JOURNAL OF VIROLOGY, Oct. 2010, p. 10907–10912 0022-538X/10/$12.00 doi:10.1128/JVI.01357-10 Copyright © 2010, American Society for Microbiology. All Rights Reserved.
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NOTES Simian Immunodeficiency Virus-Specific CD8⫹ T Cells Recognize Vpr- and Rev-Derived Epitopes Early after Infection䌤 Jonah B. Sacha,* Matthew B. Buechler, Laura P. Newman, Jason Reed, Lyle T. Wallace, John T. Loffredo, Nancy A. Wilson, and David I. Watkins Department of Pathology and Laboratory Medicine, University of Wisconsin, Madison, Wisconsin 53715 Received 27 June 2010/Accepted 22 July 2010
The kinetics of CD8ⴙ T cell epitope presentation contribute to the antiviral efficacy of these cells yet remain poorly defined. Here, we demonstrate presentation of virion-derived Vpr peptide epitopes early after viral penetration and prior to presentation of Vif-derived epitopes, which required de novo Vif synthesis. Two Rev epitopes exhibited differential presentation kinetics, with one Rev epitope presented within 1 h of infection. We also demonstrate that cytolytic activity mirrors the recognition kinetics of infected cells. These studies show for the first time that Vpr- and Rev-specific CD8ⴙ T cells recognize and kill simian immunodeficiency virus (SIV)-infected CD4ⴙ T cells early after SIV infection. The antiviral activity of AIDS virus-specific CD8⫹ T cells is well documented in both in vivo (1, 4, 21) and in vitro (8, 24, 29) studies. Accordingly, human immunodeficiency virus (HIV) vaccine modalities that focus on engendering antiviral CD8⫹ T cells are being developed (13, 26, 28). Ideally, a CD8⫹ T cell-based vaccine would stimulate responses against epitopes that are presented by major histocompatibility complex class I (MHC-I) molecules early after infection of a target cell. However, successful selection of antigenic sequences for a CD8⫹ T cell-based vaccine has been frustrated in part by an incomplete understanding of the properties of effective CD8⫹ T cell responses (25). Vpr-specific CD8ⴙ T cells recognize simian immunodeficiency virus (SIV)-infected cells early after infection. We have previously mapped the presentation kinetics of CD8⫹ T cell epitopes derived from SIVmac239 Gag, Pol, Env, Tat, and Nef (17, 18). Here we examine the presentation kinetics of CD8⫹ T cell epitopes derived from Vif, Vpr, and Rev (Fig. 1A). To this end, we performed a kinetic intracellular cytokine staining (KICS) assay, as described previously (17, 18, 20), which uses primary CD4⫹ T cell targets, SIV-specific CD8⫹ T cell clones, and a synchronous infection with SIV. A major advantage of this assay is that it does not use exogenous peptide. Rather, SIV-infected CD4⫹ T cells naturally process and present SIVderived CD8⫹ T cell peptide epitopes following a synchronous infection. Furthermore, the synchronization of infection in vitro allows the investigation of the kinetics with which MHCI-bound CD8⫹ T cell epitopes appear on the surface of infected cells. To perform the KICS assay, we first isolated and activated primary CD4⫹ T cells by using CD4 microbeads
* Corresponding author. Mailing address: Department of Pathology and Laboratory Medicine, University of Wisconsin—Madison, 555 Science Drive, Madison, WI 53711. Phone: (608) 890-0843. Fax: (608) 265-8084. E-mail:
[email protected]. 䌤 Published ahead of print on 4 August 2010.
