Journal:

Article Title: Lipopolysaccharide-Trap-Fc, a Multifunctional Agent To Battle Gram-Negative Bacteria

doi: 10.1128/IAI.00004-09

Figure Lengend Snippet: LPS-Trap-Fc1 to -Fc4 bind LPS and block LPS-mediated IL-6 production in Mono Mac 6 cells. (A) Supernatants of HEK293T cells transfected with LPS-Trap-Fc1 were incubated in the presence (lane 1) or absence (lane 3) of biotin-LPS. Additionally, supernatants were preincubated with an anti-TLR4/MD-2 MAb (lane 2) or an excess of LPS (lane 4). Complexes were immunoprecipitated with streptavidin-Sepharose, subjected to SDS-PAGE, and analyzed by Western blotting with an anti-FLAG MAb. (B) RAW 264.7 cells were incubated with LPS-Trap-Fc1 or a medium control. Cells were stimulated with 100 ng/ml LPS overnight, and IL-6 levels in supernatants were determined by ELISA. *, P < 0.002 compared to control (Mann-Whitney U test, n = 6).

Article Snippet: For specificity control, some supernatants were preincubated with either 50 μg/ml LPS ( Salmonella enterica serovar Minnesota; Sigma), which has a high binding activity for TLR4/MD-2, or 10 μg/ml anti-mouse TLR4/MD-2 MTS510 MAb (Hycult Biotechnology, Uden, The Netherlands) for 1 h at room temperature.

Techniques: Blocking Assay, Transfection, Incubation, Immunoprecipitation, SDS Page, Western Blot, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY

Journal: Immunology

Article Title: An agonistic anti‐Toll‐like receptor 4 monoclonal antibody as an effective adjuvant for cancer immunotherapy

doi: 10.1111/imm.13095

Figure Lengend Snippet: The anti‐Toll‐like receptor 4 (TLR4) monoclonal antibody (mAb) enhances the proliferation of antigen (Ag)‐specific T‐cells in vivo. 5‐Carboxyfluorescein diacetate succinimidyl ester (CFSE)‐labelled Ly5·1+ (a) OT‐I or (b) OT‐II spleen cells were adoptively transferred into C57BL/6N (Ly5·2+) mice (n = 3 per group). On the following day, transferred mice were immunized with vehicle, (a) 100 μg or (b) 10 μg ovalbumin (OVA), 3 μg of the anti‐TLR4 mAb, or both. (a) Two or (b) 3 days after immunization, spleen cells were subjected to FACS analysis to evaluate the proliferation of OT‐I and OT‐II T‐cells based on the intensity of CFSE fluorescence. Representative histograms and dot plots are shown. The absolute numbers of (a) Ly5·1+ CD8+ OT‐I and (b) Ly5·1+ CD4+ OT‐II T‐cells in the spleen, and the percentages of (a) CFSE low Ly5·1+ CD8+ OT‐I and (b) Ly5·1+ CD4+ OT‐I T‐cells are shown as means ± SEMs. One‐way ANOVA with the Tukey post hoc test; ****P < 0·0001. Data are representative of three independent experiments.

Article Snippet: The mouse anti‐mouse TLR4/MD‐2 agonistic mAb (UT12) 38 , 39 was purified from conditioned serum‐free medium (Hybridoma‐SFM; Thermo Fisher Scientific, Waltham, MA) used to culture hybridomas.

Techniques: In Vivo, Fluorescence

Journal: Immunology

Article Title: An agonistic anti‐Toll‐like receptor 4 monoclonal antibody as an effective adjuvant for cancer immunotherapy

doi: 10.1111/imm.13095

Figure Lengend Snippet: The anti‐Toll‐like receptor 4 (TLR4) monoclonal antibody (mAb) induces antigen (Ag)‐specific IFN‐γ‐producing CD8 T‐cells. C57BL/6N mice were injected i.p. with vehicle (phosphate‐buffered saline; PBS), ovalbumin (OVA; 100 μg), the anti‐TLR4 mAb (5 μg), or both. (a) Five days later, the CD44hi CD4+ and CD44hi CD8+ T‐cells in the spleen were analysed by FACS. Representative histograms and dot plots of three independent experiments are shown. (b) Five–seven days later, spleen cells were re‐stimulated with the OVA 323–339 (10 μg/ml) or OVA 257–264 (1 μg/ml) peptide for 8 hr in vitro. Intracellular IFN‐γ was stained in CD4 and CD8 T‐cells and analysed by FACS. Representative dot plots are shown. Similar results were obtained from three independent experiments.

