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Blood, 1 April 2007, Vol. 109, No. 7, pp. 2953-2960. Prepublished online as a Blood First Edition Paper on November 28, 2006; DOI 10.1182/blood-2006-07-033258.
IMMUNOBIOLOGY Immunostimulatory RNA oligonucleotides trigger an antigen-specific cytotoxic T-cell and IgG2a response1 Division of Clinical Pharmacology, Department of Internal Medicine, Ludwig-Maximilian University of Munich, Germany; 2 Division of Clinical Pharmacology, University Hospital, University of Bonn, Germany
Single-stranded RNA oligonucleotides containing an immunostimulatory motif (immunostimulatory RNA [isRNA]) are potent inducers of interferon-  via the Toll-like receptor 7. We investigated the effect of isRNA on the development of an immune response. We show that isRNA activates dendritic cells and induces production of Th1-type cytokines both in vitro and in vivo. Cytokine production led to bystander activation of T and B cells. We further demonstrate that isRNA triggers the generation of antigen-specific cytotoxic T cells and of an IgG2a-biased antibody response to antigen in a sequence-dependent manner. In summary, we provide evidence for the first time that isRNA oligonucleotides can simultaneously activate the innate and adaptive arms of the immune system.
Recognition of pathogens by the innate immune system is mediated through pattern-recognition receptors that recognize distinct microbial components.1 Nucleic acids from pathogens are recognized by several classes of receptors, including Toll-like receptors (TLRs) and cytoplasmic receptors. Microbial DNA, in particular DNA rich in unmethylated CpG motifs, is detected by TLR9 in the endosome.2,3 Long double-stranded RNA (> 30 nucleotides), a replicatory intermediate for some viruses, is detected by TLR3, by the serine-threonine kinase PKR, and by the cytoplasmic helicase proteins RIG-I and MDA5.4–9 The 5'-triphosphate end of RNA generated by viral polymerases directly binds to RIG-I.10 Single-stranded RNA (ssRNA) from ssRNA viruses has been shown to be detected through TLR7 and TLR8.11,12 Furthermore, we have recently described double-stranded, short interfering RNA (siRNA) molecules that interact with TLR7 in a sequence-specific manner to induce IFN- production in dendritic cells (DCs).13 The stimulatory activity on DCs was also observed with the corresponding single-stranded RNA oligoribonucleotides (ORNs).
DCs are professional antigen-presenting cells that express a variety of pattern-recognition receptors. Exposure of DCs to ligands for these receptors, such as microbial nucleic acids, activates intracellular signaling cascades that rapidly induce the expression of a variety of genes involved in maturation and migration of DCs.1 Mature DCs can directly interact with immune effector cells such as cytotoxic T lymphocytes (CTLs), a process that is essential for the induction of protective immunity against infectious diseases and tumors. In addition, mature, activated DCs also represent a critical source of IL-12 and IFN- The immune-activating effects of TLR ligands have prompted their use in vaccine formulations. It is well established that binding of synthetic CpG oligodeoxynucleotides (ODNs) to TLR9 both enhances the generation of an innate immune response and promotes protective Th1-type immunity in animal models.17 In humans, clinical studies have demonstrated a potential for CpG ODNs as adjuvants in antiviral vaccination.18,19 Furthermore, CpG ODNs combined with a peptide antigen promote CD8+ T-cell responses to tumor antigens in patients with melanoma.20 However, TLR9 displays a restricted expression pattern in humans, where this receptor is expressed on B cells and plasmacytoid DCs but not, as is the case in mice, on professional antigen-presenting cells that are crucial for the induction of immunity to viral and tumor antigens.21 Here we characterized the in vitro and in vivo immunostimulating potential of synthetic single-stranded ORNs that activate TLR7. We show for the first time that ORNs can be used to trigger an immune response to a model antigen in a sequence-dependent manner. This immunostimulatory effect led to the generation of antigen-specific, cytotoxic T cells and antibodies of the IgG2a isotype in the context of a Th1-type immune response. We therefore demonstrate that immunostimulatory RNA can at the same time drive both an innate immune response and an adaptive response to antigen.
