This project is based on our discovery that genetic mutations in molecules that control the programmed death, or apoptosis, of lymphocytes are responsible for the Autoimmune Lymphoproliferative Syndrome (ALPS). ALPS is a disease affecting children that leads to loss of normal lymphocyte homeostasis leading to swollen lymph glands and organs. Because lymphocytes are the primary cell mediating immune reactions, this excess of lymphocytes leads to a pathological autoimmune attack on the patients own tissues. We have identified mutations in a death-inducing cell surface receptor termed Fas (also known as APO-1 or CD95) and in other molecules that regulate apoptosis. The FAS receptor is homotrimeric and activated by the cognate FAS ligand (FASL), another homotrimeric protein complex homologous to tumor necrosis factor (TNF). Following stimulation, the intracellular death domain (DD) portion of FAS nucleates an extended helical complex of the FADD adaptor protein and caspases-8 and -10. These caspases undergo proteolytic autoprocessing and cleave, in a signaling cascade, downstream effector caspases and other targets leading to the cellular death process of apoptosis. Mutations in genes encoding FASL, FADD, and CASP10, also cause ALPS termed ALPS-FASL, ALPS-FADD, and ALPS-CASP10,respectively. Germline mutations in CASP8 or somatic mutations in NRAS and KRAS cause ALPS-related syndromes with distinct clinical phenotypes. Importantly, FAS mutations are associated with in vitro lymphocyte apoptosis defects but may show variable clinical penetrance which has not been fully defined. We completed this year, a summary of 20 years of following ALPS patients since the discovery of the disease. We highlighted the full clinical, molecular and laboratory assessments of 150 ALPS-FAS patients and 63 healthy mutations positive relatives (HMPRs) to broaden the current understanding of the diagnosis and management of ALPS-FAS. We have found that ALPS presents in childhood with nonmalignant lymphadenopathy and splenomegaly associated with the pathognomonic expansion of mature CD4 and CD8 negative (double negative) TCRalpha/beta+ T cells. Patients often have chronic multilineage cytopenias and have an increased risk of B cell lymphoma. Deleterious heterozygous mutations in the FAS gene are the most common cause of this condition. Our principal findings are that FAS mutations have a clinical penetrance of less than 60%, that elevated serum vitamin B12 is a reliable and accurate biomarker of ALPS-FAS, and the major causes of morbidity and mortality in these patients are the complications of surgical splenectomy and the development of lymphoma. The fact that 41% (27/66) of our splenectomized patients had at least one episode of post splenectomy sepsis and 6 of them have died shows the importance of avoiding splenectomy and managing chronic cytopenias with drug treatments. We observed a significantly greater relative risk of lymphoma than previously reported with a standardized incidence ratio of 149 and 61 for Hodgkin and Non-Hodgkin lymphoma, respectively, in the current report versus 51 and 14, respectively, in a previous report we published in 2001. We are presently studying a class of these ALPS-like patients that do not display mutations in the Fas receptor, its ligand (Fas ligand), or caspase-10 using a variety of molecular analyses to determine the underlying gene mutation. These experiments have been successful in uncovering the molecular basis of new diseases. Cytotoxic T lymphocyte antigen-4 (CTLA-4) is an inhibitory cell surface receptor that controls the ability of costimulatory signals to stimulate activated T cells. We identified germline heterozygous mutations in CTLA4 in subjects with severe immune dysregulation from four unrelated families. These patients manifested brain, gastrointestinal (GI), and lung lymphocytic infiltrates, autoimmune thrombocytopenia and hemolytic anemia, and hypogammaglobulinemia beginning in early childhood. These mutations cause reduced levels of cell surface CTLA4 and the resulting haploinsufficiency caused dysregulation of FoxP3(+) regulatory T (Treg) cells and hyperactivation of effector T cells accounting for the lymphocytic