Toll-like receptors in lymphoid malignancies: Double-edged sword

Critical Reviews in Oncology/Hematology 89 (2014) 262–283

Toll-like receptors in lymphoid malignancies: Double-edged sword Sara Harsini a , Maani Beigy a , Mahsa Akhavan-Sabbagh a , Nima Rezaei a,b,c,∗ a

Research Center for Immunodeficiencies, Pediatrics Center of Excellence, Children’s Medical Center, Tehran University of Medical Sciences, Tehran, Iran b Molecular Immunology Research Center, Department of Immunology, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran c Department of Infection and Immunity, School of Medicine and Biomedical Sciences, The University of Sheffield, Sheffield, UK Accepted 20 August 2013

Contents 1. 2. 3. 4.

5.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . TLR structure and ligands . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . TLR signaling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. MyD88-dependent pathway versus TRIF dependent signaling pathway . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . TLRs in lymphoid malignancies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. Lymphoma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1.1. Definition and epidemiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1.2. Lymphoma and TLRs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1.3. Clinical application of TLR agonists to lymphoma. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2. Acute lymphoblastic leukemia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2.1. Definition and epidemiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2.2. ALL and TLRs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2.3. Clinical application of TLR agonists to ALL . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3. Multiple myeloma. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3.1. Definition and epidemiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3.2. MM and TLRs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3.3. Clinical application of TLR agonists to MM . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.4. Chronic lymphocytic leukemia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.4.1. Definition and epidemiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.4.2. CLL and TLRs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.4.3. Clinical application of TLR agonists to CLL . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conflicts of interest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Reviewers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Biography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Abstract Toll-like receptors (TLRs) are the best characterized pattern recognition receptors (PRRs), which play an essential role in the recognition of invading pathogens via specific microbial molecular motifs, comprising a bridge between the innate and adaptive immune responses. Toll-like receptors expression is determined in both normal immune cells and malignant cells, with a distinctive pattern compared to each

∗ Corresponding author at: Children’s Medical Center Hospital, Dr. Qarib Street, Keshavarz Boulevard, Tehran 14194, Iran. Tel.: +98 21 6692 9234; fax: +98 21 6692 9235. E-mail address: rezaei [email protected] (N. Rezaei).

1040-8428/$ – see front matter © 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.critrevonc.2013.08.010

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other, rendering them plausible targets for cancer therapy. Improved molecular profiling of lymphoid malignancies may give new insights into pathogenesis of these cancers and pave the way for novel therapeutic agents, including TLR agonists. In the current review, we summarize the immunopathogenic roles of TLRs in B cell and T cell lymphomas, acute lymphoblastic leukemia, multiple myeloma, and chronic lymphocytic leukemia, as well as the results of studies on TLR ligands and their future implications to manage these hematologic malignancies. © 2013 Elsevier Ireland Ltd. All rights reserved. Keywords: Toll-like receptors; Pattern recognition receptors; Lymphoma; Leukemia

1. Introduction Toll-like receptors, the best characterized pattern recognition receptors (PRRs), are one of the components of the innate immunity, able to recognize conserved molecular structures of pathogens called pathogen-associated molecular patterns (PAPMs) [1–4]. These mammalian homologues of drosophila Toll proteins include a total of 11 human TLRs, detecting a wide domain of ligands from microbial patterns to endogenous and synthetic ligands [5]. The engagement of TLRs by pathogenic components culminate in the induction of interferons (IFNs), proinflammatory cytokines (IL-1, IL-6, and TNF-␣), and chemokines (which orchestrate innate immunity), and upregulation of costimulatory molecules (which promote adaptive T-cellmediated immunity) [6]. Nuclear factor-kappaB (NF-␬B) is a transcription factor that can be considered as the isthmus of TLRs signaling hourglass [7–9]. Activation of the TLRs leads not only to the induction of innate immune responses but also to the development of antigen specific adaptive immunity through activating the antigen-presenting cells (APCs) [10–12]. Most experimental models have shown that TLR agonists conduct adaptive immunity toward a predominantly T-helper1 (Th1) mode, required for efficient tumor clearance [13]. In attempt to determine the contributing roles of individual TLRs in the pathogenesis of infections, allergic diseases, autoimmune disorders, and cancers; extensive experimental studies have been described in the medical literature. Recent advances in TLR-related research have set the stage for potential therapeutic exploitation against diseases, such as lymphoid malignancies [2,14–20]. The natural immune response to the lymphoid malignancies is not optimal per se, because of weak immunogenicity of lymphoid tumor cells, a substantial misfortune due to the escape of tumor cells from the numerous activated tumorreactive cytotoxic T cells [19]. Targeting the TLRs is a rationale for producing more efficient immune response. To date, few but remarkable investigations have been managed with regard to employing TLRs in lymphoid malignancies, required to be systematically reviewed. This review is composed of three sections. In Section 1, the structure and biological functions of TLRs will be reviewed. In the second section the signaling pathway of TLRs will be briefly discussed. Then in the third section we systematically collect the recent evidence of human trials with regard to therapeutic roles of TLRs’ agonists in the field of these hematologic

malignancies: (1) B cell and T cell lymphomas, (2) acute lymphoblastic leukemia, (3) multiple myeloma, and (4) chronic lymphocytic leukemia.

2. TLR structure and ligands TLRs are type 1 integral membrane glycoproteins characterized by an ectodomain (extracellular N-terminal domain of approximately 16–28 leucine-rich repeats responsible for ligand binding and discrimination, obtaining a sufficient immune response to TLR agonists) and a cytoplasmic domain (homologous to the cytoplasmic portion of the IL-1 receptor family, also known as TIR domain) [2,5,6]. Eleven human TLRs and thirteen mouse TLRs have been discovered up to now and each of these TLRs seem to response to distinct class of either PAMPs or non-pathogenic ligands (synthetic and endogenous molecular patterns). Studies of the molecular structure of TLR ectodomains and their ligand complexes have predicted that a vast variety of ligands can be sensed through the TLRs inducing the activation of TLR signaling. Moreover, diverse dimerization of TLRs enables them in more efficient pattern recognition; examples include: the TLR3 homodimer (recognizes double-stranded RNA); TLR1–TLR2 heterodimer (discriminates triacylatedlipopeptides); TLR2–TLR6 heterodimer (discriminates diacylatedlipopeptides); TLR4-MD-2 and TLR4-CD14 heterotetramer (recognize lipopolysaccharides from Gram-negative bacteria) [2,5,6]. Although human TLR10 can be heterodimerized with TLR2 and TLR1, a true ligand for these heterodimers remains masked. Based on the literature, we can categorize the TLR ligands into these groups: (1) exogenous ligands, (2) endogenous ligands, (3) synthetic analogs, and (4) fully synthetic small molecules. Detailed list of already discovered TLR agonists is well described in previous reviews [2,21,22]. Given the location of TLRs, a group of them including TLR3, TLR7, TLR8, and TLR9, are not expressed on the cell membrane; however, they are present within one or more endosomal compartments and can sense nucleic acids [2,23]. Since this subset of TLRs (especially TLR7 and TLR9) is more involved in different aspects of hematologic malignancies discussed below, we provide a brief review of their structure and ligands. Also, Table 1 displays a comprehensive compilation of TLR ligands. TLR3 has been demonstrated to recognize viral dsRNA and the dsRNA mimic polyriboinosinic polyribocytidylic

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Table 1 Human Toll-like receptors (TLRs) and their ligands. TLRs

Major cell types

Current recognized ligands Exogenous ligands

TLR1

Myeloid cells, T and B cells, NK cells, endothelial cells [26,27] Epithelial cells, keratinocytes[28]

Endogenous ligands

Triacyl lipopeptides [65]

Soluble factors (Neisseria meningitides) [60]

Pam2Cys-␣-Gal (synthetic glycolipid) [122] Pam3CSK4 (tripalmitoyl-S-glyceryl cysteine) [66] MALP-2 (macrophage-activating lipoprotein-2) [66]

Myeloid cells, T and B cells, NK cells, endothelial cells [26,27] Epithelial cells, keratinocytes [28]

Lipoprotein/lipopeptides (Gram positive bacteria, Mycoplasma lipopeptide LAM from, Mycobacteria BCG, LTA, GXM and other fungal elements, Spirochetes) [22,67] CBLB613 (natural lipopeptide of Mycoplasma arginini) [68]

