Production of a Rat T Cell Hybridoma That Stably Expresses the T Cell Differentiation Marker RT6.2

The rat T cell alloantigen RT6.2 shows a slow rate of synthesis in isolated T cells which hampers studies on the metabolism of RT6.2 in these cells (1). In order to facilitate further molecular and functional characterization of this molecule we have established a T-T hybridoma cell line which stably expresses RT6.2. Only 1 of 102 T cell hybridomas obtained upon fusion of the rat thymoma C58NT with DA rat lymph node cells expressed this antigen. This clone - EpD3 initially showed a relatively slow rate of cell division and a highly unstable pattern of expression of RT6.2. Thus, the relative number of RT6.2 bearing cells consistently decreased during cultivation of EpD3 and of EpD3-derived subclones. Cell separation studies suggest that the switch in RT6.2 phenotype of EpD3 cultures was due to the appearance of variant cells of RT6.2- phenotype with a higher proliferative capacity and was not induced by factors in the culture medium. By repeated panning and subcloning procedures to select for RT6.2 expressing cells, a subline of EpD3 - EpD3/87 - was obtained which stably expresses high levels of RT6.2. Metabolic labeling studies of RT6.2 in EpD3 show that it is synthesized very efficiently in these cells and support our previous suggestion that RT6.2 does not bear any classic oligosaccharide side chains. Moreover, RT6.2 can be released almost completely from EpD3 cells by phosphatidylinositol-specific phospholipase C (PI-PLC).