(Miltenyi Biotec). A starting input of 1,000 SIV virions/target cell was then rendered magnetically active by incubation with ViroMag beads (OZ Biosciences) and directed to the target cell surface via magnetic force for 15 min to synchronize attachment of the virus (multiplicity of infection [MOI] of ⬇1). Next, unbound virus was washed away, and at various time points following infection, we treated the infected cells with brefeldin A (BFA) to block the transport of newly epitopeloaded MHC-I to the cell surface and incubated the infected cells with the SIV-specific CD8⫹ T cell of interest. Epitope presentation was then measured by the production of gamma interferon (IFN-␥) and/or tumor necrosis factor alpha (TNF-␣) from the CD8⫹ T cell clones (Fig. 1B to D). SIV-infected CD4⫹ T cells efficiently presented two different Vpr-derived epitopes throughout the KICS assay, with peak presentation occurring at 6 h postinfection (Fig. 1B). SIVinfected, MHC-I-mismatched targets failed to activate Vprspecific CD8⫹ T cells, and early Vpr presentation occurred in the context of two separate MHC-I molecules (Fig. 1A and data not shown). Because ⬃200 to 700 Vpr molecules are packaged into each virion (3, 14), it is likely that Vpr epitopes presented early after infection are derived from virion-associated Vpr. In contrast to the early presentation of Vpr epitopes, Vifderived epitopes appeared on the surface of SIV-infected CD4⫹ T cells late in the viral replication cycle. CD4⫹ T cells presented four different Vif-derived epitopes late in the viral replication cycle, between 18 and 24 h after SIV infection, irrespective of whether the MHC-I restricting allele was Mamu-B*08, -B*17, or -A*02 (Fig. 1C). Finally, we examined the presentation kinetics of two Mamu-B*08-restricted epitopes derived from the Rev protein. Interestingly, these two epitopes exhibited differential presentation kinetics. The Rev44–51 RL8 epitope appeared on the surface of SIV-infected CD4⫹ T cells by 6 h postinfection, while the Rev12–20 KL9 epitope first appeared at 12 h postin-
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fection (Fig. 1D). The differential presentation kinetics of the two Rev CD8⫹ T cell epitopes is not explained by differences in the functional avidities of the two Rev-specific CD8⫹ T cell clones as measured by 50% effective concentration (EC50) (Fig. 1A). A potential explanation for the differential presentation of these two Rev epitopes may be that Rev12–20 KL9 is encoded by Rev exon 1, while Rev44–51 RL8 is encoded by Rev exon 2 (10, 12). Because we observed presentation of the Vpr45–55 NL11 and Rev44–51 RL8 epitopes at 6 h postinfection, we next determined whether these epitopes were on the surface at the earliest time points following viral penetration. Both Vpr45–55 NL11 and Rev44–51 RL8 epitopes appeared on the surface of infected cells between 1 and 2 h postinfection (Fig. 1E). Thus, CD8⫹ T cells specific for these two epitopes can recognize SIVinfected cells almost immediately following infection. While it is possible that CD4⫹ T cell targets are infected with more virus in our in vitro kinetics assay than would be possible in vivo, multiple lines of evidence suggest that this is not the case. First, MHC-I-restricted presentation of virionassociated proteins has been described for many other viruses, including influenza virus (30) and cytomegalovirus (16). This phenomenon, therefore, is not unique to our in vitro system. Second, dose-response experiments demonstrate that infection with 20 to 100 input virions/cell is sufficient to trigger MHCI-restricted presentation of virion-associated proteins (17). Furthermore, only 1 in 10 virions is infectious (23), which suggests that infection with as few as 2 to 10 infectious virions is sufficient for virion-associated protein presentation. With ⬎106 virus particles/ml commonly found in the plasma of an infected individual during the acute phase of infection (15), we do not think that the amount of virus used in our in vitro assays is in excess of that which might be available in vivo. To further explore the early presentation of the Rev44–51 RL8 epitope, we synchronously infected Mamu-A*01/B*08⫹ CD4⫹ T cells with equivalent amounts of SIVmac239 or SIVsmE543-3, a virus in which the Rev44–51 RL8 epitope sequence contains a mutation at the position 2 primary anchor residue (5, 9) (Fig. 2A). Rev44–51 RL8-specific CD8⫹ T cells again efficiently recognized SIVmac239-infected CD4⫹ T cells throughout the assay but did not recognize SIVsmE543-3-infected cells (Fig. 2A). Gag181–189 CM9-specific CD8⫹ T cells, used as a positive control, recognized the SIVsmE543-3-infected CD4⫹ T cells throughout the assay (Fig. 2A). Thus, Rev44–51 RL8-specific recognition of infected cells is specifically due to presentation of the epitope from SIVmac239 Rev proteins. Early-presented Vpr CD8ⴙ T cell epitopes are virion de-
FIG. 1. Vpr-specific CD8⫹ T cells recognize SIV-infected CD4⫹ T cells prior to Vif-specific CD8⫹ T cells. (A) Information on CD8⫹ T cell epitopes used, including parent protein of epitope, amino acid sequence and position in protein, MHC-I restricting allele, and EC50. (B to D) MHC-I-matched CD4⫹ T cells were synchronously infected with SIVmac239 and cocultured at an effector-to-target ratio (E/T ratio) of 1:1 with CD8⫹ T cells specific for the Vpr epitopes NL11 and IF9 (B), the Vif epitopes RL9, RL8, HW8, and WY8 (C), and the Rev epitopes KL9 and RL8 (D). (E) MHC-I-matched CD4⫹ T cells were synchronously infected with SIVmac239 and cocultured at an E/T ratio of 1:1 with CD8⫹ T cells specific for Vpr NL11 or Rev RL8 at the time points postinfection indicated. Results are shown as the percentages of
CD8⫹ T cells producing TNF-␣ and/or IFN-␥, as detected by intracellular cytokine staining (ICS). Data are means ⫾ standard deviations from two samples and are representative of two or more independent experiments. No recognition of SIV-infected, MHC-I-mismatched CD4⫹ T cells was observed at 6 or 24 h postinfection (data not shown). All CD8⫹ T cell clones were ⬎85% positive as measured by TNF-␣ and/or IFN-␥ production when stimulated with an autologous B-lymphoblastoid cell line (BLCL) pulsed with exogenous peptide (data not shown).
