FOXP3 Mutations Research Articles

IL-2 Signaling Axis Defects: How Many Faces?

CD25, Signal transducer and activator of transcription 5B (STAT5B) and Forkhead box P3 (FOXP3) are critical mediators of Interleukin-2 (IL-2) signaling pathway in regulatory T cells (Tregs). CD25 (i.e., IL-2 Receptor α) binds with high affinity to IL-2, activating STAT5B-mediated signaling that eventually results in transcription of FOXP3, a master regulator of Treg function. Consequently, loss-of-function mutations in these proteins give rise to Treg disorders (i.e., Tregopathies) that clinically result in multiorgan autoimmunity. Immunodysregulation, Polyendocrinopathy Enteropathy X-linked (IPEX), due to mutations in FOXP3, has historically been the prototype of Tregopathies. This review describes current knowledge about defects in CD25, STAT5B, and FOXP3, highlighting that these disorders both share a common biological background and display comparable clinical features. However, specific phenotypes are associated with each of these syndromes, while certain laboratory findings could be helpful tools for clinicians, in order to achieve a prompt genetic diagnosis. Current treatment strategies will be outlined, keeping an eye on gene editing, an interesting therapeutic perspective that could definitely change the natural history of these disorders.

Development of a Treg Lineage Specific Lentiviral Vector for the Treatment of IPEX Syndrome

▪Autologous hematopoietic stem cell (HSC) transplant with gene therapy has recently demonstrated remarkable clinical success in the correction of genetic blood cell disorders. One excellent candidate disease for HSC gene therapy is Immune Dysregulation, Polyendocrinopathy, Enteropathy, X-linked (IPEX) syndrome. IPEX is a devastating autoimmune disease caused by mutations in FOXP3, a transcription factor required for the proper development and function of regulatory T (Treg) cells. Tregs are critical for providing dominant suppression of autoreactive conventional T (Tconv) cells, and thus their absence in IPEX patients causes severe and often fatal multi-organ autoimmunity. Allogeneic HSC transplant has been shown to be curative for IPEX patients, but is limited due to a lack of suitable donors and immunologic transplant complications. In order to explore an autologous HSC gene therapy strategy for IPEX, we sought to design an endogenously regulated lentiviral vector (LV) which confers targeted and developmentally appropriate FoxP3 expression. Here, we utilized three intronic regulatory regions of the FoxP3 gene which have verified enhancer/regulatory function: conserved noncoding sequence (CNS) 1, CNS2, and CNS3. These sequences were added upstream from the FoxP3 promoter and cloned into the CCL vector backbone to generate two LV expressing either mStrawberry (FoxP3 reporter vector), or a human FoxP3 cDNA and mStrawberry (FoxP3 cDNA vector). A primary screen of the FoxP3 reporter vector in human cell lines from different hematopoietic lineages (K562 [erythromyeloid, FoxP3-], Jurkat [Tconv, FoxP3-] and MT-2 [Treg-like, FoxP3+]) revealed mStrawberry expression restricted to the FoxP3+ cell line with low inherent “leakiness” of expression in FoxP3- cell types. In order to evaluate in vivo lineage specificity of the FoxP3 reporter vector, we utilized both murine congenic transplants and humanized NSG xenografts to quantify LV expression in transduced HSPC and their progeny throughout hematopoietic development. In both models, we observed high levels of mStrawberry expression in the Treg lineage with low expression in CD4 Tconv cells and CD8 T cells, and negligible expression in HSPC, B cells, myeloid cells, suggesting that the regulatory elements included in this vector confer developmentally appropriate FoxP3 expression. We next evaluated the capacity of this vector to restore Treg development when introduced into FoxP3-deficient HSC from the scurfy mouse. Scurfy HSC were transduced with the FoxP3 cDNA vector and transplanted into WT CD45.1 congenic recipients to generate corrected scurfy Tregs (cSf-Tregs). Thymic cSf-Tregs phenotypically resembled WT-Treg showing equally high expression of the Treg surface markers CD25, GITR, and CTLA-4. Furthermore, FACS-sorted cSf-Tregs were equivalent to WT-Tregs in their ability to suppress Tconv cell proliferation in vitro. We next evaluated the in vivo functionality of cSf-Tregs by assessing their ability to reverse clinical disease signs in neonatal scurfy mice. Donor CD45.2+CD4+ cells (containing either WT-Tregs, cSf-Tregs, or uncorrected Sf T cells) were purified from the spleens of WT CD45.1 transplant recipients and administered to scurfy neonates by i.p. injection. Treated scurfy mice were evaluated for disease progression at 21 days post-injection. In comparison to WT littermate controls, untreated scurfy mice showed typical signs of disease progression including scaly skin, malformed ears, splenomegaly, and a high percentage of activated (CD62L-CD44+) CD4 T cells. Administration of WT-Tregs or cSf-Tregs resulted in complete correction of all measures of the scurfy phenotype, such that these mice were indistinguishable from WT littermate controls. In contrast, scurfy mice receiving uncorrected Sf T cells showed no signs of disease amelioration, confirming that disease correction was not an artifact of the transplant/CD4 isolation procedure. These results demonstrate that correction of scurfy HSC with a lineage-specific FoxP3 cDNA LV produces CD4+ Tregs capable of controlling autoimmune T cell responses in vivo. These findings pave the way for the treatment of IPEX patients by autologous HSC transplant and may further provide valuable insights into new treatments for patients with autoimmune disorders of different origin. DisclosuresKohn:Orchard Therapeutics Ltd.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Kite Pharma: Consultancy, Membership on an entity's Board of Directors or advisory committees; BioMarin Pharmaceutical: Research Funding; Biogen IDEC: Research Funding.

