Maria Pia Cicalese

Metachromatic leukodystrophy (MLD) is an inherited lysosomal storage disease caused by arylsulfatase A (ARSA) deficiency. Patients with MLD exhibit progressive motor and cognitive impairment and die within a few years of symptom onset. We used a lentiviral vector to transfer a functional ARSA gene into hematopoietic stem cells (HSCs) from three presymptomatic patients who showed genetic, biochemical, and neurophysiological evidence of late infantile MLD. After reinfusion of the gene-corrected HSCs, the patients showed extensive and stable ARSA gene replacement, which led to high enzyme expression throughout hematopoietic lineages and in cerebrospinal fluid. Analyses of vector integrations revealed no evidence of aberrant clonal behavior. The disease did not manifest or progress in the three patients 7 to 21 months beyond the predicted age of symptom onset. These findings indicate that extensive genetic engineering of human hematopoiesis can be achieved with lentiviral vectors and that this approach may offer therapeutic benefit for MLD patients.

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.

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).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.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.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.Italian Telethon Foundation and GlaxoSmithKline.

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.

Cell-surface CD25 expression is critical for maintaining immune function and homeostasis. As in few reported cases, CD25 deficiency manifests with severe autoimmune enteritis and viral infections. To dissect the underlying immunological mechanisms driving these symptoms, we analyzed the regulatory and effector T cell functions in a CD25 deficient patient harboring a novel IL2RA mutation. Pronounced lymphoproliferation, mainly of the CD8+ T cells, was detected together with an increase in T cell activation markers and elevated serum cytokines. However, Ag-specific responses were impaired in vivo and in vitro. Activated CD8+STAT5+ T cells with lytic potential infiltrated the skin, even though FOXP3+ Tregs were present and maintained a higher capacity to respond to IL-2 compared to other T-cell subsets. Thus, the complex pathogenesis of CD25 deficiency provides invaluable insight into the role of IL2/IL-2RA-dependent regulation in autoimmunity and inflammatory diseases.

Key Points Survival was 100% for 18 patients with ADA-SCID treated with genetically modified CD34+ cells (2.3-13.4 years follow up; median, 6.9 years). Long-term engraftment, immune reconstitution, and fewer severe infections were observed in 15 out of 18 patients without leukemic transformation.

<h2>Summary</h2><h3>Background</h3> Wiskott-Aldrich syndrome is a rare, life-threatening, X-linked primary immunodeficiency characterised by microthrombocytopenia, infections, eczema, autoimmunity, and malignant disease. Lentiviral vector-mediated haemopoietic stem/progenitor cell (HSPC) gene therapy is a potentially curative treatment that represents an alternative to allogeneic HSPC transplantation. Here, we report safety and efficacy data from an interim analysis of patients with severe Wiskott-Aldrich syndrome who received lentiviral vector-derived gene therapy. <h3>Methods</h3> We did a non-randomised, open-label, phase 1/2 clinical study in paediatric patients with severe Wiskott-Aldrich syndrome, defined by either <i>WAS</i> gene mutation or absent Wiskott-Aldrich syndrome protein (WASP) expression or a Zhu clinical score of 3 or higher. We included patients who had no HLA-identical sibling donor available or, for children younger than 5 years of age, no suitable 10/10 matched unrelated donor or 6/6 unrelated cord blood donor. After treatment with rituximab and a reduced-intensity conditioning regimen of busulfan and fludarabine, patients received one intravenous infusion of autologous CD34+ cells genetically modified with a lentiviral vector encoding for human <i>WAS</i> cDNA. The primary safety endpoints were safety of the conditioning regimen and safety of lentiviral gene transfer into HSPCs. The primary efficacy endpoints were overall survival, sustained engraftment of genetically corrected HSPCs, expression of vector-derived WASP, improved T-cell function, antigen-specific responses to vaccinations, and improved platelet count and mean platelet volume normalisation. This interim analysis was done when the first six patients treated had completed at least 3 years of follow-up. The planned analyses are presented for the intention-to-treat population. This trial is registered with ClinicalTrials.gov (number NCT01515462) and EudraCT (number 2009-017346-32). <h3>Findings</h3> Between April 20, 2010, and Feb 26, 2015, nine patients (all male) were enrolled of whom one was excluded after screening; the age range of the eight treated children was 1·1–12·4 years. At the time of the interim analysis (data cutoff April 29, 2016), median follow-up was 3·6 years (range 0·5–5·6). Overall survival was 100%. Engraftment of genetically corrected HSPCs was successful and sustained in all patients. The fraction of WASP-positive lymphocytes increased from a median of 3·9% (range 1·8–35·6) before gene therapy to 66·7% (55·7–98·6) at 12 months after gene therapy, whereas WASP-positive platelets increased from 19·1% (range 4·1–31·0) to 76·6% (53·1–98·4). Improvement of immune function was shown by normalisation of in-vitro T-cell function and successful discontinuation of immunoglobulin supplementation in seven patients with follow-up longer than 1 year, followed by positive antigen-specific response to vaccination. Severe infections fell from 2·38 (95% CI 1·44–3·72) per patient-year of observation (PYO) in the year before gene therapy to 0·31 (0·04–1·11) per PYO in the second year after gene therapy and 0·17 (0·00–0·93) per PYO in the third year after gene therapy. Before gene therapy, platelet counts were lower than 20 × 10⁹ per L in seven of eight patients. At the last follow-up visit, the platelet count had increased to 20–50 × 10⁹ per L in one patient, 50–100 × 10⁹ per L in five patients, and more than 100 × 10⁹ per L in two patients, which resulted in independence from platelet transfusions and absence of severe bleeding events. 27 serious adverse events in six patients occurred after gene therapy, 23 (85%) of which were infectious (pyrexia [five events in three patients], device-related infections, including one case of sepsis [four events in three patients], and gastroenteritis, including one case due to rotavirus [three events in two patients]); these occurred mainly in the first 6 months of follow-up. No adverse reactions to the investigational drug product and no abnormal clonal proliferation or leukaemia were reported after gene therapy. <h3>Interpretation</h3> Data from this study show that gene therapy provides a valuable treatment option for patients with severe Wiskott-Aldrich syndrome, particularly for those who do not have a suitable HSPC donor available. <h3>Funding</h3> Italian Telethon Foundation, GlaxoSmithKline, and Orchard Therapeutics.

Effective treatment for metachromatic leukodystrophy (MLD) remains a substantial unmet medical need. In this study we investigated the safety and efficacy of atidarsagene autotemcel (arsa-cel) in patients with MLD.This study is an integrated analysis of results from a prospective, non-randomised, phase 1/2 clinical study and expanded-access frameworks. 29 paediatric patients with pre-symptomatic or early-symptomatic early-onset MLD with biochemical and molecular confirmation of diagnosis were treated with arsa-cel, a gene therapy containing an autologous haematopoietic stem and progenitor cell (HSPC) population transduced ex vivo with a lentiviral vector encoding human arylsulfatase A (ARSA) cDNA, and compared with an untreated natural history (NHx) cohort of 31 patients with early-onset MLD, matched by age and disease subtype. Patients were treated and followed up at Ospedale San Raffaele, Milan, Italy. The coprimary efficacy endpoints were an improvement of more than 10% in total gross motor function measure score at 2 years after treatment in treated patients compared with controls, and change from baseline of total peripheral blood mononuclear cell (PBMC) ARSA activity at 2 years after treatment compared with values before treatment. This phase 1/2 study is registered with ClinicalTrials.gov, NCT01560182.At the time of analyses, 26 patients treated with arsa-cel were alive with median follow-up of 3·16 years (range 0·64-7·51). Two patients died due to disease progression and one due to a sudden event deemed unlikely to be related to treatment. After busulfan conditioning, all arsa-cel treated patients showed sustained multilineage engraftment of genetically modified HSPCs. ARSA activity in PBMCs was significantly increased above baseline 2 years after treatment by a mean 18·7-fold (95% CI 8·3-42·2; p<0·0001) in patients with the late-infantile variant and 5·7-fold (2·6-12·4; p<0·0001) in patients with the early-juvenile variant. Mean differences in total scores for gross motor function measure between treated patients and age-matched and disease subtype-matched NHx patients 2 years after treatment were significant for both patients with late-infantile MLD (66% [95% CI 48·9-82·3]) and early-juvenile MLD (42% [12·3-71·8]). Most treated patients progressively acquired motor skills within the predicted range of healthy children or had stabilised motor performance (maintaining the ability to walk). Further, most displayed normal cognitive development and prevention or delay of central and peripheral demyelination and brain atrophy throughout follow-up; treatment benefits were particularly apparent in patients treated before symptom onset. The infusion was well tolerated and there was no evidence of abnormal clonal proliferation or replication-competent lentivirus. All patients had at least one grade 3 or higher adverse event; most were related to conditioning or to background disease. The only adverse event related to arsa-cel was the transient development of anti-ARSA antibodies in four patients, which did not affect clinical outcomes.Treatment with arsa-cel resulted in sustained, clinically relevant benefits in children with early-onset MLD by preserving cognitive function and motor development in most patients, and slowing demyelination and brain atrophy.Orchard Therapeutics, Fondazione Telethon, and GlaxoSmithKline.

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.

Abstract ARPC1B is a key factor for the assembly and maintenance of the ARP2/3 complex that is involved in actin branching from an existing filament. Germline biallelic mutations in ARPC1B have been recently described in 6 patients with clinical features of combined immunodeficiency (CID), whose neutrophils and platelets but not T lymphocytes were studied. We hypothesized that ARPC1B deficiency may also lead to cytoskeleton and functional defects in T cells. We have identified biallelic mutations in ARPC1B in 6 unrelated patients with early onset disease characterized by severe infections, autoimmune manifestations, and thrombocytopenia. Immunological features included T-cell lymphopenia, low numbers of naïve T cells, and hyper–immunoglobulin E. Alteration in ARPC1B protein structure led to absent/low expression by flow cytometry and confocal microscopy. This molecular defect was associated with the inability of patient-derived T cells to extend an actin-rich lamellipodia upon T-cell receptor (TCR) stimulation and to assemble an immunological synapse. ARPC1B-deficient T cells additionally displayed impaired TCR-mediated proliferation and SDF1-α−directed migration. Gene transfer of ARPC1B in patients’ T cells using a lentiviral vector restored both ARPC1B expression and T-cell proliferation in vitro. In 2 of the patients, in vivo somatic reversion restored ARPC1B expression in a fraction of lymphocytes and was associated with a skewed TCR repertoire. In 1 revertant patient, memory CD8+ T cells expressing normal levels of ARPC1B displayed improved T-cell migration. Inherited ARPC1B deficiency therefore alters T-cell cytoskeletal dynamics and functions, contributing to the clinical features of CID.

Recently, a novel syndrome of combined immunodeficiency, allergy, and “auto”inflammation caused by mutations in the ARPC1B gene has been reported.1Kuijpers T.W. Tool A.T.J. van der Bijl I. de Boer M. van Houdt M. de Cuyper I.M. et al.Combined immunodeficiency with severe inflammation and allergy caused by ARPC1B deficiency.J Allergy Clin Immunol. 2017; 140: 273-277.e10Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, 2Kahr W.H. Pluthero F.G. Elkadri A. Warner N. Drobac M. Chen C.H. et al.Loss of the Arp2/3 complex component ARPC1B causes platelet abnormalities and predisposes to inflammatory disease.Nat Commun. 2017; 8: 14816Crossref PubMed Scopus (133) Google Scholar, 3Somech R. Lev A. Lee Y.N. Simon A.J. Barel O. Schiby G. et al.Disruption of thrombocyte and T lymphocyte development by a mutation in ARPC1B.J Immunol. 2017; 199: 4036-4045Crossref PubMed Scopus (47) Google Scholar, 4Brigida I. Zoccolillo M. Cicalese M.P. Pfajfer L. Barzaghi F. Scala S. et al.T cell defects in patients with ARPC1B germline mutations account for their combined immunodeficiency.Blood. 2018; 132: 2362-2374PubMed Google Scholar Analysis of patient-derived hematopoietic cells has shown a defect in actin polymerization, which resulted in a wide range of clinical manifestations and immunologic-hematologic features. We report on the immunologic, cellular, and molecular phenotypes in 14 patients with biallelic ARPC1B mutations and variable clinical presentations (Fig 1, A and B; see Fig E1, A, and Table E1 in this article's Online Repository at www.jacionline.org; for case descriptions, see this article's Online Repository at www.jacionline.org), helping to delineate the broad spectrum of this novel disease and presenting unreported insights into cell-intrinsic defects involving regulatory T (Treg) cells and natural killer (NK) cells, potential players in the immune dysregulation and susceptibility to viral infections observed in these patients. The disease-causing variants are diverse and scattered throughout the gene (Fig E1, B; Table E1). Patient (P) 4, P12, and P14 have Nepalese ancestry and share the same variant, suggesting a founder mutation. In all patient samples tested, ARPC1B protein was undetectable by Western blotting and we identified an increased—although variable—expression of the ARPC1A isoform (see Fig E2 in this article's Online Repository at www.jacionline.org). The disease is characterized by a very early clinical onset (mean, 2 months; range, 1-6 months) (see Table E2 in this article's Online Repository at www.jacionline.org). Presenting symptoms included skin rash, infections, and gastrointestinal bleeding. Most patients (79%) (Fig 1, A) suffered from recurrent or severe bleeding episodes, most frequently represented by enterorrhagia. Platelet counts were reduced (see Table E3 in this article's Online Repository at www.jacionline.org), with normal volume in most cases. An increased rate and/or abnormal severity of respiratory tract infections (including pneumonia, bronchopneumonia, and bronchiolitis), and skin infections (including abscesses, erysipelas, extensive warts [Fig 1, B], and molluscum contagiosum), were observed in 71% and 50% of the patients, respectively, whereas severe, protracted bacterial gastrointestinal infections have been diagnosed in a minority of individuals (see Tables E2 and E4 in this article's Online Repository at www.jacionline.org). As summarized in Fig 1, A, and Table E5 (in this article's Online Repository at www.jacionline.org), common manifestations of immune dysregulation included moderate-to-severe eczema, which was observed in 57% of cases (Fig 1, C), associated with food allergy (anaphylactic reactions) and asthma. Cutaneous vasculitis was noted in 69% of patients, presenting as a maculopapular rash, erythema nodosum, or vasculitic purpura (Fig 1, D). In all cases investigated with a skin biopsy, leukocytoclastic vasculitis was diagnosed. Arthritis was present in 23% of patients. One child presented with 23 episodes of macrophage activation syndrome, followed by the appearance of enlarged lymph nodes, splenomegaly, and episodes of sialadenitis (Fig 1, E). Autoantibodies were absent in most patients. Growth failure was noted in all patients (Table E2), with growth hormone tests found to be impaired when performed (P2 and P3), compatible with a partial growth hormone deficiency, and no catch-up growth after hematopoietic stem cell transplantation (HSCT) (P2, P3, and P6). Immunophenotyping showed an increased number of circulating CD19+ B cells, a reduced absolute count of CD3+CD4+ and CD3+CD8+ T cells, and in 1 patient an expansion of γδ T cells, possibly driven by cytomegalovirus infection (Fig 2, A; see Table E6 in this article's Online Repository at www.jacionline.org). In vivo immunoglobulin levels were abnormal, with markedly increased IgA and IgE in almost all cases (Table E6). In contrast to Wiskott-Aldrich Syndrome or DOCK8 deficiency,5Su H.C. Jing H. Angelus P. Freeman A.F. Insights into immunity from clinical and basic science studies of DOCK8 immunodeficiency syndrome.Immunol Rev. 2019; 287: 9-19Crossref PubMed Scopus (42) Google Scholar the humoral response to polysaccharide vaccine was normal in most cases (see Table E7 in this article's Online Repository at www.jacionline.org). The T-cell subset distribution was abnormal, with low percentages of naive CD4+ and CD8+ T cells (see Fig E3, A and B, in this article's Online Repository at www.jacionline.org). In vitro T-cell proliferation in response to combination of anti-CD3+ + anti-CD28+, cytokines (IL-15, IL-2), and mitogens was largely normal, whereas response to low-dose CD3 and antigens was defective in some cases (Table E7). The TCR repertoire was persistently oligoclonal in 2 of 7 tested patients and transiently oligoclonal in 1 patient (Fig E3, C; Table E6). The proportion and phenotype of Treg cells was variable (Fig 2, B; see Fig E4, A, in this article's Online Repository at www.jacionline.org); however, in vitro expanded Treg cells showed decreased expression of all Treg-cell markers including FOXP3, Helios, CD25, and CTLA-4 (Fig 2, C; Fig E4, B). Treg-cell suppressor activity against CD4+ (Fig 2, D and E) and CD8+ T allogeneic responder cells was defective (Fig E4, C). An increase in the CD3-CD56brightCD16neg NK-cell subpopulation (27% in P2, 24% in P3, and 21% in P4) was noted when tested (P2, P3, and P4) (see Fig E5 in this article's Online Repository at www.jacionline.org; data not shown). Impaired NK-cell degranulation in the presence of K562 cells was observed and similarly to patients6Gismondi A. Cifaldi L. Mazza C. Giliani S. Parolini S. Morrone S. et al.Impaired natural and CD16-mediated NK cell cytotoxicity in patients with WAS and XLT: ability of IL-2 to correct NK cell functional defect.Blood. 2004; 104: 436-443Crossref PubMed Scopus (121) Google Scholar with Wiskott-Aldrich Syndrome IL-2 restored degranulation and killing to normal levels (Fig 2, F; data not shown). Most patients received antibiotic prophylaxis (71%). One patient with recurrent oral candidiasis remained on antifungal prophylaxis. “Auto”inflammatory manifestations appear to respond to steroids, mofetil mycophenolate, and sirolimus. The response to TNF-blocking agents was unsatisfactory. To date, 5 patients have been treated with HSCT. Two patients have a medium/long-term follow-up of 1 and 6 years, respectively, and are in good health and off all medication (P2 and P6). The other 3 patients (P3, P9, and P12) have only recently been transplanted, and they are alive and well, with resolution of all “auto”inflammatory features. In conclusion, our cohort delineates a more detailed and larger spectrum of ARPC1B deficiency phenotypes compared with previous reports. The clinical defect appears to be characterized by recurrent bacterial and viral infections, extensive eczema, allergies, thrombocytopenia, and skin vasculitis, together with bleeding often manifested as early onset gastric hemorrhage and hemorrhagic colitis. The eczematous skin phenotype can be explained by immune-mediated allergic responses and the anaphylactic reactions can be avoided by elimination of food allergens from the diet. The defective Treg-cell function is suggested to be involved in both the exaggerated TH2 responses and IgE reactivity against allergens.7Lanzi G. Moratto D. Vairo D. Masneri S. Delmonte O. Paganini T. et al.A novel primary human immunodeficiency due to deficiency in the WASP-interacting protein WIP.J Exp Med. 2012; 209: 29-34Crossref PubMed Scopus (129) Google Scholar Defects in cytoskeleton rearrangement, altered immunologic synapses formation, and reduced chemotaxis have been recently identified in ARPC1B-deficient patients' T cells,4Brigida I. Zoccolillo M. Cicalese M.P. Pfajfer L. Barzaghi F. Scala S. et al.T cell defects in patients with ARPC1B germline mutations account for their combined immunodeficiency.Blood. 2018; 132: 2362-2374PubMed Google Scholar suggesting that they may play a role in the susceptibility to infections. In addition, patients’ NK cells show a peculiar phenotypic profile and an impaired functionality including both migration defects and NK-cell dysfunction, which may well contribute to the predisposition to viral infections seen in ARPC1B-deficient patients. The neutrophil and macrophage abnormalities may explain the susceptibility of the patients to bacterial infections1Kuijpers T.W. Tool A.T.J. van der Bijl I. de Boer M. van Houdt M. de Cuyper I.M. et al.Combined immunodeficiency with severe inflammation and allergy caused by ARPC1B deficiency.J Allergy Clin Immunol. 2017; 140: 273-277.e10Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar in the presence of normal antibody levels. Although careful monitoring, antimicrobial prophylaxis, and adequate treatment are mandatory to prevent and counter infections, the immunodysregulation contributing to vasculitis and arthritis requires immunosuppression. The unique and variable combination of clinical features makes ARPC1B deficiency a complex disease entity for which HSCT is considered a curative treatment option. 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Early reports described an unexpected low number of patients affected by inborn errors of immunity (IEI) with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. However, the incidence and mortality rates in IEI are still a matter of speculation, and a detailed figure is lacking because cohorts of patients with IEI were not compared with the general population in a given country.1,2 Because of the high burden of coronavirus disease 2019 (COVID-19) in Italy, we evaluated the impact of the pandemic on patients with IEI enrolled by 21 centers in the IPINet national registry (www.ipinet.org)3 with the aim to assess SARS-CoV-2 incidence and infection-fatality rate in different IEI entities in a cohort of 3263 adult and pediatric patients for which we have the exact figure available thanks to the Italian registry for each nosological entity, to quantify the length of time of SARS-CoV-2 positivity, and to verify whether a condition of lymphopenia might be a possible predictor of COVID-19 outcome.

Abstract Purpose Deficiency of adenosine deaminase 2 (DADA2) is an inherited inborn error of immunity, characterized by autoinflammation (recurrent fever), vasculopathy (livedo racemosa, polyarteritis nodosa, lacunar ischemic strokes, and intracranial hemorrhages), immunodeficiency, lymphoproliferation, immune cytopenias, and bone marrow failure (BMF). Tumor necrosis factor (TNF-α) blockade is the treatment of choice for the vasculopathy, but often fails to reverse refractory cytopenia. We aimed to study the outcome of hematopoietic cell transplantation (HCT) in patients with DADA2. Methods We conducted a retrospective study on the outcome of HCT in patients with DADA2. The primary outcome was overall survival (OS). Results Thirty DADA2 patients from 12 countries received a total of 38 HCTs. The indications for HCT were BMF, immune cytopenia, malignancy, or immunodeficiency. Median age at HCT was 9 years (range: 2–28 years). The conditioning regimens for the final transplants were myeloablative ( n = 20), reduced intensity ( n = 8), or non-myeloablative ( n = 2). Donors were HLA-matched related ( n = 4), HLA-matched unrelated ( n = 16), HLA-haploidentical ( n = 2), or HLA-mismatched unrelated ( n = 8). After a median follow-up of 2 years (range: 0.5–16 years), 2-year OS was 97%, and 2-year GvHD-free relapse-free survival was 73%. The hematological and immunological phenotypes resolved, and there were no new vascular events. Plasma ADA2 enzyme activity normalized in 16/17 patients tested. Six patients required more than one HCT. Conclusion HCT was an effective treatment for DADA2, successfully reversing the refractory cytopenia, as well as the vasculopathy and immunodeficiency. Clinical Implications HCT is a definitive cure for DADA2 with &gt; 95% survival.

Primary immunodeficiencies (PIDs) have represented a paradigmatic model for successes and pitfalls of hematopoietic stem cells gene therapy. First clinical trials performed with gamma retroviral vectors (γ-RV) for adenosine deaminase severe combined immunodeficiency (ADA-SCID), X-linked SCID (SCID-X1), and Wiskott-Aldrich syndrome (WAS) showed that gene therapy is a valid therapeutic option in patients lacking an HLA-identical donor. No insertional mutagenesis events have been observed in more than 40 ADA-SCID patients treated so far in the context of different clinical trials worldwide, suggesting a favorable risk-benefit ratio for this disease. On the other hand, the occurrence of insertional oncogenesis in SCID-X1, WAS, and chronic granulomatous disease (CGD) RV clinical trials prompted the development of safer vector construct based on self-inactivating (SIN) retroviral or lentiviral vectors (LVs). Here we present the recent results of LV-mediated gene therapy for WAS showing stable multilineage engraftment leading to hematological and immunological improvement, and discuss the differences with respect to the WAS RV trial. We also describe recent clinical results of SCID-X1 gene therapy with SIN γ-RV and the perspectives of targeted genome editing techniques, following early preclinical studies showing promising results in terms of specificity of gene correction. Finally, we provide an overview of the gene therapy approaches for other PIDs and discuss its prospects in relation to the evolving arena of allogeneic transplant.

Abstract 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 SIR s and MVI s 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 SIR s and MVI s, to provide a reference background for future experimental therapy trials. SIR s and MVI s 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.

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. 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.

