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Toll-like receptor 3 expressing tumor parenchyma and infiltrating natural killer cells in hepatocellular carcinoma patients.

Chew V, Tow C, Huang C, Bard-Chapeau E, Copeland NG, Jenkins NA, Weber A, Lim KH, Toh HC, Heikenwalder M, Ng IO, Nardin A, Abastado JP - J. Natl. Cancer Inst. (2012)

Bottom Line: Also, expression of chemokines or treatment with poly(I:C) decreased tumor growth.TLR3 expression was also associated with longer survival in HCC patients (hazard ratio of survival = 2.1, 95% confidence interval = 1.3 to 3.4, P = .002).TLR3 is an important modulator of HCC progression and is a potential target for novel immunotherapy.

View Article: PubMed Central - PubMed

Affiliation: Singapore Immunology Network SIgN, Agency for Science, Technology and Research A*STAR, 8A Biomedical Grove, Immunos, Biopolis, Singapore 138648, Singapore.

ABSTRACT

Background: Hepatocellular carcinoma (HCC) is a highly aggressive cancer that is linked to chronically dysregulated liver inflammation. However, appropriate immune responses can control HCC progression. Here we investigated the role and underlying mechanism of toll-like receptor 3 (TLR3) in HCC.

Methods: HCC cell death, and natural killer (NK) cell activation and cytotoxicity were assessed in vitro after treatment with the TLR3 ligand poly(I:C). The effect of TLR3 on the tumor parenchyma and infiltrating immune cells was investigated in a spontaneous liver tumor mouse model and a transplanted tumor mouse model (n = 3-9 mice per group). Immunohistochemistry and quantitative polymerase chain reaction were used to analyze tumor samples from 172 HCC patients. Paired t-tests and analysis of variance tests were used to calculate P-values. The relationship between TLR3 expression and survival was determined by the Kaplan-Meier univariate survival analysis and a log-rank test. All statistical tests were two-sided.

Results: TLR3 activation increased cell death in the TLR3(+) SNU182 HCC cell line (30.5% vs 8.5%, P = .03) and promoted NK-cell activation (32.6% vs 19.4%, P < .001) and cytotoxicity (relative fourfold increase, P = .03) in vitro. In vivo, poly(I:C) treatment increased intratumoral chemokine expression, NK-cell activation and tumor infiltration, and proliferation of tumor-infiltrating T and NK cells. Proliferation of tumor parenchyma cells was decreased. Also, expression of chemokines or treatment with poly(I:C) decreased tumor growth. TLR3 expression in patient samples correlated with NK-cell activation, NK- and T-cell tumor infiltration, and inversely correlated with tumor parenchyma cell viability. TLR3 expression was also associated with longer survival in HCC patients (hazard ratio of survival = 2.1, 95% confidence interval = 1.3 to 3.4, P = .002).

Conclusions: TLR3 is an important modulator of HCC progression and is a potential target for novel immunotherapy.

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The effect of toll-like receptor 3 (TLR3) activation on natural killer (NK)-cell activation and cytotoxicity against hepatocellular carcinoma cells in vitro. A) The percentage of activated NK cells coexpressing CD56 with the early activation marker CD69 was measured by flow cytometry 24 hours after treatment with 50 µg/mL poly(I:C). Data are shown using Tukey box-and-whiskers representation from 15 independent experiments. A paired t-test was used to calculate the two-sided P-value. B) Expression of the effector genes interferon-γ (IFNG) and granzyme-B (GZB) was determined by quantitative polymerase chain reaction 24 hours after NK cells were treated with 50 µg/mL poly(I:C). The graph shows the mean fold change relative to the untreated control from 12 independent experiments. Whisker bars indicate the SD. A Wilcoxon signed-rank test was used to calculate the two-sided P-values. C) The mean interferon-γ (IFN-γ) secretion by NK cells 24 hours after treatment with 50 µg/mL poly(I:C) was measured by enzyme-linked immunosorbent assay of the culture supernatant. The data are shown using Tukey box-and-whiskers representation from eight independent experiments. A paired t-test was used to calculate the two-sided P-value. D) Cytotoxicity of poly(I:C)-activated NK cells at various ratios of effector (E) to target cells (T, SNU182 cells) was measured by determining the percentage of specific lysis in vitro. A paired t-test was used to calculate the two-sided P-value at E:T ratio of 5:1. The graph shows the means from poly(I:C)-treated and untreated NK cells and the SD (whisker bars) from three independent experiments.
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Figure 3: The effect of toll-like receptor 3 (TLR3) activation on natural killer (NK)-cell activation and cytotoxicity against hepatocellular carcinoma cells in vitro. A) The percentage of activated NK cells coexpressing CD56 with the early activation marker CD69 was measured by flow cytometry 24 hours after treatment with 50 µg/mL poly(I:C). Data are shown using Tukey box-and-whiskers representation from 15 independent experiments. A paired t-test was used to calculate the two-sided P-value. B) Expression of the effector genes interferon-γ (IFNG) and granzyme-B (GZB) was determined by quantitative polymerase chain reaction 24 hours after NK cells were treated with 50 µg/mL poly(I:C). The graph shows the mean fold change relative to the untreated control from 12 independent experiments. Whisker bars indicate the SD. A Wilcoxon signed-rank test was used to calculate the two-sided P-values. C) The mean interferon-γ (IFN-γ) secretion by NK cells 24 hours after treatment with 50 µg/mL poly(I:C) was measured by enzyme-linked immunosorbent assay of the culture supernatant. The data are shown using Tukey box-and-whiskers representation from eight independent experiments. A paired t-test was used to calculate the two-sided P-value. D) Cytotoxicity of poly(I:C)-activated NK cells at various ratios of effector (E) to target cells (T, SNU182 cells) was measured by determining the percentage of specific lysis in vitro. A paired t-test was used to calculate the two-sided P-value at E:T ratio of 5:1. The graph shows the means from poly(I:C)-treated and untreated NK cells and the SD (whisker bars) from three independent experiments.

