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Immunostimulatory Effects of Triggering TLR3 Signaling Pathway — Implication for Cancer Immunotherapy

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Mohamed Labib Salem, Said M. Hammad, Mohamed R. Elshanshory, Mohamed A. Attia and Abdel-Aziz A. Zidan

Published: 29 May 2014

DOI: 10.5772/58575

From the Edited Volume

Immune Response Activation

Edited by Guy Huynh Thien Duc

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1. Introduction

Immunostimulation based therapies hold promise of disease-specific interventions without the toxicity and other side effects, which are associated with traditional therapeutic modalities. Unfortunately, the exact manifestation of their therapeutic benefits and the mechanisms of their action are still pending for most immunotherapeutic strategies. Virtually, such kinds of treatments are ideal to establish standards of care and secure, as well as improve therapeutics outcomes. A growing interest for studying and understanding immunostimulation based therapeutics’ strategies has increased since, it could stimulate different components of innate immunity and consequently stimulation of adaptive immunity [1-3].

These immunostimulation based therapies can be divided into two categories, nonspecific and specific immunotherapies. Nonspecific immunotherapy describes therapies that are designed to enhance the immune response without deliberately seeking to modulate the response to a particular antigen. These therapies include three main categories. The first depends on administration of certain agents that induces secretion of inflammatory cytokines that nonspecifically activate the immune system [4]. The second depends on administration of certain type of cytokine that can activate specific populations of immune cells. The third category include the administration of antibodies that block immune checkpoints or other suppressive pathways that are known to shut down the activity of immune system [5]. These nonspecific activation pathways are crucial for the initiation of fully functional immune responses encompassing both the innate and adaptive immune compartments [4].

Toll like receptors (TLRs) are a group of receptors that are expressed mainly in innate immune cells, including macrophages, monocytes, dendritic cells, natural killer cells and mast cells [6]. These TLRs specifically recognize microbes as well as different microbial components called TLR ligands. Interaction of TLR and TLR ligands trigger signaling pathways that lead to activation of innate immune cells through secretion of a plethora of inflammatory mediators including cytokines and chemokines [7, 8]. As such, different TLR ligands have been designed to be used as adjuvant system,

The inclusion of TLR ligands as a potential new class of adjuvants candidates has enabled the development of clinical effective vaccination strategies against many diseases [9, 10]. Polyinosinic-polycytidylic acid [Poly(I:C)] and its clinical grade poly-L-lysine (Poly-ICLC; Hiltonol®) represent important members of these immunostimulatory vaccine adjuvants as has been shown in several preclinical and clinical studies [11, 12]. In this review, we will introduce a brief description of the TLR family followed by a description of the signaling pathway of TLR and its role as a linker between innate and adaptive immunity. The structure, expression and function of Poly(I:C) will be reviewed along with its potential application in cancer vaccination, adoptive immunotherapy and chemo-immunotherapy in preclinical and clinical trials.


2. Toll-like receptors (TLRs) and their agonists

TLRs are a class of transmembrane signaling proteins that play a critical role in initiation and acceleration of innate and adaptive immune responses against different pathogen by recognizing these pathogens themselves or their products, including proteins, carbohydrates, lipids, and nucleic acids (single-and double-stranded RNA and DNA) [13]. TLRs were discovered in 1985 by Christiane Nüsslein-Volhard as factors involved in the embryonic development and resistance of the fly Drosophila to bacterial and fungal infection [14-16]. TLRs are pattern-recognition receptors (PRR) with an extracellular leucine-rich repeats (LRRs) domain and a conserved cytoplasmic domain homologous to that of the interleukin-1 receptor (IL-1R), termed the Toll/IL-1R homology (TIR) domain [17, 18]. Such structure of TLRs recognizes pathogen-associated microbial patterns (PAMPs) encoded in various pathogens. These products include lipopolysaccharide (LPS), peptidoglycan (PGN), flagellin, bacterial DNA, viral single and double stranded RNA and synthetic double-stranded RNA such as poly(I:C). TLRs also recognize danger-associated molecular patterns (DAMPs) that are endogenous molecules including intracellular proteins such as heat shock proteins (HSPs) and protein fragments from the extracellular matrix (HMGB1) released from necrotic or dead cells [10, 19]. Given this capability of TLRs to recognize a wide range of microbial products and their synthetic mimic, modulation of these receptors can significantly play a central role in shaping the outcomes of the anti-microbial immunity. Since cancer originates from self-cells resulting in a poor anti-tumor immunity if not tolerance, triggering TLR signaling pathways during cancer immunotherapy by their specific agonists can accentuate the resultant anti-tumor immunity.

TLRs expression profiles differ among tissues and cell types of the innate and adaptive immune system. TLRs are predominantly expressed on T cells, B cells, neutrophils, monocytes, NK cells, mast cells, and antigen-presenting cells (APCs), such as macrophages (MΦ) or dendritic cells (DCs). As such induction of TLR signaling activates APCs to provoke innate immunity and to induce adaptive immunity [6, 7, 20]. More recently, TLR have been found to be expressed on endothelial, epithelial cells and tumor cells, including melanoma [21]. TLRs are fundamentally located on the plasma membrane except TLR3, TLR7 and TLR9 that are located in the endoplasmic reticulum (ER) [22-24]. Mammalian TLRs include a large family consisting of ten to thirteen different types of TLRs that named simply TLR1 to TLR13 and each one of them recognize specific microbial components [25].

To date, ten human and thirteen murine TLRs have been identified; TLR1 to TLR9 are conserved between the human and mice. However, there are TLRs found in humans and not present in all mammals. For example, TLR10 in humans is present in mice. On the other hand, TLR11, TLR12 and TLR13 in mice are functional, but there is a stop codon in the human TLR11 TLR12 and TLR13 genes, which results in a lack of production of human TLR11 TLR12 and TLR13 [26, 27]. Signaling pathways of these TLRs can be triggered by their specific ligands. TLR ligands (TLRLs) are agents that bind to and activate TLRs. They are encoded in different types of organisms at the cell surface or at the internal cell compartments and they are expressed by different types of leucocytes and other cell types [28, 29].

TLRs/TLRLs bind results in a cascade of intra-cellular signaling pathways that induce the production of inflammatory cytokines and immune response [7, 8]. TLRLs are recognized by specific TLRs that are expressed on the surface of cells, for example, TLR2 is essential for the recognition of bacterial lipoproteins, lipomannans and lipoteichoic acids [30], while TLR5 detects bacterial flagellin [31]. TLR4 has been reported to recognize bacterial cell wall component lipopolysaccharide (LPS) [30], and TLR11 which recognize a profilin-like protein of Toxoplasma gondii [32] and uropathogenic E.coli [33]. In contrast, TLRs located within the endoplasmic reticulum (ER) can detect microbial nucleic acids, for example, TLR3 is required for response to virus-derived double-stranded RNA [34] and TLR9 recognize unmethylated CpG motifs [35]. Whereas, TLR7 and TLR8 could recognize small synthetic antiviral molecules [36], and single-stranded RNA [37].


3. TLRs Signaling pathways

Recent accumulating studies showed that TLRs signaling pathways can be divided into two signaling pathways, the MyD88-dependent pathway which is common to all the TLRs that resulted in the secretion of inflammatory cytokines, and TRIF-dependent pathway that is specific for TLR3 and TLR4 which associated with the stimulation of IFN-β and the maturation of dendritic cells [38-40].

3.1. MyD88-dependent pathway

The myeloid differentiation factor 88 (MyD88) dependent responses utilized by all TLRs except TLR3 and its occurring on hetero-or homo-dimerization of the TLRs receptor, upon activation by PAMPs or DAMPs. This dimerization induces the recruitment of adaptor proteins via the cytoplasmic TIR domain. These adaptor proteins include the TIR-domain containing proteins, TIRAP (TIR-associated protein), TRIF (TIR domain-containing adaptor protein-inducing IFN-β) and TRAM (TRIF-related adaptor molecule), MyD88, Mal (MyD88 adaptor-like protein), which triggers the TLR-mediated signaling pathways [41-45].

