Journal: Experimental and Therapeutic Medicine

Article Title: IL-35 improves T reg -mediated immune suppression in atherosclerotic mice

doi: 10.3892/etm.2016.3649

Figure Lengend Snippet: Plasma levels of Foxp3. Peripheral blood mononuclear cells were collected from each group, and the proportions of CD4 + CD25 + Foxp3 + T reg /CD4 + T-cells were analyzed by flow cytometry, and quantified using FlowJo 7.6.1 software. (A) Histogram. Data are presented as the mean ± standard deviation. *P<0.01, vs. the AS group. (B) Negative group, (C) AS group, (D) atorvastatin group and (E) IL-35 group. Foxp3, forkhead box protein 3; AS, atherosclerosis; IL-35, interleukin-35; APC, allophycocyanin; PE, phycoerythrin.

Article Snippet: Anti-Foxp3 antibody was purchased from Wuhan Boster Biological Technology, Ltd. (Wuhan, China).

Techniques: Clinical Proteomics, Flow Cytometry, Software, Standard Deviation

Journal: Experimental and Therapeutic Medicine

Article Title: IL-35 improves T reg -mediated immune suppression in atherosclerotic mice

doi: 10.3892/etm.2016.3649

Figure Lengend Snippet: Levels of Foxp3 in atherosclerotic lesions. The deposition of Foxp3 in arteries from various groups was detected by immunohistochemistry (magnification, ×400). Positive Foxp3 was shown as brown nuclei. In the (A) negative and (B) AS groups, the deposition of Foxp3 in the lesions was minimal. Conversely, Foxp3 deposition was increased in the lesions of the (C) atorvastatin and (D) IL-35 groups, as compared with the AS group. (E) This was shown to be significant following quantification. There was no significant difference in the levels of Foxp3 between the atorvastatin and IL-35 groups. *P<0.01, vs. the negative control. Foxp3, forkhead box protein 3; AS, atherosclerosis; IL-35, interleukin-35.

Article Snippet: Anti-Foxp3 antibody was purchased from Wuhan Boster Biological Technology, Ltd. (Wuhan, China).

Techniques: Immunohistochemistry, Negative Control

Journal: Molecular medicine (Cambridge, Mass.)

Article Title: TWEAK/Fn14 disrupts Th17/Treg balance and aggravates conjunctivitis by inhibiting the Nrf2/HO-1 pathway in allergic conjunctivitis mice.

doi: 10.1186/s10020-024-01004-5

Figure Lengend Snippet: Fig. 3 TWEAK regulated the Th17/Treg cell ratio in AC mice. (A) The effect of TWEAK knockdown on Th17 and Treg cell ratio in spleen of AC mice was observed by flow cytometry. (B-C) WB and IHC were employed to evaluate the expression levels of FoxP3 and RORγt in mice conjunctival tissue. (D) The effect of TWEAK knockdown on the levels of Th17 and Treg cytokines in mice spleen were evaluated by ELISA. *p < 0.05, **p < 0.01, ***p < 0.001 vs. Con + AAV-shNC; #p < 0.05, ##p < 0.01, ###p < 0.001 vs. AC + AAV-shNC; ns, no significant difference

Article Snippet: After deparaffinization and rehydration, tissue slides were treated with 100 μL endogenous peroxidase blockers for 10 min. Then, the slides were incubated with primary antibody against RORγt (14-6981-82, 1:200; ThermoFisher, Waltham, MA, USA), FoxP3 (BA2032-1, 1:200; Boster, Pleasanton, CA, USA), TWEAK (BM4635, 1:200; Boster), Fn14 (GTX85216; 1:200, Genetex, Alton, CA, USA), Nrf2 (PA5-27882; 1:200, ThermoFisher) and HO-1 (PA5-77833, 1:200; ThermoFisher) overnight at 4 °C.

Techniques: Knockdown, Flow Cytometry, Expressing, Enzyme-linked Immunosorbent Assay

Journal: Molecular medicine (Cambridge, Mass.)

