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GBM Derived Gangliosides Induce T Cell Apoptosis through Activation of the Caspase Cascade Involving Both the Extrinsic and the Intrinsic Pathway.

Mahata B, Biswas S, Rayman P, Chahlavi A, Ko J, Bhattacharjee A, Li YT, Li Y, Das T, Sa G, Raychaudhuri B, Vogelbaum MA, Tannenbaum C, Finke JH, Biswas K - PLoS ONE (2015)

Bottom Line: GBM-ganglioside induced activation of the effector caspase-3 along with both initiator caspases (-9 and -8) in T cells while both the caspase-8 and -9 inhibitors were equally effective in blocking apoptosis (60% protection) confirming the role of caspases in the apoptotic process.Ganglioside-induced T cell apoptosis did not involve production of TNF-α since anti-human TNFα antibody was unable to protect T cells from nuclear blebbing and subsequent cell death.Thus, GBM-ganglioside can mediate T cell apoptosis by interacting with the TNF receptor followed by activation of both the extrinsic and the intrinsic pathway of caspases.

View Article: PubMed Central - PubMed

Affiliation: Division of Molecular Medicine, Bose Institute, Kolkata, India.

ABSTRACT
Previously we demonstrated that human glioblastoma cell lines induce apoptosis in peripheral blood T cells through partial involvement of secreted gangliosides. Here we show that GBM-derived gangliosides induce apoptosis through involvement of the TNF receptor and activation of the caspase cascade. Culturing T lymphocytes with GBM cell line derived gangliosides (10-20 μg/ml) demonstrated increased ROS production as early as 18 hrs as indicated by increased uptake of the dye H2DCFDA while western blotting demonstrated mitochondrial damage as evident by cleavage of Bid to t-Bid and by the release of cytochrome-c into the cytosol. Within 48-72 hrs apoptosis was evident by nuclear blebbing, trypan blue positivity and annexinV/7AAD staining. GBM-ganglioside induced activation of the effector caspase-3 along with both initiator caspases (-9 and -8) in T cells while both the caspase-8 and -9 inhibitors were equally effective in blocking apoptosis (60% protection) confirming the role of caspases in the apoptotic process. Ganglioside-induced T cell apoptosis did not involve production of TNF-α since anti-human TNFα antibody was unable to protect T cells from nuclear blebbing and subsequent cell death. However, confocal microscopy demonstrated co-localization of GM2 ganglioside with the TNF receptor and co-immunoprecipitation experiments showed recruitment of death domains FADD and TRADD with the TNF receptor post ganglioside treatment, suggesting direct interaction of gangliosides with the TNF receptor. Further confirmation of the interaction between GM2 and TNFR1 was obtained from confocal microscopy data with wild type and TNFR1 KO (TALEN mediated) Jurkat cells, which clearly demonstrated co-localization of GM2 and TNFR1 in the wild type cells but not in the TNFR1 KO clones. Thus, GBM-ganglioside can mediate T cell apoptosis by interacting with the TNF receptor followed by activation of both the extrinsic and the intrinsic pathway of caspases.

No MeSH data available.


Related in: MedlinePlus

T cell death mediated by GBM derived gangliosides does not involve TNF-α.T cells were treated with anti-human TNFα antibody 2hrs prior to co-culture with either CCF52 ganglioside (15μg/ml) alone or a combination of CCF52 ganglioside and recombinant human TNFα for 72hrs. T cells were then examined microscopically for nuclear blebbing (Fig 5A). While CCF52 ganglioside alone was able to induce significant T cell apoptosis (Mean = 30.21±3.27, ***p<0.001 vs Media), pre-treatment with anti-human TNFα Ab did not offer any significant protection (Mean = 28.30±4.65). Further, while recombinant human TNFα in combination with CCF52 ganglioside showed increased level of T cell apoptosis (Mean = 46.63±3.02, **p<0.01 vs CCF52 ganglioside alone), pre-treatment with anti-TNFα Ab blocked it only to the extent that was induced by exogenously added rhTNFα in combination with CCF52 ganglioside (Mean = 36.07±3.08, *p<0.05 vs CCF52 ganglioside+rhTNFα). Data is representative of at least 3 independent experiments. Data from western immunoblot analysis shows no induction of TNFα in response to CCF52 ganglioside treatment, as evidenced from the absence of any bands corresponding to human TNFα (Fig 5B).
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pone.0134425.g005: T cell death mediated by GBM derived gangliosides does not involve TNF-α.T cells were treated with anti-human TNFα antibody 2hrs prior to co-culture with either CCF52 ganglioside (15μg/ml) alone or a combination of CCF52 ganglioside and recombinant human TNFα for 72hrs. T cells were then examined microscopically for nuclear blebbing (Fig 5A). While CCF52 ganglioside alone was able to induce significant T cell apoptosis (Mean = 30.21±3.27, ***p<0.001 vs Media), pre-treatment with anti-human TNFα Ab did not offer any significant protection (Mean = 28.30±4.65). Further, while recombinant human TNFα in combination with CCF52 ganglioside showed increased level of T cell apoptosis (Mean = 46.63±3.02, **p<0.01 vs CCF52 ganglioside alone), pre-treatment with anti-TNFα Ab blocked it only to the extent that was induced by exogenously added rhTNFα in combination with CCF52 ganglioside (Mean = 36.07±3.08, *p<0.05 vs CCF52 ganglioside+rhTNFα). Data is representative of at least 3 independent experiments. Data from western immunoblot analysis shows no induction of TNFα in response to CCF52 ganglioside treatment, as evidenced from the absence of any bands corresponding to human TNFα (Fig 5B).

