RESUMEN
Emerging evidence supports that mitochondrial dysfunction contributes to systemic lupus erythematosus (SLE) pathogenesis. Here we show that programmed mitochondrial removal, a hallmark of mammalian erythropoiesis, is defective in SLE. Specifically, we demonstrate that during human erythroid cell maturation, a hypoxia-inducible factor (HIF)-mediated metabolic switch is responsible for the activation of the ubiquitin-proteasome system (UPS), which precedes and is necessary for the autophagic removal of mitochondria. A defect in this pathway leads to accumulation of red blood cells (RBCs) carrying mitochondria (Mito+ RBCs) in SLE patients and in correlation with disease activity. Antibody-mediated internalization of Mito+ RBCs induces type I interferon (IFN) production through activation of cGAS in macrophages. Accordingly, SLE patients carrying both Mito+ RBCs and opsonizing antibodies display the highest levels of blood IFN-stimulated gene (ISG) signatures, a distinctive feature of SLE.
Asunto(s)
Interferón Tipo I/metabolismo , Lupus Eritematoso Sistémico/metabolismo , Mitocondrias/metabolismo , Células Mieloides/metabolismo , Adolescente , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Niño , Preescolar , Eritroblastos/metabolismo , Eritroblastos/ultraestructura , Eritrocitos/metabolismo , Eritropoyesis , Humanos , Mitofagia , Complejo de la Endopetidasa Proteasomal/metabolismo , Ubiquitina/metabolismoRESUMEN
Group 2 innate lymphoid cells (ILC2 cells) are important for type 2 immune responses and are activated by the epithelial cytokines interleukin 33 (IL-33), IL-25 and thymic stromal lymphopoietin (TSLP). Here we demonstrated that IL-1ß was a critical activator of ILC2 cells, inducing proliferation and cytokine production and regulating the expression of epithelial cytokine receptors. IL-1ß also governed ILC2 plasticity by inducing low expression of the transcription factor T-bet and the cytokine receptor chain IL-12Rß2, which enabled the conversion of these cells into an ILC1 phenotype in response to IL-12. This transition was marked by an atypical chromatin landscape characterized by the simultaneous transcriptional accessibility of the locus encoding interferon-γ (IFN-γ) and the loci encoding IL-5 and IL-13. Finally, IL-1ß potentiated ILC2 activation and plasticity in vivo, and IL-12 acted as the switch that determined an ILC2-versus-ILC1 response. Thus, we have identified a previously unknown role for IL-1ß in facilitating ILC2 maturation and plasticity.
Asunto(s)
Plasticidad de la Célula , Inmunidad Innata , Interleucina-12/metabolismo , Interleucina-1beta/metabolismo , Linfocitos/inmunología , Animales , Diferenciación Celular , Plasticidad de la Célula/inmunología , Células Cultivadas , Citocinas/metabolismo , Humanos , Interferón gamma/metabolismo , Interleucina-17/metabolismo , Interleucina-33/metabolismo , Ratones , Ratones SCID , Receptores de Interleucina-12/genética , Receptores de Interleucina-12/metabolismo , Transducción de Señal , Proteínas de Dominio T Box/genética , Proteínas de Dominio T Box/metabolismo , Células TH1/inmunología , Balance Th1 - Th2 , Células Th2/inmunología , Linfopoyetina del Estroma TímicoRESUMEN
Human Langerhans cells (LCs) are highly efficient at priming cytolytic CD8(+) T cells compared with dermal CD14(+) dendritic cells (DCs). Here we show that dermal CD14(+) DCs instead prime a fraction of naïve CD8(+) T cells into cells sharing the properties of type 2 cytokine-secreting CD8(+) T cells (TC2). Differential expression of the CD8-antagonist receptors on dermal CD14(+) DCs, the Ig-like transcript (ILT) inhibitory receptors, explains the difference between the two types of DCs. Inhibition of CD8 function on LCs inhibited cytotoxic T lymphocytes (CTLs) and enhanced TC2 generation. In addition, blocking ILT2 or ILT4 on dermal CD14(+) DCs enhanced the generation of CTLs and inhibited TC2 cytokine production. Lastly, addition of soluble ILT2 and ILT4 receptors inhibited CTL priming by LCs. Thus, ILT receptor expression explains the polarization of CD8(+) T-cell responses by LCs vs. dermal CD14(+) DCs.
