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1.
Cancer Immunol Res ; 10(11): 1354-1369, 2022 11 02.
Artigo em Inglês | MEDLINE | ID: mdl-36095236

RESUMO

Tumor-associated macrophages (TAM) are one of the most abundant cell types in many solid tumors and typically exert protumor effects. This has led to an interest in macrophage-depleting agents for cancer therapy, but approaches developed to date have had limited success in clinical trials. Here, we report the development of a strategy for TAM depletion in mouse solid tumor models using chimeric antigen receptor (CAR) T cells targeting the macrophage marker F4/80 (F4.CAR-T). F4.CAR-T cells effectively killed macrophages in vitro and in vivo without toxicity. When injected into mice bearing orthotopic lung tumors, F4.CAR-T cells infiltrated tumor lesions and delayed tumor growth comparably with PD-1 blockade, and significantly extended mouse survival. Antitumor effects were mediated by F4.CAR-T-produced IFNγ, which promoted upregulation of MHC molecules on cancer cells and tumor-infiltrating myeloid cells. Notably, F4.CAR-T promoted expansion of endogenous CD8 T cells specific for tumor-associated antigen and led to immune editing of highly antigenic tumor cell clones. Antitumor impact was also observed in mouse models of ovarian and pancreatic cancer. These studies provide proof of principle to support CAR T-cell targeting of TAMs as a means to enhance antitumor immunity.


Assuntos
Receptores de Antígenos Quiméricos , Linfócitos T , Animais , Camundongos , Antígenos de Neoplasias , Linhagem Celular Tumoral , Modelos Animais de Doenças , Imunoterapia Adotiva , Macrófagos/metabolismo , Receptores de Antígenos Quiméricos/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto , Progressão da Doença
3.
J Immunother Cancer ; 9(5)2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33980589

RESUMO

BACKGROUND: Conventional type 1 dendritic cells (cDC1s) are central to antitumor immunity and their presence in the tumor microenvironment associates with improved outcomes in patients with cancer. DNGR-1 (CLEC9A) is a dead cell-sensing receptor highly restricted to cDC1s. DNGR-1 has been involved in both cross-presentation of dead cell-associated antigens and processes of disease tolerance, but its role in antitumor immunity has not been clarified yet. METHODS: B16 and MC38 tumor cell lines were inoculated subcutaneously into wild-type (WT) and DNGR-1-deficient mice. To overexpress Flt3L systemically, we performed gene therapy through the hydrodynamic injection of an Flt3L-encoding plasmid. To characterize the immune response, we performed flow cytometry and RNA-Seq of tumor-infiltrating cDC1s. RESULTS: Here, we found that cross-presentation of tumor antigens in the steady state was DNGR-1-independent. However, on Flt3L systemic overexpression, tumor growth was delayed in DNGR-1-deficient mice compared with WT mice. Of note, this protection was recapitulated by anti-DNGR-1-blocking antibodies in mice following Flt3L gene therapy. This improved antitumor immunity was associated with Batf3-dependent enhanced accumulation of CD8+ T cells and cDC1s within tumors. Mechanistically, the deficiency in DNGR-1 boosted an Flt3L-induced specific inflammatory gene signature in cDC1s, including Ccl5 expression. Indeed, the increased infiltration of cDC1s within tumors and their protective effect rely on CCL5/CCR5 chemoattraction. Moreover, FLT3LG and CCL5 or CCR5 gene expression signatures correlate with an enhanced cDC1 signature and a favorable overall survival in patients with cancer. Notably, cyclophosphamide elevated serum Flt3L levels and, in combination with the absence of DNGR-1, synergized against tumor growth. CONCLUSION: DNGR-1 limits the accumulation of tumor-infiltrating cDC1s promoted by Flt3L. Thus, DNGR-1 blockade may improve antitumor immunity in tumor therapy settings associated to high Flt3L expression.