Mentions: We next evaluated the antitumor properties of NK cells following TLR3 activation. NK-cell treatment with poly(I:C) led to a marked increase in expression of the early activation marker CD69 [percentage of CD56+CD69+ cells relative to all live CD45+ cells after poly(I:C) treatment vs control = 32.6% vs 19.4%, P < .001] (Figure 3, A) and in expression of cytotoxic mediators, such as IFN-γ [ninefold increase in IFNG gene expression after treatment with poly(I:C), P < .001; mean secreted IFN-γ after treatment with poly(I:C) vs control = 415 vs 179 pg/mL, P = .03] (Figure 3, B and C) and granzyme-B [threefold increase in granzyme-B gene expression after treatment with poly(I:C), P < .001] (Figure 3, B). Activated NK cells displayed up to fourfold higher cytotoxic activity (E:T ratio = 5:1, P = .03) against SNU182 cells (Figure 3, D). Thus, in vitro activation of TLR3 promotes NK-cell activation, secretion of IFN-γ, and cytotoxicity against HCC cells. This result is consistent with previous studies using lymphoma target cells (19).


Toll-like receptor 3 expressing tumor parenchyma and infiltrating natural killer cells in hepatocellular carcinoma patients.

Chew V, Tow C, Huang C, Bard-Chapeau E, Copeland NG, Jenkins NA, Weber A, Lim KH, Toh HC, Heikenwalder M, Ng IO, Nardin A, Abastado JP - J. Natl. Cancer Inst. (2012)

