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Keywords:

  • flow-based secretion assay;
  • live cells;
  • capture;
  • nanoparticles

COMMENTARY

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Flow cytometry was designed to analyze cells rather than soluble molecules. In the current issue of Cytometry, Fitzgerald and Grivel (page 205) describe a new technique that allows seeing soluble molecules secreted by the cells with flow cytometry.

We owe to flow cytometry a detailed characterization of immune cell subsets. The extensive phenotypic analysis performed by multi-parametric detection of cell surface markers allows a detailed inventory of the molecules expressed on the cell surface providing clues to understanding the mechanisms of differentiation, activation, and function. However, antigenic determinants on plasma membrane are only a fractional cellular ID. The other, not less important characteristic of immune cells is what they secrete. Indeed, secreted molecules, in particular cytokines are an important part of the regulation and coordination of various immune responses. Therefore, the analysis of cytokines, chemokines, or effector molecules that cells release is paramount in studying immune responses in vaccination or diseases (1, 2). Since flow cytometry cannot see soluble molecules, the cytokine production is evaluated by intracellular staining (3). However, this method identifies cells, which store cytokines rather than secrete them. Moreover, some cytokines, like RANTES are constitutively stored by many cells but are secreted only by those that have been adequately stimulated. Since intracellular staining of cytokines requires cell fixation and permeabilization, no further culturing of these cells or analysis of their gene expression profile can be performed. Alternatively, cytokine secretion can be studied with ELISPOT, which does not allow full characterization of the secreting cells (4).

Fitzgerald and Grivel describe a novel assay that does not have the disadvantages of the assays mentioned above. Their technique allows the capture of different secreted proteins from live cells using a versatile system of nanoparticles. This new assay is based on the use of either magnetic or quantum dots nanoparticles, that are coupled to polyclonal anti-mouse IgG antibodies to which mouse antibodies against the cytokine of interest (“capture antibody”) are bound together with cell surface-antigen-specific mouse antibody, which is used to anchor nanoparticles to the cell surface (Fig. 1). Because nanoparticles are coated with anti-mouse IgG, any mouse antibodies against human antigens can be bound to them. This system is not restricted to the use of mouse monoclonal antibodies, and can be tailored to any type of antibodies by using beads coupled to the relevant polyclonal antibodies. While incubated with cells, such nanoparticles will, through the targeting antibody, bind to the targeted receptor on the cell surface and through the capture antibody will bind cytokine secreted by this cell. Virtually any antibody can be used for targeting a cell surface marker as well as for capturing the secreted molecule.

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Figure 1. Schematic representation of the universal secretion assay using nanoparticles. The targeting antibody (yellow) binds to the targeted receptor of interest at the surface of the cells. The capture antibody (red) binds to the molecule of interest released by the cells. The captured molecule is revealed by a f luorescent-labeled antibody (black) targeting the molecule of interest. Virtually any antibody can be used for targeting a cell surface marker as well as for capturing the secreted molecule.

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Earlier, several capture assays were developed to analyze and enrich live cytokine-secreting cells (5, 6). Similar to the technique described by Grivel and Fitzgerald, in these assays a cytokine secreted by the cell is captured either by a matrix in which the cell is embedded, or by particle- attached hetero-bispecific antibodies. Both methods are cumbersome, and require skills and time. This is the reason that to date, just less than ten cytokines can be studied using commercially available cell secretion kits. In contrast, the method suggested by Grivel and Fitzgerald is easy to implement and the whole procedure requires only a few hours to perform. Nanoparticles carrying anti-mouse IgG are universal and can be used to detect any secreted molecule provided mouse antibodies against these molecules are available. The use of magnetic nanoparticles allows rapid separation of the beads coated with polyclonal anti mouse IgG antibodies from the unbound antibodies on a magnet. The coated qdots can be purified by ultrafiltration and allow the direct detection of cells targeted by the nanoparticles by flow cytometry.

The authors successfully tested their methods by comparing it with intracellular staining and the commercially available capture assay for the cytokines IL-2 and IFN-γ, for which such an assay exists. Also, the authors were able to measure the secretion of MIP-1α, MIP-1β and RANTES for which commercial capture kits are not available. I strongly believe that the new technique will have important applications. For example, it may be used to assess the cytotoxic capacity of T cells (7). To date, the surrogate marker of killing function of T cells is the detection of the degranulation marker CD107 externalized at the cell surface when granules are released (8). However, this degranulation marker does not inform on the content of these granules. Being able to quantify the secreted, rather than intracellular stored effector molecules involved in killing target cells would provide a better measure of the cytotoxic capacity of T cells. Another application of the technique would be to target the nanoparticle by using MHC-loaded peptides to detect, among antigen-specific T cells, the cells that do secrete cytokines and assess their capacity to secrete different molecules (9), as well as their clonal properties, such TCR gene usage.

Importantly, in all these and other possible applications of the new technique allowing identification of cells secreting a product of interest (not necessarily cytokines) is combined with a full phenotypic characterization of the cell population with the cutting-edge polychromatic flow cytometry. Also, the secreting cells can be sorted further for cell culture or RNA isolation, which is not the case with the classical techniques of intracellular staining or ELISPOT assays. The last but not least of the new technique's advantages is its low cost. I think that the new technique developed by Fitzgerald and Grivel will quickly become popular and will be used in different laboratories to decipher mechanisms of functioning of multicellular systems in norm and pathology.

LITERATURE CITED

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  2. COMMENTARY
  3. LITERATURE CITED