Journal: Neuro-Oncology

Article Title: Agonist OX40 immunotherapy improves survival in glioma-bearing mice and is complementary with vaccination with irradiated GM-CSF–expressing tumor cells

doi: 10.1093/neuonc/nox125

Figure Lengend Snippet: Th1 vs Th2 BILs. (A) Flow cytometry demonstrating percent expression of T-bet, IFN-γ, and IL-12R β2 on CD4+ lymphocytes 18 days after GL261 tumor implantation by treatment group. Combination immunotherapy is associated with significant increases in all 3 Th1 markers. (***P < 0.0005, **P < 0.005, *P < 0.05; absence of an asterisk denotes not significant comparison.) (B) Flow cytometry demonstrating percent expression of GATA3, the IL-4 receptor, and IL-5 on CD4+ lymphocytes 18 days after GL261 tumor implantation by treatment group. Combination immunotherapy is associated with significant decreases in all 3 Th2 markers. (**P < 0.005, *P < 0.05, absence of an asterisk denotes not significant comparison.)

Article Snippet: For the analysis of T helper cell subsets, we used Multi-Color Flow Cytometry Kits (R&D Systems) for mouse Th1 cells (conjugated antibodies to T-bet-PerCP, IFN-gamma-fluorescein, IL-12 R beta 2-APC, and CD4-PE) and Th2 cells (conjugated antibodies to CD4-PerCP, IL-4 R-fluorescein, STAT6-APC, and IL-5-PE).

Techniques: Flow Cytometry, Expressing, Tumor Implantation, Comparison

Journal: Neuro-Oncology

Article Title: Agonist OX40 immunotherapy improves survival in glioma-bearing mice and is complementary with vaccination with irradiated GM-CSF–expressing tumor cells

doi: 10.1093/neuonc/nox125

Figure Lengend Snippet: Day 18 flow cytometry of BILs shows that vaccination improves the intratumoral CD8+/FoxP3+ lymphocyte ratio, which is little affected by treatment with anti-OX40 immunotherapy. (**P < 0.005, *P < 0.05; absence of an asterisk denotes not significant comparison.)

Article Snippet: For the analysis of T helper cell subsets, we used Multi-Color Flow Cytometry Kits (R&D Systems) for mouse Th1 cells (conjugated antibodies to T-bet-PerCP, IFN-gamma-fluorescein, IL-12 R beta 2-APC, and CD4-PE) and Th2 cells (conjugated antibodies to CD4-PerCP, IL-4 R-fluorescein, STAT6-APC, and IL-5-PE).

Techniques: Flow Cytometry, Comparison

Journal: Journal of Clinical Investigation

Article Title: Callus γδ T cells and microbe-induced intestinal Th17 cells improve fracture healing in mice

doi: 10.1172/jci166577

Figure Lengend Snippet: Figure 1. Effects of fractures and Abx-induced microbiota depletion on callus and intestinal inflammatory cytokine transcripts and on the relative number of callus and intestinal γδ T cells and Th17 cells. (A) Effects of fractures on the levels of Il17a, Tnf, Il1b, and Il6 transcripts in the callus of SFB+ and SFB– JAX mice. (B) Effects of fractures on the levels of Il17a, Tnf, Il1b, and Il6 transcripts in the SI of SFB+ and SFB– JAX mice. (C) Effects of fractures on the relative frequency of γδ T cells (CD3ε+CD45+TCRγδ+) and Th17 cells (TCRβ+CD45+CD4+IL-17A+) in the callus and PPs of SFB+ and SFB– JAX mice. Femoral fractures were induced in 12-week-old female SFB+ JAX and SFB– JAX mice. Mice were treated or not with broad-spectrum Abx starting 1 week before fracture surgery. PP and callus cells were recovered daily for 3 days after fracture surgery and analyzed by flow cytometry. Time 0 indicates intact bone. n = 5 mice/group. Data are expressed as the mean ± SEM. All data were normally distributed according to the Shapiro-Wilk normality test and analyzed by 2-way ANOVA with post hoc Bonferroni correction for multiple comparisons. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 compared with the indicated group. Nonsignificant comparisons are not shown.

