Journal: Mucosal Immunology

Article Title: Lung IFNAR1 hi TNFR2 + cDC2 promotes lung regulatory T cells induction and maintains lung mucosal tolerance at steady state

doi: 10.1038/s41385-020-0254-1

Figure Lengend Snippet: a Numbers of lung CD4 + T cells at steady state in C57BL/6 J ( n = 7), Batf3 −/− ( n = 6), CCR2 −/− ( n = 4), and IRF4 fl/fl CD11c cre ( n = 7) mice. Data were compiled from two independent experiments. b Numbers of lung CD4 + T cells in WT ( n = 12), IRF4 fl/fl CD11c cre ( n = 10), RelB fl/fl CD11c cre ( n = 7), and TNFR2 fl/fl CD11c cre ( n = 5) mice at steady state. Data were representative of two independent experiments. c Image of mediastinal lymph nodes (mLNs; top) and spleens (bottom) in of 7- to 8-week-old knockout strains at steady state; n = 3 mice/group. Data are representative of two independent experiments. d Numbers of CD45 + cells in the lungs (left), mLNs (center), and spleens (right) in the indicated strains at steady state; n = 3 mice/group. Data were representative of two independent experiments. e Numbers of CD4 + T cells in IRF4 fl/fl CD11c cre mice 14 days post cell adoptive transfer. TNFR2 + or TNFR2 − cDC2 were sorted from WT mice lung and intranasally (i.n.) transferred into IRF4 fl/fl CD11c cre recipient mice; n = 4–6 mice/group. Data are representative of two independent experiments. f Numbers of lung Foxp3 + CD4 + T-reg cells at steady state in C57BL/6 J, Batf3 −/− , CCR2 −/− , and IRF4 fl/fl CD11c cre mice; n = 4–5 mice/group. Data were representative of two independent experiments. Graphs represent the mean with error bars indication s.e.m. P values determined by one-way ANOVA Tukey’s multiple comparison test. * P < 0.05, ** P < 0.001, and *** P < 0.0001.

Article Snippet: TNFR2-Fc (human IgG1) fusion protein , SinoBiological , Cat. #50128-M02H.

Techniques: Knock-Out, Adoptive Transfer Assay

Journal: Mucosal Immunology

Article Title: Lung IFNAR1 hi TNFR2 + cDC2 promotes lung regulatory T cells induction and maintains lung mucosal tolerance at steady state

doi: 10.1038/s41385-020-0254-1

Figure Lengend Snippet: a Flow cytometry plots (left) and quantification (right) of CD4 + Foxp3 + T-reg cells in in WT, Batf3 −/− , and IRF4 fl/fl CD11c cre mice treated with one dose of OVA (1 μg) intranasally (i.n.). Lungs were harvested on day 14; n = 4–6 mice/group. Data are representative of two independent experiments. b , c Flow cytometry plots (left) and quantification (right) of T-regs in TNFR2 fl/fl CD11c cre b and RelB fl/fl CD11c cre mice c treated with one dose of OVA (1 μg) i.n. Lungs were harvested on day 14; n = 3–5 mice/group. Data are representative of two independent experiments. d IRF4 fl/fl CD11c cre mice were adoptively transferred (i.n.) with lung TNFR2 + and TNFR2 − cDC2 from WT mice lung and treated with one dose of OVA (1 μg) i.n. Flow cytometry analysis (left) and quantification (right) of T-regs at day 14; n = 3–6 mice/group. Data are representative of two independent experiments. e , f Flow cytometry analysis of TNFR2 + cDC2 at steady state. Data are representative of three independent experiments; n = 3 mice/group. g IRF4 fl/fl CD11c cre mice were adoptively transferred (i.n.) with lung Mgl2 + TNFR2 + and Mgl2 − TNFR2 + cDC2 and treated with one dose of OVA (1 μg) i.n. Flow cytometry analysis (left) and quantification (right) of T-regs at day 14; n = 3–4 mice/group. Data are representative of two independent experiments. Graphs represent the mean with error bars indication s.e.m. P values determined by one-way ANOVA Tukey’s multiple comparison test a , d , g or unpaired student t -test b , c . * P < 0.05, ** P < 0.001, and *** P < 0.0001.

