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*
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
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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, 7, 8, 9, 10, 11, 12, 13). For HIV, most studies have focused on single or small combinations of epitopes, with the assumption that they 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, 19, 20, 21, 22, 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.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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 x
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.
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 µl cell lysate (equivalent to 50,000 cells) with clonotype-specific 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
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 µg/ml). 51Cr release was detected, and specific lysis was determined as: ((experimental release - spontaneous release)/(total release - spontaneous release)) x 100.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Seven HIV-infected individuals were assessed for their total
CD8+ T cell responses to HIV by ICS for IFN-
using overlapping 15-mer 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.
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To determine the clonotypic diversity of HIV-specific responses,
we sorted by flow cytometry CD8+ T cells
responding to six HIV 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 Ab-mediated 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.
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); 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
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).
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B). 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 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 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.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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(20, 21, 22, 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, 2, 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 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.
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Footnotes |
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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: ddouek{at}mail.nih.gov
3 Abbreviations used in this paper: ICS, intracellular cytokine staining; qPCR, quantitative polymerase chain reaction; CDR3, third complementarity-determining region; TCRB, TCR -chain.
Received for publication November 13, 2001. Accepted for publication January 8, 2002.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and chains of the human T-cell antigen receptor recognizing HLA-A2 and influenza A matrix peptide. Proc. Natl. Acad. Sci. USA 88:8987. usage during the primary immune response to HIV. Nature 370:463.[Medline]
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T. Furutsuki, N. Hosoya, A. Kawana-Tachikawa, M. Tomizawa, T. Odawara, M. Goto, Y. Kitamura, T. Nakamura, A. D. Kelleher, D. A. Cooper, et al. Frequent Transmission of Cytotoxic-T-Lymphocyte Escape Mutants of Human Immunodeficiency Virus Type 1 in the Highly HLA-A24-Positive Japanese Population J. Virol., August 15, 2004; 78(16): 8437 - 8445. [Abstract] [Full Text] [PDF]
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D. Meyer-Olson, N. H. Shoukry, K. W. Brady, H. Kim, D. P. Olson, K. Hartman, A. K. Shintani, C. M. Walker, and S. A. Kalams Limited T Cell Receptor Diversity of HCV-specific T Cell Responses Is Associated with CTL Escape J. Exp. Med., August 2, 2004; 200(3): 307 - 319. [Abstract] [Full Text] [PDF]
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G. Ferrari, W. Neal, J. Ottinger, A. M. Jones, B. H. Edwards, P. Goepfert, M. R. Betts, R. A. Koup, S. Buchbinder, M. J. McElrath, et al. Absence of Immunodominant Anti-Gag p17 (SL9) Responses among Gag CTL-Positive, HIV-Uninfected Vaccine Recipients Expressing the HLA-A*0201 Allele J. Immunol., August 1, 2004; 173(3): 2126 - 2133. [Abstract] [Full Text] [PDF]
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M. Lichterfeld, X. G. Yu, M. T. Waring, S. K. Mui, M. N. Johnston, D. Cohen, M. M. Addo, J. Zaunders, G. Alter, E. Pae, et al. HIV-1-specific cytotoxicity is preferentially mediated by a subset of CD8+ T cells producing both interferon-{gamma} and tumor necrosis factor-{alpha} Blood, July 15, 2004; 104(2): 487 - 494. [Abstract] [Full Text] [PDF]
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M. P. Crawford, S. X. Yan, S. B. Ortega, R. S. Mehta, R. E. Hewitt, D. A. Price, P. Stastny, D. C. Douek, R. A. Koup, M. K. Racke, et al. High prevalence of autoreactive, neuroantigen-specific CD8+ T cells in multiple sclerosis revealed by novel flow cytometric assay Blood, June 1, 2004; 103(11): 4222 - 4231. [Abstract] [Full Text] [PDF]
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M. R. Betts, D. A. Price, J. M. Brenchley, K. Lore, F. J. Guenaga, A. Smed-Sorensen, D. R. Ambrozak, S. A. Migueles, M. Connors, M. Roederer, et al. The Functional Profile of Primary Human Antiviral CD8+ T Cell Effector Activity Is Dictated by Cognate Peptide Concentration J. Immunol., May 15, 2004; 172(10): 6407 - 6417. [Abstract] [Full Text] [PDF]
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A. R. Lopes, A. Jaye, L. Dorrell, S. Sabally, A. Alabi, N. A. Jones, D. R. Flower, A. De Groot, P. Newton, R. M. Lascar, et al. Greater CD8+ TCR Heterogeneity and Functional Flexibility in HIV-2 Compared to HIV-1 Infection J. Immunol., July 1, 2003; 171(1): 307 - 316. [Abstract] [Full Text] [PDF]
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S. A. Migueles, A. C. Laborico, H. Imamichi, W. L. Shupert, C. Royce, M. McLaughlin, L. Ehler, J. Metcalf, S. Liu, C. W. Hallahan, et al. The Differential Ability of HLA B*5701+ Long-Term Nonprogressors and Progressors To Restrict Human Immunodeficiency Virus Replication Is Not Caused by Loss of Recognition of Autologous Viral gag Sequences J. Virol., June 15, 2003; 77(12): 6889 - 6898. [Abstract] [Full Text] [PDF]
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O. O. Yang, P. T. N. Sarkis, A. Ali, J. D. Harlow, C. Brander, S. A. Kalams, and B. D. Walker Determinants of HIV-1 Mutational Escape From Cytotoxic T Lymphocytes J. Exp. Med., May 19, 2003; 197(10): 1365 - 1375. [Abstract] [Full Text] [PDF]
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D. Meyer-Olson, K. W. Brady, J. T. Blackard, T. M. Allen, S. Islam, N. H. Shoukry, K. Hartman, C. M. Walker, and S. A. Kalams Analysis of the TCR {beta} Variable Gene Repertoire in Chimpanzees: Identification of Functional Homologs to Human Pseudogenes J. Immunol., April 15, 2003; 170(8): 4161 - 4169. [Abstract] [Full Text] [PDF]
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J. Michalek, R. H. Collins, H. P. Durrani, P. Vaclavkova, L. E. Ruff, D. C. Douek, and E. S. Vitetta Definitive separation of graft-versus-leukemia- and graft-versus-host-specific CD4+ T cells by virtue of their receptor beta loci sequences PNAS, February 4, 2003; 100(3): 1180 - 1184. [Abstract] [Full Text] [PDF]
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A. D. Bitmansour, D. C. Douek, V. C. Maino, and L. J. Picker Direct Ex Vivo Analysis of Human CD4+ Memory T Cell Activation Requirements at the Single Clonotype Level J. Immunol., August 1, 2002; 169(3): 1207 - 1218. [Abstract] [Full Text] [PDF]
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). The frequency of responding cells by ICS is expressed
as the percentage of IFN-
) or SLYNTIAVL-pulsed (
), or unpulsed (
) autologous B cells
by SLYNTVATL-specific (left) and SLYNTIAVL-specific
(right) T cell lines from TX7.