Assuntos
Neoplasias do Colo/terapia , Células Dendríticas/metabolismo , Terapia Genética , Lectinas Tipo C/metabolismo , Melanoma Experimental/terapia , Proteínas de Membrana/metabolismo , Receptores Imunológicos/metabolismo , Neoplasias Cutâneas/terapia , Animais , Fatores de Transcrição de Zíper de Leucina Básica/genética , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Linhagem Celular Tumoral , Quimiocina CCL5/genética , Quimiocina CCL5/metabolismo , Técnicas de Cocultura , Neoplasias do Colo/genética , Neoplasias do Colo/imunologia , Neoplasias do Colo/metabolismo , Células Dendríticas/imunologia , Regulação Neoplásica da Expressão Gênica , Lectinas Tipo C/genética , Linfócitos do Interstício Tumoral/imunologia , Linfócitos do Interstício Tumoral/metabolismo , Melanoma Experimental/genética , Melanoma Experimental/imunologia , Melanoma Experimental/metabolismo , Proteínas de Membrana/genética , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fenótipo , Receptores CCR5/genética , Receptores CCR5/metabolismo , Receptores Imunológicos/genética , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Transdução de Sinais , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/imunologia , Neoplasias Cutâneas/metabolismo , Carga Tumoral , Evasão Tumoral , Microambiente Tumoral
4.
J Immunother Cancer ; 8(1)2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32217765

RESUMO

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.


Assuntos
Neoplasias do Colo/imunologia , Citotoxicidade Imunológica/imunologia , Células Dendríticas/imunologia , Células Matadoras Naturais/imunologia , Linfoma/imunologia , Animais , Apresentação de Antígeno , Antígenos de Neoplasias/imunologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Neoplasias do Colo/patologia , Neoplasias do Colo/terapia , Feminino , Imunização , Morte Celular Imunogênica , Linfoma/patologia , Linfoma/terapia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos
5.
EMBO Mol Med ; 12(1): e10375, 2020 01 09.
Artigo em Inglês | MEDLINE | ID: mdl-31746149

RESUMO

Live 17D is widely used as a prophylactic vaccine strain for yellow fever virus that induces potent neutralizing humoral and cellular immunity against the wild-type pathogen. 17D replicates and kills mouse and human tumor cell lines but not non-transformed human cells. Intratumoral injections with viable 17D markedly delay transplanted tumor progression in a CD8 T-cell-dependent manner. In mice bearing bilateral tumors in which only one is intratumorally injected, contralateral therapeutic effects are observed consistent with more prominent CD8 T-cell infiltrates and a treatment-related reduction of Tregs. Additive efficacy effects were observed upon co-treatment with intratumoral 17D and systemic anti-CD137 and anti-PD-1 immunostimulatory monoclonal antibodies. Importantly, when mice were preimmunized with 17D, intratumoral 17D treatment achieved better local and distant antitumor immunity. Such beneficial effects of prevaccination are in part explained by the potentiation of CD4 and CD8 T-cell infiltration in the treated tumor. The repurposed use of a GMP-grade vaccine to be given via the intratumoral route in prevaccinated patients constitutes a clinically feasible and safe immunotherapy approach.


Assuntos
Imunoterapia , Neoplasias/terapia , Vacina contra Febre Amarela , Animais , Linfócitos T CD8-Positivos/imunologia , Reposicionamento de Medicamentos , Feminino , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Vacina contra Febre Amarela/uso terapêutico
6.
Cancer Cell ; 36(6): 613-629.e7, 2019 12 09.
Artigo em Inglês | MEDLINE | ID: mdl-31761658

RESUMO

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.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Imunoterapia Adotiva , Interleucina-12/genética , Linfócitos do Interstício Tumoral/imunologia , Transferência Adotiva/métodos , Animais , Anticorpos Monoclonais/farmacologia , Linhagem Celular Tumoral , Células Dendríticas/imunologia , Humanos , Imunoterapia Adotiva/métodos , Melanoma Experimental/tratamento farmacológico , Melanoma Experimental/imunologia , Camundongos , Linfócitos T Citotóxicos/imunologia
7.
Mol Ther ; 27(11): 1892-1905, 2019 11 06.
Artigo em Inglês | MEDLINE | ID: mdl-31563534

