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International audience; Long-term multilineage hematopoietic donor chimerism occurs sporadically in patients who receive a transplanted solid organ enriched in lymphoid tissues such as the intestine or liver. There is currently no evidence for the presence of kidney-resident hematopoietic stem cells in any mammal species. Graft-versus-host-reactive donor T cells promote engraftment of graft-derived hematopoietic stem cells by making space in the bone marrow. Here, we report full (over 99%) multilineage, donor-derived hematopoietic chimerism in a pediatric kidney transplant recipient with syndromic combined immune deficiency that leads to transplant tolerance. Interestingly, we found that the human kidney-derived hematopoietic stem cells took up long-term residence in the recipient's bone marrow and gradually replaced their host counterparts, leading to blood type conversion and full donor chimerism of both lymphoid and myeloid lineages. Thus, our findings highlight the existence of human kidney-derived hematopoietic stem cells with a self-renewal ability able to support multilineage hematopoiesis.

The use of chimeric antigen receptor (CAR)-engineered regulatory T cells (Tregs) has emerged as a promising strategy to promote immune tolerance. However, in conventional T cells (Tconvs), CAR expression is often associated with tonic signaling, which can induce CAR-T cell dysfunction. The extent and effects of CAR tonic signaling vary greatly according to the expression intensity and intrinsic properties of the CAR. Here, we show that the 4-1BB CSD-associated tonic signal yields a more dramatic effect in CAR-Tregs than in CAR-Tconvs with respect to activation and proliferation. Compared to CD28 CAR-Tregs, 4-1BB CAR-Tregs exhibit decreased lineage stability and reduced in vivo suppressive capacities. Transient exposure of 4-1BB CAR-Tregs to a Treg stabilizing cocktail, including an mTOR inhibitor and vitamin C, during ex vivo expansion sharply improves their in vivo function and expansion after adoptive transfer. This study demonstrates that the negative effects of 4-1BB tonic signaling in Tregs can be mitigated by transient mTOR inhibition., Chimeric antigen receptor engineering in T cells has been shown to be of great potential therapeutic benefit in a range of immune pathologies, although the functionality of such cell therapies can be limited due to tonic signalling and the induction of dysfunction. Here the authors show transient inhibition of mTOR can rescue their 41-BB-CAR-Tregs from tonic signalling-induced dysfunction.

Gene therapy (GT) has rapidly attracted renewed interest as a treatment for otherwise incurable diseases, with several GT products already on the market and many more entering clinical testing for selected indications. Clonal tracking techniques based on vector integration enable monitoring of the fate of engineered cells in the blood of patients receiving GT and allow assessment of the safety and efficacy of these procedures. However, owing to the limited number of cells that can be tested and the impracticality of studying cells residing in peripheral organs without performing invasive biopsies, this approach provides only a partial snapshot of the clonal repertoire and dynamics of genetically modified cells and reduces the predictive power as a safety readout. In this study, we developed liquid biopsy integration site sequencing, or LiBIS-seq, a polymerase chain reaction technique optimized to quantitatively retrieve vector integration sites from cell-free DNA released into the bloodstream by dying cells residing in several tissues. This approach enabled longitudinal monitoring of in vivo liver-directed GT and clonal tracking in patients receiving hematopoietic stem cell GT, improving our understanding of the clonal composition and turnover of genetically modified cells in solid tissues and, in contrast to conventional analyses based only on circulating blood cells, enabling earlier detection of vector-marked clones that are aberrantly expanding in peripheral tissues.

T-cell acute lymphoblastic leukemia (T-ALL) represents the malignant expansion of immature T cells blocked in their differentiation. T-ALL is still associated with a poor prognosis, mainly related to occurrence of relapse or refractory disease. A critical medical need therefore exists for new therapies to improve the disease prognosis. Adenylate kinase 2 (AK2) is a mitochondrial kinase involved in adenine nucleotide homeostasis recently reported as essential in normal T-cell development, as defective AK2 signaling pathway results in a severe combined immunodeficiency with a complete absence of T-cell differentiation. In this study, we show that AK2 is constitutively expressed in T-ALL to varying levels, irrespective of the stage of maturation arrest or the underlying oncogenetic features. T-ALL cell lines and patient T-ALL–derived xenografts present addiction to AK2, whereas B-cell precursor ALL cells do not. Indeed, AK2 knockdown leads to early and massive apoptosis of T-ALL cells that could not be rescued by the cytosolic isoform AK1. Mechanistically, AK2 depletion results in mitochondrial dysfunction marked by early mitochondrial depolarization and reactive oxygen species production, together with the depletion of antiapoptotic molecules (BCL-2 and BCL-XL). Finally, T-ALL exposure to a BCL-2 inhibitor (ABT-199 [venetoclax]) significantly enhances the cytotoxic effects of AK2 depletion. We also show that AK2 depletion disrupts the oxidative phosphorylation pathway. Combined with pharmaceutical inhibition of glycolysis, AK2 silencing prevents T-ALL metabolic adaptation, resulting in dramatic apoptosis. Altogether, we pinpoint AK2 as a genuine and promising therapeutic target in T-ALL.

