Publications by authors named "Laura Passerini"

38 Publications

IPEX Syndrome: Improved Knowledge of Immune Pathogenesis Empowers Diagnosis.

Front Pediatr 2021 22;9:612760. Epub 2021 Feb 22.

Mechanisms of Peripheral Tolerance Unit, San Raffaele Telethon Institute for Gene Therapy (SR-Tiget), IRCCS San Raffaele Scientific Institute, Milan, Italy.

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 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 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 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.
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http://dx.doi.org/10.3389/fped.2021.612760DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7937806PMC
February 2021

Human-engineered Treg-like cells suppress FOXP3-deficient T cells but preserve adaptive immune responses .

Clin Transl Immunology 2020 25;9(11):e1214. Epub 2020 Nov 25.

Department of Pediatrics Division of Hematology, Oncology Stem Cell Transplantation and Regenerative Medicine Stanford University School of Medicine Stanford CA USA.

Objectives: Genetic or acquired defects in FOXP3 regulatory T cells (Tregs) play a key role in many immune-mediated diseases including immune dysregulation polyendocrinopathy, enteropathy, X-linked (IPEX) syndrome. Previously, we demonstrated CD4 T cells from healthy donors and IPEX patients can be converted into functional Treg-like cells by lentiviral transfer of (CD4). These CD4 cells have potent regulatory function, suggesting their potential as an innovative therapeutic. Here, we present molecular and preclinical data supporting CD4 cell clinical progression.

Methods: The molecular characterisation of CD4 cells included flow cytometry, qPCR, RNA-seq and TCR-seq. The suppressive function of CD4 cells was assessed in xenograft-versus-host disease (xeno-GvHD) and FOXP3-deficient IPEX-like humanised mouse models. The safety of CD4 cells was evaluated using peripheral blood (PB) humanised (hu)- mice testing their impact on immune response against pathogens, and immune surveillance against tumor antigens.

Results: We demonstrate that the conversion of CD4 T cells to CD4 cells leads to specific transcriptional changes as compared to CD4 T-cell transduction in the absence of FOXP3, including upregulation of Treg-related genes. Furthermore, we observe specific preservation of a polyclonal TCR repertoire during cell production. Both allogeneic and autologous CD4 cells protect from xeno-GvHD after two sequential infusions of effector T cells. CD4 cells prevent hyper-proliferation of CD4 memory T cells in the FOXP3-deficient IPEX-like hu-mice. CD4 cells do not impede expansion of antigen-primed T cells or tumor clearance in the PB hu-mice.

Conclusion: These data support the clinical readiness of CD4 cells to treat IPEX syndrome and other immune-mediated diseases caused by insufficient or dysfunctional FOXP3 Tregs.
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http://dx.doi.org/10.1002/cti2.1214DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7688376PMC
November 2020

Induction of Antigen-Specific Tolerance in T Cell Mediated Diseases.

Front Immunol 2020 29;11:2194. Epub 2020 Sep 29.

Mechanisms of Peripheral Tolerance Unit, San Raffaele Telethon Institute for Gene Therapy (SR-Tiget), IRCCS San Raffaele Scientific Institute, Milan, Italy.

The development of novel approaches to control unwanted immune responses represents an ambitious goal in the management of a number of clinical conditions, including autoimmunity, autoinflammatory diseases, allergies and replacement therapies, in which the T cell response to self or non-harmful antigens threatens the physiological function of tissues and organs. Current treatments for these conditions rely on the use of non-specific immunosuppressive agents and supportive therapies, which may efficiently dampen inflammation and compensate for organ dysfunction, but they require lifelong treatments not devoid of side effects. These limitations induced researchers to undertake the development of definitive and specific solutions to these disorders: the underlying principle of the novel approaches relies on the idea that empowering the tolerogenic arm of the immune system would restore the immune homeostasis and control the disease. Researchers effort resulted in the development of cell-free strategies, including gene vaccination, protein-based approaches and nanoparticles, and an increasing number of clinical trials tested the ability of adoptive transfer of regulatory cells, including T and myeloid cells. Here we will provide an overview of the most promising approaches currently under development, and we will discuss their potential advantages and limitations. The field is teaching us that the success of these strategies depends primarily on our ability to dampen antigen-specific responses without impairing protective immunity, and to manipulate directly or indirectly the immunomodulatory properties of antigen presenting cells, the ultimate mediators of tolerance.
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http://dx.doi.org/10.3389/fimmu.2020.02194DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7550404PMC
April 2021

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

J Allergy Clin Immunol 2020 04 23;145(4):1262-1271.e13. Epub 2019 Dec 23.

San Raffaele Telethon Institute for Gene Therapy (SR-Tiget), IRCCS San Raffaele Scientific Institute, Milan, Italy; Department of Pediatrics, Division of Stem Cell Transplantation and Regenerative Medicine, Stanford University School of Medicine, Stanford, Calif. Electronic address:

Background: 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.

Objective: 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.

Methods: Phenotype and function of FOXP3-mutated Treg cells from rapamycin-treated patients with IPEX syndrome were tested by flow cytometry and in vitro suppression assays, and the gene expression profile of rapamycin-conditioned Treg cells by droplet-digital PCR.

Results: Clinical and histologic improvements in patients correlated with partially restored Treg function, independent of FOXP3 expression or Treg frequency. Expression of TNF-receptor-superfamily-member 18 (TNFRSF18, glucocorticoid-induced TNF-receptor-related) and EBV-induced-3 (EBI3, an IL-35 subunit) in patients' Treg cells increased during treatment as compared with that of Treg cells from untreated healthy subjects. Furthermore inhibition of glucocorticoid-induced TNF-receptor-related and Ebi3 partially reverted in vitro suppression by in vivo rapamycin-conditioned Treg cells.

