Radiation therapy induces immunosenescence mediated by p90RSK.

Radiation therapy (RT) to the chest increases the patients' risk of cardiovascular disease (CVD). A complete understanding of the mechanisms by which RT induces CVD could lead to specific preventive, therapeutic approaches. It is becoming evident that both genotoxic chemotherapy agents and radiation induce mitochondrial dysfunction and cellular senescence. Notably, one of the common phenotypes observed in cancer survivors is accelerated senescence, and immunosenescence is closely related to both cancer risk and CVD development. Therefore, suppression of immunosenescence can be an ideal target to prevent cancer treatment-induced CVD. However, the mechanism(s) by which cancer treatments induce immunosenescence are incompletely characterized. We isolated peripheral blood mononuclear cells (PBMCs) before and 3 months after RT from 16 thoracic cancer patients. We characterized human immune cell lineages and markers of senescence, DNA damage response (DDR), efferocytosis, and determinants of clonal hematopoiesis of indeterminant potential (CHIP), using mass cytometry (CyTOF). We found that the frequency of the B cell subtype was decreased after RT. Unsupervised clustering of the CyTOF data identified 138 functional subsets of PBMCs. Compared with baseline, RT increased TBX21 (T-bet) expression in the largest B cell subset of Ki67-/DNMT3a+naïve B cells, and T-bet expression was correlated with phosphorylation of p90RSK expression. CD38 expression was also increased in naïve B cells (CD27-) and CD8+ effector memory CD45RA T cells (TEMRA). In vitro, we found the critical role of p90RSK activation in upregulating (1) CD38+/T-bet+ memory and naïve B, and myeloid cells, (2) senescence-associated ß-gal staining, and (3) mitochondrial reactive oxygen species (ROS) after ionizing radiation (IR). These data suggest the crucial role of p90RSK activation in immunosenescence. The critical role of p90RSK activation in immune cells and T-bet induction in upregulating atherosclerosis formation has been reported. Furthermore, T-bet directly binds to the CD38 promoter region and upregulates CD38 expression. Since both T-bet and CD38 play a significant role in the process of immunosenescence, our data provide a cellular and molecular mechanism that links RT-induced p90RSK activation and the immunosenescence with T-bet and CD38 induction observed in thoracic cancer patients treated by RT and suggests that targeting the p90RSK/T-bet/CD38 pathway could play a role in preventing the radiation-associated CVD and improving cancer prognosis by inhibiting immunosenescence.

Full spectrum flow cytometry and mass cytometry: A 32-marker panel comparison.

High-dimensional single-cell data has become an important tool in unraveling the complexity of the immune system and its involvement in homeostasis and a large array of pathologies. As technological tools are developed, researchers are adopting them to answer increasingly complex biological questions. Up until recently, mass cytometry (MC) has been the main technology employed in cytometric assays requiring more than 29 markers. Recently, however, with the introduction of full spectrum flow cytometry (FSFC), it has become possible to break the fluorescence barrier and go beyond 29 fluorescent parameters. In this study, in collaboration with the Stanford Human Immune Monitoring Center (HIMC), we compared five patient samples using an established immune panel developed by the HIMC using their MC platform. Using split samples and the same antibody panel, we were able to demonstrate highly comparable results between the two technologies using multiple data analysis approaches. We report here a direct comparison of two technology platforms (MC and FSFC) using a 32-marker flow cytometric immune monitoring panel that can identify all the previously described and anticipated immune subpopulations defined by this panel.

Deep Analysis of the Peripheral Immune System in IBD Reveals New Insight in Disease Subtyping and Response to Monotherapy or Combination Therapy.

