RESUMEN
Allogeneic chimeric antigen receptor (CAR)-T cells hold great promise for expanding the accessibility of CAR-T therapy, whereas the risks of allograft rejection have hampered its application. Here, we genetically engineered healthy-donor-derived, CD19-targeting CAR-T cells using CRISPR-Cas9 to address the issue of immune rejection and treated one patient with refractory immune-mediated necrotizing myopathy and two patients with diffuse cutaneous systemic sclerosis with these cells. This study was registered at ClinicalTrials.gov (NCT05859997). The infused cells persisted for over 3 months, achieving complete B cell depletion within 2 weeks of treatment. During the 6-month follow-up, we observed deep remission without cytokine release syndrome or other serious adverse events in all three patients, primarily shown by the significant improvement in the clinical response index scores for the two diseases, respectively, and supported by the observations of reversal of inflammation and fibrosis. Our results demonstrate the high safety and promising immune modulatory effect of the off-the-shelf CAR-T cells in treating severe refractory autoimmune diseases.
RESUMEN
Recently, chimeric antigen receptor (CAR)-T cell therapy has shown great promise in treating haematological malignancies1-7. However, CAR-T cell therapy currently has several limitations8-12. Here we successfully developed a two-in-one approach to generate non-viral, gene-specific targeted CAR-T cells through CRISPR-Cas9. Using the optimized protocol, we demonstrated feasibility in a preclinical study by inserting an anti-CD19 CAR cassette into the AAVS1 safe-harbour locus. Furthermore, an innovative type of anti-CD19 CAR-T cell with PD1 integration was developed and showed superior ability to eradicate tumour cells in xenograft models. In adoptive therapy for relapsed/refractory aggressive B cell non-Hodgkin lymphoma (ClinicalTrials.gov, NCT04213469 ), we observed a high rate (87.5%) of complete remission and durable responses without serious adverse events in eight patients. Notably, these enhanced CAR-T cells were effective even at a low infusion dose and with a low percentage of CAR+ cells. Single-cell analysis showed that the electroporation method resulted in a high percentage of memory T cells in infusion products, and PD1 interference enhanced anti-tumour immune functions, further validating the advantages of non-viral, PD1-integrated CAR-T cells. Collectively, our results demonstrate the high safety and efficacy of non-viral, gene-specific integrated CAR-T cells, thus providing an innovative technology for CAR-T cell therapy.
Asunto(s)
Inmunoterapia Adoptiva , Linfoma de Células B , Receptores Quiméricos de Antígenos , Animales , Antígenos CD19/inmunología , Electroporación , Humanos , Inmunoterapia Adoptiva/efectos adversos , Inmunoterapia Adoptiva/métodos , Linfoma de Células B/inmunología , Linfoma de Células B/patología , Linfoma de Células B/terapia , Células T de Memoria/inmunología , Receptor de Muerte Celular Programada 1/inmunología , Receptores Quiméricos de Antígenos/inmunología , Receptores Quiméricos de Antígenos/uso terapéutico , Recurrencia , Análisis de la Célula Individual , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
BACKGROUND: Although chimeric antigen receptor T (CAR-T) cells have been proven to be an effective way of treating B cell malignancies, a lot of patients could not benefit from it because of failure in CAR-T cell manufacturing, disease progression, and unaffordable price. The study aimed to explore universal CAR-T cell products to extend the clinical accessibility. METHODS: The antitumor activity of CRISPR/Cas9-edited allogeneic anti-CD19 CAR-T (CAR-T19) cells was assessed in vitro, in animal models, and in patients with relapsed/refractory (R/R) acute B cell lymphoblastic leukemia (B-ALL) or diffuse large B cell lymphoma. RESULTS: B2M-/TRAC- universal CAR-T19 (U-CAR-T19) cells exhibited powerful anti-leukemia abilities both in vitro and in animal models, as did primary CD19+ leukemia cells from leukemia patients. However, expansion, antitumor efficacy, or graft-versus-host-disease (GvHD) was not observed in six patients with R/R B cell malignancies after U-CAR-T19 cell infusion. Accordingly, significant activation of natural killer (NK) cells by U-CAR-T19 cells was proven both clinically and in vitro. HLA-A-/B-/TRAC- novel CAR-T19 (nU-CAR-T19) cells were constructed with similar tumoricidal capacity but resistance to NK cells in vitro. Surprisingly, robust expansion of nU-CAR-T19 cells, along with rapid eradication of CD19+ abnormal B cells, was observed in the peripheral blood and bone marrow of another three patients with R/R B-ALL. The patients achieved complete remission with no detectable minimal residual disease 14 days after the infusion of nU-CAR-T19 cells. Two of the three patients had grade 2 cytokine release syndrome, which were managed using an IL-6 receptor blocker. Most importantly, GvHD was not observed in any patient, suggesting the safety of TRAC-disrupted CAR-T cells generated using the CRISPR/Cas9 method for clinical application. CONCLUSIONS: The nU-CAR-T19 cells showed a strong response in R/R B-ALL. nU-CAR-T19 cells have the potential to be a promising new approach for treating R/R B cell malignancies.