The transcription factor IRF7 is critical regulator for the production of IFN-α/β via MyD88 pathway. IRF7 presents in DCs is very low, and therefore TLR7, TLR8 and TLR9 operate mainly through the NF-κB−IRF5 pathway [10, 46, 47]. It is worth noting that the transducing ability of TLR7, TLR8 and TLR9 may change in favor of the IRF7 pathway. Whereas, TLR4 is located on the cell membrane, activation of TLR4 signal transduction through MyD88/TIRAP and TRAM/TRIF pathways leads to activation of innate immune responses, primarily through IRS serine phosphorylationI

3.2. TRIF-dependent pathway

The TLR3 ligand (dsRNA) and the TLR4 ligand (LPS) activate the TRIF-dependent pathway in which IRF-3 plays a key role. For TLR3 (dsRNA), activation of MyD88-independent pathways occurs via TRIF (TIR domain-containing adaptor protein-inducing IFN-β). Upon dsRNA binds within endosomes, TLR3 recruits the adaptor TRIF through a TIR–TIR interaction. TRIF, in turn, recruits RIP1 to activate NF-κB, via TRAFs and the IKK (IκB kinase) complex. TRIF also recruits protein kinases TBK1/IKKε and TRAF3 to activate IRF3/7. Since, IRF-3 can phosphorylate by TBK-1 and IKKε on C-terminal serines, leading to its dimerization and translocation into the nucleus. Active IRF-3 mediates transcription of genes from the IFN-β promoter. The secreted IFN-β binds to the IFN receptor (IFNR), and thus activates transcription of ISGs, such as IRF-7 [9, 48]. On the other hand, the intracellular dsRNA is recognized by the RNA helicase RIG-I (or MDA-5). Activation of the latter activates TBK-1 and IKKε via its CARD domain and activates IRF-3 as well. IRF-7 further stimulates transcription from the IFN-α and-β promoters in a positive feedback loop. Type I IFNs bind also to their receptors to trigger the JAK–STAT signaling pathway. Whereas, to induce inflammatory cytokines production. TLR4 activation is requires both the MyD88-dependent and independent pathways. This occurs via TIR domain containing adaptors TIRAP, MyD88, TRAM and TRIF, which in turn initiates activation of NFκB, MAPK, and IRF3 allowing its nuclear localization and production of IFN-β [40, 49].

The signaling pathway of TLRs is summarized in Figure 1. TLR7, TLR8 and TLR9 are present in endosomes, which are stimulated by viral ssRNA. Upon activation, TLR7, TLR8 and TLR9 signal through the adaptor MyD88, which in turn leads to phosphorylation and activation of IRF3. Following its activation, TLR3 signals through its adaptor TRIF, and thus activates non-canonical IKK kinases (TBK1/IKKε), which subsequently phosphorylate both IRF3 and IRF7. Active IRF-3 induces transcription from the IFN-β promoter. NF-κ β is also activated by TRIF mediated signaling through canonical IKK kinases (IKKα, β, and γ). RIG-I and MDA5 are located in cytoplasm and can recognize both ssRNA and dsRNA. Intracellular ssRNA and dsRNA are recognized by the RNA helicase RIG-I (or mda-5), which are expressed in most cells, activates TBK-1 and IKKε via its CARD domain through interaction with the mitochondrially located adaptor MAVS.

Different signal transducer and activator of transcription (STAT) family members can be activated by interferons (IFNs). As a response to both type I IFNs and type II IFN (IFNγ) stimulation, STAT1-STAT1 homodimers can be formed. These homodimers bind to IFNγ-activated site (GAS) enhancer elements in the promoters of IFN-stimulated genes, and these results in the induction of genes encoding pro-inflammatory cytokines and apoptotic factors. STAT1 and STAT2 heterodimers, which are activated by binding with IRF9, which in turn migrates to the nucleus to bind to IFN-stimulated response elements (ISREs) and activate antiviral and antibacterial genes.


4. Polyinosinic-polycytidylic acid [Poly(I:C)]

Although, several previous studies have been extensively studied poly(I:C) for over 35 years in humans [50, 51], it is receiving new interest as a crucial component in many new immunostimulatory combination therapeutic strategies. Poly(I:C) is a synthetic double-stranded RNA (dsRNA) that has recently been identified as a specific ligand for TLR3 [52-55]. dsRNA is a viral product produced by most viruses during their replication cycle, Both viral dsRNA and its artificial mimic, poly(I:C), are potent inducers of type I interferons (IFN-α/β) [56, 57], which is a crucial cytokines that exert anti-viral and immunostimulatory activities for both T and B lymphocytes [58, 59], DCs [11, 60] and activate monocytes to produce CSF, IL-1β, IL-12, and PGE2 [61, 62].

Several studies have been shown that Poly(I:C) induces a strong innate immune response initiated by two types of PRRs; the TLRs and the RIG-I-like receptors (RLRs) which is a family of cytoplasmic RNA helicases that includes RIG-I and MDA-5 [63-67]. In addition, several studies have shown that Poly(I:C) transduces signals which activate the NF-κB and the IFN-β promoter [53, 54] and activates several nuclear and cytoplasmic enzyme systems such as oligoadenylate synthetase (OAS), the dsRNA dependent protein kinase R (PKR), RIG-I Helicase, and MDA5 (melanoma differentiation associated gene) which are implicated in antiviral and antitumor host defenses [7].

Poly(I:C) interact with TLR3, that is the specific intracellular recognition system that responds and signals to the intracellular presence of dsRNA and RNA virus infection [68]. TLR3 is mainly expressed on a broad range of antigen-processing cells (APCs), dendritic cell (DC) subsets, fibroblasts, intestinal epithelial [54, 69, 70] monocytes, macrophage (MΦ), mast cells, NK cells [71, 72], CD4+and CD8+T cells [6, 73]. Several prior studies have shown that poly(I:C) has potent pleiotropic immunostimulatory effects on several kinds of immune cells [74-76].

Poly(I:C), a synthetic dsRNA mimic copolymer, is a specific TLR3 agonist [13, 77]. Recently, we and others have reported that in vivo administration of the TLR3 agonist poly(I:C) into naïve mice induced rapid increases in the frequencies of NK cells mainly in the liver and that these effects associate with better antigen specific responses and antitumor efficacy [6, 78-81]. Consistent with earlier studies [81], we have demonstrated that the adjuvant effects of poly(I:C) to the antigen-specific CD8+T cell responses are partially dependent on NK cells through creation of a rapid beneficial cytokine milieu [82, 83]. Furthermore, in vitro treatment of highly purified NK cells with poly(I:C) significantly augmented NK cell-mediated cytotoxicity and up-regulated their expression of the activation marker CD69 [71]. Of particular interest, similar to viral infection, poly(I:C) treatment has been found to preferentially induce recruitment and activation of hepatic NK cells [79] and their trafficking to spleen coincided with enhanced cytokine expression [80]. These studies may explain the increased antitumor effects against metastasis observed in tumor models after poly(I:C) treatment [84].


5. Poly(I:C) and anti-tumor Immunity

A fundamental difference between tumor and microbes is that only the latter encode products (signatures) that are recognized as “danger signals” by pathogen recognition receptors (PPRs) expressed in the innate immune cells. [85] In most cases, immune system can mount vigorous immune responses against microbes, but not against cancer. Therefore, the challenge in cancer immunotherapy is how to manipulate the body's own immune system to fight cancer. [86] Mimicking the anti-microbial immunity, recent preclinical and clinical studies have established that provision of TLRL adjuvant system systemically or into tumor environment itself profoundly awaken the cross talk between innate and adaptive immunity, driving generation of efficacious anti-tumor immunity. [87] For instance, the addition of CpG DNA (typical TLR9L) to a melanoma vaccine resulted in effective cytolytic responses. [88] Imiquimod, a synthetic TLR7/8L, has been successfully used in the treatment of basal cell carcinoma [89, 90] and to enhance the immunogenicity of vaccine containing Flt3 ligand and a melanoma peptide. [91] MPL, a TLR4L, has been used as an adjuvant in clinical trials of vaccines against melanoma, glioma and pancreatic and colorectal carcinoma, inducing substantial tumor-specific immunity in response to vaccination [92, 93]. We have reported recently that co-administration of the TLR3L poly(I:C) in vivo with peptide vaccination established functional effector/memory responses [94] by induction of NK-driven beneficial cytokine milieu and DC activation. [81] Although the impact of both of TLRLs at the time of Ag priming has been the focus of several studies, their impacts at the time of Ag recall on the tumor-specific memory responses have not been actively investigated.

Since, cancer cells do not encode danger signals like virus, generation of anti-tumor memory T cells in the tumor setting would require a large pool of highly activated DCs. In response to TLRLs, resident immature DCs at the site of vaccination undergo a maturation program and migrate to the draining lymph nodes (LNs) [72, 95, 96]. Therefore, utilizing TLRLs is a potential approach to induce activation of immature DCs and maximize their contribution to memory cell responses. In line with this notion, recent studies have established the adjuvant effects of several TLRLs, in particular, TLR3L (poly(I:C), TLR7/8L (Imiquimod), and TLR9L (CpG) to the anti-tumor CD8+T cell responses [81, 94, 97-101]. However, most of these studies tested the adjuvant effects of TLRLs in lymphodepleted hosts, and those few studies that utilized lymphodepleted hosts did not use adoptive cell therapy (ACT) system [98, 102-107].