infiltration of target organs. Patients also exhibited progressive loss of circulating B cells, associated with an increase of predominantly autoreactive CD21(lo) B cells and accumulation of B cells in nonlymphoid organs. Thus, gene mutations in human CTLA4 causing haploinsufficiency reveal a critical quantitative role for CTLA-4 in controlling the homeostasis of T and B lymphocytes. We call this new clinical entity CTLA-4 haploinsufficiency with autoimmune infiltration (CHAI) disease. In a related set of patients manifesting lymphocytic organ infiltration, and other immunoregulatory characteristics including autoimmunity, lymphoproliferation, and humoral immune deficiency, homozygous loss of function mutations in the LRBA gene (encoding the lipopolysaccharide-responsive and beige-like anchor protein). There was no precedent for a pathogenic role of LRBA in immunologic disease. Clinical studies led by Michael Jordan at Cincinnati Childrens Hospital established that patients with LRBA deficiency manifested a dramatic and sustained improvement in response to abatacept, a CTLA4 (cytotoxic T lymphocyte antigen-4)-immunoglobulin fusion drug. Based on this observation and the homology of LRBA to intracellular adapter proteins that guide intracellular vesicle trafficking led us to hypothesize that it regulates CTLA4. Indeed, we observed that LRBA associated with CTLA4 on early endosomal and late Golgi vesicles. There was a direct physical interaction between LRBA and the intra-cytoplasmic tail of CTLA4 that depended on a YXVM amino acid motif that is conserved in phylogeny. Molecular experiments showed that LRBA deficiency in patient cells or LRBA knockdown in cells from healthy subjects caused increased CTLA4 turnover, which depleted CTLA4 protein in FoxP3(+) regulatory and activated conventional T cells. These results explained the striking efficacy of abatacept treatment. Moreover, we observed that inhibition of lysosome degradation in LRBA-deficient cells with chloroquine prevented CTLA4 loss. Since hydroxychloroquine is an inexpensive FDA-approved medication, our results suggest that it could be a novel therapy for this disease. These findings elucidate a mechanism for CTLA4 trafficking and control of immune responses and suggest to targeted therapies for diseases involving the CTLA4 pathway. Thus, our data supports the idea of precision medicine for immunological diseases in which gene discovery guides the development of new targeted therapies.

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Buchbinder, David; Seppanen, Mikko; Rao, V Koneti et al. (2018) Clinical Challenges: Identification of Patients With Novel Primary Immunodeficiency Syndromes. J Pediatr Hematol Oncol 40:e319-e322
Lo, Bernice; Lenardo, Michael J (2017) Editorial overview: Autoimmunity: New genomics approaches are improving our understanding of autoimmunity. Curr Opin Immunol 49:iv-vi
Zheng, Lixin; Li, Jian; Lenardo, Michael (2017) Restimulation-induced cell death: new medical and research perspectives. Immunol Rev 277:44-60
Ozen, Ahmet; Comrie, William A; Ardy, Rico C et al. (2017) CD55 Deficiency, Early-Onset Protein-Losing Enteropathy, and Thrombosis. N Engl J Med 377:52-61
Abolhassani, Hassan; Edwards, Emily S J; Ikinciogullari, Aydan et al. (2017) Combined immunodeficiency and Epstein-Barr virus-induced B cell malignancy in humans with inherited CD70 deficiency. J Exp Med 214:91-106
Afzali, Behdad; Grönholm, Juha; Vandrovcova, Jana et al. (2017) BACH2 immunodeficiency illustrates an association between super-enhancers and haploinsufficiency. Nat Immunol 18:813-823
Ozen, Ahmet; Comrie, William A; Lenardo, Michael J (2017) CD55 Deficiency and Protein-Losing Enteropathy. N Engl J Med 377:1499-1500
Lenardo, Michael J (2016) Clinical Genomics - Molecular Pathogenesis Revealed. N Engl J Med 375:2117-2119
Similuk, Morgan N; Wang, Angela; Lenardo, Michael J et al. (2016) Life with a Primary Immune Deficiency: a Systematic Synthesis of the Literature and Proposed Research Agenda. J Clin Immunol 36:123-33
Lenardo, Michael; Lo, Bernice; Lucas, Carrie L (2016) Genomics of Immune Diseases and New Therapies. Annu Rev Immunol 34:121-49

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