Cytometry by Time-Of-Flight (CyTOF)

PI/Head: Olaf Rotzschke, Ph.D.
Email: Olaf_Rotzschke@immunol.a-star.edu.sg
CyTOF Services
Manager: Karen Teng
Email: Karen_Teng@immunol.a-star.edu.sg 
The SIgN CyTOF facility houses a next-generation flow cytometer that can acquire >40 independent parameters per cell allowing deep immunophenotyping of the immune response

Background

CyTOF®, or mass cytometry, uses molecularly tagged antibodies to detect and quantify specific cellular antigens, allowing for highly multiplexed assays. The SIgN Mass Cytometry facility houses a next-generation mass cytometer that employs a type of mass spectrometry approach known as Inductively Coupled Plasma – Mass Spectrometry (ICP-MS). Here, heavy-metal isotopic tags rather than fluorophores are used to tag antibodies or peptide-MHC tetramers and stain the cells. . This method of isotopic tagging incurs less crosstalk between channels than fluorophore-based tagging, and an increasing number of channels (>40) are available for detection. Once tagged, the cells are sprayed into a plasma torch to break the chemical bonds; here, the cellular contents ionizes and the elemental content of all atoms with a molecular weight between 100 and 200 atomic mass units is quantified.  The SIgN mass cytometer can discriminate between differences in atomic mass at the level of a single atomic mass unit (Figure 1).

image1


Figure 1: Mass cytometry approach

Technologies and Approach

This instrument can acquire >40 independent parameters per cell and uses custom antibody panels that are tailored to specific cellular analyses. Novel multiplexing technology in combination with this system is under development at the SIgN Mass Cytometry facility so that a large number of T-cell antigen specificities can be probed within the same sample.1 Using this approach, the phenotypic and functional characteristics of these cells can be evaluated in depth.2
Figure 2

Figure 2: CyTOF® systems installed at SIgN

There are many methods to analyse the CyTOF data output, including SPADE clustering analysis,3 Principal Component Analysis and Boolean gating (Figure 3).2
    
Figure 3

Figure 3: High-dimensional cellular analysis. (a) SPADE clustering analysis. This example plot shows CD8+ T cells from a healthy donor and each node is coloured by relative CD45RA expression. (b) Principal component analysis. High-dimensional data is compressed into composite dimensions, each of which is composed of the weighted sum of all original parameters. (c) Boolean gating. The 512-pixel heat plot summarizes the relative frequencies of all possible combinations of nine different functional capacities of CD8+ T cells.1-3

Peptide Epitope Discovery with Major Histocompatibility Complex Class I Molecules 

Peptide-Major Histocompatibility Complex Class I (pMHC-I) tetramers were first described in the 1990s, and is still widely used in cancer immunotherapy and vaccine development for epitope discovery, disease monitoring and immunotherapeutic monitoring of disease-specific T-cells, and its aforementioned isolation for adoptive T-cell transfer into patients. Based on classical streptavidin-based tetramer technology, each recombinant MHC-I protein is now enzymatically exchanged with different peptides instead of UV irradiation, to make multiple pMHC-I tetramers for identifying their corresponding CD8+ T-cells.

SIgN Mass Cytometry Platform employs these strategies to detect antigen-specific CD8+ T cells in human samples. In-depth phenotypic profiling of these cells is attainable using panels of 25-30 antibodies.
Figure 4

Figure 4: Peptide-Major Histocompatibility Complex Class I presentation is an adaptive immune response to foreign-viruses, intracellular bacteria and self-tumors or even develop peripheral immune tolerance. However, every antigen-derived peptide loaded on each MHC-I variant is unique to stage a cytotoxic CD8+ T-cell immune response. Here, to aid in vitro peptide epitope screening for frequent HLA-A+ alleles, HLA-A*11:01, HLA-A*02:01 and HLA-A*02:07, a de novo protein fragmentation approach is introduced to rapidly identify stabilizing peptide-MHC-I proteins which cannot be characterized in silico. Next, different stabilizing peptide-MHC-I protein tetramers can be made upon request for cytometry methods to identify matching CD8+ T-cells.

HyperionTM Imaging System (Imaging Mass CytometryTM)

Background

The HyperionTM Imaging System brings high-parameter CyTOF® technology together with imaging capability to enable simultaneous imaging of up to 37 protein markers using Imaging Mass CytometryTM. This system empowers deep profiling of tissues and tumors at subcellular resolution while preserving information in cell morphology and tissue architecture.
Figure 5

Figure 5: HyperionTM Imaging System installed at SIgN
Figure 6

Figure 6: Workflow of Imaging Mass CytometryTM

Tissue sections are labeled with metal-tagged antibody using IHC protocols. The tissue is then ablated in a laser ablation chamber and transported by a stream of inert gas into the CyTOF® for analysis. The measured isotope signals are plotted using the coordinates of each laser shot, resulting in a multidimensional tissue image (Giesen et al., Nature Methods, 2014; 11(4): 417-22).

Technologies and Approach

The HyperionTM Imaging System focuses a laser beam at precisely 1 micron to capture information at the pixel level from biological samples stained with heavy-metal isotopes-tagged antibodies. The system then transfers this information for analysis with the CyTOF® technology. The use of highly pure metal tags allows highly multiplexed imaging while eliminating the impact of tissue autofluorescence. Data generated from Imaging Mass CytometryTM is saved in MCD and TIFF format which can be integrated with multiple digital pathology software providers.

The Establishment of the first Center of Excellence (CoE) for Imaging Mass CytometryTM in Singapore and Southeast Asia 

Fluidigm Corporation and SIgN recently established a new CoE in Singapore as an approach to advance Imaging Mass CytometryTM. The center will focus on single-cell deep spatial immunoprofiling on a wide range of samples from blood to tissues, in order to demonstrate the utility of IMC across various diseases including cancers. This partnership is a leading step to facilitate the advancement of IMC in translational and clinical research. It also provides opportunities to researchers within and outside of Singapore to harness the power of IMC in uncovering new insights in health and diseases.

References

1.Newell, E.W., et al. Simultaneous detection of many T-cell specificities using combinatorial tetramer staining. Nat Methods. 6(7): 497-499. 2009
2.Newell, E.W., et al. Cytometry by time-of-flight shows combinatorial cytokine expression and virus-specific cell niches within a continuum of CD8+ T cell phenotypes. Immunity. 36 (1): 142-152, 2012.
3.Bendall, S.C., et al. Single-cell mass cytometry of differential immune and drug responses across a human hematopoietic continuum. Science. 332 (6030): 687-696, 2011.
4.Giesen, C., et al. Highly multiplexed imaging of tumor tissues with subcellular resolution by mass cytometry. Nat Methods. 11(4): 417-422, 2014.