Article Snippet: EGFP– naive CD4+ T cells were cultured in Th17-polarizing conditions for 4 days using the Mouse Th17 Cell Differentiation Kit (R&D Systems) to generate EGFP+CD4+ Th17 cells.

Techniques: Flow Cytometry

Journal: Journal of Clinical Investigation

Article Title: Callus γδ T cells and microbe-induced intestinal Th17 cells improve fracture healing in mice

doi: 10.1172/jci166577

Figure Lengend Snippet: Figure 2. Effects of fractures and Abx-induced microbiota depletion on callus and intestinal γδ T cells and Th17 cells, and gut permeability in WT and γδ T cell–/– mice. Femoral fractures were induced in 12-week-old female SFB+ JAX mice, SFB– JAX mice, and SFB+ Tcrd–/– mice, a strain lacking γδ T cells. SFB+ JAX mice and SFB– JAX mice were treated or not with broad-spectrum Abx starting 1 week before fracture surgery. Frequencies of (A) callus γδ T cells (CD3ε+CD45+TCRγδ+ cells), (B) PP γδ T cells, (C) callus Th17 cells (TCRβ+CD45+CD4+IL-17A+ cells), and (D) PP Th17 cells. (E) Callus and PP Th17 cells in Tcrd–/– mice. (F) Gut permeability in SFB+ WT mice and SFB+ Tcrd–/– mice. Gut permeability was assessed by serum LPS levels and FITC-dextran absorption. n = 5 mice/group. Data are expressed as the mean ± SEM. All data were normally distributed according to the Shapiro-Wilk normality test and analyzed by 2-way ANOVA and with post hoc Bonferroni correction for multiple comparisons. **P < 0.01, ***P < 0.001, and ****P < 0.0001 compared with the indicated group (A–E) or compared with intact bone (F). Nonsignificant comparisons are not shown.

Article Snippet: EGFP– naive CD4+ T cells were cultured in Th17-polarizing conditions for 4 days using the Mouse Th17 Cell Differentiation Kit (R&D Systems) to generate EGFP+CD4+ Th17 cells.

Techniques: Permeability

Journal: Journal of Clinical Investigation

Article Title: Callus γδ T cells and microbe-induced intestinal Th17 cells improve fracture healing in mice

doi: 10.1172/jci166577

Figure Lengend Snippet: Figure 3. Fractures increase homing to the callus of intestinal αβ T cells and Th17 cells but not γδ T cells. Femoral fractures were induced in 12-week- old male SFB+ Kaede mice. After 2 days, 4 PPs were surgically exposed and illuminated with a near-UV light for 2 minutes. After 1 day, mice were sacrificed, and the frequency of PPs and callus red-fluorescing αβ T cells, Th17 cells, and γδ T cells was determined by flow cytometry. PP red-flu- orescing T cells were counted in PPs from mice with fractures and PPs from uninjured control mice. Callus red-fluorescing T cells were counted in the callus tissue of fractured femurs, BM from the contralateral uninjured femur, and BM from uninjured mice. (A) Relative frequency of PP αβ T cells and relative and absolute frequency of callus αβ T cells. (B) Relative frequency of PP Th17 cells and relative and absolute frequency of callus Th17 cells. (C) Relative frequency of PP γδ T cells and relative and absolute frequency of callus γδ T cells. n = 6 mice/group. Data are expressed as the mean ± SEM. All data were normally distributed according to the Shapiro-Wilk normality test and analyzed by 2-way ANOVA with post hoc Bonfer- roni correction for multiple comparisons (callus panels), or by unpaired, 2-tailed t test (PP panels). *P < 0.05, ***P < 0.001, and ****P < 0.0001 com- pared with the indicated group. Nonsignificant comparisons are not shown.

Article Snippet: EGFP– naive CD4+ T cells were cultured in Th17-polarizing conditions for 4 days using the Mouse Th17 Cell Differentiation Kit (R&D Systems) to generate EGFP+CD4+ Th17 cells.

