Journal: PLoS ONE

Article Title: Amiselimod (MT-1303), a novel sphingosine 1-phosphate receptor-1 functional antagonist, inhibits progress of chronic colitis induced by transfer of CD4 + CD45RB high T cells

doi: 10.1371/journal.pone.0226154

Figure Lengend Snippet: MT-1303 (0.3 mg/kg) or vehicle was orally administered to C57BL/6 mice for 3 days and S1P 1 receptor expression on CD4 + T cells from mesenteric lymph nodes was determined using flow cytometry. (A) Representative histograms of S1P 1 receptor expression in each group. Black, isotype control (rat IgG); red, vehicle-treated mice (S1P 1 ); blue, MT-1303-treated mice (S1P 1 ). (B) Mean fluorescence intensity (MFI) of the S1P 1 receptor on CD4 + T cells. Data are expressed as the mean ± S.E.M. (n = 3), and statistical differences were calculated using Student’s t -test after logarithmic transformation. ** p < 0.01, compared with the vehicle-treated group. (C) The number of CD4 + T cells in peripheral blood was measured using flow cytometry. Data are expressed as the mean ± S.E.M.; n = 3. Statistical differences were calculated using Student’s t -test. ** p < 0.01, compared with the vehicle-treated group.

Article Snippet: Rat anti-mouse S1P 1 antibody (713412) and mouse CD4 + T cell enrichment column were obtained from R&D Systems, and the Lamina Propria Dissociation Kit was purchased from Miltenyi Biotec.

Techniques: Expressing, Flow Cytometry, Control, Fluorescence, Transformation Assay

Journal: PLoS ONE

Article Title: Amiselimod (MT-1303), a novel sphingosine 1-phosphate receptor-1 functional antagonist, inhibits progress of chronic colitis induced by transfer of CD4 + CD45RB high T cells

doi: 10.1371/journal.pone.0226154

Figure Lengend Snippet: SCID mice were injected with CD4 + CD45RB high T cells to induce colitis. MT-1303 or vehicle was orally administered to SCID mice every day from the day of CD4 + CD45RB high T cell transfer for 28 days. (A) Change in body weight over time. Body weight on each day is expressed as a percentage of the original weight. Each symbol represents the mean ± S.E.M. of body weight (%) in 14–15 mice (n = 14: normal group). Statistical differences were determined using Student’s t -test by comparing to the normal group (# p < 0.05, ## p < 0.01) or using Dunnett’s test by comparing with the vehicle-treated group (* p < 0.05, ** p < 0.01). (B) Clinical scores were determined the day after the final administration. Each column represents the mean ± S.E.M. of clinical scores in 14–15 mice. Statistical differences were determined using the Wilcoxon test by comparing with the normal group (## p < 0.01) or using Steel’s test by comparing with the vehicle-treated group (** p < 0.01). (C, D) Colon sections from vehicle- (C) or MT-1303 0.3 mg/kg (D)-treated mice were stained with hematoxylin-eosin.

Article Snippet: Rat anti-mouse S1P 1 antibody (713412) and mouse CD4 + T cell enrichment column were obtained from R&D Systems, and the Lamina Propria Dissociation Kit was purchased from Miltenyi Biotec.

Techniques: Injection, Staining

Journal: PLoS ONE

Article Title: Amiselimod (MT-1303), a novel sphingosine 1-phosphate receptor-1 functional antagonist, inhibits progress of chronic colitis induced by transfer of CD4 + CD45RB high T cells

