Profuse and sustained production of CXCL-9/MIG by a clinical grade dendritic cell vaccine: A feature to provide natural killer cell-dependent boosting for Th1 polarization

Karin Gustafsson,1 Madeleine Persson,2 Linda Bergqvist,3 John Alder,3 Jenny Nyström,2 Bengt Andersson,3 Vincenzo d´Angelo,4and Alex Karlsson-Parra3

Department of 1Neuroscience and Physiology, 2Medicine, 3Microbiology and Immunology, Göteborg University, Sweden, 4Neuroscience, Casa Sollievo della Sofferenza hospital, San Giovanni Rotondo, Italy.

Short title: Sustained production of CXCL9 by a human DC-vaccine

Key words: Dendritic cells, vaccine, NK cell, CXCL9, IFN-γ

* Corresponding author. Tel +46 31 342 47617 ; fax: +46 31 826 791.

E-mail address: (A. Karlsson-Parra)

Abstract

Recruitment of circulating natural killer (NK) cells into inflamed lymph nodes is known to provide a potent, interferon-γ (IFN-γ) dependent, boost for Th1-polarized immune responses in mouse models. Such NK cell recruitment into draining lymph nodes is induced by certain subcutaneously injected adjuvants, including mature vaccine dendritic cells (DCs) in a CXCR3-dependent manner. We here demonstrate that monocyte-derived immature human DCs stimulated with polyinosinic:polycytidylic acid (p-I:C), interferon (IFN)-α/tumor necrosis factor (TNFα)-/interleukin-1β(IL-1β)/IFN-γ, “α-type-1-polarized DC” (αDC1) exhibit profuse production of the CXCR3-ligand CXCL9/MIG after withdrawal of maturation stimuli. In sharp contrast, no measurable CXCL9/MIG was produced by DCs after maturation with the current “gold standard” maturation protocol for human DC-based cancer vaccines consisting of TNF-α, IL-1β, IL-6 and PGE2 (PGE2-DC). In contrast, PGE2-DC preferentially produced the Th-2 and regulatory T cell (Treg)-attracting chemokines CCL17 and CCL22, while only marginal levels were produced by αDC1. Functional studies in vitro demonstrated that αDC1-supernatants actively recruited NK cells, and that this recruitment was blocked by adding anti-CXCL9-antibodies to the αDC1-supernatant. Finally, αDC1, but not PGE2-DC significantly activated cocultured autologous NK cells as determined by CD69 expression and intracellular IFN-γ production. These novel findings indicate that fully matured and subsequently injected human αDC1-based clinical grade vaccines have the potential to recruit and activate NK cells during their arrival to draining lymph nodes and that this feature may be of relevance for efficient priming of tumor-specific Th1 cells and CTLs.

Introduction

Dendritic cells (DCs) play a central role in the initiation and regulation of innate and adaptive immune responses and have increasingly been applied as experimental vaccines for cancer patients (1, 2). Upon encountering certain pathogen-associated antigens, but also in response to pro-inflammatory cytokines, DCs become activated and start to secret inflammatory mediators and up-regulate co-stimulatory molecules as well as major histocompatibility complex class I and II molecules presenting processed antigens (3). Another functional consequence of activation in vivo is the up-regulation of the lymph node-directing chemokine receptor CCR7, enabling them to migrate in response to gradients of first CCL21 and then CCL19 to the T cell zone of the draining lymph node (4-6). These unique features of DCs are increasingly exploited for the design of DC-based vaccines in immunotherapy.

