title: Simultaneous TLR2 inhibition and TLR9 activation synergistically suppress tumor metastasis in mice.
abstract: OBJECTIVE
  To develop a rational immunotherapy against tumor metastasis by combining a Toll-like-receptor 2 (TLR2)-neutralizing antibody with a TLR9 agonist CpG ODN, and then investigate the mechanism of action for this combinational regimen.
  
  
  METHODS
  After mouse melanoma B16-F10 cell inoculation, female C57BL/6 mice were treated with either CpG ODN (0.5 mg/kg) or the anti-TLR2 antibody (200 μg/kg), or with a combination of the two agents. Pulmonary metastases were evaluated by counting metastatic nodes on the lung surface using anatomical microscopy. Flow cytometry was used to evaluate the cytotoxicity of the immune cells in tumor-draining lymph nodes, the cell population in the spleen, and the infiltration of immune cells within the lungs. Cytokine and enzyme expression in the lung tissue was evaluated using ELISA or immunostaining.
  
  
  RESULTS
  Anti-metastatic effects were detected in mice treated with either CpG ODN or the anti-TLR2 antibody alone. However, treatment with CpG ODN plus the anti-TLR2 antibody synergistically suppressed the metastasis as compared with treatment with either single agent. The combinational treatment resulted in enhanced infiltration of natural killer cells and cytotoxic T cells, reduced recruitment of type 2 macrophages and Tregs, and decreased expression of immunosuppressive factors including TGF-β1, cyclooxygenase-2 and indoleamine 2,3-dioxygenase, thus stimulated tumor cytotoxicity and suppressed metastasis. The anti-metastatic effect of the combinational regimen was further confirmed in spontaneous metastatic mouse model of Lewis lung carcinoma.
  
  
  CONCLUSIONS
  Our studies suggest that combining a TLR9 agonist with an anti-TLR2 antibody, which eliminates immunosuppressive factors from the tumor environment, is critical for an effective anti-metastatic immunotherapy.
authors.affiliation: Molecular Immunology and Pharmacology Laboratory, Institute of Meteria Medica, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, China.
authors.forename: Jun
authors.initials: J
authors.lastname: Yan
authors.forename: Fang
authors.initials: F
authors.lastname: Hua
authors.forename: Han-zhi
authors.initials: HZ
authors.lastname: Liu
authors.forename: Hong-zheng
authors.initials: HZ
authors.lastname: Yang
authors.forename: Zhuo-wei
authors.initials: ZW
authors.lastname: Hu
mesh_terms: D000276:Adjuvants, Immunologic / Q000627:therapeutic use*; D000818:Animals; D057134:Antibodies, Neutralizing / Q000276:immunology / Q000627:therapeutic use*; D005260:Female; D007167:Immunotherapy / Q000379:methods; D008168:Lung / Q000187:drug effects / Q000473:pathology; D008175:Lung Neoplasms / Q000473:pathology / Q000517:prevention & control / Q000556:secondary*; D008546:Melanoma, Experimental / Q000276:immunology / Q000473:pathology / Q000628:therapy*; D051379:Mice; D008810:Mice, Inbred C57BL; D009362:Neoplasm Metastasis / Q000517:prevention & control*; D009838:Oligodeoxyribonucleotides / Q000627:therapeutic use*; D051195:Toll-Like Receptor 2 / Q000276:immunology*; D051217:Toll-Like Receptor 9 / Q000819:agonists*
chemical_list: D000276:Adjuvants, Immunologic; D057134:Antibodies, Neutralizing; C408982:CPG-oligonucleotide; D009838:Oligodeoxyribonucleotides; D051195:Toll-Like Receptor 2; D051217:Toll-Like Receptor 9