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α-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 (DC) differentiation and maturation and to block oxidative phosphorylation. To identify the critical metabolic pathways leading to human DC functional suppression, here, we used 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 DCs were 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 DC stimulatory capacity. Tumor-derived AFP bound significantly more polyunsaturated fatty acids (PUFA) than cord blood–derived AFP. PUFAs bound to AFP increased metabolic skewing and promoted DC functional suppression. PUFAs inhibited DC differentiation in vitro, and ω-6 PUFAs 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 antitumor immunity.Significance:α-Fetoprotein (AFP) is a secreted tumor protein and biomarker with impact on immunity. Fatty acid–bound AFP promotes immune suppression by skewing human dendritic cell metabolism toward glycolysis and reduced immune stimulation.

α-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 (DC) differentiation and maturation and to block oxidative phosphorylation. To identify the critical metabolic pathways leading to human DC functional suppression, here, we used 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 DCs were 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 DC stimulatory capacity. Tumor-derived AFP bound significantly more polyunsaturated fatty acids (PUFA) than cord blood–derived AFP. PUFAs bound to AFP increased metabolic skewing and promoted DC functional suppression. PUFAs inhibited DC differentiation in vitro, and ω-6 PUFAs 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 antitumor immunity. Significance: α-Fetoprotein (AFP) is a secreted tumor protein and biomarker with impact on immunity. Fatty acid–bound AFP promotes immune suppression by skewing human dendritic cell metabolism toward glycolysis and reduced immune stimulation.

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. Significance: This work establishes direct functions for mSWI/SNF in translation and demonstrates that alterations in mSWI/SNF confer a therapeutic vulnerability to translation pathway inhibitors in cancer cells.

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.

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.

Supplementary Figure from The Interaction of SWI/SNF with the Ribosome Regulates Translation and Confers Sensitivity to Translation Pathway Inhibitors in Cancers with Complex Perturbations

Supplementary Data from The Interaction of SWI/SNF with the Ribosome Regulates Translation and Confers Sensitivity to Translation Pathway Inhibitors in Cancers with Complex Perturbations

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.Significance:This work establishes direct functions for mSWI/SNF in translation and demonstrates that alterations in mSWI/SNF confer a therapeutic vulnerability to translation pathway inhibitors in cancer cells.

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.

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.

Lymphoid specification in human hematopoietic progenitors is not fully understood. To better associate lymphoid identity with protein-level cell features, we conducted a highly multiplexed single-cell proteomic screen on human bone marrow progenitors. This screen identified 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 coincided with granulocyte-monocyte progenitor (GMP) immunophenotype, their accessible chromatin regions showed enrichment for lymphoid-associated transcription factor (TF) motifs. TdT expression on GMPs was inversely related to the SLAM family member CD84. Prospective isolation of CD84loGMPs demonstrated robust lymphoid potentialex vivo, while still retaining significant myeloid differentiation capacity, akin to LMPPs. This multi-omic study identifies previously unappreciated lymphoid-primed progenitors, redefining the lympho-myeloid axis in human hematopoiesis.

Solid organ transplant remains a life-saving therapy for children with end-stage heart, lung, liver, or kidney disease; however, ∼25% of allograft recipients experience acute rejection within the first 12 months after transplant. Our ability to detect rejection early and to develop less toxic immunosuppressive agents is hampered by an incomplete understanding of the immune changes associated with rejection, particularly in the pediatric population. Here we used high-dimensional single-cell proteomic technologies (CyTOF) to generate the first detailed, multi-lineage analysis of the peripheral blood immune composition of pediatric solid organ transplant recipients. We report that the organ transplanted impacts the immune composition post-transplant. When taking these allograft-specific differences into account, we further observed that differences in the proportion of subsets of CD8 and CD4 T cells were significantly associated with allograft health. Together, these data form the basis for mechanistic studies into the pathobiology of rejection to develop less invasive tools to identify early rejection and new immunosuppressive agents with greater specificity and less toxicity.

