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Intra-arterial transplantation of mesoangioblasts proved safe and partially efficacious in preclinical models of muscular dystrophy. We now report the first-in-human, exploratory, non-randomized open-label phase I-IIa clinical trial of intra-arterial HLA-matched donor cell transplantation in 5 Duchenne patients. We administered escalating doses of donor-derived mesoangioblasts in limb arteries under immunosuppressive therapy (tacrolimus). Four consecutive infusions were performed at 2-month intervals, preceded and followed by clinical, laboratory, and muscular MRI analyses. Two months after the last infusion, a muscle biopsy was performed. Safety was the primary endpoint. The study was relatively safe: One patient developed a thalamic stroke with no clinical consequences and whose correlation with mesoangioblast infusion remained unclear. MRI documented the progression of the disease in 4/5 patients. Functional measures were transiently stabilized in 2/3 ambulant patients, but no functional improvements were observed. Low level of donor DNA was detected in muscle biopsies of 4/5 patients and donor-derived dystrophin in 1. Intra-arterial transplantation of donor mesoangioblasts in human proved to be feasible and relatively safe. Future implementation of the protocol, together with a younger age of patients, will be needed to approach efficacy.

In the attempt to understand the origin of autoantibody (AAb) production in patients with and at-risk for T1D, multiple studies have analyzed and reported alterations in follicular helper T cells (Tfh) in presymptomatic AAb-positive subjects and patients with T1D. Yet, it is still not clear whether the regulatory counterpart of Tfh cells, represented by follicular regulatory T cells (Tfr), is similarly altered. To address this question, we performed analyses in peripheral blood, spleen and pancreatic lymph nodes (PLN) of organ donor subjects with T1D. Blood analyses were also performed in living AAb-negative and -positive subjects. While negligible differences in the frequency and phenotype of blood Tfr cells were observed between T1D, AAb-negative and AAb-positive adult subjects, the frequency of Tfr cells was significantly reduced in spleen and PLN of T1D as compared to non-diabetic controls. Furthermore, adoptive transfer of Tfr cells delayed disease development in a mouse model of T1D, a finding that could indicate that Tfr cells play an important role in peripheral tolerance and regulation of autoreactive Tfh cells. Together, our findings provide evidence of Tfr cell alterations within disease-relevant tissues in patients with T1D suggesting a role for Tfr cells in defective humoral tolerance and disease pathogenesis.

Patients with Down syndrome (DS) are characterized by increased susceptibility to autoimmunity and respiratory tract infections that are suggestive of humoral immunity impairment. Here, we sought to determine the follicular helper (Tfh) and follicular regulatory (Tfr) T cell profile in the blood of children with DS. Blood was collected from 24 children with DS, nine of which had autoimmune diseases. Children with DS showed skewed Tfh differentiation towards the CXCR3+ phenotype: Tfh1 and Tfh1/17 subsets were increased, while Tfh2 and Tfh17 subsets were reduced. While no differences in the percentage of Tfr cells were seen, the ratio of Tfh1 and CXCR3+PD-1+ subsets to Tfr cells was significantly increased in the affected children. The excessive polarization towards a CXCR3+ phenotype in children with DS suggests that re-calibration of Tfh subset skewing could potentially offer new therapeutic opportunities for these patients. Patients with Down syndrome (DS) are characterized by increased susceptibility to autoimmunity and respiratory tract infections that are suggestive of humoral immunity impairment. Here, we sought to determine the follicular helper (Tfh) and follicular regulatory (Tfr) T cell profile in the blood of children with DS. Blood was collected from 24 children with DS, nine of which had autoimmune diseases. Children with DS showed skewed Tfh differentiation towards the CXCR3+ phenotype: Tfh1 and Tfh1/17 subsets were increased, while Tfh2 and Tfh17 subsets were reduced. While no differences in the percentage of Tfr cells were seen, the ratio of Tfh1 and CXCR3+PD-1+ subsets to Tfr cells was significantly increased in the affected children. The excessive polarization towards a CXCR3+ phenotype in children with DS suggests that re-calibration of Tfh subset skewing could potentially offer new therapeutic opportunities for these patients.

