http://www.jimmunol.org/content/168/6/3099.full.pdf

This information is current as
of July 8, 2017.
A Novel Approach to the Analysis of
Specificity, Clonality, and Frequency of
HIV-Specific T Cell Responses Reveals a
Potential Mechanism for Control of Viral
Escape
Daniel C. Douek, Michael R. Betts, Jason M. Brenchley,
Brenna J. Hill, David R. Ambrozak, Ka-Leung Ngai, Nitin J.
Karandikar, Joseph P. Casazza and Richard A. Koup
References
Subscription
Permissions
Email Alerts
This article cites 32 articles, 24 of which you can access for free at:
http://www.jimmunol.org/content/168/6/3099.full#ref-list-1
Information about subscribing to The Journal of Immunology is online at:
http://jimmunol.org/subscription
Submit copyright permission requests at:
http://www.aai.org/About/Publications/JI/copyright.html
Receive free email-alerts when new articles cite this article. Sign up at:
http://jimmunol.org/alerts
The Journal of Immunology is published twice each month by
The American Association of Immunologists, Inc.,
1451 Rockville Pike, Suite 650, Rockville, MD 20852
Copyright © 2002 by The American Association of
Immunologists All rights reserved.
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
Downloaded from http://www.jimmunol.org/ by guest on July 8, 2017
J Immunol 2002; 168:3099-3104; ;
doi: 10.4049/jimmunol.168.6.3099
http://www.jimmunol.org/content/168/6/3099
A Novel Approach to the Analysis of Specificity, Clonality, and
Frequency of HIV-Specific T Cell Responses Reveals a
Potential Mechanism for Control of Viral Escape1
Daniel C. Douek,2*† Michael R. Betts,* Jason M. Brenchley,* Brenna J. Hill,*
David R. Ambrozak,* Ka-Leung Ngai,‡ Nitin J. Karandikar,§ Joseph P. Casazza,§ and
Richard A. Koup*
C
ytotoxic T lymphocytes are unable to mediate full clearance, or adequate control of viral replication in HIV and
SIV infections, in part because they select for escape
mutations within viral epitopes (1–3). However, the cellular immune response may suppress the emergence of escape variants by
targeting multiple epitopes, thereby reducing the probability of escape mutations at all epitopes occurring simultaneously in a single
virus. Indeed, the most rapid emergence of escape from CD8⫹ T
cells occurs during acute infection when fewer epitopes are targeted (1, 2, 4). After acute infection, a CD8⫹ T cell response is
generated that is directed at multiple epitopes, and a broader response is associated with better control of viral replication and
slower disease progression (2, 4, 5).
Theoretically, the immune response may inhibit the emergence
of escape by targeting individual epitopes with multiple TCR,
thereby increasing the likelihood that sequence variants may be
recognized. Recent studies have found that CD8⫹ T cells in HIV,
SIV, EBV, and influenza often target a limited range of epitopes,
and that the responses to individual epitopes are oligoclonal in
composition (6 –13). For HIV, most studies have focused on single
or small combinations of epitopes, with the assumption that they
*Vaccine Research Center, National Institute of Allergy and Infectious Diseases, and
†
Department of Experimental Transplantation and Immunology, Medicine Branch,
National Cancer Institute, National Institutes of Health, Bethesda, MD 20892; ‡MegaBases Inc., Northwestern University, Evanston Research Park, IL 60201; and §University of Texas Southwestern Medical Center, Dallas, TX 75390
Received for publication November 13, 2001. Accepted for publication January
8, 2002.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
represent the total HIV-specific response (14). However, this assumption may lead to inappropriate conclusions about the overall
breadth and clonality of the CD8⫹ T cell response (15, 16). Furthermore, the identification of TCR targeting HIV has generally
relied on in vitro selection, cloning, and long-term propagation of
cytolytic T cells (8, 17). However, the antiviral potential of CD8⫹
T cell clones is often, although not always, better associated with
cytokine production than cytotoxic activity (18 –23). Therefore,
previous studies were potentially biased toward cytotoxic clones
with better in vitro growth characteristics, resulting in an incomplete picture of responding clonotypes.
We used cytokine production to identify HIV-specific CD8⫹ T
cells, and to carry out an unbiased analysis of the number of CD8⫹
T cell epitopes recognized and the number of TCR clonotypes that
target immunodominant epitopes. We also examined the influence
of sequence variation and antiretroviral therapy on the diversity
and frequency of epitope-specific TCR clonotypes over time.
Materials and Methods
Cell stimulation and analysis
Fifteen-mer peptides overlapping by 11 residues corresponding to sequences of HXBc2/Bal chimeric HIV strain (gag, pol, env, and nef) or HIV
SF2 strain (tat, rev, vif, vpr, and vpu) were used. Responses were determined by intracellular cytokine staining (ICS)3 as previously described
(24). For sorting of unfixed HIV-specific cells, peptide-matrix analysis was
used to identify optimal gag 15-mer peptides (24). A total of 5 ⫻ 107
PBMC were stimulated with peptide and incubated at 37°C for 1 h at 107
cells/ml, then at 106/ml for 4 h. Cells were washed and incubated with
IFN-␥ catch reagent (Miltenyi Biotech, Auburn, CA) according to the manufacturer’s instructions, and stained with fluorochrome-conjugated Abs to
IFN-␥ (Miltenyi Biotech), CD69, and CD8. IFN-␥⫹CD69⫹CD8⫹ cells
were sorted by FACS. All studies were approved by the institutions’ Institutional Review Board.
