T細胞活性化における体内時計発現変化と、体内時計がT細胞の活性化にどのように関与するのかを検討するために、8~12週齢のBmal1欠損マウスおよびコントロールマウスの脾臓よりFACSを用いてナイーブT細胞を単離した。ナイーブT細胞を抗CD3・CD28抗体で24時間刺激を行った際の体内時計の発現をRT-qPCR、Western blot法で検討したところ、時計遺伝子の発現が上昇し、解糖系やアミノ酸代謝に関わる遺伝子の発現が上昇した。Bmal1欠損マウスでは、解糖系やアミノ酸代謝に関わる遺伝子の発現が野生型よりも更に上昇傾向であった。今後、RNA-seqで遺伝子発現を網羅的に検討する予定である。また、ナイーブT細胞を抗CD3・CD28抗体で72時間刺激し、細胞増殖をCelltrace Violetで評価を行ったところ、細胞増殖がBmal1欠損T細胞で亢進していた。今後、血清による同期を行い、異なる濃度の抗CD3および抗CD28抗体で刺激した細胞を、6時間ごと48時間に渡り回収し、時計遺伝子の発現をRT-qPCR法で検討するほか、ルシフェラーゼアッセイでも検討する方針である。また、これらの細胞を用いて、酸素消費量、細胞外液酸性度、NAD+/NADH比を測定するほか、メタボローム解析を実施し、体内時計の欠損がT細胞に与える代謝変化を検討する。また代謝フラックスにより解糖系、脂質酸化、グルタミン酸化、ピルビン酸の酸化を評価する。更に、活性化前後のナイーブT細胞に対しBMAL1とMYCのChIP-seqを行い、T細胞の活性化状態に起因する転写調節について、BMAL1とMYCのクロマチン結合を検討するほか、single cell RNA-seqを行い、T細胞活性化の際のpopulationを概日時計の有無で比較検討する。
To investigate whether the core clock gene expression is altered and how the clock engages in T cell activation, the naive T cells were isolated from the spleen of wild-type and Bmal1 knockout mice aged from eight to twelve weeks. The naive T cells were activated by the anti-CD3/CD28 antibody for twenty-four hours and expression of the circadian clock was explored using real-time qPCR and western blot analyses. The expression of clock genes as well as those encoding for glycolytic and amino acid metabolism was induced by T cell activation. Expression of such genes in Bmal1 knockout T cells was further upregulated in comparison to the wild-type cells. In the future, RNA-seq will be performed to understand the impact of the circadian clock on the T cell activation on a genome-wide scale. Additionally, the naive T cells was activated by anti-CD3/28 antibodies for 72 hours, and cellular proliferation was more pronounced in Bmal1 knockout T cells in the Celltrace Violet assay. In the future, the naive T cells activated by various concentration of anti-CD3/28 antibodies will be synchronized using the serum shock, and the cells will be collected every six hours over the circadian cycle for forty-eight hours. The real-time qPCR and luciferase assay will be performed to investigate the rhythmic gene expression of the circadian clock. Moreover, measurement of the oxygen consumption, extracellular acid level, NAD+/NADH ratio as well as metabolomic analysis will be performed to explore the metabolic changes upon the clock deletion. The metabolic flux analysis will be conducted to assess the metabolic pathways such as glycolysis, lipid oxidation, glutamine oxidation, and pyruvate oxidation. Chromatin immunoprecipitation followed by deep sequencing of BMAL1 and MYC will be carried out in the preactivated and activated naive T cells to understand the transcriptional regulation by these factors. Single cell RNA-seq will be also performed in the presence or absence of the clock in T cells.
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