title: Glycosylation-dependent interaction between CD69 and S100A8/S100A9 complex is required for regulatory T-cell differentiation.
abstract: Cluster of differentiation (CD)69 is a leukocyte activation receptor involved in the maintenance of immune homeostasis and is positively selected in activated regulatory T (Treg) cells, implicating its role during Treg-cell differentiation. By RNA interference, we show that CD69 is not sufficient to support the conversion of CD4(+) naive T cells into Treg cells, whereas it does that of human peripheral blood mononuclear cells (hPBMCs) (P < 0.01), suggesting that a ligand-receptor interaction is required for CD69 function. Using immunoprecipitation and mass spectrometry, we identified the S100A8/S100A9 complex as the natural ligand of CD69 in hPBMCs. CD69 specifically associates with S100A8/S100A9 complex as confirmed by in vitro binding and competition assay, and the treatment of CD69 with peptide-N-glycosidase significantly abolishes such association. In agreement, the glycomics analysis determines the glycosylation site and the N-glycan composition of CD69, and terminal removal of sialic acid from that N-linked glycans reverses the generation of forkhead box P3-positive Treg cells (23.21%; P < 0.05). More specifically, we showed that CD69-S100A8/S100A9 association is required for the up-regulation of suppressor of cytokine signaling 3 resulting in inhibited signaling of signal transducer and activator of transcription 3 (36.54% increase upon CD69 silencing; P < 0.01). This might in turn support the secretion of key regulator TGF-β (∼ 3.28-fold decrease upon CD69 silencing; P < 0.05), leading to reduced production of IL-4 in hPBMCs. Our results demonstrate the functional and mechanistic interplays between CD69 and S100A8/S100A9 in supporting Treg-cell differentiation.
authors: *Institute of Biochemical Sciences, College of Life Science, National Taiwan University, Taipei, Taiwan; and Institute of Biological Chemistry and Genomics Research Center, Academia Sinica, Taipei, Taiwan.
authors: Chih-Ru
authors: CR
authors: Lin
authors: *Institute of Biochemical Sciences, College of Life Science, National Taiwan University, Taipei, Taiwan; and Institute of Biological Chemistry and Genomics Research Center, Academia Sinica, Taipei, Taiwan.
authors: Tong-You Wade
authors: TY
authors: Wei
authors: *Institute of Biochemical Sciences, College of Life Science, National Taiwan University, Taipei, Taiwan; and Institute of Biological Chemistry and Genomics Research Center, Academia Sinica, Taipei, Taiwan.
authors: Hsien-Yu
authors: HY
authors: Tsai
authors: *Institute of Biochemical Sciences, College of Life Science, National Taiwan University, Taipei, Taiwan; and Institute of Biological Chemistry and Genomics Research Center, Academia Sinica, Taipei, Taiwan.
authors: Ying-Ta
authors: YT
authors: Wu
authors: *Institute of Biochemical Sciences, College of Life Science, National Taiwan University, Taipei, Taiwan; and Institute of Biological Chemistry and Genomics Research Center, Academia Sinica, Taipei, Taiwan gabriel@gate.sinica.edu.tw bcchen@gate.sinica.edu.tw.
authors: Pei-Yu
authors: PY
authors: Wu
authors: *Institute of Biochemical Sciences, College of Life Science, National Taiwan University, Taipei, Taiwan; and Institute of Biological Chemistry and Genomics Research Center, Academia Sinica, Taipei, Taiwan gabriel@gate.sinica.edu.tw bcchen@gate.sinica.edu.tw.
authors: Shui-Tein
authors: ST
authors: Chen
mesh_terms: D015703:Antigens, CD / Q000378:metabolism*; D000945:Antigens, Differentiation, T-Lymphocyte / Q000378:metabolism*; D040501:Calgranulin A / Q000378:metabolism*; D040502:Calgranulin B / Q000378:metabolism*; D002454:Cell Differentiation; D002478:Cells, Cultured; D006031:Glycosylation; D006801:Humans; D037181:Lectins, C-Type / Q000378:metabolism*; D009000:Monocytes / Q000166:cytology; D011485:Protein Binding; D015398:Signal Transduction; D050378:T-Lymphocytes, Regulatory / Q000166:cytology*; D000099192:CD69 Antigens
chemical_list: D015703:Antigens, CD; D000945:Antigens, Differentiation, T-Lymphocyte; D040501:Calgranulin A; D040502:Calgranulin B; D037181:Lectins, C-Type; D000099192:CD69 Antigens
keywords: hPBMCs; immune homeostasis; ligand