Human cell lines secreting lymphokines. I. Establishment of T-T hybridomas.

Lymphokines (Lks) are conventionally produced by mitogen stimulation of T lymphocytes. However, this procedure results in a mixture of factors, some of which may have antagonistic effects. An alternative method of producing Lks is the construction of monoclonal T-T hybridomas which secrete distinct Lks. By adapting the hybridoma technology for T cells, one can select distinct T hybridomas which may serve as constant sources for the production of uniform and well defined Lks. Since the compatibility of a certain cell line to serve as a fusion partner is unpredictable, several lines were tried in the hybridization procedure. An obligatory requirement of such a mutant line is its sensitivity to a selective medium, in which only the hybrid cell would survive. For this purpose three 8-azaguanine (8AG) resistant mutant lines (Jurkat/12 CEM/14 and Molt-4/10) were established from the respective T leukemic cell lines. This was achieved by culturing the cells in the presence of 200 microM 8AG. The surviving resistant cells were sensitive to aminopterin and azaserine inhibitors. Depending on the cell line and the inhibitor, death of these mutants was complete in 7 to 14 days. Non-adherent peripheral blood lymphocytes (PBL) stimulated for 48 hours with 1 microgram/ml phytohemagglutinin (PHA), were fused with the CEM/14 line. Fifteen hybridomas secreted a substance with B cell growth factor (BCGF) activity, nineteen hybridomas secreted T cell growth factor (IL-2), and eight hybridomas secreted gamma-interferon (gamma-IFN). The six lines which exhibited BCGF activity only, were expanded and cloned. The BCGF activity in the supernatant of a positive clone, designated TH-5, was found to be 3-fold more potent than a preparation of BCGF obtained by stimulation of PBL with PHA.