Stable long-term cultures of self-renewing B cells and their applications

Abstract

Monoclonal antibodies are essential therapeutics and diagnostics in a large number of diseases. Moreover, they are essential tools in all sectors of life sciences. Although the great majority of monoclonal antibodies currently in use are of mouse origin, the use of human B cells to generate monoclonal antibodies is increasing as new techniques to tap the human B cell repertoire are rapidly emerging. Cloned lines of immortalized human B cells are ideal sources of monoclonal antibodies. In this review, we summarize our studies to the regulation of the replicative life span, differentiation, and maturation of B cells that led to the development of a platform that uses immortalization of human B cells by in vitro genetic modification for antibody development. We describe a number of human antibodies that were isolated using this platform and the application of the technique in other species. We also discuss the use of immortalized B cells as antigen-presenting cells for the discovery of tumor neoantigens.

Keywords: B cells; BCL6; antibodies; germinal center; rabbit.

Figure 1

Generation of self‐renewing antibody‐producing B cells. B‐cells are isolated from selected individuals, stimulated, and transduced with a retrovirus containing BCL6 and BCL‐XL. Subsequently, transduced B cells are expanded on CD40L expressing fibroblasts in the presence of interleukin (IL)‐21. B‐cell clones are screened for production of desired antibodies.

Figure 2

Ongoing somatic hypermutation in BCL 6/ BCL ‐ XL ‐transduced cells allows in vitro affinity maturation of B‐cell clones. (A) A small number of B cells within a large clonal B‐cell pool will acquire mutations in their antibody‐coding region due to activation‐induced cytidine deaminase (AID) activity. Using fluorescently labeled antigen, B cells with enhanced or decreased antigen binding can be selected using flow cytometry. Multiple rounds of sorting can be performed before single cell cloning to achieve optimal binding affinity. (B) Example of an antigen‐specific B cell clone from which subclones with deviating antigen binding capacity were selected. B cells were stained for B‐cell receptor (BCR) expression and subclones that showed increased or decreased antigen binding were obtained after single cell sorting using a flow cytometer and expanded.

Figure 3

Transduction of antigen‐specific rabbit memory B cells. (A) Rabbit B cells were isolated from PBMCs based on immunoglobulin (Ig) expression, activated for 36–40 h on CD40L L‐cells with interleukin‐21 and transduced with a retroviral vector containing BCL6 and BCL‐XL. (B) Transduction efficiency based on green fluorescent protein expression 4 days after transduction. (C) Growth rates for rabbit B cells transduced with a retroviral vector containing BCL6 and BCL‐XL, non‐transduced rabbit B cells and human cells for comparison. (D) ELISpot analysis of the frequency of vaccine‐specific IgG B cells; B cells were incubated overnight in ELISpot plates coated with the influenza vaccine. Bound antibodies were visualized using an anti‐rabbit IgG antibody. Frequencies of vaccine‐specific cells are shown in percentages.

Figure 4

Analysis and isolation of antigen‐specific B cells. B cells from influenza H1\H3\influenza B‐vaccinated rabbits were seeded either randomly at 25 cells/well or sorted single cell using fluorescently labeled antigen. Supernatants were analyzed for binding to H1, H3, and influenza B in ELISA. The total number of positive wells is depicted in the middle of the graphs. Some clones showed reactivity to two hemagglutinin proteins.