JOURNAL OF VIROLOGY, Dec. 2005, p. 14986–14991 0022-538X/05/$08.00⫹0 doi:10.1128/JVI.79.23.14986–14991.2005 Copyright © 2005, American Society for Microbiology. All Rights Reserved.
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Tat28-35SL8-Specific CD8⫹ T Lymphocytes Are More Effective than Gag181-189CM9-Specific CD8⫹ T Lymphocytes at Suppressing Simian Immunodeficiency Virus Replication in a Functional In Vitro Assay John T. Loffredo,1 Eva G. Rakasz,1 Juan Pablo Giraldo,2 Sean P. Spencer,2 Kelly K. Grafton,1 Sarah R. Martin,2 Gnankang Napoe´,1 Levi J. Yant,2 Nancy A. Wilson,1 and David I. Watkins1,2* Wisconsin National Primate Research Center1 and Department of Pathology and Laboratory Medicine,2 University of Wisconsin, Madison, Wisconsin 53715 Received 14 July 2005/Accepted 3 September 2005
Epitope-specific CD8ⴙ T lymphocytes may play an important role in controlling human immunodeficiency virus (HIV)/simian immunodeficiency virus replication. Unfortunately, standard cellular assays do not measure the antiviral efficacy (the ability to suppress virus replication) of CD8ⴙ T lymphocytes. Certain epitopespecific CD8ⴙ T lymphocytes may be better than others at suppressing viral replication. We compared the antiviral efficacy of two immunodominant CD8ⴙ T lymphocyte responses—Tat28-35SL8 and Gag181ⴙ 189CM9—by using a functional in vitro assay. Viral suppression by Tat-specific CD8 T lymphocytes was ⴙ consistently greater than that of Gag-specific CD8 T lymphocytes. Such differences in antigen-specific CD8ⴙ-T-lymphocyte efficacy may be important for selecting CD8ⴙ T lymphocyte epitopes for inclusion in future HIV vaccines. Several lines of evidence suggest that CD8⫹ T lymphocytes are important in suppressing human immunodeficiency virus/ simian immunodeficiency virus (HIV/SIV) replication. The appearance of HIV-specific CD8⫹ T lymphocytes is correlated temporally with a precipitous reduction in viral load (8, 20) implying that these virus-specific effector cells control viral replication. However, the massive loss of memory CD4⫹ T cells during acute HIV/SIV infection also likely contributes to the initial reduction in viral replication (21, 26). Additional evidence implicating CD8⫹ T cells in the control of viral replication comes from the depletion of circulating CD8⫹ lymphocytes in SIV-infected macaques, which results in an increase in plasma viral concentrations (16, 24, 35). In addition, CD8⫹-T-lymphocytes exert selective pressure on viral sequences in vivo, selecting for immune escape variants in both the acute (3, 9, 30) and the chronic (6, 7, 10, 11, 14, 32) phase of HIV/SIV infection. Over the past decade, new methodologies have improved our ability to detect CD8⫹-T-lymphocyte responses against HIV/SIV. However, we still do not know which of these HIVspecific CD8⫹ T lymphocytes actually contributes to control of viral replication. While neutralization assays distinguish effective antibodies from ineffective ones, most current cellular assays rely on indirect readouts to measure CD8⫹-T-lymphocyte efficacy (44). Early studies demonstrated that CD8⫹ cells (38) and later virus-specific cytotoxic T lymphocytes (45) inhibited immunodeficiency virus replication in vitro. Recently, investigators using functional in vitro assays suggested that * Corresponding author. Mailing address: Department of Pathology and Laboratory Medicine, University of Wisconsin-Madison, 585 Science Dr., Madison, WI 53711. Phone: (608) 265-3380. Fax: (608) 265-8084. E-mail:
[email protected].
