Article

Price, D.A. et al. Avidity for antigen shapes clonal dominance in CD8+ T cell populations specific for persistent DNA viruses. J. Exp. Med. 202, 1349-1361

University of Oxford, Oxford, England, United Kingdom
Journal of Experimental Medicine (Impact Factor: 12.52). 12/2005; 202(10):1349-61. DOI: 10.1084/jem.20051357
Source: PubMed

ABSTRACT

The forces that govern clonal selection during the genesis and maintenance of specific T cell responses are complex, but amenable to decryption by interrogation of constituent clonotypes within the antigen-experienced T cell pools. Here, we used point-mutated peptide-major histocompatibility complex class I (pMHCI) antigens, unbiased TCRB gene usage analysis, and polychromatic flow cytometry to probe directly ex vivo the clonal architecture of antigen-specific CD8(+) T cell populations under conditions of persistent exposure to structurally stable virus-derived epitopes. During chronic infection with cytomegalovirus and Epstein-Barr virus, CD8(+) T cell responses to immunodominant viral antigens were oligoclonal, highly skewed, and exhibited diverse clonotypic configurations; TCRB CDR3 sequence analysis indicated positive selection at the protein level. Dominant clonotypes demonstrated high intrinsic antigen avidity, defined strictly as a physical parameter, and were preferentially driven toward terminal differentiation in phenotypically heterogeneous populations. In contrast, subdominant clonotypes were characterized by lower intrinsic avidities and proportionately greater dependency on the pMHCI-CD8 interaction for antigen uptake and functional sensitivity. These findings provide evidence that interclonal competition for antigen operates in human T cell populations, while preferential CD8 coreceptor compensation mitigates this process to maintain clonotypic diversity. Vaccine strategies that reconstruct these biological processes could generate T cell populations that mediate optimal delivery of antiviral effector function.

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Available from: Brenna Hill, Jun 05, 2014
The Journal of Experimental Medicine
ARTICLE
1349
JEM Vol. 202, No. 10, November 21, 2005 1349–1361 www.jem.org/cgi/doi/10.1084/jem.20051357
Avidity for antigen shapes clonal dominance
in CD8
T cell populations specific
for persistent DNA viruses
David A. Price,
1
Jason M. Brenchley,
1
Laura E. Ruff,
1
Michael R. Betts,
2
Brenna J. Hill,
1
Mario Roederer,
3
Richard A. Koup,
2
Steven A. Migueles,
4
Emma Gostick,
5
Linda Wooldridge,
5
Andrew K. Sewell,
5
Mark Connors,
4
and Daniel C. Douek
1
1
Human Immunology Section,
2
Immunology Laboratory,
3
Immunotechnology Section, Vaccine Research Center,
and
4
Laboratory of Immunoregulation, National Institute of Allergy and Infectious Diseases, National Institutes of Health,
Bethesda, MD 20892
5
Nuffield Department of Clinical Medicine, University of Oxford, John Radcliffe Hospital, Oxford OX3 9DU, England, UK
The forces that govern clonal selection during the genesis and maintenance of specific T cell
responses are complex, but amenable to decryption by interrogation of constituent clonotypes
within the antigen-experienced T cell pools. Here, we used point-mutated peptide–major
histocompatibility complex class I (pMHCI) antigens, unbiased
TCRB
gene usage analysis, and
polychromatic flow cytometry to probe directly ex vivo the clonal architecture of antigen-
specific CD8
T cell populations under conditions of persistent exposure to structurally stable
virus-derived epitopes. During chronic infection with cytomegalovirus and Epstein-Barr virus,
CD8
T cell responses to immunodominant viral antigens were oligoclonal, highly skewed, and
exhibited diverse clonotypic configurations; TCRB CDR3 sequence analysis indicated positive
selection at the protein level. Dominant clonotypes demonstrated high intrinsic antigen avidity,
defined strictly as a physical parameter, and were preferentially driven toward terminal
differentiation in phenotypically heterogeneous populations. In contrast, subdominant
clonotypes were characterized by lower intrinsic avidities and proportionately greater
dependency on the pMHCI–CD8 interaction for antigen uptake and functional sensitivity.
These findings provide evidence that interclonal competition for antigen operates in human
T cell populations, while preferential CD8 coreceptor compensation mitigates this process to
maintain clonotypic diversity. Vaccine strategies that reconstruct these biological processes
could generate T cell populations that mediate optimal delivery of antiviral effector function.
Competition for limited resources is a univer-
sal biological principle. In the case of adaptive
immunity, it is established that humoral re-
sponses are governed by Darwinian laws through
antigen-mediated positive selection of cognate
antibodies from a naturally generated diverse
peripheral repertoire with subsequent affinity
maturation. For T cell responses, the situation
is less clear. The TCR antigen recognition sys-
tem operates within a substantially lower range
of affinities and exhibits distinct kinetics com-
pared with antibody–antigen interactions (1).
Despite this, evidence from experimental manip-
ulations in murine models suggests that com-
petitive effects can influence T cell responses
to both identical and different peptide-MHC
(pMHC) antigens (2). These processes appear
to operate at the level of the APC, and are
thought to be governed by T cell avidity and
precursor frequency (2–16). However, it is un-
certain whether, and under what conditions,
such processes operate in the human immune
system.
CMV and EBV establish lifelong infection
in human hosts. The control of viral replication
in this persistent state is dependent on functional
CD8
T cell immunity (17). Several features of
the host–pathogen relationship during infection
with these herpesviruses are relevant to the issue
of interclonal competition within specific T cell
populations. First, the provision of a persistent
antigenic stimulus is an essential requirement
for progressive evolution of the cognate T cell
response. In contrast with transient perturba-
The online version of this article contains supplemental material.
CORRESPONDENCE
David A. Price:
davidprice@nih.gov
OR
Daniel C. Douek:
ddouek@nih.gov
Abbreviations used: pMHC,
peptide-MHC; pMHCI,
pMHC class I.
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INTERCLONAL COMPETITION IN CD8
T CELL RESPONSES TO CMV AND EBV | Price et al.
1350
Figure 1. Clonal analysis of CD8
T cells specific for CMV NV9
and EBV GL9. (A) CD8
T cells specific for CMV NV9. TCRBV usage,
CDR3 amino acid sequence and percent frequency, and TCRBJ usage
are shown for each clonotype defined by its CDR3. Sequences with
identical amino acid residues are shown in color. The percent fre-
quency of CD8
T cells that bound cognate pMHCI wild-type tetramer
is shown below the identifier letter code (ID) for each donor. (B) CD8
T cells specific for EBV GL9. Details as for A. (C) CDR3 codon usage
of CMV- and EBV-specific CD8
T cell clonotypes that have the same
CDR3 amino acid sequence between different donors. Each section
defines a CDR3 amino acid sequence and, below that, the CDR3 nucle-
otide alignments of all constituent clonotypes. Colors correspond to
those in A and B.
