CD40 signaling-mediated delay in terminal differentiation of B cells enables alternate fate choices during early divisions.

Memory B cells and differentiated plasma cells combine to confer sustained humoral immunity. Nonetheless, we are yet to understand how B cells decide between these fates. Although pan-T cell help augments plasma cell differentiation, signaling via CD40 alone is considered to be inhibitory. Here, we examine the capacity of CD40 signaling to interfere with lipopolysaccharide-induced differentiation. Whereas lipopolysaccharide stimulation yielded only short-lived plasmablasts, co-stimulation of CD40 enhanced activation, proliferation, survival, and isotype-switching, leading to alternate fate choices such as germinal center and memory B cells during early divisions. Contrary to the notion that CD40 signaling simply arrests differentiation, the survivors, at later time points, developed into long-lived mature plasma cells, after progressively losing their ability to get restimulated. Counterintuitively, as constitutive lipopolysaccharide stimulation itself hampered differentiation, we identified that the proliferation potential of cells acted alongside CD40 signaling. Accordingly, we propose a bi-layered regulation of differentiation - CD40 signaling and proliferation potential of cells independently inhibit the commitment to and maturation of differentiation, respectively. Elucidating such cell fate decision mechanisms will aid in better vaccine design and disease management.

Homotypic aggregates contribute to heterogeneity in B cell fates due to an intrinsic gradient of stimulant exposure.

Monocultures of several cell types result in the formation of robust clusters called homotypic aggregates (HAs). How this physical aggregation affects cell fates in immune cell cultures, is poorly understood. We studied anti-CD40-stimulated primary B cell cultures, where cells assembled into large three-dimensional LFA1-driven HAs by 72 h. The dense packing in these aggregates restricts the infiltration of stimulants, such as antibodies, to cells inside the clusters. This creates a concentration gradient of stimulant availability across the cross-section of HAs. We describe a method to retain this positional information even after the disruption of HAs, for analysis by flow cytometry. Comparison of stage-specific cell-surface markers showed that the extent of stimulant-binding affected multiple fates non-uniformly. While germinal center and lineage markers were moderately upregulated, immunoglobulins and markers associated with memory were more than doubled in the peripheral cells binding more anti-CD40. These cells also experienced a strong repression of the plasma cell regulator Prdm1 and an upregulation of the oncogene Myc. Thus, cells at different locations in HAs are subjected to unequal doses of stimulants, leading to a hitherto unreported source of heterogeneity in cell fates. These findings can be extrapolated to understand the dose-dependent effects of stimulants in other three-dimensional cell clusters.