PROJECT SUMMARY / ABSTRACT The ability of the immune system to develop immunological memory is the basis for protective immunity induced by infection and vaccination. Generation of memory T cells is governed by transcription factor networks that are different from those regulating effector T cells. Durability of T cell memory is variable, implying that memory T cells are heterogeneous at the level of transcriptional and epigenetic regulation that determine longevity. Protective immunity is increasingly compromised with age, implying a defect in the generation and survival of memory cells. In our in vitro studies of T cell responses from older adults, we found a shift in transcription factor expression that disfavored memory cell generation, in particular a lack of FOXO1 and TCF7 expression. Preliminary evidence suggests that the reduced FOXO1 expression in old activated T cells impairs lysosomal proteolytic activity and induces expansion of the compartment of late endosomes and multivesicular bodies that are able to sequestrate GSK-3β and thereby stabilize β-catenin. In parallel, older activated T cells fail to upregulate the interferon response genes SAMD9 and SAMD9L that bind to early endosomes and facilitate their fusion to mature into late endosome. Inhibition of GSK-3β activity regulates many cellular functions including WNT signaling and TCF7 activity as well as mitochondrial biogenesis, all important processes for memory cell generation and longevity. Based on these data, we propose that older T cells upon activation develop differences in their endosomal/lysosomal system compared to young individuals, with implications for GSK-3β sequestration. We will examine this hypothesis in a mechanistic in vitro Aim 1 and in vivo studies of transcription factor networks in antigen-specific memory T cells in Aim 2. In Aim 1, we will examine how age-associated differences in the expression of the transcription factor FOXO1 and the interferon response genes SAMD9/SAMD9L shape endosomal compartments and how these differences influence signaling and transcription factor pathways that control memory cell development. In Aim 2, we will examine directly ex vivo whether heterogeneity within the memory cell compartment, accounting for differences in protection, correlates with transcription factor networks. We will perform ATAC-seq on antigen-specific memory T cells to infer transcription factor binding and determine the influence of age, nature of inciting virus and vaccine status.