RESUMO
Harnessing innate immunity is emerging as a promising therapeutic approach in cancer. We report here the design of tetraspecific molecules engaging natural killer (NK) cell-activating receptors NKp46 and CD16a, the ß-chain of the interleukin-2 receptor (IL-2R), and a tumor-associated antigen (TAA). In vitro, these tetraspecific antibody-based natural killer cell engager therapeutics (ANKETs) induce a preferential activation and proliferation of NK cells, and the binding to the targeted TAA triggers NK cell cytotoxicity and cytokine and chemokine production. In vivo, tetraspecific ANKETs induce NK cell proliferation and their accumulation at the tumor bed, as well as the control of local and disseminated tumors. Treatment of non-human primates with CD20-directed tetraspecific ANKET leads to CD20+ circulating B cell depletion, with minimal systemic cytokine release and no sign of toxicity. Tetraspecific ANKETs, thus, constitute a technological platform for harnessing NK cells as next-generation cancer immunotherapies.
Assuntos
Interleucina-2 , Neoplasias , Animais , Interleucina-2/genética , Células Matadoras Naturais , Receptores de Interleucina-2/metabolismo , Citocinas , Neoplasias/genética , Quimiocinas/metabolismoRESUMO
Since their discovery, innate lymphoid cells (ILCs) have gradually been gaining greater relevance in the field of immunology due to their multiple functions in the innate immune response. They can mainly be found in mucosal and barrier organs like skin, gut, and lungs, and have been classified into five main types (NKs, ILC1s, ILC2s, ILC3s, and Lti cells) according to their function and development. They all play major roles in functions such as tissue homeostasis, early pathogen defense, regulation of inflammation, or tissue remodeling. ILCs are mostly tissue-resident cells tightly bound to the tissue structure, a fact that requires long and complex protocols that do not always provide sufficient yield for analysis. This suggests the need for optimized approaches aimed at ensuring that enriched and viable ILC samples are obtained, in order to furnish quality results. Herein a detailed protocol is established for obtaining a single-cell suspension highly enriched in lymphoid cells from mouse gut in order to identify the different subsets of ILCs by means of flow cytometry. The cell marker panel and flow cytometry gating strategies for identification and quantification of all the different ILC populations are provided for simultaneous analysis. Moreover, the protocol described includes a procedure for studying the different cytokines produced by ILC3s involved in maintaining the integrity of the gut barrier and defending against extracellular pathogens. As a result, herein an efficient method is presented for studying mouse ILCs within the lamina propria of the small intestine and colon; this can constitute a useful tool for future investigations in the field.
Assuntos
Separação Celular/métodos , Colo/imunologia , Imunidade Inata , Mucosa Intestinal/citologia , Mucosa Intestinal/imunologia , Intestino Delgado/imunologia , Linfócitos/imunologia , Animais , Citocinas/metabolismo , Citometria de Fluxo , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BLRESUMO
During embryogenesis, lymphoid tissue inducer (LTi) cells are essential for lymph node organogenesis. These cells are part of the innate lymphoid cell (ILC) family. Although their earliest embryonic hematopoietic origin is unclear, other innate immune cells have been shown to be derived from early hemogenic endothelium in the yolk sac as well as the aorta-gonad-mesonephros. A proper model to discriminate between these locations was unavailable. In this study, using a Cxcr4-CreERT2 lineage tracing model, we identify a major contribution from embryonic hemogenic endothelium, but not the yolk sac, toward LTi progenitors. Conversely, embryonic LTi cells are replaced by hematopoietic stem cell-derived cells in adults. We further show that, in the fetal liver, common lymphoid progenitors differentiate into highly dynamic alpha-lymphoid precursor cells that, at this embryonic stage, preferentially mature into LTi precursors and establish their functional LTi cell identity only after reaching the periphery.
Assuntos
Hemangioblastos/metabolismo , Hematopoese/fisiologia , Tecido Linfoide/embriologia , Receptores CXCR4/metabolismo , Animais , Desenvolvimento Embrionário/fisiologia , Hemangioblastos/citologia , Células-Tronco Hematopoéticas/metabolismo , Humanos , Imunidade Inata , Fígado/embriologia , Linfócitos/metabolismo , Linfócitos T Auxiliares-Indutores/metabolismo , Saco Vitelino/embriologiaRESUMO
In humans, psychological stress has been associated with a higher risk of infectious illness. However, the mechanisms by which the stress pathway interferes with host response to pathogens remain unclear. We demonstrate here a role for the ß2-adrenergic receptor (ß2-AR), which binds the stress mediators adrenaline and noradrenaline, in modulating host response to mouse cytomegalovirus (MCMV) infection. Mice treated with a ß2-AR agonist were more susceptible to MCMV infection. By contrast, ß2-AR deficiency resulted in a better clearance of the virus, less tissue damage, and greater resistance to MCMV. Mechanistically, we found a correlation between higher levels of IFN-γ production by liver natural killer (NK) cells and stronger resistance to MCMV. However, the control of NK cell IFN-γ production was not cell intrinsic, revealing a cell-extrinsic downregulation of the antiviral NK cell response by adrenergic neuroendocrine signals. This pathway reduces host immune defense, suggesting that the blockade of the ß2-AR signaling could be used to increase resistance to infectious diseases.
