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1.
Diabetes ; 72(11): 1629-1640, 2023 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-37625150

RESUMO

Costimulation serves as a critical checkpoint for T-cell activation, and several genetic variants affecting costimulatory pathways confer risk for autoimmune diseases. A single nucleotide polymorphism (rs763361) in the CD226 gene encoding a costimulatory receptor increases susceptibility to multiple autoimmune diseases, including type 1 diabetes. We previously found that Cd226 knockout protected NOD mice from disease, but the impact of CD226 on individual immune subsets remained unclear. Our prior reports implicate regulatory T cells (Tregs), as human CD226+ Tregs exhibit reduced suppressive function. Hence, we hypothesized that genomic Cd226 gene deletion would increase Treg stability and that Treg-specific Cd226 deletion would inhibit diabetes in NOD mice. Indeed, crossing NOD.Cd226-/- and a NOD Treg-lineage tracing strain resulted in decreased pancreatic Foxp3-deficient "ex-Tregs." We generated a novel Treg-conditional knockout (TregΔCd226) strain that displayed decreased insulitis and diabetes incidence. CD226-deficient pancreatic Tregs had increased expression of the coinhibitory counter-receptor T-cell immunoreceptor with Ig and immunoreceptor tyrosine-based inhibitory motif domains (TIGIT). Moreover, NOD splenocytes treated with TIGIT-Fc fusion protein exhibited reduced T-cell proliferation and interferon-γ production following anti-CD3/CD28 stimulation. This study demonstrates that a CD226/TIGIT imbalance contributes to Treg instability in NOD mice and highlights the potential for therapeutic targeting this costimulatory pathway to halt autoimmunity.

2.
Front Immunol ; 14: 1142648, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37325626

RESUMO

The autoimmune pathogenesis of type 1 diabetes (T1D) involves cellular infiltration from innate and adaptive immune subsets into the islets of Langerhans within the pancreas; however, the direct cytotoxic killing of insulin-producing ß-cells is thought to be mediated primarily by antigen-specific CD8+ T cells. Despite this direct pathogenic role, key aspects of their receptor specificity and function remain uncharacterized, in part, due to their low precursor frequency in peripheral blood. The concept of engineering human T cell specificity, using T cell receptor (TCR) and chimeric antigen receptor (CAR)-based approaches, has been demonstrated to improve adoptive cell therapies for cancer, but has yet to be extensively employed for modeling and treating autoimmunity. To address this limitation, we sought to combine targeted genome editing of the endogenous TCRα chain gene (TRAC) via CRISPR/Cas9 in combination with lentiviral vector (LV)-mediated TCR gene transfer into primary human CD8+ T cells. We observed that knockout (KO) of endogenous TRAC enhanced de novo TCR pairing, which permitted increased peptide:MHC-dextramer staining. Moreover, TRAC KO and TCR gene transfer increased markers of activation and effector function following activation, including granzyme B and interferon-γ production. Importantly, we observed increased cytotoxicity toward an HLA-A*0201+ human ß-cell line by HLA-A*02:01 restricted CD8+ T cells engineered to recognize islet-specific glucose-6-phosphatase catalytic subunit (IGRP). These data support the notion of altering the specificity of primary human T cells for mechanistic analyses of autoreactive antigen-specific CD8+ T cells and are expected to facilitate downstream cellular therapeutics to achieve tolerance induction through the generation of antigen-specific regulatory T cells.


Assuntos
Diabetes Mellitus Tipo 1 , Células Secretoras de Insulina , Humanos , Linfócitos T CD8-Positivos , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/metabolismo , Imunidade Celular
3.
Med ; 4(7): 457-477.e8, 2023 Jul 14.
Artigo em Inglês | MEDLINE | ID: mdl-37172578

RESUMO

BACKGROUND: The advent of chimeric antigen receptor (CAR) T cell therapies has transformed the treatment of hematological malignancies; however, broader therapeutic success of CAR T cells has been limited in solid tumors because of their frequently heterogeneous composition. Stress proteins in the MICA and MICB (MICA/B) family are broadly expressed by tumor cells following DNA damage but are rapidly shed to evade immune detection. METHODS: We have developed a novel CAR targeting the conserved α3 domain of MICA/B (3MICA/B CAR) and incorporated it into a multiplexed-engineered induced pluripotent stem cell (iPSC)-derived natural killer (NK) cell (3MICA/B CAR iNK) that expressed a shedding-resistant form of the CD16 Fc receptor to enable tumor recognition through two major targeting receptors. FINDINGS: We demonstrated that 3MICA/B CAR mitigates MICA/B shedding and inhibition via soluble MICA/B while simultaneously exhibiting antigen-specific anti-tumor reactivity across an expansive library of human cancer cell lines. Pre-clinical assessment of 3MICA/B CAR iNK cells demonstrated potent antigen-specific in vivo cytolytic activity against both solid and hematological xenograft models, which was further enhanced in combination with tumor-targeted therapeutic antibodies that activate the CD16 Fc receptor. CONCLUSIONS: Our work demonstrated 3MICA/B CAR iNK cells to be a promising multi-antigen-targeting cancer immunotherapy approach intended for solid tumors. FUNDING: Funded by Fate Therapeutics and NIH (R01CA238039).


