RESUMO
Wnt ligands oligomerize Frizzled (Fzd) and Lrp5/6 receptors to control the specification and activity of stem cells in many species. How Wnt signaling is selectively activated in different stem cell populations, often within the same organ, is not understood. In lung alveoli, we show that distinct Wnt receptors are expressed by epithelial (Fzd5/6), endothelial (Fzd4), and stromal (Fzd1) cells. Fzd5 is uniquely required for alveolar epithelial stem cell activity, whereas fibroblasts utilize distinct Fzd receptors. Using an expanded repertoire of Fzd-Lrp agonists, we could activate canonical Wnt signaling in alveolar epithelial stem cells via either Fzd5 or, unexpectedly, non-canonical Fzd6. A Fzd5 agonist (Fzd5ag) or Fzd6ag stimulated alveolar epithelial stem cell activity and promoted survival in mice after lung injury, but only Fzd6ag promoted an alveolar fate in airway-derived progenitors. Therefore, we identify a potential strategy for promoting regeneration without exacerbating fibrosis during lung injury.
Assuntos
Lesão Pulmonar , Camundongos , Animais , Proteínas Wnt , Receptores Frizzled , Via de Sinalização Wnt , Células Epiteliais Alveolares , Células-TroncoRESUMO
Dysregulated microglia are intimately involved in neurodegeneration, including Alzheimer's disease (AD) pathogenesis, but the mechanisms controlling pathogenic microglial gene expression remain poorly understood. The transcription factor CCAAT/enhancer binding protein beta (c/EBPß) regulates pro-inflammatory genes in microglia and is upregulated in AD. We show expression of c/EBPß in microglia is regulated post-translationally by the ubiquitin ligase COP1 (also called RFWD2). In the absence of COP1, c/EBPß accumulates rapidly and drives a potent pro-inflammatory and neurodegeneration-related gene program, evidenced by increased neurotoxicity in microglia-neuronal co-cultures. Antibody blocking studies reveal that neurotoxicity is almost entirely attributable to complement. Remarkably, loss of a single allele of Cebpb prevented the pro-inflammatory phenotype. COP1-deficient microglia markedly accelerated tau-mediated neurodegeneration in a mouse model where activated microglia play a deleterious role. Thus, COP1 is an important suppressor of pathogenic c/EBPß-dependent gene expression programs in microglia.
Assuntos
Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Ligases/metabolismo , Microglia/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina/genética , Doença de Alzheimer/metabolismo , Animais , Linhagem Celular , Técnicas de Cocultura/métodos , Feminino , Expressão Gênica/fisiologia , Regulação da Expressão Gênica/fisiologia , Células HEK293 , Humanos , Inflamação/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/metabolismoRESUMO
Tumor-associated macrophages are composed of distinct populations arising from monocytes or tissue macrophages, with a poorly understood link to disease pathogenesis. Here, we demonstrate that mouse monocyte migration was supported by glutaminyl-peptide cyclotransferase-like (QPCTL), an intracellular enzyme that mediates N-terminal modification of several substrates, including the monocyte chemoattractants CCL2 and CCL7, protecting them from proteolytic inactivation. Knockout of Qpctl disrupted monocyte homeostasis, attenuated tumor growth and reshaped myeloid cell infiltration, with loss of monocyte-derived populations with immunosuppressive and pro-angiogenic profiles. Antibody targeting of the receptor CSF1R, which more broadly eliminates tumor-associated macrophages, reversed tumor growth inhibition in Qpctl-/- mice and prevented lymphocyte infiltration. Modulation of QPCTL synergized with anti-PD-L1 to expand CD8+ T cells and limit tumor growth. QPCTL inhibition constitutes an effective approach for myeloid cell-targeted cancer immunotherapy.