HSP60 [82,83]

Pam2Cys-␣-Gal (synthetic glycolipid) [122]

HSP70 [84,85]

Peptidoglycan [22,69]

HSP96 [86,87]

Pam3CSK4 (tripalmitoyl-S-glyceryl cysteine) [66] MALP-2 (macrophage-activating lipoprotein-2) [66]

Lipoteichoic acid (Gram-positive bacteria) [22,69] Phenol-soluble modulin (Staphylococcus epidermidis) [69] Heat-killed bacteria (Listeria monocytogenes) [22], Porins (Neisseria) [70] FomA Porin (Fusobacterium nucleatum) [71] Soluble factors (Neisseria meningitides) [60] Atypical lipopolysaccharides (Leptospira interrogans, Porphyromonas gingivalis) [22,72,73] Outer membrane protein A [74] (Klebsiella pneumonia) [19] Glycolipids (Treponema maltophilum) [72] Lipoarabinomannan (Mycobacteria) [75], Hemagglutinin (Measles virus) [76] Structural viral proteins (Herpes simplex virus, Cytomegalovirus) [77,78] Zymosan (Saccharomyces) [79] Phospholipomannan (Candida albicans) [80] Glycoinositolphospholipids (Trypanosoma cruzi) [78] Mushroom extract (Polysaccharide krestin) [81]

HMGB1 protein [88] Hyaloronic acid [89]

Fully synthetic small molecules

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Triacyl Lipopeptides (Bacteria and Mycobacteria) [59]

Surface proteins (OSP) (Borrelia burgdorferi) [61–64]

TLR2

Synthethic analogs

TLR3

TLR4

TLR6

Myeloid cells, NK cells, mast cells, T cells, endothelial cells[26,27], epithelial cells[93], keratinocytes[28]

Myeloid cells, T cells, NK cells, endothelial cells [26,27,115] Epithelial cells, keratinocytes[28] Myeloid cells, T and B cells, NK cells, endothelial cells [26,27] Epithelial cells, keratinocytes [28]

Single-stranded viral RNA (ssRNA) [22]

mRNA [91]

Poly(I:C) [92]

Double-strand-RNA (dsRNA) [22,90]

Self-dsRNA [22]

Poly(I:C12 U) [30]

Lipopolysaccharide (Gram-negative bacteria) [22,94]

HSP60 [82,83]

Lipid A mimetics (monophosphoryl lipid A, Aminoalkyl glucosaminide 4-phosphatase) [109]

E6020 [112]

Lipoteichoic acid [22]

HSP70 [84,85]

E5531 [113]

Hsp60 (Chlamydia pneumonia) [95]

HSP22 [102]

LPS mimicking peptides [110] ER-112022 (acyclic synthetic lipid A analog) [111]

Envelope proteins (Respiratory syncytial virus and mouse mammary tumor virus) [22,96,97] Fusion protein (Respiratory syncytial virus) [22,98] Glycoinositolphospholipids (Trypanosoma cruzi) [99] Taxol (Plant product) [100] BCG [22] PTX (paclitaxel) [22] Fibronectin [22] G1-4A (polysaccharide from an Indian medicinal plant Tinospora cordifolia) [101]

HSP96 [86,87]

Flagellin (Flagellated bacteria, from Gram-positive or Gram-negative bacteria) [22,116]

Hyaluronic acid [89,103] Heparan sulfate [104] Fibrinogen [105] Surfactant-protein A [106] HMGB1 protein [22,88] ␤-Defensin 2 [107] Extradomain A of fibronectin [108] Discontinuous 13-aminoacid-peptide [65] CBLB502 [117]

Diacyl lipopetides (Mycoplasma) [22,118]

Diacyl lipopeptides [65]

RO 90-7501 [29]

E5564 [114]

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TLR5

Myeloid cells, Tcells [25], NK cells [24], endothelial cells [26,27], epithelial cells, keratinocytes[28], neurons

Lipoteichoic acid (Gram-positive bacteria) [22,69] Phenol-soluble modulin (Staphylococcus epidermidis) [118] Heat-labile soluble factor (Group B Streptococcus) [119] Zymosan (Saccharomyces) from fungal cell wall [22,120] CBLB613 (natural lipopeptide of Mycoplasma arginini) [68] 265

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Table 1 (Continued) TLRs

TLR7

Myeloid cells, NK cells [24], endothelial cell [26], T cells, B cells

Current recognized ligands Exogenous ligands

Endogenous ligands

Synthethic analogs

Fully synthetic small molecules

Single-stranded RNA (viruses) [22,121,122]

Endogenous RNA (self-ssRNA) [22,123,124]

Oligonucleotides [65]

Imidazoquinolines (Imiquimod, Resiquimod) [22,33,34]

SC-1, SC-2 [31]

852A (small-molecule imidazoquinoline [35] Guanosine nucleotides (Loxoribine, Isatoribine) [3] Bropirimine [125] 3M-007 [38]

VTX-2337 [127]

Imidazoquinolines (Resiquimod) [34]

Myeloid cells, NK cells [126] Endothelial cell [26]

Single-stranded RNA (viruses) [22,122]

TLR9

Myeloid cells, T cells, B cells, NK cells [24], endothelial cells [26,27], epithelial cells, keratinocytes [28]

Unmethylated CpG DNA (Bacteria and viruses) [22,39]

TLR10

Myeloid cells, T cells, B cells Endothelial cell[26], epithelial cells

Not determined but may interact with TLR2 [22,30]

TLR11

Dendritic Cells [134,135], macrophages, liver, kidney, and bladder epithelial cells [134]

Profillin-like molecule (Toxoplasma gondii) [135]

Endogenous RNA (self-ssRNA) [22,123,124]

VTX-1463 [128] SC-2 [31]

Hemozoin (Plasmodium) [129]

Endogenous DNA (self-DNA) [22,130]

CpG oligodeoxynucleotides (CpG 7909, CpG 10101, 1018 ISS) [30]

IC31(ODN1a + KLK) [131–133]

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TLR8

Major cell types

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acid (Poly(I:C)), so that, TLR3 is considered as one of the most important detectors of viral infections and neoplasms [2,5,18]. TLR3 is majorly expressed in myeloid cells, T cells, NK cells [24,25], endothelial cells [26,27], epithelial cells, keratinocytes [28], and neurons. Evidence supports that activation of TLR3 is encouraging as an anticancer therapy; in which Th1 immune activation, consequent cytokine and chemokine secretion, and apoptosis are paramount involved mechanisms [2,15,16,18]. Evidence supports that injection of TLR3 agonists increase survival in cancer patients [21]. It is of particular importance to note that a recent novel anticancer/antiviral approach has been introduced by using RO 90-7501 (‘2’-(4-aminophenyl)[2,59-bi-1H-benzimidazol]-5-amine) which agonizes TLR3 and RIG-I-like receptors (RLRs) [29]. This novel TLR3 agonist intrigues us to use it against hematologic malignancies which will be discussed later. IPH 3102 [21] (a high molecular mass synthetic dsRNA TLR3-specific agonist that activates NF-␬B signaling and type 1 IFN responses) and AMP-516 [30] [Ampligen or rintatolimod] (a synthetic mismatched poly I:poly C dsRNA Poly(I:C12 U), are the other TLR3 agonists that have been used as cancer drugs and vaccines, which are discussed comprehensively by Basith et al. [21]. Briefly, in vivo immunostimulator actions of IPH 3102 through myeloid dendritic cell activation are demonstrated [21]. TLR7 is the other endosomal TLR which is majorly expressed in antigen-presenting cells as in plasmacytoid dendritic cells (pDC), B-cells, and CD34+-derived dendritic cells [31]. Also it is expressed on myeloid cells, NK cells [24], endothelial cells [26], T cells, and B cells. Using small molecule ligands at TLR7 is promising in cancer research because of their profound antitumoral activity [32]. Imiquimod (Aldara), the best characterized compound of the imidazoquinoline family, is used as a topical formulation which is effective against several primary skin tumors and cutaneous metastases [32]. Resquimode (R-848) and Gardiquimod (imidazoquinolines) [33–35]; 852A (novel small molecule TLR7 agonist which is structurally related to Imiquimod); loxoribine and Isatoribine [ANA975 is its oral prodrug] (guanosine analogs) [3]; ANA773 (oral inducer of endogenous IFNs; had unacceptable toxicity via long-term in animal studies) [36]; AZD8848/DSP-3025 [37]; and 3M007 [38] (a novel TLR7 agonist) are the other TLR7 agonists (detailed review of them can be find in articles written by Basith et al. and Schön et al. [21,32]). 852A stimulates DCs to produce multiple cytokines, such as IFN␣ in vitro and in vivo [21]. R848 is a dual TLR7/TLR8 agonist that induces IFN␣, IL-12 and TNF␣. The ability of the novel TLR agonist 3M-007 to activate immune cellular responses in vitro in the peripheral blood cells of immunodepressed Sezary syndrome (a less common leukemic variant of cutaneous T-cell lymphoma associated with erythroderma, lymphadenopathy, and circulating malignant T-cells) has been demonstrated [38]; so that, NK cells, as well as CD8 T cells, became rapidly activated by 3M-007. Furthermore, the cytolytic activity of NK

