1887

Abstract

The cellular immune response plays a central role in virus clearance and pathogenesis of liver disease in hepatitis C. The study of hepatitis C virus (HCV)-specific immune responses is limited by currently available cell-culture systems. Here, the establishment and characterization of stable human HLA-A2-positive B-lymphoblastoid×T hybrid cell lines constitutively expressing either the NS3–4A complex or the entire HCV polyprotein are reported. These cell lines, termed T1/NS3-4A and T1/HCVcon, respectively, were maintained in continuous culture for more than 1 year with stable characteristics. HCV structural and non-structural proteins were processed accurately, indicating that the cellular and viral proteolytic machineries are functional in these cell lines. Viral proteins were found in the cytoplasm in dot-like structures when expressed in the context of the HCV polyprotein or in a perinuclear fringe when the NS3–4A complex was expressed alone. T1/NS3-4A and T1/HCVcon cells were lysed efficiently by HCV-specific cytotoxic T lymphocytes from patients with hepatitis C and from human HLA-A2.1 transgenic mice immunized with a liposomal HCV vaccine, indicating that viral proteins are processed endogenously and presented efficiently via the major histocompatibility complex class I pathway. In conclusion, these cell lines represent a unique tool to study the cellular immune response, as well as to evaluate novel vaccine and immunotherapeutic strategies against HCV.

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2005-06-01
2019-12-16
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References

  1. Baum, C., Forster, P., Hegewisch-Becker, S. & Harbers, K. ( 1994; ). An optimized electroporation protocol applicable to a wide range of cell lines. Biotechniques 17, 1058–1062.
    [Google Scholar]
  2. Boisvert, J., He, X.-S., Cheung, R., Keeffe, E. B., Wright, T. & Greenberg, H. B. ( 2001; ). Quantitative analysis of hepatitis C virus in peripheral blood and liver: replication detected only in liver. J Infect Dis 184, 827–835.[CrossRef]
    [Google Scholar]
  3. Brass, V., Bieck, E., Montserret, R., Wölk, B., Hellings, J. A., Blum, H. E., Penin, F. & Moradpour, D. ( 2002; ). An amino-terminal amphipathic α-helix mediates membrane association of the hepatitis C virus nonstructural protein 5A. J Biol Chem 277, 8130–8139.[CrossRef]
    [Google Scholar]
  4. Cerny, A. & Chisari, F. V. ( 1999; ). Pathogenesis of chronic hepatitis C: immunological features of hepatic injury and viral persistence. Hepatology 30, 595–601.[CrossRef]
    [Google Scholar]
  5. Cerny, A., McHutchison, J. G., Pasquinelli, C., Brown, M. E., Brothers, M. A., Grabscheid, B., Fowler, P., Houghton, M. & Chisari, F. V. ( 1995; ). Cytotoxic T lymphocyte response to hepatitis C virus-derived peptides containing the HLA A2.1 binding motif. J Clin Invest 95, 521–530.[CrossRef]
    [Google Scholar]
  6. Chen, M., Shirai, M., Liu, Z., Arichi, T., Takahashi, H. & Nishioka, M. ( 1998; ). Efficient class II major histocompatibility complex presentation of endogenously synthesized hepatitis C virus core protein by Epstein-Barr virus-transformed B-lymphoblastoid cell lines to CD4+ T cells. J Virol 72, 8301–8308.
    [Google Scholar]
  7. Cooper, S., Erickson, A. L., Adams, E. J., Kansopon, J., Weiner, A. J., Chien, D. Y., Houghton, M., Parham, P. & Walker, C. M. ( 1999; ). Analysis of a successful immune response against hepatitis C virus. Immunity 10, 439–449.[CrossRef]
    [Google Scholar]
  8. DeMars, R., Chang, C. C., Shaw, S., Reitnauer, P. J. & Sondel, P. M. ( 1984; ). Homozygous deletions that simultaneously eliminate expressions of class I and class II antigens of EBV-transformed B-lymphoblastoid cells. I. Reduced proliferative responses of autologous and allogeneic T cells to mutant cells that have decreased expression of class II antigens. Hum Immunol 11, 77–97.[CrossRef]
    [Google Scholar]
  9. De Vita, S., De Re V., Sansonno, D. & 7 other authors ( 2002; ). Lack of HCV infection in malignant cells refutes the hypothesis of a direct transforming action of the virus in the pathogenesis of HCV-associated B-cell NHLs. Tumori 88, 400–406.
