RÉSUMÉ
Respiratory viral infections cause morbidity and mortality worldwide. Despite the success of vaccines, vaccination efficacy is weakened by the rapid emergence of viral variants with immunoevasive properties. The development of an off-the-shelf, effective, and safe therapy against respiratory viral infections is thus desirable. Here, we develop NanoSTING, a nanoparticle formulation of the endogenous STING agonist, 2'-3' cGAMP, to function as an immune activator and demonstrate its safety in mice and rats. A single intranasal dose of NanoSTING protects against pathogenic strains of SARS-CoV-2 (alpha and delta VOC) in hamsters. In transmission experiments, NanoSTING reduces the transmission of SARS-CoV-2 Omicron VOC to naïve hamsters. NanoSTING also protects against oseltamivir-sensitive and oseltamivir-resistant strains of influenza in mice. Mechanistically, NanoSTING upregulates locoregional interferon-dependent and interferon-independent pathways in mice, hamsters, as well as non-human primates. Our results thus implicate NanoSTING as a broad-spectrum immune activator for controlling respiratory virus infection.
Sujet(s)
Administration par voie nasale , Nanoparticules , SARS-CoV-2 , Animaux , Nanoparticules/composition chimique , Nanoparticules/administration et posologie , Souris , Cricetinae , SARS-CoV-2/effets des médicaments et des substances chimiques , SARS-CoV-2/immunologie , Modèles animaux de maladie humaine , Humains , Protéines membranaires/agonistes , Protéines membranaires/métabolisme , Femelle , Nucléotides cycliques/pharmacologie , Rats , COVID-19/prévention et contrôle , COVID-19/immunologie , COVID-19/virologie , Infections à Orthomyxoviridae/prévention et contrôle , Infections à Orthomyxoviridae/virologie , Infections à Orthomyxoviridae/immunologie , Infections à Orthomyxoviridae/traitement médicamenteux , Mâle , Antiviraux/pharmacologie , Antiviraux/administration et posologie , Souris de lignée C57BLRÉSUMÉ
Immunization programs against SARS-CoV-2 with commercial intramuscular vaccines prevent disease but are less efficient in preventing infections. Mucosal vaccines can provide improved protection against transmission, ideally for different variants of concern (VOCs) and related sarbecoviruses. Here, we report a multi-antigen, intranasal vaccine, NanoSTING-SN (NanoSTING-Spike-Nucleocapsid), eliminates virus replication in both the lungs and the nostrils upon challenge with the pathogenic SARS-CoV-2 Delta VOC. We further demonstrate that NanoSTING-SN prevents transmission of the SARS-CoV-2 Omicron VOC (BA.5) to vaccine-naïve hamsters. To evaluate protection against other sarbecoviruses, we immunized mice with NanoSTING-SN. We showed that immunization affords protection against SARS-CoV, leading to protection from weight loss and 100% survival in mice. In non-human primates, animals immunized with NanoSTING-SN show durable serum IgG responses (6 months) and nasal wash IgA responses cross-reactive to SARS-CoV-2 (XBB1.5), SARS-CoV and MERS-CoV antigens. These observations have two implications: (1) mucosal multi-antigen vaccines present a pathway to reducing transmission of respiratory viruses, and (2) eliciting immunity against multiple antigens can be advantageous in engineering pan-sarbecovirus vaccines.
Sujet(s)
Administration par voie nasale , Anticorps antiviraux , Vaccins contre la COVID-19 , COVID-19 , SARS-CoV-2 , Animaux , SARS-CoV-2/immunologie , COVID-19/prévention et contrôle , COVID-19/immunologie , COVID-19/transmission , COVID-19/virologie , Souris , Cricetinae , Anticorps antiviraux/immunologie , Anticorps antiviraux/sang , Vaccins contre la COVID-19/immunologie , Vaccins contre la COVID-19/administration et posologie , Femelle , Souris de lignée BALB C , Humains , Mesocricetus , Antigènes viraux/immunologie , Glycoprotéine de spicule des coronavirus/immunologie , Immunoglobuline G/sang , Immunoglobuline G/immunologie , Anticorps neutralisants/immunologie , Anticorps neutralisants/sangRÉSUMÉ
Chimeric antigen receptor (CAR) T cells used for the treatment of B cell malignancies can identify T cell subsets with superior clinical activity. Here, using infusion products of individuals with large B cell lymphoma, we integrated functional profiling using timelapse imaging microscopy in nanowell grids with subcellular profiling and single-cell RNA sequencing to identify a signature of multifunctional CD8+ T cells (CD8-fit T cells). CD8-fit T cells are capable of migration and serial killing and harbor balanced mitochondrial and lysosomal volumes. Using independent datasets, we validate that CD8-fit T cells (1) are present premanufacture and are associated with clinical responses in individuals treated with axicabtagene ciloleucel, (2) longitudinally persist in individuals after treatment with CAR T cells and (3) are tumor migrating cytolytic cells capable of intratumoral expansion in solid tumors. Our results demonstrate the power of multimodal integration of single-cell functional assessments for the discovery and application of CD8-fit T cells as a T cell subset with optimal fitness in cell therapy.