We present, to our knowledge, the first case of immunosuppressive treatment (IST) application in a 12-year old child with arrhythmogenic inflammatory cardiomyopathy (AIC) resulting from the overlap between autoimmune myocarditis and primary arrhythmogenic cardiomyopathy (AC). Indication to off-lable IST was compelling, because of recurrent durg-refractory ventricular arrhythmias. We show that IST was feasible, safe, and effective on multiple clinical endpoints, including symptoms, ventricular arrhythmia recurrences, and T-troponin release. Remarkably, all diagnostic and therapeutic strategies were worked out by a dedicated multidisciplinary team, including specialized pediatric immunologists.

Gene therapy has become promising in many fields of medicine, as a single treatment could allow long-lasting and curative benefits. New medicines based on cell gene correction are expected to occur in upcoming years and will be hopefully part of the therapeutic armamentarium for inherited and acquired diseases. Issues related to the costs of these new therapies and access to care for all patients, and procedures and expertise needed to facilitate their application worldwide require to be addressed, together with long-term safety and efficacy monitoring.

Loss of adenosine deaminase activity leads to severe combined immunodeficiency (ADA-SCID); production and function of T, B, and natural killer (NK) cells are impaired. Gene therapy (GT) with an autologous CD34+-enriched cell fraction that contains CD34+ cells transduced with a retroviral vector encoding the human ADA cDNA sequence leads to immune reconstitution in most patients. Here, we report short- and medium-term safety analyses from 18 patients enrolled as part of single-arm, open-label studies or compassionate use programs. Survival was 100% with a median of 6.9 years follow-up (range, 2.3 to 13.4 years). Adverse events were mostly grade 1 or grade 2 and were reported by all 18 patients following GT. Thirty-nine serious adverse events (SAEs) were reported by 15 of 18 patients; no SAEs were considered related to GT. The most common adverse events reported post-GT include upper respiratory tract infection, gastroenteritis, rhinitis, bronchitis, oral candidiasis, cough, neutropenia, diarrhea, and pyrexia. Incidence rates for all of these events were highest during pre-treatment, treatment, and/or 3-month follow-up and then declined over medium-term follow-up. GT did not impact the incidence of neurologic/hearing impairments. No event indicative of leukemic transformation was reported., In 2016 the gene therapy, Strimvelis was approved for the treatment of patients with ADA-SCID for whom there is no suitable bone marrow donor. In this issue of Molecular Therapy, Aiuti et al. provide detailed safety data for treated patients. All patients are alive and without evidence of leukemic transformation.

Severe combined immunodeficiencies (SCIDs) are a group of inborn errors of the immune system, usually associated with severe or life-threatening infections. Due to the variability of clinical phenotypes, the diagnostic complexity and the heterogeneity of the genetic basis, they are often difficult to recognize, leading to a significant diagnostic delay (DD). Aim of this study is to define presenting signs and natural history of SCID in a large cohort of patients, prior to hematopoietic stem cell or gene therapies. To this purpose, we conducted a 30-year retro-prospective multicenter study within the Italian Primary Immunodeficiency Network. One hundred eleven patients, diagnosed as typical or atypical SCID according to the European Society for Immune Deficiencies criteria, were included. Patients were subsequently classified based on the genetic alteration, pathogenic mechanism and immunological classification. A positive relationship between the age at onset and the DD was found. SCID patients with later onset were identified only in the last decade of observation. Syndromic SCIDs represented 28% of the cohort. Eight percent of the subjects were diagnosed in Intensive Care Units. Fifty-three percent had an atypical phenotype and most of them exhibited a discordant genotype-immunophenotype. Pre-treatment mortality was higher in atypical and syndromic patients. Our study broadens the knowledge of clinical and laboratory manifestations and genotype/phenotype correlation in patients with SCID and may facilitate the diagnosis of both typical and atypical forms of the disease in countries where newborn screening programs have not yet been implemented.