Assuntos
Receptores de Antígenos Quiméricos , Humanos , Receptores de Antígenos Quiméricos/genética , Receptores de Antígenos Quiméricos/metabolismo , Linhagem Celular Tumoral , Imunoterapia Adotiva , Células Matadoras Naturais/metabolismo , Células Matadoras Naturais/transplante , Receptores Fc/metabolismo
4.
JCI Insight ; 6(14)2021 07 22.
Artigo em Inglês | MEDLINE | ID: mdl-34156979

RESUMO

Estrogen-related receptor γ (Esrrg) is a murine lupus susceptibility gene associated with T cell activation. Here, we report that Esrrg controls Tregs through mitochondria homeostasis. Esrrg deficiency impaired the maintenance and function of Tregs, leading to global T cell activation and autoimmunity in aged mice. Further, Esrrg-deficient Tregs presented an impaired differentiation into follicular Tregs that enhanced follicular helper T cells' responses. Mechanistically, Esrrg-deficient Tregs presented with dysregulated mitochondria with decreased oxygen consumption as well as ATP and NAD+ production. In addition, Esrrg-deficient Tregs exhibited decreased phosphatidylinositol and TGF-ß signaling pathways and increased mTOR complex 1 activation. We found that the expression of human ESRRG, which is high in Tregs, was lower in CD4+ T cells from patients with lupus than in healthy controls. Finally, knocking down ESRRG in Jurkat T cells decreased their metabolism. Together, our results reveal a critical role of Esrrg in the maintenance and metabolism of Tregs, which may provide a genetic link between lupus pathogenesis and mitochondrial dysfunction in T cells.


Assuntos
Lúpus Eritematoso Sistêmico/genética , Mitocôndrias/patologia , Receptores de Estrogênio/deficiência , Receptores de Estrogênio/genética , Linfócitos T Reguladores/imunologia , Animais , Modelos Animais de Doenças , Feminino , Técnicas de Silenciamento de Genes , Humanos , Células Jurkat , Lúpus Eritematoso Sistêmico/sangue , Lúpus Eritematoso Sistêmico/imunologia , Camundongos , Mitocôndrias/metabolismo , Linfócitos T Reguladores/citologia , Linfócitos T Reguladores/metabolismo
5.
Front Immunol ; 11: 2180, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33013915

RESUMO

The costimulatory molecule CD226 is highly expressed on effector/memory T cells and natural killer cells. Costimulatory signals received by T cells can impact both central and peripheral tolerance mechanisms. Genetic polymorphisms in CD226 have been associated with susceptibility to type 1 diabetes and other autoimmune diseases. We hypothesized that genetic deletion of Cd226 in the non-obese diabetic (NOD) mouse would impact type 1 diabetes incidence by altering T cell activation. CD226 knockout (KO) NOD mice displayed decreased disease incidence and insulitis in comparison to wild-type (WT) controls. Although female CD226 KO mice had similar levels of sialoadenitis as WT controls, male CD226 KO mice showed protection from dacryoadenitis. Moreover, CD226 KO T cells were less capable of adoptively transferring disease compared to WT NOD T cells. Of note, CD226 KO mice demonstrated increased CD8+ single positive (SP) thymocytes, leading to increased numbers of CD8+ T cells in the spleen. Decreased percentages of memory CD8+CD44+CD62L- T cells were observed in the pancreatic lymph nodes of CD226 KO mice. Intriguingly, CD8+ T cells in CD226 KO mice showed decreased islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP)-tetramer and CD5 staining, suggesting reduced T cell receptor affinity for this immunodominant antigen. These data support an important role for CD226 in type 1 diabetes development by modulating thymic T cell selection as well as impacting peripheral memory/effector CD8+ T cell activation and function.