Conclusions: 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.
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http://dx.doi.org/10.1016/j.jaci.2019.11.043DOI Listing
April 2020

Targeting a Pre-existing Anti-transgene T Cell Response for Effective Gene Therapy of MPS-I in the Mouse Model of the Disease.

Mol Ther 2019 07 19;27(7):1215-1227. Epub 2019 Apr 19.

San Raffaele Telethon Institute for Gene Therapy (SR-Tiget), IRCCS San Raffaele Scientific Institute, 20132 Milan, Italy. Electronic address:

Mucopolysaccharidosis type I (MPS-I) is a severe genetic disease caused by a deficiency of the alpha-L-iduronidase (IDUA) enzyme. Ex vivo hematopoietic stem cell (HSC) gene therapy is a promising therapeutic approach for MPS-I, as demonstrated by preclinical studies performed in naive MPS-I mice. However, after enzyme replacement therapy (ERT), several MPS-I patients develop anti-IDUA immunity that may jeopardize ex vivo gene therapy efficacy. Here we treat MPS-I mice with an artificial immunization protocol to mimic the ERT effect in patients, and we demonstrate that IDUA-corrected HSC engraftment is impaired in pre-immunized animals by IDUA-specific CD8 T cells spared by pre-transplant irradiation. Conversely, humoral anti-IDUA immunity does not impact on IDUA-corrected HSC engraftment. The inclusion of lympho-depleting agents in pre-transplant conditioning of pre-immunized hosts allowes rescue of IDUA-corrected HSC engraftment, which is proportional to CD8 T cell eradication. Overall, these data demonstrate the relevance of pre-existing anti-transgene T cell immunity on ex vivo HSC gene therapy, and they suggest the application of tailored immune-depleting treatments, as well as a deeper immunological characterization of patients, to safeguard the therapeutic effects of ex vivo HSC gene therapy in immunocompetent hosts.
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http://dx.doi.org/10.1016/j.ymthe.2019.04.014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6612662PMC
July 2019

Role of human forkhead box P3 in early thymic maturation and peripheral T-cell homeostasis.

J Allergy Clin Immunol 2018 12 27;142(6):1909-1921.e9. Epub 2018 Apr 27.

San Raffaele Telethon Institute for Gene Therapy, IRCCS San Raffaele Scientific Institute, Milan, Italy; Division of Stem Cell Transplantation and Regenerative Medicine, Department of Pediatrics, Stanford School of Medicine, Stanford, Calif. Electronic address:

Background: Forkhead box P3 (FOXP3) is a key transcription factor in regulatory T (Treg) cell function. FOXP3 gene mutations cause immune dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) syndrome, a fatal autoimmune syndrome. FOXP3 has also been proposed to act in effector T (Teff) cells, but to date, this role has not been confirmed.

Objective: We sought to evaluate the effect of reduced FOXP3 expression on human Treg and Teff cell development and correlate it with IPEX syndrome immune pathology.

Methods: We developed a model of humanized mice (huMice) in which the human hematopoietic system is stably knocked down or knocked out for the FOXP3 gene (knockdown [KD]/knockout [KO] huMice).

Results: Because FOXP3-KD/KO was not 100% effective, residual FOXP3 expression in hematopoietic stem progenitor cells was sufficient to give rise to Treg cells with normal expression of FOXP3. However, numerous defects appeared in the Teff cell compartment. Compared with control mice, FOXP3-KD/KO huMice showed altered thymocyte differentiation, with KD/KO thymocytes displaying significantly reduced T-cell receptor (TCR) signaling strength and increased TCR repertoire diversity. Peripheral KD/KO Teff cells were expanded and showed signs of homeostatic proliferation, such as a significantly contracted TCR repertoire, a severely reduced naive compartment, decreased telomeric repeat-binding factor 2 expression, and a skew toward a T2 profile, resembling an aged immune system. Consistent with results in FOXP3-KD/KO huMice, analysis of patients with IPEX syndrome provided evidence of defects in the Teff cell compartment at both the thymic and peripheral levels.

Conclusions: These findings support an intrinsic role for human FOXP3 in controlling thymocyte maturation and peripheral expansion of Teff cells and reveal a previously undescribed pathogenic mechanism through an altered Teff cell compartment in patients with IPEX syndrome.
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http://dx.doi.org/10.1016/j.jaci.2018.03.015DOI Listing
December 2018

Corrigendum: Monitoring T-Cell Responses in Translational Studies: Optimization of Dye-Based Proliferation Assay for Evaluation of Antigen-Specific Responses.

Front Immunol 2018 22;9:343. Epub 2018 Feb 22.

San Raffaele Telethon Institute for Gene Therapy (SR-Tiget), Division of Regenerative Medicine, Stem Cells and Gene Therapy, IRCCS San Raffaele Scientific Institute, Milan, Italy.

[This corrects the article on p. 1870 in vol. 8, PMID: 29312346.].
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http://dx.doi.org/10.3389/fimmu.2018.00343DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5827416PMC
February 2018

Monitoring T-Cell Responses in Translational Studies: Optimization of Dye-Based Proliferation Assay for Evaluation of Antigen-Specific Responses.

Front Immunol 2017 21;8:1870. Epub 2017 Dec 21.

