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Cell Biology International (2007) 31, 663–671 (Printed in Great Britain)
Silencing invariant chain of DCs enhances Th1 response using small interfering RNA
Shan Kea1, Xue‑Hua Chena1, Hao Lia, Jian‑Fang Lia, Qin‑Long Gub, Bing‑Ya Liua and Zheng‑Gang Zhua*
aDepartment of Surgery, Shanghai Institute of Digestive Surgery, Ruijin Hospital, School of Medicine, Shanghai JiaoTong University, Shanghai 200025, PR China
bDepartment of science and education, School of Medicine, Shanghai JiaoTong University, Shanghai 200025, PR China Abstract RNA interference (RNAi), which causes the degradation of any RNA in a sequence specific manner, is a posttranscriptional gene silencing mechanism. Targeting the invariant chain (Ii) in DCs has been used as an approach to enhance antitumor immunity. It is demonstrated in this article that transfection of H-2K DCs with siRNA specific for Ii gene can significantly knock down Ii. When exposed to TNF-α, immature DCs transfected with Ii siRNA can differentiate into mature DCs without reducing viability or IL-12p70 production. Ii siRNA-treated H-2K DCs exhibited an increased allostimulatory capacity in a lymphocyte proliferation assay. Furthermore, Ii siRNA-transfected H-2K DCs enhanced Th1 responses by increasing IFN-γ and decreasing IL-4 production, and much stronger cytotoxic activity was observed when DCs were co-transfected with Ii siRNA and an endogenous tumor antigen in vitro. Our findings indicate that silencing the Ii gene in DCs with siRNA may offer a potential approach to enhancing antitumor immunotherapy. Keywords: Small interfering RNA, Invariant chain, Dendritic cells, Antitumor immunity. 1Both these authors contributed equally to this study. *Corresponding author. Tel.: +86 21 6467 4654; fax: +86 21 6437 3909. 1 Introduction Dendritic cells (DCs) are highly specialized antigen presenting cells with the unique capacity to initiate and control primary immune responses (Steinman, 1991). Dendritic cells capture antigens in peripheral tissues, which are subsequently processed into small peptides as the DCs mature and move towards the secondary lymphoid organs, where the DCs present the peptides to T cells, thereby inducing a cellular immune response that involves both CD4+ type-1 helper T (Th1) cells and cytolytic CD8+T cells. The high surface density of major histocompatibility complex (MHC) and costimulatory molecules along with a high motility and the ability to produce immunostimulatory cytokines and chemokines enable DCs to be one of the most promising natural candidates against cancer (Banchereau et al., 2000; Fong and Engleman, 2000; Steinman and Dhodapkar, 2001; Guermonprez et al., 2002). Accumulating evidence indicates that inducing effective and persistent tumor-specific immune responses requires CD4+ Th1 cells as well as CD8+T cells (Pardoll and Topalian, 1998; Marzo et al., 2000; Wang, 2001; Cho et al., 2003; Smith et al., 2004). Various approaches have been proposed over the past few years to simultaneously present tumor antigens in HLA class I- and class II- restricted ways (You et al., 2001; Bonehill et al., 2004). The inhibition of invariant chain (Ii) synthesis in DCs is a feasible method among these approaches. Ii facilitates the assembly of class II dimers in the endoplasmic reticulum and provides a sorting signal for the transportation of class II-Ii complexes to the endocytic route (Bakke and Dobberstein, 1990; Anderson and Miller, 1992) where Ii molecules are degraded and one fragment of Ii, called CLIP, is replaced by antigenic peptides. CLIP could prevent premature loading of HLA class II molecules with endogenous peptides in the endoplasmic reticulum. RNA interference (RNAi) is a remarkable experimental tool that has emerged in recent years. RNAi is triggered by double-stranded RNAs (dsRNAs) that cause selective gene silencing. It is a conserved mechanism that pervades the biological world and was first discovered by Fire and his colleagues in 1998 (Fire et al., 1998). Introduction of dsRNAs into cells can elicit at least four different types of responses that can selectively suppress target gene expression. dsRNAs can induce inhibition of protein translation (Fire et al., 1998), degradation of mRNAs (Hammond et al., 2000; Yang et al., 2000; Zamore et al., 2000), transcriptional inhibition (Olsen and Ambros, 1999), and cause chromosomal rearrangements (Mochizuki et al., 2002). The RNAi approach