Assuntos
Infecções por Citomegalovirus/imunologia , Regulação para Baixo/imunologia , Imunidade Inata/imunologia , Receptores Adrenérgicos beta 2/imunologia , Transdução de Sinais/imunologia , Animais , Epinefrina/imunologia , Interferon gama/imunologia , Células Matadoras Naturais/imunologia , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Muromegalovirus/imunologia , Norepinefrina/imunologiaRESUMO
Natural killer (NK) cells are innate lymphoid cells (ILCs) involved in antimicrobial and antitumoral responses. Several NK cell subsets have been reported in humans and mice, but their heterogeneity across organs and species remains poorly characterized. We assessed the diversity of human and mouse NK cells by single-cell RNA sequencing on thousands of individual cells isolated from spleen and blood. Unbiased transcriptional clustering revealed two distinct signatures differentiating between splenic and blood NK cells. This analysis at single-cell resolution identified three subpopulations in mouse spleen and four in human spleen, and two subsets each in mouse and human blood. A comparison of transcriptomic profiles within and between species highlighted the similarity of the two major subsets, NK1 and NK2, across organs and species. This unbiased approach provides insight into the biology of NK cells and establishes a rationale for the translation of mouse studies to human physiology and disease.
Assuntos
Células Matadoras Naturais/metabolismo , Subpopulações de Linfócitos/metabolismo , Transcriptoma , Animais , Biomarcadores , Biologia Computacional/métodos , Perfilação da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Imunidade Inata , Imunofenotipagem , Células Matadoras Naturais/imunologia , Subpopulações de Linfócitos/imunologia , Camundongos , Especificidade de Órgãos/genética , Especificidade de Órgãos/imunologia , Fenótipo , Análise de Célula ÚnicaRESUMO
Biological redundancy ensures robustness in living organisms at several levels, from genes to organs. In this review, we explore the concept of redundancy and robustness through an analysis of the caecal appendix, an organ that is often considered to be a redundant remnant of evolution. However, phylogenic data show that the Appendix was selected during evolution and is unlikely to disappear once it appeared. In humans, it is highly conserved and malformations are extremely rare, suggesting a role for that structure. The Appendix could perform a dual role. First, it is a concentrate of lymphoid tissue resembling Peyer's patches and is the primary site for immunoglobulin A production which is crucial to regulate the density and quality of the intestinal flora. Second, given its shape and position, the Appendix could be a unique niche for commensal bacteria in the body. It is extremely rich in biofilms that continuously shed bacteria into the intestinal lumen. The Appendix contains a microbiota as diverse as that found in the colon and could replenish the large intestine with healthy flora after a diarrhea episode. In conditions of modern medicine hygiene, and people live healthy without their appendix. However, several reports suggest that the effects of appendectomy could be subtler and associated with the development of inflammatory conditions such as inflammatory bowel disease (IBD), heart disease but also in less expected disorders such as Parkinson's disease. Lack of an Appendix also predicts a worsen outcome for recurrent Clostridium difficile infection, which is the first nosocomial infection in hospitals. Here, we review the literature and in combination with our own data, we suggest that the Appendix might be redundant in its immunological function but unique as a reservoir of microbiota.