Assuntos
Aminoaciltransferases , Linfócitos T CD8-Positivos , Quimiocinas , Neoplasias , Aminoaciltransferases/genética , Aminoaciltransferases/metabolismo , Animais , Linfócitos T CD8-Positivos/patologia , Quimiocinas/metabolismo , Imunoterapia , Infiltração Leucêmica , Camundongos , Camundongos Knockout , Monócitos , Neoplasias/imunologiaRESUMO
Estrogen receptor-positive (ER+) breast cancers frequently remain dependent on ER signaling even after acquiring resistance to endocrine agents, prompting the development of optimized ER antagonists. Fulvestrant is unique among approved ER therapeutics due to its capacity for full ER antagonism, thought to be achieved through ER degradation. The clinical potential of fulvestrant is limited by poor physicochemical features, spurring attempts to generate ER degraders with improved drug-like properties. We show that optimization of ER degradation does not guarantee full ER antagonism in breast cancer cells; ER "degraders" exhibit a spectrum of transcriptional activities and anti-proliferative potential. Mechanistically, we find that fulvestrant-like antagonists suppress ER transcriptional activity not by ER elimination, but by markedly slowing the intra-nuclear mobility of ER. Increased ER turnover occurs as a consequence of ER immobilization. These findings provide proof-of-concept that small molecule perturbation of transcription factor mobility may enable therapeutic targeting of this challenging target class.
Assuntos
Neoplasias da Mama/metabolismo , Antagonistas do Receptor de Estrogênio/farmacologia , Fulvestranto/farmacologia , Receptores de Estrogênio/antagonistas & inibidores , Receptores de Estrogênio/metabolismo , Animais , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Proliferação de Células/efeitos dos fármacos , Cinamatos/farmacologia , Resistencia a Medicamentos Antineoplásicos , Antagonistas do Receptor de Estrogênio/uso terapêutico , Feminino , Fulvestranto/uso terapêutico , Células HEK293 , Xenoenxertos , Humanos , Indazóis/farmacologia , Ligantes , Células MCF-7 , Camundongos , Camundongos Endogâmicos NOD , Camundongos Nus , Camundongos SCID , Polimorfismo de Nucleotídeo Único , Proteólise/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Transcrição Gênica/efeitos dos fármacosRESUMO
Fibroblastic reticular cells (FRCs) are specialized stromal cells that define tissue architecture and regulate lymphocyte compartmentalization, homeostasis, and innate and adaptive immunity in secondary lymphoid organs (SLOs). In the present study, we used single-cell RNA sequencing (scRNA-seq) of human and mouse lymph nodes (LNs) to identify a subset of T cell-zone FRCs defined by the expression of Gremlin1 (Grem1) in both species. Grem1-CreERT2 knock-in mice enabled localization, multi-omics characterization and genetic depletion of Grem1+ FRCs. Grem1+ FRCs primarily localize at T-B cell junctions of SLOs, neighboring pre-dendritic cells and conventional dendritic cells (cDCs). As such, their depletion resulted in preferential loss and decreased homeostatic proliferation and survival of resident cDCs and compromised T cell immunity. Trajectory analysis of human LN scRNA-seq data revealed expression similarities to murine FRCs, with GREM1+ cells marking the endpoint of both trajectories. These findings illuminate a new Grem1+ fibroblastic niche in LNs that functions to maintain the homeostasis of lymphoid tissue-resident cDCs.
Assuntos
Células Dendríticas Foliculares/imunologia , Fibroblastos/imunologia , Linfonodos/imunologia , Células Estromais/imunologia , Idoso , Animais , Apoptose/genética , Apoptose/imunologia , Proliferação de Células/genética , Sobrevivência Celular/genética , Sobrevivência Celular/imunologia , Células Dendríticas Foliculares/metabolismo , Feminino , Fibroblastos/metabolismo , Regulação da Expressão Gênica/imunologia , Técnicas de Introdução de Genes , Humanos , Imunidade Celular/genética , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Linfonodos/citologia , Masculino , Camundongos , Camundongos Transgênicos , RNA-Seq , Análise de Célula Única , Células Estromais/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismoRESUMO
Classical type 1 dendritic cells (cDC1s) are required for antiviral and antitumor immunity, which necessitates an understanding of their development. Development of the cDC1 progenitor requires an E-protein-dependent enhancer located 41 kilobases downstream of the transcription start site of the transcription factor Irf8 (+41-kb Irf8 enhancer), but its maturation instead requires the Batf3-dependent +32-kb Irf8 enhancer. To understand this switch, we performed single-cell RNA sequencing of the common dendritic cell progenitor (CDP) and identified a cluster of cells that expressed transcription factors that influence cDC1 development, such as Nfil3, Id2 and Zeb2. Genetic epistasis among these factors revealed that Nfil3 expression is required for the transition from Zeb2hi and Id2lo CDPs to Zeb2lo and Id2hi CDPs, which represent the earliest committed cDC1 progenitors. This genetic circuit blocks E-protein activity to exclude plasmacytoid dendritic cell potential and explains the switch in Irf8 enhancer usage during cDC1 development.
