Elsevier

Vaccine

Volume 31, Issue 4, 11 January 2013, Pages 639-646
Vaccine

Comparison of clinical grade type 1 polarized and standard matured dendritic cells for cancer immunotherapy

https://doi.org/10.1016/j.vaccine.2012.11.053Get rights and content

Abstract

Monocyte-derived dendritic cells (DCs) used for immunotherapy e.g. against cancer are commonly matured by pro-inflammatory cytokines (TNF-α, IL-1β, IL-6) and prostaglandin E2 although the absence of Toll-like receptor mediated activation prevents secretion of IL-12 from DCs and subsequent efficient induction of type 1 effector T cells. Standard matured clinical grade DCs “sDCs” were compared with DCs matured with either of two type 1 polarizing maturation cocktails; the alpha-type-1 DCs “αDC1s” (TNF-α, IL-1β, IFN-γ, IFN-α, Poly(I:C)) and “mDCs” (monophosphoryl lipid A (MPL), IFN-γ) or a mixed cocktail – “mpDCs”, containing MPL, IFN-γ and PGE2. αDC1s and mDCs secreted IL-12 directly and following re-stimulation with CD40L-expressing cells and they mainly secreted the T effector cell attracting chemokines CXCL10 and CCL5 as opposed to sDCs that mainly secreted CCL22, known to attract regulatory T cells. αDC1s and mDCs were functionally superior to sDCs as they polarized naïve CD4+ T cells most efficiently into T helper type 1 effector cells and primed more functional MART-1 specific CD8+ T cells although with variation between donors. αDC1s and mDCs were transiently less capable of CCL21-directed transwell migration than standard matured DCs, likely due to their increased secretion of CCL19, which mediate internalization of CCR7. mpDCs were intermediate between standard and polarized DCs both in terms of IL-12 secretion and transwell migratory ability but functionally they resembled sDCs and strikingly had the highest expression of the inhibitory molecules PD-L1 and CD25. Thus, further studies with type 1 polarized DCs are warranted for use in immunotherapy, but when combined with PGE2 as in mpDCs, they seems to be less optimal for maturation of DCs.

Highlights

► Standard and type 1 polarized DCs were compared for use in cancer immunotherapy. ► Type 1 polarized DCs (αDC1 and mDC) induced superior T cell functionality. ► CCR7-dependent migration of type 1 polarized DCs was influenced by autocrine CCL19. ► Maturation with MPLA, IFN-γ and PGE2 (mpDC) proved to be less optimal.

Introduction

Maturation of in vitro generated monocyte-derived dendritic cells (DCs) used in immunotherapy of e.g. cancer is one of the most critical parameters for induction of effective immune responses and is still a highly debated topic [1]. The inability of standard matured DCs (sDC; TNF-α, IL-1β, IL-6 and PGE2) [2] to produce IL-12p70 (hereafter referred to as “IL-12”), a key driver of cellular immunity, is a major drawback. These DCs are widely applied in the clinic and their inability to produce IL-12 could partly explain why DC based cancer immunotherapy have so far only resulted in a limited number of clinically responding patients [3], [4], [5]. Other factors may also contribute to this limited clinical efficacy, for example the inability of DCs to home into lymph nodes [6] or if arrived, their inability to interact with appropriate T cells [7]. Patients that do mount immune responses detectable in peripheral blood upon DC immunization might still be unable to mount clinical responses because effector T cells are unable to home into the tumor site [8] or once arrived, to exert their function in a heterogeneous environment characterized by severe immune suppression [9], [10].

IL-12 is thought to directly polarize T cells and NK cells into effector subsets that secrete IFN-γ and thus improve antitumor immunity, while secreted IL-10 is believed to induce undesirable tolerance by directly inhibiting the secretion of IL-12 and thereby type 1 T cell polarization [11], [12]. The ability of type 1 polarized DCs to produce IL-12 during antigen presentation is mediated through the concomitant activation of pathogen recognition receptors by binding of pathogen-associated molecular patterns during priming or by helper T cell dependent CD40L-mediated secondary stimulation [13]. To mimic this in vitro, there seems to be a consensus to use either the Toll-like receptor (TLR) 3 ligand polyinosinic:polycytidylic acid (Poly(I:C)) [14] or the TLR4 ligand lipopolysaccharide [15], [16] or its detoxified version monophosphoryl lipid A (MPL) [17]. These TLR ligands are sometimes combined with a TLR7/8 ligand [18] but more commonly with IFN-γ, which inhibits concomitant secretion of IL-10 [19] and enable a secondary burst of IL-12 following CD40 ligation [14], [20], [21]. Alternatively, other investigators are using clinical grade products such as prophylactic vaccines, Ribomonyl or OK432 that presumably activate either TLR3 or TLR4 and thereby induce similar maturation of DCs [22], [23], [24].