CD8⫹-T-lymphocyte clones directed against early-expressed viral proteins, Nef (1, 46) and Rev (39, 40), may be particularly effective in suppressing viral replication. Another study demonstrated effective viral suppression using Pol-specific CD8⫹ T lymphocytes (37). Furthermore, dendritic cells pulsed with inactivated autologous virus can expand virus-specific CD8⫹ T cells, which are then capable of controlling HIV replication (23). While most data suggest that there are differences among the various CD8⫹-T-lymphocyte populations in their antiviral efficacy (the ability to suppress virus replication), current studies are limited by relying on a small number of well-defined clones. Moreover, the attributes of an effective CD8⫹-T-lymphocyte response are still undefined. Development of the viral suppression assay (VSA). To better address questions of antiviral efficacy, we developed a functional in vitro viral suppression assay to assess the ability of CD8⫹ T lymphocytes to control SIV replication. We depleted uninfected Indian rhesus macaque (Macaca mulatta) PBMC of CD8⫹ cells using CD8 nonhuman primate microbeads on an AutoMACS bead separation unit (Miltenyi, Auburn, CA) according to the manufacturer’s protocol. Depletions were ⬎99% effective (Fig. 1A). We stimulated the CD8⫺ fraction with 5 g/ml of phytohemagglutinin (Sigma, St. Louis, MO) for 18 to 24 h. The CD8⫺ targets were then incubated with SIVmac239 (17) at a multiplicity of infection of 5 ⫻ 10⫺5 for 4 h. This multiplicity of infection reproducibly infected the CD8⫺ peripheral blood mononuclear cells (PBMC) target cells and provided exponential SIV replication during the initial days of the assay. We used in vitro-stimulated epitope-specific CD8⫹-T-cell lines as effector cells. Cell lines were generated from fresh or CD8-enriched PBMC from SIVmac239-infected Mamu-A*01⫹
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FIG. 1. VSA schematic. (A) Target cells are freshly isolated PBMC, depleted of CD8⫹ cells, and activated with phytohemagglutinin (PHA). (B) Effector cells are SIV-specific in vitro-stimulated CD8⫹ T cells that are sorted to high specificity. (C) After a 4-h incubation of the target cells with SIVmac239, we combined target and effector cells at E:T ratios of 1:10, and 1:20. The cocultures were maintained for 8 to 11 days. 0.5 ml of supernatant was taken every 2 days for vRNA quantification. MOI, multiplicity of infection.
macaques in the chronic phase by using previously described methods (42). Intracellular cytokine staining assays verified that the in vitro cultures produced gamma interferon and tumor necrosis factor alpha in response to their cognate antigen and were not functionally impaired as has been observed in vivo (5, 13, 41). We performed Mamu-A*01 tetramer stains (2) to measure epitope specificity. Typically, after 1 to 3 months, CD8⫹-T-cell lines reached a suitable specificity (⬎50%) for cell sorting. We sorted CD3⫹, CD8⫹, and Mamu-A*01 tetramer⫹ lymphocytes using a MoFlo cell sorter (DakoCytomation, Fort Collins,
CO). Post-sort analysis was performed to assess purity (typically ⬎99%) and viability as assessed by trypan blue exclusion (Gibco, Grand Island, NY) (Fig. 1B). In cases where a cell line was ⬎94% tetramer-positive CD8⫹ T lymphocytes but in limited quantities, we used unsorted cells after verifying that other immunodominant CD8⫹-T-lymphocyte responses were absent. We added 5.0 ⫻ 105 infected CD8⫺ lymphocytes (targets) to each well of a 24-well plate (Fig. 1C). Sorted CD8⫹ T cells (effectors) were added to wells at effector to target ratios (E:T) of 1:10 and 1:20. We chose these E:Ts because early testing indicated E:Ts above 1:10 would sometimes provide viral sup-
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FIG. 2. Day 8 intracellular Gag p27 staining of representative VSA results with Tat28-35SL8- and Gag181-189CM9-specific CD8⫹ T-lymphocytes from a single macaque (animal 2125). Results for MHC class I-matched effector and target cells (A) and MHC-mismatched effector (MamuA*01⫹) and target (Mamu-A*01⫺) cells (B) at an E:T of 1:10 are shown. Viral suppression only occurred in an MHC class I-restricted fashion.
pression in our major histocompatibility complex (MHC)-mismatched controls, whereas ratios below 1:20 would not provide consistent levels of viral suppression. The final volume of cell culture medium was 2 ml and contained 50 U of interleukin-2 (NIH AIDS Research and Reference Reagent Program, Germantown, MD)/ml. The cocultures were maintained for 8 to 11
days. Every 2 days, 0.5 ml of supernatant was collected and replaced with fresh medium. We purified vRNA from 400 l of supernatant using the MagAttract vRNA minikit on a BioRobot M48 workstation (QIAGEN, Valencia, CA). Viral loads were determined by quantitative reverse transcription-PCR on a LightCycler 2.0 (Roche Diagnostics, Indianapolis, IN) as
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FIG. 3. Quantitative PCR of representative VSA results with Tat28-35SL8- and Gag181-189CM9-specific CD8⫹ T lymphocytes from macaque 2125. Results for MHC class I-matched effector and target cells (A) MHC-mismatched effector (Mamu-A*01⫹) and target (Mamu-A*01⫺) cells (B) at an E:T of 1:10 are shown. Viral suppression only occurred in an MHC class I-restricted fashion.
