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, 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 T cells to die through apoptosis. Because we were not making progress rapidly enough pursuing all three diseases at the same time, we have chosen to focus on in antigenic drug against multiple sclerosis. We would like to test antigen-induced apoptosis as a means of treating MS and other autoimmune diseases in a clinical trial. We have initiated studies of recombinant molecules containing antigens potentially involved in multiple sclerosis with the goal of establishing a Cooperative Research and Development Agreement with a large pharmaceutical company to clinically test such a form of therapy. At present there is increasing evidence that myelin proteins antigens are the target of the autoimmune attack. 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 end organ damage during autoimmune diseases to determine if these can provide an early warning system of autoimmune attack. 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 safetly and effectively ameliorate disease in preclinical models for multiple sclerosis (MS). We can 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 nave T cell activation but does not affect deletional tolerance. The second approach has been to define the genetic basis of previously unknown immune regulation disorders and then generate a molecular understanding of the biochemical pathway to develop new effective therapies. We have successfully used this approach to define a new class of disorders affecting the regulation of phosphoinositide-3 kinase (PI-3K) which is one of most important signaling systems for cell proliferation in both normal and malignant cells. We have discovered humans who 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. Our results also open the door to testing direct p110delta enzyme inhibitors in PASLI patients. 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 individuals from three different families with a PASLI-like disease who carried the identical heterozygous splice site mutation 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 causes an in-frame deletion of exon 11, resulting in the deletion of 42 amino acids in the inter-SH2 domain of the regulatory proteins. The mutant proteins are expressed, albeit at low levels, in patient T cells and are associated with increased PI3K signaling. In these new patients, we attributed their clinical findings to overactive mTOR suggesting that these patients should be treated with rapamycin to inhibit mTOR similar to PASLI patients. Thus, we have elucidated the mechanism of a novel immunodeficiency/immunoregulatory disorder at the molecular level that is caused by gain-of-function mutations in PIK3CD encoding p110delta and its regulatory subunit associated with deficiencies in antibody production combined with poor anti-viral immunity and lymphoaccumulation due to exacerbated mTOR-mediated terminal differentiation of T cells into short-lived effector cells. Our new understanding of this disease has allowed us to deploy rapamycin as an off-the-shelf intervention and initiate a clinical trial of a specific PI-3K inhibitor in PASLI patients at the NIH Clinical Center.
Showing the most recent 10 out of 23 publications