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cells was also shown to be markedly upregulated by 3M007 [38]. Since NK cells play a key role in the host immune response against the tumor cells these encouraging effects are seem to mediate a beneficial clinical effect [38]. TLR9 is majorly expressed on B cells, pDCs [16], Myeloid cells, T cells, NK cells [24], endothelial cells [26,27], epithelial cells, and keratinocytes [28]. TLR9 recognizes unmethylated cytosine-phosphate-guanine (CpG) motifs in bacterial and DNA viruses [22,39]. CpG-containing oligodeoxynucleotides (CpG ODNs) targeting TLR9 have already been studied for the treatment or vaccination of cancers and other diseases. We know CpG ODNs directly stimulate pDCs, B Cells, and NK cells [40,41]. Following stimulation of matured and well-differentiated pDCs, IL-12 and type I IFNs secretion will be increased leading to amplifying Th1 phenotype which culminates in enhanced CD8+ cell mediated cytotoxicity [42,43]. Antigen-specific humoral responses through B-cell activation [44], inhibition of Bcell apoptosis [45], and IgG class switching [46] are some other immunostimulatory actions of CpG ODNs. Furthermore, CpG ODNs can induce apoptosis in malignant cells and activate innate immunity [2,47]. Altogether, using CpG ODNs has become one of the most encouraging candidates for the development of both prophylactic and therapeutic vaccines against hematologic malignancies. Not to forget novel TLR9 agonists that are desirable to be employed against hematologic malignancies, we prefer only to briefly introduce them but refer you to the clinical trials that have employed them (as cited next to them) and to more detailed reviews written by Basith et al. [21]: IMO-2025 [48] and IMO-2125 (immune modulatory oligonucleotides) [49]; IMO-2134 [QAX935] (a lead compound) [50]; SD-101 (a novel C type TLR9 agonist) [51]; dSLIM (double stem loop DNA-based immunomodulator); MGN1703 and MGN1706 (DNA-based immunomodulators) [49]; Agatolimod, AVE0675 and SAR-21609 (CpG DNAbased TLR9 agonists) [52,53]; and DIMS 0150 [54]. Actually IMO-2125 induces high levels of IFN␣ [21]. IMO-2025 had anticancer activities in a mouse model, which was augmented in combination with chemotherapy agents [21]. SD-101 stimulates 20-fold higher levels of both IFN␣ and IFN␥ [21]. ISS1018 is another efficient TLR9 agonist in treating follicular lymphoma in combination with Rituximab (a Phase II clinical trial) and in treating non-Hodgkin’s lymphoma [21,55,56]. Immunostimulatory sequences (ISSs) are short sequences of synthetic DNA consisting of unmethylated CpG dimers that affect human immune system [21,55,56]. ISSs signal through the TLR9, that is expressed on only a few specific subsets of immune cells [44]. Immunostimulatory effects of CpG ODN exert through induction of proliferation and immunoglobulin production by B cells and the induction of IFN␣, IFN␤, IL12, and TNF␣ by pDCs [21,55,56]. IL-12, TNF␣, and, especially, IFN␣ motivate cytotoxicity of NK cells, and IL-12 induces effective IFN␥ production by NK [57]. The use of dSLIM motivates the human immune system to protect against tumor-associated

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antigens by targeting the TLR9 receptor on certain immune cells [21,48]. Tumor-associated antigens are released by cancer cells as a consequence of chemotherapy and radiotherapy, so dSLIM-activated immune system is seemed to overcome its fatal tolerance toward cancer cells and tumorassociated antigens then attacks them selectively [21,48]. Moreover, dSLIMs have been demonstrated to enhance the anti-leukemic effects of peptide vaccines accompanying with the granulocyte monocyte colony stimulating factor (GMCSF), in vivo [48,58].

3. TLR signaling Following ligand recognition, TLRs will be homodimerized or heterodimerized, which leads to motivation of intracellular signaling pathways that induce inflammatory cytokine genes and upregulate co-stimulatory molecules on dendritic cells [2,6,136]. For better understanding of the role and importance of TLRs in lymphoid malignancies, we prefer to briefly explain the TLR signaling. Downstream signaling pathways are conducted by various cytoplasmic adaptor molecules including: (1) MyD88 (myeloid differentiation primary response protein 88); (2) TIRAP (TIR-domain-containing adaptor protein) also known as MAL (MyD88-adaptor-like protein); (3) TRIF (TIRdomain-containing adaptor protein inducing IFN-␤) also known as TICAM1 (TIR-domain-containing molecule1); (4) TRAM (TRIF-related adaptor molecule) also known as TICAM2 [2,6,136]. 3.1. MyD88-dependent pathway versus TRIF dependent signaling pathway MyD88 is the universal adaptor activated by all TLRs except TLR3, leading to activation of MAPKs (mitogenactivated protein kinases) and the transcription factor NF-␬B [2,6,136]. It has been discovered that TLR types 5, 7, 8, 9, and 11 directly initiate MyD88-dependent pathway, but TLR types 1, 2, 4, and 6 employ TIRAP as the proximal signaling intermediate [6,136]. MyD88 signaling pathway functions majorly through IRAKs (IL1R-associated kinases), TAK1 (transforming growth factor-b activated kinase 1), TAB1 (TAK1-binding protein 1), TAB2 (TAK1-binding protein 2), and TRAF6 (tumor-necrosis factor-receptor-associated factor 6). TLR 3 and TLR4 initiate an alternative pathway, independent of MyD88, by recruiting TRIF; which allows the activation of NF-␬B, MAPKs and the transcription factor IRF3 (IFN-regulatory factor 3) [6,136]. In summary, MyD88 is crucial for the secretion of inflammatory cytokines triggered by all TLRs. TIRAP is involved in the MyD88-dependent pathway through TLR2 and TLR4, while TRIF is operated in TLR3 and TLR4-mediated MyD88-independent pathway. The MyD88-dependent signaling pathway conducts the early phase of NF-␬B activation, whereas the

Fig. 1. Intracellular TLR-mediated signaling pathways. All TLRs except TLR3 excite MyD88-dependent signaling pathway either directly or in combination with the additional adaptor molecule TIRAP. TLR3 and TLR4 initiate TRIF-dependent signaling pathway, so that TLR4 recruits TRAM as the proximal signaling intermediate. IRAK family and TRAF6 are recruited through MyD88 pathway, which ultimately activate TAK1/TAB1/TAB2 complex. Phosphorylation of MAPK and IKK complex by activated TAK1 leads to activation and nuclear translocation of the transcriptional factors AP-1 and NF-␬B, respectively. NF-␬B and AP-1 induce the gene transcription of inflammatory cytokines. The activated TRAF6 and RIP1 through TRIF pathway contribute to MyD88-independent/TRIF-dependent NF-␬B and AP-1 activation. Moreover, interaction of TRIF with TBK1, together with IKKi phosphorylates IRF3. Consequence is activation and nuclear translocation of IRF3 which initiates the gene transcription of type I IFNs. The schematically represented TLR4-mediated signaling pathways serve as a reference for other TLRs. TLR, Toll-like receptor; MyD88, myeloid differentiation primary response protein 88; TIRAP, TIRdomain-containing adaptor protein; TRIF, TIR-domain-containing adaptor protein inducing IFN-b; TRAM, TRIF-related adaptor molecule; IRAK, IL-1R-associated kinase; TRAF6, tumor-necrosis factor (TNF)-receptorassociated factor 6; TAK1, transforming growth factor-b (TGF-b)-activated kinase1; TAB, TAK1-binding protein; IkB, inhibitor of nuclear factorkB; IKK, IkB kinase; MAPK, mitogen-activated protein kinase; AP-1, activator protein1; RIP1, receptor-interacting protein1; TBK1, TRAFfamily-member-associated NF-␬B activator (TANK)-bindingkinase1; IKKi, inducible IKK; IRF3, IFN-regulatory factor 3.

