dry, single-tube format for staining human whole blood or PBMC. Maxpar Pathsetter automates reporting of population statistics and stain assessments for the panel. Maxpar Pathsetter showed comparable population frequencies between the two CyTOF systems and improved staining assessment on CyTOF XT. Overall, these studies find that the CyTOF XT system generates better signal resolution than the Helios system. Automated acquisition by CyTOF XT enables researchers to accurately and reproducibly streamline human immunophenotyping. For Research Use Only. Not for use in diagnostic procedures.

Maxpar® Direct™ Immune Profiling Assay™ (Cat. No. 201325) is a 30-marker panel for suspension mass cytometry. This panel provides an unprecedented sample-to-answer solution for detecting and analyzing 30 surface markers in a single experiment. The 18 open mass channels in the Maxpar Direct Assay facilitate panel expansion and enable flexibility for higher multiplexity and applications. Among the potential complementary applications, intracellular cytokine staining (ICS) is of particular interest as it may be used to assess infiltrating immune cell phenotypes in the tumor microenvironment. However, for the purpose of assessing cell viability in this workflow, the effectiveness of the Cell-ID™ Intercalator-Rh (103Rh, Cat. No. 201103) included in the Maxpar Direct Assay is in question, as cell permeabilization during ICS can potentially damage the DNA-intercalator bond. In this study, we investigated the compatibility of 103Rh with intracellular staining. We stained either human peripheral blood mononuclear cell or whole blood samples with the Maxpar Direct Assay followed by intracellular staining for the detection of expressed cytokines. We demonstrate that 103Rh provides equivalent functionality as a cell viability indicator during intracellular staining for cytoplasmic proteins compared to the benchmark Cell-ID Cisplatin-194Pt (Cat. No. 201194). This work was designed to support use of the Maxpar Direct Immune Profiling Assay in combination with additional intracellular markers. Overall, these findings expand the applicability of Cell-ID Intercalator-Rh (103Rh) to processes that involve cytoplasmic staining. For Research Use Only. Not for use in diagnostic procedures.

Imaging Mass Cytometry™ (IMC™) is a vital tool to deeply characterize the complexity and diversity of any tissue without disrupting spatial context. The Hyperion™ Imaging System utilizes IMC, based on CyTOF® technology, to assess up to 40 individual structural and functional markers in tissues, providing unprecedented insight into the organization and function of tissue microenvironment. We have previously demonstrated the application of IMC in combination with Maxpar® panel kits on human tissues. Here, we showcase Maxpar OnDemand Antibodies for IMC application including 11 new highly relevant markers to construe cellular and molecular composition of mouse tissues. We analyzed a normal mouse tissue microarray using IMC spatial proteomic analysis. Tissues were stained with a 20-marker panel designed to highlight tissue architecture and major immune lineage markers. We generated a detailed spatial map of the diverse tissue architecture and successfully identified immune, epithelial, and stromal cell populations in various mouse tissues. Additionally, we classified the activation state of immune cell populations, adhesion state of epithelial cells, and molecular composition of the extracellular matrix. This work demonstrates the capability of IMC to identify subcellular localization of cellular and structural markers in the mouse tissue microenvironment. Future studies utilizing IMC in combination with Maxpar OnDemand Antibodies will enable in-depth phenotypic characterization of the tissue microenvironment in various mouse models of development and disease, and thus provide the basis for the use of high-multiplex imaging in preclinical investigations. For Research Use Only. Not for use in diagnostic procedures.