RESUMO

Immune checkpoint blockade has shown anti-cancer efficacy, but requires systemic administration of monoclonal antibodies (mAbs), often leading to adverse effects. To avoid toxicity, mAbs could be expressed locally in tumors. We developed adeno-associated virus (AAV) and Semliki Forest virus (SFV) vectors expressing anti-programmed death ligand 1 (aPDL1) mAb. When injected intratumorally in MC38 tumors, both viral vectors led to similar local mAb expression at 24 h, diminishing quickly in SFV-aPDL1-treated tumors. However, SFV-aPDL1 induced >40% complete regressions and was superior to AAV-aPDL1, as well as to aPDL1 mAb given systemically or locally. SFV-aPDL1 induced abscopal effects and was also efficacious against B16-ovalbumin (OVA). The higher SFV-aPDL1 antitumor activity could be related to local upregulation of interferon-stimulated genes because of SFV RNA replication. This was confirmed by combining local SFV-LacZ administration and systemic aPDL1 mAb, which provided higher antitumor effects than each separated agent. SFV-aPDL1 promoted tumor-specific CD8 T cells infiltration in both tumor models. In MC38, SFV-aPDL1 upregulated co-stimulatory markers (CD137/OX40) in tumor CD8 T cells, and its combination with anti-CD137 mAb showed more pronounced antitumor effects than each single agent. These results indicate that local transient expression of immunomodulatory mAbs using non-propagative RNA vectors inducing type I interferon (IFN-I) responses represents a potent and safe approach for cancer treatment.


Assuntos
Anticorpos Monoclonais/genética , Anticorpos Monoclonais/farmacologia , Antígeno B7-H1/antagonistas & inibidores , Expressão Gênica , Vetores Genéticos/genética , Neoplasias/genética , Neoplasias/imunologia , Vírus de RNA/genética , Animais , Antígeno B7-H1/genética , Antígeno B7-H1/metabolismo , Linhagem Celular , Dependovirus/genética , Modelos Animais de Doenças , Feminino , Terapia Genética/métodos , Vetores Genéticos/administração & dosagem , Humanos , Imunomodulação/efeitos dos fármacos , Imunofenotipagem , Injeções Intralesionais , Camundongos , Neoplasias/patologia , Neoplasias/terapia , Proteínas Recombinantes de Fusão/genética , Vírus da Floresta de Semliki/genética , Taxa de Sobrevida , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Carga Tumoral
8.
Cancer Res ; 78(23): 6643-6654, 2018 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-30297531

RESUMO

: Multiple lines of evidence indicate a critical role of antigen cross-presentation by conventional BATF3-dependent type 1 classical dendritic cells (cDC1) in CD8-mediated antitumor immunity. Flt3L and XCL1, respectively, constitute a key growth/differentiation factor and a potent and specific chemoattractant for cDC1. To exploit their antitumor functions in local immunotherapy, we prepared Semliki Forest Virus (SFV)-based vectors encoding XCL1 and soluble Flt3L (sFlt3L). These vectors readily conferred transgene expression to the tumor cells in culture and when engrafted as subcutaneous mouse tumor models. In syngeneic mice, intratumoral injection of SFV-XCL1-sFlt3L (SFV-XF) delayed progression of MC38- and B16-derived tumors. Therapeutic activity was observed and exerted additive effects in combination with anti-PD-1, anti-CD137, or CTLA-4 immunostimulatory mAbs. Therapeutic effects were abolished by CD8ß T-cell depletion and were enhanced by CD4 T-cell depletion, but not by T regulatory cell predepletion with anti-CD25 mAb. Antitumor effects were also abolished in BATF3- and IFNAR-deficient mice. In B16-OVA tumors, SFV-XF increased the number of infiltrating CD8 T cells, including those recognizing OVA. Consistently, following the intratumoral SFV-XF treatment courses, we observed increased BATF3-dependent cDC1 among B16-OVA tumor-infiltrating leukocytes. Such an intratumoral increase was not seen in MC38-derived tumors, but both resident and migratory cDC1 were boosted in SFV-XF-treated MC38 tumor-draining lymph nodes. In conclusion, viral gene transfer of sFlt3L and XCL1 is feasible, safe, and biologically active in mice, exerting antitumor effects that can be potentiated by CD4 T-cell depletion. SIGNIFICANCE: These findings demonstrate that transgenic expression of sFLT3L and XCL1 in tumor cells mediates cross-priming of, and elicits potent antitumor activity from, CD8 T lymphocytes, particularly in combination with CD4 T-cell depletion.


