RESUMEN
IL-12 is a potent cytokine for cancer immunotherapy. However, its systemic delivery as a recombinant protein has shown unacceptable toxicity in the clinic. Currently, the intratumoral injection of IL-12-encoding mRNA or DNA to avoid such side effects is being evaluated in clinical trials. In this study, we aimed to improve this strategy by further favoring IL-12 tethering to the tumor. We generated in vitro transcribed mRNAs encoding murine single-chain IL-12 fused to diabodies binding to CSF1R and/or PD-L1. These targeted molecules are expressed in the tumor microenvironment, especially on myeloid cells. The binding capacity of chimeric constructs and the bioactivity of IL-12 were demonstrated in vitro and in vivo. Doses as low as 0.5 µg IL-12-encoding mRNA achieved potent antitumor effects in subcutaneously injected B16-OVA and MC38 tumors. Treatment delivery was associated with increases in IL-12p70 and IFN-γ levels in circulation. Fusion of IL-12 to the diabodies exerted comparable efficacy against bilateral tumor models. However, it achieved tethering to myeloid cells infiltrating the tumor, resulting in nearly undetectable systemic levels of IL-12 and IFN-γ. Overall, tethering IL-12 to intratumoral myeloid cells in the mRNA-transferred tumors achieves similar efficacy while reducing the dangerous systemic bioavailability of IL-12.
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Non-small-cell lung cancer (NSCLC) has become a paradigm of precision medicine, with the discovery of numerous disease subtypes defined by specific oncogenic driver mutations leading to the development of a range of molecularly targeted therapies. Over the past decade, rapid progress has also been made in the development of immune-checkpoint inhibitors (ICIs), especially antagonistic antibodies targeting the PD-L1-PD-1 axis, for the treatment of NSCLC. Although many of the major oncogenic drivers of NSCLC are associated with intrinsic resistance to ICIs, patients with certain oncogene-driven subtypes of the disease that are highly responsive to specific targeted therapies might also derive benefit from immunotherapy. However, the development of effective immunotherapy approaches for oncogene-addicted NSCLC has been challenged by a lack of predictive biomarkers for patient selection and limited knowledge of how ICIs and oncogene-directed targeted therapies should be combined. Therefore, whether ICIs alone or with chemotherapy or even in combination with molecularly targeted agents would offer comparable benefit in the context of selected oncogenic driver alterations to that observed in the general unselected NSCLC population remains an open question. In this Review, we discuss the effects of oncogenic driver mutations on the efficacy of ICIs and the immune tumour microenvironment as well as the potential vulnerabilities that could be exploited to overcome the challenges of immunotherapy for oncogene-addicted NSCLC.
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Carcinoma de Pulmón de Células no Pequeñas , Inmunoterapia , Neoplasias Pulmonares , Dependencia del Oncogén , Humanos , Antineoplásicos/uso terapéutico , Antígeno B7-H1 , Carcinoma de Pulmón de Células no Pequeñas/genética , Neoplasias Pulmonares/tratamiento farmacológico , OncogenesRESUMEN
OBJECTIVE: Liver disease is accelerated in people with HIV (PWH) with hepatitis B virus (HBV) coinfection. We hypothesized that liver fibrosis in HIV-HBV is triggered by increased hepatocyte apoptosis, microbial translocation and/or HIV/HBV viral products. DESIGN: Sera from PWH with HBV coinfection versus from those with HBV only or putative mediators were used to examine the pathogenesis of liver disease in HIV-HBV. METHODS: We applied sera from PWH and HBV coinfection versus HBV alone, or putative mediators (including HMGB1), to primary human hepatic stellate cells (hHSC) and examined pro-fibrogenic changes at the single cell level using flow cytometry. High mobility group box 1 (HMGB1) levels in the applied sera were assessed according to donor fibrosis stage. RESULTS: Quantitative flow cytometric assessment of pro-fibrogenic and inflammatory changes at the single cell level revealed an enhanced capacity for sera from PWH with HBV coinfection to activate hHSC. This effect was recapitulated by lipopolysaccharide, HIV-gp120, hepatocyte conditioned-media and the alarmin HMGB1. Induction of hepatocyte cell death increased their pro-fibrogenic potential, an effect blocked by HMGB1 antagonist glycyrrhizic acid. Consistent with a role for this alarmin, HMGB1 levels were elevated in sera from PWH and hepatitis B coinfection compared to HBV alone and higher in those with HIV-HBV with liver fibrosis compared to those without. CONCLUSIONS: Sera from PWH and HBV coinfection have an enhanced capacity to activate primary hHSC. We identified an increase in circulating HMGB1 which, in addition to HIV-gp120 and translocated microbial products, drove pro-fibrogenic changes in hHSC, as mechanisms contributing to accelerated liver disease in HIV-HBV.
