RESUMEN
Regulatory T cells (Treg) impede effective antitumor immunity. However, the role of Tregs in the clinical outcomes of patients with triple-negative breast cancer (TNBC) remains controversial. Here, we found that an immunosuppressive TNBC microenvironment is marked by an imbalance between effector αßCD8+ T cells and Tregs harboring hallmarks of highly suppressive effector Tregs (eTreg). Intratumoral eTregs strongly expressed PD-1 and persisted in patients with TNBC resistant to PD-1 blockade. Importantly, CD25 was the most selective surface marker of eTregs in primary TNBC and metastases compared with other candidate targets for eTreg depletion currently being evaluated in trials for patients with advanced TNBC. In a syngeneic TNBC model, the use of Fc-optimized, IL2 sparing, anti-CD25 antibodies synergized with PD-1 blockade to promote systemic antitumor immunity and durable tumor growth control by increasing effector αßCD8+ T-cell/Treg ratios in tumors and in the periphery. Together, this study provides the rationale for the clinical translation of anti-CD25 therapy to improve PD-1 blockade responses in patients with TNBC. SIGNIFICANCE: An imbalance between effector CD8+ T cells and CD25high effector Tregs marks immunosuppressive microenvironments in αPD-1-resistant TNBC and can be reversed through effector Treg depletion to increase αPD-1 efficacy.
Asunto(s)
Linfocitos T Reguladores , Neoplasias de la Mama Triple Negativas , Humanos , Receptor de Muerte Celular Programada 1 , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Neoplasias de la Mama Triple Negativas/patología , Microambiente TumoralRESUMEN
Interactions of developing T cells with Aire+ medullary thymic epithelial cells expressing high levels of MHCII molecules (mTEChi) are critical for the induction of central tolerance in the thymus. In turn, thymocytes regulate the cellularity of Aire+ mTEChi. However, it remains unknown whether thymocytes control the precursors of Aire+ mTEChi that are contained in mTEClo cells or other mTEClo subsets that have recently been delineated by single-cell transcriptomic analyses. Here, using three distinct transgenic mouse models, in which antigen presentation between mTECs and CD4+ thymocytes is perturbed, we show by high-throughput RNA-seq that self-reactive CD4+ thymocytes induce key transcriptional regulators in mTEClo and control the composition of mTEClo subsets, including Aire+ mTEChi precursors, post-Aire and tuft-like mTECs. Furthermore, these interactions upregulate the expression of tissue-restricted self-antigens, cytokines, chemokines, and adhesion molecules important for T-cell development. This gene activation program induced in mTEClo is combined with a global increase of the active H3K4me3 histone mark. Finally, we demonstrate that these self-reactive interactions between CD4+ thymocytes and mTECs critically prevent multiorgan autoimmunity. Our genome-wide study thus reveals that self-reactive CD4+ thymocytes control multiple unsuspected facets from immature stages of mTECs, which determines their heterogeneity.
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Autoantígenos/fisiología , Células Epiteliales/fisiología , Timocitos/fisiología , Timo , Animales , Linfocitos T CD4-Positivos , Proteínas de Unión al ADN , Epitelio/fisiología , Femenino , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Histonas , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Proteínas del Tejido Nervioso , Transducción de SeñalRESUMEN
This protocol details the step-by-step procedure for in-depth immune phenotyping of peripheral blood natural killer (NK) cells from clinical samples by mass cytometry. The protocol consists of three main steps: PBMC incubation with a mix of metal-conjugated antibodies for extracellular phenotyping followed by fixation, permeabilization and incubation with a mix of metal-conjugated antibodies for staining of intracellular proteins, and sample acquisition on a mass cytometer. High-dimensional analysis enables the visualization of NK cell subsets and their phenotypical characteristics. For complete details on the use and execution of this protocol, please refer to Chretien et al. (2021).