Wiskott Aldrich syndrome (WAS) is a rare X-linked primary immunodeficiency associated with thrombocytopenia, eczema, infectious, autoimmune complications, and lymphomas. Patients lacking an HLA-matched donor may benefit from an alternative therapeutic approach based on the infusion of autologous gene corrected CD34+ cells. We previously reported a non-randomised, open-label, phase 1/2 clinical study applying a lentiviral vector based gene therapy (GT) protocol in 7 paediatric patients with severe WAS (score ≥ 3/5) (S. Hacein-Bey Abina et al, JAMA 2015). One patient died 7 months after GT because of pre-existing severe opportunistic infections, as reported. Two additional patients have been treated since that initial report, with a follow-up of at least 4 years. We here present a comprehensive long-term study on 8 patients with a follow-up from 4 to 9 years (median 7.6). The safety and efficacy of the approach is thoroughly investigated, with a particular focus on the correction of thrombocytopenia and auto-immunity. A stable engraftment of genetically and functionally corrected lymphoid and myeloid cells was reached in all patients, with no serious treatment-associated adverse events or concerning clonal expansion. Corrected lymphoid cells displayed a selective advantage over time with increasing vector copy number (VCN) level. In turn, this led to (i) sustained expression of WAS protein (WASp) in the patients' cells and (ii) clinical resolution of severe eczema and susceptibility to recurrent infections. In line with these results, T-cell function was restored after GT, as shown by the recovery of immune synapse assembly and the normalization of naïve T cell numbers. The T-cell compartment was also reconstituted in the patient treated at the age of 30 years, suggesting that GT for WAS is a treatment option in adult patients. In parallel with the robustness of T-cell reconstitution a normalized B-cell compartment was observed after GT, as shown in particular by increasing levels of WASp + switched memory B cells over time and the age-matched levels of KRECs. Five patients out of 8 were able to discontinue Ig replacement therapy while achieving normal post-vaccination antibody titers. Autoimmune disorders and bleeding episodes were significantly less frequent, despite only partial correction of the platelet compartment. After GT, a few autoimmune manifestations were observed: the persistence of lower extremity vasculitis (P2, very severe prior to GT), the new occurrence of nephrotic syndrome (P9), and the presence of anti-platelet antibodies (P2, P4, P7). The levels of circulating autoantibodies detected before GT (including ANA and vasculitis-related autoantibodies) normalized after treatment. Following GT, platelets were found to express sub-normal levels of WASp and to only partially augment their size. Platelet function studies indicated a partial correction of the platelet compartment achieved by GT, which may be sufficient to prevent occurrence of the hemorrhagic symptoms typical of WAS. Our results suggest that lentiviral GT provides sustained clinical benefits for patients with WAS. Overall clinical remission was observed in our patients despite very severe disease scores before GT. More efficacious and more reliable transduction protocols and conditioning regimen are likely to further improve outcomes, particularly with regard to platelet recovery, where the advantages of intrinsic correction are less apparent. Disclosures Booth: Orchard Therapeutics: Consultancy, Honoraria; SOBI: Consultancy, Honoraria; Takeda: Honoraria; GSK: Honoraria; Rocket Pharmaceuticals, Inc.: Consultancy. Thrasher: Orchard Therapeutics: Consultancy, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Generation bio: Consultancy, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; 4Bio Capital: Consultancy, Membership on an entity's Board of Directors or advisory committees; Rocket Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees. Cavazzana: Smart Immune: Other: co-founder.

Pioneering gene therapy trials have shown that the genetic engineering of haematopoietic stem and progenitor cells can be an alternative to allogeneic transplantation in the treatment of primary immunodeficiencies. Early trials also highlighted the risk of insertional mutagenesis and oncogene transactivation associated with the first generation of gammaretroviral vectors. These events prompted the development of safer, self-inactivating lentiviral or gammaretroviral vectors. These lentiviral vectors have been successfully used to treat over 200 patients with 10 different haematological disorders (including primary immunodeficiencies, haemoglobinopathies and metabolic disorders) and for the generation of chimeric antigen receptor-T cells for cancer therapy. However, several challenges, such as effective reconstitution during inflammation, remain if gene therapy is to be extended to more complex diseases in which haematopoietic stem and progenitor cells can be altered by the disease environment. We discuss the progress made and future challenges for gene therapy and contrast gene therapy with gene-editing strategies. Advances in the design of vectors based on retroviruses, such as lentiviruses and gammaretroviruses, have led to improvements in the safety and stability of gene therapies directed at haematopoietic stem and progenitor cells. In this Review, Cavazzana and colleagues discuss the results from recent clinical trials of retroviral vectors for the treatment of genetic disorders, including severe combined immunodeficiencies and β-haemoglobinopathies (β-thalassaemia and sickle cell disease). They highlight the progress made and the remaining challenges in applying gene therapies more broadly.

FOXP3 deficiency in mice and in patients with immune dysregulation polyendocrinopathy enteropathy X-linked (IPEX) syndrome results in fatal autoimmunity by altering regulatory T (T-reg) cells. CD4(+) T cells in patients with IPEX syndrome and Foxp3-deficient mice were analyzed by single-cell cytometry and RNA-sequencing, revealing heterogeneous T-reg-like cells, some very similar to normal T-reg cells, others more distant. Conventional T cells showed no widespread activation or helper T cell bias, but a monomorphic disease signature affected all CD4(+) T cells. This signature proved to be cell extrinsic since it was extinguished in mixed bone marrow chimeric mice and heterozygous mothers of patients with IPEX syndrome. Normal T-reg cells exerted dominant suppression, quenching the disease signature and revealing in mutant T-reg-like cells a small cluster of genes regulated cell-intrinsically by FOXP3, including key homeostatic regulators. We propose a two-step pathogenesis model: cell-intrinsic downregulation of core FOXP3-dependent genes destabilizes T-reg cells, de-repressing systemic mediators that imprint the disease signature on all T cells, furthering T-reg cell dysfunction. Accordingly, interleukin-2 treatment improved the T-reg-like compartment and survival. FOXP3 deficiency leads to dramatic loss of immune homeostasis. This multicenter collaborative group finds that loss of FOXP3 function only disrupts a few core genes, but this unmasks a degree of systemic inflammation, and it is this environment that then strongly perturbs T-reg cells.

Immunodysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) syndrome is caused by mutations in forkhead box P3 (FOXP3), which lead to the loss of function of regulatory T cells (Tregs) and the development of autoimmune manifestations early in life. The selective induction of a Treg program in autologous CD4+ T cells by FOXP3 gene transfer is a promising approach for curing IPEX. We have established a novel in vivo assay of Treg functionality, based on adoptive transfer of these cells into scurfy mice (an animal model of IPEX) and a combination of cyclophosphamide (Cy) conditioning and interleukin-2 (IL-2) treatment. This model highlighted the possibility of rescuing scurfy disease after the latter’s onset. By using this in vivo model and an optimized lentiviral vector expressing human Foxp3 and, as a reporter, a truncated form of the low-affinity nerve growth factor receptor (ΔLNGFR), we demonstrated that the adoptive transfer of FOXP3-transduced scurfy CD4+ T cells enabled the long-term rescue of scurfy autoimmune disease. The efficiency was similar to that seen with wild-type Tregs. After in vivo expansion, the converted CD4FOXP3 cells recapitulated the transcriptomic core signature for Tregs. These findings demonstrate that FOXP3 expression converts CD4+ T cells into functional Tregs capable of controlling severe autoimmune disease.

