Immune Dysregulation, Polyendocrinopathy, Enteropathy, X-linked Research Articles

Regulatory T cells in peripheral tissue tolerance and diseases.

Maintenance of peripheral tolerance by CD4+Foxp3+ regulatory T cells (Tregs) is essential for regulating autoreactive T cells. The loss of function of Foxp3 leads to autoimmune disease in both animals and humans. An example is the rare, X-linked recessive disorder known as IPEX (Immune Dysregulation, Polyendocrinopathy, Enteropathy X-linked) syndrome. In more common human autoimmune diseases, defects in Treg function are accompanied with aberrant effector cytokines such as IFNγ. It has recently become appreciated that Tregs plays an important role in not only maintaining immune homeostasis but also in establishing the tissue microenvironment and homeostasis of non-lymphoid tissues. Tissue resident Tregs show profiles that are unique to their local environments which are composed of both immune and non-immune cells. Core tissue-residence gene signatures are shared across different tissue Tregs and are crucial to homeostatic regulation and maintaining the tissue Treg pool in a steady state. Through interaction with immunocytes and non-immunocytes, tissue Tregs exert a suppressive function via conventional ways involving contact dependent and independent processes. In addition, tissue resident Tregs communicate with other tissue resident cells which allows Tregs to adopt to their local microenvironment. These bidirectional interactions are dependent on the specific tissue environment. Here, we summarize the recent advancements of tissue Treg studies in both human and mice, and discuss the molecular mechanisms that maintain tissue homeostasis and prevent pathogenesis.

FOXP3 TSDR Measurement Could Assist Variant Classification and Diagnosis of IPEX Syndrome

Pathogenic FOXP3 variants cause immune dysregulation polyendocrinopathy enteropathy X-linked (IPEX) syndrome, a progressive autoimmune disease resulting from disruption of the regulatory T cell (Treg) compartment. Assigning pathogenicity to novel variants in FOXP3 is challenging due to the heterogeneous phenotype and variable immunological abnormalities. The number of cells with demethylation at the Treg cell-specific demethylated region (TSDR) is an independent biomarker of IPEX. We aimed to investigate if diagnosing IPEX at presentation with isolated diabetes could allow for effective monitoring of disease progression and assess whether TSDR analysis can aid FOXP3 variant classification and predict disease course. We describe a large genetically diagnosed IPEX cohort (n = 65) and 13 individuals with other monogenic autoimmunity subtypes in whom we quantified the proportion of cells with FOXP3 TSDR demethylation, normalized to the number with CD4 demethylation (%TSDR/CD4) and compare them to 29 unaffected controls. IPEX patients presenting with isolated diabetes (50/65, 77%) often later developed enteropathy (20/50, 40%) with a median interval of 23.5 weeks. %TSDR/CD4 was a good discriminator of IPEX vs. unaffected controls (ROC-AUC 0.81, median 13.6% vs. 8.5%, p < 0.0001) with higher levels of demethylation associated with more severe disease. Patients with other monogenic autoimmunity had a similar %TSDR/CD4 to controls (median 8.7%, p = 1.0). Identifying increased %TSDR/CD4 in patients with novel FOXP3 mutations presenting with isolated diabetes facilitates diagnosis and could offer an opportunity to monitor patients and begin immune modulatory treatment before onset of severe enteropathy.

Epigenetic and immunological indicators of IPEX disease in subjects with FOXP3 gene mutation

