Characterisation of a ligand for the costimulatory receptor CD28 in chickens
Characterisation of a ligand for the costimulatory receptor CD28 in chickens
The rapid development of a T cell response to invading pathogens determines the efficiency of an immune system for eliminating infection. For complete activation of T cells, two signals are required. The primary signal is provided by the antigen-MHC complex recognised by the TCR, while the costimulatory signal is provided by a T cell surface receptor, CD28, binding to its ligands on APCs.
To investigate the role of costimulation in avian species, it was necessary to clone the gene(s) for the ligands to CD28. The production of a fusion protein comprising the extracellular domain of CD28 and domains 3 and 4 of rat CD4 was incorrectly folded when analysed with the BIAcore system so could not be used to screen for CD28 ligands. The recognition of human CD86-Ig by COS cells tranfected with CD28 cDNA implied a cross-species reaction that led to the discovery that murine CTLA-4-Ig coupled to beads was able to recognise something expressed by a chicken macrophage cell line. A cDNA library from this cell line was screened with mCTLA-4-Ig and two clones were isolated, which subsequently bound to chicken CD28-Ig-coupled beads. Sequence analysis revealed these clones to be almost identical to each other, and homologous with mammalian CD28 ligand sequences, encoding an ORF of 297 amino acids. Alignments and phylogenetic analysis indicated that the isolated clones were the chicken homologue of CD80. Northern blot analysis detected one major transcript of 3.2kb which was expressed in HD11 cells and spleen and thymus tissue. Attempts were made to produce monoclonal antibodies by immunisation with transfected COS cells or plasmid DNA, but were unsuccessful. The detailed expression pattern and functional role of this ligand remains to be evaluated and could be undertaken with the availability of antibody reagents.
University of Southampton
1998
O'Regan, Michelle Nora
(1998)
Characterisation of a ligand for the costimulatory receptor CD28 in chickens.
University of Southampton, Doctoral Thesis.
Record type:
Thesis
(Doctoral)
Abstract
The rapid development of a T cell response to invading pathogens determines the efficiency of an immune system for eliminating infection. For complete activation of T cells, two signals are required. The primary signal is provided by the antigen-MHC complex recognised by the TCR, while the costimulatory signal is provided by a T cell surface receptor, CD28, binding to its ligands on APCs.
To investigate the role of costimulation in avian species, it was necessary to clone the gene(s) for the ligands to CD28. The production of a fusion protein comprising the extracellular domain of CD28 and domains 3 and 4 of rat CD4 was incorrectly folded when analysed with the BIAcore system so could not be used to screen for CD28 ligands. The recognition of human CD86-Ig by COS cells tranfected with CD28 cDNA implied a cross-species reaction that led to the discovery that murine CTLA-4-Ig coupled to beads was able to recognise something expressed by a chicken macrophage cell line. A cDNA library from this cell line was screened with mCTLA-4-Ig and two clones were isolated, which subsequently bound to chicken CD28-Ig-coupled beads. Sequence analysis revealed these clones to be almost identical to each other, and homologous with mammalian CD28 ligand sequences, encoding an ORF of 297 amino acids. Alignments and phylogenetic analysis indicated that the isolated clones were the chicken homologue of CD80. Northern blot analysis detected one major transcript of 3.2kb which was expressed in HD11 cells and spleen and thymus tissue. Attempts were made to produce monoclonal antibodies by immunisation with transfected COS cells or plasmid DNA, but were unsuccessful. The detailed expression pattern and functional role of this ligand remains to be evaluated and could be undertaken with the availability of antibody reagents.
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Published date: 1998
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Local EPrints ID: 463275
URI: http://eprints.soton.ac.uk/id/eprint/463275
PURE UUID: c82a8324-b434-44be-9234-a87bc3e9003e
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Date deposited: 04 Jul 2022 20:48
Last modified: 04 Jul 2022 20:48
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Author:
Michelle Nora O'Regan
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