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lthough it has long been
known that antigen-specific
functions of the immune
system are based on re-
sponses of T and B cells bearing clonal recep-
tors, it is only quite recently that it has
become possible to analyse polyclonal re-
sponses in vivo at this level. Initial results of
studies of clonal T-cell populations, particu-
larly in humans, have generated data that
reveal hitherto unsuspected features of T-cell
immune responses and raise new questions
relating to the life history of individual clones
and the regulation of different T-cell subsets.
T-cell receptor (TCR) and immunoglobulin (Ig) transgenic mice
provide clonal populations of lymphocytes that can be tracked using
monoclonal antibodies specific for the TCR and Ig clonotypes. These
mouse models allow experiments in which the response of a trans-
genic clonal population takes place in isolation from other respond-
ing clones and therefore under conditions of abnormal regulation.
Attempts to create more physiological conditions by transfer of a
monoclonal population into a polyclonal background still only allow
tracking of that individual clone. Experiments in which the antigen
stimulates T- or B-cell responses derived from a highly restricted
V-gene repertoire provide another opportunity for studying clonal
responses and have demonstrated the rapid clonal expansion of B
and T cells early in an immune response
13
. More recently, major histo-
compatibility complex (MHC)peptide tetramers have provided a
method for detecting the whole population of T cells capable of
binding to a particular epitope, regardless of their clonotype
49
.
However, this method requires previous identification of target
epitopes and therefore may not identify the whole response to a
pathogen.
Analysis of polyclonal responses to complex antigens, under
physiological conditions, needs sensitive methods for detecting indi-
vidual clones against a background of non-responding polyclonal
cells. Initially, expanded populations of T cells were often identified
by detecting an increased number of cells staining with a mono-
clonal antibody to a TCR V family. However, V expansions may
be polyclonal, for example, when stimulated by superantigens
10,11
.
T-cell clones within such a population could be identified by cloning
the polymerase chain reaction (PCR) product of that V family and
sequencing a representative number of cDNAs to derive data on the
frequency of clonal sequences
12
. This method is laborious and will
only detect well-expanded clones. However, developments in
methodology have provided alternatives for analysis of clonal
responding populations. One such advance is single-cell PCR of
sorted T cells to determine the TCR
sequence
13
.
Methods for more global analysis depend
on detection of variation in the CDR3 region
of TCR V genes by either measurement of
CDR3 length (spectratyping)
14,15
or detection
of specific CDR3 sequences using hetero-
duplex analysis
16,17
. Data suggest that the
heteroduplex method can detect cells at a
frequency of at least 1 in 10 000, whereas
CDR3-length analysis is one log less sensi-
tive
18
, although this can be improved by
further rounds of PCR using J-region primers
(Immunoscope)
19
. The heteroduplex tech-
nique facilitates tracking of individual clones identified by unique
molecular footprints within different lymphocyte subpopulations
20
or anatomical sites
21
. Methods relying on CDR3 length will provide
amplification of signals from different clones within a V sharing a
CDR3 length, thus facilitating detection of clonal expansions in situ-
ations where the antigen imposes CDR3 length constraints
2
.
Clonal expansions are described more commonly in healthy hu-
mans in the CD8

than in the CD4

T-cell fraction. Here, we discuss

whether these long-term differences are related to potential differ-
ences in the proliferative response to antigen of the CD8

and CD4

subsets. Alternative explanations are more efficient clonal contrac-

tion at the end of an immune response or differences in compart-
mentalization or maintenance of antigen-specific clones. Figure 1
presents a speculative scheme for the behaviour of CD4

and CD8

clones and Boxes 1 and 2 summarize possible explanations for these

differences.
Acute clonal expansions
A number of reports have addressed the size and clonality of acute
CD4

and CD8

immune responses in mice and humans. Studies of

acute viral infections in mice, using either limiting dilution analysis
(LDA)
22
or direct visualization
23
, indicate an increase in cytotoxic T
lymphocyte (CTL) frequencies in the order of 10
3
(Ref. 24). In another
study, the dramatic expansion of MHC class I restricted CD8

