A. these results show that altering the lipid composition of lipid rafts offers profound effects on T cell activation and differentiation. EXPERIMENTAL Methods Materials Anti-human LAT and anti-human Lck antibodies were purchased from Santa Cruz Biotechnology Inc. Anti-human Zap70 antibody (clone 2F3.2), anti-phosphorylated LAT (Tyr191) antibody, and anti-phosphorylated Src antibody (clone 2N8), that also recognizes phospho-Lck, were from Upstate Biotechnology Inc. Anti-phosphorylated Zap70 (Tyr292) antibody was from Sigma. Anti-GAPDH and anti-flotillin-1 antibodies were purchased from Abcam and BD Biosciences, respectively. Goat anti-rabbit IgG and anti-mouse IgG coupled with HRP were acquired from Pierce. Dynabeads coupled with anti-human CD3/CD28 or anti-mouse CD3/CD28 antibody were from Invitrogen. Soluble anti-mouse CD3? (clone 145.2C11), anti-mouse CD28 (clone 37.51), anti-human CD3? (clone OKT3), anti-CD28 (clone CD28.6), anti-CD25 (clone Personal computer.61), and anti-mouse RORt antibodies were purchased from eBioscience. All cytokines were acquired either from R&D Systems or PeproTech. Treatment of Jurkat T Cells with Genz-123346 Freshly propagated Jurkat cells were managed in RPMI 1640 medium comprising 25 mm HEPES, 1 mm sodium pyruvate, 10% FBS, 1 penicillin/streptomycin, and 50 m -mercaptoethanol at 37 C under 5% CO2. Genz-123346 ((1(16). Twelve fractions (430 l each) were collected, freezing on dry snow, and stored at ?80 C. The samples were processed for Western blot analysis, after which the blots were probed with anti-LAT, anti-phospho-LAT, or anti-flotillin-1 (a lipid raft marker) antibody. Western blot films were scanned on an HP Scanjet G3010 photo scanner, and signals of total LAT and phosphorylated LAT in fractions 2C5 (raft fractions) were quantified using NIH ImageJ software. The ratios of LAT were calculated based on the combined signals in fractions 2C5. Western Blot Analysis of TCR Signaling Molecules Frozen cell pellets were lysed in 200 l of cell lysis buffer (50 mm Tris (pH 7.5), 150 mm NaCl, 1% Triton X-100, 0.25% SDS, 1 mm EDTA, 1 mm NaF, and 5 mm sodium orthovanadate) preheated to 100 C and boiled inside a heating block at 100 C for another 5 min. Samples were sonicated briefly until no longer viscous (5 s), and the protein content was identified using the Micro BCA protein assay kit (Pierce). Approximately 25 g of total protein was subjected to electrophoresis, after which the separated proteins were transferred onto nitrocellulose membranes and probed using antibodies against Lck, Zap70, and LAT or their phosphorylated counterparts. GAPDH was used to monitor for variations in protein loading. Calcium Mobilization Assay Approximately 50 ml of Genz-123346-treated or mock-treated Jurkat T cells (total of 2 107) in medium was treated with Fura-2 acetoxymethyl ester at a final concentration of 5 m for 60 min inside a cell tradition incubator. The Fura-2 acetoxymethyl ester-treated cells were then collected by centrifugation, resuspended in 2 ml of assay buffer (140 mm NaCl, 5 mm KCl, 0. 7 mm CaCl2, 0.7 mm MgCl2, 20 mm HEPES, 10 mm glucose, and 0.1% BSA (pH 7.4)), and allowed to equilibrate for 10 min. Approximately 400 l of the labeled cells was added to a fluorometer cuvette and supplemented with CaCl2 to a final concentration of 5 mm. The base line was recorded for 1 min, after which anti-CD3/CD28 antibody-coupled Dynabeads (or additional controls) were added at a cell/bead percentage of 1 1:2, and the calcium response was recorded for up to 300 s using an RF-5301PC spectrofluorophotometer (Shimadzu Corp.). Cytoplasmic calcium levels were calculated based on the fluorescence percentage of Fura-2 acetoxymethyl ester at excitation wavelengths of 340 and 380 nm as explained previously (27). Isolation, Tradition, and Differentiation of Main T Cells Naive mouse T cells from spleens and lymph nodes of normal C57BL/6 mice and wild-type littermates of GM3 synthase knock-out (KO; Atreleuton ?