JOURNAL OF VIROLOGY, Feb. 2007, p. 1517–1523 0022-538X/07/$08.00⫹0 doi:10.1128/JVI.01780-06 Copyright © 2007, American Society for Microbiology. All Rights Reserved.
Vol. 81, No. 3
Not All Cytokine-Producing CD8⫹ T Cells Suppress Simian Immunodeficiency Virus Replication䌤 Chungwon Chung,1 Wonhee Lee,1 John T. Loffredo,1 Benjamin Burwitz,1 Thomas C. Friedrich,1 Juan Pablo Giraldo Vela,2 Gnankang Napoe,1 Eva G. Rakasz,1 Nancy A. Wilson,1 David B. Allison,3 and David I. Watkins1,2* Wisconsin National Primate Research Center1 and Department of Pathology and Laboratory Medicine,2 University of Wisconsin—Madison, Madison, Wisconsin 53715-1299, and Department of Biostatistics, University of Alabama at Birmingham, Birmingham, Alabama 352943 Received 16 August 2006/Accepted 9 November 2006
Current assays of CD8ⴙ T-lymphocyte function measure cytokine production rather than the ability of these lymphocytes to suppress viral replication. Here we show that CD8ⴙ T-cell clones recognizing the same epitope vary enormously in the ability to suppress simian immunodeficiency virus SIVmac239 replication in an in vitro suppression assay. However, all Nef165-173IW9- and Vif66-73HW8-specific clones from elite controllers effectively suppressed SIV replication. Interestingly, in vitro suppression efficacy was not always associated with the ability to produce gamma interferon, tumor necrosis factor alpha, or interleukin-2. Several lines of evidence suggest that CD8⫹ T lymphocytes are critical in controlling human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) replication. Depletion of CD8⫹ cells from SIV-infected macaques results in increased viremia (27, 37, 49). The appearance of HIV-specific CD8⫹ T lymphocytes is correlated temporally with a precipitous reduction in viremia (10, 32). Furthermore, particular major histocompatibility complex (MHC) class I alleles are associated with control of viral replication (40, 41, 43, 56). In addition, CD8⫹ T lymphocytes exert selective pressure on viral sequences in vivo, selecting for escape variants (5–7, 11, 15, 21, 24, 42, 45).
Unfortunately, T-cell correlates of control of viral replication after HIV or SIV infection are not clearly defined. Neither the magnitude nor the breadth of CD8⫹ T-lymphocyte responses is consistently correlated with clinical outcome (1). Since these quantifiable traits of CD8⫹ T-lymphocyte responses do not appear to affect disease outcome, control of viral replication might instead be affected by the “quality” of CD8⫹ T lymphocytes. Factors that may influence HIV- or SIV-specific CD8⫹ T-lymphocyte antiviral efficacy include epitope expression kinetics, evolutionary constraints on epitope sequences, T-cell receptor (TCR)
TABLE 1. In vitro antiviral efficacies of the epitope-specific CD8⫹ T-cell clones tested in this study Epitope
No. of clones tested
No. of clones with ⱖ80% suppressiona
786
Mamu-A*01-Gag181-189CM9 Mamu-A*02-Nef159-167YY9 Mamu-B*17-Nef165-173IW9
21 3 7
8 0 7
151
1,071
Mamu-B*17Env830-838FW9 Mamu-B*17-Nef195-203MW9
13 16
11 12
917
58
968
Mamu-B*17-Vif66-73HW8
7
7
r00044
70
3,060
1,765
Mamu-A*02-Nef159-167YY9
6
1
r00060
68
28,000
908
Mamu-A*01-Gag181-189CM9 Mamu-A*01-Tat28-35SL8
3 12
1 6
r2125
493
107,000
629
Mamu-A*01-Gag181-189CM9
6
0
11
1
Animal
Days postinfection
r95061
1,519
⬍50
r95071
469
r98016
r96112 a
406
Viral load (no. of copies/ml)
234,000
CD4 cell count (no. of cells/l)
*
369
Mamu-B 17-Env830-838FW9
Reduction in SIV Gag p27-positive cell frequency in day 8 VSA target.
