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1.
Blood ; 132(22): 2362-2374, 2018 Nov 29.
Artigo em Inglês | MEDLINE | ID: mdl-30254128

RESUMO

ARPC1B is a key factor for the assembly and maintenance of the ARP2/3 complex that is involved in actin branching from an existing filament. Germline biallelic mutations in ARPC1B have been recently described in 6 patients with clinical features of combined immunodeficiency (CID), whose neutrophils and platelets but not T lymphocytes were studied. We hypothesized that ARPC1B deficiency may also lead to cytoskeleton and functional defects in T cells. We have identified biallelic mutations in ARPC1B in 6 unrelated patients with early onset disease characterized by severe infections, autoimmune manifestations, and thrombocytopenia. Immunological features included T-cell lymphopenia, low numbers of naïve T cells, and hyper-immunoglobulin E. Alteration in ARPC1B protein structure led to absent/low expression by flow cytometry and confocal microscopy. This molecular defect was associated with the inability of patient-derived T cells to extend an actin-rich lamellipodia upon T-cell receptor (TCR) stimulation and to assemble an immunological synapse. ARPC1B-deficient T cells additionally displayed impaired TCR-mediated proliferation and SDF1-α-directed migration. Gene transfer of ARPC1B in patients' T cells using a lentiviral vector restored both ARPC1B expression and T-cell proliferation in vitro. In 2 of the patients, in vivo somatic reversion restored ARPC1B expression in a fraction of lymphocytes and was associated with a skewed TCR repertoire. In 1 revertant patient, memory CD8+ T cells expressing normal levels of ARPC1B displayed improved T-cell migration. Inherited ARPC1B deficiency therefore alters T-cell cytoskeletal dynamics and functions, contributing to the clinical features of CID.

2.
Front Immunol ; 5: 16, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24550909

RESUMO

T-cell therapy after hematopoietic stem cell transplantation (HSCT) has been used alone or in combination with immunosuppression to cure hematologic malignancies and to prevent disease recurrence. Here, we describe the outcome of patients with high-risk/advanced stage hematologic malignancies, who received T-cell depleted (TCD) haploidentical-HSCT (haplo-HSCT) combined with donor T lymphocytes pretreated with IL-10 (ALT-TEN trial). IL-10-anergized donor T cells (IL-10-DLI) contained T regulatory type 1 (Tr1) cells specific for the host alloantigens, limiting donor-vs.-host-reactivity, and memory T cells able to respond to pathogens. IL-10-DLI were infused in 12 patients with the goal of improving immune reconstitution after haplo-HSCT without increasing the risk of graft-versus-host-disease (GvHD). IL-10-DLI led to fast immune reconstitution in five patients. In four out of the five patients, total T-cell counts, TCR-Vß repertoire and T-cell functions progressively normalized after IL-10-DLI. These four patients are alive, in complete disease remission and immunosuppression-free at 7.2 years (median follow-up) after haplo-HSCT. Transient GvHD was observed in the immune reconstituted (IR) patients, despite persistent host-specific hypo-responsiveness of donor T cells in vitro and enrichment of cells with Tr1-specific biomarkers in vivo. Gene-expression profiles of IR patients showed a common signature of tolerance. This study provides the first indication of the feasibility of Tr1 cell-based therapy and paves way for the use of these Tr1 cells as adjuvant treatment for malignancies and immune-mediated disorders.

3.
Sci Transl Med ; 5(215): 215ra174, 2013 Dec 11.
Artigo em Inglês | MEDLINE | ID: mdl-24337481

RESUMO

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.


Assuntos
Linfócitos T CD4-Positivos/citologia , Fatores de Transcrição Forkhead/genética , Técnicas de Transferência de Genes , Animais , Proliferação de Células , Citocinas/metabolismo , Diabetes Mellitus Tipo 1/congênito , Diarreia , Feminino , Citometria de Fluxo , Doenças Genéticas Ligadas ao Cromossomo X/genética , Doenças Genéticas Ligadas ao Cromossomo X/imunologia , Engenharia Genética , Doença Enxerto-Hospedeiro , Transplante de Células-Tronco Hematopoéticas , Humanos , Doenças do Sistema Imunitário/congênito , Memória Imunológica , Inflamação , Leucócitos Mononucleares/citologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Transplante de Neoplasias , Fenótipo , Linfócitos T Reguladores/citologia
4.
Haematologica ; 95(12): 2134-43, 2010 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-20713457

