Biology Reference
In-Depth Information
tors up-regulate or down-regulate which coreceptor has generated a sense of
confusion regarding not only the relevance of the observations seen but also
in the reproducibility of the e¨ects reported. Even reproducible e¨ects such as
IL-2's up-regulation of CCR5 on primary T cells is of questionable relevance
for HIV-1 infection in vivo if such an increase in CCR5
cells does not lead to a
detectable increase in viral load ( Weissman et al., 2000). On the other hand,
endogenously regulated levels of CCR5 in vivo appear to have an e¨ect on viral
replication as individuals heterozygous for the CCR5-null mutation, ccr5D32,
clearly have a delayed progression to clinical AIDS (reviewed in Carrington et
al., 1999) and appear to have lower viral loads than their wild-type counter-
parts, at least in some studies (Katzenstein et al., 1997; Walli et al., 1998).
Furthermore, the panoply of CCR5 promoter polymorphisms with disease-
modulating e¨ects suggests that genetically determined responses of CCR5 ex-
pression levels to immunoregulatory e¨ects can dictate how well individuals
can support R5 viral replication. The problem remains that there has been no
systematic attempt to quantify the levels of coreceptor a¨ected by these genetic
and immunoregulatory factors (see below for exceptions).
FACS analysis of coreceptor expression also presents technical di½culties
not immediately obvious to the uninitiated. For example, Ficoll puri®cation of
lymphocytes acutely and dramatically down-regulates expression of some viral
coreceptors ( Lee et al., 1999a; Sharron et al., 2000; Weissman et al., 2000),
obscuring attempts to correlate chemokine receptor expression with immuno-
logical or virologic parameters in vivo. In addition, chemokine receptors can
exist in distinct conformational states, not all of which may be equally acces-
sible to the virus ( Lee et al., 1999a), making the choice of the antibody used
an important variable. Recent advances in ¯ow cytometry have suggested the
importance of quantifying cell surface antigenic density in addition to the fre-
quency of antigen-positive cells ( Lavabre-Bertrand, 1996; Poncelet et al., 1996).
Antigenic density of certain cell surface markers has been ascribed prognostic
signi®cance in some malignancies (Cabezudo et al., 1999; Kuss et al., 1999) and
in HIV infection, increased antigenic density of the activation marker CD38 on
CD8
T cells has been associated with poor prognosis (Liu et al., 1996). Given
the complexities involved in measuring coreceptor expression, we propose that
measuring the absolute antigenic density of coreceptors on relevant immune
cells may allow for comparisons of data generated from various labs and pro-
vide a standardized format to compare the myriad immunoregulatory networks
a¨ecting coreceptor expression. Quantitative FACS analysis provides a way to
measure absolute antigenic density.
QUANTITATIVE FACS (QFACS) ANALYSIS
Quantitative FACS analysis (QFACS) is based on the principle of converting
the relative mean ¯uorescence intensities (MFI ) of a particular analyte cell
population into an absolute number of antibody binding sites (ABS). This
