Our studies in FY2014 have addressed several major questions: 1. Tolerance versus autoimmunity is determined, in part, by the ratio between T conventional cells (Tconv) and Tregs specific for self-antigens. Tregs develop separately from Tconv in the thymus, contain a unique epigenetic landscape shaping their distinct gene expression profiles and continue to be marked by unique cell-surface and intracellular proteins in the periphery. Despite these differences, both Tregs and Tconv have the potential to respond to an identical array of peripheral self-antigens in a particular end-organ niche. A major unanswered question is the relationship of the T cell receptor (TCR) repertoire between Tregs and Tconv. Previous authors studying TCR repertoire in both mice and humans have estimated the overlap between Tregs and Tconv to range from as low as 3 to as high as 30%. These studies have either relied on studying rearrangement on a fixed V-alpha background and used limited V-beta recombination partners (mouse) or have focused on a limited set of endogenous V-beta family members (humans). We have improved upon these previous studies in two ways. First, we have for the first time conducted high-throughput deep sequencing of all V-beta CDR3 families. Second, we have purified thymically-derived Tregs using CD25 (high) and CD127 (low) to distinguish thymic-derived Tregs from activated Tconv. We have found that the Treg and Tconv TCR repertoires are almost completely distinct and non-overlapping. These results are most compatible with a classic threshold model for T cell selection in the thymus. If a Tconv precursor expresses a TCR that strongly binds to a self-peptide, it will be deleted, whereas if a Treg expresses a TCR with high affiinity for a self-peptide, it will be positively selected. Thus, the Treg TCR repertoire will be strongly biased for self-peptides and will favor the prevention of autoreactivity and autoimmune disease. 2. The major goals of this study were to fully characterize the gene signature of human Tregs. We have compared the transcriptomes expressed by human Treg and Tconv cells when freshly explanted and at different time points after activation in vitro. We have utilized RNA-seq for comprehensive mRNA profiling. One of the major challenges in identifying reproducible and consistent gene signatures associated with different subpopulations of highly related cells is to obtain homogeneous cell subpopulations with a high degree of purity. Although immunomagnetic bead separation technology can be used to obtain enriched populations of human Treg, in our experience cell sorting is required to achieve highly purified Foxp3+ Tregs (>90-95%). One additional variable that is difficult to control is inherent donor variability and samples obtained even after cell sorting that did not meet our criteria of purity, were not used further in these studies. Single cell transcriptome sequencing, when routinely available, would avoid some of these issues. We are confident of the appropriateness of our purification approach, as we observed conserved gene signatures among multiple healthy human donors. To the best of our knowledge, the present study is the first to extensively validate gene expression for human FoxP3+Tregs using the nCounter system. We have identified a number of genes that have not previously been shown to be Treg-specific including, LAYN, UTS2, RTKN2, F5, FCRL1, CCR3, METTL7A, CSF2RB, TRIB1, CECAM4 and several genes whose expression is enhanced in Tconv cells including ENC1, FCRL6, ZEB2, NKG7, IGFBP3 and ITGA1. One previous study has also used RNA-seq for transcriptome profiling of resting and activated human Tregs. However, the cell populations used in that study were purified by magnetic bead technology and the differential gene expression patterns were not validated further. Moreover, gene expression was only analyzed in freshly explanted cells and at single time point (16h) following activation, and cell populations from multiple individuals were not analyzed separately. We focused our attention on two of the unique genes that were highly differentially expressed in Tregs and appeared to encode cell membrane associated proteins--Rhotekin-2 (RTKN2) and Layilin (LAYN). RTKN2 belongs to the Rhotekin family of proteins containing a Rho-binding domain and pleckstrin homology domain, while LAYN, is a transmembrane glycoprotein homologous to C-type lectins. We could successfully knock down expression of both RTKN2 and LAYN using siRNA technology and could demonstrate that decreased expression of both of these genes resulted in enhanced Treg suppression. This result suggests that these proteins may act to inhibit a component of the Treg suppression cascade. In parallel studies of the differential expression of miRNAs in Tregs/Tconv cells using TaqMan technology, we observed enhanced expression of miR-146a, miR-146b, let-7g, miR-21, miR-660 and miR-598 in Treg and selective expression of miR-125a in Tconv. We were successful in our attempts to downregulate expression of the miRNAs selectively expressed in Treg using PNA inhibitors, but the decrease in expression of these miRNAs had no effect in the standard Treg inhibition of Tconv cell proliferation assay. Overall, there appears to be relatively few miRNAs that are exclusively expressed in Treg and a great deal of overlap in miRNA expression between Tregs and activated Tconv or T memory cells. 3. We have developed a CRADA with the Department of Biotherapeutics at Boehringer-Ingelheim (BI) Pharmaceuticals for studies to generate novel mAbs that will modulate the function of human Treg cells. We have prepared large numbers of expanded highly purified human Tregs that are used to immunize mice to generate the mAbs. A number of novel screening techniques have been developed by BI to identify mAbs that selectively recognize human Tregs and not activated T conventional cells or non-lymphoid cells. These novel mAbs are then tested in a panel of in vitro T suppressor cell assays that we have developed. In FY2014, we screened hundreds of supernatants and identified supernatants that selectively react with human Tregs and/or modulate the function of Tregs when added to in vitro assays. These hybridomas are now being cloned and antibodies are being purified from supernatants. Purified antibodies will be used in immunoprecipitation assays to identify the target antigen by mass spectroscopy. 4. Tregs have a diminished capacity to activate the PI3K/Akt pathway. Although blunted Akt activity is necessary to maintain Treg function, the consequences of this altered signaling are unclear. Glucose transporter 1 (Glut1) is a cell surface receptor responsible for facilitating glucose transport across plasma membranes whose expression is tightly coupled to co-stimulatory signals and Akt phosphorylation. Freshly isolated human Tregs were unable to up-regulate Glut1 in response to TCR and co-stimulatory signals as compared to conventional T cells. Consequently, the ability of Tregs to utilize glucose was also reduced. Introduction of Foxp3 into conventional T cells inhibited both Akt activation and Glut1 expression, indicating that Foxp3 can regulate Glut1. Finally, pharmacologic activation of Akt in Tregs can induce Glut1, overcoming the effects of Foxp3. Together, these results illustrate the molecular basis behind differential glucose metabolism in Tregs.