Michael D. Leipold

Genetic and Environmental Determinants of Human NK Cell Diversity Revealed by Mass Cytometry

Both genetics and environment contribute to human NK cell diversity. NK Cell Nature Versus Nurture Natural killer (NK) cells were first discovered because of their ability to kill tumor cells without any previous exposure. However, this population is actually quite heterogeneous: Different subgroups of NK cells express different combinations of activating and inhibiting receptors that govern their specificity. Now, Horowitz et al. use mass cytometry to examine NK cell diversity in humans. The authors examined 35 parameters simultaneously in 5 sets of monozygotic twins as well as 12 unrelated donors. They found up to 30,000 phenotypic NK cell populations in a given individual. What’s more, by comparing the twins versus unrelated donors, they determined that although genetics primarily determined inhibitory receptor expression, activating receptors were controlled by the environment. These data suggest that inhibitory receptors may contribute more to NK cell self-tolerance, whereas activating receptors may guide response to pathogens and tumors. Natural killer (NK) cells play critical roles in immune defense and reproduction, yet remain the most poorly understood major lymphocyte population. Because their activation is controlled by a variety of combinatorially expressed activating and inhibitory receptors, NK cell diversity and function are closely linked. To provide an unprecedented understanding of NK cell repertoire diversity, we used mass cytometry to simultaneously analyze 37 parameters, including 28 NK cell receptors, on peripheral blood NK cells from 5 sets of monozygotic twins and 12 unrelated donors of defined human leukocyte antigen (HLA) and killer cell immunoglobulin-like receptor (KIR) genotype. This analysis revealed a remarkable degree of NK cell diversity, with an estimated 6000 to 30,000 phenotypic populations within an individual and >100,000 phenotypes in the donor panel. Genetics largely determined inhibitory receptor expression, whereas activation receptor expression was heavily environmentally influenced. Therefore, NK cells may maintain self-tolerance through strictly regulated expression of inhibitory receptors while using adaptable expression patterns of activating and costimulatory receptors to respond to pathogens and tumors. These findings further suggest the possibility that discrete NK cell subpopulations could be harnessed for immunotherapeutic strategies in the settings of infection, reproduction, and transplantation.

Barcoding of Live Human Peripheral Blood Mononuclear Cells for Multiplexed Mass Cytometry

Mass cytometry is developing as a means of multiparametric single-cell analysis. In this study, we present an approach to barcoding separate live human PBMC samples for combined preparation and acquisition on a cytometry by time of flight instrument. Using six different anti-CD45 Ab conjugates labeled with Pd104, Pd106, Pd108, Pd110, In113, and In115, respectively, we barcoded up to 20 samples with unique combinations of exactly three different CD45 Ab tags. Cell events carrying more than or less than three different tags were excluded from analyses during Boolean data deconvolution, allowing for precise sample assignment and the electronic removal of cell aggregates. Data from barcoded samples matched data from corresponding individually stained and acquired samples, at cell event recoveries similar to individual sample analyses. The approach greatly reduced technical noise and minimizes unwanted cell doublet events in mass cytometry data, and it reduces wet work and Ab consumption. It also eliminates sample-to-sample carryover and the requirement of instrument cleaning between samples, thereby effectively reducing overall instrument runtime. Hence, CD45 barcoding facilitates accuracy of mass cytometric immunophenotyping studies, thus supporting biomarker discovery efforts, and it should be applicable to fluorescence flow cytometry as well.

Platinum-conjugated antibodies for application in mass cytometry

Mass cytometry has overcome limitations of fluorescent single cell cytometry by allowing for the measurement of up to currently ∼40 different parameters on a single cell level. However, the cellular proteome comprises many more potential analytes, and current mass cytometry instrumentation allows for theoretically up to 121 different mass detection channels. The labeling of specific probes with appropriate metal ions is a significant hurdle for exploiting more of mass cytometrys analytical capacity. To this end, we here describe the labeling of antibody with natural abundance or isotopically purified platinum as formulated in cisplatin and circumventing the use of chelator‐loaded polymers. We confirm the utility of cisplatin–antibody‐conjugates for surface, intracellular, and phosphoepitope‐specific immunophenotyping, as well as for application in cell surface CD45‐based barcoding. Cisplatin‐labeling of antibody increases the analytical capacity of the CyTOF® platform by two channels based on available reagents, and has the potential to add a total of six channels for detection of specific probes, thus helping to better extend the analytical mass range of mass cytometers.

Multiparameter Phenotyping of Human PBMCs Using Mass Cytometry

The standard for single-cell analysis of phenotype and function in recent decades has been fluorescence flow cytometry. Mass cytometry is a newer technology that uses heavy metal ions, rather than fluorochromes, as labels for probes such as antibodies. The binding of these ion-labeled probes to cells is quantitated by mass spectrometry. This greatly increases the number of phenotypic and functional markers that can be probed simultaneously. Here, we review topics that must be considered when adapting existing flow cytometry panels to mass cytometry analysis. We present a protocol and representative panels for surface phenotyping and intracellular cytokine staining (ICS) assays.

Another step on the path to mass cytometry standardization.

