Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Systemic Inflammatory Response Elicited by Superantigen Destabilizes T Regulatory Cells Rendering them Ineffective during Toxic Shock Syndrome ¹

doi: 10.4049/jimmunol.1400980

Figure Lengend Snippet: HLA-DR3 transgenic mice were injected with immune complexes comprising of IL-2 and anti-IL-2 antibodies on days 0, 1 and 2 (3xIL2C). On day 5, mice were killed and the distribution of various cell types in the spleens was analyzed by flow cytometry. (A) Representative dot plots and bar charts depicting distribution of CD4+CD25+FoxP3+ Tregs in naïve and 3xIL2C treated mice. (B) Bar chart depicting the distribution of CD4+ and CD8+ T cells expressing indicated TCR Vβ families in naïve and 3xIL2C treated mice (C) B cells and macrophages in naïve and 3xIL2C treated mice. Each bar represents mean±SE of data obtained from 4–6 mice in each group. * p<0.05 compared to naïve mice.

Article Snippet: Purification, expansion and adoptive transfer of in vitro expanded T regulatory cells CD4 + CD25 + T cells were isolated from naïve HLA-DR3 transgenic mice using magnetic mouse Treg purification kit following the manufacturer’s protocol (Miltenyi Biotec Inc, Auburn, CA, USA).

Techniques: Transgenic Assay, Injection, Flow Cytometry, Expressing

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Systemic Inflammatory Response Elicited by Superantigen Destabilizes T Regulatory Cells Rendering them Ineffective during Toxic Shock Syndrome ¹

doi: 10.4049/jimmunol.1400980

Figure Lengend Snippet: HLA-DR3 transgenic mice were left untreated or injected with immune complexes comprising of IL-2 and anti-IL-2 on days 0, 1 and 2 (3xIL2C). On day 5, mice were challenged with SEB (50 µg/mouse) or PBS and killed 3 days later for flow cytometric analysis. (A) Distribution of CD4+ and CD8+ T cells expressing TCR Vβ8 (SEB-responsive) and TCR Vβ6 (SEB non-responsive) in the spleens and (B) Thymus. Each bar represents mean±SE of data obtained from 6–8 mice in each group. * p<0.05 compared to naïve mice. # p<0.05 compared to SEB group.

Article Snippet: Purification, expansion and adoptive transfer of in vitro expanded T regulatory cells CD4 + CD25 + T cells were isolated from naïve HLA-DR3 transgenic mice using magnetic mouse Treg purification kit following the manufacturer’s protocol (Miltenyi Biotec Inc, Auburn, CA, USA).

Techniques: Transgenic Assay, Injection, Expressing

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Systemic Inflammatory Response Elicited by Superantigen Destabilizes T Regulatory Cells Rendering them Ineffective during Toxic Shock Syndrome ¹

doi: 10.4049/jimmunol.1400980

Figure Lengend Snippet: HLA-DR3 transgenic mice were left untreated or injected with IL-2, anti-IL-2 immune complexes on days 0, 1 and 2 (3xIL2C) to expand endogenous Tregs. On day 5, mice were challenged with SEB (50 µg/mouse) or PBS and killed 3 days later. Organs were collected in buffered formalin, paraffin-embedded and processed. Hematoxylin and Eosin stained sections were evaluated by light microscopy. Representative low and high magnification images are shown.

Article Snippet: Purification, expansion and adoptive transfer of in vitro expanded T regulatory cells CD4 + CD25 + T cells were isolated from naïve HLA-DR3 transgenic mice using magnetic mouse Treg purification kit following the manufacturer’s protocol (Miltenyi Biotec Inc, Auburn, CA, USA).

Techniques: Transgenic Assay, Injection, Staining, Light Microscopy

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Systemic Inflammatory Response Elicited by Superantigen Destabilizes T Regulatory Cells Rendering them Ineffective during Toxic Shock Syndrome ¹

doi: 10.4049/jimmunol.1400980

Figure Lengend Snippet: HLA-DR3 transgenic mice were left untreated or injected with IL-2, anti-IL-2 immune complexes on days 0, 1 and 2 to expand Tregs (3xIL2C). On day 5, mice were challenged with SEB (50 µg/mouse) or PBS and killed 3 days later. The percentage of CD25+FoxP3+ T cells in the CD4+ gated splenocytes were determined by flow cytometry and their absolute numbers were deduced. Each bar represents mean±SE of data obtained from 6–8 mice in each group. * p<0.05 compared to naïve mice. # p<0.05 compared to IL2C group.