San Raffaele Telethon Institute for Gene Therapy (SR-Tiget), Division of Regenerative Medicine, Stem Cells and Gene Therapy, IRCCS San Raffaele Scientific Institute, Milan, Italy.

Adoptive therapy with regulatory T cells or tolerance-inducing antigen (Ag)-presenting cells is innovative and promising therapeutic approach to control undesired and harmful activation of the immune system, as observed in autoimmune diseases, solid organ and bone marrow transplantation. One of the critical issues to elucidate the mechanisms responsible for success or failure of these therapies and define the specificity of the therapy is the evaluation of the Ag-specific T-cell responses. Several efforts have been made to develop suitable and reproducible assays. Here, we focus on dye-based proliferation assays. We highlight with practical examples the fundamental issues to take into consideration for implementation of an effective and sensitive dye-based proliferation assay to monitor Ag-specific responses in patients. The most critical points were used to design a road map to set up and analyze the optimal assay to assess Ag-specific T-cell responses in patients undergoing different treatments. This is the first step to optimize monitoring of tolerance induction, allowing comparison of outcomes of different clinical studies. The road map can also be applied to other therapeutic interventions, not limited to tolerance induction therapies, in which Ag-specific T-cell responses are relevant such as vaccination approaches and cancer immunotherapy.
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http://dx.doi.org/10.3389/fimmu.2017.01870DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5742609PMC
December 2017

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.

Front Immunol 2017 12;8:1282. Epub 2017 Oct 12.

Department of Stem Cell Transplantation and Regenerative Medicine, Division of Pediatrics, Stanford School of Medicine, Stanford, CA, United States.

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 and suppressive activity. The resulting CD4 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 [immune-dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX)], in whom CD4 T cell could be used as therapeutics to control autoimmunity. Here, we will discuss the potential advantages of using CD4 T cells for 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.
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http://dx.doi.org/10.3389/fimmu.2017.01282DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5643480PMC
October 2017

HIV-1-mediated insertional activation of STAT5B and BACH2 trigger viral reservoir in T regulatory cells.

Nat Commun 2017 09 8;8(1):498. Epub 2017 Sep 8.

San Raffaele Telethon Institute for Gene Therapy (SR-Tiget), IRCCS, San Raffaele Scientific Institute, Milan, 20132, Italy.

HIV-1 insertions targeting BACH2 or MLK2 are enriched and persist for decades in hematopoietic cells from patients under combination antiretroviral therapy. However, it is unclear how these insertions provide such selective advantage to infected cell clones. Here, we show that in 30/87 (34%) patients under combination antiretroviral therapy, BACH2, and STAT5B are activated by insertions triggering the formation of mRNAs that contain viral sequences fused by splicing to their first protein-coding exon. These chimeric mRNAs, predicted to express full-length proteins, are enriched in T regulatory and T central memory cells, but not in other T lymphocyte subsets or monocytes. Overexpression of BACH2 or STAT5B in primary T regulatory cells increases their proliferation and survival without compromising their function. Hence, we provide evidence that HIV-1-mediated insertional activation of BACH2 and STAT5B favor the persistence of a viral reservoir in T regulatory cells in patients under combination antiretroviral therapy.HIV insertions in hematopoietic cells are enriched in BACH2 or MLK2 genes, but the selective advantages conferred are unknown. Here, the authors show that BACH2 and additionally STAT5B are activated by viral insertions, generating chimeric mRNAs specifically enriched in T regulatory cells favoring their persistence.
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http://dx.doi.org/10.1038/s41467-017-00609-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5591266PMC
September 2017

Clinical Outlook for Type-1 and FOXP3(+) T Regulatory Cell-Based Therapy.

Front Immunol 2015 25;6:593. Epub 2015 Nov 25.

Department of Pediatric Stem Cell Transplantation and Regenerative Medicine, Stanford School of Medicine , Palo Alto, CA , USA.

T regulatory cells (Tregs) are subsets of T lymphocytes specialized in modulating antigen-specific immune responses in vivo. Hence, Tregs represent an ideal therapeutic tool to control detrimental immune reactions. Based on solid pre-clinical results, investigators started testing the safety and efficacy of Treg-based therapies in humans. Despite promising results, a number of issues remain to be solved. We will discuss the results obtained from clinical trials and the challenges and risks we are facing in the further development of Treg-based therapies.
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http://dx.doi.org/10.3389/fimmu.2015.00593DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4658444PMC
December 2015

Human in vitro induced T regulatory cells and memory T cells share common demethylation of specific FOXP3 promoter region.

Clin Transl Allergy 2015 20;5:35. Epub 2015 Oct 20.

Division of Allergy, Immunology and Rheumatology, Department of Pediatrics, Stanford University, Stanford, CA USA.

Background: The FOXP3 gene is the master regulator for T regulatory cells and is under tight DNA methylation control at the Treg specific demethylated region (TSDR) in its first intron. This said, methylation of its promoter region, the significance of which is unknown, has also been associated with various immune-related disease states such as asthma, food allergy, auto-immunity and cancer. Here, we used induced T regulatory cells (iTreg) as a target cell population to identify candidate hypomethylated CpG sites in the FOXP3 gene promoter to design a DNA methylation quantitative assay for this region.

Findings: Three CpG sites at the promoter region showed clear demethylation pattern associated with high FOXP3 expression after activation in presence of TGFβ and were selected as primary targets to design methylation-dependent RT-PCR primers and probes. We then examined the methylation of this 'inducible-promoter-demethylated-region' (IPDR) in various FOXP3+ T cell subsets. Both naïve and memory thymic-derived Treg cells were found to be fully demethylated at both the IPDR and TSDR. Interestingly, in addition to iTregs, both CD25- and CD25(lo) conventional memory CD4+CD45RA- T cells displayed a high fraction of IPDR demethylated cells in absence of TSDR demethylation.

