DOI QR코드

DOI QR Code

Enhanced Induction of T Cell Immunity Using Dendritic Cells Pulsed with HIV Tat and HCMV-pp65 Fusion Protein In Vitro

  • Park, Jung-Sun (Catholic Hematopoietic Stem Cell Bank, College of Medicine, The Catholic University of Korea) ;
  • Park, Soo-Young (Department of Microbiology and Immunology, College of Medicine, The Catholic University of Korea) ;
  • Cho, Hyun-Il (Catholic Hematopoietic Stem Cell Bank, College of Medicine, The Catholic University of Korea) ;
  • Sohn, Hyun-Jung (Catholic Hematopoietic Stem Cell Bank, College of Medicine, The Catholic University of Korea) ;
  • Kim, Tai-Gyu (Catholic Hematopoietic Stem Cell Bank, College of Medicine, The Catholic University of Korea)
  • Received : 2011.06.01
  • Accepted : 2011.06.14
  • Published : 2011.06.30

Abstract

Background: Cytotoxic T lymphocytes (CTLs) appear to play an important role in the control and prevention of human cytomegalovirus (HCMV) infection. The pp65 antigen is a structural protein, which has been defined as a potential target for effective immunity against HCMV infection. Incorporation of an 11 amino acid region of the HIV TAT protein transduction domain (Tat) into protein facilitates rapid, efficient entry into cells. Methods: To establish a strategy for the generation of HCMV-specific CTLs in vitro, recombinant truncated N- and C-terminal pp65 protein (pp65 N&C) and N- and C-terminal pp65 protein fused with Tat (Tat/pp65 N&C) was produced in E.coli system. Peripheral blood mononuclear cells were stimulated with dendritic cells (DCs) pulsed with pp65 N&C or Tat/pp65 N&C protein and immune responses induced was examined using IFN-${\gamma}$ ELISPOT assay, cytotoxicity assay and tetramer staining. Results: DCs pulsed with Tat/pp65N&C protein could induce higher T-cell responses in vitro compared with pp65N&C. Moreover, the DCs pulsed with Tat/pp65 N&C could stimulate both of $CD8^+$ and $CD4^+$ T-cell responses. The T cells induced by DCs pulsed with Tat/pp65 N&C showed higher cytotoxicity than that of pp65-pulsed DCs against autologous lymphoblastoid B-cell line (LCL) expressing the HCMV-pp65 antigen. Conclusion: Our results suggest that DCs pulsed with Tat/pp65 N&C protein effectively induced pp65-specific CTL in vitro. Tat fusion recombinant protein may be useful for the development of adoptive T-cell immunotherapy and DC-based vaccines.

