Page 1285 - Clinical Immunology_ Principles and Practice ( PDFDrive )

CHaPtEr 92 Flow Cytometry 1247

generated during cell activation, that can be measured by using approach allows for simultaneous detection of two or more
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flow cytometry. The fluorescent probe monochlorobimane is intracellular cytokines in combination with cell surface markers
commonly used for this measurement, but it is complicated by or other intracellular markers. Important aspects of intracellular
the need to determine GSH by an independent method, such as cytokine detection include use of a protein transport inhibitor
high-performance liquid chromatography (HPLC). during activation, use of proper controls, and choice of antibodies.
Additional approaches for evaluation of cellular activation As there is little or no spontaneous cytokine production in
include assessment of intranuclear markers (Ki-67, PCNA) as circulating human lymphocytes, intracellular cytokine detection
well as surface proteins that are upregulated following cellular requires in vitro activation. Initial experience was based on
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activation (e.g., CD69, CD25, CD71). Actual cell division can supraphysiological stimulation with use of PMA and ionomycin,
be evaluated by using lipophilic membrane dyes (e.g., PKH26, but antigen-specific activation systems have also proven to be
CFSE, Cell Trace Violet), also referred to as cell tracking dyes, feasible. It should be emphasized that regardless of the activation
which lose 50% of their fluorescence with each round of cell method, the duration of activation is an important variable, as
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division. This approach has become more common in the individual cells reach maximum cytokine production at different
clinical assessment of lymphocyte function because of the capacity times. In addition, different cytokines have different optimal
to evaluate specific lymphocyte subpopulations responding to periods of activation. It is recommended that a proper kinetic
mitogenic and antigenic stimuli. Lipophilic membrane dyes also profile be established for the biological system or clinical condition
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can be used to label target cells in cell-based cytotoxicity assays. being studied. 47
Recently, an approach to evaluate lymphocyte proliferation To increase the amount of intracellular cytokines, inhibitors
following cell stimulation has been described by using the modi- of intracellular protein transport (e.g., monensin or brefeldin)
fied nucleoside EdU. Detection of DNA synthesis induced by are commonly used, which leads to accumulation of proteins
the different activating agents is measured using a copper-catalyzed within the cell. Nonspecific binding of antibody reagents is an
click chemistry, which results in EdU being covalently bonded issue, as permeabilization allows access not only to the cytokine
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to a fluorescent azide. This approach allows for assessment of of interest but also to other proteins present in much greater
cell proliferation at the cell population or subpopulation (e.g., quantities within the cell than on the cell surface. In addition,
CD3, CD4, CD8) level and can be used in association with mitogen fixation further increases nonspecific binding, and use of a
and recall-antigen stimulation. negative-control sample, which contains an excess of unlabeled
Functional evaluation of cell activation can be accomplished or “cold” anticytokine antibody, as well as a subclass-matched
with flow cytometry–directed detection of the generation of or FMO-control sample provides optimal control. When the
phosphorylated intracellular proteins associated with specific conjugated anticytokine is added to the negative-control sample,
activation signals. An example of this is the detection of phos- it can only bind to other proteins in a nonspecific manner, thereby
phorylated signal transducer and activator of transcription 1 (STAT1) providing a measure to discriminate between specific and
following interferon-γ (IFN-γ) stimulation of monocytes, which nonspecific binding. The use of directly conjugated anticytokine
has been found to be equal to or more sensitive than immunoblot- antibodies not only simplifies the staining procedure but also
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ting. This type of assay requires fixation and permeabilization provides the best distinction between specific and nonspecific
to allow entry of the specific reagent and now has been extended binding. Because the fixation agent may change the native state
to a number of additional intracellular proteins that are phos- of certain epitopes, it is also important to use antibodies that
phorylated following exposure of selected cells to specific stimuli. recognize antigens after fixation when combining cell surface
Currently, a number of intracellular signaling proteins that characterization with intracellular cytokine evaluation.
undergo phosphorylation following a specific activation signal One of the main applications of intracellular cytokine detection
can be assessed with flow cytometry by using commercially by flow cytometry has been the study and refinement of the
available reagents, some of which are offered in kit form. T-helper (Th)1/Th2/Th17 paradigms. It has recently become
The assessment of oxidative burst following cell stimulation clear that the regulated secretion of cytokines can be used to
plays a central role in neutrophil function testing where the study the response of individual T cells to both polyclonal stimuli
hydrogen peroxide-sensitive dye DHR123 is used. This procedure and specific antigens. Measuring antigen-specific T-cell cytokine
involves loading granulocytes with the dye, stimulating with expression in response to specific antigen offers a useful alternative
phorbol myristate acetate (PMA), and evaluating for fluorescence to the multimer-based approach (discussed below) to quantify
with flow cytometry. 11,44 This test has proven to be extremely the frequency of antigen-specific T cells (Fig. 92.6). 48
accurate in diagnosing patients with chronic granulomatous
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disease (CGD) and carriers of X-linked CGD. A major advantage Cell Cycle Analysis
is its sensitivity, which allows for detection of one normal cell
in a population of 1000 abnormal cells. This makes assessment ClInICal rElEvanCE
of oxidative burst a useful tool in monitoring allogeneic granu-
locyte survival after transfusion into patients with CGD, as well Cell Cycle Analysis
as a means of following donor chimerism in the setting of • Useful for screening percentage of S phase and aneuploidy
allogeneic stem cell transplantation. It also provides a method • Can be combined with cell surface studies
to identify corrected cells following gene therapy in CGD and • Can be combined with markers of apoptosis
has utility in predicting outcome. 45,46
Intracellular Cytokine Detection In addition to surface immunophenotyping and cytoplasmic
Flow cytometry affords a platform to evaluate cytokine production characterization, flow cytometry is also used in cell cycle analysis.
at the single-cell level by using cytokine-specific, directly con- PI is the most commonly used fluorochrome because of its
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jugated mAbs after fixation and permeabilization of cells. This optimal linear DNA-binding capacity in a variety of different