Cancer Immunology, Immunotherapy

, Volume 56, Issue 10, pp 1577–1587 | Cite as

Simultaneous ex vivo quantification of antigen-specific CD4+ and CD8+ T cell responses using in vitro transcribed RNA

  • Sebastian Kreiter
  • Thorsten Konrad
  • Martina Sester
  • Christoph Huber
  • Özlem Türeci
  • Ugur SahinEmail author
Original Article


Assessment of antigen-specific T-cell responses has been greatly facilitated by development of ELISPOT and intracellular cytokine flow cytometry (CFC) assays. The use of autologous antigen presenting cells transfected with in vitro transcribed RNA as stimulators allows in principle quantification of antigen-specific T-cells independent of the knowledge of the epitopes. We describe here a cytokine secretion assay that enables simultaneous assessment of both antigen-specific CD4+ as well as CD8+ T-cells directly from clinical samples without the need for generation of dendritic cells. To this aim, bulk PBMCs were electroporated with RNA encoding the antigen fused to trafficking signal sequences derived from a MHC class I molecule and used as stimulators. With human cytomegalovirus (HCMV) phosphoprotein 65 (pp65) as antigen we show that for measuring ex vivo T-cell responses in ELISPOT and CFC such stimulators are superior or at least equivalent to a pool of overlapping peptides representing the entire pp65 sequence as well as to untagged pp65 encoding RNA. This approach avoids the time consuming generation of dendritic cells as immune stimulators and, in particular when used in the context of the CFC, is robust, broadly applicable and fast.


Immune monitoring IVT-RNA CFC 



This study was supported by the Combined Project Grant SFB432, by the Heisenberg scholarship TU 115/3-1 of the Deutsche Forschungsgemeinschaft and by the Immunology Cluster of Excellence Mainz.


