RESUMO
Antiviral CD8+ T cell responses are characterized by an initial activation/priming of T lymphocytes followed by a massive proliferation, subset differentiation, population contraction and the development of a stable memory pool. The transcription factor BATF3 has been shown to play a central role in the development of conventional dendritic cells, which in turn are critical for optimal priming of CD8+ T cells. Here we show that BATF3 was expressed transiently within the first days after T cell priming and had long-lasting T cell-intrinsic effects. T cells that lacked Batf3 showed normal expansion and differentiation, yet succumbed to an aggravated contraction and had a diminished memory response. Vice versa, BATF3 overexpression in CD8+ T cells promoted their survival and transition to memory. Mechanistically, BATF3 regulated T cell apoptosis and longevity via the proapoptotic factor BIM. By programing CD8+ T cell survival and memory, BATF3 is a promising molecule to optimize adoptive T cell therapy in patients.
Assuntos
Fatores de Transcrição de Zíper de Leucina Básica/genética , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Reprogramação Celular/genética , Memória Imunológica/genética , Proteínas Repressoras/genética , Animais , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Diferenciação Celular , Sobrevivência Celular/genética , Expressão Gênica , Humanos , Imunofenotipagem , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Proteínas Repressoras/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Store-operated Ca2+ entry (SOCE) is the main Ca2+ influx pathway in lymphocytes and is essential for T cell function and adaptive immunity. SOCE is mediated by Ca2+ release-activated Ca2+ (CRAC) channels that are activated by stromal interaction molecule (STIM) 1 and STIM2. SOCE regulates many Ca2+-dependent signaling molecules, including calcineurin, and inhibition of SOCE or calcineurin impairs antigen-dependent T cell proliferation. We here report that SOCE and calcineurin regulate cell cycle entry of quiescent T cells by controlling glycolysis and oxidative phosphorylation. SOCE directs the metabolic reprogramming of naive T cells by regulating the expression of glucose transporters, glycolytic enzymes, and metabolic regulators through the activation of nuclear factor of activated T cells (NFAT) and the PI3K-AKT kinase-mTOR nutrient-sensing pathway. We propose that SOCE controls a critical "metabolic checkpoint" at which T cells assess adequate nutrient supply to support clonal expansion and adaptive immune responses.
Assuntos
Canais de Cálcio/imunologia , Sinalização do Cálcio/imunologia , Cálcio/imunologia , Linfócitos T/imunologia , Animais , Calcineurina/imunologia , Calcineurina/metabolismo , Cálcio/metabolismo , Canais de Cálcio/metabolismo , Divisão Celular/imunologia , Células Cultivadas , Feminino , Glicólise/imunologia , Células HEK293 , Humanos , Immunoblotting , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Microscopia Confocal , Fatores de Transcrição NFATC/genética , Fatores de Transcrição NFATC/imunologia , Fatores de Transcrição NFATC/metabolismo , Fosfatidilinositol 3-Quinases/imunologia , Fosfatidilinositol 3-Quinases/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/imunologia , Molécula 1 de Interação Estromal/genética , Molécula 1 de Interação Estromal/imunologia , Molécula 1 de Interação Estromal/metabolismo , Molécula 2 de Interação Estromal/genética , Molécula 2 de Interação Estromal/imunologia , Molécula 2 de Interação Estromal/metabolismo , Linfócitos T/metabolismoRESUMO
T follicular helper (Tfh) cells promote affinity maturation of B cells in germinal centers (GCs), whereas T follicular regulatory (Tfr) cells limit the GC reaction. Store-operated Ca(2+) entry (SOCE) through Ca(2+) release-activated Ca(2+) (CRAC) channels mediated by STIM and ORAI proteins is a fundamental signaling pathway in T lymphocytes. Conditional deletion of Stim1 and Stim2 genes in T cells abolished SOCE and strongly reduced antibody-mediated immune responses following viral infection caused by impaired differentiation and function of Tfh cells. Conversely, aging Stim1Stim2-deficient mice developed humoral autoimmunity with spontaneous autoantibody production due to abolished Tfr cell differentiation in the presence of residual Tfh cells. Mechanistically, SOCE controlled Tfr and Tfh cell differentiation through NFAT-mediated IRF4, BATF, and Bcl-6 transcription-factor expression. SOCE had a dual role in controlling the GC reaction by regulating both Tfh and Tfr cell differentiation, thus enabling protective B cell responses and preventing humoral autoimmunity.