Mice Female Balb/c and C57BL/6 mice were purchased from Harlan-Winkelmann (Borchen, Germany). Mice were 5 to 12 weeks of age at the onset of experiments. Animal studies were approved by the local regulatory agency (Regierung von Oberbayern, Munich, Germany). Reagents The 20-mer ORNs 9.2dr (5'-UGUCCUUCAAUGUCCUUCAA-3') and PolyA in both the unmodified phosphodiester and fully phosphorothioated (PTO) forms and the ORNs TLR4.1s (5'-UACUUAGACUACUACCUCG-3'), 9.2as (5'-UUGAAGGACAGGUUAAGCU-3'), and MyD88s (5'-CAGACAAACUAUCGACUGAtt-3') in fully PTO form were from CureVac (Tübingen, Germany). The PTO-modified CpG oligodeoxyribonucleotide 1826 (5'-TCCATGACGTTCCTGACGTT-3') was obtained from the Coley Pharmaceutical Group (Langenfeld, Germany). For flow cytometry analysis, cells were stained with anti–mouse B220-PE, CD3-APC, CD4-PE, CD8-PerCP, CD11b-PerCP, CD11c-APC, CD69-FITC, and isotype controls (BD Biosciences, Heidelberg, Germany). Chicken egg ovalbumin (OVA) was purchased from Sigma-Aldrich (St Louis, MO). Bone marrow cell culture and generation of DCs Bone marrow cells were harvested from murine femur and tibia and erythrocytes were lysed with ammonium chloride buffer (BD Biosciences). For bone marrow cell cultures, cells from Balb/c mice were cultured in RPMI 1640 medium supplemented with 10% FCS, 2 mM L-glutamine, 100 µg/mL streptomycin, and 1 IU/mL penicillin (complete RPMI). To prepare bone marrow-derived DCs (BMDCs), bone marrow cells from C57BL/6 mice were cultured in complete RPMI supplemented with 20 ng/mL GM-CSF and 20 ng/mL IL-4 (Tebu Bio, Offenbach, Germany; DC medium). On day 7, loosely adherent cells were harvested and washed. DCs (CD11c+ cells) generally represented 70% of the preparation. DCs were enriched by magnetic cell sorting after labeling with anti-CD11c microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany) (CD11c+ cells above 95%). Bone marrow cells were activated with 5 µg/mL ORN complexed with N-[1-(2,3-dioleoyloxy)propyl]-N, N, N-trimethylammonium methyl-sulfate (DOTAP; Roche, Germany) according to the manufacturer's instructions or with 6 µg/mL CpG ODN 1826 or 1 µg/mL LPS. BMDCs were activated with 10 µg/mL ORN or 1 µg/mL CpG ODN complexed with DOTAP or 1 µg/mL LPS. For coculture experiments, BMDCs were stimulated with ORN or CpG ODN complexed with DOTAP for 6 hours, then washed and cocultured with splenocytes (2 x 105 BMDCs with 2 x 105 splenocytes) in 96-well U-bottom plates for an additional 18 hours. Supernatants were harvested for detection of cytokines by enzyme-linked immunosorbent assay (ELISA). For analysis of activation markers, cells were stained with fluorochrome-coupled monoclonal antibodies (mAbs) and analyzed by flow cytometry. Data were acquired on a FACSCalibur (BD Biosciences) and analyzed using CellQuest software (BD Biosciences). Quantification of cytokines in serum and supernatant