This work was supported by AMFAR Grant no. 02680-28-RGV (to D.C.D.).
2
Address correspondence and reprint requests to Dr. Daniel C. Douek, Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Room 3509, 40 Convent Drive, Bethesda, MD 20892. E-mail address:
[email protected]
Copyright © 2002 by The American Association of Immunologists
3
Abbreviations used in this paper: ICS, intracellular cytokine staining; qPCR, quantitative polymerase chain reaction; CDR3, third complementarity-determining region;
TCRB, TCR ␤-chain.
0022-1767/02/$02.00
Downloaded from http://www.jimmunol.org/ by guest on July 8, 2017
Escape from the CD8ⴙ T cell response through epitope mutations can lead to loss of immune control of HIV replication. Theoretically, escape from CD8ⴙ T cell recognition is less likely when multiple TCRs target individual MHC/peptide complexes,
thereby increasing the chance that amino acid changes in the epitope could be tolerated. We studied the CD8ⴙ T cell response to
six immunodominant epitopes in five HIV-infected subjects using a novel approach combining peptide stimulation, cell surface
cytokine capture, flow cytometric sorting, anchored RT-PCR, and real-time quantitative clonotypic TCR tracking. We found
marked variability in the number of clonotypes targeting individual epitopes. One subject recognized a single epitope with six
clonotypes, most of which were able to recognize and lyse cells expressing a major epitope variant that arose. Additionally,
multiple clonotypes remained expanded during the course of infection, irrespective of epitope variant frequency. Thus, CD8ⴙ T
cells comprising multiple TCR clonotypes may expand in vivo in response to individual epitopes, and may increase the ability of
the response to recognize virus escape mutants. The Journal of Immunology, 2002, 168: 3099 –3104.
CLONOTYPIC ANALYSIS OF HIV-SPECIFIC CD8⫹ T CELLS
3100
Identification of TCR ␤-chain (TCRB) sequences and
quantitative clonotypic PCR
Sorted peptide-specific cells (5000 from each stimulation) were lysed and
mRNA extracted (Oligotex kit, Qiagen, Valencia, CA). Anchored RT-PCR
was performed using a modified version of the SMART (switching mechanism at 5⬘ end of RNA transcript) procedure (25) and a TCRB constant
region 3⬘ primer for the PCR, to obtain TCRB PCR products from the 5⬘
end to the start of the TCRB constant region. The PCR product was ligated
into the pGEMT Easy vector (Promega, Madison, WI) and used to transform Escherichia coli. Colonies were selected, amplified by PCR with M13
primers, and sequenced. PBMC were lysed in proteinase K (Boehringer
Mannheim, Indianapolis, IN), and real-time quantitative PCR (qPCR) was
performed on 5 ␮l cell lysate (equivalent to 50,000 cells) with clonotypespecific primers and probes. Probes were labeled with FAM (6⬘ carboxyflourescene) and QSY7 quencher (MegaBases, Chicago, IL). A standard
curve was plotted for each clonotype and template copies were calculated.
Plasmid template for the standards was that used in the sequencing of each
third complementarity-determining region (CDR3). Samples were analyzed in duplicate. Full details of all primers, components, and cycling
temperatures are available upon request.
Chromium release assay
Results
Breadth and specificity of total HIV-specific CD8⫹ T cell
responses
Seven HIV-infected individuals were assessed for their total CD8⫹
T cell responses to HIV by ICS for IFN-␥ using overlapping 15mer peptide pools covering the entire HIV sequence, thus avoiding
bias to previously identified epitopes. Their virologic and clinical
characteristics around the time of the study are summarized in
Table I. Subjects TX4 –7 were noncompliant to therapy. Although
most subjects mounted their largest response to gag, there were
marked differences in the breadth and magnitude of their responses, irrespective of clinical status (Fig. 1A). We examined
their responses to the 122 individual overlapping peptides covering
HIV gag (to which all subjects mounted a response). Subjects
TX4, 6, and 7 responded to a restricted set of gag epitopes,
whereas subjects TX1, 2, 3, and 5 mounted broader responses (Fig.
1B). Subject TX7 was notable in that the total CD8⫹ T cell response to HIV was almost completely directed against one gag
epitope.
