Although IFN-γ was first described almost 40 years ago (42
) and has been well characterized at the cellular and molecular levels in in vitro studies, only a few clinical applications have been tested (14
). Specifically, its in vivo immunomodulatory effect has not been systematically analyzed in humans or in nonhuman primates. In this study, immunotherapy with rHBV-IFN-γ and Ad-IFN-γ resulted in upregulation of intrahepatic IFN-γ mRNA and the following immunologic observations. First, the frequency of CXCR3+
T cells in the peripheral blood as well as in the intrahepatic lymphocyte population increased significantly. The intrahepatic CXCR3+
T-cell population characteristically displayed a CD3dim
phenotype, indicating that it consisted either of recently activated T cells (33
) that have downregulated the T-cell receptor or of NK T cells because NKT cells express low levels of CD3 and high levels of CXCR3 on the cell surface (15
). Second, each injection of rHBV-IFN-γ resulted in a significant increase in the percentage of circulating CD3+
T cells, which coexpressed NK cell markers such as CD16. The immediate and transient nature of this increase suggests transcriptional regulation of CD16 rather than redistribution and/or migration of specific CD3+
T cells. Indeed, upregulation of NK cell markers on CD3+
T cells has previously been described in murine models of viral infections and in association with T-cell activation (33
). Consistent with these results, an increased frequency of CD16+
T cells was also observed in chimpanzees with acute HCV infection (V. Racanelli and B. Rehermann, unpublished results). Third, an increased frequency of HCV-specific T cells was detected ex vivo in the peripheral blood and in vitro in antigen-nonspecifically expanded, liver biopsy sample-derived T-cell lines. Interesting characteristics of the HCV-specific T-cell response were its narrow focus on a few epitopes within the analyzed E1 and NS5B peptide pools and its transient nature. The narrow focus of the HCV-specific T-cell response may be related to IFN-γ's capacity to induce immunoproteasomes, which differ from constitutive proteasomes in both qualitative and quantitative aspects of antigen processing. Thus, IFN-γ-induced immunoproteasomes may generate specific T-cell epitopes in larger quantities, as recently demonstrated for an immunodominant HBV core epitope (32
). The transient nature of the induced HCV-specific T-cell response (Fig. ) is interesting in the context of recent reports that IFN-γ induces activation-induced cell death of T cells (5
). It is therefore tempting to speculate that intrahepatically expressed IFN-γ does not only activate HCV-specific T cells but also induces activation-induced cell death. This hypothesis would be in line with the lack of a long-lasting virologic response in the present study.
Whereas the induction of IFN-γ mRNA in the liver was clearly shown, it was more difficult to determine whether the immunologic effects were due to human IFN-γ, transferred by the rHBV-IFN-γ vector, or to subsequently induced endogenous chimpanzee IFN-γ. Both human and chimpanzee IFN-γ mRNAs, which differ by only 3 nucleotides, were detectable in the biopsy sample of Ch1536 taken at week 9, when immunologic parameters showed maximal changes. The following mostly indirect evidence supports the notion that the immunologic effects were caused by human IFN-γ, transferred by the vector, and amplified by chimpanzee IFN-γ, produced by HCV-specific chimpanzee T cells. First, no IFN-γ mRNA was detectable in a control chimpanzee which was injected with rHBV-Luc, thereby confirming that induction of IFN-γ mRNA did not result from a nonspecific response to the vector backbone. Second, only the rHBV-IFN-γ vector and not the rHBV-Luc control vector resulted in an increase in HCV-specific CD4+ and CD8+ T-cell responses, thereby indirectly confirming the role of the vector-expressed human IFN-γ. As shown in Fig. , the frequency of HCV-specific IFN-γ-producing chimpanzee CD4+ and CD8+ T cells was increased, thereby amplifying the overall IFN-γ response.
A specific T-cell response to the HBV backbone of the vector was excluded, because there was no increase in the IFN-γ response of HBV-specific T cells in any of the three rHBV-injected chimpanzees (Fig. ). This was expected because the vector itself is devoid of all HBV open reading frames, so that there is no in vivo production of HBV antigens in transduced cells. Furthermore, a small amount of antigen, such as the HBV proteins that package the vector, typically does not induce any cellular immune response if injected intravenously and without adjuvants. In summary, we therefore conclude that the observed immunologic changes were initiated by the immune activation by the rHBV-expressed human IFN-γ and subsequently amplified by chimpanzee IFN-γ produced by HCV-specific T cells.
This interpretation applies only to the rHBV-IFN-γ experiments, because the rAd-IFN-γ experiments were not controlled by injection of a control vector into a control animal.
Despite the observed modulation of HCV-specific and nonspecific cellular immune responses, IFN-γ gene transfer did not result in a significant and long-lasting decrease of the HCV titer. An effect of species differences between human IFN-γ and chimpanzee IFN-γ receptor was excluded as explanation for this virological nonresponse, because human IFN-γ was shown to induce typical effects, such as upregulation of major histocompatibility complex class I/II and immunoproteasome subunits, in chimpanzee fibroblasts in vitro (not shown). Therefore, one alternative explanation is that IFN-γ does not exert any antiviral effects in vivo in the HCV-infected liver. Whereas this contrasts in vitro results of IFN-γ-mediated suppression of subgenomic and genomic HCV RNAs in cell culture (7
), it is consistent with the results of a recent clinical trial. In that trial, persistently HCV-infected patients who were treated with recombinant IFN-γ displayed no change in HCV titers despite hematologic changes that indicated effective IFN-γ delivery (34
). Notably, peak IFN-γ mRNA levels in the two chronically HCV-infected chimpanzees in our study were not lower than those in liver biopsies of three chimpanzees with self-limited hepatitis C, which we tested prospectively during the acute phase of HCV infection and which displayed a maximum increases in intrahepatic IFN-γ mRNA levels of 5-, 15- and 30-fold over preinfection samples (not shown). Although it is possible that a higher concentration or longer-lasting expression of IFN-γ might be required to be effective in the chronically HCV-infected liver, therapeutic delivery of higher doses of IFN-γ might be difficult to tolerate because of systemic and/or local side effects. Constitutive high-level expression of IFN-γ in the livers of transgenic mice, for example, has been associated with the development of chronic hepatitis (40
). Alternatively, the use of recently described constructs with conditional IFN-γ expression (22
) may allow restriction of IFN-γ expression to HCV-infected hepatocytes and thereby avoid an overall increase of IFN-γ expression in the liver. Finally, a combination of IFN-γ expressing vectors with vectors that encode HCV antigens and/or cytokines such as IL-15 (26
) and IL-23 (12
) might be useful for better and longer-lasting expansion of HCV-specific T cells and, ultimately, for effective HCV control.