Toll‐like receptors on B‐CLL cells: expression and functional consequences of their stimulation

Toll‐like receptor (TLR) stimulation plays a crucial role in the homeostasis of human B cells. We investigated the expression of TLRs 1–9 on the cells of B‐cell chronic lymphocytic leukemia (B‐CLL) and analyzed the functional consequences of TLR stimulation on leukemic cells. We showed that B‐CLL cells express similar set of TLRs as memory B cells of healthy donors, i.e. TLR‐1, TLR‐2, TLR‐6, TLR‐7 and TLR‐9. However, in contrast to memory B cells, B‐CLL cells lack TLR‐4 expression. Expression of TLRs correlates with their capacity to respond to specific TLR agonists. At the level of phenotype, ODN2006 (TLR‐9 agonist) is the most potent stimulus. B‐CLL cells also respond to the stimulation with loxoribine, Pam3CSK4 and MALP‐2 (TLR‐7, TLR1/TLR2 and TLR2/TLR6 agonists, respectively). TLR‐7 and TLR‐9 stimulation induces production of IL‐6 and TNFα. In 47% of tested patients, treatment with ODN2006, MALP‐2 and Pam3CSK4 reduced leukemic cells survival. Stimulation of B‐CLL cells with TLR‐9 agonists, loxoribine, MALP‐2 and Pam3CSK4 induces significant proliferation. We report that TLR stimulation induces expression of CD38, a negative prognostic marker, on B‐CLL cells. Expression of CD38 is induced by direct stimulation of B‐CLL cells through TLR‐7 and TLR‐9 or CD38 can be induced on B‐CLL cells indirectly by a soluble factor induced in non‐B‐CLL cells after stimulation with TLR‐2, TLR‐3 or TLR‐5 agonists; the nature of this factor remains unknown. Our results argue for cautious evaluation of immunointervention strategies based on the administration of TLR agonists in the treatment of B‐CLL as their effects on B‐CLL cells may be tumor promoting as well as tumor suppressing.     

Obstacles to effective Toll-like receptor agonist therapy for hematologic malignancies

It has long been noted that products of microorganisms have clinical activity against hematologic malignancies. Recent advances suggest that Toll-like receptors (TLRs) activated by ligands in the microbial preparations might account for some of this activity, and that defined TLR agonists might improve the clinical efficacy of this approach. A potentially important mechanism of action of TLR agonists is their ability to cause tumor cells to differentiate into a 'tolerized' state in which they become highly sensitive to cytotoxic effector cells and chemotherapeutic drugs. TLR agonists as single agents have strong activity against cutaneous leukemias and lymphomas but are not as effective against systemic disease. A possible reason for this discrepancy is the hypoxic internal tumor microenvironment, which promotes glycolytic metabolism, and the presence of suppressive cytokines, prostaglandins and nucleosides that prevent strong TLR signaling in cancer cells. Accordingly, concomitant use of agents to counter this intrinsic microenvironmental inhibition, together with TLR agonists, may prove to be an effective treatment strategy for the hematologic malignancies.     

Regression of lymphomatous skin deposits in a chronic lymphocytic leukemia patient treated with the Toll-like receptor-7/8 agonist, imiquimod.

The identification of clinically relevant, active immunomodulatory agents is important for developing immunotherapeutic approaches to chronic lymphocytic leukemia (CLL) and other B-cell lymphomas that are not curable with conventional chemotherapy. In this investigation, the imidazoquinoline Toll-like receptor (TLR)-7/8 agonist, imiquimod, was found to mediate the clearance of a lymphomatous skin lesion in a CLL patient. Imidazoquinolines also activated TLR-7/8 signaling pathways, resulting in increased expression of costimulatory molecules on circulating tumor cells. These observations extend the therapeutic spectrum of imiquimod to cutaneous B-cell lymphomas and suggest the use of TLR-7/8 agonists in CLL immunotherapy.     