Page 2
JEM VOL. 202, November 21, 2005
1351
ARTICLE
tions, continuous exposure to antigen theoretically favors
maturation of the T cell response while allowing equilibra-
tion of kinetic effects due to differences in clonotype pre-
cursor frequency (4, 11). Second, persistent infection is
characterized by viral latency. In addition, multiple mecha-
nisms exist to minimize the presentation of pMHC class I
(pMHCI) antigen on the surface of the infected cell. Antigen
load is therefore relatively low, and evidence of competition
for this limited resource should become most apparent under
these conditions. Third, the double-stranded DNA genomes
of herpesviruses are genetically stable. Thus, in contrast with
RNA viruses, the fundamental biology of adaptive immu-
nity is not obscured by the myriad effects of antigenic muta-
tion. Fourth, immunodominant CD8
T cell responses to
these pathogens are generated at typically high frequencies;
this condition is likely to be an essential requirement for the
observation of interclonal competition (11). In light of these
considerations, we reasoned that chronic infections with
CMV and EBV might represent the ideal human system
in which to seek evidence for competitive effects in vivo;
we therefore examined the properties of individual clono-
types specific for immunodominant pMHCI antigens derived
from these viruses to illuminate the factors that shape clonal
selection in the periphery under these conditions.
RESULTS
Clonal structure of CD8
T cell populations specific
for CMV and EBV
Immunodominant HLA A
*
0201-restricted CD8
T cells
specific for CMV pp65
495-503
(NV9 from hereon) and EBV
BMLFI
259-267
(GL9 from hereon) were identified directly ex
vivo with cognate fluorescent pMHCI tetramers and sorted
by flow cytometry. A template-switch anchored RT-PCR
was used to amplify all expressed
TCRB
gene products with-
out bias (18); at least 50 subcloned PCR products were se-
quenced for each isolated antigen-specific CD8
T cell pop-
ulation. The amino acid sequences spanning the expressed
CDR3s formed at the TCRB VDJ junctional region and the
frequency of each response within the CD8
T cell population
are shown for all 11 donors in Fig. 1 (A and B).
Three consistent features emerged from this analysis.
First, CD8
T cell responses to CMV NV9 and EBV GL9
were oligoclonal in the setting of chronic infection. Second,
a clear hierarchical structure was apparent in most cases, with
one or two clonotypes dominating the antigen-specific T
cell population. Third, in contrast with the restricted TCR
usage that characterizes responses to some immunodominant
pMHCI antigens (19–21), both the CMV NV9 and EBV
GL9 epitope-specific CD8
T cell populations exhibited
substantial clonotypic diversity. However, although no con-
sensus TCRB CDR3 motifs were identified, several themes
transpired that presumably reflect distinct modes of antigen
recognition. For example, TCRBV12-4 was associated with
TCRBJ1.2 and conserved usage of a serine residue at CDR3
position 4 in several clonotypes specific for CMV NV9; in-
terestingly, this 4S residue replaced the germline-encoded
leucine. Similarly, CMV NV9 was commonly recognized by
clontoypes expressing TCRBV6-5/TCRBJ1.2, with threo-
nine/glycine at CDR3 positions 6/7 respectively and a pre-
ferred hydrophobic residue at position 4 (Fig. 1). Dominant
CD8
T cell clonotypes specific for EBV GL9 were fre-
quently characterized by TCRBV20-1 linked to a nongerm-
line aspartate residue at CDR3 position 4 (Fig. 1); similar
themes were identified in an earlier study (22). Importantly,
these observations are representative of the in vivo situation
in peripheral blood, because no manipulation of virus-specific
CD8
T cells was undertaken in vitro.
The following observations are also noteworthy: (a) no
significant correlations were detected between the magni-
tude of the antigen-specific CD8
T cell population and the
number of constituent clonotypes (P
0.08; Spearman’s
Rank); (b) no consistent features at the level of primary se-
quence were found that distinguished dominant from sub-
dominant clonotypes; and (c) HIV coinfection in chronic
phase did not appear to affect the clonal structure of CD8
T cell responses to CMV NV9 and EBV GL9.
Evidence for TCR-mediated clonal selection
Closer inspection of the data presented in Fig. 1 (A and B)
revealed that public clonotypes bearing identical TCRB
CDR3 amino acid sequences were present in several donors.
These promiscuous CDR3s were encoded by distinct nucle-
otide sequences in each case and confined to individual anti-
gen-specific CD8
T cell populations (Fig. 1 C). Further-
more, in some instances, dominant TCRB CDR3s within
the same donor were encoded distinctly (unpublished data).
Together with the presence of characteristic themes within
the private repertoire at the level of primary TCRB CDR3
structure, these observations provide indirect evidence for
antigen-driven clonal selection mediated through the TCR.
In some cases, this was further substantiated by N-encoded
CDR3 residue conservation; for example, the serine/gluta-
mine sequence at the V–D junction is nongermline in both
public TCRBV29-1 clonotypes specific for EBV GL9 (Fig.
1 C). To extend these findings and clarify the nature of the
selection process, we studied the properties of constituent
clonotypes within these CMV- and EBV-specific CD8
T
cell responses.
Assessment of CD8
T cell avidity using point-mutated
soluble pMHCI antigens
The extent to which a CD8
T cell depends on the pMHCI–
CD8 interaction for stable pMHCI tetramer binding is a mea-
sure of intrinsic avidity for antigen and likely reflects, in a non-
linear manner, both the affinity with which TCR binds cognate
pMHCI ligand and other factors such as the distribution, den-
sity, and mobility of TCR in the cell membrane. Thus, CD8
T cells with high intrinsic avidity for a defined antigen can be
selectively identified using pMHCI tetramers with mutations in
the
3 domain that abrogate CD8 binding without affecting the
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INTERCLONAL COMPETITION IN CD8
T CELL RESPONSES TO CMV AND EBV | Price et al.
1352
integrity of the TCR interaction (23, 24). Intrinsic avidity is
likely to be a closer reflection of TCR/pMHCI affinity than
“coreceptor-compensated” avidity; from here on, the term
avidity refers to the former parameter unless otherwise stated.
In 8/8 donors studied, both wild-type and CD8-null
CMV NV9 tetramers identified cognate CD8
T cell popu-
lations of similar magnitude (Fig. 2 A and not depicted).
Molecular analysis of these respective populations demon-
strated the presence of identical constituent clonotypes at
equivalent frequencies within donors (
n
2; unpublished
data). Thus, CD8
T cell responses to CMV NV9 exhibit a
high degree of avidity for cognate pMHCI antigen. Simi-
larly, in 2/5 donors studied, both wild-type and CD8-null
EBV GL9 tetramers identified cognate CD8
T cell popula-
tions of comparable magnitude and clonotypic composition
(Fig. 2 B and not depicted).
The CD8-null pMHCI tetramers exhibited several inter-
esting features in these experiments. First, background stain-
ing of CD8
T cells was substantially reduced (Fig. 2). This
is consistent with previous studies using pMHCI tetramers
with reduced, but measurable, CD8 binding properties (25).
