Production of functional human T-T hybridomas in selection medium lacking aminopterin and thymidine.

Abstract

The production of hybridomas between immunologically activated T cells and malignant T-cell lines offers a potentially unlimited source of soluble T-cell-derived products. Recently, human T-T hybrids have been described; however, their use has been hampered by slow growth and chromosomal instability due at least in part to the presence of thymidine in the traditional hypoxanthine/aminopterin/thymidine (HAT) selection medium. In this report, we describe the development of a rapidly growing hypoxanthine phosphoribosyltransferase-deficient human T-cell line designated J3R7, the use of azaserine/hypoxanthine (AH) medium as an alternative selection medium to HAT medium, and the production of functional T-T hybrids by using the J3R7 line and the AH selection technique. Hybrids selected in AH medium were 4-fold greater in number and 3-fold faster in growth rate than hybrids grown in HAT medium. No stable clones were obtained from HAT cultures whereas AH-derived hybrids could be readily cloned by the method of limiting dilution. Evidence for hybridization included (i) the presence of approximately twice the number of chromosomes in hybrids than in J3R7 cells; (ii) the presence on hybrid cells of the Leu-3a surface antigen, present on normal helper T cells but not on J3R7 cells; (iii) the expression of HLA antigens of both the normal T-cell partner and the J3R7 line; and (iv) the constitutive secretion of interleukin 2 from multiple hybrid clones but not from the J3R7 cell line. Thus far, these clones have maintained their rapid growth, chromosome number, surface phenotype, and constitutive secretion of interleukin 2 for 4 months.T lymphocytes produce a variety of immunologically active mediators (lymphokines) on stimulation with antigens or mitogens (1, 2). These mediators can be divided into two broad categories: antigen nonspecific, such as growth factors for T cells (interleukin 2, IL-2) and B cells (3), and antigen-specific helper and suppressor factors (4). Due to the limited availability of these factors, knowledge of their structure and function lags far behind that of immunoglobulin, the major B-cell product. Typically, the T-cell products are derived from bulk cultures ofnormal lymphocytes stimulated with mitogens such as phytohemagglutinin (PHA) or concanavalin A (Con A). Although this approach has yielded biologically active factors, the presence of multiple lymphokines as well as the mitogens themselves have hindered large-scale preparation of homogeneous material. In mice, the propagation of T-cell clones in vitro has overcome some ofthese problems (5). In man, however, such clones have been difficult to maintain and have not yet proved to be reliable sources of lymphokines.The use of T-T hybridomas produced by the fusion of an activated T cell with a malignant T-cell line offers the theoretical advantages ofa single cell source offactor, rapid and continuous growth in the absence of exogenous growth promoters, and an unlimited supply ofproduct free ofmitogen. This technique has been used successfu...

Directed Ig class switch recombination in activated murine B cells.

“…Staining and sorting of cells for the direct restriction analysis and for cloning of IgH loci into X phages has been described previously (Radbruch and Sablitzky, 1983;Winter et al, 1985 andRadbruch et al, 1986b). Hybridomas were established from unselected stimulated cells by polyethylene glycol-mediated fusion to X63 Ag8 -653 cells (Kearney et al, 1979) using hypoxanthine/azaserine medium (Foung et al, 1982) in some experiments instead of HAT medium. The fused cells were cultured together overnight and then cloned in 96-well plates by limiting dilution.…”

p185HER2 monoclonal antibody has antiproliferative effects in vitro and sensitizes human breast tumor cells to tumor necrosis factor.

“…Fused cells were plated at a density of 2 x 105 cells per well in 96-well microdilution plates. The hypoxanthine-azaserine (12) selection for hybridomas was begun 24 h later. Beginning at day 10 postfusion, supernatants from hybridoma-containing wells were tested for the presence of antibodies specific for p185HER2 by an enzyme-linked immunosorbent assay with the wheat germ agglutinin chromatography-purified p185HER2 preparation (28).…”

Nucleoplasmin cDNA sequence reveals polyglutamic acid tracts and a cluster of sequences homologous to putative nuclear localization signals.

“…Four days later the animals were killed, the spleens removed and splenocytes fused with the myelomaSp2/0-Agl4 (Shulman et al, 1978) by PEG 1500treatment. Selection was achieved by azaserine treatment at 1 .tg/ml (Foung et al, 1982). Clones secreting antibodies directed against nucleoplasmin were located by EIA and single cell cloned at least three times.…”