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

Dendritic cells (DCs) comprise several subsets that are critically involved in the initiation and regulation of immunity. Clec4A4/DC immunoreceptor 2 (DCIR2) is a C-type lectin receptor (CLR) exclusively expressed on CD8α− conventional DCs (cDCs). However, how Clec4A4 controls immune responses through regulation of the function of CD8α− cDCs remains unclear. Here we show that Clec4A4 is a regulatory receptor for the activation of CD8α− cDCs that impairs inflammation and T-cell immunity. Clec4a4−/−CD8α− cDCs show enhanced cytokine production and T-cell priming following Toll-like receptor (TLR)-mediated activation. Furthermore, Clec4a4−/− mice exhibit TLR-mediated hyperinflammation. On antigenic immunization, Clec4a4−/− mice show not only augmented T-cell responses but also progressive autoimmune pathogenesis. Conversely, Clec4a4−/− mice exhibit resistance to microbial infection, accompanied by enhanced T-cell responses against microbes. Thus, our findings highlight roles of Clec4A4 in regulation of the function of CD8α− cDCs for control of the magnitude and quality of immune response.

are presented by a dot plot, and numbers represent the proportion in each quadrant. (Right panel) Total lysate were obtained from BMDCs or DC2.4 (None), BMDCs or DC2.4 expressing mock-GFP, and BMDCs or DC2.4 expressing Clec4A4-GFP, and the immunoprecipitate with anti-FLAG M2 mAb was analyzed using anti-FLAG M5 mAb. (c,d) DC2.4, DC2.4 expressing mock-GFP, and DC2.4 expressing Clec4A4-GFP were stimulated or not stimulated with the indicated TLR ligands, and the production of IL-6 (c) and TNF-α (d) was measured by ELISA. Data are the mean ± s.d. from three individual samples in a single experiment. *P < 0.01 compared with DC2.4 expressing mock-GFP (ANOVA, Bonferroni's multiple comparison test). (e) DC2.4, DC2.4 expressing mock-GFP, and DC2.4 expressing Clec4A4-GFP were stimulated or not stimulated with LPS for the period indicated, at which time cells were lysed. Total lysate was analyzed using Ab specific for p65 or phosphorylated p65. All data are representative of at least three independent experiments.

Supplementary Figure 3
Generation and identification of Clec4a4 -/mice. (a) Strategy used to produce the Clec4a4 -/mice. (1) Partial restriction map of the WT Clec4a4 gene. Exons are depicted as black boxes. The restriction site indicated is E: EcoRV (2) Targeting vector used for the introduction of the mutations in the Clec4a4 gene. A SalI site engineered in place of the start codon in exon 1 of the Clec4a4 gene was used to clone the IRES-EGFP-Cre/Neo r auto-deleter cassette. DTa: diphtheria toxin a expression cassette, I: IRES, E: EGFP, C/N auto-deleter: Cre/Neo r auto-deleter. The Cre/Neo r auto-deleter cassette is shown bracketed by Lox P sites (filled triangles); it directs its own excision as it passes through the male germline. (3) Structure of the targeted allele following homologous recombination in ESC clones. (4) Structure of the Clec4a4 allele following expression of the Cre recombinase and excision of the Neo r cassette in mutant mice. The 3' external single-copy probe (a hatched box) and the PCR primers at the 5' end (blackarrows) used to verify proper homologous recombination events are shown. (b,c) DNA-PCR (b) and Southern blot (c) analysis of WT and recombinant ESC clones. (d,e) Genotyping of tail DNA from WT mice and from heterozygous or homozygous mice for the Clec4a4 allele by DNA-PCR (d) and Southern blot (e) analysis. In the Southern blot analysis, genomic DNA was digested by EcoRV and hybridized to the 3' external single-copy probe. All data are representative of at least three independent experiments.
Supplementary Figure 5 Deficiency of Clec4A4 amplifies TLR-mediated signaling in CD8α -cDCs. CD8α -cDCs obtained from WT mice and Clec4a4 -/mice were stimulated or not stimulated with LPS (a) or CpG-B (b) for the period indicated, at which time cells were lysed. Total lysate was analyzed using Ab specific for p65 (a,b), ERK (a), JNK (a), p38 (a), IRF-3 (a), and IRF-7 (b) or for phosphorylated versions of these proteins. All data are representative of at least three independent experiments.

Supplementary Figure 6
Deficiency of Clec4A4 promotes TLR-mediated cytokine production in vivo. WT mice (n=6) and Clec4a4 -/mice (n=6) were injected with Pam3CSK4 (a), poly(I:C) (b), and CpG-B (c), 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 (ANOVA, Bonferroni's multiple comparison test). All data are representative of at least three independent experiments.

Supplementary Figure 7
Deficiency of Clec4A4 augments inflammatory response in vivo. WT mice (n=6) and Clec4a4 -/mice (n=6) were injected with or without CpG-B, and Spl were obtained 24 hrs after the injection. The weight of Spl (a, left panel), the absolute number of leukocytes (a, right panel), and the frequency of the indicated leukocytes (b) were analyzed by flow cytometry. Data are the mean ± s.d. from six individual samples in a single experiment. *P < 0.01 compared with WT mice (ANOVA, Bonferroni's multiple comparison test). All data are representative of at least three independent experiments.
Pam3CSK4 (1.25-1 µg/ml), poly(I:C) (12.5-50 µg/ml), LPS (0.25-1 µg/ml), or CpG-B (0.025-0.1 µM) in combination with OVA 323-339 peptide under T H 1 (a)-or T H 17 (b)-polarized culture conditions for 3 days, and intracellular production of IFN-γ (a) or  in the cultured CD4 + T cells was analyzed by flow cytometry. Data are the mean percentage of IFN-γ + cells or IL-17 + cells among gated CD4 + T cells ± s.d. from three individual samples in a single experiment. (c) CFSE-labeled 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 analyzed at indicated days after the immunization by flow cytometry. Data are the mean percentage of the dividing cells ± s.d. from six individual samples in a single experiment. (d-f) WT mice (n=6) and Clec4a4 -/mice (n=6) were immunized with CpG-B plus OVA protein.
At 14 days after the immunization, Spl 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 (d), and production of IFN-γ (e, left panel) and IL-17 (e, right panel) by ELISA. Data are the mean ± s.d. from six individual samples in a single experiment. (f) Intracellular production of IFN-γ and IL-17 in the cultured CD4 + T cells was analyzed by flow cytometry. Data are presented by a dot plot, and numbers represent the proportion of IFN-γ + cells and IL-17 + cells among gated CD4 + T cells in each quadrant. *P < 0.01 compared with WT mice (ANOVA, Bonferroni's multiple comparison test). All data are representative of at least three independent experiments.