1126-P: Activating SKAP2 Mutation Linked to Diabetes with Multiple Autoimmune and Inflammatory Conditions

Recent insights into human autoimmunity and immune tolerance have come from studies of rare monogenic disorders. For example, type 1 diabetes (T1D) and thyroid autoimmunity are frequent features in patients with mutations in AIRE and FOXP3. Dissecting the consequences of these mutations has improved our understanding of the role of central T cell tolerance and T regulatory cells in controlling these organ-specific autoimmune diseases. Over the last decade, rapid advances in genetic sequencing have now allowed for a deeper exploration and discovery of rare variants that are linked to disease with techniques like whole exome sequencing. Using this approach in both kindreds and outlier patients with de novo germline mutations, there has been expanding discovery of mutations in other pathways with a link to organ-specific autoimmunity that include STAT3, STAT1, CTLA4, and LRBA. Here, we utilized a similar approach on a single patient with T1D and other autoimmune and inflammatory conditions. We identified a de novo heterozygous c.457G>A/p.Gly153Arg mutation in the SKAP2 gene. SKAP2 encodes a critical mediator of integrin signaling pathways that regulate myeloid immune cell function. Monocyte-derived macrophages from the individual displayed enhanced activity of integrin pathways and a migratory phenotype in the absence of chemokine stimulation, consistent with SKAP2 p.Gly153Arg being constitutively active. The p.Gly153Arg variant, located in the well-conserved lipid-binding loop, induced similar phenotypes when expressed in a human macrophage cell line. We propose that SKAP2 p.Gly153Arg is a gain-of-function, pathogenic mutation that disrupts myeloid immune cell function, resulting in a break in immune tolerance and T1D. This particular mutation in a patient with T1D and multiple autoimmunity implicates a role for activating SKAP2 variants in autoimmune T1D. Disclosure C. Chamberlain: None. N. Rutsch: None. W. Dixon: None. L. R. Letourneau-freiberg: None. L. H. Philipson: Advisory Panel; Self; Nevro Corp., Research Support; Self; Provention Bio, Inc. M. German: Stock/Shareholder; Self; Viacyte, Inc. M. S. Anderson: Consultant; Self; Provention Bio, Inc., VielaBio, Stock/Shareholder; Self; Medtronic, Merck & Co., Inc. C. A. Lowell: None. Funding The Leona M. and Harry B. Helmsley Charitable Trust (G-2018PG-T1D018); Larry L. Hillblom Foundation (2014-D-004-NET); University of California San Francisco Parnassus Flow Core (RRID:SCR_018206); National Institutes of Health (P30 DK063720, S101S10OD021822-01); German Research Foundation (ZA428/11-1, ZA428/18-1, INST211/604-1)

