RESUMO
FOXP3 gene is a key transcription factor driving immune tolerance and its deficiency causes immune dysregulation, polyendocrinopathy, enteropathy X-linked syndrome (IPEX), a prototypic primary immune regulatory disorder (PIRD) with defective regulatory T (Treg) cells. Although life-threatening, the increased awareness and early diagnosis have contributed to improved control of the disease. IPEX currently comprises a broad spectrum of clinical autoimmune manifestations from severe early onset organ involvement to moderate, recurrent manifestations. This review focuses on the mechanistic advancements that, since the IPEX discovery in early 2000, have informed the role of the human FOXP3+ Treg cells in controlling peripheral tolerance and shaping the overall immune landscape of IPEX patients and carrier mothers, contributing to defining new treatments.
Assuntos
Doenças Genéticas Ligadas ao Cromossomo X , Doenças do Sistema Imunitário , Enteropatias , Poliendocrinopatias Autoimunes , Humanos , Doenças Genéticas Ligadas ao Cromossomo X/genética , Doenças Genéticas Ligadas ao Cromossomo X/terapia , Linfócitos T Reguladores , Enteropatias/genética , Síndrome , Fatores de Transcrição Forkhead/genética , Mutação , Poliendocrinopatias Autoimunes/genética , Doenças do Sistema Imunitário/genética , Doenças do Sistema Imunitário/terapiaRESUMO
Thirty years ago, one of the first types of CD4+ T regulatory cells was discovered and named T regulatory type 1 (Tr1) cells. Tr1 cells represent a distinct population of T cells, which are induced in the periphery upon antigen exposure under tolerogenic conditions. They produce the immunosuppressive cytokines interleukin-10 (IL-10) and transforming growth factor-beta (TGF-ß), do not constitutively express FOXP3, and suppress the function of effector immune cells. In this review, the key studies leading to the identification and biological characterization of Tr1 cells are recapitulated. The fundamental role of Tr1 cells in regulating immune responses to pathogenic and non-pathogenic antigens, as well as their use as cell therapeutics, is summarized.
Assuntos
Antígenos/imunologia , Doenças Autoimunes/imunologia , Citocinas/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Linfócitos T Reguladores/imunologia , Animais , Doenças Autoimunes/terapia , Terapia Baseada em Transplante de Células e Tecidos/métodos , Citocinas/metabolismo , Humanos , Modelos Imunológicos , Linfócitos T Reguladores/metabolismo , Linfócitos T Reguladores/transplanteRESUMO
In the past 2 decades, a significant number of studies have been published describing the molecular and clinical aspects of immune dysregulation polyendocrinopathy enteropathy X-linked (IPEX) syndrome. These studies have refined our knowledge of this rare yet prototypic genetic autoimmune disease, advancing the diagnosis, broadening the clinical spectrum, and improving our understanding of the underlying immunologic mechanisms. Despite these advances, Forkhead box P3 mutations have devastating consequences, and treating patients with IPEX syndrome remains a challenge, even with safer strategies for hematopoietic stem cell transplantation and gene therapy becoming a promising reality. The aim of this review was to highlight novel features of the disease to further advance awareness and improve the diagnosis and treatment of patients with IPEX syndrome.