  1. 1.
    Clay TM, Hobeika AC, Mosca PJ, Lyerly HK, Morse MA (2001) Assays for monitoring cellular immune responses to active immunotherapy of cancer. Clin Cancer Res 7:1127–1135PubMedGoogle Scholar
  2. 2.
    Morse MA, Clay TM, Hobeika AC, Mosca PJ, Lyerly HK (2001) Monitoring cellular immune responses to cancer immunotherapy. Curr Opin Mol Ther 3:45–52PubMedGoogle Scholar
  3. 3.
    Czerkinsky C, Andersson G, Ekre HP, Nilsson LA, Klareskog L, Ouchterlony O (1988) Reverse ELISPOT assay for clonal analysis of cytokine production. I. Enumeration of gamma-interferon-secreting cells. J Immunol Methods 110:29–36PubMedCrossRefGoogle Scholar
  4. 4.
    Brosterhus H, Brings S, Leyendeckers H, Manz RA, Miltenyi S, Radbruch A, Assenmacher M, Schmitz J (1999) Enrichment and detection of live antigen-specific CD4(+) and CD8(+) T cells based on cytokine secretion. Eur J Immunol 29:4053–4059PubMedCrossRefGoogle Scholar
  5. 5.
    Kern F, Surel IP, Brock C, Freistedt B, Radtke H, Scheffold A, Blasczyk R, Reinke P, Schneider-Mergener J, Radbruch A, Walden P, Volk HD (1998) T-cell epitope mapping by flow cytometry. Nat Med 4:975–978PubMedCrossRefGoogle Scholar
  6. 6.
    Altman JD, Moss PA, Goulder PJ, Barouch DH, McHeyzer-Williams MG, Bell JI, McMichael AJ, Davis MM (1996) Phenotypic analysis of antigen-specific T lymphocytes. Science 274:94–96PubMedCrossRefGoogle Scholar
  7. 7.
    Hobeika AC, Morse MA, Osada T, Ghanayem M, Niedzwiecki D, Barrier R, Lyerly HK, Clay TM (2005) Enumerating antigen-specific T-cell responses in peripheral blood: a comparison of peptide MHC Tetramer, ELISpot, and intracellular cytokine analysis. J Immunother 28:63–72PubMedCrossRefGoogle Scholar
  8. 8.
    Maino VC, Maecker HT (2004) Cytokine flow cytometry: a multiparametric approach for assessing cellular immune responses to viral antigens. Clin Immunol 110:222–231PubMedCrossRefGoogle Scholar
  9. 9.
    Karlsson AC, Martin JN, Younger SR, Bredt BM, Epling L, Ronquillo R, Varma A, Deeks SG, McCune JM, Nixon DF, Sinclair E (2003) Comparison of the ELISPOT and cytokine flow cytometry assays for the enumeration of antigen-specific T cells. J Immunol Methods 283:141–153PubMedCrossRefGoogle Scholar
  10. 10.
    Suni MA, Dunn HS, Orr PL, de Laat R, Sinclair E, Ghanekar SA, Bredt BM, Dunne JF, Maino VC, Maecker HT (2003) Performance of plate-based cytokine flow cytometry with automated data analysis. BMC Immunol 4:9PubMedCrossRefGoogle Scholar
  11. 11.
    Maecker HT, Auffermann-Gretzinger S, Nomura LE, Liso A, Czerwinski DK, Levy R (2001) Detection of CD4 T-cell responses to a tumor vaccine by cytokine flow cytometry. Clin Cancer Res 7:902s–908sPubMedGoogle Scholar
  12. 12.
    Suni MA, Maino VC, Maecker HT (2005) Ex vivo analysis of T-cell function. Curr Opin Immunol 17:434–440PubMedCrossRefGoogle Scholar
  13. 13.
    Kern F, Faulhaber N, Frommel C, Khatamzas E, Prosch S, Schonemann C, Kretzschmar I, Volkmer-Engert R, Volk HD, Reinke P (2000) Analysis of CD8 T cell reactivity to cytomegalovirus using protein-spanning pools of overlapping pentadecapeptides. Eur J Immunol 30:1676–1682PubMedCrossRefGoogle Scholar
  14. 14.
    Van Tendeloo VF, Ponsaerts P, Lardon F, Nijs G, Lenjou M, Van Broeckhoven C, Van Bockstaele DR, Berneman ZN (2001) Highly efficient gene delivery by mRNA electroporation in human hematopoietic cells: superiority to lipofection and passive pulsing of mRNA and to electroporation of plasmid cDNA for tumor antigen loading of dendritic cells. Blood 98:49–56PubMedCrossRefGoogle Scholar
  15. 15.
    Teufel R, Carralot JP, Scheel B, Probst J, Walter S, Jung G, Hoerr I, Rammensee HG, Pascolo S (2005) Human peripheral blood monuclear cells transfected with messenger RNA stimulate antigen-specific cytotoxic T-lymphocytes in vitro. Cell Mol Life Sci 62(15):1755–1762PubMedCrossRefGoogle Scholar
  16. 16.
    Britten CM, Meyer RG, Frankenberg N, Huber C, Wolfel T (2004) The use of clonal mRNA as an antigenic format for the detection of antigen-specific T lymphocytes in IFN-gamma ELISPOT assays. J Immunol Methods 287:125–136PubMedCrossRefGoogle Scholar
  17. 17.
    Britten CM, Meyer RG, Graf C, Huber C, Wolfel T (2005) Identification of T cell epitopes by the use of rapidly generated mRNA fragments. J Immunol Methods 299:165–175PubMedCrossRefGoogle Scholar
  18. 18.
