Elsevier

Vaccine

Volume 25, Issue 41, 10 October 2007, Pages 7145-7152
Vaccine

The clinical grade maturation cocktail monophosphoryl lipid A plus IFNγ generates monocyte-derived dendritic cells with the capacity to migrate and induce Th1 polarization

https://doi.org/10.1016/j.vaccine.2007.07.031Get rights and content

Abstract

Ex vivo generated monocyte-derived dendritic cells (DCs) are used as a cellular vaccine against cancer in clinical trials. In order to be able to induce an efficient tumour-specific CTL response during immunotherapy, DCs have to be able to migrate to the lymph node and produce the Th1 polarizing cytokine, IL-12p70, upon encounter of T cells in the lymph node. However, most clinically used DCs do not produce IL-12p70 upon T cell contact. In this study, we compared a newly developed clinical grade DC maturation cocktail consisting of MPLA and IFNγ with two clinically available maturation cocktails, the ‘gold standard’ (TNFα, IL-1β, IL-6 and PGE2) and the ‘α type 1 polarizing’ (TNFα, IL-1β, IFNα, IFNγ and pI:C) cocktail. All three cocktails induced phenotypically mature DCs. However, in contrast to ‘gold standard’ DCs, which produce no IL-12p70 and as a result induce mainly Th2 cells, DCs matured with MPLA and IFNγ produce high levels of IL-12p70 upon CD40 triggering. Subsequently, these DCs induce mainly Th1 cells in vitro, even slightly more than by the α type 1 polarized DCs. In addition, MPLA plus IFNγ matured DCs have an intermediate migratory capacity towards CCL21. In conclusion, we here present MPLA plus IFNγ as a simple clinical grade maturation cocktail to generate immunostimulatory DCs with superior capacity to induce type 1 immunity.

Introduction

Ex vivo generated monocyte-derived dendritic cells (DCs) are increasingly applied as a cellular vaccine for the treatment of cancer in clinical studies [1], [2], [3]. Clinical studies require protocols where a sufficient number of well-characterized highly immunogenic DCs are produced according to current Good Manufacturing Practice (cGMP) Guidelines. The last couple of years it has become recognized that ex vivo generated monocyte-derived DCs (moDCs) should combine a few important features for efficient induction of tumour-specific CTLs [4], [5], [6]. First of all, the DCs should be capable to migrate to lymphoid organs by responding to the lymph node chemokines. Furthermore, the DCs should produce the Th1 polarizating cytokine, IL-12p70, while encountering T cells in the lymph node, as Th1 cells are important for the induction and maintenance of CTL responses.

Immature DCs generated from monocytes in the presence of GM-CSF and IL-4 (or IL-13) can be differentiated into mature DCs through contact with maturation factors, e.g. danger signals such as toll like receptor (TLR) ligands and/or inflammatory mediators. The described clinical grade DC maturation stimuli include CD40 ligand, polyI:C, monocyte-conditioned medium (MCM)-mimic and clinically used bacterial immunomodulators, like Ribomunyl and OK-432, in some cases combined with several GMP available cytokines like IFNα, IFNγ and TNFα [5], [6], [7], [8]. As most of the studies describing these maturation cocktails do not study both the capability to migrate and the production of biologically active IL-12, but focus on only one of these expects, it is difficult to conclude how suitable the induced DCs are for immunoactivatory immunotherapy. Furthermore, often only the production of IL-12p70 during culture is reported, while physiologically it would be more important to know whether mature DCs that have already produced high amounts of bioactive IL-12 during the ex vivo culture step are still capable of secreting IL-12p70 during interaction with antigen-specific T cells upon in vivo administration. This interaction can be mimicked in vitro by ligation of CD40 on the DCs.