In-depth monitoring of the immune response to cancer and infection is vital to ascertain disease status and to assess immunotherapeutic options. Time-of-flight technology, the basis of CyTOF® mass cytometry, enables multiplex proteomic cellular phenotyping with 50 or more markers, making it ideal for comprehensive immune profiling. Unlike fluorescence-dependent approaches, which require signal compensation that makes the development of larger panels more challenging, CyTOF utilizes monoisotopic metal-tagged antibodies that exhibit minimal background signal, enabling the highest-resolution multiparametric landscape of a single cell. The Maxpar® Direct™ Immune Profiling Assay™ is a pre-titrated dried-down 30-antibody cocktail preparation for immune profiling of human whole blood or PBMC. This assay is used with Maxpar Pathsetter™ software, which resolves whole blood into 37 immune populations comprising major lineage populations and their subsets, such as CD4 Th subsets and B and T cell memory cells, as well as stratifications of myeloid populations. The resulting system is a simple sample-to-answer solution for immune monitoring studies. Here, we expanded the 30-marker assay with 14 additional antibodies comprising pertinent targets of immunotherapy, including the exhaustion markers PD-1, PD-L1, Tim-3 and CTLA-4, and co-stimulation markers 4-1BB and ICOS. We also demonstrated the compatibility of the assay with downstream intracellular staining for cytoplasmic markers IFN-γ, TNF-α, IL-2, perforin and granzyme B for assessment of cellular function in 4h PMA/ionomycin-stimulated whole blood cultures. Next, we modified the existing Pathsetter model to automate the analysis of whole blood stained with the expanded panel to generate reports on key immune cell populations, percentages of exhausted cells and cell subsets producing cytokines. Last, to demonstrate the ability of this expanded panel to identify antigen-specific T cell subsets accompanied by their in-depth phenotypic assessment, we tested this panel against CMV peptide-stimulated whole blood samples. We demonstrated the flexibility of the Maxpar Direct Immune Profiling Assay in panel customization and a streamlined workflow for automated analysis to enable comprehensive immune profiling of human whole blood. For Research Use Only. Not for use in diagnostic procedures. Citation Format: Shariq Mujib, Stephen K. Li, Nick Zabinyakov, Christina Loh. A comprehensive whole blood CyTOF immune monitoring panel with expanded surface and intracellular markers using the Maxpar Direct Immune Profiling Assay [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1676.

Monitoring the immune response in the setting of infectious disease and cancer is critical to assess disease status and targets of immune therapy. CyTOF® mass cytometry enables multiplex cellular phenotyping with more than 50 markers, making it ideal for comprehensive immune profiling. CyTOF technology utilizes antibodies tagged with unique monoisotopic metals, resulting in distinct signals that provide a high-resolution multiparametric landscape of a single cell. The Maxpar® Direct™ Immune Profiling Assay™ is a pre-titrated, dried-down, 30-marker antibody cocktail for immune profiling of human whole blood and PBMC by CyTOF. Paired with Maxpar Pathsetter™ software, stained samples are automatically resolved into 37 immune populations including major lineages and their subsets. In this study, we expanded the 30-marker assay to a 44-marker panel including exhaustion markers such as PD-1 and CTLA-4, co-stimulation markers 4-1BB and ICOS, and intracellular cytoplasmic markers IFN-γ, TNF-α, IL-2, perforin and granzyme B to assess cellular function in PMA/ionomycin-stimulated whole blood cultures. We modified the existing Maxpar Pathsetter model to automate the analysis of the expanded panel and report on additional functional parameters such as T cell exhaustion and cytokine production. Next, we applied this panel to whole blood stimulated with CMV peptides to investigate antigen-specific immune responses in a viral infection model in concert with in-depth phenotypic assessment. Collectively, we demonstrate the flexibility of the Maxpar Direct Immune Profiling Assay to incorporate additional surface and intracellular markers to study antigen-specific immunity in the context of whole blood immune profiling. For RUO.

Mass cytometry, which utilizes CyTOF® technology, is a single-cell analysis platform that uses metal-tagged antibodies. CyTOF can resolve more than 50 parameters in a single panel without the need for compensation, making it an ideal solution for routine enumeration of immune cells. The Maxpar® Direct™ Immune Profiling System is a sample-to-answer solution for human immune profiling using mass cytometry. The Maxpar®Direct™ Immune Profiling Assay™ is an optimized 30-marker panel contained in a dry single-tube format for human whole blood or PBMC staining, and samples are acquired on the Helios™ system. Maxpar Pathsetter™ is an automated software that reports cell counts, percentage calculations, and staining intensity. It also produces graphical elements such as dot plots and a Cen-se′™ graph for 37 immune cell populations. The panel can be tailored by adding markers to open channels, the Maxpar Pathsetter model can then be customized to measure expression markers on existing classified populations or identify additional immune cell subsets. We present data where the Maxpar Direct Immune Profiling Assay is used as a core immunophenotyping panel and additional markers are added to create a nearly 50 marker panel. Added markers are used to identify MDSCs, further classify existing cell populations, and measure immuno-oncology related markers including OX40, TIM-3, Fas, PD-1, PD-L1, ICOS, and TIGIT. We demonstrate how the Maxpar Pathsetter model is modified to incorporate the added markers. The ability to customize the Maxpar Pathsetter model and expand the Maxpar Direct Immune Profiling Assay allows for flexibility of the system. It allows researchers to have a streamlined solution for broad immune profiling using mass cytometry.

Understanding the consequences of tuning TCR signaling on selection, peripheral T cell function, and tolerance in the context of native TCR repertoires may provide insight into the physiological control of tolerance. In this study, we show that genetic ablation of a natural tuner of TCR signaling, mir-181a-1/b-1, in double-positive thymocytes dampened TCR and Erk signaling and increased the threshold of positive selection. Whereas mir-181a-1/b-1 deletion in mice resulted in an increase in the intrinsic reactivity of naive T cells to self-antigens, it did not cause spontaneous autoimmunity. Loss of mir-181a-1/b-1 dampened the induction of experimental autoimmune encephalomyelitis and reduced basal TCR signaling in peripheral T cells and their migration from lymph nodes to pathogenic sites. Taken together, these results demonstrate that tolerance can be modulated by microRNA gene products through the control of opposing activities in T cell selection and peripheral T cell function.

Much has been learned about the molecular and cellular components critical for the control of immune responses and tolerance. It remains a challenge, however, to control the immune response and tolerance at the system level without causing significant toxicity to normal tissues. Recent studies suggest that microRNA (miRNA) genes, an abundant class of non-coding RNA genes that produce characteristic approximately 22 nucleotides small RNAs, play important roles in immune cells. In this article, we discuss emerging knowledge regarding the functions of miRNA genes in the immune system. We delve into the roles of miRNAs in regulating signaling strength and threshold, homeostasis, and the dynamics of the immune response and tolerance during normal and pathogenic immunological conditions. We also present observations based on analyzes of miR-181 family genes that indicate the potential functions of primary and/ or precursor miRNAs in target recognition and explore the impact of these findings on target identification. Finally, we illustrate that despite the subtle effects of miRNAs on gene expression, miRNAs have the potential to influence the outcomes of normal and pathogenic immune responses by controlling the quantitative and dynamic aspects of immune responses. Tuning miRNA functions in immune cells, through gain- and loss-of-function approaches in mice, may reveal novel approach to restore immune equilibrium from pathogenic conditions, such as autoimmune disease and leukemia, without significant toxicity.

Numerous mapping studies have implicated genetic intervals from lupus-prone New Zealand Black (NZB) chromosomes 1 and 4 as contributing to lupus pathogenesis. By introgressing NZB chromosomal intervals onto a non–lupus-prone B6 background, we determined that: NZB chromosome 1 congenic mice (denoted B6.NZBc1) developed fatal autoimmune-mediated kidney disease, and NZB chromosome 4 congenic mice (denoted B6.NZBc4) exhibited a marked expansion of B1a and NKT cells in the surprising absence of autoimmunity. In this study, we sought to examine whether epistatic interactions between these two loci would affect lupus autoimmunity by generating bicongenic mice that carry both NZB chromosomal intervals. Compared with B6.NZBc1 mice, bicongenic mice demonstrated significantly decreased mortality, kidney disease, Th1-biased IgG autoantibody isotypes, and differentiation of IFN-γ–producing T cells. Furthermore, a subset of bicongenic mice exhibited a paucity of CD21+CD1d+ B cells and an altered NKT cell activation profile that correlated with greater disease inhibition. Thus, NZBc4 contains suppressive epistatic modifiers that appear to inhibit the development of fatal NZBc1 autoimmunity by promoting a shift away from a proinflammatory cytokine profile, which in some mice may involve NKT cells.

Introgression of a New Zealand Black (NZB) chromosome 13 interval onto a C57BL/6 (B6) background (B6.NZBc13) is sufficient to produce many hallmarks of lupus, including high-titre anti-chromatin antibody production, abnormal B- and T-cell activation, and renal disease. In this study we sought to characterize the immune defects leading to these abnormalities. By generating hematopoietic chimeras and BCR transgenic mice, we show that the congenic autoimmune phenotype can be transferred by BM cells and requires the presence of autoreactive B cells. Using the hen egg white lysozyme immunoglobulin transgenic mouse model, we demonstrate that B-cell anergy, deletion, and receptor editing are intact. Nevertheless, congenic B cells exhibit altered peripheral B-cell selection, as demonstrated by enhanced survival and activation of endogenous B cells with autoreactivity to chromatin and Sm/ribonucleoprotein. Given the autoantibody specificities to nuclear antigens, TLR signalling was assessed. B6.NZBc13 B cells were hyper-responsive to poly(I:C), a TLR3 ligand, demonstrating enhanced proliferation and survival as compared to B6 B cells. Our findings indicate the presence of an intrinsic B-cell defect on NZB chromosome 13 that results in hyper-responsiveness to a dsRNA analogue and implicates its potential supporting role in the generation of autoimmunity in B6.NZBc13 mice.

The development and homeostasis of multicellular organisms relies on gene regulation within individual constituent cells. Gene regulatory circuits that increase the robustness of gene expression frequently incorporate microRNAs as post-transcriptional regulators. Computational approaches, synthetic gene circuits and observations in model organisms predict that the co-regulation of microRNAs and their target mRNAs can reduce cell-to-cell variability in the expression of target genes. However, whether microRNAs directly regulate variability of endogenous gene expression remains to be tested in mammalian cells. Here we use quantitative flow cytometry to show that microRNAs impact on cell-to-cell variability of protein expression in developing mouse thymocytes. We find two distinct mechanisms that control variation in the activation-induced expression of the microRNA target CD69. First, the expression of miR-17 and miR-20a, two members of the miR-17-92 cluster, is co-regulated with the target mRNA Cd69 to form an activation-induced incoherent feed-forward loop. Another microRNA, miR-181a, acts at least in part upstream of the target mRNA Cd69 to modulate cellular responses to activation. The ability of microRNAs to render gene expression more uniform across mammalian cell populations may be important for normal development and for disease., Author Summary microRNAs are integral to many developmental processes and may 'canalise' development by reducing cell-to-cell variation in gene expression. This idea is supported by computational studies that have modeled the impact of microRNAs on the expression of their targets and the construction of artificial incoherent feedforward loops using synthetic biology tools. Here we show that this interesting principle of microRNA regulation actually occurs in a mammalian developmental system. We examine cell-to-cell variation of protein expression in developing mouse thymocytes by quantitative flow cytometry and find that the absence of microRNAs results in increased cell-to-cell variation in the expression of the microRNA target Cd69. Mechanistically, T cell receptor signaling induces both Cd69 and miR-17 and miR-20a, two microRNAs that target Cd69. Co-regulation of microRNAs and their target mRNA dampens the expression of Cd69 and forms an incoherent feedforward loop that reduces cell-to-cell variation on CD69 expression. In addition, miR-181, which also targets Cd69 and is a known modulator of T cell receptor signaling, also affects cell-to-cell variation of CD69 expression. The ability of microRNAs to control the uniformity of gene expression across mammalian cell populations may be important for normal development and for disease.

Prostaglandin E2(PGE2), a potent lipid signaling molecule, modulates inflammatory responses through activation of downstream G-protein coupled EP1–4receptors. Here, we investigated the cell-specificin vivofunction of PGE2signaling through its E-prostanoid 2 (EP2) receptor in murine innate immune responses systemically and in the CNS.In vivo, systemic administration of lipopolysaccharide (LPS) resulted in a broad induction of cytokines and chemokines in plasma that was significantly attenuated in EP2-deficient mice.Ex vivostimulation of peritoneal macrophages with LPS elicited proinflammatory responses that were dependent on EP2 signaling and that overlapped within vivoplasma findings, suggesting that myeloid-lineage EP2 signaling is a major effector of innate immune responses. Conditional deletion of the EP2 receptor in myeloid lineage cells in Cd11bCre;EP2lox/loxmice attenuated plasma inflammatory responses and transmission of systemic inflammation to the brain was inhibited, with decreased hippocampal inflammatory gene expression and cerebral cortical levels of IL-6. Conditional deletion of EP2 significantly blunted microglial and astrocytic inflammatory responses to the neurotoxin MPTP and reduced striatal dopamine turnover. Suppression of microglial EP2 signaling also increased numbers of dopaminergic (DA) neurons in the substantia nigra independent of MPTP treatment, suggesting that microglial EP2 may influence development or survival of DA neurons. Unbiased microarray analysis of microglia isolated from adult Cd11bCre;EP2lox/loxand control mice demonstrated a broad downregulation of inflammatory pathways with ablation of microglial EP2 receptor. Together, these data identify a cell-specific proinflammatory role for macrophage/microglial EP2 signaling in innate immune responses systemically and in brain.

