在活體外細胞培養系統中針對NR4A/PD-1訊號途徑調控T細胞耗竭

Targeting NR4A/PD-1 signal axis to modulate T cell exhaustion in an in vitro cell-based system

李峻緯(Jun-Wei Li)

指導教授 ： 嚴仲陽

國立臺灣大學/醫學院/分子醫學研究所/碩士(2022年)

https://doi.org/10.6342/NTU202200305

摘要

在慢性發炎和癌症中往往發現T細胞存在有效功能低下和抑制性受體表現量上升的狀態，稱為T細胞耗竭。由活體內系統生產耗竭性T細胞耗時且只能產生有限數量，因此活體外系統在近年逐漸被建立和驗證來克服使用活體內系統的缺點。我們成功建立一個為期五天的活體外系統用以產生似耗竭性小鼠CD8 T細胞。在此系統下藉由電穿孔送入小分子干擾核糖核酸進行基因敲落，達到調查該目標基因是否參與T細胞耗竭的目的。例如，我們發現三重敲落NR4A家族會部分回復細胞激素的產生。雖然並無在耗竭性T細胞中發現PD-1受到NR4A敲落的影響，但是在我們建立的EL4細胞模型中，由慢病毒轉染方式將小髮夾核糖核酸送入細胞來敲落NR4A1/3，結果發現佛波酯/離子黴素刺激後PD-1的表達不會上升。此外，我們也嘗試使用小分子藥物來測試是否有效預防T細胞耗竭。我們使用柏萊膜衣錠 (Dasatinib)，一種SRC激酶 (kinase) 抑制劑，發現能夠有效防止T細胞耗竭，同時NR4A家族的表達會受到抑制。進一步藉由質譜流式細胞技術 (CyTOF) 和全核糖核酸測序分析發現柏萊膜衣錠處理過的耗竭性T細胞與效應T細胞基因表徵特徵大部分一致。

關鍵字

T細胞耗竭 ； 活體外系統 ； NR4A ； EL4細胞 ； 慢病毒轉染 ； 小分子干擾核糖核酸 ； 柏萊膜衣錠

並列摘要

T cell exhaustion is defined by poor effector functions, expression of inhibitory receptors and is often observed in chronic infections and cancer. In recent years, in vitro system generating exhausted T cells has been established and validated to overcome the disadvantages of in vivo system which is time-consuming and yield limited numbers. In this study, we successfully produced exhausted-like murine CD8 T cells using in vitro system within five days, and tried to knockdown genes via siRNA electroporation to investigate if the target gene contributes to T cell exhaustion. For instance, we found that triple knockdown of the NR4A family partially restored the production of cytokines. Although knockdown of NR4As by siRNAs failed to affect the expression of inhibitory receptors in primary exhausted T cells, in an EL4 cell model knockdown of NR4A1/3 by lentiviral transduction of shRNA successfully showed the suppression of upregulation of PD-1 after PMA/I stimulation. In addition, we also explored the effect of a Src kinase inhibitor, dasatinib, in preventing T cell exhaustion in the in vitro system. Our data with fluorescent flow cytometry revealed that dasatinib treatment resulted in preventing expression of inhibitory receptors, increase of cytokine production, and decrease of the expression levels of NR4As. Moreover, CyTOF and RNA-seq analysis further suggested that the phenotypes of Dasatinib-treated exhausted T cells almost resembled that of conventional effector T cells. Genome-wide transcriptomic analyses of exhausted T cells also revealed several intriguing observations that warrant further discussion and investigation.

並列關鍵字

T cell exhaustion ； in vitro system ； murine CD8 T cells ； NR4A ； siRNA electroporation ； lentiviral transduction ； dasatinib

參考文獻

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