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A
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Virus
Rev44-51 RL8 sequence
SIVmac239 SIVsmE543-3 0
0.54%
TNF-α
18
2.04%
0.21%
0.53%
CTPYDINQM ---------
Hours post infection 12
2.45%
0.31%
Gag 181-189CM9 sequence
RRRWQQLL -Q----I6
0.23%
35%
10909
12.4%
24
3.87%
Rev RL8 + SIV 4.2% mac239
0.28%
Rev RL8 + SIV 0.31% smE543
6.3%
Gag CM9 + SIV 20.9% smE543
IFN-γ
CD4+ T cells pretreated with tenofovir
B % TNF-α and/or IFN-γ secretion
D
Vpr NL11
10.4 7.8
75
5.2
50
2.6
25
0
0
6
12
18
Vif RL9
C 100
0
24
0
6
12
18
24
CD4+ T cells pretreated with tenofovir Rev KL9
100
E
75
4.5
50
3.0
25
1.5
0
0
6
12
18
24
Rev RL8
6.0
0
0
6
12
18
24
Hours post infection
FIG. 2. Differential requirements for CD8⫹ T cell epitope presentation. (A) Amino acid sequences of the Rev RL8 and Gag CM9 epitopes in SIVmac239 and SIVsmE543-3. Mamu-A*01/B*08⫹ CD4⫹ T cells were synchronously infected with equivalent viral RNA genome (vRNA) copy numbers of either SIVmac239 or SIVsmE543-3 and cocultured with Rev RL8-specific CD8⫹ T cells at the indicated times postinfection. Gag181–189 CM9-specific CD8⫹ T cells, used as a positive control, recognized the SIVsmE543-3-infected CD4⫹ T cells throughout the assay. Percentages shown are of TNF-␣- and/or IFN-␥-positive CD8⫹ T cells. Data are indicative of three independent experiments. (B to E) MHC-I-matched CD4⫹ T cells were pretreated with 400 M tenofovir disoproxil fumarate for 2 h, synchronously infected with infectious SIVmac239, and cocultured with CD8⫹ T cells specific for Vpr NL11 (B), Vif RL9 (C), Rev KL9 (D), or Rev RL8 (E). Results are shown as the percentages of TNF-␣- and/or IFN-␥-positive cells detected by ICS. Vif RL9- and Rev KL9-specific CD8⫹ T cells did not recognize CD4⫹ T cells treated with tenofovir prior to infection. Data are means ⫾ standard deviations from two samples and are representative of two or more independent experiments.
rived. Incoming virions and newly synthesized viral gene products are both rich sources of CD8⫹ T cell epitopes in infected cells (31). To determine the source of CD8⫹ T cell epitope production from Vpr, Vif, and Rev, we next repeated the KICS assay using CD4⫹ T cells pretreated with the reverse transcrip-
FIG. 3. Cytolytic activity mirrors presentation kinetics. (A to C) MHC-I-matched CD4⫹ T cells were synchronously infected with SIVmac239 and cultured alone or at an E/T ratio of 1:1 with CD8⫹ T cells specific for Vpr NL11 (A), Vif RL9 (B), or Rev RL8 (C). (D) To ensure that the elimination of Gag p27⫹ CD4⫹ T cells was MHC-I restricted, MHC-I-mismatched CD4⫹ T cells were synchronously infected with SIVmac239 and cocultured either alone (black bar) or at an E/T ratio of 1:1 with the CD8⫹ T cells indicated (white bars) for 24 h. Data are representative of three independent experiments.
