Analysis of T cell receptors reactive with squamous cell carcinoma antigen SART-1 presented by the HLA-A24 molecule.
We investigated the induction of cytotoxic T lymphocytes (CTLs) specific for squamous cell carcinoma antigen SART-1 from the peripheral blood mononuclear cells (PBMCs) of an HLA-A24 healthy donor by in vitro stimulation with dendritic cells (DCs) pulsed with the antigenic peptide. Effector cells, designated as <peptide-pulsed dendritic cell-activated killer (PDAK)> cells, exhibited potent cytotoxic activity against target cells in a SART-1-specific and HLA-restricted manner. T cell receptor (TCR) gene usage of the SART-1 PDAK cells analyzed by reverse transcribed-polymerase chain reaction (RT-PCR) and Southern blot analysis showed oligoclonal usage, including TCRVbeta18, which was indicated by blocking assay to be responsible for antigen recognition and killing. Single strand conformation polymorphism (SSCP) analysis revealed clonotypic bands of the TCRVbeta18. The PCR product of the TCRVbeta18 was sequenced and complementarity determining region (CDR) 3 was determined. RT-PCR analysis using this CDR3 sequence as a primer showed, as expected, a positive band of about 30 bp smaller than shown on regular PCR analysis. This clonotypic PCR demonstrated an increasing density of PCR bands in the SART-1 PDAK cells during stimulation with SART-1 peptide plus DCs. It is suggested that clonotypic PCR using the TCR-CDR3 sequence may be useful in assessing the precursor frequency of CTLs reactive with the SART-1 peptide in vivo.
Shimizu
Katsuhiko
K
Department of Surgical Oncology, Research Institute for Radiation Biology and Medicine, Hiroshima University, Minami-ku, Hiroshima 734-8553, Japan.
Yamaguchi
Yoshiyuki
Y
Ohta
Koji
K
Miyahara
Eiji
E
Toge
Tetsuya
T
D000951:Antigens, Neoplasm / Q000378:metabolism*; D015139:Blotting, Southern; D002294:Carcinoma, Squamous Cell / Q000378:metabolism; D003713:Dendritic Cells / Q000378:metabolism; D015234:HLA-A Antigens / Q000096:biosynthesis*; D059871:HLA-A24 Antigen; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000378:metabolism*; D009363:Neoplasm Proteins / Q000378:metabolism*; D010641:Phenotype; D016133:Polymerase Chain Reaction; D018807:Polymorphism, Single-Stranded Conformational; D012333:RNA, Messenger / Q000378:metabolism; D011948:Receptors, Antigen, T-Cell / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017411:Ribonucleoproteins, Small Nuclear; D013601:T-Lymphocytes / Q000378:metabolism*; D013997:Time Factors
D000951:Antigens, Neoplasm; D015234:HLA-A Antigens; D059871:HLA-A24 Antigen; D009363:Neoplasm Proteins; D012333:RNA, Messenger; D011948:Receptors, Antigen, T-Cell; D017411:Ribonucleoproteins, Small Nuclear; C431323:SART1 protein, human