    [Google Scholar]
  10. Diepolder, H. M., Zachoval, R., Hoffmann, R. M., Wierenga, E. A., Santantonio, T., Jung, M.-C., Eichenlaub, D. & Pape, G. R. ( 1995; ). Possible mechanism involving T-lymphocyte response to non-structural protein 3 in viral clearance in acute hepatitis C virus infection. Lancet 346, 1006–1007.[CrossRef]
    [Google Scholar]
  11. Dubuisson, J., Hsu, H. H., Cheung, R. C., Greenberg, H. B., Russell, D. G. & Rice, C. M. ( 1994; ). Formation and intracellular localization of hepatitis C virus envelope glycoprotein complexes expressed by recombinant vaccinia and Sindbis viruses. J Virol 68, 6147–6160.
    [Google Scholar]
  12. Egger, D., Wölk, B., Gosert, R., Bianchi, L., Blum, H. E., Moradpour, D. & Bienz, K. ( 2002; ). Expression of hepatitis C virus proteins induces distinct membrane alterations including a candidate viral replication complex. J Virol 76, 5974–5984.[CrossRef]
    [Google Scholar]
  13. Engelhard, V. H., Brickner, A. G. & Zarling, A. L. ( 2002; ). Insights into antigen processing gained by direct analysis of the naturally processed class I MHC associated peptide repertoire. Mol Immunol 39, 127–137.[CrossRef]
    [Google Scholar]
  14. Engler, O. B., Schwendener, R. A., Dai, W. J., Wölk, B., Pichler, W., Moradpour, D., Brunner, T. & Cerny, A. ( 2004; ). A liposomal peptide vaccine inducing CD8+ T cells in HLA-A2.1 transgenic mice, which recognise human cells encoding hepatitis C virus (HCV) proteins. Vaccine 23, 58–68.[CrossRef]
    [Google Scholar]
  15. Firat, H., Garcia-Pons, F., Tourdot, S. & 11 other authors ( 1999; ). H-2 class I knockout, HLA-A2.1-transgenic mice: a versatile animal model for preclinical evaluation of antitumor immunotherapeutic strategies. Eur J Immunol 29, 3112–3121.[CrossRef]
    [Google Scholar]
  16. Forns, X., Bukh, J. & Purcell, R. H. ( 2002; ). The challenge of developing a vaccine against hepatitis C virus. J Hepatol 37, 684–695.[CrossRef]
    [Google Scholar]
  17. Gosert, R., Egger, D., Lohmann, V., Bartenschlager, R., Blum, H. E., Bienz, K. & Moradpour, D. ( 2003; ). Identification of the hepatitis C virus RNA replication complex in Huh-7 cells harboring subgenomic replicons. J Virol 77, 5487–5492.[CrossRef]
    [Google Scholar]
  18. Grakoui, A., Wychowski, C., Lin, C., Feinstone, S. M. & Rice, C. M. ( 1993; ). Expression and identification of hepatitis C virus polyprotein cleavage products. J Virol 67, 1385–1395.
    [Google Scholar]
  19. Gremion, C., Grabscheid, B., Wölk, B., Moradpour, D., Reichen, J., Pichler, W. & Cerny, A. ( 2004; ). Cytotoxic T lymphocytes derived from patients with chronic hepatitis C virus infection kill bystander cells via Fas-FasL interaction. J Virol 78, 2152–2157.[CrossRef]
    [Google Scholar]
  20. Hanada, K., Yewdell, J. W. & Yang, J. C. ( 2004; ). Immune recognition of a human renal cancer antigen through post-translational protein splicing. Nature 427, 252–256.[CrossRef]
    [Google Scholar]
  21. Kammer, A. R., van der Burg, S. H., Grabscheid, B., Hunziker, I. P., Kwappenberg, K. M. C., Reichen, J., Melief, C. J. M. & Cerny, A. ( 1999; ). Molecular mimicry of human cytochrome P450 by hepatitis C virus at the level of cytotoxic T cell recognition. J Exp Med 190, 169–176.[CrossRef]
    [Google Scholar]
  22. Kolykhalov, A. A., Agapov, E. V., Blight, K. J., Mihalik, K., Feinstone, S. M. & Rice, C. M. ( 1997; ). Transmission of hepatitis C by intrahepatic inoculation with transcribed RNA. Science 277, 570–574.[CrossRef]
    [Google Scholar]
  23. Kumar, A., Kang, Y.-H., Gowda, K., Mullen, C. A., Gordon, D. M., Kumar, S. & Hoffman, S. L. ( 1997; ). Autologous lymphoblastoid cell lines stably transfected with Plasmodium falciparum circumsporozoite protein as targets in cytotoxic T-lymphocyte assays. Immunol Lett 55, 183–187.[CrossRef]
    [Google Scholar]
