Project summary Sle1, the strongest lupus susceptibility locus in the NZM2410 mouse model, is responsible for the loss of tolerance to chromatin, including the production of autoreactive CD4+ T cells. It corresponds to a cluster of loci, including Sle1a1 that enhances effector CD4+ T cell function and reduces the number and function of peripheral (p)Treg cells. Sle1a1 corresponds to the expression of Pbx1-d, a splice isoform of the transcription factor Pbx1 that lacks the DNA- and Hox-binding domains, as compared to Pbx1-b, the common isoform in lymphocytes. Pbx1-d is expressed at a higher level in CD4+ T cells from B6.Sle1a1 and NZM2410 mice, and from SLE patients, as compared to B6 mice and healthy controls (HC). Importantly, the PBX1 isoforms and the protein sequence are identical between mice and humans. In the previous cycle of funding, we showed that either Pbx1-d Tg expression in T cells or the FOXP3-specific deletion of Pbx1 impaired the maintenance of pTregs, resulting in a global CD4+ T cell activation, which increased atherogenic lesions as well as autoimmunity in dyslipidemic mice. Our work therefore established a novel role for Pbx1 to maintain Treg homeostasis. We also showed that Pbx1 expression in CD4+ T cells is the highest in Tregs, and that Pbx1-d is a dysfunctional protein through multiple mechanisms. Our work is the first to associate a specific genetic variation to the well-documented alterations in Tregs in human and mouse lupus. A SNP decreasing PBX1 expression in B cells has also been recently associated to SLE susceptibility and a B-cell specific deletion of Pbx1 enhanced B cell responses in mice. Therefore, Pbx1 represents a unique lupus susceptibility gene in both mice and humans that impairs Tregs through the over-expression of Pbx1-d and B cells through a reduced global expression. In this competitive renewal, we propose to characterize the mechanisms by which Pbx1 regulates pTreg maintenance and function at the molecular and cellular levels, and how this process is altered by the expression of lupus-susceptibility allele Pbx1-d. In addition, we propose to characterize the combined effects of a reduced Pbx1 expression in B cells and overexpression of Pbx1-d in Tregs to comprehensively define the contribution of Pbx1 to lupus susceptibility. To achieve these goals, we propose three specific aims conducted in mice as well as in PBMCs obtained from SLE patients and HCs. Aim 1. To characterize the functional consequences of Pbx1-d expression in Tregs using cellular assays with murine cells that express only Pbx1-b or Pbx1-d, and human cells based on their PBX1-D expression. Aim 2. To define the molecular pathways regulated by Pbx1 and Pbx1-d in Tregs with gene expression profiling, Pbx1 binding sites and chromatin accessibility, scRNA-Seq, and proteomics. Aim 3. To define the consequences of Pbx1-deficiency or Pbx1-d expression in Tregs combined with Pbx1 deficiency in B cells. The proposed experiments will provide for t...