Assuntos
Autoimunidade , Linfócitos B/imunologia , Centro Germinativo/imunologia , Imunidade Humoral , Molécula 1 de Interação Estromal/metabolismo , Molécula 2 de Interação Estromal/metabolismo , Linfócitos T/imunologia , Animais , Fatores de Transcrição de Zíper de Leucina Básica/genética , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Canais de Cálcio Ativados pela Liberação de Cálcio/metabolismo , Sinalização do Cálcio , Células Cultivadas , Fatores Reguladores de Interferon/genética , Fatores Reguladores de Interferon/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fatores de Transcrição NFATC/metabolismo , Proteína ORAI1/metabolismo , Proteínas Proto-Oncogênicas c-bcl-6/genética , Proteínas Proto-Oncogênicas c-bcl-6/metabolismo , Molécula 1 de Interação Estromal/genética , Molécula 2 de Interação Estromal/genéticaRESUMO
Whereas phosphorylation of serine, threonine, and tyrosine is exceedingly well characterized, the role of histidine phosphorylation in mammalian signaling is largely unexplored. Here we show that phosphoglycerate mutase family 5 (PGAM5) functions as a phosphohistidine phosphatase that specifically associates with and dephosphorylates the catalytic histidine on nucleoside diphosphate kinase B (NDPK-B). By dephosphorylating NDPK-B, PGAM5 negatively regulates CD4(+) T cells by inhibiting NDPK-B-mediated histidine phosphorylation and activation of the K(+) channel KCa3.1, which is required for TCR-stimulated Ca(2+) influx and cytokine production. Using recently developed monoclonal antibodies that specifically recognize phosphorylation of nitrogens at the N1 (1-pHis) or N3 (3-pHis) positions of the imidazole ring, we detect for the first time phosphoisoform-specific regulation of histidine-phosphorylated proteins in vivo, and we link these modifications to TCR signaling. These results represent an important step forward in studying the role of histidine phosphorylation in mammalian biology and disease.
Assuntos
Linfócitos T CD4-Positivos/enzimologia , Ativação Linfocitária , Proteínas Mitocondriais/metabolismo , Fosfoproteínas Fosfatases/metabolismo , Animais , Linfócitos T CD4-Positivos/imunologia , Sinalização do Cálcio , Citocinas/metabolismo , Predisposição Genética para Doença , Doença Enxerto-Hospedeiro/enzimologia , Doença Enxerto-Hospedeiro/genética , Doença Enxerto-Hospedeiro/imunologia , Células HEK293 , Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Histidina , Humanos , Mediadores da Inflamação/metabolismo , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/metabolismo , Células Jurkat , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Mitocondriais/genética , Núcleosídeo-Difosfato Quinase/metabolismo , Fenótipo , Fosfoproteínas Fosfatases/deficiência , Fosfoproteínas Fosfatases/genética , Fosforilação , Interferência de RNA , Receptores de Antígenos de Linfócitos T/metabolismo , Fatores de Tempo , TransfecçãoRESUMO
Calcium (Ca2+) signals play fundamental roles in immune cell function. The main sources of Ca2+ influx in mammalian lymphocytes following antigen receptor stimulation are Ca2+ release-activated Ca2+ (CRAC) channels. These are formed by ORAI proteins in the plasma membrane and are activated by stromal interaction molecules (STIM) located in the endoplasmic reticulum (ER). Human loss-of-function (LOF) mutations in ORAI1 and STIM1 that abolish Ca2+ influx cause a unique disease syndrome called CRAC channelopathy that is characterized by immunodeficiency autoimmunity and non-immunological symptoms. Studies in mice lacking Stim and Orai genes have illuminated many cellular and molecular mechanisms by which these molecules control lymphocyte function. CRAC channels are required for the differentiation and function of several T lymphocyte subsets that provide immunity to infection, mediate inflammation and prevent autoimmunity. This review examines new insights into how CRAC channels control T cell-mediated immunity.
Assuntos
Canais de Cálcio Ativados pela Liberação de Cálcio , Sinalização do Cálcio , Linfócitos T , Animais , Canais de Cálcio Ativados pela Liberação de Cálcio/genética , Canais de Cálcio Ativados pela Liberação de Cálcio/imunologia , Sinalização do Cálcio/imunologia , Humanos , Imunidade Celular/genética , Imunidade Celular/imunologia , Proteína ORAI1/genética , Proteína ORAI1/imunologia , Molécula 1 de Interação Estromal/genética , Molécula 1 de Interação Estromal/imunologia , Linfócitos T/imunologiaRESUMO
By virtue of mitochondrial control of energy production, reactive oxygen species (ROS) generation, and maintenance of Ca2+ homeostasis, mitochondria play an essential role in modulating T cell function. The mitochondrial Ca2+ uniporter (MCU) is the pore-forming unit in the main protein complex mediating mitochondrial Ca2+ uptake. Recently, MCU has been shown to modulate Ca2+ signals at subcellular organellar interfaces, thus fine-tuning NFAT translocation and T cell activation. The mechanisms underlying this modulation and whether MCU has additional T cell subpopulation-specific effects remain elusive. However, mice with germline or tissue-specific ablation of Mcu did not show impaired T cell responses in vitro or in vivo, indicating that 'chronic' loss of MCU can be functionally compensated in lymphocytes. The current work aimed to specifically investigate whether and how MCU influences the suppressive potential of regulatory CD4 T cells (Treg). We show that, in contrast to genetic ablation, acute siRNA-mediated downregulation of Mcu in murine Tregs results in a significant reduction both in mitochondrial Ca2+ uptake and in the suppressive capacity of Tregs, while the ratios of Treg subpopulations and the expression of hallmark transcription factors were not affected. These findings suggest that permanent genetic inactivation of MCU may result in compensatory adaptive mechanisms, masking the effects on the suppressive capacity of Tregs.