Radiotherapy combined with TLR7/8 activation induces strong immune responses against gastrointestinal tumors

In addition to local cytotoxic activity, radiotherapy may also elicit local and systemic antitumor immunity, which may be augmented by immunotherapeutic agents including Toll-like receptor (TLR) 7/8 agonists. Here, we investigated the ability of 3M-011 (854A), a TLR7/8 agonist, to boost the antigen-presenting activity of dendritic cells (DC) as an adjuvant to radiotherapy. The combined treatment induced marked local and systemic responses in subcutaneous and orthotopic mouse models of colorectal and pancreatic cancer. In vitro cytotoxicity assays as well as in vivo depletion experiments with monoclonal antibodies identified NK and CD8 T cells as the cell populations mediating the cytotoxic effects of the treatment, while in vivo depletion of CD11c⁺ dendritic cells (DC) in CD11c-DTR transgenic mice revealed DC as the pivotal immune hub in this setting. The specificity of the immune reaction was confirmed by ELISPOT assays. TLR7/8 agonists therefore seem to be potent adjuvants to radiotherapy, inducing strong local and profound systemic immune responses to tumor antigens released by conventional therapy.     

TLR7/8 agonists trigger immunostimulatory properties of human 6-sulfo LacNAc dendritic cells

Imiquimod and resiquimod represent Toll-like receptor (TLR) 7 and 8 agonists, which emerged as attractive candidates for tumor therapy. To elucidate immune cells, which mainly contribute to TLR7/8-mediated antitumoral activity, we investigated the impact of imiquimod and resiquimod on native human 6-sulfo LacNAc (slan) dendritic cells (DCs). We found that both TLR7/8 agonists significantly improve the release of various proinflammatory cytokines by slanDCs and promote their tumor-directed cytotoxic activity. Furthermore, resiquimod efficiently augmented the ability of slanDCs to stimulate T cells and natural killer cells. These results indicate that imiquimod and resiquimod trigger various immunostimulatory properties of slanDCs, which may contribute to their antitumor effects.     

Toll-like receptors on tumor cells facilitate evasion of immune surveillance

The signal pathways that trigger tumor cell escape from immune surveillance are incompletely understood. Toll-like receptors (TLRs), which activate innate and adaptive immune responses, are thought to be restricted to immune cells. We show here that TLRs, including TLR4, are expressed on tumor cells from a wide variety of tissues, suggesting that TLR activation may be an important event in tumor cell immune evasion. Activation of TLR4 signaling in tumor cells by lipopolysaccharide induces the synthesis of various soluble factors and proteins including interleukin-6, inducible nitric oxide synthase, interleukin-12, B7-H1, and B7-H2, and results in resistance of tumor cells to CTL attack. In addition, lipopolysaccharide-stimulated tumor cell supernatants inhibit both T cell proliferation and natural killer cell activity. Blockade of the TLR4 pathway by either TLR4 short interfering RNA or a cell-permeable TLR4 inhibitory peptide reverses tumor-mediated suppression of T cell proliferation and natural killer cell activity in vitro, and in vivo, delays tumor growth and thus prolongs the survival of tumor-bearing mice. These findings indicate that TLR signaling results in a cascade leading to tumor evasion from immune surveillance. These novel functions of TLRs in tumor biology suggest a new class of therapeutic targets for cancer therapy.     

The use of TLR7 and TLR8 ligands for the enhancement of cancer immunotherapy

The importance of Toll-like receptors (TLRs) in stimulating innate and adaptive immunity is now well established. In view of this, TLR ligands have become interesting targets to use as stand-alone immunotherapeutics or vaccine adjuvants for cancer treatment. TLR7 and TLR8 were found to be closely related, sharing their intracellular endosomal location, as well as their ligands. In this review, we describe the agonists of TLR7 and TLR8 that are known so far, as well as their contribution to antitumor responses by affecting immune cells, tumor cells, and the tumor microenvironment. The major benefit of TLR7/8 agonists as immune response enhancers is their simultaneous stimulation of several cell types, resulting in a mix of activated immune cells, cytokines and chemokines at the tumor site. We discuss the studies that used TLR7/8 agonists as stand-alone immunotherapeutics or cancer vaccine adjuvants, as well as the potential of TLR7/8 ligands to enhance antitumor responses in passive immunotherapy approaches.     

