Project Summary T follicular helper cells (Tfh) are a specialized subset of T cells that directly provide help to B cells to form germinal centers. Within germinal centers, B cells undergo affinity maturation of antibodies, class switching, and differentiation of long-lived plasma cells and memory B cells. T follicular helper responses are vital to long-lived antibody-mediated immune protection, which is demonstrated by the absence of long-lived antibody responses and the presentation of immune deficiency in Tfh-deficient individuals and animals. Following activation of naïve CD4 T cells by viral infection or immunization, these newly activated T cells interpret various signals that induce transcription factors that direct specific gene expression changes. In coordination with these gene expression changes that occur during T helper cell differentiation, genes undergo DNA methylation changes at CpG motifs in regulatory elements that control expression or relevant genes. DNA methylation acts as a repressive epigenetic mark, and the process of demethylation is associated with the ability to express genes important for cell function. Changes in methylation are catalyzed by Tet dioxygenases that participate in the processes of demethylation, and DNA methyltransferases that promote new (de novo) methylation to turn off irrelevant genes. The broad objectives of the specific aims in this proposal are to gain understanding of how T follicular helper cells undergo DNA methylation programing, either through demethylation or de novo methylation, and whether manipulation of such programing can enhance or impair T follicular helper cell function and thus influence antibody responses to vaccination or infection. The specific aims for this proposal are: Aim 1) Determine the role of Tet2 in regulating the balance of T follicular helper and Th1 cell and memory cell differentiation; Aim 2) Define the role of de novo methylation in regulating Tfh and Th1 memory cell differentiation and lineage commitment; and Aim 3) Determine whether DNA methyltransferase inhibition can promote Tfh differentiation and enhance antibody-mediated immune protection following immunization against influenza challenge. These studies will utilize cellular immunology approaches, gene expression and whole genome DNA methylation analyses, and immunization and infectious disease challenge to evaluate the importance of DNA methylation programing in T follicular helper cell differentiation, function, and memory formation. Together, these aims will combine to provide a mechanistic evaluation of how T follicular helper cells differentiate and identify and characterize novel pathways that can be targeted to generate more effective vaccination strategies that can improve long-lasting immunity.