Assuntos
Antígenos de Diferenciação de Linfócitos T/metabolismo , Linfócitos T CD8-Positivos/imunologia , Diabetes Mellitus Tipo 1/imunologia , Timócitos/imunologia , Animais , Antígenos de Diferenciação de Linfócitos T/genética , Antígenos CD5/genética , Antígenos CD5/metabolismo , Diferenciação Celular , Células Cultivadas , Modelos Animais de Doenças , Regulação da Expressão Gênica , Glucose-6-Fosfatase/genética , Glucose-6-Fosfatase/metabolismo , Humanos , Epitopos Imunodominantes/imunologia , Memória Imunológica , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos NOD , Camundongos Knockout , Tolerância Periférica , Receptores de Antígenos de Linfócitos T/metabolismo
6.
J Autoimmun ; 108: 102417, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32035746

RESUMO

IL-12 and IL-18 synergize to promote TH1 responses and have been implicated as accelerators of autoimmune pathogenesis in type 1 diabetes (T1D). We investigated the influence of these cytokines on immune cells involved in human T1D progression: natural killer (NK) cells, regulatory T cells (Tregs), and cytotoxic T lymphocytes (CTL). NK cells from T1D patients exhibited higher surface CD226 versus controls and lower CD25 compared to first-degree relatives and controls. Changes in NK cell phenotype towards terminal differentiation were associated with cytomegalovirus (CMV) seropositivity, while possession of IL18RAP, IFIH1, and IL2RA T1D-risk variants impacted NK cell activation as evaluated by immuno-expression quantitative trait loci (eQTL) analyses. IL-12 and IL-18 stimulated NK cells from healthy donors exhibited enhanced specific killing of myelogenous K562 target cells. Moreover, activated NK cells increased expression of NKG2A, NKG2D, CD226, TIGIT and CD25, which enabled competition for IL-2 upon co-culture with Tregs, resulting in Treg downregulation of FOXP3, production of IFNγ, and loss of suppressive function. We generated islet-autoreactive CTL "avatars", which upon exposure to IL-12 and IL-18, upregulated IFNγ and Granzyme-B leading to increased lymphocytotoxicity of a human ß-cell line in vitro. These results support a model for T1D pathogenesis wherein IL-12 and IL-18 synergistically enhance CTL and NK cell cytotoxic activity and disrupt immunoregulation by Tregs.


Assuntos
Imunidade Inata , Inflamação/imunologia , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Ativação Linfocitária/imunologia , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismo , Adolescente , Adulto , Biomarcadores , Células Cultivadas , Criança , Citocinas/metabolismo , Citotoxicidade Imunológica , Diabetes Mellitus Tipo 1/etiologia , Diabetes Mellitus Tipo 1/metabolismo , Suscetibilidade a Doenças , Feminino , Humanos , Imunofenotipagem , Inflamação/metabolismo , Inflamação/patologia , Contagem de Linfócitos , Masculino , Pessoa de Meia-Idade , Modelos Biológicos , Fenótipo , Locos de Características Quantitativas , Linfócitos T Citotóxicos/imunologia , Linfócitos T Citotóxicos/metabolismo , Adulto Jovem
7.
Cell Gene Ther Insights ; 4(1): 405-429, 2018 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-34984106

RESUMO

Interest in adoptive T-cell therapies has been ignited by the recent clinical success of genetically-modified T cells in the cancer immunotherapy space. In addition to immune targeting for malignancies, this approach is now being explored for the establishment of immune tolerance with regulatory T cells (Tregs). Herein, we will summarize the basic science and clinical results emanating from trials directed at inducing durable immune regulation through administration of Tregs. We will discuss some of the current challenges facing the field in terms of maximizing cell purity, stability and expansion capacity, while also achieving feasibility and GMP production. Indeed, recent advances in methodologies for Treg isolation, expansion, and optimal source materials represent important strides toward these considerations. Finally, we will review the emerging genetic and biomaterial-based approaches on the horizon for directing Treg specificity to augment tissue-targeting and regenerative medicine.