has several advantages over conventional methods, e.g. antisense oligonucleotide technology, including high efficacy and specificity (Ichim et al., 2004). In addition, since small interfering RNA (siRNA) is quite stable, no chemical modifications are required to achieve a sufficient half-life in cell culture media (Paroo and Corey, 2004). Inhibition of Ii expression in DCs with oligonucleotides to enhance antitumor immunity has been reported (Zhao et al., 2003). However, silencing the Ii gene in DCs with siRNA was not available in the open literature. In this study, siRNA specific for the Ii gene was transfected into DCs derived from H-2K mice and its effect on the viability, differentiation, and function of DCs was investigated. In addition, the in vitro antitumor effect of Ii silenced DCs was also explored. It was found that Ii expression in Ii siRNA-treated H-2K DCs was significantly inhibited. Silencing the Ii gene in DCs had no effect on viability and maturation. Ii siRNA-treated DCs significantly increased lymphocyte proliferation when DCs were co-cultured with allogeneic lymphocytes. Furthermore, polarization of Th1 responses was observed when DCs were transfected with Ii siRNA. Much stronger antitumor ability was exhibited when DCs were co-transfected with Ii siRNA and endogenous tumor antigen in vitro. Taken together, loading Ii-silenced DCs with endogenous tumor antigen may be an effective approach to enhance antitumor immunity. 2 Materials and methods 2.1 Animals and cell lines Subject mice were 615 strain (H-2Kk, IAk), 6–8 2.2 Generation of bone marrow-derived DCs Bone marrow-derived DCs (BMDCs) were generated following the protocol described previously (Son et al., 2002). Briefly, bone marrow cells were isolated from femurs and tibiae of 615 strain mice and treated with red blood cell lysing buffer (Roche Diagnostic Systems, Indianapolis, IN, USA). Cell density was adjusted to 5 2.3 RNA isolation Total RNA of MFC was isolated from tumor cell lysates using an RNeasy® Mini kit (Qiagen) according to the protocol for isolation of total RNA from animal cells provided by the manufacturer. The quantity and purity of RNA was determined by UV spectrophotometry. RNA samples were routinely checked by agarose gel electrophoresis and stored at −80 2.4 siRNA design, synthesis and transfection The siRNA sequence used for silencing of murine Ia-associated invariant chain [GeneBank access number: NM-010545] was designed by Qiagen software. An Ii-siRNA targeting the specific sequence CCATGAGCAATTGCCCATA was selected for this study. The double-stranded RNA consisted of the sense strand 5′-CCAUGAGCAAUUGCCCAUAtt-3′ and the antisense strand 3′-tt GGUACUCGUUAACGGGUAU-5′. Searches of the murine genome database (BLAST) were carried out to ensure that the sequence would not target other gene transcripts. siRNA were facilitated by GeneSilencer (Gene Therapy Systems, San Diego, CA, USA), which was used according to the manufacturer's protocol. The non-silencing control siRNA was an irrelevant siRNA with random nucleotides and no known specificity. Its sense strand was 5′-UUCUCCGAACGUGUCACGUtt-3′ and its antisense strand was 3′-ttAAGAGGCUUGCACAGUGCA-5′. Sequences were synthesized and annealed by the manufacturer (GENECHEM, ShangHai, PR China). Immediately after transfection, DCs were further matured with TNF-α at a concentration of 50 2.5 Western blot 48 2.6 Flow cytometry analysis To analyze the expression of surface molecules on the DCs, the following monoclonal antibodies were used: anti-CD11c, anti-CD86, anti-IAk, and anti-CD40 (all from BD Pharmingen, San Diego, California, USA). The anti-CD40, anti-IAk, and anti-CD86 antibody was PE-conjugated. The anti-CD11c antibody was FITC-conjugated or PE-conjugated. Isotype-matched antibodies (BD Pharmingen) were used as controls. The annexin V-propidium iodide method of determining apoptosis/necrosis was used as previously described (Min et al., 2000). Cells were collected on day 8 and fluorescence analysis was performed with a FACScan flow cytometer (Becton Dickinson) using CELLQuest software (Becton Dickinson). 2.7 Allogeneic lymphocyte proliferation Allogeneic lymphocyte activation was set up by culturing 48 2.8 Antigen presentation assay To investigate the antigen presentation and T cell stimulatory capacity of Ii silenced DCs, allogeneic lymphocytes were isolated from spleens. DCs were co-transfected with Ii siRNA and total RNA of MFC using GeneSilencer. After transfection, DCs were incubated in IMDM complete medium containing 50 2.9 ELISA assay IL-12p70, IFN-γ, and IL-4 were measured in the culture medium using the Quantikine® ELISA sets according to the manufacturer's instructions (R&D systems, Wiesbaden, Germany). 