Anyone who follows HIV research is familiar with how a new molecule can suddenly appear in the scientific literature, generating much excitement and hype, only to subsequently shuffle quietly from the scene having failed to deliver on its supposed promise. At first blush, the ominously named "program death-1" (PD-1) seems like it might fit the profile of such molecules-de-jour. The role of PD-1 in shutting down virus-specific T cell responses was first described in a Nature paper in January of 2006, by researchers using a mouse model of chronic viral infection. Over the summer, three separate papers (with a commentary accompanying each) trumpeted data describing the high levels of expression of PD-1 on HIV-specific T cells, suggesting that targeting this molecule might be a way of boosting antiretroviral immunity in people with HIV. It's legitimate to wonder if the PD-1 palaver is justified, or if it's another case of molecular idolatry run amok.

A Murine Discovery
The initial study was led by Daniel Barber, a student in Rafi Ahmed's immunology research laboratory at Emory University. The purpose was to look for potentially important differences between CD8 T cell responses to acute and chronic viral infections using two strains of the murine virus LCMV (lymphocytic choriomeningitis virus). Previous work has shown that when mice are infected with a particular strain of LCMV (called clone 13) a chronic infection develops and LCMV-specific CD4 & CD8 T cells become "exhausted;" in immunology-speak this means that certain T cell functions are lost. In contrast, infecting mice with a different LCMV strain (the Armstrong strain) leads to a time-limited acute infection that is rapidly cleared by the immune response, with LCMV-specific CD4 & CD8 T cells playing key roles. These LCMV-specific CD4 & CD8 T cells subsequently persist for the life of the animal and retain a full spectrum of functional properties (e.g. proliferation, cytokine production and, for CD8 T cells, the ability to kill virus-infected cells).

To try and better understand the divergent T cell responses to acute and chronic LCMV the researchers used a new technology called a microarray analysis to compare the gene expression profiles of exhausted versus functional CD8 T cells (taken from mice infected with LCMV clone 13 and LCMV Armstrong strain, respectively). The most notable difference that they uncovered was that exhausted CD8 T cells strongly expressed the gene encoding the PD-1 receptor. PD-1 can interact with one of two ligands: PD-L1 (formerly called B7-H1), which can be expressed on many cells, and PD-L2 (B7-DC), which is predominantly seen on macrophages and dendritic cells. Interactions between PD-1 and PD-L1 are considered to be inhibitory, meaning that they play a role in switching off or dampening down the T cell response. The researchers found that PD-L1 was expressed at very high levels in spleen cells from mice chronically infected with LCMV clone 13, particularly on virally infected cells.

Having uncovered this evidence that the PD-1 receptor is involved CD8 T cell exhaustion, the researchers next studied the effects of blocking PD-1/PD-L1 interactions using an antibody that specifically targets PD-L1. The excitement around PD-1 primarily stems from the results of this experiment: in chronically infected mice, the function of exhausted LCMV-specific CD8 T cells improved and LCMV viral load declined. In the often arcane world of immunology, few interventions have been described that alter T cell function in a way that leads to a potent, sustained and reproducible antiviral effect.

Because CD8 T cell exhaustion has been reported in settings where CD4 T cell responses are absent or impaired, the researchers also assessed the effect of blocking PD-L1 on LCMV-specific CD8 T cells that were generated in the absence of CD4 T cell help. Again, the functionality of these CD8 T cells (as measured by proliferation, cytokine production, and the ability to kill LCMV-infected cells and reduce LCMV viral load) was restored when PD-L1 was blocked. In their paper, Barber and colleagues infer that this finding may have particular relevance to HIV where the impairment of virus-specific CD4 T cell responses has been well documented.

PD-1 Expression in HIV Infection
Unsurprisingly, the Barber paper sent several groups of HIV immunologists scurrying into their labs. Rafi Ahmed collaborated with a team led by Cheryl Day from Bruce Walker's group to analyze PD-1 expression in a large group of South African people with HIV and investigate whether blocking PD-L1 in vitro affected HIV-specific T cell responses. The findings were published online in Nature at the same time as similar work from Rafik-Pierre Sekaly's laboratory appeared on the Nature Medicine website.

