DNA METHYLATION IN T CELL DEVELOPMENT AND FUNCTION

染色质结构和胞嘧啶和CpG二核苷酸上的DNA甲基化的调节模式已经被认为为哺乳动物中稳定和可遗传的程序提供了基础，由此关于个体细胞内组织特异性基因表达的发育决定被预先编程并以表观遗传方式从一个细胞代传递到下一个细胞代。相关研究和体外实验表明，DNA甲基化参与调节T细胞发育和功能，包括T细胞受体基因排列、等位基因排斥和细胞因子基因表达差异。然而，DNA甲基化在体内T细胞发育和功能中的作用尚未直接测试。我们已经使用Cre/loxP重组系统来创建小鼠，其中在CD 4-CD 8-（DN）阶段（在lckCreDnmt/2lox小鼠中）或在随后的CD 4 + CD 8+（DP）阶段（CD 4CreDnmt/2lox小鼠）的T细胞发育期间，选择并有效地消除主要DNA甲基转移酶基因（Dnmt 1）。这些小鼠的表型明显不同。LckCreDnmt/2lox小鼠的胸腺细胞和成熟的CD 4和CD 8 T细胞、异常的CD 4和CD 8 TCR β/CD 3/低/- SP胸腺细胞和T细胞的数量显著减少，而TCR γ δ胸腺细胞的数量增加。相反，CD 4CREDnmt/2lox小鼠中的T细胞发育基本正常。我们建议使用这些独特的小鼠来解决：目的1：确定DNA甲基化在成熟CD 4和CD 8 T细胞功能中的作用。假设：来自CD 4CreDnmt/2lox小鼠的Dnmt 1缺陷型初始CD 4和CD 8 T细胞在初次活化后比来自对照的初始T细胞更容易产生效应细胞因子;在CD 4CreDnmt/2lox小鼠中，CD 4和CD 8 T细胞发展和维持可遗传的、极化的1型对2型细胞因子基因表达模式的能力将降低; CD 4CreDnmt/2lox小鼠在体内激发后将产生记忆/效应T细胞，但它们维持细胞因子产生或细胞溶解活性的极化和保护模式的能力将受到损害。目的2：通过使用lckCreDnmt/2lox小鼠确定DNA甲基化是否有助于TCR β的等位基因排斥。假设：等位基因排除在lckCreDnmt/2lox中不太完全。目标3：确定lckCre Dnmt小鼠中CD 4和CD 8单阳性（SP）胸腺细胞和成熟T细胞上TCR β/CD 3的表面表达减少以及TCR β γ胸腺细胞数量增加的基础。假设：1）SP胸腺细胞和T细胞上TCR β/CD 3表达的降低将反映这些细胞在没有阳性选择的情况下的出现。2)TCR γ胸腺细胞数量的增加将反映相关γ TCR基因的位点可接近性和重排的增加或Notch信号传导的减少。

Regulated patterns of chromatin structure and of DNA methylation on cytosines and CpG dinucleotides have been suggested to provide the basis for a stable and heritable program in mammals, whereby developmental decisions regarding expression of tissue-specific genes within individual cells are pre-programmed and passed from one cell generation to the next in an epigenetic manner. Correlative and in vitro data suggest that DNA methylation contributes to the regulation of T cell development and function, including T cell receptor gene arrangement, allelic exclusion and differential cytokine gene expression. However, the role of DNA methylation in the development and function of T cells in vivo has not been tested directly. We have used the Cre/loxP recombination system to create mice in which the major DNA methyltransferase gene (Dnmt1) is selected and efficiently ablated during T cell development either at the CD4-CD8- (DN) stage (in lckCreDnmt/2lox mice) or at the subsequent CD4+CD8+ (DP) stage (CD4CreDnmt/2lox mice). The phenotypes of these mice differ markedly. LckCreDnmt/2lox mice have a marked reduction in the numbers of thymocytes and mature CD4 and CD8 T cells, aberrant CD4 and CD8 TCRbeta/CD3/low/- SP thymocytes and T cells, and increased numbers of TCRgammadelta thymocytes. In contrast, T cell development in CD4CREDnmt/2lox mice is essentially normal. We propose to use these unique mice to address: Aim 1: Determine the role of DNA methylation in the function of mature CD4 and CD8 T cells. Hypotheses: Dnmt1-deficient naive CD4 and CD8 T cells from CD4CreDnmt/2lox mice will produce effector cytokines more readily after primary activation than naive T cells from controls; the ability of CD4 and CD8 T cells to develop and maintain a heritable, polarized type 1 versus type 2 pattern of cytokine gene expression will be reduced in CD4CreDnmt/2lox mice; CD4CreDnmt/2lox mice will develop memory/effector T cells following challenge in vivo, but their ability to maintain a polarized and protective pattern of cytokine production or cytolytic activity will be compromised. Aim 2: Determine if DNA methylation contributes to allelic exclusion of TCRbeta through the use of lckCreDnmt/2lox mice. Hypothesis: Allelic exclusion will be less complete in lckCreDnmt/2lox. Aim 3: Determine the basis for the reduced surface expression of TCRbeta/CD3 on CD4 and CD8 single- positive (SP) thymocytes and mature T cells and increased numbers of TCRbetagamma thymocytes in lckCre Dnmt mice. Hypothesis: 1) Decreased expression of TCRbeta/CD3 on SP thymocytes and T cells will reflect the emergence of these cells in the absence of positive-selection. 2) The increased numbers of TCRgammabeta thymocytes will reflect either increased locus accessibility and rearrangement of the relevant gammabeta TCR genes or reduced Notch signaling.