Because the frequency of DCs in steady state condition is low, growth factors in particular Flt3L and G-CSF have been used to mobilize DCs in vivo [108-110]. With this regard, we have recently reported a rapid induction of type I IFNs and activation of DCs with decreases in the levels of Treg cells after CTX treatment; resulting in augmented post vaccination responses which were further improved by addition of poly(I:C) to vaccination [111]. We have found also a marked expansion of immature DCs during the recovery phase after treatment with the anticancer drug cyclophosphamide (CTX), indicating to the mobilizing effect of CTX for DCs. Since DCs are equipped with TLRs that sense different microbial products such as poly(I:C), activation of these immature DCs in vivo may augment memory T cell responses. [85]. Recent studies, including ours have demonstrated the adjuvant effects of TLRLs to ACT in lymphodepleted hosts [112-114]. These studies suggest that combinatorial treatments with chemotherapy/immunotherapy and ACT can markedly improve memory T cell responses. Our observation of expansion of DCs post CTX therapy is a suitable model to dissect the optimal timing of the adjuvant effects of TLRLs to post vaccination responses of ACT in a lymphodepleted host.

Specifically, our recent studies showed that treatment of a recipient host with the non myeloablative dose of CTX augments post vaccination CD8+T cell responses, which were associated with a quick activation of DCs in the lymphopenic phase [114]. We also found that CTX induces increases in the numbers of immature DCs during the recovery phase from days 9-16, peaking on day 12 [115]. These DCs demonstrated normal phagocytic ability in vitro and antigen uptake in vivo. Administration of poly(I:C) at the peak of DC expansion resulted in induction of rapid inflammatory milieu which associated with significant increases in the numbers of activated (CCR7highCD40high) DCs in lymph nodes.

Using the pmel-1 TCR transgenic mouse model, in which CD8+T cells can recognize the natural tumor gp100 antigen on B16 melanoma, we demonstrated that vaccination of CTX-lymphodepleted mice with the MHC class-I melanoma gp100 peptide and poly(I:C) during the lymphopenic phase (day 2) and at the peak of DC expansion (day 12) resulted in significant augmentation in the number of activated DCs in lymph nodes with a temporal increase in the expansion of pmel-1 cells. Conditional depletion studies of DCs at day 12 before revaccination of CTX-treated recipient mice revealed that DCs are required for the optimal pmel-1 cell responses. Importantly, the therapeutic anti-tumor effects of the enhanced pmel-1 cell responses were demonstrated toward an advanced tumor of the poorly immunogenic B16 melanoma, indicating the clinical significance of our observation. Furthermore, we have found improvement in DCs expansion when CTX treatment was followed by a daily treatment with 5µg/mouse G-CSF for 5 days [6], without altering the efficacy of our prime-boost vaccination with hgp100/poly(I:C) [116].

Treg cells express different TLRs [117] and can be activated by certain TLR agonists such as TLR4 [118-120], and TLR5 [121], and can be inactivated by TLR2 and TLR7/8 agonists [122, 123]. Indeed, total body irradiation and CTX-preconditioned hosts demonstrated that interference with Treg cell activities can enhance tumor-specific T cell responses and anti-tumor immunity [124], [125, 126]. Our preliminary studies and previous studies [105, 125-130], however, showed that although Treg cells decrease in numbers during the lymphopenic phase after CTX treatment they recover to their normal levels at the recovery phase. These recovered Treg cells may interfere with the efficacy of vaccination at recovery phase unless the vaccination is co-administered with a potent adjuvant that can block Treg cell function. Indeed, induction of maturation of DCs by certain TLR agonists has been found to control Treg cell function in vivo [122, 131, 132]. Therefore, it is possible that the enhanced pmel-1 cell expansion and anti-tumor effects of pmel-1 cells to peptide/poly(I:C) revaccination on day 12 post CTX treatment involve blocking of Treg cell.

The potent anti-tumor efficacy of prime-boost vaccination with tumor antigen/poly(I:C) at precise time points post CTX therapy would lead to its potential application in the clinical setting. We would envision two treatment protocols for the clinical application of this prime-boost vaccination with tumor peptide and poly(I:C) on cancer (e.g. melanoma) patients. 1) Patients can be with a lymphodepletion dose of CTX followed by G-CSF to correct leucopenia, and vaccinate the patient with a candidate tumor Ag (gp100, MART, or TRP2) with co-administration of the clinical grade form of poly(I:C) such as Hiltonol®. Vaccination and Hiltonol® treatments can be repeated during the recovery from leucopenia (after 10-15 days of chemotherapy) when the numbers of DCs are increased. 2) The same as above but with adoptive transfer of peripheral blood mononuclear cells harvested from the cancer patient prior chemotherapy. These cells can be stimulated in vitro with 1ng/mL of the tumor antigens in the presence of 10ng/mL IL-12 for 3-5 days as we recently described in our preclinical model [133].


6. Poly(I:C) and antiviral immune response

The innate immune response is the first barrier against the invading pathogens and viruses and it responds through activating inflammatory and antiviral defense mechanisms by inducing IFNs-α/β against the viruses [134]. Several in vitro and in vivo studies have demonstrated that the main effects of poly(I:C) is the induction of IFNs-α/β, which play a crucial role in innate anti-viral response [135, 136].Several studies have been extensively demonstrated that poly(I:C) triggers the activation of PKR and other kinases which followed by the phosphorylation of the substrates of these enzymes that results in the subsequent translocation of transcription factors, NF-ҚB and IRF-3, to the nucleus where then they bind to the IFN-β promoter to form a transcription complex that induce IFN-β production [137-140]. It has been thought that the proximal inducer of IFNs-α/β is intracellular dsRNA generated as an intermediate during viral replication [53].

Recently, it has been shown that dsRNA and ssRNA molecules are recognized as intermediate by TLRs that is expressed on DCs, NK cells, MΦ and epithelium during virus replication [141]. Several studies have been extensively demonstrated that Poly(I:C) plays a critical role in inducing innate immune response against many viruses, such as influenza virus [142], human respiratory syncytial virus (RSV) [143], herpes simplex virus 2 (HSV-2) [144], and murine cytomegalovirus [25]. Several in vitro and in vivo studies have been shown that retinoic-acid-inducible gene I (RIG-I) and melanoma-differentiation-associated gene 5 (MDA-5), are the key in the detection of viral dsRNA and Poly(I:C) in the cytosol and subsequent eradication of the replicating viral genomes [66, 145, 146]. Treatment of animals with poly(I:C) results inactivation of the TLR3 receptor which in turn induces the activation of NF-κB and the production of type I interferons that induces strong antiviral and antineoplastic effects accompanied by activation of CD8+T and NK cells [53, 147]. In addition, poly(I:C) is shown to serve as an adjuvant to induce protective CD4+T cell responses against HIV [148].


7. Poly(I:C) and clinical trials

7.1. Hiltonol®

Hiltonol® or Poly-ICLC is a synthetic, nuclease-resistant, hydrophilic complex of polyinosinic and polycytidylic acid, stabilized with poly-L-lysine and carboxymethyl cellulose. Poly-ICLC is IFN-β inducer, which is known to steady the blood-brain-barrier (BBB) and minimize cellular infiltration into damaged brain regions following stroke [149, 150]. Several clinical trials were conducted to utilize poly-ICLC as an IFN inducer in cancer patients, the first clinical trial (phase 1) were conducted to determine the maximum tolerated dose (MTD) and it found that the MTD was (12 mg/m2), which induced effective response in cancer patients [51]. Another study was conducted to determine the immunomodulatory effect of Poly-ICLC in cancer patients and it showed that there was no detectable serum IFN in patients that received 1 mg/m2 Poly-ICLC by IM injection [151]. In contrast, IFN was detectable in the serum of patients that received 4 mg/m2 Poly-ICLC by IV injection. Then, phase I and II clinical trials were conducted in patients with many types of cancers including leukemia, lymphoma, brain tumors, myeloma, juvenile laryngeal papillomatosis, renal cell carcinoma, breast cancer, ovarian cancer, and melanoma [152-160].