Assuntos
Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Células Dendríticas/citologia , Elementos Facilitadores Genéticos/genética , Proteína 2 Inibidora de Diferenciação/metabolismo , Fatores Reguladores de Interferon/metabolismo , Homeobox 2 de Ligação a E-box com Dedos de Zinco/metabolismo , Animais , Diferenciação Celular/imunologia , Células Cultivadas , Regulação da Expressão Gênica no Desenvolvimento/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Repressoras/metabolismo , Células-Tronco/citologiaRESUMO
Myeloid cells encounter stromal cells and their matrix determinants on a continual basis during their residence in any given organ. Here, we examined the impact of the collagen receptor LAIR1 on myeloid cell homeostasis and function. LAIR1 was highly expressed in the myeloid lineage and enriched in non-classical monocytes. Proteomic definition of the LAIR1 interactome identified stromal factor Colec12 as a high-affinity LAIR1 ligand. Proteomic profiling of LAIR1 signaling triggered by Collagen1 and Colec12 highlighted pathways associated with survival, proliferation, and differentiation. Lair1-/- mice had reduced frequencies of Ly6C- monocytes, which were associated with altered proliferation and apoptosis of non-classical monocytes from bone marrow and altered heterogeneity of interstitial macrophages in lung. Myeloid-specific LAIR1 deficiency promoted metastatic growth in a melanoma model and LAIR1 expression associated with improved clinical outcomes in human metastatic melanoma. Thus, monocytes and macrophages rely on LAIR1 sensing of stromal determinants for fitness and function, with relevance in homeostasis and disease.
Assuntos
Homeostase/fisiologia , Pulmão/metabolismo , Macrófagos Alveolares/metabolismo , Monócitos/metabolismo , Receptores Imunológicos/metabolismo , Animais , Apoptose/fisiologia , Medula Óssea/metabolismo , Medula Óssea/patologia , Células COS , Diferenciação Celular/fisiologia , Linhagem Celular , Linhagem Celular Tumoral , Linhagem da Célula/fisiologia , Proliferação de Células/fisiologia , Chlorocebus aethiops , Feminino , Humanos , Pulmão/patologia , Macrófagos Alveolares/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Monócitos/patologia , Células Mieloides/metabolismo , Células Mieloides/patologia , Metástase Neoplásica/patologia , Proteômica/métodos , Transdução de Sinais/fisiologiaRESUMO
A variety of signals finely tune insulin secretion by pancreatic ß cells to prevent both hyper-and hypoglycemic states. Here, we show that post-translational regulation of the transcription factors ETV1, ETV4, and ETV5 by the ubiquitin ligase COP1 (also called RFWD2) in ß cells is critical for insulin secretion. Mice lacking COP1 in ß cells developed diabetes due to insulin granule docking defects that were fully rescued by genetic deletion of Etv1, Etv4, and Etv5. Genes regulated by ETV1, ETV4, or ETV5 in the absence of mouse COP1 were enriched in human diabetes-associated genes, suggesting that they also influence human ß-cell pathophysiology. In normal ß cells, ETV4 was stabilized upon membrane depolarization and limited insulin secretion under hyperglycemic conditions. Collectively, our data reveal that ETVs negatively regulate insulin secretion for the maintenance of normoglycemia.
Assuntos
Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Proteínas Nucleares/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Proteínas de Ligação a DNA/metabolismo , Diabetes Mellitus/metabolismo , Exocitose , Deleção de Genes , Glucose/metabolismo , Humanos , Hiperglicemia/metabolismo , Secreção de Insulina , Camundongos , Proteínas Nucleares/genética , Proteínas Proto-Oncogênicas c-ets/metabolismo , Fatores de Transcrição/metabolismo , Ubiquitina-Proteína Ligases/genéticaRESUMO
Clearance of apoptotic cells by macrophages prevents excessive inflammation and supports immune tolerance. Here, we examined the effect of blocking apoptotic cell clearance on anti-tumor immune response. We generated an antibody that selectively inhibited efferocytosis by phagocytic receptor MerTK. Blockade of MerTK resulted in accumulation of apoptotic cells within tumors and triggered a type I interferon response. Treatment of tumor-bearing mice with anti-MerTK antibody stimulated T cell activation and synergized with anti-PD-1 or anti-PD-L1 therapy. The anti-tumor effect induced by anti-MerTK treatment was lost in Stinggt/gt mice, but not in Cgas-/- mice. Abolishing cGAMP production in Cgas-/- tumor cells, depletion of extracellular ATP, or inactivation of the ATP-gated P2X7R channel also compromised the effects of MerTK blockade. Mechanistically, extracellular ATP acted via P2X7R to enhance the transport of extracellular cGAMP into macrophages and subsequent STING activation. Thus, MerTK blockade increases tumor immunogenicity and potentiates anti-tumor immunity, which has implications for cancer immunotherapy.