Molecular diagnosis of childhood immune dysregulation, polyendocrinopathy, and enteropathy, and implications for clinical management

Most patients with childhood-onset immune dysregulation, polyendocrinopathy, and enteropathy have no genetic diagnosis for their illness. These patients may undergo empirical immunosuppressive treatment with highly variable outcomes. We sought to determine the genetic basis of disease in patients referred with Immune dysregulation, polyendocrinopathy, enteropathy, X-linked-like (IPEX-like) disease, but with no mutation in FOXP3; then to assess consequences of genetic diagnoses for clinical management. Genomic DNA was sequenced using a panel of 462 genes implicated in inborn errors of immunity. Candidate mutations were characterized by genomic, transcriptional, and (for some) protein analysis. Of 123 patients with FOXP3-negative IPEX-like disease, 48 (39%) carried damaging germline mutations in 1 of the following 27 genes: AIRE, BACH2, BCL11B, CARD11, CARD14, CTLA4, IRF2BP2, ITCH, JAK1, KMT2D, LRBA, MYO5B, NFKB1, NLRC4, POLA1, POMP, RAG1, SH2D1A, SKIV2L, STAT1, STAT3, TNFAIP3, TNFRSF6/FAS, TNRSF13B/TACI, TOM1, TTC37, and XIAP. Many of these genes had not been previously associated with an IPEX-like diagnosis. For 42 of the 48 patients with genetic diagnoses, knowing the critical gene could have altered therapeutic management, including recommendations for targeted treatments and for or against hematopoietic cell transplantation. Many childhood disorders now bundled as "IPEX-like" disease are caused by individually rare, severe mutations in immune regulation genes. Most genetic diagnoses of these conditions yield clinically actionable findings. Barriers are lack of testing or lack of repeat testing if older technologies failed to provide a diagnosis.

IPEX Syndrome: Improved Knowledge of Immune Pathogenesis Empowers Diagnosis.

Immune dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) syndrome is a rare monogenic autoimmune disease with variable clinical manifestations, ranging from early-onset severe autoimmunity, including enteropathy, eczema, and type 1 diabetes, to late-onset or atypical symptoms. Despite the clinical heterogeneity, the unifying feature of IPEX is mutation of the FOXP3 gene, which encodes a transcription factor essential for maintenance of thymus-derived regulatory T cells (Tregs). In IPEX patients, Tregs can be present, although unstable and impaired in function, unable to inhibit proliferation and cytokine production of effector T (Teff) cells. Mutated FOXP3 can also disrupt other compartments: FOXP3-deficient Teff cells proliferate more than the wild-type counterpart, display altered T-cell-receptor signaling response, a reduced T-naïve compartment and a skew toward a Th2 profile. Due to FOXP3 mutations, the frequency of autoreactive B cells is increased and the IgA and IgE production is altered, together with early emergence of tissue-specific autoantibodies. Recently, the awareness of the wide clinical spectrum of IPEX improved the diagnostic tools. In cases presenting with enteropathy, histological evaluation is helpful, although there are no pathognomonic signs of disease. On the other hand, the study of FOXP3 expression and in vitro Treg function, as well as the detection of specific circulating autoantibodies, is recommended to narrow the differential diagnosis. Nowadays, Sanger sequencing should be limited to cases presenting with the classical triad of symptoms; otherwise, next-generation sequencing is recommended, given the cost-effectiveness and the advantage of excluding IPEX-like syndromes. The latter approach could be time spearing in children with severe phenotypes and candidate to advanced therapies.