Assuntos
Apêndice/imunologia , Microbioma Gastrointestinal/imunologia , Cardiopatias/imunologia , Doenças Inflamatórias Intestinais/imunologia , Tecido Linfoide/imunologia , Doença de Parkinson/imunologia , Complicações Pós-Operatórias/imunologia , Animais , Apendicectomia , Biofilmes , Evolução Biológica , Cardiopatias/etiologia , Humanos , Imunoglobulina A/biossíntese , Doenças Inflamatórias Intestinais/etiologia , Doença de Parkinson/etiologia , Nódulos Linfáticos Agregados/imunologia , FilogeniaRESUMO
Innate Lymphoid Cells (ILC) are tissue-resident effector lymphocytes that differ from the B and T lymphocytes subsets since they lack specific antigen receptors derived from gene rearrangements. They can be divided into two groups, cytotoxic ILC (NK cells) which are dependent on the transcription factors Eomes and T-bet and produce IFN-γ, and helper-like ILC (ILC1, ILC2, ILC3) which are dependent on T-bet, GATA-3 or RORγT and produce IFN-γ, IL-5/IL-13 or IL-17/IL-22 respectively. Although they were described recently, ILC are the subject of a large number of studies that characterize their role as one of the first effectors of the innate immune response as well as in the development of a wide number of inflammatory disorders.
Assuntos
Imunidade Inata/imunologia , Linfócitos/fisiologia , Animais , Humanos , Células Matadoras Naturais/fisiologia , Subpopulações de Linfócitos/fisiologiaRESUMO
Group 3 innate lymphoid cells (ILC3s) are composed of subsets that are either positive or negative for the natural cytotoxicity receptor (NCR) NKp46 (encoded by Ncr1). ILC3s are located at mucosal sites, such as in the intestine and lung, where they are exposed to billions of commensal microbes and potentially harmful pathogens. Together with T cells, the various ILC3 subsets maintain the balance between homeostasis and immune activation. Through genetic mapping, we identified a previously uncharacterized subset of NCR(-) ILC3s in mice that transiently express Ncr1, demonstrating previously undescribed heterogeneity within the ILC3 population. In addition, we showed that sustained Notch signaling was required for the maintenance of the NCR(+) phenotype and that the cytokine transforming growth factor-ß (TGF-ß) impaired the development of NCR(+) ILC3s. Thus, the plasticity of ILC3s is regulated by the balance between the opposing effects of Notch and TGF-ß signaling, maintaining homeostasis in the face of continual challenges.
Assuntos
Linfócitos/citologia , Receptor Notch1/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Animais , Antígenos Ly/metabolismo , Diferenciação Celular , Citocinas/metabolismo , Feminino , Imunidade Inata , Mucosa Intestinal/metabolismo , Ligantes , Pulmão/metabolismo , Masculino , Camundongos , Camundongos Transgênicos , Receptor 1 Desencadeador da Citotoxicidade Natural/metabolismo , Transdução de Sinais , Linfócitos T/citologia , Transcrição GênicaRESUMO
Although IL-10 promotes a regulatory phenotype of CD11c+ dendritic cells and macrophages in vitro, the role of IL-10 signaling in CD11c+ cells to maintain intestinal tolerance in vivo remains elusive. To this aim, we generated mice with a CD11c-specific deletion of the IL-10 receptor alpha (Cd11ccreIl10rafl/fl). In contrast to the colon, the small intestine of Cd11ccreIl10rafl/fl mice exhibited spontaneous crypt hyperplasia, increased numbers of intraepithelial lymphocytes and lamina propria T cells, associated with elevated levels of T cell-derived IFNγ and IL-17A. Whereas naive mucosal T-cell priming was not affected and oral tolerance to ovalbumin was intact, augmented T-cell function in the lamina propria was associated with elevated numbers of locally dividing T cells, expression of T-cell attracting chemokines and reduced T-cell apoptosis. Upon stimulation, intestinal IL-10Rα deficient CD11c+ cells exhibited increased activation associated with enhanced IL-6 and TNFα production. Following colonization with Helicobacter hepaticus Cd11ccreIl10rafl/fl mice developed severe large intestinal inflammation characterized by infiltrating T cells and increased levels of Il17a, Ifng, and Il12p40. Altogether these findings demonstrate a critical role of IL-10 signaling in CD11c+ cells to control small intestinal immune homeostasis by limiting reactivation of local memory T cells and to protect against Helicobacter hepaticus-induced colitis.