Sickle cell disease (SCD) and transfusion-dependent β-thalassemia (TDT) are the most prevalent monogenic disorders worldwide. Trial HGB-205 ( NCT02151526 ) aimed at evaluating gene therapy by autologous CD34

The use of chimeric antigen receptor (CAR)-engineered regulatory T cells (Tregs) has emerged as a promising strategy to promote immune tolerance in transplantation. However, in conventional T cells (Tconvs), CAR expression is often associated with tonic signaling resulting from ligand-independent baseline activation. Tonic signaling may cause CAR-T cell dysfunction, especially when the CAR structure incorporates the CD28 costimulatory domain (CSD) rather than the 4-1BB CSD.Here, we explored the impact of tonic signaling on human CAR-Tregs according to the type of CSD. Compared to CD28-CAR-Tregs, 4-1BB-CAR-Tregs showed enhanced proliferation and greater activation of MAP kinase and mTOR pathways but exhibited decreased lineage stability and reduced abilities to produce IL-10 and be restimulated through the CAR. Although both CAR-Treg populations were suppressive in vivo, cell tracking with bioluminescence imaging found longer persistence for CD28-CAR-Tregs than for 4-1BB-CAR-Tregs. This study demonstrates that CD28-CAR best preserves Treg function and survival in the context of tonic signaling, in contrast with previous findings for Tconvs.SUMMARYLamarthée et al investigated the impact of chimeric antigen receptor (CAR) tonic signaling on CAR-engineered Tregs according to the incorporated costimulatory domain (either CD28 or 4-1BB). CD28 ameliorated Treg stability, long-term survival and function compared to 4-1BB.

Because of the stochasticity associated with high-throughput single-cell sequencing, current methods for exploring cell-type diversity rely on clustering-based computational approaches in which heterogeneity is characterized at cell subpopulation rather than at full single-cell resolution. Here we present Cell-ID, a clustering-free multivariate statistical method for the robust extraction of per-cell gene signatures from single-cell sequencing data. We applied Cell-ID to data from multiple human and mouse samples, including blood cells, pancreatic islets and airway, intestinal and olfactory epithelium, as well as to comprehensive mouse cell atlas datasets. We demonstrate that Cell-ID signatures are reproducible across different donors, tissues of origin, species and single-cell omics technologies, and can be used for automatic cell-type annotation and cell matching across datasets. Cell-ID improves biological interpretation at individual cell level, enabling discovery of previously uncharacterized rare cell types or cell states. Cell-ID is distributed as an open-source R software package.

The exhaustive exploration of human cell heterogeneity requires the unbiased identification of molecular signatures that can serve as unique cell identity cards for every cell in the body. However, the stochasticity associated with high-throughput single-cell sequencing has made it necessary to use clustering-based computational approaches in which the characterization of cell-type heterogeneity is performed at cell-subpopulation level rather than at full single-cell resolution. We present here Cell-ID, a clustering-free multivariate statistical method for the robust extraction of per-cell gene signatures from single-cell sequencing data. Cell-ID signatures allow unbiased cell identity recognition across different donors, tissues-of-origin, model organisms and single-cell omics technologies. Cell-ID is distributed as an open-source R software package:https://github.com/RausellLab/CelliD.

FOXP3 deficiency in mice and IPEX syndrome patients results in fatal autoimmunity by altering T regulatory cells (Treg). CD4+ T cells in IPEX patients and Foxp3-deficient mice were analyzed by single-cell cytometry and RNAseq, revealing heterogeneous Treg-like cells, some very similar to normal Tregs, others more distant. Conventional T cells showed no widespread activation or Th bias, but a monomorphic disease signature affected all CD4+ T cells, cell-extrinsic signature since extinguished in mixed bone marrow chimeric mice and heterozygous mothers of IPEX patients. Normal Tregs exerted dominant suppression, extinguishing the disease signature, and revealing in mutant Treg-like cells a small cluster of genes regulated cell-intrinsically by FOXP3, including key homeostatic regulators. We propose a two-step pathogenesis model: cell-intrinsic downregulation of core genes destabilizes Tregs, de-repressing systemic mediators which imprint the disease signature on all T cells, furthering Treg dysfunction. Accordingly, IL2 treatment improved the Treg-like compartment and survival.

FOXP3 deficiency in humans with IPEX syndrome and mice results in fatal systemic autoimmunity by altering regulatory T cell (Treg) physiology, but actual cellular and molecular mechanisms of disease are unclear, part because Treg surface markers may be unreliable in disease states. We used deep profiling by flow cytometry, population and single-cell RNAseq to analyze Tregs and conventional (Tconv) CD4+ T lymphocytes in cohorts of IPEX patients with a range of genetic lesions, and in Foxp3-deficient mice. In all patients and mice, heterogeneous Treg-like cells with an active FOXP3 locus were observed, some differing very little from normal Tregs, others more distant. Tconv showed no widespread activation or Th bias. The dominant mark was a monomorphic signature equally affecting all CD4+ T cells, unexpectedly dampening tumor-Treg and cytokine-signaling modules. In mixed bone marrow chimeras, WT Tregs exerted dominant suppression, normalizing the states of mutant Treg and Tconv, extinguishing the disease signature, and revealing a small gene cluster truly regulated, cell-intrinsically, by FOXP3. These results suggest a two-step pathogenesis model, with therapeutic implications: limited downregulation of a few core Treg genes de-represses a systemic mediator(s), which imprints the disease signature on all T cells, and further dampens Treg function.