Thrombocytopenia is a serious issue for all patients with classical Wiskott-Aldrich syndrome (WAS) and X-linked thrombocytopenia (XLT) because it causes severe and life-threatening bleeding. Lentiviral gene therapy (GT) for WAS has shown promising results in terms of immune reconstitution. However, despite the reduced severity and frequency of bleeding events, platelet counts remain low in GT-treated patients.We carefully investigated platelet defects in terms of phenotype and function in untreated patients with WAS and assessed the effect of GT treatment on platelet dysfunction.We analyzed a cohort of 20 patients with WAS/XLT, 15 of them receiving GT. Platelet phenotype and function were analyzed by using electron microscopy, flow cytometry, and an aggregation assay. Platelet protein composition was assessed before and after GT by means of proteomic profile analysis.We show that platelets from untreated patients with WAS have reduced size, abnormal ultrastructure, and a hyperactivated phenotype at steady state, whereas activation and aggregation responses to agonists are decreased. GT restores platelet size and function early after treatment and reduces the hyperactivated phenotype proportionally to WAS protein expression and length of follow-up.Our study highlights the coexistence of morphologic and multiple functional defects in platelets lacking WAS protein and demonstrates that GT normalizes the platelet proteomic profile with consequent restoration of platelet ultrastructure and phenotype, which might explain the observed reduction of bleeding episodes after GT. These results are instrumental also from the perspective of a future clinical trial in patients with XLT only presenting with microthrombocytopenia.

BackgroundImmune-dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) syndrome is a lethal disease caused by mutations in a transcription factor critical for the function of thymus-derived regulatory T (Treg) cells (ie, FOXP3), resulting in impaired Treg function and autoimmunity. At present, hematopoietic stem cell transplantation is the therapy of choice for patients with IPEX syndrome. If not available, multiple immunosuppressive regimens have been used with poor disease-free survival at long-term follow-up. Rapamycin has been shown to suppress peripheral T cells while sparing Treg cells expressing wild-type FOXP3, thereby proving beneficial in the clinical setting of immune dysregulation. However, the mechanisms of immunosuppression selective to Treg cells in patients with IPEX syndrome are unclear.ObjectiveWe sought to determine the cellular and molecular basis of the clinical benefit observed under rapamycin treatment in 6 patients with IPEX syndrome with different FOXP3 mutations.MethodsPhenotype and function of FOXP3-mutated Treg cells from rapamycin-treated patients with IPEX syndrome were tested by flow cytometry and in vitro suppression assays, and the gene expression profile of rapamycin-conditioned Treg cells by droplet-digital PCR.ResultsClinical and histologic improvements in patients correlated with partially restored Treg function, independent of FOXP3 expression or Treg frequency. Expression of TNF-receptor-superfamily-member 18 (TNFRSF18, glucocorticoid-induced TNF-receptor–related) and EBV-induced-3 (EBI3, an IL-35 subunit) in patients’ Treg cells increased during treatment as compared with that of Treg cells from untreated healthy subjects. Furthermore inhibition of glucocorticoid-induced TNF-receptor–related and Ebi3 partially reverted in vitro suppression by in vivo rapamycin-conditioned Treg cells.ConclusionsRapamycin is able to affect Treg suppressive function via a FOXP3-independent mechanism, thus sustaining the clinical improvement observed in patients with IPEX syndrome under rapamycin treatment. Immune-dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) syndrome is a lethal disease caused by mutations in a transcription factor critical for the function of thymus-derived regulatory T (Treg) cells (ie, FOXP3), resulting in impaired Treg function and autoimmunity. At present, hematopoietic stem cell transplantation is the therapy of choice for patients with IPEX syndrome. If not available, multiple immunosuppressive regimens have been used with poor disease-free survival at long-term follow-up. Rapamycin has been shown to suppress peripheral T cells while sparing Treg cells expressing wild-type FOXP3, thereby proving beneficial in the clinical setting of immune dysregulation. However, the mechanisms of immunosuppression selective to Treg cells in patients with IPEX syndrome are unclear. We sought to determine the cellular and molecular basis of the clinical benefit observed under rapamycin treatment in 6 patients with IPEX syndrome with different FOXP3 mutations. Phenotype and function of FOXP3-mutated Treg cells from rapamycin-treated patients with IPEX syndrome were tested by flow cytometry and in vitro suppression assays, and the gene expression profile of rapamycin-conditioned Treg cells by droplet-digital PCR. Clinical and histologic improvements in patients correlated with partially restored Treg function, independent of FOXP3 expression or Treg frequency. Expression of TNF-receptor-superfamily-member 18 (TNFRSF18, glucocorticoid-induced TNF-receptor–related) and EBV-induced-3 (EBI3, an IL-35 subunit) in patients’ Treg cells increased during treatment as compared with that of Treg cells from untreated healthy subjects. Furthermore inhibition of glucocorticoid-induced TNF-receptor–related and Ebi3 partially reverted in vitro suppression by in vivo rapamycin-conditioned Treg cells. Rapamycin is able to affect Treg suppressive function via a FOXP3-independent mechanism, thus sustaining the clinical improvement observed in patients with IPEX syndrome under rapamycin treatment.

COVID-19 manifestations range from asymptomatic to life-threatening infections. The outcome in different inborn errors of immunity (IEI) is still a matter of debate. In this retrospective study, we describe the experience of the of the Italian Primary Immunodeficiencies Network (IPINet). Sixteen reference centers for adult or pediatric IEI were involved. One hundred fourteen patients were enrolled including 35 pediatric and 79 adult patients. Median age was 32 years, and male-to-female ratio was 1.5:1. The most common IEI were 22q11.2 deletion syndrome in children (26%) and common variable immunodeficiency (CVID) in adults (65%). Ninety-one patients did not require hospital admission, and among these, 33 were asymptomatic. Hospitalization rate was 20.17%. Older age (p 0.004) and chronic lung disease (p 0.0008) represented risk factors for hospitalization. Hospitalized patients mainly included adults suffering from humoral immunodeficiencies requiring immunoglobulin replacement therapy and as expected had lower B cell counts compared to non-hospitalized patients. Infection fatality rate in the whole cohort was 3.5%. Seroconversion was observed is 86.6% of the patients evaluated and in 83.3% of CVID patients. 16.85% of the patients reported long-lasting COVID symptoms. All but one patient with prolonged symptoms were under IgRT. The fatality rate observed in IEI was slightly similar to the general population. The age of the patients who did not survive was lower compared to the general population, and the age stratified mortality in the 50-60 age range considerable exceeded the mortality from 50 to 60 age group of the Italian population (14.3 vs 0.6%; p < 0.0001). We hypothesize that this is due to the fact that comorbidities in IEI patients are very common and usually appear early in life.

To determine whether a simplified, 1-day/week regimen of trimethoprim/sulfamethoxazole is sufficient to prevent Pneumocystis (jirovecii [carinii]) pneumonia (PCP). Current recommended regimens for prophylaxis against PCP range from daily administration to 3 consecutive days per week dosing.A prospective survey of the regimens adopted for the PCP prophylaxis in all patients treated for childhood cancer at pediatric hematology-oncology centers of the Associazione Italiana Ematologia Oncologia Pediatrica.The 20 centers participating in the study reported a total of 2466 patients, including 1093 with solid tumor and 1373 with leukemia/lymphoma (or primary immunodeficiency; n = 2). Of these patients, 1371 (55.6%) received the 3-day/week prophylaxis regimen, 406 (16.5%) received the 2-day/week regimen, and 689 (27.9%), including 439 with leukemia/lymphoma, received the 1-day/week regimen. Overall, only 2 cases of PCP (0.08%) were reported, both in the 2-day/week group. By intention to treat, the cumulative incidence of PCP at 3 years was 0.09% overall (95% CI, 0.00-0.40%) and 0.51% for the 2-day/week group (95% CI, 0.10%-2.00%). Remarkably, both patients who failed had withdrawn from prophylaxis.A single-day course of prophylaxis with trimethoprim/sulfamethoxazole may be sufficient to prevent PCP in children with cancer undergoing intensive chemotherapy regimens. This simplified strategy might have implications for the emerging need for PCP prophylaxis in other patients subjected to the increased use of biological and nonbiological agents that induce higher levels of immune suppression, such as those with rheumatic diseases.

Background: Primary Immunodeficiencies (PIDs) are a heterogeneous group of genetic immune disorders. While some PIDs can manifest with more than one phenotype, signs and symptoms of various PIDs overlap considerably. Recently, novel defects in immune-related genes and additional variants in previously reported genes responsible for PIDs have been successfully identified by Next Generation Sequencing (NGS), allowing the recognition of a broad spectrum of disorders. Objective: To evaluate the strength and weakness of targeted NGS sequencing using custom-made Ion Torrent and Haloplex (Agilent) panels for diagnostics and research purposes. Methods: Five different panels including known and candidate genes were used to screen 105 patients with distinct PID features divided in three main PID categories: T cell defects, Humoral defects and Other PIDs. The Ion Torrent sequencing platform was used in 73 patients. Among these, 18 selected patients without a molecular diagnosis and 32 additional patients were analyzed by Haloplex enrichment technology. Results: The complementary use of the two custom-made targeted sequencing approaches allowed the identification of causative variants in 28.6% (n=30) of patients. Twenty-two out of 73 (34.6%) patients were diagnosed by Ion Torrent. In this group 20 were included in the SCID/CID category. Eight out of 50 (16%) patients were diagnosed by Haloplex workflow. Ion Torrent method was highly successful for those cases with well-defined phenotypes for immunological and clinical presentation. The Haloplex approach was able to diagnose 4 SCID/CID patients and 4 additional patients with complex and extended phenotypes, embracing all three PID categories in which this approach was more efficient. Both technologies showed good gene coverage. Conclusions: NGS technology represents a powerful approach in the complex field of rare disorders but its different application should be weighted. A relatively small NGS target panel can be successfully applied for a robust diagnostic suspicion, while when the spectrum of clinical phenotypes overlaps more than one PID an in-depth NGS analysis is required, including also whole exome/genome sequencing to identify the causative gene.

Rubella virus (RuV) has recently been found in association with granulomatous inflammation of the skin and several internal organs in patients with inborn errors of immunity (IEI). The cellular tropism and molecular mechanisms of RuV persistence and pathogenesis in select immunocompromised hosts are not clear. We provide clinical, immunological, virological, and histological data on a cohort of 28 patients with a broad spectrum of IEI and RuV-associated granulomas in skin and nine extracutaneous tissues to further delineate this relationship. Combined immunodeficiency was the most frequent diagnosis (67.8%) among patients. Patients with previously undocumented conditions, i.e., humoral immunodeficiencies, a secondary immunodeficiency, and a defect of innate immunity were identified as being susceptible to RuV-associated granulomas. Hematopoietic cell transplantation was the most successful treatment in this case series resulting in granuloma resolution; steroids, and TNF-α and IL-1R inhibitors were moderately effective. In addition to M2 macrophages, neutrophils were identified by immunohistochemical analysis as a novel cell type infected with RuV. Four patterns of RuV-associated granulomatous inflammation were classified based on the structural organization of granulomas and identity and location of cell types harboring RuV antigen. Identification of conditions that increase susceptibility to RuV-associated granulomas combined with structural characterization of the granulomas may lead to a better understanding of the pathogenesis of RuV-associated granulomas and discover new targets for therapeutic interventions.

Mobilized peripheral blood is increasingly used instead of bone marrow as a source of autologous hematopoietic stem/progenitor cells for ex vivo gene therapy. Here, we present an unplanned exploratory analysis evaluating the hematopoietic reconstitution kinetics, engraftment and clonality in 13 pediatric Wiskott-Aldrich syndrome patients treated with autologous lentiviral-vector transduced hematopoietic stem/progenitor cells derived from mobilized peripheral blood (n = 7), bone marrow (n = 5) or the combination of the two sources (n = 1). 8 out of 13 gene therapy patients were enrolled in an open-label, non-randomized, phase 1/2 clinical study (NCT01515462) and the remaining 5 patients were treated under expanded access programs. Although mobilized peripheral blood- and bone marrow- hematopoietic stem/progenitor cells display similar capability of being gene-corrected, maintaining the engineered grafts up to 3 years after gene therapy, mobilized peripheral blood-gene therapy group shows faster neutrophil and platelet recovery, higher number of engrafted clones and increased gene correction in the myeloid lineage which correlate with higher amount of primitive and myeloid progenitors contained in hematopoietic stem/progenitor cells derived from mobilized peripheral blood. In vitro differentiation and transplantation studies in mice confirm that primitive hematopoietic stem/progenitor cells from both sources have comparable engraftment and multilineage differentiation potential. Altogether, our analyses reveal that the differential behavior after gene therapy of hematopoietic stem/progenitor cells derived from either bone marrow or mobilized peripheral blood is mainly due to the distinct cell composition rather than functional differences of the infused cell products, providing new frames of references for clinical interpretation of hematopoietic stem/progenitor cell transplantation outcome.

Introduction Treatment of pediatric malignancies is becoming progressively more complex, implying the adoption of multimodal therapies. A reliable, long-lasting venous access represents one of the critical requirements for the success of those treatments. Recent technical innovations—such as minimally invasive procedures for placement, new devices and novel materials—have rapidly spread for clinical use in adult patients, but are still not consistently used in the pediatric population. Methods The Supportive Therapy Working Group of Italian Association of Hematology and Oncology (AIEOP) reviewed medical literature focusing on new aspects of central venous access devices (VADs) in pediatric patients affected by oncohematological diseases. Results Appropriate recommendations for clinical use in these patients have been discussed and formulated. Conclusions The importance of the correct choice, management and use of VADs in pediatric oncohematological patients is a necessary prerequisite for an adequate standard of care, also considering the increased chances of cure and the longer life expectancy of those patients with modern therapies.

Strimvelis (autologous CD34+ cells transduced to express adenosine deaminase [ADA]) is the first ex vivo stem cell gene therapy approved by the European Medicines Agency (EMA), indicated as a single treatment for patients with ADA-severe combined immunodeficiency (ADA-SCID) who lack a suitable matched related bone marrow donor. Existing primary immunodeficiency registries are tailored to transplantation outcomes and do not capture the breadth of safety and efficacy endpoints required by the EMA for the long-term monitoring of gene therapies. Furthermore, for extended monitoring of Strimvelis, the young age of children treated, small patient numbers, and broad geographic distribution of patients all increase the risk of loss to follow-up before sufficient data have been collected. Establishing individual investigator sites would be impractical and uneconomical owing to the small number of patients from each location receiving Strimvelis. An observational registry has been established to monitor the safety and effectiveness of Strimvelis in up to 50 patients over a minimum of 15 years. To address the potential challenges highlighted above, data will be collected by a single investigator site at Ospedale San Raffaele (OSR), Milan, Italy, and entered into the registry via a central electronic platform. Patients/families and the patient’s local physician will also be able to submit healthcare information directly to the registry using a uniquely designed electronic platform. Data entry will be monitored by a Gene Therapy Registry Centre (funded by GlaxoSmithKline) who will ensure that necessary information is collected and flows between OSR, the patient/family and the patient’s local healthcare provider. The Strimvelis registry sets a precedent for the safety monitoring of future gene therapies. A unique, patient-focused design has been implemented to address the challenges of long-term follow-up of patients treated with gene therapy for a rare disease. Strategies to ensure data completeness and patient retention in the registry will help fulfil pharmacovigilance requirements. Collaboration with partners is being sought to expand from a treatment registry into a disease registry. Using practical and cost-efficient approaches, the Strimvelis registry is hoped to encourage further innovation in registry design within orphan drug development.

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.

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.

Abstract Adenosine deaminase 2 deficiency (DADA2) is a rare inherited disorder that is caused by autosomal recessive mutations in the ADA2 gene. Clinical manifestations include early-onset lacunar strokes, vasculitis/vasculopathy, systemic inflammation, immunodeficiency, and hematologic defects. Anti–tumor necrosis factor therapy reduces strokes and systemic inflammation. Allogeneic hematopoietic stem/progenitor cell (HSPC) transplantation can ameliorate most disease manifestations, but patients are at risk for complications. Autologous HSPC gene therapy may be an alternative curative option for patients with DADA2. We designed a lentiviral vector encoding ADA2 (LV-ADA2) to genetically correct HSPCs. Lentiviral transduction allowed efficient delivery of the functional ADA2 enzyme into HSPCs from healthy donors. Supranormal ADA2 expression in human and mouse HSPCs did not affect their multipotency and engraftment potential in vivo. The LV-ADA2 induced stable ADA2 expression and corrected the enzymatic defect in HSPCs derived from DADA2 patients. Patients’ HSPCs re-expressing ADA2 retained their potential to differentiate into erythroid and myeloid cells. Delivery of ADA2 enzymatic activity in patients’ macrophages led to a complete rescue of the exaggerated inflammatory cytokine production. Our data indicate that HSPCs ectopically expressing ADA2 retain their multipotent differentiation ability, leading to functional correction of macrophage defects. Altogether, these findings support the implementation of HSPC gene therapy for DADA2.

Busulphan (BU) is associated with neurotoxicity and risk of seizures. Hence, seizure prophylaxis is routinely utilized during BU administration for stem cell transplantation (SCT). We collected data on the incidence of seizures among children undergoing SCT in Italy. Fourteen pediatric transplantation centers agreed to report unselected data on children receiving BU as part of the conditioning regimen for SCT between 2005 and 2012. Data on 954 pediatric transplantation procedures were collected; of them, 66% of the patients received BU orally, and the remaining 34%, i.v. All the patients received prophylaxis of seizures, according to local protocols, consisting of different schedules and drugs. A total of 13 patients (1.3%) developed seizures; of them, 3 had a history of epilepsy (or other seizure-related pre-existing condition); 3 had documented brain lesions potentially causing seizures per se; 1 had febrile seizures, 1 severe hypo-osmolality. In the remaining 5 patients, seizures were considered not explained and, thus, potentially related to BU administration. The incidence of seizures in children receiving BU-containing regimen was very low (1.3%); furthermore, most of them had at least 1-either pre-existing or concurrent-associated risk factor for seizures.

<h3>Background</h3> Adenosine deaminase (ADA) deficiency causes severe cellular and humoral immune defects and dysregulation because of metabolic toxicity. Alterations in B-cell development and function have been poorly studied. Enzyme replacement therapy (ERT) and hematopoietic stem cell (HSC) gene therapy (GT) are therapeutic options for patients lacking a suitable bone marrow (BM) transplant donor. <h3>Objective</h3> We sought to study alterations in B-cell development in ADA-deficient patients and investigate the ability of ERT and HSC-GT to restore normal B-cell differentiation and function. <h3>Methods</h3> Flow cytometry was used to characterize B-cell development in BM and the periphery. The percentage of gene-corrected B cells was measured by using quantitative PCR. B cells were assessed for their capacity to proliferate and release IgM after stimulation. <h3>Results</h3> Despite the severe peripheral B-cell lymphopenia, patients with ADA-deficient severe combined immunodeficiency showed a partial block in central BM development. Treatment with ERT or HSC-GT reverted most BM alterations, but ERT led to immature B-cell expansion. In the periphery transitional B cells accumulated under ERT, and the defect in maturation persisted long-term. HSC-GT led to a progressive improvement in B-cell numbers and development, along with increased levels of gene correction. The strongest selective advantage for ADA-transduced cells occurred at the transition from immature to naive cells. B-cell proliferative responses and differentiation to immunoglobulin secreting IgM after B-cell receptor and Toll-like receptor triggering were severely impaired after ERT and improved significantly after HSC-GT. <h3>Conclusions</h3> ADA-deficient patients show specific defects in B-cell development and functions that are differently corrected after ERT and HSC-GT.

Objective: A series of destructive and tumefactive lesions of the midline structures have been recently added to the spectrum of IgG4-related disease (IgG4-RD). We examined the clinical, serological, endoscopic, radiological, and histological features that might be of utility in distinguishing IgG4-RD from other forms of inflammatory conditions with the potential to involve the sinonasal area and the oral cavity. Methods: We studied 11 consecutive patients with erosive and/or tumefactive lesions of the midline structures referred to our tertiary care center. All patients underwent serum IgG4 measurement, flow cytometry for circulating plasmablast counts, nasal endoscopy, radiological studies, and histological evaluation of tissue specimens. The histological studies included immunostaining studies to assess the number of IgG4 + plasma cells/HPF for calculation of the IgG4+/IgG + plasma cell ratio. Results: Five patients with granulomatosis with polyangiitis (GPA), three with cocaine-induced midline destructive lesions (CIMDL), and three with IgG4-RD were studied. We found no clinical, endoscopic, or radiological findings specific for IgG4-RD. Increased serum IgG4 and plasmablasts levels were not specific for IgG4-RD. Rather, all 11 patients had elevated blood plasmablast concentrations, and several patients with GPA and CIMDL had elevated serum IgG4 levels. Storiform fibrosis and an IgG4+/IgG + plasma cell ratio >20% on histological examination, however, were observed only in patients with IgG4-RD. Conclusions: Histological examination of bioptic samples from the sinonasal area and oral cavity represents the mainstay for the diagnosis of IgG4-RD involvement of the midline structures.

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.

Vaccines for SARS-CoV-2 currently authorised by the European Medicine Agency are effective, safe and well tolerated in practice. Awareness of rare potential vaccine-related adverse effects (AEs) is important to improve their recognition, management and reporting. An 88-year-old man attended the emergency department with incomplete palsy of the right third cranial nerve 3 days after the first administration of Moderna mRNA-1273 SARS-CoV-2 vaccine. Imaging ruled out a vascular accident and a vaccine AE was hypothesised. Two weeks of oral steroids led to the patient’s recovery, but without evidence of humoral immune response to vaccine. Thus, full immunisation with a dose of Pfizer mRNA-BNT162b2 SARS-CoV-2 vaccine in a different site was attempted. This was uneventful and followed by a robust antibody response. Empirical change of site and vaccine brand may represent a tailored option to obtain full immune protection in selected patients, after vaccine AEs.

Abstract Common variable immunodeficiency (CVID) is the most frequent primary antibody deficiency whereby follicular helper T (Tfh) cells fail to establish productive responses with B cells in germinal centers. Here, we analyzed the frequency, phenotype, transcriptome, and function of circulating Tfh (cTfh) cells in CVID patients displaying autoimmunity as an additional phenotype. A group of patients showed a high frequency of cTfh1 cells and a prominent expression of PD‐1 and ICOS as well as a cTfh mRNA signature consistent with highly activated, but exhausted, senescent, and apoptotic cells. Plasmatic CXCL13 levels were elevated in this group and positively correlated with cTfh1 cell frequency and PD‐1 levels. Monoallelic variants in RTEL1 , a telomere length‐ and DNA repair‐related gene, were identified in four patients belonging to this group. Their blood lymphocytes showed shortened telomeres, while their cTfh were more prone to apoptosis. These data point toward a novel pathogenetic mechanism in CVID, whereby alterations in DNA repair and telomere elongation might predispose to antibody deficiency. A Th1, highly activated but exhausted and apoptotic cTfh phenotype was associated with this form of CVID.

22q11.2 deletion syndrome (22q11.2DS) is a common microdeletion syndrome, which occurs in approximately 1:4000 births. Familial autosomal dominant recurrence of the syndrome is detected in about 8-28% of the cases. Aim of this study is to evaluate the intergenerational and intrafamilial phenotypic variability in a cohort of familial cases carrying a 22q11.2 deletion.Thirty-two 22q11.2DS subjects among 26 families were enrolled.Second generation subjects showed a significantly higher number of features than their transmitting parents (212 vs 129, P = 0.0015). Congenital heart defect, calcium-phosphorus metabolism abnormalities, developmental and speech delay were more represented in the second generation (P < 0.05). Ocular disorders were more frequent in the parent group. No significant difference was observed for the other clinical variables. Intrafamilial phenotypic heterogeneity was identified in the pedigrees. In 23/32 families, a higher number of features were found in individuals from the second generation and a more severe phenotype was observed in almost all of them, indicating the worsening of the phenotype over generations. Both genetic and epigenetic mechanisms may be involved in the phenotypic variability.Second generation subjects showed a more complex phenotype in comparison to those from the first generation. Both ascertainment bias related to patient selection or to the low rate of reproductive fitness of adults with a more severe phenotype, and several not well defined molecular mechanism, could explain intergenerational and intrafamilial phenotypic variability in this syndrome.

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.

Myocarditis Disease Unit (MDU) is a functional multidisciplinary network designed to offer multidisciplinary assistance to patients with myocarditis. More than 300 patients coming from the whole Country are currently followed up at a specialized multidisciplinary outpatient clinic. Following the pandemic outbreak of the SARS-CoV-2 infection in Italy, we present how the MDU rapidly evolved to a "tele-MDU", via a dedicated multitasking digital health platform.

