Anti-Cancer Vaccine RNA-Activated Dendritic Cells As an Increases ...

The Journal of Immunology

Attenuated Expression of A20 Markedly Increases the Efficacy of Double-Stranded RNA-Activated Dendritic Cells As an Anti-Cancer Vaccine1 Karine Breckpot,2,3* Cindy Aerts-Toegaert,2* Carlo Heirman,* Uschi Peeters,* Rudi Beyaert,†‡ Joeri L. Aerts,* and Kris Thielemans* A20 is a zinc finger protein with ubiquitin-modifying activity. A20 has been described as negatively regulating signaling induced by the TNF receptor and TLR family in a number of cell types, including mouse bone marrow-derived dendritic cells (DCs). However, the expression and effect of A20 in activated human monocyte-derived DCs have not been previously evaluated. We report that DCs activated with the TLR3 ligand poly(I:C) up-regulate A20. Down-regulating A20 demonstrated its role in the functional activation of DCs. A20 down-regulated DCs showed higher activation of the transcription factors NF-␬B and activator protein-1, which resulted in increased and sustained production of IL-6, IL-10, and IL-12p70. We additionally silenced the immunosuppressive cytokine IL-10 and demonstrated that IL-10 inhibits T cell proliferation. We further demonstrated that A20 down-regulated DCs skew naive CD4ⴙ T cells toward IFN-␥ producing Th1 cells, a process which is dependent on IL-12p70 and which is unaffected by IL-10. Furthermore, A20 and/or IL-10 down-regulated DCs had an enhanced capacity to prime MelanA/MART-1 specific CD8ⴙ T cells. Finally, we demonstrated that potent T cell stimulatory DCs are generated by the simultaneous delivery of poly(I:C12U), A20, or A20/IL-10 small interfering RNA and Ag-encoding mRNA, introducing a one step approach to improve DC-based vaccines. Together these findings demonstrate that A20 negatively regulates NF-␬B and activator protein-1 in DCs and that down-regulation of A20 results in DCs with enhanced T cell stimulatory capacity. The Journal of Immunology, 2009, 182: 860 – 870.

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he discovery of tumor associated Ags (TAA),4 together with the growing knowledge about dendritic cells (DCs) and the integral part they play in the initiation and regulation of the immune response, has led to the concept of harnessing the immune system against tumors through vaccination of patients with autologous TAA-loaded DCs to restimulate/prime TAA-specific CTLs. To activate naive T cells and thus mount an effective anti-tumor immune response, DCs need to be fully mature. This can be achieved by several stimuli, among which are inflammatory sig-

*Laboratory of Molecular and Cellular Therapy, Department of Physiology-Immunology, Medical School of the Vrije Universiteit Brussel, Brussels, Belgium; †Unit of Molecular Signal Transduction in Inflammation, Department for Molecular Biomedical Research, Vlaams Instituut voor Biotechnologie, Ghent, Belgium; and ‡Department for Molecular Biology, Ghent University, Ghent, Belgium Received for publication February 13, 2008. Accepted for publication November 12, 2008. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1

This work was supported by grants to K.T. from the Belgian Science Policy’s Interuniversity Attraction Poles Program, from the Foundation against Cancer, from an Integrated Project and Network of Excellence sponsored by the European Union, and from the Fund of Scientific Research Flanders (FWO-Vlaanderen). C.A.-T., J.L.A., and K.B. are funded by the FWO-Vlaanderen.

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K.B. and C.A.-T. contributed equally to this work.

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Address correspondence and reprint requests to: Dr. Karine Breckpot, Laboratory of Molecular and Cellular Therapy, Department of Physiology-Immunology, Medical School of the ‘Vrije Universiteit Brussel’, Laarbeeklaan 103/E, 1090 Brussels, Belgium. E-mail address: [email protected]

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Abbreviations used in this paper: DC, dendritic cell; AP-1, activator protein-1; GUS, ␤-glucuronidase; siRNA, small interfering RNA; TAA, tumor-associated antigen; Treg, regulatory T cell. Copyright © 2009 by The American Association of Immunologists, Inc. 0022-1767/09/$2.00 www.jimmunol.org

nals, CD40 cross-linking, and TLR ligands (1– 4). Recently, the importance of TLR-mediated DC activation has been highlighted. It has been shown that TLR ligands are necessary and can synergize to fully activate DCs to overcome tolerogenic mechanisms as well as active inhibition by regulatory CD4⫹ T cells (Treg), factors which impede a productive anti-tumor immune response (5–10). In this regard, it has been demonstrated that DCs matured with the TLR3 ligand poly(I:C) or its clinical grade analog poly(I:C12U) are potent inducers of TAA-specific CTLs (11, 12). Engagement of TLR3 results in the activation of several transcription factors, among which activator protein-1 (AP-1), IFN regulatory factor (IRF) 3/7 and NF-␬B, which in turn results in the up-regulation of several cytokines, chemokines and cell surface molecules (13–17). A number of negative regulatory mechanisms are known to attenuate TLR signaling and maintain the immunological balance (18, 19). An example hereof is the zinc finger protein A20 (20 –23). By targeting the A20 gene in mice, it has been demonstrated that A20 is essential for restricting TNF- and TLRinduced NF-␬B and AP-1 activation signals in macrophages, and that mice deficient in A20 (Tnfaip3⫺/⫺) develop profound autoimmunity, demonstrating the important role of A20 in innate and adaptive immune responses (24, 25). A20 was originally characterized as a TNF-inducible gene in HUVECs and its expression is under the control of NF-␬B (26 – 28). A20 interacts with several proteins of the TLR3 signaling pathway, i.e., Toll IL-1 receptor domain containing adapter-inducing IFN-␤, receptor-interacting protein, TNF receptor-associated factor 6, NF-␬B essential modulator, and I␬B-kinase (21–23, 25, 27, 29, 30). Recent studies demonstrated that A20 is an ubiquitinediting enzyme with de-ubiquitinase activity in the amino-terminal region and ubiquitinase activity in the zinc finger domain of the carboxy-terminal region. It is through this dual ubiquitin-editing

The Journal of Immunology function of A20 that it can modulate down-regulation of NF-␬B signaling in response to different stimuli including TNF, IL-1, and TLRs (21, 25, 31, 32). In contrast to previous data obtained with A20 overexpression, small interfering RNA (siRNA)-mediated down-regulation of A20 did not affect the TLR3-induced IRF3 pathway, which was shown to be regulated by another de-ubiquitinating enzyme deubiquinating enzyme A (33). Although A20 has been described as an effective inhibitor of NF-␬B function in many cell types, little is known about the role of A20 in human DCs (34 – 40). Therefore, we examined the expression of A20 in human monocyte-derived DCs activated with dsRNA and evaluated whether down-regulation of A20 through the mechanism of RNA interference results in DCs with enhanced T cell stimulatory capacity.