FOXP3 and Tip60 Structural Interactions Relevant to IPEX Development Lead to Potential Therapeutics to Increase FOXP3 Dependent Suppressor T Cell Functions.

Regulatory T (Treg) cells play a role in the maintenance of immune homeostasis and are critical mediators of immune tolerance. The Forkhead box P3 (FOXP3) protein acts as a regulator for Treg development and function. Mutations in the FOXP3 gene can lead to autoimmune diseases such as Immunodysregulation, polyendocrinopathy, enteropathy, and X-linked (IPEX) syndrome in humans, often resulting in death within the first 2 years of life and a scurfy like phenotype in Foxp3 mutant mice. We discuss biochemical features of the FOXP3 ensemble including its regulation at various levels (epigenetic, transcriptional, and post-translational modifications) and molecular functions. The studies also highlight the interactions of FOXP3 and Tat-interacting protein 60 (Tip60), a principal histone acetylase enzyme that acetylates FOXP3 and functions as an essential subunit of the FOXP3 repression ensemble complex. Lastly, we have emphasized the role of allosteric modifiers that help stabilize FOXP3:Tip60 interactions and discuss targeting this interaction for the therapeutic manipulation of Treg activity.

Intrauterine IPEX.

IPEX is one of the few Inborn Errors of Immunity that may manifest in the fetal period, and its intrauterine forms certainly represent the earliest human autoimmune diseases. Here, we review the clinical, histopathologic, and genetic findings from 21 individuals in 11 unrelated families, with nine different mutations, described as cases of intrauterine IPEX. Recurrent male fetal death (multigenerational in five families) due to hydrops in the midsemester of pregnancy was the commonest presentation (13/21). Noteworthy, in the affected families, there were only fetal- or perinatal-onset cases, with no affected individuals presenting milder forms with later-life manifestation. Most alive births were preterm (5/6). Skin desquamation and intrauterine growth restriction were observed in part of the cases. Fetal ultrasonography showed hyperechoic bowel or dilated bowel loops in the five cases with available imaging data. Histopathology showed multi-visceral infiltrates with T lymphocytes and other cells, including eosinophils, the pancreas being affected in most of the cases (11/21) and as early as at 18 weeks of gestational age. Regarding the nine FOXP3 mutations found in these cases, six determine protein truncation and three predictably impair protein function. Having found distinct presentations for the same FOXP3 mutation in different families, we resorted to the mouse system and showed that the scurfy mutation also shows divergent severity of phenotype and age of death in C57BL/6 and BALB/c backgrounds. We also reviewed age-of-onset data from other monogenic Tregopathies leading to IPEX-like phenotypes. In monogenic IPEX-like syndromes, the intrauterine onset was only observed in two kindreds with IL2RB mutations, with two stillbirths and two premature neonates who did not survive. In conclusion, intrauterine IPEX cases seem to constitute a particular IPEX subgroup, certainly with the most severe clinical presentation, although no strict mutation-phenotype correlations could be drawn for these cases.

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.

Regulatory T Cells: the Many Faces of Foxp3.

Regulatory T (Treg) cells expressing the transcription factor forkhead box P3 (Foxp3) play a requisite role in the maintenance of immunological homeostasis and prevention of peripheral self-tolerance breakdown. Although Foxp3 by itself is neither necessary nor sufficient to specify many aspects of the Treg cell phenotype, its sustained expression in Treg cells is indispensable for their phenotypic stability, metabolic fitness, and regulatory function. In this review, we summarize recent advances in Treg cell biology, with a particular emphasis on the role of Foxp3 as a transcriptional modulator and metabolic gatekeeper essential to an effective immune regulatory response. We discuss these findings in the context of human inborn errors of immune dysregulation, with a focus on FOXP3 mutations, leading to Treg cell deficiency. We also highlight emerging concepts of therapeutic Treg cell reprogramming to restore tolerance in the settings of immune dysregulatory disorders.