Assuntos
Quimiocinas C/genética , Apresentação Cruzada/imunologia , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Vetores Genéticos/genética , Proteínas de Membrana/genética , Vírus da Floresta de Semliki/genética , Linfócitos T/imunologia , Animais , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Imunoterapia , Camundongos , Linfócitos T/metabolismo , Microambiente Tumoral/imunologia
9.
Cancer Immunol Res ; 6(7): 798-811, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29678874

RESUMO

T and NK lymphocytes express CD137 (4-1BB), a costimulatory receptor of the TNFR family whose function is exploitable for cancer immunotherapy. Mitochondria regulate the function and survival of T lymphocytes. Herein, we show that CD137 costimulation provided by agonist mAb and CD137L (4-1BBL) induced mitochondria enlargement that resulted in enhanced mitochondrial mass and transmembrane potential in human and mouse CD8+ T cells. Such mitochondrial changes increased T-cell respiratory capacities and were critically dependent on mitochondrial fusion protein OPA-1 expression. Mass and function of mitochondria in tumor-reactive CD8+ T cells from cancer-bearing mice were invigorated by agonist mAb to CD137, whereas mitochondrial baseline mass and function were depressed in CD137-deficient tumor reactive T cells. Tumor rejection induced by the synergistic combination of adoptive T-cell therapy and agonistic anti-CD137 was critically dependent on OPA-1 expression in transferred CD8+ T cells. Moreover, stimulation of CD137 with CD137 mAb in short-term cultures of human tumor-infiltrating lymphocytes led to mitochondria enlargement and increased transmembrane potential. Collectively, these data point to a critical link between mitochondrial morphology and function and enhanced antitumor effector activity upon CD137 costimulation of T cells. Cancer Immunol Res; 6(7); 798-811. ©2018 AACR.


Assuntos
Ligante 4-1BB/metabolismo , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Linfócitos T/metabolismo , Ligante 4-1BB/genética , Animais , Biomarcadores , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Citocinas/metabolismo , Citotoxicidade Imunológica , Feminino , Inativação Gênica , Humanos , Linfócitos do Interstício Tumoral/imunologia , Linfócitos do Interstício Tumoral/metabolismo , Melanoma Experimental , Potencial da Membrana Mitocondrial , Camundongos , Camundongos Knockout , Mitocôndrias/genética , Neoplasias/imunologia , Neoplasias/metabolismo , Neoplasias/patologia , RNA Interferente Pequeno/genética , Linfócitos T/imunologia , Microambiente Tumoral/imunologia
10.
Cancer Immunol Res ; 6(1): 69-78, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-29133290

RESUMO

CD137 (4-1BB) costimulation imprints long-term changes that instruct the ultimate behavior of T cells that have previously experienced CD137 ligation. Epigenetic changes could provide a suitable mechanism for these long-term consequences. Genome-wide DNA methylation arrays were carried out on human peripheral blood CD8+ T lymphocytes stimulated with agonist monoclonal antibody to CD137, including urelumab, which is in phase I/II clinical trials for cancer immunotherapy. Several genes showed consistent methylation patterns in response to CD137 costimulation, which were confirmed by pyrosequencing in a series of healthy donors. CD96, HHLA2, CCR5, CXCR5, and CCL5 were among the immune-related genes regulated by differential DNA methylation, leading to changes in mRNA and protein expression. These genes are also differentially methylated in naïve versus antigen-experienced CD8+ T cells. The transcription factor TCF1 and the microRNA miR-21 were regulated by DNA methylation upon CD137 costimulation. Such gene-expression regulatory factors can, in turn, broaden the effects of DNA methylation by controlling expression of their target genes. Overall, chromatin remodeling is postulated to leave CD137-costimulated T lymphocytes poised to differentially respond upon subsequent antigen recognition. Accordingly, CD137 connects costimulation during priming to genome-wide DNA methylation and chromatin reprogramming. Cancer Immunol Res; 6(1); 69-78. ©2017 AACR.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Metilação de DNA , Regulação da Expressão Gênica , Membro 9 da Superfamília de Receptores de Fatores de Necrose Tumoral/metabolismo , Animais , Anticorpos Monoclonais/farmacologia , Linfócitos T CD8-Positivos/efeitos dos fármacos , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Células Cultivadas , Quimiotaxia de Leucócito/genética , Quimiotaxia de Leucócito/imunologia , Células Dendríticas/citologia , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Perfilação da Expressão Gênica , Estudo de Associação Genômica Ampla , Humanos , Camundongos , Membro 9 da Superfamília de Receptores de Fatores de Necrose Tumoral/agonistas
11.
Oncoimmunology ; 6(4): e1283468, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28507790

