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Durham e-Theses
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Ontogeny, thymus dependence and in vitro stimulation of lymphocyte subsets in Xenopus

Gravenor, Ian (1996) Ontogeny, thymus dependence and in vitro stimulation of lymphocyte subsets in Xenopus. Doctoral thesis, Durham University.



This Thesis investigates the possibility of an extrathymic pathway of T cell development in the amphibian, Xenopus laevis. Initial studies examined the ontogenetic development of T cell surface antigen expression on both splenocytes and thymocytes in larvae at 7 days through to 6-8 month old adults, using the technique of dual-colour flow cytometry and employing a panel of T and B cell specific monoclonal antibodies. Flow cytometric analysis of splenocytes from thymectomized (Tx) Xenopus was then addressed. This revealed that early (5-7 day) larval thymectomy resulted in the ablation of T cell surface antigen expression, as defined by the monoclonal antibodies 2B1 (anti- CD5), AM22 and F17 (both anti-CD8), XT-1 (anti-XTLA-1) and D4.3 (putative anti-aβ T cell receptor). Lack of these markers was still evident in 8 month old Tx frogs, confirming the effectiveness of the operation. In vitro studies showed that no T cell marker expression could be induced on the surface of splenocytes from Tx animals following stimulation with concanavalin A (ConA), phytohaemagglutinin (PHA) or the potent mitogenic agent, phorbol myristate acetate (PMA). Studies were also carried out to investigate whether in vitro stimulation induced apoptosis. Flow cytometric studies revealed that CD5(^dull) expression could be induced on splenocytes from control Xenopus following stimulation with PMA. The nature of this induced CD5(^dull) expression was investigated further in order to determine why this phenomenon was only seen in control animals. These experiments involving mixing of T and B cell populations, revealed that CD5(^dull) expression was being induced upon the surface of Xenopus B cells, and that this PMA-induced expression required the presence of T cells, and was blocked by a protein kinase C (PKC) inhibitor. Finally, an additional search for extrathymic T cells involved examining the intestine and liver of both control and Tx Xenopus, these tissues being sites of extrathymic T cell development in higher vertebrates. The intestine of control Xenopus was shown to contain T lymphocytes with a surface phenotype distinct to that found in spleen or liver. Studies in Tx Xenopus showed that although expression of some T cell markers was ablated in liver and gut, CD5(^dull) and CD8(^dull) (as determined by the mAb AM22) lymphocytes persisted in these organs. However, proliferative studies showed that these 'T-like' cells were unable to respond to mitogenic stimulation with ConA, suggesting that they are not functional T cells.

Item Type:Thesis (Doctoral)
Award:Doctor of Philosophy
Thesis Date:1996
Copyright:Copyright of this thesis is held by the author
Deposited On:24 Oct 2012 15:06

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