Most commonly, DCs are obtained through in vitro differentiation of monocytes in the presence of granulocyte-macrophage colony stimulating factor (GM-CSF) and interleukin (IL)-4, followed by exposure to inflammatory signals to induce final maturation (7). Additional signals provided by prostaglandin E2 (PGE2 ) have been shown to be crucial for inducing a substantial chemotactic response in vitro to lymph node-derived chemokines (8, 9). DC-based vaccines matured with a clinical grade “gold standard” maturation cocktail consisting of TNF-α, IL-1β, IL-6 and PGE2show impaired IL-12 production (10, 11) but has been believed to retain the property for priming Th1 CD4+ T cells and CD8+ CTLs as assessed by different in vitro assays (11).Recently however, the presence of IL-12 during CTL-priming by DCs in vitro was found to be mandatory in order to enable direct recognition of tumor cells expressing the relevant MHC class I/peptide complex (12). In line with these findings, IL-12 p70 secretion has been shown to dramatically enhance the ability of DCs to induce tumor-specific CTLs and promote tumor rejection in therapeutic mouse models (13, 14). Taken together, these observations thus provide a reasonable explanation for the difficulty in consistently generating tumor regression in patients treated with antigen-loaded DCs that have been matured with the PGE2-containing DC-maturating protocols.

IFN-gamma has been shown to facilitate the production of IL-12p70 by DCs primed by microbial products or inflammatory cytokines such as TFN-alpha and IL-1 beta (15), a feature that we recently tried to capitalize in the generation of clinical grade DCs (16). However, addition of IFN-gamma to the standard PGE2-containing maturation protocol was shown to inhibit PGE2-induced membrane expression of CCR7 and to reduce DC-migration towards lymph node chemokines (16).Recently, Mailliard et al (17) reported that the inclusion of IFN-apha and polyinosinoine: polycytidylic acid ( p-I:C) to the “original” IL12p70-inducing cytokine cocktail, composed of IL-1 beta, TNF-alpha and IFN-gamma (15), allows for the generation of clinical grade DCs (alphaDC1) with high migratory function towards lymph node chemokines combined with a strong ability to produce IL-12p70.

The responsiveness of naïve antigen-specific T cells to DC-derived IL12p70 is tightly regulated by the transcription factor T-bet, the master regulator of TH1 differentiation (18) and also CTL development (19), which is maximally induced by T cell receptor signals in conjunction with IFN-gamma (18). In vivo, the main source of IFN-gamma during the initial priming of naïve antigen-specific CD4 and CD8 T cells is supposed to be provided by NK cells within the draining lymph node. Studies have shown that circulating NK cells enter lymph nodes draining sites of immunization or infection (20, 21). The study of Martin-Fontecha et al (20)specifically demonstrated that NK cells enter lymph nodes draining sites of certain subcutaneously injected adjuvants, including mature antigen-loaded DCs, in a CXCR3-dependent manner where they provide an early source of IFN-gamma that was necessary for TH1 polarization. This unique function of certain adjuvants suggests that, for vaccines that rely on TH1 responses, adjuvants could be selected according to their capacity to induce the recruitment of NK cells in antigen-stimulated lymph nodes. In light of this important finding we investigated the ability of mature alphaDC1 to produce CXCR3-lignads.

Our data show that alpha DCsexhibit a profuse production of the CXCR3-ligand CXCL9 after withdrawal of maturation stimuli, thus at a time point when they are ready to use. In contrast, no measurable CXCL9 was produced by DCs after maturation with the current PGE2-containing “gold standard” protocol. Functional studies in vitro further demonstrate that alphaDC1-supernatants actively recruit NK-cells in a CXCL9-depentent manner. Finally, alphaDC1 were found to induce IFN-gamma production in cocultured autologous NK cells. These novel findings indicate that fully matured and subsequently injected human alphaDC1-based clinical grade vaccines have the potential to recruit and activate NK-cells during their arrival to draining lymph nodes and that this feature may be relevant for efficient Th1 priming by DC based vaccines.