8.
Front Immunol ; 8: 1313, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29123516

RESUMO

The ability to alter antigen specificity by T-cell receptor (TCR) or chimeric antigen receptor (CAR) gene transfer has facilitated personalized cellular immune therapies in cancer. Inversely, this approach can be harnessed in autoimmune settings to attenuate inflammation by redirecting the specificity of regulatory T cells (Tregs). Herein, we demonstrate efficient protocols for lentiviral gene transfer of TCRs that recognize type 1 diabetes-related autoantigens with the goal of tissue-targeted induction of antigen-specific tolerance to halt ß-cell destruction. We generated human Tregs expressing a high-affinity GAD555-567-reactive TCR (clone R164), as well as the lower affinity clone 4.13 specific for the same peptide. We demonstrated that de novo Treg avatars potently suppress antigen-specific and bystander responder T-cell (Tresp) proliferation in vitro in a process that requires Treg activation (P < 0.001 versus unactivated Tregs). When Tresp were also glutamic acid decarboxylase (GAD)-reactive, the high-affinity R164 Tregs exhibited increased suppression (P < 0.01) with lower Tresp-division index (P < 0.01) than the lower affinity 4.13 Tregs. These data demonstrate the feasibility of rapid expansion of antigen-specific Tregs for applications in attenuating ß-cell autoimmunity and emphasize further opportunities for engineering cellular specificities, affinities, and phenotypes to tailor Treg activity in adoptive cell therapies for the treatment of type 1 diabetes.

9.
J Immunol ; 195(1): 145-55, 2015 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-25994968

RESUMO

Regulatory T cells (Tregs) play a central role in counteracting inflammation and autoimmunity. A more complete understanding of cellular heterogeneity and the potential for lineage plasticity in human Treg subsets may identify markers of disease pathogenesis and facilitate the development of optimized cellular therapeutics. To better elucidate human Treg subsets, we conducted direct transcriptional profiling of CD4(+)FOXP3(+)Helios(+) thymic-derived Tregs and CD4(+)FOXP3(+)Helios(-) T cells, followed by comparison with CD4(+)FOXP3(-)Helios(-) T conventional cells. These analyses revealed that the coinhibitory receptor T cell Ig and ITIM domain (TIGIT) was highly expressed on thymic-derived Tregs. TIGIT and the costimulatory factor CD226 bind the common ligand CD155. Thus, we analyzed the cellular distribution and suppressive activity of isolated subsets of CD4(+)CD25(+)CD127(lo/-) T cells expressing CD226 and/or TIGIT. We observed TIGIT is highly expressed and upregulated on Tregs after activation and in vitro expansion, and is associated with lineage stability and suppressive capacity. Conversely, the CD226(+)TIGIT(-) population was associated with reduced Treg purity and suppressive capacity after expansion, along with a marked increase in IL-10 and effector cytokine production. These studies provide additional markers to delineate functionally distinct Treg subsets that may help direct cellular therapies and provide important phenotypic markers for assessing the role of Tregs in health and disease.


Assuntos
Antígenos de Diferenciação de Linfócitos T/imunologia , Fenótipo , Receptores Imunológicos/imunologia , Linfócitos T Reguladores/imunologia , Transcriptoma/imunologia , Adulto , Antígenos de Diferenciação de Linfócitos T/genética , Antígenos CD4/genética , Antígenos CD4/imunologia , Diferenciação Celular , Linhagem da Célula/imunologia , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/imunologia , Perfilação da Expressão Gênica , Humanos , Fator de Transcrição Ikaros/genética , Fator de Transcrição Ikaros/imunologia , Imunofenotipagem , Interleucina-10/genética , Interleucina-10/imunologia , Ligantes , Ativação Linfocitária , Pessoa de Meia-Idade , Cultura Primária de Células , Ligação Proteica , Receptores Imunológicos/genética , Receptores Virais/genética , Receptores Virais/imunologia , Linfócitos T Reguladores/citologia
10.
J Neuroimmunol ; 267(1-2): 20-7, 2014 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-24369297

RESUMO

The transcription factor Tbet is critical for the differentiation of Th1 CD4 T cells and is associated with the induction of multiple autoimmune diseases, including experimental autoimmune encephalomyelitis (EAE). Herein, we demonstrate that Tbet suppresses IL-17A and Th17 differentiation both in vitro and in vivo in a cell-intrinsic manner, and that in fact, Tbet is not necessary for EAE induction. Moreover, we find that IFNγ inhibits the production of IL-17A and IL-17F in a STAT1-dependent, Tbet-independent manner. These findings illustrate multiple mechanisms utilized by developing Th1 cells to silence the Th17 program.