Assuntos
Diabetes Mellitus Tipo 1/congênito , Doenças Genéticas Ligadas ao Cromossomo X , Doenças do Sistema Imunitário , Doenças do Sistema Imunitário/congênito , Enteropatias , Poliendocrinopatias Autoimunes , Humanos , Linfócitos T Reguladores , Diarreia , Doenças Genéticas Ligadas ao Cromossomo X/diagnóstico , Doenças Genéticas Ligadas ao Cromossomo X/genética , Doenças Genéticas Ligadas ao Cromossomo X/terapia , Enteropatias/diagnóstico , Enteropatias/genética , Doenças do Sistema Imunitário/diagnóstico , Doenças do Sistema Imunitário/genética , Doenças do Sistema Imunitário/terapia , Mutação , Fatores de Transcrição Forkhead/genética , Poliendocrinopatias Autoimunes/diagnóstico , Poliendocrinopatias Autoimunes/genética , Poliendocrinopatias Autoimunes/terapiaRESUMO
Decades of work have aimed to genetically reprogram T cells for therapeutic purposes1,2 using recombinant viral vectors, which do not target transgenes to specific genomic sites3,4. The need for viral vectors has slowed down research and clinical use as their manufacturing and testing is lengthy and expensive. Genome editing brought the promise of specific and efficient insertion of large transgenes into target cells using homology-directed repair5,6. Here we developed a CRISPR-Cas9 genome-targeting system that does not require viral vectors, allowing rapid and efficient insertion of large DNA sequences (greater than one kilobase) at specific sites in the genomes of primary human T cells, while preserving cell viability and function. This permits individual or multiplexed modification of endogenous genes. First, we applied this strategy to correct a pathogenic IL2RA mutation in cells from patients with monogenic autoimmune disease, and demonstrate improved signalling function. Second, we replaced the endogenous T cell receptor (TCR) locus with a new TCR that redirected T cells to a cancer antigen. The resulting TCR-engineered T cells specifically recognized tumour antigens and mounted productive anti-tumour cell responses in vitro and in vivo. Together, these studies provide preclinical evidence that non-viral genome targeting can enable rapid and flexible experimental manipulation and therapeutic engineering of primary human immune cells.
Assuntos
Reprogramação Celular/genética , Edição de Genes , Genoma Humano/genética , Linfócitos T/imunologia , Linfócitos T/metabolismo , Animais , Autoimunidade/genética , Sistemas CRISPR-Cas/genética , Células Cultivadas , Humanos , Subunidade alfa de Receptor de Interleucina-2/genética , Masculino , Camundongos , Transplante de Neoplasias , Engenharia de Proteínas , Receptores de Antígenos de Linfócitos T/genética , Linfócitos T/citologiaRESUMO
BACKGROUND: 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. OBJECTIVE: We sought to study the type and extent of immunologic abnormalities that remain ill-defined in IPEX, across genetic and clinical heterogeneity. METHODS: We performed Treg-cell-specific epigenetic quantification and immunologic characterization of severe "typical" (n = 6) and "atypical" or asymptomatic (n = 9) patients with IPEX. RESULTS: 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. CONCLUSIONS: 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.
Assuntos
Doenças Genéticas Ligadas ao Cromossomo X , Poliendocrinopatias Autoimunes , Humanos , Fatores de Transcrição Forkhead , Linfócitos T Reguladores , Mutação , Epigênese GenéticaRESUMO
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.
Assuntos
Diabetes Mellitus , Doenças Genéticas Ligadas ao Cromossomo X , Humanos , Linfócitos T Reguladores , Diarreia , Doenças Genéticas Ligadas ao Cromossomo X/genética , Fatores de Transcrição Forkhead/genética , MutaçãoRESUMO
Immune dysregulation polyendocrinopathy enteropathy X linked (IPEX) syndrome is an uncurable disease of the immune system, with immune dysregulation that is caused by mutations in FOXP3. Current treatment options, such as pharmacological immune suppression and allogeneic hematopoietic stem cell transplantation, have been beneficial but present limitations, and their life-long consequences are ill-defined. Other similar blood monogenic diseases have been successfully treated using gene transfer in autologous patient cells, thus providing an effective and less invasive therapeutic. Development of gene therapy for patients with IPEX is particularly challenging because successful strategies must restore the complex expression profile of the transcription factor FOXP3, ensuring it is tightly regulated and its cell subset-specific roles are maintained. This review summarizes current efforts toward achieving gene therapy to treat immune dysregulation in IPEX patients.
