Combined TLR-3/TLR-8 Signaling in the Presence of α-Type-1 Cytokines Represents a Novel and Potent Dendritic Cell Type-1, Anti-Cancer Maturation Protocol.

Tadej Fevžer,Nataša Tešić,Kaja Zajc,Primož Poženel,Urban Švajger

doi:10.3390/cells11050835

Tadej Fevžer, Nataša Tešić + Show 3 more

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https://doi.org/10.3390/cells11050835

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Abstract

During the ex vivo generation of anti-cancer dendritic cell (DC)-based vaccines, their maturation still represents one of the most crucial steps of the manufacturing process. A superior DC vaccine should: possess extensive expression of co-stimulatory molecules, have an exceptional type-1 polarization capacity characterized by their ability to produce IL-12p70 upon contact with responding T cells, migrate efficiently toward chemokine receptor 7 (CCR7) ligands, and have a superior capacity to activate cytotoxic T cell responses. A major advance has been achieved with the discovery of the next generation maturation protocol involving TLR-3 agonist (poly I:C), tumor necrosis factor (TNF)-α, interleukin (IL)-1β, interferon (IFN)-γ, and IFN-α, and has since been known as α-type-1 maturation cocktail. We demonstrate how this combination can be greatly enhanced by the inclusion of a TLR-8 stimulation (R848), thereby contributing to potentiation between different TLR signaling pathways. For maximum efficiency, TLR-3 stimulation should precede (termed pre I:C) the stimulation with the R848/TNF-α/IL-1β/IFN-α/IFN-γ cocktail. When compared to DCs matured with α-type-1 maturation cocktail (αDCs), DCs matured with pre I:C/R848/TNF-α/IL-1β/IFN-α/IFN-γ (termed zDCs) displayed higher expression of CD80 and CD86 co-stimulatory molecules. Importantly, after CD40-ligand stimulation, which simulates DC-T cell contact, zDCs were much more proficient in IL-12p70 production. In comparison to αDCs, zDCs also displayed a significantly greater migratory capacity toward chemokine ligands (CCL)19 and CCL21, and had a significantly greater allo-stimulatory capacity. Finally, zDCs were also superior in their capacity to induce melanoma-specific CD8+ T cells, CD8+ T cell proliferation, and cytotoxic T cells, which produced approximately two times more IFN-γ and more granzyme B, than those stimulated with αDCs. In conclusion, we present a novel and superior DC maturation cocktail that could be easily implemented into next generation DC vaccine manufacturing protocols in future trials.

Highlights

Dendritic cells (DCs) are regarded as one of the primary biological tools for use in cancer immunotherapy
Prior Stimulation of TLR3 Followed by Stimulation of TLR8 in Combination with α-Type-1 Cytokines Results in Extensive Capacity of DCs to Produce IL-12p70 upon CD40 Ligation in Comparison to Controls
When measured directly from supernatants of maturing DCs, the levels of IL-12p70 were significantly higher for zDCs in comparison to αDCs, with an approximately two-fold greater production capacity

Summary

Introduction

Dendritic cells (DCs) are regarded as one of the primary biological tools for use in cancer immunotherapy. Their capacity to induce powerful antigen-specific cytotoxic T cell responses is unprecedented in the immune system [1]. Their past clinical success as cellular vaccines has been modest at best [2], the variety of mechanisms that underlie their ability to efficiently uptake and present tumor-associated antigens (TAAs) still leaves a lot of opportunity for improvement. The manufacture of MoDC-based anti-tumor vaccines generally consists out of four basic steps: patient apheresis; monocyte selection; monocyte-to-DC differentiation using granulocytemacrophage colony stimulating factor (GM-CSF) and interleukin (IL)-4; and DC maturation along with TAA loading [4]. While the first three steps are more or less similar across various protocols, the maturation of DCs has varied greatly in the last few decades and most likely plays a major role in the clinical success of DC vaccines [5]

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