RESUMEN
BACKGROUND: Adoptive T-cell therapy targeting antigens expressed in glioblastoma has emerged as a potential therapeutic strategy to prevent or delay recurrence and prolong overall survival in this aggressive disease setting. Ephrin receptor A3 (EphA3), which is highly expressed in glioblastoma; in particular, on the tumor vasculature and brain cancer stem cells, is an ideal target for immune-based therapies. METHODS: We have designed an EphA3-targeted chimeric antigen receptor (CAR) using the single chain variable fragment of a novel monoclonal antibody, and assessed its therapeutic potential against EphA3-expressing patient-derived glioblastoma neurospheres, organoids and xenografted glioblastoma tumors in immunodeficient mice. RESULTS: In vitro expanded EphA3 CAR T cells from healthy individuals efficiently recognize and kill EphA3-positive glioblastoma cells in vitro. Furthermore, these effector cells demonstrated curative efficacy in an orthotopic xenograft model of glioblastoma. EphA3 CAR T cells were equally effective in targeting patient-derived neurospheres and infiltrate, disaggregate, and induce apoptosis in glioblastoma-derived organoids. CONCLUSIONS: This study provides compelling evidence supporting the therapeutic potential of EphA3 CAR T-cell therapy against glioblastoma by targeting EphA3 associated with brain cancer stem cells and the tumor vasculature. The ability to target patient-derived glioblastoma underscores the translational significance of this EphA3 CAR T-cell therapy in the pursuit of effective and targeted glioblastoma treatment strategies.
Asunto(s)
Glioblastoma , Receptor EphA3 , Glioblastoma/terapia , Glioblastoma/inmunología , Humanos , Animales , Ratones , Neoplasias Encefálicas/inmunología , Neoplasias Encefálicas/terapia , Ensayos Antitumor por Modelo de Xenoinjerto , Receptores Quiméricos de Antígenos/inmunología , Receptores Quiméricos de Antígenos/metabolismo , Inmunoterapia Adoptiva/métodos , Linfocitos T/inmunología , Línea Celular TumoralRESUMEN
BACKGROUND & AIMS: Capsaicin receptor, also known as transient receptor potential vanilloid 1 (TRPV1), is involved in pain physiology and neurogenic inflammation. Herein, we discovered the presence of TRPV1 in hepatic stellate cells (HSCs) and aimed to delineate its function in this cell type and liver fibrosis. METHODS: TRPV1 expression was examined in liver biopsies from patients with liver fibrosis using quantitative real-time PCR and immunostaining. Its contribution to liver fibrosis was examined in Trpv1-/- mice, upon lentiviral delivery of the TRPV1 gene, and in human and mouse primary HSCs, using patch clamp, intracellular Ca2+ mobilization determination, FACS analyses and gain/loss of function experiments. Binding of sterile alpha and Toll/interleukin-1 receptor motif-containing protein 1 (SARM1) to TRPV1 was determined using mass spectrometry, co-immunoprecipitation, surface plasmon resonance, bioluminescence resonance energy transfer, and NanoBiT. RESULTS: TRPV1 mRNA levels are significantly downregulated in patients with liver fibrosis and mouse models, showing a negative correlation with F stage and α-smooth muscle actin expression, a marker of HSC activation. TRPV1 expression and function decrease during HSC activation in fibrotic livers in vivo or during culture. Genetic and pharmacological inhibition of TRPV1 in quiescent HSCs leads to NF-κB activation and pro-inflammatory cytokine production. TRPV1 requires binding of its N-terminal ankyrin repeat domain to the TIR-His583 (Toll/interleukin-1 receptor) domain of SARM1 to prevent HSCs from pro-inflammatory activation. Trpv1-/- mice display increased HSC activation and more severe liver fibrosis, whereas TRPV1 overexpression is antifibrotic in various disease models. CONCLUSION: The antifibrotic properties of TRPV1 are attributed to the prevention of HSC activation via the recruitment of SARM1, which could be an attractive therapeutic strategy against liver fibrosis. IMPACT AND IMPLICATIONS: We identified the neuronal channel protein TRPV1 as a gatekeeper of quiescence in hepatic stellate cells, a key driver of liver fibrogenesis and chronic liver disease. Physiologically expressed in healthy liver and consistently downregulated during liver fibrosis development, its therapeutic re-expression is expected to have few side effects, making it an attractive target diagnostic tool and drug candidate for industry and clinicians.