Adenosine deaminase 2 (ADA2) deficiency is an auto-inflammatory disease due to mutations in cat eye syndrome chromosome region candidate 1 (CECR1) gene, currently named ADA2. The disease has a wide clinical spectrum encompassing early-onset vasculopathy (targeting skin, gut and central nervous system), recurrent fever, immunodeficiency and bone marrow dysfunction. Different therapeutic options have been proposed in literature, but only steroids and anti-cytokine monoclonal antibodies (such as tumor necrosis factor inhibitor) proved to be effective. If a suitable donor is available, hematopoietic stem cell transplantation (HSCT) could be curative. Here we describe a case of ADA2 deficiency in a 4-year-old Caucasian girl. The patient was initially classified as autoimmune neutropenia and then she evolved toward an autoimmune lymphoproliferative syndrome (ALPS)-like phenotype. The diagnosis of ALPS became uncertain due to atypical clinical features and normal FAS-induced apoptosis test. She was treated with G-CSF first and subsequently with immunosuppressive drugs without improvement. Only HSCT from a 9/10 HLA-matched unrelated donor, following myeloablative conditioning, completely solved the clinical signs related to ADA2 deficiency. Early diagnosis in cases presenting with hematological manifestations, rather than classical vasculopathy, allows the patients to promptly undergo HSCT and avoid more severe evolution. Finally, in similar cases highly suspicious for genetic disease, it is desirable to obtain molecular diagnosis before performing HSCT, since it can influence the transplant procedure. However, if HSCT has to be performed without delay for clinical indication, related donors should be excluded to avoid the risk of relapse or partial benefit due to a hereditary genetic defect.

Objective The aim of this study was to evaluate the usefulness of magnetic resonance imaging (MRI) in detecting the progression of Duchenne muscular dystrophy (DMD) by quantification of fat infiltration (FI) and muscle volume index (MVI, a residual-to-total muscle volume ratio). Methods Twenty-six patients (baseline age: 5–12 years) with genetically proven DMD were longitudinally analyzed with lower limb 3T MRI, force measurements, and functional tests (Gowers, 10-m time, North Star Ambulatory Assessment, 6-min walking test). Five age-matched controls were also examined, with a total of 85 MRI studies. Semiquantitative (scores) and quantitative MRI (qMRI) analyses (signal intensity ratio – SIR, lower limb MVI, and individual muscle MVI) were carried out. Permutation and regression analyses according to both age and functional test-outcomes were calculated. Age-related quantitative reference curves of SIRs and MVIs were generated. Results FI was present on glutei and adductor magnus in all patients since the age of 5, with a proximal-to-distal progression and selective sparing of sartorius and gracilis. Patients' qMRI measures were significantly different from controls' and among age classes. qMRI were more sensitive than force measurements and functional tests in assessing disease progression, allowing quantification also after loss of ambulation. Age-related curves with percentile values were calculated for SIRs and MVIs, to provide a reference background for future experimental therapy trials. SIRs and MVIs significantly correlated with all clinical measures, and could reliably predict functional outcomes and loss of ambulation. Interpretations qMRI-based indexes are sensitive measures that can track the progression of DMD and represent a valuable tool for follow-up and clinical studies.

Mutations in genes that control class switch recombination and somatic hypermutation during the germinal center (GC) response can cause diverse immune dysfunctions. In particular, mutations in CD40LG, CD40, AICDA, or UNG cause hyper-IgM (HIGM) syndrome, a heterogeneous group of primary immunodeficiencies. Follicular helper (Tfh) and follicular regulatory (Tfr) T cells play a key role in the formation and regulation of GCs, but their role in HIGM pathogenesis is still limited. Here, we found that compared to CD40 ligand (CD40L)- and activation-induced cytidine deaminase (AICDA)-deficient patients, circulating Tfh and Tfr cells were severely compromised in terms of frequency and activation phenotype in a child with CD40 deficiency. These findings offer useful insight for human Tfh biology, with potential implications for understanding the molecular basis of HIGM syndrome caused by mutations in CD40.

Patients with inborn error diseases can be candidates for autologous haematopoietic stem cells (HSC) gene therapies (GT) but may require negative viral screening, including Hepatitis C (HCV), to allow HSC manipulation in Good Manufacturing Practices areas. In case of HCV positivity, patients might be excluded from life-saving treatments. As HCV antibodies could be negative in young infant immunodeficient patients due to their immature/impaired immune system, or positive due to maternal-fetal antibody transmission, the risk is usually also evaluated on the basis of the HCV-RNA. This article is protected by copyright. All rights reserved.