/?) and heterozygous (+/?) mice on a C57BL/6-129/svev mixed background (28).Yamashita T., Hashiramoto A., Haluzik M., Mizukami H., Beck S., Norton A., Kono M., Tsuji S., Daniotti J. were acquired with CD4+ T cells isolated from transgenic mice genetically deficient in GM3 synthase activity. Interestingly, decreasing the GSL levels in CD4+ T cells by either pharmacological inhibition or disruption of the gene for GM3 synthase also specifically inhibited the differentiation of T cells to the Th17 lineage but not to additional Th subsets without influencing the additional Th subsets examined. Taken collectively, these results show that altering the lipid composition of lipid rafts offers profound effects on T cell activation and differentiation. EXPERIMENTAL Methods Materials Anti-human LAT and anti-human Lck antibodies were purchased from Santa Cruz Biotechnology Inc. Anti-human Zap70 antibody (clone 2F3.2), anti-phosphorylated LAT (Tyr191) antibody, and anti-phosphorylated Src antibody (clone 2N8), that also recognizes phospho-Lck, were from Upstate Biotechnology Inc. Anti-phosphorylated Zap70 (Tyr292) antibody was from Sigma. Anti-GAPDH and anti-flotillin-1 antibodies were purchased from Abcam and BD Biosciences, respectively. Goat anti-rabbit IgG and anti-mouse IgG coupled with HRP were acquired from Pierce. Dynabeads coupled with anti-human CD3/CD28 or anti-mouse CD3/CD28 antibody were from Invitrogen. Soluble anti-mouse CD3? (clone 145.2C11), anti-mouse CD28 (clone 37.51), anti-human CD3? (clone OKT3), anti-CD28 (clone CD28.6), anti-CD25 (clone Personal computer.61), and anti-mouse RORt antibodies were purchased from eBioscience. All cytokines were acquired either from R&D Systems or PeproTech. Treatment of Jurkat T Cells with Genz-123346 Freshly propagated Jurkat cells were managed in RPMI 1640 medium comprising 25 mm HEPES, 1 mm sodium pyruvate, 10% FBS, 1 penicillin/streptomycin, and 50 m -mercaptoethanol at 37 C under 5% CO2. Genz-123346 ((1(16). Twelve fractions (430 l each) were collected, freezing on dry snow, and stored at ?80 C. The samples were processed for Western blot analysis, after which the blots were probed with anti-LAT, anti-phospho-LAT, or anti-flotillin-1 (a lipid raft marker) antibody. Western blot films were scanned on an HP Scanjet G3010 photo scanner, and signals of total LAT and phosphorylated LAT in fractions 2C5 (raft fractions) were quantified using NIH ImageJ software. The ratios of LAT were calculated based on the combined signals in fractions 2C5. Western Blot Analysis of TCR Signaling Molecules Frozen cell pellets were lysed in 200 l of cell lysis buffer (50 mm Tris (pH 7.5), 150 mm NaCl, 1% Triton X-100, 0.25% SDS, 1 mm EDTA, 1 mm NaF, and 5 mm sodium orthovanadate) preheated to 100 C and boiled inside a heating block at 100 C for another 5 min. Samples were sonicated briefly until no longer viscous (5 s), and the protein content was identified using the Micro BCA protein assay kit (Pierce). Approximately 25 g of total protein was subjected to electrophoresis, after which the separated proteins were transferred onto nitrocellulose membranes and probed using antibodies against Lck, Zap70, and LAT or their phosphorylated counterparts. GAPDH was used to monitor for variations in protein loading. Calcium Mobilization Assay Approximately 50 ml of Genz-123346-treated or mock-treated Jurkat T cells (total of 2 107) in medium was treated with Fura-2 acetoxymethyl ester at a final concentration of 5 m for 60 min inside a cell tradition incubator. The Fura-2 acetoxymethyl ester-treated cells were then collected by centrifugation, resuspended in 2 ml of assay buffer (140 mm NaCl, 5 mm KCl, 0. 