Phosphorylation of a Mr 70,000 protein is associated with interleukin 2 receptor expression.

The human T cell hybrid II23 was isolated from fusions between human peripheral blood lymphocytes which had been stimulated with phytohemagglutinin (PHA) and a subline of the human T cell line CEM called CEM.TET1. This hybrid does not constitutively express detectable levels of interleukin 2 (IL 2) receptors but can be induced to express receptors by stimuli shown to activate T cells. Antibody to CD3 (a component of the T cell receptor) coupled to agarose or PHA (greater than 3 micrograms/ml) induced both IL 2 production and receptor expression on II23 cells. Phorbol 12-myristate 13-acetate (PMA) induced IL 2 receptor expression on II23 cells but not IL 2 secretion. Because PMA is a known activator of the Ca2+/phospholipid-dependent enzyme protein kinase C, proteins of stimulated and unstimulated cells were analyzed by two-dimensional gel electrophoresis for changes in phosphoprotein patterns. A Mr 70,000 protein with a pI of 6.2 was phosphorylated in hybrids stimulated by PMA, anti-CD3 antibody coupled to agarose or PHA, i.e. by the same stimuli which induce IL 2 receptors on these cells. The immunosuppressive drug cyclosporin A inhibited IL 2 release without altering induction of IL 2 receptors or phosphorylation of the Mr 70,000 protein. The 70-kDa protein was located in the cytosol, where it remained phosphorylated for at least 4 h after stimulation. A protein with the same migratory properties on two-dimensional gels was similarly phosphorylated after stimulation of normal peripheral blood T lymphocytes, indicating that the phenomenon was not due to hybridization or transformation. This 70-kDA protein may therefore be involved in the pathway which leads to the transcription and expression of IL 2 receptors.

Preferential generation of human T cell hybrids with a tetraploid fusion partner.

The factors determining successful derivation of human T lymphocyte hybrids are largely unknown. This report describes diploid and tetraploid clones of the T cell line CEM which were fused with either a human T cell line (Jurkat) or with peripheral blood lymphocytes (PBL). Fusions of all CEMR clones with the Jurkat cell line yielded hybrids at a very high frequency (1 X 10(-4)). Fusion of diploid clones of CEM with PBL yielded no hybrids, whereas fusion of tetraploid clones of CEM with PBL resulted in growth frequencies of 1 to 3 X 10(-6). Enumeration of hybrids immediately after fusion indicated that in all cases, fused cells represented 5 to 10% of the population. That the ability to yield viable hybrids after fusion was a characteristic of tetraploid cells was indicated by the finding that tetraploid variants of a diploid clone could also yield viable hybrids after fusion. Possible mechanisms for the difference in results generated with diploid and tetraploid cells, and characteristics of the hybrid cells generated, are also discussed.