Forkhead box protein 3 (FOXP3) is the master transcription factor in CD4+CD25hiCD127lo regulatory T (Treg) cells. Mutations in FOXP3 result in IPEX (immune dysregulation, polyendocrinopathy, enteropathy, X-linked) syndrome. Clinical presentation of IPEX syndrome is broader than initially described, challenging the understanding of the disease, its evolution, and treatment choice. We sought to study the type and extent of immunologic abnormalities that remain ill-defined in IPEX, across genetic and clinical heterogeneity. We performed Treg-cell-specific epigenetic quantification and immunologic characterization of severe "typical" (n= 6) and "atypical" or asymptomatic (n= 9) patients with IPEX. Increased number of cells with Treg-cell-Specific Demethylated Region demethylation in FOXP3 is a consistent feature in patients with IPEX, with (1) highest values in those with typical IPEX, (2) increased values in subjects with pathogenic FOXP3 but still no symptoms, and (3) gradual increase over the course of disease progression. Large-scale profiling using Luminex identified plasma inflammatory signature of macrophage activation and TH2 polarization, with cytokines previously not associated with IPEX pathology, including CCL22, CCL17, CCL15, and IL-13, and the inflammatory markers TNF-α, IL-1A, IL-8, sFasL, and CXCL9. Similarly, both Treg-cell and Teff compartments, studied by Mass Cytometry by Time-Of-Flight, were skewed toward the TH2 compartment, especially in typical IPEX. Elevated TSDR-demethylated cells, combined with elevation of plasmatic and cellular markers of a polarized type 2 inflammatory immune response, extends our understanding of IPEX diagnosis and heterogeneity.

Inborn errors of immunity and related microbiome.

Inborn errors of immunity (IEI) are characterized by diverse clinical manifestations that are dominated by atypical, recurrent, chronic, or severe infectious or non-infectious features, including autoimmunity, lymphoproliferative disease, granulomas, and/or malignancy, which contribute substantially to morbidity and mortality. Some data suggest a correlation between clinical manifestations of IEI and altered gut microbiota. Many IEI display microbial dysbiosis resulting from the proliferation of pro-inflammatory bacteria or a decrease in anti-inflammatory bacteria with variations in the composition and function of numerous microbiota. Dysbiosis is considered more established, mainly within common variable immunodeficiency, selective immunoglobulin A deficiency, severe combined immunodeficiency diseases, Wiskott–Aldrich syndrome, Hyper-IgE syndrome, autoimmune polyendocrinopathy–candidiasis–ectodermal-dystrophy (APECED), immune dysregulation, polyendocrinopathy, enteropathy X-linked (IPEX) syndrome, IL-10 receptor deficiency, chronic granulomatous disease, and Kostmann disease. For certain IEIs, the specific predominance of gastrointestinal, respiratory, and cutaneous involvement, which is frequently associated with dysbiosis, justifies the interest for microbiome identification. With the better understanding of the relationship between gut microbiota, host immunity, and infectious diseases, the integration of microbiota modulation as a therapeutic approach or a preventive measure of infection becomes increasingly relevant. Thus, a promising strategy is to develop optimized prebiotics, probiotics, postbiotics, and fecal microbial transplantation to rebalance the intestinal microbiota and thereby attenuate the disease activity of many IEIs.

Whole-exome sequencing as a diagnostic tool for ipex syndrome

Immune dysregulation-Polyendocrinopathy-Enteropathy-X-linked (IPEX) syndrome is a life-threatening congenital autoimmune disorder caused by mutations in the forkhead box protein 3 (FOXP3) gene. Typical clinical manifestations of IPEX patients are early onset of intractable diarrhea, type 1 diabetes mellitus, and skin diseases. However, other autoimmune types such as severe food allergies, autoimmune cytopenias, autoimmune respiratory illness, and mesangial glomerulonephritis may complicate IPEX diagnosis. In this study, we report a Vietnamese 1-year-old boy with IPEX syndrome due to a hemizygous missense mutation, c.1190G&gt;A (p.Arg397Gln), in exon 12 of the FOXP3 gene (NM_014009.4). The child had dermatitis, diarrhea, respiratory infections, and splenomegaly. The patient's serum routine test results were expected, except for white blood cells and neutrophils were higher than the normal, while IgA concentration was slightly below the normal range. However, he got no signal of diabetes or failure to thrive. Whole exome sequencing was applied to identify a genetic variant, and variant validation was examined using Sanger sequencing. The patient’s genetic mutation was inherited from his mother, an obligate carrier. His father had a normal genotype. This study is the first report of IPEX syndrome in a Vietnamese patient with a mutation in the FOXP3 gene detected by WES. This study provides further evidence for the role of mutations in the FOXP3 gene in patients with IPEX syndrome and demonstrates the need for genetic counselling and prenatal testing. Our results also show that WES sequencing is an effective tool in diagnosing genetic diseases.

Single-cell analysis of FOXP3 deficiencies in humans and mice unmasks intrinsic and extrinsic CD4+ T cell perturbations.