Article Snippet: TNFR2-Fc (human IgG1) fusion protein , SinoBiological , Cat. #50128-M02H.

Techniques: Flow Cytometry

Journal: Mucosal Immunology

Article Title: Lung IFNAR1 hi TNFR2 + cDC2 promotes lung regulatory T cells induction and maintains lung mucosal tolerance at steady state

doi: 10.1038/s41385-020-0254-1

Figure Lengend Snippet: a – c Flow cytometry analysis of TNFR2 a and pRelB expression b on lung cDC2 in TNFR2 fl/fl and TNFR2 fl/fl CD11c cre mice at steady state. WT mice were treated i.n. with anti-TNFR2 blocking antibody (20 μg) or TNFR2-Fc recombinant protein (2 μg). Lungs were harvested 24 h later; n = 3–4 mice/group. Data are representative of two independent experiments. d Quantification of Mgl2 + TNFR2 + cDC2 in the lungs of indicated groups; n = 3–4 mice/group. Data are representative of two independent experiments. e , f Flow cytometry analysis of TNF expression by anti-TNF mAb (clone D2D4) e or mouse TNFR2-Fc recombinant protein f ; n = 3 mice/group. Data are representative of three independent experiments. g – h Flow cytometry analysis of TNFR2 + cDC2 g and Ki67 expression h in mice treated i.n. with anti-TNFR2 mAb (TR75.89) and TNFR2-agonist TNF D221N/A223R (1 μg). Lungs were harvested 24 h later; n = 3 mice/group. Data are representative of two independent experiments. Graphs represent the mean with error bars indication s.e.m. P values determined by one-way ANOVA Tukey’s multiple comparison test c , d or unpaired student t -test g , h . * P < 0.05, ** P < 0.001, and *** P < 0.0001.

Article Snippet: TNFR2-Fc (human IgG1) fusion protein , SinoBiological , Cat. #50128-M02H.

Techniques: Flow Cytometry, Expressing, Blocking Assay, Recombinant

Journal: Mucosal Immunology

Article Title: Lung IFNAR1 hi TNFR2 + cDC2 promotes lung regulatory T cells induction and maintains lung mucosal tolerance at steady state

doi: 10.1038/s41385-020-0254-1

Figure Lengend Snippet: a Flow cytometry analysis (left) and quantification (right) of lung TNFR2 + cDC2 in WT mice treated i.n. with isotype (20 µg) or anti-IFNAR1 blocking antibody (20 μg). Lungs were harvested 24 h later; n = 3–4 mice/group. Data are representative of three independent experiments. b Flow cytometry analysis (left) and quantification (right) of lung T-regs in WT treated i.n. with isotype (20 µg) or anti- IFNAR1 blocking antibody (20 μg) and one dose of OVA (1 μg) i.n. Lungs were harvested on day 14; n = 4–5 mice/group. Data are representative of three independent experiments. c Flow cytometry analysis of TNFR2 + cDC2 at steady state; n = 3 mice/group. Data are representative of two independent experiments. d IFNAR1 −/− mice were adoptively transferred (i.n.) with lung TNFR2 + cDC2 and one dose of OVA (1 μg) i.n. Flow cytometry analysis of T-regs on day 14; n = 3 mice/group. Data are representative of two independent experiment. e Flow cytometry analysis (left) and numbers (right) of T-regs in mice treated i.n. with OVA (1 μg) or IFNβ (200 ng). Lungs were harvested on day 14; n = 3–5 mice/group. Data are representative of two independent experiments. f Flow cytometry analysis (left) and absolute numbers (right) of T-regs in IRF4 fl/fl and IRF4 fl/fl CD11c cre mice treated i.n. with OVA (1 μg) or OVA (1 μg) and IFNβ (200 ng). Lungs were harvested on day 14; n = 3 mice/group. Data are representative of two independent experiments. g Numbers of Mgl2 + TNFR2 + and Mgl2 − TNFR2 + cDC2 in mice treated i.n. with PBS or IFNβ (200 ng); n = 3 mice/group. Data are representative of two independent experiment. h – i Flow cytometry analysis of IFNβ expression at steady state by the anti-IFNβ mAb (clone D2J1D); n = 3 mice/group. Data are representative of two independent experiments. Graphs represent the mean with error bars indication s.e.m. P values determined by one-way ANOVA Tukey’s multiple comparison test f or unpaired student t -test a , b , e . * P < 0.05, ** P < 0.001, and *** P < 0.0001.