doi: 10.1371/journal.pone.0226154

Figure Lengend Snippet: SCID mice were injected with CD4 + CD45RB high T cells to induce colitis. MT-1303 was orally administered to SCID mice every day from day 7 after CD4 + CD45RB high T cell transfer for 21 days, and anti-mTNF-α mAb was intraperitoneally administered to SCID mice on day 7 and day 21 after cell transfer. (A, B) Change in body weight over time. Body weight on each day is expressed as a percentage of the original weight. Each symbol represents the mean ± S.E.M. of body weight (%) in 18 mice. Statistical differences were determined using Student’s t -test by comparing with the normal group (# p < 0.05, ## p < 0.01), using Dunnett’s test by comparing with the vehicle-treated group (* p < 0.05) or using Student’s t -test by comparing with the vehicle-treated group († p < 0.05, †† p < 0.01). (C) Clinical scores were determined on day 28 after cell transfer. Each column represents the mean ± S.E.M. of clinical scores in 18 mice. Statistical differences were determined using the Wilcoxon test by comparing with the normal group (## p < 0.01), using Steel’s test by comparing with the vehicle-treated group (** p < 0.01) or using the Wilcoxon test by comparing with the vehicle-treated group (†† p < 0.01). There were no significant differences between the anti-mTNF-α mAb-treated group and MT-1303-treated groups using Steel’s test.

Article Snippet: Rat anti-mouse S1P 1 antibody (713412) and mouse CD4 + T cell enrichment column were obtained from R&D Systems, and the Lamina Propria Dissociation Kit was purchased from Miltenyi Biotec.

Techniques: Injection

Journal: PLoS ONE

Article Title: Amiselimod (MT-1303), a novel sphingosine 1-phosphate receptor-1 functional antagonist, inhibits progress of chronic colitis induced by transfer of CD4 + CD45RB high T cells

doi: 10.1371/journal.pone.0226154

Figure Lengend Snippet: MT-1303 or vehicle was orally administered to SCID mice every day from week 1 after CD4 + CD45RB high T cell transfer for 3–4 weeks. (A, B) The number of lymphocytes (A) and CD4 + T cells (B) in the lamina propria of the colon was measured using flow cytometry the day after the final administration. (C–E) LPLs were stimulated with PMA and ionomycin in the presence of brefeldin A for 6 h before intracellular cytokine staining was performed using anti-IFN-γ and anti-IL-17 mAbs (C). Th1 (D) and Th17 (E) cell counts in the lamina propria of the colon were determined. (F, G) After stimulation with PMA and ionomycin, the amount of IFN-γ (F) and IL-17 (G) in the culture supernatant was determined using a BD TM Cytometric Beads Array. Each bar represents the mean ± S.E.M. (n = 11: vehicle-treated group, n = 12: MT-1303-treated group). Statistical differences were determined using Student’s t -test by comparing with the normal group (* p < 0.05, ** p < 0.01).

Article Snippet: Rat anti-mouse S1P 1 antibody (713412) and mouse CD4 + T cell enrichment column were obtained from R&D Systems, and the Lamina Propria Dissociation Kit was purchased from Miltenyi Biotec.

Techniques: Flow Cytometry, Staining

Journal: Cancer Management and Research

Article Title: LOX-1+ PMN-MDSC enhances immune suppression which promotes glioblastoma multiforme progression

doi: 10.2147/CMAR.S210545

Figure Lengend Snippet: LOX-1+ PMN is a population of PMN-MDSC in GBM patient PB. ( A, B ) Suppressive function of LOX-1+ and LOX-1− PMN sorted from PB of GBM patients in allogeneic MLR. The data indicate the mean±sem of three independent experiments. CD4+T cell ( A ) and CD8+ T cell ( B ) proliferation was determined in triplicates using 3H-thymidine uptake. ( C ) Production of ROS in LOX-1+ and LOX-1− PMN from six patients with GBM. Production of ROS was detected by staining with DCFDA. ( D, E ) ARG1 ( D ) and iNOS ( E ) expression in LOX-1- and LOX-1+ PMN from six patients with GBM detected by qPCR. ( F, G ) Effect of 1 μM of N-acetyl L-cysteine (NAC), 1,000 U/mL of catalase and 20 μM nor-NOHA on immune suppressive function of LOX-1+ PMN. The data indicate the mean±sem of three independent experiments. Allogeneic MLR and 1:1 PMN: T cell ratio was used in all experiments. CD4+T cell ( F ) and CD8+ T cell ( G ) proliferation were determined in triplicates by 3H-thymidine incorporation. ** P <0.01, *** P <0.001. Abbreviations: LOX-1, lectin-type oxidized LDL receptor 1; PMN, polymorphonuclear neutrophil; GBM, glioblastoma multiforme; PB, peripheral blood; CPM, counts per minute.

