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Induction of lymphocyte apoptosis by tumor cell secretion of FasL-bearing microvesicles.

Andreola G, Rivoltini L, Castelli C, Huber V, Perego P, Deho P, Squarcina P, Accornero P, Lozupone F, Lugini L, Stringaro A, Molinari A, Arancia G, Gentile M, Parmiani G, Fais S - J. Exp. Med. (2002)

Bottom Line: In these structures FasL colocalizes with both melanosomal (i.e., gp100) and lysosomal (i.e., CD63) antigens.We additionally show that melanosome-containing multivesicular bodies degranulate extracellularly and release FasL-bearing microvesicles, that coexpress both gp100 and CD63 and retain their functional activity in triggering Fas-dependent apoptosis of lymphoid cells.Such vesicles may form a sort of front line hindering lymphocytes and other immunocompetent cells from entering neoplastic lesions and exert their antitumor activity.

View Article: PubMed Central - PubMed

Affiliation: Unit of Immunotherapy of Human Tumors, Istituto Nazionale dei Tumori, Milan 20133, Italy.

ABSTRACT
The hypothesis that FasL expression by tumor cells may impair the in vivo efficacy of antitumor immune responses, through a mechanism known as 'Fas tumor counterattack,' has been recently questioned, becoming the object of an intense debate based on conflicting results. Here we definitely show that FasL is indeed detectable in the cytoplasm of melanoma cells and its expression is confined to multivesicular bodies that contain melanosomes. In these structures FasL colocalizes with both melanosomal (i.e., gp100) and lysosomal (i.e., CD63) antigens. Isolated melanosomes express FasL, as detected by Western blot and cytofluorimetry, and they can exert Fas-mediated apoptosis in Jurkat cells. We additionally show that melanosome-containing multivesicular bodies degranulate extracellularly and release FasL-bearing microvesicles, that coexpress both gp100 and CD63 and retain their functional activity in triggering Fas-dependent apoptosis of lymphoid cells. Hence our data provide evidence for a novel mechanism potentially operating in Fas tumor counterattack through the secretion of subcellular particles expressing functional FasL. Such vesicles may form a sort of front line hindering lymphocytes and other immunocompetent cells from entering neoplastic lesions and exert their antitumor activity.

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FasL expression on MVs purified from melanoma cell supernatant. (A) Western blot analysis. Analysis of MVs isolated from melanoma cell supernatant by serial ultracentrifugations, as performed by Western blot and staining with G247 mAb. An ∼35-kD band can be observed in MVs from melanoma lines (lanes 4, 6, and 7), resembling the one observed in MVs from PHA-activated Jurkat cells (lane 2). Only MV from PHA-activated, and not from resting Jurkat cells (lane 3) showed positivity for FasL, suggesting that this molecule unlikely derived from FBS used for cell culture. (B) Purity controls of microvesicle preparations. Controls performed to check purity of microvesicle preparations from melanoma cell supernatant revealed the expression of the melanosomal marker gp100, and no significant contamination with Golgi, ER, or mitochondria. (C) Cytofluorimetric analysis of FasL expression on purified MVs. MVs from 501mel line were stained with anti-FasL NOK-1, anti-gp100, anti–LAMP-2, and anti-CD63 mAbs, followed by incubation with FITC goat anti–mouse IgG (dark areas); an irrelevant isotype-matched Ab plus the FITC-conjugated goat anti–mouse IgG were used as negative control (clear areas). Fluorescence was analyzed by FACSCalibur™ and CELLQuest™ software. MVs were detected as granules with a size range of 100–600 nm, relative to standard beads of 6 μm size.
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fig9: FasL expression on MVs purified from melanoma cell supernatant. (A) Western blot analysis. Analysis of MVs isolated from melanoma cell supernatant by serial ultracentrifugations, as performed by Western blot and staining with G247 mAb. An ∼35-kD band can be observed in MVs from melanoma lines (lanes 4, 6, and 7), resembling the one observed in MVs from PHA-activated Jurkat cells (lane 2). Only MV from PHA-activated, and not from resting Jurkat cells (lane 3) showed positivity for FasL, suggesting that this molecule unlikely derived from FBS used for cell culture. (B) Purity controls of microvesicle preparations. Controls performed to check purity of microvesicle preparations from melanoma cell supernatant revealed the expression of the melanosomal marker gp100, and no significant contamination with Golgi, ER, or mitochondria. (C) Cytofluorimetric analysis of FasL expression on purified MVs. MVs from 501mel line were stained with anti-FasL NOK-1, anti-gp100, anti–LAMP-2, and anti-CD63 mAbs, followed by incubation with FITC goat anti–mouse IgG (dark areas); an irrelevant isotype-matched Ab plus the FITC-conjugated goat anti–mouse IgG were used as negative control (clear areas). Fluorescence was analyzed by FACSCalibur™ and CELLQuest™ software. MVs were detected as granules with a size range of 100–600 nm, relative to standard beads of 6 μm size.

Mentions: Analysis of MVs isolated from melanoma cell supernatant, as performed by Western blot, showed that FasL was indeed detectable (Fig. 9 A, lanes 4, 6, and 7) in such preparations, with an ∼35-kD band, highly resembling the one observed in MVs from PHA-activated Jurkat cells (Fig. 9 A, lane 2).