The effect of toll-like receptor 3 (TLR3) activation on natural killer (NK)-cell activation and cytotoxicity against hepatocellular carcinoma cells in vitro. A) The percentage of activated NK cells coexpressing CD56 with the early activation marker CD69 was measured by flow cytometry 24 hours after treatment with 50 µg/mL poly(I:C). Data are shown using Tukey box-and-whiskers representation from 15 independent experiments. A paired t-test was used to calculate the two-sided P-value. B) Expression of the effector genes interferon-γ (IFNG) and granzyme-B (GZB) was determined by quantitative polymerase chain reaction 24 hours after NK cells were treated with 50 µg/mL poly(I:C). The graph shows the mean fold change relative to the untreated control from 12 independent experiments. Whisker bars indicate the SD. A Wilcoxon signed-rank test was used to calculate the two-sided P-values. C) The mean interferon-γ (IFN-γ) secretion by NK cells 24 hours after treatment with 50 µg/mL poly(I:C) was measured by enzyme-linked immunosorbent assay of the culture supernatant. The data are shown using Tukey box-and-whiskers representation from eight independent experiments. A paired t-test was used to calculate the two-sided P-value. D) Cytotoxicity of poly(I:C)-activated NK cells at various ratios of effector (E) to target cells (T, SNU182 cells) was measured by determining the percentage of specific lysis in vitro. A paired t-test was used to calculate the two-sided P-value at E:T ratio of 5:1. The graph shows the means from poly(I:C)-treated and untreated NK cells and the SD (whisker bars) from three independent experiments.
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Figure 3: The effect of toll-like receptor 3 (TLR3) activation on natural killer (NK)-cell activation and cytotoxicity against hepatocellular carcinoma cells in vitro. A) The percentage of activated NK cells coexpressing CD56 with the early activation marker CD69 was measured by flow cytometry 24 hours after treatment with 50 µg/mL poly(I:C). Data are shown using Tukey box-and-whiskers representation from 15 independent experiments. A paired t-test was used to calculate the two-sided P-value. B) Expression of the effector genes interferon-γ (IFNG) and granzyme-B (GZB) was determined by quantitative polymerase chain reaction 24 hours after NK cells were treated with 50 µg/mL poly(I:C). The graph shows the mean fold change relative to the untreated control from 12 independent experiments. Whisker bars indicate the SD. A Wilcoxon signed-rank test was used to calculate the two-sided P-values. C) The mean interferon-γ (IFN-γ) secretion by NK cells 24 hours after treatment with 50 µg/mL poly(I:C) was measured by enzyme-linked immunosorbent assay of the culture supernatant. The data are shown using Tukey box-and-whiskers representation from eight independent experiments. A paired t-test was used to calculate the two-sided P-value. D) Cytotoxicity of poly(I:C)-activated NK cells at various ratios of effector (E) to target cells (T, SNU182 cells) was measured by determining the percentage of specific lysis in vitro. A paired t-test was used to calculate the two-sided P-value at E:T ratio of 5:1. The graph shows the means from poly(I:C)-treated and untreated NK cells and the SD (whisker bars) from three independent experiments.
Mentions: We next evaluated the antitumor properties of NK cells following TLR3 activation. NK-cell treatment with poly(I:C) led to a marked increase in expression of the early activation marker CD69 [percentage of CD56+CD69+ cells relative to all live CD45+ cells after poly(I:C) treatment vs control = 32.6% vs 19.4%, P < .001] (Figure 3, A) and in expression of cytotoxic mediators, such as IFN-γ [ninefold increase in IFNG gene expression after treatment with poly(I:C), P < .001; mean secreted IFN-γ after treatment with poly(I:C) vs control = 415 vs 179 pg/mL, P = .03] (Figure 3, B and C) and granzyme-B [threefold increase in granzyme-B gene expression after treatment with poly(I:C), P < .001] (Figure 3, B). Activated NK cells displayed up to fourfold higher cytotoxic activity (E:T ratio = 5:1, P = .03) against SNU182 cells (Figure 3, D). Thus, in vitro activation of TLR3 promotes NK-cell activation, secretion of IFN-γ, and cytotoxicity against HCC cells. This result is consistent with previous studies using lymphoma target cells (19).

Bottom Line: Also, expression of chemokines or treatment with poly(I:C) decreased tumor growth.TLR3 expression was also associated with longer survival in HCC patients (hazard ratio of survival = 2.1, 95% confidence interval = 1.3 to 3.4, P = .002).TLR3 is an important modulator of HCC progression and is a potential target for novel immunotherapy.

View Article: PubMed Central - PubMed

Affiliation: Singapore Immunology Network SIgN, Agency for Science, Technology and Research A*STAR, 8A Biomedical Grove, Immunos, Biopolis, Singapore 138648, Singapore.

ABSTRACT

Background: Hepatocellular carcinoma (HCC) is a highly aggressive cancer that is linked to chronically dysregulated liver inflammation. However, appropriate immune responses can control HCC progression. Here we investigated the role and underlying mechanism of toll-like receptor 3 (TLR3) in HCC.

Methods: HCC cell death, and natural killer (NK) cell activation and cytotoxicity were assessed in vitro after treatment with the TLR3 ligand poly(I:C). The effect of TLR3 on the tumor parenchyma and infiltrating immune cells was investigated in a spontaneous liver tumor mouse model and a transplanted tumor mouse model (n = 3-9 mice per group). Immunohistochemistry and quantitative polymerase chain reaction were used to analyze tumor samples from 172 HCC patients. Paired t-tests and analysis of variance tests were used to calculate P-values. The relationship between TLR3 expression and survival was determined by the Kaplan-Meier univariate survival analysis and a log-rank test. All statistical tests were two-sided.

Results: TLR3 activation increased cell death in the TLR3(+) SNU182 HCC cell line (30.5% vs 8.5%, P = .03) and promoted NK-cell activation (32.6% vs 19.4%, P < .001) and cytotoxicity (relative fourfold increase, P = .03) in vitro. In vivo, poly(I:C) treatment increased intratumoral chemokine expression, NK-cell activation and tumor infiltration, and proliferation of tumor-infiltrating T and NK cells. Proliferation of tumor parenchyma cells was decreased. Also, expression of chemokines or treatment with poly(I:C) decreased tumor growth. TLR3 expression in patient samples correlated with NK-cell activation, NK- and T-cell tumor infiltration, and inversely correlated with tumor parenchyma cell viability. TLR3 expression was also associated with longer survival in HCC patients (hazard ratio of survival = 2.1, 95% confidence interval = 1.3 to 3.4, P = .002).

Conclusions: TLR3 is an important modulator of HCC progression and is a potential target for novel immunotherapy.

Show MeSH
Related in: MedlinePlus