Assuntos
Diabetes Mellitus Tipo 1 , Doenças Genéticas Ligadas ao Cromossomo X , Doenças do Sistema Imunitário , Diabetes Mellitus Tipo 1/congênito , Diarreia , Fatores de Transcrição Forkhead/genética , Doenças Genéticas Ligadas ao Cromossomo X/genética , Doenças Genéticas Ligadas ao Cromossomo X/terapia , Terapia Genética , Humanos , Doenças do Sistema Imunitário/congênito , Doenças do Sistema Imunitário/genética , Doenças do Sistema Imunitário/terapia , Mutação , Linfócitos T ReguladoresRESUMO
BACKGROUND AIMS: Cell therapies are costlier to manufacture than small molecules and protein therapeutics because they require multiple manipulations and are often produced in an autologous manner. Strategies to lower the cost of goods to produce a cell therapy could make a significant impact on its total cost. METHODS: Borrowing from the field of bioprocess development, the authors took a design of experiments (DoE)-based approach to understanding the manufacture of a cell therapy product in pre-clinical development, analyzing main cost factors in the production process. The cells used for these studies were autologous CD4+ T lymphocytes gene-edited using CRISPR/Cas9 and recombinant adeno-associated virus (AAV) to restore normal FOXP3 gene expression as a prospective investigational product for patients with immune dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) syndrome. RESULTS: Using gene editing efficiency as the response variable, an initial screen was conducted for other variables that could influence the editing frequency. The multiplicity of infection (MOI) of AAV and amount of single guide RNA (sgRNA) were the significant factors used for the optimization step to generate a response contour plot. Cost analysis was done for multiple points in the design space to find cost drivers that could be reduced. For the range of values tested (50â000-750â000 vg/cell AAV and 0.8-4 µg sgRNA), editing with the highest MOI and sgRNA yielded the best gene editing frequency. However, cost analysis showed the optimal solution was gene editing at 193â000 vg/cell AAV and 1.78 µg sgRNA. CONCLUSIONS: The authors used DoE to define key factors affecting the gene editing process for a potential investigational therapeutic, providing a novel and faster data-based approach to understanding factors driving complex biological processes. This approach could be applied in process development and aid in achieving more robust strategies for the manufacture of cellular therapeutics.
Assuntos
Sistemas CRISPR-Cas , RNA Guia de Cinetoplastídeos , Sistemas CRISPR-Cas/genética , Terapia Baseada em Transplante de Células e Tecidos , Edição de Genes , Humanos , Estudos Prospectivos , RNA Guia de Cinetoplastídeos/genéticaRESUMO
Type 1 regulatory (Tr1) T cells induced by enforced expression of IL-10 (LV-10) are being developed as a novel treatment for chemotherapy-resistant myeloid leukemias. In vivo, LV-10 cells do not cause graft vs host disease while mediating graft vs leukemia (GvL) effect against adult acute myeloid leukemia (AML). Since pediatric AML (pAML) and adult AML are different on a genetic and epigenetic level, we investigate herein whether LV-10 cells also efficiently kill pAML cells. We show that the majority of primary pAML are killed by LV-10 cells, with different levels of sensitivity to killing. Transcriptionally, pAML sensitive to LV-10 killing expressed a myeloid maturation signature. Overlaying the signatures of sensitive and resistant pAML onto the public NCI TARGET pAML dataset revealed that sensitive pAML clustered with M5 monocytic pAML and pAML with MLL rearrangement. Resistant pAML clustered with myelomonocytic leukemias and those bearing the core binding factor translocations inv(16) or t(8;21)(RUNX1-RUNX1T1). Furthermore, resistant pAML upregulated the membrane glycoprotein CD200, which binds to the inhibitory receptor CD200R1 on LV-10 cells. To examine if CD200 expression on target cells can impair LV-10 cell function, we overexpressed CD200 in myeloid leukemia cell lines ordinarily sensitive to LV-10 killing. Indeed, LV-10 cells degranulated less and killed fewer CD200-overexpressing cells compared to controls, indicating that pAML can utilize CD200 expression for immune evasion. Altogether, the majority of pAML are killed by LV-10 cells in vitro, supporting further LV-10 cell development as an innovative cell therapy for pAML.
