Flow Cytometry: Immunophenotyping and More
Thomas W. Mc Closkey, Ph.D. l Associate Director, Cellular Immunology R & D, ICON Central Laboratories
FACS Canto II flow cytometer at ICON Central Laboratories
A growing area involves measuring intracellular antigens. For example, the protein bcl-2 is an anti-apoptotic regulatory protein which resides inside cells1. In order to measure it, the cell sample is fixed and permeabilized and then anti-bcl-2 monoclonal antibody is added. This reagent enters inside the cell through the holes created in the cell membrane and binds to bcl-2 protein in the intracellular compartment. In a similar manner, the important regulatory T cell population is defined by the presence of the intracellular protein FOX P3. The flow cytometric test for Treg cells involves labelling surface proteins first, typically CD3, CD4, CD25, and CD127. Then cells are fixed, permeabilized, and labelled for FOX P3 expression.
Another flow cytometric application uses dyes which are specific for the cellular constituent DNA. Propidium iodide [PI] is a common dye which stains nucleic acid and indicates whether a cell is in G0/ G1, S, or G2/M phase of the cell cycle. This is an important test for the assessment of tumors. The faster a tumor is growing, the more of its cells are dividing; thus, a particularly aggressive tumor will exhibit a high percentage of cells in S phase. A tumor with only few cells in S phase indicates that it is slow growing and might warrant a different therapeutic approach.
Other flow cytometric applications growing in popularity involve the use of fluorescent beads. For example, the Cytometric Bead Array [CBA] method uses beads of differing dye intensities2 which can be discriminated as distinct clusters on the flow cytometer. Each bead population has been pre-conjugated with monoclonal antibodies of differing specificities, for example, one bead population may recognize the cytokine IL-2 while another may bind _IFN. The CBA can also measure levels of molecules involved in signal transduction pathways. Molecules such as Zap-70, ERK, MEK, and FYN can be quantified to determine which are activated in response to a particular stimulus. These types of approaches can be utilized to study the efficacy of oncology therapies, for example, it is possible to determine if interference with the drugâs mechanism of action is being encountered. In addition to the CBA cytokine assay, cytokines may also be measured directly inside the cell3. The cells are fixed and permeabilized and incubated with anti-cytokine monoclonal antibody. For example, fluorochrome-conjugated anti-IL-2 could be used to measure the amount of IL-2 produced by specific cell populations such as Thelper and Tsuppressor lymphocytes.
An area of great biological interest involves the field of apoptosis4. Apoptosis is programmed cell death, a process of self destruction cells undergo in response to an external signal. Apoptosis is a normal physiological process; the cells in the leaves of trees undergo apoptosis in the autumn, the cells in the tails of tadpoles undergo apoptosis as they metamorphose into frogs, and potentially autoimmune human T lymphocytes undergo apoptosis in the thymus so that they are not released into the peripheral circulation. Pathology occurs when there is too much [e.g., HIV disease] or too little [e.g., autoimmunity or cancer] apoptosis. Flow cytometry has developed multiple assays for the assessment of programmed cell death, utilizing characteristics cells acquire as they actively undergo the apoptotic cascade.
As our knowledge of immune function has continued to increase, the ability to identify antigen specific lymphocytes has become a prominent focus of researchâ¦
Flow cytometry is capable of performing functional analysis of live cells. Calcium mobilization in the intracellular compartment can be quantified using the dye Indo-1. This is a powerful assay for assessing the immediate response of cells to a stimulus, as this change typically begins to occur in seconds. Another functional assay is the ability to measure the production of reactive oxygen intermediates such as hydrogen peroxide or superoxide5. This informative assay can provide a real time measurement of the ability of immune cells to produce a respiratory burst, which may be elevated under inflammatory conditions and may be deficient in certain individuals, e.g. subjects with Chronic Granulomatous Disease [CGD]. In addition, it is possible to measure phagocytosis by cytometry by using fluorescent beads or fluorescently labelled target cells. When the targets are engulfed by phagocytes, the cytometer can detect the associated change in fluorescence.
As our knowledge of immune function has continued to increase, the ability to identify antigen specific lymphocytes has become a prominent focus of research6 and the introduction of tetramers has played a major role in this field. Tetramers are MHC restricted multimeric compounds loaded with a particular peptide, whose binding specificity is restricted to T cells recognizing that antigen. So, for example, a fluorochrome-conjugated class I MHC tetramer loaded with the HIV antigen gag will only bind CD8 T cells which are HIV gag specific and the cytometer can identify those cells by their fluorescence.
In this article, I have attempted to illustrate the diverse application of flow cytometry technology. With the advent of digital instrumentation which has the capacity of measuring multiple fluorescent channels simultaneously, flow cytometry has become even more capable of answering complex biological questions. As is often the case with new methods, these techniques began in research laboratories and are rapidly moving into clinical testing, including incorporation into clinical trials. Combining assays is extremely powerful and can be tremendously informative. Not only can we quantify tetramer binding, but we can now assess the T cell receptor V_ repertoire of the HIV gag specific CD8+ T lymphocytes of an individual7. Not only can we measure apoptosis, but we can simultaneously measure apoptosis and proliferation of the CD4+ T lymphocytes of subjects8. Given current instrumentation and reagent availability, clinical trial investigators now have the capacity to precisely define cell populations and accurately characterize them in terms of phenotype and function in order to identify active cellular processes. Performed by sophisticated central laboratories during clinical trials, these approaches will provide the rising knowledge necessary to understand the basic science underlying cell function and may help to generate improved therapies for the diseases which afflict us.
References
1 Adachi Y, Oyaizu N, Than S, Mc Closkey TW, and Pahwa S, IL-2 rescues in vitro lymphocyte apoptosis in patients with HIV infection: correlation with its ability to block culture induced down modulation of Bcl-2, J Immunol, 157: 4184-4193, 1996.
2 Cohen RI, Tsang D, Koenig S, Wilson D, Mc Closkey T, and Chandra S, Plasma ghrelin and leptin in adult cystic fibrosis patients, J Cyst Fibrosis, 7: 398-402, 2008.
3 Pahwa R, Mc Closkey TW, Aroniadis OC, Strbo N, Krishnan S, and Pahwa S, CD8+ T cells in HIV disease exhibit cytokine receptor perturbation and poor TCR activation but are responsive to gamma-chain cytokine driven proliferation, J Infect Dis, 193: 879-887, 2006.
4 Mc Closkey TW, Phenix BN, and Pahwa S, Flow cytometric detection and quantification of apoptotic cells, in: Manual of Molecular and Clinical Laboratory Immunology [vol. 7], B Detrick, RG Hamilton, and JD Folds, eds, Washington, DC, ASM Press, 281-290, 2006.
5 Mc Closkey TW, Todaro JA, and Laskin DL, Lipopolysaccharide treatment of rats alters antigen expression and oxidative metabolism in hepatic macrophages and endothelial cells, Hepatology, 16: 191-203, 1992.
6 Mc Closkey TW, Haridas V, Pahwa R, and Pahwa S, HIV gag and pol specific CD8 T cells in perinatal HIV infection, Cytometry [Comm Clin Cytom], 46: 265-270, 2001.
7 Mc Closkey TW, Haridas V, Pahwa R, and Pahwa S, T cell receptor Vb repertoire of the antigen specific CD8 T lymphocyte subset of HIV infected children, AIDS, 16: 1459-1465, 2002.
8 Mc Closkey TW, Haridas V, Pontrelli L, and Pahwa S, Response to superantigen stimulation in PBMC from children perinatally infected with HIV and receiving HAART therapy, Clin Diag Lab Immunol, 11: 957-962, 2004.