Asunto(s)
Enfermedad Injerto contra Huésped , Trasplante de Células Madre Hematopoyéticas , Leucemia Linfocítica Crónica de Células B , Leucemia , Receptores Quiméricos de Antígenos , Animales , Humanos , Receptores Quiméricos de Antígenos/genética , Anticuerpos , Antígenos CD19 , Linfocitos T , Antígenos HLA-ARESUMEN
Tertiary lymphoid structures (TLSs) in tumor tissues facilitate immune cell trafficking and cytotoxicity, which benefits survival and favorable responses in immune therapy. Here, we observed a high correlation of tumor necrosis factor superfamily member 14 (LIGHT) expression with TLS signature genes, which are all markers for immune cell accumulation and better prognosis, through retrieving RNA sequencing (RNA-seq) data from patients with cancer, suggesting the potential of LIGHT in reconstituting a high immune-infiltrated tumor microenvironment. Accordingly, LIGHT co-expressed chimeric antigen receptor T (LIGHT CAR-T) cells not only showed enhanced cytotoxicity and cytokine production but also improved CCL19 and CCL21 expression by surrounding cells. And the supernatant of LIGHT CAR-T cells promoted T cell migration in a paracrine manner. Furthermore, LIGHT CAR-T cells showed superior anti-tumor efficacy and improved infiltration in comparison with conventional CAR-T cells in immunodeficient NSG mice. Accordingly, murine LIGHT-OT-1 T cells normalized tumor blood vessels and enforced intratumoral lymphoid structures in C57BL/6 syngeneic tumor mouse models, implying the potential of LIGHT CAR-T in clinical application. Taken together, our data revealed a straightforward strategy to optimize trafficking and cytotoxicity of CAR-T cells by redirecting TLSs through LIGHT expression, which has great potential to expand and optimize the application of CAR-T therapy in solid tumors.
Asunto(s)
Receptores Quiméricos de Antígenos , Miembro 14 de la Superfamilia de Ligandos de Factores de Necrosis Tumoral , Animales , Ratones , Línea Celular Tumoral , Inmunoterapia Adoptiva , Ratones Endogámicos C57BL , Receptores Quiméricos de Antígenos/metabolismo , Linfocitos T , Microambiente Tumoral/genéticaRESUMEN
Vesicular stomatitis virus (VSV) and rabies and Chandipura viruses belong to the Rhabdovirus family. VSV is a common laboratory virus to study viral evolution and host immune responses to viral infection, and recombinant VSV-based vectors have been widely used for viral oncolysis, vaccination, and gene therapy. Although the tropism of VSV is broad, and its envelope glycoprotein G is often used for pseudotyping other viruses, the host cellular components involved in VSV infection remain unclear. Here, we demonstrate that the host protein leucine-rich repeat-containing G protein-coupled receptor 4 (Lgr4) is essential for VSV and VSV-G pseudotyped lentivirus (VSVG-LV) to infect susceptible cells. Accordingly, Lgr4-deficient mice had dramatically decreased VSV levels in the olfactory bulb. Furthermore, Lgr4 knockdown in RAW 264.7 cells also significantly suppressed VSV infection, and Lgr4 overexpression in RAW 264.7 cells enhanced VSV infection. Interestingly, only VSV infection relied on Lgr4, whereas infections with Newcastle disease virus, influenza A virus (A/WSN/33), and herpes simplex virus were unaffected by Lgr4 status. Of note, assays of virus entry, cell ELISA, immunoprecipitation, and surface plasmon resonance indicated that VSV bound susceptible cells via the Lgr4 extracellular domain. Pretreating cells with an Lgr4 antibody, soluble LGR4 extracellular domain, or R-spondin 1 blocked VSV infection by competitively inhibiting VSV binding to Lgr4. Taken together, the identification of Lgr4 as a VSV-specific host factor provides important insights into understanding VSV entry and its pathogenesis and lays the foundation for VSV-based gene therapy and viral oncolytic therapeutics.