Assuntos
Canais de Cálcio , Linfócitos T Reguladores , Animais , Camundongos , Cálcio/metabolismo , Canais de Cálcio/metabolismo , Linfócitos T CD4-Positivos/metabolismo , Regulação para Baixo , Mitocôndrias/genética , Mitocôndrias/metabolismo , Linfócitos T Reguladores/metabolismoRESUMO
Developing precise and efficient gene editing approaches using CRISPR in primary human T cell subsets would provide an effective tool in decoding their functions. Toward this goal, we used lentiviral CRISPR/Cas9 systems to transduce primary human T cells to stably express the Cas9 gene and guide RNAs that targeted either coding or noncoding regions of genes of interest. We showed that multiple genes (CD4, CD45, CD95) could be simultaneously and stably deleted in naive, memory, effector, or regulatory T cell (Treg) subsets at very high efficiency. Additionally, nuclease-deficient Cas9, associated with a transcriptional activator or repressor, can downregulate or increase expression of genes in T cells. For example, expression of glycoprotein A repetitions predominant (GARP), a gene that is normally and exclusively expressed on activated Tregs, could be induced on non-Treg effector T cells by nuclease-deficient Cas9 fused to transcriptional activators. Further analysis determined that this approach could be used in mapping promoter sequences involved in gene transcription. Through this CRISPR/Cas9-mediated genetic editing we also demonstrated the feasibility of human T cell functional analysis in several examples: 1) CD95 deletion inhibited T cell apoptosis upon reactivation; 2) deletion of ORAI1, a Ca2+ release-activated channel, abolished Ca2+ influx and cytokine secretion, mimicking natural genetic mutations in immune-deficient patients; and 3) transcriptional activation of CD25 or CD127 expression enhanced cytokine signaling by IL-2 or IL-7, respectively. Taken together, application of the CRISPR toolbox to human T cell subsets has important implications for decoding the mechanisms of their functional outputs.
Assuntos
Sistemas CRISPR-Cas , Edição de Genes/métodos , Linfócitos T Reguladores/imunologia , Feminino , Humanos , MasculinoRESUMO
BACKGROUND: Store-operated Ca2+ entry (SOCE) through Ca2+ release-activated Ca2+ channels is an essential signaling pathway in many cell types. Ca2+ release-activated Ca2+ channels are formed by ORAI1, ORAI2, and ORAI3 proteins and activated by stromal interaction molecule (STIM) 1 and STIM2. Mutations in the ORAI1 and STIM1 genes that abolish SOCE cause a combined immunodeficiency (CID) syndrome that is accompanied by autoimmunity and nonimmunologic symptoms. OBJECTIVE: We performed molecular and immunologic analysis of patients with CID, anhidrosis, and ectodermal dysplasia of unknown etiology. METHODS: We performed DNA sequencing of the ORAI1 gene, modeling of mutations on ORAI1 crystal structure, analysis of ORAI1 mRNA and protein expression, SOCE measurements, immunologic analysis of peripheral blood lymphocyte populations by using flow cytometry, and histologic and ultrastructural analysis of patient tissues. RESULTS: We identified 3 novel autosomal recessive mutations in ORAI1 in unrelated kindreds with CID, autoimmunity, ectodermal dysplasia with anhidrosis, and muscular dysplasia. The patients were homozygous for p.V181SfsX8, p.L194P, and p.G98R mutations in the ORAI1 gene that suppressed ORAI1 protein expression and SOCE in the patients' lymphocytes and fibroblasts. In addition to impaired T-cell cytokine production, ORAI1 mutations were associated with strongly reduced numbers of invariant natural killer T and regulatory T (Treg) cells and altered composition of γδ T-cell and natural killer cell subsets. CONCLUSION: ORAI1 null mutations are associated with reduced numbers of invariant natural killer T and Treg cells that likely contribute to the patients' immunodeficiency and autoimmunity. ORAI1-deficient patients have dental enamel defects and anhidrosis, representing a new form of anhidrotic ectodermal dysplasia with immunodeficiency that is distinct from previously reported patients with anhidrotic ectodermal dysplasia with immunodeficiency caused by mutations in the nuclear factor κB signaling pathway (IKBKG and NFKBIA).