Article Snippet: The isolation of CD4+ or CD8+ T cells from the PBMC of the same patients as LOX-1+ PMN was performed using human CD4+ or CD8+ T Cell Enrichment Column Kit (Miltenyi).

Techniques: Staining, Expressing

Journal: Cancer Management and Research

Article Title: LOX-1+ PMN-MDSC enhances immune suppression which promotes glioblastoma multiforme progression

doi: 10.2147/CMAR.S210545

Figure Lengend Snippet: LOX-1+ PMN is negatively correlation with effector immune cells in GBM patient PB. ( A ) Typical example of the analysis of the proportion of LOX-1+ PMN, IFN-γ+CD4+ T cells and IFN-γ+CD8+ T cells in two patients with GBM. ( B, C ) Quantification showing the associations between LOX-1+ PMN and IFN-γ+CD4+ T cells ( B ) and IFN-γ+CD8+ T cells in 10 patients with GBM ( C ). Abbreviations: LOX-1, lectin-type oxidized LDL receptor 1; PMN, polymorphonuclear neutrophil; GBM, glioblastoma multiforme; PB, peripheral blood.

Article Snippet: The isolation of CD4+ or CD8+ T cells from the PBMC of the same patients as LOX-1+ PMN was performed using human CD4+ or CD8+ T Cell Enrichment Column Kit (Miltenyi).

Techniques:

Journal: Cancer Management and Research

Article Title: LOX-1+ PMN-MDSC enhances immune suppression which promotes glioblastoma multiforme progression

doi: 10.2147/CMAR.S210545

Figure Lengend Snippet: LOX-1 defines a subset of PMN-MDSC in GBM patient tissues. ( A) Typical example of the analysis of PMN in GBM patient tissue. ( B, C ) Suppressive function of LOX-1+ and LOX-1− PMN sorted from tumor tissue of GBM patients in allogeneic MLR. CD4+T cell ( B ) and CD8+ T cell ( C ) proliferation was determined in triplicates using 3H-thymidine uptake. ( D ) Production of ROS in LOX-1+ and LOX-1− PMN from five patients with GBM. Production of ROS was detected by staining with DCFDA. ( E, F ) ARG1 ( E ) and iNOS ( F ) expression in LOX-1- and LOX-1+ PMN from five patients with GBM detected by qPCR. * P <0.05, ** P <0.01, *** P <0.001. Abbreviations: LOX-1, lectin-type oxidized LDL receptor 1; PMN, polymorphonuclear neutrophil; GBM, glioblastoma multiforme; MDSC, myeloid-derived suppressor cell; DCFDA, dichlorodihydrofluorescein diacetate; MLR, mixed lymphocyte reaction; n.s, not significant.

Article Snippet: The isolation of CD4+ or CD8+ T cells from the PBMC of the same patients as LOX-1+ PMN was performed using human CD4+ or CD8+ T Cell Enrichment Column Kit (Miltenyi).

Techniques: Staining, Expressing, Derivative Assay

Journal: Scientific Reports

Article Title: Novel benzoxazole derivatives DCPAB and HPAB attenuate Th1 cell-mediated inflammation through T-bet suppression

doi: 10.1038/srep42144

Figure Lengend Snippet: CD4+ T cells were isolated from lymph nodes and spleen and stimulated with anti-CD3 (2 μg/mL) and anti-CD28 (1 μg/mL) Abs and subsequently incubated with DCPAB and HPAB for 48 h. ( a ) Cell supernatants were collected and used for measuring IFN-γ and IL-2 by ELISA. ( b–d ) CD4+ T cells were stimulated and additionally cultured under skewing conditions: IL-12 (2 ng/mL) and anti-IL-4 Ab (5 μg/mL) for Th1-skewing ( b ), IL-4 and anti- IFN-γ Ab (5 μg/mL) for Th2-skewing ( c ), and TGF-β (10 ng/mL) and IL-6 (10 ng/mL) for Th17-skewing ( d ) for 48 h. Total RNA was prepared and subjected to reverse transcription and real time-PCR to determine the relative expression level of IFN-γ ( b ), IL-4 ( c ), and IL-17 ( d ), respectively. Relative transcript level was calculated after normalization with actin level and is expressed as a value of fold-induction. *P < 0.05, **P < 0.005, ***P < 0.0005.

