CD4 helper T cells coordinate the immune response and are highly dependent on the expression of the Cd4 gene for proper development and function. The function of a promoter, an enhancer and a silencer have been well documented and explain how the Cd4 gene gets turned on in CD4 T cells and off in CD8 T cells. However, once turned on, the timing and level of Cd4 expression varies during CD4 T cell development and activation. This modulation of CD4 surface levels is essential for proper lineage specification and T cell function. Consistent with the notion that small changes in CD4 expression can disrupt the fine balance between immunodeficiency and autoimmunity that a healthy immune system maintains, SNPs in the regulatory regions of the Cd4 locus have been recently correlated with the development of autoimmune diseases. Yet, how the fine-tuning of CD4 expression is regulated and coordinated with the TCR signal and other environmental cues is not understood. This small-scale investigation will focus on identifying new cis-regulatory elements in the Cd4 locus in vitro and the generation of in vivo mouse models for further studies of their functional importance for CD4 T cell development and function. The in vitro identification and characterization will consist of 1) Identifying highly conserved sequences in Cd4, using the UCSC genome browser;2) Evaluation of the candidates'ability to promote or enhance transcription in vitro by transient transfection of EGFP reporter plasmids into CD4+ cell lines;and 3) Further characterization of the candidates that exhibit a significant ability to modulate reporter expression by truncation and site-directed mutagenesis to determine the minimum sufficient sequence and transcription factor binding sites necessary for function. In a preliminary experiment described in this proposal, we have successfully identified one new developmental stage-specific cis-regulatory element (NCE) that we would like to characterize in detail both in vitro and in vivo. For the in vivo characterization we will develop two separate mouse models using recombineered bacterial artificial chromosome (BAC) of the Cd4 locus with or without a deletion of NCE. The purpose of the two models will be as follows: Model 1 - Using BAC with the EGFP gene inserted in exon 2 of Cd4, generate EGFP reporter transgenic mice, in which we can compare the expression pattern of the wild type Cd4-BAC-EGFP to that of the NCE-deleted Cd4-BAC-EGFP without changing the endogenous CD4 expression pattern. Model 2- Generate wild type Cd4-BAC and NCE-deleted Cd4-BAC transgenic mice that are also deficient for endogenous Cd4 and ?2m in order to determine directly the effect of NCE deletion on positive selection, lineage commitment, activation and differentiation of MHC-II specific CD4 T cells. These in vivo models will be the stepping stone towards our long-term objective of identifying and characterizing new transcription control elements in the Cd4 locus, and understanding how they interact to modulate CD4 levels in different CD4+ cell types at different stages of their development and function.
Currently in the United States it is estimated that 1 in 31 people develop autoimmune disease, while 1 in 3 people will develop cancer and will require chemotherapy that will deplete their CD4 T cells in their lifetime. Immune defenses depend on the presence of functional CD4 T cells, the coordinators of the immune response, and amount and timing of production of the CD4 molecule is critical for proper CD4 T cell function. Conditions such as HIV infection, chemotherapy, or congenital defects cause a decrease in the number or functionality of the CD4 T cells. In this study we will investigate the regulatory mechanisms responsible for changing the timing and level of production of the CD4 molecule, which directly affects T cell development and function. Ultimately our goal is to be able to contro CD4 levels in patients to correct for congenital or induced immunodeficiency and autoimmune disease.
