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Inborn errors of immunity (IEI) are diseases caused by genetic mutations that affect the immune system’s ability to fight pathogens, cope with the microbiota or regulate autoimmunity and inflammation. More than 500 IEI have been described and many are life-threatening and require curative therapy. Allogeneic haematopoietic stem cell transplantation is an increasingly effective curative strategy, and autologous transplantation of gene-modified haematopoietic stem and progenitor cells is also a treatment option. Gene therapy was first successfully used to restore T cell development in patients with severe combined immunodeficiency, with ex vivo engineered gammaretroviral vectors enabling the sustained correction of T cell immunodeficiency more than 20 years later. The generation of safer and more potent vectors has increased the efficacy and application of this therapy to other IEI, such as Wiskott–Aldrich syndrome and chronic granulomatous disease. Nevertheless, gene therapy based on gene addition has some limitations, the greatest of which is the lack of a physiological gene expression control. This Perspective summarizes the journey of the past 25 years that has led to the successful use of gene therapy for IEI and discusses the next steps for the field.In this Perspective, Alain Fischer reflects on the development of gene therapy for patients with inborn errors of immunity. He discusses the challenges the field has faced as well as the progress seen in the past 25 years.

Key Points Cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells are armed to kill virus-infected or transformed cells through the polarized secretion of cytotoxic granules that contain perforin and granzymes. Perforin is crucial for the access of granzymes to their pro-apoptotic substrates in the target cells. Inherited deficiencies of the granule-dependent cytotoxic pathway in humans result in a severe immunopathological condition known as haemophagocytic lymphohistiocytosis (HLH). HLH is generally triggered by an infection and is associated with an overactive T cell-mediated immune response, probably resulting from the failure of activated CTLs and NK cells to clear antigen-presenting cells and thus to terminate the immune response. Characterization of the molecular causes leading to HLH in humans and mutant mice has substantially contributed to our understanding of the key steps required for the maturation and exocytosis of cytotoxic granules during target cell killing. In addition to defects in perforin, which account for the prototypical form of HLH, defects in lysosomal trafficking regulator (LYST) or adaptor protein 3 (AP3) provide evidence for the role of these proteins in cytotoxic granule biogenesis. The coordinated delivery of cytotoxic granule contents to the immunological synapse depends on additional effector proteins, which cause HLH when defective. They are involved in the docking (RAB27a), priming (MUNC13-4) and fusion (syntaxin 11 and MUNC18-2) of the cytotoxic granules that polarize at the CTL–target cell interface. The structure of the immunological synapse is strikingly similar to that of the neurological synapse. In both cases, the delivery of mediators to the intercellular cleft must be tightly regulated in a spatial and temporal manner. Several of the effector proteins that mediate vesicle exocytosis at both synapses belong to the same families of proteins. A comparison of the proteins and mechanisms involved may provide clues to uncover additional effectors that regulate the cytotoxic function of lymphocytes. A complex and highly regulated pathway ensures that the delivery of cytotoxic cargo of cytotoxic lymphocytes is appropriately aimed and timed. As reviewed here, the study of patients and mutant mice with cytotoxicity defects has revealed many of the molecules involved in this targeted exocytosis of cytotoxic granules. Cytotoxic T cells and natural killer cells are crucial for immune surveillance against virus-infected cells and tumour cells. Molecular studies of individuals with inherited defects that impair lymphocyte cytotoxic function have also highlighted the importance of cytotoxicity in the regulation and termination of immune responses. As discussed in this Review, characterization of these defects has contributed to our understanding of the key steps that are required for the maturation of cytotoxic granules and the secretion of their contents at the immunological synapse during target cell killing. This has revealed a marked similarity between cytotoxic granule exocytosis at the immunological synapse and synaptic vesicle exocytosis at the neurological synapse. We explore the possibility that comparison of these two kinetically and spatially regulated secretory pathways will provide clues to uncover additional effectors that regulate the cytotoxic function of lymphocytes.