Techniques: Flow Cytometry, Control

Journal: Journal of Clinical Investigation

Article Title: Callus γδ T cells and microbe-induced intestinal Th17 cells improve fracture healing in mice

doi: 10.1172/jci166577

Figure Lengend Snippet: Figure 4. Fractures increase the tropism of Th17 cells to the callus via a TNF-dependent mechanism. (A) Callus Ccl20 transcript levels at days 3 and 7 PF in SFB+ TAC mice. (B) Ccl20 transcript levels in purified callus cells at day 3 PF. (C) Callus Ccl20 transcript levels in SFB+ WT and Tnf–/– mice at day 3 PF. (D and E) Relative and absolute frequencies of EGFP+ Th17 cells in the callus of SFB+ WT and Tnf–/– mice subjected to fracture 2 days before adoptive transfer of IL-17A-EGFP+ cells. (F) Relative and absolute frequencies of callus Th17 cells in SFB+ WT mice and Tnf–/– mice. (G) Relative frequency of callus Vβ14+ Th17 cells in SFB+ WT and Tnf–/– mice. n = 5–6 mice/group. Data are expressed as the mean ± SEM. All data were normally distributed according to the Shap- iro-Wilk normality test and analyzed by 2-way ANOVA with post hoc Bonferroni correction for multiple comparisons. **P < 0.01, ***P < 0.001, and ****P < 0.0001 compared with the indicated group. #P < 0.001 compared with day 3 PF SCs. Nonsignificant comparisons are not shown.

Article Snippet: EGFP– naive CD4+ T cells were cultured in Th17-polarizing conditions for 4 days using the Mouse Th17 Cell Differentiation Kit (R&D Systems) to generate EGFP+CD4+ Th17 cells.

Techniques: Purification, Adoptive Transfer Assay

Journal: Journal of Clinical Investigation

Article Title: Callus γδ T cells and microbe-induced intestinal Th17 cells improve fracture healing in mice

doi: 10.1172/jci166577

Figure Lengend Snippet: Figure 6. Blockade of Th17 cell egress from the intestine prevents the fracture-induced increase in peripheral blood and callus Th17 cells and impairs fracture healing. (A) Relative frequency of PP, peripheral blood (PB), and callus Th17 cells at days 3 and 7 PF in mice treated with the S1PR1 blocker FTY720. (B) Effects of fractures on the transcript levels of Il17a in the SI and callus in mice treated with FTY720. (C and D) Callus μCT measurements at day 14 PF. Mice were treated with FTY720 for 2 weeks starting 1 week before fracture surgery. n = 5–6 mice/group. Data are expressed as the mean ± SEM. All data were nor- mally distributed according to the Shapiro-Wilk normality test and analyzed by 2-way ANOVA with post hoc Bonferroni correction for multiple comparisons, or by unpaired, 2-tailed t tests. *P < 0.05, ***P < 0.001, and ****P < 0.0001, compared with the indicated group. Nonsignificant comparisons are not shown.

Article Snippet: EGFP– naive CD4+ T cells were cultured in Th17-polarizing conditions for 4 days using the Mouse Th17 Cell Differentiation Kit (R&D Systems) to generate EGFP+CD4+ Th17 cells.

Techniques:

Journal: Journal of Clinical Investigation

Article Title: Callus γδ T cells and microbe-induced intestinal Th17 cells improve fracture healing in mice

doi: 10.1172/jci166577

Figure Lengend Snippet: Figure 7. Blockade of Th17 cell influx into callus by treatment with anti CCL20 Ab prevents the fracture-induced increase in peripheral blood and callus Th17 cells and impairs fracture healing. (A) Relative frequency of PP, peripheral blood, and callus Th17 cells at days 3 and 7 PF in mice treated with anti- CCL20 Ab. (B) Effect of fractures on transcript levels of Il17a in the SI and callus in mice treated with anti-CCL20 Ab. (C and D) Callus μCT measurements at day 14 PF. Mice were treated with anti-CCL20 Ab or irrelevant (Irr.) Ab 1 day before surgery and every other day for 7 days. n = 5–6 mice/group. Data are expressed as the mean ± SEM. All data were normally distributed according to the Shapiro-Wilk normality test and analyzed by 2-way ANOVA with post hoc Bonferroni correction for multiple comparisons. *P < 0.05, ***P < 0.001, and ****P < 0.0001, compared with the indicated group. Nonsignificant comparisons are not shown.

