Abstract
CD40L has a well-established role in enhancing the immunostimulatory capacity of normal and malignant B cells, but a formulation suitable for clinical use has not been widely available. Like other TNF family members, in vivo and in vitro activity of CD40L requires a homotrimeric configuration, and growing evidence suggests that bioactivity depends on higher-order clustering of CD40. We generated a novel formulation of human recombinant CD40L (CD40L-Tri) in which the CD40L extracellular domain and a trimerization motif are connected by a long flexible peptide linker. We demonstrate that CD40L-Tri significantly expands normal CD19+ B cells by over 20- to 30-fold over 14 days and induces B cells to become highly immunostimulatory antigen-presenting cells (APCs). Consistent with these results, CD40L-Tri-activated B cells could effectively stimulate antigen-specific T responses (against the influenza M1 peptide) from normal volunteers. In addition, CD40L-Tri could induce malignant B cells to become effective APCs, such that tumor-directed immune responses could be probed. Together, our studies demonstrate the potent immune-stimulatory effects of CD40L-Tri on B cells that enable their expansion of antigen-specific human T cells. The potent bioactivity of CD40L-Tri is related to its ability to self-multimerize, which may be facilitated by its long peptide linker.Electronic supplementary materialThe online version of this article (doi:10.1007/s00262-012-1331-4) contains supplementary material, which is available to authorized users.
Highlights
The CD40:CD40Ligand (CD40L) pathway provides essential signals for T cell help for B cell antibody production and dendritic cell priming of CD8? T cell responses
CD40 is a member of the tumor necrosis factor receptor (TNFR) superfamily and is a type I transmembrane protein that is highly expressed on diverse cell types including many antigen-presenting cells (APC), such as B cells, dendritic cells, macrophages, and endothelial cells [1, 2]
To examine the potency of CD40L-Tri, we measured its ability to enhance expression of these molecules compared with known formulations of CD40L, namely the transfected murine fibroblast line (NIH3T3/tCD40L) and commercially available epitopetagged homotrimeric forms that are formulated with a short linker and that are inactive unless cross-linked by tag-specific antibodies
Summary
The CD40:CD40Ligand (CD40L) pathway provides essential signals for T cell help for B cell antibody production and dendritic cell priming of CD8? T cell responses. CD40 is a member of the tumor necrosis factor receptor (TNFR) superfamily and is a type I transmembrane protein that is highly expressed on diverse cell types including many antigen-presenting cells (APC), such as B cells, dendritic cells, macrophages, and endothelial cells [1, 2]. The physiological ligand of CD40 is CD40L (CD154). Expressed on activated T cells, this type II transmembrane protein is a member of the TNF family of ligands and naturally forms a trimer on the cell surface. The interaction of CD40L with CD40 effectively increases function of APCs, through the induction of costimulatory molecules as Cancer Immunol Immunother (2013) 62:347–357 well as inflammatory cytokines and chemokines. Numerous reports have demonstrated that APCs that are activated as a result of the CD40L-CD40 interaction induce T cell activation [3, 4]
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