Article Snippet: TNFR2-Fc (human IgG1) fusion protein , SinoBiological , Cat. #50128-M02H.

Techniques: Flow Cytometry, Blocking Assay, Expressing

Journal: Mucosal Immunology

Article Title: Lung IFNAR1 hi TNFR2 + cDC2 promotes lung regulatory T cells induction and maintains lung mucosal tolerance at steady state

doi: 10.1038/s41385-020-0254-1

Figure Lengend Snippet: a Flow cytometry analysis of TNFR2 + cDC2 treated i.n. with PBS or H7N7-HA (1 μg). Lungs were harvested 24 h later; n = 3 mice/group. Data are representative of two independent experiments. b Flow cytometry analysis (left) and numbers (right) of T-regs in mice treated with PBS, OVA (1 μg)/isotype control, or OVA (1 μg) and anti-TGFβ1 neutralizing antibody (75 μg); n = 3–4 mice/group. Lungs were harvested on day 14. Data are representative of two independent experiments. c Flow cytometry analysis of TGFβ1 production by TNFR2 + cDC2 in mice treated i.n. with PBS, OVA (1 μg), or OVA (1 μg) and IFNβ (200 ng). Lungs were harvested 24 h later; n = 3 mice/group. Data are representative of three independent experiments. d Flow cytometry analysis of TGFβ1 production by TNFR2 + cDC2 in mice treated with PBS, OVA (1 μg)/isotype control, or OVA (1 μg) and anti-IFNAR1 blocking antibody (20 μg). Lungs were harvested 24 h later; n = 3 mice/group. Data are representative of two independent experiments. e Experimental design for adoptive transfer. f – g IFNAR1 −/− mice were adoptively transferred (i.n.) with lung TNFR2 + cDC2 and treated with PBS or IFNβ (200 ng). Flow cytometry analysis of TGFβ1 production by TNFR2 + cDC2 f and T-regs g ; n = 3 mice/group. Data are representative of two independent experiments. Graphs represent the mean with error bars indication s.e.m. P values determined by unpaired student t -test b , f , g . * P < 0.05, ** P < 0.001, and *** P < 0.0001.

Article Snippet: TNFR2-Fc (human IgG1) fusion protein , SinoBiological , Cat. #50128-M02H.

Techniques: Flow Cytometry, Blocking Assay, Adoptive Transfer Assay

Journal: Mucosal Immunology

Article Title: Lung IFNAR1 hi TNFR2 + cDC2 promotes lung regulatory T cells induction and maintains lung mucosal tolerance at steady state

doi: 10.1038/s41385-020-0254-1

Figure Lengend Snippet: a Experimental protocol for HDM-induced acute asthma. b Flow cytometry analysis (left) and numbers (right) of TNFR2 + cDC2 in PBS or HDM-induced asthmatic WT mice; n = 2–4 mice/group. Data were compiled from two independent experiments. c Flow cytometry analysis of Mgl2 + TNFR2 + cDC2 in PBS or HDM-induced asthmatic WT mice; n = 4–5 mice/group. Data are representative of three independent experiments. d Flow cytometry analysis of T-regs in OVA (1 µg) treated (left) and HDM-induced asthmatic WT mice (right); n = 3 mice/group. Data are representative of three independent experiments. e – f Flow cytometry analysis of TNFR2 + cDC2 in HDM-induced asthmatic WT mice; n = 3 mice/group. Data are representative of three independent experiments. g Experimental design for adoptive transfer (top). Flow cytometry analysis of IL-4 production by lung CD4 + T cells; n = 3mice/group. Data are representative of two independent experiments. h Flow cytometry analysis of R2D2 in OVA (1 µg) treated (left) and HDM-induced asthmatic WT mice (right); n = 3 mice/group. Data are representative of three independent experiments. Graphs represent the mean with error bars indication s.e.m. P values determined by unpaired student t -test b . * P < 0.05, ** P < 0.001, and *** P < 0.0001.

Article Snippet: TNFR2-Fc (human IgG1) fusion protein , SinoBiological , Cat. #50128-M02H.