  24. Laskus, T., Radkowski, M., Piasek, A., Nowicki, M., Horban, A., Cianciara, J. & Rakela, J. ( 2000; ). Hepatitis C virus in lymphoid cells of patients coinfected with human immunodeficiency virus type 1: evidence of active replication in monocytes/macrophages and lymphocytes. J Infect Dis 181, 442–448.[CrossRef]
    [Google Scholar]
  25. Lauer, G. M., Ouchi, K., Chung, R. T. & 8 other authors ( 2002; ). Comprehensive analysis of CD8+-T-cell responses against hepatitis C virus reveals multiple unpredicted specificities. J Virol 76, 6104–6113.[CrossRef]
    [Google Scholar]
  26. Lechner, F., Gruener, N. H., Urbani, S. & 8 other authors ( 2000; ). CD8+ T lymphocyte responses are induced during acute hepatitis C virus infection but are not sustained. Eur J Immunol 30, 2479–2487.[CrossRef]
    [Google Scholar]
  27. Lerat, H., Rumin, S., Habersetzer, F., Berby, F., Trabaud, M.-A., Trépo, C. & Inchauspé, G. ( 1998; ). In vivo tropism of hepatitis C virus genomic sequences in hematopoietic cells: influence of viral load, viral genotype, and cell phenotype. Blood 91, 3841–3849.
    [Google Scholar]
  28. Missale, G., Bertoni, R., Lamonaca, V. & 7 other authors ( 1996; ). Different clinical behaviors of acute hepatitis C virus infection are associated with different vigor of the anti-viral cell-mediated immune response. J Clin Invest 98, 706–714.[CrossRef]
    [Google Scholar]
  29. Moradpour, D., Englert, C., Wakita, T. & Wands, J. R. ( 1996; ). Characterization of cell lines allowing tightly regulated expression of hepatitis C virus core protein. Virology 222, 51–63.[CrossRef]
    [Google Scholar]
  30. Moradpour, D., Kary, P., Rice, C. M. & Blum, H. E. ( 1998; ). Continuous human cell lines inducibly expressing hepatitis C virus structural and nonstructural proteins. Hepatology 28, 192–201.
    [Google Scholar]
  31. Moradpour, D., Grabscheid, B., Kammer, A. R., Schmidtke, G., Groettrup, M., Blum, H. E. & Cerny, A. ( 2001; ). Expression of hepatitis C virus proteins does not interfere with major histocompatibility complex class I processing and presentation in vitro. Hepatology 33, 1282–1287.[CrossRef]
    [Google Scholar]
  32. Moradpour, D., Bieck, E., Hügle, T., Wels, W., Wu, J. Z., Hong, Z., Blum, H. E. & Bartenschlager, R. ( 2002; ). Functional properties of a monoclonal antibody inhibiting the hepatitis C virus RNA-dependent RNA polymerase. J Biol Chem 277, 593–601.[CrossRef]
    [Google Scholar]
  33. National Institutes of Health ( 2002; ). National Institutes of Health Consensus Development Conference statement: management of hepatitis C. Hepatology 36, S3–S20.
    [Google Scholar]
  34. Park, B., Kim, Y., Shin, J., Lee, S., Cho, K., Früh, K., Lee, S. & Ahn, K. ( 2004; ). Human cytomegalovirus inhibits tapasin-dependent peptide loading and optimization of the MHC class I peptide cargo for immune evasion. Immunity 20, 71–85.[CrossRef]
    [Google Scholar]
  35. Pietschmann, T. & Bartenschlager, R. ( 2003; ). Tissue culture and animal models for hepatitis C virus. Clin Liver Dis 7, 23–43.[CrossRef]
    [Google Scholar]
  36. Retière, C., Prod'homme, V., Imbert-Marcille, B.-M., Bonneville, M., Vié, H. & Hallet, M.-M. ( 2000; ). Generation of cytomegalovirus-specific human T-lymphocyte clones by using autologous B-lymphoblastoid cells with stable expression of pp65 or IE1 proteins: a tool to study the fine specificity of the antiviral response. J Virol 74, 3948–3952.[CrossRef]
    [Google Scholar]
  37. Rotzschke, O., Falk, K., Stevanovic, S., Jung, G., Walden, P. & Rammensee, H. G. ( 1991; ). Exact prediction of a natural T cell epitope. Eur J Immunol 21, 2891–2894.[CrossRef]
    [Google Scholar]
  38. Salter, R. D., Howell, D. N. & Cresswell, P. ( 1985; ). Genes regulating HLA class I antigen expression in T-B lymphoblast hybrids. Immunogenetics 21, 235–246.[CrossRef]
    [Google Scholar]
  39. Sansonno, D., De Vita, S., Iacobelli, A. R., Cornacchiulo, V., Boiocchi, M. & Dammacco, F. ( 1998a; ). Clonal analysis of intrahepatic B cells from HCV-infected patients with and without mixed cryoglobulinemia. J Immunol 160, 3594–3601.