tase inhibitor tenofovir prior to infection with SIVmac239. Although de novo viral protein synthesis was blocked in these experiments, CD4⫹ T cells still presented the Vpr45–55 NL11 epitope early after SIV infection, with presentation peaking at 6 h postinfection and then decaying over time (Fig. 2B). Further, blocking de novo viral protein synthesis abolished the second wave of Vpr45–55 NL11 epitope presentation, which occurred between 18 and 24 h postinfection (Fig. 1B). Thus, the early presentation of Vpr epitopes is likely due to processing and presentation of incoming virion-derived Vpr proteins, while the second, later wave of presentation was due to de novo Vpr synthesis. In contrast to Vpr, Vif-specific CD8⫹ T cell epitopes did not
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FIG. 4. Overview of CD8⫹ T cell epitope ontogeny in SIVmac239-infected CD4⫹ T cells. (A) The viral replication cycle begins at time zero, when the virus attaches to a CD4⫹ target cell, and concludes with the production of progeny virions at ⬃21 to 24 h postinfection (18). (B) Three groups of SIV viral proteins and CD8⫹ T cell epitopes, segregated based on the timing of CD8⫹ T cell epitope presentation. The relative timing and strength of CD8⫹ T cell epitope presentation are indicated by black triangles. eARF-P1 is an abbreviation for the cryptic protein Env alternate reading frame protein 1, as described by Maness et al. (10), which contains the cryptic CD8⫹ T cell response RW9 (11).
recognize infected cells when de novo viral protein synthesis was blocked (Fig. 2C). Thus, presentation of Vif-derived epitopes likely requires synthesis of Vif proteins. Finally, because we observed differential epitope presentation from the Rev protein, we examined the presentation requirements of both Rev-derived epitopes. Blocking de novo protein synthesis fully inhibited presentation of the Rev12–20 KL9 epitope (Fig. 2D). In contrast, CD4⫹ T cells still efficiently presented the Rev44–51 RL8 epitope at 6 h postinfection, with presentation then decaying over time (Fig. 2E). Early presentation of Rev44–51 RL8 in tenofovir-treated CD4⫹ T cells was surprising, as we have observed this kinetics pattern only in epitopes derived from proteins present in the virus particle, such as Gag (17), Pol (18), and Vpr (Fig. 2B). To our knowledge, there are no reports describing Rev as a virion protein. Although we cannot exclude this possibility, virionderived Rev is unlikely to be the source of the early Rev44–51 RL8 presentation. In addition to the full-length RNA genome, HIV-1 incorporates fully spliced viral mRNA species into the virus particle (6). Therefore, it is possible that SIVmac239 virions can incorporate a Rev mRNA species, which encodes the Rev44–51 RL8 epitope. Vpr-specific CD8ⴙ T cells eliminate SIV-infected CD4ⴙ T cells early after infection. Our previous assays measured cytokine secretion but not the ability of Vpr-specific CD8⫹ T cells to kill SIV-infected CD4⫹ T cells. To measure elimination of infected cells, we performed a 24-h viral elimination assay as described previously (17, 18). Vpr-specific CD8⫹ T cells began
eliminating Gag p27⫹ cells by 6 h postinfection (Fig. 3A). In contrast, Vif-specific CD8⫹ T cells did not eliminate infected CD4⫹ T cells until 18 h postinfection (Fig. 3B). Rev44–51 RL8specific CD8⫹ T cells eliminated infected cells by 6 h postinfection and then throughout the remainder of the assay (Fig. 3C). To ensure that elimination of infected cells was MHC-I restricted, we repeated the assay using CD4⫹ T cells that do not express any of the restricting MHC-I alleles. We observed no elimination of Gag p27⫹ MHC-I-mismatched CD4⫹ T cells in these experiments (Fig. 3D). Direct killing of infected cells has been postulated as the major mechanism by which CD8⫹ T cells exert their antiviral effect. However, CD8⫹ T cells exert their antiviral function in vivo without reducing the average life span of HIV-infected cells (7, 27). These findings suggest that CD8⫹ T cells might contain retroviruses through noncytolytic mechanisms, such as the secretion of antiviral cytokines and chemokines. Although the reduction of Gag p27⫹ cells was modest in the 24-h elimination assay, the kinetics of elimination were repeatable in separate assays and dependent on MHC-I (Fig. 3). Furthermore, the CD8⫹ T cells eliminated infected cells with kinetics that mirrored those of the secretion of cytokines (compare Fig. 1 and 3). Therefore, although these CD8⫹ T cells secreted cytokines more robustly in response to infected cells, the kinetics of cytokine secretion and cytotoxic activity were the same. CD8ⴙ T cell epitope presentation patterns in SIV-infected