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Coinfección , Infecciones por VIH , Proteína HMGB1 , Hepatitis B , Humanos , Virus de la Hepatitis B , Alarminas , Hepatitis B/complicaciones , Cirrosis Hepática/patologíaRESUMEN
Cancer is a leading cause of death worldwide and, despite new targeted therapies and immunotherapies, a large group of patients fail to respond to therapy or progress after initial response, which brings the need for additional treatment options. Manipulating the immune system using a variety of approaches has been explored for the past years with successful results. Sustained progress has been made to understand the T cell-mediated anti-tumor responses counteracting the tumorigenesis process. The T-lymphocyte pool, especially its capacity for antigen-directed cytotoxicity, has become a central focus for engaging the immune system in defeating cancer. The adoptive cell transfer of autologous tumor-infiltrating lymphocytes has been used in humans for over 30 years to treat metastatic melanoma. In this review, we provide a brief history of ACT-TIL and discuss the current state of ACT-TIL clinical development in solid tumors. We also discuss how key advances in understanding genetic intratumor heterogeneity, to accurately identify neoantigens, and new strategies designed to overcome T-cell exhaustion and tumor immunosuppression have improved the efficacy of the TIL-therapy infusion. Characteristics of the TIL products will be discussed, as well as new strategies, including the selective expansion of specific fractions from the cell product or the genetic manipulation of T cells for improving the in-vivo survival and functionality. In summary, this review outlines the potential of ACT-TIL as a personalized approach for epithelial tumors and continued discoveries are making it increasingly more effective against other types of cancers.
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Linfocitos Infiltrantes de Tumor , Melanoma , Traslado Adoptivo/métodos , Humanos , Inmunoterapia , Inmunoterapia Adoptiva/métodos , Linfocitos Infiltrantes de Tumor/metabolismo , Linfocitos Infiltrantes de Tumor/patología , Melanoma/genética , Melanoma/patologíaRESUMEN
CD137 (4-1BB; TNFSR9) is an activation-induced surface receptor that through costimulation effects provide antigen-primed T cells with augmented survival, proliferation and effector functions as well as metabolic advantages. These immunobiological mechanisms are being utilised for cancer immunotherapy with agonist CD137-binding and crosslinking-inducing agents that elicit CD137 intracellular signaling. In this study, side-by-side comparisons show that provision of CD137 costimulation in-cis with regard to the TCR-CD3-ligating cell is superior to that provided in-trans in terms of T cell activation, proliferation, survival, cytokine secretion and mitochondrial fitness in mouse and human. Cis ligation of CD137 relative to the TCR-CD3 complex results in more intense canonical and non-canonical NF-κB signaling and provides a more robust induction of cell cycle and DNA damage repair gene expression programs. Here we report that the superiority of cis versus trans CD137-costimulation is readily observed in vivo and is relevant for understanding the immunotherapeutic effects of CAR T cells and CD137 agonistic therapies currently undergoing clinical trials, which may provide costimulation either in cis or in trans.
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Complejo CD3/inmunología , Linfocitos T CD8-positivos/inmunología , Complejo Receptor-CD3 del Antígeno de Linfocito T/inmunología , Miembro 9 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/inmunología , Animales , Complejo CD3/genética , Proliferación Celular , Citocinas/genética , Citocinas/inmunología , Humanos , Activación de Linfocitos , Ratones , Ratones Endogámicos C57BL , Complejo Receptor-CD3 del Antígeno de Linfocito T/genética , Miembro 9 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/genéticaRESUMEN
BACKGROUND: Modified vaccinia virus Ankara (MVA) are genetically engineered non-replicating viral vectors. Intratumoral administration of MVA induces a cyclic GMP-AMP synthase-mediated type I interferon (IFN) response and the production of high levels of the transgenes engineered into the viral genome such as tumor antigens to construct cancer vaccines. Although type I IFNs are essential for establishing CD8-mediated antitumor responses, this cytokine family may also give rise to immunosuppressive mechanisms. METHODS: In vitro assays were performed to evaluate the activity of simvastatin and atorvastatin on type I IFN signaling and on antigen presentation. Surface levels of IFN α/ß receptor 1, endocytosis of bovine serum albumin-fluorescein 5 (6)-isothiocyanate, signal transducer and activator of transcription (STAT) phosphorylation, and real-time PCR of IFN-stimulated genes were assessed in the murine fibroblast cell line L929. In vivo experiments were performed to characterize the effect of simvastatin on the MVA-induced innate immune response and on the antitumor effect of MVA-based antitumor vaccines in B16 melanoma expressing ovalbumin (OVA) and Lewis lung carcinoma (LLC)-OVA tumor models. RNAseq analysis, depleting monoclonal antibodies, and flow cytometry were used to evaluate the MVA-mediated immune response. RESULTS: In this work, we identified commonly prescribed statins as potent IFNα pharmacological inhibitors due to their ability to reduce surface expression levels of IFN-α/ß receptor 1 and to reduce clathrin-mediated endocytosis. Simvastatin and atorvastatin efficiently abrogated for 8 hours the transcriptomic response to IFNα and enhanced the number of dendritic cells presenting an OVA-derived peptide bound to major histocompatibility complex (MHC) class I. In vivo, intraperitoneal or intramuscular administration of simvastatin reduced the inflammatory response mediated by peritumoral administration of MVA and enhanced the antitumor activity of MVA encoding tumor-associated antigens. The synergistic antitumor effects critically depend on CD8+ cells, whereas they were markedly improved by depletion of CD4+ lymphocytes, T regulatory cells, or NK cells. Either MVA-OVA alone or combined with simvastatin augmented B cells, CD4+ lymphocytes, CD8+ lymphocytes, and tumor-specific CD8+ in the tumor-draining lymph nodes. However, only the treatment combination increased the numbers of these lymphocyte populations in the tumor microenvironment and in the spleen. CONCLUSION: In conclusion, blockade of IFNα functions by simvastatin markedly enhances lymphocyte infiltration and the antitumor activity of MVA, prompting a feasible drug repurposing.
