Novel system for generating cytotoxic effector lymphocytes using carcinoembryonic antigen (CEA) peptide and cultured dendritic cells.
We have established a practical system for generating antitumor effector lymphocytes using the tumor antigen peptide CEA and cultured dendritic cells (DCs), and have also characterized effector cells. DCs were induced from the adherent cell population of autologous peripheral blood mononuclear cells (PBMCs) obtained from HLA-A0201 normal or tumor-bearing donors using IL-4 and GM-CSF. The cultured DCs were shown to express class I, class II, CD80 and CD86 molecules. The PBMCs were stimulated for 7 days with the DCs pulsed with the HLA-A0201-restricted CEA peptide CEA9 671 and then expanded in an anti-CD3 antibody (1 microgram/ml)-coated flask in the presence of a 80 U/ml IL-2 (IL-2/CD3 system). The effector cells, which were designated as CEA peptide-pulsed dendritic cell-activated killer (CEA-PDAK) cells, were preferentially CD3+CD8+, and capable of killing T2 cells pulsed with CEA peptide but not T2 cells alone. The CA-PDAK cells also lysed the gastric cancer cell line KATO III (HLA-A0201, CEA (+)), but not the WiDr (HLA-A2402, CEA(+)) cells. The cytotoxicity was abrogated when the CEA-PDAK cells were treated with anti-TCR alpha beta antibody or when the target cells were treated with the anti-class I antibody prior to the cytotoxicity assay. The CEA-PDAK cells exerted their cytotoxic activity even in the presence of a high amount of CEA protein at the effector phase, which mimicked the clinical setting. The CEA-PDAK cells showed approximately a hundred-fold expansion in total cell numbers yielded without any loss of the specific lysis, when stimulated with the IL-2/CD3 system compared to those stimulated with IL-2 alone. The TCR V beta gene analysis for the CEA-PDAK cells, conducted by means of RT-PCR-Southern blotting, demonstrated oligoclonal expression of TCR beta 7 and 12, and the latter was shown to be responsible for the killing activity. SSCP analysis indicated the clonotype of the TCR V beta 12 gene, indicating a selective expansion of lymphocytes bearing a limited TCR variable region by the stimulation with CEA peptide-pulsed DCs. Taken together, the effector lymphocytes reactive with the CEA antigen can be generated from PBMCs with the antigenic CEA peptide and cultured DCs. The IL-2/CD3 system is effective and practical in activating the effector cells for the clinical use of CEA-PDAK cells. Adoptive immunotherapy using this system may be promising for treating CEA-expressing tumors.
Ohta
Koji
K
Department of Surgical Oncology, Research Institute for Radiation Biology and Medicine, Hiroshima University, Kasumi 1-2-3, Minami-ku, Hiroshima 734-8553, Japan.
Yamaguchi
Yoshiyuki
Y
Shimizu
Katsuhiko
K
Miyahara
Eiji
E
Toge
Tetsuya
T
D002272:Carcinoembryonic Antigen / Q000276:immunology*; D002478:Cells, Cultured; D003110:Colonic Neoplasms / Q000276:immunology; D003602:Cytotoxicity, Immunologic; D003713:Dendritic Cells / Q000166:cytology / Q000276:immunology*; D019674:Genes, T-Cell Receptor beta / Q000502:physiology; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000276:immunology / Q000494:pharmacology; D015234:HLA-A Antigens / Q000276:immunology; D015789:HLA-A2 Antigen / Q000276:immunology; D006801:Humans; D016233:Immunotherapy, Active / Q000379:methods; D015847:Interleukin-4 / Q000276:immunology / Q000494:pharmacology; D008213:Lymphocyte Activation / Q000276:immunology; D013274:Stomach Neoplasms / Q000276:immunology; D050378:T-Lymphocytes, Regulatory / Q000166:cytology / Q000276:immunology*; D014407:Tumor Cells, Cultured
D002272:Carcinoembryonic Antigen; D015234:HLA-A Antigens; C435939:HLA-A*02:01 antigen; D015789:HLA-A2 Antigen; D015847:Interleukin-4; D016178:Granulocyte-Macrophage Colony-Stimulating Factor