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 spectra 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-μm 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 find 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.

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 4-fold. These same parameters also efficiently resolved two other anuclear particles: human red blood cells (RBCs) and platelets. The addition of palladium-based mass-tag barcoding to our approach improves mass cytometry of synaptic particles.

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.

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.

Single-cell technologies generate large, high-dimensional datasets encompassing a diversity of omics. Dimensionality reduction enables visualization of data by representing cells in two-dimensional plots that capture the structure and heterogeneity of the original dataset. Visualizations contribute to human understanding of data and are useful for guiding both quantitative and qualitative analysis of cellular relationships. Existing algorithms are typically unsupervised, utilizing only measured features to generate manifolds, disregarding known biological labels such as cell type or experimental timepoint. Here, 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 users to tailor visualizations to separate specific aspects of cellular heterogeneity. We implement feature selection by hybrid subset selection (HSS) and demonstrate that this flexible, computationally-efficient approach generates non-stochastic, interpretable axes amenable 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 exploration of single-cell mass cytometry, transcriptomics and chromatin accessibility data.

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.

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 (R

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

Summary 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., Graphical abstract, Highlights Multiplexed imaging of gastric toxicity associated with anti-PD-1 immunotherapy Analysis reveals severe mucosal injury associated with IFN-γ-expressing epithelial cells Inflamed gastric epithelium is accompanied by mixed infiltration of CD4 and CD8 T cells, Ferrian et al. leverage multiplexed ion beam imaging by time-of-flight to characterize a case of anti-PD-1-associted autoimmune gastritis. This study reveals gastritis characterized by severe mucosal injury, IFN-γ-producing gastric epithelial cells, and inflammation that includes CD8 and CD4 T cell infiltrates with reduced expression of granzyme B and FOXP3, respectively.

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

SummaryγδT cells contribute to host immune defense uniquely; but how they function in different stages (e.g., acute versus chronic) of a specific infection remains unclear. As the role ofγδT cells in early, activeMycobacterium tuberculosis(Mtb) infection is well documented, we focused on elucidating theγδT cell response in persistent or controlled Mtb infection. Systems analysis of circulatingγδT cells from a South African adolescent cohort identified a distinct population of CD8+γδT cells that expanded in this state. These cells had features indicative of persistent antigenic exposure but were robust cytolytic effectors and cytokine/chemokine producers. While theseγδT cells displayed an attenuated response to TCR-mediated stimulation, they expressed Natural Killer (NK) cell receptors and had robust CD16 (FcγRIIIA)-mediated cytotoxic response, suggesting alternative ways forγδT cells to control this stage of the infection. Despite this NK- like functionality, the CD8+γδT cells consisted of highly expanded clones, which utilized TCRs with different Vγ/δpairs. Theses TCRs could respond to an Mtb-lysate, but not to phosphoantigens, which are components of Mtb-lysate that activateγδT cells in acute Mtb infection, indicating that the CD8+γδT cells were induced in a stage-specific, antigen-driven manner. Indeed, trajectory analysis showed that theseγδT cells arose from naive cells that had traversed distinct differentiation paths in this infection stage. Importantly, increased levels of CD8+γδT cells were also found in other chronic inflammatory conditions, including cardiovascular disease and cancer, suggesting that persistent antigenic exposure may lead to similarγδT cell responses.

Regulatory B cells (Bregs) can suppress immune responses through the secretion of IL-10 and other anti-inflammatory cytokines. 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. We therefore applied mass cytometry to simultaneously quantify cytokine production and immunophenotype in human peripheral B cells across a range of stimulatory conditions and timepoints. While multiple B cell subsets produced IL-10, no phenotype uniquely identified IL-10+ B cells and a significant portion of IL-10+ B cells co-expressed the proinflammatory cytokines IL-6 and TNFα. Despite this heterogeneity, we found operationally tolerant liver transplant recipients had a unique enrichment of IL-10+, but not TNFα+ or IL-6+, B cells as compared to 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.

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.