Conclusion: This implies that the fraction of memory T cells should be taken in account when interpreting FOXP3 promoter methylation results from clinical studies. This approach, which is available for testing in clinical samples could have diagnostic and prognostic value in patients with immune or auto-inflammatory diseases.
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http://dx.doi.org/10.1186/s13601-015-0079-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4617722PMC
October 2015

Fatal autoimmunity in mice reconstituted with human hematopoietic stem cells encoding defective FOXP3.

Blood 2015 Jun 1;125(25):3886-95. Epub 2015 Apr 1.

Department of Pediatrics, Division of Gastroenterology, Hepatology and Nutrition, Boston Children's Hospital, Boston, MA; Department of Medicine, Harvard Medical School, Boston, MA; Division of Gastroenterology, Brigham and Women's Hospital, Boston, MA.

Mice reconstituted with a human immune system provide a tractable in vivo model to assess human immune cell function. To date, reconstitution of murine strains with human hematopoietic stem cells (HSCs) from patients with monogenic immune disorders have not been reported. One obstacle precluding the development of immune-disease specific "humanized" mice is that optimal adaptive immune responses in current strains have required implantation of autologous human thymic tissue. To address this issue, we developed a mouse strain that lacks murine major histocompatibility complex class II (MHC II) and instead expresses human leukocyte antigen DR1 (HLA-DR1). These mice displayed improved adaptive immune responses when reconstituted with human HSCs including enhanced T-cell reconstitution, delayed-type hypersensitivity responses, and class-switch recombination. Following immune reconstitution of this novel strain with HSCs from a patient with immune dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) syndrome, associated with aberrant FOXP3 function, mice developed a lethal inflammatory disorder with multiorgan involvement and autoantibody production mimicking the pathology seen in affected humans. This humanized mouse model permits in vivo evaluation of immune responses associated with genetically altered HSCs, including primary immunodeficiencies, and should facilitate the study of human immune pathobiology and the development of targeted therapeutics.
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http://dx.doi.org/10.1182/blood-2014-12-618363DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4473116PMC
June 2015

Gene/cell therapy approaches for Immune Dysregulation Polyendocrinopathy Enteropathy X-linked syndrome.

Curr Gene Ther 2014 ;14(6):422-8

San Raffaele Telethon Institute for Gene Therapy (HSR-TIGET), Division of Regenerative Medicine, Stem Cells and Gene Therapy, IRCCS San Raffaele Scientific Institute, Via Olgettina 58, 20131, Milan, Italy.

Immune dysregulation, Polyendocrinopathy, Enteropathy, X-linked (IPEX) syndrome is a rare autoimmune disease due to mutations in the gene encoding for Forkhead box P3 (FOXP3), a transcription factor fundamental for the function of thymus-derived (t) regulatory T (Treg) cells. The dysfunction of Treg cells results in the development of devastating autoimmune manifestations affecting multiple organs, eventually leading to premature death in infants, if not promptly treated by hematopoietic stem cell transplantation (HSCT). Novel gene therapy strategies can be developed for IPEX syndrome as more definitive cure than allogeneic HSCT. Here we describe the therapeutic approaches, alternative to HSCT, currently under development. We described that effector T cells can be converted in regulatory T cells by LV-mediated FOXP3-gene transfer in differentiated T lymphocytes. Despite FOXP3 mutations mainly affect a highly specific T cell subset, manipulation of stem cells could be required for long-term remission of the disease. Therefore, we believe that a more comprehensive strategy should aim at correcting FOXP3-mutated stem cells. Potentials and hurdles of both strategies will be highlighted here.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4443799PMC
http://dx.doi.org/10.2174/1566523214666141001123828DOI Listing
July 2015

Forkhead box P3: the peacekeeper of the immune system.

Int Rev Immunol 2014 Mar 19;33(2):129-45. Epub 2013 Dec 19.

1Division of Regenerative Medicine, Stem Cells and Gene Therapy, San Raffaele Telethon Institute for Gene Therapy, San Raffaele Scientific Institute, Milan, Italy.

Ten years ago Forkhead box P3 (FOXP3) was discovered as master gene driving CD4(+)CD25(+) T cell regulatory (Treg) function. Since then, several layers of complexity have emerged in the regulation of its expression and function, which is not only exerted in Treg cells. While the mechanisms leading to the highly selective expression of FOXP3 in thymus-derived Treg cells still remain to be elucidated, we review here the current knowledge on the role of FOXP3 in the development of Treg cells and the direct and indirect consequences of FOXP3 mutations on multiple arms of the immune response. Finally, we summarize the newly acquired knowledge on the epigenetic regulation of FOXP3, still largely undefined in human cells.
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http://dx.doi.org/10.3109/08830185.2013.863303DOI Listing
March 2014

CD4⁺ T cells from IPEX patients convert into functional and stable regulatory T cells by FOXP3 gene transfer.

Sci Transl Med 2013 Dec;5(215):215ra174

San Raffaele Telethon Institute for Gene Therapy, Division of Regenerative Medicine, Stem Cells and Gene Therapy, San Raffaele Scientific Institute, Milan 20132, Italy.