Mentions: We investigated the role of TNF-α in the induction of GBM derived ganglioside mediated T cell apoptosis. Since, in the tumor microenvironment, tumor infiltrating lymphocytes (TILs) encounter tumor antigens and undergo activation, we thought that it will be physiologically more relevant to study the potential role of TNF-α using 7-day activated T cells (Fig 5). Data shows that while recombinant hTNF-α alone did not induce any significant T cell apoptosis, CCF52 ganglioside alone did induce T cell death (Fig 5A). However, T cell apoptosis induced by CCF52 ganglioside does not likely involve TNF-α since, anti-human TNF-α antibody was unable to block T cell death (Fig 5A). This is similar to the results obtained in a previous report from our laboratory [29], which suggested that GBM cell line induced T cell apoptosis does not involve TNF-α. However, in the presence of TNF-α, T cells show heightened susceptibility to apoptosis (~45%) by GBM derived gangliosides (Fig 5A). This is coherent to the finding from a previous report demonstrating that gangliosides and TNF-α can synergize to induce T cell apoptosis [42]. Addition of TNF-α Ab to the above system blocks T cell death to the extent that was induced by exogenously added rhTNF-α, further ruling out the involvement of any induced endogenous TNF-α in GBM derived ganglioside mediated T cell death. These results indicate that two parallel mechanisms exist for ganglioside mediated T cell death. In the presence of TNF-α, gangliosides synergize with TNF-α to induce T cell apoptosis through involvement of the TNF receptor [42]. However, in the experiments reported here, gangliosides alone mediate T cell apoptosis in the absence of endogenous TNF-α, through involvement of both receptor dependent and receptor independent pathways of caspase activation. As seen in figure Fig 5B, ganglioside treatment did not lead to induction of any TNF-α production in T cells as demonstrated by western blot analysis. Recombinant hTNF-α was used as a positive control.


GBM Derived Gangliosides Induce T Cell Apoptosis through Activation of the Caspase Cascade Involving Both the Extrinsic and the Intrinsic Pathway.

Mahata B, Biswas S, Rayman P, Chahlavi A, Ko J, Bhattacharjee A, Li YT, Li Y, Das T, Sa G, Raychaudhuri B, Vogelbaum MA, Tannenbaum C, Finke JH, Biswas K - PLoS ONE (2015)

T cell death mediated by GBM derived gangliosides does not involve TNF-α.T cells were treated with anti-human TNFα antibody 2hrs prior to co-culture with either CCF52 ganglioside (15μg/ml) alone or a combination of CCF52 ganglioside and recombinant human TNFα for 72hrs. T cells were then examined microscopically for nuclear blebbing (Fig 5A). While CCF52 ganglioside alone was able to induce significant T cell apoptosis (Mean = 30.21±3.27, ***p<0.001 vs Media), pre-treatment with anti-human TNFα Ab did not offer any significant protection (Mean = 28.30±4.65). Further, while recombinant human TNFα in combination with CCF52 ganglioside showed increased level of T cell apoptosis (Mean = 46.63±3.02, **p<0.01 vs CCF52 ganglioside alone), pre-treatment with anti-TNFα Ab blocked it only to the extent that was induced by exogenously added rhTNFα in combination with CCF52 ganglioside (Mean = 36.07±3.08, *p<0.05 vs CCF52 ganglioside+rhTNFα). Data is representative of at least 3 independent experiments. Data from western immunoblot analysis shows no induction of TNFα in response to CCF52 ganglioside treatment, as evidenced from the absence of any bands corresponding to human TNFα (Fig 5B).
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Related In: Results  -  Collection