Identification of CD8⫹ T cell clones specific for
immunodominant HIV epitopes
To determine the clonotypic diversity of HIV-specific responses,
we sorted by flow cytometry CD8⫹ T cells responding to six HIV
Table I. Clinical details of subjects at time of analysis
Subject
Age
Infection
(years)
Current
Therapy
Viral Load
(copies/ml)
CD4 Count
(cells/␮l)
TX1
TX2
TX3
TX4
27
43
48
46
17
5
6
1.5
⬍50
4814
7846
⬍400
862
794
642
274
TX5
TX6
TX7
29
65
30
1
5
3
None
None
None
d4T, 3TC,
Efaverenz
d4T, 3TC
Combivir
Combivir
97662
36577
⬍400
471
462
627
FIGURE 1. Responses to HIV peptides in HIV-infected subjects by
ICS. A, CD8⫹ T cell to peptide pools are expressed as the percentage of
IFN-␥⫹CD8⫹ T cells. B, CD8⫹ T cell responses to 122 individual peptides
covering gag. Each peak represents the response to an individual peptide.
epitopes, which elicited the highest frequency responses, in five
subjects: TX1, 2, 3, 6, and 7 (indicated with an asterisk in Fig. 1).
Identification of cells by ICS requires permeabilization, rendering
RNA unamenable to analysis. Therefore, we used cell surface Abmediated capture of secreted IFN-␥, and CD69 induction. The
TCRB CDR3 of the sorted T cells were amplified by anchored
RT-PCR to ensure that all HIV-specific TCRB sequences were
amplified without bias to particular TCRBV families. Thus, all
epitope-specific clonotypes were represented in the PCR product
with a relative frequency reflecting that in the original sorted cell
population. Table II shows the sequences and frequencies of the
TCRB CDR3 that define the clonotypes responding to dominant
gag epitopes in each subject.
There was heterogeneity in the number of T cell clonotypes
elicited to the epitopes. Three of six epitopes elicited monoclonal
responses, whereas the others elicited oligo- or polyclonal responses. Such heterogeneity occurred within the response of TX2
to two gag epitopes (Fig. 1); one epitope elicited a response consisting of 15 clonotypes, while the other was monoclonal. We confirmed the frequency and TCRBV subfamily of their epitope-specific clones by flow cytometry with TCR␤ chain-specific mAbs
(data not shown). These results show that multiple T cell clonotypes can target individual epitopes during HIV infection. Furthermore, there is considerable CDR3 sequence and length diversity
among clonotypes targeting a single epitope in a subject. In addition, there was no bias to particular TCRBV families in any of
Downloaded from http://www.jimmunol.org/ by guest on July 8, 2017
PBMC were cultured with peptide-loaded autologous EBV-transformed B
cells, restimulated on day 7, and incubated for a further week. 51Cr-release
assay was performed on day 14, with 51Cr-labeled autologous EBV-transformed B cell targets loaded with SLYNTVATL (SL9) or SLYNTIAVL (1
␮g/ml). 51Cr release was detected, and specific lysis was determined as:
((experimental release ⫺ spontaneous release)/(total release ⫺ spontaneous release)) ⫻ 100.
The Journal of Immunology
3101
Table II. CDR3 sequences of HIV gag epitope-specific CD8⫹ T cells from five subjects, their frequency out of 100 bacterial clones, and their length
with respect to the full genomic sequencea
Subject
TX1
TX2
Epitope 1
TX7
a
CDR3 Amino Acid Sequence (VDJ regions)
TCRBJ Locus
% Frequency
CDR3 Length
2.1
8.2
8.2
14.1
14.1
3.1
3.1
3.1
22.1
22.1
5
18.1
18.1
2.1
7.1/3
24.1
2.1
FYICS------ARDDGP---SYEQYFGPGTRLTVTE
SAVYFCASS--TGTAM----NTEAFFGQGTRLTVVE
SAVYFCASS--QGSYGFK------YFGPGTRLTVTE
SLYFCASS---LDGQGTT----EQYFGPGTRLTVTE
SLYFCASS---TTGMTS----QPQHFGDGTRLSILE
SMYLCASS---LGLH------YEQYFGPGTRLTVTE
SMYLCASS---DGTGVIG----GYTFGSGTRLTVVE
SMYLCASS---RVGN------NEQFFGPGTRLTVLE
SAMYFCASS--RAAPYD-----EQFFGPGTRLTVLE
SAMYFCASS--IGLAGF----NEQFFGPGTRLTVLE
SALYLCASS--FGRGP----YNEQFFGPGTRLTVLE
SAAYFCASS--PLTLGTGS-TGELFFGEGSRLTVLE
SAAYFCASS--IATGT----QETQYFGPGTRLLVLE
FYICS------APGTGN----YEQYFGPGTRLTVTE
SALYLCASS--QKGLL-----GELFFGEGSRLTVLE
DAAMYLCATS-RDLTGSV----GYTFGSGTRLTVVE
FYICS------DGP------SYEQYFGPGTRLTVTE
2.7
1.1
2.7
2.7
1.5
2.7
1.2
2.1
2.1
2.1
2.1
2.2
2.5
2.7
2.2
1.2
2.7
100
25
7
16
3
6
6
3
4
4
7
6
4
3
3
3
100
2
1
–1
1
0
–1
1
–2
–1
0
0
3
1
1
–1
1
–1
25.1
2.1a
2.1b
7.1/2/3
5.1a
5.1b
8.1
6.4
7.1
17
SAVYFCASS--HNEL----TNEKLFFGSGTQLSVLE
FYICS------DSL------TGELFFGEGSRLTVLE
FYICS------DEGGRT---YNEQFFGPGTRLTVLE
SALYLCASS--QDVG-----TDTQYFGPGTRLTVLE
SALYLCASS--FDRDK--------FFGPGTRLTVLE
SALYLCASS--FDPP-----NTEAFFGQGTRLTVVE
SAVYFCAS---RYTG-------EQYFGPGTRLTVTE
SAMYLCASR--ITGG------NEQFFGPGTRLTVLE
SALYLCASS--QERTMLD----IQYFDAGTRLSVLE
TAFYLCASSI-TG-------NTEAFFGQGTRLTVVE
1.4
2.2
2.1
2.3
2.1
1.1
2.7
2.1
2.4
1.1
100
40
38
22
11
77
3
3
4
2
0
–2
1
–1
–4
0
–2
–2
0
–2
Peptides used for stimulation are marked with an asterisk in Fig. 1.
