Biology Reference
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and CXCR4 exhibit conformational heterogeneity and choosing the ``wrong''
antibody may a¨ect the ABS value obtained by several fold ( Lee et al., 1999a;
and data not shown). For CCR5, 2D7 (Pharmingen, Torrey Pines, CA) ap-
pears to recognize the greatest number of cell surface CCR5 molecules of the
antibodies we have tested ( Lee et al., 1999a). For CXCR4, although the 12G5
clone has been used extensively for FACS analysis (primarily due to its early
availability), it appears that clones 45701, 45717, and 45718 (R&D Systems,
Minneapolis, MN ) reproducibly recognize a larger number of CXCR4 mole-
cules on primary peripheral blood lymphocytes. More importantly, these clones
also appear to inhibit more completely CXCR4-mediated infection, implying
that they bind most of the CXCR4 epitopes used by X4 viruses. Thus, although
early studies on CXCR4 quanti®cation have used the 12G5 clone, we recom-
mend that future studies use the above-mentioned clones from R&D Systems.
Another important consideration in performing QFACS analysis for co-
receptor expression is that chemokine receptors are particularly susceptible to
down-regulation upon Ficoll puri®cation and their levels are altered only lym-
phocytes even when sitting in anticoagulated whole blood left at room tem-
perature ( Reynes et al., 2000; Sharron et al., 2000; Weissman et al., 2000). In
addition, there is great individual variability in the modulatory e¨ects of in
vitro culture conditions on CCR5 and CXCR4 expression on primary lym-
phocytes and macrophages. Therefore, studies attempting to correlate chemo-
kine receptor expression levels with parameters of HIV disease progression
should be performed on fastidiously fresh whole blood using the poststaining
red blood cell lysis method. Indeed, the reproducibility of the QFACS assay is
evidenced by the fact that three separate groups using the same monoclonal
antibody (2D7 for CCR5) have arrived at the same value for the antigenic
density of CCR5 on peripheral blood CD4
cells (@5,000±10,000 antibody
binding sites per cell) (Hladik et al., 1999; Lee et al., 1999b; Reynes et al.,
2000). This was true despite the fact that standardized beads from three di¨er-
ent companies were used (QSC, Sigma, direct conversion; Qi®kit, Dako, direct
conversion; RCP-30-5, Spherotech, indirect conversion).
Given the reproducibility of the technique, we believe that QFACS should
be applied to some of the problems relating to chemokine receptor expression in
HIV pathogenesis. For example, it is now a well-established fact that cellular
activation in vitro leads to increased CCR5 expression (de Roda Husman et al.,
1999; Moore, 1997; Moriuchi et al., 1997; Ostrowski et al., 1998; Wu et al.,
1997) but only recently has it been established quantitatively (via QFACS) that
in vivo activation (as judged by human leukocyte antigen DR [HLA-DR]
expression) also results in increased CCR5 expression ( Reynes et al., 2000).
However, markers of immune activation are heterogeneous and noncoincident;
it would be fruitful for future studies to determine whether CCR5 and/or
CXCR4 expression vary with other activation markers such as CD25, CD26, or
even subsets of cytokine-secreting cells ( IL-2 or IFN-g).
In vitro studies indicate that R5 and X4 viral strains can have di¨erential