Assuntos
Leucemia Mieloide Aguda , Linfócitos T Reguladores , Adulto , Linfócitos T CD4-Positivos , Criança , Efeito Enxerto vs Leucemia , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/terapia , Translocação GenéticaRESUMO
BACKGROUND AIMS: Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a curative therapeutic approach for many hematological disorders. However, allo-HSCT is frequently accompanied by a serious side effect: graft-versus-host disease (GVHD). The clinical use of allo-HSCT is limited by the inability of current immunosuppressive regimens to adequately control GvHD without impairing the graft-versus-leukemia effect (GvL) conferred by transplanted healthy immune cells. To address this, the authors have developed an engineered type 1 regulatory T-cell product called CD4IL-10 cells. CD4IL-10 cells are obtained through lentiviral transduction, which delivers the human IL10 gene into purified polyclonal CD4+ T cells. CD4IL-10 cells may provide an advantage over standard-of-care immunosuppressants because of the ability to suppress GvHD through continuous secretion of IL-10 and enhance the GvL effect in myeloid malignancies through targeted killing of malignant myeloid cells. METHODS: Here the authors established a production process aimed at current Good Manufacturing Practice (cGMP) production for CD4IL-10 cells. RESULTS: The authors demonstrated that the CD4IL-10 cell product maintains the suppressive and cytotoxic functions of previously described CD4IL-10 cells. In addition, RNA sequencing analysis of CD4IL-10 identified novel transcriptome changes, indicating that CD4IL-10 cells primarily upregulate cytotoxicity-related genes. These include four molecules with described roles in CD8+ T and natural killer cell-mediated cytotoxicity: CD244, KLRD1, KLRC1 and FASLG. Finally, it was shown that CD4IL-10 cells upregulate IL-22, which mediates wound healing and tissue repair, particularly in the gut. CONCLUSIONS: Collectively, these results pave the way toward clinical translation of the cGMP-optimized CD4IL-10 cell product and uncover new molecules that have a role in the clinical application of CD4IL-10 cells.
Assuntos
Doença Enxerto-Hospedeiro , Transplante de Células-Tronco Hematopoéticas , Linfócitos T CD4-Positivos , Doença Enxerto-Hospedeiro/genética , Doença Enxerto-Hospedeiro/terapia , Efeito Enxerto vs Leucemia , Humanos , Imunoterapia , Linfócitos T ReguladoresRESUMO
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.
Assuntos
Diabetes Mellitus Tipo 1/congênito , Diarreia/imunologia , Fatores de Transcrição Forkhead/genética , Doenças Genéticas Ligadas ao Cromossomo X/imunologia , Doenças do Sistema Imunitário/congênito , Imunossupressores/uso terapêutico , Mutação/genética , Sirolimo/uso terapêutico , Linfócitos T Reguladores/imunologia , Movimento Celular , Células Cultivadas , Criança , Diabetes Mellitus Tipo 1/tratamento farmacológico , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/imunologia , Diarreia/tratamento farmacológico , Diarreia/genética , Regulação da Expressão Gênica , Doenças Genéticas Ligadas ao Cromossomo X/tratamento farmacológico , Doenças Genéticas Ligadas ao Cromossomo X/genética , Proteína Relacionada a TNFR Induzida por Glucocorticoide/metabolismo , Humanos , Doenças do Sistema Imunitário/tratamento farmacológico , Doenças do Sistema Imunitário/genética , Doenças do Sistema Imunitário/imunologia , Tolerância Imunológica , Interleucinas/genética , Interleucinas/metabolismo , Ativação Linfocitária , Masculino , Antígenos de Histocompatibilidade Menor/genética , Antígenos de Histocompatibilidade Menor/metabolismoRESUMO
Human CD3+CD4+ Th cells, FOXP3+ T regulatory (Treg) cells, and T regulatory type 1 (Tr1) cells are essential for ensuring peripheral immune response and tolerance, but the diversity of Th, Treg, and Tr1 cell subsets has not been fully characterized. Independent functional characterization of human Th1, Th2, Th17, T follicular helper (Tfh), Treg, and Tr1 cells has helped to define unique surface molecules, transcription factors, and signaling profiles for each subset. However, the adequacy of these markers to recapitulate the whole CD3+CD4+ T cell compartment remains questionable. In this study, we examined CD3+CD4+ T cell populations by single-cell mass cytometry. We characterize the CD3+CD4+ Th, Treg, and Tr1 cell populations simultaneously across 23 memory T cell-associated surface and intracellular molecules. High-dimensional analysis identified several new subsets, in addition to the already defined CD3+CD4+ Th, Treg, and Tr1 cell populations, for a total of 11 Th cell, 4 Treg, and 1 Tr1 cell subsets. Some of these subsets share markers previously thought to be selective for Treg, Th1, Th2, Th17, and Tfh cells, including CD194 (CCR4)+FOXP3+ Treg and CD183 (CXCR3)+T-bet+ Th17 cell subsets. Unsupervised clustering displayed a phenotypic organization of CD3+CD4+ T cells that confirmed their diversity but showed interrelation between the different subsets, including similarity between Th1-Th2-Tfh cell populations and Th17 cells, as well as similarity of Th2 cells with Treg cells. In conclusion, the use of single-cell mass cytometry provides a systems-level characterization of CD3+CD4+ T cells in healthy human blood, which represents an important baseline reference to investigate abnormalities of different subsets in immune-mediated pathologies.
