DNA METHYLATION IN T CELL DEVELOPMENT AND FUNCTION

染色质结构和胞嘧啶和CpG二核苷酸上DNA甲基化的调节模式被认为为哺乳动物稳定和可遗传的程序提供了基础，从而使个体细胞内组织特异性基因的表达的发育决定被预先编程并以表观遗传方式从一代细胞传递到下一代。相关和体外数据表明，DNA甲基化参与T细胞发育和功能的调控，包括T细胞受体基因的排列、等位基因的排除和细胞因子基因的差异表达。然而，DNA甲基化在体内T细胞发育和功能中的作用尚未得到直接测试。我们使用Cre/loxP重组系统创建了在CD4-CD8- (DN)阶段（在lckCreDnmt/2lox小鼠中）或随后的CD4+CD8+ (DP)阶段（CD4CreDnmt/2lox小鼠中）T细胞发育过程中选择主要DNA甲基转移酶基因（Dnmt1）并有效切除的小鼠。这些小鼠的表型明显不同。LckCreDnmt/2lox小鼠胸腺细胞和成熟CD4和CD8 T细胞数量明显减少，CD4和CD8异常TCRbeta/CD3/low/- SP胸腺细胞和T细胞数量明显减少，TCRgammadelta胸腺细胞数量增加。相比之下，CD4CREDnmt/2lox小鼠的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CD4和CD8 T细胞功能中的作用。假设：来自CD4CreDnmt/2lox小鼠的dnmt1缺陷初始CD4和CD8 T细胞在初次激活后比来自对照的初始T细胞更容易产生效应细胞因子；在CD4CreDnmt/2lox小鼠中，CD4和CD8 T细胞发展和维持可遗传的、极化的1型与2型细胞因子基因表达模式的能力将降低；CD4CreDnmt/2lox小鼠在体内受到刺激后会产生记忆/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TCRbeta的等位基因排除。假设：lckCreDnmt/2lox的等位基因排除不完全。目的3：确定lckre Dnmt小鼠CD4和CD8单阳性（SP）胸腺细胞和成熟T细胞表面TCRbeta/CD3表达降低和tcrβ - 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TCRbeta/CD3表达的下降反映了这些细胞在没有正选择的情况下出现。2) tcrγ - β胸腺细胞数量的增加可能反映了相关γ - β - TCR基因的基因座可及性和重排的增加或Notch信号的减少。

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.