Biology Reference
In-Depth Information
used to study the diversity and dynamics of the T-cell compartment. The ap-
pearance of the peripheral TCR repertoire is closely associated with the MHC
type of the subject. Indeed, in seminal studies, Silver and colleagues have
clearly shown that TCR repertoire identity increases linearily with the num-
ber of shared MHC alleles (Gulwani-Akolkar et al., 1991). Furthermore, rep-
ertoires of monozygotic twins are more closely related than those of haplo-
identical subjects, underscoring the contribution of minor histocompatibility
antigens and/or other as yet unindenti®ed genes to the generation of the mature
T-cell pool (Akolkar et al., 1993; Hawes et al., 1993; Malthora et al., 1992). As
will be discussed below, this property of the TCR repertoire imposes stringent
limitations on the kind of study groups on which these typing experiments
can be performed. In particular, cross-sectional studies are notably di½cult to
interpret unless some measure of human leukocyte antigen ( HLA) matching
has been put in place, as idiotypic repertoire di¨erences tend to act as con-
founding factors.
The TCR repertoire can be analyzed by means of a number of molecular
biology techniques. Multiparallel semiquantitative polymerase chain reaction
( PCR) assays based on complex sets of V-gene-speci®c oligonucleotide primers
were initially introduced (Choi et al., 1989; Genevee et al., 1992; Hall and Finn,
1992; Labrecque et al., 1993; Panzara et al., 1992; Rebai et al., 1994; Soudeyns
et al., 1993), soon followed by RNase protection assays ( Baccala et al., 1991)
and automated run-o¨-based methods such as the ``Spectratyping'' (Chen et al.,
1995), ``Immunoscope'' ( Pannetier et al., 1993, 1995), and ``GeneScan'' tech-
niques (Assaf et al., 2000). High-de®nition repertoire analysis can also be per-
formed using TCR DNA heteroduplex mobility shift assays ( HMA), which
reveal information relative to the clonality of T-cell subsets expressing speci®c
TCR V-region genes and allow the detection of expanded clonotypes within
complex T-cell populations (Shen et al., 1998; Soudeyns et al., 2000; Wack et
al., 1996). Due in part to the availability of well-characterized primer sets,
PCR-based analysis furnishes a somewhat complete picture of the repertoire.
However, the speci®city, kinetics and linearity of ampli®cation need to be rig-
orously controlled to validate experimental protocols (Hall and Finn, 1992).
Alternatively, TCR repertoire typing can be performed using ¯ow cytometry
and TCR V-region-speci®c monoclonal antibodies (mAbs). This method pres-
ents several notable advantages over PCR-based techniques: a) it is rapid,
technically simple, highly reproducible, and results generated can be directly
interpreted; b) it is based on cell-surface expression of the TCR and not on
levels of TCR mRNA, which are known to ¯uctuate in concert with the state of
T-cell activation (Maguire et al., 1990); and c) multiple markers can be tested at
once, allowing the analysis of complex cellular phenotypes and characterization
of speci®c T-cell subpopulations. Close to 100 di¨erent TCRBV-speci®c mAbs
have been derived to date, and b chain repertoire coverage currently exceeds
75% with commercially available mAbs alone. A comprehensive listing of the
mAbs most commonly used in TCRBV typing can be found in Posnett et al.
(1996). TCRAV-speci®c mAbs are much less common than their anti-TCRBV