In 2016, the European Hematology Association (EHA) published the EHA Roadmap for European Hematology Research1 aiming to highlight achievements in the diagnostics and treatment of blood disorders, and to better inform European policy makers and other stakeholders about the urgent clinical and scientific needs and priorities in the field of hematology. Each section was coordinated by 1 to 2 section editors who were leading international experts in the field. In the 5 years that have followed, advances in the field of hematology have been plentiful. As such, EHA is pleased to present an updated Research Roadmap, now including 11 sections, each of which will be published separately. The updated EHA Research Roadmap identifies the most urgent priorities in hematology research and clinical science, therefore supporting a more informed, focused, and ideally a more funded future for European hematology research. The 11 EHA Research Roadmap sections include Normal Hematopoiesis; Malignant Lymphoid Diseases; Malignant Myeloid Diseases; Anemias and Related Diseases; Platelet Disorders; Blood Coagulation and Hemostatic Disorders; Transfusion Medicine; Infections in Hematology; Hematopoietic Stem Cell Transplantation; CAR-T and Other Cell-based Immune Therapies; and Gene Therapy. INTRODUCTION Genetic engineering of hematopoietic stem cells (HSCs) has progressed from early stage clinical trials providing evidence for substantial and durable benefits in some genetic deficiencies to the first Advanced Therapy Medicinal Products (ATMP) approved for the EU market (Figure 1).2 As safety and efficacy of HSC gene therapy (HSCGT) is further established by increasingly longer follow-up and a higher number of patients treated for different diseases, it may become a new pillar of treatment for several inherited monogenic affections for which allogeneic HSC transplantation (HSCT) represents a treatment option. Because HSCGT exploits autologous patient-derived cells, it has the following advantages over conventional allogeneic HSCT: (1) is in principle available to every patients; (2) does not entail the risk of graft-versus-host disease; (3) can lower substantially the risk of graft rejection, as the only potentially antigenic element in the administered cells is the therapeutic gene product; (4) can more easily establish partial chimerism with the administered HSC, which, according to disease and protocol, choice may allow lowering the requirement for myeloablative conditioning regimens and still provide substantial therapeutic benefit. Moreover, because of the genetic engineering step, gene correction may be designed to provide an even higher benefit than obtained with allogeneic normal or haploidentical carrier HSC, for instance by increasing therapeutic gene dosage in lysosomal storage diseases. HSCGT may eventually be exploited also for the treatment of some acquired affections, by instructing a new function to HSC or some of its progeny such as in gene-based delivery of a biotherapeutic or by establishing genetic resistance to an infectious agent. On the other hand, HSCGT requires ex vivo culture and manipulation of the cells to enable genetic engineering, which in turn demands for establishing a suitable GMP-compliant multistep process from cells harvest to manufacturing and delivery at the clinical point-of-care. Furthermore, the culture as well as the genetic engineering procedure may affect some fundamental HSC biological properties negatively impacting the safety and efficiency of the procedure. Although this is an active area of investigation, most current protocols for growing HSC ex vivo fail to expand or even maintain for more than a few days the most primitive and long-term engrafting cells and both gene transfer and gene editing procedures have been reported to trigger innate cellular responses that may lead to delayed or halted growth, differentiation or even apoptosis and cell death. Consequently, if these adverse effects individually or cumulatively surpass a threshold during cell manufacturing, they may cause delayed hematopoietic recovery or even failure of engraftment, exposing the patient to high risk of infections, and also compromise the clonal composition of the engineered hematopoietic graft, which may reduce its long-term resilience and safety. Last but not least, every meaningful genetic engineering of HSC requires stable, albeit local, modification of the cellular DNA sequence for ensuring life-long transmission to its cell progeny. This is a mutagenic procedure by definition, which entails a genotoxic risk dependent on the type (gene transfer versus editing), site, extent, and specificity of engineering. Whereas genotoxicity emerged as a significant risk in early HSCGT trials leading to leukemia development in some treated patients, it appears to be substantially alleviated by the new generation of lentiviral vectors currently in broader use. In any case, long-term monitoring of HSCGT patients is still required to establish the long-term safety and efficacy of these promising new therapies. In the following sections, we highlight some key features of the genetic engineering platforms under clinical development for HSCGT, the current stage and future outlook of clinical applications of HSCGT in some major diseases families, and the regulatory implications of these novel and revolutionary medicines entering the clinical arena.Figure 1.: Bone marrow-resident hematopoietic stem cells and hematopoietic progenitor cells replenish blood and tissues with new mature cells.2 Both hematopoietic stem cells and hematopoietic progenitor cells express the cell surface marker CD34, which is used to enrich a mixture of hematopoietic stem and progenitor cells for transplantation and gene therapy. Hematopoietic stem cells can be classed as long-term hematopoietic stem cells (LT-HSCs) or short-term hematopoietic stem cells (ST-HSCs). ST-HSCs progressively acquire lineage specifications to differentiate into lineage-committed progenitors and eventually terminally differentiated cells, which are released into the peripheral blood. A simplified scheme of human hematopoiesis is presented here. Alternative models have been postulated on the basis of cell surface marker analyses, in vitro and in vivo functional assays, clonal tracking by insertion analyses in hematopoietic stem and progenitor cell gene therapy studies, and single-cell RNA analyses (reviewed previously22). Mendelian genetic disorders can affect self-renewal, differentiation, and/or the function of different blood and immune cells. Examples of genetic diseases for which gene therapy is under investigation or approved are represented in white boxes below affected cell types. Wiskott–Aldrich syndrome affects platelets and other lineages. CDP = common dendritic progenitor; CID = combined immunodeficiency; CLP = common lymphoid progenitor; CMP = common myeloid progenitor; GMP = granulomonocytic progenitor; LMMP = lymphoid-myeloid primed progenitor; MEP = megakaryocytic–erythroid progenitor; MPP = multipotent progenitor; NK cell = natural killer cell; preB = pre-B cell; preT = pre-T cell; SCID = severe combined immunodeficiency. Reprinted with permission from Nat Rev Genet. 2021;22:216–234.GENE THERAPY PLATFORMS Introduction Most current genetic engineering of HSC exploits 2 major strategies, gene replacement mediated by retroviral gene transfer vectors or targeted gene editing mediated by artificial sequence-specific endonucleases. Gene transfer strategies exploit a couple of well-established platforms that were developed in research laboratories between 15 and 20 years ago. Since then, progressive clinical testing in >300 patients worldwide for the treatment of several diseases have generated 2 ATMPs approved for the EU market with several more expected in the upcoming years (Table 1).2 Gene editing strategies are still in the earliest stage of clinical testing, although they are witnessing a remarkable and constant progress advancing the versatility, precision, and scope of the technological platforms adopted.3,4 European research contributions The EU has represented a privileged theatre for the clinical development of HSC GT based on retroviral gene transfer from the earliest pioneering clinical trials until today’s most advanced stages. Retroviral vectors are replication-defective viral particles derived either from γ-retroviruses (γ-retroviral vectors [γRVs]), such as Moloney leukemia virus (MLV), or from the human lentivirus HIV-1 (lentiviral vectors [LVs]). They integrate semi-randomly a reverse transcribed RNA genome into the chromatin of the transduced cells. The vector genome comprises cis-acting signals for packaging, reverse transcription, and integration, lacks full length or open viral genes and comprises the therapeutic gene expression cassette. γRVs were the earliest to be developed, having limited efficiency of gene transfer into the more primitive HSC because productive infection is strictly dependent on concurrent replication of the targeted cell. Moreover, their early and most commonly used design exploited the strong enhancer/promoter sequences embedded in the long terminal repeats (LTRs) of the viral genome to drive transgene expression. This feature, coupled with an integration bias favoring insertion near the promoter of actively expressed genes, result in some likelihood of altering expression of endogenous genes nearby the insertion site through promoter insertion and enhancer-dependent trans-activation. Sporadic insertions near proto-oncogenes may result in activation of their oncogenic potential, thus endowing the transduced cell with a growth advantage leading to progressive clonal expansion in vivo and, in some cases, eventual transformation by further accumulation of mutations. Vector integration studies have allowed ascribing the origin of leukemia emerging in treated patients even several years post HSCGT to such types of genotoxic insertions, and longitudinal monitoring of the clonal composition of reconstituted hematopoiesis have shown the frequent expansion of clones carrying such insertions before any clinical signs of leukemia. Despite these hurdles, one of the earliest application of γRV HSCGT for the treatment of severe combined immunodeficiency caused by deficit of adenosine deaminase (ADA-SCID) has shown efficacy and better safety outcome than observed for other diseases and became the first HSCGT ATMP approved for the EU market in 2016.5 Because of the lower gene transfer efficiency and clinically relevant genotoxicity, the γRV platform has now been almost completely replaced by LV, which have improved both aspects.1,6 The key LV features underlying these improvements are: (1) exploitation of the HIV core capacity to infect nondividing cells to enhance transduction of the more primitive HSC after a short ex vivo stimulation; (2) advanced vector design which completely eliminates transcriptional activity from the vector LTRs. Expression of the therapeutic gene is driven from an internal promoter of choice, either reconstituted from the cellular promoter of the gene to be replaced, thus mimicking its physiological expression pattern, or from a moderately active house-keeping promoter, thus providing for ubiquitous expression. These features, combined with an insertion bias that favors the body rather than the promoter of expressed genes, strongly alleviate the risk of genotoxic effects at the insertion site; (3) exploitation of the HIV mechanism for packaging unspliced viral genome to transfer complex gene expression cassettes, such as used to establish erythroid specific and robust expression of a globin transgene in hemoglobinopathies. An ever-growing number of patients treated by LV HSCGT for several diseases have been showing stable and robust engineering of reconstituted hematopoiesis with highly polyclonal composition and without signs of genotoxicity such as expanding clones or enrichment for insertions at cancer gene. Depending on the disease, conditioning applied and product manufacturing, the engineered cell fraction can reach up to near completion, with evidence for distinct types of progenitors contributing to early recovery or steady-state output, which is mostly driven by engrafted self-renewing multipotent HSC.2 Gene editing strategies allow precise and targeted modification of a DNA sequence of choice, opening up novel and unique opportunities to genetic engineering. In the longest available and clinically more advanced version, it exploits an artificial endonuclease either composed of a sequence-specific DNA binding domain made of Zinc Finger or TALE protein modules coupled to a FokI nuclease half-domain (ZFN or TALEN), or of a target complementary single guide CRISPR RNA assembled with a Cas family nuclease (CRISPR/Cas).4 Either platform can deliver a DNA double-strand break (DSB) specifically in the target sequence, whose repair is exploited for the intended type of edit. Nontemplated DSB repair most commonly occurs by nonhomologous end joining (NHEJ), an error-prone process that often introduces small base insertion or deletion (indels) while sealing the break. If the break is targeted to a coding or an essential regulatory sequence the NHEJ outcome will most often be disruption or inactivation of the sequence.7 This represents the most frequent and, provided that the engineered nuclease is highly specific, better tolerated editing procedure. In most cases, electroporation is exploited to transiently introduce the nuclease mRNA or a preassembled CRISPR/Cas nucleoprotein into the cells to be edited. Transient but robust expression is required for efficient break induction while limiting toxicity. Nuclease specificity is crucial because DNA DSBs trigger a detrimental DNA damage response which, if robust and sustained, can lead to growth arrest, senescence, or apoptosis.8 Moreover, multiple DSBs increase the risk of genotoxicity and genomic rearrangements, such as translocations between different edited sites. Developing nuclease reagents with stringent specificity for the target site and comprehensively assessing off-target activity in preclinical models have been major goals of the field. A second more ambitious editing strategy exploits DNA DSB repair by homologous recombination (HR). This requires codelivery of the nuclease with a DNA template carrying the intended edit framed by homologous sequences to the DNA flanking the nuclease target site. If successful, this strategy will allow correcting mutations in situ, thus restoring both, function and physiological expression of the affected gene, and even inserting longer sequences, such as corrective cDNAs or transgene expression cassettes in safe genomic sites.2 The current challenges in applying HR-mediated editing to HSCGT is the relatively low efficiency of the process dependent on the poor permissiveness of primitive HSC to this pathway and the cumulative detrimental impact of inducing DNA DSBs and introducing an exogenous template DNA, which is best achieved by exploiting a viral vector such as adeno-associated vector.9 Proposed research for the roadmap While application of LV gene transfer can be envisaged to be tested in an increasingly larger number of diseases, the following areas of further HSCGT platform development can be proposed. Because autologous HSCGT does not require full ablation and replacement of resident HSC, alleviation of the short-term and long-term morbidity of the procedure can be sought by careful targeting of chemotherapeutic regimens to the minimal required dose and through exploring emerging nongenotoxic conditioning strategies, such as those based on antibodies or immunotoxins targeting HSC.10,11 HSCGT may provide a favorable setting for early testing of these approaches, especially in diseases not requiring high levels of chimerism with functional cells for correction. Further optimization of ex vivo HSC culture conditions, including more faithful reconstitution of signals supporting HSC self-renewal in bone marrow niches and/or their emergence from hemogenic endothelium in the embryo, testing novel combinations of transduction enhancers and uncovering their mechanism of action on HSC, and better standardization and more effective process control in ATMP manufacturing should allow improving the extent, predictability and reproducibility of gene transfer across different patients and treatments. Close monitoring of clonal composition and HSC activity in the engineered hematopoietic graft of HSCGT patients will provide not only novel information on HSC biology in living humans but also help uncover clues to the long-term safety and robustness of therapeutic correction. As we learn more on the clinical relevance of clonal shrinking and skewing in aging hematopoiesis, it will be important to investigate whether ex vivo manipulation and genetic engineering may accelerate or aggravate such events. Delayed emergence of new HSC clones after HSCGT may highlight the capacity of engineered cells to return to long-term latency in vivo, establishing a safe reservoir for sustaining the graft in the case of emergencies or aging. On the other hand, if the growing number of treated patients and increasing time of follow-up were to lead to sporadic emergence of expanding dominant clones, we could learn about the residual extent and mechanism of genotoxicity of current vector design and introduce improved versions which are currently being investigated in stressed experimental models but await strong rational for clinical testing. Gene editing strategies must be carefully monitored upon first clinical testing to uncover any adverse outcome of the procedure in terms of hematopoietic recovery and long-term engraftment, clonal composition of the engineered graft, and preserved long-term maintenance and multipotency of the edited HSC. The occurrence of large-scale genomic alterations in some edited cells and their in vivo fate will be investigated with the goal to decrease their occurrence or purge them from the product, if necessary. Protocols allowing for improved efficiency and tolerability of editing, especially when exploiting HR, will be developed for instance by adopting emerging ex vivo HSC expansion strategies and refined editors, or inhibiting or counteracting the detrimental responses induced by the procedure. New gene editing platforms that bypass the requirement for DNA DSB, such as base editors and prime editors will be tested in preclinical studies for potential application to HSCGT investigating their efficiency, tolerability, and specificity, potentially providing for more precise and uneventful genetic engineering.4 The potential adverse effects of pre-existing immunity to gene transfer and, in particular gene editing reagents, some of which are of bacterial origin, will be investigated. Residual antigenic components might be present in the cell product and trigger cellular responses leading to clearance of the administered cells. The impact of pre-existing effector or regulatory T cells specific for the viral/editor component at different levels in the recipient will be investigated. Anticipated impact of the research We are at the beginning of a new era of medicine exploiting genes and cells as novel therapeutics, leveraging on powerful genetic engineering tools to modify cell function and capture fundamental biological processes, such as HSC-driven reconstitution of intra and extravascular hematopoietic populations for therapeutic purposes. These approaches open unprecedented opportunities and largely unexplored paths for therapeutic interventions. Remarkable and durable benefits in until now orphan and otherwise severe and lethal diseases have been reported and are expected to be further achieved in growing numbers. However, this promise comes also with a tremendous challenge to the currently established framework for developing, regulating and distributing medicines while maintaining their economic sustainability by public and private healthcare providers and guaranteeing fair access to the patients. To address this challenge and attain the benefits that may come from this research, we must continue to support research establishments that produce innovation, facilitate the creation of start-ups promoting early development of the new technologies, advance education of biomedical trainees to become familiar with the new technologies and treatments, update regulatory requirements to emerging platforms and a rapidly evolving landscape, and foster engagement of pharmaceutical industries in clinical development and commercial deployment of the new ATMPs. GENE THERAPY FOR PRIMARY IMMUNODEFICIENCIES Introduction Primary immune deficiencies (PIDs) are a heterogeneous group of >400 genetic diseases characterized by poor or absent function of one or more components of the immune system, whose main clinical manifestations comprise increased frequency and severity of infection, autoimmunity, and aberrant inflammation and malignancy.12 Early diagnosis and treatment remain a mainstay for all forms of PIDs to prevent organ damage and life-threatening infections and to improve prognosis and quality of life. Allogeneic HSCT is curative for many PIDs, but still carries a risk of mortality and morbidity from rejection, toxicity, and graft-versus-host disease. SCIDs were the first monogenic disorders for which HSCGT has been successfully developed. Clinical trials with promising results are ongoing for at least 6 different PIDs due to genetic defects of adaptive and/or innate immunity while a growing number of diseases are at preclinical stage of development.2,13 European research contributions European academic research played a key role in the development of successful HSCGT approaches for PID. In the past 2 decades, the European Commission funded collaborative research networks such as CONSERT, PERSIST, Clinigene, CELL-PID, NET4CGD, SCIDNET, SUPERSIST, and UPGRADE. Over 100 PID patients have been treated in early trials of HSCGT with γRV, showing reconstitution of immunity in most patients affected by SCID-X1 due to IL2RG deficiency, ADA-deficient SCID, Wiskott–Aldrich Syndrome (WAS), and X-linked Chronic Granulomatous Disease (X-CGD).2 ADA-SCID GT was the first PID for which non myeloablative conditioning was introduced and in 2016 became the first HSCGT approved in the EU (Strimvelis, Orchard Therapeutics Ltd, London, UK), with the indication of standard treatment for ADA-SCID lacking a compatible family donor5 Infusion of ADA gene replaced HSC resulted in persistent (>15 y) engraftment of gene-marked cells ranging from 1% to 10% in the myeloid compartment and reaching up to 100% in the lymphoid compartment, correction of adenosine metabolism and improved T-cell counts, leading to discontinuation of prophylaxis and decreased incidence of severe infections.14 The promising clinical data obtained in γRV trials have been tempered by the later development of T-lymphoblastic leukemia and myelodysplasia as a result of insertional mutagenesis. The incidence varied among disease types, suggesting that disease background, transgene function, and individual genetic predisposition influence tumorigenicity. LV-based platform has been deployed in the past decade enabling more effective and safe insertion of therapeutic genes into HSC, with no evidence of leukemogenesis reported to this date. LV-HSCGT clinical trials for SCID-X1 and ADA-SCID have shown improved lymphocyte counts with clinical benefit.15 HSCGT for WAS resulted in amelioration of immune functions, including autoimmunity, and reduced incidence of severe bleeding events.3,16 Moreover, HSCGT-mediated restoration of oxidase function in X-CGD patients leading to protection from bacterial and fungal infections in most treated patients.17 Proposed research for the roadmap Continuous patients monitoring, also through analyses of vector insertion sites, will be required to confirm long-term safety and efficacy of patients who underwent HSCGT. Clinical trials for artemis deficiency, LAD-1, and osteopetrosis have recently started.2 Preclinical studies are actively pursued for other PIDs in which HSCGT could represent an alternative to allogeneic HSCT, also in the most severe or adult patients presenting with ongoing infections and/or organ damage including JAK3-SCID, PNP deficiency, RAG1/2 deficiency, ZAP70 deficiency, Munc 13-4 deficiency, and DADA2.18 HSCGT could also become available for less severe phenotypes, in which alloHSCT would not be indicated such as BTK deficiency. New gene editing technologies have the potential to circumvent some of the problems associated with viral gene addition and could be suitable for diseases requiring physiological regulation of gene expression or inactivation of dominant alleles. Preclinical proof of concept has been attained in correcting gene mutations for IL2RG, WAS, p47-CGD, CD40L deficiency, and IPEX, and clinical testing of these strategies is awaited. One of the key factors in the success of HSCGT for SCID and WAS relies on the selective advantage of functionally corrected lymphoid cells, previously observed in patients with somatic revertants and allogeneic HSCT. Thus, even in patients not receiving conditioning, active thymopoiesis was shown to be maintained for many years after treatment, suggesting durable thymic engraftment of long-lived lymphoid progenitors.2 On the other hand, conditioning is required to achieve polyclonal engraftment of gene-corrected HSC, restore normal B-cell lymphopoiesis and establish corrected myelopoiesis. SCIDs seem ideal candidates to explore the use of conditioning mediated by monoclonal antibodies or immunotoxins for HSCGT while exploiting the selective advantage in the lymphoid lineage. Preliminary results of a clinical trial in the context of allogeneic HSCT for SCID-X1 show sufficient degree of HSC engraftment.11 GT with autologous mature lymphocytes or lymphoid progenitors has also been explored preclinically with the rationale to provide in patients not eligible to allogeneic HSCT or HSCGT, immune responses to infection in diseases such as CD40L or perforin deficiency as well as control of immune dysregulation regulation in FOXP3 deficiency. Anticipated impact of the research Gene therapy for PIDs is moving from being an experimental approach to approved drug products that are routinely beneficial. The expansion of gene addition strategies and implementation of gene editing approaches, together with the standardization of the technology, will be important for increasing the armamentarium of approved therapies as an alternative to allogeneic HSCT. HEMOGLOBINOPATHIES Introduction Hemoglobinopathies are genetic defects of hemoglobin chain production, caused by mutations in the α- or β-globin gene clusters. The most frequent and severe are transfusion-dependent β-thalassemia (TDT) and sickle cell disease (SCD), characterized by a reduced or absent level of adult hemoglobin (HbA) and the production of an abnormal structural variant (HbS), respectively. In TDT, survival of patients depends on chronic blood transfusion associated to iron chelation. In SCD, sickling of deoxygenated red blood cells (RBCs) promotes painful vaso-occlusive crises, acute chest syndrome, stroke, and eventually death. Blood transfusion and fetal hemoglobin (HbF) induction by hydroxyurea represent current treatments. Allogeneic HSCT is curative for both diseases, but with limited availability of suitable donors and variability in clinical outcome depending on patient’s age. LV-mediated HSCGT relies on the erythroid specific expression of a normal gene copy and has been recently approved in Europe for patients over 12-years-old affected by less severe β-thalassemia mutations, and it is still in experimental trials for SCD.2 Gene editing strategies in the field of hemoglobinopathies have reached the stage of clinical testing, with ongoing phase 1/2 trials using ZFN or CRISPR/Cas technologies in SCD and in TDT. Reactivation of HbF synthesis by disruption of Bcl11a suppressor led to initial encouraging clinical results19 and longer follow-up will fully disclose the real potentiality as well the caveats of this novel approach.20 European research contributions European researchers have been prime actors in nonclinical and clinical research for the cure of TDT and SCD, contributing to the discovery of the disease molecular cause and the molecular mechanisms of globin genes regulation, introducing allogeneic HSCT and translating basic research to clinical application of gene therapy. Two successful clinical trials in TDT have been conducted in France21 and Italy,22 paving the way for application also in SCD.23 The results showed correction of the disease in most adult patients carrying nonsevere mutations and in young patients with severe mutations. The level of marked engrafted cells positively correlates with the clinical benefit, highlighting a threshold of genetically corrected cells to produce sufficient HbA per cell to rescue anemia and ineffective erythropoiesis in TDT, and to dilute abnormal HbS and preventing sickling in SCD. The functional status of HSC and the BM microenvironment is particularly relevant in TDT, where an impaired HSC-niche cross-talk has a negative impact on HSC functionality.24 Proposed research for the roadmap Gene therapy for hemoglobinopathies poses unique challenges, including high level of transgene expression for therapeutic correction and high global incidence worldwide. A single ATMP on the market (Zynteglo) will not be sufficient for such a prevalent disease but demonstration of dissimilarity of drug products might be challenging, thus discouraging biotech and/or pharma investments in further academic preclinical and clinical research. New rules governing the ATMP market should be introduced to favor the treatment of large p

Preliminary evidence suggests an association between obesity and gut inflammation. To evaluate the frequency of glucose abnormalities and their correlation with systemic and intestinal inflammation in severely obese children. Thirty-four children (25 males; median age 10.8 ± 3.4 yrs) with severe obesity (BMI >95%) were screened for diabetes with oral glucose tolerance test (OGTT), systemic inflammation with C-reactive protein (CRP) and gut inflammation with rectal nitric oxide (NO) and faecal calprotectin. BMI ranged from 23 to 44 kg/m2, and BMI z-score between 2.08 e 4.93 (median 2.69 ± 0.53). Glucose abnormalities were documented in 71% of patients: type 2 diabetes in 29%, impaired fasting glucose (IFG) in 58%, and impaired glucose tolerance (IGT) in 37.5%. Thirty-one patients (91%) were hyperinsulinemic. CRP was increased in 73.5% with a correlation between BMI z-score and CRP (p 0.03). Faecal calprotectin was increased in 47% patients (mean 77 ± 68), and in 50% of children with abnormal glucose metabolism (mean 76 ± 68 μg/g), with a correlation with increasing BMI z-score. NO was pathological in 88%, and in 87.5% of glucose impairment (mean 6.8 ± 5 μM). In this study, the prevalence of glucose abnormalities in obese children is higher than in other series; furthermore, a correlation is present between markers of systemic and intestinal inflammation and glucose abnormalities.