Article Snippet: Naive CD4+ T cells (CD4 + CD62L + CD44 − ) were isolated from the lymph node of C57BL6 mice (male, 6 weeks of age, Jackson Laboratories) using naïve CD4+ T cell enrichment column (R&D systems, Minneapolis, MN) and the cells (0.5 × 10 6 cells/mouse) were then adoptively transferred into RAG KO mice (male, 6 weeks of age, n = 24) via intraperitoneal injection.

Techniques: Isolation, Incubation, Enzyme-linked Immunosorbent Assay, Cell Culture, Reverse Transcription, Real-time Polymerase Chain Reaction, Expressing

Journal: Scientific Reports

Article Title: Novel benzoxazole derivatives DCPAB and HPAB attenuate Th1 cell-mediated inflammation through T-bet suppression

doi: 10.1038/srep42144

Figure Lengend Snippet: CD4+ T cells were cultured under Th1 differentiation conditions and treated with DCPAB and HPAB for 5 days. Cells were restimulated with anti-CD3 for 24 h. ( a ) Scheme for Th1 cell differentiation. ( b ) Cells were harvested and incubated with PE-conjugated anti-IFN-γ Ab, followed by flow cytometry analysis. Cell populations were quantitated using CellQuest software. ( c ) Cell supernatants were collected from the differentiated Th1 cells and used for measuring IFN-γ by ELISA. ( d ) IFN-γ production was determined in DCPAB- and HPAB-treated cells by ELISA and real time-PCR. IFN-γ suppression activity of DCPAB and HPAB was comparatively analyzed and is expressed as the percentage of suppression activity normalized to the untreated control (100%). At least three independent experiments were performed and statistically analyzed. *P < 0.05, **P < 0.005, ***P < 0.0005.

Article Snippet: Naive CD4+ T cells (CD4 + CD62L + CD44 − ) were isolated from the lymph node of C57BL6 mice (male, 6 weeks of age, Jackson Laboratories) using naïve CD4+ T cell enrichment column (R&D systems, Minneapolis, MN) and the cells (0.5 × 10 6 cells/mouse) were then adoptively transferred into RAG KO mice (male, 6 weeks of age, n = 24) via intraperitoneal injection.

Techniques: Cell Culture, Cell Differentiation, Incubation, Flow Cytometry, Software, Enzyme-linked Immunosorbent Assay, Real-time Polymerase Chain Reaction, Activity Assay

Journal: Scientific Reports

Article Title: Novel benzoxazole derivatives DCPAB and HPAB attenuate Th1 cell-mediated inflammation through T-bet suppression

doi: 10.1038/srep42144

Figure Lengend Snippet: ( a,b ) CD4+ T cells were treated with DCPAB or HPAB for 48 h under Th1-skewing conditions. Proteins were extracted and resolved by SDS-PAGE. Protein blots were incubated with anti-T-bet Ab, followed by detection using ECL and densitometry analysis ( a ). *P < 0.05, **P < 0.005, ***P < 0.0005. Total RNA was independently prepared using TRIzol reagent and subjected to reverse transcription, followed by the quantitative analysis of T-bet mRNA ( b ). ( c,d ) HEK 293 T cells were transfected with T-bet expression vector together with INF-γ promoter-linked reporter gene (pIFN-γ-luc). The β-galactosidase expression vector, pCMVβ was also transfected for normalizing transfection efficiency. Transfected cells were incubated with DCPAB or HPAB for an additional 24 h. T-bet expression was determined by immunoblot analysis ( c ). Relative luciferase activity was calculated after normalization with β-galactosidase activity and is expressed as a fold induction from four independent experiments ( d ). **P < 0.005, ***P < 0.0005.

