In vitro studies on T cell proliferation and Lymphokine factor production in the amphibian, Xenopus

Abstract

The major aim of this Thesis has been to procure and begin to characterise T cell-derived cytokines in the clawed toad, Xenopus. Recent reports have suggested that Xenopus lymphocytes, stimulated in vitro with T cell mitogens, will generate factors that achieve the enhanced proliferation and growth of assay T lymphoblasts, but not unstimulated cells; these factors have been called 'T cell growth factors' and likened to mammalian interleukin-2. In this Thesis the nature of factors released in culture supernatants (SNs) by alloantigen- and mitogen-stimulation of Xenopus leucocytes is re-examined and it is shown that cells other than T lymphoblasts and even non-T cells are responsive to such T cell-derived ‘lymphokines’. Chapter 2 revealed that SNs collected from 48 hour cocultures of splenocytes from MHC (major histocompatibility complex)-disparate Xenopus were able to achieve enhanced proliferation not only of PHA-activated splenic- lymphoid -cells, but also of -unstimulated' splenocytes. Thymic 'blasts', but not 'unstimulated' thymocytes, were also responsive to these mixed leucocyte culture (MLC)-induced factors. In Chapter 3, to further investigate lymphokine production, splenocytes were stimulated with the T cell mitogens PHA (phytohaemagglutinin) and Con A (Concanavalin A) and the activity of the culture SNs then examined after mitogen removal. SNs taken at 24 hours achieved good proliferation of both 'unstimulated' splenocytes and splenic blasts. In Chapter 4, miniaturisation of the SN screening assay was successfully achieved, using only 1.5 x 10(^4) leucocytes in a 'hanging drop' culture, in order to minimise the amount of lymphokine required in an assay, and to allow experiments on few assay lymphocytes. It was shown that 'unstimulated' splenocytes from early- thyraectomised Xenopus responded by proliferation to active supernatants (ASNs) (MLC-, PHA- or Con A-generated), indicating that a cell type other than a T cell could be induced to proliferate in the presence of ASN. Thymectomy experiments also indicated that T cells were necessary for the generation of active supernatants in vitro. The identity of the thymus-independent cells responding to ASNs was further explored in Chapter 5. Using an anti-IgM monoclonal antibody, B cells from early- thymectomised Xenopus were separated from the rest of the splenocyte population by flow cytometry. Surface IgM(^+)cells (B cells) responded mildly to ASNs, whereas the sIgM- population (and unsorted cells) responded well to both the PHA-ASN and the MLC-ASN. Work carried out at the beginning of this Ph.D., that identified splenic antigen-presenting cells and (inconclusively) explored the role of these cells as stimulators in MLC responses in Xenopus, is reported in Chapter 6. The main conclusions to be drawn from this research are briefly discussed in Chapter 7 and suggestions for future work considered.