RESUMO
The recognition of microbial patterns by Toll-like receptors (TLRs) is critical for activation of the innate immune system. Although TLRs are expressed by human CD4(+) T cells, their function is not well understood. Here we found that engagement of TLR7 in CD4(+) T cells induced intracellular calcium flux with activation of an anergic gene-expression program dependent on the transcription factor NFATc2, as well as unresponsiveness of T cells. As chronic infection with RNA viruses such as human immunodeficiency virus type 1 (HIV-1) induces profound dysfunction of CD4(+) T cells, we investigated the role of TLR7-induced anergy in HIV-1 infection. Silencing of TLR7 markedly decreased the frequency of HIV-1-infected CD4(+) T cells and restored the responsiveness of those HIV-1(+) CD4(+) T cells. Our results elucidate a previously unknown function for microbial pattern-recognition receptors in the downregulation of immune responses.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Infecções por HIV/imunologia , HIV-1/imunologia , Receptor 7 Toll-Like/imunologia , Linfócitos T CD4-Positivos/virologia , Cálcio/imunologia , Anergia Clonal/imunologia , Regulação para Baixo , Infecções por HIV/virologia , Humanos , Leucócitos Mononucleares/imunologia , Ativação Linfocitária , Fatores de Transcrição NFATC/imunologia , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/genética , Receptor 7 Toll-Like/antagonistas & inibidores , Receptor 7 Toll-Like/genéticaRESUMO
Invariant natural killer T (iNKT) cells promote immune responses to various pathogens, but exactly how iNKT cells control antiviral responses is unclear. Here, we showed that iNKT cells induced tissue-specific antiviral effects in mice infected by lymphocytic choriomeningitis virus (LCMV). Indeed, iNKT cells inhibited viral replication in the pancreas and liver but not in the spleen. In the pancreas, iNKT cells expressed the OX40 molecule and promoted type I interferon (IFN) production by plasmacytoid dendritic cells (pDCs) through OX40-OX40 ligand interaction. Subsequently, this iNKT cell-pDC cooperation attenuated the antiviral adaptive immune response in the pancreas but not in the spleen. The dampening of pancreatic anti-LCMV CD8(+) T cell response prevented tissue damage in transgenic mice expressing LCMV protein in islet beta cells. Thus, this study identifies pDCs as an essential partner of iNKT cells for mounting an efficient, nondeleterious antiviral response in peripheral tissue.
Assuntos
Células Dendríticas/imunologia , Coriomeningite Linfocítica/imunologia , Coriomeningite Linfocítica/virologia , Vírus da Coriomeningite Linfocítica/imunologia , Células T Matadoras Naturais/imunologia , Receptores OX40/imunologia , Animais , Linfócitos T CD8-Positivos/imunologia , Diabetes Mellitus/etiologia , Diabetes Mellitus/imunologia , Diabetes Mellitus/virologia , Fígado/imunologia , Fígado/virologia , Coriomeningite Linfocítica/complicações , Camundongos , Ligante OX40/imunologia , Especificidade de Órgãos/imunologia , Pâncreas/imunologia , Pâncreas/virologia , Transdução de Sinais/imunologia , Baço/imunologia , Baço/virologia , Replicação ViralRESUMO
Foxp3(+) regulatory T cells (Tregs) exhibit plasticity, which dictates their function. Secretion of the inflammatory cytokine IFNγ, together with the acquisition of a T helper 1 (Th1)-like effector phenotype as observed in cancer, infection, and autoimmune diseases, is associated with loss of Treg suppressor function through an unknown mechanism. Here, we describe the signaling events driving the generation of human Th1-Tregs. Using a genome-wide gene expression approach and pathway analysis, we identify the PI3K/AKT/Foxo1/3 signaling cascade as the major pathway involved in IFNγ secretion by human Tregs. Furthermore, we describe the opposing roles of AKT isoforms in Th1-Treg generation ex vivo Finally, we employ multiple sclerosis as an in vivo model with increased but functionally defective Th1-Tregs. We show that the PI3K/AKT/Foxo1/3 pathway is activated in ex vivo-isolated Tregs from untreated relapsing-remitting MS patients and that blockade of the pathway inhibits IFNγ secretion and restores the immune suppressive function of Tregs. These data define a fundamental pathway regulating the function of human Tregs and suggest a novel treatment paradigm for autoimmune diseases.