In most of these early clinical trials, about 6 mg/m2 Poly-ICLC was generally used intravenously. Fever, often with temperatures greater than 40°C, was a universal adverse event in the trials and was the primary dose-limiting factor. Other common adverse events reported in these trials included nausea, vomiting, hypotension, thrombocytopenia, leukopenia, arthralgia, myalgia, and fatigue. Additionally, very few objective responses were reported in these clinical trials. Due to its toxicity and relative ineffectiveness, utilizing Poly-ICLC was deserted after the availability of recombinant IFN. It was subsequently determined that lower doses (10 to 50 mg/kg) of Poly-ICLC resulted in a broader host defense stimulation, a potent adjuvant effect, and a specific antiretroviral effect mediated by the 2’5’OAS and PKR nuclear enzyme systems [161]. Consequently, Poly-ICLC is currently being developed for use only at doses up to 50 mg/kg. The poly-ICLC treatment prior to exposure to oxygen-glucose deprivation maintained the paracellular and transcellular transport across the endothelium and attenuated the drop in transendothelial electric resistance by enhancing IFN-β mRNA expression in astrocytes and microglia [162]. Recent study utilized poly-ICLC in activation of TLR3 signaling, which exerts a beneficial effect on NK cells, resulting in the increased cetuximab-dependent lysis of head and neck cancer (HNC) cells [163].

7.2. Ampligen®

Ampligen® is known as [poly(I)-poly(C12U)] that is composed of poly(IC) with a U mismatch at every 12th base of the C strand. The main effect of Ampligen® is the induction of Th1 response. This has been demonstrated in delayed-type hypersensitivity reactions and in current clinical studies with human immunodeficiency virus-infected patients. Other cells targeted are NK cells, cytotoxic CD8+T cells, and LAK-NK cells. Despite initial trials in the 1990s, little is known about Ampligen®, since; it is not routinely used in clinical practice. However, due to its notable and potent antiviral effects, Ampligen® has been generally well tolerated in randomized clinical trials [164, 165]. Since, Ampligen® did not consistently produce antiviral effects, but its antiviral activity was always seen after therapeutic treatment. Therefore, it was assumed that Ampligen have exquisite specificity for TLR3 [166, 167]. Early studies showed that treatment of chronically HBV-infected chimpanzees with polyICLC transiently reduced the levels of serum viral DNA, HBsAg and HBeAg [168]. In two independent studies, transient suppression of DHBV in ducks by Ampligen® was also observed [169, 170]. In vitro studies, Ampligen and zidovudine have combined: to act in synergy against HIV-1-infected cell lines [171, 172] and enabling restoration of the immune function in HIV-infected individuals. Wither through maintaining a stable or raising CD4 count, increasing delayed-type hypersensitivity reaction, and reducing rate of progression to AIDS [173].

More recently, Nicodemus et al (2010) evaluated polyIC12U and confirmed the potential of TLR3 stimulation with Ampligen® in enhancement bioactivity of cancer immunotherapies [166]. Their results revealed that Ampligen® is a potent inducer for the dendritic cell maturation and local cytokines producing in culture systems including IL-4, IL-6, IL-12p70, IFN g, MIP-1a and TNF α. Interestingly, treatment of wild-type mice with Ampligen® 24 hours following infection with a lethal viral inoculation was fully protective, whereas dosing 4 h prior to inoculation is ineffective in preventing mortality [167]. Intranasal Ampligen® administered to mice in conjunction with prototype avian flu vaccines, greatly enhances the cellular and humoral immunity achievable with the vaccine and also protects mice from lethal infection [174].


8. Conclusion

Since the discovery of the important role of TLRs in innate immunity and the initiation of an immune response that follows the activation of antigen-specific acquired immunity, rapid progress has been made on our understanding of the molecular mechanisms of TLRs. The inclusion of TLR ligands as a potential new class of adjuvants candidates has enabled the development of clinical effective vaccination strategies against many diseases. Poly(I:C) and Poly-ICLC represent two important member of these immunostimulatory vaccine adjuvants in mice, nonhuman primates and in human. TLR3 is specifically expressed in quite cell-type and species; it is expressed in specific myeloid cells, vascular endothelial cells and airway epithelial cells among others. Human TLR3 is highly expressed in immature DCs whereas mouse TLR3 is highly expressed in macrophages, but in both species, its expression is induced by IFN.

Unlike other TLRs, TLR3 is unique, since, it is not required MyD88 for signaling, and instead, TRIF is the critical adaptor protein for its signaling. Whereas TLR3 displays a distinct intracellular localization compared with other TLRs, TLR7 and TLR9 trigger a quick, but short IFN response, whereas TLR3 might be more important for a prolonged response and the initiation of the adaptive immune response. Poly(I:C) interact with TLR3 that responds and signals to the intracellular presence of dsRNA and RNA virus infection. Poly(I:C), is strong inducers of IFN-α/β, which is a crucial cytokines that exert anti-viral and immunostimulatory activities for both T and B lymphocytes, DCs and activate monocytes to produce CSF, IL-1β, IL-12, and PGE2. It’s also transduces signals which activate the NF-κB and the IFN-β promoter and activates several nuclear and cytoplasmic enzyme systems such as OAS, PKR, RIG-I Helicase, and MDA5 which are implicated in antiviral and antitumor host defenses.

The above mentioned pleotropic effects of the TLR3 agonist Poly(I:C) makes it as a candidate adjuvant system for anti-viral and anti-tumor immune responses, in particular its clinical forms Hiltonol® and Ampligen® induce similar immunomodulatory effects with accepted side effects.



The authors would like to thank Prof. Karolin K. Abdul-Aziz and Dr. Muobarak J. Tuorkey (Zoology Department, Faculty of Science, Damanhour University, Egypt) for their kind help during writing this chapter.


