DNA METHYLATION IN T CELL DEVELOPMENT AND FUNCTION

染色质结构以及胞嘧啶和CpG二核苷酸上DNA甲基化的调控模式被认为是哺乳动物稳定和可遗传程序的基础，在哺乳动物中，关于组织特异性基因在单个细胞内表达的发育决定是预先编程的，并以表观遗传的方式从一个细胞世代传递到下一代细胞。相关的体外实验数据表明，DNA甲基化参与了T细胞发育和功能的调节，包括T细胞受体基因的排列、等位基因排斥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。我们已经使用Cre/loxP重组系统创建了这样的小鼠，在T细胞发育过程中，主要DNA甲基转移酶基因(Dnmt1)被选择并有效地去除，无论是在CD4-CD8-(DN)阶段(在lck CreDnmt/2lox小鼠中)，还是在随后的CD4 CD8(DP)阶段(CD4CreDnmt/2lox小鼠)。这些小鼠的表型明显不同。Lck CreDnmt/2lox小鼠胸腺细胞和成熟的CD4、CD8 T细胞明显减少，TCRbeta/CD3/Low/-SP异常的CD4、CD8胸腺细胞和T细胞明显减少，TCRGammadelta胸腺细胞明显增加。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：目标1：确定DNA甲基化在成熟的CD4和CD8T细胞功能中的作用。假说：CD4CreDnmt/2lox小鼠的DNMT1缺陷的幼稚T细胞在初次激活后将比对照组的幼稚T细胞更容易产生效应性细胞因子；CD4CreDnmt/2lox小鼠的CD4CreDnmt/2lox小鼠的CD4和CD8T细胞发展和维持可遗传的、极化的1型和2型细胞因子表达模式的能力将降低；CD4CreDnmt/2lox小鼠在体内受到攻击后将产生记忆/效应T细胞，但它们维持细胞因子产生或细胞溶解活动的极化和保护性模式的能力将受到损害。目的2：通过使用lck CreDnmt/2lox小鼠，确定DNA甲基化是否有助于TCRbeta的等位基因排斥。假设：等位基因排斥反应在lck CreDnmt/2lox中不完全。目的：探讨Lck Cre Dnmt小鼠胸腺细胞表面TCRbeta/CD3表达减少和TCRbetagma细胞数量增加的原因。假设：1)在SP胸腺细胞和T细胞上TCRbeta/CD3表达的降低将反映这些细胞在缺乏阳性选择的情况下的出现。2)TCRGammabeta胸腺细胞数量的增加将反映相关Gammabeta TCR基因位点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.