RESUMEN
Discovering the role of fibroblasts residing in the tumor microenvironment (TME) requires controlled, localized perturbations because fibroblasts play critical roles in regulating immunity and tumor biology at multiple sites. Systemic perturbations can lead to unintended, confounding secondary effects, and methods to locally genetically engineer fibroblasts are lacking. To specifically investigate murine stromal cell perturbations restricted to the TME, we developed an adeno-associated virus (AAV)-based method to target any gene-of-interest in fibroblasts at high efficiency (>80%). As proof of concept, we generated single (sKO) and double gene KOs (dKO) of Osmr, Tgfbr2, and Il1r1 in cancer-associated fibroblasts (CAFs) and investigated how their cell states and those of other cells of the TME subsequently change in mouse models of melanoma and pancreatic ductal adenocarcinoma (PDAC). Furthermore, we developed an in vivo knockin-knockout (KIKO) strategy to achieve long-term tracking of CAFs with target gene KO via knocked-in reporter gene expression. This validated in vivo gene editing toolbox is fast, affordable, and modular, and thus holds great potential for further exploration of gene function in stromal cells residing in tumors and beyond.
RESUMEN
Tumor-associated macrophages (TAMs) are frequently and simplistically categorized as immunosuppressive, and one molecule prominently used to highlight their so-called 'M2' state is the surface protein CD206. However, direct evidence of the impact of macrophages remains impaired by the lack of sufficiently penetrant and specific tools to manipulate them in vivo. We thus made a novel conditional CD206 knock-in mouse to specifically visualize and/or deplete these TAMs. Early depletion of CD206+ macrophages and monocytes (here, 'MonoMacs') strikingly led to an indirect loss of a key anti-tumor network of NK cells, conventional type I dendritic cells (cDC1) and CD8 T cells. Among myeloid cells, we found that the CD206+ TAMs are the primary producers of CXCL9, the well-established chemoattractant for CXCR3-expressing NK and CD8 T cells. In contrast, a population of stress-responsive TAMs ("Hypoxic" or Spp1+) and immature monocytes, which remain following depletion, expressed vastly diminished levels of CXCL9. We confirmed that the missing NK and CD8 T cells are the primary producers of the cDC1-attracting chemokine Xcl1 and cDC1 growth factor Flt3l. Consistent with the loss of this critical network, CD206+ TAM depletion decreased tumor control in mice. Likewise, in humans, the CD206+ MonoMac signature correlated robustly with stimulatory cDC1 signature genes. Together, these findings negate the classification of CD206+ macrophages as immunosuppressive and instead illuminate the role of this majority of TAMs in organizing a critical tumor-reactive archetype of immunity.
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Altered myeloid inflammation and lymphopenia are hallmarks of severe infections. We identified the upregulated EN-RAGE gene program in airway and blood myeloid cells from patients with acute lung injury from SARS-CoV-2 or other causes across 7 cohorts. This program was associated with greater clinical severity and predicted future mechanical ventilation and death. EN-RAGEhi myeloid cells express features consistent with suppressor cell functionality, including low HLA-DR and high PD-L1. Sustained EN-RAGE program expression in airway and blood myeloid cells correlated with clinical severity and increasing expression of T cell dysfunction markers. IL-6 upregulated many EN-RAGE program genes in monocytes in vitro. IL-6 signaling blockade by tocilizumab in a placebo-controlled clinical trial led to rapid normalization of EN-RAGE and T cell gene expression. This identifies IL-6 as a key driver of myeloid dysregulation associated with worse clinical outcomes in COVID-19 patients and provides insights into shared pathophysiological mechanisms in non-COVID-19 ARDS.
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Tissue repair responses in metazoans are highly coordinated by different cell types over space and time. However, comprehensive single-cell-based characterization covering this coordination is lacking. Here, we captured transcriptional states of single cells over space and time during skin wound closure, revealing choreographed gene-expression profiles. We identified shared space-time patterns of cellular and gene program enrichment, which we call multicellular "movements" spanning multiple cell types. We validated some of the discovered space-time movements using large-volume imaging of cleared wounds and demonstrated the value of this analysis to predict "sender" and "receiver" gene programs in macrophages and fibroblasts. Finally, we tested the hypothesis that tumors are like "wounds that never heal" and found conserved wound healing movements in mouse melanoma and colorectal tumor models, as well as human tumor samples, revealing fundamental multicellular units of tissue biology for integrative studies.