T cells
was shown to consist of a few large clones
13
. Much less information
is available for acute CD4

responses: LDA of T-cell responses in

Sendai virus-infected mice shows antigen-specific CD4

T-cell ex-
pansions of a similar magnitude
25
, although total CD4

T cells un-
dergo less proliferation than CD8

T cells after lymphocytic chorio-

meningitis virus (LCMV) infection
26
. The potent CD4

response to
pigeon cytochrome c was found to be composed of a highly restricted
TCR repertoire
2
.
T-cell clonality in immune responses
Mala K. Maini, Giulia Casorati, Paolo Dellabona, Andreas Wack and
Peter C.L. Beverley
Recent methodological advances
allow the analysis of clonal
composition within T-cell subsets.
Here, Mala Maini and colleagues
review the available data on
clonality in acute immune responses
and steady-state situations. They
highlight and explore reasons for the
striking differences in clonality
between the CD4

and CD8

T-cell
subsets.
A
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Recent experiments using MHCpeptide complexes or Elispot
assays suggest that LDA estimates of antigen-specific CD8

T-cell
frequencies were at least 520 times too low
5,27
, partly because CTL
effectors undergo activation-induced cell death (AICD) in the limit-
ing dilution cultures. These experiments reveal that a high propor-
tion (up to 70%) of the CD8

T cells responding to an acute viral

infection are in fact antigen specific, rather than non-specifically ac-
tivated
5,6,27
. CD4

responses might have been similarly underesti-

mated
28
. These data indicate that there may be the potential for
equally large CD4

and CD8

clonal bursts to occur; whether these

murine models are representative of human physiological responses
remains an open question.
In humans, expanded CD8

clones are detectable in the acute

stage of both HIV and Epstein-Barr virus (EBV) infection
29,30
, sug-
gesting that the prominent lymphocytosis is antigen driven. This has
been confirmed recently by analysis with human MHC (HLA)
peptide tetramers showing, for example, that ~40% of CD8

cells in
acute infectious mononucleosis (AIM) can
be specific for a single EBV epitope
7
. In AIM,
many clones probably contain at least 10
8
cells, although this may be a considerable
underestimate because there is gross en-
largement of many lymphoid organs and
lymphocytosis. Sequencing, in combination
with V monoclonal antibody staining data,
indicate that many of the clones are present
at frequencies as high as 10% of CD8

T
cells
30
. In infections where a CD4

lympho-
cytosis has been observed, either clonal
composition has not been analysed
31
or
clonal expansion has characterized the in-
flammatory infiltrates (which represent a
highly selected phenotypic subpopulation)
and not the peripheral CD4

population
32
.
None of the above-mentioned studies sim-
ultaneously analyses the clonality of total
CD4

and CD8

populations (or subsets

within them) in acute immune responses.
Such a comparison is possible in AIM using
heteroduplex analysis. At diagnosis, clonal
bands representing clones at >1 in 10 000 fre-
quency can be seen in all V families of
freshly isolated CD8

T cells, with up to 50
separate clones being expanded. By con-
trast, no clones are detectable in freshly iso-
lated CD4

cells, although they show some

evidence of activation. In studies of human
responses to tetanus toxoid (TT), large num-
bers of responding CD4

clones are detected

after in vitro stimulation, but none of them
are visible directly ex vivo, implying that the
size of clones lies below the detection
threshold in the unstimulated peripheral
blood mononuclear cells (PBMCs)
18
. On
weekly sampling after an in vivo boost with TT vaccine, even the
most prominent heteroduplex clones cannot be detected in unstimu-
lated PBMCs. Overall, the human data available suggest smaller
clone sizes in acute responses for CD4

than CD8

T cells, which
may well contribute to the longer-term differences in clonal expan-
sions observed.
One study, on two individuals, has documented transient CD4

expansions that were only visible four days after the onset of flu-like
symptoms
33
, whereas CD8

expansions persisted longer. This is

compatible with the hypothesis generated from murine experiments
that the duration of the acute CD4

proliferative response in a viral

infection is considerably shorter than that of CTLs (Ref. 34). This
shorter duration of response might be due either to tighter con-
straints, such as CTLA-4 inhibition (as discussed below), or a
propensity of CD4

