While the human immune system is specialized and highly efficient in fighting microbial infections, certain pathogens have evolved the capacity to exploit it for their own benefit. This can have disastrous consequences, such as cancer or death. A number of viruses, in particular human immunodeficiency virus (HIV), hepatitis C virus (HCV) and herpes viruses, have evolved efficient immune escape properties, rendering them capable of persisting in the host. While the mechanisms by which immune escape occurs vary from one virus to the next, the consequence is a chronic infection which can often pose severe health problems. For example, after a hepatitis virus infection, only a fraction of the affected individuals clear the virus. The resulting chronic infection can lead to liver damage or liver cancer, particularly after HCV infection 1, 2. Other viruses, such as Epstein–Barr virus (EBV), can also cause cancer if systemic reactivation occurs 3, 4. Furthermore, the loss of immune function that systematically occurs during chronic infection with HIV impairs the capacity of infected individuals to fight other infections (such as CMV; reviewed in 5). Numerous efforts have been undertaken to increase immunity in chronic viral infections, with the goal of eliminating the pathogen or at least reducing the immunopathological consequences of viral persistence. Interferon therapy is a current standard treatment in HCV and HBV infections. Vaccination strategies have also been widely used, alone or in combination with direct antiviral drugs, such as protease inhibitors and highly active antiretroviral therapy (HAART) in HIV infection 6, and interferon or ribavirin in HCV infection 7, 8. In most of the situations where antiviral drugs are employed, viral load can be significantly reduced and, for HIV, long-term deleterious consequences of the persistent infection are decreased. However, the outcome of ribavirin administration in chronic HCV is not quite as promising, since although viral titers are lowered, liver fibrosis is enhanced, possibly as a rather direct effect of the drug. Furthermore, and most importantly, complete elimination of the pathogen has in most cases remained elusive, despite the fact that antiviral immunity can be significantly increased in many instances. It thus appears that current strategies to treat chronic infections need to be revisited, yet without losing sight of the importance of antiviral drug therapy and strengthening of T cell immunity to the virus.
Antiviral immunity and chronic infection
The host's immune response to a viral infection is orchestrated in several stages, all of which provide critical contribution to the ultimate goal of eliminating the invading pathogen. Usually, interferon production and innate immunity occur early after infection of the host and constitute the first line of defence. Systemic viral load is reduced during this stage, due largely to the antiviral effects of interferons and natural killer (NK) cells. Shortly thereafter, the adaptive immune response follows. Here, CD4+ and CD8+ T lymphocytes are induced, in most cases activated by professional antigen-presenting cells (APCs), such as dendritic cells (DCs) that provide antigenic peptide associated with major histocompatibility complex (MHC) molecules, as well as co-stimulation (Figure 1). The adaptive response can selectively eliminate virus-specific cells (CD8) and help (CD4) with the development of B cell immunity and antibody production. In addition, CD4+ T helper cells and the cytokines they produce are of essence in determining the magnitude and maintenance of CD8 memory effector responses. In most cases, a viral infection will trigger the expansion of CD8+ T cytotoxic (Tc1) and CD4+ T helper type 1 cells (Th1), which produce interferon-gamma (IFNγ), tumour necrosis factor-alpha (TNFα) and perforin. The purpose is to cleanse the host from all remaining infected cells and achieve sterile immunity. One important additional consequence of adaptive immunity is the generation of memory B and T cells. These cells are capable of responding rapidly to re-infection with the same or antigenically related viruses. Most adaptive responses are characterized by initial expansion of cells and ensuing contraction 9, 10. Contraction usually coincides with the elimination of the viral pathogen and involves apoptosis of effector cells. However, this scenario is modified in chronic infections; in this case, antigenic exposure is sustained but cytokine class switches may occur (loss of Th1/Tc1 response), and exhaustion of effector responses has been noted in a number of infections.