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.

A hemizygous mutation in the FOXP3 gene (IPEX syndrome) resulting in recurrent X-linked fetal hydrops: a case report

BackgroundFetal hydrops is excessive extravasation of fluid into the third space in a fetus, which could be due to a wide differential of underlying pathology. IPEX (immune dysregulation, polyendocrinopathy, enteropathy, X-linked) syndrome primarily affects males. It is a monogenic primary immunodeficiency syndrome of X-linked recessive inheritance due to FOXP3 gene variants. It is characterised by the development of multiple autoimmune disorders in affected individuals.Case presentationWe present a rare cause of male fetal hydrops in the context of IPEX syndrome and discuss FOXP3 gene variants as a differential for ‘unexplained’ fetal hydrops that may present after the first trimester.Discussion and conclusionsIn all similar cases, the pathological process begins during intrauterine life. Furthermore, there are no survivors described. Consequently, this variant should be considered as a severe one, associated with intrauterine life onset and fatal course, i.e., the most severe IPEX phenotype.

IPEX Syndrome: Genetics and Treatment Options.

(1) Background: IPEX (immune dysregulation, polyendocrinopathy, enteropathy, X-linked) syndrome characterizes a complex autoimmune reaction beginning in the perinatal period, caused by a dysfunction of the transcription factor forkhead box P3 (FOXP3). (2) Objectives: Studies have shown the clinical, immunological, and molecular heterogeneity of patients with IPEX syndrome. The symptoms, treatment, and survival were closely connected to the genotype of the IPEX syndrome. Recognition of the kind of mutation is important for the diagnostics of IPEX syndrome in newborns and young infants, as well as in prenatal screening. The method of choice for treatment is hematopoietic stem cell transplantation and immunosuppressive therapy. In children, supportive therapy for refractory diarrhea is very important, as well as replacement therapy of diabetes mellitus type 1 (DMT1) and other endocrinopathies. In the future, genetic engineering methods may be of use in the successful treatment of IPEX syndrome. (3) Conclusions: The genetic defects determine a diagnostic approach and prognosis, making the knowledge of the genetics of IPEX syndrome fundamental to introducing novel treatment methods.

SAT-664 Fluctuating Blood Glucose in an Infant with Newly Diagnosed IPEX Syndrome

Background: In the IPEX (immune dysregulation, polyendocrinopathy, enteropathy, X-linked) syndrome, Type 1 diabetes mellitus is the most common endocrine complication and usually occurs with a variable presentation from immediately at birth to within the first few months of life.Clinical case: A four-month-old male presented for evaluation of failure to thrive, eczema, and diarrhea. In the ED, his glucose value was 246 mg/dL with beta-hydroxybutyrate of 0.29 mmol/L (0.00-0.30). Within eight hours and without insulin, he became hypoglycemic and required dextrose-containing fluids to maintain euglycemia; he was quickly made NPO and started on TPN due to excessive stool output. For nearly two weeks he required no insulin while receiving 84g of dextrose per day (21 g/kg/day) in TPN. He developed bloody stools on the day that he started receiving Tacrolimus and IVIG and required transfer to the ICU, and an insulin need of 1 unit/kg/day developed with this worsening of his systemic illness. After the bloody stools resolved, immunosuppression with Rituximab was initiated. Once bowel function improved, Pedialyte and formula were slowly reintroduced and for three weeks his insulin requirement varied from 0.2-0.4 units/kg/day. In his seventh week of hospitalization his insulin was discontinued due to hypoglycemia, and at the time of discharge he had been without insulin for ten days on ad lib formula feeding.Hemoglobin A1c on admission was 10.2%, and repeat was 10.3%. A fructosamine level was obtained to evaluate the discrepancy between the initial HgbA1c and being euglycemic. It was 269 umol/L (190-270), equivalent to an approximate HgbA1c of 6.5%, suggesting that hyperglycemia resulting in an elevated HgbA1c occurred early in his life and had improved in the days to weeks prior to admission. Further testing revealed an elevated GAD-65 antibody of >250 IU/mL (<5) but normal ICA 512 and insulin autoantibody.His clinical picture was consistent with IPEX syndrome, confirmed with rapid whole genome sequencing showed a pathogenic hemizygous c.1010G>A p.Arg337Gln variant in the FOXP3 gene.A HgbA1c performed prior to discharge, eight weeks after the initial, was 6.6%. This spontaneous resolution of hyperglycemia in IPEX, with insulin needs developing only when he had worsening systemic illness as demonstrated by bloody stools, has yet to be described.Conclusion: Hyperglycemia fluctuated in the first few months of life in a patient with IPEX syndrome, likely related to severity of systemic illness and control of enteropathy.