In humans, mutations in the gene encoding for forkhead box P3 (FOXP3), a critically important transcription factor for CD4⁺CD25⁺ regulatory T (T(reg)) cell function, lead to a life-threatening systemic poly-autoimmune disease, known as immune dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) syndrome. Severe autoimmunity results from the inborn dysfunction and instability of FOXP3-mutated T(reg) cells. Hematopoietic stem cell transplantation is the only current curative option for affected patients. We show here that when CD4⁺ T cells are converted into T(reg) cells after lentivirus-mediated FOXP3 gene transfer, the resulting CD4(FOXP3) T cell population displays stable phenotype and suppressive function, especially when naïve T cells are converted. We further demonstrate that CD4(FOXP3) T cells are stable in inflammatory conditions not only in vitro but also in vivo in a model of xenogeneic graft-versus-host disease. We therefore applied this FOXP3 gene transfer strategy for the development of a T(reg) cell-based therapeutic approach to restore tolerance in IPEX syndrome. IPEX-derived CD4(FOXP3) T cells mirrored T(reg) cells from healthy donors in terms of cellular markers, anergic phenotype, cytokine production, and suppressive function. These findings pave the way for the treatment of IPEX patients by adoptive cell therapy with genetically engineered T(reg) cells and are seminal for future potential application in patients with autoimmune disorders of different origin.
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http://dx.doi.org/10.1126/scitranslmed.3007320DOI Listing
December 2013

Autoantibodies to harmonin and villin are diagnostic markers in children with IPEX syndrome.

PLoS One 2013 8;8(11):e78664. Epub 2013 Nov 8.

Center for Translational Genomics and Bioinformatics, San Raffaele Hospital Scientific Institute, Milan, Italy.

Autoantibodies to enterocyte antigens harmonin (75 kDa USH1C protein) and villin (actin-binding 95 kDa protein) are associated with the Immune dysregulation, Polyendocrinopathy, Enteropathy, X-linked (IPEX) syndrome. In this study we evaluated the diagnostic value of harmonin and villin autoantibodies in IPEX and IPEX-like syndromes. Harmonin and villin autoantibodies were measured by a novel Luminescent-Immuno-Precipitation-System (LIPS) quantitative assay, in patients with IPEX, IPEX-like syndrome, Primary Immunodeficiencies (PID) with enteropathy, all diagnosed by sequencing of the FOXP3 gene, and in type 1 diabetes (T1D), celiac disease and healthy blood donors as control groups. Harmonin and villin autoantibodies were detected in 12 (92%) and 6 (46%) of 13 IPEX patients, and in none of the IPEX-like, PID, T1D, celiac patients, respectively. All IPEX patients, including one case with late and atypical clinical presentation, had either harmonin and/or villin autoantibodies and tested positive for enterocyte antibodies by indirect immunofluorescence. When measured in IPEX patients in remission after immunosuppressive therapy or hematopoietic stem cell transplantation, harmonin and villin autoantibodies became undetectable or persisted at low titers in all cases but one in whom harmonin autoantibodies remained constantly high. In one patient, a peak of harmonin antibodies paralleled a relapse phase of enteropathy. Our study demonstrates that harmonin and villin autoantibodies, measured by LIPS, are sensitive and specific markers of IPEX, differentiate IPEX, including atypical cases, from other early childhood disorders associated with enteropathy, and are useful for screening and clinical monitoring of affected children.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0078664PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3826762PMC
March 2015

Accumulation of peripheral autoreactive B cells in the absence of functional human regulatory T cells.

Blood 2013 Feb 5;121(9):1595-603. Epub 2012 Dec 5.

Department of Immunobiology, Yale University School of Medicine, New Haven, CT 06511, USA.

Regulatory T cells (Tregs) play an essential role in preventing autoimmunity. Mutations in the forkhead box protein 3 (FOXP3) gene, which encodes a transcription factor critical for Treg function, result in a severe autoimmune disorder and the production of various autoantibodies in mice and in IPEX (immune dysregulation, polyendocrinopathy, enteropathy, X-linked) patients. However, it is unknown whether Tregs normally suppress autoreactive B cells. To investigate a role for Tregs in maintaining human B-cell tolerance, we tested the reactivity of recombinant antibodies isolated from single B cells isolated from IPEX patients. Characteristics and reactivity of antibodies expressed by new emigrant/transitional B cells from IPEX patients were similar to those from healthy donors, demonstrating that defective Treg function does not impact central B-cell tolerance. In contrast, mature naive B cells from IPEX patients often expressed autoreactive antibodies, suggesting an important role for Tregs in maintaining peripheral B-cell tolerance. T cells displayed an activated phenotype in IPEX patients, including their Treg-like cells, and showed up-regulation of CD40L, PD-1, and inducibl T-cell costimulator (ICOS), which may favor the accumulation of autoreactive mature naive B cells in these patients. Hence, our data demonstrate an essential role for Tregs in the establishment and the maintenance of peripheral B-cell tolerance in humans.
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http://dx.doi.org/10.1182/blood-2012-09-457465DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3587322PMC
February 2013

Immune dysregulation, polyendocrinopathy, enteropathy, x-linked syndrome: a paradigm of immunodeficiency with autoimmunity.

Front Immunol 2012 31;3:211. Epub 2012 Jul 31.

Division of Regenerative Medicine, Stem Cells and Gene Therapy, San Raffaele Telethon Institute for Gene Therapy, San Raffaele Scientific Institute Milan, Italy ; Vita Salute San Raffaele University Milan, Italy.

