Comparison of gene expression in Wild type and T cell-specific conditional Trim28 KO in TCR stimulated and un-stimulated naive CD4 positive and T regulatory cells

A microarray study was performed in unstimulated and TCR-stimulated CD4 + T cells and Treg in wild type and conditional Trim28 KO mice to identify genes that are regulated by Trim28. These experiments constitute a portion of the study described below: Paper Abstract: Peripheral T cell activation and differentiation into specialized effectors are regulated by TCR- and cytokine-mediated signals that induce clonal expansion and unique transcriptional factors. These processes may include active chromatin modification by nuclear factors. In search of such molecules, we found Trim28, a component of large nuclear chromatin-regulatory complex is tightly controlled upon TCR stimulation at the level of phosphorylation, and examined global impact of Trim28 loss in especially CD4+ T cells, by generating T cell-specific conditional Trim28 KO mice (CKO). CD4+ T cells from CKO mice showed defective IL-2 production and T cell proliferation associated with defective upregulation of cell-cycle associated proteins. Accordingly, young CKO showed T-lymphopenia. Surprisingly, Trim28 CKO mice eventually accumulated auto-reactive memory-phenotype T cells that produced inflammatory IL-17. CKO mice are also susceptible to induced auto-inflammatory disease with TH-17 dominant immune response. Loss of Trim28 showed aberrant accumulation of TH-17 and FoxP3+ T cells, two key T cells in inflammation vs. tolerance. We found CKO T cells showed a cell-extrinsic promotion of TH-17 and FoxP3+ T cell development by a mechanism involving overproduction of TGF-beta. Our study revealed unexpected roles of Trim28, a global chromatin regulator in both T cell activation and tolerance. Trim28 conditional KO mice and age-matched control mice were sacrificed, and neive CD4+ T cells (CD4+CD62+CD25-) and Treg (CD4+CD62+CD25+) were sorted. Stimulation of naive T cells was done with anti-CD3 and anti-CD28 for 13 hours. We collected quadruplicates for each group.

本研究针对野生型及条件性Trim28敲除（Trim28）小鼠中未受刺激与T细胞受体（TCR）刺激的CD4+ T细胞与调节性T细胞（Treg）开展微阵列（microarray）分析，以鉴定受Trim28调控的基因。本实验为下述研究的一部分：
论文摘要：外周T细胞活化并分化为特定效应细胞的过程，受TCR及细胞因子介导的信号调控，此类信号可诱导克隆扩增并激活独特的转录因子。该过程可能涉及核因子介导的活跃染色质修饰。为探寻此类调控分子，我们发现Trim28——大型核染色质调控复合物的组分之一——在TCR刺激后于磷酸化水平受到严格调控；随后我们构建T细胞特异性条件性Trim28敲除（CKO）小鼠，以此探究Trim28缺失对CD4+ T细胞的全局影响。
来自CKO小鼠的CD4+ T细胞呈现白细胞介素-2（IL-2）产生缺陷与T细胞增殖异常，且该异常与细胞周期相关蛋白的上调缺陷相关。据此，年轻的CKO小鼠表现出T淋巴细胞减少症。令人意外的是，Trim28 CKO小鼠最终会积累产生炎性白细胞介素-17（IL-17）的自身反应性记忆表型T细胞。CKO小鼠还易诱发以TH-17为主导免疫应答的自身炎症性疾病。
Trim28缺失会导致TH-17与FoxP3+ T细胞的异常积累，这两类细胞分别是炎症与免疫耐受中的关键T细胞亚群。我们发现，CKO T细胞可通过过量产生转化生长因子-β（TGF-β）的机制，以细胞非自主的方式促进TH-17与FoxP3+ T细胞的发育。本研究揭示了全局染色质调控因子Trim28在T细胞活化与免疫耐受中均发挥了意想不到的作用。
我们处死Trim28条件性敲除小鼠及同年龄对照组小鼠，分选初始CD4+ T细胞（CD4+CD62+CD25-）与Treg（CD4+CD62+CD25+）。我们采用抗CD3与抗CD28抗体刺激初始T细胞，刺激时长为13小时。每组设置4个生物学重复。