Mechanisms Of Transcriptional Regulation in Memory lymphocyte Response and Aging

记忆T淋巴细胞的特征在于它们对回忆抗原产生快速和强烈应答的能力。 去甲肾上腺素（NE）是交感神经系统在应激反应期间释放的主要儿茶酚胺之一，并且在调节免疫功能中起重要作用。NE与免疫细胞（包括T细胞）上的肾上腺素能受体结合，导致功能抑制或增强，这取决于细胞类型、细胞活化状态、NE暴露持续时间和NE浓度。在这里，我们的目的是分析NE对幼稚（Tn），中央记忆（Tcm）和效应记忆（Tem）CD 8 T细胞的功能的影响。我们从健康成人中分离出CD 8 T细胞亚群，并在体外用NE（1 × 10 -6 M）处理细胞16小时;然后用CD 3和CD 28抗体刺激NE处理和未处理的CD 8 T细胞亚群24和72小时。我们通过微阵列分析评估了这些细胞中β-2肾上腺素能受体（ADRB 2）的表达水平以及NE处理的Tcm细胞中的整体基因表达变化。鉴定NE处理后改变的表达基因，并通过RT-qPCR和ELISA进一步确认蛋白质变化。我们使用特异性肾上腺素能受体激动剂和拮抗剂进一步确定所观察到的NE对记忆CD 8 T细胞的作用是否由ADRB 2介导。最后，我们检查了NE诱导基因的mRNA和蛋白质水平，在健康成人与NE的高血清水平（> 150 pg/mL）相比，低水平（<150 pg/mL）。我们发现，记忆（Tcm和Tem）CD 8 T细胞表达的ADRB 2水平显着高于幼稚细胞。因此，记忆CD 8 T细胞显着更敏感的比幼稚细胞NE诱导的基因表达的变化在体外。全球基因表达分析显示，NE诱导的表达升高的炎性细胞因子和趋化因子在静息和激活的记忆CD 8 T细胞除了生长相关的细胞因子的表达减少。肾上腺素能受体激动剂和拮抗剂试验证实，NE对记忆性CD 8 T细胞的作用主要由ADRB 2介导。最后，与低NE组相比，具有高血清NE水平的个体具有在体外观察到的相似的升高的基因表达。我们的研究结果表明，NE优先调节记忆CD 8 T细胞的功能，通过诱导炎症细胞因子的产生和减少激活诱导的记忆CD 8 T细胞扩增。 趋化因子通过严格控制的表达在调节免疫应答中起关键作用。炎症趋化因子水平的升高通常随着年龄的增长而发生，但这种与年龄相关的变化的机制尚未完全了解。在这里，我们报告了microRNA-125 b（miR-125 b）在调节人类免疫细胞中炎症CC趋化因子4（CCL 4）表达中的作用及其随衰老而改变的表达。我们首先分析了年轻（42岁）和老年（70岁）成人血液中8种不同类型的免疫细胞中CCL 4的mRNA水平，包括CD 4和CD 8 T细胞亚群（原始、中枢和效应记忆）、B细胞和单核细胞。我们观察到单核细胞和幼稚CD 8 T细胞表达更高水平的CCL 4，并表现出与年龄相关的CCL 4增加。然后我们发现miR-125 b的水平与这些细胞中CCL 4的水平呈负相关，并且与年轻人相比，老年人的单核细胞和原始CD 8 T细胞中miR-125 b的水平降低。在单核细胞和幼稚CD 8 T细胞中通过shRNA敲低miR-125 b导致CCL 4蛋白增加，而在幼稚CD 8 T细胞中通过转染增强miR-125 b表达导致响应于刺激的CCL 4蛋白减少。最后，我们证明了miR-125 b的作用需要CCL 4的3UTR中的种子序列。总之，这些发现表明miR-125 b是CCL 4的负调控因子，其减少部分负责CCL 4的年龄相关性增加。 TCR库作为TCR的储库，用于识别所有潜在的病原体。 使用相同的遗传元件和过程来产生功能性TCR的两种主要类型的T细胞，CD 4+和CD 8+，在它们分别与MHC II类和I类结合的肽的识别方面不同。然而，目前尚不清楚CD 4+和CD 8 + T细胞的TCR库在多大程度上是不同的。 在这里，我们报告了使用5-RACE/PCR/测序方法的CD 4+和CD 8 + T细胞的TCR库的比较分析。我们发现，在每个研究对象中，CD 4 + T细胞的TCR多样性范围为1.8-8.2 × 105，平均比CD 8 + T细胞的TCR多样性大3-4倍。此外，在CD 4+（0.3%）和CD 8+（1.3%）T细胞之间TCR序列几乎没有重叠。 进一步的分析显示，CD 4+和CD 8 + T细胞对CDR 3中的某些氨基酸表现出不同的偏好，并且这通过支持向量机分类器进一步证实，表明CD 4+和CD 8 + T细胞中的TCR CDR 3之间存在明显且可辨别的差异。最后，我们鉴定了6-12%的独特TCR，其与不同的V基因共享相同的CDR 3。总之，我们的研究结果揭示了CD 4+和CD 8 + T细胞之间TCR库的不同特征，并可能用于评估T细胞免疫的能力。

Memory T lymphocytes are characterized by their ability to mount a rapid and robust response to the recall antigen. Norepinephrine (NE) is one of the primary catecholamines of the sympathetic nervous system released during a stress response and plays an important role in modulating immune function. NE binds to the adrenergic receptors on immune cells, including T cells, resulting in either suppressed or enhanced function depending on the type of cell, activation status of the cell, duration of NE exposure and concentration of NE. Here, we aim to analyze the effects of NE on the functionality of nave (Tn), central memory (Tcm) and effector memory (Tem) CD8 T cells. We isolated CD8 T cell subsets from healthy human adults and treated cells in vitro with NE (1x10-6 M) for 16 hours; we then stimulated NE treated and untreated CD8 T cell subsets with antibodies for CD3 and CD28 for 24 and 72 hours. We assessed the level of beta-2 adrenergic receptor (ADRB2) expression in these cells as well as global gene expression changes in NE treated Tcm cells by microarray analysis. Altered expressed genes after NE treatment were identified and further confirmed by RT-qPCR, and by ELISA for protein changes. We further determined whether the observed NE effects on memory CD8 T cells are mediated by ADRB2 using specific adrenergic receptor agonist and antagonists. Finally, we examined the levels of mRNA and protein of the NE-induced genes in healthy adults with high serum levels of NE (>150 pg/mL) compared to low levels (<150 pg/mL). We found that memory (Tcm and Tem) CD8 T cells expressed a significantly higher level of ADRB2 compared to nave cells. Consequently, memory CD8 T cells were