previously described (27). At the end of a VSA, intracellular Gag p27 staining was performed on the coculture to measure SIVmac239 infection. We first stained for the cell surface markers CD3, CD4, and CD8, followed by intracellular Gag p27 staining using Fix and Perm (CALTAG, Burlingame, CA) according to the manufacturer’s protocol with fluorescently conjugated 55-2F12 Gag p27 antibody (NIH AIDS Research and Reference Reagent Program, Germantown, MD) at ⬃0.75 mg/ ml. Differences in the antiviral efficacy of Tat28-35SL8- and Gag181-189CM9-specific CD8ⴙ T lymphocytes. Tat28-35SL8 and Gag181-189CM9 are Mamu-A*01-restricted immunodominant epitopes (6, 29, 31) often studied in macaque vaccine experiments (4, 6, 7, 15, 36, 43). These two responses account for ⬎50% of the total SIV-specific CD8⫹ responses in MamuA*01⫹ macaques (29). Despite extensive studies, the antiviral efficacies of these two high frequency CD8⫹-T-lymphocyte responses have not been addressed. We used the VSA to compare the ability of Tat28-35SL8- and Gag181-189CM9-specific CD8⫹ T lymphocytes to suppress SIVmac239 replication. Both cellular responses diminished viral replication at E:Ts of 1:10 and 1:20 as determined by intracellular Gag p27 staining (Fig. 2A) and quantitative reverse transcription-PCR (Fig. 3A) compared to wells containing only target cells. To ensure viral suppression was MHC class I-restricted, we combined Mamu-A*01⫺ target cells with Mamu-A*01-restricted effectors
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FIG. 4. Day 8 viral suppression by Tat28-35SL8- and Gag181-189CM9specific CD8⫹ T lymphocytes derived from several SIV-infected macaques (animal identification number shown below bars) at an E:T ratio of 1:10 as measured by fold reduction of intracellular Gag p27 (A) and fold reduction of vRNA copies/ml (B). Bars: ■, Tat-specific cells; 䊐, Gag-specific cells.
as controls in each assay. Using MHC-mismatched effector and target cells, we observed negligible reduction in SIV replication compared to wells that contain targets only in the absence of effective CD8⫹ T cells (Fig. 2B and 3B). At E:Ts of 1:10 and 1:20, the Tat28-35SL8-specific CD8⫹ T lymphocytes suppressed viral replication better than CD8⫹ T lymphocytes against Gag181-189CM9 did. By day 8, at an E:T of 1:10, the Tat28-35SL8-specific CD8⫹ T lymphocytes effectively suppressed viral replication, reducing the number of Gag p27positive cells to below background (Fig. 2A). Viral RNA concentrations in these wells were 240-fold lower than control wells (Fig. 3A). These results were consistent in at least two independent assays with CD8⫹-T-cell lines derived from the same animal (data not shown). Next, we examined whether suppression was animal- and/or epitope-specific using CD8⫹-T-cell lines generated from several Mamu-A*01⫹ SIV-infected macaques. In all instances, Tat-specific CD8⫹ T lymphocytes suppressed viral replication more than Gag-specific CD8⫹ T lymphocytes did (Fig. 4). Although animal 2095 had the least effective Tat28-35SL8-specific CD8⫹ T lymphocytes, there was still a 10-fold decrease in intracellular Gag p27 staining and a 47-fold decrease in viral
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RNA concentration on day 8 relative to wells without effector cells (Fig. 4). Our results displayed similar trends over time in multiple independent assays (data not shown). While Tat-specific CD8⫹ T lymphocytes cells were more effective in their ability to suppress SIV replication, we observed some animal-to-animal variability (Fig. 4). This variability may reflect differences in the immunological and virological status of animals at the time of cell line generation. For instance, studies correlate the impairment of CD8⫹-T-cell function and proliferative capacity with the loss of CD4⫹ T cells (22, 25, 28). Animal 2095 had low