Accapezzato D, Francavilla V, Paroli M, Casciaro M, Chircu LV, Cividini A, et al. Hepatic expansion of a virus-specific regulatory CD8(+) T cell population in chronic hepatitis C virus infection. J Clin Invest 2004;113:963–972. (Reprinted with permission.)
Regulatory T (T(R)) cells consist of phenotypically and functionally distinct CD4+ and CD8+ T-cell subsets engaged both in maintaining self-tolerance and in preventing anti–non–self effector responses (microbial, tumor, transplant, and so on) that may be harmful to the host. Here we propose that the proinflammatory function of virus-specific memory effector CCR7−CD8+ T cells, which are massively recruited in the liver, are inefficient (in terms of IFN-gamma production) in patients with chronic hepatitis C virus (HCV) infection because of the concomitant presence of virus-specific CCR7−CD8+ T(R) cells producing considerable amounts of IL-10. These CD8+ T(R) cells are antigen specific, as they can be stimulated by HCV epitopes and suppress T-cell responses that are in turn restored by the addition of neutralizing anti–IL-10. This study provides direct evidence of the existence of virus-specific CD8+ T(R) cells that infiltrate the livers of patients with chronic HCV infection, identifies IL-10 as a soluble inhibitory factor mediating suppression, and suggests that these cells play a pivotal role in controlling hepatic effector CD8+ T-cell responses.
Hepatitis C virus (HCV) readily sets up persistent infection, and in doing so must evade both innate and adaptive immune responses. Cellular (T-cell) immune responses are thought to play a significant role in determining clinical outcome, with strong and sustained responses typically associated with viral control.1, 2 Cellular immune responses are also potentially involved in immunomediated pathogenesis, both directly and through recruitment or modulation of other inflammatory cells in the liver. Consequently, much recent effort has been expended to attempt to analyze how the virus evades both CD8+ and CD4+ T cells and define the differences between “successful” and “unsuccessful” outcomes (i.e., spontaneous control of viremia vs. chronicity). Relatively fewer data are available describing how intrahepatic T-cell responses might be linked to pathology.
A general consensus is emerging that in acute infection, regardless of outcome, CD8+ T-cell responses can be readily detected; however, in those cases in which the virus is not controlled, such responses are not sustained at high levels in blood beyond a few weeks.3 This could be explained partly by viral escape through mutation, as has been elegantly shown in animal models.4 However, even in cases when epitopes appear to be intact, responses typically are weak or absent in blood ex vivo once chronicity is established. They do not appear to be entirely deleted, because they can be reconstituted in vitro through restimulation with antigen, or even fished out from blood directly using ultrasensitive detection techniques.5 Thus various groups have proposed that functional alterations in CD8+ T cells may be associated with persistent infection. Those include relatively weak interferon gamma (IFN-γ) production,6, 7 impaired proliferative capacity,8 and a “stunted” maturation state.6, 9, 10 It is not clear whether the lack of mature effector cells seen in the circulation is as a result of compartmentalization in the liver, (where they may be deleted) or failure to generate such cells in the first place—potentially through defects in antigen-presenting cells, inhibitory effects of viral gene products (such as core)11 or failure of CD4+ T-cell help.1
Things look slightly different in the liver itself. Early studies showed it is possible to clone out CD8+ T cells of diverse specificities from the liver of infected patients.12, 13 It appears that antigen-specific T cells exist at higher frequencies infected livers—probably a relative increase of approximately 10-fold as a proportion of CD8+ T cells.14, 15 (It should be noted that enrichment of virus-specific memory T cells—for example, those specific for cytomegalovirus and Epstein-Barr virus—is seen even in normal livers in human and murine models.16) Interestingly, such T cells in both infected and normal livers appear to be activated, as judged by expression of CD69, although this effect is not antigen specific.15 The questions that emerge then are: Are such T cells fully functional in the intrahepatic environment, and how does such activity relate to disease progression?