Treatment with rapamycin can restore regulatory T-cell function in IPEX patients

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 invitro 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 invitro suppression by invivo 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.

Enabling Gene-Edited, Regulatory-like, T Cells (edTreg) for Treatment of IPEX and Other Autoimmune Disorders

IPEX (immunedysregulation, polyendocrinopathy, enteropathy, X-linked) syndrome is a severe congenital autoimmune disorder in males resulting from hemizygous inheritance of a mutant FOXP3 allele. FOXP3 encodes a transcription factor that governs the development, maintenance, and function of regulatory T cells (Treg). We have developed a cell therapy strategy for treatment of IPEX using a gene-editing approach in which CRISPR/Cas9 RNPs are co-delivered with an AAV6 donor template designed to integrate into the FOXP3 locus an expression cassette containing the MND promoter driving expression of a functional FOXP3 cDNA and a surface LNGFR tag linked by a 2A ribosomal skip peptide. This approach enforces heterologous FOXP3 expression in IPEX CD4 effector T cells (Teff), while simultaneously eliminating expression of the endogenous FOXP3 allele. The resultant high level and stable expression of functional FOXP3 converts Teff to Treg-like cells with immunosuppressive activity. Using an optimized protocol, we obtained efficient HDR rates across multiple healthy donors. Edited cells were consistently enriched to &gt;95% purity by a magnetic LNGFR antibody selection and expanded 50-fold in a week. Expression of FOXP3 cDNA in edited cells was sufficient to enforce Treg-like phenotypes including the up-regulation of Treg-associated markers (CD25, CTLA-4, and ICOS), and down-regulation of CD127 and inflammatory cytokines (IL2, IFNgamma, TNFalpha). Importantly, we demonstrate sustained in vivo suppressive activity of these edited Treg-like cells (edTreg) in a xeno-GvHD mouse model. edTreg (as well as expanded natural Treg) limited effector T cell expansion and tissue infiltration and significantly protected mice from xeno-GvHD induced by co-transferred autologous effector T cells. Along with preliminary data showing successful editing in CD4 T cells from IPEX patients, our data provide key pre-clinical proof-of-concept and safety data supporting use of edTreg in a clinical trial for IPEX and, potentially, for use in other autoimmune diseases. Disclosures Torgerson: Shire: Consultancy; CSL Behring: Consultancy; ADMA Biosciences: Consultancy; UCB: Consultancy. Scharenberg:Casebia Therapeutics LLc: Employment, Equity Ownership; Alpine Biosciences: Consultancy, Equity Ownership; Generation Bio: Equity Ownership.

A Mutation in the Transcription Factor Foxp3 Drives T Helper 2 Effector Function in Regulatory T Cells

Regulatory T (Treg) cells maintain immune tolerance through the master transcription factor forkhead box P3 (FOXP3), which is crucial for Treg cell function and homeostasis. We identified an IPEX (immune dysregulation polyendocrinopathy enteropathy X-linked) syndrome patient with a FOXP3 mutation in the domain swap interface of the protein. Recapitulation of this Foxp3 variant in mice led to the development of an autoimmune syndrome consistent with an unrestrained T helper type 2 (Th2) immune response. Genomic analysis of Treg cells by RNA-sequencing, Foxp3 chromatin immunoprecipitation followed byhigh-throughput DNA sequencing (ChIP-sequencing), and H3K27ac-HiChIP revealed a specific de-repression of the Th2 transcriptional program leading to the generation of Th2-like Treg cells that were unable to suppress extrinsic Th2 cells. Th2-like Treg cells showed increased intra-chromosomal interactions in the Th2 locus, leading to type 2 cytokine production. These findings identify a direct role for Foxp3 in suppressing Th2-like Treg cells and implicate additional pathways that could be targeted to restrain Th2 trans-differentiated Treg cells.

