Williams, O.; (1991) Functional studies on the murine T cell accessory molecule CD8. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

The aims of the present study were to examine CD8 function by observing the effect of blocking CD8 expression and by looking at CD8 phosphorylation. Attempts were made to down-regulate expression of murine CD8 at both the mRNA and protein levels. The first tactic involved anti-sense RNA, produced by plasmid constructs. These were made using fragments of the CD8 cDNA and genomic clones, inserted into an expression vector in the 3' to 5' orientation. The second strategy involved the use of viral retention signals. Hybrids were made between mutant coronavirus El proteins and the membrane external domain of the CD8(alpha). The aim was for these hybrids to interfere with native CD8 intracellular transport. Anti-sense and dominant negative mutant constructs were transfected into CD8+ murine cell lines. Although the cells were shown to be transfected with the appropriate DNA constructs, no reduction in the surface expression of CD8 was observed and no anti-sense or dominant negative mutant RNA was detected. Possible reasons for this are discussed. CD8 phosphorylation was studied in order to address the functions of the murine CD8 molecule, especially with regard to its potential signalling roles. CD8 is phosphorylated on cytoplasmic serine residues during T cell activation. Site- directed mutagenesis was used to produce CD8 molecules lacking either or both of the two cytoplasmic serine residues, at positions 195 and 216. Mutation of serine residue 216 completely abolished CD8 phosphorylation in response to PMA. However, abolition of CD8 phosphorylation did not affect the ability of CD8 to restore the antigen responsiveness of a CD8 hybridoma, as measured in several different ways.

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