Genome-wide analysis identifies NR4A1 as a key mediator of T cell dysfunction

Abstract:

遭遇自身抗原或者暴露于肿瘤微环境后，T细胞会发生功能失调。T细胞的功能由共刺激信号调控。而dominance of negative co-stimulation 会导致T细胞功能的失调。然而，这其中的分子机制仍然不清楚。在本文中，利用小鼠体外耐受性T(tolerance T)细胞诱导系统和全基因组表观和基因表达测序发现，耐受性T细胞与效应性T和Treg细胞具有显著差异。其中，NR4A1在耐受性T细胞中稳定性高表达。NR4A1的过表达可以抑制效应细胞的功能，而delet 耐受性T细胞后，T细胞的耐受状态可以得到纠正，并增强了对抗肿瘤和慢性病毒感染的能力。在机制上，NR4A1 被招募结合于转录因子AP-1的结合位点从而通过抑制AP-1的功能抑制效应性基因的表达。NR4A1的结合也能够促进H3K27ac的乙酰化，从而导致耐受基因的表达。因此NR4A1可能能作为肿瘤免疫治疗的靶点。
 

Results:

利用之前作者已发表的小鼠体外模型获得耐受性T细胞。

• 对这些细胞进行全基因组转录组和表观遗传组的测序。对比Ttol和TH1, TH2,TH17发现了2357个在Ttol中特异性表达的基因。
  其中，无应答相关基因(Cblb, Dgka, Rnf149, Tagap1 and Nfatc1)， 转录抑制因子(Rel, Foxp1 and Tgif1) 在Ttol中上调, 而效应因子(Il2, Ifng, Gzmb, Il17a and Il21)和转录组分(核糖体基因如Rps29, Rpl35, Rpl14, Rplp2 and Rpl26)显著下调
   
• Ingenuity pathway analysis 分析显示, T 细胞耐受与不正常分化，IL-2/STAT5 信号通路，以及NR4A1调节的转录变化相关。
   
• 对Ttol，慢病毒诱导的衰竭性CD4+ T, NF-AT1-overexpressed anergic CD4+ T (TNF-AT)的转录本进行比较。
  发现有174个基因是这三种细胞共享的。其中，与TCR, JAK-STAT信号通路失调有关的基因重叠率最高。核糖体，蛋白质组和剪接体相关的基因在Ttol中变化更大。
   
• 检测Ttol细胞的组蛋白修饰状态
  Tol中，只有25%的H3K4me3定位在转录起始位点。（Trimethylation of Lys4 on histone 3 (H3K4me3)是转录基因激活的标志）。而其他T细胞中，大约有53%-65%。 而TSS区H3K27me3在各个T细胞亚群的改变则微不足道。

Ttol cells exhibit distinct transcriptional and epigenetic features
 
基因座位可视化显示，在Ttol细胞中，谱系特异性效应因子（Ifng，Il4，Il17a）和转录因子（Tbx21，Gata3，Rorc）显示出低水平或H3K4me3和H3K27me3的缺失。相反，低反应相关基因（Egr3，Dgkz, Cdkn2a）以及衰竭相关基因（Lag3，Pdcd1,Tigit）显示出更高水平的H3K4me3。
Distribution of H3K4me3 and H3K27me3 peaks

体外模型构建：

In summary, our analysis indicates that the fate of naı¨ve T cells, as they encounter an antigen, is determined by not a single ‘second’ signal, but instead, rich combinatorial costi- mulation. In the absence of positive costimulation mediated by CD28 and ICOS pathways, negative costimula- tory molecules actively instruct T cells to develop into toler- ant T cells characterized by inactivation of intrinsic signaling and transcriptional programs. Tolerant Tcells expressed Grail but not transcription factors for effector T cells—T-bet and Eomes, as well as GATA-3 for CD4 T cells. On the other hand, during infection and inflammatory responses, B7 and B7h molecules are highly upregulated on APC, which overcomes the constitutive negative costimulation signals and results in a positive outcome of T-cell activation—extensive T-cell ex- pansion and generation of effector function. In this case, T cells express transcription factors T-bet, Eomes and GATA-3 but not Grail, Itch or cbl-b
T-cell tolerance or function is determined by combinatorial costimulatory signals

 

• 体外实验验证：

  
  NR4A1 is required for T cell tolerance formation
  

   

• 体内实验：

  
  Disruption of NR4A1 prevents T cell exhaustion caused by tumour and viral infection
  

   

• ChIP–seq
  将NR4A1占据的基因与过量表达NR4A1的Ttol细胞和/或T细胞中差异表达的基因进行比较，发现约70％的Ttol基因被NR4A1结合。
  NR4A1结合区域分析显示NR4A1结合最多的motif是含有AP-1的共有序列和canonical NR4A1结合区域。为了验证，作者又做了anti-c-Jun的ChIP-seq，结果显示NR4A1结合区域与c-Jun的区域有很多程度的重叠。（c-Jun是AP-1的亚基）

  
  Correlation between NR4A1 and c-Jun
  

   
  由于NR4A1主要在非启动子区域结合，作者在H3K27ac上进行了ChIP-seq，这是一种超级增强子标记，反映了T细胞活化的表观遗传状态。 值得注意的是，NR4A1峰与NR4A1过表达T细胞中的H3K27ac位点大量重叠，并且共定位的主要区域是经典的NR4A1结合共有序列。 在存在或不存在NR4A1的情况下进一步检查H3K27ac分布表明NR4A1结合与超级增强区域中活化T细胞中H3K27ac修饰正相关。

  
  Correlation between NR4A1 and H3K27ac
   

H3K27ac is a modification to DNA packaging protein Histone H3. It involves the acetylation at the 27th lysineresidue of the histone H3 protein. H3K27ac is associated with the higher activation of transcription and therefore defined as an active enhancer mark. H3K27ac is found at both proximal and distal regions of transcription start site (TSS). Since the H3K27ac and H3K27me3 modification is at the same location on the histone tail, they antagonize each other.^[1] H3K27ac is often used to find active enhancers and poised enhancers subtracting from another enhancer mark H3K4me1 that contains all enhancers
from wikiwand

 

• 电泳迁移率变动分析（EMSAs）和荧光素酶报告分析
  结果显示NR4A1特异性拮抗AP-1介导的转录程序。此外，对全基因组NR4A1和c-Jun分布的检查显示，强制性表达NR4A1能够抑制TSSs中c-Jun的富集。

  
  NR4A1 specifically antagonized the AP-1-mediated transcriptional program

• 与抑制子间关系
  NR4A1能够与诱导性基因位点结合，包括Gata3，Nr4a1，Nt5e，Bach2和Samhd1同时伴随H3K27ac增强，表明NR4A1可能与这些抑制剂协同发挥作用。

  
  NR4A1 might coordinate the action of these inhibitors
  

   

Summary

功能失调的T细胞中NR4A1表达上调，可能能作为免疫治疗的新靶点。

Integrated network analysis that identifies NR4A1-centred transcriptional modules