Cytokine concentrations were determined by ELISA for IL-6 (BioSource, Solingen, Germany) and IL-12p40 (BD Biosciences) according to the manufacturer's protocol. IFN- Immunostimulation in vivo and immunization For in vivo immunostimulation, 100 µg DOTAP was incubated with 20 µg oligonucleotides and 20 µL HBS for 20 minutes and injected intravenously into the retro-orbital plexus. Single-cell suspensions were prepared from spleen and lymph nodes 18 hours after injection and treated with ammonium chloride buffer to lyse erythrocytes. For analysis of activation markers, cells were stained with fluorochrome-coupled mAbs and analyzed by flow cytometry. Blood was obtained by retro-orbital puncture at the indicated time points. Serum was prepared by centrifugation and stored at –20°C. For immunization, 20 to 50 µg OVA was injected subcutaneously together with oligonucleotides complexed to DOTAP on day 0, day 14, and day 21. The OVA-specific T-cell response was determined on day 21 and serum antibodies on day 28. OVA-specific immune response
Serum antibodies to OVA were determined by ELISA; 96-well plates were coated overnight with 10 µg/mL OVA in PBS and blocked 1 hour with 1% BSA in PBS. After incubation of serum samples for 1 hour, plates were washed with PBS/1% Tween 20 and goat anti–mouse IgG, IgG1, or IgG2a conjugated to HRP (Southern Biotechnology Laboratories, Birmingham, AL) was added at 1 µg/mL for 1 hour. Plates were again washed and ELISA was developed by o-phenylenediamine (Sigma-Aldrich). The reaction was stopped by 1 M H2SO4 and optical density (OD) was read by photometer at 450 nm. For the detection of OVA-specific CD8+ T cells, freshly isolated splenocytes were lysed by ammonium chloride buffer and stained with H-2kb-OVA257-264-PE pentamers (Proimmune, Oxford, United Kingdom) and anti-CD8. For the detection of intracellular IFN- In vivo cytotoxicity assay Targets were prepared from C57BL/6 splenocytes. The suspension was divided into 2 populations, pulsed or unpulsed with 100 µg/mL OVA257-264 for 1 hour at 37°C, washed extensively, and labeled with a high concentration (15 µM) or with a low concentration (1.5 µM) of CFSE (Molecular Probes, Eugene, OR), respectively. Peptide-pulsed CFSEhigh cells and unpulsed CFSElow cells were mixed and a total of 107 CFSE-labeled cells were injected intravenously into immunized mice. After 16 hours, splenocytes were isolated and analyzed by flow cytometry to detect the CFSE-labeled target cells. Specific lysis was calculated by the following formula: specific lysis (%) = 100% x [1-(CFSEhigh cells/CFSElow cells)/(CFSEhigh cells in naive mice/CFSElow cells in naive mice)]. Statistics All data are presented as mean ± SEM and were analyzed by unpaired, one-way analysis of variance (ANOVA) with the Newman-Keuls multiple-comparison test. Significance was set at a P < .05. Statistical analysis were performed using SPSS software (SPSS, Chicago, IL).
PTO RNA oligonucleotides activate DCs more efficiently than unmodified oligonucleotides in a sequence-dependent manner
We evaluated the immunostimulatory effects of the 20-mer ORN sequence 9.2dr on the activation of bone marrow cells. Freshly isolated bone marrow cells were stimulated with either the unmodified or fully PTO ORN 9.2dr. IL-12p40, IL-6, IFN-
Surface expression of the activation markers MHC-II, CD80, CD86, and CD40 was measured on CD11c+ DCs after stimulation of bone marrow cells with ORNs or CpG ODN (Figure 1B). Increased surface expression of CD86 and CD40 was seen after stimulation with 9.2dr and of all activation markers after stimulation with 9.2dr PTO. The increase in expression of the activation markers by 9.2dr PTO was similar to that seen with CpG ODN.
