Skip to main content
NCBI home page
Elsevier - PMC COVID-19 Collection logoLink to Elsevier - PMC COVID-19 Collection
. 2002 Dec 11;154(1):131–138. doi: 10.1016/0022-1759(92)90220-N

A transient transfection system for identifying biosynthesized proteins processed and presented to class I MHC restricted T lymphocytes

Laurence C Eisenlohr 1,∗, Jonathan W Yewdell 1, Jack R Bennink 1
PMCID: PMC7131252  PMID: 1401939

Abstract

CD8+ cytotoxic T lymphocytes (CTL) constitute a major portion of immune responses to foreign and self antigens. CTL recognize class I major histocompatibility complex molecules complexed to peptides of 8–10 residues derived from cytosolic proteins. To understand CTL responses to these antigens to manipulate CTL responses optimally, it is necessary to identify the specific peptides recognized by CTL. The methods currently used for this purpose have significant drawbacks. We describe a plasmid transfection method that results in significant lysis of histocompatible target cells. Influenza virus-specific CTLs specifically lysed target cells that were transfected with plasmids bearing cDNAs encoding full length gene products, fragments containing the region that encodes the CTL epitope, or even a ten residue peptide. This significantly lessens the time and effort required todefine genes, and gene segments that contain CTL epitopes.

Keywords: Cytotoxic T lymphocyte, Transfection, Vaccinia virus, Antigen presentation, T7 RNA polymerase

Abbreviations: BSS/BSA, balanced salt solution with 0.1% BSA; DNA, deoxyribonucleic acid; CTL, cytotoxic T lymphocyte; DMEM, Dulbecco's modified Eagle's medium; DPBS, Dulbecco's phosphate-buffered saline; FBS, fetal bovine serum; HA, hemagglutinin; IL-2R, interleukin-2 receptor; IMDM, Iscove's modified Dulbecco's medium; MHC, major histocompatibility complex; NP, nucleoprotein; pfu, plaque forming units; PR8, A/PR/8/34 influenza virus; Vac, vaccinia