Asunto(s)
Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Estomatitis Vesicular/metabolismo , Vesiculovirus/metabolismo , Proteínas del Envoltorio Viral/metabolismo , Internalización del Virus , Animales , Anticuerpos/farmacología , Femenino , Células HEK293 , Humanos , Ratones , Ratones Noqueados , Bulbo Olfatorio/metabolismo , Bulbo Olfatorio/virología , Células RAW 264.7 , Receptores Acoplados a Proteínas G/antagonistas & inhibidores , Estomatitis Vesicular/genética , Vesiculovirus/genética , Proteínas del Envoltorio Viral/genéticaRESUMEN
Extracellular UDP (eUDP), released as a danger signal by stressed or apoptotic cells, plays an important role in a series of physiological processes. Although the mechanism of eUDP release in apoptotic cells has been well defined, how the eUDP is released in innate immune responses remains unknown. In this study, we demonstrated that UDP was released in both Escherichia coli-infected mice and LPS- or Pam3CSK4-treated macrophages. Also, LPS-induced UDP release could be significantly blocked by selective TLR4 inhibitor Atractylenolide I and selective gap junction inhibitors carbenoxolone and flufenamic acid (FFA), suggesting the key role of TLR signaling and gap junction channels in this process. Meanwhile, eUDP protected mice from peritonitis by reducing invaded bacteria that could be rescued by MRS2578 (selective P2Y6 receptor inhibitor) and FFA. Then, connexin 43, as one of the gap junction proteins, was found to be clearly increased by LPS in a dose- and time-dependent manner. Furthermore, if we blocked LPS-induced ERK signaling by U0126, the expression of connexin 43 and UDP release was also inhibited dramatically. In addition, UDP-induced MCP-1 secretion was significantly reduced by MRS2578, FFA, and P2Y6 mutation. Accordingly, pretreating mice with U0126 and Gap26 increased invaded bacteria and aggravated mice death. Taken together, our study reveals an internal relationship between danger signals and TLR signaling in innate immune responses, which suggests a potential therapeutic significance of gap junction channel-mediated UDP release in infectious diseases.
Asunto(s)
Conexina 43/inmunología , Infecciones por Escherichia coli/inmunología , Transducción de Señal/inmunología , Receptor Toll-Like 4/inmunología , Uridina Difosfato/inmunología , Uridina Difosfato/metabolismo , Animales , Western Blotting , Conexina 43/metabolismo , Modelos Animales de Enfermedad , Ensayo de Inmunoadsorción Enzimática , Femenino , Uniones Comunicantes/inmunología , Inmunidad Innata , Macrófagos Peritoneales/inmunología , Ratones , Ratones Endogámicos C57BL , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptor Toll-Like 4/metabolismo , Receptores Toll-Like/inmunología , Receptores Toll-Like/metabolismoRESUMEN
Extracellular nucleotides that constitute a "danger signal" play an important role in the regulation of immune responses. However, the function and mechanism of extracellular UDP and P2Y6 in antiviral immunity remain unknown. In this study, we demonstrated the in vitro and in vivo protection of UDP/P2Y6 signaling in vesicular stomatitis virus (VSV) infection. First, we demonstrated that VSV-infected cells secrete UDP from the cytoplasm as a danger signal to arouse surrounding cells. Meanwhile, expression of the UDP-specific receptor P2Y6 also was enhanced by VSV. Consequently, UDP protects RAW 264.7 cells, murine embryonic fibroblasts, bone marrow-derived macrophages, and L929 cells from VSV and GFP lentivirus infection. This protection can be blocked by the P2Y6 selective antagonist MRS2578 or IFN-α/ß receptor-blocking Ab. VSV-induced cell death and virus replication were both enhanced significantly by knocking down and knocking out P2Y6 in different cells. Mechanistically, UDP facilitates IFN-ß secretion through the p38/JNK- and ATF-2/c-Jun-signaling pathways, which are crucial in promoting antiviral immunity. Interestingly, UDP was released through a caspase-cleaved pannexin-1 channel in VSV-induced apoptotic cells and protected cells from infection through P2Y6 receptor in an autocrine or paracrine manner. Furthermore, UDP also protected mice from VSV infection through P2Y6 receptors in an acute neurotropic infection mouse model. Taken together, these results demonstrate the important role of extracellular UDP and P2Y6 as a danger signal in antiviral immune responses and suggest a potential therapeutic role for UDP/P2Y6 in preventing and controlling viral diseases.