  1. 1. Pulendran, B. and R. Ahmed, Translating innate immunity into immunological memory: implications for vaccine development. Cell, 2006. 124(4): p. 849-63.
  2. 2. Reed, S.G., et al., New horizons in adjuvants for vaccine development. Trends Immunol, 2009. 30(1): p. 23-32.
  3. 3. Coffman, R.L., A. Sher, and R.A. Seder, Vaccine adjuvants: putting innate immunity to work. Immunity, 2010. 33(4): p. 492-503.
  4. 4. Johnson, C.B., et al., Tumours: Immunotherapy, in eLS. 2001, John Wiley & Sons, Ltd.
  5. 5. Belardelli, F. and M. Ferrantini, Cytokines as a link between innate and adaptive antitumor immunity. Trends Immunol, 2002. 23(4): p. 201-8.
  6. 6. Salem, M.L., et al., The TLR3 agonist poly(I:C) targets CD8+T cells and augments their antigen-specific responses upon their adoptive transfer into naive recipient mice. Vaccine, 2009. 27(4): p. 549-57.
  7. 7. Kawai, T. and S. Akira, Innate immune recognition of viral infection. Nat Immunol, 2006. 7(2): p. 131-7.
  8. 8. Pichlmair, A. and C. Reis e Sousa, Innate recognition of viruses. Immunity, 2007. 27(3): p. 370-83.
  9. 9. Beutler, B., Inferences, questions and possibilities in Toll-like receptor signalling. Nature, 2004. 430(6996): p. 257-63.
  10. 10. Kawai, T. and S. Akira, The role of pattern-recognition receptors in innate immunity: update on Toll-like receptors. Nat Immunol, 2010. 11(5): p. 373-84.
  11. 11. Longhi, M.P., et al., Dendritic cells require a systemic type I interferon response to mature and induce CD4+Th1 immunity with poly IC as adjuvant. J Exp Med, 2009. 206(7): p. 1589-602.
  12. 12. Stahl-Hennig, C., et al., Synthetic double-stranded RNAs are adjuvants for the induction of T helper 1 and humoral immune responses to human papillomavirus in rhesus macaques. PLoS Pathog, 2009. 5(4): p. e1000373.
  13. 13. Matsumoto, M. and T. Seya, TLR3: interferon induction by double-stranded RNA including poly(I:C). Adv Drug Deliv Rev, 2008. 60(7): p. 805-12.
  14. 14. Anderson, K.V., G. Jurgens, and C. Nusslein-Volhard, Establishment of dorsal-ventral polarity in the Drosophila embryo: genetic studies on the role of the Toll gene product. Cell, 1985. 42(3): p. 779-89.
  15. 15. Anderson, K.V., L. Bokla, and C. Nusslein-Volhard, Establishment of dorsal-ventral polarity in the Drosophila embryo: the induction of polarity by the Toll gene product. Cell, 1985. 42(3): p. 791-8.
  16. 16. Lemaitre, B., J.M. Reichhart, and J.A. Hoffmann, Drosophila host defense: differential induction of antimicrobial peptide genes after infection by various classes of microorganisms. Proc Natl Acad Sci U S A, 1997. 94(26): p. 14614-9.
  17. 17. Bowie, A. and L.A. O'Neill, The interleukin-1 receptor/Toll-like receptor superfamily: signal generators for pro-inflammatory interleukins and microbial products. J Leukoc Biol, 2000. 67(4): p. 508-14.
  18. 18. Thompson, M.R., et al., Pattern recognition receptors and the innate immune response to viral infection. Viruses, 2011. 3(6): p. 920-40.
  19. 19. Takahashi, K. and T. Kawai, [Pathogen recognition by Toll-like receptor]. Nihon Rinsho, 2007. 65 Suppl 2 Pt. 1: p. 53-7.
  20. 20. Uematsu, S. and S. Akira, [Innate immune recognition of viral infection]. Uirusu, 2006. 56(1): p. 1-8.
  21. 21. Salaun, B., et al., Toll-like receptor 3 expressed by melanoma cells as a target for therapy? Clin Cancer Res, 2007. 13(15 Pt 1): p. 4565-74.
  22. 22. Nishiya, T. and A.L. DeFranco, Ligand-regulated chimeric receptor approach reveals distinctive subcellular localization and signaling properties of the Toll-like receptors. J Biol Chem, 2004. 279(18): p. 19008-17.
  23. 23. He, X., et al., Recognition of pathogen-associated nucleic acids by endosomal nucleic acid-sensing toll-like receptors. Acta Biochim Biophys Sin (Shanghai), 2013. 45(4): p. 241-58.
  24. 24. Akira, S., K. Takeda, and T. Kaisho, Toll-like receptors: critical proteins linking innate and acquired immunity. Nat Immunol, 2001. 2(8): p. 675-80.
  25. 25. Tabeta, K., et al., Toll-like receptors 9 and 3 as essential components of innate immune defense against mouse cytomegalovirus infection. Proc Natl Acad Sci U S A, 2004. 101(10): p. 3516-21.
  26. 26. Ariffin, J.K. and M.J. Sweet, Differences in the repertoire, regulation and function of Toll-like Receptors and inflammasome-forming Nod-like Receptors between human and mouse. Curr Opin Microbiol, 2013.
  27. 27. Roach, J.C., et al., The evolution of vertebrate Toll-like receptors. Proc Natl Acad Sci U S A, 2005. 102(27): p. 9577-82.
  28. 28. Pulendran, B., Modulating vaccine responses with dendritic cells and Toll-like receptors. Immunol Rev, 2004. 199: p. 227-50.
  29. 29. Takeda, K. and S. Akira, Toll-like receptors. Curr Protoc Immunol, 2007. Chapter 14: p. Unit 14 12.
  30. 30. Takeuchi, O., et al., Differential roles of TLR2 and TLR4 in recognition of gram-negative and gram-positive bacterial cell wall components. Immunity, 1999. 11(4): p. 443-51.
  31. 31. Smith, K.D. and A. Ozinsky, Toll-like receptor-5 and the innate immune response to bacterial flagellin. Curr Top Microbiol Immunol, 2002. 270: p. 93-108.
  32. 32. Lauw, F.N., D.R. Caffrey, and D.T. Golenbock, Of mice and man: TLR11 (finally) finds profilin. Trends Immunol, 2005. 26(10): p. 509-11.
  33. 33. Zhang, D.M., et al., [Down-regulation of Toll-like receptor 4 is related to the tolerance to lipopolysaccharide in rat alveolar macrophages]. Zhonghua Jie He He Hu Xi Za Zhi, 2003. 26(7): p. 411-4.
  34. 34. Schulz, O., et al., Toll-like receptor 3 promotes cross-priming to virus-infected cells. Nature, 2005. 433(7028): p. 887-92.
  35. 35. Sivori, S., et al., CpG and double-stranded RNA trigger human NK cells by Toll-like receptors: induction of cytokine release and cytotoxicity against tumors and dendritic cells. Proc Natl Acad Sci U S A, 2004. 101(27): p. 10116-21.
  36. 36. Jurk, M., et al., Human TLR7 or TLR8 independently confer responsiveness to the antiviral compound R-848. Nat Immunol, 2002. 3(6): p. 499.
  37. 37. Heil, F., et al., Species-specific recognition of single-stranded RNA via toll-like receptor 7 and 8. Science, 2004. 303(5663): p. 1526-9.
  38. 38. Akira, S. and H. Hemmi, Recognition of pathogen-associated molecular patterns by TLR family. Immunol Lett, 2003. 85(2): p. 85-95.
  39. 39. Adachi, O., et al., Targeted disruption of the MyD88 gene results in loss of IL-1-and IL-18-mediated function. Immunity, 1998. 9(1): p. 143-50.
  40. 40. Kawai, T. and S. Akira, TLR signaling. Semin Immunol, 2007. 19(1): p. 24-32.
  41. 41. Horng, T., et al., The adaptor molecule TIRAP provides signalling specificity for Toll-like receptors. Nature, 2002. 420(6913): p. 329-33.
  42. 42. Fitzgerald, K.A., et al., Mal (MyD88-adapter-like) is required for Toll-like receptor-4 signal transduction. Nature, 2001. 413(6851): p. 78-83.
  43. 43. Yamamoto, M., et al., Cutting edge: a novel Toll/IL-1 receptor domain-containing adapter that preferentially activates the IFN-beta promoter in the Toll-like receptor signaling. J Immunol, 2002. 169(12): p. 6668-72.
  44. 44. Oshiumi, H., et al., TICAM-1, an adaptor molecule that participates in Toll-like receptor 3-mediated interferon-beta induction. Nat Immunol, 2003. 4(2): p. 161-7.
  45. 45. Oshiumi, H., et al., TIR-containing adapter molecule (TICAM)-2, a bridging adapter recruiting to toll-like receptor 4 TICAM-1 that induces interferon-beta. J Biol Chem, 2003. 278(50): p. 49751-62.
  46. 46. Keating, S.E., et al., IRAK-2 participates in multiple toll-like receptor signaling pathways to NFkappaB via activation of TRAF6 ubiquitination. J Biol Chem, 2007. 282(46): p. 33435-43.
  47. 47. Kanayama, A., et al., TAB2 and TAB3 activate the NF-kappaB pathway through binding to polyubiquitin chains. Mol Cell, 2004. 15(4): p. 535-48.
  48. 48. Takeda, K. and S. Akira, TLR signaling pathways. Semin Immunol, 2004. 16(1): p. 3-9.
  49. 49. Kawai, T. and S. Akira, Signaling to NF-kappaB by Toll-like receptors. Trends Mol Med, 2007. 13(11): p. 460-9.
