In the past year, we have completed the following studies in the chimpanzee model of hepatitis C virus infection: ? ? (i) Identification and characterization of novel chimpanzee MHC class I and class II alleles? ? 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. Chimpanzee (Pan troglodytes) MHC alleles have been designated Patr alleles. Here, we have continued our efforts to identify and characterize (i) the Patr class I and II alleles of chimpanzees and (ii) the HCV peptides that they present.? ? In a collaboration with extramural investigators, we identified ten new Patr class I alleles, which have been submitted to the IPD/MHC-NHP database (Robinson et al., Nucleic Acids Research 2003: 31:311-14) and to GenBank with the following alleles names: Patr-A*1703(DQ673903), Patr-A*2401(EU200940), Patr-B*0703(DQ673906), Patr-B*3501(DQ673905), Patr-B*3601(EU289158), Patr-B*0402 (EU289160), Patr-B*3701(EU289161), Patr-C*0206(EU200941), Patr-C*1501(EU200943) and Patr-C*1601(EU200948). ? ? In addition, two new Patr-DRB1 sequences were identified. In both humans and chimpanzees, the DRB1 locus (Patr-DRB1) is the most polymorphic MHC class II locus. The new Patr-DRB1 alleles carry the official designation DRB1*0214(DQ673902) and Patr-DRB1*0316(EU285279).? ? Studies to determine specific HCV epitopes that are presented by these Patr alleles are currently in progress. The identification and characterization of Patr alleles and Patr-binding viral peptides is important for the generation of immunological tools such as Patr-peptide tetramers which can then be used to characterize effective cellular immune responses ex vivo in liver biopsies and blood samples throughout the course of HCV infection in this animal model. ? ? (ii) Characterization of the kinetics of HCV-specific immune responses in blood and liver and comparison of molecular biology assays and functional immunology assays as read-outs? ? CD8 T cells are thought to be the main effector cells in acute HCV infection. Unfortunately, it is not possible to prospectively monitor HCV-specific intrahepatic T cell responses in humans because of clinical and ethical concerns, and all published studies on the immune response of patients with acute HCV infection are based on HCV-specific T cell activity in the peripheral blood. It is therefore not clear whether the results of peripheral blood T cell analysis accurately reflect the onset and vigor of intrahepatic responses, which is important for vaccination studies. Studying ten chimpanzees throughout the first 6 months of acute HCV infection we here addressed this issue by comparing (i) the results of molecular ex vivo analysis of intrahepatic T cell responses to those obtained by functional analysis of in vitro expanded intrahepatic T cells and (ii) the results of molecular and functional analyses of T cell responses in the liver to those of HCV-specific T cell responses in the blood.? ? To analyze T cell responses in the liver with ex vivo techniques that do not require weeks of in vitro stimulation, total RNA was isolated from prospectively collected chimpanzee liver biopsies, cDNA was synthesized and TaqMan real-time PCR was performed to determine the mRNA levels of IFN-g, CD8b and CD4. The amount of specific mRNA was normalized to endogenous references (b-actin, GAPDH and b7 mRNA) and presented as fold-increase over baseline, which was the median value of pre-infection samples of 5 non-vaccinated chimpanzees.? ? In contrast to CD8b mRNA levels, intrahepatic CD4 mRNA levels remained relatively stable throughout the first 6 months of HCV infection in all chimpanzees. These findings and the fact that CD8b is only expressed by T cells and not by NK cells (the latter express a CD8α/α homodimer) suggest that liver-infiltrating CD8 rather than CD4 T cells or NK cells contributed to IFN-g production in the acutely HCV-infected liver. This is consistent with the observation that the increase in intrahepatic IFN-γ and CD8β mRNA levels coincides with the first appearance of HCV-specific, tetramer+ CD8 T cells in the blood during acute hepatitis C. Indeed, intrahepatic CD8β and IFN-γ mRNA levels correlated significantly (R2=0.68, p<0.001), whereas CD4 and IFN-γ mRNA levels did not. To avoid potential effects of a large number of repeated measurements during the whole phase of acute HCV infection, we studied the correlation between intrahepatic CD8b and IFN-g mRNA levels for single time points, or for shorter time periods within the acute phase of hepatitis. In each analysis, the statistical significance of the correlation between intrahepatic CD8b and IFN-g mRNA levels was preserved. ? ? Next, the results of the molecular analysis of CD8 T cell responses in the liver were correlated to the results of a functional analysis of HCV-specific CD8 T cell responses in the liver and the blood. Specifically, intrahepatic IFN-γ and CD8b mRNA levels were compared to previously published data on the frequency of IFN-γ -producing CD8 T cells in the liver (determined by intracellular cytokine staining of in vitro expanded intrahepatic CD8 T cells) and blood (determined by ex vivo intracellular cytokine staining in response to peptides covering the HCV nonstructural regions). The concordance rate between the results obtained with different immunological readouts was higher than 80%. Taken together, these results suggest that all examined immunological readouts are adequate to determine the onset of vaccine- or infection-induced CD8 T cell responses. ? ? To analyze whether the strength of the HCV-specific CD8 T cell response in the blood correlated to the strength of the CD8 T cell response in the liver, regression analysis was performed. Indeed, the frequency of HCV-specific, IFN-g-producing CD8 T cells in the blood significantly correlated to the expression level of CD8b (R2=0.48, p<0.001) and IFN-g mRNA in the liver (R2=0.42, p<0.001). To avoid potential effects of a large number of repeated measurements during the whole phase of acute HCV infection, we also studied the correlation between both readouts for single time points, and for short time periods within the acute phase of hepatitis. In each analysis, the statistical significance of the correlation was preserved. Thus, the vigor of the HCV-specific CD8 T cell activity in the blood correlated to the vigor of the total CD8 T cell response in the liver during acute HCV infection. ? ? In summary, the present study demonstrates that the results of functional assays that monitor the onset and the vigor of HCV-specific CD8 T cell activity in the blood correlate significantly to (i) the results of ex vivo molecular analysis of the total CD8 T cell response in the liver, the site of HCV infection and to (ii) the results of functional assays with HCV-specific T cells isolated from the liver. It is important to note that the use of large pools of overlapping peptides covering the nonstructural HCV polyprotein sequences likely increased the sensitivity to detect HCV-specific CD8 T cell responses in the blood because earlier studies with smaller sets of minimal optimal peptides did not detect HCV-specific CD8 T cells without in vitro expansion of PBMC. In human studies, where liver biopsies cannot easily be obtained, quantification of IFN-g-producing CD8 T cells in the blood may be the method of choice. Thus, HCV-specific CD8 T cell activity in the blood may be monitored as a surrogate marker for intrahepatic CD8 T cell responses in natural history studies and vaccine trials.
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