The importance of human innate lymphoid cells to normal human physiology is unclear. Vivier and colleagues find that immunodeficient patients ‘rescued’ with normal bone marrow can recover their T cells but not their innate lymphoid cells, yet remain entirely asymptomatic for nearly 40 years. Innate lymphoid cells (ILCs) have potent immunological functions in experimental conditions in mice, but their contributions to immunity in natural conditions in humans have remained unclear. We investigated the presence of ILCs in a cohort of patients with severe combined immunodeficiency (SCID). All ILC subsets were absent in patients with SCID who had mutation of the gene encoding the common γ-chain cytokine receptor subunit IL-2Rγ or the gene encoding the tyrosine kinase JAK3. T cell reconstitution was observed in patients with SCID after hematopoietic stem cell transplantation (HSCT), but the patients still had considerably fewer ILCs in the absence of myeloablation than did healthy control subjects, with the exception of rare cases of reconstitution of the ILC1 subset of ILCs. Notably, the ILC deficiencies observed were not associated with any particular susceptibility to disease, with follow-up extending from 7 years to 39 years after HSCT. We thus report here selective ILC deficiency in humans and show that ILCs might be dispensable in natural conditions, if T cells are present and B cell function is preserved.

In a long-term (up to 11 years) follow-up of nine patients with X-linked severe combined immunodeficiency treated with retroviral insertion of a normal common γ-chain gene into hematopoietic progenitors, seven had durable T-cell reconstitution and long-term survival. B-cell immunity was not corrected. Acute leukemia developed in four patients. In patients without a matched marrow donor, gene therapy may be an option but is associated with serious risks. The cytokine receptor common γ chain, which is encoded by the interleukin-2 receptor subunit gamma ( IL2RG ) gene, is a critical functional component of the receptors for interleukin-2, interleukin-4, interleukin-7, interleukin-9, interleukin-15, and interleukin-21. 1 Naturally occurring mutations in IL2RG are responsible for X-linked severe combined immunodeficiency (SCID-X1) disease. This condition is characterized by the complete lack of T cells and natural killer cells, whereas B cells are present. 2 , 3 Hematopoietic stem-cell transplantation is a lifesaving therapy. Despite associated improvements in the survival rate, however, non−HLA-identical hematopoietic stem-cell transplantation has a number of drawbacks. For example, reconstitution of T-cell function . . .

Loss-of-function mutations in the human CTP synthase 1 gene cause an immunodeficiency disease with impaired T cell proliferation after antigen stimulation, revealing a potential new target for immunosuppressive drugs. A novel immune deficiency A study of several unrelated families susceptible to recurrent viral diseases but in whom known primary immunodeficiencies had been excluded has led to the identification of a novel human inherited immunodeficiency, and the recognition of a molecular pathway required to mount an efficient immune response against pathogens. Loss-of-function mutations in the gene for cytidine 5′ triphosphate synthase 1 ( CTPS1 ) are associated with severe and selective impairment of T-cell proliferation after antigen stimulation, identifying the enzyme as a critical checkpoint in adaptive immunity. CTPS1 deficiency does not induce other phenotype abnormalities, so these findings suggest that CTPS1 could be a viable target for therapeutics to inhibit unwanted adaptive responses. Lymphocyte functions triggered by antigen recognition and co-stimulation signals are associated with a rapid and intense cell division, and hence with metabolism adaptation 1 . The nucleotide cytidine 5′ triphosphate (CTP) is a precursor required for the metabolism of DNA, RNA and phospholipids 2 , 3 , 4 . CTP originates from two sources: a salvage pathway and a de novo synthesis pathway that depends on two enzymes, the CTP synthases (or synthetases) 1 and 2 (CTPS1 with CTPS2); the respective roles of these two enzymes are not known 5 , 6 , 7 . CTP synthase activity is a potentially important step for DNA synthesis in lymphocytes 8 , 9 . Here we report the identification of a loss-of-function homozygous mutation (rs145092287) in CTPS1 in humans that causes a novel and life-threatening immunodeficiency, characterized by an impaired capacity of activated T and B cells to proliferate in response to antigen receptor-mediated activation. In contrast, proximal and distal T-cell receptor (TCR) signalling events and responses were only weakly affected by the absence of CTPS1. Activated CTPS1-deficient cells had decreased levels of CTP. Normal T-cell proliferation was restored in CTPS1-deficient cells by expressing wild-type CTPS1 or by addition of exogenous CTP or its nucleoside precursor, cytidine. CTPS1 expression was found to be low in resting T cells, but rapidly upregulated following TCR activation. These results highlight a key and specific role of CTPS1 in the immune system by its capacity to sustain the proliferation of activated lymphocytes during the immune response. CTPS1 may therefore represent a therapeutic target of immunosuppressive drugs that could specifically dampen lymphocyte activation.

