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

<div><p>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 (10-20μg/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.</p></div

Involvement of mitochondria in GBM ganglioside mediated caspase activation.

<p>T cells were co-cultured with GBM derived gangliosides CCF52 and CCF4, and t-Bid induction was detected as shown in Fig 3A. Reduced fluorescence of DiOC₆ in T cells treated with U87 gangliosides (15μg/ml) for 48hrs, versus control cells is indicative of mitochondrial damage (Fig 3B). Evidence of mitochondrial damage was also observed in T cells exposed to GBM derived gangliosides as evidenced from mitochondrial cytochrome c release (Fig 3C). Time dependent induction of reactive oxygen species (ROS) was measured in purified T cells treated with 15μg/ml CCF52 and CCF4 derived gangliosides for 18hrs and 48hrs, as compared to media control for 48hrs, evidenced by H₂DCFDA staining and flow cytometric analysis (Fig 3D) (*p<0.05 vs Media, **p<0.01 vs Media, ***p<0.01 vs Media). ROS production is represented as the mean fluorescence intensity (MFI) of at least 3 independent experiments.</p

Human apoptosis proteome profiler array demonstrates ganglioside induced activation of pro-apoptotic and downregulation of anti-apoptotic proteins.

<p>Differential expression of pro- and anti-apoptotic proteins were examined in T cells cultured with CCF52 ganglioside (15μg/ml) for 48hrs using a human proteome profiler array kit (R&D Biosystems) as represented in Fig 4A and 4B. Fig 4C shows the entire apoptosis proteome profile array of T cells in presence or absence of CCF52 ganglioside. Data is representative of a single experiment out of two experiments done.</p

TALEN mediated targeted disruption of TNFR1 gene abolished the ganglioside GM2–TNFR1 interaction in Jurkat-T cells.

<p>Fig 7A shows schematic representation of TNFR1 specific TALEN pair. DNA sequence with black letters indicates TALEN target sequence against which TALEN pair has been designed, red letters represent spacer DNA sequences. TALEN modules are represented as yellow, red, green or blue boxes according to their base recognition specificity of A, T, G or C respectively. Large red box with overhanging 3 arrows indicates wild type Fok1 nuclease domain, shown in Fig 7A. Western immunoblotting was performed to check the expression level of TNFR1 transfected, G418 selected Jurkat-T cells vs wild type Jurkat-T cells. β-actin was used as loading control as shown in Fig 7B. Jurkat-T cells treated with GM2 or not for 10hrs were attached in poly-L-lysine coated coverslips and stained with GM2 specific and TNFR1 specific antibodies, counterstained with respective fluorescent tagged secondary antibodies and mounted on slides with Vectashield mounting media containing DAPI and assessed for co-localization of TNFR1 and GM2 under confocal microscope (Fig 7C). Both wild type and TNFR1 KO cells were treated with GM2 of not for 10hrs and processed as described above and visualized under confocal microscope as shown in Fig 7D. Scale bar represents 10μm.</p

Induction of T cell apoptosis by GBM derived gangliosides is mediated through caspase activation.

<p>Lysates from T cells treated with GBM derived gangliosides for 72hrs were resolved in a 12% SDS-PAGE and western immunoblot was performed to detect expression of caspases, as shown in Fig 2A. Fig 2B shows graphical representation demonstrating time dependent induction of caspases in purified T cells in response to CCF52 gangliosides for 18hrs, 48hrs & 72hrs as measured by staining the cells with fluorochrome labeled inhibitors of caspases (FLICA) (*p<0.05 vs Media, **p<0.01 vs Media, ***p<0.001 vs Media). Cells were acquired on a FACS-calibur multivariable flow cytometer and % caspase +ve T cells were analyzed using CellQuest 3.3 software. Representative density plot showing time dependent induction of caspases in T cells is shown in Fig 2C. Pre-treatment of T cells with inhibitors of caspases-3, -8, -9 (at 50μM each) and a pan caspase inhibitor (at 12.5μM) 2hrs prior to ganglioside (15μg/ml) treatment, significantly protected T cells from CCF52 ganglioside induced T cell death as evident by trypan blue exclusion in Fig 2D (*p<0.05 vs Media; **p<0.01 vs CCF52 ganglioside-15μg/ml; ***p<0.001 vs CCF52 ganglioside-15μg/ml), and by microscopic analysis of nuclear blebbing in Fig 2D (**p<0.01 vs Media; **p<0.01 vs CCF52 ganglioside-15μg/ml) as shown in Fig 2D. Data represents mean of at least 3 independent experiments unless mentioned otherwise.</p