* Corresponding author. Mailing address: Department of Pathology and Laboratory Medicine, University of Wisconsin—Madison, 555 Science Dr., Madison, WI 53711. Phone: (608) 265-3380. Fax: (608) 265-8084. E-mail:
[email protected]. 䌤 Published ahead of print on 29 November 2006. 1517
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repertoire, and functional avidity (2, 14, 19, 25, 30, 31, 35, 42, 47, 55). The SIV-infected rhesus macaque is the best animal model of HIV infection. The Mamu-Bⴱ17 allele in macaques and the HLA-Bⴱ57 allele in HIV-infected individuals appear to have similar protective benefits (40, 48, 56). However, fewer than one-third of Mamu-Bⴱ17-positive macaques become elite controllers after SIVmac239 infection (56). Since the presence of the Mamu-Bⴱ17 allele is not sufficient to confer elite control, it is likely that additional factors influence the quality of protective CD8⫹ T-lymphocyte responses. CD8⫹ T-cell clones specific for a particular epitope may differ greatly in antiviral efficacy. Epitope-specific CD8⫹ T cells in SIV or HIV infection are clonally diverse (16, 29). CD8⫹ T cells with unique TCRs may be crucial to control viral replication in longterm survivors after HIV infection (19). However, the relationship of clonal variation to antiviral efficacy has not been carefully examined. In the present study, we explored the possibility that epitope-specific CD8⫹ T cells exhibit clonal variation in antiviral efficacy and cytokine expression. Epitope-specific CD8ⴙ T-lymphocyte clones differ in antiviral efficacy. We isolated a total of 105 clones from seven different Mamu-Aⴱ01-, -Aⴱ02- or -Bⴱ17-restricted CD8⫹ T-cell lines derived from seven SIVmac239-infected rhesus macaques with differing plasma viral loads (Table 1). These clones were derived from three rounds of cloning to ensure clonality. All clones expressed gamma interferon (IFN-␥) and/or bound MHC class I tetramers in a peptide-specific manner (data not shown) (34, 51, 53). All clones that bound MHC class I tetramers expressed IFN-␥. We compared the abilities of multiple clones from each line from each animal to suppress SIVmac239 replication in an in vitro viral suppression assay (VSA). We used phytohemagglutinin-stimulated, SIVmac239-infected CD8-negative target cells and epitope-specific CD8⫹ T-cell clones at an effectorto-target ratio (E:T) of 1:10 according to a recently published method (33). The same target cells from an MHC class Imatched animal (Mamu-Aⴱ01, -Aⴱ02, and -Bⴱ17 positive) and a mismatched animal (Mamu-Aⴱ01, -Aⴱ02, and -Bⴱ17 negative) were used to test all clones in this study. Effective suppression was defined as a reduction of greater than 80% in Gag p27-positive cell frequency after 8 days in culture, equivalent to a 10-fold reduction in viral RNA copy number in the supernatant. The results of the quantitative PCR assay indicated that the virus was in an exponential growth phase until day 8. We first tested six Mamu-Aⴱ02-Nef159-167YY9 clones from a slow progressor, r00044. These YY9 clones varied in the ability to reduce the SIV-infected cell frequency (maximum of 95% to 13% by day 8) (Fig. 1B). Viral concentrations in supernatants, determined by quantitative PCR assay (33), were reduced approximately 100-fold on day 6, exhibiting clonal variation in antiviral efficacy (Fig. 1A). By day 8, only two YY9 clones maintained effective suppression compared to control cells cultured without effectors. Interestingly, seven Mamu-Bⴱ17-restricted Nef165-173IW9 clones from an elite controller, r95061, were highly efficient in reducing the frequency of SIV-infected target cells (93 to 99% reduction) compared to Nef159-167YY9 clones (Fig. 2A and B). However, there was still some variation in the suppressive efficacy of the different IW9 clones (Fig. 2A). We obtained
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FIG. 1. Clonal variation of representative Mamu-Aⴱ02-Nef159-167YY9specific CD8⫹ T cells in the ability to suppress SIVmac239 replication. Supernatants were collected on days 4, 6, and 8 from VSA duplicate wells with MHC class I-matched targets and six Nef159-167YY9-specific clones from animal r00044. The clones were used at an E:T of 1:10 and analyzed by quantitative PCR to determine average viral RNA copy numbers (A). Day 8 VSA target cells were stained with an anti-Gag p27 antibody to determine the frequency of SIV-infected target cells (B). Viral suppression only occurred in an MHC class I-restricted fashion (MHC class I-mismatched target data not shown).