Assuntos
Displasia Ectodérmica/genética , Síndromes de Imunodeficiência/genética , Proteína ORAI1/genética , Cálcio/metabolismo , Células Cultivadas , Pré-Escolar , Evolução Fatal , Feminino , Humanos , Lactente , Masculino , Modelos Moleculares , MutaçãoRESUMO
Graft-versus-host disease (GvHD) is a life-threatening immunological complication after allogenic hematopoietic stem cell transplantation (allo-HCT). The intrinsic graft-versus-leukemia (GvL) effect, however, is the desirable curative benefit. Patients with acute GvHD are treated with cyclosporine A (CsA) or tacrolimus (FK506), which not only often causes severe adverse effects, but also interferes with the anticipated GvL. Both drugs inhibit calcineurin, thus at first suppressing activation of the nuclear factor of activated T cells (NFAT). Therefore, we explored the specific contribution of individual NFAT factors in donor T cells in animal models of GvHD and GvL. Ablation of NFAT1, NFAT2, or a combination of both resulted in ameliorated GvHD, due to reduced proliferation, target tissue homing, and impaired effector function of allogenic donor T cells. In contrast, the frequency of Foxp3(+) regulatory T (Treg) cells was increased and NFAT-deficient Tregs were fully protective in GvHD. CD8(+) T-cell recall response and, importantly, the beneficial antitumor activity were largely preserved in NFAT-deficient effector T cells. Thus, specific inhibition of NFAT opens an avenue for an advanced therapy of GvHD maintaining protective GvL.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Doença Enxerto-Hospedeiro/imunologia , Efeito Enxerto vs Leucemia/imunologia , Fatores de Transcrição NFATC/imunologia , Linfócitos T Reguladores/imunologia , Aloenxertos , Animais , Linfócitos T CD8-Positivos/patologia , Inibidores de Calcineurina/farmacologia , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Ciclosporina/farmacologia , Modelos Animais de Doenças , Doença Enxerto-Hospedeiro/genética , Doença Enxerto-Hospedeiro/patologia , Efeito Enxerto vs Leucemia/genética , Transplante de Células-Tronco Hematopoéticas , Camundongos , Camundongos Knockout , Fatores de Transcrição NFATC/genética , Linfócitos T Reguladores/patologia , Tacrolimo/farmacologiaRESUMO
Store-operated Ca(2+) entry (SOCE) through Ca(2+) release-activated Ca(2+) (CRAC) channels is essential for immunity to infection. CRAC channels are formed by ORAI1 proteins in the plasma membrane and activated by stromal interaction molecule (STIM)1 and STIM2 in the endoplasmic reticulum. Mutations in ORAI1 and STIM1 genes that abolish SOCE cause severe immunodeficiency with recurrent infections due to impaired T cell function. SOCE has also been observed in cells of the innate immune system such as macrophages and dendritic cells (DCs) and may provide Ca(2+) signals required for their function. The specific role of SOCE in macrophage and DC function, as well as its contribution to innate immunity, however, is not well defined. We found that nonselective inhibition of Ca(2+) signaling strongly impairs many effector functions of bone marrow-derived macrophages and bone marrow-derived DCs, including phagocytosis, inflammasome activation, and priming of T cells. Surprisingly, however, macrophages and DCs from mice with conditional deletion of Stim1 and Stim2 genes, and therefore complete inhibition of SOCE, showed no major functional defects. Their differentiation, FcR-dependent and -independent phagocytosis, phagolysosome fusion, cytokine production, NLRP3 inflammasome activation, and their ability to present Ags to activate T cells were preserved. Our findings demonstrate that STIM1, STIM2, and SOCE are dispensable for many critical effector functions of macrophages and DCs, which has important implications for CRAC channel inhibition as a therapeutic strategy to suppress pathogenic T cells while not interfering with myeloid cell functions required for innate immunity.
Assuntos
Canais de Cálcio/metabolismo , Sinalização do Cálcio/imunologia , Células Dendríticas/imunologia , Macrófagos/imunologia , Glicoproteínas de Membrana/metabolismo , Animais , Apresentação de Antígeno/genética , Apresentação de Antígeno/imunologia , Proteínas Reguladoras de Apoptose/imunologia , Cálcio/metabolismo , Canais de Cálcio/genética , Proteínas de Ligação ao Cálcio/imunologia , Proteínas de Transporte/imunologia , Diferenciação Celular/imunologia , Células Dendríticas/citologia , Retículo Endoplasmático/metabolismo , Humanos , Imunidade Inata/imunologia , Inflamassomos/imunologia , Ativação Linfocitária/imunologia , Macrófagos/citologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteína 3 que Contém Domínio de Pirina da Família NLR , Proteína ORAI1 , Fagocitose/imunologia , Imunodeficiência Combinada Severa/genética , Molécula 1 de Interação Estromal , Molécula 2 de Interação Estromal , Linfócitos T/imunologiaRESUMO
We previously showed that the T cell activation inhibitor, mitochondrial (Tcaim) is highly expressed in grafts of tolerance-developing transplant recipients and that the encoded protein is localized within mitochondria. In this study, we show that CD11c(+) dendritic cells (DCs), as main producers of TCAIM, downregulate Tcaim expression after LPS stimulation or in vivo alloantigen challenge. LPS-stimulated TCAIM-overexpressing bone marrow-derived DC (BMDCs) have a reduced capacity to induce proliferation of and cytokine expression by cocultured allogeneic T cells; this is not due to diminished upregulation of MHC or costimulatory molecules. Transcriptional profiling also revealed normal LPS-mediated upregulation of the majority of genes involved in TLR signaling. However, TCAIM BMDCs did not induce Il2 mRNA expression upon LPS stimulation in comparison with Control-BMDCs. In addition, TCAIM overexpression abolished LPS-mediated Ca(2+) influx and mitochondrial reactive oxygen species formation. Addition of IL-2 to BMDC-T cell cocultures restored the priming capacity of TCAIM BMDCs for cocultured allogeneic CD8(+) T cells. Furthermore, BMDCs of IL-2-deficient mice showed similarly abolished LPS-induced T cell priming as TCAIM-overexpressing wild type BMDCs. Thus, TCAIM interferes with TLR4 signaling in BMDCs and subsequently impairs their T cell priming capacity, which supports its role for tolerance induction.
