ABSTRACT
Flow cytometry is a powerful technology for high-throughput protein quantification at the single-cell level. Technical advances have substantially increased data complexity, but novel bioinformatical tools often show limitations in statistical testing, data sharing, cross-experiment comparability, or clinical data integration. We developed MetaGate as a platform for interactive statistical analysis and visualization of manually gated high-dimensional cytometry data with integration of metadata. MetaGate provides a data reduction algorithm based on a combinatorial gating system that produces a small, portable, and standardized data file. This is subsequently used to produce figures and statistical analyses through a fast web-based user interface. We demonstrate the utility of MetaGate through a comprehensive mass cytometry analysis of peripheral blood immune cells from 28 patients with diffuse large B cell lymphoma along with 17 healthy controls. Through MetaGate analysis, our study identifies key immune cell population changes associated with disease progression.
ABSTRACT
Flow cytometry is a powerful technology for high-throughput protein quantification at the single-cell level, widely used in basic research and routine clinical diagnostics. Traditionally, data analysis is carried out using manual gating, in which cut-offs are defined manually for each marker. Recent technical advances, including the introduction of mass cytometry, have increased the number of proteins that can be simultaneously assessed in each cell. To tackle the resulting escalation in data complexity, numerous new analysis algorithms have been developed. However, many of these show limitations in terms of providing statistical testing, data sharing, cross-experiment comparability integration with clinical data. We developed MetaGate as a platform for interactive statistical analysis and visualization of manually gated high-dimensional cytometry data with integration of clinical meta data. MetaGate allows manual gating to take place in traditional cytometry analysis software, while providing a combinatorial gating system for simple and transparent definition of biologically relevant cell populations. We demonstrate the utility of MetaGate through a comprehensive analysis of peripheral blood immune cells from 28 patients with diffuse large B-cell lymphoma (DLBCL) along with 17 age- and sex-matched healthy controls using two mass cytometry panels made of a total of 55 phenotypic markers. In a two-step process, raw data from 143 FCS files is first condensed through a data reduction algorithm and combined with information from manual gates, user-defined cellular populations and clinical meta data. This results in one single small project file containing all relevant information to allow rapid statistical calculation and visualization of any desired comparison, including box plots, heatmaps and volcano plots. Our detailed characterization of the peripheral blood immune cell repertoire in patients with DLBCL corroborate previous reports showing expansion of monocytic myeloid-derived suppressor cells, as well as an inverse correlation between NK cell numbers and disease progression.
ABSTRACT
The Karolinska KI/K COVID-19 Immune Atlas project was conceptualized in March 2020 as a part of the academic research response to the developing SARS-CoV-2 pandemic. The aim was to rapidly provide a curated dataset covering the acute immune response towards SARS-CoV-2 infection in humans, as it occurred during the first wave. The Immune Atlas was built as an open resource for broad research and educational purposes. It contains a presentation of the response evoked by different immune and inflammatory cells in defined naïve patient-groups as they presented with moderate and severe COVID-19 disease. The present Resource Article describes how the Karolinska KI/K COVID-19 Immune Atlas allow scientists, students, and other interested parties to freely explore the nature of the immune response towards human SARS-CoV-2 infection in an online setting.
ABSTRACT
The molecular networks that regulate natural killer (NK) cell functions are not completely understood. Here, we present a workflow for efficient delivery of siRNA into human NK cells without compromising viability. This methodology represents a promising approach for rapidly interrogating gene functions in primary human NK cells.
Subject(s)
Killer Cells, Natural , Humans , RNA, Small Interfering/geneticsABSTRACT
BACKGROUND: Current prognostic variables can only partly explain the large outcome heterogeneity in diffuse large B cell lymphoma (DLBCL). We aimed to investigate the utility of systems-level protein and immune repertoire profiling for outcome prognostication in DLBCL. METHODS: In this retrospective study, we used proximity extension assay technology to quantify 81 immune-related proteins in serum or plasma in 2 independent cohorts in a total 111 DLBCL patients. Protein levels were assessed before and after treatment with rituximab and chemotherapy, and the patients were compared with 19 age- and sex-matched healthy blood donors. In a subset of the patients, we performed a broad mass cytometric characterization of immune cell repertoires in peripheral blood. FINDINGS: Patients displayed large deviations in protein profiles compared with healthy controls. Development of a systemic protein deviation (SPD) score provided a 4-protein-based metric that reflected the overall degree of protein deviations compared with age- and sex-matched healthy blood donors. The SPD score identified patients with very poor overall survival in both cohorts and correlated with increased frequencies of peripheral blood PD-1+ CD8+ T cells, and expansion of myeloid-derived suppressor cells. CONCLUSIONS: Our results show that a simple metric based on measurement of a small set of serum or plasma proteins can be used to probe systemic immune changes associated with poor survival in DLBCL. This finding warrants further investigation in larger, prospective studies to establish a clinical prognostic biomarker.