Endocrine complications in primary immunodeficiency diseases

Experimental and clinical data suggest a complex interaction between the endocrine and immune systems. However, only few epidemiological studies are available dealing with endocrine complications in different types of primary immunodeficiency diseases. It is well documented that there is a close association between immunodeficiency syndromes and the development of autoimmune disorders. Most of the endocrine dysfunctions are caused mainly by immune dysregulation and autoimmunity like in APECED (autoimmune polyendocrinopathy, candidiasis, ectodermal dystrophy) and IPEX (immune dysregulation, polyendocrinopathy, enteropathy, X-linked) syndromes. In these disorders, an immunologically mediated destructive process by autoreactive T cells damages multiple endocrine organs like the thyroid, parathyroid, adrenal cortex and endocrine pancreas. In some other forms of immunodeficiencies, like Di George syndrome, endocrine disturbances are mainly caused by the underlying genetic disorders but autoimmunity may also take part in the process. The aim of this paper is to give an overview of the different forms of endocrine disturbances and their pathological background in APECED and IPEX syndromes, Di George syndrome and ataxia telangiectasia. Orv Hetil. 2018; 159(49): 2065-2072.

Conversion of T-Effector Cells to Immunosuppressive T-Regulatory-like Cells By CRISPR/Cas9-Mediated Integration of a FOXP3 Transgene

Regulatory T cells (Treg) are distinct among T cell subtypes, having the primary role of suppressing adaptive immune responses. The importance of these cells in immune self-tolerance is underscored by the genetically inherited syndrome IPEX (immune dysregulation, polyendocrinopathy, enteropathy, X-linked), which is caused by an inactivating mutation in FOXP3. FOXP3 is a transcription factor that is a determinant of regulatory T cell function. Patients with IPEX syndrome suffer from the rapid and severe onset of multi-organ autoimmunity, including severe enteropathy, Type I diabetes, thyroiditis, skin inflammation and other features. In mouse models of IPEX, neonatal transplantation of wild-type Tregs is sufficient to prevent the development of disease. Less-severe Treg defects have also been implicated in the etiology of a variety of prevalent autoimmune diseases. It is possible that the pivotal role for Tregs in self-tolerance could be exploited clinically to improve therapies for autoimmunity and other diseases of tolerance. However, the use of autologous ex vivo expanded Treg as a clinical cell therapy is problematic: Tregs are present in low numbers in the peripheral blood, they expand slowly in culture ex vivo, and they may lack antigen specificities necessary for efficient suppression in specialized tissues. They may also down-regulate FOXP3 expression and lose functional activity in vivo in the setting of chronic inflammation. Additionally, autologous Tregs from patients with autoimmune disease may exhibit cell intrinsic dysfunction, while IPEX patients do not even have Tregs. To overcome these issues, we developed a gene editing approach to enforce stable expression of FOXP3 in primary human CD4+ peripheral blood T cells. CRISPR/Cas9 ribonucleoprotein and an AAV6-delivered donor template were developed to target a MND promoter-FOXP3 cDNA expression cassette (linked to a cell surface LNGFR tag by a 2A ribosome skip peptide) to the FOXP3 locus by homology directed repair (HDR). Highly efficient HDR rates were achieved across multiple donors (~34%; 5 donors in 9 experiments). For therapy of IPEX caused by FOXP3 missense mutations, integration of the functional coding sequence simultaneously abolishes endogenous FOXP3 expression. Following gene editing, expression of FOXP3 was sufficient to drive Treg-like phenotypic changes, including the up-regulation of CD25 and inhibitory receptors and down-regulation of CD127 and inflammatory cytokines. Further, consistent with the translatability of this approach into clinical manufacturing, FOXP3+ cells could be enriched to >90% purity by a simple LNGFR antibody column and expanded 20-fold within one week. Importantly, transfer of these edited Treg-like cells (edTreg) to NOD-scid-IL2Rγ-/- mice prevented xeno-graft vs. host disease (xeno-GvHD) mediated by co-transferred autologous effector T cells; xeno-GvHD protection correlated with long-term survival of the edTregs, and a marked reduction in effector T cell expansion and tissue infiltration. These data support the development of edited regulatory T cells for the treatment of IPEX and other autoimmune disease. DisclosuresScharenberg:Generation Bio: Equity Ownership; Casebia Therapeutics: Employment; Alpine Immune Sciences: Equity Ownership.