Immune dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) syndrome is a rare monogenic primary immunodeficiency (PID) due to mutations of FOXP3, a key transcription factor for naturally occurring (n) regulatory T (Treg) cells. The dysfunction of Treg cells is the main pathogenic event leading to the multi-organ autoimmunity that characterizes IPEX syndrome, a paradigm of genetically determined PID with autoimmunity. IPEX has a severe early onset and can become rapidly fatal within the first year of life regardless of the type and site of the mutation. The initial presenting symptoms are severe enteritis and/or type-1 diabetes mellitus, alone or in combination with eczema and elevated serum IgE. Other autoimmune symptoms, such as hypothyroidism, cytopenia, hepatitis, nephropathy, arthritis, and alopecia can develop in patients who survive the initial acute phase. The current therapeutic options for IPEX patients are limited. Supportive and replacement therapies combined with pharmacological immunosuppression are required to control symptoms at onset. However, these procedures can allow only a reduction of the clinical manifestations without a permanent control of the disease. The only known effective cure for IPEX syndrome is hematopoietic stem cell transplantation, but it is always limited by the availability of a suitable donor and the lack of specific guidelines for bone marrow transplant in the context of this disease. This review aims to summarize the clinical histories and genomic mutations of the IPEX patients described in the literature to date. We will focus on the clinical and immunological features that allow differential diagnosis of IPEX syndrome and distinguish it from other PID with autoimmunity. The efficacy of the current therapies will be reviewed, and possible innovative approaches, based on the latest highlights of the pathogenesis to treat this severe primary autoimmune disease of childhood, will be discussed.
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http://dx.doi.org/10.3389/fimmu.2012.00211DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3459184PMC
October 2012

Functional type 1 regulatory T cells develop regardless of FOXP3 mutations in patients with IPEX syndrome.

Eur J Immunol 2011 Apr 14;41(4):1120-31. Epub 2011 Mar 14.

San Raffaele Telethon Institute for Gene Therapy (HSR-TIGET), Division of Regenerative Medicine, Stem Cells and Gene Therapy, San Raffaele Scientific Institute, Milan, Italy.

Mutations of forkhead box p3 (FOXP3), the master gene for naturally occurring regulatory T cells (nTregs), are responsible for the impaired function of nTregs, resulting in an autoimmune disease known as the immune dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) syndrome. The relevance of other peripheral tolerance mechanisms, such as the presence and function of type 1 regulatory T (Tr1) cells, the major adaptive IL-10-producing Treg subset, in patients with IPEX syndrome remains to be clarified. FOXP3(mutated) Tr1-polarized cells, differentiated in vitro from CD4(+) T cells of four IPEX patients, were enriched in IL-10(+) IL-4(-) IFN-γ(+) T cells, a cytokine production profile specific for Tr1 cells, and expressed low levels of FOXP3 and high levels of Granzyme-B. IPEX Tr1 cells were hypoproliferative and suppressive, thus indicating that FOXP3 mutations did not impair their function. Furthermore, we isolated Tr1 cell clones from the peripheral blood of one FOXP3(null) patient, demonstrating that Tr1 cells are present in vivo and they can be expanded in vitro in the absence of WT FOXP3. Overall, our results (i) show that functional Tr1 cells differentiate independently of FOXP3, (ii) confirm that human Tr1 and nTregs are distinct T-cell lineages, and (iii) suggest that under favorable conditions Tr1 cells could exert regulatory functions in IPEX patients.
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http://dx.doi.org/10.1002/eji.201040909DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3107421PMC
April 2011

Point mutants of forkhead box P3 that cause immune dysregulation, polyendocrinopathy, enteropathy, X-linked have diverse abilities to reprogram T cells into regulatory T cells.

J Allergy Clin Immunol 2010 Dec 30;126(6):1242-51. Epub 2010 Oct 30.

Department of Surgery, University of British Columbia, and Immunity and Infection Research Centre, Vancouver Coastal Health Research Institute, Vancouver, British Columbia, Canada.

Background: Immune dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) is a primary immunodeficiency with autoimmunity caused by mutations in forkhead box P3 (FOXP3), which encodes a transcription factor involved in regulatory T (Treg) cell function. The mechanistic basis for how different mutations in FOXP3 cause distinct manifestations of IPEX remains unclear.

Objective: To determine whether 3 different point mutants of FOXP3 that cause severe or mild IPEX differ in their ability to reprogram conventional T cells into Treg cells.

Methods: Human CD4(+) T cells were transduced with wild-type or point mutant forms of FOXP3, and changes in cell surface marker expression, cytokine production, proliferation and suppressive capacity were assessed. Ex vivo T(H)17 cells were also transduced with different forms of FOXP3 to monitor changes in IL-17 production.

Results: The forkhead mutant F373A failed to upregulate CD25 and CCR4, did not suppress cytokine production, and induced suppressive activity less effectively than wild-type FOXP3. In contrast, although the forkhead mutant R347H was also defective in upregulation of CD25, it suppressed the production of cytokines, conferred suppressive capacity on CD4(+) T cells, and suppressed IL-17 production. F324L, a mutant outside the forkhead domain associated with mild IPEX, was equivalent to wild-type FOXP3 in all aspects tested.

Conclusion: Mutations in FOXP3 that cause IPEX do not uniformly abrogate the ability of FOXP3 to regulate transcription and drive the development of Treg cells. These data support the notion that factors in addition to functional changes in Treg cells, such as alterations in conventional T cells, are involved in the pathogenesis of IPEX.
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http://dx.doi.org/10.1016/j.jaci.2010.09.001DOI Listing
December 2010

Wild-type FOXP3 is selectively active in CD4+CD25(hi) regulatory T cells of healthy female carriers of different FOXP3 mutations.