Article Snippet: The mouse anti‐mouse TLR4/MD‐2 agonistic mAb (UT12) 38 , 39 was purified from conditioned serum‐free medium (Hybridoma‐SFM; Thermo Fisher Scientific, Waltham, MA) used to culture hybridomas.

Techniques: Injection, In Vitro, Staining

Journal: Immunology

Article Title: An agonistic anti‐Toll‐like receptor 4 monoclonal antibody as an effective adjuvant for cancer immunotherapy

doi: 10.1111/imm.13095

Figure Lengend Snippet: The agonistic anti‐Toll‐like receptor 4 (TLR4) monoclonal antibody (mAb) primes the expression of IL‐1β but does not stimulate its secretion. (a) Bone marrow‐derived macrophages (BMMs) were primed with the anti‐TLR4 mAb (0·1, 1, 10 μg/ml) or lipopolysaccharide (LPS; 10, 100, 1000 ng/ml) for 4 hr and then transfected with identical stimulants using polyethylenimine for 20 hr. (b) Pam3CSK4‐primed BMMs were transfected with the anti‐TLR4 mAb (0·1, 1, 10 μg/ml) or LPS (10, 100, 1000 ng/ml) as in (a). (a, b) The concentrations of IL‐1β in the culture supernatants were evaluated by ELISA. One‐way ANOVA with the Tukey post hoc test; *P < 0·05, ****P < 0·0001 (versus primed and mock‐transfected control). Data are means ± SDs of triplicate cultures and are representative of two independent experiments. (c) BMMs were stimulated with the anti‐TLR4 mAb or LPS for the indicated periods. (d) Pam3CSK4‐primed BMMs were transfected with the anti‐TLR4 mAb or LPS for the indicated periods. (c, d) Following stimulation, culture supernatants and whole cell lysates were analysed by Western blot. Data are representative of two independent experiments. (e) The mice (n = 3 per group) were primed with i.v. injection of poly(I:C) (200 μg) for 21 hr and then challenged i.p. with the anti‐TLR4 mAb (3 μg) or LPS (1 μg) i.p. Plasma samples were collected at 2 or 6 hr, and the concentration of IL‐1β was determined by ELISA. Data are means ± SEMs of two independent experiments.

Article Snippet: The mouse anti‐mouse TLR4/MD‐2 agonistic mAb (UT12) 38 , 39 was purified from conditioned serum‐free medium (Hybridoma‐SFM; Thermo Fisher Scientific, Waltham, MA) used to culture hybridomas.

Techniques: Expressing, Derivative Assay, Transfection, Enzyme-linked Immunosorbent Assay, Western Blot, Injection, Concentration Assay

Journal: Immunology

Article Title: An agonistic anti‐Toll‐like receptor 4 monoclonal antibody as an effective adjuvant for cancer immunotherapy

doi: 10.1111/imm.13095

Figure Lengend Snippet: The anti‐Toll‐like receptor 4 (TLR4) agonistic monoclonal antibody (mAb) suppresses tumour growth. C57BL/6N mice (n = 4–6 per group) were inoculated s.c. on the back with (a–c) EG7‐OVA, (d) EL4, (e) B16F10‐cOVA or (f) B16F10 (3 × 105); (a) 21 and 7 days prior to or (b–f) 1 day after inoculation, they were injected s.c. with vehicle (phosphate‐buffered saline; PBS), ovalbumin (OVA; 100 μg), the anti‐TLR4 mAb (3 μg), or the indicated combinations. Tumour volumes are shown as means ± SEMs. Two‐way ANOVA with the Sidak (a, c, d, f) or Tukey (b, e) post hoc test; *P < 0·05, **P < 0·01, ***P < 0·01, ****P < 0·0001 (versus PBS); †† P < 0·01, ††† P < 0·001, †††† P < 0·0001 (versus OVA). Data are representative of two or three independent experiments.

