TCR reconstitution in Jurkat reporter cells facilitates the identification of novel tumor antigens by cDNA expression cloning

Abstract

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 tumorspecific 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 TCRnegative Jurkat T-cell line was equipped with a nuclear factor of activated T cells ( Key words: T-cell receptor; tumor antigen; NFAT; CAMEL; cDNA expression cloning; CD8; luciferase; retrovirus; JurkatSince the characterization of the first human tumor-specific antigen in 1991, MAGE-1, 1 a considerable number of other targets for tumor-specific cytotoxic T cells (CTLs) have been identified. Most of these antigens have been cloned from melanoma cells 2 and less frequently from other types of cancer. 3,4 For the development of effective immunotherapy against cancer, the identification of a larger variety of tumor-associated antigens is of great importance, to broaden the range of different tumors that can be treated as well as to prevent the outgrowth of antigen-loss tumor cells after vaccination.Several strategies have led to the identification of tumor-specific T-cell epitopes. Most frequently, cDNA expression cloning has been employed, in which the gene encoding the antigen is identified by transfecting pools of tumor-derived cDNA into recipient cells, followed by screening with the tumor-specific CTL. [5][6][7][8] In another approach, the peptide epitope for the tumor-specific CTL is identified, either by peptide elution and mass spectrometry 9 or by mimicry from designated peptide libraries, 10 although with the latter method no tumor antigens have been identified yet. Several tumor antigens have been found by the method of "reverse immunology." These antigens were initially cloned on the basis of their differential expression in tumors versus healthy tissues and antigenic peptides were subsequently identified by performing in vitro T-cell inductions. 11,12 For efficient elucidation of novel physiologically relevant tumor-specific antigens, large quantities of tumor-specific CTLs are required to allow for multiple screening rounds. In practice, the limited amount of CTLs available often constitutes the major obstacle for the cloning of new tumor antigens. We have sought to circumvent this issue by cloning and transferring the T-cell receptor (TCR) of a tumor-specific T cell to an immortalized T-cell line. In this way, an unlimited source of tumor-specific T cells can be obtained, which facilitates the identification of novel tumor antigens. The feasibility of TCR cloning and transfer has already been demonstrated by others. [13][14][15][16][17] We studied the properties of a TCR, isolated from a characterized CAMEL (CTL-recognized antigen on melanoma)-specific CTL clone, 8 which was transferred into a TCR-negative human leukemia Jurkat T-cell line. To detect TCRmediated activation, the cells were tr...

A robust and scalable TCR-based reporter cell assay to measure HIV-1 Nef-mediated T cell immune evasion

“…Previous studies have reported that heterologous expression of TCR in a Jurkat-derived CD8 + NFAT-luciferase reporter T cell line (Jurkat/ MA) (Aarnoudse et al, 2002) conferred antigen-specific reactivity (Birkholz et al, 2009;Schaft et al, 2006). However, in our hands, TCRmediated luciferase expression in the Jurkat/MA cell line was consistently low, yielding signals less than 5-fold above negative controls.…”

A Screening Assay to Identify Agents That Enhance T-Cell Recognition of Human Melanomas

“…The TCR-minus cell line J.RT3-T3.5 [46][47][48] was infected with lentiviral particles containing the Melan-A/MART-1 specific TCR as previously described. 27 For transduction of the Melan-A/MART-1 specific TCR, 1·10 5 J.RT3-T3.5 cells were incubated with lentiviral vector at a MOI of 10.…”

T cell receptor-transgenic primary T cells as a tool for discovery of leukaemia-associated antigens

“…Notably, TCR-transfer on primary donor T cells offers a number of advantages compared to TCR reconstitution on Jurkat cells proposed by Aarnoudse et al [15]. First, TCRtransgenic T cells are fully functional in terms of cytokine production, permitting the use of ELISA-based screening assays with their high throughput potential, sensitivity and specificity.…”

“…First, TCRtransgenic T cells are fully functional in terms of cytokine production, permitting the use of ELISA-based screening assays with their high throughput potential, sensitivity and specificity. Moreover, use of Jurkat cells transfected with the NFATluciferase reporter construct leads to the increased risk of false-negative results due to the high background of the assay [15]. Next, the greater sensitivity of TCR-transgenic primary T cells is evident from the fact that Jurkat cells recognized melanoma cell lines only after enhancement of the antigen amount on target cells or transduction of Jurkat cells with CD8α.…”