  50. 50. Freeman, A.I., et al., Pharmacologic effects of polyinosinic-polycytidylic acid in man. J Med Virol, 1977. 1(2): p. 79-93.
  51. 51. Levine, A.S., et al., Initial clinical trials in cancer patients of polyriboinosinic-polyribocytidylic acid stabilized with poly-L-lysine, in carboxymethylcellulose [poly(ICLC)], a highly effective interferon inducer. Cancer Res, 1979. 39(5): p. 1645-50.
  52. 52. Cella, M., et al., Plasmacytoid dendritic cells activated by influenza virus and CD40L drive a potent TH1 polarization. Nat Immunol, 2000. 1(4): p. 305-10.
  53. 53. Alexopoulou, L., et al., Recognition of double-stranded RNA and activation of NF-kappaB by Toll-like receptor 3. Nature, 2001. 413(6857): p. 732-8.
  54. 54. Matsumoto, M., et al., Establishment of a monoclonal antibody against human Toll-like receptor 3 that blocks double-stranded RNA-mediated signaling. Biochem Biophys Res Commun, 2002. 293(5): p. 1364-9.
  55. 55. Meusel, T.R., K.E. Kehoe, and F. Imani, Protein kinase R regulates double-stranded RNA induction of TNF-alpha but not IL-1 beta mRNA in human epithelial cells. J Immunol, 2002. 168(12): p. 6429-35.
  56. 56. Levy, H.B., Induction of interferon in vivo and in vitro by polynucleotides and derivatives, and preparation of derivatives. Methods Enzymol, 1981. 78(Pt A): p. 242-51.
  57. 57. Meurs, E., et al., Molecular cloning and characterization of the human double-stranded RNA-activated protein kinase induced by interferon. Cell, 1990. 62(2): p. 379-90.
  58. 58. Kolumam, G.A., et al., Type I interferons act directly on CD8 T cells to allow clonal expansion and memory formation in response to viral infection. J Exp Med, 2005. 202(5): p. 637-50.
  59. 59. Le Bon, A., et al., Direct stimulation of T cells by type I IFN enhances the CD8+T cell response during cross-priming. J Immunol, 2006. 176(8): p. 4682-9.
  60. 60. Le Bon, A., et al., Type i interferons potently enhance humoral immunity and can promote isotype switching by stimulating dendritic cells in vivo. Immunity, 2001. 14(4): p. 461-70.
  61. 61. Manetti, R., et al., Polyinosinic acid: polycytidylic acid promotes T helper type 1-specific immune responses by stimulating macrophage production of interferon-alpha and interleukin-12. Eur J Immunol, 1995. 25(9): p. 2656-60.
  62. 62. Akiyama, Y., et al., Differential ability of human blood monocyte subsets to release various cytokines. J Leukoc Biol, 1985. 37(5): p. 519-30.
  63. 63. Kok, K.H., et al., The double-stranded RNA-binding protein PACT functions as a cellular activator of RIG-I to facilitate innate antiviral response. Cell Host Microbe, 2011. 9(4): p. 299-309.
  64. 64. Kawai, T. and S. Akira, Antiviral signaling through pattern recognition receptors. J Biochem, 2007. 141(2): p. 137-45.
  65. 65. Koyama, S., et al., Differential role of TLR-and RLR-signaling in the immune responses to influenza A virus infection and vaccination. J Immunol, 2007. 179(7): p. 4711-20.
  66. 66. Gitlin, L., et al., Essential role of mda-5 in type I IFN responses to polyriboinosinic:polyribocytidylic acid and encephalomyocarditis picornavirus. Proc Natl Acad Sci U S A, 2006. 103(22): p. 8459-64.
  67. 67. Kato, H., et al., Cell type-specific involvement of RIG-I in antiviral response. Immunity, 2005. 23(1): p. 19-28.
  68. 68. Matsumoto, M., et al., Subcellular localization of Toll-like receptor 3 in human dendritic cells. J Immunol, 2003. 171(6): p. 3154-62.
  69. 69. Muzio, M., et al., Differential expression and regulation of toll-like receptors (TLR) in human leukocytes: selective expression of TLR3 in dendritic cells. J Immunol, 2000. 164(11): p. 5998-6004.
  70. 70. Cario, E. and D.K. Podolsky, Differential alteration in intestinal epithelial cell expression of toll-like receptor 3 (TLR3) and TLR4 in inflammatory bowel disease. Infect Immun, 2000. 68(12): p. 7010-7.
  71. 71. Schmidt, K.N., et al., APC-independent activation of NK cells by the Toll-like receptor 3 agonist double-stranded RNA. J Immunol, 2004. 172(1): p. 138-43.
  72. 72. Banchereau, J., et al., Dendritic cells: controllers of the immune system and a new promise for immunotherapy. Novartis Found Symp, 2003. 252: p. 226-35; discussion 235-8, 257-67.
  73. 73. Gelman, A.E., et al., Toll-like receptor ligands directly promote activated CD4+T cell survival. J Immunol, 2004. 172(10): p. 6065-73.
  74. 74. Cella, M., et al., Maturation, activation, and protection of dendritic cells induced by double-stranded RNA. J Exp Med, 1999. 189(5): p. 821-9.
  75. 75. Haines, D.S., K.I. Strauss, and D.H. Gillespie, Cellular response to double-stranded RNA. J Cell Biochem, 1991. 46(1): p. 9-20.
  76. 76. Manetti, R., et al., Polyinosinic acid: polycytidylic acid promotes T helper type 1-specific immune responses by stimulating macrophage production of interferon-alpha and interleukin-12. Eur J Immunol, 1995. 25(9): p. 2656-60.
  77. 77. Zhou, Y., et al., Hepatitis C virus E2 envelope protein induces dendritic cell maturation. J Viral Hepat, 2007. 14(12): p. 849-58.
  78. 78. Wang, H., E. Rayburn, and R. Zhang, Synthetic oligodeoxynucleotides containing deoxycytidyl-deoxyguanosine dinucleotides (CpG ODNs) and modified analogs as novel anticancer therapeutics. Curr Pharm Des, 2005. 11(22): p. 2889-907.
  79. 79. Dong, Z., et al., Involvement of natural killer cells in PolyI:C-induced liver injury. J Hepatol, 2004. 41(6): p. 966-73.
  80. 80. Salazar-Mather, T.P., R. Ishikawa, and C.A. Biron, NK cell trafficking and cytokine expression in splenic compartments after IFN induction and viral infection. J Immunol, 1996. 157(7): p. 3054-64.
  81. 81. Salem, M.L., et al., The adjuvant effects of the toll-like receptor 3 ligand polyinosinic-cytidylic acid poly (I:C) on antigen-specific CD8+T cell responses are partially dependent on NK cells with the induction of a beneficial cytokine milieu. Vaccine, 2006. 24(24): p. 5119-32.
  82. 82. Salem, M.L., et al., Vaccination using Toll-like receptor ligand (TLRL)-primed dendritic cells and TLR3L poly I:C administration leads to an augmentation of antigen-specific memory CD8+T cell responses.. 97th Annual Meeting for American Association of Cancer Research (AACR), Washington, DC., 2006.
  83. 83. Alenzi, F.Q., et al., Cellular and molecular aspects of Goodpasture syndrome. Iran J Kidney Dis, 2012. 6(1): p. 1-8.
  84. 84. Yang, Q., et al., Antitumor activity of NK cells. Immunol Res, 2006. 36(1-3): p. 13-25.
  85. 85. Kowalczyk, D.W., Tumors and the danger model. Acta Biochim Pol, 2002. 49(2): p. 295-302.
  86. 86. Seya, T., et al., Role of toll-like receptors and their adaptors in adjuvant immunotherapy for cancer. Anticancer Res, 2003. 23(6a): p. 4369-76.
  87. 87. Okamoto, M. and M. Sato, Toll-like receptor signaling in anti-cancer immunity. J Med Invest, 2003. 50(1-2): p. 9-24.
  88. 88. Speiser, D.E., et al., Rapid and strong human CD8+T cell responses to vaccination with peptide, IFA, and CpG oligodeoxynucleotide 7909. J Clin Invest, 2005. 115(3): p. 739-46.
  89. 89. Smith, K.J., S. Hamza, and H. Skelton, The imidazoquinolines and their place in the therapy of cutaneous disease. Expert Opin Pharmacother, 2003. 4(7): p. 1105-19.
  90. 90. Stockfleth, E., et al., The use of Toll-like receptor-7 agonist in the treatment of basal cell carcinoma: an overview. Br J Dermatol, 2003. 149 Suppl 66: p. 53-6.
  91. 91. Shackleton, M., et al., The impact of imiquimod, a Toll-like receptor-7 ligand (TLR7L), on the immunogenicity of melanoma peptide vaccination with adjuvant Flt3 ligand. Cancer Immun, 2004. 4: p. 9.
  92. 92. Baldridge, J.R., et al., Taking a Toll on human disease: Toll-like receptor 4 agonists as vaccine adjuvants and monotherapeutic agents. Expert Opin Biol Ther, 2004. 4(7): p. 1129-38.
  93. 93. Stover, A.G., et al., Structure-activity relationship of synthetic toll-like receptor 4 agonists. J Biol Chem, 2004. 279(6): p. 4440-9.
