Stability of specific immunoglobulin secretion by EBV-transformed lymphoblastoid cells and human-murine heterohybridomas

The purpose of this project was to determine if stability of specific antibody secretion improved after fusion of Epstein-Barr virus (EBV)-transformed lymphoblastoid cells with P3X63Ag8.653 murine myeloma cells. Production of human monoclonal antibodies by Epstein-Barr virus transformation and somatic cell fusion has been used by many laboratories, however the steps involved have not been fully optimized. B lymphocytes isolated from the peripheral blood of normal donors were enriched for Thomsen-Friedenreich (T) antigen-reactive cells by panning on asialoglycophorin. The EBV-transformed lymphoblastoid cell lines generated from asialoglycophorin-adherent B lymphocytes were treated in three different manners: (1) cloned and maintained in culture as monoclonal lymphoblastoid cell lines, (2) cloned and fused with murine myeloma cells or (3) fused shortly after transfomation without prior cloning. Cloned lymphoblastoid cell lines maintained in culture without fusion either died or lost specific antibody secretion within five months. Uncloned lymphoblastoid cells remained viable for up to three months but lost specific antibody secretion within two months probably due to overgrowth by nonspecific clones. In an attempt to increase longevity and to stabilize specific antibody secretion by these cells, the cloned lymphoblastoid cells were fused with murine myeloma cells. In nine of ten fusions no hybrids were recovered. As an alternate approach, uncloned lymphoblastoid cells secreting T antigen-specific antibody were hybridized with murine myeloma cells, hybrids secreting T antigen-specific antibody were recovered in six of seven fusions. Furthermore, T antigen-specific antibodies of high titer were secreted by the heterohybridoma clones for more than five months of continuous culture. These heterohybridoma cells secreted more immunoglobulin, produced greater titers of antibody and maintained specific antibody secretion longer than either monoclonal or polyclonal EBV-transformed lymphoblastoid cells. These studies have conclusively demonstrated that fusion of polyclonal lymphoblastoid cells secreting T antigen-specific antibody with murine myeloma cells results in prolongation of human monoclonal antibody production compared with unfused monoclonal or polyclonal lymphoblastoid cell lines. This procedure should be generally applicable for the production of stable human monoclonal antibody-secreting cells lines from peripheral blood lymphocytes. ^

THE ROLE OF B CELLS IN THE T CELL DEPENDENT RELEASE OF HUMAN B CELL GROWTH FACTOR

Quiescent human B cells are postulated to go through activation and proliferation phases before undergoing differentiative phase for immunoglobulin secretion. The present studies address some of the aspects of activation and proliferation phase of normal human B cells. The definitions of signals responsible for B cell activation and proliferation resulted in the development of a highly specific, reproducible B cell growth factor (BCGF) assay. This BCGF bioassay utilizes activation by rabbit anti-human IgM-antibody. The functional specificity of this assay for measuring BCGF activity was demonstrated by the finding that target B cells proliferated but did not differentiate. The factor specificity was determined by specific absorption of BCGF by anti-IgM activated B cells. This assay was utilized for the studies of T-B cell collaboration and the essential function of monocytes in the production and/or release of B cell growth factor in a syngeneic in vitro system. It is apparent that highly purified T cells are poor producers of BCGF by themselves and require monocytes to secrete significant quantities of BCGF upon PHA stimulation. Macrophage soluble factor, Interleukin 1, is capable of replacing monocyte function for the release of BCGF by activated T cells. In our studies, B cells are incapable to function as accessory cells to replace monocyte function. Normal B cells are also not capable of producing BCGF under our experimental observations. However, the addition of these B cells at an optimum cell density (T:B ratio 1:1) doubles the monocyte dependent release of BCGF by syngeneic T cells. The augmentative role of B cells is expanded to understand the mechanism of BCGF release by T cells. It is observed from our studies that DR antigen of B cell surface is involved in the release of BCGF. The functional difference between DR of B cells and monocytes is observed as IL-1 could replace DR-treated monocytes whereas failed to replace DR-treated B cells for the release of BCGF by T cells. This functional difference may be attributed to the reported microheterogeneity in DR of B cells and monocytes. The addition of irradiated B cells increased the monocyte dependent T cell proliferation, suggesting the increase of T cell pool for BCGF release. In summary, the development of a biological assay specific for B cell growth factor led to the delineation of an interesting role of B cells in the release of its own growth factor by T cells. . . . (Author's abstract exceeds stipulated maximum length. Discontinued here with permission of author.) UMI ^