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Vacunas contra el Cáncer/uso terapéutico , Vectores Genéticos/uso terapéutico , Inhibidores de Hidroximetilglutaril-CoA Reductasas/uso terapéutico , Interferón Tipo I/antagonistas & inhibidores , Virus Vaccinia/efectos de los fármacos , Animales , Vacunas contra el Cáncer/farmacología , Modelos Animales de Enfermedad , Femenino , Vectores Genéticos/farmacología , Humanos , Inhibidores de Hidroximetilglutaril-CoA Reductasas/farmacología , RatonesRESUMEN
SCOPE: Vitamin D3 is a critical molecule for the properly controlled activity of the immune system. In myeloid-derived cells, vitamin D3 induces the production of the antimicrobial and antitumor peptide cathelicidin. In this study, the mechanism of the entry of 25-hydroxycholecalciferol (25(OH)D) in myeloid-derived cells is explored. METHODS AND RESULTS: Here, a novel regulatory pathway of vitamin D3 biology is described. Using a polyclonal antibody, two different chemical inhibitors, and a high-density lipoprotein as a competing ligand, it is demonstrated here that the 25(OH)D signaling pathway in myeloid cells depends on scavenger receptor class B type I (SR-B1). This effect is observed in the THP-1 monocytic cell line and in human primary monocytes. SR-B1 blockade abrogates the cellular uptake of 25(OH)D leading to a general shut down of the gene transcription program modulated by 25(OH)D. The results obtained at the transcriptional level are confirmed at the protein and functional level for CD14 in the THP-1 cell line. CONCLUSION: In conclusion, SR-B1 plays a critical role in vitamin D3 biology, paving the way for novel therapeutic interventions.
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Calcifediol/farmacocinética , Células Mieloides/metabolismo , Receptores Depuradores de Clase B/metabolismo , Anticuerpos/farmacología , Péptidos Catiónicos Antimicrobianos/genética , Calcifediol/metabolismo , Células Cultivadas , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Receptores de Lipopolisacáridos/metabolismo , Monocitos/efectos de los fármacos , Monocitos/metabolismo , Células Mieloides/efectos de los fármacos , Receptores Depuradores de Clase B/inmunología , Células THP-1 , CatelicidinasRESUMEN
Activation-induced cell death (AICD) is a complex immunoregulatory mechanism that causes the demise of a fraction of T-lymphocytes upon antigen-driven activation. In the present study we investigated the direct role of TNF in AICD of CD8 T lymphocytes. Methods: Human peripheral mononuclear cells were isolated from healthy donors and fresh tumor-infiltrating lymphocytes were obtained from cancer patients undergoing surgery. T cells were activated with anti-CD3/CD28 mAbs or with a pool of virus peptides, in combination with clinical-grade TNF blocking agents. Results: A portion of CD8 T cells undergoes apoptosis upon CD3/CD28 activation in a manner that is partially prevented by the clinically used anti-TNF agents infliximab and etanercept. TNF-mediated AICD was also observed upon activation of virus-specific CD8 T cells and tumor-infiltrating CD8 T lymphocytes. The mechanism of TNF-driven T cell death involves TNFR2 and production of mitochondrial oxygen free radicals which damage DNA. Conclusion: The use of TNF blocking agents reduces oxidative stress, hyperpolarization of mitochondria, and the generation of DNA damage in CD8 T celss undergoing activation. The fact that TNF mediates AICD in human tumor-reactive CD8 T cells suggests that the use of TNF-blocking agents can be exploited in immunotherapy strategies.