    Holtkamp S, Kreiter S, Selmi A, Simon P, Koslowski M, Huber C, Tureci O, Sahin U (2006) Modification of antigen encoding RNA increases stability, translational efficacy and T-cell stimulatory capacity of dendritic cells. Blood 108(13):4009–4017PubMedCrossRefGoogle Scholar
  19. 19.
    Bonehill A, Heirman C, Tuyaerts S, Michiels A, Zhang Y, Van der Bruggen BP, Thielemans K (2003) Efficient presentation of known HLA class II-restricted MAGE-A3 epitopes by dendritic cells electroporated with messenger RNA encoding an invariant chain with genetic exchange of class II-associated invariant chain peptide. Cancer Res 63:5587–5594PubMedGoogle Scholar
  20. 20.
    Kavanagh DG, Kaufmann DE, Sunderji S, Frahm N, Le Gall S, Boczkowski D, Rosenberg ES, Stone DR, Johnston MN, Wagner BS, Zaman MT, Brander C, Gilboa E, Walker BD, Bhardwaj N (2006) Expansion of HIV-specific CD4+ and CD8+ T cells by dendritic cells transfected with mRNA encoding cytoplasm- or lysosome-targeted Nef. Blood 107:1963–1969PubMedCrossRefGoogle Scholar
  21. 21.
    Su Z, Vieweg J, Weizer AZ, Dahm P, Yancey D, Turaga V, Higgins J, Boczkowski D, Gilboa E, Dannull J (2002) Enhanced induction of telomerase-specific CD4(+) T cells using dendritic cells transfected with RNA encoding a chimeric gene product. Cancer Res 62:5041–5048PubMedGoogle Scholar
  22. 22.
    Hoffmeister B, Bunde T, Rudawsky IM, Volk HD, Kern F (2003) Detection of antigen-specific T cells by cytokine flow cytometry: the use of whole blood may underestimate frequencies. Eur J Immunol 33:3484–3492PubMedCrossRefGoogle Scholar
  23. 23.
    Nomura LE, Walker JM, Maecker HT (2000) Optimization of whole blood antigen-specific cytokine assays for CD4(+) T cells. Cytometry 40:60–68PubMedCrossRefGoogle Scholar
  24. 24.
    Diamond DJ, York J, Sun JY, Wright CL, Forman SJ (1997) Development of a candidate HLA A*0201 restricted peptide-based vaccine against human cytomegalovirus infection. Blood 90:1751–1767PubMedGoogle Scholar
  25. 25.
    Gure AO, Tureci O, Sahin U, Tsang S, Scanlan MJ, Jager E, Knuth A, Pfreundschuh M, Old LJ, Chen YT (1997) SSX: a multigene family with several members transcribed in normal testis and human cancer. Int J Cancer 72:965–971PubMedCrossRefGoogle Scholar
  26. 26.
    Maecker HT, Dunn HS, Suni MA, Khatamzas E, Pitcher CJ, Bunde T, Persaud N, Trigona W, Fu TM, Sinclair E, Bredt BM, McCune JM, Maino VC, Kern F, Picker LJ (2001) Use of overlapping peptide mixtures as antigens for cytokine flow cytometry. J Immunol Methods 255:27–40PubMedCrossRefGoogle Scholar
  27. 27.
    Ponsaerts P, Van den Bosch G, Cools N, Van Driessche A, Nijs G, Lenjou M, Lardon F, Van Broeckhoven C, Van Bockstaele DR, Berneman ZN, Van Tendeloo VF (2002) Messenger RNA electroporation of human monocytes, followed by rapid in vitro differentiation, leads to highly stimulatory antigen-loaded mature dendritic cells. J Immunol 169:1669–1675PubMedGoogle Scholar
  28. 28.
    Schmittel A, Keilholz U, Bauer S, Kuhne U, Stevanovic S, Thiel E, Scheibenbogen C (2001) Application of the IFN-gamma ELISPOT assay to quantify T cell responses against proteins. J Immunol Methods 247:17–24PubMedCrossRefGoogle Scholar
  29. 29.
    Schmolke S, Drescher P, Jahn G, Plachter B (1995) Nuclear targeting of the tegument protein pp65 (UL83) of human cytomegalovirus: an unusual bipartite nuclear localization signal functions with other portions of the protein to mediate its efficient nuclear transport. J Virol 69:1071–1078PubMedGoogle Scholar
  30. 30.
    Heine A, Grunebach F, Holderried T, Appel S, Weck MM, Dorfel D, Sinzger C, Brossart P (2006) Transfection of dendritic cells with in vitro-transcribed CMV RNA induces polyclonal CD8+- and CD4+-mediated CMV-specific T cell responses. Mol Ther 13:280–288PubMedCrossRefGoogle Scholar
  31. 31.
    Sanderson S, Frauwirth K, Shastri N (1995) Expression of endogenous peptide-major histocompatibility complex class II complexes derived from invariant chain-antigen fusion proteins. Proc Natl Acad Sci USA 92:7217–7221PubMedCrossRefGoogle Scholar
  32. 32.
    Thomson SA, Burrows SR, Misko IS, Moss DJ, Coupar BE, Khanna R (1998) Targeting a polyepitope protein incorporating multiple class II-restricted viral epitopes to the secretory/endocytic pathway facilitates immune recognition by CD4+ cytotoxic T lymphocytes: a novel approach to vaccine design. J Virol 72:2246–2252PubMedGoogle Scholar
  33. 33.
    Diebold SS, Cotten M, Koch N, Zenke M (2001) MHC class II presentation of endogenously expressed antigens by transfected dendritic cells. Gene Ther 8:487–493PubMedCrossRefGoogle Scholar