Assuntos
Cálcio/metabolismo , Células Dendríticas/imunologia , Interleucina-2/biossíntese , Proteínas Mitocondriais/genética , Linfócitos T/imunologia , Linfócitos T/metabolismo , Receptores Toll-Like/metabolismo , Animais , Antígeno B7-2/metabolismo , Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Análise por Conglomerados , Expressão Gênica , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Antígenos de Histocompatibilidade Classe II/metabolismo , Interleucina-2/genética , Interleucina-2/farmacologia , Lipopolissacarídeos/imunologia , Ativação Linfocitária/efeitos dos fármacos , Ativação Linfocitária/genética , Ativação Linfocitária/imunologia , Masculino , Camundongos , Proteínas Mitocondriais/metabolismo , NF-kappa B/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transplante de Pele , Linfócitos T/efeitos dos fármacos , Transplante HomólogoRESUMO
EAE serves as an animal model for multiple sclerosis and is initiated by autoreactive T cells that infiltrate the CNS. Recognition of myelin-associated Ags within the CNS leads to activation of the transcription factor family NFAT. Here, we demonstrate an essential role for NFAT in disease induction, as the combined lack of NFAT1 (NFATc2) and NFAT2 (NFATc1) completely protected mice. Single deficiency of either NFAT1 or NFAT2 ameliorated the course of EAE, and NFAT2 ablation resulted in an obstructed proinflammatory reaction. However, NFAT1 deficit led to an anti-inflammatory response with nonpathogenic Th17 and Th2 cells concurrently secreting IL-17, IL-4, and IL-10. Both IL-4 and IL-10 contributed to disease protection. In Nfat1(-/-) CD4(+) T cells, the expression of anti-inflammatory lymphokines was mediated by NFAT2, thus directly enabling protective IL expression. Consequently, blocking NFAT in toto may be an option for immunosuppressive therapy. More importantly, selective NFAT1 blockade could represent a safe long-term immunomodulatory treatment approach for multiple sclerosis patients, potentially avoiding the adverse effects of global immunosuppression.
Assuntos
Encefalomielite Autoimune Experimental/prevenção & controle , Fatores de Transcrição NFATC/deficiência , Animais , Linfócitos T CD4-Positivos/imunologia , Citocinas/biossíntese , Encefalomielite Autoimune Experimental/etiologia , Encefalomielite Autoimune Experimental/imunologia , Humanos , Imunomodulação , Interleucina-10/biossíntese , Interleucina-4/biossíntese , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Esclerose Múltipla/imunologia , Esclerose Múltipla/terapia , Glicoproteína Mielina-Oligodendrócito/administração & dosagem , Glicoproteína Mielina-Oligodendrócito/imunologia , Fatores de Transcrição NFATC/antagonistas & inibidores , Fatores de Transcrição NFATC/genética , Fatores de Transcrição NFATC/imunologia , Fragmentos de Peptídeos/administração & dosagem , Fragmentos de Peptídeos/imunologia , Transdução de Sinais/imunologia , Células Th17/imunologia , Células Th2/imunologiaRESUMO
Anergic T cells can survive for long time periods passively in a hyporesponsive state without obvious active functions. Thus, the immunological reason for their maintenance is unclear. Here, we induced peptide-specific anergy in T cells from mice by coculturing these cells with immature murine dendritic cells (DCs). We found that these anergic, nonsuppressive IL-10(-) Foxp3(-) CTLA-4(+) CD25(low) Egr2(+) T cells could be converted into suppressive IL-10(+) Foxp3(-) CTLA-4(+) CD25(high) Egr2(+) cells resembling type-1 Treg cells (Tr1) when stimulated a second time by immature DCs in vitro. Addition of TGF-ß during anergy induction favored Foxp3(+) Treg-cell induction, while TGF-ß had little effect when added to the second stimulation. Expression of both CD28 and CTLA-4 molecules on anergic T cells was required to allow their conversion into Tr1-like cells. Suppressor activity was enabled via CD28-mediated CD25 upregulation, acting as an IL-2 sink, together with a CTLA-4-mediated inhibition of NFATc1/α activation to shut down IL-2-mediated proliferation. Together, these data provide evidence and mechanistical insights into how persistent anergic T cells may serve as a resting memory pool for Tr1-like cells.
Assuntos
Antígenos CD28/genética , Antígeno CTLA-4/genética , Anergia Clonal , Células Dendríticas/imunologia , Fatores de Transcrição Forkhead/genética , Interleucina-10/genética , Linfócitos T Reguladores/imunologia , Animais , Antígenos CD28/imunologia , Antígeno CTLA-4/imunologia , Comunicação Celular , Diferenciação Celular , Técnicas de Cocultura , Células Dendríticas/citologia , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/metabolismo , Proteína 2 de Resposta de Crescimento Precoce/genética , Proteína 2 de Resposta de Crescimento Precoce/imunologia , Fatores de Transcrição Forkhead/imunologia , Regulação da Expressão Gênica/imunologia , Interleucina-10/imunologia , Interleucina-2/genética , Interleucina-2/imunologia , Subunidade alfa de Receptor de Interleucina-2/genética , Subunidade alfa de Receptor de Interleucina-2/imunologia , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Fatores de Transcrição NFATC/genética , Fatores de Transcrição NFATC/imunologia , Transdução de Sinais , Linfócitos T Reguladores/citologia , Linfócitos T Reguladores/efeitos dos fármacos , Linfócitos T Reguladores/metabolismo , Fator de Crescimento Transformador beta/farmacologiaRESUMO
NFAT transcription factors control the proliferation and survival of peripheral lymphocytes. We have reported previously that the short isoform NFATc1/αA whose generation is induced by immune receptor stimulation supports the proliferation and inhibits the activation-induced cell death of peripheral T and B cells. We will show in this study that in novel bacterial artificial chromosome transgenic mice that express EGFP under the control of entire Nfatc1 locus the Nfatc1/Egfp transgene is expressed as early as in double-negative thymocytes and in nonstimulated peripheral T and B cells. Upon immune receptor stimulation, Nfatc1/Egfp expression is elevated in B, Th1, and Th2 cells, but only weakly in T regulatory, Th9, and Th17 cells in vitro whose generation is affected by TGFß. In naive lymphocytes, persistent immune receptor signals led to a 3-5 increase in NFATc1/αA RNA levels during primary and secondary stimulation, but a much stronger induction was observed at the protein level. Whereas anti-CD3(+)CD28 stimulation of primary T cells induces both NFATc1/αA and their proliferation and survival, anti-IgM stimulation of B cells induces NFATc1/αA and proliferation, but activation-induced cell death after 3-d incubation in vitro. The anti-IgM-mediated activation-induced cell death induction of B cells in vitro is suppressed by anti-CD40-, LPS-, and CpG-mediated signals. In addition to inducing NF-κB factors, together with anti-IgM, these signals also support the generation of NFATc1/αA. According to these data and the architecture of its promoter region, the Nfatc1 gene resembles a primary response gene whose induction is affected at the posttranscriptional level.
