Functional studies of cytokines produced by Xenopus Lymphocytes following T cell mitogen stimulation

Abstract

In vitro culture conditions for the production and assay of T cell mitogen - induced culture supernatants have been investigated in an amphibian model system, the clawed toad, Xenopus. The thesis has paid particular attention to probing the ability of such supernatants ( and T cell mitogen ) to activate lymphocytes from early ( 7 day ) - thymectomized ( Tx ) animals, in order to investigate possible thymus-independent' T - like' cell emergence. Supernatants from 24-hour-cultures of concanavalin A ( ConA )-treated Xenopus splenocytes were first generated. Following neutralization of ConA by methyl- mannopyranoside, active supematants ( ASNs ) were shown to induce proliferation of freshly-prepared adult splenocytes ( supposedly unstimulated ) from control Xenopus equally as well as in vitro pre-stimulated cells. ASNs were also able to enhance tritiated thymidine ((^3)HTdR) incorporation by larval thymocytes which had been ' co-stimulated ' with a sub-optimal concentration of phytohaemagglutinin. Elevated (^3)HTdR incorporation was also observed when such supematants were tested on splenocytes from Tx toadlets. Purification of Xenopus ASN was not attempted here, but such material has been shown elsewhere to contain cytokines such as interleukin-2 ( IL-2 ). Human recombinant IL-2 proved unable to co-stimulate larval thymocytes. The use of a panel of monoclonal antibodies investigated the possibility that T cell markers might be up-regulated on control and Tx splenocytes following in vitro incubation with ASN, or after in vivo injection with such ' cytokine ' - rich material. These flow cytometric studies on animals of various developmental stages failed toreveal experimentally-inducible expression of the T cell specific markers XTLA-1and the putative CDS marker. [ Additional studies illustrated that cultured control splenocytes display blastogenesis and increased expression of major histocompatibility complex class H molecules, following ConA stimulation. ] Flow cytometry interestingly revealed the natural, in vivo, emergence of a low percentage of XTLA-1 and CDS - expressing splenocytes removed from thymectomized Xenopus aged 6 months and older. Such presence of a few ' T - like ' cells in Tx animals of this age ( but not in younger toads ) was confuted functionally through (^3)HTdR studies on these splenocytes following ConA stimulation.