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Células Asesinas Naturales , Leucocitos Mononucleares , Humanos , Citometría de Flujo/métodos , Anticuerpos , Coloración y EtiquetadoRESUMEN
The rationale for therapeutic targeting of Vδ2+ γδ T cells in breast cancer is strongly supported by in vitro and murine preclinical investigations, characterizing them as potent breast tumor cell killers and source of Th1-related cytokines, backing cytotoxic αß T cells. Nonetheless, insights regarding Vδ2+ γδ T cell phenotypic alterations in human breast cancers are still lacking. This paucity of information is partly due to the challenging scarcity of these cells in surgical specimens. αß T cell phenotypic alterations occurring in the tumor bed are detectable in the periphery and correlate with adverse clinical outcomes. Thus, we sought to determine through an exploratory study whether Vδ2+ γδ T cells phenotypic changes can be detected within breast cancer patients' peripheral blood, along with association with tumor progression. By using mass cytometry, we quantified 130 immune variables from untreated breast cancer patients' peripheral blood. Supervised analyses and dimensionality reduction algorithms evidenced circulating Vδ2+ γδ T cell phenotypic alterations already established at diagnosis. Foremost, terminally differentiated Vδ2+ γδ T cells displaying phenotypes of exhausted senescent T cells associated with lymph node involvement. Thereby, our results support Vδ2+ γδ T cells implication in breast cancer pathogenesis and progression, besides shedding light on liquid biopsies to monitor surrogate markers of tumor-infiltrating Vδ2+ γδ T cell antitumor activity.
RESUMEN
Over the past decade, immunotherapy has become a powerful and evident tool in the fight against cancers. Notably, the rise of checkpoint blockade using monoclonal antibodies (anti-CTLA4, anti-PD1) to avoid interaction between inhibitory molecules allowed the betterment of patient care. Indeed, immunotherapies led to increased overall survival in forms of cutaneous melanoma or lung cancer. However, the percentage of patients responding varies from 20 to 40% depending on the type of cancer and on the expression of the target molecules by the tumor. This is due to the tumor microenvironment which allows the acquisition of resistance mechanisms to immunotherapies by tumor cells. These are closely linked to the architecture and cellular composition of the tumor microenvironment. This one acts on different parameters such as the immune cells infiltrate its composition and therefore, favors the recruitment of immunosuppressive cells as well as the tumor expression of checkpoint inhibitors such as Programmed Death Ligand-1 (PD-L1). Therefore, the analysis and modeling of the complexity of the microenvironment is an important parameter to consider, not only in the search for new therapies but also for the identification and stratification of patients likely to respond to immunotherapy. This is why the use of 3D culture models, reflecting the architecture and cellular composition of a tumor, is essential in immuno-oncology studies. Nowadays, there are several 3-D culture methods such as spheroids and organoids, which are applicable to immuno-oncology. In this review we evaluate 3D culture models as tools for the development of treatments in the field of immuno-oncology.
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Comunicación Celular , Inmunoterapia , Neoplasias/terapia , Microambiente Tumoral , Animales , Técnicas de Cultivo de Célula , Línea Celular Tumoral , Humanos , Dispositivos Laboratorio en un Chip , Técnicas Analíticas Microfluídicas/instrumentación , Neoplasias/inmunología , Neoplasias/metabolismo , Neoplasias/patología , Organoides , Impresión Tridimensional , Esferoides Celulares , Células Tumorales CultivadasRESUMEN
Endometrial Cancer is the most common cancer in the female genital tract in developed countries, and with its increasing incidence due to risk factors such as aging and obesity tends to become a public health issue. However, its immune environment has been less characterized than in other tumors such as breast cancers. NK cells are cytotoxic innate lymphoid cells that are considered as a major anti-tumoral effector cell type which function is drastically altered in tumors which participates to tumor progression. Here we characterize tumor NK cells both phenotypically and functionally in the tumor microenvironment of endometrial cancer. For that, we gathered endometrial tumors, tumor adjacent healthy tissue, blood from matching patients and healthy donor blood to perform comparative analysis of NK cells. First we found that NK cells were impoverished in the tumor infiltrate. We then compared the phenotype of NK cells in the tumor and found that tumor resident CD103+ NK cells exhibited more co-inhibitory molecules such as Tigit, and TIM-3 compared to recruited CD103- NK cells and that the expression of these molecules increased with the severity of the disease. We showed that both chemokines (CXCL12, IP-10, and CCL27) and cytokines profiles (IL-1ß and IL-6) were altered in the tumor microenvironment and might reduce NK cell function and recruitment to the tumor site. This led to hypothesize that the tumor microenvironment reduces resident NK cells cytotoxicity which we confirmed by measuring cytotoxic effector production and degranulation. Taken together, our results show that the tumor microenvironment reshapes NK cell phenotype and function to promote tumor progression.