T cells to more rapid apoptosis, perhaps regu-

lated by CD4 itself
35
. Available data indicate that CD4

and CD8

T cells differ in their susceptibility to Fas-mediated AICD at the

CD4
+
Clonal burst
CD8
+
Threshold of
clonal detection
Threshold of
clonal detection
Clonal
size
Clonal
size
Antigenic stimulus Time
Chronic or repeated
antigenic stimulation
breakdown of homeostasis
Clonal
burst
Clonal
burst
Fig. 1. Clonal expansions in CD4

and CD8

T cells. For the purposes of this model we show the be-

haviour of a single clone, but responses to a complex antigen must be considered in terms of the diverse
clonotypes responding to each epitope within the antigen. After acute T-cell responses, large CD8

(red) and CD4

(blue) clones are generated with postulated survival of best-fit clones when major
histocompatibility complex (MHC)antigen complexes become limiting. Escape from apoptosis and
replicative senescence (by telomerase upregulation allowing telomere length preservation despite ex-
tensive clonal division), might allow long-term clonal persistence. In CD8

T-cell responses, relatively

large clonal expansions can persist in the memory pool after acute antigenic exposure. CD4

T-cell
responses are more tightly constrained, perhaps by factors such as CTLA-4 inhibition, such that cir-
culating steady-state clones rarely reach the threshold of detection of current methods (indicated in
green). They attain a detectable size only after sustained antigenic stimulation and/or a breakdown of
normal homeostatic controls. The initial clonal burst and contraction take place within weeks of an
antigenic challenge (unbroken lines). Broken lines indicate the subsequent clonal contraction and re-
expansion that can occur over several years. Whether differences in acute burst size contribute to the
CD4

/CD8

discrepancies observed in persistent clones is unclear. There are conflicting data from
murine and human models regarding whether the acute clonal burst size is smaller for CD4

(dark
blue) than for CD8

T cells. The alternative model for CD4

T cells (light blue) is that the initial

clonal expansion is as large as in CD8

T cells, but this is followed by a more marked contraction or

less efficient maintenance phase.
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termination of an acute immune response. There is good evidence
that AICD can occur independently of Fas in CD8

T cells after anti-

gen challenge
36,37
, whereas Fas involvement in CD4

T-cell AICD is
controversial
38,39
. Therefore, brief CD4

expansions may escape de-

tection as a result of delayed sampling in human infections.
Another reason for the failure to detect expanded CD4

clones in
humans may be compartmentalization at the site where antigen
resides. Most human studies are performed on circulating T cells,
which represent just 12% of total lymphocytes. One of the few ex-
ceptions is a study showing a hepatitis C virus-specific CD4

clone
restricted to hepatic-infiltrating lymphocytes and not detectable in
peripheral blood
40
. CD4

T-cell expansions could be more restricted

in distribution because they are often in response to an extracellular
pathogen or allergen localized to a tissue, whereas CD8

T cells are
the major defence against systemic viral infections.
Persistent clonal expansions
Early studies showed that aged mice and humans frequently exhibit
expansions of one or several V families
12,41,42
. More detailed analysis
has demonstrated that such expansions are most prominent among
CD8

cells, are frequently mono- or oligoclonal and are increasingly

present with ageing
15,20,4346
. This is a quantitative rather than qualita-
tive change, because heteroduplex analysis reveals a small number of
clonal populations in freshly isolated CD8

T cells from young healthy

adults. By contrast, clonal populations are infrequently detected in
freshly isolated CD4

cells from young individuals, although they are

seen in the elderly. These observations suggest that CD4

T-cell clones
require a chronic, sustained form of antigenic stimulation over many
years to expand, whereas clonal expansions within CD8

T cells may
accumulate rapidly after antigen encounter.
The hypothesis that at least some of these expanded clones are the
residue of acute responses is consistent with the finding that sec-
ondary immune responses in mice are composed of the same clono-
types dominating primary responses
2,13
. This is supported by the
finding that many of the expanded clones seen in AIM can still be
detected one year later directly ex vivo by their molecular footprint,
using the heteroduplex technique. On restimulation in vitro with the
autologous B-lymphoblastoid cell line at this time, the CTLs gener-
ated contain clones detected at the onset of infection. Thus, memory
CTLs are generated early in the course of the acute infection and per-
sist as expanded clones for at least one year. A related study has
demonstrated the persistence of EBV-specific TCR clones in AIM
and during convalescence
47
.
Further examples of maintenance of CD8