Article Title: TWEAK/Fn14 disrupts Th17/Treg balance and aggravates conjunctivitis by inhibiting the Nrf2/HO-1 pathway in allergic conjunctivitis mice.

doi: 10.1186/s10020-024-01004-5

Figure Lengend Snippet: Fig. 6 Inhibition of Nrf2/HO-1 signaling pathway affected Th17/Treg cell ratio in AC mice with TWEAK knockdown. (A) The effect of Nrf2 inhibitor on Th17/Treg cell ratio in AC mice with TWEAK knockdown was assessed by flow cytometry. (B-C) WB and IHC assays were employed to evaluate the expres sion of FoxP3 and RORγt in conjunctival tissue of AC mice. (D) The levels of Th17 and Treg cytokines in mice spleen were detected by ELISA. *p < 0.05, **p < 0.01, ***p < 0.001 vs. AC + AAV-shNC; #p < 0.05, ##p < 0.01, ###p < 0.001 vs. AC + AAV-shTWEAK

Article Snippet: After deparaffinization and rehydration, tissue slides were treated with 100 μL endogenous peroxidase blockers for 10 min. Then, the slides were incubated with primary antibody against RORγt (14-6981-82, 1:200; ThermoFisher, Waltham, MA, USA), FoxP3 (BA2032-1, 1:200; Boster, Pleasanton, CA, USA), TWEAK (BM4635, 1:200; Boster), Fn14 (GTX85216; 1:200, Genetex, Alton, CA, USA), Nrf2 (PA5-27882; 1:200, ThermoFisher) and HO-1 (PA5-77833, 1:200; ThermoFisher) overnight at 4 °C.

Techniques: Inhibition, Knockdown, Flow Cytometry, Enzyme-linked Immunosorbent Assay

Journal: Nature immunology

Article Title: Control of peripheral tolerance by regulatory T cell-intrinsic Notch signaling

doi: 10.1038/ni.3288

Figure Lengend Snippet: ( a ) Frequencies and numbers of CD3 and CD4 T cells from the spleen of 8 weeks old Foxp3 EGFPCre , Foxp3 EGFPCre Pofut Δ/Δ , Foxp3 EGFPCre Rbpj Δ/Δ and Foxp3 EGFPCre Notch1 Δ/Δ mice. ( b ) Flow cytometric analyses of CD4 and Foxp3 markers on CD3 + T cells are shown. ( c ) Frequencies and numbers of T reg cells for each group of panel (a). ( d ) Flow cytometric analysis of CD62L and CD44 markers on CD4 + T cells are shown. ( e ) Frequencies and numbers of memory CD4 + T cells for each group. ( f ) Flow cytometric analyses of IFN-γ and IL-17 on CD4 + T cells are shown. ( g ) Frequencies and numbers of IFN-γ producing CD4 + T cells for each group. ( h ) Flow cytometric analyses of IFN-γ and IL-17 expression in CD8 + T cells. ( i ) Frequencies and numbers of IFN-γ producing CD8 + T cells shown in panel (H). ( j ) Expression of Foxp3, CD25, CTLA4, Helios and Nrp1 markers were evaluated of splenic T reg cells and expressed as mean fluorescence intensity (MFI). Results are representative of at least 3 experiments per panel. * p<0.05, ** p<0.01, *** p<0.001 and **** p<0.0001 by one way ANOVA with post test analysis.

Article Snippet: Chromatin immunoprecipitation on purified T reg cells was performed with Agarose ChIP Kit (Pierce) and anti-RBPJ (Cell signaling), anti-Notch1 (biolegend), anti- Foxp3 (MBL), anti-Histone H3 trimethylated on Lysine 4 (H3K4me3) (abcam), anti-dimethylated on Lysine 27 (H3K27me2) (Millipore) and respective isotype control antibodies.

