Cytometry for immunology: A stable and happy marriage


*Correspondence to: Andrea Cossarizza, Department of Surgery, Medicine, Dentistry and Morphological Sciences, University of Modena and Reggio Emilia, via Campi 287, Modena 41125, Italy. E-mail: (or) Andreas Radbruch, German Rheumatism Research Center, Charité University, Hannoversche Strasse 27, Berlin 10115, Germany. E-mail:

The XV International Congress of Immunology (ICI) held in Milan, Italy, August 22–27, 2013 is likely a perfect occasion to celebrate a marriage that was officiated in the mid-1970s, when monoclonal antibodies (mAbs) were created and became available for research. After a very brief period of engagement, the “official exchange of rings” was represented by the possibility to conjugate mAbs with adequate fluorochromes. This changed almost completely the main use of an instrument, that is, the flow cytometer, that had been created and commercialized few years before for the measurement of cellular DNA by fluorescent intercalating dyes such as ethidium bromide. The recognition of immune cells stained with fluorescent mAbs, typically performed by fluorescence microscopy, was then passed to complex (and at that time quite esoteric) instruments that could analyze thousands of cells in a few seconds, and could provide informative and highly repetitive results.

In the early 1980s, the dramatic epidemics of human immunodeficiency virus (HIV)/AIDS pointed out the importance of CD4+ T cells for most activities of the cognate immune system, and the importance of the loss of this cell type due to the action of the virus or excessive immune activation. The quantitation of CD4+ T cells in a growing number of patients required the development of cytofluorimeters that were much easier to use than the original instruments they derived from.

Such instruments are now more user-friendly than ever and can be used by almost all researchers after a relatively short period of training. As a result, in the last decades, immunologists have been using cytometric techniques to dissect almost all components of the immune response. The best demonstration is that the large majority, if not almost all, articles published in main immunological journals make use of flow cytometry. On the other side, in recent years, a large space in the journal Cytometry A has been dedicated to all the novel immunological techniques and approaches that are continuously developed to investigate, among others, cell phenotype, the quality or the amount of molecules produced or secreted by the cell populations of interest, different signaling processes, cell differentiation, proliferation or death, killing capacities, cell–cell interactions, response against tumour cells or to immunosuppressive drugs, transcription factors' activity, quantification of soluble molecules, drug uptake [1-9]. Clearly, flow cytometry is essential to sort the cells of interest for further investigations, such as cell cloning, analysis of gene expression, functional studies at the single cell level. It is also notable that the most recent advancements, and in particular mass cytometry [10], are allowing measurement of an unprecedented number of immune parameters at the single cell level, and to analyze in a relatively simple manner incredible amounts of data [11-14].

A rapid check in PubMed shows that, as of July 2013, typing “flow cytometry immunology” gives more than 54,000 articles, the first of which, curiously, is not related to lymphocytes [15]. However, during the 5th International Symposium of Flow Cytometry, Clinical and Biological Applications, held in Rome, Italy (December 2–5, 1980), a poster session was dedicated to Immunology [16].

Thus, on the occasion of the XV ICI, we thought it worthwhile to dedicate a large space to Immunology in the main journal in the field of Cytometry. In this issue, scientists with a great expertise in the field of cytometry provide a first set of four reviews that cope with important topics of interest for our community.

It is well known by all immunologists that in the last decades the group of Moretta has revealed fundamental aspects of NK cell biology. Here, Montaldo discuss how, after the first description of NK cells more than 30 years ago, the production of a variety of mAbs has allowed identification of several NK cell surface receptors and markers, as well as to characterize their functional properties (this issue page 702). A brief historical overview on the discovery of human NK cell receptors is provided, and the main phenotypic features of differentiating and mature NK cells in healthy donors as well as their alterations in certain pathologic conditions are evidenced.

Infection with the HIV causes dramatic changes to the immune system. The review by Cossarizza et al. describes some of the most relevant cellular and molecular alterations that can be detected by flow cytometry (this issue page 680). It is underlined how this technology has facilitated the understanding of the biology of the virus, as well as the characterization of the main mechanisms that the immune system activates to fight HIV. Finally, the role of flow cytometry in monitoring the efficacy of antiretroviral therapy is evidenced.

Mesenchymal stem/stromal cells (MSCs) are adult multipotent progenitors that mediate tissue regeneration, have immunomodulatory capabilities, and support hematopoiesis, or can differentiate into mesodermal cell lineages. MSCs are heterogeneous, and only well defined MSC subpopulations are endowed with one of the aforementioned abilities. Corselli et al. here discuss how current criteria used to define cultured polyclonal MSCs (expression of nonspecific markers and in vitro mesodermal differentiation) are not sufficient to fully understand and exploit the potential of these cells (this issue page 714). Flow cytometry can be used to identify a perivascular origin of MSCs: the purification of MSCs and specialized subsets thereof is now possible, thus opening new clinical perspectives.

Several immunologists are interested in antigen-specific T cells, whose cytometric identification was introduced more than 15 years ago. Starting from the methods of producing cell clones by limiting dilution in the precytometric era, several efforts have been made to identify and quantify such cells. Bacher and Scheffold discuss how several issues regarding the study of such rare cells still remain unsolved, mainly because of the extremely low frequency of antigen-specific lymphocyte subsets and the complexity of antigen recognition by T cells (this issue page 692). Such problems regard either “normal” CD4+ T cells or regulatory T cells. The most advanced technologies for the analysis of rare antigen-specific T cell populations, including naïve and regulatory T cells, are thus discussed.

Interestingly, a separate section of this issue of Cytometry A, another review points out the problems related to the identification of very rare cellular subsets, such as the bone marrow-residing hematopoietic stem cells (HSCs), which are of immunological interest as well (this issue page 721). Several considerations and aspects in multicolor flow cytometry, exemplified by analysis of human and mouse HSCs, are discussed, along with advantages and drawbacks of polychromatic flow cytometry. Novel strategies, such as the use of internal reference populations for sample analysis is finally underlined. Similarly, to further reinforce the strength of this long and happy marriage, and in this case the importance of Immunology for Cytometry, it is noteworthy that this issue of Cytometry A also publishes other articles of immunological interest, that cope with age- and gender-related changes in lymphocyte populations in Omani healthy adults (this issue page 739); with the use of image-based flow cytometry to divide activated granulocytes into unique subsets based on their degree of phagocytosis and oxidative burst (this issue page 745); with the standardization of intracellular cytokine staining (this issue page 728); with the quantification of human γδT-cells, iNKT cells and haematopoetic precursors (this issue page 676); and, finally, with the use of new violet dyes that can be conjugated to mAbs for a better characterization of a variety of cell subsets (this issue page 752).

In conclusion, this issue of Cytometry can just give a first and not exhaustive idea of the potential of flow cytometry, and the widespread use of this technology by the community of immunologists. In any case, we hope that our colleagues could find what is presented here of interest, and what will follow in another set of reviews dedicated to the use of cytometry by immunologists, in the January 2014 issue.