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.