In addition, the intensity of cognate staining with the CD8-
null reagents was consistently lower compared with the cor-
responding wild-type pMHCI tetramers under standardized
conditions (Fig. 2). These findings indicate a role for CD8 in
both cognate and noncognate antigen binding. Second, and
in contrast with the corresponding wild-type reagent under
identical conditions, CD8-null pMHCI tetramers failed to
stain the cognate CD8
T cell population at lower concen-
trations in several donors (Fig. 2 B and not depicted). This
observation indicates that the pMHCI–CD8 interaction as-
sumes greater relevance at low concentrations of soluble
ligand and presumably reflects a spectrum of avidities in anti-
gen-specific CD8
T cell responses. Third, in 3/5 donors,
the CD8-null reagent stained only a fraction of the CD8
T
cell population specific for EBV GL9 compared with the
corresponding wild-type tetramer even at high concentra-
tions (Figs. 2 and 3; donor T, 0.74 vs. 1.8%; donor M, 0.11
vs. 0.22%; donor F, 0.4 vs. 0.5%). These subtle differences
were exploited to separate antigen-specific CD8
T cell
clonotypes according to avidity based on differential staining
patterns with CD8-null pMHCI tetramers directly ex vivo.
Dissection of interclonal avidity relationships in CD8
T cell
populations directly ex vivo
In donor T, the cognate CD8-null pMHCI tetramer iden-
tified a substantially smaller population of CD8
T cells
specific for EBV GL9 than the corresponding wild-type
tetramer across a range of concentrations (Fig. 3). Consis-
tent with the relative clonal frequencies estimated by mo-
lecular methods, we reasoned that this high avidity popula-
tion might comprise the dominant clonotype within the
total antigen-specific CD8
T cell response (Fig. 1 B).
Analysis of the constituent clonotypes confirmed this hy-
pothesis (Fig. 3). Similarly, in donor F, the smaller EBV
GL9-specific CD8
T cell population (0.4% of total) iden-
tified with the CD8-null tetramer contained 3/4 of the
dominant clonotypes, but none of the subdominant clono-
types, that were present in the population detected using
the corresponding wild-type reagent (Fig. 1 and not de-
picted). Thus, dominant clonotypes exhibit high avidity for
cognate viral antigen.
Figure 2. Wild-type and CD8-null pMHCI tetramers stain comparable
populations of HLA A*0201-restricted CMV- and EBV-specific CD8
T cells directly ex vivo. Representative tetramer staining patterns and
titrations are shown for: (A) donor D (specificity: CMV NV9) and (B) donor
B (specificity: EBV GL9). Data plots represent live, CD3
lymphocytes.
(A and B, bottom right) The mean fluorescence intensity (MFI) of staining
with both the wild-type (red) and CD8-null (blue) tetramers at a concen-
tration of 0.5 g/l for both cognate (CD8
tet
; closed circles) and non-
cognate (CD8
tet
; open circles) binding is shown for all donors tested. In
each case, the MFI values are internally comparable. The p-values derived
from nonparametric paired t test comparisons of the MFIs for wild-type
and CD8-null tetramer binding across all donors shown were as follows:
CMV NV9 cognate, 0.078; CMV NV9 noncognate, 0.078; EBV GL9 cognate,
0.0009; and EBV GL9 noncognate, 0.0625.
Page 4
JEM VOL. 202, November 21, 2005
1353
ARTICLE
In donor F, both wild-type and CD8-null pMHCI tet-
ramers identified comparable populations of CD8
T cells
specific for CMV NV9 across a range of concentrations (Fig.
4 A); the clonotypic profile was identical in each case (Fig. 1
A and not depicted). However, a distinct staining pattern
was observed in titration experiments with the CD8-null re-
agent. A subpopulation of antigen-specific T cells, character-
ized by low intensity staining with tetramer and high levels
of CD8 coreceptor expression, became progressively more
prominent at lower concentrations (Fig. 4 A). Previous work
has shown that cognate CD8
T cells internalize pMHCI
tetramers at 37
C (26). In this light, we reasoned that the ti-
tration experiment represented an ex vivo competition assay
in which clonotypes of lower relative avidity are exposed at
limiting concentrations of antigen in the absence of a pMHCI–
CD8 interaction. The latter could potentially compensate
under physiological circumstances for a low affinity TCR–
pMHCI interaction (27–29), and the cosegregation of high
CD8 expression levels with reduced uptake of the tetramer
lends credence to this hypothesis (Fig. 4 A). Fine resolution
flow cytometric sorting and molecular analysis confirmed
that the distinct subpopulation isolated on the basis of re-
duced intensity staining with the cognate CD8-null reagent
comprised predominantly the subdominant clonotype (Fig. 4
B). Parallel studies of
TCRA
gene usage identified only two
mRNA species (unpublished data), thereby indicating that
the incomplete segregation patterns were more likely reflec-
tive of technical parameters, which were set to capture the
outcompeted population with maximal visual resolution and
distinction from background. The minority clonotype in this
subpopulation was not present in the analysis presented for
donor F in Fig. 1 A, but was identified at very low frequen-
cies in similar analyses from another time point; the rela-
tive enrichment and exclusive appearance of this clonotype
within the “low avidity” compartment is consistent with the
hypothesis that competition for antigen is a determinant of
clonal hierarchy. No such distinction in terms of clonotypic
composition was observed between populations separated
according to intensity of staining with the corresponding
wild-type tetramer, presumably because the interclonal com-
pensated avidities approximate each other (unpublished data).
Comparable experiments were performed with the CD8-
null pMHCI tetramer using PBMCs from donors K and P;
in each case, the subdominant clonotypes specific for CMV
NV9 were substantially more prevalent in the T cell popula-
tions sorted on the basis of lower intensity staining, despite
the relative lack of distinct staining patterns compared with
donor F (Fig. 4 C). As observed before (Fig. 4 B), additional
clonotypes were detected only within the population that
exhibited low avidity for cognate antigen; the finding that
even the least prevalent of these clonotypes exhibited
TCRB
gene usage patterns characteristic of the “themes” observed
for CD8
T cells that recognize CMV NV9 attests to their
specificity and argues against contamination with noncog-
nate clonotypes (Figs. 1 and 4 C). Thus, subdominant clono-
types are distinguished by lower relative avidities for cognate
pMHCI and a consequently greater dependence on CD8 for
soluble antigen uptake.
The role of CD8 in transduction from antigen avidity
to functional sensitivity
To examine the relationship between effector function and
antigen avidity directly ex vivo, C1R cells were generated
that expressed equivalent levels of either wild-type or CD8-
null HLA A
*
0201 tagged with GFP. These cells were pulsed
in parallel with exogenous CMV NV9 peptide at a range of
concentrations and used to present antigen to PBMCs from
donor F. In conjunction with a flow cytometric readout
based on functional parameters, this approach enabled the
discrimination of interclonal avidity and antigen sensitivity
relationships formally both in the presence and absence of a
pMHCI–CD8 interaction. Substantial differences in func-
tional sensitivity were observed according to the nature of the
peptide-presenting cell; for C1R cells bearing mutant CD8-
null HLA A
*
0201, the peptide concentration threshold for
detection of cytokine release in the dominant responding
CD8
T cell clonotype was two orders of magnitude higher
than that observed for the corresponding C1R cells express-
ing wild-type HLA A
*
0201 (Fig. 5 A). This is consistent
with a predominant role for CD8 at low levels of antigen
density during the process of T cell activation (30). Higher
antigen concentrations were also required to elicit functional
responses in the subdominant TCRV
27 clonotype com-
Figure 3. Interclonal avidity differences can be separated directly
ex vivo using wild-type and CD8-null pMHCI tetramers in parallel.