Blood 2009 Nov 8;114(19):4138-41. Epub 2009 Sep 8.

San Raffaele Telethon Institute for Gene Therapy (HSR-TIGET), Division of Regenerative Medicine, Stem Cells and Gene Therapy, San Raffaele Scientific Institute, 58-20132 Milan, Italy.

Forkhead box P3 (FOXP3) is constitutively expressed by CD4(+)CD25(hi) regulatory T cells (nTregs). Mutations of FOXP3 cause a severe autoimmune syndrome known as immune dysregulation polyendocrinopathy enteropathy X-linked, in which nTregs are absent or dysfunctional. Whether FOXP3 is essential for both differentiation and function of human nTreg cells remains to be demonstrated. Because FOXP3 is an X-linked gene subject to X-chromosome inactivation (XCI), we studied 9 healthy female carriers of FOXP3 mutations to investigate the role of wild-type (WT) versus mutated FOXP3 in different cell subsets. Analysis of active WT versus mutated (mut)-FOXP3 allele distribution revealed a random pattern of XCI in peripheral blood lymphocytes and in naive and memory CD4(+)T cells, whereas nTregs expressed only the active WT-FOXP3. These data demonstrate that expression of WT-FOXP3 is indispensable for the presence of a normal nTreg compartment and suggest that FOXP3 is not necessary for effector T-cell differentiation in humans.
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http://dx.doi.org/10.1182/blood-2009-04-214593DOI Listing
November 2009

Regulated and multiple miRNA and siRNA delivery into primary cells by a lentiviral platform.

Mol Ther 2009 Jun 17;17(6):1039-52. Epub 2009 Mar 17.

San Raffaele Telethon Institute for Gene Therapy, Milan, Italy.

RNA interference (RNAi) has tremendous potential for investigating gene function and developing new therapies. However, the design and validation of proficient vehicles for stable and safe microRNA (miR) and small interfering RNA (siRNA) delivery into relevant target cells remains an active area of investigation. Here, we developed a lentiviral platform to efficiently coexpress one or more natural/artificial miR together with a gene of interest from constitutive or regulated polymerase-II (Pol-II) promoters. By swapping the stem-loop (sl) sequence of a selected primary transcript (pri-miR) with that of other miR or replacing the stem with an siRNA of choice, we consistently obtained robust expression of the chimeric/artificial miR in several cell types. We validated our platform transducing a panel of engineered cells stably expressing sensitive reporters for miR activity and on a natural target. This approach allowed us to quantitatively assess at steady state the target suppression activity and expression level of each delivered miR and to compare it to those of endogenous miR. Exogenous/artificial miR reached the concentration and activity typical of highly expressed natural miR without perturbing endogenous miR maturation or regulation. Finally, we demonstrate the robust performance of the platform reversing the anergic/suppressive phenotype of human primary regulatory T cells (Treg) by knocking-down their master gene Forkhead Transcription Factor P3 (FOXP3).
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http://dx.doi.org/10.1038/mt.2009.48DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2835189PMC
June 2009

Clinical and molecular profile of a new series of patients with immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome: inconsistent correlation between forkhead box protein 3 expression and disease severity.

J Allergy Clin Immunol 2008 Dec 25;122(6):1105-1112.e1. Epub 2008 Oct 25.

Department of Pediatrics, Anna Meyer University Children's Hospital, University of Florence, Florence, Italy.

Background: Immune dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) syndrome is an autoimmune genetic disorder caused by mutation of the forkhead box protein 3 gene (FOXP3), a key regulator of immune tolerance.

Objective: We sought to provide clinical and molecular indicators that facilitate the understanding and diagnosis of IPEX syndrome.

Methods: In 14 unrelated affected male subjects who were given diagnoses of IPEX syndrome based on FOXP3 gene sequencing, we determined whether particular FOXP3 mutations affected FOXP3 protein expression and correlated the molecular and clinical data.

Results: Molecular analysis of FOXP3 in the 14 subjects revealed 13 missense and splice-site mutations, including 7 novel mutations. Enteropathy, generally associated with endocrinopathy and eczema, was reported in all patients, particularly in those carrying mutations within FOXP3 functional domains or mutations that altered protein expression. However, similar genotypes did not always result in similar phenotypes in terms of disease presentation and severity. In addition, FOXP3 protein expression did not correlate with disease severity.

Conclusion: Severe autoimmune enteropathy, which is often associated with increased IgE levels and eosinophilia, is the most prominent early manifestation of IPEX syndrome. Nevertheless, the disease course is variable and somewhat unpredictable. Therefore genetic analysis of FOXP3 should always be performed to ensure an accurate diagnosis, and FOXP3 protein expression analysis should not be the only diagnostic tool for IPEX syndrome.
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http://dx.doi.org/10.1016/j.jaci.2008.09.027DOI Listing
December 2008

STAT5-signaling cytokines regulate the expression of FOXP3 in CD4+CD25+ regulatory T cells and CD4+CD25- effector T cells.

Int Immunol 2008 Mar 12;20(3):421-31. Epub 2008 Feb 12.

San Raffaele Telethon Institute for Gene Therapy (HSR-TIGET), Via Olgettina 58, 20132 Milan, Italy.