Article Snippet: Purification, expansion and adoptive transfer of in vitro expanded T regulatory cells CD4 + CD25 + T cells were isolated from naïve HLA-DR3 transgenic mice using magnetic mouse Treg purification kit following the manufacturer’s protocol (Miltenyi Biotec Inc, Auburn, CA, USA).

Techniques: Transgenic Assay, Injection, Flow Cytometry

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Systemic Inflammatory Response Elicited by Superantigen Destabilizes T Regulatory Cells Rendering them Ineffective during Toxic Shock Syndrome ¹

doi: 10.4049/jimmunol.1400980

Figure Lengend Snippet: HLA-DR3 transgenic mice were left untreated or injected with IL-2, anti-IL-2 immune complexes on days 0, 1 and 2 to expand Tregs. On day 5, mice were challenged with SEB (50 µg/mouse) or PBS. Mice were killed 24 (day 6) or 48 hours (day 7) later, splenocytes isolated and stained with indicated antibodies. (A) Representative dot plots depicting distribution of CD4+FoxP3+ and CD4+FoxP3 T cells in different groups of mice (B) Histogram overlays depicting expression of GITR on the cells gated as above (C) Bar chart depicting percentages of GITR+ cells within the indicated gates and (D) Bar chart depicting median fluorescent intensity of GITR expression on cells within the indicated gates. Each bar represents mean±SE of data obtained from 3 mice in each group. * p<0.05 compared to naïve mice.

Article Snippet: Purification, expansion and adoptive transfer of in vitro expanded T regulatory cells CD4 + CD25 + T cells were isolated from naïve HLA-DR3 transgenic mice using magnetic mouse Treg purification kit following the manufacturer’s protocol (Miltenyi Biotec Inc, Auburn, CA, USA).

Techniques: Transgenic Assay, Injection, Isolation, Staining, Expressing

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Systemic Inflammatory Response Elicited by Superantigen Destabilizes T Regulatory Cells Rendering them Ineffective during Toxic Shock Syndrome ¹

doi: 10.4049/jimmunol.1400980

Figure Lengend Snippet: IFN-γ-sufficient and IFN-γ-deficient HLA-DR3 transgenic mice were injected with 100 µg of rat anti-mouse IL-17 antibodies or isotype controls. Ten minutes later, mice were challenged with SEB (50 µg/mouse) or PBS and killed 3 days later. Distribution of CD4+CD25+FoxP3+ Tregs was determined by flow cytometry. Each bar represents mean±SE of data obtained from 3–6 mice in each group. * p<0.05.

Article Snippet: Purification, expansion and adoptive transfer of in vitro expanded T regulatory cells CD4 + CD25 + T cells were isolated from naïve HLA-DR3 transgenic mice using magnetic mouse Treg purification kit following the manufacturer’s protocol (Miltenyi Biotec Inc, Auburn, CA, USA).

Techniques: Transgenic Assay, Injection, Flow Cytometry

Journal: Molecular Therapy. Nucleic Acids

Article Title: R88-APOBEC3Gm Inhibits the Replication of Both Drug-resistant Strains of HIV-1 and Viruses Produced From Latently Infected Cells

doi: 10.1038/mtna.2014.2

Figure Lengend Snippet: Doxycycline (Dox) regulated R88-A3G D128K (R88-A3Gm) expression and antiviral effect in pTZ-R88-A3Gm–transduced CD4 + C8166 T cell line. pTZ-control and pTZ-R88-A3Gm–transduced C8166 T cells were induced with Dox (1 µg/ml or as indicated) for 2 days and infected with pNL-4.3 virus at ( a , b ) a multiplicity of infection (MOI) of 1 or ( c ) MOI of 0.01. ( a ) Levels of HIV-1 Gag-p24 in the supernatants were measured by p24 enzyme-linked immunosorbent (ELISA) assay at 5 days postinfection. ( b ) Viruses produced from infected C8166 T cells were concentrated from the supernatant by ultracentrifugation after 5 days of infection. The presence of R88-A3G was analyzed by western blotting with anti-A3G antibody, and HIV-1 Gag protein was detected by anti-p24 antibody. ( c ) Virus replication was measured by p24 ELISA from the supernatant at different time intervals (left). HIV-1–induced syncytium formation was visualized under microscopy (right). Results were from one representative of two independent experiments. A3G, human cytidine deaminase apolipoprotein B messenger RNA editing enzyme catalytic polypeptide 3; pTZ, pTRIPZ.

