Biology Reference
In-Depth Information
CTL assays do not provide quantitative information on the frequency of anti-
gen-speci®c T cells. The readout of such assays re¯ects both the absolute num-
ber of responsive cells plated in the assay and the ability of these cells to ex-
ponentially expand over the 5±6 days of in vitro incubation. The latter may not
be constant from sample to sample or assay to assay, particularly in the setting
of HIV infection, where active disease may be associated with 1) viral infection
and consequent destruction of the cells responding in the assay; 2) an immune
hyperactivation state that predisposes T cells to apoptosis, particularly in vitro;
and 3) the presence of viral products with inhibitory/apoptosis-inducing activity
(Cohen et al., 1999; Shearer, 1998). In addition, long-term assays may under-
estimate antigen-speci®c frequencies when the percentage of CD4
cells in the
mononuclear preparations is low, decreasing the absolute number of respond-
ing cells in each analysis.
Recently, more quantitative functional assays have been developed that have
distinct advantages over the traditional proliferation and cytotoxicity assays.
Among these are cytokine ¯ow cytometry (CFC) ( Pitcher et al., 1999; Suni
et al., 1998; Waldrop et al., 1997) and enzyme-linked immunospot (ELISPOT )
assays (Czerkinsky et al., 1983; Hutchings et al., 1989) for detection of indi-
vidual cytokine-secreting cells. Immuno¯uorescent staining with major histo-
compatibility complex (MHC)-peptide molecular complexes (``tetramers'') has
also been used as a simple and rapid method for assessing the presence of T
cells speci®c for single epitopes of pathogenic importance (Altman et al., 1996;
Gray et al., 1999; Allen et al., 2000; Gray et al., 2000; Allen et al., 2001).
However, it is not a functional test, as it reveals only the speci®city of a popu-
lation of T cells as determined by their ability to recognize a particular peptide
bound to a particular MHC molecule. This limits the clinical utility of tet-
ramers as monitoring tools, because they are restricted by epitope and by MHC
allele, and because speci®city is not always correlated with function in certain
disease states (Goulder et al., 2000).
Both CD4
and CD8
memory T cells respond to appropriate antigen
stimulation with the rapid production of a number of e¨ector and/or immuno-
regulatory cytokines involved in maintaining antiviral immunity. Interferon
( IFN )-g and tumor necrosis factor (TNF )-a expression, for example, represent
canonical activation markers for identifying antigen-speci®c T cells (Maino et
al., 2000; McMichael et al., 2000; Picker and Maino, 2000; Pitcher et al., 1999).
To this end, we have taken advantage of the fact that both CD4
and CD8
memory T cells respond to appropriate antigen stimulation with the rapid pro-
duction of e¨ector and/or immunoregulatory cytokines to develop a ¯ow cyto-
metric approach for the qualitative and quantitative assessment of antigen-
speci®c cells. Flow cytometric detection of cytokine expression (CFC) by CD4
and CD8 T lymphocytes in response to speci®c antigen not only serves as a
marker for quantitative assessment of cell-mediated immunity but also enables
a qualitative assessment of cytokine expression by individual cells. More re-
cently, this approach has been adapted to allow determination of 1) complete
pathogen CD4
and CD8
T-cell responses in clinical samples (Maino and