  94. 94. Salem, M.L., et al., Defining the antigen-specific T-cell response to vaccination and poly(I:C)/TLR3 signaling: evidence of enhanced primary and memory CD8 T-cell responses and antitumor immunity. J Immunother, 2005. 28(3): p. 220-8.
  95. 95. Bachmann, M.F., M. Kopf, and B.J. Marsland, Chemokines: more than just road signs. Nat Rev Immunol, 2006. 6(2): p. 159-64.
  96. 96. Shortman, K. and Y.J. Liu, Mouse and human dendritic cell subtypes. Nat Rev Immunol, 2002. 2(3): p. 151-61.
  97. 97. Prins, R.M., et al., The TLR-7 agonist, imiquimod, enhances dendritic cell survival and promotes tumor antigen-specific T cell priming: relation to central nervous system antitumor immunity. J Immunol, 2006. 176(1): p. 157-64.
  98. 98. Weigel, B.J., et al., CpG oligodeoxynucleotides potentiate the antitumor effects of chemotherapy or tumor resection in an orthotopic murine model of rhabdomyosarcoma. Clin Cancer Res, 2003. 9(8): p. 3105-14.
  99. 99. Badovinac, V.P., et al., Accelerated CD8+T-cell memory and prime-boost response after dendritic-cell vaccination. Nat Med, 2005. 11(7): p. 748-56.
  100. 100. Davila, E. and E. Celis, Repeated administration of cytosine-phosphorothiolated guanine-containing oligonucleotides together with peptide/protein immunization results in enhanced CTL responses with anti-tumor activity. J Immunol, 2000. 165(1): p. 539-47.
  101. 101. Davila, E., R. Kennedy, and E. Celis, Generation of antitumor immunity by cytotoxic T lymphocyte epitope peptide vaccination, CpG-oligodeoxynucleotide adjuvant, and CTLA-4 blockade. Cancer Res, 2003. 63(12): p. 3281-8.
  102. 102. Craft, N., et al., The TLR7 agonist imiquimod enhances the anti-melanoma effects of a recombinant Listeria monocytogenes vaccine. J Immunol, 2005. 175(3): p. 1983-90.
  103. 103. Nowak, M., et al., LPS-induced liver injury in D-galactosamine-sensitized mice requires secreted TNF-alpha and the TNF-p55 receptor. Am J Physiol Regul Integr Comp Physiol, 2000. 278(5): p. R1202-9.
  104. 104. Berraondo, P., et al., Eradication of large tumors in mice by a tritherapy targeting the innate, adaptive, and regulatory components of the immune system. Cancer Res, 2007. 67(18): p. 8847-55.
  105. 105. Taieb, J., et al., Chemoimmunotherapy of tumors: cyclophosphamide synergizes with exosome based vaccines. J Immunol, 2006. 176(5): p. 2722-9.
  106. 106. Garay, R.P., et al., Cancer relapse under chemotherapy: why TLR2/4 receptor agonists can help. Eur J Pharmacol, 2007. 563(1-3): p. 1-17.
  107. 107. Adams, M., et al., The rationale for combined chemo/immunotherapy using a Toll-like receptor 3 (TLR3) agonist and tumour-derived exosomes in advanced ovarian cancer. Vaccine, 2005. 23(17-18): p. 2374-8.
  108. 108. Berhanu, A., et al., Combinational FLt3 ligand and granulocyte macrophage colony-stimulating factor treatment promotes enhanced tumor infiltration by dendritic cells and antitumor CD8(+) T-cell cross-priming but is ineffective as a therapy. Cancer Res, 2006. 66(9): p. 4895-903.
  109. 109. Koopman, G., et al., Increase in plasmacytoid and myeloid dendritic cells by progenipoietin-1, a chimeric Flt-3 and G-CSF receptor agonist, in SIV-Infected rhesus macaques. Hum Immunol, 2004. 65(4): p. 303-16.
  110. 110. Maraskovsky, E., et al., Dramatic numerical increase of functionally mature dendritic cells in FLT3 ligand-treated mice. Adv Exp Med Biol, 1997. 417: p. 33-40.
  111. 111. Salem, M.L., et al., Defining the Ability of Cyclophosphamide Preconditioning to Enhance the Antigen-specific CD8+T-cell Response to Peptide Vaccination: Creation of a Beneficial Host Microenvironment Involving Type I IFNs and Myeloid Cells. J Immunother, 2007. 30(1): p. 40-53.
  112. 112. Paulos, C.M., et al., Toll-like receptors in tumor immunotherapy. Clin Cancer Res, 2007. 13(18 Pt 1): p. 5280-9.
  113. 113. Paulos, C.M., et al., Microbial translocation augments the function of adoptively transferred self/tumor-specific CD8 T cells via TLR4 signaling. J Clin Invest, 2007. 117(8): p. 2197-204.
  114. 114. Salem, M.L., et al., Defining the ability of cyclophosphamide preconditioning to enhance the antigen-specific CD8+T-cell response to peptide vaccination: creation of a beneficial host microenvironment involving type I IFNs and myeloid cells. J Immunother (1997), 2007. 30(1): p. 40-53.
  115. 115. Salem, M.L., et al., Recovery from cyclophosphamide-induced lymphopenia results in expansion of immature dendritic cells which can mediate enhanced prime-boost vaccination antitumor responses in vivo when stimulated with the TLR3 agonist poly(I:C). J Immunol, 2009. 182(4): p. 2030-40.
  116. 116. Rubinstein, M.P., et al., Loss of T cell-mediated antitumor immunity after construct-specific downregulation of retrovirally encoded T-cell receptor expression in vivo. Cancer Gene Ther, 2009. 16(2): p. 171-83.
  117. 117. Kabelitz, D., Expression and function of Toll-like receptors in T lymphocytes. Curr Opin Immunol, 2007. 19(1): p. 39-45.
  118. 118. Caramalho, I., et al., Regulatory T cells selectively express toll-like receptors and are activated by lipopolysaccharide. J Exp Med, 2003. 197(4): p. 403-11.
  119. 119. Lewkowicz, P., et al., Lipopolysaccharide-activated CD4+CD25+T regulatory cells inhibit neutrophil function and promote their apoptosis and death. J Immunol, 2006. 177(10): p. 7155-63.
  120. 120. den Haan, J.M., G. Kraal, and M.J. Bevan, Cutting edge: Lipopolysaccharide induces IL-10-producing regulatory CD4+T cells that suppress the CD8+T cell response. J Immunol, 2007. 178(9): p. 5429-33.
  121. 121. Crellin, N.K., et al., Human CD4+T cells express TLR5 and its ligand flagellin enhances the suppressive capacity and expression of FOXP3 in CD4+CD25+T regulatory cells. J Immunol, 2005. 175(12): p. 8051-9.
  122. 122. Sutmuller, R.P., et al., Toll-like receptor 2 controls expansion and function of regulatory T cells. J Clin Invest, 2006. 116(2): p. 485-94.
  123. 123. Liu, P.T., et al., Toll-like receptor triggering of a vitamin D-mediated human antimicrobial response. Science, 2006. 311(5768): p. 1770-3.
  124. 124. Antony, P.A., et al., CD8+T cell immunity against a tumor/self-antigen is augmented by CD4+T helper cells and hindered by naturally occurring T regulatory cells. J Immunol, 2005. 174(5): p. 2591-601.
  125. 125. Wada, S., et al., Cyclophosphamide augments antitumor immunity: studies in an autochthonous prostate cancer model. Cancer Res, 2009. 69(10): p. 4309-18.
  126. 126. Hirschhorn-Cymerman, D., et al., OX40 engagement and chemotherapy combination provides potent antitumor immunity with concomitant regulatory T cell apoptosis. J Exp Med, 2009. 206(5): p. 1103-16.
  127. 127. Ghiringhelli, F., et al., CD4+CD25+regulatory T cells suppress tumor immunity but are sensitive to cyclophosphamide which allows immunotherapy of established tumors to be curative. Eur J Immunol, 2004. 34(2): p. 336-44.
  128. 128. Hillebrands, J.L., et al., A regulatory CD4+T cell subset in the BB rat model of autoimmune diabetes expresses neither CD25 nor Foxp3. J Immunol, 2006. 177(11): p. 7820-32.
  129. 129. Huleatt, J.W., et al., Vaccination with recombinant fusion proteins incorporating Toll-like receptor ligands induces rapid cellular and humoral immunity. Vaccine, 2007. 25(4): p. 763-75.
  130. 130. Teitz-Tennenbaum, S., et al., Radiotherapy combined with intratumoral dendritic cell vaccination enhances the therapeutic efficacy of adoptive T-cell transfer. J Immunother, 2009. 32(6): p. 602-12.
  131. 131. Fehervari, Z. and S. Sakaguchi, A paragon of self-tolerance: CD25+CD4+regulatory T cells and the control of immune responses. Arthritis Res Ther, 2004. 6(1): p. 19-25.
  132. 132. Netea, M.G., et al., Toll-like receptor 2 suppresses immunity against Candida albicans through induction of IL-10 and regulatory T cells. J Immunol, 2004. 172(6): p. 3712-8.
  133. 133. Diaz-Montero, C.M., et al., Priming of naive CD8+T cells in the presence of IL-12 selectively enhances the survival of CD8+CD62Lhi cells and results in superior anti-tumor activity in a tolerogenic murine model. Cancer Immunol Immunother, 2008. 57(4): p. 563-72.