Material and Methods

Culture Medium and Reagents

The medium used throughout was AIM-V (Invitrogen, Paisley, UK). The following factors were used to generate DCs: GM-CSF, IL-4, IFN-α, TNF-α, IL-1β, IL-6, PGE2 and IFN-γ (R&D Systems, Abingdon, UK), anti-CXCL9 (Peprotech, London, UK), and p-I:C (Sigma–Aldrich, Stenheim, Germany)

Generation of Immature Dendritic Cells

Peripheral blood mononuclear cells (PBMCs) obtained from healthy blood donors were isolated on density gradients, with Lymphoprep (Nycomed, Oslo, Norway). The use of human blood-donor cells was approved by the Human Research Ethics Committee at the Sahlgrenska Academy, Göteborg University. Isolated PBMCs were resuspended in AIM-V medium, plated in 24-well plastic culture plates at 2.5×106 cells per well and allowed to adhere for 2hours. Non-adherent cells were removed and the remaining adherent cells, were cultured in AIM-V with addition of GM-CSF and IL-4 (both 1,000U/mL) for 5 days, in order to obtain immature DCs.

Maturation of Dendritic Cells

After 5 days of culture in GM-CSF and IL-4, the culture media was supplemented with the “gold standard” maturation cocktail consisting of IL-1β (25ng/mL), TNF-α (50ng/mL), IL-6 (10ng/mL) and PGE2 (1μg/mL). Alternatively, DCs were matured by adding IFN-α (3,000 U/mL), IFN-γ (1,000 U/mL), IL-1β (25 ng/mL), TNF-α (50 ng/mL), p-I:C (20µg/mL) to obtain αDC1. When indicated, different combinations of the components included in the αDC1-maturation cocktail were added. The cells were cultivated for a further 24 hours. Cells were then washed and harvested for RNA isolation and when indicated, cells were washed twice to remove residual cytokines and replaced in the well and cultured in fresh AIM-V medium for a further 24 hours. Cells were then harvested for functional assessment and supernatants were collected and stored in –70 °C for future use in migration assays and chemokine measurements by ELISA.

Flow Cytometry Analysis

Staining of cells was performed with phycoerytrin (PE)-conjugated anti-CD56, allophycocyanin (APC)-conjugated anti-CD3 fluorescein isothiocyanate (FITC)-conjugated anti-CD14, anti-IFN-γ, and peridin chlorophyll protein (PerCP)-conjugated anti-CD69 (all from BD Biosciences, San Diego, CA). Fluorochrome-conjugated antibodies were added to DCs alone or DCs cocultured with autologous or allogeneic mononuclear cells, incubated at 4°C for 20min and washed. All flow cytometry data were acquired on a FACS-Calibur cytometer (BD Biosciences) with CellQuest software (BD Biosciences) and analyzed with FloJo software (Tree star, Inc).

Real-Time PCR

RNA was prepared from dendritic cells as described above, using the Qiagen mini kit (Qiagen, Solna, Sweden). The concentration and quality of the RNA were evaluated by Agilent 2100 Bioanalyzer (Nano LabChip, Agilent Technologies, Dalco Chromtech AB, Stockholm, Sweden). Reverse transcription of RNA and real-time PCR analysis were performed as previously described (22). In brief, 500 ngRNA was transcribed into cDNA using avian myeloblastosis virus reverse transcriptase. The mRNA level of each target gene was quantified by real-time PCR on an ABI Prism 7900 Sequence Detection System (Taqman; Applied Biosystems, Foster City, CA, USA) using low-density array (LDA). The LDA card included the target genes and endogenous controls listed in table 1.

Samples were run in duplicates of 50 ng and the comparative ΔΔCT-method of relative quantification was used to calculate for differences in gene expression between the control and the two treatment groups. GAPDH, β-actin and HPRT were used as endogenous controls to correct for variation in sample loading and efficiency of the amplification reaction.

