RÉSUMÉ
Cancer immunotherapies using chimeric antigen receptor (CAR) T cells have tremendous potential and proven clinical efficacy against a number of malignancies. Research and development are emerging to deepen the knowledge of CAR T cell efficacy and extend the therapeutic potential of this novel therapy. To this end, functional characterization of CAR T cells plays a central role in consecutive phases across fundamental research and therapeutic development, with increasing needs for standardization. The functional characterization of CAR T cells is typically achieved by assessing critical effector functions, following co-culture with cell lines expressing the target antigen. However, the use of target cell lines poses several limitations, including alterations in cell fitness, metabolic state or genetic drift due to handling and culturing of the cells, which would increase variabilities and could lead to inconsistent results. Moreover, the use of target cell lines can be work and time intensive, and introduce significant background due to the allogenic responses of T cells. To overcome these limitations, we developed a synthetic bead-based platform ("Artificial Targets") to characterize CAR T cell function in vitro. These synthetic microparticles could specifically induce CAR T cell activation, as measured by CD69 and CD137 (4-1BB) upregulation. In addition, engagement with Artificial Targets resulted in induction of multiple effector functions of CAR T cells mimicking the response triggered by target cell lines including cytotoxic activity, as assessed by exposure of CD107a (LAMP-1), expression and secretion of cytokines, as well as cell proliferation. Importantly, in contrast to target cells, stimulation with Artificial Targets showed limited unspecific CAR T cell proliferation. Finally, Artificial Targets demonstrated flexibility to engage multiple costimulatory molecules that can synergistically enhance the CAR T cell function and represented a powerful tool for modulating CAR T cell responses. Collectively, our results show that Artificial Targets can specifically activate CAR T cells for essential effector functions that could significantly advance standardization of functional assessment of CAR T cells, from early development to clinical applications.
Sujet(s)
Microparticules membranaires , Lignée cellulaire , Prolifération cellulaire , Techniques de coculture , CytokinesRÉSUMÉ
The microenvironment profoundly influences tumor initiation across numerous tissues but remains understudied in brain tumors. In the cerebellum, canonical Wnt signaling controlled by Norrin/Frizzled4 (Fzd4) activation in meningeal endothelial cells is a potent inhibitor of preneoplasia and tumor progression in mouse models of Sonic hedgehog medulloblastoma (Shh-MB). Single-cell transcriptome profiling and phenotyping of the meninges indicate that Norrin/Frizzled4 sustains the activation of meningeal macrophages (mMΦs), characterized by Lyve1 and CXCL4 expression, during the critical preneoplastic period. Depleting mMΦs during this period enhances preneoplasia and tumorigenesis, phenocopying the effects of Norrin loss. The anti-tumorigenic function of mMΦs is derived from the expression of CXCL4, which counters CXCL12/CXCR4 signaling in pre-tumor cells, thereby inhibiting cell-cycle progression and promoting migration away from the pre-tumor niche. These findings identify a pivotal role for mMΦs as key mediators in chemokine-regulated anti-cancer crosstalk between the stroma and pre-tumor cells in the control of MB initiation.
Sujet(s)
Tumeurs du cervelet , Médulloblastome , Souris , Animaux , Médulloblastome/métabolisme , Protéines Hedgehog/métabolisme , Cellules endothéliales/métabolisme , Voie de signalisation Wnt , Tumeurs du cervelet/métabolisme , Microenvironnement tumoralRÉSUMÉ
Although T cells can exert potent anti-tumor immunity, a subset of T helper (Th) cells producing interleukin-22 (IL-22) in breast and lung tumors is linked to dismal patient outcome. Here, we examined the mechanisms whereby these T cells contribute to disease. In murine models of lung and breast cancer, constitutional and T cell-specific deletion of Il22 reduced metastases without affecting primary tumor growth. Deletion of the IL-22 receptor on cancer cells decreases metastasis to a degree similar to that seen in IL-22-deficient mice. IL-22 induced high expression of CD155, which bound to the activating receptor CD226 on NK cells. Excessive activation led to decreased amounts of CD226 and functionally impaired NK cells, which elevated the metastatic burden. IL-22 signaling was also associated with CD155 expression in human datasets and with poor patient outcomes. Taken together, our findings reveal an immunosuppressive circuit activated by T cell-derived IL-22 that promotes lung metastasis.
