Mechanisms Of Transcriptional Regulation in Memory lymphocyte Response and Aging

记忆T淋巴细胞的特点是它们能够对回忆抗原产生快速而强大的反应。去甲肾上腺素（NE）是交感神经系统在应激反应中释放的主要儿茶酚胺之一，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、细胞激活状态、NE暴露的持续时间和NE的浓度。在这里，我们的目的是分析NE对中神经（Tn）、中枢记忆（Tcm）和效应记忆（Tem） CD8 T细胞功能的影响。我们从健康成人中分离出CD8 T细胞亚群，并用NE （1 × 10-6 M）体外处理细胞16小时；然后，我们用CD3和CD28抗体刺激NE处理和未处理的CD8 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（ADRB2）的表达水平以及NE处理的Tcm细胞的整体基因表达变化。通过RT-qPCR鉴定并进一步证实NE处理后表达改变的基因，并通过ELISA检测蛋白变化。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对记忆性CD8 T细胞的影响是否由ADRB2介导。最后，我们检测了血清NE水平高（bb0 150 pg/mL）和低（<150 pg/mL）的健康成人中NE诱导基因的mRNA和蛋白质水平。我们发现记忆（Tcm和Tem） CD8 T细胞表达的ADRB2水平明显高于普通细胞。因此，记忆性CD8 T细胞对NE诱导的基因表达变化明显比普通细胞更敏感。全球基因表达分析显示，NE诱导静息和激活记忆CD8 T细胞中炎症因子和趋化因子的表达升高，以及生长相关细胞因子的表达降低。肾上腺素能受体激动剂和拮抗剂实验证实，NE对记忆性CD8 T细胞的作用主要是由ADRB2介导的。最后，与低NE组相比，血清NE水平高的个体在体外观察到相似的基因表达升高。我们的研究结果表明，NE通过诱导炎症细胞因子的产生和减少激活诱导的记忆性CD8 T细胞的扩增来优先调节记忆性CD8 T细胞的功能。

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.