RESUMO

CD69 is an early activation marker on the surface of T lymphocytes undergoing activation by cognate antigen. We observed intense expression of CD69 on tumor-infiltrating T-lymphocytes that reside in the hypoxic tumor microenvironment and hypothesized that CD69 could be, at least partially, under the control of the transcriptional hypoxia response. In line with this, human and mouse CD3-stimulated lymphocytes cultured under hypoxia (1% O2) showed increased expression of CD69 at the protein and mRNA level. Consistent with these findings, mouse T lymphocytes that had recently undergone hypoxia in vivo, as denoted by pimonidazole staining, were more frequently CD69+ in the tumor and bone marrow hypoxic tissue compartments. We found evidence for HIF-1α involvement both when using T-lymphocytes from inducible HIF-1α-/- mice and when observing tumor-infiltrating T-lymphocytes in mice whose T cells are HIF-1α-/-. Direct pro-transcriptional activity of HIF-1α on a newly identified hypoxia response element (HRE) found in the human CD69 locus was demonstrated by ChIP experiments. These results uncover a connection between the HIF-1α oxygen-sensing pathway and CD69 immunobiology.

12.
J Immunol ; 198(1): 31-39, 2017 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-27994166

RESUMO

Immune mechanisms have evolved to cope with local entry of microbes acting in a confined fashion but eventually inducing systemic immune memory. Indeed, in situ delivery of a number of agents into tumors can mimic in the malignant tissue the phenomena that control intracellular infection leading to the killing of infected cells. Vascular endothelium activation and lymphocyte attraction, together with dendritic cell-mediated cross-priming, are the key elements. Intratumoral therapy with pathogen-associated molecular patterns or recombinant viruses is being tested in the clinic. Cell therapies can be also delivered intratumorally, including infusion of autologous dendritic cells and even tumor-reactive T lymphocytes. Intralesional virotherapy with an HSV vector expressing GM-CSF has been recently approved by the Food and Drug Administration for the treatment of unresectable melanoma. Immunomodulatory monoclonal Abs have also been successfully applied intratumorally in animal models. Local delivery means less systemic toxicity while focusing the immune response on the malignancy and the affected draining lymph nodes.


Assuntos
Antineoplásicos/administração & dosagem , Imunoterapia/métodos , Neoplasias/imunologia , Neoplasias/terapia , Animais , Humanos
13.
Cancer Res ; 76(20): 5994-6005, 2016 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-27550452

RESUMO

Preclinical and clinical evidence indicate that the proimmune effects of radiotherapy can be synergistically augmented with immunostimulatory mAbs to act both on irradiated tumor lesions and on distant, nonirradiated tumor sites. The combination of radiotherapy with immunostimulatory anti-PD1 and anti-CD137 mAbs was conducive to favorable effects on distant nonirradiated tumor lesions as observed in transplanted MC38 (colorectal cancer), B16OVA (melanoma), and 4T1 (breast cancer) models. The therapeutic activity was crucially performed by CD8 T cells, as found in selective depletion experiments. Moreover, the integrities of BATF-3-dependent dendritic cells specialized in crosspresentation/crosspriming of antigens to CD8+ T cells and of the type I IFN system were absolute requirements for the antitumor effects to occur. The irradiation regimen induced immune infiltrate changes in the irradiated and nonirradiated lesions featured by reductions in the total content of effector T cells, Tregs, and myeloid-derived suppressor cells, while effector T cells expressed more intracellular IFNγ in both the irradiated and contralateral tumors. Importantly, 48 hours after irradiation, CD8+ TILs showed brighter expression of CD137 and PD1, thereby displaying more target molecules for the corresponding mAbs. Likewise, PD1 and CD137 were induced on tumor-infiltrating lymphocytes from surgically excised human carcinomas that were irradiated ex vivo These mechanisms involving crosspriming and CD8 T cells advocate clinical development of immunotherapy combinations with anti-PD1 plus anti-CD137 mAbs that can be synergistically accompanied by radiotherapy strategies, even if the disease is left outside the field of irradiation. Cancer Res; 76(20); 5994-6005. ©2016 AACR.