Subject(s)
Lymphoma, Large B-Cell, Diffuse , Programmed Cell Death 1 Receptor , Biomarkers , CD8-Positive T-Lymphocytes/pathology , Humans , Lymphoma, Large B-Cell, Diffuse/drug therapy , Prospective Studies , Retrospective StudiesABSTRACT
Natural killer (NK) cells derived or isolated from different sources have been gaining in importance for cancer therapies. In this study, we evaluate and compare key characteristics between NK cells derived or isolated from umbilical cord blood, umbilical cord blood hematopoietic stem/progenitor cells, peripheral blood, and induced pluripotent stem cells (iPSCs). Specifically, we find CD56+ NK cells isolated and expanded directly from umbilical cord blood (UCB56) and NK cells derived from CD34+ hematopoietic stem/progenitors in umbilical cord blood (UCB34) differ in their expression of markers associated with differentiation including CD16, CD2, and killer Ig-like receptors (KIRs). UCB56-NK cells also displayed a more potent cytotoxicity compared to UCB34-NK cells. NK cells derived from iPSCs (iPSC-NK cells) were found to have variable KIR expression, with certain iPSC-NK cell populations expressing high levels of KIRs and others not expressing KIRs. Notably, KIR expression on UCB56 and iPSC-NK cells had limited effect on cytotoxic activity when stimulated by tumor target cells that express high levels of cognate HLA class I, suggesting that in vitro differentiation and expansion may override the KIR-HLA class I mediated inhibition when used across HLA barriers. Together our results give a better understanding of the cell surface receptor, transcriptional, and functional differences between NK cells present in umbilical cord blood and hematopoietic progenitor-derived NK cells which may prove important in selecting the most active NK cell populations for treatment of cancer or other therapies.
Subject(s)
Cell Differentiation/immunology , Fetal Blood/cytology , Hematopoietic Stem Cells/immunology , Induced Pluripotent Stem Cells/cytology , Killer Cells, Natural/immunology , Neuroblastoma/immunology , Receptors, KIR/immunology , Cell Differentiation/genetics , Cell Line, Tumor , Cell Survival/immunology , Genotype , HLA Antigens/genetics , HLA Antigens/immunology , Histocompatibility Antigens Class I/genetics , Histocompatibility Antigens Class I/immunology , Humans , Neuroblastoma/pathology , Receptors, KIR/genetics , TransfectionABSTRACT
Understanding innate immune responses in COVID-19 is important to decipher mechanisms of host responses and interpret disease pathogenesis. Natural killer (NK) cells are innate effector lymphocytes that respond to acute viral infections but might also contribute to immunopathology. Using 28-color flow cytometry, we here reveal strong NK cell activation across distinct subsets in peripheral blood of COVID-19 patients. This pattern was mirrored in scRNA-seq signatures of NK cells in bronchoalveolar lavage from COVID-19 patients. Unsupervised high-dimensional analysis of peripheral blood NK cells furthermore identified distinct NK cell immunotypes that were linked to disease severity. Hallmarks of these immunotypes were high expression of perforin, NKG2C, and Ksp37, reflecting increased presence of adaptive NK cells in circulation of patients with severe disease. Finally, arming of CD56bright NK cells was observed across COVID-19 disease states, driven by a defined protein-protein interaction network of inflammatory soluble factors. This study provides a detailed map of the NK cell activation landscape in COVID-19 disease.
Subject(s)
Betacoronavirus/genetics , Betacoronavirus/immunology , Coronavirus Infections/epidemiology , Coronavirus Infections/immunology , Killer Cells, Natural/immunology , Pneumonia, Viral/epidemiology , Pneumonia, Viral/immunology , Severity of Illness Index , Adaptive Immunity , CD56 Antigen/metabolism , COVID-19 , Coronavirus Infections/blood , Coronavirus Infections/pathology , Female , Flow Cytometry/methods , Humans , Lymphocyte Activation , Male , Middle Aged , Pandemics , Phenotype , Pneumonia, Viral/blood , Pneumonia, Viral/pathology , Polymerase Chain Reaction , Prospective Studies , Protein Interaction Maps/immunology , Receptors, KIR/metabolism , SARS-CoV-2 , Serologic Tests , Sweden/epidemiologyABSTRACT
Cytokine-inducible SH2-containing protein (CIS; encoded by the gene CISH) is a key negative regulator of interleukin-15 (IL-15) signaling in natural killer (NK) cells. Here, we develop human CISH-knockout (CISH-/-) NK cells using an induced pluripotent stem cell-derived NK cell (iPSC-NK cell) platform. CISH-/- iPSC-NK cells demonstrate increased IL-15-mediated JAK-STAT signaling activity. Consequently, CISH-/- iPSC-NK cells exhibit improved expansion and increased cytotoxic activity against multiple tumor cell lines when maintained at low cytokine concentrations. CISH-/- iPSC-NK cells display significantly increased in vivo persistence and inhibition of tumor progression in a leukemia xenograft model. Mechanistically, CISH-/- iPSC-NK cells display improved metabolic fitness characterized by increased basal glycolysis, glycolytic capacity, maximal mitochondrial respiration, ATP-linked respiration, and spare respiration capacity mediated by mammalian target of rapamycin (mTOR) signaling that directly contributes to enhanced NK cell function. Together, these studies demonstrate that CIS plays a key role to regulate human NK cell metabolic activity and thereby modulate anti-tumor activity.