In sickle cell disease (SCD), the β6 Glu→Val substitution leads to sickle hemoglobin (HbS) polymerization and red blood cell (RBC) sickling. Transplantation of autologous, genetically modified hematopoietic stem/progenitor cells (HSPCs) represents a promising therapeutic option for patients lacking a compatible donor. We previously designed a lentiviral vector (βAS3 LV) expressing a potent anti-sickling βAS3 globin and demonstrated its safety and efficacy in SCD patient cells (Weber et al., 2018). In vitro and in vivo preclinical studies demonstrated the safety and efficacy of a gene therapy (GT) protocol based on the efficient transduction of plerixafor-mobilized SCD HSPCs by βAS3 LV. Based on these results a Phase 1/2 clinical trial (NCT03964792) started in January 2020. Three homozygous SCD patients (P1, P2 and P3) were recruited. Before GT, patients suffered from severe SCD symptoms that were not controlled despite of a strict adherence to the treatment. Plerixafor-mobilized HSPCs were transduced achieving a VCN of 0.9±0.2 in liquid culture, 1.6±0.3 in BFU-E and 0.8±0.2 in CFU-GM. The patients were hypertransfused to reach HbS levels Here, for the 3 patients we report the follow-up of 18 (P1), 10 (P2) and 6 (P3) months. No drug-related severe adverse events were observed. Importantly, no sign of clonal hematopoiesis was detected by NGS sequencing performed on the graft before and after transduction. Despite of the similar VCN in the drug product, gene marking in peripheral blood mononuclear cells was variable and ranged between 0.2 and 0.6, as measured at the last follow-up (0.4 in P1, 0.6 in P2 and 0.2 in P3). These results pinpoint the difficulty to estimate the gene marking, self-renewal and engraftment potential of HSCs in the grafts. P1 and P2 clinically benefit from the GT treatment despite they presented non-severe vaso-occlusive crises (VOCs) not requiring hospitalization (two for P1 at month 6 and 14 and one in P2 at month 8 post-GT). Despite the rapid resolution of these episodes, P1 started hydroxyurea at month 10 and stopped it at month 16. P2 initiated a phlebotomy program to treat liver iron overload and decrease viscosity. Intravascular hemolysis improved in P1 and P2, as shown by a decrease in plasma hemoglobin and heme, and an increase in hemopexin (HPX). Interestingly, in P1 a mild worsening occurred at the time of VOC. In P1 and P2, total bilirubin, a marker of extravascular hemolysis, decreased post-GT. Deformability, measured using osmoscan, improved in P1 and to a lesser extent in P2; however, both still exhibited some degree of dehydration compared to normal RBCs (Fig. 1). Adhesion to thrombospondin decreased after GT with an increase during VOC in P1 or prior the VOC in P2 (Fig. 1). In both patients, markers of inflammation (e.g., C reactive protein) and reticulocyte counts decreased upon GT. An in vitro sickling assays was performed for P1 and showed significant improvement, reaching a frequency of sickling cells similar to SCD heterozygous carriers (from 88% before GT to 45% 6 months post-GT). The GT treatment was not effective in P3 due to the modest intake and rapid decline of gene modified cells. In P1 and P2, who were no longer on transfusions, HbAS3 levels correlated with the VCN (2.1 g/dl for P1 and 3.3 g/dl for P2) and the therapeutic Hb accounted for 21.5%, and 28.7% of the total hemoglobins, respectively. For P3, who remained transfusion-dependent, this frequency was Overall, these clinical data indicate a variable efficacy of the DREPAGLOBE GT treatment, which likely depends on the extent of gene marking achieved in HSCs in vivo and on the engraftment capability of genetically modified HSCs in SCD patients' bone marrow. Single-cell RNA-seq analysis of HSPCs and evaluation of the bone marrow niche in SCD patients will aid to define critical parameters for achieving successful outcomes in GT clinical trials for SCD. Figure 1 Figure 1. Disclosures Joseph: bluebird bio: Consultancy. El Nemer: Hemanext: Consultancy. Bartolucci: INNOVHEM: Other: Co-founder; AGIOS: Consultancy; GBT: Consultancy; Bluebird: Consultancy, Research Funding; Jazz Pharma: Other: Lecture fees; Fabre Foundation: Research Funding; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Lecture fees, Steering committee, Research Funding; F. Hoffmann-La Roche Ltd: Consultancy; Hemanext: Consultancy; Addmedica: Consultancy, Other: Lecture fees, Research Funding; Emmaus: Consultancy. Cavazzana: Smart Immune: Other: co-founder.

Integration of new DNA into cellular genomes mediates replication of retroviruses and transposons; integration reactions have also been adapted for use in human gene therapy. Tracking the distributions of integration sites is important to characterize populations of transduced cells and to monitor potential outgrow of pathogenic cell clones. Here, we describe a pipeline for quantitative analysis of integration site distributions named INSPIIRED (integration site pipeline for paired-end reads). We describe optimized biochemical steps for site isolation using Illumina paired-end sequencing, including new technology for suppressing recovery of unwanted contaminants, then software for alignment, quality control, and management of integration site sequences. During library preparation, DNAs are broken by sonication, so that after ligation-mediated PCR the number of ligation junction sites can be used to infer abundance of gene-modified cells. We generated integration sites of known positions insilico, and we describe optimization of sample processing parameters refined by comparison to truth. We also present a novel graph-theory-based method for quantifying integration sites in repeated sequences, and we characterize the consequences using synthetic and experimental data. In an accompanying paper, we describe an additional set of statistical tools for data analysis and visualization. Software is available at https://github.com/BushmanLab/INSPIIRED.

Although studies of mixed chimerism following hematopoietic stem cell transplantation in patients with sickle cell disease (SCD) may provide insights into the engraftment needed to correct the disease and into immunological reconstitution, an extensive multilineage analysis is lacking. We analyzed chimerism simultaneously in peripheral erythroid and granulomonocytic precursors/progenitors, highly purified B and T lymphocytes, monocytes, granulocytes and red blood cells (RBC). Thirty-four patients with mixed chimerism and ≥12 months of follow-up were included. A selective advantage of donor RBC and their progenitors/precursors led to full chimerism in mature RBC (despite partial engraftment of other lineages), and resulted in the clinical control of the disease. Six patients with donor chimerism 10 g/dL) and three with AS donors (hemoglobin

Background Mutation of the IL2RG gene results in a form of severe combined immune deficiency (SCID-X1), which has been treated successfully with hematopoietic stem cell gene therapy. SCID-X1 gene therapy results in reconstitution of the previously lacking T cell compartment, allowing analysis of the roles of T cell immunity in humans by comparing before and after gene correction. Methods Here we interrogate T cell reconstitution using four forms of high throughput analysis. (1) Estimation of the numbers of transduced progenitor cells by monitoring unique positions of integration of the therapeutic gene transfer vector. (2) Estimation of T cell population structure by sequencing of the recombined T cell receptor (TCR) beta locus. (3) Metagenomic analysis of microbial populations in oropharyngeal, nasopharyngeal, and gut samples. (4) Metagenomic analysis of viral populations in gut samples. Results Comparison of progenitor and mature T cell populations allowed estimation of a minimum number of cell divisions needed to generate the observed populations. Analysis of microbial populations showed the effects of immune reconstitution, including normalization of gut microbiota and clearance of viral infections. Metagenomic analysis revealed enrichment of genes for antibiotic resistance in gene-corrected subjects relative to healthy controls, likely a result of higher healthcare exposure. Conclusions This multi-omic approach enables the characterization of multiple effects of SCID-X1 gene therapy, including T cell repertoire reconstitution, estimation of numbers of cell divisions between progenitors and daughter T cells, normalization of the microbiome, clearance of microbial pathogens, and modulations in antibiotic resistance gene levels. Together, these results quantify several aspects of the long-term efficacy of gene therapy for SCID-X1. This study includes data from ClinicalTrials.gov numbers NCT01410019, NCT01175239, and NCT01129544. Electronic supplementary material The online version of this article (10.1186/s13073-018-0580-z) contains supplementary material, which is available to authorized users.

