Project Summary/Abstract High affinity class-switched autoantibodies (autoAb) are the major pathogenic effector molecules in lupus. They are generated by B cells that differentiate through either the germinal center (GC) or the extrafollicular (EF) pathways. The frequency of follicular helper (Tfh) cells and EF helper (eTf) CD4+ T cells are expanded in lupus in direct correlation with disease activity. The EF pathway has been best characterized in the MRL/lpr lupus- prone mice with AM14 BCR Tg B cells, which have been used as a model of autoreactive B cells activated through dual BCR and TLR9 signals. Overall, the GC and EF pathways are crucial nodes of lupus pathogenesis and lupus-prone mice present validated models for mechanistic studies of these pathways. We and others have shown that the metabolism of immune cells was altered in lupus patients and mice, and that some of these alterations offer therapeutic targets. Relatively little is known on the metabolic programs that sustain either autoreactive or immunization-induced (Imm-) GC B cells and Tfh cells, and nothing is known about the metabolism of EF activation. We have shown during the first cycle of funding that the production of anti-dsDNA IgG but not Imm-Abs, was glucose-dependent. In contrast, both autoAbs and Imm-Abs were eliminated by the inhibition of glutaminolysis. These results obtained with bulk polyclonal populations treated with metabolic inhibitors his led us to hypothesize that autoAbs and Imm-Abs have different metabolic requirements through the GC stage, which could be ultimately translated in their selective targeting. In this competitive renewal, we propose to use auto- and Imm-Ig-specific B and T cells, as well as GC B and Tact-specific deletions of metabolic enzymes to determine the intrinsic metabolic requirements of autoreactive and Imm-GC B and Tfh cells at the antigen- and cell-specific levels. We also propose to address the metabolic requirements of EF activation of autoreactive B cells. Using unique mouse models and a combination of scRNA-Seq and deep metabolomic analyses, we propose three specific aims to determine the requirements in glucose (1), glutamine (2), and FAO (3) for autoAb vs. Imm-Ab production, and to identify the corresponding mechanisms. These experiments will help to define the specific metabolic requirements of the B and CD4+ T cells that participate in the production of lupus autoAbs. This knowledge may ultimately identify means to eliminate autoAb production while preserving protective humoral autoimmunity.