Wiskott-Aldrich syndrome (WAS) is a rare primary immunodeficiency caused by mutations in Wiskott-Aldrich syndrome protein (WASp), a key regulator of cytoskeletal dynamics in hematopoietic cells. A high proportion of patients experience autoimmunity caused by a breakdown in T- and B-cell tolerance. Moreover, excessive production of type I interferon (IFN-I) by plasmacytoid dendritic cells (pDCs) contributes to autoimmune signs; however, the factors that trigger excessive innate activation have not been defined.Neutrophil extracellular traps (NETs) emerged as major initiating factors in patients with diseases such as systemic lupus erythematosus and rheumatoid arthritis. In this study we explored the possible involvement of aberrant neutrophil functions in patients with WAS.We evaluated the expression of a set of granulocyte genes associated with NETs in a cohort of patients with WAS and the presence of NET inducers in sera. Using a mouse model of WAS, we analyzed NET release by WASp-null neutrophils and evaluated the composition and homeostasis of neutrophils in vivo. By using depletion experiments, we assessed the effect of neutrophils in promoting inflammation and reactivity against autoantigens.Transcripts of genes encoding neutrophil enzymes and antimicrobial peptides were increased in granulocytes of patients with WAS, and serum-soluble factors triggered NET release. WASp-null neutrophils showed increased spontaneous NETosis, induced IFN-I production by pDCs, and activated B cells through B-cell activating factor. Consistently, their depletion abolished constitutive pDC activation, normalized circulating IFN-I levels, and, importantly, abolished production of autoantibodies directed against double-stranded DNA, nucleosomes, and myeloperoxidase.These findings reveal that neutrophils are involved in the pathogenic loop that causes excessive activation of innate cells and autoreactive B cells, thus identifying novel mechanisms that contribute to the autoimmunity of WAS.

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.

Autoantibodies (AAbs) are a hallmark of Type 1 diabetes (T1D). Alterations in the frequency and phenotype of follicular helper (Tfh) T cells have been previously documented in patients with type 1 diabetes (T1D), but the contribution of follicular regulatory T (Treg) cells, which are responsible for suppressing AAb development, is less clear. Here, we investigated the frequency and activation status of follicular (CXCR5+) and conventional (CXCR5-) Treg cells in the blood of children with new-onset T1D, and children with risk for developing T1D (AAb-positive) and compared them to AAb-negative controls. Blood follicular and conventional Treg cells were higher in frequency in children with new onset T1D, but expressed reduced amounts of PD-1 as compared to AAb-negative children. Interestingly, the proportion of circulating FOXP3+ Tregs expressing PD-1 was also reduced in AAb-positive at-risk children as compared to AAb-negative controls, suggesting its potential use as a biomarker of disease progression. Follicular Treg cells were reduced in frequency in the spleens of prediabetic NOD mice as they became older and turned diabetic. Interestingly, PD-1 expression declined also on circulating follicular and conventional Treg cells in prediabetic NOD mice as they aged. Together, these findings show that the frequency of circulating follicular and conventional Treg cells and their levels of PD-1 change with disease progression in children at-risk for developing T1D and in NOD mice.

Mutations affecting the non-canonical pathway of NF-κB were recently identified to underlie a form of common variable immunodeficiency strongly associated with autoimmunity. Although intrinsic B-cell abnormalities explain most of the humoral defects of this disease, detailed data on the impact of NFKB2 on follicular helper (Tfh) and regulatory (Tregs) T cells are scarce. Here, we show that Tfh, CXCR5+, and CXCR5− Treg cell subsets were significantly reduced in patients heterozygous for a truncating mutation of NFKB2. Plasma CXCL13 levels were reduced, underlining an important role for NFKB2 in regulating the germinal center (GC) response. Proinflammatory IFNγ, IL-17 and IL-10 cytokine production by CD4 T cells was lower in the mutated patients, but the production of IL-4 and IL-21 was not altered. Taken together, our findings show that NFKB2 influences the quality and efficiency of human GC reaction, by affecting not only the B cells but also GC-relevant T cell subsets.

In the attempt to understand the origin of autoantibody (AAb) production in patients with and at risk for type 1 diabetes (T1D), multiple studies have analyzed and reported alterations in T follicular helper (Tfh) cells in presymptomatic AAb+ subjects and patients with T1D. Yet, whether the regulatory counterpart of Tfh cells, represented by T follicular regulatory (Tfr) cells, is similarly altered is still unclear. 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- and AAb+ subjects. While negligible differences in the frequency and phenotype of blood Tfr cells were observed among T1D, AAb-, and AAb+ adult subjects, the frequency of Tfr cells was significantly reduced in spleen and PLN of T1D as compared with nondiabetic control subjects. 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.

Stem cell therapy is a promising approach to regenerate healthy tissues starting from a limited amount of self-renewing cells. Immunological rejection of cell therapy products might represent a major limitation. In this study, we investigated the immunological functional profile of mesoangioblasts, vessel-associated myogenic stem cells, currently tested in a phase 1-2a trial, active in our Institute, for the treatment of Duchenne muscular dystrophy. We report that in resting conditions, human mesoangioblasts are poorly immunogenic, inefficient in promoting the expansion of alloreactive T cells and intrinsically resistant to T-cell killing. However, upon exposure to interferon-γ or differentiation into myotubes, mesoangioblasts acquire the ability to promote the expansion of alloreactive T cells and acquire sensitivity to T-cell killing. Resistance of mesoangioblasts to T-cell killing is largely due to the expression of the intracellular serine protease inhibitor-9 and represents a relevant mechanism of stem cell immune evasion.

Corrigendum1 December 2016Open Access Intra-arterial transplantation of HLA-matched donor mesoangioblasts in Duchenne muscular dystrophy Giulio Cossu Giulio Cossu Search for more papers by this author Stefano C Previtali Stefano C Previtali Search for more papers by this author Sara Napolitano Sara Napolitano Search for more papers by this author Maria Pia Cicalese Maria Pia Cicalese Search for more papers by this author Francesco Saverio Tedesco Francesco Saverio Tedesco Search for more papers by this author Francesca Nicastro Francesca Nicastro Search for more papers by this author Maddalena Noviello Maddalena Noviello Search for more papers by this author Urmas Roostalu Urmas Roostalu Search for more papers by this author Maria Grazia Natali Sora Maria Grazia Natali Sora Search for more papers by this author Marina Scarlato Marina Scarlato Search for more papers by this author Maurizio De Pellegrin Maurizio De Pellegrin Search for more papers by this author Claudia Godi Claudia Godi Search for more papers by this author Serena Giuliani Serena Giuliani Search for more papers by this author Francesca Ciotti Francesca Ciotti Search for more papers by this author Rossana Tonlorenzi Rossana Tonlorenzi Search for more papers by this author Isabella Lorenzetti Isabella Lorenzetti Search for more papers by this author Cristina Rivellini Cristina Rivellini Search for more papers by this author Sara Benedetti Sara Benedetti Search for more papers by this author Roberto Gatti Roberto Gatti Search for more papers by this author Sarah Marktel Sarah Marktel Search for more papers by this author Benedetta Mazzi Benedetta Mazzi Search for more papers by this author Andrea Tettamanti Andrea Tettamanti Search for more papers by this author Martina Ragazzi Martina Ragazzi Search for more papers by this author Maria Adele Imro Maria Adele Imro Search for more papers by this author Giuseppina Marano Giuseppina Marano Search for more papers by this author Alessandro Ambrosi Alessandro Ambrosi Search for more papers by this author Rossana Fiori Rossana Fiori Search for more papers by this author Maria Pia Sormani Maria Pia Sormani Search for more papers by this author Chiara Bonini Chiara Bonini Search for more papers by this author Massimo Venturini Massimo Venturini Search for more papers by this author Letterio S Politi Letterio S Politi Search for more papers by this author Yvan Torrente Yvan Torrente Search for more papers by this author Fabio Ciceri Fabio Ciceri Search for more papers by this author Giulio Cossu Giulio Cossu Search for more papers by this author Stefano C Previtali Stefano C Previtali Search for more papers by this author Sara Napolitano Sara Napolitano Search for more papers by this author Maria Pia Cicalese Maria Pia Cicalese Search for more papers by this author Francesco Saverio Tedesco Francesco Saverio Tedesco Search for more papers by this author Francesca Nicastro Francesca Nicastro Search for more papers by this author Maddalena Noviello Maddalena Noviello Search for more papers by this author Urmas Roostalu Urmas Roostalu Search for more papers by this author Maria Grazia Natali Sora Maria Grazia Natali Sora Search for more papers by this author Marina Scarlato Marina Scarlato Search for more papers by this author Maurizio De Pellegrin Maurizio De Pellegrin Search for more papers by this author Claudia Godi Claudia Godi Search for more papers by this author Serena Giuliani Serena Giuliani Search for more papers by this author Francesca Ciotti Francesca Ciotti Search for more papers by this author Rossana Tonlorenzi Rossana Tonlorenzi Search for more papers by this author Isabella Lorenzetti Isabella Lorenzetti Search for more papers by this author Cristina Rivellini Cristina Rivellini Search for more papers by this author Sara Benedetti Sara Benedetti Search for more papers by this author Roberto Gatti Roberto Gatti Search for more papers by this author Sarah Marktel Sarah Marktel Search for more papers by this author Benedetta Mazzi Benedetta Mazzi Search for more papers by this author Andrea Tettamanti Andrea Tettamanti Search for more papers by this author Martina Ragazzi Martina Ragazzi Search for more papers by this author Maria Adele Imro Maria Adele Imro Search for more papers by this author Giuseppina Marano Giuseppina Marano Search for more papers by this author Alessandro Ambrosi Alessandro Ambrosi Search for more papers by this author Rossana Fiori Rossana Fiori Search for more papers by this author Maria Pia Sormani Maria Pia Sormani Search for more papers by this author Chiara Bonini Chiara Bonini Search for more papers by this author Massimo Venturini Massimo Venturini Search for more papers by this author Letterio S Politi Letterio S Politi Search for more papers by this author Yvan Torrente Yvan Torrente Search for more papers by this author Fabio Ciceri Fabio Ciceri Search for more papers by this author Author Information Giulio Cossu, Stefano C Previtali, Sara Napolitano, Maria Pia Cicalese, Francesco Saverio Tedesco, Francesca Nicastro, Maddalena Noviello, Urmas Roostalu, Maria Grazia Natali Sora, Marina Scarlato, Maurizio De Pellegrin, Claudia Godi, Serena Giuliani, Francesca Ciotti, Rossana Tonlorenzi, Isabella Lorenzetti, Cristina Rivellini, Sara Benedetti, Roberto Gatti, Sarah Marktel, Benedetta Mazzi, Andrea Tettamanti, Martina Ragazzi, Maria Adele Imro, Giuseppina Marano, Alessandro Ambrosi, Rossana Fiori, Maria Pia Sormani, Chiara Bonini, Massimo Venturini, Letterio S Politi, Yvan Torrente and Fabio Ciceri EMBO Mol Med (2016)8:1470-1471https://doi.org/10.15252/emmm.201607129 This article corrects the following: Intra-arterial transplantation of HLA-matched donor mesoangioblasts in Duchenne muscular dystrophy05 November 2015 ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The authors of the above research article noticed that an error occurred during the labeling of Figure 2B, where “Pt 05 gastrocnemius (pre-treatment)” appeared twice on the upper lane and “Pt 06 gastrocnemius (post-treatment)” twice on the lower lane. The corrected labeling is included here in the new Figure 2 and reads as follows: “Pt 05 gastrocnemius (pre-treatment)” and “Pt 05 gastrocnemius (post-treatment)” on the upper lane, and “Pt 06 gastrocnemius (pre-treatment)” and “Pt 06 gastrocnemius (post-treatment)” on the lower lane. The results and conclusions of the article remain unchanged. The authors apologize for the oversight and any inconvenience caused. Figure 2. Download figure Download PowerPoint Next ArticlePrevious Article Read MoreAbout the coverClose modalView large imageVolume 8,Issue 12,December 2016Cover: “ TECRL, a new life‐threatening inherited arrhythmia gene associated with overlapping clinical features of both LQTS and CPVT” by Harsha D Devalla, Roselle Gélinas, Elhadi H Aburawi, Abdelaziz Beqqali, Arie O Verkerk, Lihadh Al‐Gazali, John D Rioux, Zahurul A Bhuiyan, Robert Passier and colleagues, also highlighted by Perry and Vandenberg. Novel recessive mutations in TECRL gene were identified in patients with exercise‐induced arrhythmias and high risk of sudden cardiac death. Patient‐specific human induced pluripotent stem cells differentiated to cardiomyocytes provided a valuable model for studying the disease at the cellular and molecular level. Mutant cardiomyocytes recapitulated salient features of the clinical phenotype in vitro and drug screening identified flecainide as a potential therapeutic. Cover concept by Harsha D. Devalla, Leiden University Medical Center. (Artistic rendition by Uta Mackensen) Volume 8Issue 121 December 2016In this issue FiguresRelatedDetailsLoading ...

Emapalumab, a fully human anti-IFNγ monoclonal antibody, has been approved in the US as second-line treatment of primary hemophagocytic lymphohistiocytosis (HLH) patients and has shown promise in patients with graft failure (GF) requiring a second allogeneic hematopoietic stem cell transplantation (HSCT). The blockade of IFNγ activity may increase the risk of severe infections, including fatal mycobacteriosis. We report a case of secondary HLH-related GF in the context of HLA-haploidentical HSCT successfully treated with emapalumab in the presence of concomitant life-threatening infections, including disseminated tuberculosis (TB). A 4 years old girl with Adenosine Deaminase-Severe Combined Immunodeficiency complicated by disseminated TB came to our attention for ex-vivo hematopoietic stem cell-gene therapy. After engraftment failure of gene corrected cells, she received two HLA-haploidentical T-cell depleted HSCT from the father, both failed due to GF related to concomitant multiple infections and secondary HLH. Emapalumab administration allowed to control HLH, as well as to prevent GF after a third haplo-HSCT from the mother. Remarkably, all infections improved with antimicrobial medications and disseminated TB did not show any reactivation. This seminal case supports emapalumab use for treatment of secondary HLH and prevention of GF in patients undergoing haplo-HSCT even in the presence of multiple infections, including TB.

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

Vaccination with Bacillus Calmette-Guérin (BCG) can be harmful to patients with combined primary immunodeficiencies. We report the outcome of BCG vaccination in a series of twelve patients affected by adenosine deaminase deficiency (ADA-SCID). BCG vaccination resulted in a very high incidence of complications due to uncontrolled replication of the mycobacterium. All patients who developed BCG-related disease were treated successfully and remained free from recurrence of disease. We recommend the prompt initiation of enzyme replacement therapy and secondary prophylaxis to reduce the risk of BCG-related complications in ADA-SCID patients.

Abstract Purpose Inborn errors of immunity (IEI) represent a heterogeneous group of rare genetically determined diseases. In some cases, patients present with complex or atypical phenotypes, not fulfilling the accepted diagnostic criteria for IEI and, thus, at high risk of misdiagnosis or diagnostic delay. This study aimed to validate a platform that, through the opinion of immunologist experts, improves the diagnostic process and the level of care of patients with atypical/complex IEI. Methods Here, we describe the functioning of the IEI-Virtual Consultation System (VCS), an innovative platform created by the Italian Immunodeficiency Network (IPINet). Results In the validation phase, from January 2020 to June 2021, 68 cases were entered on the IEI-VCS platform. A final diagnosis was achieved in 35/68 cases (51%, 95% CI 38.7 to 64.2). In 22 out of 35 solved cases, the diagnosis was confirmed by genetic analysis. In 3/35 cases, a diagnosis of secondary immunodeficiency was made. In the remaining 10 cases, an unequivocal clinical and immunological diagnosis was obtained, even though not substantiated by genetic analysis. Conclusion From our preliminary study, the VCS represents an innovative and useful system to improve the diagnostic process of patients with complex unsolved IEI disorders, with benefits both in terms of reduction of time of diagnosis and access to the required therapies. These results may help the functioning of other international platforms for the management of complex cases.

In physiological conditions, few circulating hematopoietic stem/progenitor cells (cHSPC) are present in the peripheral blood but their contribution to hematopoietic homeostasis in humans remain unsolved. By integrating advanced immunophenotyping, cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq), functional single-cell assays and integration site (IS) clonal tracking, we unveiled the phenotypic composition, the transcriptional features and the biological role of human cHSPC subpopulations in relationship to their bone marrow (BM) counterpart. We found that cHSPC progressively reduced in cell count over aging and are enriched for primitive, lymphoid and erythroid subpopulations, showing pre-activated transcriptional and functional state. Moreover, cHSPC have low expression of multiple BM-retention molecules, but maintain their homing potential after xenotransplantation. By generating a comprehensive Human Organ-Resident HSPC (HuOR) dataset based on scRNAseq data, we detected organ-specific seeding properties of the distinct trafficking HSPC subpopulations. Of note, circulating multi-lymphoid progenitors (MLP) are primed for seeding the thymus and actively contribute to T-cell production at steady state in patients treated with HSPC-gene therapy (GT). Human clonal tracking data from GT patients also showed that cHSPC connect distant BM niches and participate to steady-state hematopoietic production, with primitive cHSPC having the highest re-circulation capability to travel in and out the BM. Finally, in case of hematopoietic impairment, cHSPC composition reflects the BM-HSPC content and might represent a biomarker of the BM state for clinical and research purposes. Overall, our comprehensive work unveiled fundamental insights into the in vivo dynamics of human HSPC trafficking and its role in sustaining hematopoietic homeostasis.

Mucopolysaccharidosis type I Hurler (MPSIH) is characterized by severe and progressive skeletal dysplasia that is not fully addressed by allogeneic hematopoietic stem cell transplantation (HSCT). Autologous hematopoietic stem progenitor cell-gene therapy (HSPC-GT) provides superior metabolic correction in patients with MPSIH compared with HSCT; however, its ability to affect skeletal manifestations is unknown. Eight patients with MPSIH (mean age at treatment: 1.9 years) received lentiviral-based HSPC-GT in a phase 1/2 clinical trial (NCT03488394). Clinical (growth, measures of kyphosis and genu velgum), functional (motor function, joint range of motion), and radiological [acetabular index (AI), migration percentage (MP) in hip x-rays and MRIs and spine MRI score] parameters of skeletal dysplasia were evaluated at baseline and multiple time points up to 4 years after treatment. Specific skeletal measures were retrospectively compared with an external cohort of HSCT-treated patients. At a median follow-up of 3.78 years after HSPC-GT, all patients treated with HSPC-GT exhibited longitudinal growth within WHO reference ranges and a median height gain greater than that observed in patients treated with HSCT after 3-year follow-up. Patients receiving HSPC-GT experienced complete and earlier normalization of joint mobility compared with patients treated with HSCT. Mean AI and MP showed progressive decreases after HSPC-GT, suggesting a reduction in acetabular dysplasia. Typical spine alterations measured through a spine MRI score stabilized after HSPC-GT. Clinical, functional, and radiological measures suggested an early beneficial effect of HSPC-GT on MPSIH-typical skeletal features. Longer follow-up is needed to draw definitive conclusions on HSPC-GT's impact on MPSIH skeletal dysplasia.

Abstract Hematopoietic stem cell gene therapy (GT) using a γ-retroviral vector (γ-RV) is an effective treatment for Severe Combined Immunodeficiency due to Adenosine Deaminase deficiency. Here, we describe a case of GT-related T-cell acute lymphoblastic leukemia (T-ALL) that developed 4.7 years after treatment. The patient underwent chemotherapy and haploidentical transplantation and is currently in remission. Blast cells contain a single vector insertion activating the LIM-only protein 2 ( LMO2) proto-oncogene, confirmed by physical interaction, and low Adenosine Deaminase (ADA) activity resulting from methylation of viral promoter. The insertion is detected years before T-ALL in multiple lineages, suggesting that further hits occurred in a thymic progenitor. Blast cells contain known and novel somatic mutations as well as germline mutations which may have contributed to transformation. Before T-ALL onset, the insertion profile is similar to those of other ADA-deficient patients. The limited incidence of vector-related adverse events in ADA-deficiency compared to other γ-RV GT trials could be explained by differences in transgenes, background disease and patient’s specific factors.

Patient’s treatment with Sofosbuvir/Ledipasvir was supported by Fondazione Telethon. Patient’s treatment with Strimvelis under exceptional circumstances was supported by Fondazione Telethon and Regione Lombardia. GSK donated Strimvelis under exceptional circumstances. Patients with inborn error diseases can be candidates for autologous hematopoietic stem cells (HSC) gene therapies (GT) but may require negative viral screening, including hepatitis C virus (HCV), to allow HSC manipulation in Good Manufacturing Practices areas. In the 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. HCV clearance can now be achieved through direct‐acting antivirals. The fixed‐dose combination of ledipasvir/sofosbuvir (Harvoni; Gilead Sciences, Foster City, CA) in children ages 12 to 17 years was approved in 2017 for HCV genotypes 1, 4, 5, and 6.1 For younger patients, a phase II study (3‐17 years) with Harvoni is ongoing (ClinicalTrials.gov identifier: NCT02249182) and an anecdotal report of viral clearance has been published in a 4‐year‐old boy with HCV genotype 1b infection.3 Growing experience on direct‐acting antivirals in children, who are more often treatment naïvely and less likely to show hepatic cirrhosis, suggest that HCV can be eradicated in a short time, thus allowing potential eligibility to GT for subjects in need of urgent treatment. Presentation of the Case We report the experience in a male Egyptian infant affected by severe combined immunodeficiency caused by adenosine deaminase deficiency (ADA‐SCID) with HCV 4a genotype infection. The patient was not eligible for GT due to the infectious risk of bone marrow cells used as starting material for the manufacture of Strimvelis, based on the current EU Cell and Tissue Directive. Harvoni was administered off‐label to the patient and HCV clearance allowed the patient to be treated with autologous HSC‐GT with Strimvelis for the correction of his immunodeficiency.4 The patient was born from consanguineous parents and delivered by way of caesarean section. His older brother died due to ADA‐SCID. Our patient was soon diagnosed with ADA‐SCID and started on polyethylene‐glycol‐conjugated bovine adenosine deaminase (PEG‐ADA). In the absence of a human leukocyte antigen–identical sibling donor, the patient was referred at 4 months of age to our institution for HSC‐GT.5 At screening (Table 1), we diagnosed HCV 4a genotype infection and suspended the GT treatment program. Table 1 - Clinical Data and Significant Laboratory Findings at the Start of Harvoni Age (months) 5 Weight (kg) (percentile) 6.5 (50th) Length (cm) (percentile) 58.7 (3rd) White blood cell (×109/L) (normal value, 6.7‐14×109/L) 3.8 Lymphocytes (×109/L) (normal value, 3.9‐9.0) 0.8 CD3+ (cells/μL) (normal value, (2500‐5600) 16 CD3+CD4+ (cells/μL) (normal value, 1800‐4000) 13 CD3+CD8+ (cells/μL) (normal value, 590‐1600) 0.4 CD4+ naïve (cells/μL) (normal value, 1300‐3600) 1.2 ALT (IU/L) (normal value, 6‐59 IU/L) 135 AST (IU/L) (normal value, 5‐35 IU/L) 85 Total bilirubin (mg/dL) (normal value 0.1‐1.0) 0.19 Direct bilirubin (mg/dL) (normal value 0.01‐0.25) 0.14 GGT (IU/L) (normal value 11‐68) 31 HCV antibody Negative* Liver ultrasound Normal *The child was on immunoglobulin supplementation, given to the known impaired antibody production in ADA SCID patients.Abbreviations: AST, aspartate aminotransferase; GGT, gamma‐glutamyl transferase. Vertical transmission from the mother was ruled out, as testing resulted in HCV‐RNA and antibody being negative. Transfusions were never administered to the child and infection from substitutive immunoglobulins was considered highly unlikely. The patient’s contagion during caesarean section or blood withdrawal in his home country, where HCV genotype 4 is endemic, was postulated. Because of the patient’s profound lymphopenia and reduced T‐cell functions, despite the ongoing enzyme replacement therapy with PEG‐ADA, spontaneous clearance of HCV was considered unlikely and the patient was judged at risk of poor control of HCV infection. Moreover, the risk/benefit of early eradication of the HCV infection was favorable because of the urgency of treatment with Strimvelis.4 Sofosbuvir/ledipasvir was started at 5 months of age at 100/22.5 mg (15/3.4 mg/kg/day) daily dose and continued for 12 weeks. The dose was extrapolated from that used in adults and children over 6 years of age1 and from treatment duration time for genotype 4a.2 Tablets were pulverized by San Raffaele Hospital pharmacy and the required daily dose was packed in single sachets. The powder was dissolved into milk and regularly administered by parents, after appropriate training. Approval of the local Ethical Committee and from the hospital management board for off‐label use and written informed consent from the parents were obtained prior to treatment. A diffuse maculo‐papular skin rash appeared after 1 week of Harvoni, but did not contraindicate the continuation of therapy and resolved in 1 month. Rapid viral clearance was observed (Fig. 1). Following consultation with European Medicines Agency (EMA), a risk‐assessment procedure was formulated for the manipulation of HSC from a patient with previous HCV infection and shared with the Competent Authority for Cells and Tissues (Centro Nazionale Trapianti). Based on the evidence that sustained virological response at week 4 (SVR4) is 95% to 100% predictive of SVR24,1 the risk assessment was favorable, and the patient underwent treatment with Strimvelis (Fig. 1) after conditioning with a low dose of busulfan.4Figure 1: HCV‐RNA and alanine aminotransferase (ALT) during treatment with Harvoni. HCV‐RNA levels persisted high for 1 month before starting Harvoni. Plasmatic viral copies were 1.85 × 106 UI/mL at diagnosis and 1.5 × 106 UI/mL in more than one evaluation when Harvoni was started. After 2 weeks of treatment, the viral load resulted in being negative, but on the following plasma evaluation, the HCV‐RNA was 19 copies/mL, probably due to a slight initial fluctuation around the lower threshold of PCR method and returned persistently negative in all of the following evaluations. Although the family did not report episodes of vomiting or lack of compliance, we cannot exclude a transient issue of administration in the first weeks of treatments. From week 5 from the start of Harvoni onward, HCV‐RNA was undetectable, allowing its suspension at week 12. ALT values remarkably decreased during treatment. A transient ALT increase with persistent HCV‐RNA negativity was detected from weeks 3 to 11 from the start of Harvoni and ascribed to insufficient detoxification; it resolved after PEG‐ADA dose adjustment. Hepatoprotective drugs (silymarin 6 mg/kg/day and ursodeoxycholic acid 20 mg/kg/day, oral formulations) were administered from week 4, according to our local protocol for hepatic prophylaxis in the context of hematopoietic stem cell transplantation. This may be more relevant for ADA‐SCID patients, who are known to experience also hepatic toxic damage due to accumulation of adenosine metabolites.6 PEG‐ADA was stopped 3 weeks before GT, as foreseen in the treatment schedule. The post‐GT slight ALT increase may be ascribed to busulfan administration and the PEG‐ADA suspension carryover effect. The threshold of less than 12 IU/mL HCV‐RNA represents our rate of undetectable HCV‐RNA levels in real‐time PCR.HCV‐RNA negative testing persisted during GT and throughout the follow‐up. Currently, the patient is +11 months from GT, free from PEG‐ADA, and shows multilinear engraftment of transduced HSC in the bone marrow, increasing lymphocyte count, and restored ADA expression. Discussion A once‐daily single dose regimen of 12‐week treatment duration with sofosbuvir/ledipasvir had an acceptable safety profile and was efficacious in the clearance of HCV infection in an ADA‐SCID patient allowing GT with Strimvelis. Moreover, in this patient, SVR4 was predictive of SVR12, SVR24, and SVR48. The occurrence of this case triggered a change in the control of procedures for Strimvelis, to allow autologous bone marrow to be used for manufacture if the HCV results from the patient are confirmed as negative by nucleic acid testing. Additional data on a cohort of HCV‐infected children younger than 3 years are needed to provide further evidence of the safety and efficacy of this treatment. Our results support the need for the development of specific recommendations regarding the use of Harvoni off‐label, while waiting for a label extension, in infants who need life‐saving treatment with advanced therapies. Potential conflict of interest Nothing to report.