Article Snippet: CD4 + T lymphocytes were isolated from peripheral blood mononuclear cells by negative selection with EasySep Human CD4 + CD25 + T Cell Isolation Kit (Stemcell Technologies, Vancouver, Canada), activated with 3 μg/ml phytohemaglutinin and maintained in RPMI-1640 supplemented with 10 U interleukin-2.

Techniques: Expressing, Control, Infection, Virus, Enzyme-linked Immunosorbent Assay, Produced, Western Blot, Microscopy

Journal: Molecular Therapy. Nucleic Acids

Article Title: R88-APOBEC3Gm Inhibits the Replication of Both Drug-resistant Strains of HIV-1 and Viruses Produced From Latently Infected Cells

doi: 10.1038/mtna.2014.2

Figure Lengend Snippet: pTZ-R88-A3Gm–transduced primary cells were significantly resistant to HIV-1 replication. ( a ) Primary CD4 + T cells and macrophages were transduced with pTZ-R88-A3Gm or pTZ-control vectors for 3 days. Total DNA was extracted from transduced cells and subjected to real-time polymerase cgain reaction analysis to quantify the level of R88-A3G. ( b ) Transduced CD4 + T cells and macrophages were induced with doxycycline (Dox) (1 µg/ml) and infected with either nevirapine-resistant or pNL4.3-Bal strains of HIV-1. Viral replication was measured by p24 enzyme-linked immunosorbent assay. Result a was from one representative of two donors. Result b was from one representative of three donors. A3G, human cytidine deaminase apolipoprotein B messenger RNA editing enzyme catalytic polypeptide 3; pTZ, pTRIPZ; R88-A3Gm, R88-A3G D128K .

Article Snippet: CD4 + T lymphocytes were isolated from peripheral blood mononuclear cells by negative selection with EasySep Human CD4 + CD25 + T Cell Isolation Kit (Stemcell Technologies, Vancouver, Canada), activated with 3 μg/ml phytohemaglutinin and maintained in RPMI-1640 supplemented with 10 U interleukin-2.

Techniques: Transduction, Control, Infection, Enzyme-linked Immunosorbent Assay

Journal: Molecular Therapy. Nucleic Acids

Article Title: R88-APOBEC3Gm Inhibits the Replication of Both Drug-resistant Strains of HIV-1 and Viruses Produced From Latently Infected Cells

doi: 10.1038/mtna.2014.2

Figure Lengend Snippet: pTZ-R88-A3Gm–mediated inhibition of HIV-1 replication in primary latently infected cells. ( a ) Unstimulated CD4 + T cells were infected with nevirapine-resistant virus (multiplicity of infection of 2.5) by spinoculation (day 0). Three days later, the infected cells were activated by phytohemaglutinin (5 µg/ml) and interleukin-2 (IL-2) (100 U/ml) treatment (day 3), and then transduced with the pTZ-R88-A3Gm or pTZ-control vectors (day 5). Viral replication was monitored by Gag-p24 enzyme-linked immunosorbent assay (ELISA) at different time points after cells were induced with doxycycline (Dox) (1 µg/ml, day 6). ( b ) C8166 T cells were infected with cell free supernatants from pTZ-R88-A3Gm and pTZ-control transduced primary CD4 + T cells collected at day 8. Viral replication in C8166 T cells was measured by Gag-p24 ELISA at 3 and 5 days postinfection. The values were the averages of triplicates with the indicated standard deviations. ( c ) Unstimulated CD4 + T cells were infected with the same viruses as for ( a ) (day 0) and transduc e d with the pTZ-R88-A3Gm or pTZ-control vectors (day 1). Two days after Dox induction, the infected cells were activated by phytohemaglutinin (5 µg/ml) and interleukin-2 (100 U/ml) treatment (day 4), and viral replication was monitored by Gag-p24 ELISA at different time points. ( d ) C8166 T cells were infected with cell free supernatants from pTZ-R88-A3Gm and pTZ-control transduced primary CD4 + T cells collected at day 7. Viral replication in C8166 T cells was measured as for ( b ). The values were the averages of triplicates with the indicated standard deviations. Statistical significance was calculated as described in . Results were from one representative of two donors. A3G, human cytidine deaminase apolipoprotein B messenger RNA editing enzyme catalytic polypeptide 3.

Article Snippet: CD4 + T lymphocytes were isolated from peripheral blood mononuclear cells by negative selection with EasySep Human CD4 + CD25 + T Cell Isolation Kit (Stemcell Technologies, Vancouver, Canada), activated with 3 μg/ml phytohemaglutinin and maintained in RPMI-1640 supplemented with 10 U interleukin-2.