Techniques: Expressing, Fluorescence

Journal: Nature immunology

Article Title: Control of peripheral tolerance by regulatory T cell-intrinsic Notch signaling

doi: 10.1038/ni.3288

Figure Lengend Snippet: ( a ) Survival and ( b ) severity score of lethally irradiated (8.5/9Gy) BALB/c mice infused with C57Bl/6 Foxp3 EGFPCre , Foxp3 EGFPCre Rbpj Δ/Δ or Foxp3 EGFPCre Notch1 Δ/Δ T cell-depleted bone marrow, either alone (BM only) or together with spleen cells of the respective genotypes. For T cell subpopulation analyses were carried out on spleen cells at day 5 post adoptive transfer. The BM only groups were not included in panel b. ( c ) Flow cytometric analyses of IFN-γ and Foxp3 markers on CD4 + T cells are shown. ( d ) Frequencies of IFN-γ producing CD4 + T conv (Foxp3 − ) and T reg (Foxp3 + ) cells for each group. ( e ) Flow cytometric analyses of IFN-γ and IL-2 markers on CD8 + T cells. ( f ) Frequencies and numbers of IFN-γ producing CD8 + T cells for each group. ( g ) Flow cytometric analyses of Foxp3 marker on CD4 + T cells are shown. ( h ) Frequencies and numbers T reg cells for each group. ( i ) Viability dye and Annexin V (AnnV) staining of T reg cells are shown ( j ) Frequencies of apoptotic (AnnV + ) T conv and T reg cells for each group. ( k ) Overlay of representative N1c expression on T conv and T reg cells are shown. ( l ) MFI of N1c expression in T conv and T reg cells for each group. Results are representative of at least 2 experiments per panel. * p<0.05, ** p<0.01, *** p<0.001 and **** p<0.0001 by log-rank test, one way ANOVA and two way ANOVA with post test analysis.

Article Snippet: Chromatin immunoprecipitation on purified T reg cells was performed with Agarose ChIP Kit (Pierce) and anti-RBPJ (Cell signaling), anti-Notch1 (biolegend), anti- Foxp3 (MBL), anti-Histone H3 trimethylated on Lysine 4 (H3K4me3) (abcam), anti-dimethylated on Lysine 27 (H3K27me2) (Millipore) and respective isotype control antibodies.

Techniques: Irradiation, Adoptive Transfer Assay, Marker, Staining, Expressing

Journal: Nature immunology

Article Title: Control of peripheral tolerance by regulatory T cell-intrinsic Notch signaling

doi: 10.1038/ni.3288

Figure Lengend Snippet: ( a ) A representative picture of spleens and peripheral lymph nodes from 6 months old Foxp3 EGFPCre and Foxp3 EGFPCre Rosa26 N1c/N1c mice. ( b ) Representative pictures of H&E staining of lung, spinal vessels, spinal cord and pancreas from 6 month old Foxp3 EGFPCre Rosa26 N1c/N1c mice. Frequencies ( c ) and numbers ( d ) of CD3, CD4, CD8 T cells, T reg cells, IFN-γ producing CD4 and CD8 T cells and memory CD62L lo CD44 hi CD4 T cells from the spleen of 8 weeks old Foxp3 EGFPCre (white circles) and Foxp3 EGFPCre Rosa26 N1c/N1c (black circles) mice. ( e ) Heatmap summarizing the expression of circulating autoantibodies significantly increased in 8 weeks old Foxp3 EGFPCre Rosa26 N1c/N1c compared to Foxp3 EGFPCre mice (serum from Foxp3 K276X was used as a positive control). ( f ) Diabetes incidence of littermate control and Foxp3 EGFPCre Rosa26 N1c/N1c female (n=18 and n=13 respectively) and male (n=16 and n=8 respectively) mice on NOD background. ( g, h ) Representative pictures of H&E staining of pancreas and histological score of 15 weeks old control females (white circles) or males (white squares) and Foxp3 EGFPCre Rosa26 N1c/N1c females (Black circles) or males (black squares) on NOD background. * p<0.05, ** p<0.01, *** p<0.001 and **** p<0.0001 by unpaired two-tailed Student’s t -test and log-rank test.

Article Snippet: Chromatin immunoprecipitation on purified T reg cells was performed with Agarose ChIP Kit (Pierce) and anti-RBPJ (Cell signaling), anti-Notch1 (biolegend), anti- Foxp3 (MBL), anti-Histone H3 trimethylated on Lysine 4 (H3K4me3) (abcam), anti-dimethylated on Lysine 27 (H3K27me2) (Millipore) and respective isotype control antibodies.

