Analyzing the Phenotypic and Functional Complexity of Lymphocytes Using Cytof (Cytometry by Time-Of-Flight)

RESEARCH HIGHLIGHT

Analyzing the phenotypic and functional complexity of lymphocytes using CyTOF (cytometry by time-of-flight)

Guobing Chen and Nan-ping Weng

Cellular & Molecular Immunology (2012) 9, 322–323; doi:10.1038/cmi.2012.16; published online 28 May 2012

he human immune system is equipped antigen peptide-MHC tetramers on human status of cytokines, cytotoxic granule T with vast numbers of lymphocytes pos- CD81 T cells. These parameters covered components and CD107, a degranulation sessing distinct antigen specificities. The nine functional attributes and distinguished marker. A shift in the proportions of enormous repertoire and heterogeneity of all reported CD81 T-cell subsets, such as cells with each phenotype was identified after lymphocytes ensures their ability to effec- naive, central memory, effector memory, in vitro stimulation with either PMA/ tively fight against external microbial inva- terminal effector, long-lived memory pre- ionomycin or anti-CD36anti-CD28. PMA/ ders, as well as internal aberrant cells. cursor effector cells and short-lived effector ionomycin induced a greater number of dis- However, the understanding of the specific cells. The authors used principal compon- tinct functional types of CD81 T cells than phenotypes and functions of lymphocytes is ent analysis (PCA) to analyze the large did anti-CD36anti-CD28. However, because hindered by the lack of tools that can be used CyTOF datasets and presented their results these studies were performed at the same to visualize this complexity. Fluorescent dye- either in two-dimensional PCA or in com- timepoint after stimulation, it is not clear based flow cytometry, which can simulta- bination with a protein structure program whether the observed difference in the num- 1 neously analyze 8–10 parameters of a single (PyMOL) as three-dimensional PCA. ber of functional types of CD8 T cells 1 cell, is commonly used in immunological CD8 T-cell subsets visualized by three- between these two stimuli is unique to the research, but further expansion of the num- dimensional PCA show a uniform ‘folded stimulators or reflects the difference in cell ber of parameters (up to 10–18) is limited by Y’ pattern. The previously defined subsets response kinetics to different stimulators, or the overlapping excitation and emission spec- are grouped in distinct locations, and the possibly represents a combination of these tra of different fluorescent dyes. Recently, interrelatedness of these subsets can be factors. Nevertheless, the functional hetero- 1 Bendall et al.1 used transition element iso- observed clearly. For example, naive T geneity of CD8 T cells can now be visualized topes as chelated antibody tags for the atomic cells are present at the base of the Y, and by the combinatorial diversity of the express- mass spectrometric analysis of single cells, short-lived effector cells and central mem- ion status of large numbers of effector mole- called cytometry by time-of-flight (CyTOF), ory cells form distinct nodes at the tips of cules. It is certain that more types of 1 which expanded the analysis to 34 or more the Y pattern (Figure 1a). Furthermore, a functional CD8 T cells will be uncovered parameters simultaneously. In addition to graded progression of some markers could as more effector molecules are included in greatly increasing the number of the para- be observed between the subsets, especially the analysis. meters that can be evaluated, these isotopes within the memory compartment, gen- Further extending this analysis to different 1 have very little crosstalk and no need for back- erating a continuum of T-cell phenotypes. viral-specific CD8 T cells, the authors made a This continuity reveals the stochastic nat- very intriguing finding, i.e., different viral- ground adjustment, thus eliminating two 1 1 other common problems associated with ure of the expression of CD8 T-cell pro- specific CD8 T cells have distinct niches in fluorescent dyes. gression markers, and the pattern might the CyTOF three-dimensional PCA. Three Using this newly developed CyTOF tech- be useful for defining the differentiation virus-specific cells were inspected: two cause nology, Newell et al.2 analyzed the pheno- state of a given cell or identifying corre- chronic infection (Epstein–Barr virus and lates of effective versus non-effective cytomegalovirus), and one causes episodic typic and functional characteristics of 1 human CD81 T cells by measuring 17 cell CD8 T cell-mediated immune responses. acute infection influenza virus (Flu). It could also be used for the classification 1 surface markers, six cytokines, two cyto- Epstein–Barr virus-specific CD8 Tcells of additional subsets of CD81 T cells, such toxic granule components and three viral had effector memory T cell-like phenotypes, as exhausted T cells and the recently whereas cytomegalovirus-specific CD81 T described memory T cells with stem cell- cells had the properties of both late-stage effec- 3 Laboratory of Molecular Biology and Immunology, like properties. tors and effector memory cell T cells. In con- 1 National Institute on Aging, National Institutes of One major contribution of this study is trast, Flu-specific CD8 T cells showed central Health, Baltimore, MD, USA its illustration of the functional complex- memory T cell-like phenotypes (Figure 1b). Correspondence: , Dr NP Weng, 251 Bayview Blvd, 1 2 Suite 100, Baltimore, Maryland 21224, USA. ity of CD8 T cells. Newell et al. iden- Furthermore, they found that none of these 1 E-mail: [email protected] tified over 200 functional phenotypes of viral antigen-specific CD8 T cells expressed Received 17 April 2012; accepted 21 April 2012 CD81 T cells defined by the expression CD49d (a4 integrin, VLA-4), which suggests Research Highlight 323