References

  1. Bennink J.R., Yewdell J.W., Smith G.L., Moss B. Recognition of cloned influenza virus hemagglutinin gene products by cytotoxic T lymphocytes. J. Virol. 1986;57:786. doi: 10.1128/jvi.57.3.786-791.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Berger E.A., Fuerst T.R., Moss B. Vol. 85. 1988. A soluble recombinant polypeptide comprising the amino-terminal half of the extracellular region of the CD4 molecule contains an active binding site for human immunodeficiency virus; p. 2357. (Proc. Natl. Acad. Sci. USA). [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Chakrabarti S., Brechling K., Moss B. Vaccinia virus expression vector: coexpression of β-galactosidase provides visual screening of recombinant virus plaques. Mol. Cell. Biol. 1985;5:3403. doi: 10.1128/mcb.5.12.3403. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Chavrier P., Parton R.G., Hauri H.P., Simons K., Zerial M. Localization of low molecular weight GTP binding proteins to exocytic and endocytic compartments. Cell. 1990;62:317. doi: 10.1016/0092-8674(90)90369-p. [DOI] [PubMed] [Google Scholar]
  5. Cochran M.S., Mackett M., Moss B. Vol. 82. 1985. Eukaryotic transient expression system dependent on transcription factors and regulatory DNA sequences of vaccinia virus; p. 19. (Proc. Natl. Acad. Sci. USA). [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Del Val M., Schlicht H.-J., Ruppert T., Reddehase M.J., Koszinowski U.H. Efficient processing of an antigenic sequence for presentation by MHC class I molecules depends on its neighboring residues in the protein. Cell. 1991;66:1145. doi: 10.1016/0092-8674(91)90037-y. [DOI] [PubMed] [Google Scholar]
  7. Eisenlohr L.C., Yewdell J.W., Bennink J.R. Flanking sequences influence the presentation of an endogenously synthesized peptide to cytotoxic T lymphocytes. J. Exp. Med. 1992;175:481. doi: 10.1084/jem.175.2.481. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Elroy-Stein O., Moss B. Vol. 87. 1990. Cytoplasmic expression system based on constitutive synthesis of bacteriophage T7 RNA polymerase in mammalian cells; p. 6743. (Proc. Natl. Acad. Sci. USA). [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Elroy-Stein O., Fuerst T.R., Moss B. Vol. 86. 1989. Cap-independent translation of mRNA conferred by encephalomyocarditis virus 5′ sequence improves the performance of the vaccinia virus/bacteriophage T7 hybrid expression system; p. 6126. (Proc. Natl. Acad. Sci. USA). [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Fuerst T.R., Niles E.G., Studier F.W., Moss B. Vol. 83. 1986. Eukaryotic transient-expression system based on recombinant vaccinia virus that synthesizes bacteriophage T7 RNA polymerase; p. 8122. (Proc. Natl. Acad. Sci. USA). [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Gallagher T.M., Escarmis C., Buchmeier M.J. Alteration of the pH dependence of coronavirus-induced cell fusion: effect of mutations in the spike glycoprotein. J. Virol. 1991;65:1916. doi: 10.1128/jvi.65.4.1916-1928.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Gerhard W., Yewdell J.W., Frankel M.E., Webster R.G. Antigenic structure of influenza virus hemagglutinin defined by hybridoma antibodies. Nature. 1981;290:713. doi: 10.1038/290713a0. [DOI] [PubMed] [Google Scholar]
  13. Giordano T., Howard T.H., Coleman J., Sakamoto K., Howard B.H. Isolation of a population of transiently transfected quiescent and senescent cells by magnetic affinity cell sorting. Exp. Cell Res. 1991;192:193. doi: 10.1016/0014-4827(91)90175-t. [DOI] [PubMed] [Google Scholar]
  14. Gould K., Cossins J., Bastin J., Brownlee G.G., Townsend A. A 15 amino acid fragment of influenza nucleoprotein synthesized in the cytoplasm is presented to class I-restricted cytotoxic T lymphocytes. J. Exp. Med. 1989;170:1051. doi: 10.1084/jem.170.3.1051. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Kozak M. The scanning model for translation: an update. J. Cell Biol. 1989;108:229. doi: 10.1083/jcb.108.2.229. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Pamer E.G., Harty J.T., Bevan M.J. Precise prediction of a dominant class I MHC-restricted epitope of Listeria monocytogenes. Nature. 1991;353:852. doi: 10.1038/353852a0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Rich D.P., Anderson M.P., Gregory R.J., Cheng S.H., Paul S., Jefferson D.M., McCann J.D., Klinger K.W., Smith A.E., Welsh M.J. Expression of cystic fibrosis transmembrane conductance regulator corrects defective chloride channel regulation in cystic fibrosis airway epithelial cells. Nature. 1990;347:358. doi: 10.1038/347358a0. [DOI] [PubMed] [Google Scholar]
  18. Rötzschke O., Falk K., Deres K., Schild H., Norda M., Metzger J., Jung G., Rammensee H.-G. Isolation and analysis of naturally processed viral peptides as recognized by cytotoxic T cells. Nature. 1990;348:252. doi: 10.1038/348252a0. [DOI] [PubMed] [Google Scholar]
  19. Rötzschke O., Falk K., Stevanovitc S., Jung G., Walden P., Rammensee H.-G. Exact prediction of a natural T cell epitope. Eur. J. Immunol. 1991;21:2891. doi: 10.1002/eji.1830211136. [DOI] [PubMed] [Google Scholar]
  20. Sweetser M.T., Morrison L., Braciale V.L., Braciale T.J. Recognition of pre-processed endogenous antigen by class 1 but not class II MHC-restricted T cells. Nature. 1989;342:180. doi: 10.1038/342180a0. [DOI] [PubMed] [Google Scholar]
  21. Tevethia S., Tevethia M., Lewis A., Reddy V., Weissman S. Biology of simian virus 40 (SV40) transplantation antigen (TrAg). IX. Analysis of TrAg in mouse cells synthesizing truncated SV40 large T antigen. Virology. 1983;128:319. doi: 10.1016/0042-6822(83)90259-3. [DOI] [PubMed] [Google Scholar]
  22. Townsend A.R.M., McMichael A.J., Carter N.P., Huddleston J.A., Brownlee G.G. Cytotoxic T cell recognition of the influenza nucleoprotein and hemagglutinin expressed in transfected mouse L cells. Cell. 1984;39:13. doi: 10.1016/0092-8674(84)90187-9. [DOI] [PubMed] [Google Scholar]
  23. Townsend A.R.M., Gotch F.M., Davey J. Cytotoxic T cells recognize fragments of the influenza nucleoprotein. Cell. 1985;42:457. doi: 10.1016/0092-8674(85)90103-5. [DOI] [PubMed] [Google Scholar]
  24. Van Bleek G.M., Nathenson S.G. Isolation of an endogenously processed immunodominant viral peptide from the class 1 H-2Kd molecule. Nature. 1990;348:213. doi: 10.1038/348213a0. [DOI] [PubMed] [Google Scholar]
  25. Van der Bruggen P., Traversari C., Chomez P., Lurquin C., De Plaen E., Van den Eynde B., Knuth A., Boon T. A gene encoding an antigen recognized by cytolytic T lymphocytes on a human melanoma. Science. 1991;254:1643. doi: 10.1126/science.1840703. [DOI] [PubMed] [Google Scholar]
  26. Whitton J.L., Oldstone M.B.A. Class 1 MHC can present an endogenous peptide to cytotoxic T lymphocytes. J. Exp. Med. 1989;170:1033. doi: 10.1084/jem.170.3.1033. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Yewdell J.W., Frank E., Gerhard W. Expression of influenza a virus internal antigens on the surface of infected P815 cells. J. Immunol. 1981;126:1814. [PubMed] [Google Scholar]
  28. Yewdell J.W., Bennink J.R., Smith G.L., Moss B. Vol. 82. 1985. Influenza a virus nucleoprotein is a major target antigen for cross-reactive anti-influenza a virus cytotoxic T lymphocytes; p. 1785. (Proc. Natl. Acad. Sci. USA). [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Zagouras P., Rose J.K. Carboxy-terminal SEKDEL sequences retard but do not retain two secretory proteins in the endoplasmic reticulum. J. Cell Biol. 1989;109:2633. doi: 10.1083/jcb.109.6.2633. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Zinkernagel R.M., doherty P.C. MHC-restriced cytotoxic T cells: studies on the biological role of polymorphic major transplantation antigens determining T-cell restriction-specificity, function, and responsiveness. Adv. Immunol. 1979;27:52. doi: 10.1016/s0065-2776(08)60262-x. [DOI] [PubMed] [Google Scholar]

Articles from Journal of Immunological Methods are provided here courtesy of Elsevier

RESOURCES