GENETIC TESTING IDENTIFIES CAUSE OF ENTEROPATHY AND GROWTH FAILURE IN A 10 YEAR OLD MALE

Introduction Enteropathies with features of celiac disease or inflammatory bowel disease (IBD) should spark consideration of an immunodeficiency when presenting at an early age. Case Description We present a 10-year old male with chronic diarrhea starting at 6 weeks of life, initially improved with TPN, steroids, and cow's milk free diet. By 2 years of age, diarrhea returned, and he required frequent courses of steroids to manage his symptoms. The patient had mild dermatitis and poor growth, with otherwise negative review of systems. Endoscopies found duodenitis with severe villous blunting and pancolitis. Laboratory evaluations were notable for negative anti-enterocyte antibodies, elevated antigliadin and tissue transglutaminase IgA levels, and normal T regulatory (Treg) cells and forkhead box P3 (FOXP3) expression. He was diagnosed with Celiac Disease and years later, Crohn's Disease. His clinical course did not improve with gluten avoidance or various immunomodulators. Eventually, sequencing of the FOXP3 gene was performed, revealing a missense mutation consistent with Immune dysregulation Polyendocrinopathy Enteropathy X-linked (IPEX) syndrome. Discussion IPEX is a rare syndrome caused by a mutation in the FOXP3 transcription factor of CD4+ Treg cells. The classic IPEX syndrome includes protracted diarrhea, dermatitis, and endocrinopathies, but phenotypes vary. Endoscopic findings of the IPEX enteropathy can mimic those seen in Celiac Disease and IBD, thus a high index of suspicion should be maintained in select patients with early onset of symptoms. Finally, this case highlights the importance of genetic analysis of the FOXP3 gene itself, as IPEX patients can have normal FOXP3 protein expression and Treg numbers.

Forkhead-Box-P3 Gene Transfer in Human CD4+ T Conventional Cells for the Generation of Stable and Efficient Regulatory T Cells, Suitable for Immune Modulatory Therapy.

The development of novel approaches to control immune responses to self- and allogenic tissues/organs represents an ambitious goal for the management of autoimmune diseases and in transplantation. Regulatory T cells (Tregs) are recognized as key players in the maintenance of peripheral tolerance in physiological and pathological conditions, and Treg-based cell therapies to restore tolerance in T cell-mediated disorders have been designed. However, several hurdles, including insufficient number of Tregs, their stability, and their antigen specificity, have challenged Tregs clinical applicability. In the past decade, the ability to engineer T cells has proven a powerful tool to redirect specificity and function of different cell types for specific therapeutic purposes. By using lentivirus-mediated gene transfer of the thymic-derived Treg transcription factor forkhead-box-P3 (FOXP3) in conventional CD4+ T cells, we converted effector T cells into Treg-like cells, endowed with potent in vitro and in vivo suppressive activity. The resulting CD4FOXP3 T-cell population displays stable phenotype and suppressive function. We showed that this strategy restores Treg function in T lymphocytes from patients carrying mutations in FOXP3 [immune-dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX)], in whom CD4FOXP3 T cell could be used as therapeutics to control autoimmunity. Here, we will discuss the potential advantages of using CD4FOXP3 T cells for in vivo application in inflammatory diseases, where tissue inflammation may undermine the function of natural Tregs. These findings pave the way for the use of engineered Tregs not only in IPEX syndrome but also in autoimmune disorders of different origin and in the context of stem cell and organ transplantation.