Assuntos
Doenças Autoimunes/imunologia , Doenças Autoimunes/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismo , Doenças Autoimunes/genética , Biomarcadores , Diferenciação Celular , Citocinas , Fatores de Transcrição Forkhead/metabolismo , Perfilação da Expressão Gênica , Inativação Gênica , Humanos , Imunomodulação , Interferon gama/metabolismo , Fenótipo , Fosfatidilinositol 3-Quinases/metabolismo , Isoformas de Proteínas , Transdução de Sinais , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Linfócitos T Reguladores/citologia , TranscriptomaRESUMO
T-cell immunoglobulin and mucin domain 3 (TIM-3) is an Ig-superfamily member expressed on IFN-γ-secreting Th1 and Tc1 cells and was identified as a negative regulator of immune tolerance. TIM-3 is expressed by a subset of activated CD4(+) T cells, and anti-CD3/anti-CD28 stimulation increases both the level of expression and the number of TIM-3(+) T cells. In mice, TIM-3 is constitutively expressed on natural regulatory T (Treg) cells and has been identified as a regulatory molecule of alloimmunity through its ability to modulate CD4(+) T-cell differentiation. Here, we examined TIM-3 expression on human Treg cells to determine its role in T-cell suppression. In contrast to mice, TIM-3 is not expressed on Treg cells ex vivo but is upregulated after activation. While TIM-3(+) Treg cells with increased gene expression of LAG3, CTLA4, and FOXP3 are highly efficient suppressors of effector T (Teff) cells, TIM-3(-) Treg cells poorly suppressed Th17 cells as compared with their suppression of Th1 cells; this decreased suppression ability was associated with decreased STAT-3 expression and phosphorylation and reduced gene expression of IL10, EBI3, GZMB, PRF1, IL1Rα, and CCR6. Thus, our results suggest that TIM-3 expression on Treg cells identifies a population highly effective in inhibiting pathogenic Th1- and Th17-cell responses.
Assuntos
Fatores de Transcrição Forkhead/imunologia , Regulação da Expressão Gênica/imunologia , Proteínas de Membrana/imunologia , Linfócitos T Reguladores/imunologia , Células Th17/imunologia , Animais , Antígenos CD/imunologia , Antígeno CTLA-4/imunologia , Diferenciação Celular/imunologia , Feminino , Granzimas/imunologia , Receptor Celular 2 do Vírus da Hepatite A , Humanos , Proteína Antagonista do Receptor de Interleucina 1/imunologia , Interleucina-10/imunologia , Interleucinas/imunologia , Masculino , Camundongos , Antígenos de Histocompatibilidade Menor , Receptores CCR6/imunologia , Fator de Transcrição STAT3/imunologia , Linfócitos T Reguladores/citologia , Células Th17/citologia , Proteína do Gene 3 de Ativação de LinfócitosRESUMO
Invariant natural killer T (iNKT) cells are a distinct lineage of innate-like T lymphocytes and converging studies in mouse models have demonstrated the protective role of iNKT cells in the development of type 1 diabetes. Recently, a new subset of iNKT cells, producing high levels of the pro-inflammatory cytokine IL-17, has been identified (iNKT17 cells). Since this cytokine has been implicated in several autoimmune diseases, we have analyzed iNKT17 cell frequency, absolute number and phenotypes in the pancreas and lymphoid organs in non-obese diabetic (NOD) mice. The role of iNKT17 cells in the development of diabetes was investigated using transfer experiments. NOD mice exhibit a higher frequency and absolute number of iNKT17 cells in the lymphoid organs as compared with C57BL/6 mice. iNKT17 cells infiltrate the pancreas of NOD mice where they express IL-17 mRNA. Contrary to the protective role of CD4(+) iNKT cells, the CD4(-) iNKT cell population, which contains iNKT17 cells, enhances the incidence of diabetes. Treatment with a blocking anti-IL-17 antibody prevents the exacerbation of the disease. This study reveals that different iNKT cell subsets play distinct roles in the regulation of type 1 diabetes and iNKT17 cells, which are abundant in NOD mice, exacerbate diabetes development.