Induction of lymphocyte apoptosis by tumor cell secretion of FasL-bearing microvesicles.

Andreola G, Rivoltini L, Castelli C, Huber V, Perego P, Deho P, Squarcina P, Accornero P, Lozupone F, Lugini L, Stringaro A, Molinari A, Arancia G, Gentile M, Parmiani G, Fais S - J. Exp. Med. (2002)

FasL expression on MVs purified from melanoma cell supernatant. (A) Western blot analysis. Analysis of MVs isolated from melanoma cell supernatant by serial ultracentrifugations, as performed by Western blot and staining with G247 mAb. An ∼35-kD band can be observed in MVs from melanoma lines (lanes 4, 6, and 7), resembling the one observed in MVs from PHA-activated Jurkat cells (lane 2). Only MV from PHA-activated, and not from resting Jurkat cells (lane 3) showed positivity for FasL, suggesting that this molecule unlikely derived from FBS used for cell culture. (B) Purity controls of microvesicle preparations. Controls performed to check purity of microvesicle preparations from melanoma cell supernatant revealed the expression of the melanosomal marker gp100, and no significant contamination with Golgi, ER, or mitochondria. (C) Cytofluorimetric analysis of FasL expression on purified MVs. MVs from 501mel line were stained with anti-FasL NOK-1, anti-gp100, anti–LAMP-2, and anti-CD63 mAbs, followed by incubation with FITC goat anti–mouse IgG (dark areas); an irrelevant isotype-matched Ab plus the FITC-conjugated goat anti–mouse IgG were used as negative control (clear areas). Fluorescence was analyzed by FACSCalibur™ and CELLQuest™ software. MVs were detected as granules with a size range of 100–600 nm, relative to standard beads of 6 μm size.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2193755&req=5

fig9: FasL expression on MVs purified from melanoma cell supernatant. (A) Western blot analysis. Analysis of MVs isolated from melanoma cell supernatant by serial ultracentrifugations, as performed by Western blot and staining with G247 mAb. An ∼35-kD band can be observed in MVs from melanoma lines (lanes 4, 6, and 7), resembling the one observed in MVs from PHA-activated Jurkat cells (lane 2). Only MV from PHA-activated, and not from resting Jurkat cells (lane 3) showed positivity for FasL, suggesting that this molecule unlikely derived from FBS used for cell culture. (B) Purity controls of microvesicle preparations. Controls performed to check purity of microvesicle preparations from melanoma cell supernatant revealed the expression of the melanosomal marker gp100, and no significant contamination with Golgi, ER, or mitochondria. (C) Cytofluorimetric analysis of FasL expression on purified MVs. MVs from 501mel line were stained with anti-FasL NOK-1, anti-gp100, anti–LAMP-2, and anti-CD63 mAbs, followed by incubation with FITC goat anti–mouse IgG (dark areas); an irrelevant isotype-matched Ab plus the FITC-conjugated goat anti–mouse IgG were used as negative control (clear areas). Fluorescence was analyzed by FACSCalibur™ and CELLQuest™ software. MVs were detected as granules with a size range of 100–600 nm, relative to standard beads of 6 μm size.
Mentions: Analysis of MVs isolated from melanoma cell supernatant, as performed by Western blot, showed that FasL was indeed detectable (Fig. 9 A, lanes 4, 6, and 7) in such preparations, with an ∼35-kD band, highly resembling the one observed in MVs from PHA-activated Jurkat cells (Fig. 9 A, lane 2).

Bottom Line: In these structures FasL colocalizes with both melanosomal (i.e., gp100) and lysosomal (i.e., CD63) antigens.We additionally show that melanosome-containing multivesicular bodies degranulate extracellularly and release FasL-bearing microvesicles, that coexpress both gp100 and CD63 and retain their functional activity in triggering Fas-dependent apoptosis of lymphoid cells.Such vesicles may form a sort of front line hindering lymphocytes and other immunocompetent cells from entering neoplastic lesions and exert their antitumor activity.

View Article: PubMed Central - PubMed

Affiliation: Unit of Immunotherapy of Human Tumors, Istituto Nazionale dei Tumori, Milan 20133, Italy.

ABSTRACT
The hypothesis that FasL expression by tumor cells may impair the in vivo efficacy of antitumor immune responses, through a mechanism known as 'Fas tumor counterattack,' has been recently questioned, becoming the object of an intense debate based on conflicting results. Here we definitely show that FasL is indeed detectable in the cytoplasm of melanoma cells and its expression is confined to multivesicular bodies that contain melanosomes. In these structures FasL colocalizes with both melanosomal (i.e., gp100) and lysosomal (i.e., CD63) antigens. Isolated melanosomes express FasL, as detected by Western blot and cytofluorimetry, and they can exert Fas-mediated apoptosis in Jurkat cells. We additionally show that melanosome-containing multivesicular bodies degranulate extracellularly and release FasL-bearing microvesicles, that coexpress both gp100 and CD63 and retain their functional activity in triggering Fas-dependent apoptosis of lymphoid cells. Hence our data provide evidence for a novel mechanism potentially operating in Fas tumor counterattack through the secretion of subcellular particles expressing functional FasL. Such vesicles may form a sort of front line hindering lymphocytes and other immunocompetent cells from entering neoplastic lesions and exert their antitumor activity.

Show MeSH
Related in: MedlinePlus