Asunto(s)
Antígenos CD/inmunología , Dermis/inmunología , Células de Langerhans/metabolismo , Receptores de Lipopolisacáridos , Glicoproteínas de Membrana/inmunología , Receptores Inmunológicos/inmunología , Linfocitos T Citotóxicos/inmunología , Antígenos CD/biosíntesis , Antígenos CD/genética , Dermis/citología , Dermis/metabolismo , Humanos , Células de Langerhans/citología , Células de Langerhans/inmunología , Receptor Leucocitario Tipo Inmunoglobulina B1 , Glicoproteínas de Membrana/biosíntesis , Glicoproteínas de Membrana/genética , Receptores Inmunológicos/biosíntesis , Receptores Inmunológicos/genética , Linfocitos T Citotóxicos/citología , Linfocitos T Citotóxicos/metabolismoRESUMEN
We recently reported that human epidermal Langerhans cells (LCs) are more efficient than dermal CD14(+) DCs at priming naive CD8(+) T cells into potent CTLs. We hypothesized that distinctive dendritic cell (DC) cytokine expression profiles (ie, IL-15 produced by LCs and IL-10 expressed by dermal CD14(+) DCs) might explain the observed functional difference. Blocking IL-15 during CD8(+) T-cell priming reduced T-cell proliferation by â¼ 50%. These IL-15-deprived CD8(+) T cells did not acquire the phenotype of effector memory cells. They secreted less IL-2 and IFN-γ and expressed only low amounts of CD107a, granzymes and perforin, and reduced levels of the antiapoptotic protein Bcl-2. Confocal microscopy analysis showed that IL-15 is localized at the immunologic synapse of LCs and naive CD8(+) T cells. Conversely, blocking IL-10 during cocultures of dermal CD14(+) DCs and naive CD8(+) T cells enhanced the generation of effector CTLs, whereas addition of IL-10 to cultures of LCs and naive CD8(+) T cells inhibited their induction. TGF-ß1 that is transcribed by dermal CD14(+) DCs further enhanced the inhibitory effect of IL-10. Thus, the respective production of IL-15 and IL-10 explains the contrasting effects of LCs and dermal CD14(+) DCs on CD8(+) T-cell priming.
Asunto(s)
Reactividad Cruzada/inmunología , Células Dendríticas/inmunología , Dermis/citología , Interleucina-10/biosíntesis , Interleucina-15/biosíntesis , Células de Langerhans/inmunología , Linfocitos T Citotóxicos/inmunología , Anticuerpos Bloqueadores/farmacología , Linfocitos T CD8-positivos/citología , Linfocitos T CD8-positivos/efectos de los fármacos , Linfocitos T CD8-positivos/inmunología , Diferenciación Celular/efectos de los fármacos , Reactividad Cruzada/efectos de los fármacos , Células Dendríticas/citología , Células Dendríticas/efectos de los fármacos , Humanos , Sinapsis Inmunológicas/efectos de los fármacos , Interleucina-10/farmacología , Interleucina-15/farmacología , Células de Langerhans/citología , Células de Langerhans/efectos de los fármacos , Receptores de Lipopolisacáridos/metabolismo , Linfocitos T Citotóxicos/citología , Linfocitos T Citotóxicos/efectos de los fármacos , Factor de Crecimiento Transformador beta1/farmacologíaRESUMEN
We evaluated human CD8(+) T-cell responses generated by targeting antigens to dendritic cells (DCs) through various lectin receptors. We found the immunoreceptor tyrosine-based inhibitory motif-containing DC immunoreceptor (DCIR) to mediate potent cross-presentation. A single exposure to a low dose of anti-DCIR-antigen conjugate initiated antigen-specific CD8(+) T-cell immunity by all human DC subsets including ex vivo-generated DCs, skin-isolated Langerhans cells, and blood myeloid DCs and plasmacytoid DCs. The delivery of influenza matrix protein (FluMP) through DCIR resulted in expansion of FluMP-specific memory CD8(+) T cells. Enhanced specific CD8(+) T-cell responses were observed when an antigen was delivered to the DCs via DCIR, compared with those induced by a free antigen, or antigen conjugated to a control monoclonal antibody or delivered via DC-SIGN, another lectin receptor. DCIR targeting also induced primary CD8(+) T-cell responses against self (MART-1) and viral (HIV gag) antigens. Addition of Toll-like receptor (TLR) 7/8 agonist enhanced DCIR-mediated cross-presentation as well as cross-priming, particularly when combined with a CD40 signal. TLR7/8 activation was associated with increased expansion of the primed CD8(+) T cells, high production of interferon-γ and tumor necrosis factor-α, and reduced levels of type 2-associated cytokines. Thus, antigen targeting via the human DCIR receptor allows activation of specific CD8(+) T-cell immunity.
