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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online January 20, 2004 as doi:10.1096/fj.03-0881fje. |
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Gene Therapy Group, Department of Microbiology and Immunology, University of Arizona, Tucson, Arizona, USA
2Correspondence: Gene Therapy Group, Department of Microbiology and Immunology, University of Arizona, Tucson, AZ 85721, USA. E-mail: davidh{at}u.arizona.edu
SPECIFIC AIMS
Traditional methods for identifying T cell-recognized tumor (Ags) are laborious and time-consuming. In an attempt to simplify the procedure, a novel strategy, SING (signal transduction molecule-mediated, NFAT-controlled GFP expression), was established as a direct approach for cloning T cell-recognized tumor Ags.
PRINCIPAL FINDINGS
1. Engineered "artificial" antigen-presenting cell line BS-4 could express green fluorescent protein (GFP) after activation with phorber ester plus ionomycin
To establish the "SING" system, mouse lymphoma cell line BW5147 was cotransduced with two vectors: a nuclear factor of activated T cell (NFAT)-controlled GFP expression vector and a vector coding for a chimeric H-2Kb molecule with the cytoplasmic domains replaced with T cell-derived signaling domains. The resulting cells were termed BS cells. A BS clone, BS-4, obtained by limiting dilution has a low background of GFP expression without stimulation and high transgenic H-2Kb expression (Fig. 1
A, B). After overnight stimulation with phorber ester (PMA, 10 ng/mL) plus ionomycin (500 ng/mL), nearly 100% of BS-4 cells became GFP+.
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2. BS-4 cells could also express GFP in response to anti-H-2Kb mAb cross-linking
As shown in Fig. 1C
, 86% of BS-4 cells expressed GFP after stimulation with immobilized anti-H-2Kb mAb (4 µg/cm2).
3. GFP expression is shut down after withdrawal of stimuli
4. Either OVA peptide-pulsed or OVA gene-transduced BS-4 cells express GFP after engagement with OVA-specific T hybridoma cells
To assess the GFP response after engagement of the chimeric H-2Kb molecule, an OVA-specific, H-2Kb-restricted T cell hybridoma, B3Z, was used to stimulate OVA257-264 peptide (5x10-5 M)-pulsed BS-4 cells. As shown in Fig. 2
B, 24% of the total B3Z-BS-4 cell mixture {
44% of BS-4 cells: =0.24/(0.24+0.31)} became GFP+; whereas in control (H-2Kb binding) mTrp2 peptide-pulsed groups only background GFP expression was observed (Fig. 2A
), suggesting the functionality and specificity of the chimeric H-2Kb molecule. The response of peptide-pulsed BS-4 cells to TCR ligation was dose dependent (Fig. 2C
). The lowest OVA peptide concentration that stimulated BS-4 cells to express significant amounts of GFP was 10–7 M. The threshold OVA peptide concentration for GFP induction in BS-4 cells after H-2Kb engagement could be lowered to <10-9 M by introducing costimulatory molecule CD28 into BS-4 cells (Fig. 2D
). Under this condition, >10% OVA gene-transduced BS-4 cells become GFP+ after coculture with OVA-specific T cells (Fig. 2E, F
).
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5. CTL can stimulate BS-4 cells to express GFP in the presence of the granule exocytosis inhibitor concanamycin A
CONCLUSIONS AND SIGNIFICANCE
To simplify and expedite the traditional laborious procedures, we designed a novel approach that allowed tumor Ag-transduced target cells to express GFP after stimulation with Ag-specific TCR using an NFAT-based inducible expression vector. Reporter gene constructs (GFP, LacZ, and luciferase) driven by multiple copies of NFAT binding sites have been used as output signals to test the activation of the NFAT pathway downstream of the TCR. However, our approach is different from previous systems in that the activation of the NFAT pathway is mediated through the chimeric H-2Kb molecule rather than the TCR. Therefore, instead of repeatedly screening expression cDNA libraries using T cells, Ag-expressing target cells could be directly enriched and isolated based on inducible GFP expression after coculture with Ag-specific T cells.
In the studies reported here, we showed that the chimeric H-2Kb molecule could transduce signals after engagement with Ag-specific TCR in the presence of Ag peptides. These results provide proof of principle for using the "SING" system to directly isolate Ag-expressing BS-4 cells from BS-4 cell repertoires that are retrovirally transduced with tumor-derived cDNA libraries. The "SING" system also provides an alternative way to measure the interaction between T cell and APCs. Unlike methods such as ELISA (detection of cytokines), ELISPOT and intracellular cytokine staining, no expensive reagents and lengthy procedures are required in the "SING" system.
The diagram in Fig. 3
shows the overall procedures using the "SING" system to clone T cell-recognized tumor antigens. First, a tumor-derived retroviral cDNA library (cDNAs are inserted into a retroviral vector) is made. Second, the cDNA library is transferred to BS-4 cells via retroviral transduction. Third, tumor-specific T cells (noncytotoxic) are cocultured with cDNA-transduced BS-4 cells (BS-4/cDNAs). Fourth, GFP+ BS-4 cells are isolated by FACS sorting and/or other selective methods. Fifth, cDNAs coding for T cell-recognized antigens are retrieved by PCR using vector-specific primers and sequenced. The cloned tumor cDNAs are then confirmed by the ability to activate antigen-specific T cells after being reintroduced to APCs.
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FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-0881fje; 
This article has been cited by other articles:
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  T. Zhang, A. Barber, and C. L. Sentman Generation of Antitumor Responses by Genetic Modification of Primary Human T Cells with a Chimeric NKG2D Receptor Cancer Res., June 1, 2006; 66(11): 5927 - 5933. [Abstract] [Full Text] [PDF]
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mAb, and subjected to 2-color FACS analysis. Sensitivity of the SING system after H-2Kb engagement was determined by peptide titration (C, D). BS-4 cells were pulsed with either OVA (•) or control Trp2 peptides (
) ranging from 10-10 to 10-4 M, then cocultured with B3Z cells at a ratio of 1:5 for 24 h (C). The percentage of GFP+ BS-4 cells was calculated as (%GFP+/%CD8–) x100. In an improved SING system, CD28 gene-transduced BS-4 (BS-4/CD28) and B7.1 gene-transduced B3Z (B3Z/B7.1) cell were used (D). Data represent the mean ± SD of 3 independent experiments. GFP expression by OVA-expressing BS-4 cells after H-2Kb engagement was determined by 2-color FACS analysis (E, F). CD28-transduced (E) or CD28+OVA doubly transduced BS-4 (F) cells were cocultured with B7.1+ B3Z T cells at a ratio of 1:5. After 24 h, cells were stained with PE anti-CD8 mAb. The result is representative of 8 independent and similar experiments.