Sujet(s)
Interleukines , Tumeurs , Récepteurs viraux , Lymphocytes T auxiliaires , Animaux , Humains , Souris , Antigènes de différenciation des lymphocytes T/métabolisme , Interleukines/génétique , Interleukines/métabolisme , Cellules tueuses naturelles/métabolisme , Tumeurs/métabolisme , Liaison aux protéines , Lymphocytes T auxiliaires/métabolisme ,RÉSUMÉ
Spontaneous mouse models of medulloblastoma (MB) offer a tractable system to study malignant progression in the brain. Mouse Sonic Hedgehog (Shh)-MB tumours first appear at postnatal stages as preneoplastic changes on the surface of the cerebellum, the external granule layer (EGL). Here we compared traditional histology and 3DISCO tissue clearing in combination with light sheet fluorescence microscopy (LSFM) to identify and quantify preneoplastic changes induced by disrupting stromal Norrin/Frizzled 4 (Fzd4) signalling, a potent tumour inhibitory signal in two mouse models of spontaneous Shh-MB. We show that 3DISCO-LSFM is as accurate as traditional histology for detecting Norrin/Fzd4-associated changes in PNL formation in Ptch+/- mice and EGL hyperplasia in Neurod2-SmoA1+/- mice. Moreover, we show that the anti-tumour effect of Norrin/Fzd4 signalling is restricted to the posterior region of the cerebellum and is characterized by defective neural progenitor migration away from the EGL. In conclusion, 3DISCO-LSFM is a valid way to monitor tumour initiation events in mouse MB models and reveals an unanticipated regional restriction of stromal signalling in constraining tumour initiation.
Sujet(s)
Tumeurs du cervelet , Médulloblastome , Animaux , Tumeurs du cervelet/génétique , Tumeurs du cervelet/anatomopathologie , Cervelet/métabolisme , Modèles animaux de maladie humaine , Protéines Hedgehog/génétique , Protéines Hedgehog/métabolisme , Imagerie tridimensionnelle , Médulloblastome/génétique , Médulloblastome/anatomopathologie , SourisRÉSUMÉ
Adoptive T cell therapy (ACT) is a therapeutic approach which employs genetically manipulated autologous T cells to target and eliminate a patient's malignancy. This novel therapeutic approach, when employing a chimeric antigen receptor (CAR) targeting CD19-expressing B cells, has shown remarkable success in treating acute B-cell lymphocytic leukemia. However, blood born malignancies represent only a fraction of cancers which affect patients. Unfortunately, the utilization of ACT to target solid malignancies has only shown marginal success rates. There are many known obstacles which hinder CAR T cell therapy in patients suffering from solid cancer, one notable obstacle is the effective trafficking of CAR T cells to the tumor site. With the rapid advancement of novel approaches and targets which may enhance CAR T cell infiltration into solid tumors, a standardized approach to assess and measure CAR T cell infiltration becomes imperative in order to compare these different approaches across platforms. Here we describe a flow cytometry method which enables the rapid detection and quantification of CAR T cells which have reached and entered the tumor mass following intravenous injection. Competence with single cell preparation and flow cytometry is required for optimal results.
