TCR reconstitution in Jurkat reporter cells facilitates the identification of novel tumor antigens by cDNA expression cloning
| Autor: | Corlien A, Aarnoudse, Margreet, Krüse, Renate, Konopitzky, Nathalie, Brouwenstijn, Peter I, Schrier |
|---|---|
| Rok vydání: | 2002 |
| Předmět: |
DNA
Complementary Skin Neoplasms Receptors Antigen T-Cell alpha-beta T-Lymphocytes Gene Expression Epitopes Jurkat Cells Antigens Neoplasm Genes Reporter Transduction Genetic HLA-A2 Antigen Animals Humans Cloning Molecular Luciferases Melanoma Gene Library NFATC Transcription Factors Nuclear Proteins Peptide Fragments DNA-Binding Proteins Retroviridae COS Cells Epitope Mapping Plasmids T-Lymphocytes Cytotoxic Transcription Factors |
| Zdroj: | International journal of cancer. 99(1) |
| ISSN: | 0020-7136 |
| Popis: | The identification of novel tumor antigens is of extreme importance for effective immunotherapy against cancer. A major obstacle in this field is the limited life span of tumor-specific cytotoxic T lymphocytes (CTLs) in vitro. Therefore we searched for a method to isolate the tumor specificity of these CTLs, i.e., their T-cell receptors (TCRs) and transfer it to an immortalized T-cell line. For this purpose, a TCR-negative Jurkat T-cell line was equipped with a nuclear factor of activated T cells (NFAT)-luciferase reporter construct to allow measurement of TCR-mediated activation. To establish the feasibility of this tumor-specific TCR transduction, we cloned the TCR genes of a known T-cell clone specific for the tumor antigen CAMEL (CTL-recognized antigen on melanoma) into a retroviral construct. Jurkat reporter cells transduced with this construct, Jrt-TCRalpha3beta5, were tested for their reactivity against CAMEL-expressing melanoma cells, peptide-loaded T2 cells and CAMEL-transfected COS-1 cells. The melanoma cell lines were poorly recognized, but peptide-pulsed and -transfected cells effectively stimulated NFAT signaling. The activation of TCR(+) Jurkat reporter cells was shown to be dependent on the antigen density on the target cells and the expression level of the coreceptor CD8 on the Jurkat cells. To verify the benefit of this TCR reconstitution method for identification of novel antigens, pools of the cDNA library from which CAMEL was originally cloned were transfected in COS-1 cells and screened with Jrt-TCRalpha3beta5. Identical cDNA pools were found that were positive with these cells and with the CAMEL-specific CTL clone. Our results illustrate that TCR-reconstituted Jurkat reporter cells are a useful tool in the identification of novel tumor antigens by cDNA expression cloning. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|
Plný text