Oligomerization of the Recombinant CD40L Increases Its Agonist Activity upon Stimulation of Human B Cells

Abstract

The CD40L molecule is primarily expressed on the activated CD4⁺ T lymphocytes and plays a key role in the immune response. Upon contact with a B lymphocyte, CD40L binds to the CD40 receptor and triggers signaling pathways, leading to B cell proliferation and differentiation and inducing a switch in Ig synthesis. When using soluble forms of CD40L as an activator, preliminary forced multimerization of CD40L is a prerequisite. A construct in which the receptor-binding domain of CD40L is fused to the collagen-like domain of adiponectin (Adn) is often used to multimerize CD40L. It is believed that Adn-CD40L forms several oligomeric forms. In our study, we obtained an Adn-CD40L preparation from which we isolated the low-molecular-weight and high-molecular-weight forms of Adn-CD40L (LMW and HMW). We next studied their biochemical, structural, and functional characteristics. It was concluded that LMW is a trimer of Adn-CD40L, while HMW is a mixture of higher-order oligomers. LMW demonstrated higher activity in the NF-κB-dependent signaling pathway activation assays compared to HMW. However, HMW induced more pronounced B-cell expansion and stimulated Ig secretion to a greater extent than LMW. These results could be used to develop new CD40L-based immunotherapeutics.