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The former US Secretary of Defense, Donald Rumsfeld, once famously divided our areas of understanding into three categories: “known knowns … things we know we know”, “known unknowns … [things we] know we … do not know”, and “unknown unknowns—the ones we don't know we don't know” [1]. In HIV immunity, the role of CD8+ cytotoxic T lymphocytes (CTLs) in controlling virus has been thought to be “known” for over two decades. The recognition of viral peptides by CD8+ T cells was initially demonstrated by showing that CTLs could lyse chromium-labeled target cells presenting viral epitopes [2]. Multiple lines of evidence have subsequently supported the important role for CTLs in controlling HIV replication [3],[4]. However, most of these studies have not explicitly examined the mechanisms by which viral control is achieved. Because of the name (“cytotoxic”) and the early assays for detection (measuring cytolysis using chromium release in vitro), it is usually assumed that CD8+ T cells control HIV infection by killing HIV-infected cells. Indeed, the use of the terms CD8+ T lymphocyte and cytotoxic T lymphocyte have become almost interchangeable in HIV. Thus, the mechanism of CTL control of infection through the cytolysis of infected cells has fallen into the category of a “known known”.

Two papers in this issue of PLoS Pathogens present a major challenge to the assumption that CD8+ T cells kill HIV-infected cells [5],[6]. Using very similar methodology, Klatt et al. and Wong et al. used an approach of depleting CD8+ T cells during simian immunodeficiency virus (SIV) infection of macaques to study the mechanisms of CD8+ T cell control of virus. What makes their studies different from previous studies of CD8+ T cell depletion is that they also treated animals with anti-retroviral therapy (ART) after CD8+ T cell depletion. Because ART blocks subsequent rounds of viral infection, it has been used extensively over the last decade to study the turnover of HIV-infected cells [7],[8]. Combining CD8 depletion and ART treatment allowed the two groups to study the death rate of SIV-infected cells in the presence and absence of CD8+ T cells. Remarkably, the groups found no difference in the decay rate of virus in CD8-depleted compared to control animals. This indicates that CD8+ T cells had no significant impact on the death rate of infected cells, suggesting that a high death rate is an intrinsic property of infected cells. Since CD8+ T cells have a clear role in viral control, they must act via some mechanism other than lysis of infected cells.

On the surface, we could simply accept that CD8+ T cell control of HIV infection acts via non-cytolytic mechanisms such as the production of cytokines and/or chemokines, and make that our new credo. Such a shift in beliefs would be supported by the demonstration that the ability of CD8+ T cells to produce multiple cytokines is associated with good viral control in HIV, suggesting an important role for cytokines [9],[10]. However, it is difficult to reconcile the results of the Klatt and Wong papers with other recent in vivo and in vitro results (see Table 1). First amongst these are studies of CD8+ T cell depletion in acute SIV infection [3],[11]. These studies demonstrate that in the absence of CD8+ T cells, the virus level rises to its “normal” peak in acute infection, but then does not decline from the peak for a prolonged period. The simplest interpretation of this result is that, in the absence of CD8+ T cells, the peak number of infected cells does not decline, but remains constant because these cells are not dying. However, two other observations make this unlikely: First, the increased loss of CD4+ T cells in CD8-depleted animals indicates that many cells are dying. Second, the Klatt and Wong results show that infected cells die at the same rate independent of the presence of CD8+ T cells in chronic infection, so why would things be so different in primary infection? How can viral load stay so high in CD8-depleted animals in acute infection if infected cells are dying at the same rate? One explanation would be that CD8+ T cell depletion leads to activation of CD4+ T cells; since SIV preferentially infects activated cells, the infected CD4+ T cells are dying, but these dying cells are being replaced because of the high level of CD4+ T cell activation. However, a recent study of CD8+ T cell depletion in acute SIV infection has excluded this possibility, by showing that CD4+ T cell activation is neither necessary nor sufficient to produce the prolonged high viral loads observed in vivo [11].

Table 1. Evidence for and against Cytolytic Control of HIV.

doi:10.1371/journal.ppat.1000728.t001

A non-cytolytic mechanism of CD8+ T cell control in vivo is also difficult to reconcile with in vitro results directly demonstrating CD8+ T cell lysis [2],[12]. Early studies of in vitro lysis relied on pulsing target cells with HIV peptides, or infecting cells with vaccinia virus bearing HIV proteins [2]. These experiments may not reflect the normal levels of peptide-MHC present on HIV-infected cells, both because they may involve unphysiologically high doses of peptide, and because HIV Nef is known to down-regulate MHC class I expression in infected cells. However, recent studies have demonstrated the recognition and lysis of HIV-infected primary target cells in vitro, presumably bearing physiological levels of peptide, and suffering any effects of Nef and other proteins on antigen presentation [12]. Notably, these studies required prior activation of the CD8+ T cells to demonstrate high levels of killing, but still suggest a role for direct killing of infected cells. A compromise between the cytolytic and non-cytolytic camps is the idea that infected cells may be killed in a “window period” before they start to produce virus [13],[14]. However, this still fails to explain all of the experimental observations.

The Klatt and Wong papers provide a direct challenge to the assumption that CD8+ T cell cytolysis of infected cells is the mechanism of viral control in HIV. However, it is difficult to reconcile the combination of in vivo and in vitro results investigating CD8+ T cell activity in HIV. Given the enormous effort invested in the development of HIV vaccines specifically directed at inducing CD8+ T cell responses, it is somewhat disconcerting to admit that CD8+ T cell function in HIV remains a “known unknown”.