Assuntos
Linfócitos B/efeitos dos fármacos , Fatores de Transcrição NFATC/genética , Células Th1/efeitos dos fármacos , Células Th2/efeitos dos fármacos , Animais , Anticorpos/farmacologia , Linfócitos B/imunologia , Linfócitos B/metabolismo , Proliferação de Células/efeitos dos fármacos , Cromossomos Artificiais Bacterianos/genética , Expressão Gênica/efeitos dos fármacos , Expressão Gênica/imunologia , Genes Reporter , Proteínas de Fluorescência Verde , Lipopolissacarídeos/farmacologia , Ativação Linfocitária/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , NF-kappa B/genética , NF-kappa B/imunologia , Fatores de Transcrição NFATC/agonistas , Fatores de Transcrição NFATC/imunologia , Regiões Promotoras Genéticas , Isoformas de Proteínas/agonistas , Isoformas de Proteínas/genética , Isoformas de Proteínas/imunologia , Linfócitos T Reguladores/efeitos dos fármacos , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismo , Células Th1/imunologia , Células Th1/metabolismo , Células Th17/efeitos dos fármacos , Células Th17/imunologia , Células Th17/metabolismo , Células Th2/imunologia , Células Th2/metabolismo , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/imunologiaRESUMO
The transcriptional co-activator BOB.1/OBF.1 was originally identified in B cells and is constitutively expressed throughout B cell development. BOB.1/OBF.1 associates with the transcription factors Oct1 and Oct2, thereby enhancing octamer-dependent transcription. In contrast, in T cells, BOB.1/OBF.1 expression is inducible by treatment of cells with PMA/Ionomycin or by antigen receptor engagement, indicating a marked difference in the regulation of BOB.1/OBF.1 expression in B versus T cells. The molecular mechanisms underlying the differential expression of BOB.1/OBF.1 in T and B cells remain largely unknown. Therefore, the present study focuses on mechanisms controlling the transcriptional regulation of BOB.1/OBF.1 and Oct2 in T cells. We show that both calcineurin- and NF-κB-inhibitors efficiently attenuate the expression of BOB.1/OBF.1 and Oct2 in T cells. In silico analyses of the BOB.1/OBF.1 promoter revealed the presence of previously unappreciated combined NFAT/NF-κB sites. An array of genetic and biochemical analyses illustrates the involvement of the Ca(2+)/calmodulin-dependent phosphatase calcineurin as well as NFAT and NF-κB transcription factors in the transcriptional regulation of octamer-dependent transcription in T cells. Conclusively, impaired expression of BOB.1/OBF.1 and Oct2 and therefore a hampered octamer-dependent transcription may participate in T cell-mediated immunodeficiency caused by the deletion of NFAT or NF-κB transcription factors.
Assuntos
NF-kappa B/metabolismo , Fatores de Transcrição NFATC/metabolismo , Fator 2 de Transcrição de Octâmero/genética , Linfócitos T/metabolismo , Transativadores/genética , Animais , Sítios de Ligação , Células Cultivadas , Humanos , Células Jurkat , Camundongos , Camundongos Endogâmicos C57BL , NF-kappa B/antagonistas & inibidores , Fatores de Transcrição NFATC/antagonistas & inibidores , Fator 2 de Transcrição de Octâmero/biossíntese , Regiões Promotoras Genéticas , Transativadores/biossíntese , Transcrição GênicaRESUMO
Several lines of evidence suggest nuclear factor of activated T-cells (NFAT) to control regulatory T cells: thymus-derived naturally occurring regulatory T cells (nTreg) depend on calcium signals, the Foxp3 gene harbors several NFAT binding sites, and the Foxp3 (Fork head box P3) protein interacts with NFAT. Therefore, we investigated the impact of NFAT on Foxp3 expression. Indeed, the generation of peripherally induced Treg (iTreg) by TGF-ß was highly dependent on NFAT expression because the ability of CD4(+) T cells to differentiate into iTreg diminished markedly with the number of NFAT family members missing. It can be concluded that the expression of Foxp3 in TGF-ß-induced iTreg depends on the threshold value of NFAT rather than on an individual member present. This is specific for iTreg development, because frequency of nTreg remained unaltered in mice lacking NFAT1, NFAT2, or NFAT4 alone or in combination. Different from expectation, however, the function of both nTreg and iTreg was independent on robust NFAT levels, reflected by less nuclear NFAT in nTreg and iTreg. Accordingly, absence of one or two NFAT members did not alter suppressor activity in vitro or during colitis and transplantation in vivo. This scenario emphasizes an inhibition of high NFAT activity as treatment for autoimmune diseases and in transplantation, selectively targeting the proinflammatory conventional T cells, while keeping Treg functional.
