Development of a human T-T cell hybridoma secreting B cell growth factor.

Abstract

The success of long-term culture of normal human and murine B cells has been hampered by the limited availability of soluble factors capable of maintaining proliferation of activated B lymphocytes. Previous experiments using various culture-derived supernatants in a human system were unable to separate the activities of B cell growth factor (BCGF) and interleukin 2 (IL-2) by immunochemical means. Thus, purified factors with BCGF activity in the absence of IL-2 activity have not been available for study. In the present study, normal human peripheral blood T cells were fused with the hypoxanthine/aminopterin/thymidine-sensitive human T-leukemic cell line, CEM-6. Supernatants from the resulting hybrid cells were tested for the ability to maintain proliferation of normal human B cells in a recently described assay system for human BCGF. Hybrids demonstrating BCGF activity were cloned by limiting dilution. One hybrid clone, 2B11, continued to support proliferation of B cells in both long-term cultures and 6-d assays at a level significantly above that seen with conventionally produced growth factors. No IL-2 activity was found in the supernatant from hybrid 2B11. The hybridoma supernatant was fractionated by gel filtration, and maximum proliferation of B cells was supported by the 18-20,000 mol wt protein fraction. Thus, a human T-T cell hybridoma that has BCGF activity in the absence of any demonstrable IL-2 activity has been developed. Human T-T cell hybridomas secreting discrete immunoregulatory factors should prove to be powerful tools in dissecting the mechanisms of immunoregulation of human lymphocyte function.

Human B lymphoma cell line producing B cell growth factor.

“…Ball-1A5 was then maintained in medium containing 10% vol/vol BCGF. 3B3, a human T-T hybridoma, which was developed in our laboratory, produces BCGF in the absence of IL 2 or B cell differentiation factor (BCDF) activity (4). The characteristics of a BCGF produced by a subclone of 3B3, 2B,1, have been previously reported (9).…”

“…Furthermore, distinct lymphokines act on the proliferating B cells to induce them to differentiate (2). The source of BCGF in human studies has been claimed to be one of the following: mitogenstimulated unfractionated peripheral lymphocytes, T cell clones (3), or T-T hybridomas (4). Long-term growth of nonmalignant and non-Epstein-Barr virus (EBV)-transformed B lymphocytes has been claimed to be dependent upon BCGF (5).…”

Interleukin 2 receptors on human B cells. Implications for the role of interleukin 2 in human B cell function.

“…Mixed lymphocyte reaction (MLR) supernatant was prepared as previously described (10). BSF derived from a human T-T hybridoma cell line was the same preparation as previously described in detail (22).…”

Effects of in vitro corticosteroids on B cell activation, proliferation, and differentiation.

“…Investigation in this and other laboratories has resulted in delineation of the sequential events in the human B cell activation cascade (4)(5)(6)(7)(8). By using this model system, it has been shown that resting B cells can be activated by a number of initial signals.…”