The consequences of recurrent hepatitis C on the survival of graft and LT recipients can only be avoided by the development of safe and effective antiviral strategies that can not only prevent initial graft infection but also eradicate established hepatitis C recurrence (3
). With regard to initial graft infection, the circulating virions infect the liver graft immediately after LT. HCV RNA concentrations usually increase a few days after LT, reflecting active HCV replication in the liver graft. In general, in such an early phase of a viral infection, the first line of host defense may be effective in removing the virus; however, recent reports have indicated that HCV effectively escapes the innate immune system comprising NK and NKT cells, resulting in persistent infection (21
). It has been reported that cross-linking of CD81 on NK cells by the major envelope protein of HCV, HCV-E2, blocks NK cell activation, IFN-γ production, cytotoxic granule release, and proliferation (21
). Engagement of CD81 on NK cells blocks tyrosine phosphorylation through a mechanism that is distinct from the negative signaling pathways associated with NK cell inhibitory receptors for major histocompatibility complex class I molecules (22
). These facts prove that HCV-E2–mediated inhibition of NK cells is an efficient HCV evasion strategy, which involves targeting the early antiviral activities of NK cells and allowing the virus to establish itself as a chronic infection.
We have explored whether CD81 cross-linking–induced inhibitory effects occur even in IL-2–stimulated NK cells. CD81 cross-linking by a mAb specific for CD81 inhibited antitumor cytotoxicity and anti-HCV activity mediated by resting NK cells, but this manipulation did not alter both these activities of IL-2–stimulated NK cells (Supplemental Figure 4). This indicated that exposure to IL-2 before CD81 cross-linking abrogates subsequent inhibitory signals in the NK cells. This would be one mechanism whereby the adoptive immunotherapy with IL-2/OKT3–treated liver lymphocytes inhibited HCV replication at the early phase of infection after LT.
Although the role of NK cells in controlling HCV infection and replication has not been completely elucidated, a recent report has indicated that NK cells do not exert a direct cytolytic effect on the HCV replicon–containing hepatic cells but release IFN-γ, suppressing HCV RNA expression (11
). The role of IFN-γ in the expression of NK cell–mediated anti-HCV activity has been proved by the observation that NK cell–conditioned media have an enhanced expression of signal transducer and activator of transcription 1, a nuclear factor that is essential in IFN-γ–mediated antiviral pathways. It has also been reported that hepatocytes cultured in NK cell–conditioned media express higher levels of IFN-α/β, IFN regulatory factor 3, and IFN regulatory factor 7, confirming that NK cells play a key role in suppressing HCV infection of and replication in human hepatocytes in an IFN-dependent manner (23
). Similar to recent reports, in the present study, we demonstrated that the NK cells among the IL-2/OKT3–treated liver lymphocytes released soluble factors, predominantly IFN-γ, thus suppressing HCV replication (Figures –).
In addition to NK cells, NKT cells are thought to be involved in eliciting innate responses against infection; however, the role of NKT cells in controlling HCV infection/replication remains unclear. One report has indicated that the number of NKT cells in patients with chronic HCV infection does not differ from that in healthy donors; however, activated NKT cells in HCV-infected patients produce higher levels of IL-13 — but comparable levels of IFN-γ — than those in healthy subjects, showing that NKT cells are biased toward T-helper 2-type responses in chronic HCV infection (24
). Another recent report has shown that the sustained response of patients with chronic hepatitis C to treatment with IFN-α and ribavirin is closely associated with increased dynamism of NK and NKT cells in the liver, implicating an NKT cell–mediated mechanism in anti-HCV activity (25
). Here, we have described that NKT as well as NK cells in the IL-2/OKT3–treated liver lymphocytes could play a vital role in controlling HCV replication in hepatic cells via an IFN-γ–associated mechanism (Figures and ).
Therefore, in the early phase of HCV reinfection after LT, the effects of IFN-γ secretion from adoptively injected liver lymphocytes may include inhibition of HCV virion production, which is probably caused by suppression of viral RNA and protein synthesis without immune lysis of intact hepatic cells. This IFN-γ secretion from both CD3+
NKT cells and CD3+
T cells was markedly upregulated after treatment with OKT3, which was originally used to prevent GVHD (Figure B). This is possibly because of the potent mitogenic activity of OKT3 that induces the activation of CD3+
NKT cells and CD3+
T cells. However, the administration of OKT3-coated cells in vivo results in the opsonization and subsequent trapping and/or lympholysis of cells by the reticuloendothelial system (26
). Thus, GVHD is prevented in LT recipients treated with adoptive immunotherapy.
Our finding that the IL-2/OKT3–treated liver lymphocytes controlled HCV replication via an IFN-γ–associated mechanism can lead to the clinical application of recombinant IFN-γ for anti-HCV treatment. However, a clinically applicable dose of recombinant IFN-γ could not induce significant inhibitory effects on HCV viremia in the previous study (29
). Based on the accumulation of adoptively injected IL-2/OKT3–treated liver lymphocytes in the liver of human hepatocyte–chimeric mice (data not shown), the immunotherapy with the liver lymphocytes would provide sufficient IFN-γ to the HCV-infected site.
It has been recently reported that HCV-specific CD8+
T cells exert strong antiviral effects by both cytopathic and IFN-γ–mediated noncytopathic effector functions (30
). However, in patients with chronic HCV infection, dysfunction and functional restoration of HCV-specific CD8+
T cell responses have been reported (31
). Since HCV-specific CD8+
T cell defects may be important in persistent HCV infections, correcting these defects is considered to our knowledge to be a novel approach to treat HCV infection. Further studies are required to investigate whether activation of NK or NKT cells functionally restores HCV-specific CD8+
In conclusion, adoptive immunotherapy using IL-2/OKT3–treated liver lymphocytes containing abundant NK and NKT cells could mount remarkable anti-HCV responses in HCV-infected LT recipients, although its effects were incomplete or transient. Treatment-related improvements, such as defining the best schedule and frequency of cell inoculation and developing more potent effectors, could improve clinical benefits.