Asunto(s)
Interferón beta/biosíntesis , Receptores Purinérgicos P2/metabolismo , Transducción de Señal , Uridina Difosfato/metabolismo , Estomatitis Vesicular/metabolismo , Vesiculovirus/fisiología , Factor de Transcripción Activador 2/metabolismo , Animales , Apoptosis , Línea Celular , Células Cultivadas , Conexinas/metabolismo , Modelos Animales de Enfermedad , Femenino , Uniones Comunicantes/metabolismo , Expresión Génica , Humanos , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Macrófagos , Ratones , Ratones Noqueados , Modelos Biológicos , Proteínas Proto-Oncogénicas c-jun/metabolismo , Receptores Purinérgicos P2/genética , Estomatitis Vesicular/genética , Estomatitis Vesicular/virología , Replicación Viral , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
The recognition of pathogen-associated molecular patterns by Toll-like receptors (TLRs) is pivotal in both innate and adaptive immune responses. Here we demonstrate that deletion of Lgr4/Gpr48 (G-protein-coupled receptor 48), a seven-transmembrane glycoprotein hormone receptor, potentiates TLR2/4-associated cytokine production and attenuates mouse resistance to septic shock. The expression of CD14, a co-receptor for TLR2/4-associated pathogen-associated molecular patterns, is increased significantly in Lgr4-deficient macrophages, which is consistent with the increased immune response, whereas the binding activity of cAMP-response element-binding protein is decreased significantly in Lgr4-deficient macrophages, which up-regulate the expression of CD14 at the transcriptional level. Together, our data demonstrate that Lgr4/Gpr48 plays a critical role in modulating the TLR2/4 signaling pathway and represents a useful therapeutic approach of targeting Lgr4/Gpr48 in TLR2/4-associated septic shock and autoimmune diseases.
Asunto(s)
Inmunidad Innata/fisiología , Receptores de Lipopolisacáridos/biosíntesis , Receptores Acoplados a Proteínas G/metabolismo , Receptor Toll-Like 2/metabolismo , Receptor Toll-Like 4/metabolismo , Regulación hacia Arriba/fisiología , Animales , Enfermedades Autoinmunes/genética , Enfermedades Autoinmunes/inmunología , Enfermedades Autoinmunes/metabolismo , Enfermedades Autoinmunes/terapia , Línea Celular , Humanos , Receptores de Lipopolisacáridos/genética , Receptores de Lipopolisacáridos/inmunología , Macrófagos/citología , Macrófagos/inmunología , Macrófagos/metabolismo , Ratones , Ratones Noqueados , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/inmunología , Choque Séptico/genética , Choque Séptico/inmunología , Choque Séptico/metabolismo , Choque Séptico/terapia , Transducción de Señal/fisiología , Receptor Toll-Like 2/genética , Receptor Toll-Like 2/inmunología , Receptor Toll-Like 4/genética , Receptor Toll-Like 4/inmunologíaRESUMEN
Extracellular ATP (eATP), released as a "danger signal" by injured or stressed cells, plays an important role in the regulation of immune responses, but the relationship between ATP release and innate immune responses is still uncertain. In this study, we demonstrated that ATP was released through Toll-like receptor (TLR)-associated signaling in both Escherichia coli-infected mice and lipopolysaccharide (LPS)- or Pam3CSK4-treated macrophages. This ATP release could be blocked completely only by N-ethylmaleimide (NEM), not by carbenoxolone (CBX), flufenamic acid (FFA), or probenecid, suggesting the key role of exocytosis in this process. Furthermore, LPS-induced ATP release could also be reduced dramatically through suppressing calcium mobilization by use of U73122, caffeine, and thapsigargin (TG). In addition, the secretion of interleukin-1ß (IL-1ß) and CCL-2 was enhanced significantly by ATP, in a time- and dose-dependent manner. Meanwhile, macrophage-mediated phagocytosis of bacteria was also promoted significantly by ATP stimulation. Furthermore, extracellular ATP reduced the number of invading bacteria and protected mice from peritonitis by activating purinergic receptors. Mechanistically, phosphorylation of AKT and ERK was overtly increased by ATP in antibacterial immune responses. Accordingly, if we blocked the P2X- and P2Y-associated signaling pathway by using suramin and pyridoxal phosphate-6-azo(benzene-2,4-disulfonic acid), tetrasodium salt (PPADS), the ATP-enhanced immune response was restrained significantly. Taken together, our findings reveal an internal relationship between danger signals and TLR signaling in innate immune responses, which suggests a potential therapeutic significance of calcium mobilization-mediated ATP release in infectious diseases.
