Fig 2: Deficiency of Clec4A4 increases Ag-specific CD4+ T-cell responses in vivo.(a–d) CD45.1+OT-II CD4+ T cells were cultured with CD8a- 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 OVA323–339 peptide under TH1 (a,b)- or TH17 (c,d)-polarized culture conditions for 3 days, and intracellular production of IFN-? (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-?+ 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 [3H]thymidine incorporation (g, left panel) and production of IFN-? (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-? 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-?+ 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.

Fig 3: Retroviral transduction of Clec4A4 suppresses the TLR-mediated activation of cDCs.(a) BMDCs expressing mock-GFP, Clec4A4-GFP, Clec4A4N186Q-GFP, Clec4A4?Y68–L236-GFP or Clec4A4?I5–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.

Fig 4: Deficiency of Clec4A4 modulates TLR-mediated activation of CD8a- cDCs.(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), CD3? (blue) and Clec4A4 (red) or CD11c (red). (d) The expression of Clec4A4 and cell-surface molecules on CD8a- 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 CD8a- cDCs were obtained 24 h after injection. The expression of cell surface molecules on CD8a- 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 CD8a- 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 CD8a- cDCs (104) obtained from WT mice and Clec4a4-/- mice in the presence or absence of OVA323–339 peptide (1 µM; f) or OVA257–264 peptide (1 nM; g), and the proliferation was measured by [3H]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.

Fig 5: Deficiency of Clec4A4 enhances the ability of CD8a- cDCs to produce cytokines in response to TLR ligands.CD8a- 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.