Dendritic Cells Matured with a TLR7/8 Agonist Induce T Helper 1 Cell Polarization, Activate NK Cells and Are Thus Highly Suitable for Application in Cancer Immunotherapy

Abstract 358^FN2

Dendritic cells (DCs) are important regulators of the human immune response. By means of direct intercellular interactions and secretion of cytokines, they can induce a stimulatory or a regulatory response, depending on the environment in which they developed. In vitro, it is possible to imitate this process by addition of various cytokines. The aim of this study was to evaluate DCs matured by different cytokine cocktails for expression of immunostimulatory and -inhibitory molecules and correspondent activation of T helper 1 (Th1) and natural killer (NK) cells. The selection of these cocktails was guided by potential clinical application and usage in a GMP setting.

We compared three different ways of DC generation from peripheral blood monocytes of healthy donors: 1) maturation by a cocktail including the TLR7/8 agonist R848 (TLR-mDCs), 2) DC generation by the standard combination of proinflammatory cytokines (IL-4, GM-CSF, IL-1β, PGE₂, TNF-α, and IL-6) applied in many clinical studies so far (cc-mDCs), and 3) addition of IL-10 in order to induce a more regulatory phenotype (IL10-mDCs). The expression of a broad range of costimulatory and coinhibitory molecules (CD80, CD86, CD273, CD274, CD275, CD276, B7-H4, HVEM, CD30L, CD70, CD134L = OX40L, CD137L = 4-1BBL) on the surface of these DC populations was analyzed by FACS. Secretion of various cytokines crucial for interaction with other immune cells (IL-12p70, IL-10, TNF-α, IFN-γ, IL-2, and TGF-β) was measured by cytometric bead array after stimulation with CD40 ligand. In order to assess the functional importance of these signals, we performed in vitro polarization assays for T helper cells after co-culture with DCs and measured the in vitro stimulatory potential of the DCs for natural killer (NK) cells by CD69 upregulation and intracellular IFN-γ staining.

We could show that TLR-mDCs were characterized by a predominance of costimulatory (e.g. CD80, CD86) relative to coinhibitory molecules (e.g. CD273, CD274, HVEM). When stimulated by CD40L, they displayed a cytokine profile with very high IL-12p70 and TNF-α, but little if any IL-10 production. In a co-culture with autologous T cells, the combination of these signals resulted in a strong polarization toward IFN-γ secreting Th1 cells, with little or no stimulation of Th2 and Th17 cells. The costimulatory profile of cc-mDCs, in comparison, was shifted toward a lower expression of costimulatory molecules and similar or higher expression of coinhibitory molecules (ratio of CD86 to CD273 expression around 40 compared to > 60 for TLR-mDCs, p < 0.005). No IL-12p70 and low levels of IL-10 were secreted. These signals were reflected in a less pronounced type 1 polarization of T helper cells. IL10-mDCs expressed very low levels of CD80 and CD86 and displayed a coinhibitory molecule pattern similar to cc-mDCs. Additionally, they secreted the immunoregulatory molecule IL-10 in higher amounts and did not activate T helper cells at all. As IL-12p70 is an important factor for NK cell activation, only TLR-mDCs were capable of upregulating the activation marker CD69 on NK cells and inducing significant secretion of IFN-γ.

Both Th1 and NK cells play an important role in tumor defense. With this set of data, we clearly showed that TLR-mDCs, in consequence of their positive costimulatory profile and their high IL-12p70 secretion, are superior with respect to type 1 polarization of T cells and activation of NK cells. They are therefore highly suitable for application in cancer immunotherapy. This DC type will be used in a phase I/II trial for postremission therapy in patients with non-favorable AML, which will start in our clinic in 2012.