861 System (Applied Biosystems) using the following parameters: 2 min at 50°C followed by 10 min at 95°C and 40 cycles of 15 s at 95°C and 1 min at 60°C. A20 primers were as follows: F, 5⬘-CGAGAAGTCCGGAAGCTTGT-3⬘; R, 5⬘-CATGTACTGAGAAGTTTCATGCA-3⬘; Probe FAM-CAATTGCCG TCACCGTTCGTTTTCA-TAMRA. Relative mRNA abundance was normalized to the expression of ␤-glucuronidase (GUS) (43). Primers and probes were purchased from Eurogentec.

Western Blot Cells were harvested and lysed 0, 1, 6, 24, and 48 h after maturation. Cell lysates were subjected to SDS-PAGE, followed by transfer of the proteins to a nitrocellulose membrane. Membranes were blocked with 5% nonfat milk and probed for 1 h at room temperature with 1 ␮g/ml anti-human A20 Ab (clone 59A426; eBioscience) and 0.1 ␮g/ml anti-actin Ab (Santa Cruz Biotechnology). The blot was developed by chemiluminescence (ECL; Pierce) and exposed to an autograph (Fuji). The signal density was determined using Photocapt MW software (Vildber Lourmat).

Panomics Procarta Transcription Factor Plex Assay

Materials and Methods In vitro generation of DCs PBMC isolated from leukapheresis products of healthy donors were used as a source of DC precursors. Both the generation and the cryopreservation of immature DCs have been described previously (41). Briefly, CD14⫹ monocytes were enriched in a semi-closed culture system using Cell Factories (Nunc). The adherent fraction was cultured in DC-complete medium, i.e., RPMI 1640 medium (Cambrex) supplemented with 1% heat-inactivated AB serum (PAA Laboratories), 20 ␮g/ml gentamicin, 1000 U/ml GM-CSF (prepared in house) and 500 U/ml IL-4 (BruCells). The cells were refreshed on day 2 and 4 of culture. On day 6, DCs were harvested and cryopreserved until further use.

DC maturation Maturation of DCs was performed at a cell density of 5 ⫻ 105/ml DCcomplete medium using 100 ng/ml LPS and 450 U/ml TNF-␣ (PeproTech), 12.5 ␮g/ml poly(I:C) (Amersham), a mixture of inflammatory cytokines containing 100 U/ml IL-1␤, 1000 U/ml IL-6 (both prepared in house), 450 U/ml TNF-␣ and 1 ␮g/ml Prostaglandin E2 (Pharmacia) or electroporation of DCs with 1 ␮g poly(I:C12U) (Ampligen; Hemispherx Biopharma).

Transfection of DC with siRNA siRNA. The siRNA sequence used for targeted down-regulation of human A20 and IL-10 was designed and synthesized by Invitrogen (stealth siRNA). The sense strand of the siRNA targeting A20 and IL-10 are 5⬘GGAGUCUCUCAAAUCUCAGGAAUUU-3⬘ and 5⬘-CCAAACCAC AAGACAGACUUGCAAA-3⬘, respectively. An irrelevant siRNA with random nucleotides and no known specificity was used as a control. Lipofection of siRNA into DCs. The delivery of siRNA by lipofection was performed as described previously (42). In brief, 12.5 ␮l of 20 ␮M annealed siRNA was incubated with 5 ␮l of Lipofectamine 2000 (Invitrogen) in a volume of 500 ␮l of serum free Optimem (Invitrogen) at room temperature for 20 min. This mixture was added to 2.5 ⫻ 106 immature DCs, which were cultured at a cell density of 1 ⫻ 106/ml DC-complete medium without antibiotics. Twenty-four hours later the lipofected DCs were matured with poly(I:C). Additional experiments were performed 1, 6, 24, or 48 h after maturation. Electroporation of siRNA into DCs. Immature DCs were coelectroporated with Melan-A/MART-1 mRNA, poly(I:C12U) and siRNA by an optimized protocol (11). Briefly, after two wash steps, 4 ⫻ 106 DCs were resuspended in a final volume of 200 ␮l Optimix Solution B (EQUIBIO, Ashford, U.K.) containing 20 ␮g mRNA, 1 ␮g poly(I:C12U) and 120 pmol siRNA. Electroporation was performed in a 4 mm gap cuvette using the EquiBio EasyjecT Plus apparatus (EquiBio) and the following electroporation conditions: voltage 300 V, capacitance 150 ␮F, and resistance 99 ⍀, resulting in a pulse time of 5 ms. Immediately after electroporation, cells were diluted to a density of 1 ⫻ 106/ml in DC-complete medium and left to recover for 1 h.

Real-time RT-PCR DCs were either left unmodified or were lipofected with irrelevant or A20 siRNA. Twenty-four hours later these cells were activated using 12.5 ␮g/ml poly(I:C). Cells were harvested 0, 1, 6, and 24 h after activation and RNA was prepared using the SV Total RNA Isolation System (Promega) followed by first-strand cDNA synthesis using oligo(dT) primers and the Revert Acid H Minus First Strand cDNA Synthesis kit (Fermentas). Samples were subjected to real-time RT-PCR analysis on an ABI PRISM 7700 Sequence Detection

To evaluate the profile of the transcription factors AP-1, IRF, and NF-␬B in mature irrelevant or A20 siRNA lipofected DCs, we used a customized Procarta Transcription Factor Plex from Panomics. The assay was performed using 2 and 0.5 ␮g of nuclear extract, which was prepared with the Panomics’ nuclear extraction kit following the manufacturer’s instructions. The nuclear extracts were incubated with 10 ␮l of probe mix containing biotin-labeled double stranded oligonucleotides (transcription factor binding site). Transcription factors bound to these oligonucleotides were recovered, the biotin-labeled oligonucleotides eluted, denatured, and subsequently hybridized to beads coated with the complementary oligonucleotides. Next, the beads were incubated with a PE-conjugated streptavidin. Sample analysis was performed with the Bioplex 200 System (Bio-Rad).