Inherited CTPS1, CD27, and CD70 deficiencies in humans have revealed key factors of T‐lymphocyte expansion, a critical prerequisite for an efficient immunity to Epstein–Barr virus (EBV) infection. RASGRP1 is a T‐lymphocyte‐specific nucleotide exchange factor known to activate the pathway of MAP kinases (MAPK). A deleterious homozygous mutation in RASGRP1 leading to the loss RASGRP1 expression was identified in two siblings who both developed a persistent EBV infection leading to Hodgkin lymphoma. RASGRP1‐deficient T cells exhibited defective MAPK activation and impaired proliferation that was restored by expression of wild‐type RASGRP1. Similar defects were observed in T cells from healthy individuals when RASGRP1 was downregulated. RASGRP1‐deficient T cells also exhibited decreased CD27‐dependent proliferation toward CD70‐expressing EBV‐transformed B cells, a crucial pathway required for expansion of antigen‐specific T cells during anti‐EBV immunity. Furthermore, RASGRP1‐deficient T cells failed to upregulate CTPS1, an important enzyme involved in DNA synthesis. These results show that RASGRP1 deficiency leads to susceptibility to EBV infection and demonstrate the key role of RASGRP1 at the crossroad of pathways required for the expansion of activated T lymphocytes. Synopsis RASGRP1 deficiency is characterized by a high susceptibility to develop Epstein‐Barr virus (EBV)‐driven B‐cell lymphoproliferative disorders such as B‐cell lymphoma like Hodgkin lymphoma. This is caused by defective expansion of activated T cells required for an efficient immune response to EBV. RASGRP1 is a critical factor of T‐cell proliferation including CD27‐, CD70‐ and CTPS1‐dependent pathways. RASGRP1 is required for expression of genes involved cell proliferation. This study emphasizes that T‐cell expansion is a critical step in immunity to EBV. Graphical Abstract RASGRP1 deficiency is characterized by a high susceptibility to develop Epstein‐Barr virus (EBV)‐driven B‐cell lymphoproliferative disorders such as B‐cell lymphoma like Hodgkin lymphoma. This is caused by defective expansion of activated T cells required for an efficient immune response to EBV.