CD8
T cells specific for EBV GL9 in PBMCs from donor T were stained
with the corresponding wild-type (top) or CD8-null (bottom) pMHCI
tetramers and sorted by flow cytometry. Constituent clonotypes within
each sorted population are shown (right; total number of clones analyzed:
wild-type, 65; CD8-null, 77). Common clonotypes were identical at the
nucleotide level.
Page 5
INTERCLONAL COMPETITION IN CD8
T CELL RESPONSES TO CMV AND EBV | Price et al.
1354
pared with the dominant clonotype; this distinction was ex-
aggerated in the absence of a pMHCI–CD8 interaction, and
reflected by interclonal differences in TCR down-regulation
under these conditions (Fig. 5 A). Comparable results were
obtained with PBMCs from donor T using exogenous EBV
GL9 peptide in this system, although the antigen density
threshold required for functional activation was substantially
higher (Fig. 5 B). Interestingly, the subdominant TCRV
25-1
clonotype exhibited minimal TCR down-regulation, even in
the presence of pMHCI–CD8 binding (Fig. 5 B). This ob-
servation is consistent with the lower avidity of this clonotype
identified by lack of staining with the cognate CD8-null tet-
ramer (Fig. 3), and suggests a complex relationship between
TCR triggering and downstream functional consequences.
The observed interclonal cosegregation of avidity and
TCR down-regulation suggests that competitive effects can
Figure 4. Differential patterns of staining with CD8-null pMHCI
tetramers can distinguish subtle variations in clonotype avidity
directly ex vivo. (A) Titration experiments with PBMCs from donor F reveal
a distinct subpopulation of CD8
T cells specific for CMV NV9 (indicated
by arrow) that is out-competed for uptake of cognate CD8-null pMHCI
tetramer at low concentrations (right); this staining pattern is not observed
with the corresponding wild-type pMHCI tetramer (left). Concentrations of
each pMHCI tetramer, from top to bottom, are: 0.5, 0.25, 0.05, 0.025, and
0.005 g/l. Plots are gated on live lymphocytes. (B) The subdominant
clonotype specific for CMV NV9 from donor F is identified by reduced
intensity staining with the corresponding CD8-null pMHCI tetramer at low
concentrations. Subpopulations of cognate CD8
T cells were sorted by
flow cytometry through gates depicted by the colored boxes (left). Constit-
uent clonotypes within each sorted population are shown (right; total
number of clones analyzed: CD8-null bright, 83; CD8-null dim, 87). The
dominant clonotype identified with the corresponding wild-type tetramer
(Fig. 1) is shown in bold. Common clonotypes were identical at the nucle-
otide level. (C) Donor K (left) and donor P (right) PBMCs were stained with
the CD8-null CMV NV9 pMHCI tetramer and sorted through the gates
indicated by the colored boxes. Clonotypes identified by molecular analysis
of these sorted cells are shown below each panel (total number of clones
analyzed: donor K, 74; donor P, 67). Clonal representation and dominance
hierarchies from parallel sorts of CD8
T cells that stained brightly with
the CD8-null reagent reflected those identified with the corresponding
wild-type tetramer in each case (Fig. 1 and not depicted); the dominant
clonotypes from these sorts are in bold. In donor P, public clonotypes were
detected within the gated CD8
T cell population; these are shown in
colored boxes that match those in Fig. 1 C.
Page 6
JEM VOL. 202, November 21, 2005
1355
ARTICLE
impair the antigen-mediated triggering process (6, 31). We
sought to confirm this with a direct examination of antigen
uptake. Previous studies have demonstrated that CD8
T
cells can internalize pMHCI complexes presented on the tar-
get cell surface (32, 33). To quantify this process at the level
of individual antigen-specific clonotypes, we measured the
frequency of GFP-expressing cells in each of the responding
TCRV
-defined CD8
T cell populations shown in Fig. 5
at the maximum peptide concentration. In all cases, the
dominant clonotype displaying IFN
production in response
to cognate antigen-bearing targets acquired GFP more effi-
ciently than the corresponding subdominant clonotype (Ta-
ble I). These findings indicate that dominant antigen-specific
CD8
T cell clonotypes internalize pMHCI complexes more
efficiently than subdominant clonotypes when presented si-
multaneously with identical antigen-bearing target cells, and
are consistent with the notion of avidity-based competitive
signal extinction as a mechanism for clonal dominance (6).
In sum, these results substantiate the avidity profiles de-
termined by physical analyses with soluble antigens (Figs. 3
and 4) and are consistent with a proportionately greater role
for CD8 in the facilitation of antigen recognition by clonotypes
with lower avidities (34).
Phenotypic properties of virus-specific CD8
T cell
clonotypes directly ex vivo
If competition for cognate antigen is a substantial formative in-
fluence in vivo, then this phenomenon might become manifest
as interclonal differences in maturation phenotype. To test this
possibility, we conducted a phenotypic analysis of individual
clonotypes contained within the CD8
T cell populations spe-
Figure 5. Functional titration experiments in the absence of pMHC-
CD8 binding expose a proportionately greater coreceptor contribution
to antigen sensitivity in low avidity clonotypes directly ex vivo. C1R
cells expressing equivalent levels of either wild-type or CD8-null HLA
A*0201 on the cell surface were pulsed with the indicated concentrations of
either CMV NV9 (A) or EBV GL9 (B) peptide for 2 h and washed thoroughly.
PBMCs from donor F (A) or donor T (B) were pulsed with the unrelated HLA
A*0201-restricted peptide SLYNTVATL to block autologous presentation of
NV9 or GL9 peptides, respectively. For each condition, 2 10
6
PBMCs were
added to 10
6
C1R cells and incubated for 6 h in the presence of brefeldin A;
cells were then analyzed for intracellular IFN production by flow cytometry
(representative plots are shown in Fig. S1, available at http://www.jem.org/
cgi/content/full/jem.20051357/DC1). Plots are gated on CD3
, CD8
lympho-
cytes. Colored dots represent T cells expressing the indicated subdominant
(blue) or dominant (red) TCRV that produce IFN; these events are super-
imposed on density plots representing the total CD8
T cell population
according to expression of subdominant (y axis) or dominant (x axis) TCRV.
The percentage of all CD8
T cells that express subdominant (blue) or domi-
nant (red) TCRV and produce IFN is indicated. Precise quantification of
the dominant clonotype, but not the subdominant clonotype, in B was ham-
pered by TCR down-regulation. Peptide concentrations are shown at top
right in each plot. Negative controls were mock-pulsed with medium. Other
effector functions showed similar patterns (not depicted).