MyD88-independent signaling pathway initiates the late phase of NF-␬B activation [2,6,8,136,137]. Fig. 1 portrays the signaling pathway of TLRs. More detailed review of TLR signaling has been provided in the articles prepared by Hedayat et al. [2], Kawai and Akira [6], and Takeda and Akira [136].

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4. TLRs in lymphoid malignancies Hematologic malignancies comprise a category of clonal diseases that arise from cells of the immune system at different stages of their differentiation process, from blasts to memory cells. It is crucial to assess the expression of TLRs on transformed cells of the immune system, as well as the biology of their signaling, so as to assume them as a target for an effective therapy in lymphoid malignancies. 4.1. Lymphoma 4.1.1. Definition and epidemiology Lymphomas are proliferative diseases of lymph nodes or extra-nodal lymphatic tissue, which include a broad area of hematologic malignancies arising from B, T or NK cells at distinct stages of their maturation and differentiation [5,138,139]. Malignant lymphomas entail considerable heterogeneity in terms not only of morphology and clinical course but also of etiological factors [140]. A considerable increase in incidence of lymphomas has been observed over the past decades [141]. Although the etiology of malignant lymphomas and underlying causes of this increase in incidence remains poorly understood, genetic and environmental factors influencing immune regulation and inflammation in addition to various infectious factors such as autoimmune and chronic inflammatory conditions have been linked [140–142]. 4.1.1.1. Non-Hodgkin’s lymphoma (NHL). Although NHL contains the most prevalent forms of lymphomas, their exact etiology is not clear for us. However, based on increased incidence of NHL among immunosuppressed individuals and after autoimmune diseases, evidence suggests that infections and immune dysregulation might play a role [143]. Furthermore, considerable associations between specific infectious agents and some NHL subtypes have been observed: (1) EBV and Burkitt lymphoma; (2) HTLV1 and adult T-cell leukemia/lymphoma; (3) HHV8 and primary effusion lymphoma, (4) Helicobacter pylori and gastric MALT lymphoma [143]. Cutaneous T-cell lymphoma (CTCL) is a heterogeneous group of lymphoproliferative disorders caused by clonally derived, skin-invasive CD4 T cells; which has vast clinical and histopathological manifestations and is characterized as extranodal non-Hodgkin lymphomas of skin [144,145]. Mycosis fungoides (MF) is the most common and well characterized type of the CTCLs [144]. MF accounts for approximately 1% of all NHLs [144] and is the most common primary cutaneous lymphoma [146]. 4.1.1.2. Hodgkin’s lymphoma (HL). HL is categorized into a classical (cHL) and a nodular lymphocyte predominant form (NLPHL). cHLs (including nodular sclerosis, mixed cellularity, lymphocyte-rich, and lymphocyte-depleted HL) represent approximately 95% of patients, while NLPHL stands for only 5% of cases [147,148]. The tumor cells are different in these

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subtypes: Hodgkin and Reed/Sternberg (HRS) cells in cHL and lymphocyte-predominant (LP) cells in NLPHL [147]. 4.1.2. Lymphoma and TLRs Since mutations of TLRs are suspected to be involved in function alterations of these pattern recognition receptors [5,141], we prefer to briefly collect recent evidence regarding TLR polymorphisms and risk of NHLs. In meta-analysis study done by Purdue et al. [143] of TLR gene polymorphisms in three case control studies of NHL, variation in the TLR10–TLR1–TLR6 region was associated with NHL risk. In particular, two SNPs within the region, rs10008492 and rs4833103, were significantly associated with NHL. TLR10, TLR1 and TLR6 are located in a gene cluster spanning 57 kb on chromosome 4p14. TLR1 and TLR6 are important elements of TLR2 signaling, which interact with TLR2 to create heterodimer receptors able to recognize a broad spectrum of pathogen ligands, so that TLR1–TLR2 heterodimer discriminates triacylatedlipopeptides and TLR2–TLR6 heterodimer distinguishes diacylatedlipopeptides [143,149]. Evidence shows that genetic variation in the TLR10–TLR1–TLR6 region may affect risk of inflammatory diseases, associations with asthma [150,151] and aspergillosis following allogeneic stem cell transplantation [152]. They did not find associations with variation in TLR2, although the variant rs3804100 was significantly associated with marginal zone lymphoma (MZL). Furthermore, TLR2-16933T > A (rs4696480) variant has been shown [141] to increase the risk of follicular lymphoma (FL) by 2.82-fold, and to decrease risk of chronic lymphocytic leukemia (CLL) (OR = 0.61) (note that this SNP was not genotyped in meta-analysis of Purdue et al.). The observed association between TLR2 and MZL can be justified by considering that specific infectious organisms have been linked to the pathogenesis of MZL, especially MALT lymphoma [153]. There is supporting evidence that H. pylori infection has a causal role in pathogenesis of gastric MALT lymphoma [154,155]. It is worthy to remind TLR2 plays a key role in initiating immune responses to H. pylori [156,157]. No associations with TLR4 variants have been found by Purdue et al. [143]. TLR4 Asp299Gly is under controversy due to inconsistent correlations to lymphoma that has been reported from small studies. This variant attenuates receptor signaling and reduces IL-12 and IFN-␥ levels, and has been shown to be linked with an 2.76-fold elevated risk for MALT lymphoma; 1.80-fold increased risk of HL (borderline non-significant); and 1.90-fold increased risk of T-NHL (non-significant) [141]. Oppositely, decreased risk of MALT lymphoma [158] and diffuse large B-cell lymphoma (DLBCL) [159] has been found in two other studies. Furthermore, it has been recently indicated that TLR4 signaling triggers a cascade resulting in Mantle cell lymphoma (MCL) growth and evasion from immune system [160]. MCL cells express various TLRs, but TLR4 is among the highest expressed TLRs [160]. We know MCL is an incurable B-cell malignancy, and MCL patients have the poorest prognosis among all B-cell lymphomas [160]. The activation

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of TLR4 signaling in MCL cells by LPS (well-known TLR4 ligand) is shown to induce MCL proliferation and up-regulate the secretion of cytokines like IL-6, IL-10, and vascular endothelial growth factor (VEGF) [160]. Actually, LPS-pretreated MCL cells have been shown to inhibit the proliferation and cytolytic activity of T cells by releasing IL-10 and VEGF [160]. It has been shown that knockdown of TLR4 on MCL cells inhibits the effect of LPS on MCL cells regarding cell growth or secretion of the cytokines and evasion of the immune system [160]. While meta-analysis of Purdue et al. has found no associations between SNPs of TLR4 and MCL, it is of particular importance to find out how expression of TLR4 is increased in MCL cells regardless of no variations in TLR4 gene polymorphisms. However, study of Wang et al. [160] instructs us that TLR4 signaling molecules may be novel therapeutic targets for MCL. TLR9 rs5743836 (−1237 T/C) is associated with an elevated risk for NHL in Portuguese and Italians [161]. The C allele of rs5743836 has been recently reported [47] to be linked with lack of cell death upon TLR9 triggering observed in the Mutu-I and BJAB BL cell lines. Also, non-significant association between TLR9 rs352140 (2848 G/A) and CpG2006-induced cell death responses of BL cells has been shown [47]. Thus, these data encourages us to evaluate TLR9 SNPs before initiation of therapy in BL patients, because they may do as potential biological markers for the response to treatment [47]. MF cases did not show alterations in pattern of TLRs expression in different stages, however, increased epidermal staining of TLRs 2, 3, 4, 5, 6, and 8 with higher scores was associated with more aggressive stages [162]. Endothelial cells show increased expression of TLR 4 and 6. Tumor infiltrate staining exhibited strongest expression of TLRs 5 and 7 [162]. CTCL show profound defects in cell-mediated immunity [145]. It has been shown that increased production of IL-4, IL-5, and IL-10 (inhibitors of Th1-type cytokine production) by the malignant T cells leads to depressed Th1 responses. Also it has been found that number of DCs and their cytokine secretions (IL-12 and IFN-␣) can be decreased, just the same as decreased number of CD8 T cells [145]. Fewer studies have been done for detecting associations between TLRs and pathogenesis of HL, however, there are important lessons in current evidence worthy to be learned for arranging research strategies in future. Deregulated activity of multiple signaling pathways and transcription factors in HRS cells has been shown to be involved in the pathophysiology of these cells [163]. NF-␬B function is presumably the paramount pathway for the survival of HRS cells [147]. As we mentioned above, the activation of NF-␬B is mediated through TLR signaling pathway, just as important as other cell-surface receptors, e.g. CD40 and RANK [147]. Notably, in nearly 40% of cHLs the HRS cells are latently infected by EBV which activates NF-␬B by expression of EBV-encoded latent membrane protein 1 (LMP1) [147]. Recently, a global gene expression study [164] of isolated HRS cells and other

