Journal: Nature Communications

Article Title: Clec4A4 is a regulatory receptor for dendritic cells that impairs inflammation and T-cell immunity

doi: 10.1038/ncomms11273

Figure Lengend Snippet: ( a ) BMDCs expressing mock-GFP, Clec4A4-GFP, Clec4A4 N186Q -GFP, Clec4A4 ΔY68–L236 -GFP or Clec4A4 ΔI5–V10 -GFP were stimulated or not stimulated with the indicated LPS, and the production of cytokines was measured by enzyme-linked immunosorbent assay (ELISA). Data are the mean±s.d. from three individual samples in a single experiment. * P <0.01 compared with BMDCs expressing mock-GFP or among groups (analysis of variance (ANOVA), Bonferroni's multiple comparison test). ( b ) BMDCs expressing mock-GFP or Clec4A4-GFP were stimulated or not stimulated with LPS for the period indicated, at which time cells were lysed. Total lysate was analysed using Ab specific for p65, ERK, JNK, p38 and IRF-3 or for phosphorylated versions of these proteins. ( c , d ) BMDCs expressing mock-GFP or Clec4A4-GFP were stimulated or not stimulated with LPS in combination with or without crosslinking of Clec4A4 with anti-FLAG M2 mAb for 30 min. ( c ) The immunoprecipitate with anti-FLAG M2 mAb was analysed using anti-phosphotyrosine (p-Tyr) mAb, anti-SHP-1 mAb, anti-SHP-2 mAb or anti-FLAG M5 mAb. ( d ) The immunoprecipitate with anti-FLAG M2 mAb or control IgG was analysed for protein tyrosine phosphatase activity. Data are the mean±s.d. from three individual samples in a single experiment. * P <0.01 compared with the immunoprecipitate with anti-FLAG M2 mAb obtained from unstimuated cells (ANOVA, Bonferroni's multiple comparison test). All data are representative of at least three independent experiments.

Article Snippet: After restriction enzyme digestion, the synthesized DNA sequence was cloned into the sites of pFUSE-hIgG2-Fc2 (Invivogen), and transfected into FreeStyle 293-F cells (Life Technologies) using 293fectin Transfection Reagent (Life Technologies) according to the manufacturers' instructions. huIgFc or Clec4A4-huIgFc fusion protein was purified from the culture supernatant using HiTrap Protein G HP (GE Healthcare Life Sciences), and the purified fusion protein was separated by 10% SDS–PAGE under non-reducing or reducing conditions, respectively, and stained with a Silver Stain kit (Bio-Rad Laboratories) to confirm the protein products.

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Activity Assay

Journal: Nature Communications

Article Title: Clec4A4 is a regulatory receptor for dendritic cells that impairs inflammation and T-cell immunity

doi: 10.1038/ncomms11273

Figure Lengend Snippet: ( a ) Schematic diagram of structures of the oligosaccharides in NGL. Gal, D -galactose; Glc, D -glucose. ( b ) The binding of huIgFc, Clec4A4-huIgFc, SIGNR1-huIgFc or Clec9A-huIgFc to glycans was measured by ELISA. Data are the mean±s.d. from three individual samples in a single experiment. * P <0.01 compared with DEEG or among groups (analysis of variance (ANOVA), Bonferroni's multiple comparison test). ( c ) The binding of huIgFc, Clec4A4-huIgFc, SIGNR1-huIgFc or Clec9A-huIgFc to DC2.4, DC2.4-expressing mock-GFP, and DC2.4-expressing Clec4A4-GFP was analysed by flow cytometry with anti-huIgFc-PE. Data are presented by a dot plot, and numbers represent the proportion of the binding of huIgFc, Clec4A4-huIgFc, SIGNR1-huIgFc or Clec9A-huIgFc in each quadrant. All data are representative of at least three independent experiments.