Table I. Antigen-specific uptake of GFP-labelled pMHCI
complexes from the surface of C1R target cells
HLA A*0201 heavy chain
Antigen TCRV IFN
a
Wild type
b
D227K/T228A
b
CMV-NV9
6-5
4.5 8.9
1.7 3.0
27
1.3 1.7
0.8 1.3
EBV-GL9
20-1
9.6 16.4
3.3 2.6
25-1
4.8 11.2
1.7 3.0
a
CD8
T cells that do not secrete IFN in response to stimulation () represent the
internal negative control for each assay.
b
Values represent the percentage of CD8
T cells that contain GFP for each experi-
mental condition.
Page 7
INTERCLONAL COMPETITION IN CD8
T CELL RESPONSES TO CMV AND EBV | Price et al.1356
cific for EBV GL9 in donor T and CMV NV9 in donor F us-
ing polychromatic flow cytometry (Fig. 6). For these experi-
ments, only the relevant wild-type tetramers were used in
conjunction with TCR V-specific monoclonal antibodies to
distinguish each antigen-specific clonotype; this approach stan-
dardizes the experimental conditions and obviates any biologi-
cal anomalies that might be induced by reagents with differ-
ential CD8 binding properties. In donor T, dominant and
subdominant clonotypes specific for EBV GL9 displayed al-
most identical terminally differentiated (CD27
CD45RO
CD57
) phenotypes (Fig. 6 A); this relatively unusual pheno-
type suggests an ongoing response to active viral replication
(35). Interestingly, however, when the light scatter profile was
adjusted to incorporate larger and more granular cells, a distinct
subpopulation of CD3
dim
CD8
dim
CD57
TCRV20-1
anti-
gen-specific T cells was identified with the EBV GL9 tetramer
after doublet exclusion; this is consistent with preferential anti-
gen-derived signal sequestration by this clonotype even in the
presence of a pMHCI–CD8 interaction (unpublished data).
Terminally differentiated phenotypes also dominated the
CMV- and EBV-specific CD8
T cell populations in other
donors (unpublished data). In donor F, however, clear inter-
clonal phenotypic differences were observed in the CD8
T
cell population specific for CMV NV9. The dominant
clonotype was terminally differentiated as defined by the ex-
pression of CD57 on the cell surface; the principal sub-
dominant clonotype, in contrast, exhibited a less differenti-
ated CD45RO
dim
CD57
effector phenotype (Fig. 6 B). Thus,
avidity differences can be reflected in cellular differentiation
phenotype, with high avidity clonotypes preferentially driven
toward replicative senescence.
Functional properties of virus-specific CD8
T cells
clonotypes directly ex vivo
To determine whether interclonal avidity differences are asso-
ciated with functional heterogeneity in the CD8
T cell re-
sponse to cognate antigen, we developed a flow cytometric
panel that enables the independent assessment of five distinct
effector functions simultaneously at the single cell level. In do-
nor F, the functional profile of the antigen-specific CD8
T
cell population in response to exogenous CMV NV9 peptide
was dominated by 4/32 possible permutations defined on the
basis of individual effector readouts; both the dominant and
subdominant clonotypes, differentiated on the basis of their
distinct phenotypic profiles, were equally distributed in each
of these subsets (Fig. 7). Thus, although the nature of the
CD8
T cell response in the presence of saturating concentra-
tions of exogenous peptide is not necessarily reflective of the
in vivo situation, these results do demonstrate that clonotypes
with different avidities for cognate antigen can mediate identi-
cal effector functions in the presence of a sufficient stimulus.
This constitutes an important control and is consistent with
the concept that interclonal differences in the strength of the
antigen-derived signal, which can contribute to overall T cell
fitness (36), occur at the limiting concentrations of antigen
that are likely to be encountered in vivo and are not represen-
tative of inherent differences between T cell clonotypes.
DISCUSSION
In this study, we used a combination of point-mutated pMHCI
antigens in both soluble and cell-associated forms, a modified
template-switch anchored RT-PCR that detects all ex-
pressed TCRBV genes without bias, and polychromatic flow
cytometric analysis of cellular function and phenotype to dis-
sect directly ex vivo factors that contribute to the clonal
structure of antigen-specific CD8
T cell populations during
persistent infection with CMV and EBV. A number of con-
sistent features emerged from this analysis.
First, CD8
T populations specific for CMV NV9 and
EBV GL9 are oligoclonal and generally dominated by one or
two prevalent clonotypes (Fig. 1, A and B). Furthermore, no
consistent TCRBV CDR3 motifs were apparent in either
Figure 6. CD8
T cell clonotypes specific for the same viral anti-
gen can have similar or different memory phenotypes. PBMCs were
stained with the cognate wild-type pMHCI tetramer and mAbs specific for
CD8, CD27, CD45RO, CD57, and the indicated TCRV. The memory pheno-
type of the total CD8
population is depicted as a black density plot, and
the antigen-specific cells separated according to TCRV expression are
shown superimposed as colored dots. (A) Distinct CD8
T cell clonotypes
specific for EBV GL9 in donor T exhibit similar memory phenotypes despite
differences in avidity. (B) Distinct CD8
T cell clonotypes specific for CMV
NV9 in donor F exhibit different memory phenotypes.
Page 8
JEM VOL. 202, November 21, 2005 1357
ARTICLE
case. Thus, in contrast with some immunodominant CD8
T cell responses, the peripheral TCR repertoire that can
serve as a reservoir for recruitment of antigen-specific clono-
types is diverse and does not appear to be limited by the ex-
acting structural constraints that can be imperative for func-
tional pMHCI engagement (20). The potential for a given
pMHCI complex to be recognized by TCRs with diverse
structural features is likely to be a prerequisite for the occur-
rence of interclonal competition within an antigen-specific
CD8
T cell response.
Second, common antigen-specific clonotypes were ob-
served in different individuals for each of the CMV- and
EBV-specific CD8
T cell populations (Fig. 1, A and B).
Importantly, these public clonotypes were encoded distinctly
at the nucleotide level in different donors and were confined
by antigen specificity (Fig. 1 C). In addition, several recur-
rent patterns were observed at the level of primary TCRB
CDR3 structure in the private repertoire specific for each
antigen. These data provide evidence that cognate TCR se-
lection in the periphery operates at the protein level as pre-
viously described for antibody responses, and implies that
ongoing selection for optimal fit guides convergent clonal
evolution in vivo.
Third, CD8
T cells specific for CMV NV9 were char-
acterized by high levels of avidity for cognate antigen in all
donors tested (Figs. 2 A and 4; not depicted). This property
is consistent with ongoing competition for antigen in vivo.
Curiously, the same did not appear to hold for CD8
T
cells specific for EBV GL9, which exhibited comparatively
greater dependency on the pMHCI–CD8 interaction for
stable binding and uptake of soluble tetrameric antigen com-
plexes (Figs. 2 B and 3; not depicted) and for activation (Fig.
