Project Summary Amino acids are crucial nutrients that are also important to support immunity. Yet, we have limited understanding with regard to how immune challenges modulate amino acid availability, and how immune cells sense amino acid and transduce the signals to execute immune reponses. Rag-GTPase has recently been identified as a key amino acid sensor that mostly transduce signals from amino acids to mechanistic target of rapamycin (mTOR) complex 1 (mTORC1) in non-hematopoietic cells. However, Rag-GTPase also modulates transcription factor TFEB, a member of the microphthalmia (MiT/TFE) family of HLH-leucine zipper transcription factors, whose functions in B cells remain unknown. Moreover, Rag-GTPase independent mTORC1 activation has been identified. How Rag-GTPase and mTORC1 coordinates to regulate humoral immunity has not been addressed. We compared the functions of Rag-GTPase and mTORC1 in B cell response in vivo using genetic knockout models. Our data showed that while both Rag-GTPase and mTORC1 are required for systemic immune challenges, Rag-GTPase, but not mTORC1, is critical for humoral immune response towards respiratory influenza infection. This divergent requirement between Rag-GTPase and mTORC1 is associated with differential amino acid availability between systemic immunization and airway influenza infection. Furthermore, we showed that Rag-GTPase suppresses TFEB and promotes autophagy, which is associated with ERK activation, but largely independent of mTORC1. Thus, we hypothesize that reduced availability of specific amino acids during respiratory viral infection renders B cells dependent on Rag- GTPase-TFEB pathway, for GC reaction and anti-influenza antibody production. In Aim 1, we will first test whether the respiratory route of live virus immune challenge is the determining factor for Rag-GTPase dependent, but mTORC1 independent, humoral immunity. Second, we will further investigate the temporal and spatial dynamics of amino acid availability during immune challenges. Finally, we will test whether dietary amino acid intervention can improve humoral immunity against respiratory viral infection. In Aim 2, we will utilize complementary loss-of-function and gain-of-function approaches to elucidate the downstream signaling mechanisms by which Rag-GTPase promotes GC reaction and humoral immunity. We will further characterize the Rag-GTPase interactome in B cells using unbiased proteomics approach. Our study will define a novel Rag-GTPase-ERK-TFEB signaling axis that respond to amino acid availability to promote B cell activation and antibody production against airway viral infection.