Asunto(s)
Adenosina Trifosfato/metabolismo , Calcio/metabolismo , Infecciones por Escherichia coli/inmunología , Infecciones por Escherichia coli/prevención & control , Inmunidad Innata , Transducción de Señal , Receptores Toll-Like/inmunología , Animales , Modelos Animales de Enfermedad , Escherichia coli/inmunología , Femenino , Lipopolisacáridos/inmunología , Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Ratones Endogámicos C57BL , Peritonitis/inmunología , Peritonitis/prevención & controlRESUMEN
BACKGROUND: Emerging evidences suggest that aberrant metabolites contributes to the immunosuppressive microenvironment that leads to cancer immune evasion. Among tumor immunosuppressive cells, myeloid-derived suppressor cells (MDSCs) are pathologically activated and extremely immunosuppressive, which are closely associated with poor clinical outcomes of cancer patients. However, the correlation between MDSCs mediated immunosuppression and particular cancer metabolism remained elusive. METHODS: Spontaneous lung adenocarcinoma and subcutaneous mouse tumor models, gas chromatography-mass spectrometry (GC-MS) and immunofluorescence assay of patient-derived lung adenocarcinoma tissues, and flow cytometry, RNA sequencing and Western blotting of immune cells, were utilized. RESULTS: Metabolite profiling revealed a significant accumulation of acetic acids in tumor tissues from both patients and mouse model, which contribute to immune suppression and cancer progression significantly through free fatty acid receptor 2 (FFAR2). Furthermore, FFAR2 is highly expressed in the myeloid-derived suppressor cells (MDSCs) from the tumor of lung adenocarcinoma (LUAD) patients which is greatly associated with poor prognosis. Surprisingly, whole or myeloid Ffar2 gene deletion markedly inhibited urethane-induced lung carcinogenesis and syngeneic tumor growth with reduced MDSCs and increased CD8+ T cell infiltration. Mechanistically, FFAR2 deficiency in MDSCs significantly reduced the expression of Arg1 through Gαq/Calcium/PPAR-γ axis, which eliminated T cell dysfunction through relieving L-Arginine consumption in tumor microenvironment. Therefore, replenishment of L-Arginine or inhibition to PPAR-γ restored acetic acids/FFAR2 mediated suppression to T cells significantly. Finally, FFAR2 inhibition overcame resistance to immune checkpoint blockade through enhancing the recruitment and cytotoxicity of tumor-infiltrating T cells. CONCLUSION: Altogether, our results demonstrate that the acetic acids/FFAR2 axis enhances MDSCs mediated immunosuppression through Gαq/calcium/PPAR-γ/Arg1 signaling pathway, thus contributing to cancer progression. Therefore, FFAR2 may serve as a potential new target to eliminate pathologically activated MDSCs and reverse immunosuppressive tumor microenvironment, which has great potential in improving clinical outcomes of cancer immunotherapy.