Thus, the development of immunity as it is ‘ideally’ seen in acute infections that are being resolved is not always the outcome. Frequently, viruses are able to persist by using a variety of strategies to avoid recognition by the immune system, as well as mechanisms to suppress the antiviral immune response. For example, viral antigen can become sequestered in immune-privileged sites, or viral proteins inside the cells can directly interfere with antigen presentation by modulating MHC expression, and certain viruses such as herpes simplex virus (HSV) and CMV can become latent 11 (reviewed in 12–14). In addition, although functional effector T cells are initially generated during the early stages of infection, there can be a gradual decrease in their number and function during the course of chronic infection. This has notably been described initially by the group of Rolf Zinkernagel in persistent lymphocytic choriomeningitis virus (LCMV) infection of mice 15. LCMV is an arenavirus that is a natural pathogen for both humans and mice 16. Infection with LCMV induces a potent Th1-type antiviral immune response, including powerful virus-specific CD8+ and CD4+ T cell responses that peak around days 8 and 10 post-infection, respectively 17. During their expansion, virus-specific CD8+ and CD4+ T cells rapidly acquire effector functions, including the ability to produce cytokines such as IFNγ, TNFα and IL-2 18–21. Viral clearance is dependent on the presence of virus-specific CD8+ cytotoxic T lymphocytes (CTLs) 22–25 and usually occurs in the blood and spleen within 10–12 days of infection. Whereas adult mice inoculated with LCMV Armstrong rapidly clear the infection and remain immune-competent, inoculation with the LCMV variant Clone 13, which differs from its parent (Armstrong) virus at only two amino acid positions 26–28, results in a protracted infection that persists for several months. The variant LCMV Clone 13 was originally isolated from the spleen of a 2 month-old mouse infected at birth with the Armstrong strain 29. While LCMV Armstrong and Clone 13 differ in two amino acid positions in the viral glycoprotein, the molecular basis of persistence and suppression of the anti-LCMV CTL response to this strain has been mapped to a single amino acid change in the glycoprotein of LCMV 27, 29, 30. LCMV Clone 13 chronic infection is associated with both the functional impairment and deletion of virus-specific CD8+ T cells associated with general immunosuppression. It is established that sustained exposure of CD8+ T cells to viral antigen leads to a certain state of ‘exhaustion’ of these initially active cells, rendering them incapable of further controlling the infection 15. Recently, however, it has become clear that this exhausted or impaired state is not irreversible, and that it is possible to resuscitate the antiviral response through immune modulation targeting certain signalling pathways, such as those of programmed death-1 (PD-1) and interleukin-10 (IL-10).
IL-10 production in viral persistence
While impairment of CD8+ CTL responses has been described in immune responses against LCMV Clone 13 in the mouse as well as HCV and HIV in humans, the underlying mechanisms accounting for this loss of immune function have not been fully elucidated. Interestingly, in certain persistent viral infections, an increase in systemic IL-10 production can be observed. This has been reported for various human chronic viral infections, in particular HCV 31, 32, but also HIV (and SIV) 33–35 and EBV 36. IL-10 is an immune-modulatory cytokine that can be produced by different cell types in humans and mice, including monocytes, macrophages, B cells, CD4+ and CD8+ T cells (reviewed in 37–39). Importantly, while APCs and T cells are the main producers of IL-10, they are also important targets of this cytokine, which has been shown to play its immunosuppressive role through interplay between these two cell types. IL-10 can directly inhibit cytokine production by T cells in vitro40, but also prevents the maturation of DCs, rendering these cells ineffective in activating T and other immune cells. IL-10 plays a role in blocking pro-inflammatory cytokine production, co-stimulation, MHC class II expression and chemokine secretion. By acting on APCs, IL-10 regulates the proliferation and differentiation of Th1 cells, which are helper T cells that control not only host defence, but also many crucial effector immune responses in vivo, such as anti-tumour immunity and auto-immunity 41. In the case of HIV, IL-10-mediated hampering of APC maturation reduces the efficacy of antigen presentation from these cells and induces T cell-dependent suppression of antiviral responses 42, 43. Interestingly, a number of viruses are capable of expressing IL-10 homologues that often bear strong sequence homology with host cellular IL-10. This is notably the case of EBV, which expresses an IL-10 protein that is 84% identical to human IL-10 44.
