Page 1295 - Clinical Immunology_ Principles and Practice ( PDFDrive )

CHaPter 93 Assessment of Functional Immune Responses in Lymphocytes 1257

T-cell subsets or alternatively in the culture supernatant of (CTV) and Cell Proliferation Dye eFluor 670 (CPD), which have
activated cells. Typically, the cytokines measured in in vitro different excitation and emission spectra compared with CFSE.
stimulation assays include IL-2, interferon (IFN)-γ, IL-4, IL-5, These dyes also can be used for tracking lymphocyte proliferation
IL-6, IL-17, and tumor necrosis factor (TNF)-α. status in vivo, in animal models, permitting measurement of up
The process of T-cell activation as described above results in to 11 cell divisions.*
IL-2 production that drives T-cell proliferation. Measuring T-cell Another alternative to tritiated thymidine is the use of a
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proliferation after stimulation in vitro with various specific and thymidine analog, 5-ethynyl-2′-deoxyuridine (Edu ), which can
nonspecific stimuli has been a staple of the diagnostic immunol- combine with a fluorescent azide in a copper-catalyzed cycload-
ogy repertoire for several decades. T-cell proliferation assays can dition reaction (referred to as “Click” chemistry) and permits
be divided into three categories: use of nonspecific mitogenic flow cytometric evaluation of lymphocyte proliferative responses
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stimuli such as PHA, pokeweed mitogen (PWM), and concana- by assessing its incorporation into cellular DNA. Edu - labeling
valin A (Con A); physiological global stimuli based on CD3 has been shown to be a fast and sensitive method for measuring
coreceptor cross-linking (using anti-CD3, either soluble or cell proliferation and also facilitates identification of dividing
bead-bound) with or without CD28 cross-linking (using cells. It is relatively more photo-stable than CFSE and is added
anti-CD28, either soluble or bead-bound); and with or without to cells after completion of the stimulation period with the
exogenous IL-2. Antigen-specific stimulation depends on the measurement being comparable to the thymidine method in
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use of common recall antigens. regards to a gain of signal is the endpoint. The Edu assay has
The commonly used method for assessing T-cell proliferation not shown the limitations of the CFSE method in the clinical
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for decades involved measurement of H-thymidine ( H-T) laboratory and has been used to evaluate lymphocyte proliferation
incorporated into the DNA of proliferating cells. The results are in a spectrum of patients, including those with severe combined
expressed as either counts per minute (cpm) or disintegrations immunodeficiencies (SCIDs). In fact, this assay has been par-
per minute (dpm) of both activated and nonactivated cells ticularly useful in discriminating between functional T cells and
(background) cultured for a fixed period of time, usually 72 nonfunctional T cells in the context of severe T cell lymphopenia,
hours for mitogens. A reference range based on proliferation which cannot be achieved with the standard thymidine assay
results from a group of control subjects should be provided (Figs. 93.4-93.6). All the proliferation data shown in this chapter
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along with the patients’ results (both background and poststimula- utilize this Edu -based measurement of T cell proliferation, and
tion results). Although this method remains widely employed, results are typically provided for both CD45 bright positive (total
several disadvantages exist, including but not limited to the use lymphocytes) and CD3 T cells, with the former being more
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of radioactive material ( H-T); its inability to discriminate representative of the data generated with the standard thymidine
responder cell subsets or to account for cellular dilution, which assay.*
is particularly relevant in the context of T-cell lymphopenia;
and the lack of information on the contribution of cell death MEASUREMENT OF CELL-MEDIATED
after stimulation and its impact on the final result. To overcome CYTOTOXICITY
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the intrinsic shortcomings of the H-T method, newer flow
cytometry assays are currently being used in the clinical diagnostic CD8 T cells are considered the representative cytotoxic T cell in
setting and are gaining popularity because of the additional the immune system, and cellular cytotoxicity is a mechanism to
information they provide. eliminate cells infected with intracellular pathogens, allogeneic
The flow cytometric methods for measuring cell prolifera- cells, or tumor cells. CD8 T cells, like CD4 T cells, recognize
tion include the use of fluorescent dyes to identify proliferating antigen via the TCR and kill target cells via cytotoxic protein
cells. One of the more commonly used dyes, carboxyfluorescein granule exocytosis and/or cytokine production. Over the past few
diacetate succinimidyl ester (CFSE), must be carefully used when decades, the cytotoxic potential of CD4 T cells has been described,
measuring lymphocyte proliferation in vitro since it can be toxic particularly in viral infections. Although cytotoxic CD4 T cells
to cells and nonoptimal labeling conditions can impact measure- are rare in the circulation of healthy individuals (<2%), they
ment of cell proliferation and interpretation of results. CFSE is can account for substantial proportions of total CD4 T cells in
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a fluorescent cell-membrane permeable dye similar in physical certain viral infections, including but not limited to HIV. The
properties to the commonly used fluorochrome, fluorescein CD4 cytotoxic T cells appear to be a lineage of memory CD4 T
isothiocyanate (FITC). During cell proliferation, the intensity cells that contain cytotoxic granules with perforin and granzymes,
of staining in daughter cells is half that of parent cells, allowing and they are thought to arise in the context of chronic or potent
visualization of the number of rounds of cell divisions associated activation with viral infections such as cytomegalovirus (CMV),
with the successive decrease in fluorescence. The disadvantages Epstein-Barr virus (EBV), human immunodeficiency virus (HIV),
to using CFSE in the clinical laboratory are its photo-instability, or certain autoimmune diseases. 11
limitation in the number of cell divisions being identified Cellular cytotoxic activity has been conventionally measured
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(≤7 cell divisions), and interpretation requires measuring loss of by release of chromium ( Cr) from labeled target (T) cells
signal rather than gain of signal. In addition, as noted above, CFSE cultured with various ratios of effector (E) cells (varied E:T
at concentrations of 37 nM to 10 µM can be toxic to cells resulting ratio). The percentage lysis is developed using the level of
in increased cell death. Furthermore, it can modulate expression radioactivity in the supernatant of target cells in comparison to
of activation markers, resulting in a decrease in CD69, HLA-DR,
and CD25 expression. Finally, it has been reported that there is
an increase in the number of false-positive results with CFSE
making it suboptimal for measuring lymphocyte proliferation *This section has been reproduced in part with permission from ASM
in patients with severe cellular immunodeficiencies. Alternatives Press (Abraham RS, Lymphocyte Activation) (copyright permission
to CFSE include related compounds, such as Cell Trace Violet obtained)