Assuntos
Diferenciação Celular/efeitos dos fármacos , Interleucina-17/farmacologia , Proteínas com Domínio T/metabolismo , Células Th17/efeitos dos fármacos , Animais , Antígenos CD4/metabolismo , Polaridade Celular/efeitos dos fármacos , Sistema Nervoso Central/patologia , Modelos Animais de Doenças , Encefalomielite Autoimune Experimental/imunologia , Encefalomielite Autoimune Experimental/patologia , Regulação da Expressão Gênica/efeitos dos fármacos , Proteínas de Homeodomínio/genética , Subunidade p40 da Interleucina-12/genética , Interleucina-17/metabolismo , Interleucinas/metabolismo , Antígenos Comuns de Leucócito/genética , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/metabolismo , Fator de Transcrição STAT1/genética , Proteínas com Domínio T/genética
11.
J Immunol ; 190(10): 5000-11, 2013 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-23606540

RESUMO

Experimental autoimmune encephalomyelitis (EAE) is a rodent model of multiple sclerosis (MS), a debilitating autoimmune disease of the CNS, for which only limited therapeutic interventions are available. Because MS is mediated in part by autoreactive T cells, particularly Th17 and Th1 cells, in the current study, we tested whether inhibitors of glycogen synthase kinase-3 (GSK3), previously reported to reduce Th17 cell generation, also alter Th1 cell production or alleviate EAE. GSK3 inhibitors were found to impede the production of Th1 cells by reducing STAT1 activation. Molecularly reducing the expression of either of the two GSK3 isoforms demonstrated that Th17 cell production was sensitive to reduced levels of GSK3ß and Th1 cell production was inhibited in GSK3α-deficient cells. Administration of the selective GSK3 inhibitors TDZD-8, VP2.51, VP0.7, or L803-mts significantly reduced the clinical symptoms of myelin oligodendrocyte glycoprotein35-55-induced EAE in mice, nearly eliminating the chronic progressive phase, and reduced the number of Th17 and Th1 cells in the spinal cord. Administration of TDZD-8 or L803-mts after the initial disease episode alleviated clinical symptoms in a relapsing-remitting model of proteolipid protein139-151-induced EAE. Furthermore, deletion of GSK3ß specifically in T cells was sufficient to alleviate myelin oligodendrocyte glycoprotein35-55-induced EAE. These results demonstrate the isoform-selective effects of GSK3 on T cell generation and the therapeutic effects of GSK3 inhibitors in EAE, as well as showing that GSK3 inhibition in T cells is sufficient to reduce the severity of EAE, suggesting that GSK3 may be a feasible target for developing new therapeutic interventions for MS.


Assuntos
Encefalomielite Autoimune Experimental/tratamento farmacológico , Quinase 3 da Glicogênio Sintase/antagonistas & inibidores , Células Th1/metabolismo , Células Th17/metabolismo , Animais , Encefalomielite Autoimune Experimental/imunologia , Ativação Enzimática , Feminino , Quinase 3 da Glicogênio Sintase/metabolismo , Glicogênio Sintase Quinase 3 beta , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Esclerose Múltipla , Glicoproteína Mielina-Oligodendrócito , Oligopeptídeos/farmacologia , Fragmentos de Peptídeos , Isoformas de Proteínas/metabolismo , Fator de Transcrição STAT1/metabolismo , Medula Espinal/citologia , Medula Espinal/imunologia , Células Th1/efeitos dos fármacos , Células Th1/imunologia , Células Th17/efeitos dos fármacos , Células Th17/imunologia , Tiadiazóis/farmacologia
12.
Proc Natl Acad Sci U S A ; 109(13): 5004-9, 2012 Mar 27.
Artigo em Inglês | MEDLINE | ID: mdl-22411837

RESUMO

Suppressor of cytokine signaling (SOCS) proteins are feedback inhibitors of the JAK/STAT pathway. SOCS3 has a crucial role in inhibiting STAT3 activation, cytokine signaling, and inflammatory gene expression in macrophages/microglia. To determine the role of SOCS3 in myeloid cells in neuroinflammation, mice with conditional SOCS3 deletion in myeloid cells (LysMCre-SOCS3(fl/fl)) were tested for experimental autoimmune encephalomyelitis (EAE). The myeloid-specific SOCS3-deficient mice are vulnerable to myelin oligodendrocyte glycoprotein (MOG)-induced EAE, with a severe, nonresolving atypical form of disease. In vivo, enhanced infiltration of inflammatory cells and demyelination is prominent in the cerebellum of myeloid-specific SOCS3-deficient mice, as is enhanced STAT3 signaling and expression of inflammatory cytokines/chemokines and an immune response dominated by Th1 and Th17 cells. In vitro, SOCS3-deficient macrophages exhibit heightened STAT3 activation and are polarized toward the classical M1 phenotype. SOCS3-deficient M1 macrophages provide the microenvironment to polarize Th1 and Th17 cells and induce neuronal death. Furthermore, adoptive transfer of M2 macrophages into myeloid SOCS3-deficient mice leads to delayed onset and reduced severity of atypical EAE by decreasing STAT3 activation, Th1/Th17 cells, and proinflammatory mediators in the cerebellum. These findings indicate that myeloid cell SOCS3 provides protection from EAE through deactivation of neuroinflammatory responses.