these responses, and no clear correlation between disease state and
the number of clonotypes elicited by the epitopes.
Longitudinal analysis of epitope sequence variability
To assess whether multiple CD8⫹ T cell clonotypes specific for a
particular HIV epitope influence the ability of epitope variants to
escape a CD8⫹ T cell response, we studied subject TX7 who recognized only one epitope in gag (SL9) during a 3-year period. This
epitope has been shown to resist escape from CD8⫹ T cells, partially because CD8⫹ T cells can recognize multiple variants of this
epitope (26, 27). Six different TCR clonotypes targeted this epitope
in subject TX7. Therefore, we could address whether epitope variants arose and dominated the viral quasispecies, whether the multiple TCR clonotypes could recognize these variants, and whether
multiple clonotypes remained expanded in vivo over time.
TX7 was initially treated with antiretroviral therapy and his viral
load was suppressed; however, he subsequently became noncompliant, and his viral load rebounded. Plasma and PBMC virus were
sequenced at the SL9 epitope region at multiple time points before
and after therapy began. Initially, the SL9 sequence SLYNTVATL
was present in all viruses sequenced. Then during therapy, three
variants emerged, SLYNTVAVL, SLYNTIATL, and SLYNTIAVL, the latter being predominant. None of the amino acid
changes were at MHC anchor residues, and all four peptides can
bind to HLA-A2, but have markedly variable recognition by different T cell clones (27). Later, the original sequence reemerged
and persisted. This could simply be a result of the loss of selective
advantage of the variants due to intermittent compliance with therapy (28). However, these particular variant epitopes have been
shown to arise in the absence of therapy (27), and thus, they may
have failed to escape recognition by the CD8⫹ T cell response.
Quantitative clonotype PCR of epitope-specific CD8⫹ T cells
To determine whether the different CD8⫹ T cell clonotypes
showed differential recognition of the variant epitope sequences,
we stimulated PBMC from TX7 at 156 wk after therapy initiation
with either the original SL9 or the predominant variant epitope,
SLYNTIAVL. The frequencies of responding CD8⫹ T cells were
2.8 and 2.6%, respectively, suggesting that both peptides stimulated a CD8⫹ T cell response, and that neither epitope escaped
immune recognition. We sorted cells responding to each peptide
and probed for the presence of the clonotypes by qPCR. We designed CDR3-specific primers and probes for each of the six T cell
clonotypes identified in subject TX7. The sensitivity of this assay
was one clonotype cell in 100,000 PBMC (data not shown). The
specificity of qPCR for HIV-specific clonotypes of TX7 was
confirmed as follows: 1) no amplification of clonotype sequences was seen in PBMC from five uninfected adults; 2) no
amplification was seen in the PBMC of TX7 when probed for
the two predominant HIV-specific clonotypes of TX6; and 3) no
HIV-specific clonotypes were detected in sorted CMV-specific CD8⫹
T cells from TX7 (Fig. 2A).
As expected, SL9-specific CD8⫹ T cells contained all clonotypes (except clone BV6.4, which was no longer detectable by
qPCR at this time point, as discussed in the next section). SLYN
TIAVL-specific T cells contained the same clonotypes at the same
relative frequencies as SL9-specific T cells, except that clone
BV17 was undetectable (Fig. 2B). Thus, the TCR expressed by
BV17 only recognized SL9, whereas the TCR expressed by the
four other clones were more promiscuous and could respond to
both SL9 and the variant epitopes. To confirm that this promiscuity
was not due to the high peptide concentration used in the assay, we
stimulated with suboptimal peptide concentrations. We found that
Downloaded from http://www.jimmunol.org/ by guest on July 8, 2017
TX2
Epitope 2
TX3
TX6
TCRBV Locus
3102
CLONOTYPIC ANALYSIS OF HIV-SPECIFIC CD8⫹ T CELLS
magnitude of the CD8⫹ T cell response to SL9 correlated positively with fluctuations in viral load resulting from nonadherence
to therapy, and multiple T cell clonotypes were present at each
time point analyzed, although their relative frequencies changed
over the 3-year study period. Clone BV6, while initially well represented, steadily decreased in frequency and was undetectable
after 96 wk. Notably, clone BV17, which recognized SL9 but not
the variant SLYNTIAVL epitope (Fig. 2A) became undetectable
20 wk after therapy initiation, when SLYNTIAVL was transiently
predominant, yet subsequently expanded to become the second
most frequent clonotype. Thus, the absolute and relative frequencies of the clonotypes changed not only in relation to viral load, but
also with respect to each other and to viral epitope variation.
