Circadian Clock Activity in Mouse and Human CD4+ T Cells

Though it is well established that immunological functions of CD4+ T cells are time of day-dependent, the underlying molecular mechanisms remain largely obscure. To address the question whether T cells themselves harbor a functional clock driving circadian rhythms of immune function, we analyzed clock gene expression and immune responses of CD4+ T cells purified from blood of healthy subjects at different time points throughout the day. Circadian clock function as well as immune function was further analyzed in cultivated T cells and circadian clock reporter systems. We found robust rhythms of clock gene expression as well as, after stimulation, of IFN-g production and CD40L expression in both freshly isolated and in cultured CD4+ T cells. Moreover, circadian luciferase reporter activities in CD4+ T cells and in thymic sections from PER2::LUCIFERASE reporter mice suggest that endogenous T cell clock rhythms are self-sustained under constant culture conditions. Microarray analysis of stimulated CD4+ T cell cultures revealed a rhythmic regulation of the NF-kB pathway as a candidate mechanism regulating circadian immune responses. Collectively, these data demonstrate for the first time that CD4+ T cell responses are regulated by an intrinsic cellular circadian oscillator capable of driving rhythmic adaptive immune responses in vitro and in vivo. The study is designed with 3 biological replicates from three different time points.

尽管已有研究证实CD4阳性T细胞（CD4+ T cells）的免疫功能具有昼夜依赖性，但其背后的分子机制在很大程度上仍不明确。为探究T细胞自身是否携带驱动免疫功能昼夜节律的功能性时钟，我们于一日内不同时间点从健康受试者血液中纯化得到CD4阳性T细胞，对其时钟基因表达与免疫应答进行了分析。我们还在培养的T细胞以及昼夜节律报告基因系统中进一步分析了昼夜时钟功能与免疫功能。我们发现，无论是新鲜分离的还是体外培养的CD4阳性T细胞，其时钟基因表达均呈现显著节律性；在受到刺激后，干扰素γ（IFN-γ）的产生与CD40配体（CD40L）的表达同样呈现节律性。此外，在CD4阳性T细胞以及来自PER2::荧光素酶（PER2::LUCIFERASE）报告基因小鼠的胸腺组织切片中检测到的昼夜节律荧光素酶报告活性表明，内源性T细胞时钟节律在恒定培养条件下可自主维持。对受刺激的CD4阳性T细胞培养物进行基因芯片分析后，我们发现核因子κB（NF-κB）通路存在节律性调控，这可能是调控昼夜节律免疫应答的候选机制。综上，本研究首次证实CD4阳性T细胞应答受内在细胞昼夜节律振荡器调控，该振荡器可在体外与体内驱动节律性适应性免疫应答。本研究设置了来自三个不同时间点的3次生物学重复。