Techniques: Inhibition, Infection, Virus, Transduction, Control, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Immunology

Article Title: Shifting gears: Study of immune system parameters of male habitual marathon runners

doi: 10.3389/fimmu.2022.1009065

Figure Lengend Snippet: Gating strategy for isolation of CD3+CD4+CD25+ T cells and CD3+CD4+CD25- T cells from PBMCs. Characteristic dot plots showing purity after cell sorting using BD FACS Aria III Cell Sorter.

Article Snippet: CD3+CD4+CD25+ and CD3+CD4+CD25- T cells were isolated from PBMCs using the BD FACS Aria III Cell Sorter.

Techniques: Isolation, FACS

Journal: Frontiers in Immunology

Article Title: The Long Noncoding RNA MALAT1 Induces Tolerogenic Dendritic Cells and Regulatory T Cells via miR155/Dendritic Cell-Specific Intercellular Adhesion Molecule-3 Grabbing Nonintegrin/IL10 Axis

doi: 10.3389/fimmu.2018.01847

Figure Lengend Snippet: Ectopic MALAT1 favors tolerogenic dendritic cells (tDCs) by inducing T cell hyporesponsiveness and Tregs expansion. Dendritic cells (DCs) were transfected with a MALAT1 pcDNA3.1 vector (pMALAT1, 2.5 µg/ml), a control vector (Vector, 0.625 µg/ml), MALAT1 siRNA (siMALAT1, 100 nM), or siRNA control (siNC, 25 nM) for 6 h before LPS treatment. (A) The expression of MALAT1 was confirmed by quantitative real-time reverse transcription PCR in DCs receiving the different treatments. Expression of MALAT1 was effectively upregulated by pMALAT1, whereas MALAT1 siRNA suppressed MALAT1 expression in DCs. (B) Expression of the costimulatory markers CD80, CD86, and MHCII was analyzed by flow cytometry and is shown as the percentage of CD11c + cells. MALAT1-suppressed DCs displayed increases in CD80, CD86, and MHCII expression levels compared with those of control DCs treated with LPS. (C) The cytokine secretion levels of IL10, TGFβ, IL12, IFNγ, and IL6 in the DC supernatants were analyzed by ELISA. The induced expression of MALAT1 in LPS-stimulated DCs resulted in reduced levels of IL6, IL12, and IFNγ, whereas levels of IL10 were significantly increased, but no significant effect was observed on TGF-β. (D) DC-triggered T cell proliferation was evaluated by BrdU-ELISA. For this, DCs were treated with mitomycin C (10 mg/ml, 2 h) and cocultured with allogeneic T cells for 48 h at a DC:T cell ratio of 1:20. T cells cocultured with MALAT1-overexpressing DCs exhibited reduced proliferative ability compared with those cocultured with LPS-DCs. (E,F) In the cocultured T cells, the numbers of Tregs (CD4 + , CD25 + , and Foxp3 + ) cells were assessed by flow cytometry. Boxes depict gates and numbers correspond to the percentage of cells in each gate. The data are shown with a representative flow cytometry (E) and the percentages (F) . The number of Tregs was significantly increased when T cells were cocultured with MALAT1-overexpressing DCs compared with LPS-DCs. (G–I) Tregs (CD4 + CD25 + ) isolated from T cells cocultured with pMALAT1-conditioned DCs by MACS were added into coculture with DCs (from BALB/c, third-party mice or both types ratio of 1:1) and T cells, with a Treg:T cell:DC ratio of 10:10:1. Then, the suppressive ability of the Tregs was assessed by T cell proliferation assays using BrdU-ELISA. Tregs derived from MALAT1-overexpressing DC (from BALB/c mice) cocultures suppressed T cell proliferation in the presence of BALB/c DCs as stimulators but not in the presence of DCs from third-party mice, whereas there was no significant difference in T cell proliferation between both types of LPS-induced DCs. The data are presented as the mean ± SD from at least three independent experiments. * P < 0.05; ** P < 0.01. pMALAT1-Treg, Tregs induced by pMALAT1-conditioned DCs in MLRs; No-Treg, without Tregs.

Article Snippet: Then, the isolated CD4 + T cell population was positively selected with anti-CD25 mouse microbeads into CD4 + CD25 + and CD4 + CD25 − T cell fractions (Miltenyi Biotec).

