The ability of the adaptive immune system to develop immunological memory is central to immune health and is the foundation for the great success of vaccination programs in preventive medicine. With increasing age, the proficiency for forming immunological memory dramatically declines, resulting in incomplete protection after vaccination. The cause for this decline is multifactorial, with an inability in maintaining a nave T cell compartment as well as a failure of T cells to respond and differentiate upon antigenic stimulation. In our current studies, we have found that epigenetic signatures are sensitive and robust to identify T cell-intrinsic changes as they occur with aging and allow conclusion on the transcription factor networks involved. Age-associated changes, both at the transcriptional as well as epigenetic level, fall into two dimensions; failure to maintain stem-like features resulting in the initiation of differentiation programs, and defects in cell maintenance pathways. Interestingly, CD8 T cells are more sensitive to these changes than CD4 T cells. In Aim 1, we will use this difference between CD4 and CD8 T cells to examine the influence of age on T cell homeostasis. We will define nave T cell population heterogeneity at the single cell level and examine the hypothesis that transcription factor networks in CD4 T cells are protective to aging. In Aim 2, we will study the influence of age on TCR signaling and analyze how TCR signal strength and age-associated changes in microRNA expression shape activation-induced modifications in the epigenome of T cells from young and older individuals. Aim 3 will examine the epigenetic basis for the preservation of functional long-lived memory cells. Based on longitudinal studies of chromatin accessibility in antigen-specific CD8 T cells, we have inferred transcription factor networks that characterize memory cells years after yellow fever vaccination. We will now extend these studies to address the hypothesis that transcription factor networks in memory T cells change with age. We will analyze CD4 and CD8 memory T cells to different viruses, which are known to behave differently to aging, such as varicella zoster and cytomegalovirus. In addition, we will examine in single cell studies whether aging influences the cellular heterogeneity in these virus-specific memory T cells. We will compare our results in the aged host to those generated in Project 1 on tissue-resident T cells or CD28+ and CD28- effector and memory T cells after yellow fever vaccination. Collectively, our studies will define the epigenetic landscape in antigen-specific T cells that confer effector or long-lived memory function and relate epigenetic changes to mechanisms relevant to T cell aging.