Sujet(s)
Lymphocytes T CD8+ , Immunothérapie adoptive , Lymphome B diffus à grandes cellules , Récepteurs chimériques pour l'antigène , Analyse sur cellule unique , Humains , Lymphome B diffus à grandes cellules/immunologie , Lymphome B diffus à grandes cellules/thérapie , Analyse sur cellule unique/méthodes , Immunothérapie adoptive/méthodes , Lymphocytes T CD8+/immunologie , Récepteurs chimériques pour l'antigène/immunologie , Sous-populations de lymphocytes T/immunologie , Produits biologiquesRÉSUMÉ
Chimeric antigen receptor (CAR) T cell show promise in cancer treatments, but their mechanism of action is not well understood. Decoding the mechanisms used by individual T cells can help improve the efficacy of T cells while also identifying mechanisms of T cell failure leading to tumor escape. Here, we used a suite of assays including dynamic single-cell imaging of cell-cell interactions, dynamic imaging of fluorescent reporters to directly track cytotoxin activity in tumor cells, and scRNA-seq on patient infusion products to investigate the cytotoxic mechanisms used by individual CAR T cells in killing tumor cells. We show that surprisingly, overexpression of the Granzyme B (GZMB) inhibitor, protease inhibitor-9 (PI9), does not alter the cytotoxicity mediated by CD19-specific CAR T cells against either the leukemic cell line, NALM6; or the ovarian cancer cell line, SkOV3-CD19. We designed and validated reporters to directly assay T cell delivered GZMB activity in tumor cells and confirmed that while PI9 overexpression inhibits GZMB activity at the molecular level, this is not sufficient to impact the kinetics or magnitude of killing mediated by the CAR T cells. Altering cytotoxicity mediated by CAR T cells required combined inhibition of multiple pathways that are tumor cell specific: (a) B-cell lines like NALM6, Raji and Daudi were sensitive to combined GZMB and granzyme A (GZMA) inhibition; whereas (b) solid tumor targets like SkOV3-CD19 and A375-CD19 (melanoma) were sensitive to combined GZMB and Fas ligand inhibition. We realized the translational relevance of these findings by examining the scRNA-seq profiles of Tisa-cel and Axi-cel infusion products and show a significant correlation between GZMB and GZMA expression at the single-cell level in a T cell subset-dependent manner. Our findings highlight the importance of the redundancy in killing mechanisms of CAR T cells and how this redundancy is important for efficacious T cells.
Sujet(s)
Tumeurs , Récepteurs chimériques pour l'antigène , Humains , Récepteurs chimériques pour l'antigène/génétique , Granzymes/génétique , Lymphocytes T , Immunothérapie adoptive/méthodesRÉSUMÉ
BACKGROUND: Adoptive cell therapy based on the infusion of chimeric antigen receptor (CAR) T cells has shown remarkable efficacy for the treatment of hematologic malignancies. The primary mechanism of action of these infused T cells is the direct killing of tumor cells expressing the cognate antigen. However, understanding why only some T cells are capable of killing, and identifying mechanisms that can improve killing has remained elusive. METHODS: To identify molecular and cellular mechanisms that can improve T-cell killing, we utilized integrated high-throughput single-cell functional profiling by microscopy, followed by robotic retrieval and transcriptional profiling. RESULTS: With the aid of mathematical modeling we demonstrate that non-killer CAR T cells comprise a heterogeneous population that arise from failure in each of the discrete steps leading to the killing. Differential transcriptional single-cell profiling of killers and non-killers identified CD137 as an inducible costimulatory molecule upregulated on killer T cells. Our single-cell profiling results directly demonstrate that inducible CD137 is feature of killer (and serial killer) T cells and this marks a different subset compared with the CD107apos (degranulating) subset of CAR T cells. Ligation of the induced CD137 with CD137 ligand (CD137L) leads to younger CD19 CAR T cells with sustained killing and lower exhaustion. We genetically modified CAR T cells to co-express CD137L, in trans, and this lead to a profound improvement in anti-tumor efficacy in leukemia and refractory ovarian cancer models in mice. CONCLUSIONS: Broadly, our results illustrate that while non-killer T cells are reflective of population heterogeneity, integrated single-cell profiling can enable identification of mechanisms that can enhance the function/proliferation of killer T cells leading to direct anti-tumor benefit.
