In Vitro Induction of Carcinoembryonic Antigen (CEA) Specific Cytotoxic T Lymphocytes Using Dendritic Cells Pulsed with CEA Peptide.
- Author:
Min Ho PARK
1
;
Chang Seok LEE
;
Mun Hyeong CHO
;
Chul HAN
;
Yang Seok KOH
;
Jung Chul KIM
;
Jung Han YOON
;
Young Jong JEGAL
;
Jong Hee NAM
Author Information
1. Department of Surgery, Chonnam National University Medical School, Gwangju, Korea. jhyoon@jnu.ac.kr
- Publication Type:In Vitro ; Original Article
- Keywords:
Dendritic cells;
Carcinoembryonic antigen;
Specific cytotoxic T lymphocytes;
Cancer immunotherapy
- MeSH:
Carcinoembryonic Antigen*;
Cell Line;
Dendritic Cells*;
Enzyme-Linked Immunospot Assay;
Flow Cytometry;
Foundations;
Granulocyte-Macrophage Colony-Stimulating Factor;
HLA-DR Antigens;
Humans;
Immunotherapy;
Immunotherapy, Active;
Interleukin-4;
Lymphocytes;
Monocytes;
Phenotype;
T-Lymphocytes;
T-Lymphocytes, Cytotoxic*;
Tissue Donors
- From:Journal of the Korean Surgical Society
2005;69(5):359-366
- CountryRepublic of Korea
- Language:Korean
-
Abstract:
PURPOSE: Dendritic cells (DCs) are the most potent antigen- presenting cells for initiating the T cell immune response in vivo. Recent studies have shown that active immunotherapy with tumor antigen pulsed DC tumor antigen specific cytotoxic T lymphocyte (CTL) response. The aim of this study was to establish clinically compatible procedures for generating human DCs and to determine if the CEA peptide- pulsed DCs can activate the CEA specific CTL responses in vitro. METHODS: DCs were generated from the peripheral blood monocytes (PBMCs) of HLA A2+ healthy donors using GM-CSF and IL-4. Phenotypic analysis was performed using flow cytometry with FITC- or PE-conjugated Abs against CD1a, CD14, CD80, HLA-DR, CD83 and CD86. The immature DCs were pulsed with a CEA peptide (HLA A2 epitope, [YLSGANLNL]) and the tumor lysates isolated from HLA A2+ CEA positive cell line, NCI-H498, and were incubated with the autologous PBMCs in order to generate an antigen specific CTLs in vitro. After three rounds of stimulation, the presence of a CEA-specific CTL response was determined using a CEA positive cell line as the specific targets with the standard 51Cr release assay, the ELISPOT assay, and the flow cytometry using CEA peptide-MHC tetramer. RESULTS: The DCs obtained after 6 days of culture expressed high levels of CD1a, HLA-DR, and CD80, which corresponded to the immature DC phenotype. The 51Cr- release assay showed that DCs pulsed with the CEA peptide or the lysates of the CEA-positive NCI-H498 cell line could stimulate the CEA-specific CTL responses. The CTL response to DCs pulsed with the CEA peptide was also generated using the DCs pulsed with the CEA peptide. In the ELISPOT assay, the number of CEA peptide-specific, INF-gamma-secreting spots were increased in the CTLs generated by DCs pulsed with the CEA pepide and the tumor lysates. In the peptide-MHC tetramer assay, the CD8+ T cells with the receptors specific to CEA-peptide were increased by stimulation with the DCs pulsed with the CEA peptide and the tumor lysates. CONCLUSION: These findings show that the CEA peptide pulsed DCs can generate CEA specific CTL responses and antigen bearing DCs can be used as the target cells for a cytotoxicity assay. This study provides the foundations for DC-based cancer immunotherapy for CEA expressing solid tumors.