References Top

1. US Department of Defense (2002) DoD news briefing - Secretary Rumsfeld and Gen. Myers. Available: http://www.defense.gov/transcripts/trans​cript.aspx?transcriptid=2636 . Accessed 29 December 2009.
2. Nixon DF, Townsend AR, Elvin JG, Rizza CR, Gallwey J, et al. (1988) HIV-1 gag-specific cytotoxic T lymphocytes defined with recombinant vaccinia virus and synthetic peptides. Nature 336: 484–487. Find this article online
3. Schmitz JE, Kuroda MJ, Santra S, Sasseville VG, Simon MA, et al. (1999) Control of viremia in simian immunodeficiency virus infection by CD8+ lymphocytes. Science 283: 857–860. Find this article online
4. Phillips RE, Rowland-Jones S, Nixon DF, Gotch FM, Edwards JP, et al. (1991) Human immunodeficiency virus genetic variation that can escape cytotoxic T cell recognition. Nature 354: 453–459. Find this article online
5. Wong JK, Strain MC, Porrata R, Reay E, Sankaran-Walters S, et al. (2010) In vivo CD8+ T-cell suppression of SIV viremia is not mediated by CTL clearance of productively infected cells. PLoS Pathog 6: e1000748. doi:10.1371/journal.ppat.1000748.
6. Klatt NR, Shudo E, Ortiz AM, Engram JC, Paiardini M, et al. (2010) CD8+ lymphocytes control viral replication in SIVmac239-infected rhesus macaques without decreasing the lifespan of productively infected cells. PLoS Pathog 6: e1000747. doi:10.1371/journal.ppat.1000747.
7. Ho DD, Neumann AU, Perelson AS, Chen W, Leonard JM, et al. (1995) Rapid turnover of plasma virions and CD4 lymphocytes in HIV-1 infection. Nature 373: 123–126. Find this article online
8. Wei X, Ghosh SK, Taylor ME, Johnson VA, Emini EA, et al. (1995) Viral dynamics in human immunodeficiency virus type 1 infection. Nature 373: 117–122. Find this article online
9. Betts MR, Nason MC, West SM, De Rosa SC, Migueles SA, et al. (2006) HIV nonprogressors preferentially maintain highly functional HIV-specific CD8+ T cells. Blood 107: 4781–4789. Find this article online
10. Almeida JR, Price DA, Papagno L, Arkoub ZA, Sauce D, et al. (2007) Superior control of HIV-1 replication by CD8+ T cells is reflected by their avidity, polyfunctionality, and clonal turnover. J Exp Med 204: 2473–2485. Find this article online
11. Okoye A, Park H, Rohankhedkar M, Coyne-Johnson L, Lum R, et al. (2009) Profound CD4+/CCR5+ T cell expansion is induced by CD8+ lymphocyte depletion but does not account for accelerated SIV pathogenesis. J Exp Med 206: 1575–1588. Find this article online
12. Migueles SA, Osborne CM, Royce C, Compton AA, Joshi RP, et al. (2008) Lytic granule loading of CD8+ T cells is required for HIV-infected cell elimination associated with immune control. Immunity 29: 1009–1021. Find this article online
13. Sacha JB, Chung C, Rakasz EG, Spencer SP, Jonas AK, et al. (2007) Gag-specific CD8+ T lymphocytes recognize infected cells before AIDS-virus integration and viral protein expression. J Immunol 178: 2746–2754. Find this article online
14. Davenport MP, Ribeiro RM, Zhang L, Wilson DP, Perelson AS (2007) Understanding the mechanisms and limitations of immune control of HIV. Immunol Rev 216: 164–175. Find this article online
15. Jin X, Bauer DE, Tuttleton SE, Lewin S, Gettie A, et al. (1999) Dramatic rise in plasma viremia after CD8(+) T cell depletion in simian immunodeficiency virus-infected macaques. J Exp Med 189: 991–998. Find this article online
16. Davenport MP, Ribeiro RM, Perelson AS (2004) Kinetics of virus-specific CD8+ T cells and the control of human immunodeficiency virus infection. J Virol 78: 10096–10103. Find this article online
17. Davenport MP, Zhang L, Bagchi A, Fridman A, Fu TM, et al. (2005) High-potency human immunodeficiency virus vaccination leads to delayed and reduced CD8+ T-cell expansion but improved virus control. J Virol 79: 10059–10062. Find this article online
18. Chea S, Dale CJ, De Rose R, Ramshaw IA, Kent SJ (2005) Enhanced cellular immunity in macaques following a novel peptide immunotherapy. J Virol 79: 3748–3757. Find this article online
19. Geiben-Lynn R, Kursar M, Brown NV, Kerr EL, Luster AD, et al. (2001) Noncytolytic inhibition of X4 virus by bulk CD8(+) cells from human immunodeficiency virus type 1 (HIV-1)-infected persons and HIV-1-specific cytotoxic T lymphocytes is not mediated by beta-chemokines. J Virol 75: 8306–8316. Find this article online
20. Walker CM, Erickson AL, Hsueh FC, Levy JA (1991) Inhibition of human immunodeficiency virus replication in acutely infected CD4+ cells by CD8+ cells involves a noncytotoxic mechanism. J Virol 65: 5921–5927. Find this article online
21. Tomaras GD, Lacey SF, McDanal CB, Ferrari G, Weinhold KJ, et al. (2000) CD8(+) T cell-mediated suppressive activity inhibits HIV-1 after virus entry with kinetics indicating effects on virus gene expression. Proc Natl Acad Sci U S A 97: 3503–3508. Find this article online