similar results (87 to 99% reduction in Gag p27-positive cell frequency) from the analysis of seven Vif66-73 HW8-specific clones from another elite controller, r98016 (Table 1). When we carried out the same analysis with clones from other lines, there was again extensive evidence of clonal variation in Mamu-Aⴱ01 (Gag181-189 CM9 and Tat28-35SL8)-, Mamu-Aⴱ02 (Nef159-167YY9)-, and Mamu-Bⴱ17 (Env830-838FW9 and Nef195-203MW9)-restricted clones (Table 1). Together, our data show that variation in suppressive efficacy occurred in T-cell clones against five of the seven epitopes tested and was independent of both MHC class I restriction and viral protein. Effective suppression of SIVmac239 replication in vitro is not always associated with cytokine responses. None of the Mamu-Aⴱ02-Nef159-167YY9-specific clones from elite control-
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FIG. 2. Effective suppressions of SIVmac239 replication by all Mamu-Bⴱ17-Nef165-173IW9-specific clones. Supernatants were collected on days 4, 6, and 8 from VSA duplicate wells with MHC class I-matched target cells. Seven Nef165-173IW9 clones from animal r95061 were used at an E:T of 1:10 and analyzed by quantitative PCR to determine average viral RNA copy numbers (A). Day 8 VSA target cells were stained with an anti-Gag p27 antibody to determine the frequency of SIV-infected cells (B). Viral suppression only occurred in an MHC class I-restricted fashion (MHC class I-mismatched target data not shown).
ler r95061 exhibited effective suppression (Table 1). However, all of these ineffective clones produced IFN-␥ and tumor necrosis factor alpha (TNF-␣) (Fig. 3A). Furthermore, both Nef195-203MW9 clones with effective (98% reduction in Gagp27-positive cell frequency) and ineffective (24.6% reduction) suppression of SIVmac239 replication had robust IFN-␥, TNF-␣, and interleukin-2 (IL-2) responses to 10 M cognate peptide stimulation (Fig. 3B). A less effective Env830-838FW9 clone had stronger IFN-␥, TNF-␣, and IL-2 responses than an effective FW9 clone (Fig. 3C). In addition, two Vif66-73HW8 clones with highly effective suppression exhibited notable dif-
ferences in IFN-␥, TNF-␣, and IL-2 responses (Fig. 3D). Interestingly, an effective HW8 clone had no IL-2 response after stimulation with 10 M cognate peptide. In t-test statistical analyses with log-transformed data from both suppressive and nonsuppressive groups of clones, there was no significant correlation between the TNF-␣ or IL-2 response and virus suppression efficacy (P ⫽ 0.94 and 0.44, respectively) (Fig. 3E and G). Interestingly, the IFN-␥ response was significantly correlated with the ability of clones to suppress viral replication (P ⫽ 0.002) (Fig. 3F). It should be noted, however, that there were many suppressive clones with very low IFN-␥ responses.
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FIG. 3. IFN-␥, TNF-␣, and IL-2 responses were not always associated with the ability to suppress SIVmac239 replication. Gag p27 staining was carried out with day 8 VSA target cells to determine the frequency of SIV-infected target cells. Clones were stimulated with autologous B-lymphoblastoid cell lines pulsed with cognate peptide and stained with anti-IFN-␥, anti-TNF-␣, and/or anti-IL-2 antibodies. Nef159-167YY9specific clones from r95061 (A), Nef195-203MW9- and Env830-838FW9-specific clones from r95071 (B and C), and Vif66-73HW8-specific clones from r98016 (D) were compared. Analyses showing the statistical significance of differences between SIV suppression and cytokine expression with log-transformed data from suppressive and nonsuppressive groups are shown in panels E, F, and G.
