Establishment of a human B cell line that proliferates in response to B cell growth factor

Abstract

A human B cell line which shows a marked dose dependence on B cell growth factor (BCGF) when cultured in less than or equal to 2% serum has been established. Human B lymphocytes were obtained from peripheral blood of normal donors and cultured in the presence of anti-IgM (mu chain specific) and BCGF. Frequent refeedings with fresh medium containing BCGF and anti-IgM led to the establishment of a long term cultured human B cell line, HAB-40. Phenotyping of HAB-40 revealed that the cell population consisted predominantly of IgM-bearing (72%) and B1 (100%) positive cells. This B cell line consistently secreted IgM and IgG when co-cultured in the presence of PMA, anti-IgM and beta or gamma interferon (IFN). Also, it was Epstein-Barr virus nuclear antigen (EBNA) positive (100%). HAB-40 cells have been successfully maintained in the presence of BCGF without anti-IgM for over a year. Removal of BCGF led to the rapid loss of viable cells in cultures containing less than 2% serum. HAB-40 cells in microassays exhibited a marked dose-dependent incorporation of [3H]thymidine in response to BCGF in the absence of any exogenous stimulants such as anti-IgM or Staphylococcus aureus Cowan I (SAC). Recombinant interleukin 2 (IL-2) failed to augment the [3H]thymidine uptake by these B cells despite the low density expression of Tac antigen (IL-2 receptor) on their cell surface, or even when the cells were stimulated with phorbol myristate acetate (PMA) to express higher density of Tac antigen (48%). HAB-40 cells could be maintained in BCGF which was partially purified to deplete it of other contaminating proteins. None of the seven well established EBNA-positive human B cell lines nor two chronic B lymphocytic leukemia (B-CLL) cell lines that were tested showed BCGF dependence. The same BCGF-active chromatographic fractions that were active on HAB-40 cells also stimulated BCL1 and normal human B cells stimulated with anti-IgM. In the presence of less than or equal to 2% serum proteins this cell line provides a simple, reproducible assay for BCGF even in the presence of contaminant IL-2.