TCR clonotypes at high

circulating frequency come from chronic HIV infection (Ref. 48),
human cytomegalovirus infection
49
and observations after influenza
infection
50
. Thus, it is clear that multiple CD8

T-cell clones can per-

sist at a relatively high frequency in vivo in humans. By contrast,
clonal expansion of CD4

cells appears to be much more tightly regu-

lated and clonal frequency is generally below the threshold of detec-
tion currently possible for global repertoire analysis. In general, per-
sistence of intracellular pathogens recognized by CD8

T cells is more
common than extracellular antigens recognized by CD4

T cells.
Even in the case of repeated CD4

antigenic stimulation with allergen

in an atopic individual, clone frequency has been estimated to remain
as low as 1 in 10
5
(L. Wedderburn, pers. commun.). However, there is
evidence for very low level persistence of antigen-specific CD4

clones for up to five or six years (only detectable with the very sensi-
tive method of N-region clonotypic probing or after in vitro expan-
sion)
17,51
, and we have found that the hierarchy of the whole TT-
restimulated repertoire of clones remains stable for over one year
18
.
Thus, there is a profound difference in the size of persistent CD4

and CD8

T-cell clones, which may relate to differences in long-term

maintenance, as well as to the discrepancies in the acute phase dis-
cussed above. For example, an important role for CTLA-4 in the
regulation of clonal homeostasis of CD4

but not CD8

T cells has
been shown in a CTLA-4
/
mouse model
52
. It is of interest to see
whether clonal expansions of CD4

T cells seen in the elderly are ac-

companied by deficits in CTLA-4 expression and/or function. Simi-
larly, CD4

T cells become refractory to interleukin 2 (IL-2)-induced

proliferation more rapidly
53
and are less susceptible to type I inter-
feron and IL-15-mediated bystander proliferation
26,54
than are CD8

T cells. Upregulation of telomerase in proliferating T cells may result

in preservation of telomere length and thus protect against replica-
tive senescence
55
. This process may also be regulated differentially in
CD8

compared with CD4

T-cell clones and contribute to their

capacity to persist and expand.
Phenotype and clinical correlates of persistent clones
The differences between CD4

and CD8

T cells in clonal persistence

described above become even more striking when the age-dependent
Box 2. Postulated reasons why clonal expansions
are less frequent in CD4

than CD8

T cells on
long-term follow-up
Rescue from apoptosis or replicative senescence is less ef-
ficient in CD4

than CD8

T cells so that fewer clones persist

and are capable of substantial re-expansion
There are stronger homeostatic controls over the size of
CD4

memory clones; for example, by CTLA-4

Cytokine-driven proliferation is less efficient for CD4

than
CD8

clones
Strong, persistent antigenic drive is less frequent for CD4

than CD8

clones (responding to chronic systemic intra-

cellular pathogens)
Box 1. Postulated reasons why clonal expansions
are less frequent in CD4

than CD8

T cells in
acute responses
CD4

bursts are smaller than CD8

bursts in common
human immune responses
CD4

bursts have a shorter duration than CD8

bursts (more
susceptible to Fas-mediated apoptosis or inhibition by
CTLA-4) and are therefore often missed in sampling
CD4

expansions are more restricted in distribution, reflect-

ing the localization of extracellular pathogens
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accumulation of expanded clones in CD45RA

and R0

subsets
is analysed
20
. CD4

CD45RA

T cells remain largely polyclonal

throughout life, whereas clonal expansions are detectable mainly
within CD4

CD45R0

cells of very old donors (>80 years old).

By contrast, CD8

T-cell clonal expansions are regularly detected

both in CD45RA

and CD45R0

compartments in 7075-year-old
donors, an age at which the corresponding CD4

T cells are still poly-

clonal. Moreover, the same clone can frequently be demonstrated
both in separated CD45R0

and RA

CD8

T-cell subsets, indicating

that CD8

T cells from one clone that have been exposed to antigen

may express either CD45R0 or RA (Ref. 20). This is in accord
with studies suggesting that the high molecular weight CD45 iso-
form is not a reliable marker for naive CD8

T cells in mice
23
or
humans
46,5658
.
At least some of the expansions seen in elderly humans exhibit
the CD8