2.10 Induction of tumor-specific CTL using Ii-silenced DCs DCs were co-transfected with Ii siRNA and total RNA of MFC using GeneSilencer. After transfection, DCs were incubated in IMDM complete medium containing 50 2.11 CTL assay To determine the cytolytic activity of induced CTL, we used the cytotox 96® non-radioactive cytotoxicity assay based on the calorimetric detection of the released enzyme LDH. Briefly, target cells (tumor cell lines) were harvested, washed, counted, and diluted to 2 Only targets with spontaneous release of LDH less than or equal to 10% of the maximum release were considered. 2.12 Statistical analysis Statistical analysis was performed using SAS 6.0 (from School of Medicine, Shanghai JiaoTong University). One way ANOVA followed by the Newman–Keuls test was used to evaluate statistical significance and a value of P 3 Results 3.1 DCs are efficiently transfected with siRNA and Ii expression is significantly down-regulated To establish a protocol for Ii RNAi in H-2K BMDCs using the GeneSilencer siRNA transfection reagent, the efficacy of siRNA transfection was evaluated by a FITC-labeled control siRNA. The transfection efficiency was measured using a flow cytometer (Fig. 1A). More than 80% of DCs were efficiently transfected. The specificity of siRNA inhibition in DCs after transfection was investigated. Immature DCs were collected on culture day 6 and were transfected with 200 
Fig. 1 Efficacy of siRNA transfection of DCs and specific inhibition of Ii expression. A, DCs (1 3.2 siRNA transfection does not reduce the viability of DCs To assess the toxicity of siRNA and the transfection reagent, the viability of DCs was measured. On day 6 of culture, BMDCs derived from H-2K mice were treated with transfection reagent alone (Mock), transfected with non-silencing siRNA, or with Ii siRNA. After 48 
Fig. 2 siRNA transfection does not affect the viability of DCs. DCs were cultured and treated as indicated in materials and methods. Percentage apoptosis and necrosis was evaluated using annexin V and propidium iodide by flow cytometry. Data are representative of three independent experiments. 3.3 IL-12p70 production of DCs after siRNA transfection The maturation of DCs could be partially characterized by their IL-12p70 production after antigen or TNF-α stimulation. Thus, the IL-12p70 concentration in the culture medium of immature and mature DCs treated with transfection reagent alone, non-silencing siRNA, or Ii siRNA after 48 
Fig. 3 siRNA transfection does not influence IL-12p70 production by DCs. DCs were treated as indicated in materials and methods. Supernatants were harvested from cultures and analyzed for IL-12p70 production using ELISA. The results are the mean 3.4 Cell surface phenotype analysis after Ii siRNA transfection To evaluate the effects of Ii siRNA transfection on DC phenotype, a homogenous population of immature DCs derived from H-2K mice was used after having been cultured with GM-CSF and IL-4 for 6 
Fig. 4 siRNA transfection neither alters nor induces DC maturation. DCs were treated as indicated in materials and methods. DC phenotype was assessed by expression analysis of CD40, CD86, and IAk by flow cytometry. A, mature DCs; B, immature DCs. Data are representative of three independent experiments. 3.5 Lymphocyte stimulatory ability of DCs after Ii siRNA transfection To determine the allostimulatory ability of DCs transfected with Ii siRNA, an allogeneic lymphocyte activation experiment was set up. BMDCs derived from H-2K mice were transfected with Ii siRNA, non-silencing control siRNA, transfection reagent alone, or were left untreated on culture day 6. These DCs were matured with 50 
Fig. 5 Ii siRNA transfection increases DC-induced allogeneic lymphocyte proliferation. DCs were transfected and matured as indicated in Section 3.6 Ii siRNA-treated DCs polarize naïve T cells toward a Th1 response In this study, it was observed that Ii expression in H-2K BMDCs was significantly inhibited by Ii siRNA. When these cells were loaded with endogenous antigen and co-cultured with allogeneic lymphocytes, they appeared optimally to induce a Th1 response. To examine the function of Ii siRNA-transfected DCs, DCs were co-transfected with Ii siRNA and total RNA of MFC initially, then allogeneic lymphocytes isolated from spleens were stimulated with these cells. After 6 