Assuntos
Macrófagos/imunologia , Proteínas de Membrana/metabolismo , Neoplasias/imunologia , Nucleotídeos Cíclicos/metabolismo , Receptores Purinérgicos P2X7/metabolismo , c-Mer Tirosina Quinase/imunologia , Trifosfato de Adenosina/metabolismo , Animais , Apoptose , Antígeno B7-H1/imunologia , Células Cultivadas , Feminino , Imunidade Inata , Imunoterapia , Interferon Tipo I/metabolismo , Macrófagos/metabolismo , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Neoplasias/metabolismo , Neoplasias/patologia , Neoplasias/terapia , Nucleotidiltransferases/deficiência , Nucleotidiltransferases/metabolismo , Fagocitose , Receptor de Morte Celular Programada 1/imunologia , Receptores Purinérgicos P2X7/deficiência , Transdução de Sinais/imunologia , Ensaios Antitumorais Modelo de Xenoenxerto , c-Mer Tirosina Quinase/genéticaRESUMO
The immune phenotype of a tumour is a key predictor of its response to immunotherapy1-4. Patients who respond to checkpoint blockade generally present with immune-inflamed5-7 tumours that are highly infiltrated by T cells. However, not all inflamed tumours respond to therapy, and even lower response rates occur among tumours that lack T cells (immune desert) or that spatially exclude T cells to the periphery of the tumour lesion (immune excluded)8. Despite the importance of these tumour immune phenotypes in patients, little is known about their development, heterogeneity or dynamics owing to the technical difficulty of tracking these features in situ. Here we introduce skin tumour array by microporation (STAMP)-a preclinical approach that combines high-throughput time-lapse imaging with next-generation sequencing of tumour arrays. Using STAMP, we followed the development of thousands of arrayed tumours in vivo to show that tumour immune phenotypes and outcomes vary between adjacent tumours and are controlled by local factors within the tumour microenvironment. Particularly, the recruitment of T cells by fibroblasts and monocytes into the tumour core was supportive of T cell cytotoxic activity and tumour rejection. Tumour immune phenotypes were dynamic over time and an early conversion to an immune-inflamed phenotype was predictive of spontaneous or therapy-induced tumour rejection. Thus, STAMP captures the dynamic relationships of the spatial, cellular and molecular components of tumour rejection and has the potential to translate therapeutic concepts into successful clinical strategies.
Assuntos
Neoplasias , Linfócitos T , Microambiente Tumoral , Humanos , Imunoterapia , Neoplasias/imunologia , Neoplasias/patologia , Neoplasias/terapia , Linfócitos T/imunologia , Fenótipo , Fibroblastos , Monócitos , Inibidores de Checkpoint Imunológico/farmacologia , Inibidores de Checkpoint Imunológico/uso terapêuticoRESUMO
Recent single-cell studies of cancer in both mice and humans have identified the emergence of a myofibroblast population specifically marked by the highly restricted leucine-rich-repeat-containing protein 15 (LRRC15)1-3. However, the molecular signals that underlie the development of LRRC15+ cancer-associated fibroblasts (CAFs) and their direct impact on anti-tumour immunity are uncharacterized. Here in mouse models of pancreatic cancer, we provide in vivo genetic evidence that TGFß receptor type 2 signalling in healthy dermatopontin+ universal fibroblasts is essential for the development of cancer-associated LRRC15+ myofibroblasts. This axis also predominantly drives fibroblast lineage diversity in human cancers. Using newly developed Lrrc15-diphtheria toxin receptor knock-in mice to selectively deplete LRRC15+ CAFs, we show that depletion of this population markedly reduces the total tumour fibroblast content. Moreover, the CAF composition is recalibrated towards universal fibroblasts. This relieves direct suppression of tumour-infiltrating CD8+ T cells to enhance their effector function and augments tumour regression in response to anti-PDL1 immune checkpoint blockade. Collectively, these findings demonstrate that TGFß-dependent LRRC15+ CAFs dictate the tumour-fibroblast setpoint to promote tumour growth. These cells also directly suppress CD8+ T cell function and limit responsiveness to checkpoint blockade. Development of treatments that restore the homeostatic fibroblast setpoint by reducing the population of pro-disease LRRC15+ myofibroblasts may improve patient survival and response to immunotherapy.