5). Although the relationship between antigen avidity and
affinity is complex, it seems feasible that these EBV-specific
CD8
T cells exhibit weaker TCR–pMHCI interaction af-
finities. There are several potential explanations for this ob-
servation. For example, it is known that high avidity CD8
T cells are susceptible to apoptosis and clonal deletion under
conditions of excess antigen load (37–39). This process con-
tributes to the pattern and quality of the CD8
T cell re-
Figure 7. Individual CD8
T cell clonotypes specific for the same
antigen can display similar functionality despite differences in pheno-
type and avidity. (A) PBMCs from donor F were stimulated for 5 h with CMV
NV9 peptide and stained with a panel of mAbs to examine degranulation
(CD107a), cytokine production (IFN, TNF, IL2), and chemokine production
(MIP1). The 31 possible positive responses that can be discerned from
simultaneous examination of these five functional parameters are shown
on the x axis. The total frequency of CD8
T cells displaying each particular
functional profile is shown on the y axis. The four major responses are colored
in blue, red, green, and purple. (B) The memory phenotypes of specific T cells
expressing one of the four major functional patterns shown in A are overlaid
on the phenotype of the total CD8
T cell population. Two memory popula-
tions are apparent for each functional response, corresponding directly to the
phenotypes of the two major CD8
T cell clonotypes specific for CMV NV9
(Fig. 6). All events shown are CD3
, CD8
, CD4
, CD14
, CD19
, with a small
lymphocyte forward/side scatter profile excluding doublets and aggregates.
Page 9
INTERCLONAL COMPETITION IN CD8
T CELL RESPONSES TO CMV AND EBV | Price et al.1358
sponse to persistent viral infections that evolves over time
(40). Thus, it is reasonable to postulate that high levels of
viremia during the acute phase of EBV infection might lead
to a predominantly low avidity CD8
T cell response during
the chronic phase, especially given that GL9 is a lytic epitope
preferentially expressed in the early stages. In contrast, the
early period of viral dissemination during CMV infection
might be associated with fewer negative effects on the reac-
tive immune repertoire, thereby allowing the continuous se-
lection and accrual of high avidity clonotypes during the
chronic phase of infection (41, 42). Further studies will be
needed to distinguish whether the observed differences in
CD8
T cell avidity for CMV- and EBV-derived epitopes
reflect a function of virus biology or an intrinsic property of
the epitopes themselves.
Fourth, clonal dominance within the hierarchical struc-
ture of CD8
T cell populations specific for CMV NV9 and
EBV GL9 is a function of avidity for antigen. Thus, domi-
nant clonotypes exhibited high levels of avidity directly ex
vivo coupled to more sensitive functional response profiles,
whereas the inverse applied to subdominant clonotypes
(Figs. 3–5). These data provide evidence that interclonal
competition for antigen is a formative influence during the
maintenance of CD8
T cell populations specific for persis-
tent DNA viruses. In addition, this process can be reflected
by interclonal phenotypic dissociations, with high avidity
clonotypes preferentially driven toward senescence presum-
ably as a consequence of successful antigen sequestration
(Fig. 6). This latter point is reinforced by the finding that
CD8
T cell clonotypes with identical specificities can be
equivalent in terms of their potential to respond functionally
when presented with sufficient densities of cognate antigen
(Fig. 7). Studies in murine models indicate that the sensitiv-
ity with which specific T cell populations recognize antigen
can evolve during the development of an effector response
independent of changes in TCR usage; the mechanisms in-
volved include changes in membrane composition and the
basal phosphorylation status of signaling molecules, and al-
tered topographical organization of TCR and coreceptor
(43–47). There is also some evidence for avidity maturation
within antigen-specific T cell populations due to positive
selection of cognate TCRs with both optimal affinity for
pMHC and slower dissociation kinetics resulting in longer
dwell times (8, 9, 12). In the present study, the observation
that intrinsic avidity properties cosegregate with distinct
clonotypes suggests that TCR-dependent mechanisms are
the principal determinants of response optimization during
antigen-driven CD8
T cell expansion in chronic infection.
Although the relationship between antigen avidity and func-
tional sensitivity is nonlinear, such progressive evolution to-
ward high avidity CD8
T cell usage within the confines of
peripheral T cell repertoire is potentially advantageous in
terms of improved effector function delivery and control of
viral replication (48–50).
Fifth, the data indicate that the differential compensatory
role of CD8 enables the recruitment and maintenance of low
avidity clonotypes within the antigen-specific CD8
T cell
response, albeit at mostly subdominant frequencies, as sug-
gested previously by studies of clones derived in vitro (27,
51). Indeed, it is apparent that this process, which affects
both overall avidity and functional sensitivity in a manner
dependent on CD8 expression levels and other variables
(29), can enable low avidity clonotypes to coexist quite
competitively in some instances with their higher avidity as-
sociates (Figs. 1, 3, and 5). Such coreceptor-mediated effects
could serve to promote clonotypic diversity within antigen-
experienced memory T cell pools and confer potential evo-
lutionary advantages in the face of pathogens with variable
antigenicity (21). Interestingly, a recent study concluded that
an affinity threshold mechanism operates during the periph-
eral selection and expansion of antigen-specific CD4
T cell
populations to limit the competitive advantage of clonotypes
with the highest avidity and prevent monopolization of the
response (12). It is possible that these distinct mechanisms for
engendering clonotypic diversity reflect differences in the
biophysical properties of the corresponding T cell corecep-
tors; thus, whereas CD8 has been shown to stabilize TCR–
pMHCI complexes at the cell surface (52), the affinity of the
pMHCII–CD4 interaction is substantially lower. Presum-
ably, there is also a threshold in CD8
T cell populations be-
yond which further increases in avidity either fail to confer
any additional competitive advantage or actually become
deleterious. The present study does not examine these issues,
but it seems likely that such a threshold might occur to limit
peripheral antigen-driven selection when the optimal activa-
tion window is exceeded by clonotypes with excessively
high avidity that escape thymic editing; under these circum-
stances, clonotypes with lower avidities might become dom-
inant. Other scenarios can also be envisaged in which low
avidity clonotypes might predominate. For example, a cross-
reactive clonotype driven to proliferate by high avidity rec-
ognition of the true cognate antigen might be detected with
wild-type pMHCI tetrameric complexes bearing the partial
agonist peptide due to the compensatory capacity of the
CD8 coreceptor. Further work is required to dissect these
complex relationships that are created by the existence of an-
tigen-specific CD8
T cells in the setting of a complex and
dynamic system rather than in isolation.
Many different selection forces can potentially interact to
define the clonotypic structure of a T cell population acti-
vated in response to a specific antigen. These formative pro-
cesses include antigen-induced apoptosis, avidity-mediated
competition, precursor frequency, senescence and exhaus-
tion, and structural constraints (2–13, 20, 21, 37–39, 53).
The relative impact of each of these factors under any given
circumstance will likely vary according to the nature and
dose of antigen, the duration of exposure, and the conditions
under which it is encountered. In this study, we provide di-
rect ex vivo evidence that competitive effects operate within
CD8
T cell populations specific for epitopes derived from
persistent DNA viruses to generate hierarchies that are dom-
inated by clonotypes with high avidity for antigen during
Page 10
JEM VOL. 202, November 21, 2005 1359
ARTICLE
human infection; concurrently, compensatory mechanisms
mediated through the CD8 coreceptor act to mitigate this
competitive advantage. These coincident processes likely
serve to optimize the delivery of antiviral effector function
while maintaining clonotypic diversity within the reactive
memory T cell pool.