Techniques: Staining, Expressing, Positive Control, Two Tailed Test

Journal: Nature immunology

Article Title: Control of peripheral tolerance by regulatory T cell-intrinsic Notch signaling

doi: 10.1038/ni.3288

Figure Lengend Snippet: (a) Expression of Foxp3, CD25, CTLA4, OX40, Helios, Nrp1 and Eos markers were evaluated in splenic T reg cells of Foxp3 EGFPCre (Dashes lines and white circles) and Foxp3 EGFPCre Rosa26 N1c/N1c (Solid lines and black circles) mice. Gray plains represent expression of those markers in T conv of Foxp3 EGFPCre mice. Cell turn-over was assessed in T reg cells of Foxp3 EGFPCre (Dashes lines and white circles), Foxp3 EGFPCre Rosa26 N1c/N1c (Solid black lines and black circles) and Foxp3 EGFPCre Rbpj Δ/Δ (Solid gray lines and Gray triangles) by Ki67 staining (b) for active cell cycle phases and by AnnV staining (c) for apoptosis. (d) In vitro suppression of responder CD4 + T cell proliferation (T Eff ) was assessed by evaluation of proliferation dye dilution upon anti- CD3/C28 stimulation in the presence of various number of T reg cells from each genotype. In vivo suppressive capacity of T reg cells from Foxp3 EGFPCre (white circles), Foxp3 EGFPCre Rosa26 N1c/N1c (black circles) was assessed in the CD4 T cell transfer-induced colitis model. (e) Changes in body weight over time are shown (n=4–7). One representative experiment of 3 is shown. (f–h) Disease severity was evaluated by H&E staining of colon sections and end point colon length. (i) Absolute number of total, IFN-γ and IL-17-producing CD45.1 + CD4 + T cells from the naïve T cell input was quantified within the entire colon. (j) Stability of T reg cell lineage was shown by percent of EGFP expression of the CD45.2 T cell compartment. * p<0.05, ** p<0.01, *** p<0.001 and **** p<0.0001 by unpaired two-tailed Student’s t -test, One way ANOVA with post test analysis and two way ANOVA.

Article Snippet: Chromatin immunoprecipitation on purified T reg cells was performed with Agarose ChIP Kit (Pierce) and anti-RBPJ (Cell signaling), anti-Notch1 (biolegend), anti- Foxp3 (MBL), anti-Histone H3 trimethylated on Lysine 4 (H3K4me3) (abcam), anti-dimethylated on Lysine 27 (H3K27me2) (Millipore) and respective isotype control antibodies.

Techniques: Expressing, Staining, In Vitro, In Vivo, Two Tailed Test

Journal: Nature immunology

Article Title: Control of peripheral tolerance by regulatory T cell-intrinsic Notch signaling

doi: 10.1038/ni.3288

Figure Lengend Snippet: (a) Frequencies and numbers of CD3, CD4 and T reg cells from the spleen of 8 weeks old Foxp3 EGFPcre , Foxp3 EGFPCre Rosa26 N1c/N1c , Foxp3 EGFPCre Rbpj Δ/Δ and Foxp3 EGFPCre Rosa26 N1c/N1c Rbpj Δ/Δ mice. (b) Flow cytometric analysis of CD62L and CD44 markers on CD4 + T cells. (c) Scatter plots represent frequencies and numbers of memory CD4 + T cells for each group. (d) Flow cytometric analysis of IFN-γ and IL-17 secretion by CD4 + T cells are shown. (e) Scatter plots represent frequencies and numbers of IFN-γ producing CD4 + T cells for each group. (f, g) Expression of Foxp3, CD25, Helios, Nrp1 markers were evaluated in splenic T reg cells of each group. n=4–5 per group. (h) In vitro suppression of responder CD4 + T cell proliferation (T Eff ) was assessed by evaluation of proliferation dye dilution upon anti-CD3/C28 stimulation in the presence of various number of T reg cells from each genotype. (I) Heatmap summarizing the expression of significantly modulated circulating autoantibodies in 8 weeks old Foxp3 EGFPCre , Foxp3 EGFPcre Rosa26 N1c/N1c and Foxp3 EGFPcre RBPJ Δ/Δ and Foxp3 EGFPCre Rosa26 N1c/N1c Rbpj Δ/Δ mice (serum from Foxp3 K276X was used as positive control). One representative experiment of 2 or 3 is shown for panels (a-h). * p<0.05 and ** p<0.01 by One way ANOVA with post test analysis and two way ANOVA.