7 mm CaCl2, 0.7 mm MgCl2, 20 mm HEPES, 10 mm glucose, and 0.1% BSA (pH 7.4)), and allowed to equilibrate for 10 min. Approximately 400 l of the labeled cells was added to a fluorometer cuvette and supplemented with CaCl2 to a final concentration of 5 mm. The base line was recorded for 1 min, after which anti-CD3/CD28 antibody-coupled Dynabeads (or additional controls) were added at a cell/bead percentage of 1 1:2, and the calcium response was recorded for up to 300 s using an RF-5301PC spectrofluorophotometer (Shimadzu Corp.). Cytoplasmic calcium levels were calculated based on the fluorescence ratio of Fura-2 acetoxymethyl ester at excitation wavelengths of 340 and 380 nm as explained previously (27). Isolation, Culture, and Differentiation.Natl. were attenuated by treatment with Genz-122346. Concomitant with these events were significant reductions in IL-2 production and T cell proliferation. Similar findings were obtained with CD4+ T cells isolated from transgenic mice genetically deficient in GM3 synthase activity. Interestingly, lowering the GSL levels in CD4+ T cells by either pharmacological inhibition or disruption of the gene for GM3 synthase also specifically inhibited the differentiation of T cells to the Th17 lineage but not to other Th subsets without affecting the other Th subsets examined. Taken together, these results show that altering the lipid composition of lipid rafts has profound effects on T cell activation and differentiation. EXPERIMENTAL PROCEDURES Materials Anti-human LAT and anti-human Lck antibodies were purchased from Santa Cruz Biotechnology Inc. Anti-human Zap70 antibody (clone 2F3.2), anti-phosphorylated LAT (Tyr191) antibody, and anti-phosphorylated Src antibody (clone 2N8), that also recognizes phospho-Lck, were obtained from Upstate Biotechnology Inc. Anti-phosphorylated Zap70 (Tyr292) antibody was from Sigma. Anti-GAPDH and anti-flotillin-1 antibodies were purchased from Abcam and BD Biosciences, respectively. Goat anti-rabbit IgG and anti-mouse IgG coupled with HRP Atreleuton were acquired from Pierce. Dynabeads coupled with anti-human CD3/CD28 or anti-mouse CD3/CD28 antibody were obtained from Invitrogen. Soluble anti-mouse CD3? (clone 145.2C11), anti-mouse CD28 (clone 37.51), anti-human CD3? (clone OKT3), anti-CD28 (clone CD28.6), anti-CD25 (clone PC.61), and anti-mouse RORt antibodies were purchased from eBioscience. All cytokines were obtained either from R&D Systems or PeproTech. Treatment of Jurkat T Cells with Genz-123346 Freshly propagated Jurkat cells were managed in RPMI 1640 medium made up of 25 mm HEPES, 1 mm sodium pyruvate, 10% FBS, 1 penicillin/streptomycin, and 50 m -mercaptoethanol at 37 C under 5% CO2. Genz-123346 ((1(16). Twelve fractions (430 l each) were collected, frozen on dry ice, and stored at ?80 C. The samples were processed for Western blot analysis, after which the blots were probed with anti-LAT, anti-phospho-LAT, or anti-flotillin-1 (a lipid raft marker) antibody. Western blot films were scanned on an HP Scanjet G3010 photo scanner, and signals of total LAT and phosphorylated LAT in fractions 2C5 (raft fractions) were quantified using NIH ImageJ software. The ratios of LAT were calculated based on the combined signals in fractions 2C5. Western Blot Analysis of TCR Signaling Molecules Frozen cell pellets were lysed in 200 l of cell lysis buffer (50 mm Tris (pH 7.5), 150 mm NaCl, 1% Triton X-100, 0.25% SDS, 1 mm EDTA, 1 mm NaF, and 5 mm sodium orthovanadate) preheated to 100 C and boiled in a heating block at 100 C for another 5 min. Samples were sonicated briefly until no longer viscous (5 s), and the protein content was decided using the Micro BCA protein assay kit (Pierce). Approximately 25 g of total protein was subjected to