Both studies reported similar results: In untreated HIV infection, levels of PD-1 were significantly higher on CD8 T cells compared to uninfected individuals. More remarkably, PD-1 expression also correlated with other surrogate markers of disease progression: there was an inverse correlation with CD4 T cell counts and a positive correlation with HIV viral load levels. The strength of these correlations was very similar to those reported for the immune activation marker CD28 (the r values were >0.70, where an r value of 1 is a perfect correlation). PD-1 expression was analyzed just on HIV-specific CD8 T cells, and the same correlations emerged. Cheryl Day and colleagues also looked at expression of PD-1 on HIV-specific CD4 T cells and found a similar picture to that seen for CD8 T cells, including the significant correlations with CD4 T cell counts and viral load. PD-1 expression was also significantly higher on HIV-specific CD8 T cells compared to those specific for CMV in the same individuals.

Both research groups also looked at the effects on HIV-specific T cells of blocking PD-1/PD-L1 interactions with a PD-L1-specific antibody in vitro. They found that the antibody enhanced production of cytokines and also increased the ability of HIV-specific T cells (both CD4 and CD8) to proliferate (copy themselves) in response to stimulation with HIV antigens. Although a variety of in vitro interventions can affect HIV-specific T cell function to some degree (the cytokine IL-12, for example), blocking PD-1 appeared to exert much more dramatic restorative effects.

Following quickly on the heels of these two papers was a study from researchers at the NIH's Vaccine Research Center. Although broadly in line with the prior results, the researchers reported that, in their hands, the impact of elevated PD-1 expression was most notable for its negative effect on CD8 T cell proliferation and survival. They found that blocking PD-1/PD-L1 interactions in vitro increased HIV-specific CD8 T cell proliferation and thereby increased the number of HIV-specific CD8 T cells capable of making a broad array of cytokines, but it did not necessarily restore cytokine production capacity to exhausted CD8 T cells.

Taken together, these studies offer strong support for the idea that PD-1 plays a key role in T cell exhaustion, and suggest that this may be particularly important in HIV infection. The association between PD-1 expression and disease progression in HIV also suggests that accumulating T cell dysfunction contributes to the pathogenesis of the disease, a suggestion consistent with data obtained as long ago as the late 1980s using older functional assays that evaluated T cell proliferation to well-characterized antigens and mitogens over the course of HIV infection.

The Therapeutic Horizon
The most promising aspect of the PD-1 data – and, inevitably, the aspect that has stirred most interest – is the implication that blocking PD-L1 could have therapeutic potential in HIV and other chronic viral infections (a paper showing elevated PD-1 expression on hepatitis C-specific CD8 T cells has just appeared in the Journal of Virology, demonstrating that this is a burgeoning area of study). However, there is a mammoth caveat to bear in mind: PD-1 may play an important role in switching off potentially harmful T cell responses that target body tissues and cause autoimmune disease. While it might be a useful strategy to revive exhausted virus-specific T cell responses, a concomitant outbreak of autoimmunity would obviously be disastrous. Rather than simply administering an antibody to block PD-L1, some researchers suspect it will be necessary to limit the blocking strategy to just the virus-specific T cells of interest, perhaps using a combination of vaccination and anti-PD-L1 approaches.

To gain insights into the potential risks and benefits of PD-1 and PD-L1 inhibition, several groups are now planning studies in non-human primates. Rafi Ahmed's group is collaborating with Chris Walker to explore the approach in HCV-infected chimpanzees, while the laboratories of Genoveffa Franchini at the NCI and Rama Rao Amara at Emory University are planning to study anti-PD-1 approaches in SIV-infected macaques.

Pharmaceutical companies have also become interested in this area of research; a US-based biotech company called Medarex has collaborated with the Japanese pharmaceutical company Ono to produce an anti-PD-L1 antibody (dubbed MDX-1106) for human use. A phase I trial of MDX-1106 in people with refractory cancers is now being conducted by Nicholas Restivo at the National Cancer Institute, based on the possibility that cancer-fighting T cells might be revived by the approach. Suffice it to say that the safety of MDX-1106 will need to be carefully evaluated, particularly given the recent shocking outcome of a trial that was also looking at an antibody targeting an immunological receptor (TGN1412, which targeted CD28 and led to severe inflammation and life-threatening illness in several phase I study participants).

Compared with some of the forgotten molecules of yesteryear, it does appear that PD-1 is underpinned by solid science. But whether this research heralds a new era in T cell-based immunotherapies? That remains to be seen.