Assuntos
Antígeno CD11c/metabolismo , Colite/prevenção & controle , Infecções por Helicobacter/prevenção & controle , Imunidade nas Mucosas , Interleucina-10/metabolismo , Mucosa Intestinal/metabolismo , Intestino Grosso/metabolismo , Intestino Delgado/metabolismo , Linfócitos T/metabolismo , Animais , Antígeno CD11c/deficiência , Antígeno CD11c/genética , Antígeno CD11c/imunologia , Colite/imunologia , Colite/metabolismo , Colite/microbiologia , Modelos Animais de Doenças , Predisposição Genética para Doença , Infecções por Helicobacter/imunologia , Infecções por Helicobacter/metabolismo , Infecções por Helicobacter/microbiologia , Helicobacter hepaticus/imunologia , Helicobacter hepaticus/patogenicidade , Homeostase , Memória Imunológica , Interferon gama/imunologia , Interferon gama/metabolismo , Interleucina-10/imunologia , Subunidade alfa de Receptor de Interleucina-10/genética , Subunidade alfa de Receptor de Interleucina-10/imunologia , Subunidade alfa de Receptor de Interleucina-10/metabolismo , Interleucina-17/imunologia , Interleucina-17/metabolismo , Mucosa Intestinal/imunologia , Mucosa Intestinal/microbiologia , Intestino Grosso/imunologia , Intestino Grosso/microbiologia , Intestino Delgado/imunologia , Intestino Delgado/microbiologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fenótipo , Transdução de Sinais , Linfócitos T/imunologia , Linfócitos T/microbiologiaRESUMO
Intestinal T cells and group 3 innate lymphoid cells (ILC3 cells) control the composition of the microbiota and gut immune responses. Within the gut, ILC3 subsets coexist that either express or lack the natural cytoxicity receptor (NCR) NKp46. We identified here the transcriptional signature associated with the transcription factor T-bet-dependent differentiation of NCR(-) ILC3 cells into NCR(+) ILC3 cells. Contrary to the prevailing view, we found by conditional deletion of the key ILC3 genes Stat3, Il22, Tbx21 and Mcl1 that NCR(+) ILC3 cells were redundant for the control of mouse colonic infection with Citrobacter rodentium in the presence of T cells. However, NCR(+) ILC3 cells were essential for cecal homeostasis. Our data show that interplay between intestinal ILC3 cells and adaptive lymphocytes results in robust complementary failsafe mechanisms that ensure gut homeostasis.
Assuntos
Imunidade Inata , Interleucinas/biossíntese , Linfócitos/imunologia , Linfócitos/metabolismo , Animais , Citrobacter rodentium/imunologia , Análise por Conglomerados , Modelos Animais de Doenças , Infecções por Enterobacteriaceae/genética , Infecções por Enterobacteriaceae/imunologia , Infecções por Enterobacteriaceae/metabolismo , Infecções por Enterobacteriaceae/mortalidade , Infecções por Enterobacteriaceae/patologia , Feminino , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Homeostase , Subpopulações de Linfócitos/imunologia , Subpopulações de Linfócitos/metabolismo , Masculino , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Proteína de Sequência 1 de Leucemia de Células Mieloides/deficiência , Proteína de Sequência 1 de Leucemia de Células Mieloides/genética , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Receptor 1 Desencadeador da Citotoxicidade Natural/metabolismo , Transdução de Sinais , Proteínas com Domínio T/deficiência , Proteínas com Domínio T/genética , Proteínas com Domínio T/metabolismo , Transcriptoma , Interleucina 22Assuntos
Células Dendríticas/imunologia , Memória Imunológica/imunologia , Interleucina-10/metabolismo , Leishmania major/imunologia , Leishmaniose Cutânea/imunologia , Animais , Modelos Animais de Doenças , Interleucina-10/imunologia , Leishmania major/patogenicidade , Leishmaniose Cutânea/fisiopatologia , Camundongos , Camundongos Endogâmicos C57BL , Distribuição Aleatória , Transdução de Sinais/imunologiaRESUMO
Interleukin-10 (IL-10) is a potent immunomodulatory cytokine, whose cellular targets have not yet been precisely identified. Dendritic cell (DC)-specific IL-10 receptor knockout mice exhibit exaggerated T-cell reactivation in the skin, highlighting a key role of DCs in the maintenance of local immune homeostasis, beyond their classical function as regulators of T-cell priming in lymph nodes.