significantly more sensitive than nave cells to NE induced changes in gene expressions in vitro. Global gene expression analysis revealed that NE induced an elevated expression of inflammatory cytokines and chemokines in resting and activated memory CD8 T cells in addition to a reduced expression of growth-related cytokines. The effects of NE on memory CD8 T cells were primarily mediated by ADRB2 as confirmed by the adrenergic receptor agonist and antagonist assays. Finally, individuals with high serum levels of NE had similar elevated gene expressions observed in vitro compared to the low NE group. Our results demonstrate that NE preferentially modulates the functions of memory CD8 T cells by inducing inflammatory cytokine production and reducing activation-induced memory CD8 T cell expansion. Chemokines play a pivotal role in regulating the immune response through a tightly controlled expression. Elevated levels of inflammatory chemokines commonly occur with aging but the mechanism underlying this age-associated change is not fully understood. Here we report the role of microRNA-125b (miR-125b) in regulating inflammatory CC chemokine 4 (CCL4) expression in human immune cells and its altered expression with aging. We first analyzed the mRNA level of CCL4 in 8 different types of immune cells including CD4 and CD8 T cell subsets (nave, central and effector memory), B cells and monocytes in blood from both young (&#8804; 42 yr) and old (&#8805; 70 yr) adults. We observed that monocytes and nave CD8 T cells expressed higher levels of CCL4 and exhibited an age-related increase in CCL4. We then found the level of miR-125b was inversely correlated with the level of CCL4 in these cells, and the level of miR-125b was reduced in monocytes and nave CD8 T cells of the old compared to the young adults. Knock-down of miR-125b by shRNA in monocytes and nave CD8 T cells led to an increase of CCL4 protein whereas enhanced miR-125b expression by transfection in nave CD8 T cells resulted in a reduction of the CCL4 protein in response to stimulation. Finally, we demonstrated that miR-125b action requires the seed sequence in 3UTR of CCL4. Together these findings demonstrated that miR-125b is a negative regulator of CCL4 and its reduction is partially responsible for the age-related increase of CCL4. The TCR repertoire serves as a reservoir of TCRs for recognizing all potential pathogens. Two major types of T cells, CD4+ and CD8+, that use the same genetic elements and process to generate a functional TCR, differ in their recognition of peptide bound to MHC class II and I, respectively. However, it is currently unclear to what extent the TCR repertoire of CD4+ and CD8+ T cells is different. Here, we report a comparative analysis of the TCR&#946; repertoires of CD4+ and CD8+ T cells using a 5-RACE/PCR/sequencing method. We found that TCR&#946; diversity of CD4+ T cells ranges from 1.8-8.2 x105 and is 3-4 times greater on average than that of CD8+ T cells in each study subject. Furthermore, there was little overlap in TCR&#946; sequences between CD4+ (0.3%) and CD8+ (1.3%) T cells. Further analysis showed that CD4+ and CD8+ T cells exhibited distinct preferences for certain amino acids in the CDR3, and this was further confirmed by a support vector machine classifier, suggesting there are distinct and discernible differences between TCR&#946; CDR3 in CD4+ and CD8+ T cells. Finally, we identified 6-12% of the unique TCR&#946;s that share an identical CDR3 with different V genes. Together, our findings reveal the distinct features of the TCR&#946; repertoire between CD4+ and CD8+ T cells and could potentially be used to evaluate the competency of T cell immunity.