Journal: The Journal of Biological Chemistry

Article Title: Identification of Tribbles-1 as a Novel Binding Partner of Foxp3 in Regulatory T Cells *

doi: 10.1074/jbc.M112.448654

Figure Lengend Snippet: A, sort parameters for isolation of the human CD4+CD25− (up) and CD4+CD25+CD127− (down) T cells also called regulatory T cells. The left panels show the gate eliminating the CD4− T cells and selected specifically the CD4+ T cells (FSC mean Forward Scatter). The right top panel shows the gate for selecting the CD4+CD25− T cells from the CD4+ fraction previously selected. The right bottom panel shows the gate for selecting the CD4+CD25+CD127− T cells from the CD4+ fraction previously selected. B, sort parameters for isolation of the mouse CD4+CD25− and CD4+CD25high T cells (int mean intermediate). The different populations were isolated after a CD4+ T cell enrichment of mouse splenocytes. The panel displays only CD4+ T cells. CD4− T cells were eliminated from this population by the enrichment procedure and then the population was gated for CD4+ T cells. The left gate shows the sorting of the CD4+CD25− T cells. The right gate shows the sorting of the CD4+CD25high T cells.

Article Snippet: CD4+ T cells were isolated using the biotinylated mouse CD4 T lymphocyte enrichment mixture (BD Biosciences) according to the manufacturer's instructions.

Techniques: Isolation

Journal: The Journal of Biological Chemistry

Article Title: Identification of Tribbles-1 as a Novel Binding Partner of Foxp3 in Regulatory T Cells *

doi: 10.1074/jbc.M112.448654

Figure Lengend Snippet: A, quantification of the TRIB1 and Foxp3 mRNA in human CD4+CD25− and CD4+CD25+CD127− T cells. Real-time quantitative RT-PCR analysis for quantification of TRIB1 mRNA (left) and Foxp3 mRNA (middle) in CD4+CD25− and CD4+CD25+CD127− human T cells from seven healthy individuals. Statistical analyses were performed using a non parametric Mann-Whitney test. Analysis of correlation between TRIB1 and FOXP3 in CD4+CD25+CD127− T cells and statistical analysis of correlation were performed with a nonparametric Spearman test (right). B, quantification of the TRIB1 and Foxp3 mRNA in mouse CD4+CD25− and CD4+CD25high T cells. Real-time quantitative RT-PCR analysis of TRIB1 (left) and FOXP3 (right) mRNA in CD4+CD25− and CD4+CD25high mouse T cells of six normal C57Bl6 mice. Statistical analyses were performed using a nonparametric Mann-Whitney test.

Article Snippet: CD4+ T cells were isolated using the biotinylated mouse CD4 T lymphocyte enrichment mixture (BD Biosciences) according to the manufacturer's instructions.

Techniques: Quantitative RT-PCR, MANN-WHITNEY

Journal: The Journal of Biological Chemistry

Article Title: Identification of Tribbles-1 as a Novel Binding Partner of Foxp3 in Regulatory T Cells *

doi: 10.1074/jbc.M112.448654

Figure Lengend Snippet: Quantification of TRIB1 and Foxp3 mRNA in human CD4+CD25+CD127− T cells freshly isolated or after activation. Real-time quantitative RT-PCR analysis for quantification of TRIB1 mRNA (A) and FOXP3 mRNA (B) in CD4+CD25+CD127− human T cells from healthy individuals after 0 to 24 h of culture in complete medium associated with recombinant IL-2 (rIL-2), anti-CD3, and anti-CD28 antibodies. Analysis of correlation between TRIB1 and Foxp3 expression with statistical analyses performed using a parametric Pearson test.

Article Snippet: CD4+ T cells were isolated using the biotinylated mouse CD4 T lymphocyte enrichment mixture (BD Biosciences) according to the manufacturer's instructions.

Techniques: Isolation, Activation Assay, Quantitative RT-PCR, Recombinant, Expressing

Journal:

Article Title: Epinephrine-primed murine bone marrow-derived dendritic cells facilitate production of IL-17A and IL-4 but not IFN-? by CD4 + T cells

doi: 10.1016/j.bbi.2010.05.003

Figure Lengend Snippet: CD4+ T cell fate-determining cytokine expression by BMDCs is predicted by exposure to epinephrine. Total RNA was extracted from BMDCs. mRNA transcription of IL-12p40, IL-12p35 and IL-23p19 was analyzed by quantitative real-time RT-PCR technique. Relative fold difference of mRNA transcription in epinephrine only group, epinephrine-LPS treated (EL) group and LPS only group was compared to un-stimulated control BMDC group as 1. Asterisk (*) indicates significant group differences in control/epinephrine only vs other groups and sharp (#) indicates significant differences in epinephrine pre-treated (EL) vs LPS only group (A). BMDCs were also treated by epinephrine (10−6 M) with or without various concentration (10−4 ~ 10−7 M) of butoxamine for 2 hr followed by LPS (1 μg/ml) challenge for additional 3 hr. All data represent mean (N=3) ± standard error. Asterisk (*) indicates significant difference in control vs other groups and sharp (#) indicates significant differences in EL vs butoxamine treated and LPS only group. To present group differences, one-way ANOVA was used followed by Student-Newman posthoc test.