Techniques: Flow Cytometry, Adoptive Transfer Assay

Journal: Mucosal Immunology

Article Title: Lung IFNAR1 hi TNFR2 + cDC2 promotes lung regulatory T cells induction and maintains lung mucosal tolerance at steady state

doi: 10.1038/s41385-020-0254-1

Figure Lengend Snippet: a Flow cytometry analysis of pulmonary cDC2 subpopulation in healthy human lungs. Data are representative of eight independent experiments. b Flow cytometry analysis of TNFR2 + cDC2 in donor, emphysema, and chronic-rejected human lungs. Data are representative of three independent experiments. c Frequency of TNFR2 + cDC2 in diseased human lungs. Data were compiled from multiple independent experiments. Graphs represent the mean with error bars indication s.e.m.

Article Snippet: TNFR2-Fc (human IgG1) fusion protein , SinoBiological , Cat. #50128-M02H.

Techniques: Flow Cytometry

Journal: Mucosal Immunology

Article Title: Lung IFNAR1 hi TNFR2 + cDC2 promotes lung regulatory T cells induction and maintains lung mucosal tolerance at steady state

doi: 10.1038/s41385-020-0254-1

Figure Lengend Snippet:

Article Snippet: TNFR2-Fc (human IgG1) fusion protein , SinoBiological , Cat. #50128-M02H.

Techniques: Recombinant, Activation Assay, Staining, Mutagenesis, Software

Journal: Mucosal immunology

Article Title: Immature lung TNFR2 − conventional DC 2 subpopulation activates moDCs to promote cyclic di-GMP mucosal adjuvant responses in vivo

doi: 10.1038/s41385-018-0098-0

Figure Lengend Snippet: A. C57BL/6 (WT), TNFR1 −/− and TNFR2 −/− mice were immunized (i.n.) with two doses of PspA or PspA plus CDG (5ug). Serum anti-PspA IgG and BALF IgA were determined by ELISA. n=3. B. WT and TNFR2 −/− mice were treated (i.n.) with saline or CDG (5µg) for 16 hours. CD86 expression in lung cDC2 were determined by Flow cytometry. n=3. C. IRF4 fl/fl CD11c cre mice were adoptively transferred (i.n.) with lung cDC2 sorted from WT or TNFR2 −/− mice lung. The recipient mice were administered (i.n.) with saline or CDG (5µg) for 16hrs. CD86 expression in lung cDC2 were determined by Flow cytometry. n=3. D. Flow cytometry analysis of TNFR2 expression on lung cDC2 in WT mice administered (i.n) with saline or CDG for 16hrs. n>3. E. Flow cytometry analysis of TNFR2 expression on pRelB + cDC2 from mice treated with saline or CDG. n=3. F. Flow cytometry analysis of pRelB expression on TNFR2 + cDC2 from mice treated with CDG. n=3. G. Flow cytometry analysis of pRelB expression on cDC2 from WT and TNFR2 −/− mice. n=3. H. Flow cytometry analysis of CD86 expression on lung cDC2 in RelB fl/fl and RelB fl/fl CD11C Cre mice treated with CDG. n=3. Graphs represent means ± standard error from three independent experiments. The significance is represented by and asterisk (*) where p<0.05 (unpaired Student’s t test).

Article Snippet: The following reagents were from Sino Biological: TNFR2 Ab (cat# 50128-R112-A), TNFR2-Fc mouse TNFR2 extracellular domain, Met 1-Gly 258, was fused with the Fc region of human IgG1 at the C-terminus, cat# 50128-M02H), human IgG1 Fc (cat# 10702-HNAH).

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

Journal: Mucosal immunology

Article Title: Immature lung TNFR2 − conventional DC 2 subpopulation activates moDCs to promote cyclic di-GMP mucosal adjuvant responses in vivo

doi: 10.1038/s41385-018-0098-0

Figure Lengend Snippet: A. IRF4 fl/fl CD11c cre mice were adoptively transferred (i.n.) with lung cDC2 sorted from WT or TNFR2 −/− mice lung and immunized (i.n.) with PspA or CDG/PspA. Serum anti-PspA IgG and BALF anti-PspA IgA were determined by ELISA. n=3. B. RelB fl/fl and RelB fl/fl CD11C Cre mice were immunized (i.n) with CDG/PspA or PspA alone as before. Serum anti-PspA IgG and BALF anti-PspA IgA were determined by ELISA. n=3. C. Lung cells from immunized RelB fl/fl and RelB fl/fl CD11C Cre mice were stimulated with 5∝g/ml PspA for 4 days in culture. Cytokines were measured in the supernatant by ELISA. n>3. Graphs represent means ± standard error from three independent experiments. The significance is represented by and asterisk (*) where p<0.05 (unpaired Student’s t test).

