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Immune evasion by murine melanoma mediated through CC chemokine receptor-10.

Murakami T, Cardones AR, Finkelstein SE, Restifo NP, Klaunberg BA, Nestle FO, Castillo SS, Dennis PA, Hwang ST - J. Exp. Med. (2003)

Bottom Line: Compared with B16 murine melanoma, cells rendered more immunogenic via overexpression of luciferase, B16 cells that overexpressed both luciferase and CCR10 resisted host immune responses and readily formed tumors.In vivo, cutaneous injection of neutralizing antibodies to endogenous CCL27 blocked growth of CCR10-expressing melanoma cells.We propose that CCR10 engagement by locally produced CCL27 allows melanoma cells to escape host immune antitumor killing mechanisms (possibly through activation of PI3K/Akt), thereby providing a means for tumor progression.

View Article: PubMed Central - PubMed

Affiliation: Dermatology Branch, CCR, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA.

ABSTRACT
Human melanoma cells frequently express CC chemokine receptor (CCR)10, a receptor whose ligand (CCL27) is constitutively produced by keratinocytes. Compared with B16 murine melanoma, cells rendered more immunogenic via overexpression of luciferase, B16 cells that overexpressed both luciferase and CCR10 resisted host immune responses and readily formed tumors. In vitro, exposure of tumor cells to CCL27 led to rapid activation of Akt, resistance to cell death induced by melanoma antigen-specific cytotoxic T cells, and phosphatidylinositol-3-kinase (PI3K)-dependent protection from apoptosis induced by Fas cross-linking. In vivo, cutaneous injection of neutralizing antibodies to endogenous CCL27 blocked growth of CCR10-expressing melanoma cells. We propose that CCR10 engagement by locally produced CCL27 allows melanoma cells to escape host immune antitumor killing mechanisms (possibly through activation of PI3K/Akt), thereby providing a means for tumor progression.

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Involvement of PI3K and Akt in CCR10-mediated protection from apoptosis. (A) CCR10-B16 cells were exposed to PBS or CCL27 (1 μg/ml) for the indicated time in the presence of cell signaling inhibitors (wortmannin (200 nM, WM), LY294002 (10 μM, LY), pertussis toxin (200 ng/ml, PTX), and PD98059 (20 μM, PD), lysed, and analyzed by Western blot for p-Akt and total Akt. (B) 14 d after inoculation into the dermis, CCR10-B16 and residual pLNCX2 tumors in the ears were probed for p-Akt expression by IHC. (C) CCR10-B16 cells that had been pretreated for 18 h with the PI3K inhibitor, LY294002 (10 μM), PTX (200 ng/ml), or DMSO alone were exposed to multimerized FasL in the presence and absence of CCL27 (1 μg/ml) for 16 h at 37°C. As a negative control, cells were exposed to the anti-FLAG epitope antibody without prior exposure to FasL (α-FLAG). One of three experiments with similar results.
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fig5: Involvement of PI3K and Akt in CCR10-mediated protection from apoptosis. (A) CCR10-B16 cells were exposed to PBS or CCL27 (1 μg/ml) for the indicated time in the presence of cell signaling inhibitors (wortmannin (200 nM, WM), LY294002 (10 μM, LY), pertussis toxin (200 ng/ml, PTX), and PD98059 (20 μM, PD), lysed, and analyzed by Western blot for p-Akt and total Akt. (B) 14 d after inoculation into the dermis, CCR10-B16 and residual pLNCX2 tumors in the ears were probed for p-Akt expression by IHC. (C) CCR10-B16 cells that had been pretreated for 18 h with the PI3K inhibitor, LY294002 (10 μM), PTX (200 ng/ml), or DMSO alone were exposed to multimerized FasL in the presence and absence of CCL27 (1 μg/ml) for 16 h at 37°C. As a negative control, cells were exposed to the anti-FLAG epitope antibody without prior exposure to FasL (α-FLAG). One of three experiments with similar results.

Mentions: To increase Fas expression, CCR10-B16 cells were treated with recombinant murine IFN-γ (50 ng/ml; Peprotech) for 12–16 h in DME/0.5% FCS. To confirm Fas expression, cells were harvested and washed, and then stained with PE-conjugated anti–mouse Fas mAb (BD Biosciences) (see Fig. 5 A) for flow cytometric analysis using FloJo analysis software (Tree Star). For induction of apoptosis, IFN-γ–treated CCR10-B16 cells were exposed to 10 ng/ml recombinant human FLAG-tagged FasL (Apotech) in combination with 1 μg/ml anti-FLAG M2 mAb (Sigma-Aldrich) for 16 h plus 1 μg/ml chemokine (when indicated) at 37°C in the presence of 0.5% FCS as described (21). VAD-fmk (CLONTECH Laboratories) was used at 1 μM as an inhibitor of Fas-mediated cell death. IFN-γ–treated CCR10-B16 cells that had not been exposed to FasL were stained with annexin V for baseline assessment of apoptosis. After exposure of B16 cells to apoptosis-inducing conditions for 16 h, attached (and detached) cells were collected from tissue culture plates for annexin V staining according to manufacturer's instructions (BD Biosciences). Analysis was performed by flow cytometry with FloJo software. P values were based on two-sided, nonparametric Student's t tests (unless otherwise specified).