Assuntos
Fibroblastos Associados a Câncer , Proteínas de Membrana , Miofibroblastos , Neoplasias Pancreáticas , Células Estromais , Animais , Humanos , Camundongos , Fibroblastos Associados a Câncer/metabolismo , Linfócitos T CD8-Positivos/imunologia , Proteínas de Membrana/metabolismo , Miofibroblastos/metabolismo , Neoplasias Pancreáticas/imunologia , Neoplasias Pancreáticas/patologia , Receptores de Fatores de Crescimento Transformadores beta , Fator de Crescimento Transformador beta/metabolismo , Microambiente Tumoral , Antígeno B7-H1RESUMO
Fibroblasts are non-haematopoietic structural cells that define the architecture of organs, support the homeostasis of tissue-resident cells and have key roles in fibrosis, cancer, autoimmunity and wound healing1. Recent studies have described fibroblast heterogeneity within individual tissues1. However, the field lacks a characterization of fibroblasts at single-cell resolution across tissues in healthy and diseased organs. Here we constructed fibroblast atlases by integrating single-cell transcriptomic data from about 230,000 fibroblasts across 17 tissues, 50 datasets, 11 disease states and 2 species. Mouse fibroblast atlases and a DptIRESCreERT2 knock-in mouse identified two universal fibroblast transcriptional subtypes across tissues. Our analysis suggests that these cells can serve as a reservoir that can yield specialized fibroblasts across a broad range of steady-state tissues and activated fibroblasts in disease. Comparison to an atlas of human fibroblasts from perturbed states showed that fibroblast transcriptional states are conserved between mice and humans, including universal fibroblasts and activated phenotypes associated with pathogenicity in human cancer, fibrosis, arthritis and inflammation. In summary, a cross-species and pan-tissue approach to transcriptomics at single-cell resolution has identified key organizing principles of the fibroblast lineage in health and disease.
Assuntos
Fibroblastos/citologia , Transcriptoma , Animais , Células Cultivadas , Doença , Feminino , Fibroblastos/classificação , Técnicas de Introdução de Genes , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neoplasias , Especificidade de Órgãos , Fenótipo , RNA-Seq , Análise de Célula Única , Células EstromaisRESUMO
Plasma membrane rupture (PMR) is the final cataclysmic event in lytic cell death. PMR releases intracellular molecules known as damage-associated molecular patterns (DAMPs) that propagate the inflammatory response1-3. The underlying mechanism of PMR, however, is unknown. Here we show that the cell-surface NINJ1 protein4-8, which contains two transmembrane regions, has an essential role in the induction of PMR. A forward-genetic screen of randomly mutagenized mice linked NINJ1 to PMR. Ninj1-/- macrophages exhibited impaired PMR in response to diverse inducers of pyroptotic, necrotic and apoptotic cell death, and were unable to release numerous intracellular proteins including HMGB1 (a known DAMP) and LDH (a standard measure of PMR). Ninj1-/- macrophages died, but with a distinctive and persistent ballooned morphology, attributable to defective disintegration of bubble-like herniations. Ninj1-/- mice were more susceptible than wild-type mice to infection with Citrobacter rodentium, which suggests a role for PMR in anti-bacterial host defence. Mechanistically, NINJ1 used an evolutionarily conserved extracellular domain for oligomerization and subsequent PMR. The discovery of NINJ1 as a mediator of PMR overturns the long-held idea that cell death-related PMR is a passive event.
