Mass cytometry study on the immune responses of human and mouse peripheral blood mononuclear cells

Abstract

The immune system is comprised of heterogeneous mixture of immune cells with various phenotypes and expressions of numerous proteins. The complex network of these immune cells and related proteins protects our body against foreign pathogens via coordinated biological processes (i.e., an immune response) occurring within a living organism. Among many components of immune system, the peripheral blood mononuclear cells (PBMCs), comprised of a diverse mixture of highly specialized immune cells, are known to play a key role in fighting against infection. Therefore, PBMCs have been considered as an important tool for researchers and clinicians working on human health and disease, since they can be used to test immune responses of our body to various external stimuli and improve our understanding of various diseases, help us to develop new therapeutic treatments, and assess the efficacy of newly developed drugs. Currently, fluorescence-based flow cytometry, which differentiate each immune cell based on their expressions of surface proteins, is the widely-used method for the monitoring of immune response. However, the fluorescence-based flow cytometry has drawbacks in providing sufficient information on the immune systems comprised of highly heterogeneous cells with multiple phenotypes and expressions of various proteins, due to its limited number of detection channels caused by the spectral overlaps between individual fluorescence channels. Mass cytometry (CyTOF), a novel flow cytometry technique with metal isotope detection by time-of-flight ICP-MS instead of light scattering and fluorescence detection, was recently developed and its ability to detect multiple metal-tagged biomarkers with minimal overlap and cellular background interference makes it become widely used in biomedical research, from the deep phenotyping of heterogeneous cells, mapping of cell differentiation and disease progression to high-resolution profiling of the cell cycle, differential cytokine expression and signaling response. The current mass cytometry instrument (i.e., the Helios system) allows up to 50 metal isotope labels (with atomic weights ranging from 75 to 209) to be detected simultaneously at a single-cell resolution. However, the high dimensional data produced by the mass cytometry instrument is difficult to visualize and interpret. Therefore, it is necessary to adapt advanced data analysis algorithms for dimensionality reduction or automated clustering. For instance, the dimensionality reduction algorithms, such as t-SNE (t-distributed stochastic neighbor embedding) or UMAP (uniform manifold approximation and projection) help us to recognize high-dimensional mass cytometry data in easy-to-understand 2-dimensional plot and the automated clustering algorithms, such as PhenoGraph, can be used to distinguish cellular clusters based on their attribute characteristics. New clues can be obtained about information that was difficult to grasp from a human perspective Therefore, mass cytometry combined with high-dimensional data analysis algorithms provide insights on the complex immune systems and enables us to have a more comprehensive understanding of their heterogeneity. In this thesis, using single-cell based mass cytometry, I have investigated the immune responses of human and mouse peripheral blood mononuclear cells affected by various kind of stress or stimulation, such as liver fibrosis and exposure to silver nanoparticles. In the Chapter 1 of this thesis, mass cytometry was used to investigate immune response of mouse peripheral blood mononuclear cells (mPBMCs) affected by liver fibrosis. The number of liver disease, especially liver fibrosis, patients has increased continuously due to the development of modern society. However, there are lack of researches or simple diagnostic method for liver fibrosis, and to determine the target treatment of drugs and effects due to the complex factors of the liver disease. Here, mPBMC of liver fibrosis mice model (normal chow:7, liver fibrosis mice induced by thioacetamide (TAA): 8, and drug treated mice:8) was analyzed, and population % and phenotypic changes were assessed to demonstrate the liver fibrosis related immune response using mass cytometry with various computational analysis. This chapter investigated the immunological reactions in mouse immune blood cells that play a crucial role in liver fibrosis diagnosis. Furthermore, the target efficiency of the drug was investigated by analyzing recovery of mPBMC population % to the normal state when the drug was administered. This research provided one of the methodological approaches applying mass cytometry to make a diagnosis of liver fibrosis as well as to test efficiency of drug for the clinical or usages. In the Chapter 2 of this thesis, mass cytometry was used to explore size-dependent immune response of human peripheral blood mononuclear cells (hPBMCs) exposed poly-vinyl-pyrrolidone (PVP) coated Ag NPs(PVPAg10, PVPAg20, and PVPAg40) for a high-dimensional single-cell analysis technique capable of phenotyping immune cells populations. Silver nanoparticles (Ag NPs) have been widely used in consumer products and biomedical applications due to their antiseptic properties1,2. The increasing production and use of Ag NPs have raised concerns about their adverse effects on human health, especially on the immune system. Although, there have been many interesting studies on the immune response of Ag NPs in recent years1–3, comprehensive understandings on their immunomodulatory responses are still lacking, mainly due to the complexity of immune system. Here, the smaller-sized Ag NPs, PVPAg10 and PVPAg20, and the larger sized Ag NP, PVPAg40 were ex vivo exposed to human PBMCs. This research indicate that the PVPAg10, PVPAg20 and PVPAg40 exposure makes different aspect of inflammatory responses depending on their size range. Our study provides appropriate ex-vivo model using mass cytometry to predict size-dependent immune responses in hPBMC exposed to different sizes of Ag NP at single cell level as well as the better understanding of the immunomodulatory response of nanotechnology-based applications.