electrophoresis, after which the separated proteins were transferred onto nitrocellulose membranes and probed using antibodies against Lck, Zap70, and LAT or their phosphorylated counterparts. GAPDH was used to monitor for variations in protein loading. Calcium Mobilization Assay Approximately 50 ml of Genz-123346-treated or mock-treated Jurkat T cells (total of 2 107) in medium was treated with Fura-2 acetoxymethyl ester at a final concentration of 5 m for 60 min in a cell culture incubator. The Fura-2 acetoxymethyl ester-treated cells were then collected by centrifugation, resuspended in 2 ml of assay buffer (140 mm NaCl, 5 mm KCl, 0. 7 mm CaCl2, 0.7 mm MgCl2, 20 mm HEPES, 10 mm glucose, and 0.1% BSA (pH 7.4)), and allowed to equilibrate for 10 min. Approximately 400 l of the labeled cells was added to a fluorometer cuvette and supplemented with CaCl2 to a final concentration of 5 mm. The base line was recorded for 1 min, after which anti-CD3/CD28 antibody-coupled Dynabeads (or other controls) were added at a cell/bead ratio of 1 1:2, and the calcium response was recorded for up to 300 s using an RF-5301PC spectrofluorophotometer (Shimadzu Corp.). Cytoplasmic calcium levels were calculated based on the fluorescence ratio of Fura-2 acetoxymethyl ester at excitation wavelengths of 340 and 380 nm as explained previously (27). Isolation, Culture, and Differentiation of Main T Cells Naive.L., Sandhoff K. proliferation. Comparable findings were obtained with CD4+ T cells isolated from transgenic mice genetically deficient in GM3 synthase activity. Interestingly, lowering the GSL levels in CD4+ T cells by either pharmacological inhibition or disruption of the gene for GM3 synthase also specifically inhibited the differentiation of T cells to the Th17 lineage but not to other Th subsets without affecting the other Th subsets examined. Taken together, these results show that altering the lipid composition of lipid rafts has profound effects on T cell activation and differentiation. EXPERIMENTAL PROCEDURES Materials Anti-human LAT and anti-human Lck antibodies were purchased from Santa Cruz Biotechnology Inc. Anti-human Zap70 antibody (clone 2F3.2), anti-phosphorylated LAT (Tyr191) antibody, and anti-phosphorylated Src antibody (clone 2N8), that also recognizes phospho-Lck, were obtained from Upstate Biotechnology Inc. Anti-phosphorylated Zap70 (Tyr292) antibody was from Sigma. Anti-GAPDH and anti-flotillin-1 antibodies were purchased from Abcam and BD Biosciences, respectively. Goat anti-rabbit IgG and anti-mouse IgG coupled with HRP were acquired from Pierce. Dynabeads coupled with anti-human CD3/CD28 or anti-mouse CD3/CD28 antibody were obtained from Invitrogen. Soluble anti-mouse CD3? (clone 145.2C11), anti-mouse CD28 (clone 37.51), anti-human CD3? (clone OKT3), anti-CD28 (clone CD28.6), anti-CD25 (clone Personal computer.61), and anti-mouse RORt antibodies were purchased from eBioscience. All cytokines had been acquired either from R&D Systems or PeproTech. Treatment of Jurkat T Cells with Genz-123346 Freshly propagated Jurkat cells had been taken care of in RPMI 1640 moderate including 25 mm HEPES, 1 mm sodium pyruvate, 10% FBS, 1 penicillin/streptomycin, and 50 m -mercaptoethanol at 37 C under 5% CO2. Genz-123346 ((1(16). Twelve fractions (430 l each) had been collected, freezing on dry snow, and kept at ?80 C. The examples had been processed for Traditional western blot analysis, and the blots had been probed with anti-LAT, anti-phospho-LAT, or anti-flotillin-1 (a lipid raft marker) antibody. Traditional western blot films had been scanned with an Horsepower Scanjet G3010 photo scanning device, and indicators of total LAT and phosphorylated LAT in fractions 2C5 (raft fractions) had been quantified using NIH ImageJ software program. The ratios