  34. 34.
    Bonehill A, Heirman C, Tuyaerts S, Michiels A, Breckpot K, Brasseur F, Zhang Y, Van Der Bosch P, Thielemans K (2004) Messenger RNA-electroporated dendritic cells presenting MAGE-A3 simultaneously in HLA class I and class II molecules. J Immunol 172:6649–6657PubMedGoogle Scholar
  35. 35.
    Rowell JF, Ruff AL, Guarnieri FG, Staveley-O’Carroll K, Lin X, Tang J, August JT, Siliciano RF (1995) Lysosome-associated membrane protein-1-mediated targeting of the HIV-1 envelope protein to an endosomal/lysosomal compartment enhances its presentation to MHC class II-restricted T cells. J Immunol 155:1818–1828PubMedGoogle Scholar
  36. 36.
    Wu TC, Guarnieri FG, Staveley-O’Carroll KF, Viscidi RP, Levitsky HI, Hedrick L, Cho KR, August JT, Pardoll DM (1995) Engineering an intracellular pathway for major histocompatibility complex class II presentation of antigens. Proc Natl Acad Sci USA 92:11671–11675PubMedCrossRefGoogle Scholar
  37. 37.
    Bonini C, Lee SP, Riddell SR, Greenberg PD (2001) Targeting antigen in mature dendritic cells for simultaneous stimulation of CD4+ and CD8+ T cells. J Immunol 166:5250–5257PubMedGoogle Scholar
  38. 38.
    Bonehill A, Heirman C, Thielemans K (2005) Genetic approaches for the induction of a CD4+ T cell response in cancer immunotherapy. J Gene Med 7:686–695PubMedCrossRefGoogle Scholar
  39. 39.
    Lizee G, Basha G, Jefferies WA (2005) Tails of wonder: endocytic-sorting motifs key for exogenous antigen presentation. Trends Immunol 26:141–149PubMedCrossRefGoogle Scholar
  40. 40.
    Lizee G, Basha G, Tiong J, Julien JP, Tian M, Biron KE, Jefferies WA (2003) Control of dendritic cell cross-presentation by the major histocompatibility complex class I cytoplasmic domain. Nat Immunol 4:1065–1073PubMedCrossRefGoogle Scholar
  41. 41.
    Day PM, Yewdell JW, Porgador A, Germain RN, Bennink JR (1997) Direct delivery of exogenous MHC class I molecule-binding oligopeptides to the endoplasmic reticulum of viable cells. Proc Natl Acad Sci USA 94:8064–8069PubMedCrossRefGoogle Scholar
  42. 42.
    Amoscato AA, Prenovitz DA, Lotze MT (1998) Rapid extracellular degradation of synthetic class I peptides by human dendritic cells. J Immunol 161:4023–4032PubMedGoogle Scholar
  43. 43.
    Luft T, Rizkalla M, Tai TY, Chen Q, MacFarlan RI, Davis ID, Maraskovsky E, Cebon J (2001) Exogenous peptides presented by transporter associated with antigen processing (TAP)-deficient and TAP-competent cells: intracellular loading and kinetics of presentation. J Immunol 167:2529–2537PubMedGoogle Scholar
  44. 44.
    Holmes KV, Enjuanes L (2003) The SARS coronavirus: a postgenomic era. Science 300:1377–1378PubMedCrossRefGoogle Scholar
  45. 45.
    Scarselli M, Giuliani MM, Adu-Bobie J, Pizza M, Rappuoli R (2005) The impact of genomics on vaccine design. Trends Biotechnol 23:84–91PubMedCrossRefGoogle Scholar
  46. 46.
    Pizza M, Scarlato V, Masignani V, Giuliani MM, Arico B, Comanducci M, Jennings GT, Baldi L, Bartolini E, Capecchi B, Galeotti CL, Luzzi E, Manetti R, Marchetti E, Mora M, Nuti S, Ratti G, Santini L, Savino S, Scarselli M, Storni E, Zuo PJ, Broeker M, Hundt E, Knapp B, Blair E, Mason T, Tettelin H, Hood DW, Jeffries AC, Saunders NJ, Granoff DM, Venter JC, Moxon ER, Grandi G, Rappuoli R (2000) Identification of vaccine candidates against serogroup B meningococcus by whole-genome sequencing. Science 287:1816–1820PubMedCrossRefGoogle Scholar
  47. 47.
    Milstien J, Lambert S (2002) Emergency response vaccines—a challenge for the public sector and the vaccine industry. Vaccine 21:146–154PubMedCrossRefGoogle Scholar
  48. 48.
    Sturniolo T, Bono E, Ding JY, Raddrizzani L, Tuereci O, Sahin U, Braxenthaler M, Gallazzi F, Protti MP, Sinigaglia F, Hammer J (1999) Generation of tissue-specific and promiscuous HLA ligand databases using DNA microarrays and virtual HLA class II matrices. Nat Biotechnol 17:555–561PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2007

Authors and Affiliations

  • Sebastian Kreiter
    • 1
  • Thorsten Konrad
    • 1
  • Martina Sester
    • 2
  • Christoph Huber
    • 1
  • Özlem Türeci
    • 1
  • Ugur Sahin
    • 1
    Email author
  1. 1.Division of Experimental and Translational Oncology, Department of Internal Medicine IIIJohannes Gutenberg UniversityMainzGermany
  2. 2.Department of NephrologySaarland UniversityHomburg/SaarGermany

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