Limiting dilution assay for human B cells based on their activation by mutant EL4 thymoma cells: total and antimalaria responder B cell frequencies

The analysis of human B cell responses at the clonal level (limiting dilution assay) is still technically difficult. In the present study we report on a culture system that leads to activation, proliferation and differentiation into antibody-secreting cells (ASC) of about 90% of B cells from peripheral blood or spleen. In this system, B cells are cultured in the presence of a mutant subclone of the mouse thymoma EL4 for B cell activation and human T cell plus macrophage supernatant as source of proliferation and differentiation factors. ASC precursors generating clonal responses of IgM only, IgM plus IgG, or IgG only occurred at a ratio of about 6:3:1. The mean clone size was 380 cytoplasmic Ig+ cells; the mean amount of Ig secreted per clone was 20 ng. Furthermore, it has been found using this system that a considerable proportion of peripheral blood B cells from individuals with a history of malaria infection could generate clones of anti-malaria (Plasmodium falciparum) ASC (range of 0.1 to 1%, n = 6). In a control group of blood donors the corresponding frequencies were 10 times lower (range of 0.01 to 0.1%, n = 9). These results show that the EL4 culture system can be applied to the investigation of the human B cell specificity repertoire and of priming effects such as result from infectious disease.