Assuntos
Anticorpos Monoclonais/uso terapêutico , Linfócitos T CD8-Positivos/imunologia , Neoplasias Experimentais/radioterapia , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Membro 9 da Superfamília de Receptores de Fatores de Necrose Tumoral/antagonistas & inibidores , Animais , Fatores de Transcrição de Zíper de Leucina Básica/fisiologia , Linhagem Celular Tumoral , Feminino , Humanos , Linfócitos do Interstício Tumoral/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Neoplasias Experimentais/imunologia , Receptor de Morte Celular Programada 1/análise , Receptor de Morte Celular Programada 1/imunologia , Receptor de Interferon alfa e beta/fisiologia , Proteínas Repressoras/fisiologia , Microambiente Tumoral , Membro 9 da Superfamília de Receptores de Fatores de Necrose Tumoral/análise , Membro 9 da Superfamília de Receptores de Fatores de Necrose Tumoral/imunologia
14.
Oncoimmunology ; 5(1): e1062967, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26942078

RESUMO

Hypoxia is a common feature in solid tumors that has been implicated in immune evasion. Previous studies from our group have shown that hypoxia upregulates the co-stimulatory receptor CD137 on activated T lymphocytes and on vascular endothelial cells. In this study, we show that exposure of mouse and human tumor cell lines to hypoxic conditions (1% O2) promotes CD137 transcription. However, the resulting mRNA is predominantly an alternatively spliced form that encodes for a soluble variant, lacking the transmembrane domain. Accordingly, soluble CD137 (sCD137) is detectable by ELISA in the supernatant of hypoxia-exposed cell lines and in the serum of tumor-bearing mice. sCD137, as secreted by tumor cells, is able to bind to CD137-Ligand (CD137L). Our studies on primed T lymphocytes in co-culture with stable transfectants for CD137L demonstrate that tumor-secreted sCD137 prevents co-stimulation of T lymphocytes. Such an effect results from preventing the interaction of CD137L with the transmembrane forms of CD137 expressed on T lymphocytes undergoing activation. Indeed, silencing CD137 with shRNA renders more immunogenic tumor-cell variants upon inoculation to immunocompetent mice but which readily grafted on immunodeficient or CD8+ T-cell-depleted mice. These mechanisms are interpreted as a molecular strategy deployed by tumors to repress lymphocyte co-stimulation via CD137/CD137L.

15.
J Thorac Oncol ; 11(4): 524-36, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26845193

RESUMO

INTRODUCTION: Anti-programmed cell death 1 (anti-PD-1) and anti-programmed cell death ligand 1 (PD-L1) antagonist monoclonal antibodies (mAbs) against metastatic non-small cell lung cancer with special efficacy in patients with squamous cell lung cancer are being developed in the clinic. However, robust and reliable experimental models to test immunotherapeutic combinations in squamous lung tumors are still lacking. METHODS: We generated a transplantable squamous cell carcinoma cell line (UN-SCC680AJ) from a lung tumor induced by chronic N-nitroso-tris-chloroethylurea mutagenesis in A/J mice. Tumor cells expressed cytokeratins, overexpressed p40, and lacked thyroid transcription factor 1, confirming the squamous lineage reported by histological analysis. More than 200 mutations found in its exome suggested potential for antigenicity. Immunocompetent mice subcutaneously implanted with this syngeneic cell line were treated with anti-CD137 and/or anti-PD-1 mAbs and monitored for tumor growth/progression or assessed for intratumoral leukocyte infiltration using immunohistochemical analysis and flow cytometry. RESULTS: In syngeneic mice, large 12-day-established tumors derived from the transplantable cell line variant UN-SCC680AJ were amenable to curative treatment with anti-PD-1, anti-PD-L1, or anti-CD137 immunostimulatory mAbs. Single-agent therapies lost curative efficacy when treatment was started beyond day +17, whereas a combination of anti-PD-1 plus anti-CD137 achieved complete rejections. Tumor cells expressed weak baseline PD-L1 on the plasma membrane, but this could be readily induced by interferon-γ. Combined treatment efficacy required CD8 T cells and induced a leukocyte infiltrate in which T lymphocytes co-expressing CD137 and PD-1 were prominent. CONCLUSIONS: These promising results advocate the use of combined anti-PD-1/PD-L1 plus anti-CD137 mAb immunotherapy for the treatment of squamous non-small cell lung cancer in the clinical setting.


