J.E. Cook, P.C.L. Beverley / Experimental Gerontology 36 (2001) 583±589

Experimental Gerontology 36 (2001) 583±589

583

www.elsevier.nl/locate/expgero

Analysis of lymphocyte diversity in the elderly:
heteroduplex analysis and alternative techniques
Joanne E. Cook*, Peter C.L. Beverley
The Edward Jenner Institute for Vaccine Research, Compton, Berkshire, RG20 7NN, UK
Received 20 November 2000; accepted 14 December 2000

Abstract
Heteroduplex analysis allows global analysis of T cell receptor clonality. This review outlines the
method, compares it to other available techniques for the study of clonality and reviews current
literature on how these are being used to investigate alterations in the T cell repertoire within elderly
individuals. q 2001 Elsevier Science Inc. All rights reserved.
Keywords: Heteroduplex analysis; Ageing; Immune system; T cell receptor; Clonality

1. Introduction
In developed countries, the average age of the population is increasing and infection
in the elderly is one of the leading causes of mortality and morbidity (Dodet, 2000). In
immunosenescence, defects in the function of most components of the immune system
have been documented, though antigen-presenting cell function exhibits only minor
changes (Steger et al., 1996). T cells show abnormalities of signalling, response to
speci®c recall antigens and production of many cytokines (Miller, 1996). Altered B cell
function is re¯ected in an increase in autoantibody production (Bovbjerg et al., 1991), but
the elderly respond poorly to vaccination (Govaert et al., 1994). These changes are thought
to lead to an increased prevalence of infection, tumours and autoimmunity (Wick et al.,
2000).
È
The naive T cell repertoire is determined by selection in the thymus. In the periphery of
È
young adults, naive T cells have a diverse repertoire while antigen-experienced memory
and effector cells are much less diverse (Arstila et al., 1999), but in aged individuals very
large monoclonal populations of T cells are frequently found (Posnett et al., 1994; Schwab
* Corresponding author. Tel.: 144-1635-577927; fax: 144-1635-577901.
E-mail address: joanne.cook@jenner.ac.uk (J.E. Cook).
0531-5565/01/$ - see front matter q 2001 Elsevier Science Inc. All rights reserved.
PII: S 0531-556 5(00)00260-6

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J.E. Cook, P.C.L. Beverley / Experimental Gerontology 36 (2001) 583±589

et al., 1997; Wack et al., 1998). The signi®cance of these changes in the T cell repertoire
has yet to be fully elucidated, but global analysis of young and elderly T cell repertoires
will be required to resolve outstanding issues. In this article, we review methods for
studying T cell repertoires, particularly the heteroduplex method and discuss ®ndings in
the elderly.
2. Heteroduplex analysis
Heteroduplex analysis (HDA) provides a rapid, global method for detection of
expanded populations of T cells expressing identical Vb, T cell receptor (TCR) chains.
With the proviso that more than one a-chain may be associated with a single a-chain
(Annels et al., 2000), the method allows tracking of individual clones de®ned by their
TCR.
The method depends on the fact that when a mixture of similar DNA strands are allowed
to anneal and run in polyacrylamide gels, strand duplices containing mismatched base
pairs (heteroduplices) are retarded compared to duplices containing completely complementary strands (homoduplices). Retardation in the gel correlates with the extent of
mismatching between the two DNA strands. Inclusion of an extended C-region monoclonal `carrier' DNA, speci®c for each variable region family of the b chain of the TCR,
makes it possible to use a probe speci®c for the C-region to detect carrier DNA by
Southern blotting, thereby greatly increasing the sensitivity of the method (Wack et al.,
1996).
In practice RNA is extracted from the test T cell population and reverse transcribed
using random primers. Aliquots of sample cDNA are ampli®ed by polymerane chain
reaction (PCR) using primers speci®c for each Vb family, 25 in human. Monoclonal
carrier cDNA is similarly ampli®ed using primers that generate a fragment extended at
the TCR C-region end. Vb-ampli®ed test samples are mixed with an excess of carrier of
the same Vb family, denatured at 958C to separate the strands, and re-annealed at 508C.
Four types of duplices are produced, although the excess of monoclonal carrier minimises
the formation of test sample homoduplices or heteroduplices (Fig. 1).
Samples are separated by polyacrylamide gel electrophoresis, transferred to nylon
membrane and Southern blotted using radioactive or chemoluminescent labelled external
C-region probe. Carrier homoduplices are identi®ed as a strong band running furthest in
the gel. Heteroduplices formed between test sample cDNA product and carrier indicate the
presence of clonally-expanded populations (Wack et al., 1996). They are retarded in the
gel and are seen as weaker bands above the carrier homoduplex against the background of
a polyclonal smear.
3. Comparison of methods
Commercially available monoclonal antibodies cover the majority of human and
murine Vb families and can be used to enumerate each family. Detection of an increased
percentage of cells expressing a particular Vb does not necessarily indicate the presence of
clonal expansions, but could be due to a polyclonal response of cells of that Vb, family

