Biology Reference
In-Depth Information
struction of new peptide/MHC tetramers for use in regions where nonsubtype B
HIV-1 infections predominate.
Peptide/MHC tetramers were ®rst described by Altman et al. (Altman et al.,
1996), who used them to label HIV-1 peptide-speci®c populations of CD8
T
cells. Syntheses of MHC class I/peptide monomers are created by reacting
Escherichia coli-expressed MHC class I molecules with a speci®c viral pep-
tide. The monomers are then biotinylated and made into tetramers with
¯uorochrome-coupled avidin to form soluble complexes that stably bind to the
TCR speci®c for the viral peptide ( Boniface et al., 1998).
Figure 15.1A shows a cartoon diagram of a heterogeneous population of CD8
T cells expressing T-cell receptors ( TCR1±4) recognizing a number of HIV-1
epitopes. Only one TCR ( TCR 2) can recognize and bind to the epitope presented
by the HLA-A2.1 tetramer. Because the core of the peptide/MHC tetramer is
avidin, its use after conjugation with biotin-streptavidin-phycoerythrin ( PE)
will allow TCR-antigen engagement to be detected using ¯uorescence anlaysis.
Thus, staining a population of PBMC consisting of oligoclonally expanded
CD8
cells recognizing HIV-1 epitopes can be rapidly identi®ed using a pre-
determined peptide/MHC tetramer complex in combination with monoclonal
antibodies to CD3 and CD8 surface antigens. HLA class I-restricted epitope
responses can then be visualized using standard ¯ow cytometry procedures.
Figure 15.1A shows a typical three-color staining scenario, with a region (R1)
placed around bright CD8
cells on a forward scatter/CD8-Cy5 plot and gated
to a dot-plot of ¯uorescein isothiocyanate ( FITC) (any choice of monoclonal
antibody) vs. peptide/MHC PE ¯uorescence. After collection of 50,000±100,000
events in the CD8
region, surface expression of an array of di¨erent antigens
(adhesion molecules or activation markers) may be assessed. In this generic exam-
ple, 1.2% of CD8
peptide/MHC tetramer positive cells are dim for CD45RA
( Fig. 15.1A).
WHAT CAN TETRAMER ANALYSIS TELL US?
The use of peptide/MHC tetramers has allowed a reappraisal of the magnitude
of CD8
T-cell responses to a speci®ed epitope. For example, the magnitude of
CD8
antigen-speci®c responses to one epitope is much higher than anticipated
from killing assays in acute viral infections: LCMV infection in mice (Murali-
Krishna et al., 1998) and EBV infection in humans (Callan et al., 1998). It has
been shown that 50% of spleen CD8
cells were speci®c for LCMV during the
acute phase of infectionÐsome 40 times higher than would be accounted for
using LDA (Murali-Krishna et al., 1998). It is possible to track ex vivo the
frequency of epitope-speci®c CD8
cells using a wide range of MHC class I-
restricted epitopes during HIV-1 infection. Table 15.1 shows a list of peptide/
MHC tetramers that have been used to track antigen-speci®c responses either in
humans or monkeys. The list is not exclusive to HIV-1/SIV and includes EBV
