Development of a human T-cell hybridoma secreting separate B-cell growth and differentiation factors.

Abstract

A cloned human T-cell hybridoma (7D5) secreting B-cell growth factor (BCGF) and B-cell differentiation factor (BCDF) was established. Supernatant from this hybrid was capable of maintaining proliferation in anti-IgM-activated normal human B cells. In addition, the hybridoma supernatant induced differentiation and antibody secretion in Staphylococcus aureus Cowan I-stimulated B cells. No interleukin 2 was present in supernatant from this hybridoma. Molecular size of the hybridoma-derived BCGF and BCDF was determined by gel filtration chromatography. BCGF activity was present in the 20-kDa fractions, and BCDF activity eluted in the 30-to 35-kDa fractions. The isoelectric points of the factors, determined by chromatofocusing, were 6.6 for BCGF and 5.9 for BCDF. Finally, absorption experiments were performed using specific target cells. Phytohemagglutinin-stimulated T-cell blasts did not remove either BCGF or BCDF activity. Anti-IgM-activated B cells absorbed BCGF but not BCDF. In contrast, CESS cells removed BCDF but not BCGF. Thus, a human T-cell hybridoma secreting two distinct B-cell lymphokines was developed. Further immunochemical and functional studies of these immunoregulatory molecules should greatly enhance our understanding of the regulation of human B-cell function in normal and disease states.The production and characterization of immunoregulatory factors acting on B lymphocytes has been an area of recent interest and investigation (1-6). The technology of cloning and hybridization of human and murine T cells has provided renewable sources of monoclonal B-cell-specific lymphokines (7-10). These factors have proven to be powerful tools in delineating the complex mechanisms involved in the regulation of human immune responses.In a previous report, we described the development of a human T-cell hybridoma (2B11) secreting monoclonal B-cell growth factor (BCGF) (11). The hybridoma supernatant was devoid of both interleukin 2 (IL-2) and B-cell differentiation factor (BCDF) activities. The hybridoma-derived BCGF was capable of maintaining proliferation in normal and human B cells that had been activated by Staphylococcus aureus Cowan strain I (SAC) or anti-Ig (12). However, this factor was not able to induce differentiation in preactivated B cells. This finding correlated with earlier studies by ourselves and others that showed Ig production by B cells to be dependent on a sequential presentation of an activation signal, growth factor, and differentiation factor (13-15).In the present study, we report the development of a human T-cell hybridoma secreting BCGF and BCDF Hybridization. T-cell hybridomas were established as described (11). Normal T cells were obtained from human peripheral blood lymphocytes and stimulated with mitogen as described above. These activated T cells were fused with CEM-6 cells (an azaguanine-resistant mutant of human Tlymphoblastoid origin) in a 2:1 ratio in the presence of polyethylene glycol and were suspended in 96-well, flat-bottomed microtiter plates (Costar 3596). Hybri...

Purification to homogeneity of a high molecular weight human B cell growth factor; demonstration of specific binding to activated B cells; and development of a monoclonal antibody to the factor.

“…This supports the model of B cell function in which resting B cells must be activated by antigen or a surface crosslinking signal before they can respond to BCGF (20,32,33). Furthermore, the lack of binding of our labeled HMW-BCGF to resting or activated T cells parallels previous studies (39,40) demonstrating that anti-#-activated B cells but not T cells absorb BCGF activity from culture supernatants. The existence of purified HMW-BCGF in sufficient quantities to perform binding studies has allowed us to put these previous phenomenologic observations on a quantitative, biochemical basis.…”

Long-Lived Plasma Cells in Mice and Men

“…IL-5 and IL-6 were among the earliest cytokines shown to have important roles in PC biology in both mice and humans ( 47 – 50 ). IL-5 was originally identified as a B-cell growth and differentiation factor ( 51 ) as well as an eosinophil differentiation and IgA-enhancing factor ( 52 , 53 ).…”

Interleukin 2-dependent and interleukin 2-independent pathways of regulation of thymocyte function by interleukin 6.

“…Interleukin 6 (IL-6), also termed B-cell-stimulatory factor 2 (BSF-2), is a lymphokine originally identified as a B-cell differentiation factor capable of inducing the secretion of immunoglobulins in Staphylococcus aureus Cowan Istimulated normal B lymphocytes and a transformed B-cell line (CESS) (1,2). IL-6 cDNA was isolated from a human T-cell line (TCL-Nal) encoding a 212-amino acid precursor, cleavage ofwhich yields the mature form ofthe IL-6 molecule consisting of 184 amino acids (3).…”