Quantification of cytokine production The presence of IL-6 (eBioscience), IL-10 (R&D Systems), IL-12p70, and IFN-␣ (Bender MedSystems) in supernatants was examined by ELISA according to the manufacturer’s instructions.

Flow cytometric analysis Cells were stained on ice in PBS containing 1% BSA, 0.02% NaN3, and 10% normal goat serum to block Fc receptors for 30 min. Allophycocyanin- or PE-conjugated mAbs against CD25, CD40, CD80, CD83, CD86, HLA class I, and CCR7 were purchased from BD Pharmingen. The antiHLA-DR Ab (clone L243, prepared in house) was affinity purified, biotinylated, and finally detected with PE-conjugated streptavidin. To determine cell viability, cells were collected in 250 ␮l of BD FACSFlow containing 50 ␮g/ml propidium iodide. Data were collected on a FACSCanto flow cytometer and analyzed using Diva software (BD Biosciences).

Allogeneic MLR Graded numbers of DCs were cocultured with 2 ⫻ 105 T cell enriched cells in triplicate in round-bottom 96-wells in RPMI 1640 medium supplemented with 1% human AB serum for 5 days. The last 16 h of the coculture were performed in the presence of 1 ␮Ci [3H-methyl]thymidine (Amersham). Cells were harvested on glass filter paper and thymidine uptake was quantified by liquid scintillation counting (Wallac Microbeta). To neutralize IL-10 bioactivity, we used 0.5 ␮g/ml monoclonal anti-human IL-10 Ab (clone 25209; R&D Systems).

CD4⫹ T cell polarization Coculture. Naive CD4⫹ T cells were isolated from PBMC by immunomagnetic sorting using the CD4⫹ T cell isolation kit II (Miltenyi Biotec), after which CD45RA⫹ T cells were positively selected using anti-CD45RA coated beads (Miltenyi Biotec). These allogeneic CD4⫹ CD45RA⫹ T cells (5 ⫻ 104) were cocultured with siRNA lipofected DCs (1 ⫻ 104) in roundbottom 96-wells (36⫻) in IMDM medium (Invitrogen) supplemented with 1% human AB serum. Where indicated 0.5 ␮g/ml monoclonal anti-human IL-10 Ab or anti-human IL-12 Ab (clone 24910; R&D Systems) was added. Proliferation. See section allogeneic MLR. Analysis of the CD4⫹ T cell phenotype. CD4⫹ T cells were harvested 6 days after priming and were divided as follows: one-third were not restimulated and served for evaluation of the Treg phenotype and two-thirds were restimulated at 1 ⫻ 106/ml with anti-CD3/CD28 beads (Dynal, Invitrogen) for 24 h either or not in the presence of 10 ng/ml Brefeldin A (Golgi Plug;

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RNA INTERFERENCE IN THE TLR3 PATHWAY

FIGURE 1. A20 down-regulation in human DCs by siRNA. Immature DCs were matured for 0, 6, 24, or 48 h with poly(I:C), after which the DCs were harvested for real-time RTPCR determination of A20 and GUS mRNA (control gene) (a) or A20 and ␤-actin protein (control) detection in Western blot (b). RNA interference was applied to suppress A20 expression. Therefore, immature DCs were lipofected with irrelevant or A20 siRNA for 24 h. Subsequently, these cells were not matured or matured for 1, 6, or 24 h with poly(I:C), after which immature and mature DCs were used for real-time RT-PCR determination of A20 and GUS mRNA (0 and 1 h post activation) (c) or A20 and ␤-actin protein detection in Western blot (24 h post activation) (d). a and c represent summary data of three independent experiments. The horizontal line indicates the mean of the three experiments. b and d are representative for three independent experiments. irr., Irrelevant; iDC, immature DC; mDC, mature DC.

BD Biosciences). Supernatants of restimulated cells (no Brefeldin A) were harvested and analyzed by ELISA for the presence of IL-4 (Pierce), IL-10 (R&D Systems) and IFN-␥ (Endogen) following the manufacturer’s instructions. Cells that were not restimulated were harvested and stained for the surface markers PE-cyanin7 conjugated CD4, PE conjugated CD25, and PE-cyanin5.5 conjugated CD127 (eBioscience), after which the cells were permeabilized using the FoxP3 Fix/Perm Buffer Set (eBioscience) and subsequently stained with allophycocyanin conjugated FoxP3 (eBioscience). Cells restimulated in the presence of Brefeldin A were harvested and stained with PE-cyanin7 conjugated CD4 and PE-cyanin5.5 conjugated CD127, after which they were permeabilized with the BD Cytofix/Cytoperm Kit (BD Pharmingen) (44) and stained with PE conjugated IFN-␥ (eBioscience), FITC conjugated IL-4 (R&D Systems), and Alexa Fluor 647 conjugated IL-17 (eBioscience). Nonbinding isotype-matched Abs (BD Pharmingen) were used as controls. Fluorescence analysis was performed with a FACSCanto flow cytometer using FACSDiva software for data analysis (BD Biosciences).

immunodominant peptide (prepared in house), in combination with a FITClabeled anti-CD8 mAb (BD Pharmingen), was used for extracellular staining of in vitro primed CD8⫹ T cells. As a negative control, a PE-labeled HLA-A2 tetramer loaded with MAGE-A3 peptide, amino acid 271–279 (FLWGPRALV) (prepared in house) was used. All stainings were performed at 4°C for 15 min and evaluated by flow cytometry.

