CRISPR-targeted MAGT1 insertion restores XMEN patient hematopoietic stem cells and lymphocytes.

XMEN disease, defined as "X-linked MAGT1 deficiency with increased susceptibility to Epstein-Barr virus infection and N-linked glycosylation defect," is a recently described primary immunodeficiency marked by defective T cells and natural killer (NK) cells. Unfortunately, a potentially curative hematopoietic stem cell transplantation is associated with high mortality rates. We sought to develop an ex vivo targeted gene therapy approach for patients with XMEN using a CRISPR/Cas9 adeno-associated vector (AAV) to insert a therapeutic MAGT1 gene at the constitutive locus under the regulation of the endogenous promoter. Clinical translation of CRISPR/Cas9 AAV-targeted gene editing (GE) is hampered by low engraftable gene-edited hematopoietic stem and progenitor cells (HSPCs). Here, we optimized GE conditions by transient enhancement of homology-directed repair while suppressing AAV-associated DNA damage response to achieve highly efficient (>60%) genetic correction in engrafting XMEN HSPCs in transplanted mice. Restored MAGT1 glycosylation function in human NK and CD8+ T cells restored NK group 2 member D (NKG2D) expression and function in XMEN lymphocytes for potential treatment of infections, and it corrected HSPCs for long-term gene therapy, thus offering 2 efficient therapeutic options for XMEN poised for clinical translation.

Infection by Leishmania (Leishmania) infantum chagasi causes intestinal changes B-1 cells dependent.

Evaluating the histopathological and morphometric changes caused by Leishmania (Leishmania) infantum chagasi infection either in the presence or absence of B-1 cells. Wild-type Balb/c and XID mice were used. Half of XID mice received B-1 cells adoptive transfer (XID + B1). Five animals from each group were infected (Balb/c I, XID I and XID + B1 I), totalizing six groups (n = 5). After 45 days of infection, the ileum was collected for histological processing and analysis. After infection, the XID animals showed an increase in the thickness of the intestinal layers, in the depth and width of the crypt and in the villi width. However, the Balb/c I group showed a reduction in almost all these parameters, whereas the villi width was increased. The villi height decreased in the infected XID animals; however, it was increased in the XID + B1 I group. Leishmania (L) infantum chagasiinfection caused a decrease in the number of Paneth cells; however, their area was increased. Finally, goblet cells and enterocytes presented different change profiles among groups. This study showed that the parasite infection causes structural and histopathological alterations in the intestine. These changes might be influenced by the absence of B-1 cells.

Novel IL2RG Mutation Causes Leaky TLOWB+NK+ SCID With Nodular Regenerative Hyperplasia and Normal IL-15 STAT5 Phosphorylation.

X-linked severe combined immunodeficiency disease (SCID) is caused by mutations in the interleukin (IL)-2 receptor γ (IL2RG) gene and patients usually present with a TBNK SCID phenotype. Nevertheless, a minority of these patients present with a TBNK phenotype, similar to the IL-7R-deficient patients. We report a patient with a novel missense p.Glu297Gly mutation in the IL2RG gene presenting with a leaky TBNK SCID with delayed onset, moderate susceptibility to infections, and nodular regenerative hyperplasia. He presents with preserved STAT5 tyrosine phosphorylation in response to IL-15 stimulation but not in response to IL-2 and IL-7, resulting in the NK phenotype.

Impact of Day 14 Peripheral Blood Chimerism after Allogeneic Hematopoietic Stem Cell Bone Transplantation on the Treatment Outcome of Non-Malignant Disease.

BACKGROUND: The impact of early peripheral blood chimerism on the outcome of allogeneic hematopoietic stem cell transplantation (allo-HSCT) is unclear. We aimed to determine whether day 14 peripheral blood chimerism after allo-HSCT predicts outcomes in patients with non-malignant diseases. METHODS: Data from 56 patients who received allo-HSCT between April 2007 and March 2016 were retrospectively analyzed. Chimerism was evaluated using short-tandem repeat polymerase chain reaction, with mixed chimerism (MC) defined as greater than 1% recipient cells which was further categorized into low-level MC (> 1% and < 15% of recipient-derived cells) and high-level MC (≥ 15% of the recipient-derived cells). RESULTS: Thirty-six patients showed complete donor chimerism (CC), 14 low-level MC, and 6 high-level MC at day 14 post-transplant. The estimated 5-year event-free survival (EFS) was higher in the CC or low-level MC groups than in the high-level MC group (86.1% vs. 71.4% vs. 33.3%; P = 0.001). In BM or peripheral blood stem cell (BM/PBSC) transplants, the 5-year EFS was higher in the CC or low-level MC group than in the high-level MC group (93.1% vs. 66.7% vs. 0%; P < 0.001). However, in cord blood transplants, the 5-year OS and EFS according to the day 14 peripheral blood chimerism did not reach statistical significance. CONCLUSION: Although CC is not always necessary after allo-HSCT for non-malignant diseases, our data suggest that day 14 peripheral blood chimerism may predict outcomes in patients with non-malignant diseases who underwent BM/PBSC transplants.