Immunoregulatory cell therapy with lentiviral-mediated FOXP3 converted CD4+ T cells into Treg cells: towards the proof-of-concept application in IPEX syndrome

Background & Aim FOXP3 is an essential transcription factor for the regulatory T cell (Treg) function and therefore is a key regulator for the tolerance maintenance. Treg cell therapy, either with freshly isolated or expanded in vitro, has been proven to be safe but still challenging because of difficulties in isolating sufficient number of pure Treg cells, stability of the expanded preparations and Treg plasticity in vivo under pro-inflammatory conditions. The key role of FOXP3 and Treg cells in immune function is exemplified by immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome (IPEX), a monogenic primary immune regulatory disease caused by mutations in FOXP3, that lead to impaired Treg cell function and early onset autoimmune manifestations. Our recent international retrospective study demonstrated suboptimal disease-free survival from current therapeutic options such as immunosuppression and allogeneic hematopoietic stem cell transplantation. Therefore, there is a great unmet medical need in patients with IPEX syndrome. Methods, Results & Conclusion We investigated lentiviral FOXP3 gene transfer (LV-FOXP3) in CD4+ T cells as an innovative approach to generate in large number stable Treg cells that could provide therapeutic benefit to IPEX patients lacking functional Treg. We have shown that LV-FOXP3 can converts IPEX patients-derived CD4+ effector T cells into Treg-like cells (CD4LV-FOXP3 T cells). To better investigate the safety and efficacy of this approach and to make it suitable for large scale GMP generation of CD4LV-FOXP3 T cells, we optimized the original lentiviral vector and we assessed the phenotype and function of CD4LV-FOXP3 T cells both in vitro using transcriptome analysis and in vivo in different humanized mice models. CD4LV-FOXP3 T cells express key molecules in common with freshly isolated Treg cells. CD4LV-FOXP3 T cells can significantly extend the survival of xeno GVHD model mice, both in autologous and allogenic conditions and they can suppress xeno GVHD reaction caused by secondary infusion of responder cells. These data indicated CD4LV-FOXP3 T cells have a sustained suppressive function in the various autologous in vivo mice models. In conclusion, we completed the preclinical requirement for the use of the CD4LV-FOXP3 cell product in IPEX syndrome. The Proof-of-concept trial in a severe monogenic pediatric disease will open to future clinical applications of CD4LV-FOXP3 cell therapy in other autoimmune and immunodysregulatory disorders of different origin.

The autoimmune targets in IPEX are dominated by gut epithelial proteins

The autoimmune targets in IPEX are dominated by gut epithelial proteins

052 Regulatory T cell-mediated, OX40-dependent peripheral tolerance to autoantigen, desmoglein 3

Peripheral immunological tolerance is critical to prevent autoimmunity but detail of the mechanism has not been exactly understood. The purpose of this study is to unveil the mechanisms of peripheral tolerance to a pemphigus autoantigen, desmoglein 3 (Dsg3). To achieve this, Dsg3-specific TCR transgenic (Dsg3H1) CD4+ T cell was used. This T cell was deleted by central tolerance, but developed into the periphery in the absence of Dsg3 as in Dsg3-/- mice. When Dsg3H1 T cells were adoptively transferred, the peripheral Dsg3H1 T cells disappeared in skin-draining LNs in WT mice by Day14, but survived in Dsg3-/- mice, indicating antigen-specific elimination. To identify crucial cell subsets and molecules involved in this process, various gene-engineered or antibody-treated mice were used as recipients. The elimination was affected in MHC II-/- (n=6), but not in Aire-/- (n=3) or anti-PD-1 treated mice (n=3), indicating an essential role of MHC II+ antigen presenting cells, but not of Aire or PD-1. Moreover, under in vivo ablation of Foxp3+ Treg in DEREG mice, Dsg3H1 T cells were not eliminated, but expanded and induced dermatitis (n=12). Furthermore, Dsg3H1 T cells were eliminated even in three different Foxp3 mutant knock-in mice which have functionally-affected Treg to various degrees. Therefore, Treg-specific molecules that remain functional in those mutants can be responsible for the elimination. Transcriptome analysis of the mutant Treg identified OX40, co-stimulatory molecule involved in lymphocyte survival, as a candidate molecule. In fact, anti-OX40L blocking antibody partially restored the elimination of Dsg3H1 T cells in the absence of Treg in DEREG mice (n=3). Thus, these findings indicated Treg-mediated, OX40-dependent elimination of autoreactive CD4+ T cells as a novel skin peripheral tolerance mechanism.

