Induction and clonal expansion of tumor-specific cytotoxic T lymphocytes from renal cell carcinoma patients after stimulation with autologous dendritic cells loaded with tumor cells.
Melanoma and renal cell carcinoma (RCC) are considered to be the most immunogenic tumors in humans. To generate conditions to induce primary T-cell responses against RCC and to allow further expansion of tumor-specific cytotoxic T lymphocytes (CTL) for adoptive transfer, peripheral blood mononuclear cells from RCC patients were stimulated with primary autologous tumor cells or monocyte-derived dendritic cells (DC) loaded with either tumor lysate (TU-LY) or apoptotic tumor cells (TU-AP). Whereas repetitive stimulation (4x) with tumor cells alone induced a predominant population of CD3(-) natural killer cells, 4 weeks of stimulation with tumor-loaded DC favored induction and expansion of CD4+ T cells (>80%). However, 2 weekly stimulation cycles with tumor-loaded DC followed by restimulation with autologous irradiated tumor cells alone were optimal for induction of tumor-specific CTL responses in vitro. Using these culture conditions a marked increase of CD4+ T cells was observed during the first 2 weeks of stimulation with tumor-loaded DC. Subsequent restimulation with autologous tumor cells alone gave rise to 500-fold expansion of CD8+ T cells. These CD8+ T cells were shown to exhibit strong major histocompatibility complex class I-restricted cytotoxic activity against the autologous tumor. Comparison of TU-LY and TU-AP as a source of tumor antigen for loading DC did not show any difference in stimulating tumor-specific CTL. Length pattern analysis of the complementary determining region 3 (CDR3) of the T-cell receptor Vbeta chain revealed expansion of oligoclonal CTL populations with outgrowth of 1 or 2 clones after prolonged stimulation with autologous tumor cells. Our study demonstrated an efficient method for generating tumor-specific CTL in vitro that may be used to identify tumor cell antigens or that can be expanded for adoptive T-cell transfer in tumor immunotherapy.
Department of Hematology/Oncology, Freiburg University Medical Center, Freiburg, Germany.
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T
Kurokawa
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M
Oelke
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A
Mackensen
D018528:ATP-Binding Cassette Transporters / Q000032:analysis; D019264:Adoptive Transfer; D000368:Aged; D000951:Antigens, Neoplasm / Q000276:immunology; D017209:Apoptosis; D015153:Blotting, Western; D015496:CD4-Positive T-Lymphocytes / Q000276:immunology; D016827:CD8 Antigens / Q000276:immunology; D018414:CD8-Positive T-Lymphocytes / Q000276:immunology; D002292:Carcinoma, Renal Cell / Q000276:immunology* / Q000628:therapy; D022801:Complementarity Determining Regions / Q000032:analysis; D003713:Dendritic Cells / Q000276:immunology*; D005260:Female; D005434:Flow Cytometry; D005805:Genes, MHC Class I / Q000276:immunology; D006801:Humans; D007376:Interleukin-2 / Q000378:metabolism; D007680:Kidney Neoplasms / Q000276:immunology* / Q000628:therapy; D008213:Lymphocyte Activation / Q000276:immunology; D008297:Male; D008875:Middle Aged; D009000:Monocytes / Q000276:immunology; D016133:Polymerase Chain Reaction; D011948:Receptors, Antigen, T-Cell / Q000378:metabolism; D013602:T-Lymphocytes, Cytotoxic / Q000166:cytology / Q000276:immunology*; D014407:Tumor Cells, Cultured
D018528:ATP-Binding Cassette Transporters; D000951:Antigens, Neoplasm; D016827:CD8 Antigens; D022801:Complementarity Determining Regions; D007376:Interleukin-2; D011948:Receptors, Antigen, T-Cell