Article Snippet: Naive CD4+ T cells (CD4 + CD62L + CD44 − ) were isolated from the lymph node of C57BL6 mice (male, 6 weeks of age, Jackson Laboratories) using naïve CD4+ T cell enrichment column (R&D systems, Minneapolis, MN) and the cells (0.5 × 10 6 cells/mouse) were then adoptively transferred into RAG KO mice (male, 6 weeks of age, n = 24) via intraperitoneal injection.

Techniques: SDS Page, Incubation, Reverse Transcription, Transfection, Expressing, Plasmid Preparation, Western Blot, Luciferase, Activity Assay

Journal: Scientific Reports

Article Title: Novel benzoxazole derivatives DCPAB and HPAB attenuate Th1 cell-mediated inflammation through T-bet suppression

doi: 10.1038/srep42144

Figure Lengend Snippet: CD4+ T cells were isolated from WT, T-bet KO, and DTg/KO mice and stimulated with anti-CD3 and anti-CD28 Abs. CD4+ T cells of DTg/KO mice were additionally treated with doxycycline (dox, 1 μg/ml) for retrieving T-bet expression (T-bet KO+ T-bet). Cells were incubated with DCPAB (10 μM) or HPAB (10 μM) during Th1 cell differentiation and restimulated with anti-CD3 (1 μg/ml) according to the experimental scheme ( a ). ( b ) Protein extracts were resolved by SDS-PAGE and analyzed by immunoblotting with anti-T-bet Ab. ( c ) Cell supernatants were harvested at day 6 and used for the ELISA measuring IFN-γ. **P < 0.005. ( d ) Cells were separately stained with PE-conjugated anti-IFN-γ Ab and quantitatively analyzed using flow cytometry and CellQuest program.

Article Snippet: Naive CD4+ T cells (CD4 + CD62L + CD44 − ) were isolated from the lymph node of C57BL6 mice (male, 6 weeks of age, Jackson Laboratories) using naïve CD4+ T cell enrichment column (R&D systems, Minneapolis, MN) and the cells (0.5 × 10 6 cells/mouse) were then adoptively transferred into RAG KO mice (male, 6 weeks of age, n = 24) via intraperitoneal injection.

Techniques: Isolation, Expressing, Incubation, Cell Differentiation, SDS Page, Western Blot, Enzyme-linked Immunosorbent Assay, Staining, Flow Cytometry

Journal: Scientific Reports

Article Title: Novel benzoxazole derivatives DCPAB and HPAB attenuate Th1 cell-mediated inflammation through T-bet suppression

doi: 10.1038/srep42144

Figure Lengend Snippet: Naive CD4+ T cells were isolated from WT mice and adoptively transferred into RAG KO mice by intraperitoneal injection. After 6 weeks, CD4+ T/RAG KO mice were daily administered with DCPAB (5 mg/kg) or HPAB (5 mg/kg) for 2 weeks through an intraperitoneal injection. ( a ) Disease activity index was determined in CD4+ T/RAG KO mice treated with vehicle, DCPAB, or HPAB and is expressed as mean ± SD of 8 mice. *P < 0.05, **p < 0.005. ( b ) Colon tissues were sectioned and visualized using H&E staining. Representative images are shown. ( c,d ) Spleen cells were stimulated with anti-CD3 (1 μg/ml) for 24 h. Cells were stained with anti-IFN-γ Ab followed by flow cytometry analysis ( c ). Cell supernatants were collected and used for measuring IFN-γ ( d ). **P < 0.005, ***p < 0.0005.

Article Snippet: Naive CD4+ T cells (CD4 + CD62L + CD44 − ) were isolated from the lymph node of C57BL6 mice (male, 6 weeks of age, Jackson Laboratories) using naïve CD4+ T cell enrichment column (R&D systems, Minneapolis, MN) and the cells (0.5 × 10 6 cells/mouse) were then adoptively transferred into RAG KO mice (male, 6 weeks of age, n = 24) via intraperitoneal injection.

Techniques: Isolation, Injection, Activity Assay, Staining, Flow Cytometry