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cells are divisible into three groups. With this new data on the presentation kinetics of Vpr, Vif, and Rev epitopes, we can divide epitope expression patterns into three distinct groups. This is possible because we have now mapped the kinetics of CD8⫹ T cell epitope presentation for at least one epitope from all of the SIVmac239 proteins except Vpx. While we have observed differential antigen presentation kinetics for epitopes in Gag (19) and Rev (Fig. 1D), the majority of CD8⫹ T cell epitopes derived from the same viral protein appear on the surface of infected cells with similar kinetics. The viral replication cycle begins when an infectious virion attaches to a target cell and concludes when progeny virions bud, approximately 21 to 24 h later (Fig. 4A) (25). Following virion penetration into the cytoplasm, MHC-I-bound CD8⫹ T cell epitopes from SIV proteins appear on the cell surface with kinetics that fall into one of the following three groups (Fig. 4B): (i) group ␣ kinetics, where the epitope first appears ⬃2 h postinfection, decays over time, and then rebounds late in the replication cycle; (ii) group  kinetics, where the epitope appears with intermediate kinetics at ⬃12 h postinfection and increases in magnitude throughout the replication cycle; and (iii) group ␥ kinetics, where the epitope first appears at ⬃18 h postinfection and then increases in magnitude throughout the replication cycle. Kinetics of epitope presentation is only one of many factors contributing to the antiviral efficacy of CD8⫹ T cells. Indeed, some CD8⫹ T cell epitopes expressed late in the replication cycle, such as epitopes derived from Env or Vif, may still be particularly efficacious targets (22, 25). This is especially true if the CD8⫹ T cell has a high killing efficiency (2) or recognizes an epitope under functional constraints. Nevertheless, the earlier in the replication cycle an epitope is presented by MHC-I on the surface of an infected cell, the more likely it is to reduce the ability of the infected cell to produce progeny virions. Therefore, while exceptions to the hierarchy depicted in Fig. 4 certainly exist, we propose that a CD8⫹ T cell-based vaccine elicit responses directed primarily at the proteins in the ␣ and  kinetics groups. This work was supported by NIH grants RO1 AI076114 and RO1 AI049120 to D.I.W. This publication was also made possible in part by NCRR grant P51 RR000167. This research was conducted in part at a facility constructed with support from Research Facilities Improvement Program grant numbers RR15459-01 and RR020141-01. The reagent tenofovir disoproxil fumarate was obtained through the AIDS Research and Reference Reagent Program, NIAID, NIH. J.B.S. thanks Louise Sacha for ongoing support. We thank Francesca Norante for technical assistance and Vanessa Hirsch for kindly providing SIVsmE543-3. 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, 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. 2. Bennett, M. S., H. L. Ng, M. Dagarag, A. Ali, and O. O. Yang. 2007. Epitope-dependent avidity thresholds for cytotoxic T-lymphocyte clearance of virus-infected cells. J. Virol. 81:4973–4980. 3. Briggs, J. A., M. N. Simon, I. Gross, H. G. Krausslich, S. D. Fuller, V. M. Vogt, and M. C. Johnson. 2004. The stoichiometry of Gag protein in HIV-1. Nat. Struct. Mol. Biol. 11:672–675. 4. Friedrich, T. C., L. E. Valentine, L. J. Yant, E. G. Rakasz, S. M. Piaskowski, J. R. Furlott, K. L. Weisgrau, B. Burwitz, G. E. May, E. J. Leon, T. Soma, G. Napoe, S. V. Capuano III, N. A. Wilson, and D. I. Watkins. 2007. Subdomi-
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