Article Snippet: After restriction enzyme digestion, the synthesized DNA sequence was cloned into the sites of pFUSE-hIgG2-Fc2 (Invivogen), and transfected into FreeStyle 293-F cells (Life Technologies) using 293fectin Transfection Reagent (Life Technologies) according to the manufacturers' instructions. huIgFc or Clec4A4-huIgFc fusion protein was purified from the culture supernatant using HiTrap Protein G HP (GE Healthcare Life Sciences), and the purified fusion protein was separated by 10% SDS–PAGE under non-reducing or reducing conditions, respectively, and stained with a Silver Stain kit (Bio-Rad Laboratories) to confirm the protein products.

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

Journal: Nature Communications

Article Title: Clec4A4 is a regulatory receptor for dendritic cells that impairs inflammation and T-cell immunity

doi: 10.1038/ncomms11273

Figure Lengend Snippet: ( a , b ) The expression of Clec4A4 and cell surface molecules on splenocytes ( a ) and CD11c + DCs ( b ) obtained from WT mice and Clec4a4 −/− mice was analysed by flow cytometry. Data are presented by a dot plot, and numbers represent the proportion in each quadrant. ( c ) Immunofluorescent microscopic analysis was performed on frozen horizontal sections. Sections were stained for CD19 (green), CD3ɛ (blue) and Clec4A4 (red) or CD11c (red). ( d ) The expression of Clec4A4 and cell-surface molecules on CD8α − cDCs obtained from WT mice and Clec4a4 −/− mice was analysed by flow cytometry. Data are presented by a histogram, and numbers represent mean fluorescence intensity (MFI). ( e ) WT mice ( n =6) and Clec4a4 −/− mice ( n =6) were injected with or without CpG-B, and CD8α − cDCs were obtained 24 h after injection. The expression of cell surface molecules on CD8α − cDCs was analysed by flow cytometry. Data are MFI±s.d. from six individual samples in a single experiment. ( f , g ) WT mice ( n =6) and Clec4a4 −/− mice ( n =6) were not injected (left panel) or injected (right panel) with CpG-B, and CD8α − cDCs were obtained 24 h after injection. CD45.1 + OT-II CD4 + T cells ( f ) or CD45.1 + OT-I CD8 + T cells ( g ) were cultured with CD8α − cDCs (10 4 ) obtained from WT mice and Clec4a4 −/− mice in the presence or absence of OVA 323–339 peptide (1 μM; f ) or OVA 257–264 peptide (1 nM; g ), and the proliferation was measured by [ 3 H]thymidine incorporation. Data are the mean±s.d. from six individual samples in a single experiment. * P <0.01 compared with WT mice (analysis of variance (ANOVA), Bonferroni's multiple comparison test). All data are representative of at least three independent experiments.

Article Snippet: After restriction enzyme digestion, the synthesized DNA sequence was cloned into the sites of pFUSE-hIgG2-Fc2 (Invivogen), and transfected into FreeStyle 293-F cells (Life Technologies) using 293fectin Transfection Reagent (Life Technologies) according to the manufacturers' instructions. huIgFc or Clec4A4-huIgFc fusion protein was purified from the culture supernatant using HiTrap Protein G HP (GE Healthcare Life Sciences), and the purified fusion protein was separated by 10% SDS–PAGE under non-reducing or reducing conditions, respectively, and stained with a Silver Stain kit (Bio-Rad Laboratories) to confirm the protein products.

Techniques: Expressing, Flow Cytometry, Staining, Fluorescence, Injection, Cell Culture

Journal: Nature Communications

Article Title: Clec4A4 is a regulatory receptor for dendritic cells that impairs inflammation and T-cell immunity

doi: 10.1038/ncomms11273

Figure Lengend Snippet: CD8α − cDCs obtained from WT mice and Clec4a4 −/− mice were stimulated or not stimulated with Pam3CSK4 ( a ), poly(I:C) ( b ), LPS ( c ) and CpG-B ( d ), and the production of cytokines was measured by ELISA. Data are the mean±s.d. from three individual samples in a single experiment. * P <0.01 compared with WT mice (analysis of variance (ANOVA), Bonferroni's multiple comparison test). All data are representative of at least three independent experiments.