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Macaques with CD8⫹ T-lymphocyte responses to similar epitope sets after infection with molecularly cloned SIVmac239 have variable disease courses, suggesting that epitope specificity alone cannot account for effective control of viremia (3, 4, 28, 52). The ability of a clone to reduce SIV replication in vitro likely depends upon its cytolytic mechanism, as has been demonstrated previously (22, 54). Loss of T cells with an effective TCR repertoire in an epitope-specific CD8⫹ T-lymphocyte population may result in a poor clinical outcome for HIVinfected humans or SIV-infected macaques (14, 19). However, other findings suggested that CD8⫹ T cells from healthier individuals might be functional, independent of TCR expression (39, 54). In previous studies, only one or two CD8⫹ T-cell clones specific for a few HIV or SIV epitope-specific CD8⫹ T-cell lines were used to determine the ability to suppress virus replication (50, 55). The variation in antiviral efficacy among epitope-specific CD8⫹ T cells in HIV or SIV infection has not, until now, been comprehensively assessed. Using a recently developed in vitro functional assay to evaluate the antiviral efficacy of epitope-specific CD8⫹ T-cell clones, we identified clonal variation in the ability to suppress virus replication in five of the seven CD8⫹ T-cell specificities tested. Some epitope-specific CD8⫹ T cells derived from particular animals all had effective clones with relatively minor variation in clonal efficacy. Clones with effective suppression of SIV replication could diminish the frequency of SIV-infected cells by ⬎99% in our 8-day coculture assay. Therefore, variations in antiviral efficacy among certain epitope-specific CD8⫹ T lymphocytes may result in different disease courses in MHC class I-matched animals. The association between cytokine-secreting T-cell responses and HIV or SIV control remains controversial. HIV-1-specific IFN-␥-secreting T-cell responses were significantly and inversely correlated with viral load in previous studies (8, 9, 12, 13, 17, 20, 26, 36, 44, 46), whereas others showed no clear correlation (1, 18, 23, 38). In this study with SIV epitopespecific clones, all of the ineffective clones in the VSA secreted IFN-␥ and TNF-␣ and/or were positive for tetramer staining. Furthermore, some effective suppressor clones had no IL-2 response after cognate peptide stimulation. In addition, functional avidities of suppressive and nonsuppressive clones were not associated with viral suppression efficacy (data not shown). Therefore, current assays using cytokine secretion may not actually measure CD8⫹ T-cell efficacy. The specificity and magnitude of HIV- or SIV-specific CD8⫹ T lymphocytes can be identified by using MHC class I tetramers. The function of these antigen-specific T lymphocytes is currently assessed by enzyme-linked immunospot and intracellular cytokine staining assays that measure the ability of these cells to secrete a range of cytokines. Unfortunately, tetramer-positive epitope-specific CD8⫹ T cells varied in the ability to suppress SIV replication. These results suggest that currently available cytokine-based assays, including enzyme-linked immunospot and intracellular cytokine staining assays, may not be reliable tools to evaluate protective CD8⫹ T-lymphocyte responses. We thank Jason Reed, Shari Piaskowski, and Jason Stephany for technical assistance. We also appreciate David O’Connor and Jonah Sacha for helpful discussions.
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The NIH AIDS Research and Reference Reagent Program provided recombinant human IL-2. This research was supported by National Institutes of Health grants R01 AI049120, R24 RR015371, R01 AI052056, and P51 RR000167. This work was conducted in part at a facility constructed with support from Research Facility Improvement grants RR15459-01 and RR020141-01 to the Wisconsin National Primate Research Center. REFERENCES 1. Addo, M. M., X. G. Yu, A. Rathod, D. Cohen, R. L. Eldridge, D. Strick, M. N. Johnston, C. Corcoran, A. G. Wurcel, C. A. Fitzpatrick, M. E. Feeney, W. R. Rodriguez, N. Basgoz, R. Draenert, D. R. Stone, C. Brander, P. J. Goulder, E. S. Rosenberg, M. Altfeld, and B. D. Walker. 2003. Comprehensive epitope analysis of human immunodeficiency virus type 1 (HIV-1)-specific T-cell responses directed against the entire expressed HIV-1 genome demonstrate broadly directed responses, but no correlation to viral load. J. Virol. 77:2081– 2092. 2. Ali, A., R. Lubong, H. Ng, D. G. Brooks, J. 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