123. Gene Editing as a Therapeutic Approach to Treat IPEX Syndrome

Site-specific gene editing combined with autologous stem cell transplantation holds tremendous potential to treat both inherited and acquired diseases. However, the translational applications of gene editing are limited by the challenge of achieving sufficient levels of gene targeting in human primary cells. Recent advances in gene editing, including use of the CRISPR-Cas9 system, nuclease-resistant chemically modified small guide RNAs (sgRNAs), and serotype-optimized AAV delivery of donor repair DNA templates, are enabling clinically relevant levels of gene editing in human primary cells. Here, we build upon these advances to develop a gene editing strategy to treat the immune-dysregulation polyendocrinopathy-enteropathy-X-linked (IPEX) syndrome, a severe primary immune deficiency manifesting with life-threatening multi-organ autoimmunity in children. IPEX is caused by mutations in the forkhead box protein 3 gene (FOXP3), resulting in dysfunction of T regulatory cells (Tregs) and T effector cell lymphoproliferation. Due to the presence of disease causative mutations throughout the entire FOXP3 gene and the complex nature of FOXP3 regulatory elements, we designed a strategy to functionally correct the gene in IPEX patient cells using on-target, homology directed repair (HDR)-mediated insertion of the FOXP3 coding sequence into the gene locus. By targeting the FOXP3 gene using a CRISPR system comprised of Cas9 mRNA and chemically modified sgRNAs, we were able to attain high targeting frequencies, reaching 70-80% in human primary CD4+ T cells. We use this CRISPR system in combination with a repair donor DNA template delivered with AAV to demonstrate FOXP3 gene correction in human CD34+ hematopoietic stem and progenitor cells (HSPCs). In addition to gene correction of FOXP3, we use a similar strategy in parallel to knockout FOXP3 in wild-type human CD34+ HSPCs. We use the knockout HSPCs to generate a humanized mouse model of FOXP3 dysfunction, which enables us to study the pathophysiology of IPEX and to ultimately test the efficacy of gene-corrected Treg cell-based therapies. These results will help demonstrate the feasibility of FOXP3 gene editing, which we propose for the translational application of autologous transplant of Tregs and HSPCs as a therapy for IPEX syndrome.

Early-onset autoimmune disease as a manifestation of primary immunodeficiency.

Autoimmune disorders (AID) have been increasingly observed in association with primary immunodeficiencies (PIDs). Here, we discuss the interface between PID and AID, focusing on autoimmune manifestations early in life, which can be diagnostic clues for underlying PIDs. Inflammatory bowel disease in infants and children has been associated with IL-10 and IL-10R deficiencies, chronic granulomatous disease, immunedysregulation-polyendocrinopathy-enteropathy-X-linked syndrome (IPEX), autoinflammatory disorders, and others. Some PIDs have been identified as underlying defects in juvenile systemic lupus erythematosus: C1q-, IgA-, IgM deficiencies, alterations of the IFN-α pathway (in Aicardi–Goutières syndrome due to TREX1 mutation). IPEX (due to FOXP3 mutation leading to Treg cell deficiency), usually appearing in the first months of life, was recently observed in miscarried fetuses with hydrops who presented with CD3+ infiltrating lymphocytes in the pancreas. Hemophagocytic lymphohistiocytosis due to perforin deficiency was also identified as a cause of fetal hydrops. In conclusion, PID should be suspected in any infant with signs of autoimmunity after excluding transferred maternal effects, or in children with multiple and/or severe AID.

Robust Immune Reconstitution in Children with Severe Primary Immunodeficiency after Reduced-Intensity Conditioning Hematopoietic Stem Cell Transplantation