Article Snippet: After restriction enzyme digestion, the synthesized DNA sequence was cloned into the sites of pFUSE-hIgG2-Fc2 (Invivogen), and transfected into FreeStyle 293-F cells (Life Technologies) using 293fectin Transfection Reagent (Life Technologies) according to the manufacturers' instructions. huIgFc or Clec4A4-huIgFc fusion protein was purified from the culture supernatant using HiTrap Protein G HP (GE Healthcare Life Sciences), and the purified fusion protein was separated by 10% SDS–PAGE under non-reducing or reducing conditions, respectively, and stained with a Silver Stain kit (Bio-Rad Laboratories) to confirm the protein products.

Techniques: Enzyme-linked Immunosorbent Assay

Journal: Nature Communications

Article Title: Clec4A4 is a regulatory receptor for dendritic cells that impairs inflammation and T-cell immunity

doi: 10.1038/ncomms11273

Figure Lengend Snippet: ( a ) WT mice ( n =6) and Clec4a4 −/− mice ( n =6) were injected with LPS, and serum production of cytokines was measured at the indicated time after injection by ELISA. Data are the mean±s.d. from six individual samples in a single experiment. * P <0.01 compared with WT mice (analysis of variance (ANOVA), Bonferroni's multiple comparison test). ( b , c ) WT mice ( n =10) and Clec4a4 −/− mice ( n =10) were injected with heat-killed E. coli . ( b ) Survival rate was monitored at the indicated times for 60 h after injection of heat-killed E. coli . * P <0.01 compared with WT mice (Kaplan–Meier log-rank test). ( c ) Serum production of cytokines was measured 24 h after injection with heat-killed E. coli by ELISA. Data are the mean±s.d. from 10 individual samples in a single experiment. * P <0.01 compared with WT mice (analysis of variance (ANOVA), Bonferroni's multiple comparison test). All data are representative of at least three independent experiments.

Article Snippet: After restriction enzyme digestion, the synthesized DNA sequence was cloned into the sites of pFUSE-hIgG2-Fc2 (Invivogen), and transfected into FreeStyle 293-F cells (Life Technologies) using 293fectin Transfection Reagent (Life Technologies) according to the manufacturers' instructions. huIgFc or Clec4A4-huIgFc fusion protein was purified from the culture supernatant using HiTrap Protein G HP (GE Healthcare Life Sciences), and the purified fusion protein was separated by 10% SDS–PAGE under non-reducing or reducing conditions, respectively, and stained with a Silver Stain kit (Bio-Rad Laboratories) to confirm the protein products.

Techniques: Injection, Enzyme-linked Immunosorbent Assay

Journal: Nature Communications

Article Title: Clec4A4 is a regulatory receptor for dendritic cells that impairs inflammation and T-cell immunity

doi: 10.1038/ncomms11273

Figure Lengend Snippet: ( a – d ) CD45.1 + OT-II CD4 + T cells were cultured with CD8α − cDCs obtained from WT mice and Clec4a4 −/− mice in the presence or absence of Pam3CSK4 (1 μg ml −1 ), poly(I:C) (50 μg ml −1 ), LPS (1 μg ml −1 ) or CpG-B (0.1 μM) in combination with OVA 323–339 peptide under T H 1 ( a , b )- or T H 17 ( c , d )-polarized culture conditions for 3 days, and intracellular production of IFN-γ ( a , b ) or IL-17 ( c , d ) in the cultured CD4 + T cells was analysed by flow cytometry. ( a , c ) Data are presented by a dot plot, and numbers represent the proportion of IFN-γ + cells ( a ) and IL-17 + cells ( c ) among gated CD4 + T cells in each quadrant. ( b , d ) Data are the mean percentage of positive cells±s.d. from three individual samples in a single experiment. ( e , f ) CFSE-labelled CD45.1 + OT-II CD4 + T cells were transferred into WT mice ( n =6) and Clec4a4 −/− mice ( n =6), and then the mice were immunized with OVA protein in combination with or without the indicated TLR ligands. Ag-specific division of CD45.1 + OT-II CD4 + T cells was analysed 3 days after the immunization by flow cytometry. ( e ) Data are presented by a histogram, and numbers represent the proportion of CFSE dilution among gated CD45.1 + OT-II CD4 + T cells in each histogram. ( f ) Data are the mean percentage of positive cells±s.d. from six individual samples in a single experiment. ( g – i ) WT mice ( n =6) and Clec4a4 −/− mice ( n =6) were immunized with CFA plus OVA protein. At 14 days after the immunization, spleen CD4 + T cells were isolated then cultured with WT CD11c + DCs in the presence or absence of OVA protein for the measurement of proliferative responses by [ 3 H]thymidine incorporation ( g , left panel) and production of IFN-γ ( g , right panel) by ELISA. Data are the mean±s.d. from six individual samples in a single experiment. ( h , i ) Intracellular production of IFN-γ in the cultured CD4 + T cells was analysed by flow cytometry. ( h ) Data are presented by a dot plot, and numbers represent the proportion of IFN-γ + cells among gated CD4 + T cells in each quadrant. ( i ) Data are the mean percentage of positive cells±s.d. from six individual samples in a single experiment. * P <0.01 compared with WT mice (analysis of variance (ANOVA), Bonferroni's multiple comparison test). All data are representative of at least three independent experiments.