Discussion
the responses to both epitopes declined, and that the response to
the variant peptide was also apparent at lower peptide concentrations (Fig. 2C). Furthermore, short-term lines stimulated with either SL9 or SLYNTIAVL lysed autologous B cells pulsed with
either peptide (Fig. 2D), indicating that at least a proportion of the
cells mediated cytotoxicity.
Longitudinal analysis of clonotype frequency
To assess changes in the frequencies of the SL9-specific clonotypes with respect to viral load and epitope sequence, we performed clonotypic qPCR on longitudinal samples of the PBMC of
TX7. Fig. 3 shows the changes in viral load, epitope sequence,
epitope-specific CD8⫹ T cell frequency by ICS (lower panel), and
epitope-specific clonotype frequency by qPCR (upper panel). The
Downloaded from http://www.jimmunol.org/ by guest on July 8, 2017
FIGURE 2. Responses to original and variant epitopes. A, PBMC from
TX7 (96 wk after therapy initiation) and from five healthy donors (nos.
1–5) were probed by qPCR for the six HIV-specific TX7 clones (Table I).
CMV-specific CD8⫹ T cells from TX7 were also probed. PBMC from
subjects TX6 and TX7 were probed for two of TX6’s HIV-specific clones.
B, Relative frequencies, by qPCR, of five TX7 clones in sorted CD8⫹ T
cells specific for SLYNTVATL (filled bars) and SLYNTIAVL (open bars).
C, TX7 PBMC stimulated with different concentrations of SLYNTVATL
(F) or SLYNTIAVL (E). The frequency of responding cells by ICS is
expressed as the percentage of IFN-␥⫹CD8⫹ T cells. D, Percentage of
specific lysis of SLYNTVATL- (f) or SLYNTIAVL-pulsed (Œ), or
unpulsed (⽧) autologous B cells by SLYNTVATL-specific (left) and
SLYNTIAVL-specific (right) T cell lines from TX7.
In this study we identified, characterized, and quantified HIV-specific CD8⫹ T cell clones in an unbiased manner using a novel
approach that combines in vitro peptide stimulation, cell surface
cytokine capture, flow cytometric sorting, anchored TCRB locus
RT-PCR, and clonotypic qPCR. This approach obviated skewing
of the clonotype population by prolonged stimulation and propagation. Furthermore, we used an anchored RT-PCR step to amplify
all TCRB CDR3 of all T cell clones present in the sorted population, without the incomplete coverage of, and bias to, particular
TCRBV families conferred by TCRBV-specific PCR primers. This
approach requires no prior knowledge of either the specific epitope
or the MHC restricting element, and when combined with qPCR,
it allows for sensitive and specific quantification of clonotypes at
any point, before or after the time at which clonotypes were originally identified. It should be noted that recent papers have demonstrated heterogeneity in cytokine production by HIV-specific T
cells and that not all secrete IFN-␥ (20 –23). Therefore, we may be
underestimating the frequency of responding T cells. However,
although not absolutely complete, this type of approach offers a
broader analysis than more limited studies performed in the past
with limited sets of peptides or Ags.
We found marked heterogeneity in the epitope breadth and magnitude of HIV-specific CD8⫹ T cell responses, with no apparent
relation to disease state, which could arise as a result of attempts
by the immune system to control multiple epitope variants (1–3).
Escape of an individual epitope would be more difficult in the
presence of multiple specific T cell clonotypes, which could arise
de novo and may act to limit virus escape (27, 29, 30), or may arise
as a result of recruitment of new clonotypes in response to epitope
variants (31, 32). Thus, we observed marked heterogeneity in the
clonality of T cells responding to immunodominant HIV gag
epitopes. Indeed, one epitope was targeted by 15 TCR clonotypes,
indicating that multiple TCR may expand in vivo in response to a
single HIV epitope.
The diversity of clonotypes specific for an HIV epitope could
influence the appearance and effects of viral escape mutations. In
subject TX7, whose CD8⫹ T cell response to HIV consisted almost entirely of six clonotypes directed against one gag epitope,
there was transient expression of two nonsynonymous mutations in
that epitope during antiretroviral therapy. Although these mutations can affect T cell recognition (26, 27), this polyclonal response
persisted. Furthermore, even though one clonotype did not respond
to the mutated epitope, the viral variants failed to escape the CD8⫹
T cell response of the four remaining clonotypes, which were able
to recognize, produce cytokine in response to, and lyse target cells
expressing the mutated epitope. It should be noted that our analysis
The Journal of Immunology
3103
of recognition of the variant epitope depended on the use of peptide-pulsed targets. To rule out aberrant processing and presentation of the variant epitopes, one would ideally perform this analysis using the Ags encoded in minigenes, or vaccinia virus vectors.