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Apoptosis/efectos de los fármacos , Linfocitos T CD8-positivos/citología , Etanercept/farmacología , Infliximab/farmacología , Inhibidores del Factor de Necrosis Tumoral/farmacología , Anciano , Anciano de 80 o más Años , Linfocitos T CD8-positivos/efectos de los fármacos , Femenino , Voluntarios Sanos , Humanos , Inmunoterapia , Leucocitos Mononucleares/citología , Activación de Linfocitos/efectos de los fármacos , Linfocitos Infiltrantes de Tumor/citología , Linfocitos Infiltrantes de Tumor/efectos de los fármacos , Masculino , Persona de Mediana Edad , Mitocondrias/efectos de los fármacos , Neoplasias/tratamiento farmacológico , Neoplasias/inmunología , Estrés Oxidativo/efectos de los fármacosRESUMEN
Neutrophils are expanded and abundant in cancer-bearing hosts. Under the influence of CXCR1 and CXCR2 chemokine receptor agonists and other chemotactic factors produced by tumors, neutrophils, and granulocytic myeloid-derived suppressor cells (MDSCs) from cancer patients extrude their neutrophil extracellular traps (NETs). In our hands, CXCR1 and CXCR2 agonists proved to be the major mediators of cancer-promoted NETosis. NETs wrap and coat tumor cells and shield them from cytotoxicity, as mediated by CD8+ T cells and natural killer (NK) cells, by obstructing contact between immune cells and the surrounding target cells. Tumor cells protected from cytotoxicity by NETs underlie successful cancer metastases in mice and the immunotherapeutic synergy of protein arginine deiminase 4 (PAD4) inhibitors, which curtail NETosis with immune checkpoint inhibitors. Intravital microscopy provides evidence of neutrophil NETs interfering cytolytic cytotoxic T lymphocytes (CTLs) and NK cell contacts with tumor cells.
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Trampas Extracelulares/metabolismo , Neoplasias Experimentales/terapia , Receptores de Quimiocina/agonistas , Receptores de Interleucina-8A/agonistas , Receptores de Interleucina-8B/agonistas , Animales , Línea Celular Tumoral , Citotoxicidad Inmunológica/inmunología , Células HT29 , Humanos , Microscopía Intravital/métodos , Células Asesinas Naturales/inmunología , Ligandos , Ratones , Neoplasias Experimentales/inmunología , Neoplasias Experimentales/metabolismo , Receptores de Quimiocina/inmunología , Receptores de Quimiocina/metabolismo , Receptores de Interleucina-8A/inmunología , Receptores de Interleucina-8A/metabolismo , Receptores de Interleucina-8B/inmunología , Receptores de Interleucina-8B/metabolismo , Linfocitos T Citotóxicos/inmunologíaRESUMEN
BACKGROUND: The immune response to cancer is often conceptualized with the cancer immunity cycle. An essential step in this interpretation is that antigens released by dying tumors are presented by dendritic cells to naive or memory T cells in the tumor-draining lymph nodes. Whether tumor cell death resulting from cytotoxicity, as mediated by T cells or natural killer (NK) lymphocytes, is actually immunogenic currently remains unknown. METHODS: In this study, tumor cells were killed by antigen-specific T-cell receptor (TCR) transgenic CD8 T cells or activated NK cells. Immunogenic cell death was studied analyzing the membrane exposure of calreticulin and the release of high mobility group box 1 (HMGB1) by the dying tumor cells. Furthermore, the potential immunogenicity of the tumor cell debris was evaluated in immunocompetent mice challenged with an unrelated tumor sharing only one tumor-associated antigen and by class I major histocompatibility complex (MHC)-multimer stainings. Mice deficient in Batf3, Ifnar1 and Sting1 were used to study mechanistic requirements. RESULTS: We observe in cocultures of tumor cells and effector cytotoxic cells, the presence of markers of immunogenic cell death such as calreticulin exposure and soluble HMGB1 protein. Ovalbumin (OVA)-transfected MC38 colon cancer cells, exogenously pulsed to present the gp100 epitope are killed in culture by mouse gp100-specific TCR transgenic CD8 T cells. Immunization of mice with the resulting destroyed cells induces epitope spreading as observed by detection of OVA-specific T cells by MHC multimer staining and rejection of OVA+ EG7 lymphoma cells. Similar results were observed in mice immunized with cell debris generated by NK-cell mediated cytotoxicity. Mice deficient in Batf3-dependent dendritic cells (conventional dendritic cells type 1, cDC1) fail to develop an anti-OVA response when immunized with tumor cells killed by cytotoxic lymphocytes. In line with this, cultured cDC1 dendritic cells uptake and can readily cross-present antigen from cytotoxicity-killed tumor cells to cognate CD8+ T lymphocytes. CONCLUSION: These results support that an ongoing cytotoxic antitumor immune response can lead to immunogenic tumor cell death.
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Neoplasias del Colon/inmunología , Citotoxicidad Inmunológica/inmunología , Células Dendríticas/inmunología , Células Asesinas Naturales/inmunología , Linfoma/inmunología , Animales , Presentación de Antígeno , Antígenos de Neoplasias/inmunología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Neoplasias del Colon/patología , Neoplasias del Colon/terapia , Femenino , Inmunización , Muerte Celular Inmunogénica , Linfoma/patología , Linfoma/terapia , Ratones , Ratones Endogámicos C57BL , Ratones TransgénicosRESUMEN
Retroviral gene transfer of interleukin-12 (IL-12) into T cells markedly enhances antitumor efficacy upon adoptive transfer but has clinically shown unacceptable severe side effects. To overcome the toxicity, we engineered tumor-specific CD8+ T cells to transiently express IL-12. Engineered T cells injected intratumorally, but not intravenously, led to complete rejections not only of the injected lesion but also of distant concomitant tumors. Efficacy was further enhanced by co-injection with agonist anti-CD137 mAb or by transient co-expression of CD137 ligand. This treatment induced epitope spreading of the endogenous CD8+ T cell immune response in a manner dependent on cDC1 dendritic cells. Mouse and human tumor-infiltrating T lymphocyte cultures can be transiently IL-12 engineered to attain marked immunotherapeutic effects.
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Linfocitos T CD8-positivos/inmunología , Inmunoterapia Adoptiva , Interleucina-12/genética , Linfocitos Infiltrantes de Tumor/inmunología , Traslado Adoptivo/métodos , Animales , Anticuerpos Monoclonales/farmacología , Línea Celular Tumoral , Células Dendríticas/inmunología , Humanos , Inmunoterapia Adoptiva/métodos , Melanoma Experimental/tratamiento farmacológico , Melanoma Experimental/inmunología , Ratones , Linfocitos T Citotóxicos/inmunologíaRESUMEN
The TNF blockade therapy is currently a well-established treatment option for a variety of autoimmune diseases such as rheumatoid arthritis (RA), psoriasis or Crohn's disease, given the proinflammatory role of TNF in the course of these diseases. Importantly, TNF neutralization is also used for the treatment of corticosteroid-refractory immune-related adverse events (irAEs) induced by the combined anti-PD-1 and anti-CTLA-4 immunotherapy. The manifestation of these toxicities is an important limiting factor for the successful implementation of the inhibitory checkpoint blockade therapy (ICB), restraining its anti-tumor efficacy. In our recent study (Perez-Ruiz et al., Nature 569(7756): 428-432.), we analyzed the potential impact of prophylactic TNF neutralization therapy in the anti-PD1/CTLA-4 efficacy. Through several mouse models, we demonstrated that TNF neutralization ameliorated ICB-exacerbated colitis in addition to improving ICB-dependent anti-tumor efficacy. Similar results were obtained after prophylactic TNF blockade in graft vs host xenografted mouse models with human immune cells, which showed a reduction in colitis and hepatitis. Importantly, there was a preservation of the immunotherapeutic control of xenografted tumors after ICB treatment. Moreover, TNF and TNF-dependent gene expression is upregulated in the colon mucosa from patients affected by colitis as a side effect of ipilimumab and nivolumab. Our results, thus, provide evidence of the successful combination of prophylactic TNF neutralization with ICB therapy strategy to ameliorate toxicities, while keeping or even ameliorating anti-tumor efficacy. The prophylactic TNF blockade strategy is clinically feasible since excellent TNF inhibitors have been approved for the treatment of autoimmunity and are used for the immune-related serious adverse events in immunotherapy.
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Previous shortcomings of CD137-targeted immunotherapy may be overcome by engineered bispecific agents (Claus et al, this issue).
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Anticuerpos Biespecíficos , Neoplasias , Humanos , Inmunoterapia , Linfocitos TRESUMEN
Combined PD-1 and CTLA-4-targeted immunotherapy with nivolumab and ipilimumab is effective against melanoma, renal cell carcinoma and non-small-cell lung cancer1-3. However, this comes at the cost of frequent, serious immune-related adverse events, necessitating a reduction in the recommended dose of ipilimumab that is given to patients4. In mice, co-treatment with surrogate anti-PD-1 and anti-CTLA-4 monoclonal antibodies is effective in transplantable cancer models, but also exacerbates autoimmune colitis. Here we show that treating mice with clinically available TNF inhibitors concomitantly with combined CTLA-4 and PD-1 immunotherapy ameliorates colitis and, in addition, improves anti-tumour efficacy. Notably, TNF is upregulated in the intestine of patients suffering from colitis after dual ipilimumab and nivolumab treatment. We created a model in which Rag2-/-Il2rg-/- mice were adoptively transferred with human peripheral blood mononuclear cells, causing graft-versus-host disease that was further exacerbated by ipilimumab and nivolumab treatment. When human colon cancer cells were xenografted into these mice, prophylactic blockade of human TNF improved colitis and hepatitis in xenografted mice, and moreover, immunotherapeutic control of xenografted tumours was retained. Our results provide clinically feasible strategies to dissociate efficacy and toxicity in the use of combined immune checkpoint blockade for cancer immunotherapy.
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Anticuerpos Monoclonales/uso terapéutico , Antígeno CTLA-4/antagonistas & inhibidores , Inmunoterapia/efectos adversos , Receptor de Muerte Celular Programada 1/antagonistas & inhibidores , Inhibidores del Factor de Necrosis Tumoral/farmacología , Inhibidores del Factor de Necrosis Tumoral/uso terapéutico , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/farmacología , Linfocitos T CD8-positivos/efectos de los fármacos , Linfocitos T CD8-positivos/inmunología , Antígeno CTLA-4/inmunología , Colitis/tratamiento farmacológico , Neoplasias del Colon/tratamiento farmacológico , Neoplasias del Colon/inmunología , Sulfato de Dextran/farmacología , Femenino , Enfermedad Injerto contra Huésped , Hepatitis/tratamiento farmacológico , Humanos , Ipilimumab/efectos adversos , Masculino , Ratones , Ratones Endogámicos C57BL , Nivolumab/efectos adversos , Receptor de Muerte Celular Programada 1/inmunología , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
CD137 (4-1BB, Tnsfr9) is a member of the TNF-receptor (TNFR) superfamily without known intrinsic enzymatic activity in its cytoplasmic domain. Hence, akin to other members of the TNFR family, it relies on the TNFR-Associated-Factor (TRAF) family of adaptor proteins to build the CD137 signalosome for transducing signals into the cell. Thus, upon CD137 activation by binding of CD137L trimers or by crosslinking with agonist monoclonal antibodies, TRAF1, TRAF2, and TRAF3 are readily recruited to the cytoplasmic domain of CD137, likely as homo- and/or heterotrimers with different configurations, initiating the construction of the CD137 signalosome. The formation of TRAF2-RING dimers between TRAF2 molecules from contiguous trimers would help to establish a multimeric structure of TRAF-trimers that is probably essential for CD137 signaling. In addition, available studies have identified a large number of proteins that are recruited to CD137:TRAF complexes including ubiquitin ligases and proteases, kinases, and modulatory proteins. Working in a coordinated fashion, these CD137-signalosomes will ultimately promote CD137-mediated T cell proliferation and survival and will endow T cells with stronger effector functions. Current evidence allows to envision the molecular events that might take place in the early stages of CD137-signalosome formation, underscoring the key roles of TRAFs and of K63 and K48-ubiquitination of target proteins in the signaling process. Understanding the composition and fine regulation of CD137-signalosomes assembly and disassembly will be key to improve the therapeutic activities of chimeric antigen receptors (CARs) encompassing the CD137 cytoplasmic domain and a new generation of CD137 agonists for the treatment of cancer.
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Complejos Multiproteicos/metabolismo , Neoplasias/tratamiento farmacológico , Factor 1 Asociado a Receptor de TNF/metabolismo , Factor 2 Asociado a Receptor de TNF/metabolismo , Factor 3 Asociado a Receptor de TNF/metabolismo , Miembro 9 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/metabolismo , Animales , Proliferación Celular , Humanos , Activación de Linfocitos , Terapia Molecular Dirigida , Transducción de Señal , Linfocitos T/inmunología , Factor de Necrosis Tumoral alfa/metabolismo , UbiquitinaciónRESUMEN
Checkpoint inhibitors and adoptive cell therapy provide promising options for treating solid cancers such as HBV-related HCC, but they have limitations. We tested the potential to combine advantages of each approach, genetically reprogramming T cells specific for viral tumor antigens to overcome exhaustion by down-modulating the co-inhibitory receptor PD-1. We developed a novel lentiviral transduction protocol to achieve preferential targeting of endogenous or TCR-redirected, antigen-specific CD8 T cells for shRNA knockdown of PD-1 and tested functional consequences for antitumor immunity. Antigen-specific and intrahepatic CD8 T cells transduced with lentiviral (LV)-shPD-1 consistently had a marked reduction in PD-1 compared to those transduced with a control lentiviral vector. PD-1 knockdown of human T cells rescued antitumor effector function and promoted killing of hepatoma cells in a 3D microdevice recapitulating the pro-inflammatory PD-L1hi liver microenvironment. However, upon repetitive stimulation, PD-1 knockdown drove T cell senescence and induction of other co-inhibitory pathways. We provide the proof of principle that T cells with endogenous or genetically engineered specificity for HBV-associated HCC viral antigens can be targeted for functional genetic editing. We show that PD-1 knockdown enhances immediate tumor killing but is limited by compensatory engagement of alternative co-inhibitory and senescence program upon repetitive stimulation.