It is therefore intriguing to hypothesize that IL-10 may play a role in maintaining chronicity and pathogenicity in chronic infections (Figure 1). However, absolute proof of concept in humans has not yet been achieved, and solid evidence is not currently available for certain chronic situations, such as HIV infection. Importantly, however, it has been shown that the significant amounts of IL-10 produced during HCV infection can been attributed to the generation of a subset of suppressor cells thought to hamper viral resolution by Th1/Tc1 cells 31. In addition, administration of IL-10 to HCV patients with the intention of dampening chronic immune pathology has been found to actually increase viral loads, thus not proving beneficial 45. Furthermore, single nucleotide polymorphisms in the IL-10 region have been associated with natural clearance of HCV in some populations 46. Thus, the overall concept emerging from clinical studies examining chronic HCV infection supports the hypothesis that influencing IL-10 signalling may have an impact on this chronic viral infection and its deleterious secondary consequences, such as liver cancer, immunodeficiency and lymphomas. This may be achieved by using monoclonal antibodies (mAbs), neutralizing IL-10 function. Of note, it has been shown that in vitro use of anti-IL-10 monoclonal antibodies (mAbs) can restore immune responses mounted by T cells isolated from patients harbouring chronic infection. For example, defective T cell immunity in asymptomatic HIV-infected patients was found to be enhanced in vitro by neutralizing mAbs to IL-10 34. Similarly, IL-10 production was found to correlate with the pathology of the parasitic infection leishmaniasis in humans 47, and the associated loss of function of anti-parasitic T cells was restored in vitro by anti-IL-10 blocking mAbs 48. In vivo, a comparable therapeutic approach consisting of neutralizing IL-10 signalling has provided a sterile cure from leishmaniasis 49, as well as a number of intracellular bacterial infections (reviewed in 44). Based on these considerations and previous findings in the LCMV Clone 13 system, we hypothesized that blockade of IL-10 signalling could be a means of resolving viral persistence in vivo. In the murine model of protracted viral infection induced by LCMV Clone 13, we observed that a significant amount of IL-10 was produced systemically, which interestingly coincided with the loss of CTL responses directed against the virus 50. In our published studies, systemic administration of a blocking mAb against the IL-10 receptor (IL-10R) led to rapid resolution of persistent LCMV Clone 13 infection, as demonstrated by weight gain and reduced viral load in treated mice. Importantly, similar results were obtained simultaneously in an independent study by the group of Michael Oldstone 51. In our study, successful anti-IL-10R therapy occurred in the absence of systemic side-effects or immunopathology, and showed efficiency in mice in which the persistent infection was already established. This novel approach to treating a persistent viral infection constitutes a departure from classical vaccine strategies which have attempted to enhance antiviral responses by directly inducing or amplifying antiviral effector T cells. Indeed, such conventional approaches have failed to resolve LCMV Clone 13 infection in previous studies.
Tr1 cells in chronic infection
The cells that come into play during adaptive immune responses are usually under the control of particular T cells with regulatory function. Such regulatory T cells (Tregs) have been classified in different subsets according to the specific markers they express and/or the cytokines they produce, but their common function is to control tolerance to self as well as foreign antigens in the periphery. Indeed, they have been shown to play a crucial role in auto-immunity and transplantation. However, loss of immune function during chronic infection is sometimes the result of direct induction of immune suppression by the emergence of Tregs, such as natural Tregs generated in the thymus, or adaptive Tregs generated in response to antigen 52–55. In contrast to the natural subset of Tregs, which is CD4+CD25+ and of thymic origin, inducible Tregs, which have been more recently described, are usually generated in response to antigen in the periphery. Notably, CD4+ T cells have been shown to differentiate into Tr1 cells capable of suppressing the establishment of colitis in a IL-10-dependent fashion 56. Our results in the LCMV Clone 13 system indicate that virus-specific CD4+ T cells are the main source of IL-10 during persistent viral infection. Importantly, we found that these cells can also produce IFNγ and no IL-4. While the cytokine profile of Tr1 cells has been shown to vary with regards to cytokines such as IFNγ or TGFβ, the hallmark of these cells is elevated IL-10 production and absence of IL-4 production (reviewed in 57). Thus, these cells constitute a class of their own which differs from classical Th1 or Th2, and our results suggest that LCMV Clone 13 persistence may be attributable to such Tr1 or Tr1-like cells (Figure 1). Importantly, these cells appear to be skewed to IL-10 production as a consequence of changes in DC subsets. DCs are the most potent APCs within the immune system and have the capacity to efficiently activate CD4+ and CD8+ T cells in vivo58, 59. In particular, they are critical for the efficient activation of antiviral T cells during LCMV infection 60. DCs are subdivided into five subsets of conventional DCs and a sixth subset of plasmacytoid DCs, based on expression of different surface markers, such as CD8α and CD11b 61, 62. Previous reports suggest that not only do these DC subsets differ in their capacity to polarize effector CD4+ T cells, but they also display a functional plasticity that enables each subtype to define the class of CD4+ T cell responses, depending on the nature of the immune environment and the microbial stimulus. It has been reported that antigen-pulsed CD8α+ DCs produce high amounts of IL-12 and induce a Th1 response upon adoptive transfer, while CD8α− DCs induce IL-4 and IL-10 production 63, 64. Further studies have shown that DC-derived IL-12 and IFNγ are required for Th1 priming by CD8α+ DCs, whereas IL-10 is required for the induction of a Th2-type response by CD8α− DCs 65. Importantly, we found that virus-specific CD4+ T cells produced IL-10 in vitro after interaction with DCs belonging to the CD8α− subset from LCMV Clone 13-infected mice 50. Furthermore, persistence of LCMV was linked to a decline in the number of CD8α+ DCs. IL-10 priming in CD4+ T cells by the remaining CD8α− DCs prevented viral clearance and therefore enabled viral persistence. Anti-IL-10R therapy aborted the capacity of CD8α− DCs to induce IL-10-secreting Tr1-like cells, in this way enhancing Th1/Tc1 immunity and resolving persistent infection.