Adenosine deaminase-deficient severe combined immunodeficiency disease (ADA-SCID) is a primary immune deficiency characterized by mutations in the ADA gene resulting in accumulation of toxic compounds affecting multiple districts. Hematopoietic stem cell transplantation (HSCT) from a matched donor and hematopoietic stem cell gene therapy are the preferred options for definitive treatment. Enzyme replacement therapy (ERT) is used to manage the disease in the short term, while a decreased efficacy is reported in the medium-long term. To date, eight cases of lymphomas have been described in ADA-SCID patients. Here we report the first case of plasmablastic lymphoma occurring in a young adult with ADA-SCID on long-term ERT, which turned out to be Epstein-Barr virus associated. The patient previously received infusions of genetically modified T cells. A cumulative analysis of the eight published cases of lymphoma from 1992 to date, and the case here described, reveals a high mortality (89%). The most common form is diffuse large B-cell lymphoma, which predominantly occurs in extra nodal sites. Seven cases occurred in patients on ERT and two after haploidentical HSCT. The significant incidence of immunodeficiency-associated lymphoproliferative disorders and poor survival of patients developing this complication highlight the priority in finding a prompt curative treatment for ADA-SCID.

The immune system is a complex system able to recognize a wide variety of host agents, through different biological processes. For example, controlled changes in the redox state are able to start different pathways in immune cells and are involved in the killing of microbes. The generation and release of ROS in the form of an “oxidative burst” represent the pivotal mechanism by which phagocytic cells are able to destroy pathogens. On the other hand, impaired oxidative balance is also implicated in the pathogenesis of inflammatory complications, which may affect the function of many body systems. NADPH oxidase (NOX) plays a pivotal role in the production of ROS, and the defect of its different subunits leads to the development of chronic granulomatous disease (CGD). The defect of the different NOX subunits in CGD affects different organs. In this context, this review will be focused on the description of the effect of NOX2 deficiency in different body systems. Moreover, we will also focus our attention on the novel insight in the pathogenesis of immunodeficiency and inflammation-related manifestations and on the protective role of NOX2 deficiency against the development of atherosclerosis.

Summary Hematopoietic stem/progenitor cells (HSPCs) are capable of supporting the lifelong production of blood cells exerting a wide spectrum of functions. Lentiviral vector HSPC gene therapy generates a human hematopoietic system stably marked at the clonal level by vector integration sites (ISs). Using IS analysis, we longitudinally tracked >89,000 clones from 15 distinct bone marrow and peripheral blood lineages purified up to 4 years after transplant in four Wiskott-Aldrich syndrome patients treated with HSPC gene therapy. We measured at the clonal level repopulating waves, populations' sizes and dynamics, activity of distinct HSPC subtypes, contribution of various progenitor classes during the early and late post-transplant phases, and hierarchical relationships among lineages. We discovered that in-vitro-manipulated HSPCs retain the ability to return to latency after transplant and can be physiologically reactivated, sustaining a stable hematopoietic output. This study constitutes in vivo comprehensive tracking in humans of hematopoietic clonal dynamics during the early and late post-transplant phases., Graphical Abstract, Highlights • Hematopoietic reconstitution occurs in two distinct clonal waves • A few thousand HSPC clones stably sustain multilineage blood cell production • Steady-state hematopoiesis after transplant is maintained by both HSCs and MPPs • Natural killer clones have closer relationships to myeloid cells than to lymphoid cells, Biasco et al. report a clonal tracking study on the dynamics and nature of hematopoietic reconstitution in humans after transplant. Using integration sites as molecular tags, they measured, in gene therapy patients, repopulating waves, population size and dynamics, activity of progenitor subtypes during the early and late post-transplant phases, and hierarchical relationships among lineages.