Metachromatic Leukodystrophy (MLD; MIM# 250100) is a rare inherited lysosomal storage disorder caused by the deficiency of Arylsulfatase A (ARSA). The enzymatic defect results in the accumulation of the ARSA substrate that is particularly relevant in myelin forming cells and leads to progressive dysmyelination and dysfunction of the central and peripheral nervous system. Sulfatide accumulation has also been reported in various visceral organs, although little is known about the potential clinical consequences of such accumulation. Different forms of MLD-associated gallbladder disease have been described, and there is one reported case of an MLD patient presenting with functional consequences of sulfatide accumulation in the kidney. Here we describe a wide cohort of MLD patients in whom a tendency to sub-clinical metabolic acidosis was observed. Furthermore in some of them we report episodes of metabolic acidosis of different grades of severity developed in acute clinical conditions of various origin. Importantly, we finally show how a careful acid-base balance monitoring and prompt correction of imbalances might prevent severe consequences of acidosis.

Wiskott-Aldrich syndrome (WAS) is an X-linked disorder characterized by thrombocytopenia, eczema, and immunodeficiency. Up to 70% of patients with WAS present with at least 1 autoimmune or autoinflammatory episode, and many of them suffer from recurrent or multiple events.1Dupuis-Girod S. Medioni J. Haddad E. Quartier P. Cavazzana-Calvo M. Le Deist F. et al.Autoimmunity in Wiskott-Aldrich syndrome: risk factors, clinical features, and outcome in a single-center cohort of 55 patients.Pediatrics. 2003; 111: e622-e627Crossref PubMed Scopus (268) Google Scholar, 2Chen N. Zhang Z.Y. Liu D.W. Liu W. Tang X.M. Zhao X.D. The clinical features of autoimmunity in 53 patients with Wiskott-Aldrich syndrome in China: a single-center study.Eur J Pediatr. 2015; 174: 1311-1318Crossref PubMed Scopus (13) Google Scholar, 3Imai K. Morio T. Zhu Y. Jin Y. Itoh S. Kajiwara M. et al.Clinical course of patients with WASP gene mutations.Blood. 2004; 103: 456-464Crossref PubMed Scopus (304) Google Scholar IL-1 new-generation blockers have been used in patients exhibiting clinical symptoms compatible with an autoinflammatory condition,4Geusau A. Mothes-Luksch N. Nahavandi H. Pickl W.F. Wise C.A. Pourpak Z. et al.Identification of a homozygous PSTPIP1 mutation in a patient with a PAPA-like syndrome responding to canakinumab treatment.JAMA Dermatol. 2013; 149: 209-215Crossref PubMed Scopus (59) Google Scholar but have not been reported in WAS. Here, we describe a patient with WAS with a peculiar large genomic inversion presenting with multiple manifestations of immune dysregulation, in whom autoinflammatory manifestations improved after the use of anakinra (IL-1 receptor antagonist, Kineret). A 11.6-year-old boy was referred to our center for suspected immunodeficiency. The patient presented with a history of microthrombocytopenia since birth and eczema in the first years of life, suggestive of WAS. Analysis of WAS protein (WASp) expression was reported abnormal, but Sanger sequencing on DNA did not reveal mutations. From 1.5 years of age he underwent recurrent episodes of postinfectious vasculitis of the lower limbs and arthritis. At 7.5 years, he presented with a bilateral pneumonia that triggered Schonlein-Henoch purpura with fever and arthritis, managed with oral steroids. Subsequently, a nephritic-nephrotic syndrome was treated with antihypertensive treatment and high-dose corticosteroids (CCS), with partial response. Cyclosporin A (CyA) and CCS led to remission of renal disease, which relapsed after CyA was stopped. Intravenous high-dose CCS and anti-CD20 mAb did not lead to substantial improvement. CyA and low-dose prednisone were restarted with partial benefit. However, the patient experienced varicella zoster reactivation on his half-right-face, with sequelae to the right eye (anterior and posterior uveitis with acute retinitis) requiring a vitrectomy, and severe impairment of visual function. An anterior uveitis at the left eye was treated with steroids. At the age of 9.8 years, he developed clinical and histological features of pancolitic Crohn disease, managed with an increase in CCS, as well as arthritis and histologically confirmed vasculitis and eventually pyoderma gangrenosum (PG) on the hips, buttocks, and upper and lower limbs. Crohn disease was not responsive to infliximab, thalidomide, cyclophosphamide, or high-dose intravenous steroids, while adalimumab (Humira) resulted in an initial benefit (see Table E1 in this article's Online Repository at www.jacionline.org). The patient presented with fistulas and perianal abscesses when he was 10.7 years old and he underwent several fistulectomies and removal of granulation tissue in the perianal area by “cone-like technique.” For the poor control of the enterocolitis, a subtotal colectomy with terminal ileostomy was performed at age 11 years. When the patient was referred to our center, he was on adalimumab and low-dose CCS with a good control of bowel disease, but still showed severe manifestations of PG on the upper limbs and in the perianal area (Fig 1, A; see Table E2 in this article's Online Repository at www.jacionline.org). His parents signed informed consent for research investigations (protocol Tiget06). Because of the strong suspicion of WAS, whole-genome sequencing was performed and an inversion of 6kb spanning from the promoter to the intronic region between exons 7 and 8 was detected (see Fig E1 in this article's Online Repository at www.jacionline.org). Specific primers in this region identified the precise breaking points (see Tables E3 and E4 in this article's Online Repository at www.jacionline.org; Fig 2, B). The rearranged allele was present in the patient and his mother, whereas the patient's aunt was unaffected (data not shown and Fig 2, A-C). RNA analyses showed an aberrant transcript produced from the inverted region (Fig 2, D). WASp expression, analyzed by flow cytometry (see Fig E2, A, in this article's Online Repository at www.jacionline.org), was deeply reduced in peripheral blood T-, B-, and natural killer lymphocytes and monocytes (data not shown) while it was undetectable by Western blot performed with an antibody recognizing the N-terminal portion of WASp including exons 7 and 8 (Fig E2, B). WASp expression was restored in the patient's T-cell line transduced with a lentiviral vector expressing WASp under the control of the autologous 1.6-kb long promoter5Scaramuzza S. Biasco L. Ripamonti A. Castiello M.C. Loperfido M. Draghici E. et al.Preclinical safety and efficacy of human CD34(+) cells transduced with lentiviral vector for the treatment of Wiskott-Aldrich syndrome.Mol Ther. 2013; 21: 175-184Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar (Fig E2, C). The start of low-dose methotrexate (Reumaflex) and the increase in prednisone led to a moderate improvement in the PG after 3 months (Fig 1, B), but shortly after the patient underwent a reactivation of vasculitis and arthritis with systemic inflammation that was not controlled by multiple immunosuppressive and anti-inflammatory drugs. On the basis of the reported efficacy of IL-1 blockers in the treatment of autoinflammatory manifestations and of PG,4Geusau A. Mothes-Luksch N. Nahavandi H. Pickl W.F. Wise C.A. Pourpak Z. et al.Identification of a homozygous PSTPIP1 mutation in a patient with a PAPA-like syndrome responding to canakinumab treatment.JAMA Dermatol. 2013; 149: 209-215Crossref PubMed Scopus (59) Google Scholar anakinra was started as an off-label drug titrating the dose from 1 up to 3 mg/kg/day. This led to a resolution of vasculitis and arthritis and to a decrease in the inflammation indexes within few days (Fig 1, D) with dramatic improvement in the PG skin lesions during the following 5 months (Fig 1, C). The patient was enrolled in a gene therapy trial based on autologous gene-corrected hematopoietic stem cells (clinicaltrials.gov #NCT01515462), mobilized with G-CSF and plerixafor. Treatment with anakinra was discontinued 48 hours before mobilization, but was soon restarted because of the increase in white blood cells and inflammation indexes with exacerbation of skin lesions, arthralgia, and hematuria, and led again to a rapid laboratory and clinical remission (data not shown). Notably, the use of anakinra allowed a successful mobilization with G-CSF without the occurrence of other autoinflammatory manifestations. To our knowledge, this is the first reported case of use of IL-1R blocker in a patient with WAS, with clinical benefit. This case is very emblematic for several reasons. Whole-genome sequencing complemented by specific breakpoint sequencing allowed the identification of the inversion with intact exon sequences, elucidating the previous normal genetic analysis. Complex genomic rearrangements involving inversions are generally noncanonical gene conversion events6Chen J.M. Chuzhanova N. Stenson P.D. Férec C. Cooper D.N. Intrachromosomal serial replication slippage in trans gives rise to diverse genomic rearrangements involving inversions.Hum Mutat. 2005; 26: 362-373Crossref PubMed Scopus (58) Google Scholar and could have occurred in an ancestor allele in the family through a de novo mutation occurring in the mother. Autoimmune and autoinflammatory manifestations in patients with WAS typically present early in life, are often refractory to therapy, and are associated with a worse clinical prognosis and an increased risk of developing a malignancy.3Imai K. Morio T. Zhu Y. Jin Y. Itoh S. Kajiwara M. et al.Clinical course of patients with WASP gene mutations.Blood. 2004; 103: 456-464Crossref PubMed Scopus (304) Google Scholar, 7Moratto D. Giliani S. Bonfim C. Mazzolari E. Fischer A. Ochs H.D. et al.Long-term outcome and lineage-specific chimerism in 194 patients with Wiskott-Aldrich syndrome treated by hematopoietic cell transplantation in the period 1980-2009: an international collaborative study.Blood. 2011; 118: 1675-1684Crossref PubMed Scopus (232) Google Scholar Our patient's autoinflammatory manifestations were resistant to several immunosuppressive drugs and the use of CyA was associated with a severe viral complication. Anakinra dramatically improved PG, vasculitis, and arthritis, showed a good safety profile, and allowed stabilization of the patient for definitive treatment. The response to anakinra suggests that the dysregulation of the innate immune system is involved in the genesis of autoinflammatory manifestations in patients with WAS and shows that IL-1 may serve in selected cases as a target for therapy, avoiding the use of other classes of immunosuppressors that can increase the risk for severe infections. It has been hypothesized that defects in chemotaxis and podosomes formation in WASp-deficient cells may favor the onset of autoinflammatory manifestations. In addition, a recent study in a patient with aggressive PG showed a critical role for proline-serine-threonine phosphatase interacting protein 1, which is involved in cytoskeletal regulatory functions through interaction with WASp, in the Pyogenic Arthritis, Pyoderma gangrenosum, and Acne syndrome.8Starnes T.W. Bennin D.A. Bing X. Eickhoff J.C. Grahf D.C. Bellak J.M. et al.The F-BAR protein PSTPIP1 controls extracellular matrix degradation and filopodia formation in macrophages.Blood. 2014; 123: 2703-2714Crossref PubMed Scopus (63) Google Scholar A greater understanding of the role of WASp in inflammation and of potential pathways that may be targeted therapeutically to modulate immunity in WAS is desirable to improve the management of the affected patients while waiting for definitive treatment by stem cell transplantation or gene therapy. Fig E2WASp expression. A, Flow cytometry characterization of WASp expression in patient and HC lymphocytes. Percentage of WASp+ cells is reported on histograms. Detection of WASp was performed after permeabilization (Cytofix/Cytoperm kit; BD Biosciences, San Jose, Calif) by a noncommercial rabbit polyclonal antibody (“EFOB,” generated against a WASp peptide), kindly provided by GSK. Gray line: negative control. B, Western blot performed on untransformed T-cell line generated in vitro from patient and his parents. Antibody for detection: polyclonal H250 (BD). C, Restoration of WASp expression in a patient's T-cell line after transduction with LV. GAPDH, Glyceraldehyde 3-phosphate dehydrogenase; HC, healthy control; LV, lentiviral vector; Pt, patient.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Table E1Summary of treatment of possible autoimmune and autoinflammatory manifestationsTreatmentResponseNephritic/nephrotic syndrome SteroidsPartial Steroids + CyARemission, then relapse when CyA stopped Steroids + anti-CD20 mAbNo Steroids + CyAPartial; infectious complicationsCrohn disease Steroids + CyANo Steroids + CyA + IVIGNo Steroids + CyA + infliximabPartial, then relapse Steroids + CyA + talidomideNo Steroids + cyclophosphamideNo Steroids + adalimumabYesPG Steroids + dapsone + adalimumabNo Steroids + dapsone + adalimumab + MTXPartial Steroids + dapsone + anakinraYesArthritis and vasculitis Steroids + CyARemission, then relapse Steroids + CyA + infliximabNo Steroids + dapsone + adalimumab + MTXNo Steroids + dapsone + anakinraYesIVIG, Intravenous immunoglobulin; MTX, methotrexate. Open table in a new tab Table E2Clinical data and significant laboratory findings at WAS diagnosisAge of onset/diagnosis of diseaseNeonatal/11.6 yGenetic analyses (whole-genome sequencing)inv(X)g.5721-11840Clinical manifestationsMicrothrombocytopeniaMild eczema, severe infections, arthritis, vasculitisMultiple Henoch-Schonlein purpura episodesNephritic-nephrotic syndrome, panuveitisSteroid-resistant Chron enterocolitisPerianal fistulas and abscessesPGZhu score5AE1Zhu Q. Watanabe C. Liu T. Hollenbaugh D. Blaese R.M. Kanner S.B. et al.Wiskott-Aldrich syndrome/X-linked thrombocytopenia: WASP gene mutations, protein expression, and phenotype.Blood. 1997; 90: 2680-2689PubMed Google ScholarWBC (×109/L) (normal value, 4.8-10.8 × 109/L)14.4Neutrophils (×109/L) (normal value, 1.8-7.7 × 109/L)12.7Lymphocytes (×109/L)1Platelets range (×109/L)20-40MPV (fl) (normal value, 9.1-12.5)8CD3+ (cells/μL) (normal value, 1000-2100)E2Comans-Bitter W.M. de Groot R. van den Beemd R. Neijens H.J. Hop W.C. Groeneveld K. et al.Immunophenotyping of blood lymphocytes in childhood: reference values for lymphocyte subpopulations.J Pediatr. 1997; 130: 388-393Abstract Full Text Full Text PDF PubMed Scopus (613) Google Scholar498CD3+CD4+ (cells/μL) (normal value, 500-1300)E2Comans-Bitter W.M. de Groot R. van den Beemd R. Neijens H.J. Hop W.C. Groeneveld K. et al.Immunophenotyping of blood lymphocytes in childhood: reference values for lymphocyte subpopulations.J Pediatr. 1997; 130: 388-393Abstract Full Text Full Text PDF PubMed Scopus (613) Google Scholar364CD3+CD8+ (cells/μL) (normal value, 220-950)E2Comans-Bitter W.M. de Groot R. van den Beemd R. Neijens H.J. Hop W.C. Groeneveld K. et al.Immunophenotyping of blood lymphocytes in childhood: reference values for lymphocyte subpopulations.J Pediatr. 1997; 130: 388-393Abstract Full Text Full Text PDF PubMed Scopus (613) Google Scholar91CD19+ (cells/μL) (normal value, 180-600)E2Comans-Bitter W.M. de Groot R. van den Beemd R. Neijens H.J. Hop W.C. Groeneveld K. et al.Immunophenotyping of blood lymphocytes in childhood: reference values for lymphocyte subpopulations.J Pediatr. 1997; 130: 388-393Abstract Full Text Full Text PDF PubMed Scopus (613) Google Scholar101CD16+CD56+ (cells/μL) (normal value, 170-670)E2Comans-Bitter W.M. de Groot R. van den Beemd R. Neijens H.J. Hop W.C. Groeneveld K. et al.Immunophenotyping of blood lymphocytes in childhood: reference values for lymphocyte subpopulations.J Pediatr. 1997; 130: 388-393Abstract Full Text Full Text PDF PubMed Scopus (613) Google Scholar338CD4+CD45RA+ (cells/μL) (normal value, 320-1000)E3Shearer W.T. Rosenblatt H.M. Gelman R.S. Oyomopito R. Plaeger S. Stiehm R. et al.Lymphocyte subsets in healthy children from birth through 18 years of age: the Pediatric AIDS Clinical Trials Group P1009 study.J Allergy Clin Immunol. 2003; 112: 973-980Abstract Full Text Full Text PDF PubMed Scopus (634) Google Scholar152CD4+CD45RO+ (cells/μL) (normal value, 230-630)E3Shearer W.T. Rosenblatt H.M. Gelman R.S. Oyomopito R. Plaeger S. Stiehm R. et al.Lymphocyte subsets in healthy children from birth through 18 years of age: the Pediatric AIDS Clinical Trials Group P1009 study.J Allergy Clin Immunol. 2003; 112: 973-980Abstract Full Text Full Text PDF PubMed Scopus (634) Google Scholar201CD8+CD45RA+ (cells/μL) (normal value, 310-900)E3Shearer W.T. Rosenblatt H.M. Gelman R.S. Oyomopito R. Plaeger S. Stiehm R. et al.Lymphocyte subsets in healthy children from birth through 18 years of age: the Pediatric AIDS Clinical Trials Group P1009 study.J Allergy Clin Immunol. 2003; 112: 973-980Abstract Full Text Full Text PDF PubMed Scopus (634) Google Scholar159CD8+CD45RO+ (cells/μL) (normal value, 70-390)E3Shearer W.T. Rosenblatt H.M. Gelman R.S. Oyomopito R. Plaeger S. Stiehm R. et al.Lymphocyte subsets in healthy children from birth through 18 years of age: the Pediatric AIDS Clinical Trials Group P1009 study.J Allergy Clin Immunol. 2003; 112: 973-980Abstract Full Text Full Text PDF PubMed Scopus (634) Google Scholar40Vβ repertoirePolyclonal on CD3+IgG (g/L) (normal value, 7.07-19.19)E4Burgio GR, Perinotto G, Ugazio AG. Pediatria essenziale. UTET editore. 1991.Google Scholar7.69 (on IVIG supplementation)IgA (g/L) (normal value, 0.60-2.7)E4Burgio GR, Perinotto G, Ugazio AG. Pediatria essenziale. UTET editore. 1991.Google Scholar6.68IgM (g/L) (normal value, 0.61-2.76)E4Burgio GR, Perinotto G, Ugazio AG. Pediatria essenziale. UTET editore. 1991.Google Scholar0.33Autoimmunity screening∗Including Coombs test, ANA, ENA, AMA, ASMA, anti–LKM1 antibody, anti–dS-DNA antibody, ASCA, cANCA, pANCA, ACA, and anti–β2 glicoprotein antibody.NegativeAntiplatelets antibody (indirect test)PositiveLLACNegativeACA, Anticardiolipin antibody; AMA, antimitochondrial antibody; ANA, antinuclear antibody; ASCA, anti–Saccharomyces cerevisiae antibody; ASMA, anti–smooth muscle antibody; cANCA, cytoplasmic antineutrophilic cytoplasmic antibodies; ds-DNA, double-strand DNA antibody; ENA, extractable nuclear antibodies; IVIG, intravenous immunoglobulin; LLAC, lupus-like anticoagulant; LKM1, liver kidney microsomal type 1 antibody; pANCA, protoplasmic antineutrophilic cytoplasmic antibodies; WBC, white blood cell.∗ Including Coombs test, ANA, ENA, AMA, ASMA, anti–LKM1 antibody, anti–dS-DNA antibody, ASCA, cANCA, pANCA, ACA, and anti–β2 glicoprotein antibody. Open table in a new tab Table E3Primers for the detection of DNA inversionPrimerOrientationSequencePCR cyclesAFORCTGGAGATACGCCTAGCACA94°C 3 min, 94°C 30 s, 55°C 30 s, 72°C 1 min × 40 cycles, 72°C 10 minREVTCTGAGGGCTGTAGGGTTTGBFORAAGGTGGGCACATGGGTAG94°C 3 min, 94°C 30 s, 55°C 30 s, 72°C 1 min × 40 cycles, 72°C 10 minREVTCTCAGCAGTGGTCTTTGGA Open table in a new tab Table E4Primers for amplification of cDNARegionOrientationSequencePCR cyclesEXON1-4 (279bp)FORCGAAAATGCTTGACGCTGGC94°C 3 min, 94°C 30 s, 54.6°C 30 s, 72°C 1 min × 40 cycles, 72°C 10 minREVCTCGTCCTCGTCTGCAAAGTEXON7-10 (303bp)FORACCCAGTGGATTCAAGCATGT94°C 3 min, 94°C 30 s, 54.6°C 30 s, 72°C 1 min × 40 cycles, 72°C 10 minREVGGACCAGAACGACCCTTGTTEXON2-7 (449bp)FORGTCCTACTTCATCCGCCTTTAC94°C 3 min, 94°C 30 s, 55.8°C 30 s, 72°C 1 min × 40 cycles, 72°C 10 minREVTCTTCCCTGAGCGTTTCTTATCEXON7-8 (73bp)FORGTGGATTCAAGCATGTCAGCC94°C 3 min, 94°C 30 s, 55.8°C 30 s, 72°C 1 min × 40 cycles, 72°C 10 minREVTCTGGGTCGAGGTTGTTCAC Open table in a new tab IVIG, Intravenous immunoglobulin; MTX, methotrexate. ACA, Anticardiolipin antibody; AMA, antimitochondrial antibody; ANA, antinuclear antibody; ASCA, anti–Saccharomyces cerevisiae antibody; ASMA, anti–smooth muscle antibody; cANCA, cytoplasmic antineutrophilic cytoplasmic antibodies; ds-DNA, double-strand DNA antibody; ENA, extractable nuclear antibodies; IVIG, intravenous immunoglobulin; LLAC, lupus-like anticoagulant; LKM1, liver kidney microsomal type 1 antibody; pANCA, protoplasmic antineutrophilic cytoplasmic antibodies; WBC, white blood cell. CorrectionJournal of Allergy and Clinical ImmunologyVol. 139Issue 5PreviewWith regard to the article in the August 2016 issue entitled “A novel genomic inversion in Wiskott-Aldrich–associated autoinflammation” (J Allergy Clin Immunol 2016;138:619-22.e7), the authors wish to amend the Acknowledgment section. The authors would like to thank the Italian Ministero della Salute (Programma di rete, NET-2011-02350069) and the European Commission (ERARE-3-JTC 2015 EUROCID). I.B. is supported by the Italian Ministero della Salute (GR-2011-02346985). The authors regret the omission. Full-Text PDF Open Access

EDITORIAL article Front. Immunol., 29 May 2020Sec. T Cell Biology Volume 11 - 2020 | https://doi.org/10.3389/fimmu.2020.01042

Medical advances have dramatically improved the long-term prognosis of children and adolescents with inborn errors of immunity (IEIs). Transfer of the medical care of individuals with pediatric IEIs to adult facilities is also a complex task because of the large number of distinct disorders, which requires involvement of patients and both pediatric and adult care providers. To date, there is no consensus on the optimal pathway of the transitional care process and no specific data are available in the literature regarding patients with IEIs. We aimed to develop a consensus statement on the transition process to adult health care services for patients with IEIs. Physicians from major Italian Primary Immunodeficiency Network centers formulated and answered questions after examining the currently published literature on the transition from childhood to adulthood. The authors voted on each recommendation. The most frequent IEIs sharing common main clinical problems requiring full attention during the transitional phase were categorized into different groups of clinically related disorders. For each group of clinically related disorders, physicians from major Italian Primary Immunodeficiency Network institutions focused on selected clinical issues representing the clinical hallmark during early adulthood.