J.E. Cook, P.C.L. Beverley / Experimental Gerontology 36 (2001) 583±589

Polyclonal
cDNA sample
5’

V

D

Vβ primer
PCR

J

585

Monoclonal
carrier cDNA
C

Internal
Cβ

3’

5’

V

D

J

C

3’

External Cβ
primer

Vβ primer
PCR

Mix, denature
and re-anneal
Separate by PAGE,
Southern blot with C region probe
Heteroduplex between
sample TCR and
carrier

*

Heteroduplex between
two sample TCRs
Heteroduplex between
sample TCR and
carrier

*
Bands
detected
by labelled
C region
probe

Heteroduplex between
two sample TCRs

Carrier homoduplex
Sample TCR
homoduplex

*
*

Fig. 1. HDA showing the formation of sample and carrier heteroduplices against the background of a polyclonal
smear. Sample DNA strands are indicated by dashed lines and carrier strands by solid lines. Black lines indicate
duplices detected by labelled C region probe, indicated by *, and grey lines indicate unlabelled hetero- or
homoduplices.

(Krakauer, 1999), nor is this a sensitive method for detecting small clonal expansions
within a Vb family. Nevertheless, increasing distortions of the distribution of Vb families
correlate with increasing age (Ricalton et al., 1998).
Major histocompatibility complex (MHC) Class I tetramers allow the identi®cation
and enumeration of speci®c CD8 1 T cells if the target epitope is known (Altman

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J.E. Cook, P.C.L. Beverley / Experimental Gerontology 36 (2001) 583±589

et al., 1996). Multi-parameter ¯ow cytometry can be used to determine the phenotype of
the antigen-binding cells, which can be separated for functional analysis. Tetramers have
the advantage of identifying epitope-speci®c cells, but the disadvantage is that they do not
re¯ect the breadth of the T cell response to the whole antigen or organism, nor do they
identify the clonal diversity of the T cell response. Methods such as HDA allow global
analysis of the repertoire of T cells responding to a single epitope, whole antigen or microorganism.
Molecular methods for identifying T cell clones detect differences in the CDR3 region
of the TCR. As there are less TCR Vb than Va gene families, clonality is usually studied
by identifying unique Vb CDR3 sequences and a chains do not always exhibit allelic
exclusion (Gascoigne and Alam, 1999). All the methods for analysis of CDR3 sequences
depend on PCR ampli®cation of TCR Vb sequences using family speci®c primers and
separation of products.
CDR3 length determination is termed spectratyping (Cochet et al., 1992) or immunoscope (Pannetier et al., 1993). CDR3 length within a Vb family depends on D- and Jregion usage as well as on N-region diversity. Spectratyping analyses distortion of the
Gaussian distribution of TCR CDR3 lengths. Abnormal peaks are usually due to the
presence of a clonal population, although different TCRs with the same CDR3 length
may be within the same peak. HDA is approximately ten-fold more sensitive than spectratyping and can detect cells at a frequency of 1:10 000±1:100 000, although heteroduplex bands are very faint at 1:100 000, while spectratyping cannot detect cells reliably at a
frequency lower than 1:1000 (Maini et al., 1998). Increased sensitivity, however, can be
achieved using run-off nested PCR reactions with joining region primers (Pannetier et al.,
1995), although the complexity of the latter makes this a cumbersome assay for rapid
global analysis. Alternatively, single strand conformation polymorphism analysis (SSCP)
can be used to detect alterations in the sequence of CDR3 (Masuko-Hongo et al., 1998).
However, no comparisons between this and other methods for detecting TCR diversity
have been performed, nor has SSCP analysis been applied to the elderly.
Single cell PCR analysis has also been used to study T cell repertoires. Single T cells
can be separated from cell suspension or be isolated from tissue sections. Both the b and
the a chains can be ampli®ed and sequenced to provide unambiguous identi®cation of T
cell clones. The method avoids the problems of skewed PCR ampli®cation and varying
TCR mRNA expression in different cells (Kurokawa et al., 1999). A large number of cells,
however, need to be analysed to ensure detection of rare clones and to provide a global
representation of the T cell repertoire.
4. Clonality in the elderly
Initial studies in aged mice and humans using antibodies to TCR Vbs demonstrated the
presence of large oligoclonal expansions (Posnett et al., 1994; Ku et al., 1997), and in both
species they were shown to be predominantly within the CD8 subset. The frequency and
size of expansions appear broadly to correlate with increasing age. Further studies in
elderly humans have reached slightly varying conclusions. Schwab et al. (1997) used
spectratyping to detect the presence of expanded T cell clones in young and old healthy

