Targeted activation of dendritic cells via the CD40 receptor

Wingham, Brittany

Abstract

Dendritic cell (DC) activation underpins the patient immune response in multiple cancers, including ovarian cancer. While DCs are detected within the margins of ovarian tumours, DC populations are maintained in an immature state, displaying low levels of co-stimulatory receptor expression and cytokine production, with impaired antigen presentation capacity.
Engagement of the DC surface receptor CD40 (CD40R) is required for efficient immune activation. This thesis investigated the potential of a Monovalent Targeted Peptide targeting CD40R (MTP40) to bind the CD40R of a murine DC cell line (tsDC), and whether the interaction could be enhanced by multivalent presentation.
This work aimed to demonstrate the suitability of two potential scaffolds for multivalent presentation. The first system investigated the activity of MTP40 presented on streptavidin as a tetramer (Tet40), which improved the binding kinetics of MTP40 (KD 1.32μM) when presented as Tet40 (KD 0.072μM). Binding capacity was confirmed as MTP40 conjugated to streptavidin Dynabeads successfully isolated the CD40R by pull down assay. The second system explored MTP40 attached to the surface of gold nanoparticles through thiol-Au bonds, to produce targeted polyvalent gold nanoparticles (TPN40) presenting up to 2000 peptide units per molecule, which demonstrated binding to CD40R by coomassie stain.
Changes in outputs associated with tsDC activation were observed in response to CD40R targeted treatments. Surface expression of CD40R was upregulated in response to Tet40 or TPN40, while TPN40 induced increased expression of activating receptors CD86 and CD54. tsDC upregulated expression of IL-2 and IL-12, with a corresponding down-regulation of IL-10, in response to Tet40 or TPN40. TPN40 treatments also stimulated uptake of the model antigen FITC-Ova by tsDC.
After confirming the tsDC cell line induced functional changes in response to CD40R targeted activation, second goal was to determine whether the “activated” tsDCs were able to induce priming of effector T cells in an antigen specific manner. T cell lines significantly upregulated production of IL-2 and IL-12 in response to TPN40, while the cytotoxic T cell line specifically upregulated IFN-. T cell induced cytotoxicity and proliferation were not significantly affected by the addition of ID8 tumour cell lysates, suggesting further optimizations are needed to produce an antigen-specific response.
This study provided evidence that multivalent engagement of CD40R is required to induce effective tsDC maturation, highlighting the importance of ligand presentation in immunity. The work also provides a multistep model of immune activation as stimulated tsDCs were capable of inducing changes in T cell lines. This demonstrates proof-of-principle that multivalent activation at the DC level is sufficient to prime an effector T cell response.
These conclusions have wider significance in the field of immunology, where they suggest multivalent interactions can produce optimised immune responses. Gold nanoparticles provided a stable, well-tolerated platform for multivalent drug delivery. Future work should investigate the bimodal application of gold nanoparticles for targeted drug delivery with tumour imaging.

Citation

Wingham, B. (2018). Targeted activation of dendritic cells via the CD40 receptor. (Thesis). University of Hull. Retrieved from https://hull-repository.worktribe.com/output/4224089

• Thesis Type: Thesis
• Deposit Date: Mar 24, 2022
• Publicly Available Date: Feb 24, 2023
• Keywords: Biomedical sciences
• Public URL: https://hull-repository.worktribe.com/output/4224089
• Additional Information: Department of Biomedical Sciences, The University of Hull
• Award Date: Apr 1, 2018