MATERIALS AND METHODS
Donors. Peripheral blood samples were obtained by apheresis from
healthy volunteers and HIV-infected donors, selected according to CMV/
EBV serostatus and HLA genotype, with appropriate Institutional Review
Board approval. Donors D, H, K, R, S, and T were coinfected with HIV-1.
Plasma virus load (copies HIV RNA/ml)/total CD4
T cell count (cells/l
blood) measurements at the time of study were: 50/1,329; 2,473/616;
25,902/502; 50/958; 261/937; and 3,198/452, respectively.
Peptides. The immunodominant HLA A*0201-restricted CMV and
EBV epitopes used in this study are derived from the pp65 (NLVPM-
VATV; residues 495–503) and BMLFI (GLCTLVAML; residues 259–
267) proteins, respectively. The corresponding peptides were synthesized
to 95% purity (BioSynthesis).
Tetrameric pMHCI complexes. Tetrameric recombinant pMHCI anti-
gens were produced as described previously (54). In brief, biotin-tagged
HLA-A*0201 heavy chains and mutants thereof were expressed under the
control of a T7 promoter as insoluble inclusion bodies in Escherichia coli
strain BL21(DE3)pLysS (Novagen). IPTG-induced E. coli were lysed by re-
peated freeze/thaw cycles to release inclusion bodies that were subsequently
purified by washing with a 0.5% Triton X-100 buffer (Sigma-Aldrich). The
compound D227K/T228A mutation in the 3 domain of HLA A*0201 has
been shown to abrogate CD8 binding without affecting the biophysical
properties of the TCR docking platform (30). HLA A*0201 heavy chain
and
2
m inclusion body preparations were denatured separately in 8 M of
urea buffer (Sigma-Aldrich) and mixed at a 1:1 molar ratio; pMHCI was re-
folded in 2-mercaptoethylamine/cystamine (Sigma-Aldrich) redox buffer
with the appropriate synthetic peptide (BioSynthesis). After buffer exchange
into 10 mM Tris, pH 8.1, refolded monomer was purified by anion ex-
change. Purified monomers were biotinylated using d-biotin (Sigma-
Aldrich) and BirA enzyme. Excess biotin was removed by gel filtration.
Biotinylated pMHCI monomers were conjugated by addition of fluoro-
chrome-conjugated streptavidin at a 4:1 molar ratio, respectively, to pro-
duce tetrameric pMHCI complexes. All pMHCI tetramers were freshly
prepared for each experiment from pMHCI monomers stored at 80C to
avoid effects due to differences in protein stability (54). The concentration
of tetramer as expressed throughout this work refers to the pMHCI compo-
nent and was standardized for each comparative experiment. Once prepared,
tetramers were stored in the dark at 4C. Tetramer stains were performed at
37C for 20 min as described previously (26). For competition assays, protease
inhibitor mixes were excluded from the tetramer preparations.
Antibodies. Directly conjugated mAbs specific for the antigens listed were
used in the following fluorochrome combinations available from commercial
sources: (a) IL-2–allophycocyanin (APC); CD3-Cy7APC, IFN-FITC,
MIP1-PE, CD57-FITC, IFN-Cy7PE, and TNF-Cy7PE (BD Bio-
sciences); (b) CD4-Cy5.5PE (Caltag); (c) CD45RO-Texas red–PE (TRPE)
and TCR V-PE (Beckman Coulter). The following mAbs were conjugated
in our laboratory according to standard protocols: CD4–cascade blue, CD14-
Cy5PE, CD19-Cy5PE, CD27–cascade blue, CD107a-Alexa 680, CD8-
quantum dot (Qdot) 655, CD8-Qdot 705, CD57-Qdot 565, and TCR V-
APC. Unconjugated mAbs were obtained from BD Biosciences (CD4,
CD14, CD19, CD107a, CD8, CD57) or Beckman Coulter (TCR V). Flu-
orochromes were obtained from the following vendors: (a) cascade blue and
Alexa 680 (Invitrogen); (b) Cy5 (GE Healthcare); (c) APC (ProZyme); and
(d) Qdot 565, Qdot 655, and Qdot 705 (Quantum Dot Corp.).
Cell culture. All experiments were performed directly ex vivo on fresh or
frozen PBMCs isolated by standard Ficoll-Hypaque density gradient centrifu-
gation (GE Healthcare). Cryopreserved samples were thawed rapidly and re-
covered overnight at 37C/5% CO
2
in RPMI 1640 medium supplemented
with 10% heat-inactivated FCS, 100 U/ml penicillin, 100 g/ml streptomy-
cin, and 2 mM L-glutamine (R10) before use. C1R cells expressing GFP-
tagged, full-length wild-type HLA A*0201 and mutant D227K/T228A HLA
A*0201 were produced according to previously described protocols (30).
Functional analysis of memory T cells. Purified PBMCs were ad-
justed to 10
6
cells/ml in R10. After addition of anti-CD28 and anti-CD49d
costimulatory antibodies (1 g/ml each; BD Biosciences), 0.7 g/ml mo-
nensin (BD Biosciences), 10 g/ml brefeldin A (Sigma-Aldrich), and preti-
tred anti-CD107a mAb conjugated to Alexa 680, 1 ml of the cell suspension
was used for each experimental condition. Peptides were used at a final con-
centration of 2 M. Negative controls that contained no added peptide,
and positive controls that contained Staphylococcus enterotoxin B (Sigma-
Aldrich) at 1 g/ml were included in parallel for each assay. Cells were in-
cubated for 5 h at 37C/5% CO
2
, washed once (1% bovine serum albumin/
0.1% sodium azide in PBS), and stained with directly conjugated mAbs spe-
cific for the cell surface antigens CD4, CD8, CD14, CD19, CD27,
CD45RO, and CD57 for 20 min in the dark at room temperature. After a
further wash, cells were permeabilized using the Cytofix/Cytoperm kit (BD
Biosciences) according to the manufacturer’s instructions. After permeabili-
zation, the cells were washed twice in the supplied buffer, and then stained
with mAbs specific for CD3, IFN, MIP1, TNF, and IL-2 for 20 min in
the dark at room temperature. Cells were washed one further time and
fixed in PBS containing 1% paraformaldehyde.
Flow cytometry and cell sorting. Six-parameter analysis was conducted
using a FACSCalibur flow cytometer (BD Immunocytometry Systems).
Polychromatic analysis of phenotype and function was performed using a
modified LSR II flow cytometer (BD Immunocytometry Systems), equipped
for the detection of 17 fluorescent parameters. A minimum of 750,000 total
events was collected for each sample. Data analysis was performed in all cases
using FlowJo version 6.0 (TreeStar Inc.). Cells stained for analysis of clo-
notype composition were sorted at 25 PSI using a modified FACS DIVA
(Becton Dickinson). Instrument set-up was performed according to the man-
ufacturer’s instructions. Electronic compensation was conducted with anti-
body-capture beads (BD Biosciences) stained separately with individual mAbs
used in the test samples. Post-sort purity was consistently 99%.
