The main results of the study performed in FY24 have been the following: 1) Identification and characterization of the molecular and cellular bases of novel forms of IEI. In a collaborative study with colleagues from the Imagine Institute in Paris (France), we have identified a novel form of IEI due to mutations of the PTCRA gene, encoding for the invariant pre-TCR alpha chain of the pre-T cell receptor. We established that bi-allelic loss of function variants of the gene compromise generation of T cells expression the TCR alpha/beta heterodimer. On the other hand, hypomorphic variants of PTCRA are associated with increased risk of autoimmune disease (1). In another series of collaborative studies, we have provided fundamental contribution to the identification of the molecular and cellular bases of FLT3L deficiency (2) and autoinflammatory disease due to mutations of SHARPIN, a component of the LUBAC complex (3). Finally, in an internal collaboration with Dr. Rosenzweig’s team at the NIH, we have expanded the phenotype and molecular pathology of AIOLOS deficiency (4) 2) Characterization of the molecular and cellular bases of immunopathology, and definition of the clinical spectrum of known forms of IEI In collaboration with colleagues at the Imagine Institute (Paris, France) and at the University of California at San Francisco, we have obtained fundamental novel insights into the mechanisms that govern production of anti-type I interferon (IFN-I) antibodies. Previous studies from our and other groups had shown that neutralizing anti-IFN-I antibodies cause increased susceptibility to life-threatening viral infections. Through studies in patients and mice, we have now demonstrated that genetic defects that affect the non canonical NF-kB signaling pathway in the thymic medulla are associated with production of anti-IFN-I antibodies (5), indicating an important role of central thymic tolerance in preventing this manifestation of immunopathology. In another set of studies, we have expanded on our previous observations, and reported on the clinical features, treatment, and outcomes of autoimmune hemolytic anemia in patients with RAG deficiency (6). We have also shown that this condition is associated with perturbation of the diversity and composition of the skin microbiome (7). Furthermore, we have contributed novel clinical and laboratory data on a rare form of IEI, CD40 deficiency (8), and described a new case and performed literature review of Mulibrey syndrome (9). We have also reported on new cases of PAX1 deficiency, which causes a developmental defect of the thymus and other cervical-cranial-facies structures (10). The overall status of knowledge of primary and secondary forms of human thymic defects have been reviewed (11,12). We have also reported on the first case of DNA polymerase delta 3 (POLD3) manifesting as Omenn syndrome (13). We have collaborated with the Lionakis laboratories in a study that has established a previously unrecognized role for IFN-gamma in driving candidiasis and tissue damage at mucosal and cutaneous surfaces in patients with Autoimmune Polyglandular Syndrome type 1 (14). These observations have fundamental implications for the development of a targeted therapeutic approach in this disease. Finally, we have investigated the immunopathology of visceral leishmaniasis in otherwise healthy individuals (15). 3) Relevance of laboratory biomarkers in the evaluation of patients with IEI In a collaborative study, we have demonstrated that choosing the correct laboratory assay and interpreting its results in a critical manner if of fundamental importance during the investigations of patients with severe combined immune deficiency and other forms of severe T-cell lymphopenia (16). 4) Development of novel therapeutic approaches for RAG deficiency During FY24, we have continued to explore novel therapeutic approaches to RAG deficiency, In particular, in a series of studies in mice, including generation of humanized mouse models of RAG1 and RAG2 deficiency, we have provided preclinical evidence that gene editing can rescue the T- and B-cell differentiation potential of RAG-mutated human hematopoietic stem and progenitor cells (HSPC) (17-19). In parallel, we have tested a novel antibody drug conjugate (ADC) immunotoxin, in which anti-CD45 monoclonal antibody was conjugated with the teserin payload. We have shown that use of this ADC alone induces efficient myeloablation and depletion of more mature and dysreactive hematopoietic cells in a mouse model of hypomorphic Rag1 deficiency. This approach can be used to allow efficient engraftment of wild-type donor HSPC and immune reconstitution, even across MHC barrier (20). 5) Evaluation of current therapeutic approaches and development of novel forms of treatment in other forms of IEI In a series of collaborative studies with other members of the primary Immune Deficiency Treatment Consortium (PIDTC), we have reported on the effective outcome of hematopoietic cell transplantation (HCT) in patients with chronic granulomatous disease (21) and analyzed the impact of patient genotype, neutrophil oxidase activity, and pre-transplant infection and inflammatory status (22). We have also analyzed the outcome, and discussed the factors that may be associated with suboptimal reconstitution of platelet count, after gene therapy in patients with Wiskott-Aldrich syndrome (23).