Synthetic peptides and peptide pulsing

To our knowledge there are no prior publications on the expression of A20 in human DCs. Therefore, we decided to follow A20 mRNA and protein expression upon maturation with poly(I:C) using real-time RT-PCR and Western blotting, respectively. We demonstrated the presence of A20 mRNA in immature DCs and its up-regulation upon maturation with poly(I:C). This up-regulation was already observed 6 h (3.7 ⫾ 1.0) after activation and reached a peak at 24 h (5.4 ⫾ 0.8), after which the A20 mRNA decreased to, on average, 2.7 ⫾ 0.3-fold when compared with the levels observed in immature DCs (n ⫽ 3, Fig. 1a). A similar pattern was observed for the A20 protein up-regulation, which was on average 3.1 ⫾ 1.2-, 3.8 ⫾ 1.5- and 2.8 ⫾ 0.9-fold increased 6, 24, and 48 h after DC activation, respectively (n ⫽ 3, Fig. 1b). We further demonstrated that the A20 protein up-regulation observed upon activation with poly(I:C) was similar to that observed upon activation with LPS/TNF-␣, which was more pronounced than the A20 protein up-regulation observed upon activation with a mixture of inflammatory cytokines (n ⫽ 2, data not shown).

The lyophilized synthetic optimized Melan-A/MART-1 immunodominant epitope (Melan-A peptide, A27L, amino acid 26 –35, sequence ELAGIGILTV; Thermo Electron) presented in the context of HLA-A2 was dissolved in 100% DMSO (Sigma) at 20 mg/ml, further diluted in 10 mM of acetic acid to a final concentration of 2 mg/ml, and stored in aliquots at ⫺20°C. DCs were loaded at a cell density of 2 ⫻ 106/ml in serum-free RPMI 1640 medium with 10 ␮g/ml peptide for 2 h at 37°C.

Induction of HLA-A2 restricted Melan-A/MART-1 specific CD8⫹ T cells T cells and DCs were obtained from healthy HLA-A2⫹ donors. siRNA lipofected and Melan-A/MART-1 peptide loaded DCs or DCs coelectroporated with Melan-A/MART-1 encoding mRNA, poly(I:C12U), and A20 or A20/ IL-10 siRNA (5 ⫻ 105) were added to autologous CD8⫹ T cells (5 ⫻ 106) purified using MACS (Miltenyi Biotec) in 5 ml of IMDM supplemented with 1% human AB serum in a 6-well. CD8⫹ T cells were restimulated weekly with the same stimulator DCs as used in the primary stimulation. Seven days after the third stimulation, CD8⫹ T cells were harvested and their Ag specificity was analyzed. A PE-labeled HLA-A2 tetramer (10 nM) containing the Melan-A

Statistical analysis Where applicable and depending on the type of experiment, we conducted a two-tailed paired t test or a Kruskal-Wallis test. Differences were considered significant at p ⬍ 0.05. Statistical significance is indicated in the figures.

Results A20 expression in monocyte-derived DCs and down-regulation of A20 by RNA interference


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FIGURE 2. Enhanced up-regulation of the transcription factors AP-1 and NF-␬B in activated DCs lipofected with A20 siRNA. Immature DCs were lipofected with irrelevant or A20 siRNA, followed by maturation with poly(I:C) 24 h after lipofection. Cells were harvested 6, 24, and 48 h after maturation and analyzed for the presence of AP-1 and NF-␬B. The scatter chart represents the relative up-regulation of AP-1 (a) and NF-␬B (b) in DCs lipofected with A20 siRNA as opposed to DCs lipofected with irrelevant siRNA. The mean of the three experiments is indicated as a horizontal line. The column graph represents the mean ⫾ SD of the data collected for all three donors for DCs lipofected with irrelevant or A20 siRNA 6 h after maturation.

Next, immature DCs were left untreated or were lipofected with irrelevant or A20 siRNA. These cells were harvested as immature DCs or as DCs activated for 1, 6, or 24 h with poly(I:C). A20 mRNA levels were evaluated in real-time RT-PCR, demonstrating similar A20 mRNA up-regulation in unmodified DCs and DCs lipofected with irrelevant siRNA (data not shown). In contrast, DCs lipofected with A20 siRNA exhibited reduced levels of A20 mRNA at all evaluated time points, with a respective reduction of 4.3 ⫾ 1.7 (76.7 ⫾ 9.1%), 5.1 ⫾ 1.1 (80.4 ⫾ 4.7%), 2.9 ⫾ 1.0 (65.5 ⫾ 12.6%) and 2.5 ⫾ 0.3 (60.2 ⫾ 4.1%) compared with irrelevant siRNA lipofected cells (n ⫽ 3, Fig. 1c). As demonstrated in Western blot, this was translated into a down-regulation of A20 protein levels at 0, 1, 6 (data not shown), and 24 h after activation (n ⫽ 3, Fig. 1d). Effect of A20 down-regulation on TLR3 activated transcription factors in DCs To evaluate the effect of A20 down-regulation on the transcription factor profile in human DCs, we performed a bioplex assay, evaluating the presence of AP-1, IRF, and NF-␬B in the nucleus of DCs. Therefore, immature DCs were lipofected with irrelevant or A20 siRNA and stimulated with poly(I:C). The cells were harvested 6, 24, and 48 h after maturation, after which nuclear lysates were prepared and analyzed. The signals obtained for IRF were similar to the background signals in all tested samples, demonstrating that IRF was not up-regulated at the evaluated time points neither in DCs lipofected with irrelevant siRNA, nor in DCs lipofected with A20 siRNA (data not shown).

In contrast, the signals obtained for AP-1 and NF-␬B were at least five times higher than the background signals. A similar kinetics, a decrease of the transcription factors over time, was observed for AP-1 and NF-␬B in both DCs lipofected with irrelevant siRNA and A20 siRNA (data not shown). However, when compared with DCs lipofected with irrelevant siRNA, the levels of AP-1 and NF-␬B were enhanced at all evaluated time points in DCs lipofected with A20 siRNA (n ⫽ 3, Fig. 2). A20 down-regulation in DCs activated with poly(I:C) results in enhanced and sustained production of IL-6, IL-10, and IL-12p70 Immature DCs were not modified, lipofected with irrelevant or A20 siRNA for 24 h. These cells were left immature or were matured for 24 h with poly(I:C), after which the supernatants were harvested for evaluation in ELISA. In addition, part of the DCs that were activated for 24 h were washed and divided over two fractions, one that was immediately restimulated with poly(I:C) and one that was restimulated with poly(I:C) after a resting period of 8 h in the absence of poly(I:C). Again the supernatants were harvested for evaluation in ELISA. No detectable amounts of IL-6, IL-10, or IL-12p70 were produced by the unmodified, irrelevant siRNA or A20 siRNA lipofected and unactivated DCs (data not shown). Furthermore, no differences in cytokine production were observed between unmodified and irrelevant siRNA lipofected DCs (data not shown). These DCs produced on average 1321 ⫾ 339, 433 ⫾ 504, and 1459 ⫾ 852 pg/ml IL-6, IL-10, and IL-12p70 respectively, upon 24 h of poly(I:C) maturation (Fig. 3). These