CD4 counts (⬍200 cells/l) when PBMC were obtained to generate the Tat28-35SL8-specific CD8⫹-T-cell line. Interestingly, this animal produced the least effective Tat-specific cell line (Fig. 4). While epitope-specific CD8⫹ T-lymphocytes can inhibit viral replication by cytolytic and noncytolytic mechanisms, the reduction in SIV replication by our epitope-specific CD8⫹ T lymphocytes is likely occurring by cytolytic activity in a MHC class I-restricted fashion. Previous findings demonstrated that epitope-specific CD8⫹-T-lymphocytes suppressed viral replication primarily through direct cytolytic activity (12, 45). Although it appears that noncytolytic effects may play a role, it is likely minor and contingent on direct cell-to-cell interactions via MHC-TCR recognition. Although these studies demonstrated that soluble factors inhibit HIV/SIV replication in a MHC class I-unrestricted fashion (12, 45), we observed minimal, if any, viral suppression when the effector and target cells were MHC mismatched (Fig. 2B and 3B). Nevertheless, we cannot rule out the involvement of noncytolytic activity in our assay. When performing fluorescence-activated cell sorting analysis at the end of the assay, we found that the percentages of effector cells directed against Tat28-35SL8 and Gag181-189CM9 in the cocultures were comparable in most cases. There were some instances in which Gag181-189CM9-specific CD8⫹ T lymphocytes persisted in two- to fourfold greater numbers than those against Tat28-35SL8. However, even in these cases, the greater abundance of Gag181-189CM9-specific CD8⫹ T lymphocytes did not suppress of SIVmac239 replication as well as Tat28-35SL8-specific CD8⫹ T lymphocytes. This suggests that a difference in antiviral efficacy is not simply due to lymphocyte longevity. Previous studies have failed to address the antiviral efficacy of Tat28-35SL8- and Gag181-189CM9-specific CD8⫹ T-lymphocytes, although they have suggested possible reasons for the observed efficacy of the Tat-specific CD8⫹ T cells. Tat-specific mRNA is detected as early as 4 h after HIV infection, whereas Gag-specific mRNA appears approximately 20 h postinfection (18, 19, 33, 34). In addition, Tat28-35SL8-specific cells appear to have a higher functional avidity (⬃0.18 nM) compared to that of the Gag181-189CM9-specific cells (⬃13.3 nM) (30). These differences might enable the Tat-specific CD8⫹ T cells to recognize cells early in the virus replication cycle and those that present only small amounts of antigen. Our viral suppression assay measures the ability of antigenspecific CD8⫹ T lymphocytes to suppress SIV replication in vitro. We used this assay to show that Tat28-35SL8-specific CD8⫹ T lymphocytes are consistently more effective at suppressing SIVmac239 replication than Gag181-189CM9-specific CD8⫹ T lymphocytes. Using this assay, we hope to define
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additional potentially protective CD8⫹-T-lymphocyte responses. It is plausible that findings from our functional in vitro assay can be extrapolated to the ability to suppress HIV/SIV replication in vivo. Therefore, differences in CD8⫹-T-lymphocyte suppressive efficacy may be critically important when choosing epitopes for future HIV vaccines. We thank William Rehrauer, Jess Maxwell, and Tim Jacoby for MHC class I PCR-SSP typing and gratefully acknowledge Laura Valentine, Alex Ko, and Andrea Weiler for immunological assay assistance. David O’Connor and Thomas Friedrich provided helpful discussions. We also thank the Immunology and Virology Core Laboratories at the National Primate Research Center, University of Wisconsin-Madison for technical assistance. This research was supported by National Institutes of Health grants R01 AI049120-04 to D.I.W. and P51 RR 000167 to the Wisconsin National Primate Research Center (WNPRC). This study was conducted in part at a facility constructed with support from Research Facilities Improvement grant numbers RR15459-01 and RR020141-01 (WNPRC). REFERENCES 1. Ali, A., R. Lubong, H. Ng, D. G. Brooks, J. A. Zack, and O. O. Yang. 2004. Impacts of epitope expression kinetics and class I downregulation on the antiviral activity of human immunodeficiency virus type 1-specific cytotoxic T lymphocytes. J. Virol. 