Article Snippet: The mouse anti‐mouse TLR4/MD‐2 agonistic mAb (UT12) 38 , 39 was purified from conditioned serum‐free medium (Hybridoma‐SFM; Thermo Fisher Scientific, Waltham, MA) used to culture hybridomas.

Techniques: Injection

Journal: Immunology

Article Title: An agonistic anti‐Toll‐like receptor 4 monoclonal antibody as an effective adjuvant for cancer immunotherapy

doi: 10.1111/imm.13095

Figure Lengend Snippet: The antitumour effect of the anti‐Toll‐like receptor 4 (TLR4) monoclonal antibody (mAb) is mediated by CD8 T‐cells in vivo. C57BL/6N mice (n = 5 per group) were inoculated s.c. on the back with EG7‐OVA (3 × 105), and on the following day they were injected s.c. with vehicle (phosphate‐buffered saline; PBS) or the combination of ovalbumin (OVA; 100 μg) and the anti‐TLR4 mAb (3 μg). In mice immunized with OVA and the anti‐TLR4 mAb, an anti‐CD4 (GK1·5), ‐CD8 (YTS169·4·2·1) or rat isotype control mAb (100 μg) was injected i.v. on day −2 and i.p. on days −1 and 6. (a) On day 6, the percentages of CD4 and CD8 T‐cells in the peripheral blood were analysed by FACS and are shown as the means ± SEMs of five mice per group. One‐way ANOVA with the Tukey post hoc test; **P < 0·01, ***P < 0·001, ****P < 0·0001. (b) Tumour volumes are shown as means ± SEMs. Two‐way ANOVA with the Tukey post hoc test; ****P < 0·0001 (versus PBS). Data are representative of two independent experiments.

Article Snippet: The mouse anti‐mouse TLR4/MD‐2 agonistic mAb (UT12) 38 , 39 was purified from conditioned serum‐free medium (Hybridoma‐SFM; Thermo Fisher Scientific, Waltham, MA) used to culture hybridomas.

Techniques: In Vivo, Injection

Journal: Immunology

Article Title: An agonistic anti‐Toll‐like receptor 4 monoclonal antibody as an effective adjuvant for cancer immunotherapy

doi: 10.1111/imm.13095

Figure Lengend Snippet: The anti‐Toll‐like receptor 4 (TLR4) agonistic monoclonal antibody (mAb) enhances the therapeutic efficacy of anti‐PD‐1 mAb. C57BL/6N mice (n = 5 per group) were inoculated s.c. on the back with MC38‐OVA (5 × 105). One day after inoculation, the mice were injected s.c. with vehicle (phosphate‐buffered saline; PBS) or the combination of ovalbumin (OVA; 100 μg) and the anti‐TLR4 mAb (3 μg), followed by i.p. injection of the anti‐PD‐1 mAb (100 μg) or vehicle on days 6 and 9. Tumour volumes are shown as means ± SEMs. Two‐way ANOVA with the Tukey post hoc test; **P < 0·01, ****P < 0·0001. Data are representative of three independent experiments.

Article Snippet: The mouse anti‐mouse TLR4/MD‐2 agonistic mAb (UT12) 38 , 39 was purified from conditioned serum‐free medium (Hybridoma‐SFM; Thermo Fisher Scientific, Waltham, MA) used to culture hybridomas.

