In this project, we are investigating the development of new diagnostic and therapeutic approaches to immune dysregulatory diseases. First we have pursued deletional therapy in three immunological diseases that share a common pathogenesis: multiple sclerosis (MS), type I diabetes, and the development of inhibitory antibodies against FVIII in the treatment of hemophilia. The key feature of our approach is that the antigen itself will be used to program the specific cognate T cells to die through apoptosis via an internal regulatory program of T cells termed restimulation-induced cell death (RICD). The death is clonally specific and represents a way to eliminate disease-causing T cells by hyper-vaccinating with their specific antigen. We would like to use antigen to treat autoimmune diseases in a clinical trial and have chosen to focus on an antigenic drug against MS. Evidence suggests that myelin proteins antigens are targets of the autoimmune attack, but how the specificities determine disease outcome in progressive and relapsing-remitting MS is unclear. By programming the T cells that recognize such antigens to die, the effect of eliminating these cells on the disease can be demonstrated. We are also studying new highly sensitive diagnostic tests to detect reactive T cells against these antigens to determine if these can provide an early warning system of autoimmune attack. We have initiated studies of recombinant molecules containing antigens potentially involved in MS with the goal of establishing a Cooperative Research and Development Agreement (CRADA) with a large pharmaceutical company to clinically test such a form of therapy. We have been fortunate in achieving this goal in the past year through being selected as the lead project in a new agreement between the NIH NCATS and the Center for Therapeutic Innovation of Pfizer. Our studies have shown that antigen-specific deletion of T cell using an easily expressed 56 kD recombinant therapeutic tolerogenic protein, MMPt, comprising immunogenic segments of myelin basic protein (MBP), myelin oligodedrocyte glycoprotein (MOG) and proteolipid protein (PLP), can safely and effectively ameliorate disease in preclinical models for multiple sclerosis (MS). we find that T cells are specifically eliminated from the spinal cord and the inflammatory cell infiltrate and cytokine production is decreased. We plan to stratify patients based on T cell reactivity to the drug and then administer the drug in a repeated fashion to remove autoreactive disease-causing T cells without causing a general immunosuppression a widely applicable therapeutic strategy for immune diseases. Safety against disease exacerbation can be achieved by co-administration of immunosupressants in short term which blocks naive T cell activation but does not affect deletional tolerance. We expect to formally launch the CRADA work in fall, 2016. In order to understand the molecular underpinnings of this regular mechanism, we have carried out extensive molecular investigations of how TCR stimulation directs cells to a death pathway instead of simply activation. We have found that cycling T cell blasts become susceptible to restimulation-induced cell death (RICD) by redirecting the NF-B-activating pathway. We carried out a chemical genetics screen that revealed a critical role for PI-3K directly a novel pathway to the induction of I-kappaB kinase (IKK) in transducing a death signal independently of Fas or other tumor necrosis factor superfamily receptors. Interestingly, NF-B itself and new transcription/translation were found to be dispensable for RICD and that activated but not nave T cells are susceptible to death. This is apparently due to the fact that the caspase-3 dimer is partially cleaved but not fully processed in activated cycling T cells blasts suggesting the molecular pathway has been engaged. TCR restimulation then introduces the full processing of caspase-3. Further experiments are being directed at determining the precise substrate of IKK. This may shed light on a key molecular process that will be useful for antigen-induced treatment of autoimmune diseases and, potentially, immunotherapy for cancer. We have also discovered a genetic cause of deficient RICD involving a new class of disorders affecting the regulation of phosphoinositide-3 kinase (PI-3K),a key regulator of cell proliferation in both normal and malignant cells. We have discovered patientswho are heterozygous for mutations in the leukocyte-restricted PIK3CD gene encoding the p110delta catalytic PI3K subunit and suffer from a unique disorder we have termed p110delta activating mutations causing Senescent T cells, Lymphadenopathy, and Immunodeficiency (PASLI) disease. The p110delta catalytic subunit of phosphoinositide 3-kinase (PI3K) has been found to be selectively expressed in leukocytes and critical for lymphocyte differentiation, growth, and survival by pharmacologic and genetic inactivation in experimental animals. We discovered the first germline, heterozygous, dominant, gain-of-function mutations in the p110delta catalytic subunit of PI3K in 9 patients from 7 unrelated families. These patients' clinical presentation comprised sinopulmonary infections, EBV viremia, lymphadenopathy, nodular lymphoid hyperplasia at mucosal surfaces, and lymphoma. Our laboratory studies showed that patient T cells exhibited defective response to antigen receptor stimulation despite constitutive activation of the PI3K signalling system. We found that patients were lacking in long-lived central memory T cells and instead exhibited A surfeit of short-lived effector/TEM cells. As expected from a TEM phenotype, proliferation and IL-2 secretion were diminished while effector functions, including granzyme expression, IFN-gamma secretion, and degranulation, were elevated. TCR signaling was intact; however, hyper-activation of mTOR caused changes in cellular metabolism that were characteristic of terminal differentiation and senescence. Importantly, treatment of patients with The FDA-approved drug rapamycin to inhibit mTOR activity in vivo partially restored appearance of naive T cells in the peripheral blood, decreased the number of senescent T cells, and largely rescued the T cell activation defects. we next wanted to test specific p110delta enzyme inhibitors in PASLI patients and we have entered into a Cooperative Research and Development Agreement with Novartis to carry out a clinical trial testing their inhibitor of p110delta, a compound called CDZ173, in the NIH clinical Center. Preliminary evidence shows that CDZ173 is a potent and selective inhibitor of p110delta and can inhibit the wild-type as well as overactive forms of the enzyme. A clinical trial is currently ongoing at NIH. PI3K exists in the cell as a holocomplex of a p110 catalytic subunit bound to a various regulatory subunits: p85alpha, p55alpha, and p50alpha, which are important for p110 stability, inhibition, and recruitment to signaling locations on inner leaflet of the membrane. We recently discovered 4 patients with a PASLI-like disease with heterozygous splice site mutations in PIK3R1, a ubiquitously expressed gene encoding the PI3K regulatory subunits. Similar to other PASLI patients, PIK3R1 mutant patients suffer from recurrent sinopulmonary infections and lymphoproliferation, have increased PI3K signaling, and have expansion and skewing of peripheral blood CD8+ T cells towards a terminally differentiated, senescent effector T cell phenotype. The PIK3R1 splice mutation resulting in the deletion of 42 amino acids in a critical region of the protein. The mutant proteins are expressed, albeit at low levels, in patient T cells and are associated with increased PI3K signaling. These results suggest that this new group of patients may also benefit from CDZ173.