Although the viral variants that emerged failed to persist as dominant species, genuine viral escape mutants may not always reach
fixation because the selective advantage of the variant might be
lost (28). This is especially the case in a subject such as TX7, who
was intermittently compliant with therapy. However, these particular variant epitopes have been observed in the absence of therapy
and so may represent escape from immunologic pressure (27). Although the epitope variants described in this subject are genuine
escape mutants, the ultimate effect of mounting a broad CTL response which can recognize those epitopes is to abrogate the immunologic and virologic consequences of such escape. Thus, while
a multiclonal CTL response would not affect the probability of
viral escape (indeed, it might increase), it does increase the probability of recognition of these escape mutants.
It is of note that when viral load and HIV-specific CD8⫹ T cell
frequencies were high, the frequency of clonotypes detected by
qPCR was higher than that of responding T cells detected by ICS
(Fig. 3). Yet when virus was suppressed, the frequencies were
equal and the ratio was one. During any time interval, it is likely
that only a fraction of all epitope-specific CD8⫹ T cells will produce an optimal intracellular response, even when exposed to optimal levels of Ag and costimulation (33). Thus, while ICS may
only detect those CD8⫹ T cells which can respond under the specific conditions of the assay, qPCR may detect all clonotype cells,
irrespective of whether they are in a state of responsiveness to
peptide stimulation.
Our findings indicate that an understanding of HIV epitope escape from CD8⫹ T cells must take into account the plasticity of the
response against particular epitopes. Recognition of one or multiple epitopes, use of multiple TCR clonotypes, and HLA associations may all play roles in controlling HIV replication. Furthermore, in subjects immunized with HIV vaccines, it is possible that
the infecting virus would rapidly mutate away from the epitope
sequences used to elicit immunity. Our data suggest that strategies
which elicit multiple T cell clonotypes against HIV epitopes may
potentially provide immunity that can tolerate such mutations.
References
1. Allen, T. M., D. H. O’Connor, P. Jing, J. L. Dzuris, B. R. Mothe, T. U. Vogel,
E. Dunphy, M. E. Liebl, C. Emerson, N. Wilson, et al. 2000. Tat-specific cytotoxic T lymphocytes select for SIV escape variants during resolution of primary
viraemia. Nature 407:386.
2. Price, D. A., P. J. Goulder, P. Klenerman, A. K. Sewell, P. J. Easterbrook,
M. Troop, C. R. Bangham, and R. E. Phillips. 1997. Positive selection of HIV-1
cytotoxic T lymphocyte escape variants during primary infection. Proc. Natl.
Acad. Sci. USA 94:1890.
3. Wolinsky, S. M., B. T. M. Korber, A. U. Neumann, M. Daniels, K. J. Kunstman,
A. J. Whetsell, M. R. Furtado, Y. Cao, D. D. Ho, J. T. Safrit, and R. A. Koup.
1996. Adaptive evolution of human immunodeficiency virus type 1 during the
natural course of infection. Science 272:537.
4. Altfeld, M., E. S. Rosenberg, R. Shankarappa, J. S. Mukherjee, F. M. Hecht,
R. L. Eldridge, M. M. Addo, S. H. Poon, M. N. Phillips, G. K. Robbins, et al.
2001. Cellular immune responses and viral diversity in individuals treated during
acute and early HIV-1 infection. J. Exp. Med. 193:169.
5. Harrer, T., E. Harrer, S. A. Kalams, P. Barbosa, A. Trocha, R. P. Johnson,
T. Elbeik, M. B. Feinberg, S. P. Buchbinder, and B. D. Walker. 1996. Cytotoxic
T lymphocytes in asymptomatic long-term nonprogressing HIV-1 infection:
breadth and specificity of the response and relation to in vivo viral quasispecies
in a person with prolonged infection and low viral load. J. Immunol. 156:2616.
6. Moss, P. A., R. J. Moots, W. M. Rosenberg, S. J. Rowland-Jones, H. C. Bodmer,
A. J. McMichael, and J. I. Bell. 1991. Extensive conservation of ␣ and ␤ chains
of the human T-cell antigen receptor recognizing HLA-A2 and influenza A matrix peptide. Proc. Natl. Acad. Sci. USA 88:8987.
7. Pantaleo, G., J. F. Demarest, H. Soudeyns, C. Graziosi, F. Denis,
J. W. Adelsberger, P. Borrow, M. S. Saag, G. M. Shaw, R. P. Sekaly, and
A. S. Fauci. 1994. Major expansion of CD8⫹ T lymphocytes with a predominant
V␤ usage during the primary immune response to HIV. Nature 370:463.