Techniques: Transfection, Plasmid Preparation, Control, Expressing, Reverse Transcription, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Isolation, Derivative Assay

Journal: Frontiers in Immunology

Article Title: The Long Noncoding RNA MALAT1 Induces Tolerogenic Dendritic Cells and Regulatory T Cells via miR155/Dendritic Cell-Specific Intercellular Adhesion Molecule-3 Grabbing Nonintegrin/IL10 Axis

doi: 10.3389/fimmu.2018.01847

Figure Lengend Snippet: Dendritic cell-specific intercellular adhesion molecule-3 grabbing nonintegrin (DC-SIGN) is essential for the maintenance of MALAT1-conditioned dendritic cell (DC) tolerogenic functions. (A–C) DCs were transfected with siMALAT1 or pMALAT1 for 6 h before LPS stimulation. (A) The expression of DC-SIGN was detected by quantitative real-time reverse transcription PCR (qRT-PCR). The mRNA expression of DC-SIGN was significantly increased in DCs with overexpressed MALAT1 but decreased in those with MALAT1 shRNA compared with control DCs. (B) The level of DC-SIGN was assessed by flow cytometry and shown as percentages. (C) The DC-SIGN expression in DCs was significantly upregulated by MALAT1 overexpression, while MALAT1 downregulation counteracted this effect. (D) The expression of DC-SIGN was detected by qRT-PCR in DCs transfected with siDC-SIGN or the control (25 nM, 6 h). DC-SIGN-targeted shRNA transfection obviously downregulated the expression of DC-SIGN in DCs. (E–K) DCs were pre-incubated with or without shDC-SIGN for 6 h and subsequently cultured with the pMALAT1 vector before LPS stimulation. (E) Expression of the costimulatory markers CD80, CD86, and MHCII was assessed by flow cytometry. In DCs pretreated with the pMALAT1 vector plus DC-SIGN shRNA, the costimulatory molecules were significant increased compared with DCs only treated with the pMALAT1 vector, with no obvious difference compared to control non-treated DCs. (F) The cytokine secretion levels of IL10 in the DC supernatants were analyzed by ELISA. (G) The mRNA expression of IL10 in the DCs was analyzed by qRT-PCR. DC-SIGN knockdown by DC-SIGN shRNA impaired the MALAT1-induced upregulation of IL10 protein and mRNA expression in DCs. (H) The cytokine secretion levels of IL6, IL12, and IFNγ in the DC supernatants were analyzed by ELISA. (I) Allogeneic T cells were cocultured with these DCs and incubated with BrdU (10 mM, 24 h) to quantify T cell proliferation by BrdU-ELISA. Knockdown of DC-SIGN blocked the lower T cell proliferative activity induced by ectopic MALAT1. (J) Flow cytometry assessment of the number of Tregs (CD4 + , CD25 + , and Foxp3 + ) in these cocultures, shown as percentages. (K) Knockdown of DC-SIGN blocked the increased Tregs numbers induced by ectopic MALAT1. The data are presented as the mean ± SD from at least three independent experiments.* vs control group, P < 0.05; # vs pMALAT1 group, P < 0.05.

Article Snippet: Then, the isolated CD4 + T cell population was positively selected with anti-CD25 mouse microbeads into CD4 + CD25 + and CD4 + CD25 − T cell fractions (Miltenyi Biotec).

Techniques: Transfection, Expressing, Reverse Transcription, Quantitative RT-PCR, shRNA, Control, Flow Cytometry, Over Expression, Incubation, Cell Culture, Plasmid Preparation, Enzyme-linked Immunosorbent Assay, Knockdown, Activity Assay

Journal: Frontiers in Immunology

Article Title: The Long Noncoding RNA MALAT1 Induces Tolerogenic Dendritic Cells and Regulatory T Cells via miR155/Dendritic Cell-Specific Intercellular Adhesion Molecule-3 Grabbing Nonintegrin/IL10 Axis