Assuntos
Anticorpos Monoclonais/farmacologia , Carcinoma de Células Escamosas/tratamento farmacológico , Imunoterapia/métodos , Neoplasias Pulmonares/tratamento farmacológico , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Membro 9 da Superfamília de Receptores de Fatores de Necrose Tumoral/antagonistas & inibidores , Animais , Anticorpos Monoclonais/imunologia , Carcinoma de Células Escamosas/imunologia , Feminino , Neoplasias Pulmonares/imunologia , Camundongos , Camundongos Endogâmicos A , Camundongos Transgênicos , Transplante de Neoplasias , Receptor de Morte Celular Programada 1/imunologia , Membro 9 da Superfamília de Receptores de Fatores de Necrose Tumoral/imunologia
16.
Eur J Immunol ; 46(3): 513-22, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26773716

RESUMO

CD137 (4-1BB, TNF-receptor superfamily 9) is a surface glycoprotein of the TNFR family which can be induced on a variety of leukocyte subsets. On T and NK cells, CD137 is expressed following activation and, if ligated by its natural ligand (CD137L), conveys polyubiquitination-mediated signals via TNF receptor associated factor 2 that inhibit apoptosis, while enhancing proliferation and effector functions. CD137 thus behaves as a bona fide inducible costimulatory molecule. These functional properties of CD137 can be exploited in cancer immunotherapy by systemic administration of agonist monoclonal antibodies, which increase anticancer CTLs and enhance NK-cell-mediated antibody-dependent cell-mediated cytotoxicity. Reportedly, anti-CD137 mAb and adoptive T-cell therapy strongly synergize, since (i) CD137 expression can be used to select the T cells endowed with the best activities against the tumor, (ii) costimulation of the lymphocyte cultures to be used in adoptive T-cell therapy can be done with CD137 agonist antibodies or CD137L, and (iii) synergistic effects upon coadministration of T cells and antibodies are readily observed in mouse models. Furthermore, the signaling cytoplasmic tail of CD137 is a key component of anti-CD19 chimeric antigen receptors that are used to redirect T cells against leukemia and lymphoma in the clinic. Ongoing phase II clinical trials with agonist antibodies and the presence of CD137 sequence in these successful chimeric antigen receptors highlight the importance of CD137 in oncoimmunology.


Assuntos
Anticorpos Monoclonais/uso terapêutico , Imunoterapia Adotiva , Neoplasias/imunologia , Neoplasias/terapia , Linfócitos T Citotóxicos/imunologia , Membro 9 da Superfamília de Receptores de Fatores de Necrose Tumoral/imunologia , Membro 9 da Superfamília de Receptores de Fatores de Necrose Tumoral/metabolismo , Animais , Citotoxicidade Celular Dependente de Anticorpos , Ensaios Clínicos Fase II como Assunto , Humanos , Imunoterapia Adotiva/métodos , Células Matadoras Naturais/imunologia , Ativação Linfocitária , Camundongos , Transdução de Sinais
17.
Cancer Discov ; 6(1): 71-9, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26493961

RESUMO

UNLABELLED: Weak and ineffective antitumor cytotoxic T lymphocyte (CTL) responses can be rescued by immunomodulatory mAbs targeting PD-1 or CD137. Using Batf3(-/-) mice, which are defective for cross-presentation of cell-associated antigens, we show that BATF3-dependent dendritic cells (DC) are essential for the response to therapy with anti-CD137 or anti-PD-1 mAbs. Batf3(-/-) mice failed to prime an endogenous CTL-mediated immune response toward tumor-associated antigens, including neoantigens. As a result, the immunomodulatory mAbs could not amplify any therapeutically functional immune response in these mice. Moreover, administration of systemic sFLT3L and local poly-ICLC enhanced DC-mediated cross-priming and synergized with anti-CD137- and anti-PD-1-mediated immunostimulation in tumor therapy against B16-ovalbumin-derived melanomas, whereas this function was lost in Batf3(-/-) mice. These experiments show that cross-priming of tumor antigens by FLT3L- and BATF3-dependent DCs is crucial to the efficacy of immunostimulatory mAbs and represents a very attractive point of intervention to enhance their clinical antitumor effects. SIGNIFICANCE: Immunotherapy with immunostimulatory mAbs is currently achieving durable clinical responses in different types of cancer. We show that cross-priming of tumor antigens by BATF3-dependent DCs is a key limiting factor that can be exploited to enhance the antitumor efficacy of anti-PD-1 and anti-CD137 immunostimulatory mAbs.