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pone.0134425.g005: T cell death mediated by GBM derived gangliosides does not involve TNF-α.T cells were treated with anti-human TNFα antibody 2hrs prior to co-culture with either CCF52 ganglioside (15μg/ml) alone or a combination of CCF52 ganglioside and recombinant human TNFα for 72hrs. T cells were then examined microscopically for nuclear blebbing (Fig 5A). While CCF52 ganglioside alone was able to induce significant T cell apoptosis (Mean = 30.21±3.27, ***p<0.001 vs Media), pre-treatment with anti-human TNFα Ab did not offer any significant protection (Mean = 28.30±4.65). Further, while recombinant human TNFα in combination with CCF52 ganglioside showed increased level of T cell apoptosis (Mean = 46.63±3.02, **p<0.01 vs CCF52 ganglioside alone), pre-treatment with anti-TNFα Ab blocked it only to the extent that was induced by exogenously added rhTNFα in combination with CCF52 ganglioside (Mean = 36.07±3.08, *p<0.05 vs CCF52 ganglioside+rhTNFα). Data is representative of at least 3 independent experiments. Data from western immunoblot analysis shows no induction of TNFα in response to CCF52 ganglioside treatment, as evidenced from the absence of any bands corresponding to human TNFα (Fig 5B).
Mentions: We investigated the role of TNF-α in the induction of GBM derived ganglioside mediated T cell apoptosis. Since, in the tumor microenvironment, tumor infiltrating lymphocytes (TILs) encounter tumor antigens and undergo activation, we thought that it will be physiologically more relevant to study the potential role of TNF-α using 7-day activated T cells (Fig 5). Data shows that while recombinant hTNF-α alone did not induce any significant T cell apoptosis, CCF52 ganglioside alone did induce T cell death (Fig 5A). However, T cell apoptosis induced by CCF52 ganglioside does not likely involve TNF-α since, anti-human TNF-α antibody was unable to block T cell death (Fig 5A). This is similar to the results obtained in a previous report from our laboratory [29], which suggested that GBM cell line induced T cell apoptosis does not involve TNF-α. However, in the presence of TNF-α, T cells show heightened susceptibility to apoptosis (~45%) by GBM derived gangliosides (Fig 5A). This is coherent to the finding from a previous report demonstrating that gangliosides and TNF-α can synergize to induce T cell apoptosis [42]. Addition of TNF-α Ab to the above system blocks T cell death to the extent that was induced by exogenously added rhTNF-α, further ruling out the involvement of any induced endogenous TNF-α in GBM derived ganglioside mediated T cell death. These results indicate that two parallel mechanisms exist for ganglioside mediated T cell death. In the presence of TNF-α, gangliosides synergize with TNF-α to induce T cell apoptosis through involvement of the TNF receptor [42]. However, in the experiments reported here, gangliosides alone mediate T cell apoptosis in the absence of endogenous TNF-α, through involvement of both receptor dependent and receptor independent pathways of caspase activation. As seen in figure Fig 5B, ganglioside treatment did not lead to induction of any TNF-α production in T cells as demonstrated by western blot analysis. Recombinant hTNF-α was used as a positive control.

Bottom Line: GBM-ganglioside induced activation of the effector caspase-3 along with both initiator caspases (-9 and -8) in T cells while both the caspase-8 and -9 inhibitors were equally effective in blocking apoptosis (60% protection) confirming the role of caspases in the apoptotic process.Ganglioside-induced T cell apoptosis did not involve production of TNF-α since anti-human TNFα antibody was unable to protect T cells from nuclear blebbing and subsequent cell death.Thus, GBM-ganglioside can mediate T cell apoptosis by interacting with the TNF receptor followed by activation of both the extrinsic and the intrinsic pathway of caspases.

View Article: PubMed Central - PubMed

Affiliation: Division of Molecular Medicine, Bose Institute, Kolkata, India.

ABSTRACT
Previously we demonstrated that human glioblastoma cell lines induce apoptosis in peripheral blood T cells through partial involvement of secreted gangliosides. Here we show that GBM-derived gangliosides induce apoptosis through involvement of the TNF receptor and activation of the caspase cascade. Culturing T lymphocytes with GBM cell line derived gangliosides (10-20 μg/ml) demonstrated increased ROS production as early as 18 hrs as indicated by increased uptake of the dye H2DCFDA while western blotting demonstrated mitochondrial damage as evident by cleavage of Bid to t-Bid and by the release of cytochrome-c into the cytosol. Within 48-72 hrs apoptosis was evident by nuclear blebbing, trypan blue positivity and annexinV/7AAD staining. GBM-ganglioside induced activation of the effector caspase-3 along with both initiator caspases (-9 and -8) in T cells while both the caspase-8 and -9 inhibitors were equally effective in blocking apoptosis (60% protection) confirming the role of caspases in the apoptotic process. Ganglioside-induced T cell apoptosis did not involve production of TNF-α since anti-human TNFα antibody was unable to protect T cells from nuclear blebbing and subsequent cell death. However, confocal microscopy demonstrated co-localization of GM2 ganglioside with the TNF receptor and co-immunoprecipitation experiments showed recruitment of death domains FADD and TRADD with the TNF receptor post ganglioside treatment, suggesting direct interaction of gangliosides with the TNF receptor. Further confirmation of the interaction between GM2 and TNFR1 was obtained from confocal microscopy data with wild type and TNFR1 KO (TALEN mediated) Jurkat cells, which clearly demonstrated co-localization of GM2 and TNFR1 in the wild type cells but not in the TNFR1 KO clones. Thus, GBM-ganglioside can mediate T cell apoptosis by interacting with the TNF receptor followed by activation of both the extrinsic and the intrinsic pathway of caspases.

No MeSH data available.


Related in: MedlinePlus