Biology Reference
In-Depth Information
of the intitial expansion. The remaining antigen-speci®c memory CD8
T cells
are maintained throughout the life of the host.
In HIV disease, HIV-speci®c CD8
T cells play a major role in the control
of virus during HIV primary infection but in most infected individuals do not
completely prevent viral replication. The recent introduction of more quantita-
tive methods for measuring antigen-speci®c CD8
T cells, including the use
of MHC-peptide complexes and cytokine ¯ow cytometry, has enabled a more
precise analysis of the CD8
T-cell response in HIV disease. A number of re-
cent studies have examined CD8
T-cell cytokine responses to speci®c peptides
or peptide mixtures (Appay et al., 2000; Betts et al., 2000; Dalod et al., 1999;
Goulder et al., 2000; Kern et al., 1998) to de®ne speci®c epitopes targeted by
CTL ( Dalod et al., 1999; Donahoe et al., 2000; Kern et al., 1998; Maecker et
al., 2001) and to characterize CD8
T-cell repertoires in HIV disease (Dalod
et al., 1999; Goulder et al., 2000; McMichael et al., 2000; Sousa et al., 1999).
These studies have taken advantage of the multiparameter capability of ¯ow
cytometry for simultaneous quanti®cation of HIV-speci®c CD4
and CD8
T
cells. Figure 18.1 illustrates four-color ¯ow cytometric analysis of both CD4
and CD8
T-cell responses to mixtures of 15-mer overlapping peptides derived
from the sequences of p55 Gag protein of HIV or pp65 protein from human
cytomegalovirus (HCMV ). This analysis allows the comparison of the relative
contribution of CD8
and CD4
T cells to the maintenance of virus-speci®c T
cell immunity. In asymptomatic HIV seropositive individuals, for example, high
frequencies (5±20%) of HIV antigen-speci®c CD8
T cells were observed when
compared with the lower frequencies (0.1±3%) of speci®c CD4
T-cell responses.
The CD8
T-cell responses, however, were more variable over time compared
with the more stable CD4
T-cell responses (unpublished observations).
By using overlapping peptides spanning an entire protein sequence, CD8
T-cell responses can be detected to multiple epitopes, regardless of human
leukocyte antigen ( HLA) type. Although 9 amino acid peptides produce the
strongest CD8 responses, 15 amino acid peptides can produce similar levels of
CD8 responses, while also generating CD4 responses. An algorithm developed
by Florian Kern ( Kern et al., 1998), in which consecutive 15-mer peptides
overlapping by 11 amino acids, has been used to e¨ectively detect epitopes
recognized by both CD4
and CD8
T cells and to detect T-cell responses
to speci®c viral proteins (Kern et al., 1998; Maecker et al., 2001; Picker and
Maino, 2000). Figure 18.2 shows a typical whole blood cytometric analysis of
a CD8
T-cell response against peptide mixtures derived from the major HIV
proteins from an HIV-seropositive individual.
Using this approach, investigators can assess a number of important features
of CD8
e¨ector/memory T-cell responses to HIV. Thus, not only can the
magnitude of the total CD8
T-cell response be quantitated, but, in addition,
comparison of responses to individual subunits and epitopes can be analyzed.
Goulder and colleagues (Goulder et al., 2001b) recently demonstrated, using
CFC in longitudinal studies, that CTL responses that are present in chronic
HIV infection may di¨er substantially from those that constitute the initial
antiviral responses. Appay and co-workers (Appay et al., 2000) combined the