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Carcinoma Hepatocelular/terapia , Hepatitis B Crónica/terapia , Neoplasias Hepáticas/terapia , Receptor de Muerte Celular Programada 1/inmunología , Receptores de Antígenos de Linfocitos T/uso terapéutico , Antígenos de Neoplasias/inmunología , Antígenos de Neoplasias/uso terapéutico , Antígenos Virales/inmunología , Linfocitos T CD8-positivos/inmunología , Carcinoma Hepatocelular/inmunología , Carcinoma Hepatocelular/patología , Carcinoma Hepatocelular/virología , Vectores Genéticos/genética , Virus de la Hepatitis B/inmunología , Virus de la Hepatitis B/patogenicidad , Hepatitis B Crónica/inmunología , Hepatitis B Crónica/patología , Hepatitis B Crónica/virología , Humanos , Inmunoterapia Adoptiva/métodos , Lentivirus/genética , Hígado/inmunología , Hígado/metabolismo , Neoplasias Hepáticas/inmunología , Neoplasias Hepáticas/patología , Neoplasias Hepáticas/virología , Receptor de Muerte Celular Programada 1/genética , Receptor de Muerte Celular Programada 1/uso terapéutico , Receptores de Antígenos de Linfocitos T/inmunología , Microambiente Tumoral/genética , Microambiente Tumoral/inmunologíaRESUMEN
The TRAIL pathway can mediate apoptosis of hepatic stellate cells to promote the resolution of liver fibrosis. However, TRAIL has the capacity to bind to regulatory receptors in addition to death-inducing receptors; their differential roles in liver fibrosis have not been investigated. Here we have dissected the contribution of regulatory TRAIL receptors to apoptosis resistance in primary human hepatic stellate cells (hHSC). hHSC isolated from healthy margins of liver resections from different donors expressed variable levels of TRAIL-R2/3/4 (but negligible TRAIL-R1) ex vivo and after activation. The apoptotic potential of TRAIL-R2 on hHSC was confirmed by lentiviral-mediated knockdown. A functional inhibitory role for TRAIL-R3/4 was revealed by shRNA knockdown and mAb blockade, showing that these regulatory receptors limit apoptosis of hHSC in response to both oligomerised TRAIL and NK cells. A close inverse ex vivo correlation between hHSC TRAIL-R4 expression and susceptibility to apoptosis underscored its central regulatory role. Our data provide the first demonstration of non-redundant functional roles for the regulatory TRAIL receptors (TRAIL-R3/4) in a physiological setting. The potential for these inhibitory TRAIL receptors to protect hHSC from apoptosis opens new avenues for prognostic and therapeutic approaches to the management of liver fibrosis.
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Apoptosis , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/metabolismo , Anticuerpos Monoclonales/inmunología , Apoptosis/efectos de los fármacos , Células Cultivadas , Proteínas Ligadas a GPI/antagonistas & inhibidores , Proteínas Ligadas a GPI/genética , Proteínas Ligadas a GPI/metabolismo , Células Estrelladas Hepáticas/citología , Células Estrelladas Hepáticas/metabolismo , Humanos , Células Asesinas Naturales/inmunología , Hígado/citología , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/antagonistas & inhibidores , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/genética , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/inmunología , Miembro 10c de Receptores del Factor de Necrosis Tumoral/antagonistas & inhibidores , Miembro 10c de Receptores del Factor de Necrosis Tumoral/genética , Miembro 10c de Receptores del Factor de Necrosis Tumoral/metabolismo , Ligando Inductor de Apoptosis Relacionado con TNF/farmacología , Receptores Señuelo del Factor de Necrosis Tumoral/antagonistas & inhibidores , Receptores Señuelo del Factor de Necrosis Tumoral/genética , Receptores Señuelo del Factor de Necrosis Tumoral/metabolismoRESUMEN
Recent studies have found that tumor-infiltrating lymphocytes (TIL) expressing PD-1 can recognize autologous tumor cells, suggesting that cells derived from PD-1+ TILs can be used in adoptive T-cell therapy (ACT). However, no study thus far has evaluated the antitumor activity of PD-1-selected TILs in vivo In two mouse models of solid tumors, we show that PD-1 allows identification and isolation of tumor-specific TILs without previous knowledge of their antigen specificities. Importantly, despite the high proportion of tumor-reactive T cells present in bulk CD8 TILs before expansion, only T-cell products derived from sorted PD-1+, but not from PD-1- or bulk CD8 TILs, specifically recognized tumor cells. The fold expansion of PD-1+ CD8 TILs was 10 times lower than that of PD-1- cells, suggesting that outgrowth of PD-1- cells was the limiting factor in the tumor specificity of cells derived from bulk CD8 TILs. The highly differentiated state of PD-1+ cells was likely the main cause hampering ex vivo expansion of this subset. Moreover, PD-1 precisely identified marrow-infiltrating, myeloma-specific T cells in a mouse model of multiple myeloma. In vivo, only cells expanded from PD-1+ CD8 TILs contained tumor progression, and their efficacy was enhanced by PDL-1 blockade. Overall, our data provide a rationale for the use of PD-1-selected TILs in ACT. Cancer Res; 77(13); 3672-84. ©2017 AACR.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Inmunoterapia Adoptiva/métodos , Linfocitos Infiltrantes de Tumor/inmunología , Receptor de Muerte Celular Programada 1/biosíntesis , Animales , Línea Celular Tumoral , Humanos , Melanoma Experimental/inmunología , Melanoma Experimental/terapia , Ratones , Ratones Endogámicos C57BL , Receptor de Muerte Celular Programada 1/inmunologíaRESUMEN
