Ontogeny, thymus dependence and in vitro stimulation of lymphocyte subsets in Xenopus

Abstract

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.