Article Snippet: The following reagents were from Sino Biological: TNFR2 Ab (cat# 50128-R112-A), TNFR2-Fc mouse TNFR2 extracellular domain, Met 1-Gly 258, was fused with the Fc region of human IgG1 at the C-terminus, cat# 50128-M02H), human IgG1 Fc (cat# 10702-HNAH).

Techniques: Enzyme-linked Immunosorbent Assay

Journal: Mucosal immunology

Article Title: Immature lung TNFR2 − conventional DC 2 subpopulation activates moDCs to promote cyclic di-GMP mucosal adjuvant responses in vivo

doi: 10.1038/s41385-018-0098-0

Figure Lengend Snippet: A. Flow cytometry analysis of TNFR2 expression on lung CDG-FITC + cDC2 from WT mice. n=3. B. Lung TNF production in CDG treated (i.n.) MPYS −/− mice adoptively transferred with WT TNFR2 + or TNFR2 − lung cDC2. n=3 C. Sorted TNFR2 + and TNFR2 − lung cDC2 from WT mice were labelled with CFSE and adoptively transferred into the MPYS −/− mice. Recipient mice were treated with CDG (i.n.) for 16hrs. CD86 and CCR7 expression on CFSE positive lung cells were examined by Flow cytometry. n=3. D. Sorted TNFR2 + and TNFR2 − lung cDC2 from WT mice were adoptively transferred into the MPYS −/− PspA or CDG/PspA twice. Serum anti-PspA mice. Recipient mice were immunized (i.n.) with IgG were determined by ELISA. n=3. E. Sorted TNFR2 + and TNFR2 − lung cDC2 from WT mice were adoptively transferred into IRF4 fl/fl CD11c cre mice. Recipient mice were immunized (i.n.) with PspA or CDG/PspA. Serum anti-PspA IgG were determined by ELISA. n=3. F. Lung cells from recipient IRF4 fl/fl CD11c cre mice were stimulated with 5∝g/ml PspA for 4 days in culture. Cytokines were measured in the supernatant by ELISA. G. A cartoon illustrating following CDG administration, TNFR2 + cDC2 activate RelB to induce Th1, Th2 and Th17 responses while TNFR2 − cDC2 mediate the antibody response. Graphs represent means ± standard error from three independent experiments. The significance is represented by and asterisk (*) where p<0.05 (unpaired Student’s t test).

Article Snippet: The following reagents were from Sino Biological: TNFR2 Ab (cat# 50128-R112-A), TNFR2-Fc mouse TNFR2 extracellular domain, Met 1-Gly 258, was fused with the Fc region of human IgG1 at the C-terminus, cat# 50128-M02H), human IgG1 Fc (cat# 10702-HNAH).

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

Journal: Mucosal immunology

Article Title: Immature lung TNFR2 − conventional DC 2 subpopulation activates moDCs to promote cyclic di-GMP mucosal adjuvant responses in vivo

doi: 10.1038/s41385-018-0098-0

Figure Lengend Snippet: A. Flow cytometry analysis of lung TNFR2 + vs TNFR2 − cDC2 at steady-state. n=3. B-C. Flow cytometry analysis of IRF4 and IRF8 ( B ) and Zbtb46 ( C ) in TNFR2 + and TNFR2 − cDC2. n=3. D. Flow cytometry analysis in lungs of IRF4 fl/fl CD11c cre mice reconstituted with CD45.1 + pre-cDC2. n=3. pre-cDC2 were sorted from the bone marrow of B6.CD45.1 mice and transferred (i.n.) into IRF4 fl/fl CD11c cre mice. n=3. E. Phenotypic analysis of CD45.1 TNFR2 + and TNFR2 − cDC2 transferred into IRF4 fl/fl CD11c cre mice. n=3. F. Flow cytometry analysis of TNFR2 expression on cDC2 subpopulations transferred into MPYS −/− mice treated with CDG (i.n.) for 16hrs. n=3.