Clonotype analysis. The relevant antigen-specific CD8
T cell popula-
tions were sorted directly into 1.5 ml microtubes containing 100 l
RNAlater (Ambion Inc.). For the purposes of this study, clonotype analysis
was performed only on cells labeled physically with cognate pMHCI tetra-
mers; capture assays based on biological readouts potentially misrepresent the
repertoire due to functional impairment of CD8
T cells during persistent
viral infections. At least 5,000 cells were collected for each experimental
condition. After cell lysis, mRNA was extracted (Oligotex Kit; QIAGEN)
and subjected to a nonnested, template-switch anchored RT-PCR using a
3 TCRB constant region primer (5-GCTTCTGATGGCTCAAACA-
CAGCGACCTC-3) as described previously (18). Amplified products were
ligated into pGEM-T Easy vector (Promega) and cloned by transformation
of competent DH5 E. coli. Selected colonies were amplified by PCR using
standard M13 primers and then sequenced from an insert-specific primer us-
ing fluorescent dye terminator chemistry (Applied Biosystems). A minimum
of 50 clones was generated and analyzed per sample. Dominant and subdom-
inant assignments as used in this work refer to clonotype prevalence, which is
accurately reproduced by the molecular method described. Pseudogenes and
“nonfunctional” sequences that could not be resolved after inspection of the
individual chromatograms were discarded from the analysis. Nucleotide
comparisons were used to establish clonal identity. Data analysis was per-
formed using Sequencher Version 4.2 (Gene Codes Corporation). The
IMGT nomenclature system is used throughout this work (55).
Page 11
INTERCLONAL COMPETITION IN CD8
T CELL RESPONSES TO CMV AND EBV | Price et al.1360
Online supplemental material. Fig. S1 shows representative intracellu-
lar IFN plots and the gating strategy used to generate Fig. 5. Online sup-
plemental material is available at http://www.jem.org/cgi/content/full/
jem.20051357/DC1.
We thank D. Ambrozak for cell sorting.
D.A. Price is a Medical Research Council (UK) Clinician Scientist.
The authors have no conflicting financial interests.
Submitted: 7 July 2005
Accepted: 19 September 2005
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  • Source
    • "Fluorochrome-conjugated ELA/HLA-A2 tetramers were produced and used as described previously (Price et al., 2005). The D227K/T228A compound mutation was introduced into the a3 domain of HLA-A2 to generate CD8-null tetramers, which enable the selective identification of high-avidity antigen-specific CD8 + T-cells (Purbhoo et al., 2001; Wooldridge et al., 2009). "
    [Show abstract] [Hide abstract] ABSTRACT: Aging is associated with impaired vaccine efficacy and increased susceptibility to infectious and malignant diseases. CD8(+) T-cells are key players in the immune response against pathogens and tumors. In aged mice, the dwindling naïve CD8(+) T-cell compartment is thought to compromise the induction of de novo immune responses, but no experimental evidence is yet available in humans. Here, we used an original in vitro assay based on an accelerated dendritic cell coculture system in unfractioned peripheral blood mononuclear cells to examine CD8(+) T-cell priming efficacy in human volunteers. Using this approach, we report that old individuals consistently mount quantitatively and qualitatively impaired de novo CD8(+) T-cell responses specific for a model antigen. Reduced CD8(+) T-cell priming capacity in vitro was further associated with poor primary immune responsiveness in vivo. This immune deficit likely arises as a consequence of intrinsic cellular defects and a reduction in the size of the naïve CD8(+) T-cell pool. Collectively, these findings provide new insights into the cellular immune insufficiencies that accompany human aging.
    Full-text · Article · Oct 2015 · Aging cell
  • Source
    • "In this regard, the T cell receptor (TCR) is an excellent marker that allows antigen-specific responses to be followed along T cell differentiation and over time [11]. Thorough analyses of both viral and tumor antigen-specific CD8 T cell responses have revealed that the antigen-specific T cell repertoire is generally composed of highly frequent (also defined as dominant) as well as less frequent (defined as non-dominant) T cell clonotypes [12,13]. Through a process known as TCR clonotype selection, certain clonotypes may become more dominant along T cell differentiation [14-16] or throughout the time course of infection [17]. "
    [Show abstract] [Hide abstract] ABSTRACT: Persistent viruses are kept in check by specific lymphocytes. The clonal T cell receptor (TCR) repertoire against Epstein-Barr virus (EBV), once established following primary infection, exhibits a robust stability over time. However, the determinants contributing to this long-term persistence are still poorly characterized. Taking advantage of an in vivo clinical setting where lymphocyte homeostasis was transiently perturbed, we studied EBV antigen-specific CD8 T cells before and after non-myeloablative lympho-depleting chemotherapy of melanoma patients. Despite more advanced T cell differentiation, patients T cells showed clonal composition comparable to healthy individuals, sharing a preference for TRBV20 and TRBV29 gene segment usage and several co-dominant public TCR clonotypes. Moreover, our data revealed the presence of relatively few dominant EBV antigen-specific T cell clonotypes, which mostly persisted following transient lympho-depletion (TLD) and lymphocyte recovery, likely related to absence of EBV reactivation and de novo T cell priming in these patients. Interestingly, persisting clonotypes frequently co-expressed memory/homing-associated genes (CD27, IL7R, EOMES, CD62L/SELL and CCR5) supporting the notion that they are particularly important for long-lasting CD8 T cell responses. Nevertheless, the clonal composition of EBV-specific CD8 T cells was preserved over time with the presence of the same dominant clonotypes after non-myeloablative chemotherapy. The observed clonotype persistence demonstrates high robustness of CD8 T cell homeostasis and reconstitution.
    Full-text · Article · Oct 2013 · PLoS ONE
  • Source
    • "However, the terms 'public' and 'private' are used variably by different investigators, and have set up a false dichotomy in the spectrum of inter-individual frequencies of different TCR sequences. The term 'public' may refer to a TCR sequence seen in most individuals (as is the case for the influenza D b NP 366 -specific TCRb sequences in mice [54]), or simply to a TCR sequence seen in more than one individual [55]. Moreover, whether a TCR is classified as 'public' or 'private' is heavily dependent on the depth of sequencing (how many TCR sequences are obtained per individual), as well as the number of individuals compared [19 ]. "
    [Show abstract] [Hide abstract] ABSTRACT: The immune system is comprised of various immune cell populations that utilize a spectrum of immunoreceptors characterized by different levels of specificity, diversity, and prevalence within a host and across a population. These range from the universal receptors employed by both innate cells and innate-like cells, such as NKT and MAIT cells, through to receptors expressed on T cells with sporadic incidence. Here we review recent advances in understanding the molecular mechanisms that drive the observed spectra of T cell receptors in vivo.
    Full-text · Article · Jul 2013 · Current opinion in immunology
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