doi: 10.3389/fimmu.2018.01847

Figure Lengend Snippet: DC-SIGN + subsets induced by enforced MALAT1 exert a more potent tolerogenic ability. After pMALAT1 transfection and LPS stimulation, dendritic cells (DCs) were sorted by MACS into DC-SIGN + DCs and DC-SIGN − DCs. Then, these DCs were conditioned with mitomycin C (10 mg/ml, 2 h) and cocultured with allogeneic T cells for 48 h. (A) T cell proliferation initiated by DCs was assessed by BrdU-ELISA. DC-SIGN + DCs exhibited significantly more suppressive effects on primed T cell responses than did LPS-stimulated DCs and DC-SIGN − DCs. (B,C) The numbers of Tregs (CD4 + CD25 + Foxp3 + ) in T cell cocultures were assessed by flow cytometry (B) and are shown as percentages (C) . DC-SIGN + subsets, but not DC-SIGN − subsets, induced the generation of Tregs compared with both LPS-DCs and DC-SIGN − DCs. (D) IL10 mRNA expression levels in these isolated Tregs were assessed by quantitative real-time reverse transcription PCR. IL10 mRNA levels in Tregs induced by DC-SIGN + populations were significantly higher than those in Tregs induced by LPS-DCs or DC-SIGN − populations. (E,F) Tregs (CD4 + CD25 + ) isolated from different DC and T cell cocultures by MACS were added to a new coculture of mitomycin C-conditioned DCs (syngeneic or third party) and T cells (Treg:T cell:DC ratio of 10:10:1). The suppressive functions of Tregs on DC-primed T cell responses were assessed by BrdU-ELISA. The DC-SIGN + subpopulation-induced Tregs, but not those induced by DC-SIGN − subsets, showed marked inhibition of DC-primed T cell proliferation. The data are presented as the mean ± SD from at least three independent experiments. * P < 0.05; ** P < 0.01.

Article Snippet: Then, the isolated CD4 + T cell population was positively selected with anti-CD25 mouse microbeads into CD4 + CD25 + and CD4 + CD25 − T cell fractions (Miltenyi Biotec).

Techniques: Transfection, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Expressing, Isolation, Reverse Transcription, Inhibition

Journal: Frontiers in Immunology

Article Title: The Long Noncoding RNA MALAT1 Induces Tolerogenic Dendritic Cells and Regulatory T Cells via miR155/Dendritic Cell-Specific Intercellular Adhesion Molecule-3 Grabbing Nonintegrin/IL10 Axis

doi: 10.3389/fimmu.2018.01847

Figure Lengend Snippet: Adoptive transfer of MALAT1-overexpressing dendritic cells (DCs) protected mice from acute transplant rejection and induced Tregs expression. Transplant-recipient mice were transfused with phosphate-buffered saline (PBS), LPS-stimulated DCs, MALAT1-overexpressing DCs (pMALAT1-DCs), or sorted DC-SIGN + cells from MALAT1-overexpressing DCs (pMALAT1 + DC-SIGN + ) before transplantation or oral treatment with 1 mg/kg/day of tacrolimus for 7 days. (A) Graft survival times of recipients. Abbreviation: MST, median survival time. The survival times in the groups were compared using Mann–Whitney U testing. Transfusion with MALAT1-overexpressing DCs or DC-SIGN + DC populations significantly prolonged cardiac allograft survival compared with LPS-DC and PBS transfusion. (B) Histologic studies of cardiac allografts harvested 7 days after transplantation were stained with hematoxylin and eosin (H&E) (left) and immunohistochemically stained for Foxp3 (right) [ (B) , original magnification 20×]. (C) Assessment of H&E staining by grading according to the 2005 classification of the International Society for Heart and Lung Transplantation for acute cellular rejection. (D) Cell counts of infiltrating Foxp3 + cells in cardiac allografts from each group 7 days post-transplantation by immunohistochemical staining. Cardiac allografts from recipients transfused with MALAT1-overexpressing DCs and DC-SIGN + subpopulations had more Foxp3-positive staining cells than did those from recipients transfused with LPS-DC and PBS. The data indicate mean ± SD values derived from five samples in each group and were compared using analysis of variance with the Ryan method. (E,F) Tregs (CD4 + CD25 + Foxp3 + ) in splenic T cells were assessed by flow cytometry (E) and are shown as percentages (F) . Treg numbers were significantly elevated in the spleens of recipient mice injected with pMALAT1-DCs or pMALAT1-DC-SIGN + DCs. Filled histograms represent isotype-matched irrelevant specificity controls. (G,H) Tregs (CD4 + CD25 + ) isolated from recipients transfused with pMALAT1-conditioned DCs by MACS were added to cocultures with mitomycin C-treated splenic cells (from BALB/c mice or third-party mice) and T cells. The suppressive ability of Tregs was assessed by T cell proliferation using BrdU-ELISA. Tregs significantly decreased T cell proliferation when cocultured with donor splenic cells, but not when cocultured with third-party splenic cells. (I) Splenic T cells were separated from recipient mice at day 7 post-transplantation. The proliferating activity of splenic T cells was assessed by BrdU-ELISA. In the recipients transfused with pMALAT1-DCs or pMALAT1-DC-SIGN + DCs, splenic T cell proliferation was significantly suppressed. (J) IL10, IL12, and IL6 concentrations in the sera of recipient mice were measured by ELISA. The IL12 and IL6 in serum were significantly decreased and IL10 was significantly increased in mice transfused with pMALAT1-DCs or pMALAT1-DC-SIGN + DCs. The data are presented as the mean ± SD from at least five independent experiments. * P < 0.05; ** P < 0.01. pMALAT1-DCs, MALAT1-overexpressing DCs; pMALAT1 + DC-SIGN + , DC-SIGN + subsets from MALAT1-overexpressing DCs.