Article Snippet: The following reagents were from Sino Biological: TNFR2 Ab (cat# 50128-R112-A), TNFR2-Fc mouse TNFR2 extracellular domain, Met 1-Gly 258, was fused with the Fc region of human IgG1 at the C-terminus, cat# 50128-M02H), human IgG1 Fc (cat# 10702-HNAH).

Techniques: Flow Cytometry, Expressing

Journal: Mucosal immunology

Article Title: Immature lung TNFR2 − conventional DC 2 subpopulation activates moDCs to promote cyclic di-GMP mucosal adjuvant responses in vivo

doi: 10.1038/s41385-018-0098-0

Figure Lengend Snippet: A-B. Indicated mice were administered (i.n.) with saline or CDG for 5hrs. TNF production was measured in lung homogenates by ELISA. n>3. C. WT and TNFR2 −/− mice were treated with saline or CDG for 16 hours. TNF in lung cDC2 was determined by an intracellular cytokine stain. n=3. D. TBK1 fl/fl and TBK1 fl/fl Vav cre mice were administered (i.n.) with saline or CDG for 5hrs. TNF production was measured in lung homogenates by ELISA. n=3. E. Flow cytometry analysis of p-TBK1 expression in lung cDC2 from WT and TNFR2 −/− mice treated with saline or CDG for 16 hours. n=3. Graphs represent means ± standard error from three independent experiments. The significance is represented by and asterisk (*) where p<0.05 (unpaired Student’s t test).

Article Snippet: The following reagents were from Sino Biological: TNFR2 Ab (cat# 50128-R112-A), TNFR2-Fc mouse TNFR2 extracellular domain, Met 1-Gly 258, was fused with the Fc region of human IgG1 at the C-terminus, cat# 50128-M02H), human IgG1 Fc (cat# 10702-HNAH).

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

Journal: Mucosal immunology

Article Title: Immature lung TNFR2 − conventional DC 2 subpopulation activates moDCs to promote cyclic di-GMP mucosal adjuvant responses in vivo

doi: 10.1038/s41385-018-0098-0

Figure Lengend Snippet: A-B. WT, IRF4 fl/fl CD11c cre ( A ) and Batf3 −/− ( B ) mice were treated (i.n.) with saline or CDG (5µg) for 16 hours. CD86 expression in lung moDC were determined by Flow cytometry. n=3. C. WT and TNFR2 −/− cDC2 were adoptively transferred into the IRF4 fl/fl CD11c cre mice. The mice were treated with CDG (i.n.) for 16hrs. CD86 expression on endogenous moDC were examined by Flow cytometry. n=3. D. Flow cytometry analysis of CD86 expression on lung moDCs in RelB fl/fl and RelB fl/fl CD11c Cre mice treated with CDG for 16hrs. n=3. E. Flow cytometry analysis of TNFR2 expression on lung moDCs of WT mice treated (i.n.) with saline or CDG for 16hrs.n=3. F. Flow cytometry analysis of CD86 expression on lung moDCs from WT or TNFR2 −/− mice treated (i.n.) with saline or CDG for 16hrs. n=3. G. WT, TNFR2 −/− or TNFR2 −/− mice receiving (i.n.) WT monocytes or TNFR2 −/− monoctyes were immunized with CDG/PspA. Serum anti-PspA IgG and BALF anti-PspA IgA were determined by ELISA. n=3. H. WT cDC2 adoptively transferred into TNFR2 −/− mice were immunized with CDG/PspA. Serum anti-PspA IgG were determined by ELISA. n=3. Graphs represent means ± standard error from three independent experiments. The significance is represented by and asterisk (*) where p<0.05 (unpaired Student’s t test).

Article Snippet: The following reagents were from Sino Biological: TNFR2 Ab (cat# 50128-R112-A), TNFR2-Fc mouse TNFR2 extracellular domain, Met 1-Gly 258, was fused with the Fc region of human IgG1 at the C-terminus, cat# 50128-M02H), human IgG1 Fc (cat# 10702-HNAH).

