Biology Reference
In-Depth Information
association, these cells are implicated in controlling the initial burst of viremia.
Antiviral pressure from CTL has been demonstrated inferentially in both early
( Borrow et al., 1997) and late stages (Goulder et al., 1997a; McMichael and
Phillips, 1997) of disease by the degree of variant epitope sequences coinciding
with speci®c CTL responses. Although recent data question the pro®ciency of
immunodominant CTL responses in control of viral replication during chronic
HIV-1 infection ( Brander et al., 1998), CTL are considered to represent an
important arm of protective immunity in the early stages of infection. The
thesis that CTL may be ine¨ective during chronic (asymptomatic) infection will
be further explored in this chapter.
Until recently, it was not possible to identify the frequency and phenotypic
nature of HLA class I-restricted CTL in the blood circulation. Classical meth-
ods for measuring antigen-speci®c CTL included direct lysis (Dyer et al., 1999),
bulk cultures ( Lamhamedi-Cherradi et al., 1992), or limiting dilution assays
( LDA) (Moss et al., 1995). These methods allow for quantitative (direct lysis or
LDA) or qualitative assessment of CTL killing function and rely on either an ex
vivo hyperactive state (direct lysis) or in vitro expansion of e¨ector cells (bulk
culture or LDA). Although these methods may allow for important functional
information to be assessed, they all limit the accurate appraisal of circulating
e¨ector cells due to a lack of sensitivity. For example, it has been estimated that
to detect a direct anti-HIV CTL response from fresh peripheral blood lympho-
cytes (peripheral blood mononuclear cells, or PBMC), a frequency of >1/50
CTL precursor cells is required to be present in the culture. This is invariably
not the case, and bulk cultures or LDA have been successfully used to expand
low e¨ector cell frequencies in an antigen-speci®c manner (Moss et al., 1995).
It is highly likely that stimulation-induced cell death occurs during culture
( Klenerman and Zinkernagel, 1997) and will result in lower estimations of
CTL activity or precursor frequencies. Data reporting the use of peptide/MHC
tetramers to measure the frequency of circulating antigen-speci®c CD8
cells
indicate that the magnitude of anti-HIV-speci®c T cells is many-fold greater
than previously estimated using conventional killing assays (Altman et al., 1996;
Murali-Krishna et al., 1998; Whitmire et al., 2000). Furthermore, peptide/MHC
tetramer analysis has allowed the direct visualization and phenotypic character-
ization of antigen-speci®c T cells (Gray et al., 1999; Ogg et al., 1998).
This chapter will focus on the use of peptide/MHC tetramers and ¯ow
cytometry to visualize and measure the frequency of class I-restricted anti-
HIV-speci®c CD8
cells in the blood circulation of HIV-1-infected individuals.
We will also discuss a re-evaluation of the functional role of CTL during viral
persistence.
PRINCIPLE OF DETECTION
The most frequently used peptide/MHC tetramers used for HIV studies are
made with HLA-A2 folded around immunodominant epitopes (9 mers) in Gag