Auto‐antibodies Targeting Components of Sarcolemma Repair Represent a Pathogenic Mechanism in Idiopathic Immune Myopathies

Idiopathic immune myopathies (IIM) represent a group of disorders causing chronic inflammation and significant damage to skeletal muscle due to an unchecked autoimmune response. The main challenge slowing study of IIM is an understanding of the pathogenic mechanism responsible for initiation and progression of skeletal muscle inflammation. Two mouse models of IIM have recently provided new insights into the pathogenesis of the disease. The synaptotagmin VII null (Syt VII−/−) mouse is characterized by defects in membrane resealing and presents with a mild form myositis at 2 months of age. A more robust model of IIM combines knock‐out of Syt VII with a FoxP3 mutation resulting in a mouse with impaired membrane resealing and regulatory T‐cell deficiency. Adoptive transfer of lymphocyte preparations isolated from this double mutant mouse model to recombination‐activating gene 1 (RAG‐1) null mice results in severe skeletal muscle inflammation. Plasma membrane resealing is a highly conserved mechanism allowing an injured cell to survive following a disruption to the lipid bilayer that compromises barrier function. Given the importance of plasma membrane barrier function in preventing exposure of intracellular antigens to the immune system, specifically in an immune‐compromised environment, it is possible that compromised sarcolemma resealing could drive pathogenesis of IIM. We show, for the first time, that a deficiency in T‐regulatory cells is not sufficient to induce sarcolemma fragility, however, purified antibodies against critical proteins facilitating the sarcolemma repair process are sufficient to reduce membrane integrity. We also demonstrate that sarcolemma integrity is reduced in distal skeletal muscle in the absence of inflammation in our novel murine model of IIM. We have established by direct ELISA that auto‐antibodies against multiple proteins involved in sarcolemma repair are elevated in IIM patient sera and find that exogenous delivery of IIM positive patient serum can compromise sarcolemma resealing in healthy skeletal muscle. These findings represent a novel mechanism that drives the progression of IIM when decreased sarcolemma integrity induces a vicious cycle of antigen presentation that directly contributes to the pathophysiology of idiopathic immune myopathies.Support or Funding InformationNIH, National Institute of Arthritis and Musculoskeletal and Skin Diseases Award Number F31AR071745This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

21st Century FOX: Toward Gene Therapy for the Regulatory T Cell Deficiency Syndrome IPEX.

IPEX is a fatal X-linked autoimmune disorder that results from mutations in the canonical regulatory Tcell transcription factor FOXP3. In this issue of Cell Stem Cell, Masiuk etal. (2019) demonstrate an effective and potentially durable approach to eliminate the IPEX phenotype in murine models.

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.

Lentiviral Gene Therapy in HSCs Restores Lineage-Specific Foxp3 Expression and Suppresses Autoimmunity in a Mouse Model of IPEX Syndrome.