The development and progression of systemic lupus erythematosus is mediated by the complex interaction of genetic and environmental factors. To decipher the genetics that contribute to pathogenesis and the production of pathogenic autoantibodies, our lab has focused on the generation of congenic lupus-prone mice derived from the New Zealand Black (NZB) strain. Previous work has shown that an NZB-derived chromosome 4 interval spanning 32 to 151 Mb led to expansion of CD5+ B and Natural Killer T (NKT) cells, and could suppress autoimmunity when crossed with a lupus-prone mouse strain. Subsequently, it was shown that CD5+ B cells but not NKT cells derived from these mice could suppress the development of pro-inflammatory T cells. In this paper, we aimed to further resolve the genetics that leads to expansion of these two innate-like populations through the creation of additional sub-congenic mice and to characterize the role of IL-10 in the suppression of autoimmunity through the generation of IL-10 knockout mice. We show that expansion of CD5+ B cells and NKT cells localizes to a chromosome 4 interval spanning 91 to 123 Mb, which is distinct from the region that mediates the majority of the suppressive phenotype. We also demonstrate that IL-10 is critical to restraining autoantibody production and surprisingly plays a vital role in supporting the expansion of innate-like populations.

New Zealand Black (NZB) mice spontaneously develop a lupus-like autoimmune disease. Since CD40-CD40L interactions are important for B cell class-switch recombination and germinal center formation, we sought to understand the impact of these interactions on the immune abnormalities in NZB CD40L gene-deleted (CD40L(-/-)) mice in vivo. NZB.CD40L(-/-) mice demonstrated abrogation of all IgG autoantibodies tested and attenuated kidney disease. However, polyclonal B cell activation in vivo and B cell proliferation and class-switching in response to TLR ligands in vitro were preserved in the absence of CD40L in NZB mice. Although, plasmacytoid dendritic cell expansion and elevated BAFF production were unaffected by the absence of CD40L, there was some evidence that IFN-α-induced gene expression was reduced in the bone marrow of NZB.CD40L(-/-) mice. Our results suggest that CD40-CD40L interactions play an important role in promoting pathogenic IgG autoantibody production and kidney disease in NZB mice.

Systemic lupus erythematosus (SLE) is a multisystem autoimmune disease characterized by production of autoantibodies directed against nuclear antigens resulting in formation of immune complexes that deposit in multiple organs causing tissue damage. SLE is a complex genetic disease in which variations in multiple genes, each with a modest effect size, contribute to disease genesis. Given this genetic complexity, identification of the role of individual polymorphisms is challenging. In this context, studies of mouse models of lupus have been particularly informative. Here we review the findings arising from the study of gene deleted, transgenic and congenic lupus-prone mouse models.

Previous mapping studies have linked New Zealand Black (NZB) chromosome 4 to several lupus traits, including autoantibody production, splenomegaly, and glomerulonephritis. To confirm the presence of these traits, our laboratory introgressed homozygous NZB chromosome 4 intervals extending from either 114 to 149 Mb or 32 to 149 Mb onto the lupus-resistant C57BL/6 background (denoted B6.NZBc4S and B6.NZBc4L, respectively). Characterization of aged cohorts revealed that B6.NZBc4L mice exhibited a striking increase in splenic B1a and NKT cells in the absence of high titer autoantibody production and significant renal disease. Tissue-specific expansion of these subsets was also seen in the peritoneum and liver for B1a cells and in the bone marrow for NKT cells. Staining with CD1d tetramers loaded with an α-galactosylceramide analog (PBS57) demonstrated that the expanded NKT cell population was mainly CD1d-dependent NKT cells. The lack of both cellular phenotypes in B6.NZBc4S mice demonstrates that the genetic polymorphism(s) that result in these phenotypes are on the proximal region of NZB chromosome 4. This study confirms the presence of a locus that promotes the expansion of B1a cells and newly identifies a region that promotes CD1d-restricted NKT cell expansion on NZB chromosome 4. Taken together, the data indicate that neither an expansion of B1a cells and/nor NKT cells is sufficient to promote autoantibody production and ultimately, renal disease.