PGE2 combined with inflammatory cytokines have mostly been employed to improve CCR7-mediated migration of DCs [25], [26], but PGE2 has also been shown to induce the expression of the co-stimulatory molecules CD70, 4-BBL and OX40L, thus amplifying proliferation of activated T cells [27]. However, PGE2 is considered as a potent inhibitor of IL-12 [28], [29], which in turn inhibits functional properties of activated, antigen-specific T cells [14], [30]. In addition, cell surface and soluble CD25 as well as indoleamine 2,3-dioxygenase (IDO) is induced by PGE2 and expressed by tumor-associated DCs providing additional mechanisms of T cell inhibition [31]. A few groups have combined PGE2 with TLR based maturation of DCs and documented an ability of DCs to simultaneously migrate and produce IL-12 [18], [22], [32].

In the current study, sDCs were compared to DCs matured with two type 1 polarizing maturation cocktails previously described [14], [17], and a mixed cocktail containing MPL and IFN-γ together with PGE2. Our results confirm the T helper type 1 (Th1) polarizing ability of alpha-type-1-polarized DCs (αDC1s) and mDCs (MPL + IFN-γ). These DCs are transiently incapable of CCL21-directed migration, likely due to their increased secretion of CCL19 that mediates internalization of CCR7. The mixed cocktail of MPL, IFN-γ and PGE2 resulted in DCs that were intermediate between standard and polarized DCs both in terms of IL-12 secretion and migratory ability. However, strikingly these DCs expressed significantly more of the inhibitory molecules PD-L1 and CD25 compared with standard DCs. Thus, even though they are able to produce some IL-12, they are likely not an optimal replacement of the current golden standard for DC maturation.

Section snippets

Generation and maturation of monocyte-derived dendritic cells

Leukapheresis was performed on healthy blood donors after informed consent followed by separation using elutriation (Terumo BCT, Lakewood, CO) and subsequent freezing of CD14+ monocytes that were 80–90% pure as determined by flow cytometry (data not shown). Thawed monocytes were >99% live and cultured for five days in Nunclon culture plates (Ø 21 cm) at 5·105 cells/ml in CellGro DC medium (CellGenix, Freiburg, Germany) supplemented with 1000 U/ml GM-CSF and 250 U/ml IL-4 (both CellGenix).

Phenotype, viability and yield of mature DCs

Clinical grade DCs were generated in large culture plates from six independent, healthy donors and frozen in aliquots. The expression of stimulatory and inhibitory maturation markers of DCs were measured and scored according to their ratio with isotype-matched antibodies. Although donor variability occurred, statistical differences were observed between sDCs and polarizing maturation cocktails (αDC1, mDC and mpDC) (Fig. 1 and Supplementary Fig. S1). The sDCs showed the highest expression of

Discussion

In this study we compared clinical grade polarized and standard matured DCs in terms of phenotype, cytokine secretion profile, ability to promote type 1 polarization of CD4+ and CD8+ effector T cells and ability to migrate toward CCL21. These are all key aspects of optimally produced DCs for cancer immunotherapy [39]. The mDCs developed by Ten Brinke et al. [17] and the αDC1s developed by Mailliard et al. [14] both secreted high levels of IL-12 during maturation and following CD40L ligation.

Acknowledgments

Charlotte Vajhøj is acknowledged for laboratory assistance. The studies were supported by grants from Aase and Ejnar Danielsens Foundation, The Danish Cancer Society, The Lundbeck Foundation, and The Danish Council for Independent Research, Technology and Production Sciences, Grant# 09-070021.

Conflict of interest statement: No competing conflict of interest exists for any of the authors.

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