FIGURE 3. Down-regulation of A20 in DCs results in enhanced and sustained secretion of IL-6 (a), IL-10 (b), and IL-12p70 (c). Immature DCs were lipofected with irrelevant or A20 siRNA for 24 h, followed by 24 h activation with poly(I:C). Subsequently, the supernatant was harvested and the activated DCs washed and divided in two conditions to evaluate the stability of the functional phenotype. In the first condition, the DCs were cultured for 24 h in medium containing poly(I:C), whereas in the second condition the DCs were cultured for 8 h in the absence of poly(I:C), followed by a restimulation with poly(I:C) for 16 h, after which supernatants of these DCs were harvested. ELISA was performed to evaluate the presence of IL-6, IL-10, and IL-12p70 in these supernatants. The chart shows the mean ⫾ SD of three independent experiments. irr., Irrelevant.

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FIGURE 4. DCs lipofected with A20/IL-10 siRNA produce high amounts of IL-6 (a) and IL-12p70 (b), but not IL-10 (c). Immature DCs were lipofected with irrelevant, IL-10, A20, or a combination of A20 and IL-10 siRNA for 24 h. Subsequently these DCs were activated with poly(I: C). Supernatants were harvested 24 h after activation and were evaluated for the presence of IL-6, IL-10, and IL-12p70. The scatter charts represents the results of eight independent experiments. The mean is indicated with a horizontal line.

amounts were decreased with a factor 2.4 ⫾ 0.5, unchanged and 2.6 ⫾ 1.2 between 24 and 48 h of poly(I:C) maturation and decreased further, 5.4 ⫾ 0.3-, 2.5 ⫾ 0.9-, and 12 ⫾ 1.8-fold respectively, when the DCs were restimulated after a resting period of 8 h. In contrast, A20 siRNA lipofected DCs produced higher amounts of IL-6, IL-10, and IL-12p70, 6866 ⫾ 1404, 4037 ⫾ 1712, and 6093 ⫾ 811 pg/ml respectively, upon 24 h of activation. The A20 siRNA lipofected DCs stimulated for 24 h with poly(I:C) followed by immediate restimulation with poly(I:C) produced on average 2.7 ⫾ 0.9-, 45 ⫾ 25-, and 3.6 ⫾ 1.9-fold more IL-6, IL-10, and IL-12p70, respectively, when compared with the irrelevant siRNA modified DCs. More importantly, the A20 siRNA modified DCs, which had undergone a resting period of 8 h followed by restimulation with poly(I:C), produced similar cytokine amounts as those that were

immediately restimulated, demonstrating not only an enhanced, but also sustained cytokine production by A20 down-regulated DCs upon rechallenge with poly(I:C) (n ⫽ 3, Fig. 3). Because we previously demonstrated up-regulation of A20 upon maturation of human DCs with LPS/TNF-␣ or a mixture of inflammatory cytokines, we further evaluated whether the enhanced cytokine production observed with poly(I:C) as a maturation stimulus was also observed in DCs lipofected with A20 siRNA, but matured with the aforementioned stimuli. Maturation of DCs with LPS/TNF-␣ or a mixture of inflammatory cytokines resulted in up-regulation of all tested cytokines, which was similar for both stimuli, but was less pronounced for IL-10 when compared with poly(I:C) as a maturation stimulus (n ⫽ 3, data not shown).

FIGURE 5. Allogeneic stimulatory capacity of A20 and/or IL-10 silenced DCs. Unmodified DCs or DCs lipofected with irrelevant, A20, IL-10, or the combination A20/IL-10 siRNA were matured using poly(I:C). Allogeneic CD4⫹ and CD8⫹ T cells were incubated with these DCs at the indicated stimulator-to-responder ratios for 5 days, in the presence of either isotype matched but irrelevant Ab (a) or a neutralizing anti-human IL-10 Ab (b). Subsequently, proliferation was measured. The results are represented as means of triplicate samples from the same experiment. Data are representative of three independent experiments.


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FIGURE 6. CD4⫹ T cell priming capacity of A20 and IL-10 silenced DCs. DCs were lipofected with irrelevant (irr.), A20, and/or IL-10 siRNA for 24 h. These DCs were matured with poly(I:C) and cocultured with allogeneic naive CD4⫹ T cells in the presence of control Ab, anti-human IL-10, or anti-human IL-12 Ab. a, At day 4 we measured the naive CD4⫹ T cell proliferation. At day 6, the cells were restimulated for 24 h with anti-CD3/CD28 Abs, after which production of IFN-␥ was evaluated in either ELISA (b) or together with IL-17 (c) or IL-4 (d) in intracytoplasmic flow cytometry staining. a, c, and d are representative for three independent experiments. b, Scatter chart representing the results of three independent experiments. The mean is indicated with a horizontal line.