Asunto(s)
Neoplasias Endometriales/inmunología , Células Asesinas Naturales/inmunología , Microambiente Tumoral/inmunología , Antígenos CD/metabolismo , Quimiocina CCL27/sangre , Quimiocina CXCL10/sangre , Quimiocina CXCL12/sangre , Neoplasias Endometriales/patología , Endometrio/patología , Femenino , Receptor 2 Celular del Virus de la Hepatitis A/metabolismo , Humanos , Inmunidad Innata/inmunología , Cadenas alfa de Integrinas/metabolismo , Interleucina-1beta/sangre , Interleucina-6/sangre , Receptores Inmunológicos/metabolismoRESUMEN
Tropheryma whipplei, the agent of Whipple's disease, inhibits phago-lysosome biogenesis to create a suitable niche for its survival and replication in macrophages. To understand the mechanism by which it subverts phagosome maturation, we used biochemical and cell biological approaches to purify and characterise the intracellular compartment where Tropheryma whipplei resides using mouse bone-marrow-derived macrophages. We showed that in addition to Lamp-1, the Tropheryma whipplei phagosome is positive for Rab5 and Rab7, two GTPases required for the early to late phagosome transition. Unlike other pathogens, inhibition of PI(3)P production was not the mechanism for Rab5 stabilisation at the phagosome. Overexpression of the inactive, GDP-bound form of Rab5 bypassed the pathogen-induced blockade of phago-lysosome biogenesis. This suggests that Tropheryma whipplei blocks the switch from Rab5 to Rab7 by acting on the Rab5 GTPase cycle. A bio-informatic analysis of the Tropheryma whipplei genome revealed a glyceraldehyde-3-phosphate dehydrogenase (GAPDH) homologous with the GAPDH of Listeria monocytogenes, and this may be the bacterial protein responsible for blocking Rab5 activity. To our knowledge, Tropheryma whipplei is the first pathogen described to induce a "chimeric" phagosome stably expressing both Rab5 and Rab7, suggesting a novel and specific mechanism for subverting phagosome maturation.
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Fagosomas/metabolismo , Tropheryma/metabolismo , Enfermedad de Whipple/metabolismo , Proteínas de Unión al GTP rab/metabolismo , Proteínas de Unión al GTP rab5/metabolismo , Animales , Médula Ósea/metabolismo , Gliceraldehído-3-Fosfato Deshidrogenasa (Fosforilante)/metabolismo , Lisosomas/metabolismo , Macrófagos/metabolismo , Ratones , Proteínas de Unión a GTP rab7RESUMEN
To survive in macrophages, Coxiella burnetii hijacks the activation pathway of macrophages. Recently, we have demonstrated that C. burnetii, via its lipopolysaccharide (LPS), avoids the activation of p38α-MAPK through an antagonistic engagement of Toll-like receptor (TLR)-4. We investigated the fine-tuned mechanism leading to the absence of activation of the p38α-MAPK despite TLR-4 engagement. In macrophages challenged with LPS from the avirulent variants of C. burnetii, TLR-4 and TLR-2 co-immunoprecipitated. This association was absent in cells challenged by the LPS of pathogenic C. burnetii. The disruption makes TLRs unable to signal during the recognition of the LPS of pathogenic C. burnetii. The disruption of TLR-2 and TLR-4 was induced by the re-organization of the macrophage cytoskeleton by C. burnetii LPS. Interestingly, blocking the actin cytoskeleton re-organization relieved the disruption of the association TLR-2/TLR-4 by pathogenic C. burnetii and rescued the p38α-MAPK activation by C. burnetii. We elucidated an unexpected mechanism allowing pathogenic C. burnetii to avoid macrophage activation by the disruption of the TLR-2 and TLR-4 association.