The study by Accepezzato et al. is a large-scale analysis of intrahepatic CD8+ T-cell populations using class I tetramers and intracellular cytokine staining to better understand exactly the functionality of the intrahepatic virus-specific T cells. These analyses are technically very demanding because of the very low cell yields available, and previously have only examined very limited patient numbers, compared with nearly 50 in this study.14, 15 As previously, Accepezzato et al. found low frequencies of virus-specific T cells in blood in most cases but found generally higher overall frequencies in liver. The maturation state in the liver appears to be more advanced than in blood, reflecting accumulation of “effector memory” (CCR7−) cells in the liver. For example, some cells appear to be high in perforin, a situation that is very rarely found in blood among HCV-specific populations.6, 10
The most interesting results, however, relate to the functionality of the cells. In addition to tetramer staining, the authors stimulated the T cells in vitro with peptide and analyzed the release of IFN-γ or interleukin 10 (IL-10). IFN-γ is a classical antiviral cytokine that is believed to be of special relevance in the clearance of infected hepatocytes.17 IL-10 is a multifunctional cytokine associated with suppression of T helper 1–type responses. In the present study, three secretion patterns seemed to emerge. In some patients, there appeared to be very weak secretion of either cytokine, while in a few patients there appeared to be a dominance of IFN-γ; several other patients exhibited a dominant IL-10 secretory response. Interestingly, the overall proportion of cells secreting IFN-γ in response to viral peptides correlated positively with the level of intrahepatic inflammation (histological activity index score); conversely, the frequency of IL-10 secreting cells correlated inversely with the histological activity index score.
What is the role of intrahepatic virus-specific CD8+ T cells in determining acute viral clearance and immunopathology? With regard to acute viral clearance, this is still very unclear and unlikely to be easily addressed in human studies. It would, for example, be very interesting to know whether the early emergence of IL-10–secreting populations in the liver was associated with failure to initially control the virus. As with many studies, it is very difficult to disentangle cause and effect—IL-10–secreting cells might reasonably emerge as a consequence of long-term inflammation. The observation that even in these chronically infected patients, several had intrahepatic T-cell populations that expressed either no cytokine or largely IFN-γ suggests that induction of IL-10 secretion cannot alone explain the propensity of HCV to evade CD8+ T cells.
Regarding pathology, IL-10–secreting CD8+ T cells may play an important role in suppression or regulation of inflammation—a feature that actually might represent an appropriate adaptation of T-cell responses to chronic antigen exposure. In this respect, they may be akin to the more classical CD4+ T regulatory cells. Such populations may have anti–self specificities; however, persistent stimulation may induce similar regulatory activity even in CD4+ T cells specific for foreign antigens. A role for CD4+CD25+ T cells in regulation of peripheral CD8+ T-cell responses has recently been proposed.18
Why, then, might some patients generate IL-10–secreting T-cell populations and others not? One question arising from the study is whether the phenotype observed reflects an adaptation only of HCV-specific T cells, or whether it is a feature of the overall infiltrate. HCV-specific T cells appear to represent only a relatively small fraction of the overall intrahepatic CD8+ T-cell response (about 1% in this study, although perhaps slightly higher than this if other epitopes were included). Simply analyzing the overall cytokine preferences of intrahepatic T-cell infiltrates might be very informative. There are suggestions that polymorphisms in chemokine and chemokine receptor genes might influence intrahepatic pathology, so a genetic basis (e.g., in cytokine/receptor genes) for these distinct responses might be relevant.19 Alternatively, viral factors might lead to diverse outcomes; genotype did not appear to play an obvious role, although this issue is confounded by the fact that some of the peptides used are poorly cross-reactive. Viral mutants (altered peptide ligands) emerging in vivo have been associated with modulation of cytokine secretion of T cells.20
Finally—looking forward—could the cytokine secretion profiles of intrahepatic T cells be potentially linked to treatment response? This is an intriguing question, given that it is observed that combination therapy for chronic disease boosts previously weak or undetectable T-cell responses in the periphery.21, 22 It is still not clear to what extent such responses are reflected in the liver and how they link ultimately with sustained virological responses. Nevertheless, it has been shown that the addition of ribavirin to treatment regimens reduces the IL-10 secretion of recovered antiviral T cells.22 Thus, it seems plausible that treatment will shift the cytokine balance in the liver, which could influence significantly the overall success of therapy.
One possible message from this study is that T cells in HCV appear to be able to adapt in the face of a persisting virus, and in doing so modulate the intrahepatic environment to one that is less inflammatory (Fig. 1). The relationship between host and virus in HCV infection is potentially a long-term one. As in human relationships, each partner is capable of making compromises to minimize confrontation. In some cases this behavior modification is more successful than others. It remains to be seen whether—in cases where the relationship is breaking down—we can but watch from the sidelines, or could possibly intervene to re-educate T cells into a more appropriate response.