Analysis of cd40l and il-10 induced ig class switch in naive human b cells

Cytokines have been shown to differentially regulate the expression of specific Ig flassos in the human. Recently, IL-10 has been implicated as a possible switch factor for human B cells. We have investigated the effect of CD40 engagement and IL-10 on Ig class switch in naive human peripheral blood B cells. sIgD⁺ B cells, isolated from the peripheral blood of healthy donors using positive selection with immunomagnetic beads (MACS, Miltenyi Biotech), were cultured in the presence of FcγRII transfected Ltk^- mouse fibroblasts (CI)w32 L cells), anti-CD40 mAb 89, and IL-10 (CD40 system). The IgG produced in response to these signals was analyzed using IgG subclass specific KLISAs. Increased production of IgG1 and IgG3, but not IgG2 and IgG4 was detected after 14 days in culture. Neither IL-10 nor CD40 engagement alone wore able to increase IgG production by naive human sIgD⁺ B cells. In recent experiments, substitution of mAb89 and CDw32 L cells with TBAM-transfected human kidney cell line 293 (obtained from Dr. S. Ledennan, Columbia University), yielded comparable results. The ongoing analysis of the expression of Cγ specific germline transcripts in naive B cells in response to CD40 activation and IL-10 should provide further insight into the mechanisms operating at the early steps of human IgG class switch.