Laurie Penix (hereafter referred to as I) completed residency training in Pediatrics two and one-half years ago, and since that time has been a fellow in Pediatric Infectious Diseases at the University of Washington. I have worked in the laboratory of Dr. Christopher B. Wilson, seeking to explore the basis for the increased susceptibility of the human neonate to infections with intracellular pathogens. Although considerable progress has been made in acquiring the skills necessary to establish an independent laboratory career, an additional period of training supported by the proposed CIA is critical. I believe that Dr. Wilson's laboratory, and the environment provided by its relationship to other investigators in the Department of Immunology and the overall scientific community at the University of Washington, will foster my development into an independent investigator during the tenure of this Clinical Investigator Award. Previous data from our laboratory and others, indicated that a profound deficit in the production of interferon-gamma (IFN-gamma) by T cells from neonates, was a contributing factor to this increased susceptibility. This defect was relatively selective in that the production of IL-2 did not differ between adult and neonatal T cells. this reflected the lack of memory T cells in neonates, since putative naive and memory T cells produce comparable amounts of IL-2, whereas naive T cells (comprising 50- 65% of adult but > 95% of neonatal T cells) produce 10-fold less IFN- gamma. The differences between memory and naive T cells were due to diminished initiation of transcription of the IFN-gamma gene. I joined the laboratory and began to investigate the transcriptional regulation of the IFN-gamma gene, with the long term goal of identifying cis- elements and factors binding thereto, which account for the markedly different expression of this gene in naive T cells. My preliminary data strongly suggest that elements, which are necessary and sufficient for T cell- and activation-specific expression of lacZ reporter constructs are present within 111 bp of the IFN-gamma transcription start site; included therein are elements which bind the transcription factor GATA-3. I will examine the hypothesis that differences in expression/activation of proteins binding to the key regulatory elements in the -111 to +61 region of the IFN-gamma gene, contribute to the differential expression of this gene in naive and memory T cells. This will be done by two complementary approaches (Aims 1 and 2), pursued in parallel. to define the minimal cis-elements in the IFN-gamma promoter that confer T cell specific expression following activation, I will use transient transfection assays in Jurkat T cells. To identify the trans-acting factors interacting with the key cis-elements, I will use electrophoretic mobility shift assays, UV cross-linking, methylation interference and partial affinity purification. Once these elements and factors are delineated, in Aim 3 I will explore their role in the differential expression of IFN-gamma. This will be done by comparing trans-acting factors present in extracts from adult memory T cells to those present in extracts from naive T cells from neonates and adults. This information will enhance our understanding of the molecular basis of T cell development and may provide insight into potential therapeutic approaches to enhance cell-mediated immunity in neonates.