BACKGROUND: Inflammatory bowel disease (IBD) is a complex disease with variable presentation, progression, and response to therapies. Current disease classification is based on subjective clinical phenotypes. The peripheral blood immunophenome can reflect local inflammation, and thus we measured 39 circulating immune cell types in a large cohort of IBD and control subjects and performed immunotype:phenotype associations. METHODS: We performed fluorescence-activated cell sorting or CyTOF analysis on blood from 728 Crohn's disease, 464 ulcerative colitis, and 334 non-IBD patients, with available demographics, endoscopic and clinical examinations and medication use. RESULTS: We observed few immune cell types commonly affected in IBD (lowered natural killer cells, B cells, and CD45RA- CD8 T cells). Generally, the immunophenome was distinct between ulcerative colitis and Crohn's disease. Within disease subtype, there were further distinctions, with specific immune cell types associating with disease duration, behavior, and location. Thiopurine monotherapy altered abundance of many cell types, often in the same direction as disease association, while anti-tumor necrosis factor (anti-TNF) monotherapy demonstrated an opposing pattern. Concomitant use of an anti-TNF and thiopurine was not synergistic, but rather was additive. For example, thiopurine monotherapy use alone or in combination with anti-TNF was associated with a dramatic reduction in major subclasses of B cells. CONCLUSIONS: We present a peripheral map of immune cell changes in IBD related to disease entity and therapies as a resource for hypothesis generation. We propose the changes in B cell subsets could affect antibody formation and potentially explain the mechanism behind the superiority of combination therapy through the impact of thiopurines on pharmacokinetics of anti-TNFs.

Multimodal Mapping of the Tumor and Peripheral Blood Immune Landscape in Human Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDA) is characterized by an immune-suppressive tumor microenvironment that renders it largely refractory to immunotherapy. We implemented a multimodal analysis approach to elucidate the immune landscape in PDA. Using a combination of CyTOF, single-cell RNA sequencing, and multiplex immunohistochemistry on patient tumors, matched blood, and non-malignant samples, we uncovered a complex network of immune-suppressive cellular interactions. These experiments revealed heterogeneous expression of immune checkpoint receptors in individual patient's T cells and increased markers of CD8+ T cell dysfunction in advanced disease stage. Tumor-infiltrating CD8+ T cells had an increased proportion of cells expressing an exhausted expression profile that included upregulation of the immune checkpoint TIGIT, a finding that we validated at the protein level. Our findings point to a profound alteration of the immune landscape of tumors, and to patient-specific immune changes that should be taken into account as combination immunotherapy becomes available for pancreatic cancer.

Single-cell immune landscape of human atherosclerotic plaques.

Atherosclerosis is driven by multifaceted contributions of the immune system within the circulation and at vascular focal sites. However, specific characteristics of dysregulated immune cells within atherosclerotic lesions that lead to clinical events such as ischemic stroke or myocardial infarction are poorly understood. Here, using single-cell proteomic and transcriptomic analyses, we uncovered distinct features of both T cells and macrophages in carotid artery plaques of patients with clinically symptomatic disease (recent stroke or transient ischemic attack) compared to asymptomatic disease (no recent stroke). Plaques from symptomatic patients were characterized by a distinct subset of CD4+ T cells and by T cells that were activated and differentiated. Moreover, some T cell subsets in these plaques presented markers of T cell exhaustion. Additionally, macrophages from these plaques contained alternatively activated phenotypes, including subsets associated with plaque vulnerability. In plaques from asymptomatic patients, T cells and macrophages were activated and displayed evidence of interleukin-1ß signaling. The identification of specific features of innate and adaptive immune cells in plaques that are associated with cerebrovascular events may enable the design of more precisely tailored cardiovascular immunotherapies.

A Modified Injector and Sample Acquisition Protocol Can Improve Data Quality and Reduce Inter-Instrument Variability of the Helios Mass Cytometer.

Mass cytometry is a powerful tool for high-dimensional single cell characterization. Since the introduction of the first commercial CyTOF mass cytometer by DVS Sciences in 2009, mass cytometry technology has matured and become more widely utilized, with sequential platform upgrades designed to address specific limitations and to expand the capabilities of the platform. Fluidigm's third-generation Helios mass cytometer introduced a number of upgrades over the previous CyTOF2. One of these new features is a modified narrow bore sample injector that generates smaller ion clouds, which is expected to improve sensitivity and throughput. However, following rigorous testing, we find that the narrow-bore sample injector may have unintended negative consequences on data quality and result in lower median and higher coefficients of variation in many antibody-associated signal intensities. We describe an alternative Helios acquisition protocol using a wider bore injector, which largely mitigates these data quality issues. We directly compare these two protocols in a multisite study of 10 Helios instruments across 7 institutions and show that the modified protocol improves data quality and reduces interinstrument variability. These findings highlight and address an important source of technical variability in mass cytometry experiments that is of particular relevance in the setting of multicenter studies. © 2019 International Society for Advancement of Cytometry.