Assuntos
Inflamação/patologia , Células Mieloides/metabolismo , Sistema Nervoso/metabolismo , Sistema Nervoso/patologia , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais , Proteínas Supressoras da Sinalização de Citocina/metabolismo , Animais , Contagem de Células , Polaridade Celular , Citoproteção , Encefalomielite Autoimune Experimental/complicações , Encefalomielite Autoimune Experimental/imunologia , Encefalomielite Autoimune Experimental/metabolismo , Encefalomielite Autoimune Experimental/patologia , Deleção de Genes , Inflamação/complicações , Inflamação/imunologia , Inflamação/metabolismo , Integrases/metabolismo , Macrófagos/metabolismo , Macrófagos/patologia , Camundongos , Camundongos Endogâmicos C57BL , Proteínas da Mielina/imunologia , Glicoproteína Mielina-Oligodendrócito , Células Mieloides/imunologia , Sistema Nervoso/imunologia , Neurônios/metabolismo , Neurônios/patologia , Fenótipo , Transdução de Sinais/imunologia , Proteína 3 Supressora da Sinalização de Citocinas , Células Th1/citologia , Células Th1/imunologia , Células Th17/citologia , Células Th17/imunologia
13.
J Immunol ; 187(10): 4998-5006, 2011 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-21984703

RESUMO

Many autoimmune chronic inflammatory diseases, including multiple sclerosis, are associated with the presence of Th1 and Th17 effector CD4 T cells. Paradoxically, the principal Th1 cytokine IFN-γ does not appear necessary for disease, but the key Th1-associated transcription factor Tbet has been reported to be essential for disease development. This conundrum propelled us to investigate the regulation of this transcription factor during autoimmunity. Following the onset of experimental autoimmune encephalomyelitis, we observed a preferential upregulation of Tbet by CD4 T cells within the CNS, but not the secondary lymphoid organs. These Tbet-positive CD4 T cells were capable of producing the cytokine IFN-γ, and a proportion of these cells produced both IFN-γ and IL-17A. Interestingly, these Tbet-positive cells were present in high frequencies during disease in IFN-γ-deficient mice. Moreover, we found that CD4 T cells from IFN-γ-deficient/IFN-γ reporter mice upregulated the Thy1.1 reporter, indicating the presence of Th1 or Th1-like, Tbet-positive CD4 T cells even in the absence of the cardinal Th1 cytokine IFN-γ. These IFN-γ-deficient Th1-like cells not only maintain multiple Th1 properties but also exhibit increased expression of genes associated with the Th17 phenotype. We further examined the requirement of other Th1-associated molecules in controlling Tbet expression during experimental autoimmune encephalomyelitis and noted that STAT1, IL-12, and IFN-γ were dispensable for the induction of Tbet in vivo. Hence, this study highlights the complex regulation of Tbet and the potential unrecognized role for Th1 cells during autoimmunity.


Assuntos
Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/patologia , Diferenciação Celular/imunologia , Citocinas/fisiologia , Encefalomielite Autoimune Experimental/imunologia , Transdução de Sinais/imunologia , Proteínas com Domínio T/biossíntese , Células Th1/imunologia , Animais , Linfócitos T CD4-Positivos/metabolismo , Encefalomielite Autoimune Experimental/metabolismo , Encefalomielite Autoimune Experimental/patologia , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Células Th1/metabolismo , Células Th1/patologia
14.
J Immunol ; 186(3): 1391-8, 2011 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-21191064