Article Snippet: Then, the isolated CD4 + T cell population was positively selected with anti-CD25 mouse microbeads into CD4 + CD25 + and CD4 + CD25 − T cell fractions (Miltenyi Biotec).

Techniques: Adoptive Transfer Assay, Expressing, Saline, Transplantation Assay, MANN-WHITNEY, Staining, Immunohistochemical staining, Derivative Assay, Flow Cytometry, Injection, Isolation, Enzyme-linked Immunosorbent Assay, Activity Assay

Journal: Frontiers in Immunology

Article Title: The Long Noncoding RNA MALAT1 Induces Tolerogenic Dendritic Cells and Regulatory T Cells via miR155/Dendritic Cell-Specific Intercellular Adhesion Molecule-3 Grabbing Nonintegrin/IL10 Axis

doi: 10.3389/fimmu.2018.01847

Figure Lengend Snippet: In vivo transfer of MALAT1-overexpressing dendritic cells (DCs) deferred autoimmune myocarditis progression. After induction of experimental autoimmune myocarditis (EAM) (immunization with α-myosin H-chain peptide), mice were transfused with MALAT1-overexpressing DCs, LPS-DC, or phosphate-buffered saline (PBS), respectively. Hearts were collected on day 21 post-immunization. (A) Consecutive cardiac sections were stained with hematoxylin and eosin (H&E) (original magnification 20×). (B) Analysis of H&E staining by grading as described in Section “ .” Transfusion with MALAT1-overexpressing DCs significantly alleviated acute myocardial inflammation in EAM mice compared with PBS transfusion. (C–E) Myocardial function was evaluated by echocardiography (C) on day 42 post-immunization, and the parameters of LVEDDs (E) and EF (D) were as shown. Animals injected with MALAT1-overexpressing DCs showed less LV and LVEDDs and more EF than did those that received LPS-DCs and PBS transfusion. (F–I) Splenic T cells were separated from EAM mice at day 21 post-immunization. (F) IL10, IL12, and IL6 production in the serum of EAM mice was detected by ELISA. The expressions of IL12 and IL6 were significantly decreased and that of IL10 was increased in mice injected with pMALAT1-DCs. (G) BrdU-ELISA determined the proliferating activity of splenic T cells. (H,I) Tregs (CD4 + CD25 + Foxp3 + ) in splenic T cells were assessed by flow cytometry (H) and are shown as percentages (I) . Filled histograms represent isotype-matched irrelevant specificity controls. The data are presented as the mean ± SD from at least three independent experiments. * P < 0.05; ** P < 0.01.

Article Snippet: Then, the isolated CD4 + T cell population was positively selected with anti-CD25 mouse microbeads into CD4 + CD25 + and CD4 + CD25 − T cell fractions (Miltenyi Biotec).

Techniques: In Vivo, Saline, Staining, Injection, Enzyme-linked Immunosorbent Assay, Activity Assay, Flow Cytometry

Journal: Journal of Immunology Research

Article Title: Analysis of the Heterogeneity of CD4 + CD25 + T Cell TCR β CDR3 Repertoires in Breast Tumor Tissues, Lung Metastatic Tissues, and Spleens from 4T1 Tumor-Bearing BALB/c Mice

doi: 10.1155/2020/3184190

Figure Lengend Snippet: FCM detection of breast tumor tissues, lung metastatic tissues, and spleens sorted CD4 + CD25 + T cells in three 4T1 tumor-bearing BALB/c mice.