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

Journal: Mucosal immunology

Article Title: Immature lung TNFR2 − conventional DC 2 subpopulation activates moDCs to promote cyclic di-GMP mucosal adjuvant responses in vivo

doi: 10.1038/s41385-018-0098-0

Figure Lengend Snippet: A. WT mice were administered with saline or CDG (5µg) for 16hrs. mTNF expression was determined by Flow cytometry using mouse TNFR2-Fc recombinant protein. n=3. B-C . Flow cytometry analysis of mTNF expression in lung DCs ( B ) and cDC2 ( C ) n=3. D. Flow cytometry analysis of antigen uptake and processing in lung CD11b + DC from WT mice treated (i.n.) with DQ-OVA (20ug) and CDG (5ug) for 16 hours. n=3. Graphs represent means ± standard error from three independent experiments. The significance is represented by and asterisk (*) where p<0.05 (unpaired Student’s t test).

Article Snippet: The following reagents were from Sino Biological: TNFR2 Ab (cat# 50128-R112-A), TNFR2-Fc mouse TNFR2 extracellular domain, Met 1-Gly 258, was fused with the Fc region of human IgG1 at the C-terminus, cat# 50128-M02H), human IgG1 Fc (cat# 10702-HNAH).

Techniques: Expressing, Flow Cytometry, Recombinant

Journal: Mucosal immunology

Article Title: Immature lung TNFR2 − conventional DC 2 subpopulation activates moDCs to promote cyclic di-GMP mucosal adjuvant responses in vivo

doi: 10.1038/s41385-018-0098-0

Figure Lengend Snippet: A-B. WT mice were administered with Ea-OVA (10µg) or Ea-OVA/CDG (5µg) for 16hrs. YAE + moDCs (A) and CD86 + YAE + moDCs were determined by Flow cytometry. n=3. C-D. WT, TNFR2 −/− or TNFR2 −/− mice receiving (i.n.) WT monocytes were immunized with CDG/PspA. At Day 14, CD4 + PD1 + CXCR5 + Tfh ( C ) and CD19 + Bcl6 + B cells ( D ) were determine in lung by flow cytometry. n=3. E. TNFR2 + and TNFR2 − cDC2 were adoptively transferred into IRF4 fl/fl CD11c cre mice and immunized with CDG/PspA. At Day 14, CD4 + Bcl6 + Tfh were determine by flow cytometry. n=3. F. Model: following CDG administration, TNFR2 − cDC2 produce mTNF to activate moDCs, which will generate Tfh to mediate the antibody response. Graphs represent means ± standard error from three independent experiments. The significance is represented by and asterisk (*) where p<0.05 (unpaired Student’s t test).

Article Snippet: The following reagents were from Sino Biological: TNFR2 Ab (cat# 50128-R112-A), TNFR2-Fc mouse TNFR2 extracellular domain, Met 1-Gly 258, was fused with the Fc region of human IgG1 at the C-terminus, cat# 50128-M02H), human IgG1 Fc (cat# 10702-HNAH).

Techniques: Flow Cytometry

Journal: Journal of Cachexia, Sarcopenia and Muscle

Article Title: SIRT6 Ameliorates Cancer Cachexia–Associated Adipose Wasting by Suppressing TNFR2 Signalling in Mice