Asunto(s)
Adenocarcinoma del Pulmón , Células Supresoras de Origen Mieloide , Neoplasias , Humanos , Ratones , Animales , Calcio/metabolismo , Receptores Activados del Proliferador del Peroxisoma/metabolismo , Adenocarcinoma del Pulmón/metabolismo , Arginina/metabolismo , Acetatos/metabolismo , Microambiente TumoralRESUMEN
Emerging evidence suggests that physiological distress is highly correlated with cancer incidence and mortality. However, the mechanisms underlying psychological challenges-mediated tumor immune evasion are not systematically explored. Here, it is demonstrated that acute restraint (AR) increases the level of the plasma neuropeptide hormones, kisspeptin, and the expression levels of its receptor, Gpr54, in the hypothalamus, splenic and tumor-infiltrating T cells, suggesting a correlation between the neuroendocrine system and tumor microenvironment. Accordingly, administration of kisspeptin-10 significantly impairs T cell function, whereas knockout of Gpr54 in T cells inhibits lung tumor progression by suppressing T cell dysfunction and exhaustion with or without AR. In addition, Gpr54 defective OT-1 T cells show superior antitumor activity against OVA peptide-positive tumors. Mechanistically, ERK5-mediated NR4A1 activation is found to be essential for kisspeptin/GPR54-facilitated T cell dysfunction. Meanwhile, pharmacological inhibition of ERK5 signaling by XMD8-92 significantly reduces the tumor growth by enhancing CD8+ T cell antitumor function. Furthermore, depletion of GPR54 or ERK5 by CRISPR/Cas9 in CAR T cells intensifies the antitumor responses to both PSMA+ and CD19+ tumor cells, while eliminating T cell exhaustion. Taken together, these results indicate that kisspeptin/GPR54 signaling plays a nonredundant role in the stress-induced tumor immune evasion.
Asunto(s)
Kisspeptinas , Neoplasias Pulmonares , Humanos , Kisspeptinas/metabolismo , Monitorización Inmunológica , Sistemas Neurosecretores/metabolismo , Receptores Acoplados a Proteínas G , Receptores de Kisspeptina-1 , Transducción de Señal/fisiología , Microambiente TumoralRESUMEN
P2X7, a crucial sensor of extracellular ATP, is widely distributed in different immune cells as a potent stimulant of inflammation and immunity. P2X7 is also highly expressed in immunosuppressive cells such as tumor-associated macrophages (TAM) and even tumor cells. However, the function and potential applications of P2X7-mediated immunosuppressive responses in the tumor microenvironment remain unclear. Here, we demonstrated that P2X7 was highly expressed in TAMs and that P2X7 deficiency impaired the "M2-like" polarization of TAMs via downregulation of STAT6 and IRF4 phosphorylation both in vivo and in vitro P2X7 deficiency restricted the progression of urethane-induced lung carcinogenesis and Lewis lung cancer by decreasing tumor cell proliferation and angiogenesis, promoting T-cell mobilization, and reversing M2-like TAM polarization. Thus, deletion or blockade of P2X7 was therapeutic for lung cancer. Furthermore, resistance to both immunotherapy (anti-PD-1 antibody) and chemotherapy (cisplatin) was overcome by coadministration of the P2X7 inhibitors O-ATP, A-438079 hydrochloride, and A-740003. Therefore, our data revealed a vital role of P2X7 in tumor formation through regulating TAM polarization, suggesting the therapeutic potential of P2X7 blockade in patients with lung cancer.
Asunto(s)
Neoplasias Pulmonares/terapia , Activación de Macrófagos/fisiología , Receptores Purinérgicos P2X7/metabolismo , Animales , Modelos Animales de Enfermedad , Humanos , Ratones , Ratones NoqueadosRESUMEN
Tumor-associated macrophages (TAMs) are one of the prominent components of the tumor microenvironment (TME). The polarization peculiarity of TAMs drives them to infiltrate and active with states between M1 (anti-tumor) and M2 (pro-tumor) phenotypes in cancers. Exploiting small molecular drugs through targeting TAMs to repolarize them into an antitumor phenotype is considered as a novel strategy for cancer treatments in recent years. For discovering novel compounds that target TAMs, a series of ureido tetrahydrocarbazole derivatives were designed, synthesized and evaluated both in vitro and in vivo. Among them, compound 23a was found to dose-dependently repolarize TAMs from M2 to M1 both in vitro and in vivo. And more importantly, the in vivo experiments also revealed that compound 23a was capable of remarkably inhibiting tumor growth of the LLC mouse model. Moreover, the synergy of compound 23a with anti-PD-1 antibody had more superior antineoplastic effects than the exclusive use of either in vivo.