To assess the effect of 9.2dr-activated DCs on immune effector cells, purified BMDCs were activated with the PTO ORN 9.2dr or PolyA, washed, and cocultured with splenocytes. The early activation marker CD69 was up-regulated on CD3+ and CD8+ T cells as well as on B cells after coculture with 9.2dr PTO-activated DCs (Figure 2A). In contrast, DCs incubated with the PolyA PTO ORN did not activate splenocytes. Furthermore, splenocytes cocultured with 9.2dr PTO-activated DCs produced the effector cytokine IFN-
PTO RNA oligonucleotides activate innate immunity and induce Th1-type cytokines in vivo To characterize the immune effect of ORN in vivo, C57BL/6 mice were injected intravenously with either unmodified or PTO ORNs complexed with DOTAP. As shown in Figure 3A-B, 9.2dr PTO application resulted in a strong up-regulation of the early activation molecule CD69 on the surface of splenic CD4+ and CD8+ T cells as well as on B cells. CD69 expression was up-regulated on a small proportion of splenocytes in mice injected with the unmodified 9.2dr ORN. The increase was, however, below the level of significance. No increase in CD69 expression was seen in mice injected with either form of PolyA. CD69 was also up-regulated on both myeloid DC (MDC) and plasmacytoid DC (PDC) subpopulations in the spleen of mice treated with 9.2dr PTO, indicating in vivo activation of DCs. Neither the unmodified 9.2dr ORN nor the PolyA ORN significantly increased CD69 expression on DCs. As positive control, CD69 expression was measured in mice injected with CpG ODN (CD8+ T cells, 77%; CD4+ T cells, 40%; B cells, 51%; MDCs, 19%; PDCs, 54%).
In the serum of ORN-injected mice, IL-12p40 was increased 2 hours after injection with 9.2dr and 9.2dr PTO and the levels remained high at 6 hours after injection (Figure 4). At 22 hours after injection, IL-12p40 had returned to baseline in mice injected with unmodified 9.2dr but was still above baseline in mice treated with 9.2dr PTO. At no time point was IL-12p40 increased in mice injected with either form of PolyA. An increase in serum IL-6 was seen at the earliest time point of 2 hours in mice injected with 9.2dr PTO. IL-6 was no longer detectable in serum 6 hours after injection. IFN- serum levels were increased after 2 hours and 6 hours in mice treated with 9.2dr PTO. No increase in either IL-6 or IFN- was detected in mice injected with unmodified 9.2dr ORN or with PolyA ORN at any time point. As positive control, serum cytokines were measured in mice injected with CpG (IL-12p40, 2450 pg/mL; IL-6, 1390 pg/mL; IFN-, 1015 U/mL).
PTO RNA oligonucleotides induce an antigen-specific IgG2a immune response To evaluate the effect of immunostimulatory ORNs on the development of an immune response to antigen, mice were immunized with OVA together with PTO ORNs twice at a 14-day interval. Seven days after the second immunization, serum levels of OVA-specific antibodies were measured. In mice immunized with OVA and 9.2dr PTO, levels of OVA-specific IgG were increased compared to mice immunized with either OVA alone or OVA and PolyA (Figure 5). OVA-specific IgG1 was increased in all groups having received OVA, so that no difference between groups treated with OVA alone or OVA with ORNs was detected. In contrast, an increase in OVA-specific antibodies of the IgG2a isotype, indicative of a Th1-type response, was seen in mice immunized with OVA together with 9.2dr PTO compared to mice immunized with OVA alone. No increase was seen in mice treated with OVA and PolyA ORN. Assessment of total serum IgG1 and IgG2a revealed no change in the treated groups compared to control mice.
PTO RNA oligonucleotides trigger the induction of antigen-specific cytotoxic T cells
OVA-specific CD8 T cells were assessed in mice immunized with OVA. Whereas a small increase in pentamer-positive OVA-specific CD8+ T cells was detected in mice treated with OVA alone or with OVA together with PolyA PTO, over 4% of OVA-specific CD8+ T cells were detected in mice having received OVA together with 9.2dr PTO (Figure 6A,C). Splenocytes from immunized mice were restimulated with the MHC-I–restricted peptide OVA257-264 and assessed for IFN-
An in vivo cytotoxicity assay was performed to assess the functionality of the OVA-specific CD8+ T cells. Immunized mice were injected with CFSE-stained splenocytes from naive mice preincubated with OVA257-264 peptide. Unloaded splenocytes stained with a lower CFSE concentration served as reference. While low levels of specific lysis were detected in mice immunized with OVA alone or with OVA and PolyA, the OVA-specific lysis was increased to 60% in mice immunized with 9.2dr PTO (Figure 7).