Immune evasion by murine melanoma mediated through CC chemokine receptor-10.

Murakami T, Cardones AR, Finkelstein SE, Restifo NP, Klaunberg BA, Nestle FO, Castillo SS, Dennis PA, Hwang ST - J. Exp. Med. (2003)

Involvement of PI3K and Akt in CCR10-mediated protection from apoptosis. (A) CCR10-B16 cells were exposed to PBS or CCL27 (1 μg/ml) for the indicated time in the presence of cell signaling inhibitors (wortmannin (200 nM, WM), LY294002 (10 μM, LY), pertussis toxin (200 ng/ml, PTX), and PD98059 (20 μM, PD), lysed, and analyzed by Western blot for p-Akt and total Akt. (B) 14 d after inoculation into the dermis, CCR10-B16 and residual pLNCX2 tumors in the ears were probed for p-Akt expression by IHC. (C) CCR10-B16 cells that had been pretreated for 18 h with the PI3K inhibitor, LY294002 (10 μM), PTX (200 ng/ml), or DMSO alone were exposed to multimerized FasL in the presence and absence of CCL27 (1 μg/ml) for 16 h at 37°C. As a negative control, cells were exposed to the anti-FLAG epitope antibody without prior exposure to FasL (α-FLAG). One of three experiments with similar results.
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Related In: Results  -  Collection

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fig5: Involvement of PI3K and Akt in CCR10-mediated protection from apoptosis. (A) CCR10-B16 cells were exposed to PBS or CCL27 (1 μg/ml) for the indicated time in the presence of cell signaling inhibitors (wortmannin (200 nM, WM), LY294002 (10 μM, LY), pertussis toxin (200 ng/ml, PTX), and PD98059 (20 μM, PD), lysed, and analyzed by Western blot for p-Akt and total Akt. (B) 14 d after inoculation into the dermis, CCR10-B16 and residual pLNCX2 tumors in the ears were probed for p-Akt expression by IHC. (C) CCR10-B16 cells that had been pretreated for 18 h with the PI3K inhibitor, LY294002 (10 μM), PTX (200 ng/ml), or DMSO alone were exposed to multimerized FasL in the presence and absence of CCL27 (1 μg/ml) for 16 h at 37°C. As a negative control, cells were exposed to the anti-FLAG epitope antibody without prior exposure to FasL (α-FLAG). One of three experiments with similar results.
Mentions: To increase Fas expression, CCR10-B16 cells were treated with recombinant murine IFN-γ (50 ng/ml; Peprotech) for 12–16 h in DME/0.5% FCS. To confirm Fas expression, cells were harvested and washed, and then stained with PE-conjugated anti–mouse Fas mAb (BD Biosciences) (see Fig. 5 A) for flow cytometric analysis using FloJo analysis software (Tree Star). For induction of apoptosis, IFN-γ–treated CCR10-B16 cells were exposed to 10 ng/ml recombinant human FLAG-tagged FasL (Apotech) in combination with 1 μg/ml anti-FLAG M2 mAb (Sigma-Aldrich) for 16 h plus 1 μg/ml chemokine (when indicated) at 37°C in the presence of 0.5% FCS as described (21). VAD-fmk (CLONTECH Laboratories) was used at 1 μM as an inhibitor of Fas-mediated cell death. IFN-γ–treated CCR10-B16 cells that had not been exposed to FasL were stained with annexin V for baseline assessment of apoptosis. After exposure of B16 cells to apoptosis-inducing conditions for 16 h, attached (and detached) cells were collected from tissue culture plates for annexin V staining according to manufacturer's instructions (BD Biosciences). Analysis was performed by flow cytometry with FloJo software. P values were based on two-sided, nonparametric Student's t tests (unless otherwise specified).

Bottom Line: Compared with B16 murine melanoma, cells rendered more immunogenic via overexpression of luciferase, B16 cells that overexpressed both luciferase and CCR10 resisted host immune responses and readily formed tumors.In vivo, cutaneous injection of neutralizing antibodies to endogenous CCL27 blocked growth of CCR10-expressing melanoma cells.We propose that CCR10 engagement by locally produced CCL27 allows melanoma cells to escape host immune antitumor killing mechanisms (possibly through activation of PI3K/Akt), thereby providing a means for tumor progression.

View Article: PubMed Central - PubMed

Affiliation: Dermatology Branch, CCR, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA.

ABSTRACT
Human melanoma cells frequently express CC chemokine receptor (CCR)10, a receptor whose ligand (CCL27) is constitutively produced by keratinocytes. Compared with B16 murine melanoma, cells rendered more immunogenic via overexpression of luciferase, B16 cells that overexpressed both luciferase and CCR10 resisted host immune responses and readily formed tumors. In vitro, exposure of tumor cells to CCL27 led to rapid activation of Akt, resistance to cell death induced by melanoma antigen-specific cytotoxic T cells, and phosphatidylinositol-3-kinase (PI3K)-dependent protection from apoptosis induced by Fas cross-linking. In vivo, cutaneous injection of neutralizing antibodies to endogenous CCL27 blocked growth of CCR10-expressing melanoma cells. We propose that CCR10 engagement by locally produced CCL27 allows melanoma cells to escape host immune antitumor killing mechanisms (possibly through activation of PI3K/Akt), thereby providing a means for tumor progression.

Show MeSH
Related in: MedlinePlus