FOR over 20 years, flow cytometry has been used to analyze samples on a single-cell basis. While six-or eight-color assays are sufficient for many routine tasks, more recent work has led to the improvement of both the hardware and reagents used in flow cytometry. The development of four-laser flow cytometers and probes with narrower, less-overlapping spectra has allowed up to at least 17-color experiments (1). More recently, mass cytometry has entered the research field, with commercial mass cytometers becoming available in the last five years. Instead of fluorescent probes, mass cytometry uses probes labeled with high atomic weight elements, such as the lanthanide series metal ions, which are then detected using inductively-coupled plasma mass spectrometry (ICP-MS). It is important to note that a cell must have at least one elemental probe in order to be detected: there is no mass cytometry analog to scatter. The first generation of mass cytometers had an acquisition mass window of approximately 93 mass units (typically AW 103–195), with second-generation machines now having an increased window (typically AW 78–212). Experiments comparing mass cytometry with flow cytometry generally agree well for “frequency of parent” and similar measures (2,3). As flow cytometry became used more in research settings, interest was raised for standardization between machines and locations (4,5). This was particularly driven by the onset of multicenter research and clinical studies, where it is often advantageous to run samples close in location and time to where they were collected, as well as addressing throughput bottlenecks for a single-center analysis unit. Several international multicenter studies have established parameters for unifying analysis capabilities for a single study (6–8). These include the initial work using reagents and cell samples in common to establish the settings necessary to harmonize individual machines’ performance to a common performance standard of signal intensities and detection limits. Mass cytometry is only now starting to address these topics. Initially, all antibodies had to be conjugated, tested, and validated in-house by the end user. The greater availability of commercial mass cytometry reagents has begun to reduce the variability that results from separate user-made batches of antibody. Similarly, calibration beads have been developed that contain various metals spanning most of the acquired mass window (9). Finck et al. demonstrated that these beads can be used to help normalize any decrease in signal intensity originating from machine-performance issues over long sample running times. While Finck et al. observed some differences in tests between two different mass cytometers, the article by Tricot et al. (10) is the first to investigate this machine-based variation in detail. In this study, the authors first used two different commercially available metal-containing solution standards to investigate whether they could observe differences in sensitivity across the mass window for three different mass cytometers (instrument generation 1). The first major point is that each of the three instruments had a slightly different ion sensitivity pattern. Two instruments were fairly similar (F and C), with signal peaking around mass 160–165. The third instrument (N) had optimal sensitivity around mass 155–160, and had a more significant drop-off in sensitivity at the higher

Computationally efficient multidimensional analysis of complex flow cytometry data using second order polynomial histograms

Many methods have been described for automated clustering analysis of complex flow cytometry data, but so far the goal to efficiently estimate multivariate densities and their modes for a moderate number of dimensions and potentially millions of data points has not been attained. We have devised a novel approach to describing modes using second order polynomial histogram estimators (SOPHE). The method divides the data into multivariate bins and determines the shape of the data in each bin based on second order polynomials, which is an efficient computation. These calculations yield local maxima and allow joining of adjacent bins to identify clusters. The use of second order polynomials also optimally uses wide bins, such that in most cases each parameter (dimension) need only be divided into 4–8 bins, again reducing computational load. We have validated this method using defined mixtures of up to 17 fluorescent beads in 16 dimensions, correctly identifying all populations in data files of 100,000 beads in <10 s, on a standard laptop. The method also correctly clustered granulocytes, lymphocytes, including standard T, B, and NK cell subsets, and monocytes in 9‐color stained peripheral blood, within seconds. SOPHE successfully clustered up to 36 subsets of memory CD4 T cells using differentiation and trafficking markers, in 14‐color flow analysis, and up to 65 subpopulations of PBMC in 33‐dimensional CyTOF data, showing its usefulness in discovery research. SOPHE has the potential to greatly increase efficiency of analysing complex mixtures of cells in higher dimensions.

Comparison of CyTOF assays across sites: Results of a six-center pilot study

For more than five years, high-dimensional mass cytometry has been employed to study immunology. However, these studies have typically been performed in one laboratory on one or few instruments. We present the results of a six-center study using healthy control human peripheral blood mononuclear cells (PBMCs) and commercially available reagents to test the intra-site and inter-site variation of mass cytometers and operators. We used prestained controls generated by the primary center as a reference to compare against samples stained at each individual center. Data were analyzed at the primary center, including investigating the effects of two normalization methods. All six sites performed similarly, with CVs for both Frequency of Parent and median signal intensity (MSI) values < 30%. Increased background was seen when using the premixed antibody cocktail aliquots at each site, suggesting that cocktails are best made fresh. Both normalization methods tested performed adequately for normalizing MSI values between centers. Clustering algorithms revealed slight differences between the prestained and the sites-stained samples, due mostly to the increased background of a few antibodies. Therefore, we believe that multicenter mass cytometry assays are feasible.

Cytokine and Leukocyte Profiling Reveal Pro-Inflammatory and Autoimmune Features in Frontotemporal Dementia Patients

The growing link between systemic environment and brain function opens the possibility that cellular communication and composition in blood are correlated with brain health. We tested this concept in frontotemporal dementia with novel, unbiased tools that measure hundreds of soluble signaling proteins or characterize the vast immune cell repertoire in blood. With these tools we discovered complementary abnormalities indicative of abnormal T cell populations and autoimmunity in frontotemporal dementia.

The anatomy of single cell mass cytometry data: The data scientist's primer to CyTOF®

Mass cytometry enables the measurement of up to 50 features on single cell. This has catalyzed a shift toward multidimensional data analysis methods, rather than the manual gating strategies as traditionally for in flow cytometry data. This shift means that data scientists are involved in the analysis process to an increasing degree. As the data is analyzed in a more unbiased fashion, where noisy or uninformative observations are not easily excluded, a deeper knowledge of the origin, noise, and modalities of the data is therefore needed to embark on useful data analysis. In this primer, we introduce the idiosyncrasies of mass cytometry data with a focus on the technical properties of how data generated with the CyTOF® system, and the characteristics of protein expression in the cells of the hematopoietic continuum, specifically targeted toward data scientists. We also provide a comprehensive online repository of scripts, tutorials, and example data.