normal and malignant B cells showed us that, interestingly, EBV infection has inconsiderable impact on gene expression of HRS cells. Thus the destroyed B cell phenotype of HRS cells is not associated with obtaining a plasma celllike gene expression program, so that HL cell lines and HRS cells are actually different in gene expression (i.e. in vitro studies of HL cell lines cannot reflect the gene expression of HRS cells appropriately). A study of TLR and MyD88 gene polymorphisms have found that TLR9-1237C, TLR9-2848A were associated with an increased risk for HL (OR = 2.53 (1.36–4.71) and OR = 6.20 (1.3–28.8), respectively) [165]. Although current evidence, altogether, cannot lead us to understand the probable role of EBV-related TLRs (or other TLRs) and EBV in HL pathogenesis, it is worthy to construct studies considering TLR in HL to find out its pathogenesis and discover therapeutic strategies. This fact that TLR9 haplotypes are related to the development of HL [165] should be considered in future studies. Table 2 summarizes the information about expression of various TLRs in lymphoid malignancies and interpretation of the therapeutic approaches employed by TLR agonists. After considering TLR expressions and TLR polymorphisms in NHL and HL lymphomas, we prefer to review some of the recent therapies conducted by employing TLR agonists in lymphoma. 4.1.3. Clinical application of TLR agonists to lymphoma 4.1.3.1. TLR9 agonists. To the best of our knowledge, the pattern of TLR expression rest upon the cell subset and differentiation status of transformed cells [47]. TLR9 is preferentially expressed by B cells and plasmacytoid dendritic cells, especially by Burkitt’s lymphoma (BL) cells with the highest level of TLR9 [5,15,47,166]. CpG ODNs activate TLR9, leading to firing of MyD88 pathway and motivation of the subsequent signaling cascade that arrives to the nuclear translocation of the transcription factor NF-␬B [47]. Moreover, CpG ODNs initiate secondary effects such as secretion of proinflammatory cytokines (TNF␣, IL-6), IL-10, and chemokines; and also portray the phenotype of APCs in these BL cell lines [5,166]. Although synthetic TLR9 agonists have been already considered as potential anti-cancer agents, TLR9 stimulation with ODN CpG-2006 suppresses lytic reactivation of Epstein–Barr virus (EBV: the B-cell tropic gamma-herpes virus) and, therefore, it was firstly assumed that consequent promotion of latent EBV (that is associated with several types of B-cell lymphomas including BL) is disappointing in cancer therapy. A novel observation to be considered in the recent study of Noack et al. [47] is distinct cell survival following TLR9 triggering in B-cells with different origins. Although it was hypothesized by Noak et al. that lymphoproliferation following TLR9induced latent EBV may lead to catastrophic results in cancer therapy, one paramount result of their study is that the EBV status does not predict the possible B-cell lymphoproliferation due to TLR9 triggering. However, personalized in vitro pre-examinations (mostly considering various SNPs) for BL

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Table 2 TLR expression, and the advantages and disadvantages of TLR agonists administration in lymphoid malignancies. Lymphoid malignancy Lymphoma Burkitt lymphoma Akata BL cell lines (in vitro) [47]

TLR expression

TLR agonists used as therapeutic agents

Advantages of TLR agonists administration

Disadvantages of TLR agonists administration

TLR9 agonists

• Cytokine expression and malignant cell death

TLR9 agonists

• • Highest level of TLR9 expression (20-fold greater than spleen)

• Up-regulation of human IL-10 expression leading to escape from immune system • –

Mostly TLR9

Namalwa cell lines (in vitro) [265]

• Increased amounts of TNF, IL-6, and IL-10 and expression of the costimulatory molecules CD40, CD80, and CD86 Non-Hodgkin lymphoma Mantle cell Mostly TLR4 lymphoma TLR5 and TLR7 Cutaneous (in tumor T-cell infiltrating cells) lymphoma

Peripheral T-cell lymphoma Diffuse large B-cell lymphoma Follicular lymphoma

– TLR7 and TLR9 agonists

–

–

• Increase induction of IFN-␣ release following treatment with CpG-A in Peripheral blood mononuclear cells (PBMCs). • Activation of NK cells and CD8 T cells [145] • Augmented production of IFN-␣ and some other cytokines through an intracellular level [167] following imiquimod. Activation of T-lymphocytes, increasing the activity of NK cells, tumor necrosis factor, and APCs of the skin [146,168] –

-

TLR1, TLR2, TLR4

–

TLR2, TLR8 [266], TLR9 [267]

–

• High proportion of DLBCLs are activated by CpG stimulation (in vitro) [267]

–

TLR9 agonist vaccination adjuvant with low-dose radiotherapy [174]

• B cells become more immunogenic and vulnerable to radiotherapy following TLR9 agonist exploitation

• T-reg induction in some patients, leading to tolerance to tumor cells

Heterogeneous expression

Marginal zone lymphoma

Not specified

Hodgkin lymphoma Acute lymphoblastic leukemia

Mostly TLR9 [165] TLR1, 2, 3, 4, 5, 6, 7, and 9

Multiple myeloma

TLR1, TLR3, TLR7 and TLR9

TLR9 agonist vaccination adjuvant with low-dose radiotherapy [174] – TLR2 and TLR9 agonists

TLR3 agonist

• The intratumoral injection of the CpG ODN motivates the host APCs and B cells • Induction of sufficient immune response against tumor cells following activation of APCs and B cells Same as above

– • Augmentation of IL-6, IL-8, IL-10 and IFN-␥ production and expression of the costimulatory molecules CD40 and CD86 [174,188]

• Skewing immunity toward a Th1 response [165] • Induce apoptosis in tumoral cells [213]

–

T-reg induction in some patients, leading to tolerance to tumor cells – • T-reg induction in some patients, leading to tolerance to tumor cells [188] • Induction of NF-␬B pathway in MM cells, resulting in MM cell growth and proliferation [213]

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Table 2 (Continued) Lymphoid malignancy

TLR expression

TLR agonists used as therapeutic agents TLR9 agonist

Chronic lymphocytic leukemia

TLR7 and TLR9

TLR7 and TLR9

Advantages of TLR agonists administration

Disadvantages of TLR agonists administration

• Increasing the level of costimulatory molecules (CD80, CD86) and major histocompatibility complexes (MHC I & II) on HMCL [214]

• Induction of MM proliferation through autocrine IL-6 production [195]

• Promotion of allogenic T cells proliferation [213] • Restitution of functionality of T cells and pDCs and dispending MM cell growth mediated by pDC • Production of pro-inflammatory cytokines, chemokines and costimulatory molecules which enhance CTL responses against tumor cells [241,242]

• Proliferation of CLL cells (caused by CpG ODNs) [235,268]