Polyclonal B cell activation is a prominent feature of the lupus-prone New Zealand Black (NZB) mouse strain. We have previously demonstrated linkage between a region on NZB chromosome 13 and increased costimulatory molecule expression on B cells. In this study we have produced C57BL/6 congenic mice with an introgressed homozygous NZB interval extending from ∼24 to 73 cM on chromosome 13 (denoted B6.NZBc13). We show that B6.NZBc13 female mice not only have enhanced B cell activation but also share many other B cell phenotypic characteristics with NZB mice, including expansion of marginal zone and CD5+ B cell populations, increased numbers of IgM ELISPOTs, and increased serum levels of total IgM and IgM autoantibodies. In addition these mice have increased T cell activation, increased numbers of germinal centers, mild glomerulonephritis, and produce high-titer IgM and IgG anti-chromatin Abs. Male B6.NZBc13 mice have a less pronounced cellular phenotype, lacking expansion of the marginal zone B cell population and IgG anti-chromatin Ab production, indicating the presence of gender dimorphism for this locus. Thus, we have identified a genetic locus that recapitulates with fidelity the B cell phenotypic abnormalities in NZB mice, and we demonstrate that this locus is sufficient to induce an autoimmune phenotype. The data provide further support to the contention that immune abnormalities leading to altered B cell activation and selection contribute to the development of autoimmunity in NZB mice.

The presence of autoantibodies in New Zealand Black (NZB) mice suggests a B cell tolerance defect however the nature of this defect is unknown. To determine whether defects in B cell anergy contribute to the autoimmune phenotype in NZB mice, soluble hen egg lysozyme (sHEL) and anti-HEL Ig transgenes were bred onto the NZB background to generate double transgenic (dTg) mice. NZB dTg mice had elevated levels of anti-HEL antibodies, despite apparently normal B cell functional anergy in-vitro. NZB dTg B cells also demonstrated increased survival and abnormal entry into the follicular compartment following transfer into sHEL mice. Since this process is dependent on BAFF, BAFF serum and mRNA levels were assessed and were found to be significantly elevated in NZB dTg mice. Treatment of NZB sHEL recipient mice with TACI-Ig reduced NZB dTg B cell survival following adoptive transfer, confirming the role of BAFF in this process. Although NZB mice had modestly elevated BAFF, the enhanced NZB B cell survival response appeared to result from an altered response to BAFF. In contrast, T cell blockade had a minimal effect on B cell survival, but inhibited anti-HEL antibody production. The findings suggest that the modest BAFF elevations in NZB mice are sufficient to perturb B cell tolerance, particularly when acting in concert with B cell functional abnormalities and T cell help.

Introgression of a New Zealand Black (NZB) chromosome 13 (c13) interval onto a lupus-resistant C57BL/6 (B6) background (denoted, B6.NZBc13) is sufficient to produce many of the hallmarks of lupus. To characterize the immune defects leading to these abnormalities observed in B6.NZBc13 mice, bone marrow (BM) chimeras and BCR transgenic mice were produced. In BM chimeras, transfer of B6.NZBc13 BM cells was sufficient to transfer autoimmunity. Interestingly, in mixed BM chimeras the abnormal T and B cell activation as well as DC expansion was observed in both B6 and B6.NZBc13 derived cells; but with greater B cell activation in B6.NZBc13 derived cells. When an anti-HEL Ig transgene was crossed onto the congenic background disease was abrogated and the abnormal cellular phenotypes normalized. Although tolerance was retained in anti-HEL Ig/soluble HEL double transgenic mice, increased numbers of 'edited' cells were seen in the periphery. B cell function studies revealed altered phosphorylation of signaling molecules downstream of the BCR. These findings indicate the presence of a BM-cell intrinsic defect on NZB c13 that can be localized to a B cell defect, which is necessary to initiate the autoimmune phenotype in B6.NZBc13 mice.