The current ‘gold standard’ DC maturation cocktail for clinical trials, a MCM-mimic consisting of IL-1β, TNFα, IL-6 and PGE2, first reported by Jonuleit et al. [7], induces high migratory potential towards the lymph node expressed CCR7 ligands. This cocktail however, does not induce the production of IL-12p70 [9], [10], important for the induction of a proper CTL response. This might at least in part explain why less than 10% of cancer patients vaccinated with MCM-mimic-treated DCs displayed favourable clinical responses [11], [12]. Other maturation cocktails, which include TLR ligands, induce IL-12p70 production, but render these DCs less migratory [13], [14]. Thus, IL-12p70 production and migration by DCs seem to be inversely correlated processes. Mailliard et al. [5] however have developed a multiple component cocktail, containing IL-1β, TNFα, IFNα, IFNγ and pI:C, which induces α-type-1 polarized DCs (αDC1) that produce IL-12p70 and show intermediate migratory capacity. Furthermore, recently additional maturation cocktails, which include bacterial preparations, OK-432 and Ribomunyl, have been described to generate DCs which also produce IL-12p70 and show intermediate migratory capacity [6], [15], [16].

Lipopolysaccharide, LPS, has often been described to be a good IL-12p70 inducer. The use of LPS as a clinical grade maturation stimulus however is excluded because of its high toxicity. A chemically modified LPS, monophosphoryl lipid A (MPLA), has been shown to exhibit potent adjuvant activity, while exhibiting essentially no toxicity [17] and has been safely used as an adjuvant in various vaccine trials in human [18], [19]. Although it is shown that DCs matured with MPLA can produce IL-12p70 during culture, its usefulness for the maturation of DCs for clinical application has not been analyzed in depth [8], [20], [21]. Therefore, we studied the applicability of MPLA for DC vaccine therapy in more detail and combined MPLA with IFNγ, as is shown that IFNγ can enhance IL-12p70 production by DCs upon CD40 ligation [5], [22]. We found that the maturation cocktail consisting of MPLA and IFNγ meets the requirements for induction of type 1 immunity, by combining the capability to migrate with very high IL-12p70 production levels upon CD40L stimulation.

Section snippets

Reagents

Cellgro DC serum-free medium and IL-4, GM-CSF, IL-1β, TNFα and IL-6 were all obtained from CellGenix (Freiburg, Germany). PGE2 was obtained from Sigma (Steinheim, Germany). IFN-α and IFN-γ were obtained from Prepotech (Rock Hill, USA) and MPLA and pI:C from Invivogen (San Diego, USA). Medium used for read-out assays was Iscoves modified Dulbecco's medium (IMDM, Bio Whittaker, Verviers, Belgium) containing 10% FCS (Bodinco, Alkmaar, The Netherlands), 20 μg/ml human transferrin (Sigma–Aldrich), 100

The different cocktails induce a comparable mature DC phenotype

To study the potential use of MPLA as a clinical grade maturation stimulus for moDCs to be used for immunotherapy, we compared DCs matured with MPLA plus IFNγ with the well-known ‘gold standard’ DCs [7] and the promising αDC1 [5]. Regardless of the maturation used, all DCs had a mature phenotype and expressed the maturation marker CD83 to the same extent, while it was not expressed by imDCs (Fig. 1). Furthermore, all types of DCs upregulated the expression of the co-stimulatory molecules, CD40,

Discussion

In this study, we describe a detailed comprehensive and functional comparison of our newly developed clinical grade DC maturation cocktail consisting of MPLA and IFNγ with two types of maturation cocktails that are recently used to mature moDCs for immunotherapy trials: the PGE2 containing ‘gold standard’ cocktail and the ‘α type 1 polarizing’ cocktail. DCs matured with MPLA and IFNγ produce even higher levels of IL-12p70 upon CD40 ligation, as compared to the Th1-inducing cocktail described by

Acknowledgements

We thank Hans Vrielink and employees of Sanquin Blood bank division Northwest for optimalization of the aphaeresis settings and operation of the Elutra™. We thank Martien Kapsenberg for help in setting up the T cell polarization assay and valuable discussions.

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