Adjuvant for vaccine immunotherapy of cancer – focusing on Toll‐like receptor 2 and 3 agonists for safely enhancing antitumor immunity

Immune‐enhancing adjuvants usually targets antigen (Ag)‐presenting cells to tune up cellular and humoral immunity. CD141⁺ dendritic cells (DC) represent the professional Ag‐presenting cells in humans. In response to microbial pattern molecules, these DCs upgrade the maturation stage sufficient to improve cross‐presentation of exogenous Ag, and upregulation of MHC and costimulators, allowing CD4/CD8 T cells to proliferate and liberating cytokines/chemokines that support lymphocyte attraction and survival. These DCs also facilitate natural killer‐mediated cell damage. Toll‐like receptors (TLRs) and their signaling pathways in DCs play a pivotal role in DC maturation. Therefore, providing adjuvants in addition to Ag is indispensable for successful vaccine immunotherapy for cancer, which has been approved in comparison with antimicrobial vaccines. Mouse CD8α⁺ DCs express TLR7 and TLR9 in addition to the TLR2 family (TLR1, 2, and 6) and TLR3, whereas human CD141⁺ DCs exclusively express the TLR2 family and TLR3. Although human and mouse plasmacytoid DCs commonly express TLR7/9 to respond to their agonists, the results on mouse adjuvant studies using TLR7/9 agonists cannot be simply extrapolated to human adjuvant immunotherapy. In contrast, TLR2 and TLR3 are similarly expressed in both human and mouse Ag‐presenting DCs. Bacillus Calmette–Guerin peptidoglycan and polyinosinic–polycytidylic acid are representative agonists for TLR2 and TLR3, respectively, although they additionally stimulate cytoplasmic sensors: their functional specificities may not be limited to the relevant TLRs. These adjuvants have been posted up to a certain achievement in immunotherapy in some cancers. We herein summarize the history and perspectives of TLR2 and TLR3 agonists in vaccine‐adjuvant immunotherapy for cancer.     

Differential MyD88/IRAK4 requirements for cross-priming and tumor rejection induced by heat shock protein 70-model antigen fusion protein

Priming of CD8(+) T cells requires two signals, one produced by T-cell receptor recognition of antigen, and a second that is often provided by the innate immune response. In this context, antigens non-covalently or covalently associated with heat shock proteins (HSP) are internalized and processed in antigen-presenting cells (APC) to be presented by MHC I molecules to CD8(+) T cells, thus, signal 1 has been well characterized in this pathway of cross-presentation. Signal 2 is not fully understood, although there are reports that Toll-like receptors (TLRs) interact with HSP and activate APC. The ability of HSP to activate APC through TLRs is, however, controversial because of the possibility of endotoxin contamination. Using a variety of TLR KO mice, we present evidence that TLRs (TLR2, 3, 4, 7, and 9) and their adaptor molecules MyD88 and IRAK4 are dispensable in cross-priming by a mycobacterial HSP70-antigen (ovalbumin as a model antigen) fusion protein; in contrast, MyD88/IRAK4, but not TLRs, are required for tumor rejection induced by the same reagent. Our results indicate that HSP-mediated cross-priming uses a second signal produced by mechanisms other than TLR cascades. We hypothesize that efficient cross-priming by HSP70 alone is insufficient for tumor rejection and that MyD88/IRAK4-dependent inflammatory stimulation, which might contribute to maintenance of the initially primed effector cells, is required to eradicate tumor burden.     