Sujet(s)
Tumeurs , Récepteurs chimériques pour l'antigène , Cytométrie en flux , Humains , Immunothérapie adoptive/méthodes , Tumeurs/thérapie , Récepteurs aux antigènes des cellules T , Lymphocytes TRÉSUMÉ
The efficacy of adoptive cell therapy for solid tumours is hampered by the poor accumulation of the transferred T cells in tumour tissue. Here, we show that forced expression of C-X-C chemokine receptor type 6 (whose ligand is highly expressed by human and murine pancreatic cancer cells and tumour-infiltrating immune cells) in antigen-specific T cells enhanced the recognition and lysis of pancreatic cancer cells and the efficacy of adoptive cell therapy for pancreatic cancer. In mice with subcutaneous pancreatic tumours treated with T cells with either a transgenic T-cell receptor or a murine chimeric antigen receptor targeting the tumour-associated antigen epithelial cell adhesion molecule, and in mice with orthotopic pancreatic tumours or patient-derived xenografts treated with T cells expressing a chimeric antigen receptor targeting mesothelin, the T cells exhibited enhanced intratumoral accumulation, exerted sustained anti-tumoral activity and prolonged animal survival only when co-expressing C-X-C chemokine receptor type 6. Arming tumour-specific T cells with tumour-specific chemokine receptors may represent a promising strategy for the realization of adoptive cell therapy for solid tumours.
Sujet(s)
Immunothérapie adoptive , Tumeurs du pancréas , Récepteurs CXCR6/métabolisme , Lymphocytes T , Animaux , Thérapie cellulaire et tissulaire , Mésothéline , Souris , Tumeurs du pancréas/thérapie , Récepteurs aux chimiokines/génétiqueRÉSUMÉ
BACKGROUND: CD16-chimeric antigen receptors (CAR) T cells recognise the Fc-portion of therapeutic antibodies, which can enable the selective targeting of different antigens. Limited evidence exists as to which CD16-CAR design and antibody partner might be most effective. We have hypothesised that the use of high-affinity CD16 variants, with increased Fc-terminus antibody affinity, combined with Fc-engineered antibodies, would provide superior CD16-CAR T cell efficacy. METHODS: CD16-CAR T (wild-type or variants) cells were co-cultured with Panc-1 pancreatic cancer, Raji lymphoma or A375 melanoma cells in the presence or absence of anti-CD20, anti-MCSP, wild-type or the glycoengineered antibody variants. The endpoints were proliferation, activation, and cytotoxicity in vitro. RESULTS: The CD16 158 V variant of CD16-CAR T cells showed increased cytotoxic activity against all the tested cancer cells in the presence of the wild-type antibody directed against MCSP or CD20. Glycoengineered antibodies enhanced CD16-CAR T cell activity irrespective of CD16 polymorphisms as compared with the wild-type antibody. The combination of the glycoengineered antibodies with the CD16-CAR 158 V variant synergised as seen by the increase in all endpoints. CONCLUSION: These results indicate that CD16-CAR with the high-affinity CD16 variant 158 V, combined with Fc-engineered antibodies, have high anti-tumour efficacy.
Sujet(s)
Immunothérapie adoptive , Immunothérapie , Tumeurs/thérapie , Récepteurs chimériques pour l'antigène/immunologie , Animaux , Anticorps monoclonaux/effets indésirables , Anticorps monoclonaux/pharmacologie , Cytotoxicité à médiation cellulaire dépendante des anticorps/génétique , Lignée cellulaire tumorale , Prolifération cellulaire/effets des médicaments et des substances chimiques , Récepteurs ErbB/génétique , Humains , Fragments Fc des immunoglobulines/génétique , Fragments Fc des immunoglobulines/pharmacologie , Cellules tueuses naturelles/effets des médicaments et des substances chimiques , Cellules tueuses naturelles/immunologie , Activation des lymphocytes/effets des médicaments et des substances chimiques , Activation des lymphocytes/immunologie , Souris , Tumeurs/génétique , Tumeurs/immunologie , Tumeurs/anatomopathologie , Polymorphisme génétique , Récepteurs chimériques pour l'antigène/usage thérapeutique , Récepteurs du fragment Fc des IgG/immunologie , Rituximab/pharmacologie , Lymphocytes T/effets des médicaments et des substances chimiques , Lymphocytes T/immunologieRÉSUMÉ
Adoptive T cell therapy (ACT) refers to the therapeutic use of T cells. T cells genetically engineered to express chimeric antigen receptors (CAR) constitute the most clinically advanced form of ACT approved to date for the treatment of CD19-positive leukaemias and lymphomas. CARs are synthetic receptors that are able to confer antigen-binding and activating functions on T cells with the aim of therapeutically targeting cancer cells. Several factors are essential for CAR T cell therapy to be effective, such as recruitment, activation, expansion and persistence of bioengineered T cells at the tumour site. Despite the advances made in CAR T cell therapy, however, most tumour entities still escape immune detection and elimination. A number of strategies counteracting these problems will need to be addressed in order to render T cell therapy effective in more situations than currently possible. Non-haematological tumours are also the subject of active investigation, but ACT has so far shown only marginal success rates in these cases. New approaches are needed to enhance the ability of ACT to target solid tumours without increasing toxicity, by improving recognition, infiltration, and persistence within tumours, as well as an enhanced resistance to the suppressive tumour microenvironment.