• Production of angiogenesis inhibitors including type 1 IFNs [234] • Apoptosis of CLL cells (caused by CpG ODNs) [234,235] • Enhancing sensitivity of CLL cells to cycle-active cytotoxic drugs (caused by loxoribine, a TLR7 agonist) [260] • Augmentation of the efficacy of cytotoxic drugs targeting surface molecules on CLL cells (caused by CpG ODNs) [235]

responses to CpG ODNs can help us to predict the therapeutic outcome of TLR9 triggering and to modify adjuvant molecular treatment of BL. Aforesaid In vitro study for B-cell tumors by Noack et al. [47] elucidated the impact of TLR9 agonists on Akata BL cell lines. Following treatment with TLR9 ligands, specific cytokine expression and cell death is induced in specific BL cell lines. This cell death has features considerable for clinical decisions: (1) cell death exerts TLR9-MyD88 signaling, (2) cell death can be suppressed by pan caspase inhibitors, (3) it is independent of the presence or absence of EBV in the tumor cells, and (4) is associated with certain SNPs in the TLR9 gene [47]. The effects of synthetic CpG ODN, TLR9 agonist, is well examined as a therapeutic strategy in treatment of CTCls. Patients’ peripheral blood mononuclear cells (PBMCs) significantly increase induction of IFN␣ release following treatment with CpG-A [145]. Also considerable activation of NK cells and CD8 T cells have been observed [145]. Moreover, the combined effects of CpG-A plus IL-15 causes maximal activation of NK cells and enhanced activation of CD8 T cells [145]. Topical imiquimod (TLR7 agonist) is an endogenous mediator inducer with antiviral and antitumor activity which augments the production of IFN␣ and some other cytokines through an intracellular level [167]. Imiquimod plays an important role in activation of T-lymphocytes, increasing the activity of NK cells, tumor necrosis factor, and antigen-presenting cells in the skin [168]. We know that systemic interferon is an effective treatment for MF patients and because imiquimod acts by augmenting intracellular interferon production, it has been shown that imiquimod (as a TLR7 and TLR8 agonist) has good results

for MF plaques, especially in localized lesions that do not respond to regular therapies [146]. However, more studies with more patients are required. 4.1.3.2. Role of human IL-10 (hIL-10) following TLR9 triggering. Up-regulation of hIL-10 expression in TLR9triggered BL cells has been consistently observed, which influences the development and growth of B cells [169,170] and, therefore, functions as an autocrine growth factor for various B-cell lymphomas [171–173]. hIL-10 is an antiinflammatory cytokine that inhibits the Th1 immune response majorly through inhibiting TNF␣, and this anti-inflammatory process would be catastrophic in cancer immunotherapy, because unwanted proliferation of the malignant cells could be provoked [47]. Altogether, employing TLR9 agonists for lymphoma therapy seems to be a double-edged sword that may induce cell death, or enhance lymphoproliferation, which offers more researches to be done in this field. 4.1.3.3. Combination therapy. The major barrier for cancer immunotherapy is tumor-induced immunosuppression that induces tolerance to the tumor [166]. Although passive immunotherapy with monoclonal antibodies has been an important therapeutic regimen of lymphoma, the clinical course of this disease is still described by a continuous pattern of relapse and progressive decrease in response to therapy [166,174,175]. To the best of our knowledge, there are many strategies for cancer vaccination that are being developed to target dendritic cells, but B-cell strategies have been relatively neglected [166,174]. Brody and colleagues [174] treated 15 patients with relapsed, low-grade B-cell

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lymphoma using low-dose radiotherapy applied to a single tumor site with combined injection of a CpG ODN TLR9 agonist PF-3512676TLR. This in situ vaccination maneuver has been shown to induce systemic anti-lymphoma responses [166,174]. Klein-gonzález and colleagues explain the rational for encouraging outcomes of this strategy: (1) B cells are very vulnerable to radiotherapy and undergo apoptosis; also become more immunogenic after radiotherapy, which makes the tumor cells more susceptible to TLR9-induced responses, (2) the intratumoral route of injecting the CpG ODN acts as a ‘danger signal’ that motivates the host APCs and B cells, and (3) both APCs and lymphoma B cells are target cells, leading to sufficient immune response against tumor cells. Despite these motivating outcomes regarding in situ vaccination with CpG ODN TLR9 agonist PF-3512676TLR, these activated B cells are shown they induced T-regs in some patients which had poorer clinical outcomes compared to Treg non-inducers [166,174]. Nevertheless, this problematic condition of counter-regulatory effects of T-regs induction in some patients vaccinated with CpG ODN TLR9 agonist PF-3512676TLR, is not as disappointing as to dislodge this combinational paradigm from therapeutic plans, but of course instructs us and warns us in these ways: (1) Patients with highly T-reg infiltrated tumors are reported to have favorable clinical outcomes after conventional treatments [174]. This remarkable fact reminds that we should pay more attention toward individual features of these patients both in clinical researches and in practical therapies. (2) T-reg induction can be a particular predictor of response to in situ vaccination [174]. (3) Monoclonal antibodies (mAbs) can fire at tumor antigens on the surface of cancer cells and have an encouraging toxicity profile compared to cytotoxic chemotherapy alone [176]. It has been shown that expression of tumor antigens is dynamically inducible through agents such as TLR agonists [176]. Following binding of the mAbs to the tumor antigens, NK cells and other effector cells can recognize tumor cells, which leads to a more potent cell medicated cytotoxity which is called antibodydependent cell-mediated cytotoxicity (ADCC), These potential therapeutic actions (such as specific mAbs against malignant cells or ADCC inducer mAbs) can be applied as accompanying steps in these combinational strategies so as to overcome some of these pitfalls. For instance, stimulation of NK cells by CpG ODN enhances rituximab (anti CD20) dependent cellular cytotoxity against B-cell lymphomas [177]. 4.2. Acute lymphoblastic leukemia 4.2.1. Definition and epidemiology Acute lymphoblastic leukemia (ALL), a malignant disorder of lymphoid progenitor cells, is a heterogeneous disease with subtypes that differ substantially in their biological

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and clinical characteristics as well as their response to therapy [178,179]. ALL is considered to originate from various significant genetic lesions in B/T-precursor-stage lymphoid cells, including mutations that impart the capacity for aberrant cell proliferation and those that lead to stagespecific lymphoid differentiation arrest [180,181]. ALL affects all age groups, with greatest prevalence between the ages of 2 and 5 years. In children, ALL has become the commonest malignancy accounting for roughly 25% of childhood cancer with 5-year event-free survival rates ranging from 76% to 86% in patients being treated with protocol-based therapy and an overall cure rate of more than 80%. In adults, ALL is less common and generally carries a long-term survival probability that hardly exceeds 40% [182–185]. 4.2.2. ALL and TLRs B-cell precursor acute lymphoblastic leukemia (BCPALL) cells express TLR1, 2, 3, 4, 5, 6, 7, and 9; however, it is unclear whether the aberrant expression of TLR3, 4, and 5 occurs as a consequence of malignant differentiation or just reflects the normal B-cell precursor phenotype [1,186]. Ligation of TLR2, TLR7, and TLR9 culminates in alterations in costimulatory molecule expression on ALL cells but only TLR2 and TLR9 stimulation ameliorate T-cell responses through IFN-␥ production [1,185,187]. On the other hand, unlike other B-cell leukemic cell types, the self-renewal rate of BCP-ALL cells shows no change in the presence of ligands for TLR2, 7, and 9 despite changes in cell-surface marker expression [1,187,188]. 4.2.3. Clinical application of TLR agonists to ALL Although there is noticeable progress made in the treatment of ALL, relapses remain a prominent clinical challenge. Consequently, there is a need in developing innovative approaches that would improve past gains in leukemia-free survival. Here we consider recent advances in the treatment of ALL using TLR agonists, an issue that may cause the treatment outcome to improve further. Since triggering TLRs induces dendritic cell (DC) maturation and enhances their stimulatory properties, several studies have evaluated the use of TLR ligands as a therapeutic tool to improve the immunostimulatory potential of leukemic B cells. BCP-ALL cells express low levels of costimulatory molecules and are poor stimulators of T-cellmediated immune responses as a result [188–191]. Reid et al. assessed the impact of CpG stimulation on BCP-ALL cell lines. The competency to respond to CpG ODNs was determined by TLR9 expression level. In contrast to mature B cells and nonleukemic B-cell precursors, the response of BCP-ALL cells was characterized by increased levels of CD40 expression, tiny changes in CD86 levels, and no stimulation of CD80 expression. Moreover, CpG stimulation of ALL blasts resulted in increased secretion of IL-6, IL-8, IL-10, and IFN-␥, as well as reduced levels of IL-5 production by T cells. Reid and his companions proposed further