a PC2 Despite the weaknesses of the CyTOF Tcm technique, which include: (i) the lower sig- nal from metal-labeled probes compared with fluorescent probes; (ii) its inability to Naive Tem be used for cell sorting; and (iii) the requirement for specific instruments, CyTOF technology can detect more than twice the maximum number of tags avail- able for fluorescence-based flow cytometry, PC1 Tslec and it offers an unprecedented depth for analyzing the complexity of the phenotypes

PC3 and functions of lymphocytes, as well as other types of cells. It will likely become b an essential tool in immunology and in other fields that currently use flow cytome- CMV EBV Flu try. In CD81 T cells, this study has revealed 104 104 104 the scale of phenotypic and functional com- 103 103 103 plexity of viral specific CD81 Tcells. 102 102 102 Further characterization of these different 101 101 101 CD81 T cells will provide new insights into differentiation 0 0 0 their relative contributions to an effective 1 2 3 4 1 2 3 4 1 2 3 4 0 10 10 10 10 0 10 10 10 10 0 10 10 10 10 immune response. TN, Tcm, Tem, Teff

Figure 1 Visualization of CD81 T-cell subsets identified by CyTOF. (a) A uniform ‘folded Y’-shaped pattern 1 was established using CyTOF PCA. (b) The distinct niche occupation (red) of different viral specific CD8 T 1 Bendall SC, Simonds EF, Qiu P, Amir E, Krutzik PO, cells was demonstrated by CyTOF two-dimensional PCA. CMV, cytomegalovirus; CyTOF, cytometry by time- Finck R et al. Single-cell mass cytometry of of-flight; EBV, Epstein–Barr virus; PCA, principal component analysis; Tcm, central memory T cell; Teff, differential immune and drug responses across a terminal effector T cell; Tem, effector memory T cell; Tn, naive T cell; Tslec, short-lived effector T cell. human hematopoietic continuum. Science 2011; 332: 687–696. 2 Newell EW, Sigal N, Bendall SC, Nolan GP, Davis MM. that these cells may share a cell trafficking/ virus-specific antigen exposure and epitope Cytometry by time-of-flight shows combinatorial cytokine expression and virus-specific cell niches migration pattern that does not include the placement within a dominance hierarchy within a continuum of CD81 T cell phenotypes. participation of CD49d. However, the cause may provide some explanation. Further Immunity 2012; 36: 142–152. of these distinct characteristic responses of study will be needed to elucidate the dis- 3 Gattinoni L, Lugli E, Ji Y, Pos Z, Paulos CM, Quigley 1 MF et al. A human memory T cell subset with stem CD8 T cells to different viruses is cur- tinct and shared characteristics of these cell-like properties. Nat Med 2011; 17: 1290– 1 rently unknown. The authors suggest that viral-specific CD8 T cells. 1297.

Cellular & Molecular Immunology