Article Snippet: CD4 + T cells were purified and enriched from total splenocytes using IMagnet CD4 + T Lymphocyte Enrichment Set (BD PharMingen Inc., San Jose, CA) as described in procedures from the manufacturer.

Techniques: Expressing, Quantitative RT-PCR, Concentration Assay

Journal:

Article Title: Epinephrine-primed murine bone marrow-derived dendritic cells facilitate production of IL-17A and IL-4 but not IFN-? by CD4 + T cells

doi: 10.1016/j.bbi.2010.05.003

Figure Lengend Snippet: Epinephrine supports IL-4 and IL-17A production, but not IFN-γ by CD4 + T cells with β2 adrenergic receptor-dependent manner. Purified CD4 + T cells were cocultured with epinephrine (with or without butoxamine (10 −5 M))-pretreated BMDCs in epinephrine-free new culture media for 96 hr with or without anti-CD3 monoclonal antibody (2.5 μg/ml). Protein expression of IFN-γ, IL-4 and IL-17A was determined from culture media collected each condition including control T cell only culture. Data has been selected as representative set from 3 independent experiements.

Article Snippet: CD4 + T cells were purified and enriched from total splenocytes using IMagnet CD4 + T Lymphocyte Enrichment Set (BD PharMingen Inc., San Jose, CA) as described in procedures from the manufacturer.

Techniques: Purification, Expressing

Journal:

Article Title: Epinephrine-primed murine bone marrow-derived dendritic cells facilitate production of IL-17A and IL-4 but not IFN-? by CD4 + T cells

doi: 10.1016/j.bbi.2010.05.003

Figure Lengend Snippet: Hypothetical model of epinephrine-mediated regulation of dendritic cell function to generate adaptive immunity. Diagram represents our hypothetical model that stress-induced epinephrine impacts dendritic cells through β2-adrenergic receptor to generate dominant IL-23 and IL-10 production in response to pathogenic activation, and as a consequence, drive CD4+ T cell-mediated adaptive immunity into Th2/Th17 type.

Article Snippet: CD4 + T cells were purified and enriched from total splenocytes using IMagnet CD4 + T Lymphocyte Enrichment Set (BD PharMingen Inc., San Jose, CA) as described in procedures from the manufacturer.

Techniques: Cell Function Assay, Activation Assay

Journal: Pharmaceuticals

Article Title: Valproic Acid Suppresses Autoimmune Recurrence and Allograft Rejection in Islet Transplantation through Induction of the Differentiation of Regulatory T Cells and Can Be Used in Cell Therapy for Type 1 Diabetes

doi: 10.3390/ph14050475

Figure Lengend Snippet: The effect of VPA treatment in the activation of lymphocyte in NOD recipients. ( A ) Representative plot of the expression of CD69 in CD4 or CD8 T cells and B cells (B220). ( B ) There was no significant difference in the percentage of CD69 in the CD4, CD8 T cells or B cells.

Article Snippet: The BD IMagTM Mouse CD4 T Lymphocyte Enrichment set-DM (BD Biosciences, San Jose, CA, USA) was used for negative selection of CD4 T lymphocyte by removing non-CD4 T cells from splenocytes.

Techniques: Activation Assay, Expressing

Journal: Pharmaceuticals

Article Title: Valproic Acid Suppresses Autoimmune Recurrence and Allograft Rejection in Islet Transplantation through Induction of the Differentiation of Regulatory T Cells and Can Be Used in Cell Therapy for Type 1 Diabetes

doi: 10.3390/ph14050475

Figure Lengend Snippet: The effect of VPA treatment in the subsets of helper T cells in the spleen of NOD mouse after islet transplantation. The population of Th1, Th2, IL-10-producing and Th17 CD4 T cells in the PBS-treated or VPA-treated NOD recipients was analyzed by flow cytometry. ( A ) No significant difference in the population of Th1 cells. ( B ) A significant increase was observed in the percentage of Th2 (IL-4-producing CD4 T) cells in the VPA-treated recipients. No significant difference in the percentage of ( C ) IL-10-producing CD4 T cells and ( D ) Th17 (IL-17-producing CD4 T) cells. Data are expressed as the mean ± SEM (* = p < 0.05).

Article Snippet: The BD IMagTM Mouse CD4 T Lymphocyte Enrichment set-DM (BD Biosciences, San Jose, CA, USA) was used for negative selection of CD4 T lymphocyte by removing non-CD4 T cells from splenocytes.

Techniques: Transplantation Assay, Flow Cytometry

Journal: Pharmaceuticals

Article Title: Valproic Acid Suppresses Autoimmune Recurrence and Allograft Rejection in Islet Transplantation through Induction of the Differentiation of Regulatory T Cells and Can Be Used in Cell Therapy for Type 1 Diabetes

doi: 10.3390/ph14050475

Figure Lengend Snippet: The effect of VPA treatment in the proliferation and apoptosis of Treg cells in the NOD recipients. ( A ) The proliferation of Treg cell was analyzed by the staining of proliferation marker Ki67. The percentage in the Ki67 + cells showed no significant difference. Data are expressed as the mean ± SEM. ( B ) The apoptosis of CD4 or Treg cells was analyzed by Annexin V staining. The ratio of Annexin V positive Treg cell and CD4 T cell showed no significant difference between control and VPA-treated group. Data are expressed as the mean ± SEM.