RESUMO

BACKGROUND: CD4(+) regulatory T cells are a specialized subset of T cells that actively control immune responses. Several experimental protocols have been used to expand natural regulatory T cells and to generate adaptive type 1 regulatory T cells for regulatory T-cell-based therapies. DESIGN AND METHODS: The ability of exogenous recombinant human interleukin-10 to induce alloantigen-specific anergy in T cells was investigated and compared to that of interleukin-10 derived from tolerogenic dendritic cells, in mixed lymphocyte cultures. A detailed characterization of the effector functions of the resulting anergized T cells is reported. RESULTS: Interleukin-10, whether exogenous or derived from tolerogenic dendritic cells, induces a population of alloantigen-specific T cells (interleukin-10-anergized T cells) containing type 1 regulatory T cells, which are anergic and actively suppress alloantigen-specific effector T cells present within the mixed population. Interleukin-10-induced anergy is transforming growth factor-ß independent, and is associated with a decreased frequency of alloantigen-specific cytotoxic T lymphocyte precursors, but interleukin-10-anergized T cells are still responsive to third-party, bacterial, and viral antigens. Tolerogenic dendritic cells are more powerful than exogenous interleukin-10 in generating type 1 regulatory T-cell precursors, and are also effective in the context of HLA-matched donors. CONCLUSIONS: Based on these studies, we have developed an efficient and reproducible in vitro method to generate antigen-specific type 1 regulatory T-cell precursors starting from total peripheral blood cells with minimal cell manipulation and suitable for generating type 1 regulatory T cells for regulatory T-cell-based therapies.


Assuntos
Terapia Baseada em Transplante de Células e Tecidos/métodos , Anergia Clonal/imunologia , Isoantígenos/imunologia , Linfócitos T/imunologia , Candida albicans/imunologia , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Anergia Clonal/efeitos dos fármacos , Citomegalovirus/imunologia , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Interferon gama/metabolismo , Interleucina-10/farmacologia , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismo , Teste de Cultura Mista de Linfócitos , Monócitos/imunologia , Monócitos/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Linfócitos T/efeitos dos fármacos , Linfócitos T/metabolismo , Linfócitos T Citotóxicos/imunologia , Linfócitos T Citotóxicos/metabolismo , Toxoide Tetânico/imunologia , Fator de Crescimento Transformador beta/farmacologia
5.
J Immunol ; 177(6): 4178-86, 2006 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-16951383

RESUMO

Celiac disease (CD) results from a permanent intolerance to dietary gluten and is due to a massive T cell-mediated immune response to gliadin, the main component of gluten. In this disease, the regulation of immune responses to dietary gliadin is altered. Herein, we investigated whether IL-10 could modulate anti-gliadin immune responses and whether gliadin-specific type 1 regulatory T (Tr1) cells could be isolated from the intestinal mucosa of CD patients in remission. Short-term T cell lines were generated from jejunal biopsies, either freshly processed or cultured ex vivo with gliadin in the presence or absence of IL-10. Ex vivo stimulation of CD biopsies with gliadin in the presence of IL-10 resulted in suppression of Ag-specific proliferation and cytokine production, indicating that pathogenic T cells are susceptible to IL-10-mediated immune regulation. T cell clones generated from intestinal T cell lines were tested for gliadin specificity by cytokine production and proliferative responses. The majority of gliadin-specific T cell clones had a Th0 cytokine production profile with secretion of IL-2, IL-4, IFN-gamma, and IL-10 and proliferated in response to gliadin. Tr1 cell clones were also isolated. These Tr1 cells were anergic, restricted by DQ2 (a CD-associated HLA), and produced IL-10 and IFN-gamma, but little or no IL-2 or IL-4 upon activation with gliadin or polyclonal stimuli. Importantly, gliadin-specific Tr1 cell clones suppressed proliferation of pathogenic Th0 cells. In conclusion, dietary Ag-specific Tr1 cells are present in the human intestinal mucosa, and strategies to boost their numbers and/or function may offer new therapeutic opportunities to restore gut homeostasis.


Assuntos
Doença Celíaca/imunologia , Gliadina/imunologia , Tolerância Imunológica , Mucosa Intestinal/citologia , Mucosa Intestinal/imunologia , Linfócitos T Reguladores/imunologia , Adolescente , Adulto , Doença Celíaca/metabolismo , Doença Celíaca/patologia , Linhagem Celular , Proliferação de Células , Criança , Células Clonais , Citocinas/biossíntese , Proteínas na Dieta/imunologia , Feminino , Inibidores do Crescimento/imunologia , Humanos , Interleucina-10/fisiologia , Mucosa Intestinal/metabolismo , Masculino , Pessoa de Meia-Idade , Técnicas de Cultura de Órgãos , Linfócitos T Reguladores/classificação , Linfócitos T Reguladores/metabolismo
6.
J Clin Invest ; 116(6): 1713-22, 2006 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-16741580

RESUMO

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.