Summary Background Metachromatic leukodystrophy (a deficiency of arylsulfatase A [ARSA]) is a fatal demyelinating lysosomal disease with no approved treatment. We aimed to assess the long-term outcomes in a cohort of patients with early-onset metachromatic leukodystrophy who underwent haemopoietic stem-cell gene therapy (HSC-GT). Methods This is an ad-hoc analysis of data from an ongoing, non-randomised, open-label, single-arm phase 1/2 trial, in which we enrolled patients with a molecular and biochemical diagnosis of metachromatic leukodystrophy (presymptomatic late-infantile or early-juvenile disease or early-symptomatic early-juvenile disease) at the Paediatric Clinical Research Unit, Ospedale San Raffaele, in Milan. Trial participants received HSC-GT, which consisted of the infusion of autologous HSCs transduced with a lentiviral vector encoding ARSA cDNA, after exposure-targeted busulfan conditioning. The primary endpoints of the trial are safety (toxicity, absence of engraftment failure or delayed haematological reconstitution, and safety of lentiviral vector-tranduced cell infusion) and efficacy (improvement in Gross Motor Function Measure [GMFM] score relative to untreated historical controls, and ARSA activity, 24 months post-treatment) of HSC-GT. For this ad-hoc analysis, we assessed safety and efficacy outcomes in all patients who had received treatment and been followed up for at least 18 months post-treatment on June 1, 2015. This trial is registered with ClinicalTrials.gov, number NCT01560182. Findings Between April, 2010, and February, 2013, we had enrolled nine children with a diagnosis of early-onset disease (six had late-infantile disease, two had early-juvenile disease, and one had early-onset disease that could not be definitively classified). At the time of analysis all children had survived, with a median follow-up of 36 months (range 18–54). The most commonly reported adverse events were cytopenia (reported in all patients) and mucositis of different grades of severity (in five of nine patients [grade 3 in four of five patients]). No serious adverse events related to the medicinal product were reported. Stable, sustained engraftment of gene-corrected HSCs was observed (a median of 60·4% [range 14·0–95·6] lentiviral vector-positive colony-forming cells across follow-up) and the engraftment level was stable during follow-up; engraftment determinants included the duration of absolute neutropenia and the vector copy number of the medicinal product. A progressive reconstitution of ARSA activity in circulating haemopoietic cells and in the cerebrospinal fluid was documented in all patients in association with a reduction of the storage material in peripheral nerve samples in six of seven patients. Eight patients, seven of whom received treatment when presymptomatic, had prevention of disease onset or halted disease progression as per clinical and instrumental assessment, compared with historical untreated control patients with early-onset disease. GMFM scores for six patients up to the last follow-up showed that gross motor performance was similar to that of normally developing children. The extent of benefit appeared to be influenced by the interval between HSC-GT and the expected time of disease onset. Treatment resulted in protection from CNS demyelination in eight patients and, in at least three patients, amelioration of peripheral nervous system abnormalities, with signs of remyelination at both sites. Interpretation Our ad-hoc findings provide preliminary evidence of safety and therapeutic benefit of HSC-GT in patients with early-onset metachromatic leukodystrophy who received treatment in the presymptomatic or very early-symptomatic stage. The results of this trial will be reported when all 20 patients have achieved 3 years of follow-up. Funding Italian Telethon Foundation and GlaxoSmithKline.

Next-Generation Gene Therapy Few disciplines in contemporary clinical research have experienced the high expectations directed at the gene therapy field. However, gene therapy has been challenging to translate to the clinic, often because the therapeutic gene is expressed at insufficient levels in the patient or because the gene delivery vector integrates near protooncogenes, which can cause leukemia (see the Perspective by Verma ). Biffi et al. ( 1233158 , published online 11 July) and Aiuti et al. ( 1233151 ; published online 11 July) report progress on both fronts in gene therapy trials of three patients with metachromatic leukodystrophy (MLD), a neurodegenerative disorder, and three patients with Wiskott-Aldrich syndrome (WAS), an immunodeficiency disorder. Optimized lentiviral vectors were used to introduce functional MLD or WAS genes into the patients' hematopoietic stem cells (HSCs) ex vivo, and the transduced cells were then infused back into the patients, who were then monitored for up to 2 years. In both trials, the patients showed stable engraftment of the transduced HSC and high expression levels of functional MLD or WAS genes. Encouragingly, there was no evidence of lentiviral vector integration near proto-oncogenes, and the gene therapy treatment halted disease progression in most patients. A longer follow-up period will be needed to further validate efficacy and safety.