Isolation of Encephalitogenic CD4+ T Cell Clones in the Rat. Cloning Methodology and Interferon-gamma Secretion

In this report we detail a procedure for the cloning of a rat encephalitogenic T cell line and show that the methods normally employed for other species may not always be applicable. The two important differences to be described are, (i) that in these experiments where the parent T cell lines were generated with thymocytes as presenting cells, splenocytes were not suitable as a source of antigen-presenting or stimulator cells and (ii) semipurified forms of IL-2, specifically that derived from EL4 lymphoma cells, resulted in a much reduced cloning frequency and rate of T cell growth compared with cruder mixtures such as that derived from mitogen-stimulated splenocytes. Functional studies with clones derived from a strongly encephalitogenic (experimental autoimmune encephalomyelitis (EAE)-inducing) T cell line revealed that the clones had a reduced capacity to mediate EAE in recipient rats but were otherwise comparable to the parent line in terms of surface phenotype and fine antigen specificity. In an attempt to begin to identify the type of CD4+ T cells that may induce EAE we tested the clones and lines for secreted interferon-gamma by a sensitive ELISA, and showed that all clones secreted high levels of this factor.