ssRNA and ssRNA viruses such as vesicular stomatitis virus and influenza virus are recognized by immune cells through TLR7 and TLR8,11,12,22 but specific sequence motifs responsible for viral RNA recognition have not been described. We have recently characterized an immunostimulatory RNA sequence consisting of 9 bases (GUCCUUCAA), which needs, however, to be part of a longer oligonucleotide to become active.13 The TLR7 dependence of the immunostimulatory activity of this RNA sequence was demonstrated in vitro and in vivo using TLR7-deficient mice. The sequence used in the present study, 9.2dr, contains the 9-mer immunostimulatory motif twice, separated by a uridine base. In this study we characterized the potential of 9.2dr to stimulate the innate immune system and describe for the first time the ability of ORNs to potentiate a Th1-type response to a model antigen in a sequence-dependent manner.
Analysis of the immunostimulatory activity of 9.2dr on the innate immune response showed that this ORN activated both primary bone marrow cultures and purified DCs to produce the proinflammatory cytokines IL-12, IFN-
Unlike immunostimulatory DNA sequences, single-stranded RNA molecules are highly susceptible to degradation by RNases. Encapsulation in liposomes, complexing to a cationic peptide, or chemical modifications are necessary to enhance RNA stability and provide immunostimulatory activity.11–13,24 Cationic liposomes not only protect the ORNs from degradation, but also increase the uptake into cells and target oligonucleotides to the endosomal compartment containing TLR7.25 In the present study, ORNs were complexed with cationic liposomes (DOTAP), as we have previously shown that in the absence of DOTAP, no immunologic activity is observed.13 Furthermore, we assessed in this study the influence of the ORN backbone on the immunostimulatory activity by comparing 2 ORNs with the same nucleotide sequence 9.2dr containing either an unmodified phosphodiester backbone or a PTO-modified backbone. In vitro, cytokine induction was consistently higher using the PTO-modified ORN. In vivo, injection of unmodified 9.2dr only increased serum levels of IL-12p40. In contrast, production of IFN- The generation of an antigen-specific CTL response is of paramount importance for the development of an effective protective antiviral or antitumor immune response. The formation of CTLs is potentiated by DC activation and production of Th1-associated cytokines that enhance antigen presentation and priming of T cells.14 In particular, selective activation of IL-12–producing DCs may be beneficial for developing a vaccine directed at inducing Th1-dependent cellular immunity.26,27 Here we show that coinjection of ORNs with the model protein OVA effectively promoted the generation of antigen-specific cytotoxic T cells. We thus demonstrate for the first time that an ORN not only promotes innate immunity, but also stimulates the adaptive arm of the immune response in a sequence-dependent manner. One of the few vaccine adjuvants in clinical trials that promote the generation of CTLs are CpG oligonucleotides, which bind to TLR9.28,29 In our model, the percentage of CTLs induced by ORNs was similar to that induced by CpG ODNs. Immunostimulatory ORNs triggered the generation of a specific antibody response to OVA. While antibodies of the IgG1 isotype were induced by immunization with OVA alone, OVA-specific antibodies of the IgG2a isotype were only induced by immunostimulatory ORNs in a sequence-dependent manner. The generation of IgG2a antibodies is indicative of a bias toward a Th1-like response.30 In a similar way, CpG ODNs have been described to promote a Th1-type response associated with induction of IgG2a antibodies.31
Whereas the function of pattern recognition receptors is the stimulation of an immune response to protect the host against invading pathogens, inappropriate stimulation through these receptors can lead to autoimmunity. In systemic lupus erythematosus, the uncontrolled activity of self-reactive T and B cells leads to the sustained production of tissue-damaging autoantibodies against nuclear antigens. Interestingly, the RNA component within the prototype autoantigen U1 small nuclear ribonucleoprotein (U1snRNP) is in itself immunostimulatory, inducing IFN- A promising strategy in the immunotherapy of tumors is the use of mRNA-encoding tumor antigens to induce T-and B-cell immunity to the encoded antigens. In vivo application of mRNA induced cytotoxic T-cell activity and specific antibodies in mice.34 Furthermore, human DCs transfected ex vivo with mRNA induced an antigen-specific immune response both in vitro to a viral antigen and in vivo to a tumor-associated antigen in patients with prostate cancer.34,35 In some studies, mRNA transfection also contributed to activate DCs and enhance maturation.35,36 The sequence-dependent adjuvant effect of ORNs we describe here may play a role in enhancing the effect of mRNA-based vaccines. Furthermore, the addition of immunostimulatory RNA sequences to mRNA vaccines could be used to enhance the potency of the vaccines.