Assuntos
Diabetes Mellitus Tipo 1/imunologia , Interleucina-17/imunologia , Células T Matadoras Naturais/imunologia , Animais , Doenças Autoimunes/genética , Doenças Autoimunes/imunologia , Doenças Autoimunes/metabolismo , Antígenos CD4/imunologia , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/metabolismo , Feminino , Interleucina-17/biossíntese , Interleucina-17/genética , Ativação Linfocitária/genética , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Células T Matadoras Naturais/metabolismo , Pâncreas/imunologia , Pâncreas/metabolismo , Fenótipo , RNA Mensageiro/genéticaRESUMO
Invariant NKT cells are CD1d-restricted T cells specific for glycolipid Ags. Their activation or transgenic enrichment abrogates the development of experimental autoimmune encephalomyelitis (EAE). Herein, we demonstrate that in NKT-enriched mice the protection from EAE is associated with the infiltration of NKT cells in the CNS and the local expression of CD1d. This indicates that the CNS acquires the potential for local glycolipid presentation when exposed to inflammatory stress, permitting the triggering of NKT cells. To address the importance of CD1d-mediated Ag presentation, we used transgenic mice that express CD1d solely in the thymus. Interestingly, enrichment of NKT cells in these mice also conferred resistance to EAE, with an efficacy indistinguishable from that of NKT-enriched CD1d-sufficient mice. This protection was due to an abrogation of the encephalitogenic Th1 and Th17 response in the spleen, revealing that endogenous glycolipid presentation is dispensable for the regulatory function of NKT cells in EAE. Moreover, abrogating extrathymic CD1d expression failed to affect both the recruitment of NKT cells and their effector phenotype. CNS-infiltrating NKT cells were characterized by a cytotoxic IFN-gamma(high)IL-4(low)IL-10(low)granzyme B(high) profile, irrespective of the local expression of CD1d. Glycolipid Ag presentation is therefore dispensable for the control of autoimmune demyelination by NKT cells, underlining the importance of alternative cognate and/or soluble factors in the control of NKT cell function.
Assuntos
Antígenos CD1/fisiologia , Movimento Celular/imunologia , Encefalomielite Autoimune Experimental/imunologia , Encefalomielite Autoimune Experimental/prevenção & controle , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/patologia , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/patologia , Sequência de Aminoácidos , Animais , Antígenos CD1/biossíntese , Antígenos CD1/genética , Antígenos CD1d , Técnicas de Cocultura , Modelos Animais de Doenças , Encefalomielite Autoimune Experimental/patologia , Humanos , Células Matadoras Naturais/metabolismo , Camundongos , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos Transgênicos , Dados de Sequência Molecular , Subpopulações de Linfócitos T/metabolismoRESUMO
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMO
Human blood CD14+ monocytes are bone marrow-derived white blood cells that sense and respond to pathogens. Although innate immune activation by RNA viruses preferentially occurs through intracellular RIG-I-like receptors, other nucleic acid recognition receptors, such as Toll-like receptors (TLRs), play a role in finely programming the final outcome of virus infection. Here, we dissected how human monocytes respond to infection with either Coxsackie (CV), encephalomyocarditis (EMCV), influenza A (IAV), measles (MV), Sendai (SV), or vesicular stomatitis (VSV) virus. We found that in monocytes, type I interferon (IFN) and cytokine responses to infection were RNA virus specific and differentially involved TLR7 and TLR8, which sense single-stranded RNA. These TLRs activated distinct signaling cascades in monocytes, which correlated with differences in the production of cytokines involved in the polarization of CD4+ T helper cells. Furthermore, we found that TLR7 signaling specifically increased expression of the transcription factor FOSL1, which reduced IL-27 and TNFα production by monocytes. TLR7, but not TLR8, activation of monocytes also stimulated Ca2+ flux that prevented type I IFN responses. Our work demonstrates that in human monocytes, TLR7 and TLR8 triggered different signaling pathways that contribute to distinct phenotypes during RNA virus infection. In addition, we defined individual targets within these pathways that promoted specific T helper and antiviral responses.