ABSTRACT Background and Aims: Octreotide reduces splanchnic blood flow and is effective in controlling gastrointestinal bleeding (GIB) caused by portal hypertension. Monthly long‐acting octreotide (OCT‐LAR) with an efficacy and safety profile similar to subcutaneous daily administration presents an attractive option for long‐term therapy. We report our experience with OCT‐LAR for severe/recurrent GIB in children with portal hypertension secondary to chronic liver disease or portal vein thrombosis who were unresponsive to standard interventions. Methods: A total of 9 patients, 7 boys, who received OCT‐LAR between 2000 and 2009 were studied retrospectively (median age at first bleeding 21 months, range 1 month–14.5 years). The dose (2.5–20 mg intramuscularly monthly) was extrapolated from that used in adult acromegaly and neuroendocrine tumours (10–60 mg/mo). Response to treatment was assessed by comparing the number of bleeding events, hospital admissions for acute bleeding, and number of blood units required during the year before and year after starting OCT‐LAR. Results: OCT‐LAR led to a reduction in the number of bleeding episodes in all of the children and to cessation of bleeding in 7. Two children listed for transplantation because of severe GIB were removed from the list. No serious adverse effects immediately attributable to OCT‐LAR were observed. One child developed growth hormone deficiency and hypothyroidism during a prolonged period of treatment with subcutaneous octreotide before commencing OCT‐LAR. Conclusions: OCT‐LAR can control severe intractable recurrent GIB in children with portal hypertension. Prospective randomised controlled trials and pharmacokinetic studies are indicated to establish the optimum dose and length of treatment of OCT‐LAR and confirm its efficacy and long‐term safety in children.

Hematopoietic stem and progenitor cell (HSPC)-based gene therapy (GT) requires the collection of a large number of cells. While bone marrow (BM) is the most common source of HSPCs in pediatric donors, the collection of autologous peripheral blood stem cells (PBSCs) is an attractive alternative for GT. We present safety and efficacy data of a 10-year cohort of 45 pediatric patients who underwent PBSC collection for backup and/or purification of CD34+ cells for ex vivo gene transfer. Median age was 3.7 years and median weight 15.8 kg. After mobilization with lenograstim/plerixafor (n = 41) or lenograstim alone (n = 4) and 1−3 cycles of leukapheresis, median collection was 37 × 106 CD34+ cells/kg. The procedures were well tolerated. Patients who collected ≥7 and ≥13 × 106 CD34+ cells/kg in the first cycle had pre-apheresis circulating counts of at ≥42 and ≥86 CD34+ cells/μL, respectively. Weight-adjusted CD34+ cell yield was positively correlated with peripheral CD34+ cell counts and influenced by female gender, disease, and drug dosage. All patients received a GT product above the minimum target, ranging from 4 to 30.9 × 106 CD34+ cells/kg. Pediatric PBSC collection compares well to BM harvest in terms of CD34+ cell yields for the purpose of GT, with a favorable safety profile. Hematopoietic stem and progenitor cell (HSPC)-based gene therapy (GT) requires the collection of a large number of cells. While bone marrow (BM) is the most common source of HSPCs in pediatric donors, the collection of autologous peripheral blood stem cells (PBSCs) is an attractive alternative for GT. We present safety and efficacy data of a 10-year cohort of 45 pediatric patients who underwent PBSC collection for backup and/or purification of CD34+ cells for ex vivo gene transfer. Median age was 3.7 years and median weight 15.8 kg. After mobilization with lenograstim/plerixafor (n = 41) or lenograstim alone (n = 4) and 1−3 cycles of leukapheresis, median collection was 37 × 106 CD34+ cells/kg. The procedures were well tolerated. Patients who collected ≥7 and ≥13 × 106 CD34+ cells/kg in the first cycle had pre-apheresis circulating counts of at ≥42 and ≥86 CD34+ cells/μL, respectively. Weight-adjusted CD34+ cell yield was positively correlated with peripheral CD34+ cell counts and influenced by female gender, disease, and drug dosage. All patients received a GT product above the minimum target, ranging from 4 to 30.9 × 106 CD34+ cells/kg. Pediatric PBSC collection compares well to BM harvest in terms of CD34+ cell yields for the purpose of GT, with a favorable safety profile.

Wiskott-Aldrich syndrome (WAS) is a rare X-linked primary immunodeficiency, characterized by micro-thrombocytopenia, recurrent infections, and eczema. This study aims to describe common oral manifestations and evaluate oral microbioma of WAS patients.In this cohort study, 11 male WAS patients and 16 male healthy controls were evaluated in our Center between 2010 and 2018. Data about clinical history, oral examination, Gingival Index (GI) and Plaque Index (PI) were collected from both groups. Periodontal microbiological flora was evaluated on samples of the gingival sulcus.WAS subjects presented with premature loss of deciduous and permanent teeth, inclusions, eruption disturbance, and significantly worse GI and PI. They also showed a trend toward a higher total bacterial load. Fusobacterium nucleatum, reported to contribute to periodontitis onset, was the most prevalent bacteria, together with Porphyromonas gingivalis and Tannerella forsythia.Our data suggest that WAS patients are at greater risk of alterations in the oral cavity. The statistically higher incidence of periodontitis and the trend to higher prevalence of potentially pathological bacterial species in our small cohort, that should be confirmed in future in a larger population, underline the importance of dentistry monitoring as part of the multidisciplinary management of WAS patients.

Collection of an adequate amount of autologous haematopoietic stem progenitor cells (HSPC) is required for ex vivo manipulation and successful engraftment for certain inherited disorders. Fifty-seven paediatric patients (age 0.5-11.4 years) underwent a bone marrow harvest for the purpose of HSPC gene therapy (GT), including adenosine deaminase-severe combined immunodeficiency (ADA-SCID), Wiskott-Aldrich syndrome (WAS) and metachromatic leukodystrophy (MLD) patients. Total nucleated cells and the percentage and absolute counts of CD34+ cells were calculated at defined steps of the procedure (harvest, CD34+ cell purification, transduction with the gene transfer vector and infusion of the medicinal product). A minimum CD34+ cell dose for infusion was 2 × 106/kg, with an optimal target at 5-10 × 106/kg. Median volume of bone marrow harvested was 34.2 ml/kg (range 14.2-56.6). The number of CD34+ cells collected correlated inversely with weight and age in all patients and particularly in the MLD children group. All patients reached the minimum target dose for infusion: median dose of CD34+ cells/kg infused was 10.3 × 106/kg (3.7-25.9), with no difference among the three groups. Bone marrow harvest of volumes > 30 ml/kg in infants and children with ADA-SCID, WAS and MLD is well tolerated and allows obtaining an adequate dose of a medicinal product for HSPC-GT.

Abstract 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.

In this work we present the case of SARS-CoV-2 infection in a 1.5-year-old boy affected by severe Wiskott-Aldrich Syndrome with previous history of autoinflammatory disease, occurring 5 months after treatment with gene therapy. Before SARS-CoV-2 infection, the patient had obtained engraftment of gene corrected cells, resulting in WASP expression restoration and early immune reconstitution. The patient produced specific immunoglobulins to SARS-CoV-2 at high titer with neutralizing capacity and experienced a mild course of infection, with limited inflammatory complications, despite pre-gene therapy clinical phenotype.

Dear Editor, T-follicular helper (Tfh) cells have been implicated in the pathogenesis of a variety of autoimmune disorders because of their central role in adaptive immune responses [1]. In particular, Tfh cells orchestrate germinal centre reactions by recruiting CXCR5-expressing B-lymphocytes via CXCL13 secretion, and control antigen-dependent maturation, isotype class-switching, and somatic hypermutation of naïve B-cells [1]. IgG4-related disease (IgG4-RD) is an emerging fibro-inflammatory condition characterized by relapsing–remitting tumour-like lesions and increased serum IgG4 concentration [2, 3]. B-lymphocytes play a central pathogenic role in IgG4-RD because active disease is associated with oligoclonal expansion of fibrogenic IgG4+ plasmablasts [4]. In addition, B-cell depletion therapy with rituximab typically leads to rapid clinical improvement and flares occur in parallel with re-emergence of clonally divergent plasmablasts [2–5]. Of note, these B-cell clones are marked by enhanced somatic hypermutation consistent with iterative rounds of...

Hemophagocytic inflammatory syndrome (HIS) is a rare form of secondary hemophagocytic lymphohistiocytosis caused by an impaired equilibrium between natural killer and cytotoxic T-cell activity, evolving in hypercytokinemia and multiorgan failure. In the context of inborn errors of immunity, HIS occurrence has been reported in severe combined immunodeficiency (SCID) patients, including two cases of adenosine deaminase deficient-SCID (ADA-SCID). Here we describe two additional pediatric cases of ADA-SCID patients who developed HIS. In the first case, HIS was triggered by infectious complications while the patient was on enzyme replacement therapy; the patient was treated with high-dose corticosteroids and intravenous immunoglobulins with HIS remission. However, the patient required HLA-identical sibling donor hematopoietic stem cell transplantation (HSCT) for a definitive cure of ADA-SCID, without HIS relapse up to 13 years after HSCT. The second patient presented HIS 2 years after hematopoietic stem cell gene therapy (GT), secondarily to Varicella-Zoster vaccination and despite CD4+ and CD8+ lymphocytes’ reconstitution in line with other ADA SCID patients treated with GT. The child responded to trilinear immunosuppressive therapy (corticosteroids, Cyclosporine A, Anakinra). We observed the persistence of gene-corrected cells up to 5 years post-GT, without HIS relapse. These new cases of children with HIS, together with those reported in the literature, support the hypothesis that a major dysregulation in the immune system can occur in ADA-SCID patients. Our cases show that early identification of the disease is imperative and that a variable degree of immunosuppression could be an effective treatment while allogeneic HSCT is required only in cases of refractoriness. A deeper knowledge of immunologic patterns contributing to HIS pathogenesis in ADA-SCID patients is desirable, to identify new targeted treatments and ensure patients’ long-term recovery.

[This corrects the article DOI: 10.3389/fimmu.2019.00316.].

Abstract Wiskott-Aldrich Syndrome (WAS) is an X-linked primary immunodeficiency characterized by thrombocytopenia, recurrent infections, eczema, autoimmunity and increased susceptibility to malignancies. Allogeneic hematopoietic stem cell transplantation (HSCT) is a recognized curative treatment for WAS, but is still associated with transplant-related complications and long-term morbidity, particularly in the absence of fully matched donors. In April 2010, we initiated a phase I/II clinical trial with hematopoietic stem cell (HSC) gene therapy (GT) for WAS. The investigational medicinal product (IMP) consists of autologous CD34+ HSC engineered with a lentiviral vector (LV) driving the expression of WAS cDNA from an endogenous 1.6 kb human WAS promoter (LV-WAS), infused after a reduced intensity conditioning (RIC) based on anti-CD20 mAb, targeted busulfan and fludarabine. We previously reported early follow up (FU) results from the first 3 patients (Aiuti et al., Science 2013). Seven patients (Zhu score ≥3) have now been treated at a median age of 1.9 years (1.1 - 11.1). As of May 2015, all patients are alive with a median FU of 3.2 years (0.7 - 5.0). CD34+ cell source was bone marrow (BM) (n=5), mobilized peripheral blood (MPB) (n=1) or both (n=1). IMP dose ranged between 7.0 and 14.1 x106 CD34+/kg, containing on average 94.4 ± 3.5% transduced clonogenic progenitors and a mean vector copy number (VCN)/genome in bulk CD34+ cells of 2.7 ± 0.8. No adverse reactions were observed after IMP infusion and RIC was well tolerated. Median duration of severe neutropenia was 19 days; granulocyte-colony stimulating factor was administered to 1 patient. In the first 6 treated patients with FU &gt;2 years, we observed robust and persistent engraftment of gene corrected cells. At the most recent FU, transduced BM progenitors ranged between 20.7 and 59.7%, and LV-transduced cells were detected in multiple lineages, including PB granulocytes (VCN 0.34 - 0.93) and lymphocytes (VCN 1.18 - 2.73). WAS protein expression, measured by flow-cytometry, was detected in the majority of PB platelets [mean ± standard deviation (SD), 71.4 ± 14.0%], monocytes (63.3 ± 18.5%) and lymphocytes (78.9 ± 14.9%). Lymphocyte subset counts were normal in most patients and proliferative response to anti-CD3 mAb was in the normal range in all 6 patients. After immune reconstitution, a marked reduction in the annualized estimated rate of severe infections was observed, as compared with baseline (figure 1A). The first 6 treated patients discontinued anti-infective prophylaxis and no longer require a protected environment. Four patients stopped immunoglobulin supplementation and 2 of them developed specific antibodies after vaccination. Eczema resolved in 4 patients and remains mild in 2. No clinical manifestations of autoimmunity were observed ≥1 year after GT in accordance with improved B-cell development and decreased autoantibody production. All patients became platelet transfusion independent at a median of 4 months after GT (range: 1.0 - 8.7). Mean platelet counts progressively increased after treatment (mean ± SD: before GT, 13.4 ± 7.8 x109/l; 24-30 month FU, 45.8 ± 22.0 x109/l; 36-42 month FU, 57.0 ± 18.7 x109/l). The frequency and the severity of bleeding events decreased after the 1st year of FU. No severe bleedings were recorded after treatment (figure 1B). Quality of life improved in all patients after GT. From the 2nd year of FU, the number of hospitalizations for infections decreased and no hospitalizations due to bleeding were observed after treatment. The seventh patient treated, who received MPB derived CD34+ cells only, showed the fastest platelet recovery with the highest level of transduced myeloid cell engraftment, and is clinically well. No Serious Adverse Events (SAE) related to the IMP were observed. The most frequent SAE were related to infections (85%), occuring mainly during the 1st year of FU. Importantly, no evidence of abnormal clonal proliferations emerged after GT and the LV integration profile show a polyclonal pattern, with no skewing for proto-oncogenes. In conclusion, this updated report in 7 WAS patients show that GT is well tolerated and leads to a sustained clinical benefit. The high level of gene transfer obtained with LV-WAS results in robust engraftment of transduced HSC, even when combined with RIC. Prolonged FU will provide additional information on the long-term safety and clinical efficacy of this treatment. Figure 1. Figure 1. Disclosures Villa: Fondazione Telethon: Research Funding. Dott:GlaxoSmithKline: Consultancy. van Rossem:GlaxoSmithKline: Employment. Naldini:Salk Institute: Patents &amp; Royalties: Lentiviral vectors; San Raffaele Telethon Institute: Patents &amp; Royalties: Lentiviral vector technology; GlaxoSmithKline: Other: GSK licensed gene therapies developed at my Institute and the Institute receives milestone payments; Sangamo Biosciences: Research Funding; Biogen: Research Funding; Genenta Sciences: Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Aiuti:GlaxoSmithKline (GSK): Other: PI of clinical trial which is financially sponsored by GSK; Fondazione Telethon: Research Funding.

Background: Wiskott-Aldrich syndrome (WAS) is a rare, X-linked, life-threatening primary immunodeficiency caused by mutations in the gene encoding the WAS protein (WASP). WASP-deficient immune cells have compromised immunological synapse formation, cell migration and cytotoxicity. Thus, WAS is characterized by development of recurrent or severe infections, eczema, and increased risk of autoimmunity and malignancies. In addition, WASP deficiency results in microthrombocytopenia, leading to severe bleeding episodes. When a suitable donor is available, WAS can be treated by hematopoietic stem cell transplant (HSCT), but HSCT can be impeded by complications such as graft versus host disease, rejection and autoimmunity. Importantly, HSCT may carry higher risks in older children (&gt;2-5 yrs) [Shin et al, 2012; Moratto et al, 2011]. An alternative approach is gene therapy (GT). We previously reported interim results of a Phase I/II clinical trial (NCT01515462) in 8 subjects treated with OTL-103, a drug product composed of autologous CD34+ hematopoietic stem and progenitor cells (HSPCs) transduced ex vivo with a lentiviral vector (LV) encoding human WASP cDNA under the control of the endogenous promoter [Ferrua et al, 2019]. We now report updated results on the safety and efficacy of OTL-103 in 17 subjects treated at San Raffaele Hospital as part of the same clinical trial or expanded access programs (EAP) with up to 8 yrs follow up (FU). Methods: NCT01515462: As described in Ferrua et al, 8 male subjects (mean age at GT: 4.8 yrs, range 1.1-12.4) were treated with OTL-103. The source of autologous CD34+ HSPCs was bone marrow (BM; n=5), mobilized peripheral blood (mPB; n=2) or both (n=1). As part of a reduced-intensity conditioning regimen, rituximab was given 22 days prior and busulfan + fludarabine during the week before OTL-103 infusion. At time of reporting, all subjects had ≥3 yrs FU (range: 3-8 yrs). EAP: 9 male subjects (11.2 yrs, 1.4-35.1) received identical treatment to subjects in the clinical trial; autologous CD34+ HSPCs source was mPB in all subjects. At time of reporting, subjects had a median of 1.4 yrs FU (range: 0.1-3.0 yrs) with 6/9 having ≥1 yr FU. Results: At last FU for all subjects (median: 3.0 yrs, range 0.1-8.0), overall survival was 94% (16/17). One EAP subject died 4.5 mo post-GT, due to deterioration of an underlying neurodegenerative condition considered unrelated to OTL-103 by investigator. To date, there have been no reports of insertional oncogenesis or replication-competent LV. While most subjects experienced adverse events (AEs) due to the reduced-intensity conditioning regimen (mainly mild or moderate), there were no reports of AEs related to OTL-103. Efficacy endpoints analyses were performed on surviving patients with ≥1 yr FU. Evidence of engraftment of genetically corrected HSPCs and LV+ colonies in BM was observed within 3 mo and persisted up to 8 yrs - the longest published FU of LV vector durability to date (Figure). WASP expression was restored after GT, shown by increases in the fraction of WASP+ lymphocytes and platelets (PLT) within 3 mo and maintained thereafter (Table). After GT, PLT counts improved, leading to a reduction of frequency and severity of bleeding events. Independence from PLT transfusions and absence of severe bleeding events were observed in all subjects by 9 mo FU (Table). Immune function improved; all evaluable patients discontinued immunoglobulin supplementation after GT (median time to discontinuation: 0.9 years after GT, range: 0.2-5 years). Furthermore, reduction in severe infection rate was observed post-GT, suggestive of immune reconstitution (Table). The decrease in bleeding events and severe infection rates occurred despite the integration of subjects into normal daily activities. Eczema progressively resolved or was reduced compared to baseline. Conclusions: This combined analysis of 17 subjects treated in a clinical trial or EAP with up to 8 yrs FU demonstrates that GT continues to be an effective treatment for WAS. All surviving subjects achieved high levels of multilineage engraftment, sustained restoration of WASP expression in lymphocytes and PLTs, improved PLT counts, and fewer bleeding events. A significant reduction in severe infection rate suggests reconstitution of immune function. Importantly, clinical benefit was also attained in older subjects (&gt;5 yrs), a group considered at higher risk when treated with allogeneic HSCT. Disclosures Jones: Orchard Therapeutics: Employment, Equity Ownership. Dott:Orchard Therapeutics: Employment, Equity Ownership. Naldini:Genenta Science: Consultancy, Equity Ownership; Magenta Therapeutics: Equity Ownership; San Raffaele Telethon Institute for Gene Therapy (SR-TIGET), a joint venture between Fondazione Telethon and Ospedale San Raffaele (OSR): Other: Wiskott-Aldrich Syndrome (WAS) gene therapy was licensed to GlaxoSmithKline (GSK) in 2014. It was then licensed to Orchard Therapeutics (OTL) in April 2018. OTL is the current sponsor of the clinical trial.. Aiuti:San Raffaele Telethon Institute for Gene Therapy (SR-TIGET), a joint venture between Fondazione Telethon and Ospedale San Raffaele (OSR): Other: Wiskott-Aldrich Syndrome (WAS) gene therapy was licensed to GlaxoSmithKline (GSK) in 2014. It was than licensed to Orchard Therapeutics (OTL) in April 2018. OTL is the current sponsor of the clinical trial.; San Raffaele Telethon Institute for Gene Therapy (SR-TIGET), a joint venture between Fondazione Telethon and Ospedale San Raffaele (OSR): Other: Study PI.

Abstract Background Improved survival in ADA-SCID patients is revealing new aspects of the systemic disorder. Although increasing numbers of reports describe the systemic manifestations of adenosine deaminase deficiency, currently there are no studies in the literature evaluating genital development and pubertal progress in these patients. Methods We collected retrospective data on urogenital system and pubertal development of 86 ADA-SCID patients followed in the period 2000–2017 at the Great Ormond Street Hospital (UK) and 5 centers in Italy. In particular, we recorded clinical history and visits, and routine blood tests and ultrasound scans were performed as part of patients’ follow-up. Results and Discussion We found a higher frequency of congenital and acquired undescended testes compared with healthy children (congenital, 22% in our sample, 0.5–4% described in healthy children; acquired, 16% in our sample, 1–3% in healthy children), mostly requiring orchidopexy. No urogenital abnormalities were noted in females. Spontaneous pubertal development occurred in the majority of female and male patients with a few cases of precocious or delayed puberty; no patient presented high FSH values. Neither ADA-SCID nor treatment performed (PEG-ADA, BMT, or GT) affected pubertal development or gonadic function. Conclusion In summary, this report describes a high prevalence of cryptorchidism in a cohort of male ADA-SCID patients which could represent an additional systemic manifestation of ADA-SCID. Considering the impact urogenital and pubertal abnormalities can have on patients’ quality of life, we feel it is essential to include urogenital evaluation in ADA-SCID patients to detect any abnormalities, initiate early treatment, and prevent long-term complications.

