DNA METHYLATION IN T CELL DEVELOPMENT AND FUNCTION

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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.