• Ejrnaes M, Filippi CM, Martinic MM, Ling EM, Togher LM, Crotty S, von Herrath MG. Resolution of a chronic viral infection after interleukin-10 receptor blockade.
  J Exp Med. 2006 Oct 30;203(11):2461-72. Epub 2006 Oct 9.

  Brooks DG, Trifilo MJ, Edelmann KH, Teyton L, McGavern DB, Oldstone MB. Interleukin-10 determines viral clearance or persistence in vivo.

  Nat Med. 2006 Nov;12(11):1301-9. Epub 2006 Oct 15.

  Key PD-1 Papers

  Sharpe AH, Wherry EJ, Ahmed R, Freeman GJ. The function of programmed cell death 1 and its ligands in regulating autoimmunity and infection.

  Nat Immunol. 2007 Mar;8(3):239-45.

  Velu V, Kannanganat S, Ibegbu C, Chennareddi L, Villinger F, Freeman GJ, Ahmed R, Amara RR. Elevated Expression of the Inhibitory Receptor Programmed Death-1 on Simian Immunodeficiency Virus-Specific CD8 T Cells During Chronic Infection but not Following Vaccination.

  J Virol. 2007 Mar 21; [Epub ahead of print]

  Radziewicz H, Ibegbu CC, Fernandez ML, Workowski KA, Obideen K, Wehbi M, Hanson HL, Steinberg JP, Masopust D, Wherry EJ, Altman JD, Rouse BT, Freeman GJ, Ahmed R, Grakoui A. Liver-infiltrating lymphocytes in chronic human hepatitis C virus infection display an exhausted phenotype with high levels of PD-1 and low levels of CD127 expression.

  J Virol. 2007 Mar;81(6):2545-53. Epub 2006 Dec 20.

  Urbani S, Amadei B, Tola D, Massari M, Schivazappa S, Missale G, Ferrari C. PD-1 expression in acute hepatitis C virus (HCV) infection is associated with HCV-specific CD8 exhaustion.

  J Virol. 2006 Nov;80(22):11398-403. Epub 2006 Sep 6.

  Freeman GJ, Wherry EJ, Ahmed R, Sharpe AH. Reinvigorating exhausted HIV-specific T cells via PD-1-PD-1 ligand blockade.

  J Exp Med. 2006 Oct 2;203(10):2223-7. Epub 2006 Sep 25. Review.

  Petrovas C, Casazza JP, Brenchley JM, Price DA, Gostick E, Adams WC, Precopio ML, Schacker T, Roederer M, Douek DC, Koup RA. PD-1 is a regulator of virus-specific CD8+ T cell survival in HIV infection.

  J Exp Med. 2006 Oct 2;203(10):2281-92. Epub 2006 Sep 5.

  Trautmann L, Janbazian L, Chomont N, Said EA, Gimmig S, Bessette B, Boulassel MR, Delwart E, Sepulveda H, Balderas RS, Routy JP, Haddad EK, Sekaly RP. Upregulation of PD-1 expression on HIV-specific CD8+ T cells leads to reversible immune dysfunction.

  Nat Med. 2006 Oct;12(10):1198-202. Epub 2006 Aug 20.

  Day CL, Kaufmann DE, Kiepiela P, Brown JA, Moodley ES, Reddy S, Mackey EW, Miller JD, Leslie AJ, DePierres C, Mncube Z, Duraiswamy J, Zhu B, Eichbaum Q, Altfeld M, Wherry EJ, Coovadia HM, Goulder PJ, Klenerman P, Ahmed R, Freeman GJ, Walker BD. PD-1 expression on HIV-specific T cells is associated with T-cell exhaustion and disease progression.

  Nature. 2006 Sep 21;443(7109):350-4. Epub 2006 Aug 20.

  Okazaki T, Honjo T. The PD-1-PD-L pathway in immunological tolerance.

  Trends Immunol. 2006 Apr;27(4):195-201. Epub 2006 Feb 24. Review.

  Barber DL, Wherry EJ, Masopust D, Zhu B, Allison JP, Sharpe AH, Freeman GJ, Ahmed R. Restoring function in exhausted CD8 T cells during chronic viral infection.

  Nature. 2006 Feb 9;439(7077):682-7. Epub 2005 Dec 28.