RESUMO

CD4(+) T cells are critical for host defense but are also major drivers of immune-mediated diseases. The classical view of Th1 and Th2 subtypes of CD4(+) T cells was recently revised by the identification of the Th17 lineage of CD4(+) T cells that produce IL-17, which have been found to be critical in the pathogenesis of autoimmune and other diseases. Mechanisms controlling the differentiation of Th17 cells have been well described, but few feasible targets for therapeutically reducing Th17 cells are known. The generation of Th17 cells requires IL-6 and activation of STAT3. During polarization of CD4(+) T cells to Th17 cells, we found that inhibition of glycogen synthase kinase-3 (GSK3) blocked IL-6 production, STAT3 activation, and polarization to Th17 cells. Polarization of CD4(+) T cells to Th17 cells increased by 10-fold the expression of GSK3ß protein levels in Th17 cells, whereas GSK3ß was unaltered in regulatory T cells. Diminishing GSK3 activity either pharmacologically or molecularly blocked Th17 cell production, and increasing GSK3 activity promoted polarization to Th17 cells. In vivo inhibition of GSK3 in mice depleted constitutive Th17 cells in intestinal mucosa, blocked Th17 cell generation in the lung after Francisella tularensis infection, and inhibited the increase in spinal cord Th17 cells and disease symptoms in the experimental autoimmune encephalomyelitis mouse model of multiple sclerosis. These findings identify GSK3 as a critical mediator of Th17 cell production and indicate that GSK3 inhibitors provide a potential therapeutic intervention to control Th17-mediated diseases.


Assuntos
Diferenciação Celular/imunologia , Quinase 3 da Glicogênio Sintase/fisiologia , Interleucina-17/biossíntese , Linfócitos T Auxiliares-Indutores/imunologia , Linfócitos T Auxiliares-Indutores/metabolismo , Animais , Células da Medula Óssea/enzimologia , Células da Medula Óssea/imunologia , Células da Medula Óssea/metabolismo , Diferenciação Celular/genética , Polaridade Celular/imunologia , Células Cultivadas , Células Dendríticas/enzimologia , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Encefalomielite Autoimune Experimental/enzimologia , Encefalomielite Autoimune Experimental/imunologia , Encefalomielite Autoimune Experimental/patologia , Feminino , Francisella tularensis/imunologia , Técnicas de Introdução de Genes , Quinase 3 da Glicogênio Sintase/antagonistas & inibidores , Inibidores do Crescimento/antagonistas & inibidores , Inibidores do Crescimento/fisiologia , Interleucina-6/antagonistas & inibidores , Interleucina-6/biossíntese , Interleucina-6/fisiologia , Mucosa Intestinal/enzimologia , Mucosa Intestinal/imunologia , Mucosa Intestinal/patologia , Pulmão/enzimologia , Pulmão/microbiologia , Pulmão/patologia , Camundongos , Camundongos Endogâmicos C57BL , Linfócitos T Auxiliares-Indutores/enzimologia , Tularemia/enzimologia , Tularemia/imunologia , Tularemia/patologia
15.
AIDS Res Ther ; 5: 12, 2008 Jun 10.
Artigo em Inglês | MEDLINE | ID: mdl-18544167

RESUMO

Human T-cell leukemia virus type-1 (HTLV-1) induces adult T-cell leukemia/lymphoma (ATL/L), a fatal lymphoproliferative disorder, and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP), a chronic progressive disease of the central nervous system after a long period of latent infection. Although the mechanism of transformation and leukemogenesis is not fully elucidated, there is evidence to suggest that the viral oncoprotein Tax plays a crucial role in these processes through the regulation of several pathways including NF-kappaB and the cell cycle pathways. The observation that NF-kappaB, which is strongly induced by Tax, is indispensable for the maintenance of the malignant phenotype of HTLV-1 by regulating the expression of various genes involved in cell cycle regulation and inhibition of apoptosis provides a possible molecular target for these infected cells. To develop potential new therapeutic strategies for HTLV-1 infected cells, in this present study, we initially screened a battery of NF-kappaB and CDK inhibitors (total of 35 compounds) to examine their effects on the growth and survival of infected T-cell lines. Two drugs namely BMS-345541 and Purvalanol A exhibited higher levels of growth inhibition and apoptosis in infected cell as compared to uninfected cells. BMS-345541 inhibited IKKbeta kinase activity from HTLV-1 infected cells with an IC50 (the 50% of inhibitory concentration) value of 50 nM compared to 500 nM from control cells as measured by in vitro kinase assays. The effects of Purvalanol A were associated with suppression of CDK2/cyclin E complex activity as previously shown by us. Combination of both BMS-345541 and Purvalanol A showed a reduced level of HTLV-1 p19 Gag production in cell culture. The apparent apoptosis in these infected cells were associated with increased caspase-3 activity and PARP cleavage. The potent and selective apoptotic effects of these drugs suggest that both BMS-345541 and Purvalanol A, which target both NF-kappaB and CDK complex and the G1/S border, might be promising new agents in the treatment of these infected patients.