of LAT had been calculated predicated on the mixed indicators in fractions 2C5. Traditional western Blot Evaluation of TCR Signaling Substances Frozen cell pellets had been lysed in 200 l of cell lysis Atreleuton buffer (50 mm Tris (pH 7.5), 150 mm NaCl, 1% Triton X-100, 0.25% SDS, 1 mm EDTA, 1 mm NaF, and 5 mm sodium orthovanadate) preheated to 100 C and boiled inside a heating block at 100 C for another 5 min. Examples had been sonicated briefly until no more viscous (5 s), as well as the proteins content was established using the Micro BCA proteins assay package (Pierce). Around 25 g of total proteins was put through electrophoresis, and the separated protein had been moved onto nitrocellulose membranes and probed using antibodies against Lck, Zap70, and LAT or their phosphorylated counterparts. GAPDH was utilized to monitor for variants in proteins loading. Calcium mineral Mobilization Assay Around 50 ml of Genz-123346-treated or mock-treated Jurkat Rabbit Polyclonal to FOXO1/3/4-pan (phospho-Thr24/32) T cells (total of 2 107) in moderate was treated with Fura-2 acetoxymethyl ester at your final focus of 5 m for 60 min inside a cell tradition incubator. The Fura-2 acetoxymethyl ester-treated cells had been then gathered by centrifugation, resuspended in 2 ml of assay buffer (140 mm NaCl, 5 mm KCl, 0. 7 mm CaCl2, 0.7 mm MgCl2, 20 mm HEPES, 10 mm blood sugar, and 0.1% BSA (pH 7.4)), and permitted to equilibrate for 10 min. Around 400 l from the tagged cells was put into a fluorometer cuvette and supplemented with CaCl2 to your final focus of 5 mm. The bottom line was documented for 1 min, and anti-CD3/Compact disc28 antibody-coupled Dynabeads (or additional controls) had been added at a cell/bead percentage of just one 1:2, as well as the calcium mineral response was documented for 300 s using an RF-5301PC spectrofluorophotometer (Shimadzu Corp.). Cytoplasmic calcium mineral levels had been calculated predicated on the fluorescence percentage of Fura-2 acetoxymethyl ester at excitation wavelengths of 340 and 380 nm as referred to previously (27). Isolation, Tradition, and Differentiation of Major T Cells Naive mouse T cells from spleens and lymph nodes of regular C57BL/6 mice and wild-type littermates of GM3 synthase knock-out (KO; ?/?) and heterozygous (+/?) mice on the C57BL/6-129/svev mixed history (28) had been.For instance, GM3 continues to be reported to connect to the epidermal development element receptor via the (37) teaching a deficiency in the raft-resident proteins Raftlin also specifically reduces Th17 differentiation however, not the additional Th subsets. synthase activity. Oddly enough, decreasing the GSL amounts in Compact disc4+ T cells by either pharmacological inhibition or disruption from the gene for GM3 synthase also particularly inhibited the differentiation of T cells towards the Th17 lineage however, not to additional Th subsets without influencing the additional Th subsets analyzed. Taken collectively, these results reveal that changing the lipid structure of lipid rafts offers profound results on T cell activation and differentiation. EXPERIMENTAL Methods Components Anti-human LAT and anti-human Lck antibodies had been bought from Santa Cruz Biotechnology Inc. Anti-human Zap70 antibody (clone 2F3.2), anti-phosphorylated LAT (Tyr191) antibody, and anti-phosphorylated Src antibody (clone 2N8), that also recognizes phospho-Lck, were from Upstate Biotechnology Inc. Anti-phosphorylated Zap70 (Tyr292) antibody was from Sigma. Anti-GAPDH and anti-flotillin-1 antibodies had been bought from Abcam and BD Biosciences, respectively. Goat anti-rabbit IgG and anti-mouse IgG in conjunction with HRP had been obtained from Pierce. Dynabeads in conjunction with anti-human Compact disc3/Compact disc28 or anti-mouse Compact