Keywords

References

  1. Ho M: Epidemiology of cytomegalovirus infections. Rev Infect Dis 12(Suppl 7);S701-S710, 1990 https://doi.org/10.1093/clinids/12.Supplement_7.S701
  2. Taylor-Wiedeman J, Sissons JG, Borysiewicz LK, Sinclair JH: Monocytes are a major site of persistence of human cytomegalovirus in peripheral blood mononuclear cells. J Gen Virol 72;2059-2064, 1991 https://doi.org/10.1099/0022-1317-72-9-2059
  3. Minton EJ, Tysoe C, Sinclair JH, Sissons JG: Human cytomegalovirus infection of the monocytes/macrophages lineage in bone marrow. J Virol 68;4017-4021, 1994
  4. Soderberg-Naucler C, Fish KN, Nelson JA: Reactivation of latent human cytomegalovirus by allogeneic stimulation of blood cells from healthy donors. Cell 91;119-126, 1997 https://doi.org/10.1016/S0092-8674(01)80014-3
  5. Mindelson M, Monard S, Sissons P, Sinclair J: Detection of endogenous human cytomegalovirus in CD34+ bone marrow progenitors. J Gen Virol 77;3099-3102, 1996 https://doi.org/10.1099/0022-1317-77-12-3099
  6. Michelson S: Interaction of human cytomegalovirus with monocytes/ macrophages: a love-hate relationship. Pathol Biol (Paris) 45;146-158, 1997
  7. Meyers JD, Flournoy N, Thomas ED: Risk factors for cytomegalovirus infection after human marrow transplantation. J Infect Dis 153;478-488, 1986 https://doi.org/10.1093/infdis/153.3.478
  8. Meyers JD, Ljungman P, Fisher LD: Cytomegalovirus excretion as a predictor of cytomegalovirus disease after marrow transplantation: importance of cytomegalovirus viraemia. J Infect Dis 162;373-380, 1990 https://doi.org/10.1093/infdis/162.2.373
  9. Banchereau J, Steinman RM: Dendritic cells and the control of immunity. Nature 392;245, 1998 https://doi.org/10.1038/32588
  10. Foster AE, Forrester K, Gottlieb DJ, Barton GW, Romagnoli JA, Bradstock KF: Large-scale expansion of cytomegalovirus- specific cytotoxic T cells in suspension culture. Biotechnol Bioeng 85;138-146, 2004 https://doi.org/10.1002/bit.10801
  11. Verma IM, Somia N: Gene therapy - promises, problems and prospects. Nature 389;239-242, 1997 https://doi.org/10.1038/38410
  12. Nagahara H, Vocero-Akbani AM, Snyder EL, Ho A, Latham DG, Lissy NA, Becker-Hapak M, Ezhevski SA, Dowdy SF: Transduction of full-length TAT fusion proteins into mammalian cells: TAT-p27Kip1 induces cell migration. Nat Med 41;449-452, 1998
  13. Schwarze SR, Ho A, Vocero-Akbani A, Dowdy SF: In vivo protein transduction: delivery of a biologically active protein into the mouse. Science 285;1569-1572, 1999 https://doi.org/10.1126/science.285.5433.1569
  14. Pande H, Baak SW, Riggs AD, Clark BR, Shively JE, Zaia JA: Cloning and physical mapping of a gene fragment coding for a 64-kilodalton major late antigen of human cytomegalovirus. Proc Natl Acad Sci USA 81;4965-4969, 1984 https://doi.org/10.1073/pnas.81.15.4965
  15. Kern F, Bunde T, Faulhaber N, Kiecker F, Khatamzas E, Rudawski IM, Pruss A, Gratama JW, Volkmer-Engert R, Ewert R, Reinke P, Volk HD, Picker LJ: Cytomegalovirus (CMV) phosphoprotein 65 makes a large contribution to shaping the T cell repertoire in CMV-exposed individuals. J Infect Dis 185;1709-1716, 2002 https://doi.org/10.1086/340637
  16. Bradford MA: A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72;248-254, 1976 https://doi.org/10.1016/0003-2697(76)90527-3
  17. Gavin MA, Gilbert MJ, Riddell SR, Greenberg PD, Bevan MJ: Alkali hydrolysis of recombinant proteins allows for the rapid identification of class I MHC-restricted CTL epitopes. J Immunol 151;3971-3980, 1993
  18. Wills MR, Carmichael AJ, Mynard K, Jin X, Weekes MP, Plachter B, Sissons JG: The human cytotoxic T-lymphocyte (CTL) response to cytomegalovirus is dominated by structural protein pp65: frequency, specificity, and T-cell receptor usage of pp65-specific CTL. J Virol 70;7569-7579, 1996
  19. Cho HI, Han H, Kim CC, Kim TG: Generation of cytotoxic T lymphocytes specific for human cytomegalovirus using dendritic cells in vitro. J Immunother 24;242-249, 2001 https://doi.org/10.1097/00002371-200105000-00008
  20. Paston SJ, Dodi IA, Madrigal JA: Progress made towards the development of a CMV peptide vaccine. Human Immunology 65;544-549, 2004 https://doi.org/10.1016/j.humimm.2004.02.005
  21. Fittipaldi A, Giacca M: Transcellular protein transduction using the Tat protein of HIV-1. Adv Drug Deliv Rev 57; 597-608, 2005 https://doi.org/10.1016/j.addr.2004.10.011
  22. Wadia JS, Dowdy SF: Transmembrane delivery of protein and peptide drugs by TAT-mediated transduction in the treatment of cancer. Adv Drug Deliv Rev 57;579-596, 2005 https://doi.org/10.1016/j.addr.2004.10.005

Cited by

  1. Expression, purification and characterization of a recombinant Tat47-57-Oct4 fusion protein in Pichia pastoris vol.9, pp.2, 2011, https://doi.org/10.3892/mmr.2013.1857