Sujet(s)
Immunothérapie adoptive/tendances , Leucémies/thérapie , Récepteurs chimériques pour l'antigène/usage thérapeutique , Lymphocytes T/immunologie , Antigènes CD19/génétique , Antigènes néoplasiques/immunologie , Humains , Leucémies/immunologie , Récepteurs chimériques pour l'antigène/immunologie , Microenvironnement tumoral/immunologieRÉSUMÉ
The tumor microenvironment is a critical modulator of carcinogenesis; however, in many tumor types, the influence of the stroma during preneoplastic stages is unknown. Here we explored the relationship between pre-tumor cells and their surrounding stroma in malignant progression of the cerebellar tumor medulloblastoma (MB). We show that activation of the vascular regulatory signalling axis mediated by Norrin (an atypical Wnt)/Frizzled4 (Fzd4) inhibits MB initiation in the Ptch+/- mouse model. Loss of Norrin/Fzd4-mediated signalling in endothelial cells, either genetically or by short-term blockade, increases the frequency of pre-tumor lesions and creates a tumor-permissive microenvironment at the earliest, preneoplastic stages of MB. This pro-tumor stroma, characterized by angiogenic remodelling, is associated with an accelerated transition from preneoplasia to malignancy. These data expose a stromal component that regulates the earliest stages of tumorigenesis in the cerebellum, and a novel role for the Norrin/Fzd4 axis as an endogenous anti-tumor signal in the preneoplastic niche.
Sujet(s)
Carcinogenèse , Protéines de l'oeil/métabolisme , Récepteurs Frizzled/métabolisme , Médulloblastome/physiopathologie , Protéines de tissu nerveux/métabolisme , Transduction du signal , Animaux , Modèles animaux de maladie humaine , Régulation de l'expression des gènes , SourisRÉSUMÉ
Chromatin compaction mediates progenitor to post-mitotic cell transitions and modulates gene expression programs, yet the mechanisms are poorly defined. Snf2h and Snf2l are ATP-dependent chromatin remodelling proteins that assemble, reposition and space nucleosomes, and are robustly expressed in the brain. Here we show that mice conditionally inactivated for Snf2h in neural progenitors have reduced levels of histone H1 and H2A variants that compromise chromatin fluidity and transcriptional programs within the developing cerebellum. Disorganized chromatin limits Purkinje and granule neuron progenitor expansion, resulting in abnormal post-natal foliation, while deregulated transcriptional programs contribute to altered neural maturation, motor dysfunction and death. However, mice survive to young adulthood, in part from Snf2l compensation that restores Engrailed-1 expression. Similarly, Purkinje-specific Snf2h ablation affects chromatin ultrastructure and dendritic arborization, but alters cognitive skills rather than motor control. Our studies reveal that Snf2h controls chromatin organization and histone H1 dynamics for the establishment of gene expression programs underlying cerebellar morphogenesis and neural maturation.