Asunto(s)
Antígenos/inmunología , Linfocitos T CD8-positivos/inmunología , Células Dendríticas/inmunología , Lectinas Tipo C/inmunología , Glicoproteínas de Membrana/inmunología , Receptores Inmunológicos/inmunología , Animales , Anticuerpos Monoclonales/inmunología , Antígenos de Neoplasias/inmunología , Linfocitos B/citología , Linfocitos B/inmunología , Linfocitos B/metabolismo , Linfocitos T CD8-positivos/citología , Linfocitos T CD8-positivos/metabolismo , Células Cultivadas , Reactividad Cruzada/efectos de los fármacos , Reactividad Cruzada/inmunología , Células Dendríticas/citología , Células Dendríticas/metabolismo , Citometría de Flujo , Humanos , Células de Langerhans/citología , Células de Langerhans/inmunología , Células de Langerhans/metabolismo , Lectinas Tipo C/metabolismo , Antígeno MART-1 , Glicoproteínas de Membrana/metabolismo , Ratones , Ratones Endogámicos BALB C , Monocitos/citología , Monocitos/inmunología , Monocitos/metabolismo , Proteínas de Neoplasias/inmunología , Quinolinas/farmacología , Receptores Inmunológicos/metabolismo , Tiazoles/farmacología , Receptor Toll-Like 7/agonistas , Receptor Toll-Like 8/agonistas , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/inmunologíaRESUMEN
Common approaches for monitoring T cell responses are limited in their multiplexity and sensitivity. In contrast, deep sequencing of the T Cell Receptor (TCR) repertoire provides a global view that is limited only in terms of theoretical sensitivity due to the depth of available sampling; however, the assignment of antigen specificities within TCR repertoires has become a bottleneck. This study combines antigen-driven expansion, deep TCR sequencing, and a novel analysis framework to show that homologous 'Clusters of Expanded TCRs (CETs)' can be confidently identified without cell isolation, and assigned to antigen against a background of non-specific clones. We show that clonotypes within each CET respond to the same epitope, and that protein antigens stimulate multiple CETs reactive to constituent peptides. Finally, we demonstrate the personalized assignment of antigen-specificity to rare clones within fully-diverse uncultured repertoires. The method presented here may be used to monitor T cell responses to vaccination and immunotherapy with high fidelity.
Asunto(s)
Separación Celular/métodos , Técnicas Inmunológicas/métodos , Receptores de Antígenos de Linfocitos T/análisis , Receptores de Antígenos de Linfocitos T/inmunología , Linfocitos T/inmunología , HumanosRESUMEN
BACKGROUND: Dendritic cells localize throughout the body, where they can sense and capture invading pathogens to induce protective immunity. Hence, harnessing the biology of tissue-resident dendritic cells is fundamental for the rational design of vaccines against pathogens. METHODS: Herein, we characterized the transcriptomes of four antigen-presenting cell subsets from the human vagina (Langerhans cells, CD14(-) and CD14(+) dendritic cells, macrophages) by microarray, at both the transcript and network level, and compared them to those of three skin dendritic cell subsets and blood myeloid dendritic cells. RESULTS: We found that genomic fingerprints of antigen-presenting cells are significantly influenced by the tissue of origin as well as by individual subsets. Nonetheless, CD14(+) populations from both vagina and skin are geared towards innate immunity and pro-inflammatory responses, whereas CD14(-) populations, particularly skin and vaginal Langerhans cells, and vaginal CD14(-) dendritic cells, display both Th2-inducing and regulatory phenotypes. We also identified new phenotypic and functional biomarkers of vaginal antigen-presenting cell subsets. CONCLUSIONS: We provide a transcriptional database of 87 microarray samples spanning eight antigen-presenting cell populations in the human vagina, skin and blood. Altogether, these data provide molecular information that will further help characterize human tissue antigen-presenting cell lineages and their functions. Data from this study can guide the design of mucosal vaccines against sexually transmitted pathogens.
RESUMEN
Dendritic cells (DCs) loaded with killed allogeneic tumors can cross-prime tumor-specific naive CD8 T cells in vitro, thereby providing an option to overcome human leukocyte antigen restriction inherent to loading DC vaccines with peptides. We have vaccinated 20 patients with stage IV melanoma with autologous monocyte-derived DCs loaded with killed allogeneic Colo829 melanoma cell line. DCs were generated by culturing monocytes with granulocyte macrophage-colony stimulating factor (granulocyte macrophage-colony stimulating factor) and interleukin (IL-4) and activated by additional culture with tumor necrosis factor and CD40 ligand. A total of 8 vaccines were administered at monthly intervals. The first patient was accrued December 2002 and the last November 2003. Fourteen patients were alive at 12 months, 9 patients were alive at 24 months, and 8 patients are alive as of January 2006. The estimated median overall survival is 22.5 months with a range of 2 to 35.5 months. Vaccinations were safe and tolerable. They induced, in 2 patients who failed previous therapy, durable objective clinical responses, 1 complete regression (CR) and 1 partial regression (PR) lasting 18 and 23 months, respectively. Three out of 13 analyzed patients showed T-cell immunity to melanoma antigen recognized by autologous T cells (MART-1) tissue differentiation antigen. Two of 3 patients showed improved immune function after vaccinations demonstrated by improved secretion of interferon (IFN)-gamma or T-cell proliferation in response to MART-1 derived peptides. In one of these patients, vaccination led to elicitation of CD8 T-cell immunity specific to a novel peptide-derived from MART-1 antigen, suggesting that cross-priming/presentation of melanoma antigens by DC vaccine had occurred. Thus, the present results justify the design of larger follow-up studies to assess the clinical response to DC vaccines loaded with killed allogeneic tumor cells in patients with metastatic melanoma.
Asunto(s)
Antígenos de Neoplasias/inmunología , Linfocitos T CD8-positivos/inmunología , Vacunas contra el Cáncer/uso terapéutico , Células Dendríticas/inmunología , Melanoma/terapia , Proteínas de Neoplasias/inmunología , Neoplasias Cutáneas/terapia , Adulto , Anciano , Vacunas contra el Cáncer/efectos adversos , Diferenciación Celular , Línea Celular Tumoral , Reactividad Cruzada , Citotoxicidad Inmunológica , Células Dendríticas/citología , Antígeno HLA-A2/inmunología , Humanos , Inmunoterapia Adoptiva , Interferón gamma/metabolismo , Antígeno MART-1 , Melanoma/inmunología , Melanoma/patología , Persona de Mediana Edad , Monocitos/citología , Monocitos/inmunología , Metástasis de la Neoplasia , Péptidos/inmunología , Neoplasias Cutáneas/inmunología , Neoplasias Cutáneas/patología , Tasa de Supervivencia , Trasplante HomólogoRESUMEN
Cytokines, most particularly TNF and type I IFN (IFN-alphabeta), have been long considered essential elements in the development of autoimmunity. Identification of TNF in the pathogenesis of rheumatoid arthritis and TNF antagonist therapy represent successes of immunology. IFN-alphabeta plays a major role in systemic lupus erythematosus (SLE), a prototype autoimmune disease characterized by a break of tolerance to nuclear components. Here, we show that TNF regulates IFN-alpha production in vitro at two levels. First, it inhibits the generation of plasmacytoid dendritic cells (pDCs), a major producer of IFN-alphabeta, from CD34+ hematopoietic progenitors. Second, it inhibits IFN-alpha release by immature pDCs exposed to influenza virus. Neutralization of endogenous TNF sustains IFN-alpha secretion by pDCs. These findings are clinically relevant, as five of five patients with systemic juvenile arthritis treated with TNF antagonists display overexpression of IFN-alpha-regulated genes in their blood leukocytes. These results, therefore, might provide a mechanistic explanation for the development of anti-dsDNA antibodies and lupus-like syndrome in patients undergoing anti-TNF therapy.