Techniques: Injection

Journal: Immunology

Article Title: An agonistic anti‐Toll‐like receptor 4 monoclonal antibody as an effective adjuvant for cancer immunotherapy

doi: 10.1111/imm.13095

Figure Lengend Snippet: The agonistic anti‐Toll‐like receptor 4 (TLR4) monoclonal antibody (mAb) induces an inflammatory reaction of lesser magnitude than does lipopolysaccharide (LPS). (a) Mouse TLR4/MD‐2‐expressing Ba/F3‐transfected cells carrying NF‐κB‐responsive luciferase reporter genes were stimulated with the anti‐TLR4 mAb or LPS at the indicated concentrations for 5–7 hr. Luciferase activities are shown as mean ± SD fold increases of triplicate cultures compared with non‐stimulated cells. One‐way ANOVA with the Tukey post hoc test; *P < 0·05, **P < 0·01, ***P < 0·001, ****P < 0·0001 (versus the control). Data are representative of three independent experiments. (b, c) Bone marrow‐derived macrophages (BMMs) were stimulated with the anti‐TLR4 mAb or LPS at the indicated concentrations for 24 hr. The concentrations of (b) TNF‐α and (c) IL‐6 in the culture supernatants were evaluated by ELISA. One‐way ANOVA with the Tukey post hoc test; **P < 0·01, ***P < 0·001, ****P < 0·0001 (versus the control). Data are means ± SDs of triplicate cultures and are representative of three independent experiments. (d, e) The mice (n = 3 per group) were administered the anti‐TLR4 mAb or LPS i.p. One or three hours later, plasma was collected, and the concentrations of (d) TNF‐α and (e) IL‐6 were determined by ELISA. Two‐way ANOVA with the Tukey post hoc test; ****P < 0·0001 (versus the anti‐TLR4 mAb). Data are means ± SEMs of two independent experiments.

Article Snippet: The mouse anti‐mouse TLR4/MD‐2 agonistic mAb (UT12) 38 , 39 was purified from conditioned serum‐free medium (Hybridoma‐SFM; Thermo Fisher Scientific, Waltham, MA) used to culture hybridomas.

Techniques: Expressing, Transfection, Luciferase, Derivative Assay, Enzyme-linked Immunosorbent Assay

Journal: Immunology

Article Title: An agonistic anti‐Toll‐like receptor 4 monoclonal antibody as an effective adjuvant for cancer immunotherapy

doi: 10.1111/imm.13095

Figure Lengend Snippet: The antitumour effect of the anti‐Toll‐like receptor 4 (TLR4) monoclonal antibody (mAb) is more potent than that of lipopolysaccharide (LPS). 5‐Carboxyfluorescein diacetate succinimidyl ester (CFSE)‐labelled Ly5·1+ (a) OT‐I or (b) OT‐II spleen cells were adoptively transferred into C57BL/6N (Ly5·2+) mice (n = 3 per group). On the following day, the mice were immunized with the anti‐TLR4 mAb or LPS at the indicated doses with (a) 100 μg or (b) 10 μg ovalbumin (OVA). (a) Two or (b) three days after immunization, spleen cells were subjected to FACS analysis to evaluate the proliferation of transferred OT‐I and OT‐II T‐cells. Representative histograms and dot plots are shown. The absolute numbers of (a) Ly5·1+ CD8+ OT‐I and (B) Ly5·1+ CD4+ OT‐II T‐cells in the spleen, and the percentages of (a) CFSE low Ly5·1+ CD8+ OT‐I and (B) Ly5·1+ CD4+ OT‐I) T‐cells are shown as means ± SEMs. One‐way ANOVA with the Tukey post hoc test; *P < 0·05, ***P < 0·001, ****P < 0·0001. Data are representative of two independent experiments. (c) C57BL/6N mice (n = 5 per group) were inoculated s.c. on the back with EG7‐OVA (3 × 105 cells), and the following day they were injected s.c. with vehicle (phosphate‐buffered saline; PBS), the anti‐TLR4 mAb (3 μg), or LPS (0·1 μg) with OVA (100 μg). Tumour volumes are shown as means ± SEMs. Two‐way ANOVA with the Tukey post hoc test; *P < 0·05, ***P < 0·001 (versus OVA); † P < 0·05 (versus OVA/LPS). Data are representative of two independent experiments.

Article Snippet: The mouse anti‐mouse TLR4/MD‐2 agonistic mAb (UT12) 38 , 39 was purified from conditioned serum‐free medium (Hybridoma‐SFM; Thermo Fisher Scientific, Waltham, MA) used to culture hybridomas.

Techniques: Injection