Assuntos
Anticorpos Monoclonais/administração & dosagem , Fatores de Transcrição de Zíper de Leucina Básica/genética , Células Dendríticas/imunologia , Melanoma Experimental/terapia , Proteínas Repressoras/genética , Animais , Anticorpos Monoclonais/uso terapêutico , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Linhagem Celular Tumoral , Células Dendríticas/citologia , Humanos , Imunoterapia/métodos , Ativação Linfocitária/imunologia , Melanoma Experimental/genética , Melanoma Experimental/metabolismo , Melanoma Experimental/patologia , Camundongos , Camundongos Transgênicos , Receptor de Morte Celular Programada 1/imunologia , Proteínas Repressoras/metabolismo , Membro 9 da Superfamília de Receptores de Fatores de Necrose Tumoral/imunologia
18.
Oncoimmunology ; 4(12): e1054597, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26587331

RESUMO

CD137 (4-1BB) is a surface protein initially discovered to mark activated T lymphocytes. However, its broader expression pattern also encompasses activated NK cells, B cells and myeloid cells, including mature dendritic cells. In this study, we have immunostained for CD137 on paraffin-embedded lymphoid tissues including tonsils, lymph nodes, ectopic tertiary lymphoid tissue in Hashimoto thyroiditis and cancer. Surprisingly, immunostaining mainly decorated intrafollicular lymphocytes in the tissues analyzed, with only scattered staining in interfollicular areas. Moreover, pathologic lymphoid follicles in follicular lymphoma and tertiary lymphoid tissue associated with non-small cell lung cancer showed a similar pattern of immunostaining. Multispectral fluorescence cytometry demonstrated that CD137 expression was restricted to CD4+ CXCR5+ follicular T helper lymphocytes (TFH cells) in tonsils and lymph nodes. Short-term culture of lymph node cell suspensions in the presence of either an agonistic anti-CD137 monoclonal antibody (mAb) or CD137-ligand stimulated the functional upregulation of TFH cells in 3 out of 6 cases, as indicated by CD40L surface expression and cytokine production. As a consequence, immunostimulatory monoclonal antibodies targeting CD137 (such as urelumab and PF-05082566) should be expected to primarily act on this lymphocyte subset, thus modifying ongoing humoral immune responses in patients with autoimmune disease and cancer.

19.
Cancer Immunol Res ; 3(5): 449-54, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25691326

RESUMO

Virotherapy and checkpoint inhibitors can be combined for the treatment of cancer with complementarity and potential for synergistic effects. We have developed a cytolytic but nonreplicative viral vector system based on Semliki Forest virus that encodes IL12 (SFV-IL12). Following direct intratumoral injection, infected cells release transgenic IL12, die, and elicit an inflammatory response triggered by both abundantly copied viral RNA and IL12. In difficult-to-treat mouse cancer models, such as those derived from MC38 and bilateral B16-OVA, SFV-IL12 synergized with an anti-PD-1 monoclonal antibody (mAb) to induce tumor regression and prolong survival. Similar synergistic effects were attained upon PD-L1 blockade. Combined SFV-IL12 + anti-PD-1 mAb treatment only marginally increased the elicited cytotoxic T-lymphocyte response over SFV-IL12 as a single agent, at least when measured by in vivo killing assays. In contrast, we observed that SFV-IL12 treatment induced expression of PD-L1 on tumor cells in an IFNγ-dependent fashion. PD-L1-mediated adaptive resistance thereby provides a mechanistic explanation of the observed synergistic effects achieved by the SFV-IL12 + anti-PD-1 mAb combination.


Assuntos
Anticorpos Monoclonais/uso terapêutico , Interleucina-12/genética , Neoplasias/terapia , Terapia Viral Oncolítica , Receptor de Morte Celular Programada 1/imunologia , Vírus da Floresta de Semliki/genética , Animais , Linhagem Celular Tumoral , Feminino , Vetores Genéticos , Interleucina-12/imunologia , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Neoplasias/imunologia , Baço/citologia
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