International audience; The hierarchy of human hemopoietic progenitor cells that produce lymphoid and granulocytic–monocytic (myeloid) lineages is unclear. Multiple progenitor populations produce lymphoid and myeloid cells, but they remain incompletely characterized. Here we demonstrated that lympho-myeloid progenitor populations in cord blood — lymphoid-primed multi-potential progenitors (LMPPs), granulocyte-macrophage progenitors (GMPs) and multi-lymphoid progenitors (MLPs) — were functionally and transcriptionally distinct and heterogeneous at the clonal level, with progenitors of many different functional potentials present. Although most progenitors had the potential to develop into only one mature cell type (‘uni-lineage potential’), bi- and rarer multi-lineage progenitors were present among LMPPs, GMPs and MLPs. Those findings, coupled with single-cell expression analyses, suggest that a continuum of progenitors execute lymphoid and myeloid differentiation, rather than only uni-lineage progenitors’ being present downstream of stem cells.

Analysis of sites of newly integrated DNA in cellular genomes is important to several fields, but methods for analyzing and visualizing these datasets are still under development. Here, we describe tools for data analysis and visualization that take as input integration site data from our INSPIIRED pipeline. Paired-end sequencing allows inference of the numbers of transduced cells as well as the distributions of integration sites in target genomes. We present interactive heatmaps that allow comparison of distributions of integration sites to genomic features and that support numerous user-defined statistical tests. To summarize integration site data from human gene therapy samples, we developed a reproducible report format that catalogs sample population structure, longitudinal dynamics, and integration frequency near cancer-associated genes. We also introduce a novel summary statistic, the UC50 (unique cell progenitors contributing the most expanded 50% of progeny cell clones), which provides a single number summarizing possible clonal expansion. Using these tools, we characterize ongoing longitudinal characterization of a patient from the first trial to treat severe combined immunodeficiency-X1 (SCID-X1), showing successful reconstitution for 15 years accompanied by persistence of a cell clone with anintegration site near the cancer-associated gene CCND2. Software is available at https://github.com/BushmanLab/INSPIIRED.

International audience; More than 20 years ago, X-linked severe combined immunodeficiency (SCID-X1) appeared to be the best condition to test the feasibility of hematopoietic stem cell gene therapy. The seminal SCID-X1 clinical studies, based on first-generation gammaretroviral vectors, demonstrated good long-term immune reconstitution in most treated patients despite the occurrence of vector-related leukemia in a few of them. This gene therapy has successfully enabled correction of the T cell defect. Natural killer and B cell defects were only partially restored, most likely due to the absence of a conditioning regimen. The success of these pioneering trials paved the way for the extension of gene-based treatment to many other diseases of the hematopoietic system, but the unfortunate serious adverse events led to extensive investigations to define the retrovirus integration profiles. This review puts into perspective the clinical experience of gene therapy for SCID-X1, with the development and implementation of new generations of safer vectors such as self-inactivating gammaretroviral or lentiviral vectors as well as major advances in integrome knowledge.

Slow T-cell reconstitution is a major clinical concern after transplantation of cord blood (CB)-derived hematopoietic stem cells. Adoptive transfer of in vitro-generated T-cell progenitors has emerged as a promising strategy for promoting de novo thymopoiesis and thus accelerating T-cell reconstitution. Here, we describe the development of a new culture system based on the immobilized Notch ligand Delta-like-4 (DL-4). Culture of human CD34+ CB cells in this new DL-4 system enabled the in vitro generation of large amounts of T-cell progenitor cells that (a) displayed the phenotypic and molecular signatures of early thymic progenitors and (b) had high T lymphopoietic potential. When transferred into NOD/SCID/γc−/− (NSG) mice, DL-4 primed T-cell progenitors migrated to the thymus and developed into functional, mature, polyclonal αβ T cells that subsequently left the thymus and accelerated T-cell reconstitution. T-cell reconstitution was even faster and more robust when ex vivo-manipulated and nonmanipulated CB samples were simultaneously injected into NSG mice (i.e., a situation reminiscent of the double CB transplant setting). This work provides further evidence of the ability of in vitro-generated human T-cell progenitors to accelerate T-cell reconstitution and also introduces a feeder-cell-free culture technique with the potential for rapid, safe transfer to a clinical setting.

An important proof of principle has been achieved with the development of an in vitro T-cell differentiation assay based on the coculture of hematopoietic progenitors with the OP9-Delta1 stromal cell line. The original murine T cell differentiation assay has since been adapted for human T-cell differentiation, however with lower efficiency. The choice of both medium and cytokines is crucial in this assay, therefore our work has been focused on these two factors. The use of freshly reconstituted medium, the optimization of interleukine-7 (IL-7) concentration, and the addition of stem cell factor (SCF) have allowed to improve the proliferation of progenitors and T-cell precursors as well as the yield of double positive CD4+CD8+ T cells, and mature γδ and αβ T cells. These optimizations make the OP9-Delta1 system sensitive enough to perform both quantitative and qualitative assays with various type of progenitors, including those transduced by a retroviral vector. The improved OP9-Delta1 assay therefore constitutes an extremely useful test for basic research purposes and for translational medicine.

Human adenylate kinase 2 deficiency causes the most profound human haematopoietic defect associated with sensorineural deafness. Reticular dysgenesis (RD) is an autosomal recessive form of human severe combined immunodeficiency (SCID) characterized by an early differentiation block in the myeloid lineage and a profound impairment in lymphoid maturation associated with bilateral sensorineural deafness. The lack of polymorphonuclear neutrophils in affected newborns is responsible for the occurrence of severe infections earlier than usually observed in the other forms of SCID. Furthermore, RD associated neutropenia is characterized by the lack of responsiveness to G-CSF. We have identified bi-allelic mutations in the adenylate kinase 2 (AK2) gene is seven patients affected with RD. These mutations resulted in the absence or a strong decrease in protein expression. Restoration of AK2 expression in the bone marrow cells of RD patients overcomes the neutrophil differentiation arrest, underlying its specific requirement in the development of a restricted set of haematopoietic lineages. Lastly, we established that AK2 is specifically expressed in the stria vascularis region of the inner ear. The function of this gene in the differentiation of a given set of cell lineages is rapidly in progress, showing that studies of primary immunodeficiencies continue to provide key information on human lympho-haematopoietic development. Moreover the AK2 enzyme seems a key molecule in different biological systems such as the lympho-haematopoiesis and the brain development.

