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Preparation of CD4+ T Cells for Analysis of GD3 and GD2 Ganglioside Membrane Expression by Microscopy
Published on: November 8, 2016
GBM Derived Gangliosides Induce T Cell Apoptosis through Activation of the Caspase Cascade Involving Both the
Barun Mahata1, Soumika Biswas2, Patricia Rayman2
1Division 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.
Insights
Glioblastoma-derived gangliosides trigger T cell death by directly engaging the TNF receptor, activating caspase cascades. This interaction initiates apoptosis through both extrinsic and intrinsic pathways, highlighting a novel mechanism in cancer immune evasion.
Area of Science:
- Immunology
- Cancer Biology
- Cellular Biology
Background:
- Human glioblastoma (GBM) cell lines can induce apoptosis in peripheral blood T cells.
- Secreted gangliosides play a partial role in this T cell apoptosis.
- The precise molecular mechanisms by which GBM gangliosides induce T cell apoptosis remain incompletely understood.
Purpose of the Study:
- To elucidate the specific mechanisms by which glioblastoma (GBM)-derived gangliosides induce apoptosis in T cells.
- To investigate the role of the TNF receptor and caspase cascade in GBM-ganglioside-mediated T cell death.
- To determine if GBM gangliosides directly interact with the TNF receptor.
Main Methods:
- Culturing T lymphocytes with GBM cell line-derived gangliosides.
- Assessing reactive oxygen species (ROS) production using H2DCFDA.
- Western blotting to detect mitochondrial damage (Bid cleavage, cytochrome-c release).
- Apoptosis assessment via nuclear blebbing, trypan blue positivity, and AnnexinV/7AAD staining.
- Measuring caspase activation (caspase-3, -8, -9) and assessing inhibitor efficacy.
- Evaluating the role of TNF-α using anti-human TNFα antibody.
- Confocal microscopy to visualize ganglioside-TNF receptor co-localization.
- Co-immunoprecipitation to identify interacting death domain proteins (FADD, TRADD).
- Confocal microscopy on wild-type and TNFR1 knockout Jurkat cells.
Main Results:
- GBM-derived gangliosides increased ROS production and induced mitochondrial damage (Bid cleavage, cytochrome-c release) in T cells.
- Apoptosis was confirmed within 48-72 hours by nuclear blebbing, trypan blue positivity, and AnnexinV/7AAD staining.
- GBM-gangliosides activated effector caspase-3 and initiator caspases -8 and -9, with inhibitors providing 60% protection.
- Anti-TNFα antibody did not prevent ganglioside-induced T cell apoptosis, ruling out TNF-α production as the mediator.
- Confocal microscopy and co-immunoprecipitation revealed co-localization of GM2 ganglioside with the TNF receptor and recruitment of FADD and TRADD.
- Experiments with TNFR1 knockout cells confirmed the specific interaction between GM2 and TNFR1.
Conclusions:
- GBM-derived gangliosides induce T cell apoptosis via direct interaction with the TNF receptor.
- This interaction activates both the extrinsic (caspase-8) and intrinsic (caspase-9) caspase pathways, leading to effector caspase-3 activation.
- GBM gangliosides represent a novel mechanism for glioblastoma to evade T cell-mediated immune responses by inducing T cell apoptosis through the TNF receptor pathway.
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