Project Summary Influenza is a leading cause of death in the elderly and current vaccines only protect a fraction of this population despite widespread vaccination programs and specialized formulations. A better understanding of the molecular mechanisms that control immunological aging is urgently needed so that newly designed vaccines, adjuvants and pharmacologic interventions can be employed to restore youthful antibody (Ab) responses in the older population. Our overarching goals are focused on determining the effects of aging on immune cells that mediate the anti-influenza Ab vaccine response. We will perform an integrated and comprehensive evaluation of circulating T follicular helper (Tfh), T follicular regulatory (Tfr) cells and B cells as well as their subsets from young and older individuals prior to and after seasonal influenza vaccination. We will test the hypothesis that a consequence of advancing age is an altered distribution of individual subsets of Tfh/Tfr cells resulting in metabolic reprogramming and defective B cell elicited influenza vaccine responses. Our integrated analysis includes studies to investigate at the organismal and cellular level age-related declines in metabolic pathways, such as defective mitochondrial biogenesis and one carbon metabolism and to determine if these changes contribute to the immune dysfunction. Finally, knowledge gained from these studies will be used to perform proof of concept rescue experiments in vitro to restore optimal influenza vaccine responsiveness. To support these studies, samples will be used from a seasonal influenza vaccinee cohort of 153 participants (ages 21-76, including 15 individuals ≥ 65yr) who have donated blood at days 0, 7 and 32 after quadrivalent hemagglutinin (HA) vaccination. We plan a targeted expansion of this cohort to recruit an additional 100 donors ≥ 65 years of age and a group of 25 younger adults (≤ 40yr, n=25) for single cell genomic and metabolomic studies that require fresh blood samples. We will temporally follow the influenza vaccine response and compare young and older groups. Specifically, in Aim 1 we will examine alterations in subset composition and metabolic reprogramming in follicular T cells in young and aged individuals. The completion of these studies will allow us to understand whether defects in humoral immunity associated with aging are mediated by changes in Tfh and Tfr subsets, intrinsic functionality, or both. In Aim 2 we will assess characteristics of the Ab response and determine composition and metabolic changes in B cell populations in young and aged individuals. At the completion of these studies we will also understand changes that result in immune imprinting. In Aim 3 we will analyze age- related dysfunction of Tfh, Tfr and B cells in vitro in response to influenza vaccine antigens. Tfh/B cell co-cultures will be treated with immune modulators and small molecules to restore immunologic and metabolic function to overcome age-related def...