Journal of Clinical Immunology

, Volume 24, Issue 6, pp 653–663 | Cite as

Monitoring of CD4+ and CD8+ T-Cell Responses After Dendritic Cell-Based Immunotherapy Using CFSE Dye Dilution Analysis



CFSE dye dilution analysis and [3H] thymidine incorporation were used side by side to assess proliferative responses of peripheral blood mononuclear cells (PBMCs) after vaccination of renal cell carcinoma patients (n=6) with antigen-loaded dendritic cells. Immune responses against the control antigen keyhole limpet hemocyanin (KLH) were induced in all patients. While [3H] thymidine incorporation revealed a 4 to 977-fold increase in KLH-induced proliferation (mean: 209-fold), CFSE-labeling experiments demonstrated that the KLH-responsive population of postvaccination PBMCs represented 7–53% (mean: 23%). Combining CFSE-labeling with T-cell subset analysis confirmed the presence of CD4+ KLH-reactive T cells but also revealed a substantial population of CD8+ KLH-reactive T cells in one patient as well as minor populations of CD8+ KLH-reactive T cells in three other patients. Our data indicate that CFSE dye dilution analysis is a valuable tool for immune monitoring after dendritic cell vaccination.

CFSE vaccination dendritic cells KLH renal cell carcinoma 


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  1. 1.
    Schuler G, Schuler-Thurner B, Steinman RM: The use of dendritic cells in cancer immunotherapy. Curr Opin Immunol15:138–147, 2003Google Scholar
  2. 2.
    Harris JR, Markl J: Keyhole limpet hemocyanin (KLH): A biomedical review. Micron30:597–623, 1999Google Scholar
  3. 3.
    Rieser C, Ramoner R, Höltl L, Rogatsch H, Papesh C, Stenzl A, Bartsch G, Thurnher M: Mature dendritic cells induce T-helper type-1-dominant immune responses in patients with metastatic renal cell carcinoma. Urol Int63:151–159, 1999Google Scholar
  4. 4.
    Höltl L, Zelle-Rieser C, Gander H, Papesh C, Ramoner R, Bartsch G, Rogatsch H, Barsoum AL, Coggin JH, Thurnher M: Immunotherapy of metastatic renal cell carcinoma with tumor lysate-pulsed autologous dendritic cells. Clin Cancer Res8:3369–3376, 2002Google Scholar
  5. 5.
    Gilliet M, Kleinhans M, Lantelme E, Schadendorf D, Burg G, Nestle FO: Intranodal injection of semimature monocyte-derived dendritic cells induces T helper type 1 responses to protein neoantigen. Blood102:36–42, 2003Google Scholar
  6. 6.
    Dhodapkar MV, Steinman RM, Sapp M, Desai H, Fossella C, Krasovsky J, Donahoe SM, Dunbar PR, Cerundolo V, Nixon DF, Bhardwaj N: Rapid generation of broad T-cell immunity in humans after a single injection of mature dendritic cells. J Clin Invest104:173–180, 1999Google Scholar
  7. 7.
    Palucka AKD, Dhodapkar MV, Paczesny S, Burkeholder S, Wittkowski KM, Steinman RM, Fay J, Banchereau J: Single injection of CD34+ progenitor-derived dendritic cell vaccine can lead to induction of T-cell immunity in patients with stage IV melanoma. J Immunother 26(5):432–439, 2003Google Scholar
  8. 8.
    Guermonprez P, Valladeau J, Zitvogel L, Thery C, Amigorena S: Antigen presentation and T cell stimulation by dendritic cells. Annu Rev Immunol20:621–667, 2002Google Scholar
  9. 9.
    Lyons AB, Parish CR: Determination of lymphocyte division by flow cytometry. J Immunol Methods171:131–137, 1994Google Scholar
  10. 10.
    Lyons AB: Divided we stand: Tracking cell proliferation with carboxyfluorescein diacetate succinimidyl ester. Immunol Cell Biol77:509–515, 1999Google Scholar
  11. 11.
    Popma SH, Krasinskas AM, McLean AD, Szeto WY, Kreisel D, Moore JS, Rosengard BR: Immune monitoring in xenotransplantation: The multiparameter flow cytometric mixed lymphocyte culture assay. Cytometry42:277–283, 2000Google Scholar
  12. 12.
    Fulcher D, Wong S: Carboxyfluorescein succinimidyl ester-based proliferative assays for assessment of T cell function in the diagnostic laboratory. Immunol Cell Biol77:559–564, 1999Google Scholar
  13. 13.
    Rieser C, Bock G, Klocker H, Bartsch G, Thurnher M: Prostaglandin E2 and tumor necrosis factor alpha cooperate to activate human dendritic cells: Synergistic activation of interleukin 12 production. J Exp Med186:1603–1608, 1997Google Scholar
  14. 14.
    Schuler-Thurner B, Schultz ES, Berger TG, Weinlich G, Ebner S, Woerl P, Bender A, Feuerstein B, Fritsch PO, Romani N, Schuler G: Rapid induction of tumor-specific type 1 T helper cells in metastatic melanoma patients by vaccination with mature, cryopreserved, peptide-loaded monocyte-derived dendritic cells. J Exp Med195:1279–1288, 2002Google Scholar
  15. 15.
    Schnurr M, Galambos P, Scholz C, Then F, Dauer M, Endres S, Eigler A: Tumor cell lysate-pulsed human dendritic cells induce a T-cell response against pancreatic carcinoma cells: An in vitro model for the assessment of tumor vaccines. Cancer Res61:6445–6450, 2001Google Scholar
  16. 16.
    Mannering SI, Morris JS, Jensen KP, Purcell AW, Honeyman MC, van Endert PM, Harrison LC: A sensitive method for detecting proliferation of rare autoantigen-specific human T cells. J Immunol Methods283:173–183, 2003Google Scholar
  17. 17.
    Santana MA, Rosenstein Y: What it takes to become an effector T cell: The process, the cells involved, and the mechanisms. J Cell Physiol195:392–401, 2003Google Scholar
  18. 18.
    Apostolopoulos V, McKenzie IF, Lees C, Matthaei KI, Young IG: A role for IL-5 in the induction of cytotoxic T lymphocytes in vivo. Eur J Immunol30:1733–1739, 2000Google Scholar
  19. 19.
    Takatsu K, Kikuchi Y, Takahashi T, Honjo T, Matsumoto M, Harada N, Yamaguchi N, Tominaga A: Interleukin 5, a T-cell-derived B-cell differentiation factor also induces cytotoxic T lymphocytes. Proc Natl Acad Sci USA84:4234–4238, 1987Google Scholar
  20. 20.
    Ramos T: Interleukin 5 is a differentiation factor for cytotoxic T lymphocytes. Immunol Lett21:277–283, 1989Google Scholar
  21. 21.
    Bercovici N, Givan AL, Waugh MG, Fisher JL, Vernel-Pauillac F, Ernstoff MS, Abastado JP, Wallace PK: Multiparameter precursor analysis of T-cell responses to antigen. J Immunol Methods 276:5–t17, 2003Google Scholar
  22. 22.
    Putz T, Gander H, Ramoner R, Zelle-Rieser C, Rahm A, Nussbaumer W, Bartsch G, Höltl L, Thurnher M: Generation of clinical grade monocyte-derived dendritic cells using the CliniMACS system. Methods Mol Med, in press.Google Scholar

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© Springer Science+Business Media, Inc. 2004

Authors and Affiliations

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    • 1
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    • 1
    • 1
  1. 1.Department of UrologyMedical University of InnsbruckAnichstrasseAustria

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