Asunto(s)
Neoplasias , Cicatrización de Heridas , Ratones , Animales , Humanos , Cicatrización de Heridas/genética , Piel/patología , Neoplasias/patología , Macrófagos/metabolismo , Fibroblastos/fisiología , Células del EstromaRESUMEN
Although infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has pleiotropic and systemic effects in some individuals1-3, many others experience milder symptoms. Here, to gain a more comprehensive understanding of the distinction between severe and mild phenotypes in the pathology of coronavirus disease 2019 (COVID-19) and its origins, we performed a whole-blood-preserving single-cell analysis protocol to integrate contributions from all major immune cell types of the blood-including neutrophils, monocytes, platelets, lymphocytes and the contents of the serum. Patients with mild COVID-19 exhibit a coordinated pattern of expression of interferon-stimulated genes (ISGs)3 across every cell population, whereas these ISG-expressing cells are systemically absent in patients with severe disease. Paradoxically, individuals with severe COVID-19 produce very high titres of anti-SARS-CoV-2 antibodies and have a lower viral load compared to individuals with mild disease. Examination of the serum from patients with severe COVID-19 shows that these patients uniquely produce antibodies that functionally block the production of the ISG-expressing cells associated with mild disease, by activating conserved signalling circuits that dampen cellular responses to interferons. Overzealous antibody responses pit the immune system against itself in many patients with COVID-19, and perhaps also in individuals with other viral infections. Our findings reveal potential targets for immunotherapies in patients with severe COVID-19 to re-engage viral defence.
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Anticuerpos Antivirales/inmunología , COVID-19/inmunología , COVID-19/fisiopatología , Interferones/antagonistas & inhibidores , Interferones/inmunología , SARS-CoV-2/inmunología , SARS-CoV-2/patogenicidad , Anticuerpos Antivirales/sangre , Formación de Anticuerpos , Secuencia de Bases , COVID-19/sangre , COVID-19/virología , Femenino , Humanos , Inmunoglobulina G/inmunología , Interferones/metabolismo , Masculino , Neutrófilos/inmunología , Neutrófilos/patología , Dominios Proteicos , Receptor de Interferón alfa y beta/antagonistas & inhibidores , Receptor de Interferón alfa y beta/inmunología , Receptor de Interferón alfa y beta/metabolismo , Receptores de IgG/inmunología , Análisis de la Célula Individual , Carga Viral/inmunologíaRESUMEN
While effective in specific settings, adoptive chimeric antigen receptor (CAR) T cell therapy for cancer requires further improvement and optimization. Our previous results show that CD40L-overexpressing CAR T cells mobilize endogenous immune effectors, resulting in improved antitumor immunity. However, the cell populations required for this protective effect remain to be identified. Here we show, by analyzing Batf3-/- mice lacking the CD103+ conventional dendritic cell type 1 (cDC1) subpopulation important for antigen cross-presentation, that CD40L-overexpressing CAR T cells elicit an impaired antitumor response in the absence of cDC1s. We further find that CD40L-overexpressing CAR T cells stimulate tumor-resident CD11b-CD103- double-negative (DN) cDCs to proliferate and differentiate into cDC1s in wild-type mice. Finally, re-challenge experiments show that endogenous CD8+ T cells are required for protective antitumor memory in this setting. Our findings thus demonstrate the stimulatory effect of CD40L-overexpressing CAR T cells on innate and adaptive immune cells, and provide a rationale for using CD40L-overexpressing CAR T cells to improve immunotherapy responses.