Sujet(s)
Ligand de 4-1BB/génétique , Analyse de profil d'expression de gènes , Immunothérapie adoptive , Leucémies/thérapie , Tumeurs de l'ovaire/thérapie , Récepteurs chimériques pour l'antigène/génétique , Analyse sur cellule unique , Lymphocytes T/transplantation , Transcriptome , Ligand de 4-1BB/métabolisme , Animaux , Cytotoxicité immunologique/génétique , Femelle , Récepteur cellulaire-2 du virus de l'hépatite A/génétique , Récepteur cellulaire-2 du virus de l'hépatite A/métabolisme , Humains , Immunophénotypage , Cellules K562 , Leucémies/génétique , Leucémies/immunologie , Leucémies/métabolisme , Souris de lignée NOD , Souris SCID , Tumeurs de l'ovaire/génétique , Tumeurs de l'ovaire/immunologie , Tumeurs de l'ovaire/métabolisme , Phénotype , Récepteurs chimériques pour l'antigène/métabolisme , Lymphocytes T/immunologie , Lymphocytes T/métabolisme , Tests d'activité antitumorale sur modèle de xénogreffeRÉSUMÉ
The protein p53 is a crucial tumor suppressor, often called "the guardian of the genome"; however, mutations transform p53 into a powerful cancer promoter. The oncogenic capacity of mutant p53 has been ascribed to enhanced propensity to fibrillize and recruit other cancer fighting proteins in the fibrils, yet the pathways of fibril nucleation and growth remain obscure. Here, we combine immunofluorescence three-dimensional confocal microscopy of human breast cancer cells with light scattering and transmission electron microscopy of solutions of the purified protein and molecular simulations to illuminate the mechanisms of phase transformations across multiple length scales, from cellular to molecular. We report that the p53 mutant R248Q (R, arginine; Q, glutamine) forms, both in cancer cells and in solutions, a condensate with unique properties, mesoscopic protein-rich clusters. The clusters dramatically diverge from other protein condensates. The cluster sizes are decoupled from the total cluster population volume and independent of the p53 concentration and the solution concentration at equilibrium with the clusters varies. We demonstrate that the clusters carry out a crucial biological function: they host and facilitate the nucleation of amyloid fibrils. We demonstrate that the p53 clusters are driven by structural destabilization of the core domain and not by interactions of its extensive unstructured region, in contradistinction to the dense liquids typical of disordered and partially disordered proteins. Two-step nucleation of mutant p53 amyloids suggests means to control fibrillization and the associated pathologies through modifying the cluster characteristics. Our findings exemplify interactions between distinct protein phases that activate complex physicochemical mechanisms operating in biological systems.
Sujet(s)
Amyloïde/composition chimique , Mutation faux-sens , Protéine p53 suppresseur de tumeur/composition chimique , Substitution d'acide aminé , Amyloïde/génétique , Amyloïde/métabolisme , Humains , Cellules MCF-7 , Protéine p53 suppresseur de tumeur/génétique , Protéine p53 suppresseur de tumeur/métabolismeRÉSUMÉ
BACKGROUND: Despite approval of immunotherapy for a wide range of cancers, the majority of patients fail to respond to immunotherapy or relapse following initial response. These failures may be attributed to immunosuppressive mechanisms co-opted by tumor cells. However, it is challenging to use conventional methods to systematically evaluate the potential of tumor intrinsic factors to act as immune regulators in patients with cancer. METHODS: To identify immunosuppressive mechanisms in non-responders to cancer immunotherapy in an unbiased manner, we performed genome-wide CRISPR immune screens and integrated our results with multi-omics clinical data to evaluate the role of tumor intrinsic factors in regulating two rate-limiting steps of cancer immunotherapy, namely, T cell tumor infiltration and T cell-mediated tumor killing. RESULTS: Our studies revealed two distinct types of immune resistance regulators and demonstrated their potential as therapeutic targets to improve the efficacy of immunotherapy. Among them, PRMT1 and RIPK1 were identified as a dual immune resistance regulator and a cytotoxicity resistance regulator, respectively. Although the magnitude varied between different types of immunotherapy, genetically targeting PRMT1 and RIPK1 sensitized tumors to T-cell killing and anti-PD-1/OX40 treatment. Interestingly, a RIPK1-specific inhibitor enhanced the antitumor activity of T cell-based and anti-OX40 therapy, despite limited impact on T cell tumor infiltration. CONCLUSIONS: Collectively, the data provide a rich resource of novel targets for rational immuno-oncology combinations.