Article Snippet: Chromatin immunoprecipitation on purified T reg cells was performed with Agarose ChIP Kit (Pierce) and anti-RBPJ (Cell signaling), anti-Notch1 (biolegend), anti- Foxp3 (MBL), anti-Histone H3 trimethylated on Lysine 4 (H3K4me3) (abcam), anti-dimethylated on Lysine 27 (H3K27me2) (Millipore) and respective isotype control antibodies.

Techniques: Expressing, In Vitro, Positive Control

Journal: Nature immunology

Article Title: Control of peripheral tolerance by regulatory T cell-intrinsic Notch signaling

doi: 10.1038/ni.3288

Figure Lengend Snippet: (a) Microarray analysis of gene expression (mean) of Foxp3 EGFPCre Rosa26 N1c/N1c (n = 4) (X axis) versus Foxp3 EGFPCre T reg cells (n=4). Numbers in plots indicate total genes downregulated (red) or upregulated (blue) in N1c overexpressing T reg cells relative to their expression in Foxp3 EGFPCr e T reg cells (cutoff of a two fold change). (b) Comparison of changes in gene expression induced by Notch1 signaling ( Foxp3 EGFPCre Rosa26 N1c/N1c vs Foxp3 EGFPCre ; horizontal axis) and those induced by Notch gain of function in absence of canonical pathway ( Foxp3 EGFPCre Rosa26 N1c/N1c Rbpj Δ/Δ vs Foxp3 EGFPCre ; vertical axis) within T reg cells. Blue lines mark a fold change of 2. (c, d) Genes whose expressions are significantly modulated (p<0.05 by one way ANOVA) were segregated in 2 clusters based on the pattern of modulation (canonical and non-canonical) and representative heat maps for each cluster are shown.

Article Snippet: Chromatin immunoprecipitation on purified T reg cells was performed with Agarose ChIP Kit (Pierce) and anti-RBPJ (Cell signaling), anti-Notch1 (biolegend), anti- Foxp3 (MBL), anti-Histone H3 trimethylated on Lysine 4 (H3K4me3) (abcam), anti-dimethylated on Lysine 27 (H3K27me2) (Millipore) and respective isotype control antibodies.

Techniques: Microarray, Expressing

Journal: Nature immunology

Article Title: Control of peripheral tolerance by regulatory T cell-intrinsic Notch signaling

doi: 10.1038/ni.3288

Figure Lengend Snippet: (a) qPCR analysis of Il12rb2 and Ifng transcripts in T reg cells isolated from Foxp3 EGFPCre , Foxp3 EGFPCre Rosa26 N1c/N1c and Foxp3 EGFPCre Rosa26 N1c/N1c Rbpj Δ/Δ mice (n=4–6 per group). Results represent mean fold change + S.E.M. compared to mean of Foxp3 EGFPCre T reg cells. Representative flow cytometry dot plots (b) and histograms (c) of IFN-γ production by sorted T reg cells after ex-vivo IL-12 stimulation (n=3 per group). Representative histograms (d) and dot plots (e) of P-STAT4 on gated Foxp3 − CXCR3 + and CD4 + Foxp3 + CXCR3 + cells after CD4 enrichment and IL-12 stimulation (n=3–8 per group). ChIP graphs represent quantitative PCR analysis of the ratio of enriched Ifng CNS22 binding site immuneprecipitated with anti-RBPJ (f) and anti-N1c (g) to the input DNA on T reg cells isolated from Foxp3 EGFPCre , Foxp3 EGFPCre Rosa26 N1c/N1c and Foxp3 EGFPCre Rosa26 N1c/N1c Rbpj Δ/Δ mice (n=3 per group). * p<0.05, ** p<0.01 and *** p<0.001 by unpaired two-tailed Student’s t -test and One Way ANOVA with post test analysis.