T cells undergo profound metabolic changes to meet the increased energy demands of maintaining an antiviral response. We postulated that differences in metabolic reprogramming would shape the efficacy of CD8 T cells mounted against persistent viral infections. We found that the poorly functional PD-1(hi) T cell response against hepatitis B virus (HBV) had upregulated the glucose transporter, Glut1, an effect recapitulated by oxygen deprivation to mimic the intrahepatic environment. Glut1(hi) HBV-specific T cells were dependent on glucose supplies, unlike the more functional cytomegalovirus (CMV)-specific T cells that could utilize oxidative phosphorylation in the absence of glucose. The inability of HBV-specific T cells to switch to oxidative phosphorylation was accompanied by increased mitochondrial size and lower mitochondrial potential, indicative of mitochondrial dysfunction. Interleukin (IL)-12, which recovers HBV-specific T cell effector function, increased their mitochondrial potential and reduced their dependence on glycolysis. Our findings suggest that mitochondrial defects limit the metabolic plasticity of exhausted HBV-specific T cells.
Asunto(s)
Linfocitos T CD8-positivos/fisiología , Linfocitos T CD8-positivos/virología , Adolescente , Adulto , Linfocitos T CD8-positivos/metabolismo , Citomegalovirus/patogenicidad , Femenino , Glucosa/metabolismo , Transportador de Glucosa de Tipo 1/metabolismo , Glucólisis/fisiología , Virus de la Hepatitis B/patogenicidad , Humanos , Interleucina-12/metabolismo , Masculino , Persona de Mediana Edad , Mitocondrias/metabolismo , Mitocondrias/fisiología , Mitocondrias/virología , Fosforilación Oxidativa , Receptor de Muerte Celular Programada 1/metabolismo , Virosis/metabolismo , Virosis/fisiopatología , Virosis/virología , Adulto JovenRESUMEN
UNLABELLED: In chronic hepatitis B (CHB), failure to control hepatitis B virus (HBV) is associated with T cell dysfunction. HBV transgenic mice mirror many features of the human disease, including T cell unresponsiveness, and thus represent an appropriate model in which to test novel therapeutic strategies. To date, the tolerant state of CD8(+) T cells in these animals could be altered only by strong immunogens or by immunization with HBV antigen-pulsed dendritic cells; however, the effectors induced were unable to suppress viral gene expression or replication. Because of the known stimulatory properties of alpha interferon (IFN-α) and interleukin-15 (IL-15), this study explored the therapeutic potential of liver-directed gene transfer of these cytokines in a murine model of CHB using adeno-associated virus (AAV) delivery. This combination not only resulted in a reduction in the viral load in the liver and the induction of an antibody response but also gave rise to functional and specific CD8(+) immunity. Furthermore, when splenic and intrahepatic lymphocytes from IFN-α- and IL-15-treated animals were transferred to new HBV carriers, partial antiviral immunity was achieved. In contrast to previous observations made using either cytokine alone, markedly attenuated PD-L1 induction in hepatic tissue was observed upon coadministration. An initial study with CHB patient samples also gave promising results. Hence, we demonstrated synergy between two stimulating cytokines, IL-15 and IFN-α, which, given together, constitute a potent approach to significantly enhance the CD8(+) T cell response in a state of immune hyporesponsiveness. Such an approach may be useful for treating chronic viral infections and neoplastic conditions. IMPORTANCE: With 350 million people affected worldwide and 600,000 annual deaths due to HBV-induced liver cirrhosis and/or hepatocellular carcinoma, chronic hepatitis B (CHB) is a major health problem. However, current treatment options are costly and not very effective and/or need to be administered for life. The unprecedented efficacy of the strategy described in our paper may offer an alternative and is relevant for a broad spectrum of readers because of its clear translational importance to other chronic viral infections in which a hyporesponsive antigen-specific T cell repertoire prevents clearance of the pathogen.