Asunto(s)
Células Estrelladas Hepáticas , Canales Catiónicos TRPV , Humanos , Ratones , Animales , Canales Catiónicos TRPV/genética , Canales Catiónicos TRPV/metabolismo , Canales Catiónicos TRPV/farmacología , Células Estrelladas Hepáticas/metabolismo , Hígado/patología , Cirrosis Hepática/patología , Regulación de la Expresión Génica , Proteínas del Citoesqueleto/metabolismo , Proteínas del Citoesqueleto/farmacología , Proteínas del Dominio Armadillo/genética , Proteínas del Dominio Armadillo/metabolismoRESUMEN
There is now convincing evidence that the successful development of an effective CMV vaccine will require improved formulation and adjuvant selection that is capable of inducing both humoral and cellular immune responses. Here, we have designed a novel bivalent subunit vaccine formulation based on CMV-encoded oligomeric glycoprotein B (gB) and polyepitope protein in combination with human compatible TLR9 agonist CpG1018. The polyepitope protein includes multiple minimal HLA class I-restricted CD8+ T cell epitopes from different antigens of CMV. This subunit vaccine generated durable anti-viral antibodies, CMV-specific CD4+ and CD8+ T cell responses in multiple HLA expressing mice. Antibody responses included broad TH1 isotypes (IgG2a, IgG2b and IgG3) and potently neutralized CMV infection in fibroblasts and epithelial cells. Furthermore, polyfunctional antigen-specific T cell immunity and antiviral antibody responses showed long-term memory maintenance. These observations argue that this novel vaccine strategy, if applied to humans, could facilitate the generation of robust humoral and cellular immune responses which may be more effective in preventing CMV-associated complications in various clinical settings.
Asunto(s)
Infecciones por Citomegalovirus , Inmunidad Humoral , Adyuvantes Inmunológicos , Animales , Anticuerpos Antivirales , Linfocitos T CD8-positivos , Epítopos de Linfocito T/metabolismo , Humanos , Inmunidad Celular , Ratones , Oligodesoxirribonucleótidos , Receptor Toll-Like 9/metabolismo , Vacunas Combinadas , Vacunas de SubunidadRESUMEN
SARM1 (sterile alpha and TIR motif containing 1) is responsible for depletion of nicotinamide adenine dinucleotide in its oxidized form (NAD+) during Wallerian degeneration associated with neuropathies. Plant nucleotide-binding leucine-rich repeat (NLR) immune receptors recognize pathogen effector proteins and trigger localized cell death to restrict pathogen infection. Both processes depend on closely related Toll/interleukin-1 receptor (TIR) domains in these proteins, which, as we show, feature self-association-dependent NAD+ cleavage activity associated with cell death signaling. We further show that SARM1 SAM (sterile alpha motif) domains form an octamer essential for axon degeneration that contributes to TIR domain enzymatic activity. The crystal structures of ribose and NADP+ (the oxidized form of nicotinamide adenine dinucleotide phosphate) complexes of SARM1 and plant NLR RUN1 TIR domains, respectively, reveal a conserved substrate binding site. NAD+ cleavage by TIR domains is therefore a conserved feature of animal and plant cell death signaling pathways.
Asunto(s)
Proteínas del Dominio Armadillo/química , Proteínas del Citoesqueleto/química , NAD+ Nucleosidasa/química , NAD/metabolismo , Proteínas de Plantas/química , Dominios Proteicos , Receptores Inmunológicos/química , Animales , Proteínas del Dominio Armadillo/metabolismo , Axones/enzimología , Axones/patología , Sitios de Unión , Muerte Celular , Secuencia Conservada , Cristalografía por Rayos X , Proteínas del Citoesqueleto/metabolismo , Células HEK293 , Humanos , Ratones , NAD+ Nucleosidasa/metabolismo , NADP/metabolismo , Neuronas/enzimología , Proteínas de Plantas/metabolismo , Multimerización de Proteína , Receptores Inmunológicos/metabolismo , Degeneración Walleriana/enzimología , Degeneración Walleriana/patologíaRESUMEN
Toll-like receptor (TLR) signaling is a key innate immunity response to pathogens. Recruitment of signaling adapters such as MAL (TIRAP) and MyD88 to the TLRs requires Toll/interleukin-1 receptor (TIR)-domain interactions, which remain structurally elusive. Here we show that MAL TIR domains spontaneously and reversibly form filaments in vitro. They also form cofilaments with TLR4 TIR domains and induce formation of MyD88 assemblies. A 7-Å-resolution cryo-EM structure reveals a stable MAL protofilament consisting of two parallel strands of TIR-domain subunits in a BB-loop-mediated head-to-tail arrangement. Interface residues that are important for the interaction are conserved among different TIR domains. Although large filaments of TLR4, MAL or MyD88 are unlikely to form during cellular signaling, structure-guided mutagenesis, combined with in vivo interaction assays, demonstrated that the MAL interactions defined within the filament represent a template for a conserved mode of TIR-domain interaction involved in both TLR and interleukin-1 receptor signaling.