RESUMO
IL-10 is an anti-inflammatory cytokine that regulates the extent of host immunity to infection by exerting suppressive effects on different cell types. Herpes viruses induce IL-10 to modulate the virus-host balance towards their own benefit, resulting in prolonged virus persistence. To define the cellular and molecular players involved in IL-10 modulation of herpes virus-specific immunity, we studied mouse cytomegalovirus (MCMV) infection. Here we demonstrate that IL-10 specifically curtails the MCMV-specific CD4 T cell response by suppressing the bidirectional crosstalk between NK cells and myeloid dendritic cells (DCs). In absence of IL-10, NK cells licensed DCs to effectively prime MCMV-specific CD4 T cells and we defined the pro-inflammatory cytokines IL-12, IFN-γ and TNF-α as well as NK cell activating receptors NKG2D and NCR-1 to regulate this bidirectional NK/DC interplay. Consequently, markedly enhanced priming of MCMV-specific CD4 T cells in Il10(-/-) mice led to faster control of lytic viral replication, but this came at the expense of TNF-α mediated immunopathology. Taken together, our data show that early induction of IL-10 during MCMV infection critically regulates the strength of the innate-adaptive immune cell crosstalk, thereby impacting beneficially on the ensuing virus-host balance for both the virus and the host.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Células Dendríticas/imunologia , Infecções por Herpesviridae/imunologia , Interleucina-10/imunologia , Células Matadoras Naturais/imunologia , Muromegalovirus/fisiologia , Replicação Viral/imunologia , Animais , Linfócitos T CD4-Positivos/patologia , Citocinas/genética , Citocinas/imunologia , Células Dendríticas/patologia , Infecções por Herpesviridae/genética , Infecções por Herpesviridae/patologia , Interleucina-10/genética , Células Matadoras Naturais/metabolismo , Camundongos , Camundongos KnockoutRESUMO
BACKGROUND: IL-10 is a pleiotropic cytokine and potent negative regulator of both innate and adaptive immune responses. Consequently, IL-10-deficient (IL-10(-/-)) mice have enhanced contact hypersensitivity (CHS) to topical hapten. OBJECTIVE: Although the importance of IL-10 production by (regulatory) T cells and Langerhans cells in regulating CHS has been established by cell type-specific il-10 gene targeting, it remains elusive to what extent IL-10 controls dendritic cell (DC) function in vivo. METHODS: To this aim, we generated mice with a DC-specific deletion of the IL-10 receptor (IL-10R). RESULTS: Despite the ability of IL-10 to inhibit DC maturation in vitro, DCs of resting DC-IL10R(-/-) mice retained their immature phenotype in vivo. In contrast, IL-10R(-/-) DCs produced increased levels of proinflammatory cytokines and IL-10 after in vitro stimulation. Induction of CHS was indistinguishable from that seen in control mice at 24 hours after hapten challenge but resulted in increased ear swelling at 48 hours and delayed resolution of the inflammatory reaction. Adoptive T-cell transfer experiments revealed that only T-cell reactivation and not sensitization by IL-10R(-/-) DCs leads to enhanced CHS. Accordingly, the expression of proinflammatory cytokines and IL-10 was augmented in the skin of DC-IL10R(-/-) mice after hapten challenge. CONCLUSION: Our data demonstrate that IL-10 signaling in DCs is dispensable during naive T-cell priming but is critical to prevent an exaggerated effector T-cell response in the skin.
Assuntos
Células Dendríticas/imunologia , Dermatite de Contato/imunologia , Interleucina-10/fisiologia , Ativação Linfocitária/imunologia , Pele/imunologia , Linfócitos T/imunologia , Animais , Citocinas/biossíntese , Células Dendríticas/metabolismo , Dermatite de Contato/genética , Deleção de Genes , Imunidade Inata , Camundongos , Camundongos Knockout , Fenótipo , Receptores de Interleucina-10/genética , Transdução de Sinais , Pele/metabolismoRESUMO
The pivotal role of TGF-beta in Langerhans cell (LC) development has been previously established in TGF-beta-deficient mice, which lack epidermal LCs. As to whether TGF-beta also governs LC homeostasis and function remains elusive. To assess the role of TGF-beta-mediated control of cutaneous dendritic cells (DCs) in vivo, we generated mice with a conditional knockout of the TGF-beta receptor 1 (TbetaR1) under a DC-specific promoter (DC-TbetaR1(del) mice). While initial LC seeding occurred in DC-TbetaR1(del) mice, the cells disappeared from the epidermis during the first week of life. TbetaR1-deficient LCs demonstrated spontaneous maturation and gained migratory potential based on increased surface expression of MHC class II, costimulatory molecules, and CCR7 and downregulation of E-cadherin. In parallel to their early loss from the epidermis, migrating LCs were reduced in the dermis and skin-draining lymph nodes of adult DC-TbetaR1(del) mice, whereas the number of Langerin(+) dermal DCs was similar to wild-type. In the absence of LCs, low-dose contact hypersensitivity in DC-TbetaR1(del) mice was significantly diminished. In contrast, ear swelling was restored to wild-type levels when a higher hapten dose was applied to efficiently target TbetaR1-deficient dermal DCs. In conclusion, TGF-beta inhibits in vivo LC maturation and migratory phenotype, identifying TGF-beta as a critical factor controlling LC homeostasis in the steady state.