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investigations regarding these agents for use in the treatment of ALL [188]. On the other hand, it should be noted that inappropriate T-cell activation can induce anergy and regulatory T cells [192]. Following this experiment, Corthals et al. suggested the TLR-2 ligand, Pam3CysSerLys4, as a potential therapeutic tool in addition to TLR-9 agonists, to improve anti-ALL immune activity by promoting Th1 response [187]. Another research conducted by Fabricius and his colleagues, demonstrated the combination of CpG, IL-4 and CD40L as a more effective way to enhance immunogenicity of BCPALL cells in comparison to CpG alone. They showed the aforementioned combination as a potent therapeutic strategy, which resulted in increased expression of immunogenic molecules and induction of both allogenic and autologous cytotoxic T lymphocytes (CTLs) against BCP-ALL cells, as a consequence [193]. These findings reveal that blasts own the capability to respond to certain stimulatory agents and this may become a target for novel therapies, but requires further assessments. 4.3. Multiple myeloma 4.3.1. Definition and epidemiology Multiple myeloma (MM) is a fatal B cell malignancy determined by accumulation of neoplastic plasma cells in the bone marrow [194–197]. MM is a part of spectrum of hematologic tumors which range from monoclonal gammopathy of undetermined significance (MGUS) to plasma cell leukemia. MM accounts for 10% of all hematologic malignancies [198,199]. The incidence rate of MM accounts for 0.8% of all cancers and its death rate indicates 1.0% of all cancer deaths per year worldwide [200]. The 5-year survival rate for MM is about 35%. This rate is higher in the younger and lower in the elderly [201]. Although the exact etiology of MM is not yet well understood, a variety of risk factors have been suggested to be related to MM including genetic factors, environmental factors, MGUS, chronic inflammation, radiation and infection. 4.3.2. MM and TLRs Like normal B cells, malignant plasma cells are also sensitive to pathogens through TLRs, but polyclonal activation of tumor cells due to stimulation of TLRs results in the remarkable reduction in the production of normal immunoglobulin [195,202] and inhibition of normal T cell function [5,201]. Thus, MM patients are vulnerable to recurrent infections [5,195,203,204]. While only a limited range of TLRs are expressed in plasma cells from bone marrow of healthy donors, a broad spectrum of TLRs is expressed in the human multiple myeloma cell lines (HMCL) and primary myeloma cells. Bone marrow (BM) microenvironment might involve in myeloma cells proliferation and survival [205–208]. In fact, bone marrow stromal cells (BMSCs) produce several cytokines including IL-6, IL-10, IL-21, 1L-15, TNF␣ and IGF-1 (insulin-like growth factor-1) that contribute to the

growth and survival of malignant cells [208,209]. Therefore, increased level of these cytokines in the BM of myeloma cells suggests a pivotal role of bone marrow in frequent TLR expression in MM cells [210,211]. The most frequently expressed TLRs among HMCL and primary myeloma cells are TLR1, TLR7 and TLR9. Here, we discuss some of the tumoral effects of TLRs and then proceed to their anti-tumoral benefits. 4.3.2.1. TLRs induce MM proliferation through autocrine IL-6 production. Several studies have shown pro-tumoral effects of TLR2, 4, 5, 6, 7 and 9 in HMCL. TLR ligands mediate secretion of autocrine IL-6 from BM stromal cells which is the most important growth and survival factor of MM cells [194,195]. Thus, TLR activation allows more IL-6 autocrine loop, suggesting a key role of this factor in MM pathogenesis through survival, growth and drug resistance of MM cells [213]. 4.3.2.2. TLRs induce immune escape of MM cells through B7H1. Some surface molecules over-express on tumoral cells. Among them, expression of B7H1 (a ligand for PD-1) is significantly increased in myeloma cell lines. Expression of B7H1 is due to triggering of TLR signaling pathway or with IFN-␥, resulting in T cell inactivation [5,212]. Indeed, TLR signaling could induce immune escape of MM cells through inhibition of NK cells and CTLs [214]. 4.3.2.3. TLRs induce NF-κB pathway in MM. Although TLR3 and TLR9 show anti-tumoral effects either by enhancing the immune system effectors or by inducing cell apoptosis, they are potent stimulators of NF-␬B pathway causing proliferation of myeloma cells. NF-␬B-dependent proliferation of myeloma cells suggests that triggering these TLRs can result in MM cell growth and proliferation [213]. 4.3.3. Clinical application of TLR agonists to MM One of the best characteristics of TLRs is their capacity to stimulate adaptive immune system [215]. As immune system effectors, such as T-cells, B-cells, or DCs depict functional abnormalities [216], so strategies contributing to immunostimulation should be considered [213]. 4.3.3.1. TLR9 agonists and anti-tumoral responses. pDCs in the BM of MM patients not only inhibit proper function of T cell, but also mediate MM cell proliferation and drug resistance [217]. Triggering MM cells with CpG ODNs both restitute functionality of T cells and pDCs and dispense MM cell growth mediated by pDC. In addition, targeting TLR9 with CpG ODNs increases the level of costimulatory molecules (CD80, CD86) and antigen-presenting molecules (MHC I & II) on the TLR9 positive HMCL RPMI8226 [218]. This HMCL also promotes allogenic T-cells proliferation treated by CpG ODNs [213]. Exposure of HMCL self-antigens to immune system through MHC molecules all

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mediated by CpG ODNs, suggest that CpG ODNs can make MM cells more sensitive to immune system. 4.3.3.2. TLR3 agonists and anti-tumoral responses. Through several TLRs with apoptotic features, TLR3 is the best defined. TLR3 agonists not only directly induce apoptosis in many tumoral cells [213], but also stimulate the immunity [219], which has highlighted their capacity as therapeutic targets. According to data collected from studies, TLR signaling show dual responses in MM cells. Actually, although TLRs can improve immunity, tumor cells can also turn these pathways to their own advantages [213]. Thus, more investigation is required to determine whether TLRs as therapeutic adjuvants can be exploited. This goal may be achievable by concurrent separating the tumoral effects of TLRs from adjuvants effects. 4.4. Chronic lymphocytic leukemia 4.4.1. Definition and epidemiology B-cell chronic lymphocytic leukemia (B-CLL) is one of the most entirely studied form of hematologic malignancies with the highest prevalence in patients older than 50 years. The disease presents with progressive accumulation of monoclonal CD5+/CD19+, CD23+, CD21+, CD40+ B cells with prolonged cell survival and low proliferative index in the peripheral blood, bone marrow, and lymphoid organs [220–222]. Irrespective of its phenotypic homogeneity, BCLL is characterized by extremely heterogenous clinical course with survival from months to decades [222]. The cellular origin of CLL remains unclear. However, numerous experimental data suggest that CLL results from a multiplication of B lymphocytes selected during clonal expansion through multiple exposures to antigens [223]. 4.4.2. CLL and TLRs Data regarding TLR expression spectrum in B-CLL is limited to TLR7, 8, and 9. Eventhough differences exist among cell lines, It has been established through several studies that TLR7 and TLR9 mRNA are expressed in most samples [230–236], whereas published information indicate that TLR8 was not normally expressed by CLL cells [234], as well as TLR3 and TLR4 proteins [230]. TLR9, expressed by B-CLL cells, have similar levels to those of peripheral blood B cells, while TLR7 expression is generally lower than in human peripheral blood mononuclear cells [230,234]. 4.4.3. Clinical application of TLR agonists to CLL Treatment with conventional therapies including chemotherapy (alkylating agents, fludarabine), radiotherapy and even monoclonal antibodies cannot eliminate tumor cells completely, as these standard methods put residual tumor cells resulting in disease relapse [1,237], hence there is a need to assess the effectiveness of other potential treatment strategies.