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Ligando de CD40/genética , Linfocitos T CD8-positivos/inmunología , Células Dendríticas/inmunología , Inmunoterapia Adoptiva/métodos , Linfoma de Células B/terapia , Receptores Quiméricos de Antígenos/genética , Inmunidad Adaptativa , Animales , Presentación de Antígeno , Antígenos CD/genética , Antígenos CD/inmunología , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/deficiencia , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/genética , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/inmunología , Antígeno CD11b/deficiencia , Antígeno CD11b/genética , Antígeno CD11b/inmunología , Ligando de CD40/inmunología , Linfocitos T CD8-positivos/citología , Células Dendríticas/citología , Femenino , Expresión Génica , Inmunidad Innata , Inmunofenotipificación , Cadenas alfa de Integrinas/deficiencia , Cadenas alfa de Integrinas/genética , Cadenas alfa de Integrinas/inmunología , Linfoma de Células B/genética , Linfoma de Células B/inmunología , Linfoma de Células B/patología , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Trasplante de Neoplasias , Receptores Quiméricos de Antígenos/inmunología , Proteínas Represoras/deficiencia , Proteínas Represoras/genética , Proteínas Represoras/inmunologíaRESUMEN
While SARS-CoV-2 infection has pleiotropic and systemic effects in some patients, many others experience milder symptoms. We sought a holistic understanding of the severe/mild distinction in COVID-19 pathology, and its origins. We performed a wholeblood preserving single-cell analysis protocol to integrate contributions from all major cell types including neutrophils, monocytes, platelets, lymphocytes and the contents of serum. Patients with mild COVID-19 disease display a coordinated pattern of interferonstimulated gene (ISG) expression across every cell population and these cells are systemically absent in patients with severe disease. Severe COVID-19 patients also paradoxically produce very high anti-SARS-CoV-2 antibody titers and have lower viral load as compared to mild disease. Examination of the serum from severe patients demonstrates that they uniquely produce antibodies with multiple patterns of specificity against interferon-stimulated cells and that those antibodies functionally block the production of the mild disease-associated ISG-expressing cells. Overzealous and autodirected antibody responses pit the immune system against itself in many COVID-19 patients and this defines targets for immunotherapies to allow immune systems to provide viral defense.
RESUMEN
While SARS-CoV-2 infection has pleiotropic and systemic effects in some patients, many others experience milder symptoms. We sought a holistic understanding of the severe/mild distinction in COVID-19 pathology, and its origins. We performed a whole-blood preserving single-cell analysis protocol to integrate contributions from all major cell types including neutrophils, monocytes, platelets, lymphocytes and the contents of serum. Patients with mild COVID-19 disease display a coordinated pattern of interferon-stimulated gene (ISG) expression across every cell population and these cells are systemically absent in patients with severe disease. Severe COVID-19 patients also paradoxically produce very high anti-SARS-CoV-2 antibody titers and have lower viral load as compared to mild disease. Examination of the serum from severe patients demonstrates that they uniquely produce antibodies with multiple patterns of specificity against interferon-stimulated cells and that those antibodies functionally block the production of the mild disease-associated ISG-expressing cells. Overzealous and auto-directed antibody responses pit the immune system against itself in many COVID-19 patients and this defines targets for immunotherapies to allow immune systems to provide viral defense. ONE SENTENCE SUMMARY: In severe COVID-19 patients, the immune system fails to generate cells that define mild disease; antibodies in their serum actively prevents the successful production of those cells.
RESUMEN
Chimeric antigen receptor (CAR) T cells provide great efficacy in B cell malignancies. However, improved CAR T cell therapies are still needed. Here, we engineered tumor-targeted CAR T cells to constitutively express the immune-stimulatory molecule CD40 ligand (CD40L) and explored efficacy in different mouse leukemia/lymphoma models. We observed that CD40L+ CAR T cells circumvent tumor immune escape via antigen loss through CD40/CD40L-mediated cytotoxicity and induction of a sustained, endogenous immune response. After adoptive cell transfer, the CD40L+ CAR T cells displayed superior antitumor efficacy, licensed antigen-presenting cells, enhanced recruitment of immune effectors, and mobilized endogenous tumor-recognizing T cells. These effects were absent in Cd40-/- mice and provide a rationale for the use of CD40L+ CAR T cells in cancer treatment.
Asunto(s)
Ligando de CD40/inmunología , Inmunoterapia Adoptiva/métodos , Neoplasias/terapia , Receptores Quiméricos de Antígenos/inmunología , Animales , Células Presentadoras de Antígenos/inmunología , Ligando de CD40/genética , Línea Celular Tumoral , Humanos , Ratones , Ratones Endogámicos BALB C , Neoplasias/inmunología , Receptores Quiméricos de Antígenos/genética , Escape del Tumor , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Current approaches to cancer immunotherapy aim to engage the natural T cell response against tumors. One limitation is the elimination of self-antigen-specific T cells from the immune repertoire. Using a system in which precursor frequency can be manipulated in a murine melanoma model, we demonstrated that the clonal abundance of CD4(+) T cells specific for self-tumor antigen positively correlated with antitumor efficacy. At elevated precursor frequencies, intraclonal competition impaired initial activation and overall expansion of the tumor-specific CD4(+) T cell population. However, through clonally derived help, this population acquired a polyfunctional effector phenotype and antitumor immunity was enhanced. Conversely, development of effector function was attenuated at low precursor frequencies due to irreversible T cell exhaustion. Our findings assert that the differential effects of T cell clonal abundance on phenotypic outcome should be considered during the design of adoptive T cell therapies, including use of engineered T cells.