Assuntos
Moléculas de Adesão Celular Neuronais/metabolismo , Morte Celular , Membrana Celular/metabolismo , Fatores de Crescimento Neural/metabolismo , Animais , Apoptose , Moléculas de Adesão Celular Neuronais/química , Moléculas de Adesão Celular Neuronais/genética , Morte Celular/genética , Feminino , Humanos , Macrófagos , Masculino , Camundongos , Mutação , Necrose , Fatores de Crescimento Neural/química , Fatores de Crescimento Neural/genética , Multimerização Proteica , Piroptose/genéticaRESUMO
Although RAF monomer inhibitors (type I.5, BRAF(V600)) are clinically approved for the treatment of BRAFV600-mutant melanoma, they are ineffective in non-BRAFV600 mutant cells1-3. Belvarafenib is a potent and selective RAF dimer (type II) inhibitor that exhibits clinical activity in patients with BRAFV600E- and NRAS-mutant melanomas. Here we report the first-in-human phase I study investigating the maximum tolerated dose, and assessing the safety and preliminary efficacy of belvarafenib in BRAFV600E- and RAS-mutated advanced solid tumours (NCT02405065, NCT03118817). By generating belvarafenib-resistant NRAS-mutant melanoma cells and analysing circulating tumour DNA from patients treated with belvarafenib, we identified new recurrent mutations in ARAF within the kinase domain. ARAF mutants conferred resistance to belvarafenib in both a dimer- and a kinase activity-dependent manner. Belvarafenib induced ARAF mutant dimers, and dimers containing mutant ARAF were active in the presence of inhibitor. ARAF mutations may serve as a general resistance mechanism for RAF dimer inhibitors as the mutants exhibit reduced sensitivity to a panel of type II RAF inhibitors. The combination of RAF plus MEK inhibition may be used to delay ARAF-driven resistance and suggests a rational combination for clinical use. Together, our findings reveal specific and compensatory functions for the ARAF isoform and implicate ARAF mutations as a driver of resistance to RAF dimer inhibitors.
Assuntos
Resistencia a Medicamentos Antineoplásicos/genética , Melanoma/tratamento farmacológico , Melanoma/genética , Mutação , Proteínas Proto-Oncogênicas A-raf/antagonistas & inibidores , Proteínas Proto-Oncogênicas A-raf/genética , Quinases raf/antagonistas & inibidores , Animais , Linhagem Celular , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Feminino , Humanos , Melanoma/patologia , Camundongos , Multimerização Proteica/efeitos dos fármacos , Proteínas Proto-Oncogênicas A-raf/química , Quinases raf/químicaRESUMO
Despite the resounding clinical success in cancer treatment of antibodies that block the interaction of PD1 with its ligand PDL11, the mechanisms involved remain unknown. A major limitation to understanding the origin and fate of T cells in tumour immunity is the lack of quantitative information on the distribution of individual clonotypes of T cells in patients with cancer. Here, by performing deep single-cell sequencing of RNA and T cell receptors in patients with different types of cancer, we survey the profiles of various populations of T cells and T cell receptors in tumours, normal adjacent tissue, and peripheral blood. We find clear evidence of clonotypic expansion of effector-like T cells not only within the tumour but also in normal adjacent tissue. Patients with gene signatures of such clonotypic expansion respond best to anti-PDL1 therapy. Notably, expanded clonotypes found in the tumour and normal adjacent tissue can also typically be detected in peripheral blood, which suggests a convenient approach to patient identification. Analyses of our data together with several external datasets suggest that intratumoural T cells, especially in responsive patients, are replenished with fresh, non-exhausted replacement cells from sites outside the tumour, suggesting continued activity of the cancer immunity cycle in these patients, the acceleration of which may be associated with clinical response.
Assuntos
Linfócitos do Interstício Tumoral/citologia , Linfócitos do Interstício Tumoral/metabolismo , Neoplasias/patologia , Variantes Farmacogenômicos , Receptores de Antígenos de Linfócitos T/genética , Linfócitos T/citologia , Anticorpos Monoclonais Humanizados/uso terapêutico , Antineoplásicos/uso terapêutico , Células Clonais , Humanos , Neoplasias/tratamento farmacológico , Neoplasias/imunologia , Linfócitos T/metabolismo , TranscriptomaRESUMO
Type 2 innate lymphoid cells (ILC2 cells) participate in host defense against helminth parasites and in allergic inflammation. Given their functional relatedness to type 2 helper T cells (T(H)2 cells), we explored whether Gfi1 acts as a shared transcriptional determinant in ILC2 cells. Gfi1 promoted the development of ILC2 cells and controlled their responsiveness during infection with Nippostrongylus brasiliensis and protease allergen-induced lung inflammation. Gfi1 'preferentially' regulated the responsiveness of ILC2 cells to interleukin 33 (IL-33) by directly activating Il1rl1, which encodes the IL-33 receptor (ST2). Loss of Gfi1 in activated ILC2 cells resulted in impaired expression of the transcription factor GATA-3 and a dysregulated genome-wide effector state characterized by coexpression of IL-13 and IL-17. Our findings establish Gfi1 as a shared determinant that reciprocally regulates the type 2 and IL-17 effector states in cells of the innate and adaptive immune systems.