16.
Ann Surg Oncol ; 14(8): 2250-6, 2007 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-17520317

RESUMO

PURPOSE: Heme oxygenase-1 (HO-1) gene, which encodes an oxidative response protein, plays a role in cytoprotection. A (GT)n dinucleotide repeat in HO-1 promoter is polymorphic and modulates the transcriptional activity of the gene. A HO-1 gene promoter polymorphism was reported to be associated with the risks of lung adenocarcinoma and oral squamous cancer. In this study, the correlation between the HO-1 gene promoter polymorphism and the clinicopathological characteristics, along with the risk of gastric cancer, was analyzed. EXPERIMENTAL DESIGN: We examined the genotypic frequencies of (GT)n repeats in 183 gastric cancer patients and 250 control subjects by PCR-based genotyping and DNA sequencing. The length polymorphisms of (GT)n repeats were classified into short (S) component (n or= 31). The distribution of S, M and L components in patient and control groups were evaluated to determine the correlation with susceptibility and clinicopathological characteristics of gastric adenocarcinoma. RESULTS: Higher frequencies of L-allele, L-allele carrier (S/L, M/L, L/L) and S/L genotype were found in gastric cancer patients. The frequencies of M-allele, M-allele carrier (M/M, M/L, M/S) and M/M genotype were significantly lower in patients with gastric cancer than controls. Furthermore, the frequency of lymphovascular tumor invasion was significantly lower in M-allele carriers compared to non-M-allele carriers (S/S, S/L, L/L) (p = 0.009). CONCLUSIONS: These findings suggest that the long (GT)n repeat of HO-1 gene promoter was associated with a higher frequency of gastric adenocarcinoma, and the medium (GT)n repeat might possess protective effect against gastric adenocarcinoma with a lower frequency of lymphovascular invasion in tumors.


Assuntos
Adenocarcinoma/genética , Heme Oxigenase-1/genética , Polimorfismo Genético , Regiões Promotoras Genéticas , Neoplasias Gástricas/genética , Adenocarcinoma/patologia , Adenocarcinoma/cirurgia , Adulto , Idoso , Alelos , Estudos de Casos e Controles , Intervalos de Confiança , Feminino , Frequência do Gene , Heterozigoto , Humanos , Metástase Linfática , Masculino , Repetições de Microssatélites , Pessoa de Meia-Idade , Invasividade Neoplásica , Razão de Chances , Reação em Cadeia da Polimerase , Estudos Retrospectivos , Fatores de Risco , Análise de Sequência de DNA , Neoplasias Gástricas/patologia , Neoplasias Gástricas/cirurgia
17.
Cancer Lett ; 243(1): 47-54, 2006 Nov 08.
Artigo em Inglês | MEDLINE | ID: mdl-16387424

RESUMO

Areca (betel)-chewing is tightly associated with the high prevalence of oral squamous cell carcinoma (OSCC) in Asians. NFKB1 encodes a 105kDa protein that can be processed to produce p50 subunit of nuclear factor-kappaB protein complex. A insertion (ins)/deletion (del) polymorphism (-94ins/delATTG) in NFKB1 promoter, which may drive the ins allele two-fold increase in NFKB1 transcription relative to del allele, was recently found. This study identified that the odds ratio in OSCC carrying ins allelotype were 1.78 relative to controls (56.7 vs 41.8%) in subjects more than 50 years old. L allelotype of Heme oxygenase-1 (HO-1), accounting for a long (GT)(n) repeat in HO-1 promoter, is associated with the risks of areca-related OSCC. Subjects carried both NFKB1 ins and HO-1 L allelotypes had significant risks for various subsets of OSCC. OSCC with lymph node metastasis or advanced stage had significantly higher frequency of NFKB1 ins and HO-1 L allelotypes. This study suggested that the functional NFKB1promoter polymorphism could be valuable for assessment of cancer risk.


Assuntos
Areca/efeitos adversos , Carcinoma de Células Escamosas/etiologia , Neoplasias Bucais/etiologia , Subunidade p50 de NF-kappa B/genética , Polimorfismo Genético/genética , Regiões Promotoras Genéticas/genética , Adulto , Alelos , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patologia , Repetições de Dinucleotídeos/genética , Frequência do Gene , Genótipo , Heme Oxigenase-1/genética , Humanos , Metástase Linfática , Masculino , Pessoa de Meia-Idade , Neoplasias Bucais/genética , Neoplasias Bucais/patologia , Mutagênese Insercional/genética , Estadiamento de Neoplasias , Razão de Chances , Fatores de Risco , Deleção de Sequência/genética
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