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CLL cells are potentially sensitive to T cell-based immunotherapies [237] but because of inherently poor immunogenicity, even tumor-specific CTLs cannot clear tumor cells completely [1,19,238]. So, methods to enhance immunogenicity of CLL cells and resultant increased adaptive immune responses might improve immunotherapies against CLL cells [230,239]. TLR agonists as novel adjuvants may provide achievements to this aim, either by enhancing DCs’ function and resultant T cell activity [104,240] or by increasing antigen presenting capability of CLL cells [13,239,240]. Advances in our knowledge of TLRs and their agonists have directed us to exploit TLR agonists as potential therapeutic components in novel treatment strategies for CLL [13,104]. TLR7 and TLR9 agonists are the most being used agonists as potential adjuvants in CLL cells [19,230,241,242]. Triggering TLRs with their agonists culminates in production of pro-inflammatory cytokines, chemokines and costimulatory molecules which enhance CTL responses against tumor cells [242,243]. Furthermore, TLR agonists can inhibit tumor progression indirectly through production of angiogenesis inhibitors including type 1 IFNs [244] as CLL proliferation depends on angiogenesis [245]. 4.4.3.1. Impact of TLR7 and TLR9 agonists on CLL cells. TLR9 agonists (CpG ODN) cause CLL cells proliferation in vitro and increased expression of MHC class 1 and costimulatory molecules including B7-family (CD80 and CD86) and TNF receptor family (CD40) [235,242]. Taken together, overexpression of these molecules results in an immunogenic phenotype of CLL cells, rendering them more sensitive to killing by cytotoxic T cells and chemotherapeutic agents in vitro [246,247]. Similar findings have been made by Jahrsdörfer et al. on costimulatory molecules expression [231,236]. Also, Docker et al. proved that CLL cells treated with CpG could trigger the proliferation of T cells in mixed lymphocytic responses (MLR) [235]. CpG ODNs also induced production of IL-6 and TNF-␣ in some samples of patients [222,242,248]. TLR7 agonist, imidazoquinoline, also made similar observations on leukemic cells but compared with CpG ODNs, TLR-7 activation stimulates to a higher extent of TNF-␣ and IL-6 [230,235,249]. 4.4.3.2. TLR, proliferation and apoptosis of leukemic cells. Previous studies showed that TLR9 agonist causes CLL cells proliferation [235,236]. However, it was reported that CpG ODN might induce apoptosis in CLL cells [242,250]. In fact, different groups of patients had heterogeneous reactions to TLR9 agonist [250,251]. It was observed that CpG caused apoptosis in mutated, but proliferation in unmutated patient samples [232,252]. The newest clinical trial [253] of CpG 7909 on patients with CLL suggested astonishing results. The phase I clinical trial in patients with relapsed CLL showed immunologic changes consistent with prior in vitro studies and findings

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[253]. The cases enrolled in this trial were divided into two groups according to the injection route: (1) subcutaneous (SC) and (2) intravascular (IV). Based on the obtained data, CLL cells count in the peripheral blood had a significant but transient depletion in patients with SC administration [253]. Increased T cell activity, NK, and T cell counts were observed in some IV administration cohorts. Compared to the IV injection, SC administration of CpG 7909 just provoked T cell activity [253]. Phenotypic changes including increased levels of CD86 and CD20 markers in IV route was also observed. All the effects mentioned above were dose and route dependent [253]. Though this trial showed no therapeutic effect of CpG on CLL cells, these findings can be informative to set the scene for future studies on CpG as a therapeutic goal in treatment of CLL patients [253]. 4.4.3.3. Combination therapy of CLL. CLL cells seem to proliferate strongly under treatment with both CpG ODN and IL-2, as a consequence of increased expression of cyclins D2 and D3, and loss of p27 (a cell-cycle inhibitor) [254,255]. It is in line with other experiments of CLL cells treated by both IL2 and TLR7 agonists including imidazoquinolines [256] and the oxidized guanosine analog, loxoribine [257]. TLR7 agonists also increases expression of the high-affinity IL-2R component, CD25 [256]. On the other hand, while CLL cells treated with both IL-2 and TLR7 agonists show upregulation in expression of CD80, they do not become able in T-cell stimulatory function, which is demonstrated to be linked with strong activation of STAT3 (a well-known immunosuppressive transcription factor) [258] and augmentation of IL10 (the immunosuppressive cytokine) [256]. These CLL cells may behave like T-regs due to production of IL-10 [259]. On the contrary, treatment of TLR7-activated CLL cells with protein kinase C (PKC) induced overexpression of CD83 (a DC marker), together with CD80, CD54 and CD86; blocked STAT3 activation, stimulated T-cell proliferation and finally culminated in gaining the phenotype of dendritic cell surface [230]. However, CLL cells treated only with TLR7 and PKC agonists appear to stay weak stimulators of T-cell proliferation [256]. Fortunately, it has been discovered that coupling IL-2 with the previous TLR7 agonist-PKC treatment cause CLL cells to exit cell-cycle, inhibits IL-10 production, and gaining both the phenotypic and functional characteristic of highly immunostimulatory DCs [256]. This combinational regimen suggests potentially novel immunotherapeutic strategies for creating endogenous vaccines in order to prevent disease progression in untreated patients [19,247]. 4.4.3.4. Promotion of CLL cell death by TLR agonists. CpG ODNs can directly kill CLL cells (especially with 13q abnormalities) which can be initiated by excessive proliferation of these cells [260]. Moreover, CpG ODNs have been reported to increase the expression of some surface molecules on CLL cells. For instance, it has been found that pre-treating CLL cells with CpG ODNs can improve IL-2R toxins-induced cell

death, due to augmented expression of CD25 which forces CLL cells to uptake more toxins. As a straight conclusion of what have been discovered, CpG ODNs can augment the efficacy of cytotoxic drugs targeting surface molecules on CLL cells [261]. Loxoribine, a TLR7 agonist [262], was formerly found to make CLL cells to move into the cell-cycle, and to enhance sensitivity of CLL cells to cycle-active cytotoxic drugs, such as doxorubicin and etoposide [263]. It is obvious that TLR agonists will not be, therefore, highly effective as single agents but their potential important role in CLL have been shown to exert through their tumorsensitizing ability for cytotoxic agents e.g. CTLs [261]. Based on our recent knowledge, combinational regimens including both CpG ODNs and imidazoquinolines, accompanying with radiotherapies, and conventional chemotherapeutic designs such as denileukindiftitox (ONTAK) [264], rituximab and alemtuzumab seem to surpass single treatments alone. Also, IL-2 can be employed in these neo-adjuvant designs to increase susceptibility of CLL cells to cytotoxic agents [257].

5. Conclusions Hematologic malignancies treatment has been improved over the last years, leading to increased complete remission rates, although the high incidence of relapse is still observed, since treatment with conventional therapies including chemotherapy, radiotherapy and even monoclonal antibodies cannot eliminate tumor cells thoroughly. Therefore, the introduction of novel therapeutics as adjuvants for conventional therapies has been a topic of numerous investigations. In this regard, TlRs has been of immense significance as they are the most thoroughly studied pattern recognition receptors, comprising a bridge between the innate and adaptive immunity, either by increasing antigen presenting capability of malignant cells or by enhancing antigen presenting cells’ function and resultant T cell activation. The ever expanding knowledge of the molecular basis of TLR signaling may help us elicit a potent immune response to anti-tumor therapies, although we still have to shed the light on the shadows existing in the exploitation of TLRs in the treatment of lymphoid malignancies. For instance, the associations between certain TLR polymorphism and specific hematologic malignancies still need to be addressed so as to both evaluate the risk of these malignancies and to use them as potential biological markers for the response to treatment. It is also crucial to adequately assess the dual role of certain TLR agonists (like TLR9 agonist) in mediating both beneficial and detrimental effects, including induction of cell death and enhancement of lymphoproliferation. In the present review, the current knowledge on the immunopathogenic roles of TLRs in B cell and T cell lymphomas, acute lymphoblastic leukemia, multiple myeloma, and chronic lymphocytic leukemia, as well as the results of studies on TLR ligands and their future implications to manage the aforementioned hematologic malignancies have been

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summarized. In order to delineate the therapeutic capacity of TLRs in lymphoid malignancies, continuous research is needed.

Conflicts of interest All authors declare that there are no conflicts of interest.

Reviewers Dr. Mona Hedayat, Boston Children’s Hospital, Harvard Medical School, United States. Professor Kiyoshi Takeda, Immunology Frontier Research Center, Osaka University, Japan.

Acknowledgment The authors would like to acknowledge Prof. Ronald Levy, Stanford University School of Medicine, Stanford, CA for critical reading of this paper.

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Biography Dr. Nima Rezaei received his Medical Degree from Tehran University of Medical Sciences in 2002. He received his M.Sc. and Ph.D. degrees in the field of Clinical Immunology and Genetic Medicine from the University of Sheffield in 2007 and 2009, respectively, as well as a short-term fellowship, awarded by the ESID, in the Pediatric Immunology and BMT Unit of the Newcastle General Hospital. He is already the Chief Executive Director of the Children’s Medical Center Hospital, the Pediatrics Center of Excellence in Iran as well as the Deputy President of the Research Center for Immunodeficiencies. He has presented more than 270 lectures/posters in congresses and has published more than 340 articles in international scientific journals during last decade.