Simultaneous A20 and IL-10 down-regulation in dendritic cells We demonstrated above that down-regulation of A20 in DCs results in elevated levels of IL-10, which is an immunosuppressive cytokine (45). Therefore, we tested whether the elevated IL-10 secretion could be abrogated by IL-10 siRNA. Immature DCs were lipofected with irrelevant, IL-10, A20, or A20/IL-10 siRNA. The secretion of IL-6, IL-10, and IL-12p70 was measured 24 h after maturation with poly(I: C), demonstrating reduced levels of IL-10 in the supernatants of DCs lipofected with IL-10 siRNA, whereas the levels of IL-6 and IL12p70 were not affected. DCs lipofected with both A20 and IL-10 siRNA produced low levels of IL-10 and high levels of the immunostimulatory cytokines IL-6 and IL-12p70 (n ⫽ 8, Fig. 4). Phenotype and allostimulatory capacity of A20 and IL-10 down-regulated DCs The phenotype (CD25, CD40, CD80, CD83, MHC I, MHC II, PD-L1, PD-L2 and CCR7) and viability (propidium iodide) of un-

modified DCs and DCs lipofected with irrelevant, A20, IL-10 or A20/IL-10 siRNA was evaluated 24 h after maturation by flow cytometry. No phenotypical changes or differences in cell viability were observed between the different conditions (data not shown). Mixed lymphocyte reactions were performed to determine the effect of A20 and/or IL-10 down-regulation on the allostimulatory capacity of DCs. Unmodified, irrelevant, and A20 siRNA lipofected DCs had a similar capacity to induce allogeneic T cell proliferation, whereas IL-10 and A20/IL-10 siRNA lipofected DCs showed an enhanced allostimulatory capacity, suggesting that the presence of IL-10 is the limiting factor in the former cocultures (n ⫽ 5, Fig. 5a). Therefore, we evaluated the effect of IL-10 by using a neutralizing anti-human IL-10 Ab in the different cocultures. In the presence of this Ab, unmodified, irrelevant, and A20 siRNA lipofected DCs had the same allostimulatory capacity as IL-10 and A20/IL-10 siRNA lipofected DCs, demonstrating the suppressive role of IL-10 on T cell proliferation (n ⫽ 3, Fig. 5b).

a HLA-A2⫹ CD8⫹ T cells of different donors were stimulated with Melan-A peptide pulsed mature DCs that were lipofected with irrelevant, A20, or/and IL-10 siRNA. After three rounds of antigen stimulation, the cultures were evaluated in terms of number of T cells and percentage of CD8⫹/Melan-A⫹ T cells. The fold increase was calculated and irrelevant siRNA lipofected DCs served as control (fold increase ⫽ 1). b Irr., Irrelevant.

0.1 ⫻ 105 6.4 ⫻ 105 0.1 ⫻ 105 5.4 ⫻ 105 0.31% 32% 0.55% 18% 1.6 ⫻ 106 2.0 ⫻ 106 1.9 ⫻ 106 3.0 ⫻ 106 1 50 2.1 24 0.1 ⫻ 105 5.0 ⫻ 105 0.3 ⫻ 105 3.5 ⫻ 105 0.65% 15% 1.69% 10% 2.3 ⫻ 106 3.4 ⫻ 106 1.9 ⫻ 106 3.5 ⫻ 106 1 27 2.1 33 0.3 ⫻ 106 7.2 ⫻ 106 0.6 ⫻ 106 8.9 ⫻ 106 13% 65% 27% 74% 2.1 ⫻ 106 1.1 ⫻ 106 2.1 ⫻ 106 12 ⫻ 106 1 36 1.9 8.5 0.06% 1.82% 0.14% 0.51% Irr. siRNAb A20 siRNA IL-10 siRNA A20/IL-10 siRNA

1.0 ⫻ 106 1.2 ⫻ 106 0.8 ⫻ 106 1.0 ⫻ 106

0.6 ⫻ 105 2.1 ⫻ 106 1.1 ⫻ 105 5.2 ⫻ 105

No. of CD8 Melan-A⫹ cells % CD8 tetramer⫹ cells No. of T cells

% CD8 tetramer⫹ cells

No. of CD8 Melan-A⫹ cells

Fold increase

No. of T cells

% CD8 tetramer⫹ cells


Fold increase

No. of T cells

Donor 4 Donor 3 Donor 2

Fold increase No. of CD8 Melan-A⫹ cells % CD8 tetramer⫹ cells

We next evaluated the ability of A20 and/or IL-10 siRNA lipofected DCs to stimulate naive TAA specific CD8⫹ T cells. Purified CD8⫹ T cells from HLA-A2⫹ healthy donors were stimulated with mature DCs, which were lipofected with irrelevant, A20, and/or IL-10 siRNA and loaded with the HLA-A2 restricted Melan-A/MART-1 peptide. T cells were stimulated three times at weekly intervals without additional cytokines. Seven days after the last stimulation, the Melan-A/MART-1 specific CD8⫹ T cell response was measured by tetramer staining. Based on the number of viable T cells, assessed with trypan blue, and the percentage of CD8⫹/Melan-A tetramer⫹ cells, measured by flow cytometry, we calculated the fold increase in numbers of TAA specific T cells as a measure of the T cell stimulatory capacity of the DCs (n ⫽ 4, Table I). The absolute number of CD8⫹/Melan-A tetramer⫹ cells was consistently higher in the cultures stimulated with A20 or A20/IL-10 siRNA lipofected DCs when compared with the cultures stimulated with irrelevant siRNA lipofected DCs. In comparison, IL-10 siRNA lipofected DCs had a marginally increased capacity to induce Melan-A/MART-1 specific T cell responses.

Donor 1

Effect of A20 and IL-10 down-regulation on priming of Melan-A specific CD8⫹ T cells

Table I. Melan-A-specific CD8⫹ T cell priming capacity of A20 and/or IL-10 silenced DCsa