Assuntos
Fatores de Transcrição Forkhead , Doenças Genéticas Ligadas ao Cromossomo X/imunologia , Poliendocrinopatias Autoimunes/imunologia , Enteropatias Perdedoras de Proteínas/imunologia , Subpopulações de Linfócitos T/imunologia , Linfócitos T Reguladores/imunologia , Animais , Biomarcadores/metabolismo , Linfócitos T CD4-Positivos/imunologia , Pré-Escolar , Citocinas/imunologia , Modelos Animais de Doenças , Feminino , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/imunologia , Doenças Genéticas Ligadas ao Cromossomo X/genética , Humanos , Lactente , Interleucina-2/genética , Interleucina-2/imunologia , Células Jurkat , Leucócitos Mononucleares/metabolismo , Masculino , Camundongos , Mutação de Sentido Incorreto , Fenótipo , Poliendocrinopatias Autoimunes/genética , Regiões Promotoras Genéticas , Enteropatias Perdedoras de Proteínas/genética
7.
J Exp Med ; 196(10): 1335-46, 2002 Nov 18.
Artigo em Inglês | MEDLINE | ID: mdl-12438424

RESUMO

T regulatory (Tr) cells are essential for the induction of peripheral tolerance. Several types of Tr cells exist, including CD4(+) T cells which express CD25 constitutively and suppress immune responses via direct cell-to-cell interactions, and type 1 T regulatory (Tr1) cells, which function via secretion of interleukin (IL)-10 and transforming growth factor (TGF)-beta. The relationship between CD25(+)CD4(+) T cells and Tr1 cells remains unclear. Here, we demonstrate at the clonal level that Tr1 and CD25(+)CD4(+) T cells are two distinct subsets of regulatory cells with different cytokine production profiles. Furthermore, CD25(-)CD4(+) T cells can be rendered anergic by IL-10 and differentiated into Tr1 cells in the absence of CD25(+)CD4(+) T cells. Cloned human CD25(+)CD4(+) T cell populations are heterogeneous and only a subset of clones continues to express high levels of CD25 and is suppressive. The intensity of CD25, cytotoxic T lymphocyte antigen (CTLA)-4, and glucocorticoid-induced tumor necrosis factor (TNF) receptor expression correlates with the suppressive capacity of the T cell clones. None of the CD25(+)CD4(+) T cell clones with suppressive function produce IL-10, but all produce TGF-beta. Suppression mediated by CD25(+)CD4(+) T cell clones is partially dependent on TGF-beta, but not on constitutive high expression of CD25. Together these data indicate that naturally occurring human CD25(+)CD4(+) T cells are distinct from IL-10-producing Tr1 cells.


Assuntos
Antígenos CD4/imunologia , Imunoconjugados , Interleucina-10/biossíntese , Receptores de Interleucina-2/imunologia , Linfócitos T Reguladores/metabolismo , Fator de Crescimento Transformador beta/biossíntese , Abatacepte , Antígenos CD , Antígenos de Diferenciação/imunologia , Antígeno CTLA-4 , Anergia Clonal/imunologia , Células Clonais , Técnicas de Cocultura , Ensaio de Imunoadsorção Enzimática , Humanos , Interleucina-10/imunologia , Linfócitos T Reguladores/imunologia
8.
Eur J Immunol ; 32(8): 2237-45, 2002 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-12209636

RESUMO

Cloned T regulatory type 1 (Tr1) cells produce IL-10, TGF-beta, IFN-gamma, and very low or non-detectable levels of IL-2 and IL-4, following TCR-mediated activation. In addition, upon TCR stimulation, Tr1 cell clones up-regulate activation markers but show low proliferative responses, partially due to the suppressive effect of autocrine IL-10 and TGF-beta. Here we show that Tr1 cells have growth requirements different from those of Th1 and Th2 cells. Exogenous IL-15, and to a lesser extent IL-2, induce and support the proliferation of Tr1 cells in the absence of TCR activation. This strong cytokine response correlates with high constitutive levels of the IL-2/15Rbeta and common gamma chains expressed by Tr1 cell clones. Furthermore, suboptimal doses of IL-15, in combination with IL-2, induce a significant growth (median value: 25-fold increase in cell number) of Tr1 cell clones during a culture period of 11 days, which leads to an in vitro expansion of Tr1 cell clones comparable to that of Th1 and Th2 cell clones. Tr1 cell clones cultured in IL-15 continue to secrete immunosuppressive cytokines and to proliferate poorly upon reactivation via TCR. These findings indicate that Tr1 cells are constitutively capable of responding to cytokines and mainly to IL-15. This growth factor enables a significant in vitro expansion of Tr1 cells facilitating further biological and biochemical characterization of this unique T cell subset.


Assuntos
Citocinas/farmacologia , Ativação Linfocitária , Receptores de Antígenos de Linfócitos T/fisiologia , Linfócitos T/fisiologia , Células Clonais , Citocinas/biossíntese , Humanos , Interleucina-15/farmacologia , Interleucina-2/farmacologia , Interleucina-4/farmacologia , Interleucina-7/farmacologia , Receptores de Interleucina-2/análise
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