A novel deletion mutation in IL2RG gene results in X-linked severe combined immunodeficiency with an atypical phenotype.

Severe combined immunodeficiency (SCID) is the most serious disorder among primary immunodeficiency diseases threatening children's life. Atypical SCID variant, presenting with mild reduced T cells subsets, is often associated with infection susceptibility but poor clinical diagnosis. The atypical X-SCID patient in the present study showed a mild clinical presentation with a TlowNK+B+ immunophenotype. The patient has reduced T- cell subpopulations with a subdued thymic output measured by sjTRECs. Further analysis showed that T cells maintained a normal proliferation and a broad Vß repertoire. NK cells, however, exhibited a skewed development toward immature CD3-CD16+CD56- cells. Genetic analysis revealed a novel deletion at nucleotide 52 in exon 1 of IL2RG gene. Sequence alignment predicted a truncated IL2RG protein missing signal peptide derived from a possible alternative reading frame. The novel mutation in IL2RG gene identified in our study may help the early diagnosis of atypical X-SCID.

Faster T-cell development following gene therapy compared with haploidentical HSCT in the treatment of SCID-X1.

During the last decade, gene therapy via ex vivo gene transfer into autologous hematopoietic stem cells has emerged as a convincing therapy for severe combined immunodeficiency caused by ILR2G mutation (SCID-X1) despite the occurrence of genotoxicity caused by the integration of first-generation retroviral vectors. However, the place of gene therapy among the therapeutic armamentarium remains to be defined. We retrospectively analyze and compare clinical outcomes and immune reconstitution in 13 consecutive SCID-X1 patients having undergone haploidentical hematopoietic stem cell transplantation (HSCT) and 14 SCID-X1 patients treated with gene therapy over the same period at a single center level: the Necker Children's Hospital (Paris, France). Our results show a clear advantage in terms of T-cell development of gene therapy over HSCT with a mismatched donor. Patients treated with gene therapy display a faster T-cell reconstitution and a better long-term thymic output. Interestingly, this advantage of gene therapy vs haploidentical HSCT seems to be independent of the existence of clinical graft-versus-host disease in the latter condition. If data of safety are confirmed over the long term, gene therapy for SCID-X1 appears to be an equal, if not superior, alternative to haploidentical HSCT.

Accumulation of peripheral autoreactive B cells in the absence of functional human regulatory T cells.

Regulatory T cells (Tregs) play an essential role in preventing autoimmunity. Mutations in the forkhead box protein 3 (FOXP3) gene, which encodes a transcription factor critical for Treg function, result in a severe autoimmune disorder and the production of various autoantibodies in mice and in IPEX (immune dysregulation, polyendocrinopathy, enteropathy, X-linked) patients. However, it is unknown whether Tregs normally suppress autoreactive B cells. To investigate a role for Tregs in maintaining human B-cell tolerance, we tested the reactivity of recombinant antibodies isolated from single B cells isolated from IPEX patients. Characteristics and reactivity of antibodies expressed by new emigrant/transitional B cells from IPEX patients were similar to those from healthy donors, demonstrating that defective Treg function does not impact central B-cell tolerance. In contrast, mature naive B cells from IPEX patients often expressed autoreactive antibodies, suggesting an important role for Tregs in maintaining peripheral B-cell tolerance. T cells displayed an activated phenotype in IPEX patients, including their Treg-like cells, and showed up-regulation of CD40L, PD-1, and inducibl T-cell costimulator (ICOS), which may favor the accumulation of autoreactive mature naive B cells in these patients. Hence, our data demonstrate an essential role for Tregs in the establishment and the maintenance of peripheral B-cell tolerance in humans.