Asunto(s)
Coxiella burnetii/metabolismo , Lipopolisacáridos/metabolismo , Proteína Quinasa 14 Activada por Mitógenos/metabolismo , Fiebre Q/metabolismo , Receptor Toll-Like 2/metabolismo , Receptor Toll-Like 4/metabolismo , Animales , Células Cultivadas , Coxiella burnetii/genética , Activación Enzimática , Interacciones Huésped-Patógeno , Humanos , Macrófagos/enzimología , Macrófagos/metabolismo , Macrófagos/microbiología , Ratones , Ratones Endogámicos C57BL , Proteína Quinasa 14 Activada por Mitógenos/genética , Unión Proteica , Fiebre Q/enzimología , Fiebre Q/genética , Fiebre Q/microbiología , Receptor Toll-Like 2/genética , Receptor Toll-Like 4/genéticaRESUMEN
Dugesia japonica planarian flatworms are naturally exposed to various microbes but typically survive this challenge. We show that planarians eliminate bacteria pathogenic to Homo sapiens, Caenorhabditis elegans, and/or Drosophila melanogaster and thus represent a model to identify innate resistance mechanisms. Whole-transcriptome analysis coupled with RNAi screening of worms infected with Staphylococcus aureus or Legionella pneumophila identified 18 resistance genes with nine human orthologs, of which we examined the function of MORN2. Human MORN2 facilitates phagocytosis-mediated restriction of Mycobacterium tuberculosis, L. pneumophila, and S. aureus in macrophages. MORN2 promotes the recruitment of LC3, an autophagy protein also involved in phagocytosis, to M. tuberculosis-containing phagosomes and subsequent maturation to degradative phagolysosomes. MORN2-driven trafficking of M. tuberculosis to single-membrane, LC3-positive compartments requires autophagy-related proteins Atg5 and Beclin-1, but not Ulk-1 and Atg13, highlighting the importance of MORN2 in LC3-associated phagocytosis. These findings underscore the value of studying planarian defenses to identify immune factors.
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Proteínas del Helminto/inmunología , Legionella pneumophila/fisiología , Proteínas Asociadas a Microtúbulos/inmunología , Fagocitosis , Planarias/inmunología , Planarias/microbiología , Staphylococcus aureus/fisiología , Animales , Infecciones Bacterianas/genética , Infecciones Bacterianas/inmunología , Infecciones Bacterianas/microbiología , Modelos Animales de Enfermedad , Proteínas del Helminto/genética , Humanos , Legionella pneumophila/inmunología , Proteínas Asociadas a Microtúbulos/genética , Planarias/genética , Staphylococcus aureus/inmunologíaRESUMEN
Macrophages are critical components of the innate and adaptive immune responses, and they are the first line of defense against foreign invaders because of their powerful microbicidal activities. Macrophages are widely distributed throughout the body and are present in the lymphoid organs, liver, lungs, gastrointestinal tract, central nervous system, bone, and skin. Because of their repartition, they participate in a wide range of physiological and pathological processes. Macrophages are highly versatile cells that are able to recognize microenvironmental alterations and to maintain tissue homeostasis. Numerous pathogens have evolved mechanisms to use macrophages as Trojan horses to survive, replicate in, and infect both humans and animals and to propagate throughout the body. The recent explosion of interest in evolutionary, genetic, and biochemical aspects of host-pathogen interactions has renewed scientific attention regarding macrophages. Here, we describe a procedure to isolate and cultivate macrophages from murine bone marrow that will provide large numbers of macrophages for studying host-pathogen interactions as well as other processes.