8. Kalams, S. A., R. P. Johnson, A. K. Trocha, M. J. Dynan, S. Ngo, R. T. D’Aquila,
J. T. Kurnick, and B. D. Walker. 1994. Longitudinal analysis of T cell receptor
(TCR) gene usage by human immunodeficiency virus 1 envelope-specific cytotoxic T lymphocyte clones reveals a limited TCR repertoire. J. Exp. Med. 179:
1261.
9. Kalams, S. A., R. P. Johnson, M. J. Dynan, K. E. Hartman, T. Harrer, E. Harrer,
A. K. Trocha, W. A. Blattner, S. P. Buchbinder, and B. D. Walker. 1996. T cell
receptor usage and fine specificity of human immunodeficiency virus 1-specific
cytotoxic T lymphocyte clones: analysis of quasispecies recognition reveals a
dominant response directed against a minor in vivo variant. J. Exp. Med. 183:
1669.
Downloaded from http://www.jimmunol.org/ by guest on July 8, 2017
FIGURE 3. Clonotype frequency in HIV
infection. The frequency of six SLYNT
VATL-specific clones of TX7 was measured
in PBMC ⬎3 years by qPCR. The frequency
of each clone as a percentage of PBMC is
represented by a different colored bar (top).
The arrow shows when HIV-specific clones
were identified. Middle, Plasma and PBMC
virus sequence. Blue boxes denote SLYNTVATL, red boxes denote SLYNTIAVL,
hatched box denotes the variant mix. Up to 20
sequences were analyzed at each time point.
Lower, Frequency of SLYNTVATL-specific
IFN-␥⫹CD8⫹ T cells in TX7 PBMC (filled
bars) determined by ICS, and viral load (red
circles).
3104
23.
24.
25.
26.
27.
28.
29.
30.
31.
32.
33.
CD8⫹ T cells produce antiviral cytokines but are impaired in cytolytic function.
J. Exp. Med. 192:63.
Kostense, S., G. S. Ogg, E. H. Manting, G. Gillespie, J. Joling, K. Vandenberghe,
E. Z. Veenhof, D. van Baarle, S. Jurriaans, M. R. Klein, and F. Miedema. 2001.
High viral burden in the presence of major HIV-specific CD8⫹ T cell expansions:
evidence for impaired CTL effector function. Eur. J. Immunol. 31:677.
Betts, M. R., D. A. Ambrozak, D. C. Douek, S. E. Bonhoeffer, J. M. Brenchley,
J. P. Casazza, R. A. Koup, and L. J. Picker. 2001. Analysis of total human
immunodeficiency virus (HIV)-specific CD4 and CD8 T-cell responses: relationship to viral load in untreated HIV infection. J. Virol. 75:11983.
Chenchik, A., Y. Zhu, L. Diatchenko, R. Li, J. Hill, and P. Siebert. 1998. Generation and use of high quality cDNA from small amounts of total RNA by
SMART PCR. In RT-PCR Methods for Gene Cloning and Analysis.
P. Siebert and J. Larrick, eds. BioTechniques Books, Natick, p. 305.
Sewell, A. K., G. C. Harcourt, P. J. Goulder, D. A. Price, and R. E. Phillips. 1997.
Antagonism of cytotoxic T lymphocyte-mediated lysis by natural HIV-1 altered
peptide ligands requires simultaneous presentation of agonist and antagonist peptides. Eur. J. Immunol. 27:2323.
Brander, C., K. E. Hartman, A. K. Trocha, N. G. Jones, R. P. Johnson, B. Korber,
P. Wentworth, S. P. Buchbinder, S. Wolinsky, B. D. Walker, and S. A. Kalams.
1998. Lack of strong immune selection pressure by the immunodominant, HLAA*0201-restricted cytotoxic T lymphocyte response in chronic human immunodeficiency virus-1 infection. J. Clin. Invest. 101:2559.
Kelleher, A. D., C. Long, E. C. Holmes, R. L. Allen, J. Wilson, C. Conlon,
C. Workman, S. Shaunak, K. Olson, P. Goulder, et al. 2001. Clustered mutations
in HIV-1 gag are consistently required for escape from HLA-B27-restricted cytotoxic T lymphocyte responses. J. Exp. Med. 193:375.
Haas, G., U. Plikat, P. Debre, M. Lucchiari, C. Katlama, Y. Dudoit, O. Bonduelle,
M. Bauer, H. G. Ihlenfeldt, G. Jung, and M. B. Meyer. 1996. Dynamics of viral
variants in HIV-1 Nef and specific cytotoxic T lymphocytes in vivo. J. Immunol.
157:4212.
Weidt, G., W. Deppert, O. Utermohlen, J. Heukeshoven, and F. Lehmann-Grube.
1995. Emergence of virus escape mutants after immunization with epitope vaccine. J. Virol. 69:7147.
Goulder, P. J., A. K. Sewell, D. G. Lalloo, D. A. Price, J. A. Whelan, J. Evans,
G. P. Taylor, G. Luzzi, P. Giangrande, R. E. Phillips, and A. J. McMichael. 1997.
Patterns of immunodominance in HIV-1-specific cytotoxic T lymphocyte responses in two human histocompatibility leukocyte antigens (HLA)-identical siblings with HLA-A*0201 are influenced by epitope mutation. J. Exp. Med. 185:
1423.