Disseminated BCG infection mimicking metastatic nasopharyngeal carcinoma in an immunodeficient child with a novel hypomorphic NEMO mutation.

BACKGROUND: Nuclear factor-κB essential modulator (NEMO) deficiency is a developmental and immunological disorder. The genetic and phenotypic correlation has been described. METHODS: We report a unique clinical presentation and the identification of a novel missense mutation in the NEMO gene in a 3-year-old boy with bacillus Calmette-Guerin (BCG) infection. RESULTS: The patient presented with fever, cervical lymphadenopathy, and abnormal anti-Epstein-Barr virus (EBV) antibody titers, suggestive of EBV-related diseases including chronic active EBV infection, X-linked lymphoproliferative syndrome, or nasopharyngeal carcinoma. Although the biopsy specimen from a nasopharyngeal lesion was initially diagnosed as squamous cell carcinoma, this was changed to disseminated BCG infection involving the nasopharynx, multiple systemic lymph nodes, and brain. A novel mutation (designated D311E) in the NEMO gene, located in the NEMO ubiquitin-binding (NUB) domain, was identified as the underlying cause of the immunodeficiency. Impaired immune responses which are characteristic of patients with NEMO deficiency were demonstrated. The patient underwent successful unrelated bone marrow transplantation at 4.9 years of age. CONCLUSION: This study suggests the importance of the NUB domain in host defense against mycobacteria. The unique presenting features in our patient indicate that a hypomorphic NEMO mutation can be associated with atypical pathological findings of the epithelial tissues in patients with BCG infection.

The cellular amount of the common gamma-chain influences spontaneous or induced cell proliferation.

Mutations of the IL2RG encoding the common gamma-chain (gamma(c)) lead to the X-linked SCID disease. Gene correction through ex vivo retroviral transduction restored the immunological impairment in the most of treated patients, although lymphoproliferative events occurred in five of them. Even though in two cases it was clearly documented an insertional mutagenesis in LMO2, it is conceivable that gamma(c) could have a role per se in malignant lymphoproliferation. The gamma(c) is a shared cytokine receptor subunit, involved also in growth hormone (GH) receptor signaling. Through short interfering RNA or using X-linked SCID B lymphoblastoid cell lines lacking gamma(c), we demonstrate that self-sufficient growth was strongly dependent on gamma(c) expression. Furthermore, a correlation between gamma(c) amount and the extent of constitutive activation of JAK3 was found. The reduction of gamma(c) protein expression also reduced GH-induced proliferation and STAT5 nuclear translocation in B lymphoblastoid cell lines. Hence, our data demonstrate that gamma(c) plays a remarkable role in either spontaneous or GH-induced cell cycle progression depending on the amount of protein expression, suggesting a potential role as enhancing cofactor in lymphoproliferation.

Development of human hepatocellular carcinoma in X-linked severe combined immunodeficient pigs: An orthotopic xenograft model.

Hepatocellular carcinoma (HCC) is the fifth most common primary tumor and the third leading cause of cancer-related deaths worldwide. Rodent models of HCC have contributed to the advancement of studies investigating liver carcinogenesis, tumor-host interactions, and drug screening. However, their small size renders them unsuitable for surgical or clinical imaging studies, necessitating the development of larger-size HCC models. Here, we developed a xenograft model of human HCC in X-linked interleukin-2 receptor gamma chain gene (Il2rg)-targeted severe combined immunodeficient (SCID) pigs. HepG2 cell suspension in serum-free medium containing 50% membrane matrix was directly injected into the liver parenchyma of eight X-linked Il2rg-targeted SCID pigs (6.6-15.6 kg) via ultrasonography-guided percutaneous puncture. Tumor engraftment was evaluated weekly using ultrasonography, and cone-beam computed tomography was performed during arterial portography (CTAP) and hepatic arteriography (CTHA) to evaluate the hemodynamics of engrafted tumors. The engrafted tumors were histologically analyzed following necropsy and assessed for pathological similarities to human HCCs. Macroscopic tumor formation was observed in seven of the eight pigs (simple nodular tumors in three and multinodular tumors in four). Engrafted tumors were identified as low-echoic upon ultrasonography and as perfusion-defect nodules on the CTAP images. Meanwhile, CTHA showed that the tumors were hyperattenuating. Further, histopathological findings of the engrafted tumors were consistent with those of human HCC. In conclusion, the porcine model of human HCC, successfully generated herein, might help develop more effective therapeutic strategies for HCC.