Article Snippet: EGFP– naive CD4+ T cells were cultured in Th17-polarizing conditions for 4 days using the Mouse Th17 Cell Differentiation Kit (R&D Systems) to generate EGFP+CD4+ Th17 cells.

Techniques:

Journal: Frontiers in Immunology

Article Title: Plasma Extracellular Vesicles Enhance HIV-1 Infection of Activated CD4 + T Cells and Promote the Activation of Latently Infected J-Lat10.6 Cells via miR-139-5p Transfer

doi: 10.3389/fimmu.2021.697604

Figure Lengend Snippet: Increased inflammatory responses by PBMCs and T cells treated with HIV ART-naïve pEVs. (A) Representative images of stimulated CD4 + and CD8 + T cells incubated with CFSE labelled pEVs. (B) Internalisation score of HC, ART-naïve and ART pEV uptake by CD4 + and CD8 + T cells. (C) Representative flow cytometry plots, and (D) cumulative data of the frequency of ki67 expressing CD4 + and CD8 + T cells left untreated (No EVs) or pre-incubated with pEVs from HC or HIV ART-naïve patients. (E) Representative flow plots, and (F) cumulative data of the proliferation of stimulated CD4 + and CD8 + T cells left untreated (No EVs) or pre-incubated with pEVs from HCs or HIV ART-naïve patients, indicated by CFSE dilution. (G) Concentrations of IFN-γ produced by PBMCs pre-incubated with pEVs (100 μg, 200 μg or 400 μg) from HCs or HIV ART-naïve patients before stimulation with SEB (100 ng/ml) for 72hrs. Dotted lines indicate the levels of IFN-γ produced by untreated PBMCs. (H) Concentrations of IL-2 produced by PBMCs pre-incubated with pEVs (100 μg, 200 μg or 400 μg) from HCs or HIV ART-naïve patients. (I) Fold percentage of IFN-γ expression, and (J) co-expression of IFN-γ and T-Bet+ by naïve CD4 + T cells pre-incubated with pEVs from HCs or HIV ART-naïve patients and stimulated under Th1-polarising conditions. Representative data from 3 independent experiments shown. Data points within pEV groups represent individual pEV samples. Error bars indicate mean±SEM. Indicated p-values were obtained from the Dunnett’s multiple comparisons test.

Article Snippet: Isolated naïve CD4 + T cells were plated with pEVs (400 μg) and polarised for 5 days using the Human Th1 Cell Differentiation Kit (R&D Systems) according to manufacturer’s instructions.

Techniques: Incubation, Flow Cytometry, Expressing, Produced

Journal: Stem Cell Research

Article Title: Derivation of human induced pluripotent stem cell line EURACi004-A from skin fibroblasts of a patient with Arrhythmogenic Cardiomyopathy carrying the heterozygous PKP2 mutation c.2569_3018del50

doi: 10.1016/j.scr.2018.09.003

Figure Lengend Snippet: Characterization and validation.

Article Snippet: SOX2 and SSEA4 expression in iPSCs adapted to feeder-free condition was analyzed by flow cytometry using Multi-Color Flow Cytometry Kit (R&D System) following manufacturer's instruction.

Techniques: Biomarker Discovery, Light Microscopy, Immunocytochemistry, Expressing, Flow Cytometry, Gene Expression, Mutagenesis, Sequencing, Southern Blot

Journal: Stem Cell Research

Article Title: Derivation of human induced pluripotent stem cell line EURACi004-A from skin fibroblasts of a patient with Arrhythmogenic Cardiomyopathy carrying the heterozygous PKP2 mutation c.2569_3018del50

doi: 10.1016/j.scr.2018.09.003

Figure Lengend Snippet: Reagents details.

Article Snippet: SOX2 and SSEA4 expression in iPSCs adapted to feeder-free condition was analyzed by flow cytometry using Multi-Color Flow Cytometry Kit (R&D System) following manufacturer's instruction.

Techniques: Immunocytochemistry, Flow Cytometry, Control