Asunto(s)
Enfermedades Autoinmunes/inmunología , Interferón-alfa/fisiología , Factor de Necrosis Tumoral alfa/fisiología , Humanos , Transcripción GenéticaRESUMEN
Twenty-two HLA A*0201 patients with stage IV melanoma were enrolled in a phase 1 safety and feasibility trial using a composite dendritic cell (DC) vaccine generated by culturing CD34 hematopoietic progenitors and activated with IFN-alpha. The DC vaccine was loaded with peptides derived from four melanoma tissue differentiation antigens (MART-1, tyrosinase, MAGE-3, and gp100) and influenza matrix peptide (Flu-MP). Twenty patients were evaluable, 14 of whom received vaccination with peptide-pulsed DCs without keyhole limpet hemocyanin (KLH) and 6 of whom received vaccination with KLH-loaded DCs. Patients were vaccinated until disease progression or until they had received eight vaccinations. None of the analyzed patients showed the expansion of melanoma-peptide-specific circulating effector memory T cells that secrete IFN-gamma in direct ELISPOT. Melanoma-peptide-specific recall memory CD8 T cells able to secrete IFN-gamma and to proliferate could be detected in six of the seven analyzed patients. There were no objective clinical responses. The estimated median overall survival was 12 months (range 2-38), and the median event-free survival was 4 months (range 1-12). There was no statistically significant survival advantage in patients who received KLH-loaded vaccines. As of March 2005, four patients remained alive, 26+, 28+, 28+, and 36+ months. Three of them had received KLH-loaded vaccines and all of them had had additional therapy. Overall, these results suggest that IFN-alpha-activated CD34-DCs are safe but elicit only limited immune responses, underscoring the need to test different DC maturation factors.
Asunto(s)
Antígenos CD34/biosíntesis , Vacunas contra el Cáncer , Células Dendríticas/citología , Interferón Tipo I/uso terapéutico , Melanoma/terapia , Células Madre/citología , Adulto , Antígenos de Neoplasias/biosíntesis , Antígenos de Neoplasias/química , Proliferación Celular , Progresión de la Enfermedad , Supervivencia sin Enfermedad , Ensayo de Inmunoadsorción Enzimática , Antígenos HLA-A/biosíntesis , Antígeno HLA-A2 , Humanos , Inmunoterapia Adoptiva/métodos , Virus de la Influenza A/química , Interferón-alfa/metabolismo , Interferón gamma/metabolismo , Antígeno MART-1 , Melanoma/inmunología , Glicoproteínas de Membrana/biosíntesis , Persona de Mediana Edad , Monofenol Monooxigenasa/biosíntesis , Proteínas de Neoplasias/biosíntesis , Fragmentos de Péptidos/química , Péptidos/uso terapéutico , Factores de Tiempo , Resultado del Tratamiento , Proteínas de la Matriz Viral/química , Antígeno gp100 del MelanomaRESUMEN
Dendritic cells (DCs) initiate and control immune responses. Plasmacytoid DCs (pDCs) represent a unique DC subset able to promptly release large amounts of type I interferon (IFN-alphabeta) upon viral encounter. Here we report that depletion of pDCs from human blood mononuclear cells abrogates the secretion of specific and polyclonal IgGs in response to influenza virus. Furthermore, purified pDCs triggered with virus induce CD40-activated B cells to differentiate into plasma cells. Two pDC cytokines act sequentially, with IFN-alphabeta generating non-Ig-secreting plasma blasts and IL-6 inducing their differentiation into Ig-secreting plasma cells. These plasma cells display the high levels of CD38 found on tissue plasma cells. Thus, pDCs are critical for the generation of plasma cells and antibody responses.
Asunto(s)
Células Dendríticas/citología , Células Dendríticas/inmunología , Interferón Tipo I/metabolismo , Interleucina-6/metabolismo , Células Plasmáticas/citología , Células Plasmáticas/inmunología , Adulto , Linfocitos B/inmunología , Antígenos CD40/metabolismo , Comunicación Celular , Diferenciación Celular , Humanos , Inmunoglobulinas/biosíntesis , Técnicas In Vitro , Activación de LinfocitosRESUMEN
Distinct human dendritic cell (DC) subsets differentially control immunity. Thus, insights into their in vivo functions are important to understand the launching and modulation of immune responses. We show that nonobese diabetic/LtSz-scid/scid (NOD/SCID) mice engrafted with human CD34+ hematopoietic progenitors develop human myeloid and plasmacytoid DCs. The skin displays immature DCs expressing Langerin, while other tissues display interstitial DCs. Myeloid DCs from these mice induce proliferation of allogeneic CD4 T cells in vitro, and bone marrow human cells containing plasmacytoid DCs release interferon-alpha (IFN-alpha) upon influenza virus exposure. Injection of influenza virus into reconstituted mice triggers IFN-alpha release and maturation of mDCs. Thus, these mice may provide a model to study the pathophysiology of human DC subsets.