In chronic granulomatous disease allogeneic haemopoietic stem-cell transplantation (HSCT) in adolescents and young adults and patients with high-risk disease is complicated by graft-failure, graft-versus-host disease (GVHD), and transplant-related mortality. We examined the effect of a reduced-intensity conditioning regimen designed to enhance myeloid engraftment and reduce organ toxicity in these patients. This prospective study was done at 16 centres in ten countries worldwide. Patients aged 0–40 years with chronic granulomatous disease were assessed and enrolled at the discretion of individual centres. Reduced-intensity conditioning consisted of high-dose fludarabine (30 mg/m2 [infants <9 kg 1·2 mg/kg]; one dose per day on days −8 to −3), serotherapy (anti-thymocyte globulin [10 mg/kg, one dose per day on days −4 to −1; or thymoglobuline 2·5 mg/kg, one dose per day on days −5 to −3]; or low-dose alemtuzumab [<1 mg/kg on days −8 to −6]), and low-dose (50–72% of myeloablative dose) or targeted busulfan administration (recommended cumulative area under the curve: 45–65 mg/L × h). Busulfan was administered mainly intravenously and exceptionally orally from days −5 to −3. Intravenous busulfan was dosed according to weight-based recommendations and was administered in most centres (ten) twice daily over 4 h. Unmanipulated bone marrow or peripheral blood stem cells from HLA-matched related-donors or HLA-9/10 or HLA-10/10 matched unrelated-donors were infused. The primary endpoints were overall survival and event-free survival (EFS), probabilities of overall survival and EFS at 2 years, incidence of acute and chronic GVHD, achievement of at least 90% myeloid donor chimerism, and incidence of graft failure after at least 6 months of follow-up. 56 patients (median age 12·7 years; IQR 6·8–17·3) with chronic granulomatous disease were enrolled from June 15, 2003, to Dec 15, 2012. 42 patients (75%) had high-risk features (ie, intractable infections and autoinflammation), 25 (45%) were adolescents and young adults (age 14–39 years). 21 HLA-matched related-donor and 35 HLA-matched unrelated-donor transplants were done. Median time to engraftment was 19 days (IQR 16–22) for neutrophils and 21 days (IQR 16–25) for platelets. At median follow-up of 21 months (IQR 13–35) overall survival was 93% (52 of 56) and EFS was 89% (50 of 56). The 2-year probability of overall survival was 96% (95% CI 86·46–99·09) and of EFS was 91% (79·78–96·17). Graft-failure occurred in 5% (three of 56) of patients. The cumulative incidence of acute GVHD of grade III–IV was 4% (two of 56) and of chronic graft-versus-host disease was 7% (four of 56). Stable (≥90%) myeloid donor chimerism was documented in 52 (93%) surviving patients. This reduced-intensity conditioning regimen is safe and efficacious in high-risk patients with chronic granulomatous disease. None.

Loss of NBEAL2 function leads to grey platelet syndrome (GPS), a bleeding disorder characterized by macro-thrombocytopenia and α-granule-deficient platelets. A proportion of patients with GPS develop autoimmunity through an unknown mechanism, which might be related to the proteins NBEAL2 interacts with, specifically in immune cells. Here we show a comprehensive interactome of NBEAL2 in primary T cells, based on mass spectrometry identification of altogether 74 protein association partners. These include LRBA, a member of the same BEACH domain family as NBEAL2, recessive mutations of which cause autoimmunity and lymphocytic infiltration through defective CTLA-4 trafficking. Investigating the potential association between NBEAL2 and CTLA-4 signalling suggested by the mass spectrometry results, we confirm by co-immunoprecipitation that CTLA-4 and NBEAL2 interact with each other. Interestingly, NBEAL2 deficiency leads to low CTLA-4 expression in patient-derived effector T cells, while their regulatory T cells appear unaffected. Knocking-down NBEAL2 in healthy primary T cells recapitulates the low CTLA-4 expression observed in the T cells of GPS patients. Our results thus show that NBEAL2 is involved in the regulation of CTLA-4 expression in conventional T cells and provide a rationale for considering CTLA-4-immunoglobulin therapy in patients with GPS and autoimmune disease. NBEAL2 loss of function mutations lead to grey platelet syndrome, a condition characterised by α-granule-deficient platelets and, in a proportion of cases, by autoimmunity. Here authors show that NBEAL2 physically interacts with CTLA-4 in human T cells, and NBEAL2 deficiency leads to reduced CTLA-4 surface expression in effector T cells, but not regulatory T cells, thus tipping the balance towards autoimmunity.