CD4⫹ T cell help, provided by Th1 cells, for the priming of CTLs imprints the capacity for optimal secondary expansion upon re-encounter with Ag. To evaluate the role of A20 in Th cell polarization, we cultured purified CD4⫹ CD45RA⫹ T cells with allogeneic DCs lipofected with irrelevant, A20, and/or IL-10 siRNA (n ⫽ 3). These cultures were performed in the presence of irrelevant, neutralizing anti-human IL-10 or anti-human IL-12 Ab to further elucidate the role of IL-10 and IL-12 in this system. Proliferation of the CD4⫹ T cells was measured 4 days after the start of the cocultures, demonstrating that T cells cocultured with DCs with down-regulated IL-10 secretion proliferated more extensively than those cocultured with DCs lipofected with irrelevant or A20 siRNA. Addition of anti-human IL-10 Ab to the latter cocultures resulted in enhanced T cell proliferation, whereas addition of antihuman IL-12 Ab had no effect on proliferation (Fig. 6a). After 6 days, part of the CD4⫹ T cells were harvested and evaluated in flow cytometry for the presence of Treg, defined as CD4⫹CD25⫹CD127⫺FoxP3⫹ T cells. The percentage of Treg was comparable in the different cocultures (data not shown). The remaining CD4⫹ T cells were restimulated with anti-CD3/CD28 mAb coated beads for another 24 h with or without the addition of Brefeldin A to determine the cytokine profile in flow cytometry or ELISA, respectively. In ELISA, we demonstrated that the CD4⫹ T cells secreted low levels of IL-4 and IL-10 with no differences between the differently stimulated T cells (data not shown). In contrast, the production of IFN-␥ was enhanced upon stimulation with IL-10 siRNA lipofected DCs (1.4 ⫾ 0.1-fold) and was even more pronounced upon stimulation with DCs lipofected with A20 (5.1 ⫾ 0.1-fold) or A20/IL-10 siRNA (4.5 ⫾ 1.2-fold). Addition of anti-human IL-10 Ab did not influence IFN-␥ secretion (data not shown), whereas addition of anti-human IL-12 in these cocultures resulted in decreased IFN-␥ secretion (Fig. 6b). Intracellular staining for IL-17 did not reveal the induction of Th17 cells, whereas the staining for IL-4 and IFN-␥ was in line with the results obtained in ELISA (Fig. 6, c and d).

Fold increase

Effect of A20 and IL-10 down-regulation on CD4⫹ T cell polarization

1 128 2.1 108


No. of T cells

866


867

FIGURE 7. DCs simultaneously electroporated with Melan-A mRNA, poly(I:C12U), and A20 or A20/IL-10 siRNA are phenotypically and functionally activated. a, The staining for the maturation marker CD83 for one representative experiment is shown (n ⫽ 5). MFI, Mean fluorescence intensity. b, The secretion of IFN-␣, IL-6, IL-10, and IL-12p70 by irrelevant, A20, or A20/IL-10 siRNA electroporated DCs is shown (n ⫽ 5).

Electroporation of DCs with Melan-A/MART-1 encoding mRNA, poly(I:C12U), and A20 or A20/IL-10 siRNA We previously demonstrated that electroporation is a powerful technique for the delivery of both TAA (mRNA) and activation stimulus (poly(I:C12U)) to DCs (11). It has also been demonstrated that similar electroporation conditions can be applied for the introduction of siRNA into DCs (46). Because we observed that DCs electroporated with poly(I:C12U) up-regulate A20 to similar levels as DCs passively pulsed with poly(I:C) (n ⫽ 2, data not shown), we evaluated whether the simultaneous electroporation of MelanA/MART-1 mRNA, poly(I:C12U), and A20 or A20/IL-10 siRNA resulted in potent stimulatory DCs. To evaluate the phenotypic

maturation of these DCs, we measured the expression of CD83 by flow cytometry, 24 h after electroporation. Immature DCs served as a control. We demonstrated that the proposed one-step electroporation resulted in phenotypically mature DCs and that the electroporation of A20 or A20/IL-10 siRNA did not influence the expression of the evaluated maturation marker (n ⫽ 5, Fig. 7a). Furthermore, we evaluated the secretion of IL-6, IL-10, and IL12p70, as well as IFN-␣, a cytokine produced upon electroporation with poly(I:C12U), as a measure of their functionality upon siRNA modification. When compared with DCs coelectroporated with irrelevant siRNA, DCs coelectroporated with A20 siRNA secreted higher levels of IFN-␣, IL-6, IL-10, and IL-12p70, whereas DCs

a HLA-A2⫹ CD8⫹ T cells of healthy donors were stimulated with DCs coelectroporated with Melan-A mRNA poly(I:C12U) and irrelevant, A20 or A20/IL-10 siRNA. Three stimulations at weekly intervals were performed. Seven days after the last stimulation, the cultures were evaluated in terms of number of T cells and percentage of CD8⫹/Melan-A⫹ T cells. The fold increase was calculated against irrelevant siRNA coelectroporated DCs (fold increase ⫽ 1). b Irr., Irrelevant.

1 6 7 0.4 ⫻ 105 2.0 ⫻ 105 2.5 ⫻ 105 1.42% 32% 17% Irr. siRNAb A20 siRNA A20/IL-10 siRNA

1.2 ⫻ 106 1.3 ⫻ 106 2.8 ⫻ 106

0.2 ⫻ 105 4.2 ⫻ 105 4.7 ⫻ 105

1 24 28

1.3 ⫻ 106 2.0 ⫻ 106 2.1 ⫻ 106

0.08% 1.00% 1.34%

0.1 ⫻ 104 2.0 ⫻ 104 2.8 ⫻ 104

1 20 29

2.9 ⫻ 106 4.0 ⫻ 106 4.1 ⫻ 106

1.23% 5.05% 6.13%

Fold increase % CD8 tetramer⫹ cells No. of T cells Fold increase # CD8 Melan-A⫹ cells % CD8 tetramer⫹ cells % CD8 tetramer⫹ cells No. of T cells


Fold increase

No. of T cells

Donor 2 Donor 1

Table II. Priming of naive Melan-A specific CD8⫹ T cells by DCs coelectroporated with Melan-A mRNA, poly(I:C12U) and irrelevant, A20 or A20/IL-10 siRNAa



Donor 3

868

coelectroporated with A20/IL-10 siRNA secreted high amounts of IFN-␣, IL-6, and IL-12p70, but not IL-10 (n ⫽ 5, Fig. 7b). DCs modified as described above were used to prime autologous HLAA2/Melan-A specific CD8⫹ T cells by three weekly stimulations. Seven days after the last stimulation the CD8⫹ T cells were harvested and evaluated as described above. We demonstrated that DCs coelectroporated with A20 or A20/IL-10 siRNA have a similar capacity to stimulate Melan-A/MART-1 specific T cells and that these DCs induced 17-fold (⫾9) and 21-fold (⫾12) more Melan-A specific T cells than DCs coelectroporated with irrelevant siRNA, respectively (n ⫽ 3, Table II).