CD28

CD11b

phenotype, which has been suggested to

be incapable of further proliferation
12,59
, perhaps as a result of criti-
cal telomeric shortening
60
. If this is the case, the clonal expansions
must also contain other cells capable of division because they persist
over time. The observation that expanded CD8

CD28

TCR V
clonotypes could often also be detected within the CD28

compart-
ment of the same donor, suggests a possible role for the CD28

com-
partment as a proliferative reservoir
20
. Interestingly, there are more
expanded clones in the CD28

than the CD45R0

compartment of
both CD4

and CD8

T cells, reinforcing the concept that, for the de-

tection of clonally expanded T cells, the loss of CD28 expression is a
more stringent marker than the acquisition of CD45R0.
CD8

CD28
-
cells are found in the response to acute viral infec-
tions and may be an effector population, so that expanded clones in
the elderly might represent responses directed to common
pathogens, particularly those like EBV and other herpes viruses,
which are carried for long periods of time and provide chronic anti-
genic stimulation. Although cells of this phenotype are difficult to
grow in vitro, this hypothesis might be tested using MHC tetramers
for dominant EBV peptides
7
to stain expanded V populations in the
elderly. Alternatively, a correlation could be sought between the TCR
clonotypes detected by heteroduplex analysis in the periphery in the
elderly, and those present in these individuals EBV-specific memory
CTLs restimulated in vitro.
Clonal expansions in elderly humans appear to be benign in most
cases; monoclonal T-cell proliferation is rarely an indication of ma-
lignant transformation. Conversely, indolent lymphoproliferative
disorders, which are frequent events in elderly individuals, could
themselves stimulate and sustain the expansion of tumour-specific
T-cell clones over time
61,62
. What seems likely is that because clonal
expansions make up an increasing proportion of the T-cell pool in
the elderly, the remaining naive repertoire will be narrowed, perhaps
accounting for the inability of elderly individuals to respond well to
neoantigens
63
. This shrinkage in the available TCR repertoire would
be predicted to be more pronounced for CD8

T cells, whereas
the CD4

CD45RA

T-cell repertoire would tend to remain more

diverse.
Another example of apparent dysregulation is autoimmunity,
where clonal populations have been observed frequently among
CD4

T cells. For example oligoclonal CD4

expansions were
detected in synovial fluid and also in peripheral blood of patients
with rheumatoid arthritis and their unaffected siblings, but not in
healthy controls
64
. In multiple sclerosis, a dramatic oligoclonal V5.3
expansion detected in circulating CD4

T cells was conserved

between HLA-DR2 patients
65
.
Conclusions
Analysis of immune responses at a clonal level is just beginning. So
far, responses to a limited number of infectious agents have been
studied in depth. The data available suggest that CD4

and CD8

T
cells differ in the way in which they respond to antigenic stimuli in
humans. Whereas CD8

T cells generate large clones rapidly, CD4

clones appear not to expand to the same extent. This is congruent

with their differing effector functions, with larger CD8

clones being
required to achieve efficient direct CTL killing, whereas the effects of
CD4

T cells are potentiated via cytokines. The most potent stimuli

regulating the amount of expansion appear to be simply antigenic
dose and availability. When antigen becomes limited, competition
for MHCpeptide complexes by T cells with receptors of differing
affinities would be expected to limit individual clone size
66
. A better
knowledge of the mechanism and timing of clonal size control will
help to explain how the balance between a polyclonal repertoire and
antigen-specific expansions is regulated.
This work was partially funded by ICRF; M.K.M. was funded by an MRC
Clinical Training Fellowship. We thank L. Wedderburn for useful discussion
and critical reading of the manuscript.
Mala Maini (m.maini@ucl.ac.uk) is at the Dept of Sexually Transmitted
Diseases, Royal Free and University College Medical School, Mortimer
Market off Capper St, London, UK WC1E 6AU; Giulia Casorati and
Paolo Dellabona are at Unita di Immunochimica, DIBIT, Instituto Sci-
entifico H.S. Raffaele, via Olgettina 58, I-20132 Milan, Italy; Andreas
Wack is at NIMR, The Ridgeway, Mill Hill, London, UK NW7 1AA;
Peter Beverley is at The Edward Jenner Institute for Vaccine Research,
Compton, Berkshire, UK RG20 7NN.
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