Article Snippet: The following reagents and instruments were used: CD4 + CD25 + T cells Isolation Kit, CD4-PC5.5, CD3 ε -FITC, and CD25-PE antibody (mouse, Miltenyi Biotec, Germany); Fatal bovine serum (FBS), trypsin, RPMI-1640 medium (Gibco, USA); DNA QIAamp DNA Mini Kit, Dneasy® Blood & Tissue Kit (Qiagen, Germany); Ficoll Lymphocyte Separation Solution (Beijing Solaibao Technology Co., Ltd.); and Gentle MACS dissociator (Miltenyi Biotec, Germany).

Techniques:

Journal: Journal of Immunology Research

Article Title: Analysis of the Heterogeneity of CD4 + CD25 + T Cell TCR β CDR3 Repertoires in Breast Tumor Tissues, Lung Metastatic Tissues, and Spleens from 4T1 Tumor-Bearing BALB/c Mice

doi: 10.1155/2020/3184190

Figure Lengend Snippet: Clonotypes distribution plots (1/DS) (a–f) and statistical analysis of 1/DS (g) of CD4 + CD25 + T cell TCR β CDR3 repertoires among tissues from three 4T1 tumor-bearing BALB/c mice; statistical analysis (h) of the five/ten/twenty most highly expanded clones of CDR3 repertoires in three 4T1 tumor-bearing BALB/c mice ( n = 3, ∗ p ≤ 0.05).

Article Snippet: The following reagents and instruments were used: CD4 + CD25 + T cells Isolation Kit, CD4-PC5.5, CD3 ε -FITC, and CD25-PE antibody (mouse, Miltenyi Biotec, Germany); Fatal bovine serum (FBS), trypsin, RPMI-1640 medium (Gibco, USA); DNA QIAamp DNA Mini Kit, Dneasy® Blood & Tissue Kit (Qiagen, Germany); Ficoll Lymphocyte Separation Solution (Beijing Solaibao Technology Co., Ltd.); and Gentle MACS dissociator (Miltenyi Biotec, Germany).

Techniques: Clone Assay

Journal: Journal of Immunology Research

Article Title: Analysis of the Heterogeneity of CD4 + CD25 + T Cell TCR β CDR3 Repertoires in Breast Tumor Tissues, Lung Metastatic Tissues, and Spleens from 4T1 Tumor-Bearing BALB/c Mice

doi: 10.1155/2020/3184190

Figure Lengend Snippet: The number of public sequences of CD4 + CD25 + T cells TCR β CDR3 repertoires among three tissues of each 4T1 tumor-bearing BALB/c mice (a); the number and ratio (Supplement Table ) and the statistical analysis (b) of public CDR3 sequences from their respective unique productive CDR3 repertoires in each type of tissue ( n = 3, ∗∗ p < 0.01).

Article Snippet: The following reagents and instruments were used: CD4 + CD25 + T cells Isolation Kit, CD4-PC5.5, CD3 ε -FITC, and CD25-PE antibody (mouse, Miltenyi Biotec, Germany); Fatal bovine serum (FBS), trypsin, RPMI-1640 medium (Gibco, USA); DNA QIAamp DNA Mini Kit, Dneasy® Blood & Tissue Kit (Qiagen, Germany); Ficoll Lymphocyte Separation Solution (Beijing Solaibao Technology Co., Ltd.); and Gentle MACS dissociator (Miltenyi Biotec, Germany).

Techniques:

Journal: Journal of Immunology Research

Article Title: Analysis of the Heterogeneity of CD4 + CD25 + T Cell TCR β CDR3 Repertoires in Breast Tumor Tissues, Lung Metastatic Tissues, and Spleens from 4T1 Tumor-Bearing BALB/c Mice

doi: 10.1155/2020/3184190

Figure Lengend Snippet: The number of public CDR3 sequences from CD4 + CD25 + T cells TCR β CDR3 repertoires among same tissues from three 4T1 tumor-bearing BALB/c mice (a); the number and ratio (b) and the statistical analysis (c) of public CDR3 sequences from their respective unique productive CDR3 repertoires and total productive CDR3 repertoires, respectively.

Article Snippet: The following reagents and instruments were used: CD4 + CD25 + T cells Isolation Kit, CD4-PC5.5, CD3 ε -FITC, and CD25-PE antibody (mouse, Miltenyi Biotec, Germany); Fatal bovine serum (FBS), trypsin, RPMI-1640 medium (Gibco, USA); DNA QIAamp DNA Mini Kit, Dneasy® Blood & Tissue Kit (Qiagen, Germany); Ficoll Lymphocyte Separation Solution (Beijing Solaibao Technology Co., Ltd.); and Gentle MACS dissociator (Miltenyi Biotec, Germany).

Techniques: