T cells recognize viral peptides in the context of MHC class I and II molecules on the surface of cells that are either infected by the virus or take up and process exogenous viral antigens. Here, we have (i) characterized chimpanzee MHC class I alleles and the viral peptides that they present and (ii) analyzed mechanisms that affect and modulate the processing and presentation of viral antigens in acute hepatitis C.? ? (i) Characterization of chimpanzee MHC class I alleles and the viral peptides that they present:? ? Chimpanzee (Pan troglodytes) MHC alleles have been designated Patr alleles. Patr maps to chromosome 5, the homologue of human chromosome 6. Similar to the human HLA system, Patr has been divided into class I, II and III regions. The class I region encodes molecules that are expressed on all cells, whereas the class II regions encodes molecules that are expressed only on specific cells of the immune system, such as B cells, macrophages and dendritic cells. The class III region encodes several members of the innate immune system, which include complement components and tumor necrosis factor.? ? MHC molecules display certain common features that determine the nature of binding peptides, i.e., specific anchor amino acid residues that interact with the polymorphic complementary pockets of the HLA peptide-binding grooves. However, only a limited set of peptides that are presented by both HLA alleles and Patr-orthologues have been identified to date. Molecular characterization of Patr alleles is the first step to identify HLA orthologues and to select chimpanzees for vaccine studies based on their MHC homology to common HLA haplotypes.? ? In collaborations with Drs. De Re and Caggliari in Aviano, Italy, Patr class I and class II alleles of seventeen chimpanzees were typed using a novel sequence-based typing (SBT) strategy. Thirteen novel Patr alleles were identified. Studies to determine specific HCV peptides that are presented by these Patr alleles are currently in progress. Understanding the molecular and functional similarities between chimpanzee and human MHC alleles may aid the characterization of effective cellular immune responses and the identification of peptides that are presented in the context of these MHC molecules, which is helpful for vaccine development.? ? ? (ii) Virus-induced type I interferon regulates the antigen-processing machinery at the site of viral infection:? ? In virus-infected cells, viral proteins are degraded to small peptides by the proteasome complex, further trimmed by aminopeptidases in the endoplasmic reticulum, and presented by MHC class I molecules to CD8 T cells. Many components of the MHC class I antigen-processing pathway are thought to be regulated solely by IFN-gamma. Recently, however, we reported type I IFN-mediated induction of immunoproteasomes, a more efficient form of proteasomes, in a viral infection of the liver. In the present study, we extended this analysis to additional components of the MHC class I antigen-processing machinery. Specifically, we investigated the induction of the proteasome activator 28 (PA28) subunits alpha and beta, the ER aminopeptidases-1 (ERAP1), -2 (ERAP2) and leucine aminopeptidase (LAP) in vivo and in vitro during hepatitis C virus (HCV) infection.? ? For the in vivo study, five chimpanzees were studied during the first 6 months of HCV infection and serial liver biopsies were analyzed for mRNA levels of PA28 subunits and aminopeptidases. In the early phase of HCV infection (2-8 weeks post infection) intrahepatic mRNA levels of all PA28 subunits and aminopeptidases increased significantly. Unexpectedly, this increased expression occurred much earlier than the intrahepatic IFN-gamma response and instead, coincided with the intrahepatic type I IFN response. This temporal relation suggested a role of type I IFN in the up-regulation of PA28 subunits and aminopeptidases. ? ? To further study this mechanism in vitro, human hepatoma Huh-7 cells were treated with either consensus sequence IFN-alpha (IFN-alpha-con1), IFN-beta or, as a positive control, IFN-gamma. All cytokines induced the expression of PA28a, PA28b, ERAP1, ERAP2 and LAP at the mRNA and protein level. The induction of these components of the antigen processing machinery by type I IFN was confirmed in primary human hepatocytes. In contrast, the expression levels of the control enzyme prolyl endopeptidase, and the constitutive proteasome 4 subunit remained stable.? ? Next, we asked whether endogenously induced type I IFN had the same effect as exogenously added type I IFN on PA28 and aminopeptidase expression. Endogenous induction of type I IFN in response to a viral infection occurs by sensing of dsRNA by retinoic acid-inducible gene I (RIG-I), which results in the phophorylation and activation of interferon response factor 3 (IRF-3) and subsequent production of IFN-beta. Secreted IFN-beta binds to the IFN-alpha/beta receptor in an autocrine and paracrine manner, resulting in the activation of the JAK/STAT signaling pathway, and ultimately in transcription of IFN-stimulated genes and production of various IFN-alpha subtypes. To mimic viral infection, Huh-7 cells were first transfected with the synthetic double-stranded RNA poly(I-C), which resulted in an increase of PA28alpha, PA28beta, ERAP1, ERAP2 and LAP mRNA levels, but not PREP mRNA levels. Poly(I-C) also induced the expression of IFN-beta and downstream 2,5-oligoadenylate synthetase (2,5-OAS-1), suggesting that the induction of the components of the antigen processing machinery was mediated by type I-IFN. ? ? To prove this hypothesis, we performed the same experiment in the presence of the vaccinia virus-encoded B18 receptor protein (VV B18R), which competes with the IFN-alpha/beta receptor for IFN-binding. The addition of VV B18R abrogated the induction of PA28a, PA28b, ERAP1, ERAP2 and LAP along with abrogation of 2,5-OAS-1 induction. Thus, induction of PA28 subunits and ER aminopeptidases in response to intracellular dsRNA was indeed mediated by endogenously produced type I IFN.? ? Furthermore, transfection of Huh-7 cells with HCV RNA (pHCV-H77) increased mRNA levels of PA28alpha, PA28beta, ERAP1, ERAP2 and LAP, but not that of the control enzyme PREP, and induced IFN-beta production and the downstream response of 2,5-OAS-1 and the induction was abrogated by the addition of VV B18R. These data demonstrate that PA28 and ER aminopeptidases were not only induced by synthetic dsRNA but also by HCV RNA itself in a type I IFN-dependent manner. ? ? In summary, viral RNA-induced type I IFN does not only exert direct antiviral functions but also enhances antigen processing in virus-infected cells to facilitate recognition by effector CD8 T cells. The duration of type I IFN-dependent induction of PA28 subunits and ER aminopeptidases during natural infection was transient in vitro as well as in vivo, in the liver of HCV-infected chimpanzees. It is therefore conceivable that prolonged treatment with high doses of exogenous interferon may extend the upregulation of PA28 subunits and ER aminopeptidases and reduce the gap in peak time between increased expression of antigen processing enzymes in the liver (peak at 2-8 weeks after infection) and infiltration of HCV-specific CD8 T cells (peak at 12-16 weeks after infection). Because an effective response of HCV-specific CD8+ T cells against HCV-infected hepatocytes is an important determinant for the outcome of HCV infection, prolonged maximal expression of antigen-processing enzymes may contribute to the high (95%) effectiveness of type I IFN based therapies in acute HCV infection. Thus, type I IFN plays a previously unknown role in the induction of components of the antigen-processing machinery in vitro and in vivo.
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