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Células de la Médula Ósea/citología , Técnicas Citológicas/métodos , Macrófagos/citología , Animales , Células de la Médula Ósea/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/fisiología , Fibroblastos/citología , Factor Estimulante de Colonias de Granulocitos y Macrófagos/farmacología , Macrófagos/efectos de los fármacos , RatonesRESUMEN
Q fever is a disease caused by Coxiella burnetii, an obligate intracellular bacterium. Acute Q fever is characterized by efficient immune response, whereas chronic Q fever is characterized by dysregulated immune response as demonstrated by the lack of granulomas, the failure of C. burnetii to induce lymphoproliferation, and interferon-γ production. The mitogen-activated protein kinase (MAPK) signaling pathway plays crucial roles in innate immune responses and control of bacterial infections. However, its role in Q fever has not been addressed. First, we investigated the activation of MAPKs p38, c-jun N-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK) 1/2 in murine macrophages stimulated with C. burnetii. Coxiella burnetii NM phase I (virulent) and NM phase II (avirulent) induced the activation of JNK and ERK1/2. Avirulent C. burnetii activate p38, whereas C. burnetii did not induce the phosphorylation of p38. Second, the level of p38 activation was studied in Q fever patients. We found that p38 was activated in monocyte-derived macrophages from healthy donors and patients with acute Q fever in response to a potent agonist such as lipopolysaccharide. Interestingly, p38 was not activated in patients with active chronic Q fever and was activated in patients with cured chronic Q fever. These results suggest that the determination of p38 activation may serve as a tool for measuring Q fever activity.
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Coxiella burnetii/patogenicidad , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Biomarcadores , Perfilación de la Expresión Génica , Humanos , Macrófagos/inmunología , FosforilaciónRESUMEN
Q fever is a disease caused by Coxiella burnetii, an obligate intracellular bacterium. Acute Q fever is spontaneously resolutive and is characterized by an efficient immune response. In contrast, chronic Q fever is characterized by dysregulated immune response, as demonstrated by the failure of C. burnetii to induce lymphoproliferation and the lack of granulomas. Recently, it has been demonstrated that when co-expressed in heterologous mammalian cell lines, the ligands of Numb proteins X1 and X2 (LNX1 and LNX2) regulate the level of the T-cell co-receptor CD8, which plays an essential role in T-cell-mediated immune response. We decided to investigate the expression of LNX1 and LNX2 genes in patients with acute or chronic Q fever. Interestingly, we found a high level of LNX1 and LNX2 mRNAs in endocarditis, the principal manifestation of chronic Q fever, but not in acute Q fever. Our data suggest that LNXs may be used as complementary biomarkers to follow the prognosis of chronic Q fever.
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Proteínas Portadoras/biosíntesis , Coxiella burnetii/patogenicidad , Fiebre Q/diagnóstico , Fiebre Q/patología , Ubiquitina-Proteína Ligasas/biosíntesis , Biomarcadores , Linfocitos T CD8-positivos/inmunología , Perfilación de la Expresión Génica , Humanos , Pronóstico , ARN Mensajero/biosíntesisRESUMEN
Variations in lipopolysaccharide (LPS), a bacterial outer membrane component, determine virulence of the obligate intracellular bacterium Coxiella burnetii, but the underlying mechanisms are unknown. We find that while avirulent C. burnetii LPS (avLPS) stimulates host p38α-MAPK signaling required for proper trafficking of bacteria containing compartments to lysosomes for destruction, pathogenic C. burnetii LPS (vLPS) does not. The defect in vLPS and pathogenic C. burnetii targeting to degradative compartments involves an antagonistic engagement of TLR4 by vLPS, lack of p38α-MAPK-driven phosphorylation, and block in recruitment of the homotypic fusion and protein-sorting complex component Vps41 to vLPS-containing vesicles. An upstream activator of p38α-MAPK or phosphomimetic mutant Vps41-S796E expression overrides the inhibition, allowing vLPS and pathogenic C. burnetii targeting to phagolysosomes. Thus, p38α-MAPK and its crosstalk with Vps41 play a central role in trafficking bacteria to phagolysosomes. Pathogenic C. burnetii has evolved LPS variations to evade this host response and thrive intracellularly.