Detection of T lymphocytes with a second-site mutation in skin lesions of atypical X-linked severe combined immunodeficiency mimicking Omenn syndrome.

X-linked severe combined immunodeficiency (XSCID) is caused by mutations of the common gamma chain (gammac) and usually characterized by the absence of T and natural killer (NK) cells. Here, we report an atypical case of XSCID presenting with autologous T and NK cells and Omenn syndrome-like manifestations. The patient carried a splice-site mutation (IVS1+5G>A) that caused most of the mRNA to be incorrectly spliced but produced normally spliced transcript in lesser amount, leading to residual gammac expression and development of T and NK cells. The skin biopsy specimen showed massive infiltration of revertant T cells. Those T cells were found to have a second-site mutation and result in complete restoration of correct splicing. These findings suggest that the clinical spectrum of XSCID is quite broad and includes atypical cases mimicking Omenn syndrome, and highlight the importance of revertant mosaicism as a possible cause for variable phenotypic expression.

Clinical and immunologic consequences of a somatic reversion in a patient with X-linked severe combined immunodeficiency.

X-linked severe combined immunodeficiency is a life-threatening disorder caused by mutations in the gene encoding the interleukin-2 receptor gamma chain (IL2RG). Hypomorphic mutations and reversion of mutations in subpopulations of cells can result in variant clinical phenotypes, making diagnosis and treatment difficult. We describe a 5-year-old boy with mild susceptibility to infection who was investigated for a mutation in IL2RG due to persistent natural killer (NK)- and T-cell lymphopenia. A functionally relevant novel T466C point mutation was found in B, NK, and epithelial cells, whereas alpha/beta and gamma/delta T cells showed the normal gene sequence, suggesting reversion of the mutation in a common T-cell precursor. This genetic correction in T cells resulted in a diverse T-cell repertoire and significant immunity despite failure to produce specific antibodies linked to an intrinsic defect of mutant B cells. These observations confirm the potential of revertant T-cell precursors to reconstitute immune function, but questions remain on the longevity of revertant cells implicating the need for careful follow up and early consideration of hematopoietic stem cell transplantation (HSCT).

Chronic noninfectious necrotizing granulomas in a child with Nijmegen breakage syndrome.

Nijmegen breakage syndrome (NBS) is a chromosomal breakage disorder with characteristic physical features, chromosomal instability, and combined immunodeficiency. It is closely related to other chromosomal breakage disorders like ataxia telangiectasia. Noninfectious granulomatous inflammation refractory to treatment is a relatively common feature in ataxia telangiectasia. Herein we report a patient with NBS who developed chronic refractory necrotizing granulomatous ulcerations and review the pathophysiology of NBS and noninfectious granulomas in primary immunodeficiency syndromes.

Defective glycosylation and multisystem abnormalities characterize the primary immunodeficiency XMEN disease.

X-linked immunodeficiency with magnesium defect, EBV infection, and neoplasia (XMEN) disease are caused by deficiency of the magnesium transporter 1 (MAGT1) gene. We studied 23 patients with XMEN, 8 of whom were EBV naive. We observed lymphadenopathy (LAD), cytopenias, liver disease, cavum septum pellucidum (CSP), and increased CD4-CD8-B220-TCRαß+ T cells (αßDNTs), in addition to the previously described features of an inverted CD4/CD8 ratio, CD4+ T lymphocytopenia, increased B cells, dysgammaglobulinemia, and decreased expression of the natural killer group 2, member D (NKG2D) receptor. EBV-associated B cell malignancies occurred frequently in EBV-infected patients. We studied patients with XMEN and patients with autoimmune lymphoproliferative syndrome (ALPS) by deep immunophenotyping (32 immune markers) using time-of-flight mass cytometry (CyTOF). Our analysis revealed that the abundance of 2 populations of naive B cells (CD20+CD27-CD22+IgM+HLA-DR+CXCR5+CXCR4++CD10+CD38+ and CD20+CD27-CD22+IgM+HLA-DR+CXCR5+CXCR4+CD10-CD38-) could differentially classify XMEN, ALPS, and healthy individuals. We also performed glycoproteomics analysis on T lymphocytes and show that XMEN disease is a congenital disorder of glycosylation that affects a restricted subset of glycoproteins. Transfection of MAGT1 mRNA enabled us to rescue proteins with defective glycosylation. Together, these data provide new clinical and pathophysiological foundations with important ramifications for the diagnosis and treatment of XMEN disease.

