Long-term growth of human B cells and their use in a microassay for B-cell growth factor.

Abstract

Normal human B lymphocytes, prepared from peripheral venous blood, have been stimulated with intact antiIgM (,u chain specific) bound to an insoluble matrix. The activation event, in a subfraction of human B cells, was associated with subsequent receptivity to the mitogenic effects of exogenously added B-cell growth factor. The ability of the cell population to specifically absorb the B-cell growth factor was dependent upon the time of stimulation with the anti-IgM. Continuous replenishment of the growth factor resulted in the ability to maintain long-term growth-factor-dependent human B-cell populations. These cultured B lymphocytes were shown to specifically absorb the B-cell growth factor, suggesting the presence of membrane receptors for it. The cultured B lymphocytes were routinely maintained in logarithmic-phase growth, in the presence of growth factor, with a population doubling time of 36 hr. These cultured B cells have been utilized in a microassay for the assessment of B-cell growth factor activity that is accurate, sensitive, and precise.Experimentation has demonstrated that an activated human B lymphocyte may be stimulated to enter the S phase of the cell cycle by factor(s) present in media conditioned by lectin-stimulated peripheral blood lymphocytes (1-4). One factor capable of stimulating human B cell proliferation, presently known as B-cell growth factor (BCGF), is a trypsin-sensitive protein with a molecular weight of 12,000-13,000 (5, 6). The protein has a mildly acidic isoelectric point (pH 6.3-6.6), is relatively unstable at 56°C (6, 7), is unaffected by low concentrations of reducing agents, and is stable between pH 3 and pH 8 (7). The human BCGF possesses functional specificity in that its action is strictly proliferative. The factor also has target cell specificity in that its scope is limited to B lymphocytes (3, 5).Further experimentation on many aspects of the biological and biochemical characterization of the growth factor would be enhanced by the establishment of long-term cultures of normal human B cells dependent upon BCGF. In this report we describe a procedure for the polyclonal activation of human B lymphocytes by using anti-IgM (,. chain specific) bound to an insoluble matrix. Anti-immunoglobulin antibodies have previously been utilized in several experimental systems because of their efficacy in human lymphocyte activation; such studies have suggested an important role for the immunoglobulin receptor in B-lymphocyte regulation (8-11). This activation procedure is shown in the present paper to render a subset of cells subsequently responsive to the effects of BCGF and to provide conditions supporting the long-term growth of these B lymphocytes. The long-term BCGF-dependent populations provide an effective system for the sensitive, accurate assessment of BCGF activity and provide evidence for the presence of growth factor receptors on these cells. MATERIALS AND METHODSPreparation of Growth Factors. BCGF-containing growth factor preparations were prepared essentially as d...

Interleukin 4 (B-cell growth factor II/eosinophil differentiation factor) is a mitogen and differentiation factor for preactivated murine B lymphocytes.

“…The interrelationships of BSF-1 and I3CGF II in the stimulation of B cells also remain to be elucidated. Since BSF-1 with anti-Ig (presumably mimicking the effects of antigen) is sufficient to cause B cells to synthesize DNA (3,(6)(7)(8), the role of BCGF II is puzzling. Earlier studies had suggested that proliferation and maturation to antibody secretion in B lymphocytes are controlled by different lymphokines (9).…”

“…Murine B-cell-stimulating factor type 1 (BSF-1) acts on resting B cells and drives them to a transitional activated state characterized by the expression of high levels of Ia antigens (4,5). In combination with antibodies to surface Ig receptors (anti-Ig), it induces B cells to synthesize DNA (3,(6)(7)(8).…”

Antigen-independent regulation of cytoplasmic calcium in B cells with a 12-kDa B-cell growth factor and anti-CD19.

“…In general, the signals for B cells mediated by these factors and antibodies have not been biochemically identified. One of these factors, 12-kDa or low molecular weight B-cell growth factor (LMW-BCGF), produced by lectin-activated T cells, is able to support clonal B-cell proliferation and is a predominant factor affecting B cells (12,21).…”

“…However, a second signal, complementary to the sIg-mediated signal, is required to drive resting B cells into S phase (4)(5)(6). The complementary or "progression" signal can be provided by a variety of growth factors (y interferon, interleukins 1, 2, and 4, 12-and 60-kDa B-cell growth factors), complement components (C3d), or monoclonal antibodies (mAbs) binding to B-cell receptors (anti-CDw40, anti-CD23, and anti-CD22) (7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20). In general, the signals for B cells mediated by these factors and antibodies have not been biochemically identified.…”

Growth factor-mediated tumor cell proliferation in hairy cell leukemia.

“…BCGF activity was selectively enriched ~50-fold after DEAE chromatography and further enriched >100-fold after hydroxylapatite chromatography. BCGF was added to culture wells in unit (U) amounts determined on long-term cultured B cells as previously described (11). The TCGF used was purified, cloned material kindly provided by Cetus Corp., Emeryville, CA (lot No.…”