J.E. Cook, P.C.L. Beverley / Experimental Gerontology 36 (2001) 583±589

587

donors. The young donors displayed a heterogeneous pattern of CDR3 lengths within each
Vb family. In individuals over the age of 65, the heterogeneity of CDR3 lengths was
reduced with single dominant peaks observed. In contrast to other studies, equal numbers
of clonal expansions were observed within the CD4 1 and CD8 1 subset. Most of the
expansions were CD45RO 1 although some were within the CD45RA 1 population. In
another study, HDA was used to study the Vb repertoire in individuals of 20±100 years
(Wack et al., 1998). A few clonal expansions were seen in young individuals, but with
advancing years increasing numbers of large CD8 1 clonal expansions were seen. Clones
were largely seen not only within the CD45RO 1 subset in the young, but also with
increasing age within the CD45RA 1 subset. CD4 1 T cells showed a more polyclonal
repertoire with only a small number of expansions observed within the CD45RO 1 subset
of centenarians.
These observations in the elderly should be set against the background of data obtained
from young healthy adults and patients with viral infections. Although clonal expansions
are seen in the healthy young, sensitive methods are needed to detect them and when they
are observed, they are generally present in the CD8 subset (Shen et al., 1998). In acute
viral infections clonal expansions are readily detected ex vivo and are commonly CD8
positive (Maini et al., 2000). CD4 clones can usually be detected only after in vitro restimulation with recall antigens (Maini et al., 1998). These data suggest that CD8 are
generally larger than CD4 clones and the modi®cation of the repertoire that occurs with
ageing may be an exaggerated re¯ection of differences in the control of the repertoire in
CD4 1 and CD8 1 T cells, which already exist in young individuals.
5. Signi®cance of oligoclonality
In addition to the development of clonal expansions, ®rst in the CD45RO and later in the
CD45RA subset, thymic output decreases rapidly with increasing age such that there are
È
fewer naive cells (Mackall and Gress, 1997). This suggests that there must be a narrowing
È
È
of the naive T cell repertoire because of the recruitment of naive T cells into memory and
the lack of replacement from the thymus. Concurrently, diversity of the memory compartment is reduced because large clonal expansions develop, although a formal comparison of
TCR diversity in the old with that of the young (Arstila et al., 1999) has yet to be carried
out. The increasing oligoclonality of T cells observed with ageing is probably related to
antigen exposure during the lifetime of an individual and perhaps particularly to chronic
viral carriage as viral infections have been shown to induce large oligoclonal CD8 1
clonal responses (Sourdive et al., 1998; Weekes et al., 1999; Maini et al., 2000).
Clonally restricted populations were ®rst documented in humans by Hingorani et al.
(1993). In the elderly, very large clonal populations described by Posnett et al. (1994) were
at ®rst considered to be equivalent to `benign monoclonal gammopathy' of B cells
suggesting that they might have escaped from normal regulation. The ®nding that many
expansions in the elderly contain a high proportion of cells that have lost CD28 was
considered to support this view. It is, however, now apparent that loss of CD28 is part
of the normal differentiation of CD8 T cell clones (Posnett et al., 1999). These cells may
therefore most likely be differentiated effectors related to the effector-memory population

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J.E. Cook, P.C.L. Beverley / Experimental Gerontology 36 (2001) 583±589

described by Lanzavecchia (Sallusto et al., 1999), although it remains to be determined
whether they are fully functional. Although it has been reported that T cells from aged
humans or mice are relatively refractory to acute stimuli in vitro, in mice these cells
continue to divide in vivo (Ku et al., 1997) and in humans, clonal expansions have
been shown to persist for at least two years (Schwab et al., 1997).
Whatever the exact functional capabilities of the clonal expansions, the resulting
È
narrowing of the naive T cell repertoire will produce a decrease in the capacity of the
immune system to respond to neo-antigens. The predominance of a narrow range of
memory clones in the elderly immune system will also predispose the elderly to immune
deviation. These two factors, quite apart from intrinsic abnormalities of lymphocytes, will
lead to poor and inappropriate response to newly encountered pathogens and may largely
account for the increase in infections observed in the elderly. If the diversity of the
repertoire in elderly humans could be reconstituted by improving thymic output (Aspinall
and Andrew, 2000), the ageing immune system might be able to ®ght infections better.

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