McNeill, Louise; (1999) Functional analysis of the CD8[beta] polypeptide and its role in T cell differentiation. Doctoral thesis (Ph.D), UCL (University College London).

Preview

Text Functional_analysis_of_the_CD8.pdf Download (13MB) | Preview

Abstract

The CD8 molecule is a cell surface glycoprotein that is expressed predominantly on class I MHC-restricted T cells. CD8 is expressed as a disulphide-linked a heterodimer on the surface of thymocytes and peripheral T cells. Much of the work establishing the role of CD8 as a coreceptor has concentrated on CD8a, which interacts with the a3 region of MHC class I molecules and intracellularly with the protein tyrosine kinase p56lck. Recently an important role for the CD8β chain in thymic development and antigen responsiveness has been suggested. Previous biochemical analysis of the CD8β chain has shown that there is a decrease in the sialylation of O-linked sugars during T cell maturation and on T cell activation. The alteration in sialic acid levels is specific for the CD8β chain, and is predicted to occur within the connective peptide region, between the external and transmembrane domains. Glycosylation analysis of the CD8 complex from thymocytes and resting T cells show the O-glycans have a core 1 structure with sialic acid joined in the a2-6 linkage to galNAc. On activation the O-glycans appear to become hyposialylated and change their core structure, influenced by changes in the O-glycan biosynthetic pathway. To examine the role of the connecting peptide region of CD8β, transgenic CD8βa Hinge mice have been constructed with a hybrid molecule in which the connecting peptide region of CD8β has been exchanged with the corresponding region of CD8a. The transgenic mice were backcrossed onto the CD8β knockout to allow the role of the hybrid molecule to be studied in the absence of the endogenous CD8β molecule. The CD8βaH transgenic mice show a partial block in T cell differentiation subsequent to the double positive stage in the thymus which results in reduced CD8 T cell numbers in the periphery. This phenotype seems to mirror the phenotype of the CD8β knockout suggesting the hybrid molecule was unable to restore CD8β function in these mice. Biochemical analysis of the transgenic mice shows the hinge region of the CD8βaH molecule is more heavily glycosylated than either CD8β or CD8a. Two additional polypeptides, of 14kD and 26kD molecular weight, were shown to associate with the CD8 complex. The 14kD molecule is a novel polypeptide which associates with CD8 complex in thymocytes and resting T lymphocytes, but not activated T lymphocytes. The presence of the molecule was thought to be linked to the changes in glycosylation of CD8β, but was found to co-precipitate with the CD8 complex from CD8β-deficient mice, suggesting that it does not associate with CD8β. The 26kD molecule was found only within the cell and was not present in CD8β-deficient mice. It is thought that this novel polypeptide may be involved in the developmentally regulated transport of CD8, since in the absence of CD8β and p26 a block in the cellular transport of CD8 was found with a build up of partially glycosylated precursors held in the ER. These results suggest CD8[beta] has a role in T cell differentiation and that part of its function may be mediated through the glycosylated hinge region, which may have a unique role within the TCR/MHC complex.

Download activity - last month

Export as XMLCSVJSON

Download activity - last 12 months

Export as CSVXMLJSON

Downloads by country - last 12 months

Export as CSVXMLJSON

Archive Staff Only

View Item