Article Snippet: Chromatin immunoprecipitation on purified T reg cells was performed with Agarose ChIP Kit (Pierce) and anti-RBPJ (Cell signaling), anti-Notch1 (biolegend), anti- Foxp3 (MBL), anti-Histone H3 trimethylated on Lysine 4 (H3K4me3) (abcam), anti-dimethylated on Lysine 27 (H3K27me2) (Millipore) and respective isotype control antibodies.

Techniques: Isolation, Flow Cytometry, Ex Vivo, Real-time Polymerase Chain Reaction, Binding Assay, Two Tailed Test

Journal: Nature immunology

Article Title: Control of peripheral tolerance by regulatory T cell-intrinsic Notch signaling

doi: 10.1038/ni.3288

Figure Lengend Snippet: ( a ) Flow cytometric analysis and ( b ) scatter plot analysis of MFI of unstimulated and anti-CD3/anti-CD28 stimulated T reg cells from Foxp3 EGFPCre , Foxp3 EGFPCre Rosa26 N1c/N1c , Foxp3 EGFPCre Rosa26 N1c/N1c Rictor Δ/Δ and Foxp3 EGFPCre Rosa26 N1c/N1c Rbpj Δ/Δ mice. ( c ) N1c augments anti-CD3 mAb-induced translocation of nuclear Foxo1 to the cytosol in a Rictor dependent manner. Unstimulated and anti-CD3 mAb stimulated T reg cells were stained for nuclear DNA (DAPI) and Foxo1 and examined by confocal microscopy for Foxo1 distribution in the nucleus vs. cytosol. ( d ) Quantitation of percent Foxo1 nuclear expression. Each point represents one cell. ( e ) Flow cytometric analyses of Foxp3 and EGFP expression in peripheral T reg cells from Foxp3 EGFPCre Rosa26 N1c/N1c , Foxp3 EGFPCre Rosa26 N1c/N1c Rictor Δ/Δ and Foxp3 EGFPCre Rosa26 N1c/N1c Rbpj Δ/Δ mice. ( f ) Fractions of EGFP + cells among Foxp3 + T reg cells from panel (e). ( g ) Methylation status of individual CpG motifs within the TSDR of CNS2 in Foxp3 . Individual CpG motifs are numbered with reference to the transcription initiation site of Foxp3. ( h ) Global methylation status of the TSDR of CNS2 in Foxp3 . * p<0.05, ** p<0.01 and *** p<0.001 by unpaired two-tailed Student’s t -test and One Way ANOVA with post test analysis.

Article Snippet: Chromatin immunoprecipitation on purified T reg cells was performed with Agarose ChIP Kit (Pierce) and anti-RBPJ (Cell signaling), anti-Notch1 (biolegend), anti- Foxp3 (MBL), anti-Histone H3 trimethylated on Lysine 4 (H3K4me3) (abcam), anti-dimethylated on Lysine 27 (H3K27me2) (Millipore) and respective isotype control antibodies.

Techniques: Translocation Assay, Staining, Confocal Microscopy, Quantitation Assay, Expressing, Methylation, Two Tailed Test

Journal: Cancer Cell International

Article Title: Dendritic cells matured with recombinant human sperm associated antigen 9 (rhSPAG9) induce CD4 + , CD8 + T cells and activate NK cells: a potential candidate molecule for immunotherapy in cervical cancer

doi: 10.1186/s12935-021-01951-7

Figure Lengend Snippet: FACS analysis of CD3 + /FoxP3 + Tregs in ( a ) Cisplatin untreated SPDCs stimulated co-cultures ( b ) Cisplatin treated (150 μM) SPDCs stimulated co-cultures ( c ) Bar graphs indicate CD3 + / FoxP3 + Tregs ratio in SPDCs stimulated co-cultures treated with three different doses of cisplatin treated SPDCs ( P = 0.340; P10–P12)

Article Snippet: The cells were further probed with anti-CD4-PC5, anti-CD8-APC, anti-CD56-PE, anti-CD25-ECD, anti-FOXP3-PE antibodies and Propidium iodide (1 µg/ml; Himedia, India) to verify the response of the autologous or allogeneic PBMCs upon stimulation with SPDCs.

Techniques: