RESUMEN
Cytomegalovirus (CMV) induces a unique T cell response, where antigen-specific populations do not contract, but rather inflate during viral latency. It has been proposed that subclinical episodes of virus reactivation feed the inflation of CMV-specific memory cells by intermittently engaging T cell receptors (TCRs), but evidence of TCR engagement has remained lacking. Nuclear factor of activated T cells (NFAT) is a family of transcription factors, where NFATc1 and NFATc2 signal downstream of TCR in mature T lymphocytes. We show selective impacts of NFATc1 and/or NFATc2 genetic ablations on the long-term inflation of MCMV-specific CD8+ T cell responses despite largely maintained responses to acute infection. NFATc1 ablation elicited robust phenotypes in isolation, but the strongest effects were observed when both NFAT genes were missing. CMV control was impaired only when both NFATs were deleted in CD8+ T cells used in adoptive immunotherapy of immunodeficient mice. Transcriptome analyses revealed that T cell intrinsic NFAT is not necessary for CD8+ T cell priming, but rather for their maturation towards effector-memory and in particular the effector cells, which dominate the pool of inflationary cells.
Asunto(s)
Infecciones por Citomegalovirus , Muromegalovirus , Animales , Ratones , Muromegalovirus/fisiología , Linfocitos T CD8-positivos , Citomegalovirus , Receptores de Antígenos de Linfocitos T , Memoria InmunológicaRESUMEN
During chronic stimulation, CD8+ T cells acquire an exhausted phenotype characterized by expression of inhibitory receptors, down-modulation of effector function, and metabolic impairments. T cell exhaustion protects from excessive immunopathology but limits clearance of virus-infected or tumor cells. We transcriptionally profiled antigen-specific T cells from mice infected with lymphocytic choriomeningitis virus strains that cause acute or chronic disease. T cell exhaustion during chronic infection was driven by high amounts of T cell receptor (TCR)-induced transcription factors IRF4, BATF, and NFATc1. These regulators promoted expression of inhibitory receptors, including PD-1, and mediated impaired cellular metabolism. Furthermore, they repressed the expression of TCF1, a transcription factor required for memory T cell differentiation. Reducing IRF4 expression restored the functional and metabolic properties of antigen-specific T cells and promoted memory-like T cell development. These findings indicate that IRF4 functions as a central node in a TCR-responsive transcriptional circuit that establishes and sustains T cell exhaustion during chronic infection.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Memoria Inmunológica , Factores Reguladores del Interferón/inmunología , Coriomeningitis Linfocítica/inmunología , Virus de la Coriomeningitis Linfocítica/inmunología , Receptores de Antígenos de Linfocitos T/inmunología , Animales , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/genética , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/inmunología , Linfocitos T CD8-positivos/virología , Diferenciación Celular , Regulación de la Expresión Génica , Células HEK293 , Factor Nuclear 1-alfa del Hepatocito/genética , Factor Nuclear 1-alfa del Hepatocito/inmunología , Humanos , Factores Reguladores del Interferón/deficiencia , Factores Reguladores del Interferón/genética , Activación de Linfocitos , Depleción Linfocítica , Coriomeningitis Linfocítica/genética , Coriomeningitis Linfocítica/virología , Virus de la Coriomeningitis Linfocítica/crecimiento & desarrollo , Ratones , Ratones Noqueados , Factores de Transcripción NFATC/genética , Factores de Transcripción NFATC/inmunología , Receptor de Muerte Celular Programada 1/genética , Receptor de Muerte Celular Programada 1/inmunología , Receptores de Antígenos de Linfocitos T/genética , Transducción de SeñalRESUMEN
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.
Asunto(s)
Canales de Calcio/inmunología , Señalización del Calcio/inmunología , Calcio/inmunología , Linfocitos T/inmunología , Animales , Calcineurina/inmunología , Calcineurina/metabolismo , Calcio/metabolismo , Canales de Calcio/metabolismo , División Celular/inmunología , Células Cultivadas , Femenino , Glucólisis/inmunología , Células HEK293 , Humanos , Immunoblotting , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Microscopía Confocal , Factores de Transcripción NFATC/genética , Factores de Transcripción NFATC/inmunología , Factores de Transcripción NFATC/metabolismo , Fosfatidilinositol 3-Quinasas/inmunología , Fosfatidilinositol 3-Quinasas/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/inmunología , Molécula de Interacción Estromal 1/genética , Molécula de Interacción Estromal 1/inmunología , Molécula de Interacción Estromal 1/metabolismo , Molécula de Interacción Estromal 2/genética , Molécula de Interacción Estromal 2/inmunología , Molécula de Interacción Estromal 2/metabolismo , Linfocitos T/metabolismoRESUMEN
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.
Asunto(s)
Autoinmunidad , Linfocitos B/inmunología , Centro Germinal/inmunología , Inmunidad Humoral , Molécula de Interacción Estromal 1/metabolismo , Molécula de Interacción Estromal 2/metabolismo , Linfocitos T/inmunología , Animales , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/genética , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/metabolismo , Canales de Calcio Activados por la Liberación de Calcio/metabolismo , Señalización del Calcio , Células Cultivadas , Factores Reguladores del Interferón/genética , Factores Reguladores del Interferón/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Factores de Transcripción NFATC/metabolismo , Proteína ORAI1/metabolismo , Proteínas Proto-Oncogénicas c-bcl-6/genética , Proteínas Proto-Oncogénicas c-bcl-6/metabolismo , Molécula de Interacción Estromal 1/genética , Molécula de Interacción Estromal 2/genéticaRESUMEN
CD56+ T cells are a group of pro-inflammatory CD3+ lymphocytes with characteristics of natural killer cells, being involved in antimicrobial immune defense. Here, we performed deep phenotypic profiling of CD3+ CD56+ cells in peripheral blood of normal human donors and individuals sensitized to birch-pollen or/and house dust mite by high-dimensional mass cytometry combined with manual and computational data analysis. A co-regulation between major conventional T-cell subsets and their respective CD3+ CD56+ cell counterparts appeared restricted to CD8+ , MAIT, and TCRγδ+ T-cell compartments. Interestingly, we find a co-regulation of several CD3+ CD56+ cell subsets in allergic but not in healthy individuals. Moreover, using FlowSOM, we distinguished a variety of CD56+ T-cell phenotypes demonstrating a hitherto underestimated heterogeneity among these cells. The novel CD3+ CD56+ subset description comprises phenotypes superimposed with naive, memory, type 1, 2, and 17 differentiation stages, in part represented by a phenotypical continuum. Frequencies of two out of 19 CD3+ CD56+ FlowSOM clusters were significantly diminished in allergic individuals, demonstrating less frequent presence of cells with cytolytic, presumably protective, capacity in these donors consistent with defective expansion or their recruitment to the affected tissue. Our results contribute to defining specific cell populations to be targeted during therapy for allergic conditions.
Asunto(s)
Complejo CD3/inmunología , Antígeno CD56/inmunología , Subgrupos de Linfocitos T/inmunología , Antígenos de Diferenciación de Linfocitos T/inmunología , Diferenciación Celular/inmunología , Humanos , Células Asesinas Naturales/inmunología , Fenotipo , Receptores de Antígenos de Linfocitos T gamma-delta/inmunologíaRESUMEN
BACKGROUND: Effector functions of IgG Abs are regulated by their Fc N-glycosylation pattern. IgG Fc glycans that lack galactose and terminal sialic acid residues correlate with the severity of inflammatory (auto)immune disorders and have also been linked to protection against viral infection and discussed in the context of vaccine-induced protection. In contrast, sialylated IgG Abs have shown immunosuppressive effects. OBJECTIVE: We sought to investigate IgG glycosylation programming during the germinal center (GC) reaction following immunization of mice with a foreign protein antigen and different adjuvants. METHODS: Mice were analyzed for GC T-cell, B-cell, and plasma cell responses, as well as for antigen-specific serum IgG subclass titers and Fc glycosylation patterns. RESULTS: Different adjuvants induce distinct IgG+ GC B-cell responses with specific transcriptomes and expression levels of the α2,6-sialyltransferase responsible for IgG sialylation that correspond to distinct serum IgG Fc glycosylation patterns. Low IgG Fc sialylation programming in GC B cells was overall highly dependent on the Foxp3- follicular helper T (TFH) cell-inducing cytokine IL-6, here in particular induced by water-in-oil adjuvants and Mycobacterium tuberculosis. Furthermore, low IgG Fc sialylation programming was dependent on adjuvants that induced IL-27 receptor-dependent IFN-γ+ TFH1 cells, IL-6/IL-23-dependent IL-17A+ TFH17 cells, and high ratios of TFH cells to Foxp3+ follicular regulatory T cells. Here, the 2 latter were dependent on M tuberculosis and its cord factor. CONCLUSION: This study's findings regarding adjuvant-dependent GC responses and IgG glycosylation programming may aid in the development of novel vaccination strategies to induce IgG Abs with both high affinity and defined Fc glycosylation patterns in the GC.
Asunto(s)
Adyuvantes Inmunológicos/administración & dosificación , Antígenos/administración & dosificación , Centro Germinal/inmunología , Inmunoglobulina G/inmunología , Compuestos de Alumbre/administración & dosificación , Animales , Linfocitos B/efectos de los fármacos , Linfocitos B/inmunología , Citocinas/inmunología , Femenino , Adyuvante de Freund/administración & dosificación , Glicosilación , Lipopolisacáridos/administración & dosificación , Ratones Endogámicos C57BL , Ratones Noqueados , Aceite Mineral/administración & dosificación , Mycobacterium tuberculosis/inmunología , Ovalbúmina/administración & dosificación , Polisorbatos/administración & dosificación , Escualeno/administración & dosificación , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunología , VacunaciónRESUMEN
Follicular T helper (Tfh) cells are key regulators of the germinal center reaction and long-term humoral immunity. Tfh cell differentiation requires the sustained expression of the transcriptional repressor Bcl6; however, its regulation in CD4(+)T cells is incompletely understood. Here, we report that the transcriptional coactivator Bob1, encoded by thePou2af1gene, promotes Bcl6 expression and Tfh cell development. We found that Bob1 together with the octamer transcription factors Oct1/Oct2 can directly bind to and transactivate theBcl6andBtlapromoters. Mixed bone marrow chimeras revealed that Bob1 is required for the expression of normal levels of Bcl6 andBTLA, thereby controlling the pool size and composition of the Tfh compartment in a T cell-intrinsic manner. Our data indicate that T cell-expressed Bob1 is directly involved in Tfh cell differentiation and required for mounting normal T cell-dependent B-cell responses.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Linfocitos T Colaboradores-Inductores/citología , Transactivadores/metabolismo , Animales , Sitios de Unión , Linfocitos T CD4-Positivos/inmunología , Diferenciación Celular , Proteínas de Unión al ADN/genética , Femenino , Regulación de la Expresión Génica , Inmunización , Masculino , Ratones Endogámicos C57BL , Ratones Mutantes , Regiones Promotoras Genéticas , Proteínas Proto-Oncogénicas c-bcl-6 , Receptores Inmunológicos/genética , Receptores Inmunológicos/metabolismo , Receptores de Interleucina-21/genética , Receptores de Interleucina-21/metabolismo , Receptores de Interleucina-6/genética , Receptores de Interleucina-6/metabolismo , Linfocitos T Colaboradores-Inductores/fisiología , Transactivadores/genéticaRESUMEN
The mannose receptor (MR) is an endocytic receptor involved in serum homeostasis and antigen presentation. Here, we identify the MR as a direct regulator of CD8(+) T-cell activity. We demonstrate that MR expression on dendritic cells (DCs) impaired T-cell cytotoxicity in vitro and in vivo. This regulatory effect of the MR was mediated by a direct interaction with CD45 on the T cell, inhibiting its phosphatase activity, which resulted in up-regulation of cytotoxic T-lymphocyte-associated Protein 4 (CTLA-4) and the induction of T-cell tolerance. Inhibition of CD45 prevented expression of B-cell lymphoma 6 (Bcl-6), a transcriptional inhibitor that directly bound the CTLA-4 promoter and regulated its activity. These data demonstrate that endocytic receptors expressed on DCs contribute to the regulation of T-cell functionality.
Asunto(s)
Antígeno CTLA-4/genética , Lectinas Tipo C/genética , Antígenos Comunes de Leucocito/genética , Activación de Linfocitos/genética , Lectinas de Unión a Manosa/genética , Receptores de Superficie Celular/genética , Animales , Presentación de Antígeno/genética , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Antígeno CTLA-4/inmunología , Regulación de la Expresión Génica/genética , Humanos , Tolerancia Inmunológica/genética , Lectinas Tipo C/inmunología , Antígenos Comunes de Leucocito/inmunología , Activación de Linfocitos/inmunología , Receptor de Manosa , Lectinas de Unión a Manosa/inmunología , Ratones , Proteínas Proto-Oncogénicas c-bcl-6/genética , Receptores de Superficie Celular/inmunología , Linfocitos T Citotóxicos/inmunología , Activación Transcripcional/genéticaRESUMEN
BACKGROUND: Wilms' tumor 1-associating protein (WTAP) is a nuclear protein, which is ubiquitously expressed in many tissues. Furthermore, in various types of malignancies WTAP is overexpressed and plays a role as an oncogene. The function of WTAP in diffuse large B-cell lymphoma (DLBCL), however, remains unclear. METHODS: Immunohistochemistry was applied to evaluate the levels of WTAP expression in DLBCL tissues and normal lymphoid tissues. Overexpression and knock-down of WTAP in DLBCL cell lines, verified on mRNA and protein level served to analyze cell proliferation and apoptosis in DLBCL cell lines by flow cytometry. Finally, co-immunoprecipitation (Co-IP), IP, and GST-pull down assessed the interaction of WTAP with Heat shock protein 90 (Hsp90) and B-cell lymphoma 6 (BCL6) as well as determined the extend of its ubiquitinylation. RESULTS: WTAP protein levels were consistently upregulated in DLBCL tissues. WTAP promoted DLBCL cell proliferation and improved the ability to confront apoptosis, while knockdown of WTAP in DLBCL cell lines allowed a significant higher apoptosis rate after treatment with Etoposide, an anti-tumor drug. The stable expression of WTAP was depended on Hsp90. In line, we demonstrated that WTAP could form a complex with BCL6 via Hsp90 in vivo and in vitro. CONCLUSION: WTAP is highly expressed in DLBCL, promoting growth and anti-apoptosis in DLBCL cell lines. WTAP is a client protein of Hsp90 and can appear in a complex with BCL6 and Hsp90 in DLBCL. Down-regulation of WTAP could improve the chemotherapeutic treatments in DLBCL.
Asunto(s)
Proteínas HSP90 de Choque Térmico/metabolismo , Linfoma de Células B Grandes Difuso/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Proto-Oncogénicas c-bcl-6/metabolismo , Apoptosis , Proteínas de Ciclo Celular , Línea Celular Tumoral , Proliferación Celular , Regulación Neoplásica de la Expresión Génica , Humanos , Linfoma de Células B Grandes Difuso/patología , Unión Proteica , Estabilidad Proteica , Factores de Empalme de ARNRESUMEN
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.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Enfermedad Injerto contra Huésped/inmunología , Efecto Injerto vs Leucemia/inmunología , Factores de Transcripción NFATC/inmunología , Linfocitos T Reguladores/inmunología , Aloinjertos , Animales , Linfocitos T CD8-positivos/patología , Inhibidores de la Calcineurina/farmacología , Proliferación Celular/efectos de los fármacos , Proliferación Celular/genética , Ciclosporina/farmacología , Modelos Animales de Enfermedad , Enfermedad Injerto contra Huésped/genética , Enfermedad Injerto contra Huésped/patología , Efecto Injerto vs Leucemia/genética , Trasplante de Células Madre Hematopoyéticas , Ratones , Ratones Noqueados , Factores de Transcripción NFATC/genética , Linfocitos T Reguladores/patología , Tacrolimus/farmacologíaRESUMEN
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.
Asunto(s)
Calcio/metabolismo , Células Dendríticas/inmunología , Interleucina-2/biosíntesis , Proteínas Mitocondriales/genética , Linfocitos T/inmunología , Linfocitos T/metabolismo , Receptores Toll-Like/metabolismo , Animales , Antígeno B7-2/metabolismo , Proteína beta Potenciadora de Unión a CCAAT/metabolismo , Análisis por Conglomerados , Expresión Génica , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Antígenos de Histocompatibilidad Clase II/metabolismo , Interleucina-2/genética , Interleucina-2/farmacología , Lipopolisacáridos/inmunología , Activación de Linfocitos/efectos de los fármacos , Activación de Linfocitos/genética , Activación de Linfocitos/inmunología , Masculino , Ratones , Proteínas Mitocondriales/metabolismo , FN-kappa B/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Trasplante de Piel , Linfocitos T/efectos de los fármacos , Trasplante HomólogoRESUMEN
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.
Asunto(s)
Encefalomielitis Autoinmune Experimental/prevención & control , Factores de Transcripción NFATC/deficiencia , Animales , Linfocitos T CD4-Positivos/inmunología , Citocinas/biosíntesis , Encefalomielitis Autoinmune Experimental/etiología , Encefalomielitis Autoinmune Experimental/inmunología , Humanos , Inmunomodulación , Interleucina-10/biosíntesis , Interleucina-4/biosíntesis , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Esclerosis Múltiple/inmunología , Esclerosis Múltiple/terapia , Glicoproteína Mielina-Oligodendrócito/administración & dosificación , Glicoproteína Mielina-Oligodendrócito/inmunología , Factores de Transcripción NFATC/antagonistas & inhibidores , Factores de Transcripción NFATC/genética , Factores de Transcripción NFATC/inmunología , Fragmentos de Péptidos/administración & dosificación , Fragmentos de Péptidos/inmunología , Transducción de Señal/inmunología , Células Th17/inmunología , Células Th2/inmunologíaRESUMEN
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.
Asunto(s)
Antígenos CD28/genética , Antígeno CTLA-4/genética , Anergia Clonal , Células Dendríticas/inmunología , Factores de Transcripción Forkhead/genética , Interleucina-10/genética , Linfocitos T Reguladores/inmunología , Animales , Antígenos CD28/inmunología , Antígeno CTLA-4/inmunología , Comunicación Celular , Diferenciación Celular , Técnicas de Cocultivo , Células Dendríticas/citología , Células Dendríticas/efectos de los fármacos , Células Dendríticas/metabolismo , Proteína 2 de la Respuesta de Crecimiento Precoz/genética , Proteína 2 de la Respuesta de Crecimiento Precoz/inmunología , Factores de Transcripción Forkhead/inmunología , Regulación de la Expresión Génica/inmunología , Interleucina-10/inmunología , Interleucina-2/genética , Interleucina-2/inmunología , Subunidad alfa del Receptor de Interleucina-2/genética , Subunidad alfa del Receptor de Interleucina-2/inmunología , Activación de Linfocitos , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Factores de Transcripción NFATC/genética , Factores de Transcripción NFATC/inmunología , Transducción de Señal , Linfocitos T Reguladores/citología , Linfocitos T Reguladores/efectos de los fármacos , Linfocitos T Reguladores/metabolismo , Factor de Crecimiento Transformador beta/farmacologíaRESUMEN
Human brain microvascular endothelial cells forming the blood-brain barrier (BBB) release soluble vascular cell adhesion molecule-1 (sVCAM-1) under inflammatory conditions. Furthermore, sVCAM-1 serum levels in untreated patients with multiple sclerosis (MS) correlate with a breakdown of the BBB as measured by gadolinium-enhanced MRI. To date, it is unknown whether sVCAM-1 itself modulates BBB permeability. Here, we provide evidence that human brain endothelium expresses integrin α-4/ß-1, the molecular binding partner of sVCAM-1, and that sVCAM-1 directly impairs BBB function by inducing intracellular signalling events through integrin α-4. Primary human brain microvascular endothelial cells showed low to moderate integrin α-4 and strong ß-1 but no definite ß-7 expression in vitro and in situ. Increased brain endothelial integrin α-4 expression was observed in active MS lesions in situ and after angiogenic stimulation in vitro. Exposure of cultured primary brain endothelial cells to recombinant sVCAM-1 significantly increased their permeability to the soluble tracer dextran, which was paralleled by formation of actin stress fibres and reduced staining of tight junction-associated molecules. Soluble VCAM-1 was also found to activate Rho GTPase and p38 MAP kinase. Chemical inhibition of these signalling pathways partially prevented sVCAM-1-induced changes of tight junction arrangement. Importantly, natalizumab, a neutralising recombinant monoclonal antibody against integrin α-4 approved for the treatment of patients with relapsing-remitting MS, partially antagonised the barrier-disturbing effect of sVCAM-1. In summary, we newly characterised sVCAM-1 as a compromising factor of brain endothelial barrier function that may be partially blocked by the MS therapeutic natalizumab.
Asunto(s)
Barrera Hematoencefálica/efectos de los fármacos , Barrera Hematoencefálica/fisiopatología , Células Endoteliales/metabolismo , Integrina alfa4/metabolismo , Esclerosis Múltiple/metabolismo , Molécula 1 de Adhesión Celular Vascular/farmacología , Adulto , Anciano , Anciano de 80 o más Años , Autopsia , Técnicas de Cultivo de Célula , Células Endoteliales/citología , Células Endoteliales/efectos de los fármacos , Endotelio Vascular/citología , Femenino , Humanos , Factores Inmunológicos/farmacología , Integrina beta1/metabolismo , Masculino , Persona de Mediana Edad , Esclerosis Múltiple/tratamiento farmacológico , Esclerosis Múltiple/patología , Esclerosis Múltiple/fisiopatología , Natalizumab/farmacología , Resultado del Tratamiento , Molécula 1 de Adhesión Celular Vascular/metabolismoRESUMEN
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.
Asunto(s)
Linfocitos B/efectos de los fármacos , Factores de Transcripción NFATC/genética , Células TH1/efectos de los fármacos , Células Th2/efectos de los fármacos , Animales , Anticuerpos/farmacología , Linfocitos B/inmunología , Linfocitos B/metabolismo , Proliferación Celular/efectos de los fármacos , Cromosomas Artificiales Bacterianos/genética , Expresión Génica/efectos de los fármacos , Expresión Génica/inmunología , Genes Reporteros , Proteínas Fluorescentes Verdes , Lipopolisacáridos/farmacología , Activación de Linfocitos/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , FN-kappa B/genética , FN-kappa B/inmunología , Factores de Transcripción NFATC/agonistas , Factores de Transcripción NFATC/inmunología , Regiones Promotoras Genéticas , Isoformas de Proteínas/agonistas , Isoformas de Proteínas/genética , Isoformas de Proteínas/inmunología , Linfocitos T Reguladores/efectos de los fármacos , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/metabolismo , Células TH1/inmunología , Células TH1/metabolismo , Células Th17/efectos de los fármacos , Células Th17/inmunología , Células Th17/metabolismo , Células Th2/inmunología , Células Th2/metabolismo , Factor de Crecimiento Transformador beta/genética , Factor de Crecimiento Transformador beta/inmunologíaRESUMEN
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.
Asunto(s)
FN-kappa B/metabolismo , Factores de Transcripción NFATC/metabolismo , Factor 2 de Transcripción de Unión a Octámeros/genética , Linfocitos T/metabolismo , Transactivadores/genética , Animales , Sitios de Unión , Células Cultivadas , Humanos , Células Jurkat , Ratones , Ratones Endogámicos C57BL , FN-kappa B/antagonistas & inhibidores , Factores de Transcripción NFATC/antagonistas & inhibidores , Factor 2 de Transcripción de Unión a Octámeros/biosíntesis , Regiones Promotoras Genéticas , Transactivadores/biosíntesis , Transcripción GenéticaRESUMEN
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.
Asunto(s)
Enfermedades Autoinmunes/inmunología , Factores de Transcripción Forkhead/metabolismo , Factores de Transcripción NFATC/metabolismo , Linfocitos T Reguladores/metabolismo , Linfocitos T/metabolismo , Traslado Adoptivo , Animales , Inmunoprecipitación de Cromatina , Colitis/inmunología , Ciclosporina , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Proteínas de Homeodominio/genética , Humanos , Immunoblotting , Activación de Linfocitos/inmunología , Ratones , Factores de Transcripción NFATC/inmunología , Linfocitos T/inmunología , Linfocitos T Reguladores/inmunología , Factor de Crecimiento Transformador betaRESUMEN
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.
Asunto(s)
Anergia Clonal , AMP Cíclico/metabolismo , Linfocitos T Reguladores/inmunología , Animales , Proliferación Celular , Humanos , Ratones , Factores de Transcripción NFATC/metabolismo , Linfocitos T Reguladores/citología , Regulación hacia ArribaRESUMEN
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.
Asunto(s)
Núcleo Celular/inmunología , Modulador del Elemento de Respuesta al AMP Cíclico/inmunología , Activación de Linfocitos/inmunología , Factores de Transcripción NFATC/inmunología , Elementos de Respuesta/inmunología , Linfocitos T Reguladores/inmunología , Transporte Activo de Núcleo Celular/efectos de los fármacos , Transporte Activo de Núcleo Celular/genética , Transporte Activo de Núcleo Celular/inmunología , Animales , Anticuerpos/inmunología , Anticuerpos/farmacología , Antígenos CD28/genética , Antígenos CD28/inmunología , Antígenos CD28/metabolismo , Núcleo Celular/genética , Núcleo Celular/metabolismo , AMP Cíclico/genética , AMP Cíclico/inmunología , AMP Cíclico/metabolismo , Modulador del Elemento de Respuesta al AMP Cíclico/genética , Modulador del Elemento de Respuesta al AMP Cíclico/metabolismo , Interleucina-2/biosíntesis , Interleucina-2/genética , Interleucina-2/inmunología , Activación de Linfocitos/efectos de los fármacos , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Factores de Transcripción NFATC/genética , Factores de Transcripción NFATC/metabolismo , Elementos de Respuesta/genética , Linfocitos T Reguladores/metabolismoRESUMEN
Neutrophil-derived bactericidal/permeability-increasing protein (BPI) is known for its bactericidal activity against gram-negative bacteria and neutralization of lipopolysaccharide. Here, we define BPI as a potent activator of murine dendritic cells (DCs). As shown in GM-CSF-cultured, bone-marrow-derived cells (BMDCs), BPI induces a distinct stimulation profile including IL-2, IL-6, and tumor necrosis factor expression. Conventional DCs also respond to BPI, while M-CSF-cultivated or peritoneal lavage macrophages do not. Subsequent to BPI stimulation of BMDCs, CD4+ T cells predominantly secrete IL-22 and, when naive, preferentially differentiate into T helper 22 (Th22) cells. Congruent with the tissue-protective properties of IL-22 and along with impaired IL-22 induction, disease severity is significantly increased during dextran sodium sulfate-induced colitis in BPI-deficient mice. Importantly, physiological diversification of intestinal microbiota fosters BPI-dependent IL-22 induction in CD4+ T cells derived from mesenteric lymph nodes. In conclusion, BPI is a potent activator of DCs and consecutive Th22 cell differentiation with substantial relevance in intestinal homeostasis.