Characterization of the cellular and molecular bases of inborn errors of immunity

National Institute Of Allergy And Infectious Diseases

Investigators

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Abstract

The main results of the study performed in FY23 have been the following: 1) Identification and characterization of the molecular and cellular bases of novel forms of IEI. We have participated at a multi-center study that has identified a novel IEI due to ARPC5 mutations, which result in disruption of the actin cytoskeleton causing impaired cell motility and defective cytokine production (1). We have continued to investigate potential genetic defects associated with herpes simplex encephalitis in humans, and identified RIPK3 deficiency as a novel basis for this condition (2). In another collaborative study, we have demonstrated that gain-of-function (GOF) mutations of JAK1 cause severe allergic inflammation associated with dysregulated myelopoiesis (3). By performing whole genome sequencing on newborns with low levels of T-cell receptor excision circles (TRECs) and T-cell lymphopenia, we have shown that haploinsufficiency for the FOXI3 transcription factor affect T-cell development. Using an in vitro assay of T-cell differentiation on an artificial thymic organoid platform, we have shown that this condition is not due to a hematopoietic cell-autonomous defect, but rather to a thymic intrinsic defect. However, individuals with FOXI3 haploinsufficiency often improve their T-cell count with time, so that thymus transplantation is not required (4). 2) Characterization of the molecular and cellular bases underlying phenotypic heterogeneity in known forms of IEI We have demonstrated that a heterozygous GOF mutation in IKBKB is responsible for a clinical condition characterized by autoimmunity and hyperinflammation (5), reviewed the occurrence of granulomatous lesions and EBV-induced lymphoproliferative disease in various forms of IEI (6,7) and demonstrated that haploinsufficiency for DNA ligase 4 (LIG4) may cause immunodeficiency with associated autoimmunity (8). Finally, we have continued our studies aimed at understanding the pathophysiology of various clinical manifestations of human RAG deficiency, including chronic lung disease (9) and autoimmune hemolytic anemia (10). 3) Understanding the basis of immune dysregulation in trisomy 21and the role of neutralizing anti-type I Interferon antibodies in life-threatening influenza Individuals with trisomy 21 are at increased risk of autoimmunity. By performing a multi-omic analysis of their immune system, we have shown that trisomy 21 is characterized by disruption of B-cell tolerance, with expansion of CD11c+ T-bet+ CD21lo B cells expressing VH4-34 and enhanced production of cytokines by CD4+ T cells (11). In addition, we have reviewed and discussed recent evidence from the literature whereby the presence of a third copy of IFNAR1 and IFNAR2 genes (both located on chromosome 21) translates into enhanced IFN signaling, which on one hand may facilitate autoimmunity and help reduce the incidence of viral infections in subjects with trisomy 21, but at the same time leads to increased expression of USP18, which negatively regulates type I interferon signaling upon a second challenge with viruses (12). This may help explain why individuals with trisomy 21 have reduced incidence of viral infections, but these (when present) more frequently lead to severe complications. We had previously demonstrated that neutralizing autoantibodies against type I Interferons (IFN-I) are associated with increased risk of life-threatening COVID-19. WE have now expanded this finding and shown that the same is true for the risk of developing severe influenza (13). 4) Understanding human thymic defects and development of guidelines for the management of individuals with 22q11del syndrome We have reviewed the biology and clinical aspects of primary thymic defects in humans (14) and participated at the development of guidelines for the management of immunological problems associated with 22q11del syndrome, based on expert opinion (15). 5) Definition of revised criteria for the diagnosis of SCID, analysis of outcome after HSCT and development of novel therapeutic approaches to this condition As part of the PIDTC Steering Committee, we have revised the diagnostic criteria for SCID (16) and evaluated the results of the application of these criteria to the cohort of SCID patients enrolled in PIDTC protocols (17). Late-onset hepatitis is not an uncommon complication after HSCT or gene therapy for SCID. We have studied 11 patients with such complication, and found that it was associated with enteric virus (Aich virus, norovirus, sapovirus) infection in stool and/or liver, and expansion of effector memory CD8+ T cells in the liver. We also found that use of non-myeloablative conditioning, incomplete immune reconstitution and split chimerism were risk factors (18). Hypomorphic RAG mutations are often associated with delayed-onset (and consequently delayed diagnosis) of the disease, that frequently manifests with inflammation (granulomas) and autoimmunity in addition to infections. We have participated at an international retrospective study of the outcome of HSCT in these patients. The results have shown relatively poor outcome, (67.5% survival at 4 years after HSCT). Active infections at the time of transplantation, organ damage and T-cell depletion for haploidentical HSCT were identified as major risk factors. Improved outcome was observed in patients identified after newborn screening, transplanted within 3.5 months of life, and without organ damage (19). Poor T-cell reconstitution is associated with worse quality of life and higher incidence of complications after HSCT for SCID. Analysis of the T-cell phenotype in these patients demonstrated T-cell exhaustion, which was especially frequent in recipients of unconditioned HSCT (20). Gene therapy represents an attractive form of treatment for patients with SCID and other genetic disorders. Lentivirus-mediated gene addition has been already successfully used in patients with X-linked SCID. In a pre-clinical study, we compared this approach to CRISPR-Cas9-mediated gene editing. Both approaches overcame the differentiation block in T-cell development in vitro, and allowed robust engraftment upon transplantation of treated stem cells into immunodeficient mice (21). These results support the development of gene-editing treatment for X-linked SCID. More in general, gene editing (including base editing) represents an attractive option for the development of clinical trials for various forms of SCID (22). Finally, newborn screening for SCID is available in all 50 states in the USA since December 2018, however definitive evidence that its implementation translates into improved survival for babies with SCID was lacking. We have reviewed outcome of HSCT for SCID across decades in North America, and demonstrated that only in the last decade (2010-2018) a significant improvement in survival has been observed (87% compared to 72-73% in previous decades). Newborn screening has allowed infants with SCID to be diagnosed and treated promptly, avoiding pre-transplant infections (23). These data strongly suggest that public health programmes worldwide can benefit from the implementation of population-wide newborn screening for SCID.