Elucidation of the Effects of a Current X-SCID Therapy on Intestinal Lymphoid Organogenesis Using an In Vivo Animal Model.

BACKGROUND & AIMS: Organ-level research using an animal model lacking Il2rg, the gene responsible for X-linked severe combined immunodeficiency (X-SCID), is clinically unavailable and would be a powerful tool to gain deeper insights into the symptoms of patients with X-SCID. METHODS: We used an X-SCID animal model, which was first established in our group by the deletion of Il2rg gene in pigs, to understand the clinical signs from multiple perspectives based on pathology, immunology, microbiology, and nutrition. We also treated the X-SCID pigs with bone marrow transplantation (BMT) for mimicking a current therapeutic treatment for patients with X-SCID and investigated the effect at the organ-level. Moreover, the results were confirmed using serum and fecal samples collected from patients with X-SCID. RESULTS: We demonstrated that X-SCID pigs completely lacked Peyer's patches (PPs) and IgA production in the small intestine, but possessed some dysfunctional intestinal T and B cells. Another novel discovery was that X-SCID pigs developed a heterogeneous intestinal microflora and possessed abnormal plasma metabolites, indicating that X-SCID could be an immune disorder that affects various in vivo functions. Importantly, the organogenesis of PPs in X-SCID pigs was not promoted by BMT. Although a few isolated lymphoid follicles developed in the small intestine of BMT-treated X-SCID pigs, there was no evidence that they contributed to IgA production and microflora formation. Consistently, most patients with X-SCID who received BMT possessed abnormal intestinal immune and microbial environments regardless of the presence of sufficient serum IgG. CONCLUSIONS: These results indicate that the current BMT therapies for patients with X-SCID may be insufficient to induce the organogenesis of intestinal lymphoid tissues that are associated with numerous functions in vivo.

X-linked primary immunodeficiencies as a bridge to better understanding X-chromosome related autoimmunity.

Recent studies indicate that genes located on the X-chromosome play a major and unique role in autoimmunity. The fact that most X-linked primary immune deficiencies carry significant autoimmune manifestations greatly supports this notion. Autoimmunity and immune deficiency have been considered two opposite extremes resulting from immune dysregulation and failure of immune development and/or function, respectively. Growing evidence has been accumulating to indicate that autoimmune phenomena occur in patients suffering from primary immune deficiency (PID), and the molecular and cellular mechanisms that interconnect these conditions are being unraveled. The study of rare single-gene disorders associated with significant autoimmunity may shed light on the pathophysiology of more complex multifactorial and polygenic autoimmune disorders. In this regard, primary immunodeficiencies represent unique "experiments of Nature" that illustrate the critical role played by single-gene products in the development, function and homeostasis of the immune system. In this review we will focus on the clinical features and on the cellular and molecular pathophysiology of the known X-linked PID in which autoimmune manifestations are more common, in the attempt to understand what single-gene defects can teach us on the role that key immune pathways and cellular processes may play to prevent autoimmunity.

A synonymous splice site mutation in IL2RG gene causes late-onset combined immunodeficiency.

X-Linked severe combined immunodeficiency (X-SCID) is a severe form of primary immunodeficiency characterized by absence of T cells and NK cells. X-SCID is caused by a loss-of-function mutation in the IL2RG gene that encodes common gamma chain (γc), which plays an essential role in lymphocyte development. We report the first case of hypomorphic X-SCID caused by a synonymous mutation in the IL2RG gene leading to a splice anomaly, in a family including two patients with diffuse cutaneous warts, recurrent molluscum contagiosum, and mild respiratory infections. The mutation caused aberrant splicing of IL2RG mRNA, subsequently resulted in reduced γc expression. The leaky production of normally spliced IL2RG mRNA produced undamaged protein; thus, T cells and NK cells were generated in the patients. Functional assays of the patients' T cells and NK cells revealed diminished cytokine response in the T cells and absent cytokine response in the NK cells. In addition, the TCR repertoire in these patients was limited. These data suggest that a fine balance between aberrant splicing and leaky production of normally spliced IL2RG mRNA resulted in late-onset combined immunodeficiency in these patients.