The immunostimulatory effects of TLR ligands on both the innate and the adaptive arms of the immune system have prompted the investigation of their therapeutic potential in vaccine formulations. In particular, CpG ODNs, like immunostimulatory RNA, promote innate immune responses characterized by DC activation associated with IL-12 and IFN- Although preliminary results from clinical trials with CpG ODNs are encouraging, the restricted expression pattern in humans of TLR9, the receptor for CpG ODNs, may represent an additional difficulty. We have now shown that a similar Th1-type immune response can be induced by ORNs and thus that ORNs can serve as vaccine adjuvant to induce a CTL immune response. The immunostimulating activity of ORNs and ssRNA is mediated through TLR7 and TLR8,11–13 which, in contrast to TLR9, are expressed in humans on a broad range of immune cells, including professional antigen-presenting cells such as MDCs and monocytes.40,41 Indeed, ligands for TLR7 and TLR8 stimulate human monocytes as well as PDCs and B cells.42,43 Therefore, a ligand for TLR7 and TLR8 such as an immunostimulatory ORN may, in patients, show a therapeutic efficacy in vaccine formulations that is superior to TLR9 ligands.
Contribution: C.B., V.H., G.H., and S.E. designed research; L.S., C.W., N.S., S.S., and A.V. performed research; V.H. and G.H. contributed vital new reagents; L.S., C.W., D.A., and N.S. collected data; C.B., L.S., D.A., and S.E. analyzed data; and C.B. and S.E. wrote the paper. Conflict-of-interest disclosure: G.H. is a consultant for Coley Pharmaceuticals, whose product was studied in the present work. The other authors declare no competing financial interests. Correspondence: Carole Bourquin, Division of Clinical Pharmacology, Department of Internal Medicine, Ludwig-Maximilian University of Munich, 80336 Munich, Germany; e-mail: carole.bourquin{at}med.uni-muenchen.de.
This work was supported by grants from the Deutsche Krebshilfe (10-2214-En3) (S.E.), from the Else-Kröner Fresenius Stiftung and the Deutsche Forschungsgemeinschaft (En 169/7-2 and Graduiertenkolleg 1202) (C.B. and S.E.), and from the Bundesministerium für Bildung und Forschung (Biofuture 0311896) (G.H.). This work is part of the thesis of L.S. and C.W. at the University of Munich. We thank Susanne Wenk, Rosemarie Kiefl, and Julia Vorac for technical assistance.
Submitted July 3, 2006; accepted November 13, 2006.
Prepublished online as Blood First Edition Paper, November 28, 2006
DOI: 10.1182/blood-2006-07-033258
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Abstract
Introduction
, cells were restimulated for 1 hour with 5 µg/mL OVA257-264 peptide, brefeldin A was added at a concentration of 1 µg/mL, and cells were incubated for a further 4 hours. Cells were stained with anti-CD8, then fixed with 2% paraformaldehyde and treated with permeabilizing solution (0.5% bovine serum albumin, 0.5% saponin, 0.02% sodium azide in PBS). The fixed cells were stained with FITC-conjugated anti–IFN-