Assuntos
Doenças Autoimunes/imunologia , Fatores de Transcrição Forkhead/metabolismo , Fatores de Transcrição NFATC/metabolismo , Linfócitos T Reguladores/metabolismo , Linfócitos T/metabolismo , Transferência Adotiva , Animais , Imunoprecipitação da Cromatina , Colite/imunologia , Ciclosporina , Citometria de Fluxo , Imunofluorescência , Proteínas de Homeodomínio/genética , Humanos , Immunoblotting , Ativação Linfocitária/imunologia , Camundongos , Fatores de Transcrição NFATC/imunologia , Linfócitos T/imunologia , Linfócitos T Reguladores/imunologia , Fator de Crescimento Transformador betaRESUMO
The main molecular mechanism of human regulatory T cell (Treg)-mediated suppression has not been elucidated. We show in this study that cAMP represents a key regulator of human Treg function. Repression of cAMP production by inhibition of adenylate cyclase activity or augmentation of cAMP degradation through ectopic expression of a cAMP-degrading phosphodiesterase greatly reduces the suppressive activity of human Treg in vitro and in a humanized mouse model in vivo. Notably, cAMP repression additionally abrogates the anergic state of human Treg, accompanied by nuclear translocation of NFATc1 and induction of its short isoform NFATc1/αA. Treg expanded under cAMP repression, however, do not convert into effector T cells and regain their anergic state and suppressive activity upon proliferation. Together, these findings reveal the cAMP pathway as an attractive target for clinical intervention with Treg function.
Assuntos
Anergia Clonal , AMP Cíclico/metabolismo , Linfócitos T Reguladores/imunologia , Animais , Proliferação de Células , Humanos , Camundongos , Fatores de Transcrição NFATC/metabolismo , Linfócitos T Reguladores/citologia , Regulação para CimaRESUMO
Inducible cAMP early repressor (ICER) is a transcriptional repressor, which, because of alternate promoter use, is generated from the 3' region of the cAMP response modulator (Crem) gene. Its expression and nuclear occurrence are elevated by high cAMP levels in naturally occurring regulatory T cells (nTregs). Using two mouse models, we demonstrate that nTregs control the cellular localization of ICER/CREM, and thereby inhibit IL-2 synthesis in conventional CD4(+) T cells. Ablation of nTregs in depletion of regulatory T-cell (DEREG) mice resulted in cytosolic localization of ICER/CREM and increased IL-2 synthesis upon stimulation. Direct contacts between nTregs and conventional CD4(+) T cells led to nuclear accumulation of ICER/CREM and suppression of IL-2 synthesis on administration of CD28 superagonistic (CD28SA) Ab. In a similar way, nTregs communicated with B cells and induced the cAMP-driven nuclear localization of ICER/CREM. High levels of ICER suppressed the induction of nuclear factor of activated T cell c1 (Nfatc1) gene in T cells whose inducible Nfatc1 P1 promoter bears two highly conserved cAMP-responsive elements to which ICER/CREM can bind. These findings suggest that nTregs suppress T-cell responses by the cAMP-dependent nuclear accumulation of ICER/CREM and inhibition of NFATc1 and IL-2 induction.
