Volkman, Ariane; (1996) The influence of dendritic cells on tolerance induction to circulating and extrathymic proteins. Doctoral thesis (Ph.D.), University College London.

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Abstract

This thesis examines the role of antigen presentation in negative selection of MHC class II restricted T cells specific for the complement component C5. C5 is an extracellular self protein, which only reaches the thymus via the blood circulation. Thymic antigen presenting cells were introduced into fetal thymic reaggregation culture with thymocytes from C5 specific T cell receptor transgenic mice, to follow the development of T cells in the presence or absence of self antigen presented by different antigen presenting cells. In order to mimic the physiological distribution of C5 peptide/MHC class II complexes on the antigen presenting cells as closely as possible, they were isolated from thymi of C5 sufficient mice, so that the amount of C5 peptide bound to MHC class II on their surface would reflect the amount of self antigen they have access to and process normally in vivo. The results showed that not only thymic dendritic cells, but also cortical and medullary epithelial cells were able to induce negative selection of C5 specific thymocytes with similar efficiency. In contrast, thymic macrophages were unable to influence the development of C5 specific T cells, which suggests that the main function of macrophages in the thymus is the disposal of dying thymocytes. In the second part, a conditionally immortalized dendritic cell line (tsDC) was established from bone marrow of mice transgenic for a temperature sensitive mutant of the simian virus 40 large T antigen under the control of the MHC class I (Kb) promoter. At the permissive temperature of 33–37°C tsDC divide in the absence of growth factors. They share a number of cell surface markers with bone marrow macrophages, but unlike macrophages constitutively express MHC class II, are negative for non-specific esterase and are unable to phagocytose sheep red blood cells. TsDC show characteristic dendrites, an abundance of acidic vesicles and are highly active in endocytosis. If maintained at 33°C, tsDC process and present exogenous protein to MHC class II restricted T cell hybridomas and act as potent mixed leukocyte reaction stimulators, but fail to activate naive T cells. Transfer to 39°C arrests growth and results in upregulation of surface markers such as B7.1, CD40 and ICAM-1. Further upregulation of cell surface markers and acquisition of functional maturity occur following contact with T cells and their cognate antigen or in culture with a cytokine mixture derived from activated T cells.

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