Nowak, M. A., R. M. May, R. E. Phillips, S. Rowland-Jones, D. G. Lalloo,
S. McAdam, P. Klenerman, B. Koppe, K. Sigmund, C. R. Bangham, et al. 1995.
Antigenic oscillations and shifting immunodominance in HIV-1 infections. Nature 375:606.
Germain, R. N. 2001. The art of the probable: system control in the adaptive
immune system. Science 293:240.
Downloaded from http://www.jimmunol.org/ by guest on July 8, 2017
10. Callan, M. F., N. Steven, P. Krausa, J. D. Wilson, P. A. Moss, G. M. Gillespie,
J. I. Bell, A. B. Rickinson, and A. J. McMichael. 1996. Large clonal expansions
of CD8⫹ T cells in acute infectious mononucleosis. Nat. Med. 2:906.
11. Sourdive, D. J., K. Murali-Krishna, J. D. Altman, A. J. Zajac, J. K. Whitmire,
C. Pannetier, P. Kourilsky, B. Evavold, A. Sette, and R. Ahmed. 1998. Conserved
T cell receptor repertoire in primary and memory CD8 T cell responses to an
acute viral infection. J. Exp. Med. 188:71.
12. Wilson, J. D., G. S. Ogg, R. L. Allen, P. J. Goulder, A. Kelleher, A. K. Sewell,
C. A. O’Callaghan, S. L. Rowland-Jones, M. F. Callan, and A. J. McMichael.
1998. Oligoclonal expansions of CD8⫹ T cells in chronic HIV infection are
antigen specific. J. Exp. Med. 188:785.
13. Chen, Z. W., Y. Li, X. Zeng, M. J. Kuroda, J. E. Schmitz, Y. Shen, X. Lai,
L. Shen, and N. L. Letvin. 2001. The TCR repertoire of an immunodominant
CD8⫹ T lymphocyte population. J. Immunol. 166:4525.
14. Ogg, G. S., X. Jin, S. Bonhoeffer, P. R. Dunbar, M. A. Nowak, S. Monard,
J. P. Segal, Y. Cao, S. L. Rowland-Jones, V. Cerundolo, et al. 1998. Quantitation
of HIV-1-specific cytotoxic T lymphocytes and plasma load of viral RNA. Science 279:2103.
15. Dalod, M., M. Dupuis, J. C. Deschemin, D. Sicard, D. Salmon, J. F. Delfraissy,
A. Venet, M. Sinet, and J. G. Guillet. 1999. Broad, intense anti-human immunodeficiency virus (HIV) ex vivo CD8⫹ responses in HIV type 1-infected patients: comparison with anti-Epstein-Barr virus responses and changes during
antiretroviral therapy. J. Virol. 73:7108.
16. Betts, M. R., J. P. Casazza, B. A. Patterson, S. Waldrop, W. Trigona, T. M. Fu,
F. Kern, L. J. Picker, and R. A. Koup. 2000. Putative immunodominant human
immunodeficiency virus-specific CD8⫹ T-cell responses cannot be predicted by
major histocompatibility complex class I haplotype. J. Virol. 74:9144.
17. Walker, B. D., C. Flexner, K. Birch-Limberger, L. Fisher, T. J. Paradis,
A. Aldovini, R. Young, B. Moss, and R. T. Schooley. 1989. Long-term culture
and fine specificty of human cytotoxic T lymphocyte clones reactive with human
immunodeficiency virus type 1. Proc. Natl. Acad. Sci. USA 86:9514.
18. Martz, E., and D. M. Howell. 1989. CTL: virus control cells first and cytolytic
cells second? DNA fragmentation, apoptosis and the prelytic halt hypothesis.
Immunol. Today 10:79.
19. Guidotti, L. G., R. Rochford, J. Chung, M. Shapiro, R. Purcell, and F. V. Chisari.
1999. Viral clearance without destruction of infected cells during acute HBV
infection. Science 284:825.
20. Sandberg, J. K., N. M. Fast, and D. F. Nixon. 2001. Functional heterogeneity of
cytokines and cytolytic effector molecules in human CD8⫹ T lymphocytes. J. Immunol. 167:181.
21. Goulder, P. J., Y. Tang, C. Brander, M. R. Betts, M. Altfeld, K. Annamalai,
A. Trocha, S. He, E. S. Rosenberg, G. Ogg, et al. 2000. Functionally inert HIVspecific cytotoxic T lymphocytes do not play a major role in chronically infected
adults and children. J. Exp. Med. 192:1819.
22. Appay, V., D. F. Nixon, S. M. Donahoe, G. M. Gillespie, T. Dong, A. King,
G. S. Ogg, H. M. Spiegel, C. Conlon, C. A. Spina, et al. 2000. HIV-specific
CLONOTYPIC ANALYSIS OF HIV-SPECIFIC CD8⫹ T CELLS