S180骨肉瘤细胞TLRs表达及其与肿瘤耐受的相关性

目的探讨TLRs在诱导S180骨内瘤细胞免疫耐受中的作用,为肿瘤临床的免疫治疗提供新的思路。方法建立S180腹水癌早晚期模型,采用RT-PCR方法检测体外S180骨肉瘤细胞和荷瘤鼠体内早晚期S180骨肉瘤细胞TLR1-9 mRNA的表达水平,以及LPS体外刺激S180骨肉瘤细胞前后TGF-β和IL-10 mRNA表达变化,ELISA方法检测LPS体外刺激S180骨肉瘤细胞前后肿瘤上清液中TGF-β蛋白分泌的含量。结果体外S180骨肉瘤细胞表达TLR1-7和TLR9,TLR8不表达。荷瘤鼠体内早晚期S180骨肉瘤细胞TLRs的表达不同,TLR4、9表达早期高于晚期,其中TLR4的高表达有统计学意义（P<0.05);TLR1、2、3、5、6和7表达晚期高于早期,但差异无统计学意义(P>0.05)。10μg/ml 的LPS在不同时间点体外刺激S180骨肉瘤细胞后IL-10 mRNA表达水平均上调,且有时间依赖关系,TGF-β在mRNA和蛋白水平也均有增加。结论体外S180骨肉瘤细胞表达多种TLRs,荷瘤鼠体内早晚期S180骨肉瘤细胞TLRs表达的不同在肿瘤发展的不同时期可能发挥不同的作用,S180骨肉瘤细胞TLR4信号通路活化后可能通过促进TGF-β和IL-10的分泌从而参与肿瘤的免疫耐受。     

Targeting Toll-Like Receptors for Cancer Therapy

The immune system encompasses a broad array of defense mechanisms against foreign threats, including invading pathogens and transformed neoplastic cells. Toll-like receptors (TLRs) are critically involved in innate immunity, serving as pattern recognition receptors whose stimulation leads to additional innate and adaptive immune responses. Malignant cells exploit the natural immunomodulatory functions of TLRs, expressed mainly by infiltrating immune cells but also aberrantly by tumor cells, to foster their survival, invasion, and evasion of anti-tumor immune responses. An extensive body of research has demonstrated context-specific roles for TLR activation in different malignancies, promoting disease progression in certain instances while limiting cancer growth in others. Despite these conflicting roles, TLR agonists have established therapeutic benefits as anti-cancer agents that activate immune cells in the tumor microenvironment and facilitate the expression of cytokines that allow for infiltration of anti-tumor lymphocytes and the suppression of oncogenic signaling pathways. This review focuses on the clinical application of TLR agonists for cancer treatment. We also highlight agents that are undergoing development in clinical trials, including investigations of TLR agonists in combination with other immunotherapies.     

Phosphorothioate modification of the TLR9 ligand CpG ODN inhibits poly(I:C)-induced apoptosis of hepatocellular carcinoma by entry blockade

Toll-like receptors (TLRs) play a crucial role in the innate immune response and subsequent induction of adaptive immune responses. Recently, it has been noted that TLRs on tumor cells are involved in tumor development, and several TLR agonists, such as the TLR3 agonist poly(I:C) and the TLR9 agonist CpG ODN, are being developed as vaccine adjuvants and cancer immunotherapeutics. In this study, we investigated whether combining poly(I:C) with a TLR9 agonist CpG ODN would result in a stronger anti-tumor effect on hepatocellular carcinoma cells (HCCs). Surprisingly, we found that simultaneous transfection of poly(I:C) and ODN M362 exhibited a lower pro-apoptotic effect on HCCs than transfection with poly(I:C) alone. Simultaneous co-transfection was accompanied by down-regulation of poly(I:C)-related innate receptors, pro-inflammatory cytokines and apoptotic genes induced by poly(I:C), indicating that ODN M362 blocked the activation of poly(I:C)-triggered intrinsic immune responses and cellular apoptosis. Further studies indicated that these effects were partly due to the phosphorothioate-modification of CpG ODN, which blocked the entry of poly(I:C) into tumor cells. This entry blockade was avoided by administering poly(I:C) after CpG ODN. Moreover, poly(I:C)-mediated pro-apoptotic effects were enhanced in vitro and in vivo by pre-treating HCC cells with CpG ODN. Our findings thus suggest that when combining poly(I:C) and CpG ODN for cancer therapy, these agents should be used in an alternating rather than simultaneous manner to avoid the blocking effect of phosphorothioate-modified TLR9 ligands.     