Migration assays

DCs matured with either the αDC1 or the PGE2-DC maturation cocktail for 24hours were washed twice and replaced in their wells in fresh AIM-V medium. After 24 hours, supernatants were collected and stored in –70 °C for future use in migration assays. Chemotaxis of NK cells toward chemokines produced by DCs matured with the indicated stimuli was tested by using a transwell assay. Briefly, lower chambers of transwell plates (5.0μm pore size, BD Biosciences, Erembodegen, Belgium) were filled with 500μL of supernatants. 500 μL of medium only was used as a control. To one well containing αDC1-supernatant, anti-CXCL9 was added. About 1×106 PBMC were added in 200μL AIM-V medium in the upper chamber, and cells were incubated for 90min. Cells in the lower chambers were harvested and stained with PE-conjugated anti-CD56 and APC-conjugated anti-CD3. NK cells were subsequently defined and counted using flow cytometry. Specific migration was calculated by subtracting the mean number of spontaneously migrated cells (migration to medium only) from the number of cells that migrated in response to the chemokine containing supernatants.

Natural killer cell Activation

To evaluate DC-mediated IFN-γ production and CD69 expression by NK cells PBMCs were incubated with autologous monocyte-derived DCs (mononuclear cell: DC ratio 5:1) in AIM-V medium supplemented with 5 % autologous plasma in the presence of10 µg/mL brefeldin A (GolgiPlugg, Sigma). DCs used in this experiment were stimulated with either the “gold standard” maturation cocktail or the “αDC1” maturation cocktail for 24 hours, washed twice and replaced in the well and cultured in fresh medium for a further 24 hours before harvested and added to the mononuclear cells. After 6 hours of coculture, cells were harvested, fixed, permeabilized with the Cytofix/Cytoperm reagent (BD Biosciences), and stained with PE-conjugated anti-CD56, APC-conjugated anti-CD3, PerCP-conjugated anti-CD69 and FITC-conjugated anti-IFN-γ.

Chemokine Detection

For measurement of chemokines, 24 hours culture supernatants from previously washed mature DCs were collected and stored at –70°C until chemokine concentrations were determined by specific ELISAs. The commercially available ELISAs for CXCL9, CXCL10, CXCL11, CCL17, and CCL22 (R&D system, Minneapolis, USA) were performed according to the manufacturer’s instructions. To get insight to the kinetics of the chemokine production supernatants were collected after 12 hours, cells were then washed and replaced for a further 12 hours before final supernatant were collected. The lower limits of detection were X pg/mL for CXCL9, X pg/mL for CXCL10, X pg/mL for CXCL11, X pg/mL for CCL17 and X pg/mL for CCL22

Statistical analysis

The statistical significance of differences between experimental samples was determined using the Student's t-test for paired samples.

Results

Mature alphaDCs produce extremely high levels of CXCL9 in a sustained fashion.

Several studies have been conducted in order to evaluate chemokine production in different types of DCs, including monocyte-derived DCs. I most of these studies, however, FCS have been included during the generation of fully mature DCs and chemokine production has been measured during the course of maturation. To the best of our knowledge no reports on chemokine production by fully mature “effector” DCs (generated in clinically relevant serum-free medium and subsequently washed) has earlier been reported. Initial real-time PCR analysis revealed that mRNA expression of the CXCR3-ligands CXCL9/MIG, CXCL10/IP-10 and CXCL11/TARC were highly expressed in alphaDCs after incubation in the maturation-cocktail for 24 h compared to PGE2-DC (data not shown). In order to verify if protein production from that time point (after maturation for 24 h) and onward correlated with mRNA data, standard ELISA was used to measure the amount of chemokine production in 24 h culture supernatants of previously washed mature DCs. Measurement of chemokine production during maturation was further conducted in order to get insight to the kinetics of chemokine production. As shown in Fig. 1 the most remarkable finding was the extremely high and sustained production of CXCL9/MIG by alpha DCs. In contrast, PGE2-DC preferentially produced the Th-2 and regulatory T cell (Treg)-attracting chemokines CCL17 and CCL22, while only marginal levels were produced by αDC1. In order to delineate factors of importance for CXCL9/MIG production, each component was abolished from the cocktail. As seen in Fig. 2 IFN-gamma was the most critical factor in the maturation cocktail for the induction of high production of MIG. Maturation with one single factor in no case induced significant production ( > 1 ng) of MIG in mature alphaDC1 (data not shown).