Article Snippet: After restriction enzyme digestion, the synthesized DNA sequence was cloned into the sites of pFUSE-hIgG2-Fc2 (Invivogen), and transfected into FreeStyle 293-F cells (Life Technologies) using 293fectin Transfection Reagent (Life Technologies) according to the manufacturers' instructions. huIgFc or Clec4A4-huIgFc fusion protein was purified from the culture supernatant using HiTrap Protein G HP (GE Healthcare Life Sciences), and the purified fusion protein was separated by 10% SDS–PAGE under non-reducing or reducing conditions, respectively, and stained with a Silver Stain kit (Bio-Rad Laboratories) to confirm the protein products.

Techniques: Cell Culture, Flow Cytometry, Isolation, Enzyme-linked Immunosorbent Assay

Journal: Nature Communications

Article Title: Clec4A4 is a regulatory receptor for dendritic cells that impairs inflammation and T-cell immunity

doi: 10.1038/ncomms11273

Figure Lengend Snippet: ( a , b ) CFSE-labelled CD45.1 + OT-I CD8 + T cells were transferred into WT mice ( n =6) and Clec4a4 −/− mice ( n =6), and then the mice were immunized with OVA protein in combination with or without the indicated TLR ligands. Ag-specific division of CD45.1 + OT-I CD8 + T cells was analysed 3 days after the immunization by flow cytometry. ( a ) Data are presented by a histogram, and numbers represent the proportion of CFSE dilution among gated CD45.1 + OT-I CD8 + T cells in each quadrant. ( b ) Data are the mean percentage of positive cells±s.d. from six individual samples in a single experiment. ( c – f ) WT mice ( n =6) and Clec4a4 −/− mice ( n =6) were immunized with the indicated TLR ligands, anti-CD40 mAb, and OVA protein. At 6 days after the immunization, splenocytes were analysed for the generation of MHC I-OVA tetramer + CD44 high CD8 + T cells ( c , d ) and for intracellular IFN-γ-producing CD8 + T cells ( e , f ) by flow cytometry. Data are presented by a dot plot ( c , e ) and numbers represent the proportion of MHC I-OVA tetramer + CD44 high cells ( c ) and IFN-γ + cells ( e ) among gated CD8 + T cells in each quadrant. ( d , f ) Data are the mean percentage of positive cells±s.d. from six individual samples in a single experiment. * P <0.01 compared with WT mice (analysis of variance (ANOVA), Bonferroni's multiple comparison test). All data are representative of at least three independent experiments.

Article Snippet: After restriction enzyme digestion, the synthesized DNA sequence was cloned into the sites of pFUSE-hIgG2-Fc2 (Invivogen), and transfected into FreeStyle 293-F cells (Life Technologies) using 293fectin Transfection Reagent (Life Technologies) according to the manufacturers' instructions. huIgFc or Clec4A4-huIgFc fusion protein was purified from the culture supernatant using HiTrap Protein G HP (GE Healthcare Life Sciences), and the purified fusion protein was separated by 10% SDS–PAGE under non-reducing or reducing conditions, respectively, and stained with a Silver Stain kit (Bio-Rad Laboratories) to confirm the protein products.

Techniques: Flow Cytometry