Cross-linking CD40 on human B cell precursors inhibits or enhances growth depending on the stage of development and the IL costimulus

The function of the cell surface molecule CD40 on B cell precursors (BCP) is not well understood. We now report studies using the L cell/CD40 system (anti-CD40 mAb immobilized on CD32⁺ mouse L cells) to assess the potential function of CD40 during human B cell ontogeny. Stimulation of human B lineage cells with IL-4 in the L cell/CD40 system yielded a hierarchy of responsiveness: high density tonsillar B cells > fetal splenic B cells > fetal bone marrow surface Ig⁺ immature B cells > fetal bone marrow surface Ig^- BCP. Using a microsphere/flow cytometry growth quantitation assay, we found that substituting IL-3 for IL-4 in the L cell/CD40 system provided a stronger growth stimulus for fetal bone marrow BCP and immature B cells. We also found that FACS-purified fetal bone marrow CD10⁺/CD34⁺/CD40⁺/cytoplasmic μ^- pro-B cells responded maximally to IL-3 plus IL-7. Surprisingly, anti-CD40 inhibited the pro-B cell response to IL-7. In contrast, FACS-purified fetal bone marrow CD10⁺/CD34^-/CD40⁺/cytoplasmic μ⁺ pre-B cells were essentially nonresponsive to IL-3, IL-7, or anti-CD40 alone, but were uniquely responsive to IL-3 plus anti-CD40. B-lineage cells derived after 14 days from IL-7-stimulated pre-B cells were predominantly CD19⁺/L chain^-, whereas pre-B cells stimulated with IL-3 plus anti-CD40 were predominantly CD19⁺/L chain⁺. The L chain⁺ cells from pre-B cell cultures were both μ⁺/δ⁺ and μ^-/δ⁺. Our results demonstrate that the response to CD40 signaling depends upon the BCP developmental stage and the IL costimulus, and indicate that normal human pro-B cells and pre-B cells have different growth factor requirements.