Subject(s)
Induced Pluripotent Stem Cells , Cell Line, Tumor , Cytokines , Humans , Interleukin-15 , Killer Cells, NaturalABSTRACT
Natural killer (NK) cell repertoires are made up of phenotypically distinct subsets with different functional properties. The molecular programs involved in maintaining NK cell repertoire diversity under homeostatic conditions remain elusive. Here, we show that subset-specific NK cell proliferation kinetics correlate with mTOR activation, and global repertoire diversity is maintained through a high degree of intra-lineage subset plasticity during interleukin (IL)-15-driven homeostatic proliferation in vitro. Slowly cycling sorted KIR+CD56dim NK cells with an induced CD57 phenotype display increased functional potential associated with increased transcription of genes involved in adhesion and immune synapse formation. Rapidly cycling cells upregulate NKG2A, display a general loss of functionality, and a transcriptional signature associated with increased apoptosis/cellular stress, actin-remodeling, and nuclear factor κB (NF-κB) activation. These results shed light on the role of intra-lineage plasticity during NK cell homeostasis and suggest that the functional fate of the cell is tightly linked to the acquired phenotype and transcriptional reprogramming.
Subject(s)
Killer Cells, Natural/metabolism , Apoptosis/genetics , Apoptosis/physiology , CD56 Antigen/metabolism , CD57 Antigens/metabolism , Humans , Interleukin-15/metabolism , Kinetics , NF-kappa B/metabolism , NK Cell Lectin-Like Receptor Subfamily C/metabolism , Phenotype , Sequence Analysis, RNA , Synapses/metabolismABSTRACT
Inhibitory signaling during natural killer (NK) cell education translates into increased responsiveness to activation; however, the intracellular mechanism for functional tuning by inhibitory receptors remains unclear. Secretory lysosomes are part of the acidic lysosomal compartment that mediates intracellular signalling in several cell types. Here we show that educated NK cells expressing self-MHC specific inhibitory killer cell immunoglobulin-like receptors (KIR) accumulate granzyme B in dense-core secretory lysosomes that converge close to the centrosome. This discrete morphological phenotype is independent of transcriptional programs that regulate effector function, metabolism and lysosomal biogenesis. Meanwhile, interference of signaling from acidic Ca2+ stores in primary NK cells reduces target-specific Ca2+-flux, degranulation and cytokine production. Furthermore, inhibition of PI(3,5)P2 synthesis, or genetic silencing of the PI(3,5)P2-regulated lysosomal Ca2+-channel TRPML1, leads to increased granzyme B and enhanced functional potential, thereby mimicking the educated state. These results indicate an intrinsic role for lysosomal remodeling in NK cell education.
Subject(s)
Killer Cells, Natural/metabolism , Lysosomes/metabolism , Aminopyridines/pharmacology , Animals , Granzymes/metabolism , Heterocyclic Compounds, 3-Ring/pharmacology , Humans , K562 Cells , Killer Cells, Natural/drug effects , Lysosomal-Associated Membrane Protein 1/genetics , Lysosomal-Associated Membrane Protein 1/metabolism , Lysosomes/drug effects , Mice , Receptors, KIR/metabolism , Signal Transduction/drug effects , Signal Transduction/physiologyABSTRACT
The functional capacity of NK cells is dynamically tuned by integrated signals from inhibitory and activating cell surface receptors in a process termed NK cell education. However, the understanding of the cellular and molecular mechanisms behind this functional tuning is limited. In this study, we show that the expression of the adhesion molecule and activation receptor DNAX accessory molecule 1 (DNAM-1) correlates with the quantity and quality of the inhibitory input by HLA class I-specific killer cell Ig-like receptors and CD94/NKG2A as well as with the magnitude of functional responses. Upon target cell recognition, the conformational state of LFA-1 changed in educated NK cells, associated with rapid colocalization of both active LFA-1 and DNAM-1 at the immune synapse. Thus, the coordinated expression of LFA-1 and DNAM-1 is a central component of NK cell education and provides a potential mechanism for controlling cytotoxicity by functionally mature NK cells.