  134. 134. Le Bon, A. and D.F. Tough, Links between innate and adaptive immunity via type I interferon. Curr Opin Immunol, 2002. 14(4): p. 432-6.
  135. 135. Koyama, S., et al., Innate immune response to viral infection. Cytokine, 2008. 43(3): p. 336-41.
  136. 136. Kumar, A., J. Zhang, and F.S. Yu, Toll-like receptor 3 agonist poly(I:C)-induced antiviral response in human corneal epithelial cells. Immunology, 2006. 117(1): p. 11-21.
  137. 137. Yoneyama, M., et al., Direct triggering of the type I interferon system by virus infection: activation of a transcription factor complex containing IRF-3 and CBP/p300. EMBO J, 1998. 17(4): p. 1087-95.
  138. 138. Wathelet, M.G., et al., Virus infection induces the assembly of coordinately activated transcription factors on the IFN-beta enhancer in vivo. Mol Cell, 1998. 1(4): p. 507-18.
  139. 139. Lin, R., et al., Virus-dependent phosphorylation of the IRF-3 transcription factor regulates nuclear translocation, transactivation potential, and proteasome-mediated degradation. Mol Cell Biol, 1998. 18(5): p. 2986-96.
  140. 140. Juang, Y.T., et al., Primary activation of interferon A and interferon B gene transcription by interferon regulatory factor 3. Proc Natl Acad Sci U S A, 1998. 95(17): p. 9837-42.
  141. 141. Sen, G.C. and S.N. Sarkar, Transcriptional signaling by double-stranded RNA: role of TLR3. Cytokine Growth Factor Rev, 2005. 16(1): p. 1-14.
  142. 142. Guillot, L., et al., Involvement of toll-like receptor 3 in the immune response of lung epithelial cells to double-stranded RNA and influenza A virus. J Biol Chem, 2005. 280(7): p. 5571-80.
  143. 143. Rudd, B.D., et al., Differential role for TLR3 in respiratory syncytial virus-induced chemokine expression. J Virol, 2005. 79(6): p. 3350-7.
  144. 144. Ashkar, A.A., et al., Toll-like receptor (TLR)-3, but not TLR4, agonist protects against genital herpes infection in the absence of inflammation seen with CpG DNA. J Infect Dis, 2004. 190(10): p. 1841-9.
  145. 145. Yoneyama, M., et al., The RNA helicase RIG-I has an essential function in double-stranded RNA-induced innate antiviral responses. Nat Immunol, 2004. 5(7): p. 730-7.
  146. 146. Yoneyama, M., et al., Shared and unique functions of the DExD/H-box helicases RIG-I, MDA5, and LGP2 in antiviral innate immunity. J Immunol, 2005. 175(5): p. 2851-8.
  147. 147. Akazawa, T., et al., Antitumor NK activation induced by the Toll-like receptor 3-TICAM-1 (TRIF) pathway in myeloid dendritic cells. Proc Natl Acad Sci U S A, 2007. 104(1): p. 252-7.
  148. 148. Trumpfheller, C., et al., The microbial mimic poly IC induces durable and protective CD4+T cell immunity together with a dendritic cell targeted vaccine. Proc Natl Acad Sci U S A, 2008. 105(7): p. 2574-9.
  149. 149. Veldhuis, W.B., et al., Interferon-beta blocks infiltration of inflammatory cells and reduces infarct volume after ischemic stroke in the rat. J Cereb Blood Flow Metab, 2003. 23(9): p. 1029-39.
  150. 150. Kraus, J., et al., Interferon-beta stabilizes barrier characteristics of brain endothelial cells in vitro. Ann Neurol, 2004. 56(2): p. 192-205.
  151. 151. Maluish, A.E., et al., Immunomodulatory effects of poly(I,C)-LC in cancer patients. J Biol Response Mod, 1985. 4(6): p. 656-63.
  152. 152. Droller, M.J., Immunotherapy of metastatic renal cell carcinoma with polyinosinic-polycytidylic acid. J Urol, 1987. 137(2): p. 202-6.
  153. 153. Hawkins, M.J., M. Levin, and E.C. Borden, An Eastern Cooperative Oncology Group phase I-II pilot study of polyriboinosinic-polyribocytidylic acid poly-L-lysine complex in patients with metastatic malignant melanoma. J Biol Response Mod, 1985. 4(6): p. 664-8.
  154. 154. Krown, S.E., et al., Phase I trials of poly(I,C) complexes in advanced cancer. J Biol Response Mod, 1985. 4(6): p. 640-9.
  155. 155. Lampkin, B.C., et al., Phase II trial of a complex polyriboinosinic-polyribocytidylic acid with poly-L-lysine and carboxymethyl cellulose in the treatment of children with acute leukemia and neuroblastoma: a report from the Children's Cancer Study Group. Cancer Res, 1985. 45(11 Pt 2): p. 5904-9.
  156. 156. Levine, A.S. and H.B. Levy, Phase I-II trials of poly IC stabilized with poly-L-lysine. Cancer Treat Rep, 1978. 62(11): p. 1907-12.
  157. 157. Nakamura, O., et al., Phase I-II trials of poly(ICLC) in malignant brain tumor patients. J Interferon Res, 1982. 2(1): p. 1-4.
  158. 158. Rettenmaier, M.A., M.L. Berman, and P.J. DiSaia, Treatment of advanced ovarian cancer with polyinosinic-polycytidylic lysine carboxymethylcellulose (poly(ICLC)). Gynecol Oncol, 1986. 24(3): p. 359-61.
  159. 159. Stevenson, H.C., et al., A phase I evaluation of poly(I,C)-LC in cancer patients. J Biol Response Mod, 1985. 4(6): p. 650-5.
  160. 160. Theriault, R.L., et al., Evaluation of polyinosinic-polycytidylic and poly-L-lysine in metastatic breast cancer. Cancer Treat Rep, 1986. 70(11): p. 1341-2.
  161. 161. Ewel, C.H., et al., Polyinosinic-polycytidylic acid complexed with poly-L-lysine and carboxymethylcellulose in combination with interleukin 2 in patients with cancer: clinical and immunological effects. Cancer Res, 1992. 52(11): p. 3005-10.
  162. 162. Gesuete, R., et al., Poly-ICLC preconditioning protects the blood-brain barrier against ischemic injury in vitro through type I interferon signaling. J Neurochem, 2012. 123 Suppl 2: p. 75-85.
  163. 163. Ming Lim, C., et al., TLR3 agonists improve the immunostimulatory potential of cetuximab against EGFR head and neck cancer cells. Oncoimmunology, 2013. 2(6): p. e24677.
  164. 164. Strayer, D.R., et al., A controlled clinical trial with a specifically configured RNA drug, poly(I).poly(C12U), in chronic fatigue syndrome. Clin Infect Dis, 1994. 18 Suppl 1: p. S88-95.
  165. 165. Thompson, K.A., et al., Results of a double-blind placebo-controlled study of the double-stranded RNA drug polyI:polyC12U in the treatment of HIV infection. Eur J Clin Microbiol Infect Dis, 1996. 15(7): p. 580-7.
  166. 166. Nicodemus, C.F. and J.S. Berek, TLR3 agonists as immunotherapeutic agents. Immunotherapy, 2010. 2(2): p. 137-40.
  167. 167. Gowen, B.B., et al., TLR3 is essential for the induction of protective immunity against Punta Toro Virus infection by the double-stranded RNA (dsRNA), poly(I:C12U), but not Poly(I:C): differential recognition of synthetic dsRNA molecules. J Immunol, 2007. 178(8): p. 5200-8.
  168. 168. Purcell, R.H., et al., Modification of chronic hepatitis-B virus infection in chimpanzees by administration of an interferon inducer. Lancet, 1976. 2(7989): p. 757-61.
  169. 169. Ijichi, K., et al., In vivo antiviral effects of mismatched double-stranded RNA on duck hepatitis B virus. J Med Virol, 1994. 43(2): p. 161-5.
  170. 170. Niu, J., et al., The use of ampligen alone and in combination with ganciclovir and coumermycin A1 for the treatment of ducks congenitally-infected with duck hepatitis B virus. Antiviral Res, 1993. 21(2): p. 155-71.
  171. 171. O'Marro, S.D., et al., The effect of combinations of ampligen and zidovudine or dideoxyinosine against human immunodeficiency viruses in vitro. Antiviral Res, 1992. 17(2): p. 169-77.
  172. 172. Gillespie, D., et al., Synergistic inhibition of AZT-resistant HIV by AZT combined with poly(I):poly(C12U), without synergistic toxicity to bone marrow progenitor cell elements. In Vivo, 1994. 8(3): p. 375-81.
  173. 173. Carter, W.A., et al., Mismatched double-stranded RNA, Ampligen (poly(I): poly(C12U), demonstrates antiviral and immunostimulatory activities in HIV disease. Int J Immunopharmacol, 1991. 13 Suppl 1: p. 69-76.
  174. 174. Ichinohe, T., et al., Intranasal immunization with H5N1 vaccine plus Poly I:Poly C12U, a Toll-like receptor agonist, protects mice against homologous and heterologous virus challenge. Microbes Infect, 2007. 9(11): p. 1333-40.

Written By

Mohamed Labib Salem, Said M. Hammad, Mohamed R. Elshanshory, Mohamed A. Attia and Abdel-Aziz A. Zidan

Published: 29 May 2014