Fig. 6 Ii siRNA-treated DCs promote Th1 polarization. Allogeneic lymphocytes isolated from spleens were stimulated with Mock, non-silencing siRNA, or Ii siRNA-treated mature DCs loaded with total RNA of MFC. After 6 3.7 DCs transfected with Ii siRNA and total RNA of MFC may enhance the MFC-specific CTL response The cytotoxic activity of CTL induced by Ii siRNA-treated H-2K DCs was analyzed 7 
Fig. 7 DCs transfected with Ii siRNA and MFC RNA enhance the tumor-specific CTL response in vitro. Allogeneic lymphocytes isolated from spleens were stimulated with DCs co-transfected with Ii siRNA and MFC RNA (lymphocyte/DC was 10:1). After 7 4 Discussion It is well accepted that a successful vaccine should induce antigen specific CD4+Th1 as well as CD8+T cells. The antigen specific CD4+Th1 cells are needed for the induction and maintenance of CD8+T cell responses and may directly or indirectly contribute to tumor cell destruction (Bennett et al., 1998; Ridge et al., 1998; Schoenberger et al., 1998; Toes et al., 1999). One of the major functions of Ii is to protect the antigenic peptide binding site on MHC class II molecules from binding to endogenously derived antigenic peptides in the endoplasmic reticulum (Bertolino and Rabourdin-Combe, 1996), thus preventing the presentation of endogenous antigen to CD4+T cells. In addition, the diversity of MHC class II presented antigenic peptides is severely restricted by the Ii protein (Bodmer et al., 1994). Because of the pivotal role of Ii in endogenous antigen cross-presentation aforementioned, one of the effective approaches to simultaneously activate CD4+ and CD8+T cells is to knock Ii down. Although antisense oligonucleotides have been used to inhibit Ii expression (Qiu et al., 1999), RNAi appears to be more attractive due to several distinct advantages. Firstly, it is extremely efficient: only a few copies of dsRNA are enough to activate the RNA-induced silencing complex. Once the RNA-induced silencing complex is activated, multiple rounds of gene specific mRNA cleavage will be conducted (Martinez et al., 2002). Secondly, RNAi is very specific, i.e., only the sequences with identity to one of the strands of dsRNA will be cleaved (Hannon, 2002). Although both antisense oligonucleotides and RNAi could efficiently knock target gene down, there are still unspecific effects of antisense oligonucleotides. One problem is toxicity: antisense oligonucleotides cause concentration- and cell type-dependent cell death. The other problem is non-sequence-specific binding to proteins. Antisense oligonucleotides have been shown to bind to a number of different proteins and to thereby cause significant non-specific effects (Stein, 1995; Benimetskaya et al., 1997). On the contrary, three observations demonstrated that siRNA was highly specific: the effect was concentration dependent, it was saturable, and no cellular toxicity was observed (Senn et al., 2005). Thirdly, the effect of RNAi is long lasting and can be spread to progeny cells after replication (Fire et al., 1998). Finally, this technique is not difficult to perform and its effect can be confirmed within days using simple detection systems, such as RT-PCR, ELISA, and western blot (Hill et al., 2003). As depicted in our study, Ii expression in Ii siRNA-treated H-2K DCs was significantly inhibited at the protein level which was estimated by the western blot technique (Fig. 1B). However, whether siRNA transfection alters the viability and maturation of H-2K DCs cannot be ignored for gene therapy. Our study revealed that no variations in the viability and immunophenotypes of Ii siRNA-transfected DCs and the parallel control DCs were detected (Figs. 2 and 4). This demonstrated that siRNA did not affect the viability and maturation of H-2K DCs. The Ii gene encodes two major isoforms, p31 and p41 (Weber et al., 1996). These two isoforms are produced by differential RNA splicing. In this study, p41 expression was found to be down-regulated much more obviously than p31. It may be partially attributed to the longer half-life of p31 (Kampgen et al., 1991; Arunachalam et al., 1994). Although a functional role of Ii in class II antigen presentation is now well established, the role of the Ii isoforms remains controversial. Peterson and Miller reported in 1992 (Peterson and Miller, 1992) that