Comprehensive Immune Monitoring of Clinical Trials to Advance Human Immunotherapy.

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.

Development of a Comprehensive Antibody Staining Database Using a Standardized Analytics Pipeline.

Large-scale immune monitoring experiments (such as clinical trials) are a promising direction for biomarker discovery and responder stratification in immunotherapy. Mass cytometry is one of the tools in the immune monitoring arsenal. We propose a standardized workflow for the acquisition and analysis of large-scale mass cytometry experiments. The workflow includes two-tiered barcoding, a broad lyophilized panel, and the incorporation of a fully automated, cloud-based analysis platform. We applied the workflow to a large antibody staining screen using the LEGENDScreen kit, resulting in single-cell data for 350 antibodies over 71 profiling subsets. The screen recapitulates many known trends in the immune system and reveals potential markers for delineating MAIT cells. Additionally, we examine the effect of fixation on staining intensity and identify several markers where fixation leads to either gain or loss of signal. The standardized workflow can be seamlessly integrated into existing trials. Finally, the antibody staining data set is available as an online resource for researchers who are designing mass cytometry experiments in suspension and tissue.

An integrated framework using high-dimensional mass cytometry and fluorescent flow cytometry identifies discrete B cell subsets in patients with red meat allergy.

BACKGROUND: B cells play a critical role in the development and maintenance of food allergy by producing allergen-specific IgE. Despite the importance of B cells in IgE-mediated food allergy, the identity of sIgE-producing human B cells and how IgE is regulated are poorly understood. OBJECTIVE: To identify the immunophenotypes of circulating B cells associated with the production of galactose-alpha-1,3-galactose-specific IgE production in patients with red meat allergy. METHODS: B cells in PBMC samples obtained from 19 adults with physician-diagnosed red meat allergy and 20 non-meat allergic healthy controls were assessed by mass cytometry along with a bioinformatics analysis pipeline to identify discrete B cell phenotypes that associated with serum sIgE. Fluorescent flow cytometry was then applied to sort purify discrete B cell subsets, and B cells were functionally evaluated on an individual cell level for the production of sIgE by ELISPOT. RESULTS: Discrete B cell phenotypes abundant in meat allergic subjects compared to non-meat allergic controls were found in peripheral blood that do not share typical characteristics of classical isotype-switched memory B cells that express high levels of CD27. These B cell subsets shared higher IgD and lower IgM expression levels coupled with CXCR4, CCR6 and CD25 expression. In vitro polyclonal stimulation of purified B cell subsets from meat allergic subjects demonstrated that these subsets were enriched for cells induced to secrete sIgE. CONCLUSIONS AND CLINICAL RELEVANCE: Circulating B cells display increased abundance of discrete B cell subsets in meat allergic subjects. This observation, coupled with the capacity of individual B cell subsets to produce sIgE following activation, implicates these novel B cell phenotypes in promoting IgE in meat allergy.

Protein Barcodes Enable High-Dimensional Single-Cell CRISPR Screens.

CRISPR pools are being widely employed to identify gene functions. However, current technology, which utilizes DNA as barcodes, permits limited phenotyping and bulk-cell resolution. To enable novel screening capabilities, we developed a barcoding system operating at the protein level. We synthesized modules encoding triplet combinations of linear epitopes to generate >100 unique protein barcodes (Pro-Codes). Pro-Code-expressing vectors were introduced into cells and analyzed by CyTOF mass cytometry. Using just 14 antibodies, we detected 364 Pro-Code populations; establishing the largest set of protein-based reporters. By pairing each Pro-Code with a different CRISPR, we simultaneously analyzed multiple phenotypic markers, including phospho-signaling, on dozens of knockouts. Pro-Code/CRISPR screens found two interferon-stimulated genes, the immunoproteasome component Psmb8 and a chaperone Rtp4, are important for antigen-dependent immune editing of cancer cells and identified Socs1 as a negative regulator of Pd-l1. The Pro-Code technology enables simultaneous high-dimensional protein-level phenotyping of 100s of genes with single-cell resolution.

