Modified CD40L‐Activated B‐Cell Proliferation Model for Validating the Suppressive Activity of CD40‐CD154 Pathway Inhibitors

ABSTRACT Background CD40‐CD154 pathway inhibitors are considered indispensable immunosuppressive drugs in xenotransplantation. At present, novel anti‐CD154 and anti‐CD40 monoclonal antibodies (mAbs) are continuously being developed. It is important to establish a simple and efficient in vitro method to evaluate the effectiveness of these therapeutic mAbs. Methods A modified CD40L‐activated B‐cell proliferation model was established using irradiated NIH/3T3 cells transfected with human CD40 ligand (hCD40L‐NIH/3T3) as stimulator cells and human or rhesus monkey peripheral blood mononuclear cells (PBMCs) as responder cells. After 8 days of culture, B‐cell proliferation was detected by flow cytometry. Various concentrations of anti‐CD40 or anti‐CD154 mAbs were added to the co‐culture system as an intervention. The inhibitory effects of anti‐CD154 and anti‐CD40 mAbs on the proliferation of B cells from humans and rhesus monkeys were studied and compared. Results After 8 days of co‐culture, the proliferation rate of B cells in both human and rhesus monkey PBMCs was more than 80%, and the expression of MHC‐II and the co‐stimulatory molecules CD80, CD86, and CD40 on B cells was significantly up‐regulated. All three anti‐CD154 mAbs showed a similar strong inhibitory effect on human B‐cell proliferation, but the inhibitory effect on the proliferation of rhesus monkey B cells was weaker than that on human B cells, which showed a typical dose‐dependent inhibition. The three anti‐CD40 mAbs from different sources had different effects. One mAbs potently inhibited both human and monkey B‐cell proliferation, whereas the other two mAbs exhibited strong or moderate inhibitory effects on human B‐cell proliferation but had little inhibitory effect on monkey B‐cell proliferation. Conclusion We have successfully established a modified CD40L‐activated B‐cell proliferation model for the in vitro evaluation of CD40‐CD154 pathway inhibitors, which may provide important evidence for the selection of appropriate therapeutic antibodies and their dose determination for xenotransplantation.