The Role of Toll-Like Receptors in Oncotherapy

Toll-like receptors (TLRs) are associated with tumor growth and immunosuppression, as well as apoptosis and immune system activation. TLRs can activate apoptosis and innate and adaptive immunity pathways, which can be pharmacologically targeted for the development of anticancer oncotherapies. Several studies and clinical trials indicate that TLR agonists are promising adjuvants or elements of novel therapies, particularly when used in conjunction with chemotherapy or radiotherapy. An increasing number of studies suggest that the activation of TLRs in various cancer types is related to oncotherapy; however, before this finding can be applied to clinical practice, additional studies are required. Research suggests that TLR agonists may have potential applications in cancer therapy; nevertheless, because TLR signaling can also promote tumorigenesis, a critical and comprehensive evaluation of TLR action is warranted. This review focuses on recent studies that have assessed the strengths and weaknesses of utilizing TLR agonists as potential anticancer agents.     

Cell mediated immune responses through TLR4 prevents DMBA-induced mammary carcinogenesis in mice

Toll-like receptors (TLRs) activate signals that are critically involved in the initiation of adaptive immune responses and many tumorigenic chemicals have been associated with activation of those pathways. To determine the role of TLR-4 (TLR4) in mammary carcinogenesis, we subjected TLR4 deficient and wild type (WT) mice to oral gavage with carcinogenic polyaromatic hydrocarbon 7,12-dimethylbenz(a)anthracene (DMBA). TLR4 deficient mice developed more tumors relative to the WT mice. T cells of TLR4 deficient mice produced elevated levels of IL-17 and lower levels of IFN-γ relative to WT mice. IL-12 secreted by CD11c(+) cells was higher in WT mice, whereas greater amounts of IL-23 were produced by CD11c(+) cells from TLR4 deficient mice. Moreover, there was higher incidence of regulatory T cells in TLR4 deficient mice than WT mice. Similarly, various markers of angiogenesis [matrix metalloproteinases (MMP)-2 and MMP-9, CD31 and vascular endothelial growth factor] were highly expressed in tumors from TLR4 deficient mice than WT mice. The results of this study indicate that TLR4 plays an important role in the prevention of DMBA induced mouse mammary tumorigenesis and efforts to divert the cell-mediated immune response may, therefore, prove to be beneficial in the prevention of mammary tumors.     

Inhibition and promotion of tumor growth with adeno-associated virus carcinoembryonic antigen vaccine and Toll-like receptor agonists

Carcinoembryonic antigen (CEA) is a cancer vaccines' target. Several features of recombinant adeno-associated virus (rAAV) are attractive for vaccine applications. Combining other viral vector vaccines with Toll-like receptor (TLR) agonists enhances antitumor immunity. Wild-type and CEA transgenic (Tg) mice were immunized with rAAV-expressing CEA, the TLR9 agonist, oligodinucleotide (ODN)1826 and the TLR7 agonist, imiquimod. Mice were challenged with MC38 colon tumor cells and MC38 cells expressing CEA. rAAV-CEA immunization combined with ODN1826 or imiquimod enhanced CEA-specific T-helper 1 immunity and protected against tumor challenge in wild-type but not in CEA-Tg mice. In contrast, immunization with rAAV-CEA in CEA-Tg mice could abrogate the antitumor effects of ODN1826 and promote tumor growth. Compared to wild-type, CEA-Tg mice were characterized by a greater myeloid suppressor cell and T-helper 2 response to TLR agonists and to syngeneic tumors. Depleting PDCA1(+) plasmacytoid dendritic cells and Gr1(+) myeloid cells increased anti-CEA immune responses in CEA-Tg mice to rAAV-CEA-ODN1826 immunization, whereas depleting CD25(+) T cells did not. There are differences in the response of wild-type and CEA-Tg mice to rAAV-CEA, TLR agonists and syngeneic tumor. In CEA-Tg mice, tumor growth can be promoted with rAAV-CEA and TLR agonists. Dendritic and myeloid cells play a regulatory role.     

