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Type: Journal article
Title: Ovine dendritic cells transduced with an adenoviral CTLA4eEGPF fusion protein construct induce hyporesponsiveness to allostimulation
Author: Newland, A.
Kireta, S.
Russ, G.
Krishnan, R.
Citation: Immunology, 2004; 113(3):310-317
Publisher: Blackwell Publishing Ltd
Issue Date: 2004
ISSN: 0019-2805
Abstract: CTLA4 (CD152) is a transmembrane molecule expressed on activated T cells and functions as a negative regulator of T cell activation upon binding to the costimulatory molecules CD80/86. In this study, CTLA4eEGFP constructs were engineered by cloning the extracellular domains of ovine and human CTLA4 (CTLA4e) 'in frame' with the enhanced green fluorescent protein (EGFP). Recombinant adenoviral vectors were generated by incorporation of the CTLA4eEGFP sequence into the adenoviral genome using homologous recombination in Esherichia coli. The functional activity of the adenoviral vectors was shown by the secretion of the CTLA4eEGFP upon infection of ovine fibroblasts and the binding of the fusion protein to the target ovine and human dendritic cells expressing CD80/86 receptors by flow cytometry. The EGFP tag facilitated molecular size determinations and quantification of the secreted ovine CTLA4 fusion protein by immunoprecipitation and enzyme-linked immunosorbent assay (ELISA), respectively, using anti-GFP mAbs. Ovine dendritic cells obtained from pseudoafferent lymphatic cannulation of sheep were characterized based on high major histocompatibility complex (MHC) class II expression and cross-reactivity with monoclonal antibodies to the human dendritic cell markers, CD83 and CMRF-56. In addition, ovine dendritic cells (DC) were transfected with the adenoviral CTLA4eEGFP and when used as stimulators in a mixed lymphocyte reaction showed a reduced capacity to induce allogeneic lymphocyte proliferation. This study verifies that the ovine CTLA4eEGFP fusion protein functions similarly to its human homologue and that DC modified with adenoviral CTLA4-EGFP may provide an effective therapeutic approach in targeting alloreactive T cells to prolong allograft acceptance in a preclinical ovine model of renal transplantation.
Keywords: Dendritic Cells
CHO Cells
Sheep, Domestic
Green Fluorescent Proteins
Recombinant Fusion Proteins
Antigens, Differentiation
Antigens, CD
Lymphocyte Culture Test, Mixed
Transduction, Genetic
Lymphocyte Activation
Immune Tolerance
Genetic Vectors
CTLA-4 Antigen
DOI: 10.1111/j.1365-2567.2004.01966.x
Appears in Collections:Aurora harvest
Medicine publications

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