Maintenance of human germinal center B cells in vitro

Abstract

The ability to maintain germinal center (GC) B cells in culture should facilitate studies on the molecular and cellular events which accompany affinity maturation and the generation of memory in T-dependent responses. We have investigated the ability of cytokines to maintain human tonsillar GC B cells (IgD-/CD39-/CD38+/CD77+) in the 'CD40 culture system.' In the absence of added cytokines, CD40 monoclonal antibody held on CD32-transfected L cells effectively sustained DNA synthesis in GC B cells for a maximum 3 to 4 days. Of the following cytokines (interleukin-1β [IL-1β], IL-2, IL-3, IL- 4, IL-6, IL-7, IL-10, and stem cell factor), only IL-2 and IL-4 provided a significant enhancement to DNA synthesis in the CD40 culture system; this was modest and short-term. Following a study on the cooperative activity between pairs of cytokines, triple combinations were identified that could maintain high levels of GC B-cell stimulation for at least 10 days. IL-10 was a common component of these synergistic cytokine cocktails, which were IL-10 + IL-4 + IL-7; IL-10 + IL-3 + IL-7; IL-10 + IL-1β + IL-2; IL-10 + IL-1β + IL-3, and IL-10 + IL-3 + IL-6. Culture of GC B cells with these cytokine combinations resulted in a net increase in viable cell numbers of 50% to 100% whereas total cell numbers increased up to fourfold. Cells recovered from these cultures retained a GC B-cell phenotype with a significant proportion being CD38+/CD44-, features characteristic of centroblasts. Studies with metabolically inactive CD32-L cells supported a role for stromal cell- derived soluble factors in maintaining GC B cells in vitro.