Assuntos
Núcleo Celular/imunologia , Modulador de Elemento de Resposta do AMP Cíclico/imunologia , Ativação Linfocitária/imunologia , Fatores de Transcrição NFATC/imunologia , Elementos de Resposta/imunologia , Linfócitos T Reguladores/imunologia , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Transporte Ativo do Núcleo Celular/genética , Transporte Ativo do Núcleo Celular/imunologia , Animais , Anticorpos/imunologia , Anticorpos/farmacologia , Antígenos CD28/genética , Antígenos CD28/imunologia , Antígenos CD28/metabolismo , Núcleo Celular/genética , Núcleo Celular/metabolismo , AMP Cíclico/genética , AMP Cíclico/imunologia , AMP Cíclico/metabolismo , Modulador de Elemento de Resposta do AMP Cíclico/genética , Modulador de Elemento de Resposta do AMP Cíclico/metabolismo , Interleucina-2/biossíntese , Interleucina-2/genética , Interleucina-2/imunologia , Ativação Linfocitária/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Fatores de Transcrição NFATC/genética , Fatores de Transcrição NFATC/metabolismo , Elementos de Resposta/genética , Linfócitos T Reguladores/metabolismoRESUMO
Candida albicans, an opportunistic fungal pathogen, produces the quorum-sensing molecule farnesol, which we have shown alters the transcriptional response and phenotype of human monocyte-derived dendritic cells (DCs), including their cytokine secretion and ability to prime T cells. This is partially dependent on the nuclear receptor peroxisome proliferator-activated receptor gamma (PPAR-γ), which has numerous ligands, including the sphingolipid metabolite sphingosine 1-phosphate. Sphingolipids are a vital component of membranes that affect membrane protein arrangement and phagocytosis of C. albicans by DCs. Thus, we quantified sphingolipid metabolites in monocytes differentiating into DCs by High-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). Farnesol increased the activity of serine palmitoyltransferase, leading to increased levels of 3-keto-dihydrosphingosine, dihydrosphingosine, and dihydrosphingosine 1-phosphate and inhibited dihydroceramide desaturase by inducing oxidative stress, leading to increased levels of dihydroceramide and dihydrosphingomyelin species and reduced ceramide levels. Accumulation of dihydroceramides can inhibit mitochondrial function; accordingly, farnesol reduced mitochondrial respiration. Dihydroceramide desaturase inhibition increases lipid droplet formation, which we observed in farnesol-treated cells, coupled with an increase in intracellular triacylglycerol species. Furthermore, inhibition of dihydroceramide desaturase with either farnesol or specific inhibitors impaired the ability of DCs to prime interferon-γ-producing T cells. The effect of farnesol on sphingolipid metabolism, triacylglycerol synthesis, and mitochondrial respiration was not dependent on PPAR-γ. In summary, our data reveal novel effects of farnesol on sphingolipid metabolism, neutral lipid synthesis, and mitochondrial function in DCs that affect their instruction of T cell cytokine secretion, indicating that C. albicans can manipulate host cell metabolism via farnesol secretion.IMPORTANCECandida albicans is a common commensal yeast, but it is also an opportunistic pathogen which is one of the leading causes of potentially lethal hospital-acquired infections. There is growing evidence that its overgrowth in the gut can influence diseases as diverse as alcohol-associated liver disease and COVID-19. Previously, we found that its quorum-sensing molecule, farnesol, alters the phenotype of dendritic cells differentiating from monocytes, impairing their ability to drive protective T cell responses. Here, we demonstrate that farnesol alters the metabolism of sphingolipids, important structural components of the membrane that also act as signaling molecules. In monocytes differentiating to dendritic cells, farnesol inhibited dihydroceramide desaturase, resulting in the accumulation of dihydroceramides and a reduction in ceramide levels. Farnesol impaired mitochondrial respiration, known to occur with an accumulation of dihydroceramides, and induced the accumulation of triacylglycerol and oil bodies. Inhibition of dihydroceramide desaturase resulted in the impaired ability of DCs to induce interferon-γ production by T cells. Thus, farnesol production by C. albicans could manipulate the function of dendritic cells by altering the sphingolipidome.
Assuntos
Candida albicans , Células Dendríticas , Farneseno Álcool , Monócitos , Percepção de Quorum , Esfingolipídeos , Farneseno Álcool/farmacologia , Farneseno Álcool/metabolismo , Humanos , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/metabolismo , Células Dendríticas/imunologia , Candida albicans/efeitos dos fármacos , Candida albicans/metabolismo , Esfingolipídeos/metabolismo , Percepção de Quorum/efeitos dos fármacos , Monócitos/metabolismo , Monócitos/efeitos dos fármacos , Monócitos/microbiologia , Monócitos/imunologia , PPAR gama/metabolismo , PPAR gama/genética , Espectrometria de Massas em Tandem , Citocinas/metabolismoRESUMO
Objectives: Sjögren's Disease (SjD) is an autoimmune disorder characterized by progressive dysfunction, inflammation and destruction of salivary and lacrimal glands, and by extraglandular manifestations. Its etiology and pathophysiology remain incompletely understood, though a role for autoreactive B cells has been considered key. Here, we investigated the role of effector and regulatory T cells in the pathogenesis of SjD. Methods: Histological analysis, RNA-sequencing and flow cytometry were conducted on glands, lungs, eyes and lymphoid tissues of mice with regulatory T cell-specific deletion of stromal interaction proteins (STIM) 1 and 2 ( Stim1/2 Foxp3 ), which play key roles in calcium signaling and T cell function. The pathogenicity of T cells from Stim1/2 Foxp3 mice was investigated through adoptively transfer into lymphopenic host mice. Additionally, single-cell transcriptomic analysis was performed on peripheral blood mononuclear cells (PBMCs) of patients with SjD and control subjects. Results: Stim1/2 Foxp3 mice develop a severe SjD-like disorder including salivary gland (SG) and lacrimal gland (LG) inflammation and dysfunction, autoantibodies and extraglandular symptoms. SG inflammation in Stim1/2 Foxp3 mice is characterized by T and B cell infiltration, and transcriptionally by a Th1 immune response that correlates strongly with the dysregulation observed in patients with SjD. Adoptive transfer of effector T cells from Stim1/2 Foxp3 mice demonstrates that the SjD-like disease is driven by interferon (IFN)-γ producing autoreactive CD4 + T cells independently of B cells and autoantiboodies. scRNA-seq analysis identifies increased Th1 responses and attenuated memory Treg function in PBMCs of patients with SjD. Conclusions: We report a more accurate mouse model of SjD while providing evidence for a critical role of Treg cells and IFN-γ producing Th1 cells in the pathogenesis of SjD, which may be effective targets for therapy.