Discussion In this study, we have demonstrated, to our knowledge for the first time, that A20 is up-regulated upon TLR activation of human monocyte-derived DCs and that it acts as a negative regulator of DC function. We demonstrated by RNA interference that A20 negatively regulates the transcription factors NF-␬B and AP-1, resulting in negative regulation of cytokine production by mature DCs and hence negative regulation of the immunostimulatory potency of these DCs, as shown in the stimulation of naive CD4⫹ T cells and Melan-A/MART-1 specific CD8⫹ T cells. Thus, this study provides important new insights into the regulation of TLR3-mediated DC activation and adaptive immune responses in humans. Because A20 has been described to terminate TLR-mediated NF-␬B up-regulation in mice (25), we first examined the effect of A20 down-regulation on the levels of selected transcription factors, demonstrating enhanced levels of AP-1 and NF-␬B 6 h after stimulation of human DCs with poly(I:C). Recently, Werner et al. (47) demonstrated in murine embryonic fibroblasts that NF-␬B activation follows a particular dynamic, characterized by a rapid up-regulation (minutes), followed by a postinduction repression (after 1 h) and subsequent plateau of late activity (up to 3 h). Interestingly, it was demonstrated that the postinduction repression was delayed in A20 knock-out cells and that in these cells NF-␬B activity was elevated for prolonged periods (3– 6 h), after which NF-␬B, probably under the control of other regulators, gradually decreased (47). We observed a similar pattern, enhanced levels of NF-␬B 6 h after stimulation followed by a decreased level of NF-␬B at 24 and 48 h after stimulation in A20 silenced DCs. Recently, Song et al. (40) silenced A20 in mouse DCs and demonstrated that this down-regulation results in up-regulation of different cell surface Ags associated with DC activation and the increased production of IL-6, IL-12p40 and TNF-␣ even in the absence of an activation stimulus. This is in contrast with our observations and our view on the role of A20, as a brake on TLR activation, in DCs. We propose that taking away A20 (the brake) without providing acceleration (TLR activation) will have no consequence, whereas taking away A20 and providing the necessary stimulation results in an enhanced DC function. This metaphor supports our observations, i.e., no enhanced cytokine secretion in the absence of an activation stimulus and enhanced production of IL-6, IL-10, and IL-12p70 upon stimulation with poly(I:C) or other maturation stimuli, such as LPS/TNF-␣ or a mixture of inflammatory cytokines. More importantly, we observed that down-regulation of A20 results in DCs that, in contrast to irrelevant siRNA lipofected DCs, produce high amounts of cytokines upon rechallenge with the same activation stimulus, demonstrating that A20 plays a role in DC exhaustion. This phenomenon, described as the transient maturation of DCs upon stimulation with TLR stimuli, is characterized by an elevated production of IL-12 early after stimulation, followed by high IL-10 production in the so called exhaustion phase. This cytokine profile cannot be reversed by rechallenge with the same stimulus and results in suboptimal T cell stimulation (48 –50). The fact that A20 silenced

The Journal of Immunology human DCs produce high amounts of cytokines and are not tolerated to rechallenge with the same stimulus is an important observation, which has been previously described in mice, not only in view of A20 (47), but also other NF-␬B regulators such as TRIM30␣ (51). It further has an important impact on anti-tumor immunotherapy, where it has been repeatedly demonstrated that breaking tolerance of tumor specific CTLs requires persistent TLR ligation alongside Ag-loaded DCs (8). Here, persistent TLR ligation is mimicked by A20 down-regulation. In our in vitro stimulations, A20-silenced DCs skewed naive CD4⫹ T cells toward a Th1 phenotype, but not a Treg, Th2, or Th17 phenotype. The requirement for CD4⫹ Th1 cells in the field of cancer immunotherapy is well accepted. They have been shown in some instances to induce tumor eradication on their own (52, 53), but their main importance lies in their ability to activate other cells (e.g., licensing of DCs) along with generation of a long-lasting memory CTL response (54 –56). In this regard, we demonstrated that A20 downregulated DCs were more potent in the induction of TAA specific CD8⫹ T cells, than irrelevant siRNA modified DCs, suggesting that these DCs could make up a potent anti-tumor vaccine, fulfilling the requirement of stimulating both Th1 cells and CTLs, but not Treg or potentially harmful Th17 cells (57). Furthermore, the data published by Song et al. (40) on A20-silenced mouse DCs, further strengthen this suggestion by demonstrating that these DCs hyperactivate OVAspecific T cells and inhibit Treg function in vivo. Because A20-silenced DCs produced high amounts of IL-10, a well-known immune suppressor, we further evaluated whether additional down-regulation of IL-10 resulted in DCs with enhanced T cell stimulatory capacity, as previously described by Liu et al. (58) and Chhabra et al. (59). In accordance with the publication of Liu et al. (58), we were able to demonstrate that IL-10 impacts on the proliferation of T cells and that down-regulation of IL-10 results in enhanced secretion of IFN-␥ by allogeneic naive T cells. Furthermore, we observed that IL-10 silenced DCs were on average two times better in the stimulation of TAA-specific T cells, which is in line with the data published by Chhabra et al. (59). Simultaneous down-regulation of A20 and IL-10 only had a beneficial effect on T cell proliferation, whereas it did not result in DCs with higher capacity to drive a Th1 response, which was dependent on the secretion of IL-12p70, as shown by the addition of anti-human IL-12 Ab in these cocultures. Moreover, the additional down-regulation of IL-10 did not have an effect on the stimulation of TAA-specific CD8⫹ T cells, suggesting that in these cultures other factors, such as IL-6 and IL-12, determine the outcome of the stimulation. Taken together, our data indicate that A20 is a negative regulator of NF-␬B and AP-1 activation in monocyte-derived DCs and that its down-regulation results in DCs with enhanced stimulatory capacity. This study has implications not only for understanding the regulation of adaptive immunity, but also for the development of effective vaccines against cancer and perhaps infectious diseases by enhancing the stimulatory potential of DCs.

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2. 3. 4. 5. 6.

7.

8.

9.

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11.

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14.

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16.

17. 18. 19. 20. 21.

22.

23.

24.

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Acknowledgments We thank Aude Bonehill, Jurgen Corthals, Sandra Tuyaerts and An Van Nuffel for their help with DC cultures; Elsy Vaeremans and Sandra Van Lint for the mRNA preparations; and Inken Sehrsam and Karel Fostier for the helpful technical suggestions during the transcription factor plex.

26.

27.

Disclosures The authors have no financial conflict of interest.

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