Comprehensive innate immune profiling of chikungunya virus infection in pediatric cases.

Chikungunya virus (CHIKV) is a mosquito-borne alphavirus that causes global epidemics of debilitating disease worldwide. To gain functional insight into the host cellular genes required for virus infection, we performed whole-blood RNA-seq, 37-plex mass cytometry of peripheral blood mononuclear cells (PBMCs), and serum cytokine measurements of acute- and convalescent-phase samples obtained from 42 children naturally infected with CHIKV Semi-supervised classification and clustering of single-cell events into 57 sub-communities of canonical leukocyte phenotypes revealed a monocyte-driven response to acute infection, with the greatest expansions in "intermediate" CD14++CD16+ monocytes and an activated subpopulation of CD14+ monocytes. Increases in acute-phase CHIKV envelope protein E2 expression were highest for monocytes and dendritic cells. Serum cytokine measurements confirmed significant acute-phase upregulation of monocyte chemoattractants. Distinct transcriptomic signatures were associated with infection timepoint, as well as convalescent-phase anti-CHIKV antibody titer, acute-phase viremia, and symptom severity. We present a multiscale network that summarizes all observed modulations across cellular and transcriptomic levels and their interactions with clinical outcomes, providing a uniquely global view of the biomolecular landscape of human CHIKV infection.

Average Overlap Frequency: A simple metric to evaluate staining quality and community identification in high dimensional mass cytometry experiments.

High dimensional cytometry now allows measurement of over 50 parameters in a single sample, and is typically visualized using sophisticated dimensionality-reducing methods and analyzed with automated clustering algorithms. While these tools facilitate the identification and presentation of key findings, it remains challenging to effectively monitor and report the staining quality of individual markers. We present the Average Overlap Frequency (AOF), a simple and efficient metric to evaluate and quantify the robustness of staining and clustering quality in high-dimensional data. We leverage the AOF to compare and determine the optimal storage conditions for stained whole blood samples prior to mass cytometry analysis. We also show that the AOF can be easily incorporated as part of automated analysis pipelines in large scale immune monitoring studies and used to flag and exclude samples with poor staining quality. We propose that the AOF may be incorporated as an essential quality control metric to better identify and report the underlying sample quality in all CyTOF and other high-dimensional cytometry experiments.

High-dimensional CyTOF analysis of dengue virus-infected human DCs reveals distinct viral signatures.

Dengue virus (DENV) is the most prevalent mosquito-borne virus causing human disease. Of the 4 DENV serotypes, epidemiological data suggest that DENV-2 secondary infections are associated with more severe disease than DENV-4 infections. Mass cytometry by time-of-flight (CyTOF) was used to dissect immune changes induced by DENV-2 and DENV-4 in human DCs, the initial targets of primary infections that likely affect infection outcomes. Strikingly, DENV-4 replication peaked earlier and promoted stronger innate immune responses, with increased expression of DC activation and migration markers and increased cytokine production, compared with DENV-2. In addition, infected DCs produced higher levels of inflammatory cytokines compared with bystander DCs, which mainly produced IFN-induced cytokines. These high-dimensional analyses during DENV-2 and DENV-4 infections revealed distinct viral signatures marked by different replication strategies and antiviral innate immune induction in DCs, which may result in different viral fitness, transmission, and pathogenesis.

Innate Immune Landscape in Early Lung Adenocarcinoma by Paired Single-Cell Analyses.