Assuntos
Proteínas de Ligação a DNA/imunologia , Imunidade Inata/imunologia , Células Th2/imunologia , Fatores de Transcrição/imunologia , Transcriptoma/imunologia , Animais , Líquido da Lavagem Broncoalveolar/química , Líquido da Lavagem Broncoalveolar/imunologia , Células Cultivadas , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Citometria de Fluxo , Fator de Transcrição GATA3/genética , Fator de Transcrição GATA3/imunologia , Fator de Transcrição GATA3/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Proteína 1 Semelhante a Receptor de Interleucina-1 , Interleucina-13/genética , Interleucina-13/imunologia , Interleucina-13/metabolismo , Interleucina-17/genética , Interleucina-17/imunologia , Interleucina-17/metabolismo , Interleucina-33 , Interleucinas/farmacologia , Pulmão/imunologia , Pulmão/metabolismo , Ativação Linfocitária/efeitos dos fármacos , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos , Camundongos Knockout , Camundongos Transgênicos , Nippostrongylus/imunologia , Nippostrongylus/fisiologia , Análise de Sequência com Séries de Oligonucleotídeos , Receptores de Interleucina/genética , Receptores de Interleucina/imunologia , Receptores de Interleucina/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Infecções por Strongylida/imunologia , Infecções por Strongylida/parasitologia , Células Th2/metabolismo , Células Th2/parasitologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transcriptoma/genéticaRESUMO
Preclinical and clinical studies demonstrate that T cell-dependent bispecific antibodies (TDBs) induce systemic changes in addition to tumor killing, leading to adverse events. Here, we report an in-depth characterization of acute responses to TDBs in tumor-bearing mice. Contrary to modest changes in tumors, rapid and substantial lymphocyte accumulation and endothelial cell (EC) activation occur around large blood vessels in normal organs including the liver. We hypothesize that organ-specific ECs may account for the differential responses in normal tissues and tumors, and we identify a list of genes selectively upregulated by TDB in large liver vessels. Using one of the genes as an example, we demonstrate that CD9 facilitates ICAM-1 to support T cell-EC interaction in response to soluble factors released from a TDB-mediated cytotoxic reaction. Our results suggest that multiple factors may cooperatively promote T cell infiltration into normal organs as a secondary response to TDB-mediated tumor killing. These data shed light on how different vascular beds respond to cancer immunotherapy and may help improve their safety and efficacy.
Assuntos
Anticorpos Biespecíficos , Neoplasias , Camundongos , Animais , Linfócitos T , Anticorpos Biespecíficos/farmacologia , Anticorpos Biespecíficos/uso terapêutico , Neoplasias/tratamento farmacológico , Comunicação Celular , Células EndoteliaisRESUMO
Caspase-8 is a protease with both pro-death and pro-survival functions: it mediates apoptosis induced by death receptors such as TNFR11, and suppresses necroptosis mediated by the kinase RIPK3 and the pseudokinase MLKL2-4. Mice that lack caspase-8 display MLKL-dependent embryonic lethality4, as do mice that express catalytically inactive CASP8(C362A)5. Casp8C362A/C362AMlkl-/- mice die during the perinatal period5, whereas Casp8-/-Mlkl-/- mice are viable4, which indicates that inactive caspase-8 also has a pro-death scaffolding function. Here we show that mutant CASP8(C362A) induces the formation of ASC (also known as PYCARD) specks, and caspase-1-dependent cleavage of GSDMD and caspases 3 and 7 in MLKL-deficient mouse intestines around embryonic day 18. Caspase-1 and its adaptor ASC contributed to the perinatal lethal phenotype because a number of Casp8C362A/C362AMlkl-/-Casp1-/- and Casp8C362A/C362AMlkl-/-Asc-/- mice survived beyond weaning. Transfection studies suggest that inactive caspase-8 adopts a distinct conformation to active caspase-8, enabling its prodomain to engage ASC. Upregulation of the lipopolysaccharide sensor caspase-11 in the intestines of both Casp8C362A/C362AMlkl-/- and Casp8C362A/C362AMlkl-/-Casp1-/- mice also contributed to lethality because Casp8C362A/C362AMlkl-/-Casp1-/-Casp11-/- (Casp11 is also known as Casp4) neonates survived more often than Casp8C362A/C362AMlkl-/-Casp1-/- neonates. Finally, Casp8C362A/C362ARipk3-/-Casp1-/-Casp11-/- mice survived longer than Casp8C362A/C362AMlkl-/-Casp1-/-Casp11-/- mice, indicating that a necroptosis-independent function of RIPK3 also contributes to lethality. Thus, unanticipated plasticity in death pathways is revealed when caspase-8-dependent apoptosis and MLKL-dependent necroptosis are inhibited.