膀胱癌细胞钟声蛋白样受体表达与卡介苗诱导

[目的]探讨膀胱癌细胞钟声蛋白样受体(TLRs)在卡介苗(BCG)诱导的抗膀胱癌免疫反应中的作用.[方法]以不同剂量(菌数/细胞数比值)的BCG和大肠杆菌分别刺激培养人膀胱癌T24细胞株.1小时后提取细胞总RNA,用半定量RT-PCR技术检测T24细胞中TLR-2和TLR-4的表达水平.12小时后提取各孔细胞的上清液,用ELISA试剂盒测定IL-4和IL-12的浓度.[结果](1)BCG刺激后T24细胞中TLR-2和TLR-4的表达水平及IL-12浓度随着BCG剂量增加而逐渐升高,在菌数/细胞数比值为10时达到最高,随后逐渐降低,但仍高于未刺激组.(2)BCG刺激后T24细胞中IL-4的浓度与未刺激组相比无明显差别.(3)大肠杆菌刺激后各剂量组T24细胞中TLR-2和TLR-4的表达水平以及IL-4和IL-12浓度与未刺激组相比无明显差别.[结论]膀胱癌细胞TLRs表达可能与BCG诱导的抗膀胱癌免疫反应有关.     

Expression of Toll-like Receptor 9 Increases with Progression of Cervical Neoplasia in Tunisian Women - A Comparative Analysis of Condyloma,Cervical Intraepithelial Neoplasia and Invasive Carcinoma

Toll-like receptors (TLRs) are expressed in immune and tumor cells and recognize pathogen-associated molecular patterns. Cervical cancer (CC) is directly linked to a persistent infection with high risk human papillomaviruses (HR-HPVs) and could be associated with alteration of TLRs expression. TLR9 plays a key role in the recognition of DNA viruses and better understanding of this signaling pathway in CC could lead to the development of novel immunotherapeutic approaches. The present study was undertaken to determine the level of TLR9 expression in cervical neoplasias from Tunisian women with 53 formalin-fixed and paraffin-embedded specimens, including 22 samples of invasive cervical carcinoma (ICC), 18 of cervical intraepithelial neoplasia (CIN), 7 of condyloma and 6 normal cervical tissues as control cases. Quantification of TLR9 expression was based on scoring four degrees of extent and intensity of immunostaining in squamous epithelial cells. TLR9 expression gradually increased from CIN1 (80% weak intensity) to CIN2 (83.3% moderate), CIN3 (57.1%strong) and ICC (100% very strong). It was absent in normal cervical tissue and weak in 71.4% of condyloma. The mean scores of TLR9 expression were compared using the Kruskall-Wallis test and there was a statistical significance between normal tissue and condyloma as well as between condyloma, CINs and ICC. These results suggest that TLR9 may play a role in progression of cervical neoplasia in Tunisian patients and could represent a useful biomarker for malignant transformation of cervical squamous cells.     

Expressions of Toll-like receptors 3, 4, 7, and 9 in cervical lesions and their correlation with HPV16 infection in Uighur women

Recent findings show that Toll-like receptors(TLRs) expressed in immune cells play a crucial role in the innate immune response and the subsequent induction of adaptive immune responses against microbial infection on tissue injury.Furthermore,expression of TLRs in cancer cells is associated with tumor proliferation and invasion.To explore the role of TLRs expression in cervical carcinogenesis in Uighur women,we detected the expressions of TLR3,TLR4,TLR7,and TLR9 in 25 normal cervical tissues,64 cervical int...     

Toll-like receptors mediate proliferation and survival of multiple myeloma cells.

Multiple myeloma (MM) is an incurable B-cell malignancy characterized by accumulation of malignant plasma cells in bone marrow (BM) and recurrent or persistent infections. Toll-like receptors (TLRs) are essential in the host defense against infections and today 10 human TLRs (TLR1-TLR10) and one TLR-homolog (RP105) have been characterized. B cells express several TLRs (mainly TLR1, 6, 7, 9, 10 and RP105) and TLR-initiated responses in B cells include proliferation, anti-apoptosis effect and plasma cell (PC) differentiation. The present study was designed to analyze the role of TLRs in MM. We show that frequent expressions of TLRs were detected in cell lines from MM patients (minimum six TLRs in each). In comparison, only few TLRs (mainly TLR1 and or RP105) were found expressed in PCs from BM of healthy donors. In addition, TLR-specific ligands induce increased proliferation and survival of the MM cell lines, partially due to an autocrine interleukin-6 production. Importantly, we demonstrate that also PC from MM patients proliferates in response to TLR-specific ligands. In conclusion, TLR-ligands may contribute to increased growth and survival of MM cells in MM patients.