To guide the design of immunotherapy strategies for patients with early stage lung tumors, we developed a multiscale immune profiling strategy to map the immune landscape of early lung adenocarcinoma lesions to search for tumor-driven immune changes. Utilizing a barcoding method that allows a simultaneous single-cell analysis of the tumor, non-involved lung, and blood cells, we provide a detailed immune cell atlas of early lung tumors. We show that stage I lung adenocarcinoma lesions already harbor significantly altered T cell and NK cell compartments. Moreover, we identified changes in tumor-infiltrating myeloid cell (TIM) subsets that likely compromise anti-tumor T cell immunity. Paired single-cell analyses thus offer valuable knowledge of tumor-driven immune changes, providing a powerful tool for the rational design of immune therapies. VIDEO ABSTRACT.

Host-Protozoan Interactions Protect from Mucosal Infections through Activation of the Inflammasome.

While conventional pathogenic protists have been extensively studied, there is an underappreciated constitutive protist microbiota that is an integral part of the vertebrate microbiome. The impact of these species on the host and their potential contributions to mucosal immune homeostasis remain poorly studied. Here, we show that the protozoan Tritrichomonas musculis activates the host epithelial inflammasome to induce IL-18 release. Epithelial-derived IL-18 promotes dendritic cell-driven Th1 and Th17 immunity and confers dramatic protection from mucosal bacterial infections. Along with its role as a "protistic" antibiotic, colonization with T. musculis exacerbates the development of T-cell-driven colitis and sporadic colorectal tumors. Our findings demonstrate a novel mutualistic host-protozoan interaction that increases mucosal host defenses at the cost of an increased risk of inflammatory disease.

Data-Driven Phenotypic Dissection of AML Reveals Progenitor-like Cells that Correlate with Prognosis.

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.

Highly multiplexed profiling of single-cell effector functions reveals deep functional heterogeneity in response to pathogenic ligands.

Despite recent advances in single-cell genomic, transcriptional, and mass-cytometric profiling, it remains a challenge to collect highly multiplexed measurements of secreted proteins from single cells for comprehensive analysis of functional states. Herein, we combine spatial and spectral encoding with polydimethylsiloxane (PDMS) microchambers for codetection of 42 immune effector proteins secreted from single cells, representing the highest multiplexing recorded to date for a single-cell secretion assay. Using this platform to profile differentiated macrophages stimulated with lipopolysaccharide (LPS), the ligand of Toll-like receptor 4 (TLR4), reveals previously unobserved deep functional heterogeneity and varying levels of pathogenic activation. Uniquely protein profiling on the same single cells before and after LPS stimulation identified a role for macrophage inhibitory factor (MIF) to potentiate the activation of LPS-induced cytokine production. Advanced clustering analysis identified functional subsets including quiescent, polyfunctional fully activated, partially activated populations with different cytokine profiles. This population architecture is conserved throughout the cell activation process and prevails as it is extended to other TLR ligands and to primary macrophages derived from a healthy donor. This work demonstrates that the phenotypically similar cell population still exhibits a large degree of intrinsic heterogeneity at the functional and cell behavior level. This technology enables full-spectrum dissection of immune functional states in response to pathogenic or environmental stimulation, and opens opportunities to quantify deep functional heterogeneity for more comprehensive and accurate immune monitoring.

Palladium-based mass tag cell barcoding with a doublet-filtering scheme and single-cell deconvolution algorithm.

Mass-tag cell barcoding (MCB) labels individual cell samples with unique combinatorial barcodes, after which they are pooled for processing and measurement as a single multiplexed sample. The MCB method eliminates variability between samples in antibody staining and instrument sensitivity, reduces antibody consumption and shortens instrument measurement time. Here we present an optimized MCB protocol. The use of palladium-based labeling reagents expands the number of measurement channels available for mass cytometry and reduces interference with lanthanide-based antibody measurement. An error-detecting combinatorial barcoding scheme allows cell doublets to be identified and removed from the analysis. A debarcoding algorithm that is single cell-based rather than population-based improves the accuracy and efficiency of sample deconvolution. This debarcoding algorithm has been packaged into software that allows rapid and unbiased sample deconvolution. The MCB procedure takes 3-4 h, not including sample acquisition time of ∼1 h per million cells.

Single-cell trajectory detection uncovers progression and regulatory coordination in human B cell development.

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.

viSNE enables visualization of high dimensional single-cell data and reveals phenotypic heterogeneity of leukemia.

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.