Glycosylation-dependent interaction between CD69 and S100A8/S100A9 complex is required for regulatory T-cell differentiation
Glycosylation-dependent interaction between CD69 and S100A8/S100A9 complex is required for regulatory T-cell differentiation
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DOI：
10.1096/fj.15-273987
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发表时间：
2015-12-01
期刊：
影响因子：
4.8
通讯作者：
Chen, Shui-Tein
中科院分区：
文献类型：
--
作者：
Lin, Chih-Ru;Wei, Tong-You Wade;Chen, Shui-Tein
分化簇(CD)69是一种参与维持免疫内稳态的白细胞激活受体，在激活的调节性T(T-reg)细胞中呈阳性选择，提示其在T-reg-细胞分化中的作用。通过RNA干扰，我们发现CD69不足以支持CD4(+)初始T细胞向Treg细胞的转化，而它能支持人外周血单个核细胞(HPBMC)的转化(P<；0.01)，这表明CD69的功能需要配体-受体的相互作用。通过免疫沉淀和质谱分析，我们确定S100A8/S100A9复合体是人外周血单核细胞中CD69的天然配体。体外结合和竞争实验证实CD69与S100A8/S100A9复合体特异性结合，用N-糖苷酶处理CD69可显著消除这种结合。一致的是，糖组分析确定了CD69的糖基化位点和N-糖链组成，从N-连接的糖链上末端去除唾液酸可以逆转叉头盒P3阳性T-reg细胞的产生(23.21%；P<；0.05)。更具体地说，我们发现CD69-S100A8/S100A9联合作用于细胞因子信号转导抑制因子3的上调，导致信号转导和转录激活因子3信号的抑制(CD69沉默增加36.54%；P<；0.01)。这可能反过来支持关键调节因子转化生长因子-β的分泌(类似于CD69沉默后3.28倍的下降；P<；0.05)，导致hPBMC产生IL-4的减少。我们的结果证明CD69和S100A8/S100A9在支持T-reg细胞分化方面存在功能和机制上的相互作用。
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 (T-reg)cells, implicating its role during T-reg-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 T-reg 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-beta (similar to 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 T-reg-cell differentiation.