Assuntos
Subpopulações de Linfócitos T/imunologia , Linfócitos T Auxiliares-Indutores/imunologia , Linfócitos T Reguladores/imunologia , Adulto , Idoso , Biodiversidade , Feminino , Fatores de Transcrição Forkhead/metabolismo , Homeostase , Humanos , Tolerância Imunológica , Masculino , Espectrometria de Massas/métodos , Pessoa de Meia-Idade , Análise de Célula ÚnicaRESUMO
Monogenic diseases of the immune system, also known as inborn errors of immunity, are caused by single-gene mutations resulting in immune deficiency and dysregulation. More than 350 diseases have been described to date, and the number is rapidly expanding, with increasing availability of next-generation sequencing facilitating the diagnosis. The spectrum of immune dysregulation is wide, encompassing deficiencies in humoral, cellular, innate, and adaptive immunity; phagocytosis; and the complement system, which lead to autoinflammation and autoimmunity. Multiorgan autoimmunity is a dominant symptom when genetic mutations lead to defects in molecules essential for the development, survival, and/or function of regulatory T (Treg) cells. Studies of "Tregopathies" are providing critical mechanistic information on Treg cell biology, the role of Treg cell-associated molecules, and regulation of peripheral tolerance in human subjects. The pathogenic immune networks underlying these diseases need to be dissected to apply and develop immunomodulatory treatments and design curative treatments using cell and gene therapy. Here we review the pathogenetic mechanisms, clinical presentation, diagnosis, and current and future treatments of major known Tregopathies caused by mutations in FOXP3, CD25, cytotoxic T lymphocyte-associated antigen 4 (CTLA4), LPS-responsive and beige-like anchor protein (LRBA), and BTB domain and CNC homolog 2 (BACH2) and gain-of-function mutations in signal transducer and activator of transcription 3 (STAT3). We also discuss deficiencies in genes encoding STAT5b and IL-10 or IL-10 receptor as potential Tregopathies.
Assuntos
Síndromes de Imunodeficiência/imunologia , Linfócitos T Reguladores/imunologia , Animais , Fatores de Transcrição Forkhead/imunologia , Humanos , Síndromes de Imunodeficiência/diagnóstico , Interleucina-10/imunologia , Receptores de Interleucina-10/imunologiaRESUMO
BACKGROUND: Peanut allergy (PA) is a life-threatening condition that lacks regulator-approved treatment. Regulatory T type 1 (TR1) cells are potent suppressors of immune responses and can be induced in vivo upon repeated antigen exposure or in vitro by using tolerogenic dendritic cells. Whether oral immunotherapy (OIT) leads to antigen-specific TR1 cell induction has not been established. OBJECTIVES: We sought to determine whether peanut-specific TR1 cells can be generated in vitro from peripheral blood of patients with PA at baseline or during OIT and whether they are functional compared with peanut-specific TR1 cells induced from healthy control (HC) subjects. METHODS: Tolerogenic dendritic cells were differentiated in the presence of IL-10 from PBMCs of patients with PA and HC subjects pulsed with the main peanut allergens of Arachis hypogaea, Ara h 1 and 2, and used as antigen-presenting cells for autologous CD4+ T cells (CD4+ T cells coincubated with tolerogenic dendritic cells pulsed with the main peanut allergens [pea-T10 cells]). Pea-T10 cells were characterized by the presence of CD49b+ lymphocyte-activation gene 3 (LAG3)+ TR1 cells, antigen-specific proliferative responses, and cytokine production. RESULTS: CD49b+LAG3+ TR1 cells were induced in pea-T10 cells at comparable percentages from HC subjects and patients with PA. Despite their antigen specificity, pea-T10 cells of patients with PA with or without OIT, as compared with those of HC subjects, were not anergic and had high TH2 cytokine production upon peanut-specific restimulation. CONCLUSIONS: Peanut-specific TR1 cells can be induced from HC subjects and patients with PA, but those from patients with PA are functionally defective independent of OIT. The unfavorable TR1/TH2 ratio is discussed as a possible cause of PA TR1 cell impairment.
