Combined immune deficiencies (CID) include a heterogeneous group of genetic conditions characterized by impaired development and/or function of T and B cells. Infants affected with the most severe form of these disorders, also known as severe combined immune deficiency (SCID), are highly prone to serious infections since early after birth, and die within the first few years of life unless immune reconstitution is obtained, usually after hematopoietic cell transplantation (HCT). However, significant phenotypic heterogeneity has been reported in CID, with some patients presenting later in life, and whose clinical manifestations also include autoimmunity. Defects of the RAG1 and RAG2 genes represent a prototypical example of such clinical heterogeneity, but the molecular and cellular bases underlying phenotypic diversity remain ill defined. Finally, in some cases, SCID and CID may associate with other developmental defects, including skeletal and neurological defects. Genetic characterization of the molecular bases of CID has helped identify molecules and pathways that play a critical role in immune system development and function. During the last year, the following main achievements have been obtained: 1) Using a combination of next generation sequencing, biochemical investigations, cellular and animal modeling with patient-derived induced pluripotent stem cells (iPSCs) and zebrafish, we have identified a novel form of SCID associated with skeletal dysplasia and severe neurological problems. Whole exome sequencing revealed biallelic mutations in the Exosostin-Like 3 (EXTL3) gene, encoding for an enzyme involved in heparin sulfate (HS) biosynthesis. Biochemical studies have demonstrated abnormal length and composition of HS chains in patients cells. HS proteoglycans (HSPG) play a critical role in modulating the activity of morphogenetic proteins, cytokines and growth factors, including fibroblast growth factors (FGFs), BMP4 and IL-7, which are involved in skeletal and hematolymphoid differentiation. The patients fibroblasts showed increased signaling in response to FGF2 in vitro, resembling what observed in patients with skeletal defects due to gain-of-function mutations of FGF receptor 2 (FGFR2) and FGFR3 genes. Stable transfection of a normal copy of the EXTL3 gene into patients cells rescued composition and length of HS chains and signaling in response to FGF2. A natural model of extl3 deficiency in zebrafish (the boxer mutant, box) has short pectoral fins and neuronal defects, resembling the short limbed dwarfism and the neurological phenotype seen in the patients. Upon intercrossing of box with rag2:gfp transgenic fish, decreased thymopoiesis was observed also in the mutant fish. Hematopoietic differentiation of CD34+ hematopoietic progenitor cells. Furthermore, defective differentiation of patient-derived iPSCs into thymic epithelial progenitor cells was also observed. These data indicate that EXTL3 mutations affecting HSPG biosynthesis compromise both hematolymphoid differentiation and thymic development, in addition to skeletal and neuronal development. 2) We had previously shown that the severity of the clinical and immunological phenotype of patients with RAG1 mutations correlates with the recombination activity of the mutant protein in vitro. To investigate the in vivo effects of RAG mutations and to shed light on phenotypic heterogeneity of the disease, we have studied diversity and composition of T and B cell repertoire in 12 patients with RAG mutations presenting with various clinical phenotypes. Next generation sequencing showed that restriction of repertoire diversity correlates with the severity of the clinical phenotype. Primary component analysis clearly distinguished patients from controls, and in the case of B cell repertoire, it also distinguished between different groups of RAG-mutated patients. Abnormalities in the composition and length of the CDR3 region of T cell receptor (TCR) and B cell receptor (BCR) transcripts were more prominent in patients with a more severe phenotype. Immunoglobulin transcripts from patients with RAG mutations showed a marked reduction in the frequency of Tyrosine residues, reflecting reduced usage of the IGHJ6 gene during V(D)J recombination. These abnormalities were also present in unique sequences (indicating a primary restriction of the immune repertoire) and may contribute to the autoimmunity frequently associated with this condition. Finally, although not required for NK cell development, RAG expression may initiate in common lymphoid progenitors. It had been recently shown that NK cells from Rag-/- mice have a mature and activated phenotype, reduced cellular fitness, and enhanced cellular cytotoxicity. By studying 66 patients with RAG mutations and variable clinical phenotypes, we have demonstrated that NK cells from RAG-mutated patients have an immature phenotype, yet display increased perforin content and enhanced degranulation capacity. Furthermore, the degree of NK cell abnormalities correlated with the severity of the clinical and immunological phenotype. These data suggest that NK cell abnormalities may play a role in the increased rate of graft rejection observed after unconditioned hematopoietic stem cells transplantation (HSCT) in patients with RAG deficiency, and indicate that serotherapy targeting NK cells should be considered in the HSCT preparative regimen.
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