Asunto(s)
Antineoplásicos/síntesis química , Carbazoles/síntesis química , Macrófagos/efectos de los fármacos , Urea/análogos & derivados , Urea/síntesis química , Animales , Anticuerpos Monoclonales/administración & dosificación , Antineoplásicos/farmacología , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Apoptosis/efectos de los fármacos , Carbazoles/administración & dosificación , Carbazoles/farmacología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Descubrimiento de Drogas/métodos , Sinergismo Farmacológico , Femenino , Humanos , Macrófagos/citología , Ratones , Ratones Endogámicos C57BL , Células RAW 264.7 , Relación Estructura-Actividad , Microambiente Tumoral , Urea/administración & dosificación , Urea/farmacologíaRESUMEN
Therapies targeting immune checkpoints have shown great clinical potential in a subset of patients with cancer but may be hampered by a failure to reverse the immunosuppressive tumor microenvironment (TME). As the most abundant immune cells in TME, tumor-associated macrophages (TAM) play nonredundant roles in restricting antitumor immunity. The leucine-rich repeat-containing G-protein-coupled receptor 4 (Lgr4, also known as Gpr48) has been associated with multiple physiologic and pathologic functions. Lgr4 and its ligands R-spondin 1-4 have been shown to promote the growth and metastasis of tumor cells. However, whether Lgr4 can promote tumor progression by regulating the function of immune cells in the tumor microenvironment remains largely unknown. Here, we demonstrate that Lgr4 promotes macrophage M2 polarization through Rspo/Lgr4/Erk/Stat3 signaling. Notably, urethane-induced lung carcinogenesis, Lewis lung carcinoma (LLC), and B16F10 melanoma tumors were all markedly reduced in Lgr4fl/flLyz2cre/+ mice, characterized by fewer protumoral M2 TAMs and increased CD8+ T lymphocyte infiltration in the TME. Furthermore, LLC tumor growth was greatly depressed when Rspo/Lgr4/Erk/Stat3 signaling was blocked with either the LGR4 extracellular domain or an anti-Rspo1 antibody. Importantly, blocking Rspo-Lgr4 signaling overcame LLC resistance to anti-PD-1 therapy and improved the efficacy of PD-1 immunotherapy against B16F10 melanoma, indicating vital roles of Rspo-Lgr4 in host antitumor immunity and a potential therapeutic target in cancer immunotherapy.Significance: This study identifies a novel receptor as a critical switch in TAM polarization whose inhibition sensitizes checkpoint therapy-resistant lung cancer to anti-PD-1 therapy.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/78/17/4929/F1.large.jpg Cancer Res; 78(17); 4929-42. ©2018 AACR.
Asunto(s)
Carcinogénesis/genética , Carcinoma Pulmonar de Lewis/inmunología , Melanoma Experimental/inmunología , Receptores Acoplados a Proteínas G/genética , Trombospondinas/genética , Animales , Linfocitos T CD8-positivos/inmunología , Carcinogénesis/inmunología , Carcinoma Pulmonar de Lewis/genética , Carcinoma Pulmonar de Lewis/patología , Carcinoma Pulmonar de Lewis/terapia , Línea Celular Tumoral , Polaridad Celular/genética , Genes cdc/inmunología , Humanos , Ligandos , Macrófagos/inmunología , Macrófagos/patología , Melanoma Experimental/genética , Melanoma Experimental/patología , Melanoma Experimental/terapia , Ratones , Receptor de Muerte Celular Programada 1/antagonistas & inhibidores , Receptor de Muerte Celular Programada 1/inmunología , Receptores Acoplados a Proteínas G/inmunología , Transducción de Señal/inmunología , Trombospondinas/inmunología , Microambiente Tumoral/genética , Microambiente Tumoral/inmunologíaRESUMEN
As the most prominent clinical drug targets for the inhibition of platelet aggregation, P2Y12 and P2Y13 have been found to be highly expressed in both platelets and macrophages. However, the roles and function of P2Y12/13 in the regulation of macrophage-mediated innate immune responses remain unclear. Here, we demonstrate that adenosine 5'-diphosphate (ADP), the endogenous ligand of P2Y1, P2Y12 and P2Y13, was released both in E. coli-infected mice and from macrophages treated with either lipopolysaccharide (LPS) or Pam3CSK4. Furthermore, the expression of P2Y13 was clearly increased in both LPS-treated macrophages and tuberculosis patients. ADP protected