The delayed reconstitution of the T-lymphoid compartment represents a major clinical challenge after HLA-mismatched hematopoietic stem cell transplantation. The generation of new T lymphocytes deriving from transplanted hematopoietic stem cells requires several months, a period associated with an increased risk of opportunistic infections and relapses. Recently, the early steps of human lymphopoiesis and the nature of the thymus-seeding progenitors were described. Moreover several scientific groups succeeded to generate T-cell precursors from murine and human hematopoietic stem cells in vitro by transitory exposition to Notch-ligands. Here we summarize and discuss these results and their possible usage in the development of new cell therapies to shorten the immunodeficient period following hematopoietic stem cell transplantation.

To address the role of chromatin structure in the establishment of hematopoietic stem cell (HSC) multilineage potential and commitment to the lymphoid lineage, we have analyzed histone modifications at a panel of lymphoid- and myeloid-affiliated genes in multipotent and lineage-committed hematopoietic cells isolated from human cord blood. Our results show that many B- and T-lymphoid genes, although silent in HSCs, are associated with acetylated histones H3 and H4. We also detected histone H3 lysine 4 methylation but not repressive lysine 9 or 27 methylation marks at these loci, indicative of an open chromatin structure. Interestingly, the relative level of H3 lysine 4 dimethylation to trimethylation at B-specific loci was high in multipotent CD34+CD38lo progenitors and decreased as they become actively transcribed in B-lineage cells. In vitro differentiation of CD34+ cells toward the erythroid, granulocyte, and T-cell lineages resulted in a loss of histone acetylation at nonlineage-associated genes. This study provides evidence that histone modifications involved in chromatin decondensation are already in place at lymphoid-specific genes in primary human HSCs, supporting the idea that these genes are “primed” for expression before lineage commitment. This permissive chromatin structure is progressively lost as the stem cell differentiates.

Identification of a thymus-seeding progenitor originating from human bone marrow (BM) constitutes a key milestone in understanding the mechanisms of T cell development and provides new potential for correcting T cell deficiencies. We report the characterization of a novel lymphoid-restricted subset, which is part of the lineage-negative CD34(+)CD10(+) progenitor population and which is distinct from B cell-committed precursors (in view of the absence of CD24 expression). We demonstrate that these Lin(-)CD34(+)CD10(+)CD24(-) progenitors have a very low myeloid potential but can generate B, T, and natural killer lymphocytes and coexpress recombination activating gene 1, terminal deoxynucleotide transferase, PAX5, interleukin 7 receptor alpha, and CD3epsilon. These progenitors are present in the cord blood and in the BM but can also be found in the blood throughout life. Moreover, they belong to the most immature thymocyte population. Collectively, these findings unravel the existence of a postnatal lymphoid-polarized population that is capable of migrating from the BM to the thymus.

International audience; Activation of mammalian Notch receptor by its ligands induces TNFalpha-converting enzyme-dependent ectodomain shedding, followed by intramembrane proteolysis due to presenilin (PS)-dependent gamma-secretase activity. Here, we demonstrate that a new modification, a monoubiquitination, as well as clathrin-dependent endocytosis, is required for gamma-secretase processing of a constitutively active Notch derivative, DeltaE, which mimics the TNFalpha-converting enzyme-processing product. PS interacts with this modified form of DeltaE, DeltaEu. We identified the lysine residue targeted by the monoubiquitination event and confirmed its importance for activation of Notch receptor by its ligand, Delta-like 1. We propose a new model where monoubiquitination and endocytosis of Notch are a prerequisite for its PS-dependent cleavage, and discuss its relevance for other gamma-secretase substrates.

Introduction: Patients with sickle cell disease (SCD) may develop a persistent mixed chimerism (MC) after hematopoietic stem cell transplantation (HSCT) associated with clinical control of the disease. In order to further investigate this condition we analyzed the chimerism in sorted myeloid and lymphoid subpopulations, and erythroid progenitors.Methods: Between 1990 and 2013, 112 sickle cell disease (SCD) patients underwent allogeneic HSCT after myeloablative conditioning. Among the 107 patients with available chimerism at 2 years, 55.1% had a MC, i.e. 9590nana6AA9111.893.83.13.10191223311337.5577AS15513.556.5412.60583471na4975604AS13210.65433.3nana5344444658323717AS1311.547.841.23.21.48575na9273837618A/Dpubjab8012.647.90nana887795959494.59912A/b0Thal579.69503291689698999910011AA5413.49703088781001009998985AA3410.978.38nana444641na5058432AS468.42468.1nana21709.45.43112.5na8AS1339.734.7632.401631121013089AS1539.552.145.920183026161117153AA3310.189.553.22.330631718231317

Notch signaling is involved in numerous cell fate decisions in invertebrates and vertebrates. The Notch receptor is a type I transmembrane (TM) protein that undergoes two proteolytic steps after ligand binding, first by an ADAM (a distintegrin and metalloprotease) in the extracellular region, followed by γ-secretase-mediated cleavage inside the TM domain. We demonstrate here that the murine ligand Delta1 (Dll1) undergoes the same sequence of cleavages, in an apparently signal-independent manner. Identification of the ADAM-mediated shedding site localized 10 aa N-terminal to the TM domain has enabled us to generate a noncleavable mutant. Kuzbanian/ADAM10 is involved in this processing event, but other proteases can probably substitute for it. We then show that Dll1 is part of a high-molecular-weight complex containing presenilin1 and undergoes further cleavage by a γ-secretase-like activity, therefore releasing the intracellular domain that localizes in part to the nucleus. Using the shedding-resistant mutant, we demonstrate that this γ-secretase cleavage depends on prior ectodomain shedding. Therefore Dll1 is a substrate for regulated intramembrane proteolysis, and its intracellular region possibly fulfills a specific function in the nucleus.