Assuntos
Caspase 8/metabolismo , Morte Celular/genética , Mucosa Intestinal/citologia , Animais , Proteínas Adaptadoras de Sinalização CARD/metabolismo , Caspase 8/genética , Feminino , Regulação da Expressão Gênica , Células HEK293 , Humanos , Mucosa Intestinal/enzimologia , Estimativa de Kaplan-Meier , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mutação , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismoRESUMO
BACKGROUND: Cancer immunotherapy approaches that elicit immune cell responses, including T and NK cells, have revolutionized the field of oncology. However, immunosuppressive mechanisms restrain immune cell activation within solid tumors so additional strategies to augment activity are required. METHODS: We identified the co-stimulatory receptor NKG2D as a target based on its expression on a large proportion of CD8+ tumor infiltrating lymphocytes (TILs) from breast cancer patient samples. Human and murine surrogate NKG2D co-stimulatory receptor-bispecifics (CRB) that bind NKG2D on NK and CD8+ T cells as well as HER2 on breast cancer cells (HER2-CRB) were developed as a proof of concept for targeting this signaling axis in vitro and in vivo. RESULTS: HER2-CRB enhanced NK cell activation and cytokine production when co-cultured with HER2 expressing breast cancer cell lines. HER2-CRB when combined with a T cell-dependent-bispecific (TDB) antibody that synthetically activates T cells by crosslinking CD3 to HER2 (HER2-TDB), enhanced T cell cytotoxicity, cytokine production and in vivo antitumor activity. A mouse surrogate HER2-CRB (mHER2-CRB) improved in vivo efficacy of HER2-TDB and augmented NK as well as T cell activation, cytokine production and effector CD8+ T cell differentiation. CONCLUSION: We demonstrate that targeting NKG2D with bispecific antibodies (BsAbs) is an effective approach to augment NK and CD8+ T cell antitumor immune responses. Given the large number of ongoing clinical trials leveraging NK and T cells for cancer immunotherapy, NKG2D-bispecifics have broad combinatorial potential.
Assuntos
Neoplasias da Mama , Linfócitos T CD8-Positivos , Células Matadoras Naturais , Subfamília K de Receptores Semelhantes a Lectina de Células NK , Humanos , Animais , Subfamília K de Receptores Semelhantes a Lectina de Células NK/metabolismo , Subfamília K de Receptores Semelhantes a Lectina de Células NK/imunologia , Camundongos , Linfócitos T CD8-Positivos/imunologia , Células Matadoras Naturais/imunologia , Feminino , Neoplasias da Mama/imunologia , Neoplasias da Mama/terapia , Receptor ErbB-2/imunologia , Linhagem Celular Tumoral , Imunoterapia/métodos , Ativação Linfocitária/imunologia , Linfócitos do Interstício Tumoral/imunologia , Linfócitos do Interstício Tumoral/metabolismoRESUMO
Genetic and environmental cues shape the evolution of the B cell Ig repertoire. Activation-induced cytidine deaminase (AID) is essential to generating Ig diversity through isotype class switching and somatic mutations, which then directly influence clonal selection. Impaired B cell development in AID-knockout mice has made it difficult to study Ig diversification in an aging repertoire. Therefore, in this report, we used a novel inducible AID-knockout mouse model and discovered that deleting AID in adult mice caused spontaneous germinal center formation. Deep sequencing of the IgH repertoire revealed that Ab diversification begins early in life and evolves over time. Our data suggest that activated B cells form germinal centers at steady state and facilitate continuous diversification of the B cell repertoire. In support, we identified shared B cell lineages that were class switched and showed age-dependent rates of mutation. Our data provide novel context to the genesis of the B cell repertoire that may benefit the understanding of autoimmunity and the strength of an immune response to infection.