Assuntos
Alérgenos/imunologia , Antígenos de Plantas/imunologia , Arachis/imunologia , Hipersensibilidade a Amendoim/imunologia , Linfócitos T Reguladores/imunologia , Adolescente , Adulto , Biomarcadores , Criança , Pré-Escolar , Citocinas/biossíntese , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Feminino , Humanos , Ativação Linfocitária , Masculino , Hipersensibilidade a Amendoim/diagnóstico , Hipersensibilidade a Amendoim/metabolismo , Especificidade do Receptor de Antígeno de Linfócitos T , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Linfócitos T Reguladores/metabolismo , Adulto JovemRESUMO
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 TH2 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.
Assuntos
Diabetes Mellitus Tipo 1/congênito , Diarreia/imunologia , Fatores de Transcrição Forkhead/imunologia , Doenças Genéticas Ligadas ao Cromossomo X/imunologia , Doenças do Sistema Imunitário/congênito , Linfócitos T/imunologia , Timo/imunologia , Adolescente , Adulto , Animais , Diferenciação Celular , Criança , Pré-Escolar , Diabetes Mellitus Tipo 1/imunologia , Homeostase , Humanos , Doenças do Sistema Imunitário/imunologia , Lactente , Recém-Nascido , Masculino , Camundongos Transgênicos , Adulto JovemRESUMO
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.
Assuntos
Autoimunidade/imunologia , Modelos Animais de Doenças , Fatores de Transcrição Forkhead/imunologia , Síndromes de Imunodeficiência/imunologia , Animais , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Fatores de Transcrição Forkhead/deficiência , Fatores de Transcrição Forkhead/genética , Transplante de Células-Tronco Hematopoéticas , Humanos , Imuno-Histoquímica , Imunofenotipagem , Camundongos , Camundongos Knockout , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transplante HeterólogoRESUMO
The role of forkhead box P3 (FOXP3) is well-established in T-regulatory cells, but the function of transient FOXP3 expression in activated human conventional T (Tconv) cells is unknown. In the present study, we used 2 approaches to determine the role of FOXP3 in human Tconv cells. First, we obtained Tconv clones from a female subject who is hemizygous for a null mutation in FOXP3, allowing the comparison of autologous T-cell clones that do or do not express FOXP3. Second, we knocked down activation-induced FOXP3 in Tconv cells from healthy donors with small interfering RNAagainst FOXP3. We found that FOXP3-deficient Tconv cells proliferate more and produce more cytokines than wild-type Tconv cells and have differential expression of 274 genes. We also investigated the role of FOXP3 in Th1 and Th17 cells and found that the expression of activation-induced FOXP3 was higher and more sustained in Th17 cells compared with Th1 cells. Knocking down FOXP3 expression in Th17 cells significantly increased the production of IFN-γ and decreased the expression of CCR4, but had no effect on IL-17 expression. These data reveal a novel function of FOXP3 in Tconv cells and suggest that expression of this protein is important in the function of multiple CD4(+) T-cell lineages.
Assuntos
Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/fisiologia , Células Th1/fisiologia , Células Th17/fisiologia , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD4-Positivos/fisiologia , Linhagem da Célula/imunologia , Proliferação de Células , Células Clonais/citologia , Células Clonais/fisiologia , Feminino , Fatores de Transcrição Forkhead/metabolismo , Regulação da Expressão Gênica/imunologia , Humanos , Interferon gama/metabolismo , Interleucina-2/metabolismo , Masculino , RNA Interferente Pequeno , Receptores CCR4/genética , Receptores CCR4/metabolismo , Células Th1/metabolismo , Células Th17/metabolismo , TranscriptomaRESUMO
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.