doi: 10.1002/jcsm.13734

Figure Lengend Snippet: SIRT6 functions in lipolysis mediated by TNFR2. (A–B) Tumour necrosis factor receptor (TNFR1 and TNFR2) protein levels in the eWAT from WT + PBS, WT + LLC, SIRT6 TG + PBS and SIRT6 TG + LLC mice were determined by western blot ( n = 6 per group). (C) Serum TNFR2 concentrations in mice were measured by ELISA (PBS, n = 7 per group; LLC, n = 11 per group). (D) cAMP levels in WT + DMEM, WT + LLC‐CM, TG + DMEM, TG + LLC‐CM adipocytes ( n = 3 per group) or WT + DMEM, WT + LLC‐CM, KO + DMEM, KO + LLC‐CM adipocytes ( n = 4 per group) were measured. WT + DMEM (WT adipocytes were treated with DMEM medium), WT + LLC‐CM (WT adipocytes were treated with LLC cell‐conditioned medium), TG + DMEM (SIRT6 TG adipocytes were treated with DMEM medium), TG + LLC‐CM (SIRT6 TG adipocytes were treated with LLC cell‐conditioned medium), KO + DMEM (SIRT6 KO adipocytes were treated with DMEM medium) and KO + LLC‐CM (SIRT6 KO adipocytes were treated with LLC cell‐conditioned medium). (E) MEFs isolated from KO and WT embryos were induced differentiation into mature adipocytes. Adipocytes were treated for 24 h with DMEM, LLC cell‐conditioned medium and a combination of LLC cell‐conditioned medium and 12.5 μg/mL TNFR2 antagonist. Representative images of oil red O staining and the quantification of lipid content were shown ( n = 4 per group). (F) Serum TNFα and TNFR2 concentrations of cachectic cancer patients ( n = 12) and non‐cachectic cancer patients ( n = 10) were compared. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: And then, the medium was changed to maintain medium with 5 μg/mL insulin and 1‐μM rosiglitazone for 48 h. Finally, mature adipocytes were maintained in control high glucose DMEM medium or LLC‐conditioned‐medium supplemented with 10% FBS and 1% penicillin/streptomycin for 24 h. TNFR2 antagonist (anti mouse TNFR2/CD120b/TNFRSR1B Neutralizing Antibody, 50128‐RN204, Sino Biological) (12.5 μg/mL) or 20‐μM MDL800 (SML2925, Sigma) were used to treat cells for 24 h. Medium supernatant and cells were collected for further analysis, individually.

Techniques: Western Blot, Enzyme-linked Immunosorbent Assay, Isolation, Staining

Journal: Journal of Cachexia, Sarcopenia and Muscle

Article Title: SIRT6 Ameliorates Cancer Cachexia–Associated Adipose Wasting by Suppressing TNFR2 Signalling in Mice

doi: 10.1002/jcsm.13734

Figure Lengend Snippet: MDL800 reverses LLC‐induced adipocytes lipolysis. (A) Western blot analysis of SIRT6 and its substrates H3K9ac and H3K56ac in WT adipocytes with or without 20‐μM MDL‐800 treatment for 24 h ( n = 3 per group). (B) MEFs isolated from WT embryos were induced differentiation into mature adipocytes. Adipocytes were treated for 24 h with DMEM, LLC cell‐conditioned medium and a combination of LLC cell‐conditioned medium and 20‐μM MDL800. Representative images of oil red O staining and the quantification of lipid content were shown ( n = 3 per group). (C) Glycerol levels in medium supernatant were measured ( n = 6 per group). (D) The expression and phosphorylation levels of lipolysis associated proteins in the WT + DMEM, WT + LLC‐CM and WT + LLC‐CM + 20‐μM MDL800 were determined by western blot ( n = 4–6 per group). (E) cAMP levels in WT + DMEM, WT + LLC‐CM and WT + LLC‐CM + 20 μM MDL800 adipocytes were measured ( n = 3 per group). (F) Role of SIRT6 in cancer cachexia. TNFα, secreted from tumour cell, interacts with membrane TNFR2 and then induces the activation of lipolysis signalling pathway, mainly indicated by increased expression of ATGL and over‐phosphorylation of HSL and Perilipin 1. Without SIRT6, lipolysis is enhanced due to increased expression of TNFR2 and interaction of TNFα and TNFR2, which led to adipose wasting in tumour‐bearing mice. When SIRT6 is overexpressed or pharmaceutically activated, decreased expression of TNFR2 attenuates the activation of lipolysis signalling, which led to adipose homeostasis and then metabolic balance in mice. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: And then, the medium was changed to maintain medium with 5 μg/mL insulin and 1‐μM rosiglitazone for 48 h. Finally, mature adipocytes were maintained in control high glucose DMEM medium or LLC‐conditioned‐medium supplemented with 10% FBS and 1% penicillin/streptomycin for 24 h. TNFR2 antagonist (anti mouse TNFR2/CD120b/TNFRSR1B Neutralizing Antibody, 50128‐RN204, Sino Biological) (12.5 μg/mL) or 20‐μM MDL800 (SML2925, Sigma) were used to treat cells for 24 h. Medium supernatant and cells were collected for further analysis, individually.

Techniques: Western Blot, Isolation, Staining, Expressing, Membrane, Activation Assay