Gene therapy for transfusion dependent beta-thalassemia is based on the autologous transplantation of hematopoietic stem cells (HSCs) engineered by lentiviral vectors expressing a transcriptionally regulated human beta-globin gene.Gene therapy (GT) could represent an alternative to HSCT with the following potential advantages: use of autologous stem cells, tailored conditioning with no need for immune suppression post GT, no risk of GVHD or rejection.Our contribution to this field was devoted to the clinical development of a gene therapy protocol based on the high-titer vector GLOBE, a 3 rd generation self-inactivating lentiviral vector encoding for the human beta globin gene. Transfusion dependent beta thalassemia patients (any genotype) undergo peripheral blood stem cell harvest following mobilization with the lenograstim and plerixafor. After transduction of immune-selected autologous CD34+ cells and successful release of the frozen drug substance, patient undergo a conditioning regimen based on myeloablative treosulfan and thiotepa favoring efficient engraftment of corrected cells with reduced extra-medullary toxicity (TIGET-BTHAL; EudraCT number 2014‐004860‐39). The route of administration of gene modified HSCs is intraosseous in the posterior-superior iliac crests with the aim of enhancing engraftment and minimizing first-pass intravenous filter. Three days after gene therapy, previously collected unstimulated autologous peripheral blood leucocytes (1-10 x107 CD3+/kg) are reinfused intravenously to favor immune-reconstitution. After 2 years follow-up, patients will be followed up for a further six years in a long-term follow-up study. On the basis of extensive efficacy and safety preclinical studies the clinical trial TIGET-BTHAL was approved and started in 2015 at Scientific Institute San Raffaele, Milan, Italy. The clinical study foresees treatment of 10 patients: 3 adults (group 1) followed by 3 patients aged 8-17 years (group 2) and 4 patients aged 3-7 years (group 3), with a staggered enrolment strategy based on evaluation of safety and preliminary efficacy in adult patients by an independent data safety monitoring board (DSMB) before inclusion of pediatric subjects. In March 2016 the DSMB approved enrolment of group 2 patients and, in September 2016, of group 3 patients. As of August 2017, seven patients (3 adults aged 31-35 years and 4 pediatric patients aged 6-13 years) with different genotypes (β 0 /β 0 , β + /β + and β 0 /β + ) have been treated with GLOBE-transduced CD34 + cells at a dose of 16x10 6 -19.5x10 6 cells/kg and a vector copy number (VCN)/cell ranging from 0.7 to 1.5. Median follow-up is 13 months (range 8-22). The procedure was well tolerated by all patients, with no product-related adverse events, no evidence of replication competent lentivirus nor of abnormal clonal proliferation on regular peripheral blood and bone marrow analyses. Grade 3-4 adverse events or serious adverse events were principally of infectious origin as expected after a myeloablative autograft. Median time to neutrophil engraftment was 19 days (range 17-25) and to platelet engraftment 15 days (range 10-21). Multilineage engraftment of gene-marked cells was observed in peripheral blood and bone marrow, with a median of 0.58 (range 0.37-1.55) vector copy number/cell in GlyA+ bone marrow erythroid cells at 6 months post GT.Polyclonal vector integrations profiles have been detected in the first 3 patients tested. The three adult patients had a reduction of transfusion requirement but are still transfusion dependent at the last follow-up (22, 18 and 16 months respectively). Among the 4 pediatric patients, 3 have discontinued transfusion shortly after gene therapy and are transfusion independent at the last follow-up (13, 10 and 8 months respectively). One pediatric patient is still receiving regular blood transfusions. A correlation was observed between level of engraftment of gene-marked cells in peripheral blood and bone marrow and transfusion requirement. Preliminary data suggest that the applied clinical protocol for gene therapy with GLOBE LV is well tolerated and leads to significantly reduced transfusion requirement. Follow up analysis are ongoing and updated clinical outcome will be presented. Disclosures Marktel: GSK: Other: B-thalassemia gene therapy was developed by Fondazione Telethon and Ospedale San Raffaele and has been inlicenced by GSK that provides funding for the clinical trial, Research Funding. Naldini: San Raffaele Institute: Employment, Patents & Royalties; Telethon Foundation: Consultancy, Patents & Royalties; Bioverativ: Research Funding; Editas: Research Funding; Genenta Sciences: Equity Ownership, Honoraria, Membership on an entity9s Board of Directors or advisory committees, Research Funding; Magenta: Equity Ownership, Membership on an entity9s Board of Directors or advisory committees; Oncorus: Membership on an entity9s Board of Directors or advisory committees; Sangamo Biosciences: Membership on an entity9s Board of Directors or advisory committees. Cappellini: Sanofi-Genzyme: Honoraria, Research Funding, Speakers Bureau; Celgene: Honoraria; Vifor: Honoraria; Novartis: Speakers Bureau. Ciceri: GSK: Other: B-thalassemia gene therapy was developed by Fondazione Telethon and Ospedale San Raffaele and has been inlicenced by GSK that provides funding for the clinical trial, Research Funding. Aiuti: GSK: Other: B-thalassemia gene therapy was developed by Fondazione Telethon and Ospedale San Raffaele and has been inlicenced by GSK that provides funding for the clinical trial, Research Funding. Ferrari: GSK: Other: B-thalassemia gene therapy was developed by Fondazione Telethon and Ospedale San Raffaele and has been inlicenced by GSK that provides funding for the clinical trial, Research Funding.

Pediatric Allergy and ImmunologyVolume 33, Issue 8 e13833 LETTER TO THE EDITOROpen Access SARS-CoV-2 infection and treatment in a cohort of patients with inborn errors of immunity Francesca Conti, Francesca Conti Pediatric Unit, IRCCS Azienda Ospedaliero-Universitaria di Bologna, Bologna, ItalySearch for more papers by this authorLucia Pacillo, Lucia Pacillo orcid.org/0000-0002-7950-8848 Academic Department of Pediatrics (DPUO), Research Unit of Clinical Immunology and Vaccinology, Bambino Gesù Children's Hospital, Istituto di Ricovero e Cura a Carattere Scientifico (IRCCS), Rome, Italy PhD Program in Immunology, Molecular Medicine and Applied Biotechnology, University of Rome Tor Vergata, Rome, ItalySearch for more papers by this authorDonato Amodio, Corresponding Author Donato Amodio donato.amodio@opbg.net orcid.org/0000-0003-4550-3018 Academic Department of Pediatrics (DPUO), Research Unit of Clinical Immunology and Vaccinology, Bambino Gesù Children's Hospital, Istituto di Ricovero e Cura a Carattere Scientifico (IRCCS), Rome, Italy Correspondence Donato Amodio, Academic Department of Pediatrics (DPUO), Research Unit of Clinical Immunology and Vaccinology, Bambino Gesù Children's Hospital, Istituto di Ricovero e Cura a Carattere Scientifico (IRCCS), Rome, Italy. Email: donato.amodio@opbg.netSearch for more papers by this authorBeatrice Rivalta, Beatrice Rivalta Academic Department of Pediatrics (DPUO), Research Unit of Clinical Immunology and Vaccinology, Bambino Gesù Children's Hospital, Istituto di Ricovero e Cura a Carattere Scientifico (IRCCS), Rome, Italy PhD Program in Immunology, Molecular Medicine and Applied Biotechnology, University of Rome Tor Vergata, Rome, ItalySearch for more papers by this authorMattia Moratti, Mattia Moratti Specialty School of Paediatrics, University of Bologna, Bologna, ItalySearch for more papers by this authorCaterina Campoli, Caterina Campoli Infectious Diseases Unit, Department of Medical and Surgical Sciences, IRCCS, Azienda Ospedaliero-Universitaria di Bologna, Alma Mater Studiorum, University of Bologna, Bologna, ItalySearch for more papers by this authorDaniele Zama, Daniele Zama Paediatric Emergency Unit, Scientific Institute for Research and Healthcare (IRCCS) Azienda Ospedaliero-Universitaria di Bologna, Bologna, ItalySearch for more papers by this authorIlaria Corsini, Ilaria Corsini Paediatric Emergency Unit, Scientific Institute for Research and Healthcare (IRCCS) Azienda Ospedaliero-Universitaria di Bologna, Bologna, ItalySearch for more papers by this authorCarmela Giancotta, Carmela Giancotta Academic Department of Pediatrics (DPUO), Research Unit of Clinical Immunology and Vaccinology, Bambino Gesù Children's Hospital, Istituto di Ricovero e Cura a Carattere Scientifico (IRCCS), Rome, ItalySearch for more papers by this authorStefania Bernardi, Stefania Bernardi Academic Department of Pediatrics (DPUO), Immune and Infectious Diseases Division, Research Unit of Primary Immunodeficiencies, Bambino Gesù Children's Hospital, Istituto di Ricovero e Cura a Carattere Scientifico (IRCCS), Rome, ItalySearch for more papers by this authorSamuele Naviglio, Samuele Naviglio orcid.org/0000-0002-1915-9965 Pediatric Hematology-Oncology, Institute for Maternal and Child Health IRCCS "Burlo Garofolo", Trieste, ItalySearch for more papers by this authorMaria Pia Cicalese, Maria Pia Cicalese San Raffaele Telethon Institute for Gene Therapy (SR-Tiget) and Pediatric Immunohematology and Bone Marrow Transplantation Unit, IRCCS San Raffaele Scientific Institute, Milan, Italy Vita-Salute San Raffaele University, Milan, ItalySearch for more papers by this authorMarco Rabusin, Marco Rabusin Pediatric Hematology-Oncology, Institute for Maternal and Child Health IRCCS "Burlo Garofolo", Trieste, ItalySearch for more papers by this authorAlessandro Aiuti, Alessandro Aiuti orcid.org/0000-0002-5398-1717 San Raffaele Telethon Institute for Gene Therapy (SR-Tiget) and Pediatric Immunohematology and Bone Marrow Transplantation Unit, IRCCS San Raffaele Scientific Institute, Milan, Italy Vita-Salute San Raffaele University, Milan, ItalySearch for more papers by this authorCaterina Cancrini, Caterina Cancrini Academic Department of Pediatrics (DPUO), Research Unit of Clinical Immunology and Vaccinology, Bambino Gesù Children's Hospital, Istituto di Ricovero e Cura a Carattere Scientifico (IRCCS), Rome, Italy Department of Systems Medicine, Chair of Pediatrics, University of Rome "Tor Vergata", Rome, ItalySearch for more papers by this authorMarcello Lanari, Marcello Lanari Paediatric Emergency Unit, Scientific Institute for Research and Healthcare (IRCCS) Azienda Ospedaliero-Universitaria di Bologna, Bologna, ItalySearch for more papers by this authorPierluigi Viale, Pierluigi Viale Infectious Diseases Unit, Department of Medical and Surgical Sciences, IRCCS, Azienda Ospedaliero-Universitaria di Bologna, Alma Mater Studiorum, University of Bologna, Bologna, ItalySearch for more papers by this authorPaolo Palma, Paolo Palma orcid.org/0000-0002-3066-4719 Academic Department of Pediatrics (DPUO), Research Unit of Clinical Immunology and Vaccinology, Bambino Gesù Children's Hospital, Istituto di Ricovero e Cura a Carattere Scientifico (IRCCS), Rome, Italy Department of Systems Medicine, Chair of Pediatrics, University of Rome "Tor Vergata", Rome, ItalySearch for more papers by this authorAndrea Pession, Andrea Pession Pediatric Unit, IRCCS Azienda Ospedaliero-Universitaria di Bologna, Bologna, ItalySearch for more papers by this authorAndrea Finocchi, Andrea Finocchi Academic Department of Pediatrics (DPUO), Research Unit of Clinical Immunology and Vaccinology, Bambino Gesù Children's Hospital, Istituto di Ricovero e Cura a Carattere Scientifico (IRCCS), Rome, Italy Department of Systems Medicine, Chair of Pediatrics, University of Rome "Tor Vergata", Rome, ItalySearch for more papers by this author Francesca Conti, Francesca Conti Pediatric Unit, IRCCS Azienda Ospedaliero-Universitaria di Bologna, Bologna, ItalySearch for more papers by this authorLucia Pacillo, Lucia Pacillo orcid.org/0000-0002-7950-8848 Academic Department of Pediatrics (DPUO), Research Unit of Clinical Immunology and Vaccinology, Bambino Gesù Children's Hospital, Istituto di Ricovero e Cura a Carattere Scientifico (IRCCS), Rome, Italy PhD Program in Immunology, Molecular Medicine and Applied Biotechnology, University of Rome Tor Vergata, Rome, ItalySearch for more papers by this authorDonato Amodio, Corresponding Author Donato Amodio donato.amodio@opbg.net orcid.org/0000-0003-4550-3018 Academic Department of Pediatrics (DPUO), Research Unit of Clinical Immunology and Vaccinology, Bambino Gesù Children's Hospital, Istituto di Ricovero e Cura a Carattere Scientifico (IRCCS), Rome, Italy Correspondence Donato Amodio, Academic Department of Pediatrics (DPUO), Research Unit of Clinical Immunology and Vaccinology, Bambino Gesù Children's Hospital, Istituto di Ricovero e Cura a Carattere Scientifico (IRCCS), Rome, Italy. Email: donato.amodio@opbg.netSearch for more papers by this authorBeatrice Rivalta, Beatrice Rivalta Academic Department of Pediatrics (DPUO), Research Unit of Clinical Immunology and Vaccinology, Bambino Gesù Children's Hospital, Istituto di Ricovero e Cura a Carattere Scientifico (IRCCS), Rome, Italy PhD Program in Immunology, Molecular Medicine and Applied Biotechnology, University of Rome Tor Vergata, Rome, ItalySearch for more papers by this authorMattia Moratti, Mattia Moratti Specialty School of Paediatrics, University of Bologna, Bologna, ItalySearch for more papers by this authorCaterina Campoli, Caterina Campoli Infectious Diseases Unit, Department of Medical and Surgical Sciences, IRCCS, Azienda Ospedaliero-Universitaria di Bologna, Alma Mater Studiorum, University of Bologna, Bologna, ItalySearch for more papers by this authorDaniele Zama, Daniele Zama Paediatric Emergency Unit, Scientific Institute for Research and Healthcare (IRCCS) Azienda Ospedaliero-Universitaria di Bologna, Bologna, ItalySearch for more papers by this authorIlaria Corsini, Ilaria Corsini Paediatric Emergency Unit, Scientific Institute for Research and Healthcare (IRCCS) Azienda Ospedaliero-Universitaria di Bologna, Bologna, ItalySearch for more papers by this authorCarmela Giancotta, Carmela Giancotta Academic Department of Pediatrics (DPUO), Research Unit of Clinical Immunology and Vaccinology, Bambino Gesù Children's Hospital, Istituto di Ricovero e Cura a Carattere Scientifico (IRCCS), Rome, ItalySearch for more papers by this authorStefania Bernardi, Stefania Bernardi Academic Department of Pediatrics (DPUO), Immune and Infectious Diseases Division, Research Unit of Primary Immunodeficiencies, Bambino Gesù Children's Hospital, Istituto di Ricovero e Cura a Carattere Scientifico (IRCCS), Rome, ItalySearch for more papers by this authorSamuele Naviglio, Samuele Naviglio orcid.org/0000-0002-1915-9965 Pediatric Hematology-Oncology, Institute for Maternal and Child Health IRCCS "Burlo Garofolo", Trieste, ItalySearch for more papers by this authorMaria Pia Cicalese, Maria Pia Cicalese San Raffaele Telethon Institute for Gene Therapy (SR-Tiget) and Pediatric Immunohematology and Bone Marrow Transplantation Unit, IRCCS San Raffaele Scientific Institute, Milan, Italy Vita-Salute San Raffaele University, Milan, ItalySearch for more papers by this authorMarco Rabusin, Marco Rabusin Pediatric Hematology-Oncology, Institute for Maternal and Child Health IRCCS "Burlo Garofolo", Trieste, ItalySearch for more papers by this authorAlessandro Aiuti, Alessandro Aiuti orcid.org/0000-0002-5398-1717 San Raffaele Telethon Institute for Gene Therapy (SR-Tiget) and Pediatric Immunohematology and Bone Marrow Transplantation Unit, IRCCS San Raffaele Scientific Institute, Milan, Italy Vita-Salute San Raffaele University, Milan, ItalySearch for more papers by this authorCaterina Cancrini, Caterina Cancrini Academic Department of Pediatrics (DPUO), Research Unit of Clinical Immunology and Vaccinology, Bambino Gesù Children's Hospital, Istituto di Ricovero e Cura a Carattere Scientifico (IRCCS), Rome, Italy Department of Systems Medicine, Chair of Pediatrics, University of Rome "Tor Vergata", Rome, ItalySearch for more papers by this authorMarcello Lanari, Marcello Lanari Paediatric Emergency Unit, Scientific Institute for Research and Healthcare (IRCCS) Azienda Ospedaliero-Universitaria di Bologna, Bologna, ItalySearch for more papers by this authorPierluigi Viale, Pierluigi Viale Infectious Diseases Unit, Department of Medical and Surgical Sciences, IRCCS, Azienda Ospedaliero-Universitaria di Bologna, Alma Mater Studiorum, University of Bologna, Bologna, ItalySearch for more papers by this authorPaolo Palma, Paolo Palma orcid.org/0000-0002-3066-4719 Academic Department of Pediatrics (DPUO), Research Unit of Clinical Immunology and Vaccinology, Bambino Gesù Children's Hospital, Istituto di Ricovero e Cura a Carattere Scientifico (IRCCS), Rome, Italy Department of Systems Medicine, Chair of Pediatrics, University of Rome "Tor Vergata", Rome, ItalySearch for more papers by this authorAndrea Pession, Andrea Pession Pediatric Unit, IRCCS Azienda Ospedaliero-Universitaria di Bologna, Bologna, ItalySearch for more papers by this authorAndrea Finocchi, Andrea Finocchi Academic Department of Pediatrics (DPUO), Research Unit of Clinical Immunology and Vaccinology, Bambino Gesù Children's Hospital, Istituto di Ricovero e Cura a Carattere Scientifico (IRCCS), Rome, Italy Department of Systems Medicine, Chair of Pediatrics, University of Rome "Tor Vergata", Rome, ItalySearch for more papers by this author First published: 01 August 2022 https://doi.org/10.1111/pai.13833 Associate Editor: Fabio Candotti Francesca Conti, Lucia Pacillo contributed equally to this work and share first authorship. 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Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat To the Editor, Many authors recently reported the effect of the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) pandemic on patients affected by Inborn Errors of Immunity (IEI).1, 2 Although mostly experiencing a mild disease and the course of the infection is comparable or even less symptomatic than the general population, young male IEIs patients were more severely affected and more frequently admitted to intensive care unit compared with the age-matched healthy peers.3 Most viral variants were actually first described in immunocompromised patients.4, 5 Indeed, impaired B and/or T cell function can be responsible for persisting viral shedding often observed in IEI patients with subsequent higher risk of persistent viral replication and mutation within the host.6 Noteworthily, in the last 2 years Inborn Errors of type I IFN immunity and autoantibody-mediated phenocopies of IEIs were identified as responsible for life-threatening COVID-19. These conditions were not considered in the studies about IEIs and SARS-CoV-2 infection.7, 8 Predisposition to increased risk and most severe infection in IEI, despite a lack of clear data in favor of a high risk for progression to severe COVID-19, led the scientific community to recommend the use of early SARS-CoV2-directed treatments to potentially reduce the incidence of long-lasting infection, as well as morbidity and mortality risk.9 Casirivimab–imdevimab, also known as REGEN-COV, is an anti-spike mAb that has been authorized for the treatment of high-risk patients (>12 years of age, >40 kg) with mild-to-moderate COVID-19.10 Bamlanivimab, named LY-CoV555, then combined with etesevimab, showed a beneficial effect for accelerating the natural decline of the viral load over time. In December 2021, it was approved by FDA for patients<12 years.10 Sotrovimab, formerly known as VIR-7831, is an engineered human mAb that neutralizes SARS-CoV-2.10 There are several data confirming the safety and efficacy in both adults and pediatric population.11 We describe a cohort of 63 SARS-CoV-2-infected IEI patients, providing the first case series report on the early employment of anti-SARS-CoV-2 treatments with antiviral drugs and MAbs in these patients (Table S1). Thirty-nine patients were younger than 18 years (median age 9.5 years), whereas 24 were adults (median age 33 years). Male-to-female ratio was 1:1. Seven individuals have been previously reported.2 The distribution of SARS-CoV-2-infected IEI patients into International Union of Immunological Societies (IUIS) categories was similar to those described by Meyts et al.1 and Milito et al.2 (Figure 1) and also reflected the frequencies of each disease. In fact, 71% of patients belonged to second and third IUIS categories. Interestingly, 58% of IEIs were genetically determined and the most frequently involved pathways were those affecting T cell homeostasis: 22q11.2 deletion, STAT3 and ATM variants were addressed together as responsible for 25% of molecular IEI diagnoses in our cohort. Comorbidities were variable and, in some cases, influenced the clinical course of coronavirus infection and the therapeutic choice. FIGURE 1Open in figure viewerPowerPoint Distribution of SARS-CoV-2-infected patients by (A) IEI entities and (B) treatment According to current guidelines, all patients affected by IEI are eligible for SARS-COV2 treatment. At the time of this manuscript, specific guidelines about the choice of the most suitable treatment for each patient were lacking. The criteria for treatment choice were based on the underlying IEI, the patient clinical conditions, the variety of concern (VOC), when available, or the current prevalence of resistant variants and drug's availability in each center and patient's age. SARS-CoV-2 infection prevalence and incidence dramatically increased since the last months of 2021. Indeed, the number of SARS-CoV-2-positive IEI individuals reported in this manuscript is much higher compared with the cases previously reported. This mirrors the current epidemiology of coronavirus disease 2019 (COVID-19) in general population, characterized by a huge spread due to both the increasing prevalence of Omicron variant over Delta and the gradual softening of social restriction measures. As reported in the first pandemic phase, most of our IEI patients did not develop severe COVID-19. In most of our patients, SARS-CoV-2 infection dated back no more than October 2021, and 15/17 tested subjects were infected by the Omicron variant. Therefore, it is possible to speculate that most cases were sustained by this variant, which has been reported to be less commonly related to severe complications.12 Indeed, 90% of our patients experienced a mild clinical course and only 6/63 (9,5%) showed a moderate/severe course: 4/6 were under 18 years and 5/6 presented important comorbidities that may have influenced the clinical course. 5/6 presented a moderate course including a del22 syndrome with associated CID and cardiopathy, a APDS patient with an ongoing lymphoma relapse, a CVID patient with IBD, a patient with CVID and associated osteochondrosis and one post-HSCT and kidney transplantation for Schimke syndrome with epilepsy and ischemic/hemorrhagical neurological events. Only a 4-year-old male child with an unclassified IEI and a previous history of hemolytic uremic syndrome died 6 days after SARS-CoV-2 infection due to CMV-related acute respiratory distress syndrome, acute renal failure and cerebral edema despite MAbs administration. Remarkably, 3 patients had SARS-CoV-2 infection after gene therapy (one after experimental gene therapy for WAS13, and two after approved gene therapy for ADA deficiency); all patients experienced a favorable clinical course. A novel issue in our study was the cohort's high anti-SARS-CoV-2 vaccination coverage: 59% of individuals had received at least two vaccine doses, with no significant adverse effects, while 38% had not received any vaccine. Nevertheless, vaccinal campaign did not seem to impact substantially on infection frequency and severity among the studied population, probably due both to Omicron vaccine-escaping ability and to IEI-related defective vaccine immunogenicity14. Indeed, patients affected by IEI are able to mount a specific antibody response following vaccination although at a lower magnitude compared with healthy peers. On the contrary, the proportion of those who are not capable of Ag-specific T cells generation at all is higher compared with healthy individuals. Given this evidence, it is getting clearer and clearer the importance of developing vaccination schedules tailored for vulnerable populations such as IEI subjects or patient with secondary immunodeficiencies. Antivirals or neutralizing monoclonal antibodies were employed in 29/63 patients (46%). Antivirals and MAbs were used in 5/29 and 25/29 cases, respectively. No serious adverse effects were observed. The therapy was proposed to each patient for which the medical staff was aware according to the eligibility criteria.9 Variability in the therapeutic strategies approach was mainly determined by drugs' availability in each center and patients' age. Notably, our report is also the first to describe the off-label use of MAbs in pediatric patients below the age for which they were licensed (8/25 patients treated with MAbs were < 12 years of age). Brown et al.15 recently described a cohort of 31 IEI patients, predominantly affected by humoral defects, with chronic or relapsing COVID-19, and showed a better response to combination of remdesivir plus MAbs. In our study, non-standardized timing and antigenic/PCR methods determination for SARS-CoV-2 testing along with the absence of a long-term follow-up did not allow an unbiased impact assessment of the efficacy of these early treatments. However, we did not observe substantial differences in terms of infection clearance (2 days spared) and symptom recovery (1 day spared) between treated and untreated patients (p-value 0.86); nevertheless, we observed a faster mean time clearance in patients who had received at least 2 vaccination doses respect to the rest of our IEI cohort (17 versus 24 days). Overall, our experience confirms that COVID-19 has a mild clinical course in most patients with IEIs, yet a significant minority remains at a risk of severe disease. Early preventive therapies are frequently employed in this population, with good tolerance, even when used off-label for children under 12 years of age. Standardized protocol specifically set on IEI patients and information on pediatric doses, however, are lacking, possibly hindering access to these treatments. ACKNOWLEDGMENTS We would like to thank the patients, caregivers and clinic teams for their participation and kind support of this study. Open access funding provided by Ospedale Pediatrico Bambino Gesu. FUNDING INFORMATION This research received no external funding. CONFLICT OF INTEREST The authors declare no conflict of interest. ETHICAL APPROVAL All patients or caregivers gave their informed consent prior to their inclusion in the study. PEER REVIEW The peer review history for this article is available at https://publons.com/publon/10.1111/pai.13833. Supporting Information Filename Description pai13833-sup-0001-TableS1.docxWord 2007 document , 26.4 KB TableS1 Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article. REFERENCES 1Meyts I, Bucciol G, Quinti I, et al. Coronavirus disease 2019 in patients with inborn errors of immunity: an international study. J Allergy Clin Immunol. 2021; 147(2): 520- 531. doi:10.1016/j.jaci.2020.09.010 2Milito C, Lougaris V, Giardino G, et al. Clinical outcome, incidence, and SARS-CoV-2 infection-fatality rates in Italian patients with inborn errors of immunity. J Allergy Clin Immunol Pract. 2021; 9(7): 2904- 2906. doi:10.1016/j.jaip.2021.04.017 3Choi B, Choudhary MC, Regan J, et al. Persistence and evolution of SARS-CoV-2 in an immunocompromised host. N Engl J Med. 2020; 383(23): 2291- 2293. doi:10.1056/NEJMc2031364 4McCormick KD, Jacobs JL, Mellors JW. The emerging plasticity of SARS-CoV-2. Science. 2021; 371(6536): 1306- 1308. doi:10.1126/science.abg4493 5Jayk Bernal A, Gomes da Silva MM, Musungaie DB, et al. Molnupiravir for Oral treatment of Covid-19 in nonhospitalized patients. N Engl J Med. 2022; 386(6): 509- 520. doi:10.1056/NEJMoa2116044 6Haidar G, Mellors JW. Improving the outcomes of immunocompromised patients with coronavirus disease 2019. Clin Infect Dis off Publ Infect Dis Soc Am. 2021; 73(6): e1397- e1401. doi:10.1093/cid/ciab397 7Zhang Q, Bastard P, Liu Z, et al. Inborn errors of type I IFN immunity in patients with severe COVID-19. Science. 2020; 370(6515): eabd4570. doi:10.1126/science.abd4570 8Bastard P, Rosen LB, Zhang Q, et al. Auto-antibodies against type I IFNs in patients with life-threatening COVID-19. Science. 2020; 370(6515): eabd4585. doi:10.1126/science.abd4585 9 Centers for Disease Control and Prevention. Prioritization of anti-SARS-CoV-2 therapies for the treatment and prevention of COVID-19 when there are logistical or supply constraints. 2022. Available at: https://www.covid19treatmentguidelines.nih.gov/overview/prioritization-of-therapeutics 10Romani L, Calò Carducci FI, Chiurchiù S, et al. Safety of monoclonal antibodies in children affected by SARS-CoV-2 infection. Children (Basel). 2022; 9(3): 369. doi:10.3390/children9030369 11Mak G, Dassner AM, Hammer BM, Hanisch BR. Safety and tolerability of monoclonal antibody therapies for treatment of COVID-19 in pediatric patients. Pediatr Infect Dis J. 2021; 40: e507- e509. 12Wang L, Berger NA, Kaelber DC, Davis PB, Volkow ND, Xu R. Comparison of outcomes from COVID infection in pediatric and adult patients before and after the emergence of omicron. MedRxiv Prepr Serv Health Sci. 2022;2021.12.30.21268495. doi:10.1101/2021.12.30.21268495 13Cenciarelli S, Calbi V, Barzaghi F, et al. Mild SARS-CoV-2 infection after gene therapy in a child with Wiskott-Aldrich syndrome: a case report. Front Immunol. 2020; 11:603428. 14Amodio D, Ruggiero A, Sgrulletti M, et al. Humoral and cellular response following vaccination with the BNT162b2 mRNA COVID-19 vaccine in patients affected by primary immunodeficiencies. Front Immunol. 2021; 12: 727850. doi:10.3389/fimmu.2021.727850 15Brown LK, Moran E, Goodman A, et al. Treatment of chronic or relapsing COVID-19 in immunodeficiency. J Allergy Clin Immunol. 2022; 149(2): 557- 561.e1. Volume33, Issue8August 2022e13833 This article also appears in:COVID-19 and Allergic Diseases FiguresReferencesRelatedInformation