Forkhead box P3 (FOXP3) is considered a specific marker for CD4(+)CD25(+) regulatory T (Treg) cells, but increasing evidence suggests that human CD4(+)CD25(-) effector T (Teff) cells can transiently express FOXP3 upon activation. We demonstrate that the signal transducer and activator of transcription 5 (STAT5)-signaling cytokines, IL-2, IL-15 and to a lesser extent IL-7, induce FOXP3 up-regulation in vitro in activated human Teff cells. In contrast, cytokines which do not activate STAT5, such as IL-4 or transforming growth factor-beta alone, do not directly induce FOXP3 expression in activated Teff cells. Moreover, expression of a constitutively active form of STAT5a is sufficient to induce FOXP3 expression in Teff cells. Expression of FOXP3 in activated Teff cells requires both TCR-mediated activation and endogenous IL-2, but is not dependent on cell division and does not induce suppressive function. The presence of STAT5-activating cytokines is also required to maintain high FOXP3 expression and suppressive activity of Treg cells in vitro. These data indicate that activation of STAT5 sustains FOXP3 expression in both Treg and Teff cells and contribute to our understanding of how cytokines affect the expression of FOXP3.
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http://dx.doi.org/10.1093/intimm/dxn002DOI Listing
March 2008

Isolation, expansion, and characterization of human natural and adaptive regulatory T cells.

Methods Mol Biol 2007 ;380:83-105

San Raffaele Telethon Institute for Gene Therapy, Milan, Italy.

Regulatory T cells play a central role in controlling homeostasis, and in inducing and maintaining tolerance to both foreign and self-antigens. Several types of T cells with regulatory activity have been described both in mice and humans, and those within the CD4+ subset have been extensively studied. Among them, the best characterized are the naturally occurring CD4+CD25+ regulatory T (Treg) cells, and the adaptive type 1 regulatory T (Tr1) cells. Natural Treg cells can arise directly from the thymus, are characterized by the constitutive expression of the transcription factor Foxp3, and suppress T cell responses in a cell-cell contact mediated mechanism. On the contrary, adaptive Tr1 cells arise in the periphery upon encountering antigen in a tolerogenic environment, produce high levels of interleukin (IL)-10 and mediate suppression via IL-10. During the last decade, much effort has been placed on developing protocols to generate regulatory T-cell lines and clones, to further define the similarities and differences between various regulatory T-cell subsets. In this chapter, we will outline protocols to expand naturally occurring Treg cells, to differentiate homogeneous population of Tr1 cells in vitro, and to generate natural Treg and Tr1 cell clones and cell lines.
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http://dx.doi.org/10.1007/978-1-59745-395-0_6DOI Listing
April 2008

Activation-induced FOXP3 in human T effector cells does not suppress proliferation or cytokine production.

Int Immunol 2007 Apr 27;19(4):345-54. Epub 2007 Feb 27.

Department of Surgery, University of British Columbia, and Immunity and Infection Research Centre, Vancouver Coastal Health Research Institute, 2660 Oak Street, Vancouver, Canada.

Forkhead box P3 (FOXP3) is currently thought to be the most specific marker for naturally occurring CD4(+)CD25(+) T regulatory cells (nTregs). In mice, expression of FoxP3 is strictly correlated with regulatory activity, whereas increasing evidence suggests that in humans, activated T effector cells (Teffs) may also express FOXP3. In order to better define the role of FOXP3 in human Teff cells, we investigated the intensity and kinetics of expression in ex vivo Teff cells, suppressed Teff cells and Teff cell lines. We found that all dividing Teff cells expressed FOXP3, but only transiently, and at levels that were significantly lower than those in suppressive nTregs. This temporary expression in Teff cells was insufficient to suppress expression of reported targets of FOXP3 repressor activity, including CD127, IL-2 and IFN-gamma, and was not correlated with induction of a nTreg phenotype. Thus expression of FOXP3 is a normal consequence of CD4(+) T cell activation and, in humans, it can no longer be used as an exclusive marker of nTregs. These data indicate that our current understanding of how FOXP3 contributes to immune tolerance in humans needs to be re-evaluated.
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http://dx.doi.org/10.1093/intimm/dxm014DOI Listing
April 2007

Defective regulatory and effector T cell functions in patients with FOXP3 mutations.

J Clin Invest 2006 Jun;116(6):1713-22

San Raffaele Telethon Institute for Gene Therapy (HSR-TIGET), Milan, Italy.

The autoimmune disease immune dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) is caused by mutations in the forkhead box protein P3 (FOXP3) gene. In the mouse model of FOXP3 deficiency, the lack of CD4+ CD25+ Tregs is responsible for lethal autoimmunity, indicating that FOXP3 is required for the differentiation of this Treg subset. We show that the number and phenotype of CD4+ CD25+ T cells from IPEX patients are comparable to those of normal donors. CD4+ CD25high T cells from IPEX patients who express FOXP3 protein suppressed the in vitro proliferation of effector T cells from normal donors, when activated by "weak" TCR stimuli. In contrast, the suppressive function of CD4+ CD25high T cells from IPEX patients who do not express FOXP3 protein was profoundly impaired. Importantly, CD4+ CD25high T cells from either FOXP3+ or FOXP3- IPEX patients showed altered suppression toward autologous effector T cells. Interestingly, IL-2 and IFN-gamma production by PBMCs from IPEX patients was significantly decreased. These findings indicate that FOXP3 mutations in IPEX patients result in heterogeneous biological abnormalities, leading not necessarily to a lack of differentiation of CD4+ CD25high Tregs but rather to a dysfunction in these cells and in effector T cells.
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http://dx.doi.org/10.1172/JCI25112DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1472239PMC
June 2006