The generation of stable human T-cell hybridomas which constitutively produce interleukin-2 and chemotactic factor.
We report the successful generation of human T-cell hybridomas that constitutively secrete lymphokines. An acute lymphoblastic leukemia T-cell line, CCRF-H-SB2, free of reverse transcriptase and mycoplasma, was sensitized to hypoxanthine, aminopterin, and thymidine (HAT) by selecting out a mutant deficient in hypoxanthine guanine phosphoribosyl transferase (HGPRT) in 8-azaguanine. Peripheral blood T lymphocytes from normal donors were incubated in vitro with 10 micrograms/ml of concanavalin A for 48 h and subsequently fused with the CCRF-H-SB2 HAT-sensitive cell line. Following 5 weeks in culture, 38 of 440 wells (8.6%) demonstrated hybridoma growth. Supernatants of these cultures were screened for interleukin-2 (IL-2), chemotactic factor, interferon, migration inhibition factor, and macrophage-activating factor activities. Twelve (of 38) hybrids exhibited IL-2 activity, and eight of these were successfully cloned. The highest secreting clone was demonstrated to have mRNA to IL-2 while the parent CCRF-H-SB2 had no detectable mRNA to IL-2. Three hybrid cultures produced chemotactic factor; one was successfully cloned and grown in serum-free medium, where it continued to constitutively produce chemotactic factor as well as IL-2 activity. The chemotactic factor was determined to have the same molecular weight (12,500 daltons) as leukocyte-derived chemotactic factor. Constitutive IL-2 production remained stable for over 12 months. None of the hybridomas tested produced detectable levels of gamma interferon, migration inhibition factor, or macrophage activation factor. Because these T-cell hybridomas produce lymphokines constitutively and this phenotype is stable, they can be an important source of highly purified human lymphokines for clinical and laboratory investigations.
D000954:Antigens, Surface / Q000032:analysis; D002460:Cell Line; D002630:Chemotactic Factors / Q000096:biosynthesis*; D002634:Chemotaxis, Leukocyte; D004247:DNA / Q000032:analysis; D006801:Humans; D006822:Hybrid Cells / Q000276:immunology; D006825:Hybridomas / Q000276:immunology*; D007371:Interferon-gamma / Q000032:analysis; D007376:Interleukin-2 / Q000096:biosynthesis* / Q000235:genetics; D007621:Karyotyping; D007945:Leukemia, Lymphoid / Q000276:immunology*; D009000:Monocytes / Q000502:physiology; D012333:RNA, Messenger / Q000235:genetics; D013601:T-Lymphocytes / Q000276:immunology*
D000954:Antigens, Surface; D002630:Chemotactic Factors; D007376:Interleukin-2; D012333:RNA, Messenger; D007371:Interferon-gamma; D004247:DNA