The immunological events leading to type 1 diabetes (T1D) are complex and heterogeneous, underscoring the necessity to study rare cases to improve our understanding. Here, we report the case of a 16-year-old patient who showed glycosuria during a regular checkup. Upon further evaluation, stage 2 T1D, autoimmune thrombocytopenic purpura (AITP), and common variable immunodeficiency (CVID) were diagnosed. The patient underwent low carb diet, losing &amp;gt; 8 kg, and was placed on Ig replacement therapy. Anti-CD20 monoclonal antibody (Rituximab, RTX) was administered 2 years after diagnosis to treat peripheral polyneuropathy, whereas an atypical mycobacteriosis manifested 4 years after diagnosis and was managed with prolonged antibiotic treatment. In the fifth year of monitoring, the patient progressed to insulin dependency despite ZnT8A autoantibody resolution and IA-2A and GADA autoantibody decline. The patient had low T1D genetic risk score (GRS = 0.22817) and absence of human leukocyte antigen (HLA) DR3/DR4-DQ8. Genetic analysis identified the monoallelic mutation H159Y in TNFRSF13C , a gene encoding B-cell activating factor receptor (BAFFR). Significant reduced blood B-cell numbers and BAFFR levels were observed in line with a dysregulation in BAFF–BAFFR signaling. The elevated frequency of PD-1 + dysfunctional Tfh cells composed predominantly by Th1 phenotype was observed at disease onset and during follow-up. This case report describes a patient progressing to T1D on a BAFFR-mediated immunodysregulatory background, suggesting a role of BAFF–BAFFR signaling in islet-specific tolerance and T1D progression.

Proper regulation of the immune system is required for protection against pathogens and preventing autoimmune disorders. Inborn errors of the immune system due to inherited or de novo germline mutations can lead to the loss of protective immunity, aberrant immune homeostasis, and the development of autoimmune disease, or combinations of these. Forward genetic screens involving clinical material from patients with primary immunodeficiencies (PIDs) can vary in severity from life-threatening disease affecting multiple cell types and organs to relatively mild disease with susceptibility to a limited range of pathogens or mild autoimmune conditions. As central mediators of innate and adaptive immune responses, T cells are critical orchestrators and effectors of the immune response. As such, several PIDs result from loss of or altered T cell function. PID-associated functional defects range from complete absence of T cell development to uncontrolled effector cell activation. Furthermore, the gene products of known PID causal genes are involved in diverse molecular pathways ranging from T cell receptor signaling to regulators of protein glycosylation. Identification of the molecular and biochemical cause of PIDs can not only guide the course of treatment for patients, but also inform our understanding of the basic biology behind T cell function. In this chapter, we review PIDs with known genetic causes that intrinsically affect T cell function with particular focus on perturbations of biochemical pathways.

Term and preterm neonates have very few circulating Tfh-like cells (cTfh), and no circulating Tfr-like cells. Neonatal cTfh are CXCR5loPD-1loCD45RAhi, suggestive of a naive, possibly recently activated phenotype. CXCL13 is high at birth, but decreases rapidly in the first weeks of life. Overall, signs of GC activity in human neonates are weak, even in those born prematurely or after sepsis. Neonates, especially those born prematurely, are highly susceptible to systemic infections, and mount ineffective responses to most vaccines [1]. CD4+ T-follicular helper (Tfh) cells express the B-cell-specific chemokine receptor CXCR5 and support the activity of B lymphocytes, but some of them express the FoxP3 transcription factor and are endowed with regulatory activity (T-follicular regulatory [Tfr] cells) [2]. Blood CXCR5+CD4+ T cells represent the circulating counterparts of Tfh and Tfr cells found in secondary lymphoid organs (SLO). Changes in circulating Tfh (cTfh) and Tfr (cTfr) cells frequency and phenotype seem to recapitulate the events taking place in the germinal centers (GCs) of SLO, and are currently considered putative disease biomarkers in several clinical settings [3]. Here, we analyzed circulating CXCR5+CD4+ T cells during the first 5 weeks of life in term and preterm neonates, compared them to older children and adults, and investigated their longitudinal changes in preterm neonates affected by late-onset neonatal sepsis (LOS). We analyzed CXCR5+ T cells from 19 full-term infants and 21 very preterm infants. Demographic data are summarized in Supporting Information Table S1. Three different groups of healthy controls (HCs) were also enrolled: young children (1–9 years old) (n = 9), older children—adolescents (9–18 years old) (n = 29), and adults (n = 27). Groups were balanced for sex (Supporting Information Table S2). Both the percentage and absolute number of CXCR5+CD4+ cells were significantly lower in full-term newborns as compared to HCs, while almost no CXCR5+ CD4+FOXP3+ cTfr cells were detected (Figs. 1A–D) (see Supporting Information for detailed methodology and gating strategy, Figs. S1 and S2). Despite weaker CXCR5 expression, 89% of neonatal CXCR5+ cells expressed CD45RA, suggesting they were activated naive cells. Instead, CXCR5+CD4+ T cells in children and older adults expressed lower levels of CD45RA as compared to naïve T cells, suggesting they were antigen-experienced Tfh cells’ counterparts (Fig. 1E and F). Less than 6% of CXCR5+CD4+ cTfh-like cells in neonates expressed programmed cell death protein 1 (PD-1), in line with their naïve phenotype (Fig. 1G and H). Thus, neonatal CXCR5+CD4+ did not exhibit the canonical phenotype of cTfh cells (CD45RA– /loCXCR5highPD-1high) [4, 5], but were consistent with the immaturity of adaptive immune system typical of early life, and with the known predominance of naïve over memory T cells [6]. CXCL13, the natural ligand of CXCR5, is considered a soluble marker of GC activity [7]. Surprisingly, plasma levels of CXCL13 were higher in full-term newborns at birth compared to HCs, but dropped rapidly after birth (Fig. 1I and Supporting Information Fig. S3C). Considered this and previous evidence [8], we speculate that either a passive transfer from maternal circulation or the local, temporary production by cord macrophages or dendritic stromal cells during the peri-partum inflammatory response may justify the increased CXCL13 levels in neonatal plasma. In very preterm neonates, the absolute number and proportion of CXCR5+CD4+ cTfh-like cells was lower than in term neonates (Fig. 2–C). Conversely, the expression of PD-1 was higher (Fig. 2D), and inversely correlated with gestational age (GA) (Fig. 2E). Similarly, we found higher plasma levels of CXCL13 in preterm neonates (Fig. 2F), which also correlated negatively with GA (Fig. 2G). Thus, despite lower absolute numbers of CXCR5+CD4+ cTfh-like cells, very preterm neonates showed signs of GC activation at birth. These phenomena may be correlated with the pathological mechanisms frequently underlying preterm birth (e.g., intrauterine inflammation). Moreover, we compared Tfh-like associated cell markers in preterm neonates affected or not by LOS. In subsequently septic preterm neonates, the frequency of CXCR5+CD4+ cTfh-like cells at birth was not significantly different from those who did not develop LOS, but an increased proportion of them expressed PD-1 (Fig. 2H and I). However, as septic neonates had lower mean GA compared to nonseptic ones (Supporting information, Table S3), GA may have been the main determinant of the PD-1 high expression on CXCR5+CD4+ cTfh-like cells. Longitudinal analysis showed that the proportion of CXCR5+CD4+ Tfh-like cells increased 2 weeks after neonatal sepsis, while the expression of PD-1 decreased more sharply in those neonates not affected by LOS (Fig. 2J and K and Supporting information Fig. S3). These data suggest that the phenomenon of postsepsis immunoparalysis, typical of adult patients and involving a defective function of T cells, may not affect septic neonates [9]. In summary, our data possibly reflect the absence of active GCs within the human neonatal secondary lymphoid organs, as suggested by previous studies in animal models [6]. This may in turn contribute to the weak response to most vaccines in early life. Nonetheless, the unique phenotype of these neonatal CXCR5+CD4+ cells raises uncertainty about their ontogeny and function, prompting further investigation. Our data provide important clues underlying GC dysfunction in early life, and inform on novel elements to take into account for developing effective vaccines for term and preterm neonates. We thank Serena Scala and Luca Basso-Ricci for their contribution to the study design and for the processing of samples. This work was supported from grants 5×1000 OSR PILOT & SEED GRANT and GR-2016-02365089 to G.F. & M.P.C. The authors declare no commercial or financial conflict of interest. The peer review history for this article is available at https://publons.com/publon/10.1002/eji.202048831. The data that support the findings of this study are available from the corresponding author upon reasonable request. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

Islet autoantibodies (AAbs) are detected in >90% of individuals with clinically suspected type 1 diabetes at disease onset. A single AAb, sometimes at low titre, is often detected in some individuals, making their diagnosis uncertain. Type 1 diabetes genetic risk scores (GRS) are a useful tool for discriminating polygenic autoimmune type 1 diabetes from other types of diabetes, particularly the monogenic forms, but testing is not routinely performed in the clinic. Here, we used a type 1 diabetes GRS to screen for monogenic diabetes in individuals with weak evidence of autoimmunity, i.e. with a single AAb at disease onset.In a pilot study, we genetically screened 142 individuals with suspected type 1 diabetes, 42 of whom were AAb-negative, 27 of whom had a single AAb (single AAb-positive) and 73 of whom had multiple AAbs (multiple AAb-positive) at disease onset. Next-generation sequencing (NGS) was performed in 41 AAb-negative participants, 26 single AAb-positive participants and 60 multiple AAb-positive participants using an analysis pipeline of more than 200 diabetes-associated genes.The type 1 diabetes GRS was significantly lower in AAb-negative individuals than in those with a single and multiple AAbs. Pathogenetic class 4/5 variants in MODY or monogenic diabetes genes were identified in 15/41 (36.6%) AAb-negative individuals, while class 3 variants of unknown significance were identified in 17/41 (41.5%). Residual C-peptide levels at diagnosis were higher in individuals with mutations compared to those without pathogenetic variants. Class 3 variants of unknown significance were found in 11/26 (42.3%) single AAb-positive individuals, and pathogenetic class 4/5 variants were present in 2/26 (7.7%) single AAb-positive individuals. No pathogenetic class 4/5 variants were identified in multiple AAb-positive individuals, but class 3 variants of unknown significance were identified in 19/60 (31.7%) patients. Several patients across the three groups had more than one class 3 variant.These findings provide insights into the genetic makeup of patients who show weak evidence of autoimmunity at disease onset. Absence of islet AAbs or the presence of a single AAb together with a low type 1 diabetes GRS may be indicative of a monogenic form of diabetes, and use of NGS may improve the accuracy of diagnosis.

Topic: 23. Hematopoiesis, stem cells and microenvironment Background: Although most hematopoietic stem/progenitor cells (HSPC) reside in the bone marrow (BM), few circulating HSPC (cHSPC) can be also found in the peripheral blood (PB) at steady state. Aims: The aim of our project is to unveil the contribution of cHSPC to hematopoietic homeostasis and their relationship with BM-resident counterpart. Methods: We phenotypically characterized cHSPC composition by applying multi-parametric flow cytometry on 110 PB and, as control, 48 BM samples of healthy donors (HD) of different age groups. These analyses were combined with single cell RNA sequencing (scRNAseq) and ad hoc designed in vitro and in vivo assays to investigate the transcriptional and functional properties of steady-state cHSPC subpopulations. To study cHSPC vs BM-HSPC differentiation in vivo in humans, we exploited integration site (IS) clonal tracking of cHSPC, BM HSPC and mature PB lineages isolated from 8 HSPC-gene therapy (GT) treated patients once steady-state hematopoiesis is established. Results: cHSPC show a progressive reduction in number during aging and a different composition than BM counterpart, with Multi Lymphoid Progenitors (MLP) displaying the highest PB circulation propensity. Applying scRNAseq on PB- and BM-derived HSPC, we identified a unique transcriptional profile of both primitive and lineage-committed cHSPC subpopulations, characterized by lower replicative, metabolic and transcriptional activity, but increased differentiation-, adhesion- and immune response-priming than BM counterpart. From a functional point of view, cHSPC were endowed with BM homing capability and multilineage differentiation potential both in vitro and in vivo. We also detected a reduced long-term human cell engraftment in cHSPC- than BM HSPC-transplanted mice. This latter finding might be explained by the low primitive Hematopoietic Stem Cell (HSC) content and the transcriptional pre-activated state observed in steady-state PB HSC. Finally, in line with the enrichment of lymphoid transcriptional signature observed in trafficking HSPC, we found a higher IS sharing between PB mature lymphoid compartment and steady-state PB-derived HSPC. Moreover, a substantial fraction of trafficking HSPC displayed an enriched expression of the gene signature associated with thymus seeding progenitor type 1 (TSP1), a group of low-cycling immature lymphoid progenitors with thymus-emigrant properties. Although at lower level with respect to PB cells, we detected a TSP1 signature also in BM dataset, thus suggesting that few thymus-emigrant lymphoid progenitors originate in the BM and preferentially egress into the PB in order to seed lymphoid organs. Summary/Conclusion: Altogether, our findings indicate PB trafficking HSPC as a peculiar steady-state reservoir of low-cycling, pre-activated hematopoietic progenitors, which are endowed with BM homing and repopulation potential. Furthermore, our results of IS analyses combined with the higher expression of TSP1 signature in PB vs. BM HSPC indicate the key function of steady-state trafficking HSPC in the seeding of the thymus, with the aim of locally differentiating into lymphoid progeny. Overall, our work represents one of the most comprehensive studies on cHSPC, unveiling fundamental insights on their biological role in humans. Keywords: Stem cell mobilization, Stem cell gene therapy, Hematopoietic stem and progenitor cells

The severe acute respiratory syndrome coronavirus-2 pandemic significantly affected clinical practice, also in pediatric oncology units. Cancer patients needed to be treated with an adequate dose density despite the SARS-CoV-2 infection, balancing risks of developing severe COVID-19 disease.Although the pandemic spread worldwide, the prevalence of affected children was low. The percentage of children with severe illness was approximately 1-6%. Pediatric cancer patients represent a prototype of a previously healthy immune system that is hampered by the tumor itself and treatments, such as chemotherapy and steroids. Through a review of the literature, we reported the immunological basis of the response to SARS-CoV-2 infection, the existing antiviral treatments used in pediatric cancer patients, and the importance of vaccination. In conclusion, we reported the real-life experience of our pediatric oncology unit during the pandemic period.Starting from the data available in literature, and our experience, showing the rarity of severe COVID-19 disease in pediatric patients with solid tumors, we recommend carefully tailoring all the oncological treatments (chemotherapy/targeted therapy/stem cell transplantation/radiotherapy). The aim is the preservation of the treatment's timing, balanced with an evaluation of possible severe COVID-19 disease.

X-linked chronic granulomatous disease is a rare disease caused by mutations in the CYBB gene. While more extensive knowledge is available on genetics, pathogenesis, and possible therapeutic options, mitochondrial activity and its implications on patient monitoring are still not well-characterized. We have developed a novel protocol to study mitochondrial activity on whole blood of XCGD patients before and after transplantation, as well as on XCGD carriers. Here we present results of these analyses and of the restoration of mitochondrial activity in hyperinflamed X-linked Chronic Granulomatous Disease after hematopoietic stem cell transplantation. Moreover, we show a strong direct correlation between mitochondrial activity, chimerism, and DHR monitored before and after transplantation and in XCGD carriers. In conclusion, based on these findings, we suggest testing this new ready-to-use marker to better characterize patients before and after treatment and to investigate disease expression in carriers.

Deficiency of adenosine deaminase 2 (DADA2) is an autosomal recessive disease associated with a highly variable clinical presentation, including vasculitis, immunodeficiency, and hematologic manifestations, potentially progressing over time. The present study describes the long-term evolution of the immuno-hematological features and therapeutic challenge of two identical adult twin sisters affected by DADA2. The absence of plasmatic adenosine deaminase 2 (ADA2) activity in both twins suggested the diagnosis of DADA2, then confirmed by genetic analysis. Exon sequencing revealed a missense (p.Leu188Pro) mutation on the paternal ADA2 allele. While, whole genome sequencing identified an unreported deletion (IVS6_IVS7del*) on the maternal allele predicted to produce a transcript missing exon 7. The patients experienced the disease onset during childhood with early strokes (Patient 1 at two years, Patient 2 at eight years of age), subsequently followed by other shared DADA2-associated features, including neutropenia, hypogammaglobulinemia, reduced switched memory B cells, inverted CD4:CD8 ratio, increased naïve T cells, reduced follicular regulatory T cells, the almost complete absence of NK cells, T-large granular cell leukemia, and osteoporosis. Disease evolution differed: clinical manifestations presented several years earlier and were more pronounced in Patient 1 than in Patient 2. Due to G-CSF refractory life-threatening neutropenia, Patient 1 successfully underwent an urgent hematopoietic stem cell transplantation (HSCT) from a 9/10 matched unrelated donor. Patient 2 experienced a similar, although delayed, disease evolution and is currently on anti-TNF therapy and anti-infectious prophylaxis. The unique cases confirmed that heterozygous patients with null ADA2 activity deserve deep investigation for possible structural variants on a single allele. Moreover, this report emphasizes the importance of timely recognizing DADA2 at the onset to allow adequate follow-up and detection of disease progression. Finally, the therapeutic management in these identical twins raises significant concerns as they share a similar phenotype, with a delayed but almost predictable disease evolution in one of them, who could benefit from a prompt definitive treatment like elective allogeneic HSCT. Additional data are required to assess whether the absence of enzymatic activity at diagnosis is associated with hematological involvement and is also predictive of bone marrow dysfunction, encouraging early HSCT to improve functional outcomes.

See “Autoimmune Hepatitis in Children in Eastern Denmark” by Vitfell-Pedersen et al on page 376. Autoimmune hepatitis (AIH) may be particularly aggressive in children and induce severe liver damage, if untreated. On the contrary, immunosuppressive therapy, with prednisolone alone or in combination with azathioprine, leads to remission in 80% of cases resulting in a normal life expectancy, good quality of life (1), and a transplant-free survival rate of 90% (2). In this issue of JPGN, Vitfell-Pedersen et al report the features and long-term outcome of children with AIH enrolled in a paediatric population-based survey from the eastern part of Denmark (3). This retrospective study, if compared with previous European series, shows some unusual findings such as male preponderance, extremely low rate of anti-liver kidney microsomal type 1 antibody–positive patients, and absence of fulminant cases (4,5). Despite these peculiarities, which could be due to bias of the study, it is noteworthy that liver cirrhosis, present in 69.6% of patients at diagnosis, did not affect either the overall survival or the survival free of liver transplantation. This favourable outcome could suggest the possibility, reported in the literature, but still a matter of debate among experts, of reversibility of liver fibrosis in some chronic liver diseases (6,7). Obviously, this is a simple hypothesis because histological evaluation of the liver was not performed in Danish patients with sustained biochemical remission. Another relevant finding of the Danish study is that 84% of patients achieved remission in a median of 5.4 months from the start of treatment (60.6% on treatment with prednisolone and azathioprine and 24% on prednisolone alone). Consequently, treatment with prednisolone and azathioprine seemed to be more effective than prednisolone alone in inducing remission. Unfortunately, the authors did not clarify why a single treatment schedule was not used and azathioprine added only to some patients (poor control of transaminases, steroid adverse effects?) or why so many patients were not controlled by treatment. At the end of follow-up, 15% of the patients were in stable remission and off therapy. Even for this issue, no information about the putative predictors of such a good outcome was provided. Actually, despite the availability of many studies and guidelines for AIH (8), many aspects concerning children with AIH remain unclear. In this context, it would have been useful to have more details about clinical features of patients treated with steroid alone versus those treated with steroid and azathioprine, doses of immunosuppressant drugs during the different phases of the treatment, duration of each phase, and minimum required doses to maintain remission to draw conclusions about the best mode of treatment for AIH in children. Other limitations of the study are represented by the scarce use of the available autoantibody repertoire for the diagnosis of AIH (8,9) absence of indication of the autoantibodies’ serum levels considered positive for children (8), and limited use of cholangiography, which was not performed in all children with AIH to exclude biliary involvement according to American Association for the Study of Liver Diseases guidelines (8). Even with these limitations, we think that Vitfell-Pedersen study is valuable because it draws attention to many unclarified points of AIH in childhood. To date, because of the relative rarity of this condition, in common clinical practice the management of children with AIH often reflects the preferences of individual centres (8). It is probably time to initiate large multicentre prospective studies to reach common agreement on the management of children with AIH.

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.

During my PhD program in Human Reproduction, Development and Growth (XXVII Cycle, years 2011-2014), I contributed to the development of new approaches for the treatment of PIDs and other genetic diseases by advanced therapies, such as gene and cellular therapy. Moreover, I participated in the clinical and biological characterization of patients with immunodeficiencies and in the implementation of new approaches for the clinical management of children affected by hematological and immunological disorders undergoing bone marrow transplantation. My other lines of research have been focused on the use of new drugs in pediatric population, characterization of severe and unusual conditions and description of the State of the Art of known patologies in childhood.