Bell, Sarah Elizabeth; (1991) The role of differentiation antigens in T cell ontogeny and immune function. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Using the technique of murine foetal thymus organ culture as an in vitro model of thymic ontogeny, the role of the differentiation antigens CD4 and CD5, during development of the T cell repertoire has been examined. The developing subpopulations have been phenotyped in this culture system, and the in vitro recolonisation potential of purified progenitor populations from mouse foetal liver investigated. Selective cell depletion by density gradient centrifugation, and anti-heat stable antigen (HSA) antibody plus complement treatment, or cell sorting, has enabled a progenitor population to be identified within the HS A-negative subset of foetal liver. Antisense oligodeoxynucleotides corresponding to the 5' region of the genes encoding CD4, or CD5 (a and p chains) have been used to specifically inhibit cell surface expression of these molecules in foetal thymus organ culture. The cellular uptake and stability of these oligonucleotides was assessed by 5' end labelling using [7P]-ATP, and analysis following various times of incubation with the labelled oligonucleotide. By incubation of foetal thymus lobes with a biotinylated oligonucleotide, and staining tissue sections with a streptavidin reagent, the oligonucletides were demonstrated to penetrate an intact thymus explant. The addition of CD4 antisense oligonucleotides appears to cause a specific decrease in cell surface expression of this molecule and the appearance of a population of CD4-CD8+ cells. The functional importance of the domains of CD5, CD4, and CD8 has been investigated by constructing chimaeric molecules comprising the extracellular, transmembrane, and cytoplasmic domains of CD4, or CD5, and the y chain of CD3. Hybrid constructs comprising the extracellular domain of CDS and the transmembrane and cytoplasmic domains of CD3y were successfully transfected into a T cell hybridoma. Although these molecules can be expressed, they appear to be retained within the cell. This is in agreement with recent studies which have identified a retention signal in the transmembrane domain of components of the TCR/CD8 complex, which targets individual components, particularly CDBy, for ER degradation.

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