Assuntos
Monócitos/imunologia , Vírus de RNA/imunologia , Transdução de Sinais/imunologia , Receptor 7 Toll-Like/imunologia , Receptor 8 Toll-Like/imunologia , Cálcio/imunologia , Cálcio/metabolismo , Células Cultivadas , Citocinas/genética , Citocinas/imunologia , Citocinas/metabolismo , Regulação da Expressão Gênica/imunologia , Interações Hospedeiro-Patógeno/imunologia , Humanos , Interferon Tipo I/genética , Interferon Tipo I/imunologia , Interferon Tipo I/metabolismo , Monócitos/metabolismo , Monócitos/virologia , Proteínas Proto-Oncogênicas c-fos/genética , Proteínas Proto-Oncogênicas c-fos/imunologia , Proteínas Proto-Oncogênicas c-fos/metabolismo , Interferência de RNA , Vírus de RNA/classificação , Vírus de RNA/fisiologia , Transdução de Sinais/genética , Receptor 7 Toll-Like/genética , Receptor 7 Toll-Like/metabolismo , Receptor 8 Toll-Like/genética , Receptor 8 Toll-Like/metabolismoRESUMO
Endowing chimeric antigen receptor (CAR) T cells with additional potent functionalities holds strong potential for improving their antitumor activity. However, because potency could be deleterious without control, these additional features need to be tightly regulated. Immune pathways offer a wide array of tightly regulated genes that can be repurposed to express potent functionalities in a highly controlled manner. Here, we explore this concept by repurposing TCR, CD25 and PD1, three major players of the T cell activation pathway. We insert the CAR into the TCRα gene (TRACCAR), and IL-12P70 into either IL2Rα or PDCD1 genes. This process results in transient, antigen concentration-dependent IL-12P70 secretion, increases TRACCAR T cell cytotoxicity and extends survival of tumor-bearing mice. This gene network repurposing strategy can be extended to other cellular pathways, thus paving the way for generating smart CAR T cells able to integrate biological inputs and to translate them into therapeutic outputs in a highly regulated manner.
Assuntos
Sistema Imunitário/metabolismo , Receptores de Antígenos Quiméricos/imunologia , Linfócitos T/imunologia , Animais , Linhagem Celular Tumoral , Edição de Genes , Humanos , Interleucina-12/genética , Ativação Linfocitária/imunologia , Camundongos , Neoplasias/imunologia , Neoplasias/patologia , Receptores de Antígenos de Linfócitos T/metabolismo , Nucleases dos Efetores Semelhantes a Ativadores de Transcrição/metabolismoRESUMO
Using a TALEN-mediated gene-editing approach, we have previously described a process for the large-scale manufacturing of "off-the-shelf" CAR T cells from third-party donor T cells by disrupting the gene encoding TCRα constant chain (TRAC). Taking advantage of a previously described strategy to control TALEN targeting based on the exclusion capacities of non-conventional RVDs, we have developed highly efficient and specific nucleases targeting a key T cell immune checkpoint, PD-1, to improve engineered CAR T cells' functionalities. Here, we demonstrate that this approach allows combined TRAC and PDCD1 TALEN processing at the desired locus while eliminating low-frequency off-site processing. Thus, by replacing few RVDs, we provide here an easy and rapid redesign of optimal TALEN combinations. We anticipate that this method can greatly benefit multiplex editing, which is of key importance especially for therapeutic applications where high editing efficiencies need to be associated with maximal specificity and safety.
RESUMO
NKT cells are innate-like αß T cells that are conserved between humans and mice. They are distinct from conventional T cells as they recognize lipid antigens presented by the CD1d molecule. Most NKT cells expressed a highly restricted TCR repertoire and can be activated by α-galactosylceramide (α-GalCer) and detected by α-GalCer-loaded-CD1d tetramers. Upon activation, NKT cells respond in few hours by producing cytokines and stimulating many other cells of the innate and adaptive immune system. Over the last decade, many studies have analyzed the regulatory role of NKT cells that can either suppress or exacerbate immune functions. This chapter describes the tools and techniques required to study in vivo and in vitro the regulatory role of NKT cells in mouse as well as from human blood.