    [Google Scholar]
  40. Sansonno, D., Lotesoriere, C., Cornacchiulo, V., Fanelli, M., Gatti, P., Iodice, G., Racanelli, V. & Dammacco, F. ( 1998b; ). Hepatitis C virus infection involves CD34+ hematopoietic progenitor cells in hepatitis C virus chronic carriers. Blood 92, 3328–3337.
    [Google Scholar]
  41. Schirle, M., Keilholz, W., Weber, B., Gouttefangeas, C., Dumrese, T., Becker, H. D., Stevanovic, S. & Rammensee, H. G. ( 2000; ). Identification of tumor-associated MHC class I ligands by a novel T cell-independent approach. Eur J Immunol 30, 2216–2225.[CrossRef]
    [Google Scholar]
  42. Schmidt-Mende, J., Bieck, E., Hügle, T., Penin, F., Rice, C. M., Blum, H. E. & Moradpour, D. ( 2001; ). Determinants for membrane association of the hepatitis C virus RNA-dependent RNA polymerase. J Biol Chem 276, 44052–44063.[CrossRef]
    [Google Scholar]
  43. Shoukry, N. H., Cawthon, A. G. & Walker, C. M. ( 2004; ). Cell-mediated immunity and the outcome of hepatitis C virus infection. Annu Rev Microbiol 58, 391–424.[CrossRef]
    [Google Scholar]
  44. Sung, V. M.-H., Shimodaira, S., Doughty, A. L., Picchio, G. R., Can, H., Yen, T. S. B., Lindsay, K. L., Levine, A. M. & Lai, M. M. C. ( 2003; ). Establishment of B-cell lymphoma cell lines persistently infected with hepatitis C virus in vivo and in vitro: the apoptotic effects of virus infection. J Virol 77, 2134–2146.[CrossRef]
    [Google Scholar]
  45. Takaki, A., Wiese, M., Maertens, G., Depla, E., Seifert, U., Liebetrau, A., Miller, J. L., Manns, M. P. & Rehermann, B. ( 2000; ). Cellular immune responses persist and humoral responses decrease two decades after recovery from a single-source outbreak of hepatitis C. Nat Med 6, 578–582.[CrossRef]
    [Google Scholar]
  46. Thimme, R., Bukh, J., Spangenberg, H. C., Wieland, S., Pemberton, J., Steiger, C., Govindarajan, S., Purcell, R. H. & Chisari, F. V. ( 2002; ). Viral and immunological determinants of hepatitis C virus clearance, persistence, and disease. Proc Natl Acad Sci U S A 99, 15661–15668.[CrossRef]
    [Google Scholar]
  47. Ureta-Vidal, A., Firat, H., Pérarnau, B. & Lemonnier, F. A. ( 1999; ). Phenotypical and functional characterization of the CD8+ T cell repertoire of HLA-A2.1 transgenic, H-2Kb0Db0 double knockout mice. J Immunol 163, 2555–2560.
    [Google Scholar]
  48. Wertheimer, A. M., Miner, C., Lewinsohn, D. M., Sasaki, A. W., Kaufman, E. & Rosen, H. R. ( 2003; ). Novel CD4+ and CD8+ T-cell determinants within the NS3 protein in subjects with spontaneously resolved HCV infection. Hepatology 37, 577–589.[CrossRef]
    [Google Scholar]
  49. Whitmore, M. M., Li, S., Falo, L., Jr & Huang, L. ( 2001; ). Systemic administration of LPD prepared with CpG oligonucleotides inhibits the growth of established pulmonary metastases by stimulating innate and acquired antitumor immune responses. Cancer Immunol Immunother 50, 503–514.[CrossRef]
    [Google Scholar]
  50. Wölk, B., Sansonno, D., Kräusslich, H.-G., Dammacco, F., Rice, C. M., Blum, H. E. & Moradpour, D. ( 2000; ). Subcellular localization, stability, and trans-cleavage competence of the hepatitis C virus NS3-NS4A complex expressed in tetracycline-regulated cell lines. J Virol 74, 2293–2304.[CrossRef]
    [Google Scholar]
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