Human apoptosis proteome profiler array demonstrates ganglioside induced activation of pro-apoptotic and downregulation of anti-apoptotic proteins.

<p>Differential expression of pro- and anti-apoptotic proteins were examined in T cells cultured with CCF52 ganglioside (15μg/ml) for 48hrs using a human proteome profiler array kit (R&D Biosystems) as represented in Fig 4A and 4B. Fig 4C shows the entire apoptosis proteome profile array of T cells in presence or absence of CCF52 ganglioside. Data is representative of a single experiment out of two experiments done.</p

GBM derived gangliosides induce apoptosis of T cells.

<p>Peripheral blood T lymphocytes were isolated and purified from blood of healthy volunteers by negative selection. T lymphocytes were co-cultured with 15μg/ml of CCF52 derived gangliosides for 18–72 hrs, followed by annexinV-PE/7AAD staining for flow cytometric estimation of apoptosis, as shown in Fig 1A (*p<0.05 vs Media, ***p<0.001 vs Media). Purified T cells were incubated with different concentration (1–10μg/ml) of CCF52 gangliosides for 48hrs and 72hrs and apoptosis was measured by flow cytometric analysis using annexinV-PE/7AAD staining as indicated in Fig 1B (*p<0.05 vs Media, ***p<0.001 vs Media). Fig 1C shows graphical representation showing the percentage of nuclear blebbing event in T cells treated with gangliosides (15μg/ml) isolated from different glioblastoma cell lines for 72hrs (*p<0.05 vs Media, ***p<0.001 vs Media). Fig 1D shows representative photomicrograph demonstrating induction of apoptosis in T cells following 72hrs treatment with three different GBM derived gangliosides (15μg/ml) as evidenced by microscopic evaluation of nuclear blebbing by staining with DAPI. Data represents mean of at least 3 independent experiments unless mentioned otherwise.</p

GBM derived gangliosides interact with TNFR1 to induce recruitment of FADD and TRADD leading eventually to downstream activation of caspases.

<p>Differential profiles of ganglioside isolates from GBM cell lines CCF52 and CCF4 were obtained by HPTLC using procedure described earlier, as shown in Fig 6A. ELISA assay was also used to detect GM2 and GD1a in extracted gangliosides from CCF52 cell line (Fig 6B). Wells of a 96 well ELISA plate were coated with varying concentrations of CCF52 gangliosides as indicated in the figure legend (Fig 6B). Ganglioside GM2 shed by tumors are taken up by T cells (around 35% GM2-+ve T cells) when cultured with CCF52 supernatants as shown in Fig 6C. Co-localization of GM2 (green) and TNFR1 (red) in T cells treated with CCF52 ganglioside (15μg/ml) for 24hrs was demonstrated from overlay by confocal microscopy (Fig 6D). T cells from normal volunteers were co-cultured with CCF52 gangliosides for 6, 12, 24 and 48hrs. Cells were washed, lysed and co-immunoprecipitated with anti-human TNFR1 Ab. Western immunoblot analysis confirmed that TRADD and FADD co-immunoprecipitated with TNFR1 (Fig 6E), indicating recruitment of the DISC. Representative data from one of 3 experiments is shown.</p