Assuntos
Autoimunidade , Linfócitos B/citologia , Linfócitos B/imunologia , Linfócitos T Reguladores/citologia , Linfócitos T Reguladores/fisiologia , Doenças Autoimunes/imunologia , Doenças Autoimunes/patologia , Autoimunidade/imunologia , Linfócitos B/patologia , Estudos de Casos e Controles , Células Cultivadas , Pré-Escolar , Humanos , Lactente , Recém-Nascido , Contagem de Linfócitos , Tolerância Periférica/imunologia , Síndrome , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/patologia , Doenças por Imunodeficiência Combinada Ligada ao Cromossomo X/imunologia , Doenças por Imunodeficiência Combinada Ligada ao Cromossomo X/patologiaRESUMO
B-cell responses are guided by the integration of signals through the B-cell receptor (BCR), CD40, and cytokine receptors. The common γ chain (γc)-binding cytokine interleukin (IL)-21 drives humoral immune responses via STAT3-dependent induction of transcription factors required for plasma cell generation. We investigated additional mechanisms by which IL-21/STAT3 signaling modulates human B-cell responses by studying patients with STAT3 mutations. IL-21 strongly induced CD25 (IL-2Rα) in normal, but not STAT3-deficient, CD40L-stimulated naïve B cells. Chromatin immunoprecipitation confirmed IL2RA as a direct target of STAT3. IL-21-induced CD25 expression was also impaired on B cells from patients with IL2RG or IL21R mutations, confirming a requirement for intact IL-21R signaling in this process. IL-2 increased plasmablast generation and immunoglobulin secretion from normal, but not CD25-deficient, naïve B cells stimulated with CD40L/IL-21. IL-2 and IL-21 were produced by T follicular helper cells, and neutralizing both cytokines abolished the B-cell helper capacity of these cells. Our results demonstrate that IL-21, via STAT3, sensitizes B cells to the stimulatory effects of IL-2. Thus, IL-2 may play an adjunctive role in IL-21-induced B-cell differentiation. Lack of this secondary effect of IL-21 may amplify the humoral immunodeficiency in patients with mutations in STAT3, IL2RG, or IL21R due to impaired responsiveness to IL-21.
Assuntos
Linfócitos B/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Interleucina-2/farmacologia , Interleucinas/farmacologia , Plasmócitos/efeitos dos fármacos , Fator de Transcrição STAT3/genética , Linfócitos B/citologia , Linfócitos B/metabolismo , Ligante de CD40/farmacologia , Diferenciação Celular/genética , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Expressão Gênica/efeitos dos fármacos , Humanos , Interleucina-10/farmacologia , Subunidade alfa de Receptor de Interleucina-2/genética , Subunidade alfa de Receptor de Interleucina-2/metabolismo , Mutação , Análise de Sequência com Séries de Oligonucleotídeos , Plasmócitos/citologia , Plasmócitos/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Transcriptoma/efeitos dos fármacosRESUMO
T regulatory Type 1 (Tr1) cells are adaptive T regulatory cells characterized by the ability to secrete high levels of IL-10 and minimal amounts of IL-4 and IL-17. Recently, CD49b and LAG-3 have been identified as Tr1-cell-specific biomarkers in mice and humans. Tr1 cells suppress T-cell- and antigen-presenting cell- (APC) responses primarily via the secretion of IL-10 and TGF-ß. In addition, Tr1 cells release granzyme B and perforin and kill myeloid cells. Tr1 cells inhibit T cell responses also via cell-contact dependent mechanisms mediated by CTLA-4 or PD-1, and by disrupting the metabolic state of T effector cells via the production of the ectoenzymes CD39 and CD73. Tr1 cells were first described in peripheral blood of patients who developed tolerance after HLA-mismatched fetal liver hematopoietic stem cell transplant. Since their discovery, Tr1 cells have been proven to be important in maintaining immunological homeostasis and preventing T-cell-mediated diseases. Furthermore, the possibility to generate and expand Tr1 cells in vitro has led to their utilization as cellular therapy in humans. In this chapter we summarize the unique and distinctive biological properties of Tr1 cells, the well-known and newly discovered Tr1-cell biomarkers, and the different methods to induce Tr1 cells in vitro and in vivo. We also address the role of Tr1 cells in infectious diseases, autoimmunity, and transplant rejection in different pre-clinical disease models and in patients. Finally, we highlight the pathological settings in which Tr1 cells can be beneficial to prevent or to cure the disease.