Therefore, blockade of IL-10 signalling in protracted LCMV Clone 13 infection may act directly on DCs, providing a central switch in immunity where the overall antiviral immune response is orchestrated. Because of such a role on immune function through modulation of DCs, IL-10 has been used in different systems as a means to induce tolerance. The clinical use of immature DCs or DCs grown in vitro in the presence of IL-10 can in this way be of significant interest, since such cells can induce antigen-specific tolerance if they have been pulsed with a cognate antigen prior to in vivo injection 66, 67. IL-10-modulated DCs have been associated with tolerance in asthma, and the induction of Tr1 cells depends on the presence of IL-10. Many in vivo models of tolerance (in some cases induced by transfer of Tregs) are ultimately dependent on the action of IL-10 56, 66, 68–70. The flipside of its beneficial effect in auto-immunity and graft rejection, however, is that IL-10 can hamper immunity not only in host defence but also in cancer 71.
The recent observation by us and others that persistent viral infection can be resolved by neutralizing the effect of IL-10 in vivo suggests that successful treatment of other chronic infections also associated with high IL-10 production may be achieved using the same approach. It is possible to enhance IL-10 function with no loss of function of the host defence system. It thus appears feasible to lower detrimental IL-10 effects in chronic infection without immediately facing auto-immunity as a consequence, especially if the treatment is temporary. Conventional immunomodulatory therapies consist of enhancing antiviral immunity by acting directly on the number of virus-specific T cells. Such an approach, while effective in some cases, has mostly failed to resolve chronic viral infections. Viruses are capable of escaping recognition by the immune system, using various strategies, such as the active induction of immune suppression leading to the loss of T cell function. One of these mechanisms is the induction of immunomodulatory cytokines such as IL-10, which ultimately leads to the impairment of T cell immunity. Interestingly, it appears that induction of IL-10 production is a strategy shared by different viruses in order to achieve immune suppression and escape antiviral immunity. In particular, the elevated levels of IL-10 observed in chronic HCV infection suggest that targeting IL-10 signalling may enhance antiviral immunity in this scenario. Importantly, however, current therapies such as interferon or antiviral drugs have proved to be efficient in lowering viral titers and should thus not be abandoned. However, these strategies could be revised to incorporate an additional component with the goal of modulating the class of the antiviral response. Disruption of IL-10-mediated class switching may be an essential ingredient for a complete cure (Figure 1). In particular, it may enable the successful treatment of patients who do not respond to conventional therapy. Based on current knowledge, it should thus be determined whether anti-IL-10R blockade restores the function of antiviral T cells from patients with chronic infection, such as HCV. This has been successfully achieved in vitro for HIV. The next step would be to assess anti-IL-10R therapy in clinical trials.
However, while IL-10 production constitutes a crucial element in the impairment of antiviral immunity, it is not the only component enabling viral persistence. In particular, in the case of persistent infection with LCMV Clone 13, recent work has shown that antiviral CD8+ T cell exhaustion operates through induction of PD-1 expression. PD-1 is an inhibitory receptor of the B7–CD28 family, identified by subtractive hybridization of a T cell hybridoma undergoing programmed cell death ( 72, reviewed in 73, 74). PD-1 was found to be highly up-regulated on CD8+ T cells from mice persistently infected with LCMV Clone 13, and blockade of PD-1 interaction with its ligand PD-L1 restored CD8+ T cell function and resulted in viral clearance 75. Therefore, it appears an interesting strategy to neutralize the function of IL-10, along with that of molecules such as PD-1, in order to enhance viral clearance in chronic infections. In this way, synergy might be achieved in combating the viral disease with minimal side-effects. Importantly, similar to LCMV infection, it has recently been demonstrated that exhaustion of HCV-specific CD8+ T cells in chronic HCV infection is associated with PD-1 expression 76, 77. In addition, in vitro blockade of the PD-1 interaction with PD-L1 restored the function of anti-HCV CD8+ T cells 78. Furthermore, PD-1 up-regulation has also been associated with exhaustion of HIV-specific T cells and immune dysfunction 79, 80. Therefore, combination of therapeutic agents that block the IL-10 and PD-1 signalling pathways may hold great promise for the treatment of persistent viral infections in humans, such as HCV, HIV and possibly CMV. In addition, this strategy may prove even more effective when used in combination with conventional antiviral strategies, vaccines and/or other immunomodulatory agents which show efficacy in chronic situations, such as HCV and HIV infections. Such an approach to treating chronic viral infections may represent a novel alternative to classical vaccine strategies that have not been successful in enhancing antiviral T cell responses. Importantly, targeting host factors that do not directly interact with the virus will limit the possibility that mutated resistant viral strains emerge, as has been the case, for example, with HIV.