mice from E. coli 0111-induced peritonitis by recruiting more macrophages to the infected sites. Consistent with this, ADP and ADP-treated cell culture medium attracted more macrophages in the transwell assay by enhancing the expression of MCP-1. Nevertheless, P2Y1 is dispensable for ADP-mediated protection against bacterial infection. However, either P2Y12/P2Y13 deficiency or blocking the downstream signaling of P2Y12/P2Y13 blocked the ADP-mediated immune response and allowed more bacteria to persist in the infected mice. Furthermore, extracellular signal-regulated kinase (ERK) phosphorylation was clearly increased by ADP, and this type of activation could be blocked by either forskolin or analogs of cyclic AMP (cAMP) (for example, 8-bromo-cAMP). Accordingly, ADP-induced MCP-1 production and protection against bacterial infection could also be reduced by U0126, forskolin and 8-bromo-cAMP. Overall, our study reveals a relationship between danger signals and innate immune responses, which suggests the potential therapeutic significance of ADP-mediated purinergic signaling in infectious diseases.
Asunto(s)
Adenosina Difosfato/farmacología , Infecciones Bacterianas/enzimología , Infecciones Bacterianas/inmunología , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Espacio Extracelular/química , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Monocitos/metabolismo , Animales , Movimiento Celular/efectos de los fármacos , Quimiocina CCL2/metabolismo , AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Escherichia coli/efectos de los fármacos , Escherichia coli/fisiología , Femenino , Interacciones Huésped-Patógeno/efectos de los fármacos , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Ratones , Ratones Endogámicos C57BL , Modelos Biológicos , Monocitos/efectos de los fármacos , Fosforilación/efectos de los fármacos , Células RAW 264.7 , Receptores Purinérgicos P2/metabolismoRESUMEN
A new spectral distribution data file is set up by handling Arai's experimental data of spectral distribution, through the procession of interpolation and fitting. The distance of wavelength is 0.002 nm. The data file is applied to our FPM program and then to the analysis of refractory steels GH131. Correlation between theoretical and measured intensities is good. Results of theoretical influence coefficient method and fundamental parameter method are consistent with certified values; the relative error is smaller than 1% to Cr and Ni. The results show the accuracy is improved and the application range of FP and SFP is enlarged.
RESUMEN
Norcantharidin (NCTD), a demethylated analog of cantharidin, is a common used clinical drug to inhibit proliferation and metastasis of cancer cells. But the role of NCTD in modulating immune responses remains unknown. Here, we investigated the function and mechanism of NCTD in regulation of TLR4 associated immune response in macrophages. We evaluated the influence of NCTD on host defense against invaded pathogens by acute peritonitis mouse model, ELISA, Q-PCR, nitrite quantification, phagocytosis assay and gelatin zymography assay. Our data showed that the survival and the serum concentrations of IL-6 and TNF-α were all enhanced by NCTD significantly in peritonitis mouse model. Accordingly, LPS-induced cytokine, nitric oxide and MMP-9 production as well as the phagocytosis of bacteria were all up-regulated by NCTD in a dose dependent manner in both RAW264.7 cells and bone marrow-derived macrophages (BMMs). Then we further analyzed TLR4 associated signaling pathway by Western blot, Immunofluorescence and EMSA in the presence or absence of LPS. The phosphorylation of AKT and p65 at serine 536 but not serine 468 was enhanced obviously by NCTD in a dose dependent manner, whereas the degradation of IκBα was little effected. Consequently, the nuclear translocation and DNA binding ability of NF-κB was also increased by NCTD obviously in RAW264.7 cells. Our results demonstrated that NCTD could facilitate LPS-mediated immune response through promoting the phosphorylation of AKT/p65 and transcriptional activity of NF-κB, thus reprofiling the traditional anti-tumor drug NCTD as a novel immune regulator in promoting host defense against bacterial infection.