BACKGROUND In previous clinical trials involving children with X-linked severe combined immunodeficiency (SCID-X1), a Moloney murine leukemia virus–based γ-retrovirus vector expressing interleukin-2 receptor γ-chain (γc) complementary DNA successfully restored immunity in most patients but resulted in vector-induced leukemia through enhancermediated mutagenesis in 25% of patients. We assessed the efficacy and safety of a self-inactivating retrovirus for the treatment of SCID-X1. METHODS We enrolled nine boys with SCID-X1 in parallel trials in Europe and the United States to evaluate treatment with a self-inactivating (SIN) γ-retrovirus vector containing deletions in viral enhancer sequences expressing γc (SIN-γc). RESULTS All patients received bone marrow–derived CD34+ cells transduced with the SIN-γc vector, without preparative conditioning. After 12.1 to 38.7 months of follow-up, eight of the nine children were still alive. One patient died from an overwhelming adenoviral infection before reconstitution with genetically modified T cells. Of the remaining eight patients, seven had recovery of peripheral-blood T cells that were functional and led to resolution of infections. The patients remained healthy thereafter. The kinetics of CD3+ T-cell recovery was not significantly different from that observed in previous trials. Assessment of insertion sites in peripheral blood from patients in the current trial as compared with those in previous trials revealed significantly less clustering of insertion sites within LMO2, MECOM, and other lymphoid proto-oncogenes in our patients. CONCLUSIONS This modified γ-retrovirus vector was found to retain efficacy in the treatment of SCID-X1. The long-term effect of this therapy on leukemogenesis remains unknown. (Funded by the National Institutes of Health and others; ClinicalTrials.gov numbers, NCT01410019, NCT01175239, and NCT01129544.)

Within the human TCR-α/δ locus, ordered rearrangements requires RUNX1, which binds to the Dδ2-23RSS and interacts with RAG1 to enhance RAG1 deposition at this site. Absence of this RUNX1 binding site in the homologous murine Dδ1-23RSS offers an explanation for the lack of ordered TCR-δ gene assembly in mice., V(D)J recombination of TCR loci is regulated by chromatin accessibility to RAG1/2 proteins, rendering RAG1/2 targeting a potentially important regulator of lymphoid differentiation. We show that within the human TCR-α/δ locus, Dδ2-Dδ3 rearrangements occur at a very immature thymic, CD34+/CD1a−/CD7+dim stage, before Dδ2(Dδ3)-Jδ1 rearrangements. These strictly ordered rearrangements are regulated by mechanisms acting beyond chromatin accessibility. Importantly, direct Dδ2-Jδ1 rearrangements are prohibited by a B12/23 restriction and ordered human TCR-δ gene assembly requires RUNX1 protein, which binds to the Dδ2-23RSS, interacts with RAG1, and enhances RAG1 deposition at this site. This RUNX1-mediated V(D)J recombinase targeting imposes the use of two Dδ gene segments in human TCR-δ chains. Absence of this RUNX1 binding site in the homologous mouse Dδ1-23RSS provides a molecular explanation for the lack of ordered TCR-δ gene assembly in mice and may underlie differences in early lymphoid differentiation between these species.

International audience; BACKGROUND: The genotoxicity of zidovudine has been established in experimental models. The objective of the study was to identify genotoxicity markers in cord blood cells from newborns exposed in utero to antiretroviral (ARV) combinations containing zidovudine. METHODS: Cells were investigated by karyotyping and gene expression analysis of the CD34(+) hematopoietic stem/progenitor cell (HPC) compartment. RESULTS: Karyotyping of the cord blood cells from 15 ARV-exposed newborns and 12 controls revealed a higher proportion of aneuploid cells in the exposed group (median, 18.8% [interquartile range, 10.0%-26.7%] vs 6.6% [interquartile range, 3.1%-11.7%]; P < .001). All chromosomes were involved, with a random distribution of these alterations. Gene expression profiling of CD34(+) HPCs from 7 ARV-exposed and 6 control newborns revealed that >300 genes were significantly upregulated or downregulated by at least 1.5-fold in the exposed group (P < .05 for all comparisons). Significant alterations of genes involved in cell cycle control, mitotic checkpoints, and DNA repair were identified. Although this study does not allow discrimination between the roles of each of the 3 drugs, both cytogenetic and transcriptional findings are similar to those in cellular experiments that used zidovudine alone. CONCLUSIONS: The cord blood cells, including hematopoietic stem cells, from newborns exposed in utero to a zidovudine-based ARV combination present cytogenetic and transcriptional abnormalities compatible with DNA damage.

IPEX (Immunodysregulation Polyendocrinopathy Enteropathy X-linked) syndrome is the prototype of primary immunodeficiency with prevailing autoimmunity. The disease is caused by mutations in the gene encoding the transcription factor forkhead box P3 (FOXP3), which leads to the loss of function of thymus-derived CD4+CD25+ regulatory T (tTreg) cells. In IPEX patients, the absence of a functional Treg cell compartment leads to the development of multiple autoimmune manifestations (including severe enteropathy, type 1 diabetes and eczema) usually in the first months or years of life. The current treatments for IPEX syndrome include immunosuppressive, hormone replacement therapies. Unfortunately, immunosuppressive treatments are usually only partially effective and their dose is often limited because of the occurrence of infectious complications and toxicity. Currently, the only curative treatment for IPEX syndrome is allogeneic hematopoietic stem cell transplantation (HSCT). The absence of an HLA-compatible donor for all patients and their poor clinical condition particularly expose them to a risk of mortality when HLA partially compatible donors are used. For all these reasons, effective alternative therapeutic approaches are urgently needed. Various preclinical studies have shown that partial donor chimerism is sufficient for complete remission meaning that a small number of functional natural Treg is sufficient to restore immune tolerance. This suggests that a gene therapy approach designed to selectively induce a Treg program in T cells by expressing FOXP3 could be a promising potential cure for IPEX. However, several issues might compromise the success of this strategy: (i) will the introduction of FOXP3 alone be sufficient to induce a stable Treg program or will it require additional transcription factors to lock the Treg function and sustain the stability of transduced cells? (ii) Targeting effector CD4+ T cells might be an issue in terms of T-cell receptor repertoire, since the TCR repertoire of nTregs is different from the one of effector CD4+ T cells, (iii) will FOXP3-transduced T cells be able to migrate to appropriate tissues to control auto-immune reactions?, (iv) infusion of nTreg prevents the appearance of some autoimmune manifestations in murine models, however the infusion was done in prophylaxis before the appearance of the symptoms. In order to address these questions, we have developed a mouse scurfy model to evaluate the functional and stability of the correction in vivo in parallel to the characterization of gene corrected human CD4 T cells from IPEX patients. Scurfy mice develop a disease very close to human pathology due to a spontaneous mutation of Foxp3 gene. We improved Scurfy mice model to improve animal production and increase the timeline of treatement. We demonstrated that FOXP3 gene transfer into murine CD4+ T cells enable the generation of potent regulatory T cells. Indeed we showed the functional suppressive properties of the generated CD4-FOXP3 cells in an optimized flow-cytometry-based in vitro suppression assay. The ability of CD4-FOXP3 to prevent Scurfy disease by adoptive transfer in the first days of life is currently under evaluation. Similarly in humans, we demonstrated that FOXP3 gene transfer into CD4+ T cells from IPEX patients enable the generation of potent regulatory T cells, as shown through the functional in vitro suppressive properties of the generated CD4IPEX-FOXP3. Moreover comparison of the transcriptional profile of these regulatory CD4IPEX-FOXP3 cells to natural Treg by RNA-seq analysis demonstrated a good repression of cytokine transcripts (IL4/5/13/CSF2, CD40L), a strong repression of IL7R, a strong induction of IL1R2, and a moderate activation of typical Treg genes (IL2RA, IKZF2, CTLA4). Therefore, the introduction of a functional copy of the FOXP3 gene into an IPEX patient's T cells may be enough to restore immune tolerance and thus avoid the complications of allogenic HSCT. We will also discuss the challenge of generating a large, homogenous and stable population of cells in vitro for adoptive transfer and whether it can ensure long-term disease correction without generating a context of generalized immunosuppression. Disclosures No relevant conflicts of interest to declare.

