Molecular Mechanisms Of The Autoimmune Lymphoproliferative Syndrome And Other Immunoregulatory Disorders

National Institute Of Allergy And Infectious Diseases

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Abstract

This project was originally 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 congenital disease-causing loss of normal lymphocyte homeostasis manifested as swollen lymph glands and organs. The 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, India, and Turkey, providing many patients to study at the cellular and genomic level. Here we focus on three projects this reporting period. We have discovered a very interesting genetic disease causing severe abdominal pain and diarrhea due to 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 previously 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. We named this disease CD55 deficiency with hyperactivation of complement, angiopathic thrombosis, and PLE (CHAPLE disease). To establish an effective treatment, we first studied the monoclonal antibody eculizumab, which targets complement Factor C5, to treat 16 CHAPLE cases with distinct CD55 gene mutations. We were interested to understand how pharmacological inhibition downstream of C5 will affect immune dysregulatory disease caused by CD55 loss by affecting upstream control at the C3 convertase. We demonstrated that eculizumab is broadly effective in patients with CHAPLE disease with different mutations. Recovery from complement damage to gut lymphatics was achieved rapidly in 100% of cases. The loss of immunoglobulins, infections, and long-standing functional GI abnormalities were substantially reversed. However, the effects of the drug were temporary. We saw an immediate flare-up of symptoms and serum albumin and immunoglobulin loss when the medication was withdrawn. This observation implies that complement and its innate immune and inflammatory effector mechanisms are constantly provoked, and that patients will require continuous treatment. Thus, eculizumab effectively treats, but does not cure, CHAPLE disease. Aside from the high cost, other drawbacks of eculizumab include a requirement for IV drug administration and adverse side effects. Currently, we are conducting a clinical trial with collaborators Dr. Ivan Fuss in NIAID and scientists from Regeneron Pharmaceuticals, using the monoclonal antibody Pozelimab. This antibody is also directed against the terminal complement protein C5, blocking the formation of the membrane-attack complex that mediates cell lysis and preventing the formation of specific anaphylatoxins. Pozelimab can be given by intravenous or subcutaneous administration and binds to polymorphic variations in C5 that are not recognized by eculizumab. This trial offers a promising opportunity to treat CHAPLE disease patients for which there are no approved targeted therapies. We have recruited a patient with this rare disease to the NIH Clinical Center and he is currently undergoing therapy. Our second project focuses on a new inborn error of immunity in which autophagic flux, metabolism, redox state, and antibacterial activity are deranged in patients, who present with an autoimmune lymphoproliferative syndrome-like disease, recurrent infections, and vasculitis. All patients shared germline mutations of the gene encoding GTPase of the immunity-associated protein (GIMAP) family member 6, GIMAP6, which we determined to be a small GTPase expressed in lymphocytes that regulates cell survival and autophagy. Using a GIMAP6 knockout mouse model, we found that GIMAP6 deficiency results in defective autophagy in T cells and interferes with the formation of a heterotrimeric complex with binding partners GIMAP7 and GABARAPL2. Gimap6-deficient mice also exhibited kidney disease and increased mortality. Loss of Gimap6 results in increased host susceptibility to infections, which may be explained by its ability to be induced by IFN. There may be key roles of GIMAP6 in selected cell-types outside of the immune system, suggesting an important new horizon for understanding GIMAPs. Our final project focused on a pseudoenzyme that facilitates trafficking, stabilization, and cell surface processing of key regulatory proteins, called iRHOM. Our study describes a new immunodeficiency caused by mutations in the RHBDF2 gene, which encodes iRHOM protein 2. One enzyme dependent iRHOM2 protein is ADAM17, which processes signaling molecules, including TNF, TNF receptors, and members of the epidermal growth factor family. The complete loss of iRHOM2 protein expression led to markedly different clinical phenotypes of respiratory and intestinal infections, so we examined these mucosal interfaces and their distinct microbial environment. Our experiments revealed that CD62L, a cell adhesion molecule processed by ADAM17 that is normally reduced by anti-CD3 stimulation, remained high for patient T cells, indicating ADAM17 could impact our patients by adversely affecting leukocyte migration. Additionally, soluble TNF was decreased in the serum from all patients, and regulation of TNF most likely contributes as the chief cytokine causing this disease. Our investigation showed that heterozygous humans had roughly half the level of iRHOM2 but processed ADAM17 compared to controls. Our transcriptomic analysis of whole blood showed a reduction in transcription of genes involved in leukocyte migration and neutrophil mediated immunity. Time-series gene expression data after stimulation showed that patient T cells had major decreases in the genes for cytokine production and response to TNF as compared to controls. This result indicates that the lack of TNF secretion by iRHOM2 deficiency had a major impact on patient T cells resulting in decreased expression of gene sets normally responding to TNF. These findings were consistent with the concept that the lack of TNF secretion has the greatest role in shaping this human disease. Since TNF is the driver of rheumatoid arthritis and other inflammatory disease, iRHOM2 may be an attractive new therapeutic target.