presentation was only facilitated by the alternatively spliced form p41. However, some evidence exists that both Ii forms promote antigen processing equally well (Stockinger et al., 1989; Serwe et al., 1997). IL-12p70 is an inducible cytokine which is crucial to promoting the development of Th1 cells and cell-mediated immunity (Trinchieri and Scott, 1995; Magram et al., 1996; Murphy et al., 2000). It is mainly released by DCs. Although silencing Ii gene in H-2K DCs cannot directly enhance IL-12p70 production in our study (Fig. 3), it may enhance cross-presentation of an endogenous antigen, such as total RNA of tumor cells, to CD4+T cells (Zhao et al., 2003). Activated CD4+T cells will greatly increase their interaction with conditioned DCs. This interaction via CD40 and CD40 ligand can trigger DC production of IL-12p70 and is critical for generating Th1-cell help for cytotoxic responses (Bennett et al., 1998; Ridge et al., 1998; Schoenberger et al., 1998; Lanzavecchia and Sallusto, 2000). After antigen stimulation, CD4+T cells differentiate into at least two types of effector cells that differ as to the pattern of cytokines they produce upon restimulation. Th1 cells are defined by the production of IFN-γ and mediate predominantly cellular immune responses, whereas the signature cytokines of Th2 cells (IL-4, IL-5, IL-13) are involved in allergic reactions (Abbas et al., 1996; Murphy, 1998; O'Garra, 1998). In our study, when the Ii gene was silenced, H-2K DCs significantly enhanced allogeneic lymphocyte proliferation (Fig. 5). In addition, H-2K DCs polarized a Th1 response which was confirmed by high IFN-γ production and much lower IL-4 production from allogeneic lymphocytes (Fig. 6). For CD4+T cells, IFN-γ directs Th1 differentiation and blocks Th2 differentiation by modulating the expression of transcription factors and cytokine receptors (Stark et al., 1998; Murphy and Reiner, 2002; Maldonado et al., 2004; Hwang et al., 2005). It has been shown that tumor-specific type-1 immunity, which is manipulated mainly by tumor antigen-specific Th1 and type-1 cytotoxic T (Tc1) cells, plays a critical role in tumor eradication (Nishimura et al., 1999; Chamoto et al., 2003; Trinchieri, 2003). Tumor-specific Tc1 cells can directly destroy tumor cells when they recognize tumor antigenic peptides bound by MHC class I molecules. On the other hand, Th1 cells produce IL-2 and IFN-γ by recognition of antigenic peptides presented on MHC class II molecules. Although few tumor cells express MHC class II molecules essential for Th cell activation, it is widely accepted that Th cells, especially Th1 cells, play an important role in the eradication of tumor cells via cellular immunity (Pardoll, 1998; Pardoll and Topalian, 1998; Fallarino et al., 2000). This may be because tumor-specific Th1 cells provide local help to enhance antitumor cellular immunity by interacting with MHC class II-bound tumor antigens on antigen presenting cells such as DCs. In addition, direct interaction of Th cells with MHC class II molecules expressed on tumor cells may result in stronger induction of tumor-specific Tc cells (Armstrong et al., 1997; Dissanayake et al., 2004; Rimsza et al., 2004). To further assess the function of Ii silenced H-2K DCs, an initial observation was conducted on the antitumor effect of these cells in vitro. Interestingly, much stronger cytotoxic activity was detected when the gastric cancer cell line MFC was co-cultured with CTL induced by Ii siRNA and MFC RNA co-transfected DCs (Fig. 7). It indicated that silencing the Ii gene in DCs may enhance their antitumor ability in vitro. Our results are consistent with previous reports (Zhao et al., 2003). Another interesting finding was that low cytotoxic activity was detected in the control cell lines CT26 and B16 (Fig. 7). One likely reason for this observation is that these tumor cell lines may share some antigens encoded by the MFC RNA to which the cytotoxic response is directed. In conclusion, we showed that silencing the Ii gene did not affect the viability and maturation of DCs derived from H-2K mice. Furthermore, Ii siRNA-treated H-2K DCs significantly increased allogeneic lymphocyte proliferation and polarized allogeneic lymphocytes toward the Th1 response. 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ISSN Print: 1065-6995
ISSN Electronic: 1095-8355 Published by Portland Press Limited on behalf of the International Federation for Cell Biology (IFCB) |
Abstract
