This project is based on our discovery that genetic mutations that affect 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 causing swollen lymph glands and organs. This excess of lymphocytes leads to a pathological autoimmune attack on the patients own tissues. During the clinical investigations on ALPS, many patients have been referred to our program with other immunoregulatory and immunodeficiency syndromes for evaluation. Therefore we launched a clinical genomics program to identify the genetic causes of these diseases. In addition to NIH patients, we have also established clinical research centers in China, Turkey, and India, providing many patients to study. We are presently studying ALPS-like patients to determine the underlying gene mutations. Cytotoxic T lymphocyte antigen-4 (CTLA-4) is an inhibitory cell surface receptor that controls costimulatory signals that stimulate activated T cells. We identified germline heterozygous mutations in CTLA4 in subjects with severe immune dysregulation. These mutations cause reduced cell surface CTLA4 leading to dysregulation of FoxP3(+) regulatory T (Treg) cells and hyperactivation of effector T cells. Patients also exhibited progressive loss of circulating B cells associated with an increase of predominantly autoreactive CD21(lo) B cells. Thus, gene mutations in human CTLA4 causing haploinsufficiency reveal a critical quantitative role for CTLA-4 in controlling the lymphocyte homeostasis. We call this new clinical entity CTLA-4 haploinsufficiency with autoimmune infiltration (CHAI) disease. In another set of patients, who manifested similar lymphocytic organ infiltration, autoimmunity, lymphoproliferation, and humoral immune deficiency, we detected homozygous loss of function mutations in the gene encoding the lipopolysaccharide-responsive and beige-like anchor (LRBA) protein. Clinical studies led by our collaborator, Dr. Michael Jordan at Cincinnati Children's Hospital, established that patients with LRBA deficiency were greatly improved with abatacept, a CTLA4-immunoglobulin fusion drug. Based on this observation and the homology of LRBA to adapter proteins that guide intracellular vesicle trafficking, we hypothesized that LRBA regulates CTLA4. Indeed, we observed that LRBA associated with CTLA4 on early endosomal and late Golgi vesicles. LRBA directly bound the intra-cytoplasmic tail of CTLA4, which depended on a YXVM amino acid motif. 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. We call this disease LRBA deficiency with autoantibodies, T regulatory (Treg) cell defects, autoimmune infiltration, and enteropathy (LATAIE). Our results explained the striking efficacy of abatacept treatment. Moreover, we observed that inhibition of lysosomal deposition of the CTLA4 protein in LRBA-deficient cells with chloroquine prevented CTLA4 loss. Since hydroxychloroquine is an inexpensive FDA-approved medication, we are testing it as a novel therapy for this disease. Both treatment approaches may also be useful for CHAI disease patients. These findings elucidate a mechanism for CTLA4 trafficking and how this contributes to the control of immune responses. Importantly, our discovery reveals targeted therapies for diseases involving the CTLA4 pathway. The Ras-associated autoimmune leukoproliferative disorder (RALD) is a chronic, nonmalignant condition we have defined that presents with persistent monocytosis and is often associated with leukocytosis, lymphoproliferation, and autoimmune phenomena. We have shown that RALD is invariably associated with activating, generally de novo, somatic mutations of the KRAS or NRAS genes in hematopoietic progenitors. RALD has clinical and laboratory features that overlap with those of juvenile myelomonocytic leukemia (JMML) and chronic myelomonocytic leukemia (CMML), including identical somatic mutations in KRAS or NRAS genes noted in peripheral blood mononuclear cells. Long-term follow-up of these patients suggests that RALD has an indolent clinical course whereas JMML is fatal if left untreated. Ongoing work suggests that there are other genetic mutations that differentiate RALD from JMML and CMML. We have recently published a summary of this topic to improve clinical care and prognosis. We recently discovered a very interesting, apparently de novo, mutation in the gene encoding a key signaling protein that provides insights into immune regulation in humans. Signal transducer and activator transcription (STAT) proteins are a family of transcription factors that regulate gene expression programs underlying key immunological processes. We have studied a patient with a unique heterozygous missense mutation in STAT5B, which generates a variant protein with defective intrinsic function and the ability to dominantly interfere with STAT5-driven transcriptional activity. Phenotypically, the patient presented with immune thrombocytopenia, lymphadenopathy, splenomegaly, aberrant antibody titers, granulocytosis and necrotizing granulomas associated with a B cell-intrinsic antibody class switch defect and a prominent accumulation of CD4+ T effector memory(TEM) cells. RNA sequencing showed a reduction in IL-2/STAT5-dependent transcriptional activity. In addition, ex vivo analysis of the memory T cell compartment revealed a selective defect in cytokine-driven, T cell receptor restimulation-induced cell death (RICD) within TEM cells, a finding we confirmed in animal models of STAT5 deficiency. Thus, we gained important insights from intensive immunological and biochemical investigations of this single patient that reveal the role of STAT5B as a central orchestrator of lymphocyte homeostasis and function in humans. We studied 10 patients, mostly coming from our center in Istanbul, Turkey, suffering from abdominal pain and diarrhea caused by early-onset protein-losing enteropathy (PLE) with lymphangiectasia, edema due to hypoproteinemia, malabsorption, and, less frequently, bowel inflammation, recurrent infections, and angiopathic thromboembolic disease. We identified autosomal recessive mutations leading to loss of protein expression in the gene encoding CD55/Decay accelerating factor. Patient T lymphocytes and CD55-deficient cell lines displayed abnormally increased deposition of complement factor C3d. Genetic reconstitution of CD55 prevented C3d deposition. Stimulation of anaphylatoxin receptors on patient T lymphocytes produced increased tumor necrosis factor alpha, which caused a decreased ratio of the anti-coagulatory protein thrombomodulin to the pro-coagulatory protein tissue factor. CD55 costimulation by CD97, and the contingent production of interleukin-10, was defective in patient T lymphocytes. Hence, CD55 deficiency with hyperactivation of complement, angiopathic thrombosis, and PLE (CHAPLE) disease is caused by abnormal complement deposition due to autosomal recessive loss-of-function (LOF) mutations in the CD55 gene. Our results suggest that eculizumab (Soliris, Alexion), an FDA-approved, complement-inhibiting therapeutic may benefit these patients. We are currently pursuing a clinical trial in collaboration with Alexion Inc. to test this therapy. In summary, this project achieves precision medicine for immunological diseases by gene discovery that guides the development of new targeted therapies.

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2016
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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
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
Lo, Bernice; Lenardo, Michael J (2017) Editorial overview: Autoimmunity: New genomics approaches are improving our understanding of autoimmunity. Curr Opin Immunol 49:iv-vi
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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