78:561–567. 2. Allen, T. M., B. R. Mothe, J. Sidney, P. Jing, J. L. Dzuris, M. E. Liebl, T. U. Vogel, D. H. O’Connor, X. Wang, M. C. Wussow, J. A. Thomson, J. D. Altman, D. I. Watkins, and A. Sette. 2001. CD8⫹ lymphocytes from simian immunodeficiency virus-infected rhesus macaques recognize 14 different epitopes bound by the major histocompatibility complex class I molecule mamu-A*01: implications for vaccine design and testing. J. Virol. 75:738–749. 3. 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, K. J. Kunstman, X. Wang, D. B. Allison, A. L. Hughes, R. C. Desrosiers, J. D. Altman, S. M. Wolinsky, A. Sette, and D. I. Watkins. 2000. Tat-specific cytotoxic T lymphocytes select for SIV escape variants during resolution of primary viraemia. Nature 407: 386–390. 4. Amara, R. R., F. Villinger, J. D. Altman, S. L. Lydy, S. P. O’Neil, S. I. Staprans, D. C. Montefiori, Y. Xu, J. G. Herndon, L. S. Wyatt, M. A. Candido, N. L. Kozyr, P. L. Earl, J. M. Smith, H. L. Ma, B. D. Grimm, M. L. Hulsey, J. Miller, H. M. McClure, J. M. McNicholl, B. Moss, and H. L. Robinson. 2001. Control of a mucosal challenge and prevention of AIDS by a multiprotein DNA/MVA vaccine. Science 292:69–74. 5. 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, D. V. Havlir, D. D. Richman, A. Waters, P. Easterbrook, A. J. McMichael, and S. L. RowlandJones. 2000. HIV-specific CD8⫹ T cells produce antiviral cytokines but are impaired in cytolytic function. J. Exp. Med. 192:63–75. 6. Barouch, D. H., J. Kunstman, J. Glowczwskie, K. J. Kunstman, M. A. Egan, F. W. Peyerl, S. Santra, M. J. Kuroda, J. E. Schmitz, K. Beaudry, G. R. Krivulka, M. A. Lifton, D. A. Gorgone, S. M. Wolinsky, and N. L. Letvin. 2003. Viral escape from dominant simian immunodeficiency virus epitopespecific cytotoxic T lymphocytes in DNA-vaccinated rhesus monkeys. J. Virol. 77:7367–7375. 7. Barouch, D. H., J. Kunstman, M. J. Kuroda, J. E. Schmitz, S. Santra, F. W. Peyerl, G. R. Krivulka, K. Beaudry, M. A. Lifton, D. A. Gorgone, D. C. Montefiori, M. G. Lewis, S. M. Wolinsky, and N. L. Letvin. 2002. Eventual AIDS vaccine failure in a rhesus monkey by viral escape from cytotoxic T lymphocytes. Nature 415:335–339. 8. Borrow, P., H. Lewicki, B. H. Hahn, G. M. Shaw, and M. B. Oldstone. 1994. Virus-specific CD8⫹ cytotoxic T-lymphocyte activity associated with control of viremia in primary human immunodeficiency virus type 1 infection. J. Virol. 68:6103–6110. 9. Borrow, P., H. Lewicki, X. Wei, M. S. Horwitz, N. Peffer, H. Meyers, J. A. Nelson, J. E. Gairin, B. H. Hahn, M. B. Oldstone, and G. M. Shaw. 1997. Antiviral pressure exerted by HIV-1-specific cytotoxic T lymphocytes (CTLs) during primary infection demonstrated by rapid selection of CTL escape virus. Nat. Med. 3:205–211. 10. Chen, Z. W., A. Craiu, L. Shen, M. J. Kuroda, U. C. Iroku, D. I. Watkins, G. Voss, and N. L. Letvin. 2000. Simian immunodeficiency virus evades a dominant epitope-specific cytotoxic T lymphocyte response through a mutation resulting in the accelerated dissociation of viral peptide and MHC class I. J. Immunol. 164:6474–6479. 11. Evans, D. T., D. H. O’Connor, P. Jing, J. L. Dzuris, J. Sidney, J. da Silva, T. M. Allen, H. Horton, J. E. Venham, R. A. Rudersdorf, T. Vogel, C. D. Pauza, R. E. Bontrop, R. DeMars, A. Sette, A. L. Hughes, and D. I. Watkins. 1999. Virus-specific cytotoxic T-lymphocyte responses select for amino-acid
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