Hematopoietic stem cell transplantation (HSCT) is a potentially curative therapy for severe primary immunodeficiency diseases (PID), but it is still unclear what is the optimal transplant approach. To help answer this question, the stem cell transplant team at Ann & Robert H. Lurie Children’s Hospital of Chicago (formerly Children’s Memorial Hospital) evaluated the risks and benefits of a reduced-intensity conditioning (RIC) transplant approach for treatment of severe PID. Between 2000 and 2013, 42 children with severe PID were treated with allogeneic HSCT following a RIC regimen consisting of fludarabine (30mg/m2/day x 6), rabbit anti-thymocyte globulin (once daily x 4), and intravenous busulfan (once daily x 2). Total busulfan exposure, as measured by the plasma concentration-time area-under-the-curve (AUC), was targeted individually for each patient at 4000 µM*min/day in an earlier cohort (n=14), and 5000 in a later cohort (n=28). Patients were treated in an ambulatory setting and were hospitalized only for specific medical or social reasons. There were 31 male and 11 female patients. The median age at the time of transplant was 8.2 months (range 1 month-17.4 years). The median weight was 7.6 kg (range 2.5-81.5 kg). Patients were diagnosed with severe combined immunodeficiency (n=17), Wiscott-Aldrich syndrome (n=7), hyper-IgM syndrome (n=5), major histocompatibility complex II deficiency (n=3), X-linked lymphoproliferative disease (n=3), NEMO syndrome (n=2), Omenn syndrome, reticular dysgenesis, Chediak-Higashi syndrome, IPEX (immune dysregulation, polyendocrinopathy, enteropathy, X-linked) syndrome, and IPEX-like syndrome (n=1 each). Fourteen patients had related donors and 28 had unrelated donors. Twenty-eight patients received peripheral blood HSC, 12 received cord blood, and 2 received bone marrow as the source of HSC. All patients tolerated the conditioning regimen with minimal nausea, vomiting, or pain. No severe mucositis, sinusoidal occlusion syndrome, seizure, grade III/IV acute GVHD or extensive chronic GVHD were seen. In most patients, the engraftment was rapid, with ANC ≥ 500 at a median of +15 days post-transplant, following a nadir at day +10 with a median ANC nadir of 60. In the AUC 4000 cohort, 2 patients (14%) developed primary graft failure and 2 patients (14%) developed immune-mediated secondary graft failure; in the AUC 5000 cohort, 2 patient (7%) developed primary graft failure and 1 patient developed secondary graft failure after primary EBV infection. Two patients died from complications associated with primary graft failure, 2 from complications caused by EBV infections, and 2 from complications related to their underlying diseases in the early post-transplant period. Kaplan–Meier analysis shows overall and event-free survival rates of 81.9% and 73.6%, respectively, at a median follow-up of 4.3 years. Most engrafted patients have stable donor chimerism over time. At a mean of 3.5 years post-transplant, the median donor chimerism in total white blood cells was 97% and the first and third quartiles were 77% and 99%, respectively. Engrafted patients demonstrated robust immune reconstitution with B and T cell counts normalized by 3 months and 9 months post-transplant, and CD45RA+ naive T cells by 1 year. Between 1 to 4 years post-transplant, the median absolute CD4+, CD8+ and CD19+ cell counts were 1388, 976 and 720 per mm3, respectively, percentage of CD45RA+ naive CD4 and CD8 cells were 68% and 81%, and endogenous IgG and IgM levels were 840 and 97 mg/dL. T cells showed regular responses to antigen and mitogen stimulation and exhibited normal TCRVβ repertoires. Endogenous IgG levels returned to normal by day +100, and no intravenous immunoglobulin infusion was required after day +100 in all but 1 patients. Positive vaccine-specific antibody responses were seen in patients after reimmunization at 12-24 months post-transplant. Clinically, patients have normal growth and development and do not exhibit increased susceptibility to infection. Our data showed that HSCT after RIC offers an optimal treatment for severe PID by providing robust post-transplant immune reconstitution while minimizing side effects. To reduce the risk of graft failure, we recommend a targeted busulfan AUC of 5000 µM*min/day for 2 days. DisclosuresNo relevant conflicts of interest to declare.

B cells are critical for autoimmune pathology in Scurfy mice

Impaired regulatory T-cell function results in a severe chronic autoimmune disease affecting multiple organs in Scurfy mice and humans with the immune dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) syndrome. Previous studies have shown that T helper cells but not cytotoxic T cells are critical for the disease pathology. Whether this T-cell subset is responsible directly for tissue inflammation or rather indirectly via the interaction with B cells or myeloid cells is largely unknown. To study this and to identify potential therapeutic targets for this lethal disease we investigated the contribution of B cells to this complex autoimmune phenotype. We show that B cells and the production of autoantibodies plays a major role for skin, liver, lung, and kidney inflammation and therapeutic depletion of B cells resulted in reduced tissue pathology and in prolonged survival. In contrast, the absence of B cells did not impact systemic T-cell activation and hyperreactivity, indicating that autoantibody production by B cells may be a major factor for the autoimmune pathology in mice deficient for regulatory T cells.