Article Snippet: The BD IMagTM Mouse CD4 T Lymphocyte Enrichment set-DM (BD Biosciences, San Jose, CA, USA) was used for negative selection of CD4 T lymphocyte by removing non-CD4 T cells from splenocytes.

Techniques: Staining, Marker

Journal: Pharmaceuticals

Article Title: Valproic Acid Suppresses Autoimmune Recurrence and Allograft Rejection in Islet Transplantation through Induction of the Differentiation of Regulatory T Cells and Can Be Used in Cell Therapy for Type 1 Diabetes

doi: 10.3390/ph14050475

Figure Lengend Snippet: The effect of VPA treatment in the induction of Treg cells’ differentiation from the naive CD4 T cells of NOD mouse. Naive CD4 T cells were cultured with differential concentrations of VPA solutions (0 mM, 1 mM, 2 mM, and 4 mM) for different incubation times (0 h, 12 h, and 24 h). After VPA solution treatment, the percentage of lymphocytes were analyzed via flow cytometry. ( A ) The percentage of Treg cells differentiated from naïve CD4 T cells was significantly increased following VPA treatment at different concentrations of VPA at 24 h. ( B ) The absolute number of Treg cells differentiated from naïve CD4 T cells was calculated by multiplying the total number of the cells with the percentage of Treg cells. It also showed a significantly increased following VPA treatment at different concentrations of VPA at 24 h. Data are expressed as the mean ± SEM (n = 6; * p < 0.05, ** p <0.01).

Article Snippet: The BD IMagTM Mouse CD4 T Lymphocyte Enrichment set-DM (BD Biosciences, San Jose, CA, USA) was used for negative selection of CD4 T lymphocyte by removing non-CD4 T cells from splenocytes.

Techniques: Cell Culture, Incubation, Flow Cytometry

Journal: Pharmaceuticals

Article Title: Valproic Acid Suppresses Autoimmune Recurrence and Allograft Rejection in Islet Transplantation through Induction of the Differentiation of Regulatory T Cells and Can Be Used in Cell Therapy for Type 1 Diabetes

doi: 10.3390/ph14050475

Figure Lengend Snippet: The molecular mechanism of VPA treatment in the induction of Treg cells’ differentiation from naïve CD4 T cells of NOD mouse. The CD4 naive T lymphocytes were cultured with various concentrations of VPA solutions (0 mM, 1 mM, 2 mM, and 4 mM) at different incubation times. ( A ) The levels of STAT5 and ( B ) phosphorylated STAT5 (p-STAT5) were measured by Western blot. ( C , D ) The levels of histone acetylation (acetyl-H3) were assessed by Western blot. Data are expressed as the mean ± SEM (* = p < 0.05).

Article Snippet: The BD IMagTM Mouse CD4 T Lymphocyte Enrichment set-DM (BD Biosciences, San Jose, CA, USA) was used for negative selection of CD4 T lymphocyte by removing non-CD4 T cells from splenocytes.

Techniques: Cell Culture, Incubation, Western Blot

Journal: Pharmaceuticals

Article Title: Valproic Acid Suppresses Autoimmune Recurrence and Allograft Rejection in Islet Transplantation through Induction of the Differentiation of Regulatory T Cells and Can Be Used in Cell Therapy for Type 1 Diabetes

doi: 10.3390/ph14050475

Figure Lengend Snippet: The effect of adoptively transferring VPA-induced Treg cells in the islet graft survival in syngeneic islet transplantation. ( A ) Naïve CD4 T cells were harvested from the spleen of the NOD mice. The naïve naive CD4 T cells were cultured in the medium with 2 mM VPA or PBS. A total of 1 × 10 6 PBS-treated or VPA-treated cells were collected and adoptively transferred into the NOD mice twice at day 1 and day 3 after islet transplantation. ( B ) The islet graft survival was significant longer in the VPA-treated group compared to the PBS-treated group (* p < 0.05; n = 6). ( C , D ) The infiltration of lymphocytes in islet grafts was lower in the VPA-treated Treg transfer into NOD recipients at day 6 post-islet transplantation. In contrast, more islet grafts were presented in the VPA-treated Treg transferred NOD recipients. The secretion of insulin was assessed by immunohistochemical staining. Red arrow indicates the insulin staining in the ( E ) PBS-treated group and the ( F ) VPA-treated Treg transferred NOD recipients.

Article Snippet: The BD IMagTM Mouse CD4 T Lymphocyte Enrichment set-DM (BD Biosciences, San Jose, CA, USA) was used for negative selection of CD4 T lymphocyte by removing non-CD4 T cells from splenocytes.

Techniques: Transferring, Transplantation Assay, Cell Culture, Immunohistochemical staining, Staining