Project 1: Adenosine Deaminase Deficiency Genetic deficiency of adenosine deaminase (ADA) can result in severe T, B and NK cell lymphopenia, but also non-immunological abnormalities of the skeleton, liver, lungs, kidney and central nervous system. We evaluated patients for signs of liver disease. Findings included fatty liver and/or hepatomegaly, increased fat content, histologic abnormalities (steatosis, non-alcoholic steatohepatitis, fibrosis, and cirrhosis), and elevated liver stiffness. Three children showed features of metabolic syndrome. Liver abnormalities correlated with ongoing enzyme replacement therapy and were less pronounced in patients successfully treated with cell therapy. We also observed a spectrum of lung pathologies including hyperreactive and obstructive airways disease, granulomatous inflammation, fibrosis and subpleural cysts. In 7 out of 8 patients, impulse oscillometry (IOS) evaluation showed increased baseline reactance (X) to levels 119-254% above reference. Only two patients had significant response to bronchodilators. These data suggest a pattern of small airway disease only partially reversible. Notably, changes occurred in patients with otherwise adequate red blood cell deoxynucleotide levels and independent from measures of immune function. Also concerning is the association between ADA deficiency and dermatofibrosarcoma protuberans (DFSP), a rare skin malignancy that we have diagnosed in 9 patients. Follow-up by serial exam and photography for 2.5-7 years showed that most lesions enlarged, but none metastasized. Lesions that evolved from plaque to protuberant morphology or presented subcutaneous nodular/infiltrative pattern were resected. None recurred with a follow-up to 7 years. This conservative management approach may contain morbidity without compromising oncologic outcome. In summary, non-immunologic manifestations of ADA deficiency are emerging that may be life-limiting and warrant comprehensive assessment in all patients. Project 2: Wiskott-Aldrich Syndrome (WAS) WAS is a rare X-linked disorder caused by mutations of the WAS gene and presenting with life-threatening hemorrhages, infections, autoimmunity and cancer. Autoimmune disorders are diagnosed in 40-70% of WAS patients and complicate post-HSC transplant outcome in >20% of cases. We and others have characterized the CD4+CD25+FOXP3+ natural regulatory T cells (nTregs) in Was KO mice and WAS patients and shown abnormal capabilities of WAS nTreg to suppress T and B cell proliferation, which may contribute to autoimmunity. In addition, in a series of 18 WAS patients, we have found molecular and phenotypic abnormalities of conventional B-lymphocytes, including reduced mutation frequency of C-amma transcripts, skewed usage of VH3 and VH4 gene families and increased representation of CD19hiCD21lo B-cells (Simon KL, Anderson SM, Garabedian EK, Moratto D, Sokolic RA, Candotti F: Molecular and phenotypic abnormalities of B lymphocytes in patients with Wiskott-Aldrich syndrome. J Allergy Clin Immunol. 2014 Mar;133(3):896-9). These findings provide insights that are particularly relevant due to the high prevalence of autoantibody-mediated disorders that complicate the clinical phenotype of WAS patients. In collaboration with Dr. Yatin Vyas of the University of Iowa and following up of our recent discovery that the WAS protein (WASp) is present in the nucleus where it play a transcriptional role, we have now shown that the nuclear effects of WASp on reprogramming transcription are uncoupled from its cytoplasmic signaling and actin effects, thus demonstrating an ARP2/3-independent action outside of the cytoplasm. Specifically, we have shown that that only nuclear WASp can function as a transcriptional cofactor, a role that cannot be substituted for by the actions of cytosol-constrained WASp. Consequently, expressing WASp mutants expressed only in the cytosol disallows the acquisition of TH1 functions in human T-cells. These chromatin defects occur despite preserved cytosolic function of the WASp mutant. Significantly, this uncoupling of compartment-specific roles is immediately relevant to the development of TH cellmediated immune dysfunction (e.g. susceptibility to severe infections from intracellular pathogens, predisposition to hematologic malignancies, increased levels of IgE, atopic eczema, and autoimmune colitis) in WAS (Sadhukhan S, Sarkar K, Taylor M, Candotti F, Vyas YM: Nuclear role of WASp in gene transcription is uncoupled from its ARP2/3-dependent cytoplasmic role in actin polymerization. J Immunol. 2014 Jul 1;193(1):150-60). Project 3: Reticular Dysgenesis (RD) Reticular Dysgenesis (RD) is an autosomal recessive form of SCID and is characterized by virtual absence of circulating lymphocytes and neutrophils, as well as sensory neural hearing loss. The lack of immune function in patients with RD leads to extreme susceptibility to infections and early death, unless hematopoietic and immune reconstitution is achieved by allogeneic hematopoietic stem cell transplantation (HSCT). We have recently shown that RD is caused by mutations of the adenylate kinase-2 (AK2) gene (Lagresle-Peyrou C., Six E.M., Picard C., Rieux-Laucat F., Michel V., Ditadi A., Demerens-de Chappedelaine C., Morillon E., Valensi F., Simon-Stoos K.L., Mullikin J.C., Noroski L.M., Besse C., Wulffraat N.M., Ferster A., Abecasis M.M., Calvo F., Petit C., Candotti F., Abel L., Fischer A., Cavazzana-Calvo M.: Human adenylate kinase 2 deficiency causes a profound hematopoietic defect associated with sensorineural deafness. Nat Genet 2009;41:106-11). AK-2 is a mithocondrial phosphotransferase that plays an important role in cellular and mitochondrial energy homeostasis. Since its first description, RD was assumed to be a disorder of primitive multipotent reticular progenitor cells primarily failing to develop into granulocytic and lymphoid precursors. The pathophysiology of the concurrent myeloid and lymphoid defects in RD, however, has long been unresolved. To explore this topic, we studied the effects of AK2 deficiency in zebrafish embryos and human iPSCs derived from an RD patient. Our results showed severe impairment of HSC development associated with increased oxidative stress and apoptosis in ak2 deficient zebrafish. Directed myeloid differentiation of AK2 deficient iPSC resulted in the characteristic promyelocyte maturation arrest observed in RD patients and was associated with an energy depleted adenine nucleotide profile. Notably, we also found that treatment with antioxidants rescued the hematopoietic abnormalities in ak2- mutant zebrafish and restored granulocytic differentiation of AK2-deficient iPSCs. Overall, our results support a mechanistic hypothesis involving energy depletion and abnormal redox state in AK2 deficiency and suggest the potential use of antioxidants as supportive therapeutic modality in RD.
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