Generation of a lymphocyte growth factor by treatment of human cells with neuraminidase and galactose oxidase.

Abstract

The oxidizing mitogens, sodium periodate and galactose oxidase, induce proliferation ofT cells by generation of aldehyde moieties on cell surface glycoproteins (1-3). A brief (30-min) incubation of lymphocytes with these agents is sufficient to generate the mitogenic signal. The oxidizing agent can then easily be removed by washing the cells. These procedures provide a useful tool for investigation of soluble factors that are released from activated lymphocytes and that may play a role in mediating lymphocyte activation. [8,9]). These factors have a variety of biological activities, the most striking being the induction of proliferation in activated lymphocytes, the activation of cytotoxic T cells (CTL), and the maintenance of such cells in long-term tissue culture. We report here that a mitogenic or growth factor is produced by human lymphocytes activated by a brief treatment of the cells with neuraminidase and galactose oxidase (NAGO). The soluble factors produced by NAGO-stimulated lymphocytes are'mitogenic for nonproliferating cells that have previously been exposed to mitogens or allogeneic cells, or that have been incubated without mitogens for 7-14 d. The NAGO-induced factors do not produce proliferation in freshly isolated, unprimed peripheral blood lymphocytes. Moreover, these soluble factors induce differentiation of MLC memory cells to secondary CTL. Materials and MethodsHuman peripheral blood mononuclear cells (PBL) were obtained from healthy normal volunteers, age 21-47 yr, by Ficoll (Pharmacia Fine Chemicals, Div. of Pharmacia, Inc., Piscataway, N. J.) -Hypaque (Winthrop Laboratories, New York) gradient centrifugation as previously described (10). Lectin-induced memory cells were prepared by adding PHA (2 #g/ ml) or Con A (2 pg/ml) to PBL (10e/ml) that were suspended in RPMI-1640 (Grand Island Biological Co., Grand Island, N. Y.) that contained 5% fetal calf serum, 100 U/ml of penicillin, and 100 #g/ml of streptomycin (RPMI-1640 medium). NAGO-induced memory cells were prepared by exposing PBL (10-20 × 10e/ml), suspended in phosphate-buffered saline (PBS), to 50 U/ml of neuraminidase and 2.6 U/ml of galactose oxidase for 30 min at 37°C in a shaking water bath. The treated PBL were then washed twice with PBS and resuspended in RPMI-1640 medium that contained D-galactose (5 mg/ml). The mitogen-treated memory cells

Biochemical and biological characterization of lymphocyte regulatory molecules. V. Identification of an interleukin 2-producing human leukemia T cell line.

“…Activated T cells have been grown in culture only by the continual addition of IL-2. In all cases, such cell lines have been shown to mediate effector T cell function and have not been found to demonstrate capacity for IL-2 production (7,9,15). The notion that I1-2producing cells die upon factor release is also supported from our previous studies conducted on an IL-2-producing murine T cell lymphoma.…”

Interleukin‐2 Dependent Culture of Cytolytic T Cell Lines

“…It should be stressed that an excellent source of mitogen-free IL-2 could be obtained via neuraminidase/galactose oxidase (NaGO) treatment of human peripheral blood cells. Originally described by Novogrodsky and his co-workers (Novogrodsky et al 1980) as a procedure for triggering human T cell proliferation, we have recently found that NaGO treatment of human PBL produces supernates with considerable IL-2 activity.…”

“…As is clear from examination of the data displayed in Table V, harvest of conditioned medium with peak IL-2 activity was cell concentration and culture duration dependent. This was most likely due to the well documented observation that IL-2 supernate titer depends upon two competing reactions: the first being IL-2 production by a subpopulation of T cells and the second being the binding of IL-2 by a proliferating mitogen-ac- Novogrodsky et al 1980). tivated T cell subset in the same culture (Smith et al 1979a, Bonnard et al 1979, Coutinho et al 1979.…”