Recent publications demonstrate that low dose IL-2 given to patients is therapeutic in both chronic GVHD and HCV vasculitis, disorders with clinical similarities to SLE. In each instance, there was an increase in circulating Tregs and a marked reduction in inflammatory cytokines in the serum of treated patients. Our recent studies have shown that the E3 ligase GRAIL is both necessary and sufficient to generate CD4 Tregs. Additional unpublished work from our group has recently demonstrated that 75% of a cohort of 20 SLE patients lacked GRAIL expression in their Tregs. GRAIL is highly expressed in Tregs from normal subjects and mice with a disrupted gene have ineffective Treg activity. This proposal is based on unpublished studies from my lab on the relationship of IL-2 engagement of the IL-2R in Tregs and is intended to study the mechanism of action (MoA) of low dose IL-2 in Treg induction or activation to ask if it might work in the treatment of SLE. In these studies, we employed two new innovative proteomics-based approaches to identify proteins that were associated with or ubiquitinated by GRAIL: 1 APEX and 2) QUAINT. APEX is a proximity labeling strategy that is based on genetically engineered target constructs containing a modified ascorbate peroxidase enzyme that biotinylates nearby proteins in the presence of biotin-phenol and H2O2. Following treatment, transfected cells can be lysed, and biotin tagged proteins can be recovered with streptavidin-coated beads and identified by MS. The spatial limitations of the APEX enzymatic activity are restricted to within a radius of less than 20 nm. The QUAINT proteomic assay is based on the fact that tryptic cleavage of ubiquitinated proteins results in the presence of a remnant diglycine (GG) tag on the ubiquitin-modified lysine of digested peptides. Antibody generated against this specific K-?-GG site can be used for enrichment of these tryptic peptides followed by identification of ubiquitinated sites by MS. Notch and Notch ligands, are targets of GRAIL and GRAIL ubiquitinates Cullin5 at Lys 724, the same residue that needs to be neddylated to allow formation of the SOCS3-Elongin-Cullin5 complex that ultimately degrades JAK1 and stops IL-2R signaling. Our current data demonstrate that low dose IL-2 preferentially activates Stat5b signaling from the IL-2R of Tregs driving the transcription, among others, of Treg genes FoxP3 and CD25, and GRAIL prevents the normal negative regulator of cytokine signaling protein (SOCS3) blockade of JAK1 kinase activity as well as disrupts the formation of the SOCS3-Elongin-Cullin5 E3 ligase formation allowing continuation of the IL-2R Stat5b signaling in Tregs from normal donors. We need to demonstrate that similar signaling cascades from the IL-2R, Notch pathways and Stat5b phosphorylation, are present in Tregs from SLE patients to support low dose IL-2 in therapy of SLE or to allow identification of alternative drugable targets to restore Treg function.