Immune dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) syndrome is a devastating autoimmune disease caused by mutations in FoxP3, a transcription factor required for the development and function of regulatory Tcells (Treg cells). Allogeneic hematopoietic stem cell transplant (HSCT) can be curative, but suitable donors are often unavailable. Here, we demonstrate a strategy for autologous HSCT and gene therapy utilizing a lentiviral vector (LV) to restore FoxP3 expression under the control of endogenous human FOXP3 regulatory elements. Both murine transplant models and humanized mice engrafted with LV-modified HSCs show high levels of LV expression selective for CD4+CD25+FoxP3+ Treg cells. LV transduction of scurfy (FoxP3mut) HSCs restores development of functional FoxP3+ Treg cells that suppress Tcell proliferation invitro and rescue the scurfy autoimmune phenotype invivo. These findings demonstrate preclinical efficacy for the treatment of IPEX patients by autologous HSC transplant and may provide valuable insights into new cell therapies for autoimmunity.

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.

Two case reports about immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome and literature review

Objective To explore the clinical manifestation, laboratory findings, treatment and prognosis of immune dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) syndrome, and to improve pediatricians′ knowledge of this disease. Methods Clinical data of two cases of IPEX were retrospectively analyzed, and related literatures were reviewed. Results One of the two male children showed severe and early-onset enteropathy, another showed insulin-dependent diabetes onset.Both of them complicated with sepsis.DNA sequencing of whole-genome exon group showed a mutation in FOXP3 gene.Finally, one of the two IPEX children accepted allogeneic hematopoietic stem cell transplantation(HSCT). Another one was waiting for the treatment of HSCT. Conclusion IPEX should be considered also in infants with typical symptoms including early-onset refractory diarrhea, multiple endocrine disease and severe recurring infections.Gene sequencing mayl help diagnose the disease.Early HSCT can improve the patients′ outcomes. Key words: Immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome; Immuno-deficiency; FOXP3 mutation; Regulatory T cells

Clinical Heterogeneity of Immune Dysregulation, Polyendocrinopathy, Enteropathy, X-Linked Syndrome: A French Multicenter Retrospective Study.

ObjectiveImmune dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) syndrome is an autoimmune disease caused by mutations in the forkhead box protein 3 gene (FOXP3), which encodes a key regulator of immune tolerance. The aim of this study was to describe the clinical heterogeneity of the disease in a national French cohort.MethodsMulticenter retrospective study of patients diagnosed with IPEX syndrome caused by mutations in FOXP3.ResultsThirty children from 26 families were included. Age at disease onset (median [first to third quartile]) was 1.5 mo [0–84] and at death 3.5 years [0–10.5] (n = 15) indicating a high heterogeneity. Initial presentation was diarrhoea (68%), type 1 diabetes (T1D; 25%), skin lesions (7%) and nephropathy (3%). During the course of the disease the following main symptoms were observed: diarrhoea (100%), skin lesions (85%), T1DM (50%), severe food allergies (39%), haematological disorders (28%), nephropathies (25%), hepatitis (14%) as well as the presence of a variety of autoantibodies. Immunosuppressive mono- or combination therapy led to improvement in eight children. Three boys displayed a stable disease course without any immunosuppressive medication. Overall 10-year survival rate was 43% (42% in transplanted patients and 52% in patients on immunosuppressive therapy). Five out of 22 identified FOXP3 mutations have not been described yet: c.−23 + 1G > A, c.−23 + 5G > A, c.264delC, c.1015C > T and c.1091A > G. The first two produced atypical, attenuated phenotypes. Missense and frameshift mutations affecting the forkhead domain were associated with poor survival (Gehan–Wilcoxon p = 0.002).ConclusionThe broad phenotypic heterogeneity of IPEX raises questions about modifying factors and justifies early FOXP3 sequencing in suspected cases.

FOXP3 mutations causing early-onset insulin-requiring diabetes but without other features of immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome

FOXP3 mutations causing early-onset insulin-requiring diabetes but without other features of immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome