PROJECT SUMMARY/ABSTRACT The γδNKT (also known as NKγδT or Vδ6.3 T) lineage represents an innate type of γδT cells that is generated in the neonatal thymus and subsequently maintained throughout life in tissues such as the thymus, spleen, and liver. These cells are closely related to αβ lineage iNKT cells, which express a semi-invariant αβTCR, and together they constitute the main populations of innate-like T lymphocytes. A major function of innate-like T lymphocytes is to modulate the development and function of other immune cells including conventional T lymphocytes. γδNKT cells use a stereotyped Vγ1.1Vδ6.3 TCR; however, their ligand(s) remains unknown. The generation of γδNKT cells in the thymus is tightly controlled by the activity of E box family of DNA-binding proteins (E proteins), which play profound roles in T cell development. The activity of E proteins is inhibited by Id proteins. The Id family protein, Id3, is induced by TCR signaling in proportion to TCR signal strength and it is the Id3-E protein balance that critically regulates the generation of γδNKT cells. Work completed by this Program Project has revealed that TCR signals orchestrate genome-wide remodeling of E protein binding to a constellation of enhancers during γδT lineage specification, differentiation, and expansion. However, the identity of the critical E protein targets that control the development of individual γδ T sublineages remains a major gap in knowledge. We, together with Project 1, have identified two critical E protein targets that regulate the development of γδNKT cells: the genomic elements encoding the stereotyped Vγ1.1Vδ6.3 TCR and a transcription factor that plays a crucial role in executing the differentiation program initiated by the TCR. We now propose to elucidate the mechanism by which E-proteins regulate γδNKT development and restrict it to neonatal life. We will do so through the following aims. In aim 1, we will investigate E-protein function in regulating the recombination events responsible for generation of the Vδ6.3 TCR chain. In aim 2, we will examine the unique nature of the stereotyped Vγ1.1Vδ6.3 TCR and how E-proteins coordinate the changes in gene expression triggered by Vγ1.1Vδ6.3 TCR signals. The current proposal is built upon key observations derived from the Program Project. The proposed experiments will continue to draw the complementary expertise in the areas of TCR signaling (Project 1), mouse genetics (Project 2), in vitro modeling of mouse and human T cell development (Project 3), and genomics and molecular imaging technologies (Project 4). Our specific aims will not only lead to a better understanding of the mechanism of γδNKT development but will also provide unique insight into the general mechanisms through which E proteins control cell fate.