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The analysis of costimulatory receptor signaling cascades in normal T lymphocytes using in vitro gene transfer and reporter gene analysis

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Abstract

Ligation of the antigen receptor and costimulatory receptors on the surface of T lymphocytes initiates intracellular signals that regulate cell-cycle progression and cell differentiation. To effectively manipulate the activation of T cells for immunotherapeutic applications, it will be important to understand how these signaling pathways are integrated to control specific gene transcription events. Here we describe a novel transient transfection procedure that efficiently introduces DNA into non-dividing normal human and murine T lymphocytes while maintaining high cell viability. Using this technique, reporter genes can be introduced to characterize intracellular signaling pathways that regulate specific gene transcription events in normal T-lymphocyte populations. We show that the CD28 receptor can be differentially coupled to downstream signaling pathways in different T-lymphocyte populations. In addition, we demonstrate that a gene encoding a tagged constitutively active mitogen-activated kinase kinase-1 protein can be transfected and rapidly expressed to regulate the expression of Bcl-2 in normal thymocytes.

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Figure 1: Electroporation of GFP expression vectors produces high levels of GFP expression in murine DP thymocytes and human Jurkat T cells.
Figure 2: Transient expression of FLAG–MEKca increases the expression of Bcl-2 in DP thymocytes.
Figure 3: NF-κB–luc reporter gene analysis in Jurkat T cells, murine DP thymocytes and preactivated DO11.10 T cells shows CD28 receptors are differentially coupled to the NF-κB signaling pathway.

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Acknowledgements

This work was supported by funding from the American Cancer Society and the Mayo Foundation.

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Authors and Affiliations

  1. Department of Immunology, Mayo Clinic, Rochester, Minnesota, USA

    Michael P. Bell, Catherine J. Huntoon, Daniel Graham & David J. McKean

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  1. Michael P. Bell
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  2. Catherine J. Huntoon
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  3. Daniel Graham
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  4. David J. McKean
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Correspondence to David J. McKean.

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Bell, M., Huntoon, C., Graham, D. et al. The analysis of costimulatory receptor signaling cascades in normal T lymphocytes using in vitro gene transfer and reporter gene analysis. Nat Med 7, 1155–1158 (2001). https://doi.org/10.1038/nm1001-1155

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