Biology Reference
In-Depth Information
and CMVÐallowing the possibility of simultaneously tracking antigen-speci®c
CD8
T cells to more than one virus within the same individual.
FUNCTION
It may be intuitive to make the extrapolation between quantitative assessments
of tetramer-positive cells with functional CTL epitope recognition. However,
many studies have noted a discrepancy between tetramer staining and CTL
function (Appay et al., 2000; Gray et al., 1999; Zajac et al., 1998), suggesting
that these cells are functionally defective. Lack of function (CTL activity; IFN-
g release or perforin content) may be related to loss of CD4
T-cell help in the
case of anti-LCMV CD8
cells in CD4-de®cient mice (Zajac et al., 1998) and
HIV-1 infected patients (Spiegel et al., 2000a), due to anergy (Lee et al., 1999),
or due to impaired maturation (Appay et al., 2000). It is likely during the
asymptomatic stage of HIV-1 infection that tetramer-positive cells are marking
either memory cells (Gray et al., 1999) or defective e¨ector CTL. In the acute
stage of HIV-1 infection, a recent study has shown that high frequencies of
HLA-B27-restricted tetramer-positive cells exist prior to seroconversion and are
closely related to the temporal appearance of viral load (Wilson et al., 2000).
Moreover, a prior study (Ogg et al., 1999b) shows that tetramer-positive cells
during acute HIV-1 infection are probably marking functional CTL that can
control viral replication. It may be postulated that at some stage after early
HIV infection, antigen-speci®c cells cease to be functionally competent. This is
highlighted by a study that we undertook to examine function of tetramer-
positive cells in chronically HIV-1-infected individuals (Gray et al., 1999).
Freshly stained tetramer positive cells were readily detectable in the peripheral
blood ( Fig. 15.1B) with Gag-speci®c cells invariably at higher frequencies
than Pol-speci®c cells. Concomitant direct CTL lysis of PBMC revealed no
appreciable CTL activity (Fig. 15.1B) (Gray et al., 1999). CTL activity was
only observed after exposure of cells to antigen in culture that resulted in a 20-
to 40-fold expansion of tetramer-positive CD8
cells ( Fig. 15.2, A and B).
PHENOTYPE
Flow cytometry o¨ers a valuable tool to simultaneously measure the expression
of multiple surface and cytoplasmic antigens and has been used to charac-
terize di¨erent cell populations within a heterogeneous mix of PBMC. Peptide/
tetramer complexes o¨er a further tool that allows the phenotypic expression of
epitope-speci®c CD8
cells.
Early tetramer studies showed that epitope-speci®c cells were largely to
be found in the CD45RA negative population and predominantly expressed
CD45RO, compatible with our knowledge of antigen-committed cells losing
CD45RA and gaining the CD45RO isoform (Michie et al., 1992). Recent evi-