disc3/Compact disc28 antibody had been from Invitrogen. Soluble anti-mouse Compact disc3? (clone 145.2C11), anti-mouse Compact disc28 (clone 37.51), anti-human Compact disc3? (clone OKT3), anti-CD28 (clone Compact disc28.6), anti-CD25 (clone Personal computer.61), and anti-mouse RORt antibodies were purchased from eBioscience. All cytokines had been acquired either from R&D Systems or PeproTech. Treatment of Jurkat T Cells with Genz-123346 Freshly propagated Jurkat cells had been taken care of in RPMI 1640 moderate including 25 mm HEPES, 1 mm sodium pyruvate, 10% FBS, 1 penicillin/streptomycin, and 50 m -mercaptoethanol at 37 C under 5% CO2. Genz-123346 ((1(16). Twelve fractions (430 l each) had been collected, freezing on dry snow, and kept at ?80 C. The examples had been processed for Traditional western blot analysis, and the blots had been probed with anti-LAT, anti-phospho-LAT, or anti-flotillin-1 (a lipid raft marker) antibody. Traditional western blot films had been scanned with an Horsepower Scanjet G3010 photo scanning device, and indicators of total LAT and phosphorylated LAT in fractions 2C5 (raft fractions) had been quantified using NIH ImageJ software program. The ratios of LAT had been calculated predicated on the mixed indicators in fractions 2C5. Traditional western Blot Evaluation of TCR Signaling Substances Frozen cell pellets had been lysed in 200 l of cell lysis buffer (50 mm Tris (pH 7.5), 150 mm NaCl, 1% Triton X-100, 0.25% SDS, 1 mm EDTA, 1 mm NaF, and 5 mm sodium orthovanadate) preheated to 100 C and boiled inside a heating block at 100 C for another 5 min. Examples had been sonicated briefly until no more viscous (5 s), as well as the protein content was identified using the Micro BCA protein assay kit (Pierce). Approximately 25 g of total protein was subjected to electrophoresis, after which the separated proteins were transferred onto nitrocellulose membranes and probed using antibodies against Lck, Zap70, and LAT or their phosphorylated counterparts. GAPDH was used to monitor for variations in protein loading. Calcium Mobilization Assay Approximately 50 ml of Genz-123346-treated or mock-treated Jurkat T cells (total of 2 107) in medium was treated with Fura-2 acetoxymethyl ester at a final concentration of 5 m for 60 min inside a cell tradition incubator. The Fura-2 acetoxymethyl ester-treated cells were then collected by centrifugation, resuspended in 2 ml of assay buffer (140 mm NaCl, 5 mm Atreleuton KCl, 0. 7 mm CaCl2, 0.7 mm MgCl2, 20 mm HEPES, 10 mm glucose, and 0.1% BSA (pH 7.4)), and allowed to equilibrate for 10 min. Approximately 400 l of the labeled cells was added to a fluorometer cuvette and supplemented with CaCl2 to a final concentration of 5 mm. The base line was recorded for 1 min, after which anti-CD3/CD28 antibody-coupled Dynabeads (or additional controls) were added at a cell/bead percentage of 1 1:2, and the calcium response was recorded for up to 300 s using an RF-5301PC spectrofluorophotometer (Shimadzu Corp.). Cytoplasmic calcium levels were calculated based on the fluorescence percentage of Fura-2 acetoxymethyl ester at excitation wavelengths of 340 Atreleuton and 380 nm as explained previously (27). Isolation, Tradition, and Differentiation of Main T Cells Naive mouse T cells from spleens and lymph nodes of normal C57BL/6 mice and wild-type littermates of GM3 synthase knock-out (KO; ?/?) and heterozygous (+/?) mice on a C57BL/6-129/svev mixed background (28) were purified using the CD4+CD62Lhigh cell isolation kit from Miltenyi Biotech. In some experiments, CD4+CD62LhighCD44lowCD25? cells were further purified by cell sorting on a FACSAria cell sorter (BD Biosciences). Approximately 2 105 cells were plated onto U-bottom 96-well plates precoated with 1 g/ml anti-CD3? antibody in a total volume.
PTP