AlphaDC1-supernatants recruit NK cells in a CXCL9 dependent manner.

As shown in Fig. 3, alphaDC1-SN efficiently induced recruitment of NK cells in transwell experiments. Since circulating human NK cells has been shown to express functional CXCR3, we tested the possibility that the high level of CXCL9 in alphaDC1-SNs, which is one out of three known ligands for CXCR3, was responsible for the observed NK cell recruitment. Adding anti-CXCL9 antibodies to the alphaDC1-supernatats led to a marked reduction in the number of NK cells that migrated to the lower chamber.

AlphaDC1 activate cocultured autologous NK cells as determined by CD69 expression and intracellular IFN-gamma production.

As NK cells were shown to be recruited toward supernatants from alphDC1 we next investigated whether NK cells might become activated, as determined by expression of CD69 and intracellular IFN-γ, upon interaction with αDC1or PGE2-DC. Before coculturing with autologous PBMCs, previously washed mature DCs were replaced in the well and cultured in fresh medium for a further 24 hours. Activation of NK cells was analyzed by flow cytometry after 6 hours of coculture. Onlt DCs matured with the αDC1 maturation cocktail were able to induce substantial IFN-γ production (Fig. 4A) and upregulation of CD69 (Fig. 4B).

Discussion

Our results assign an additional novel feature of mature clinical grade (propagated in serum-free media) alphaDC1 that is of potential importance for their efficacy as carriers of anticancer vaccines. In addition to confirming recent data provided by Mailliard et al (17) as to phenotype, migratory capacity and IL-12 production, we have shown that alphaDC1, in contrast to DCs matured with the “gold standard” cocktail, exhibit a profuse production of the CXCR3-ligand CXCL9 after withdrawal of maturating stimuli. Supernatants collected 24 h after washing of mature alphaDC1 were further shown to efficiently recruit NK cells in a CXCL9-dependent manner. Finally, coculture of alphaDC1 with non-adherent autologous or allogeneic PBMCs induced IFN-gamma production by NK-cells in an IL-12-dependent manner.

NK cells have been to shown to control anti-tumor (23-25), allogeneic (26), xenogeneic (27) and virus-specific CTL (28) responses, and emerging evidence from rodent models further shows that DC-based cancer vaccines appear to collaborate with NK cells in the activation of tumor-specific CTL responses (29-31). In vitro studies have shown that one of the basic requirements for DC-NK interactions is proximity or direct cognate interactions (32, 33), but there has been little data demonstrating NK cells and DCs contact in vivo. Two recent papers have however focused on interaction between DCs and NK cells in lymph nodes draining subcutaneous inflammatory sites (20, 21). Injection of bone marrow−derived, lipopolysaccharide-matured DCs subcutaneously in mice was shown to induce a rapid increase in draining lymph node cellularity (20). Although the proportion of T and B cells was found to be similar in DC-draining and control lymph nodes, the frequency of NK cells was up to tenfold higher in DC-draining than in control lymph nodes (20). The increase in NK cell numbers peaked within 2 days, was dependent on the DC dose, and was not due to cell proliferation but rather to enhanced cell recruitment (20). A similar increase of draining lymph node NK cell number was shown after subcutaneous infection of mice with Leishmania major(21). Confocal microscopy of lymph node sectionsfrom L. major–infected mice revealed that NK cellsfrom infected mice tended to accumulate in the T cell area which is the site where antigen-loaded DCs are known to settle in order to communicate with recruited T cells entering via high endothelial venules (34).

Of fundamental importance, Martin-Fontecha et al (20) demonstrated that the chemokine receptor CXCR3 exhibited a nonreduntant role in the NK cell recruitment induced by injecting mature syngeneic DCs. The novel feature of high and sustained production of the NK cell-recruiting CXCR3-ligand CXCL9 by mature alphaDC1, as revealed in the present study, thus predict a similar NK cell recruiting capacity when injected into human patients.