The historical model of hematopoiesis, mainly derived from murine studies, is based on the existence of a single hematopoietic stem cell (HSC) capable of generating all blood cell lineages. However this model has been challenged by the proposal of four types of murine HSC, which differ by their relative contribution to the myeloid and lymphoid lineages. Here we have used data from a gene therapy trial to treat Wiskott-Aldrich syndrome (WAS) to explore hematopoiesis in humans. In the trial, the therapeutic vector (lentivirus) integrates into the genome at unique positions in each hematopoietic stem and progenitor cell (HSPC) and is consequently transmitted to all its progeny. Thus hematopoietic ontogeny in humans can be inferred by tracking the appearance of unique integration sites in fractionated blood cell populations. This provides a unique opportunity to model the developmental complexity of the human haematological system. Considerable effort over the last 15 years has been devoted to optimizing retroviral integration sites (RIS) analysis using ligation mediated PCR (LM-PCR), combined with acoustic shearing and high-throughput Illumina sequencing. Acoustic shearing enables more precise quantification of RIS abundance through the enumeration of the various sizes of shear fragments (SonicAbundance) containing a given RIS, which correspond to individual ancestor HSPC and its blood progeny. In four WAS patients treated by gene therapy, we have sorted peripheral blood samples for 5 cell types: myeloid (granulocytes and monocytes) and lymphoid subpopulations (T, B and NK cells), and analysed their RIS profile. Each RIS corresponds to a particular stem/progenitor cell clone, with a particular pattern defined by its presence or absence in each of the 5 lineages. These data are then use to reconstruct aspects of the hematopoietic hierarchy. In order to face the challenging issue of cell sorting contamination we have been using a stringent sort precision mode and we treat residual contamination explicitly in downstream statistical models. Using these approaches, we have characterized up to tens of thousands RIS per patients with a follow up of 4 years. In order to minimize biases due to sparse sampling, we concentrate on the more abundant RIS clones that can be more easily caught and are therefore analysed more reliably. We showed that a significant fraction of RIS clones are detected in a single lineage, while other RIS clones are characterized by different levels of contribution to the myeloid and lymphoid lineages, highlighting the heterogeneity of human HSC. Clones contributing to all 5 lineages are readily recovered but this study also unravels a diversity of inferred hematopoietic programs with various potentials contributing to human blood homeostasis. Longitudinal analysis of clonal dynamics is ongoing, with preliminary results highlighting the maintenance of this heterogeneity of HSPC over time. These new findings provide unique data on human hematopoiesis based on gene corrected WAS patients. Disclosures No relevant conflicts of interest to declare.

IPEX (Immunodysregulation Polyendocrinopathy Enteropathy X-linked) syndrome is the prototype of primary immunodeficiency with prevailing autoimmunity. The disease is caused by mutations in the gene encoding the transcription factor forkhead box P3 (FOXP3), which leads to the loss of function of thymus-derived CD4+CD25+ regulatory T (tTreg) cells. In IPEX patients, the absence of a functional Treg cell compartment leads to the development of multiple autoimmune manifestations (including severe enteropathy, type 1 diabetes and eczema) usually in the first months or years of life. The current treatments for IPEX syndrome include immunosuppressive, hormone replacement therapies. Unfortunately, immunosuppressive treatments are usually only partially effective and their dose is often limited because of the occurrence of infectious complications and toxicity. Currently, the only curative treatment for IPEX syndrome is allogeneic hematopoietic stem cell transplantation (HSCT). The absence of an HLA-compatible donor for all patients and their poor clinical condition particularly expose them to a risk of mortality when HLA partially compatible donors are used. For all these reasons, effective alternative therapeutic approaches are urgently needed. Various preclinical studies have shown that partial donor chimerism is sufficient for complete remission meaning that a small number of functional natural Treg is sufficient to restore immune tolerance. This suggests that T cell gene therapy approaches designed to selectively restore the repertoire of Treg cells is a promising potential cure for IPEX. We demonstrated that FOXP3 gene transfer into CD4+ T cells from IPEX patients enable the generation of potent and stable regulatory T cells. Indeed we showed the functional suppressive properties of the generated CD4IPEX-FOXP3 cells in an optimized flow-cytometry-based in vitro suppression assay. We are currently comparing the transcriptional profile of these regulatory CD4IPEX-FOXP3 cells in comparison to natural Treg by RNA-seq analysis. Therefore, the introduction of a functional copy of the FOXP3 gene into an IPEX patient's T cells may be enough to restore immune tolerance and thus avoid the complications of allogenic HSCT. We will also discuss the challenge of generating a large, homogenous and stable population of cells in vitro for adoptive transfer and whether it can ensure long-term disease correction without generating a context of generalized immunosuppression.