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1.
Cell ; 160(1-2): 269-84, 2015 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-25594183

RESUMO

The stem cells that maintain and repair the postnatal skeleton remain undefined. One model suggests that perisinusoidal mesenchymal stem cells (MSCs) give rise to osteoblasts, chondrocytes, marrow stromal cells, and adipocytes, although the existence of these cells has not been proven through fate-mapping experiments. We demonstrate here that expression of the bone morphogenetic protein (BMP) antagonist gremlin 1 defines a population of osteochondroreticular (OCR) stem cells in the bone marrow. OCR stem cells self-renew and generate osteoblasts, chondrocytes, and reticular marrow stromal cells, but not adipocytes. OCR stem cells are concentrated within the metaphysis of long bones not in the perisinusoidal space and are needed for bone development, bone remodeling, and fracture repair. Grem1 expression also identifies intestinal reticular stem cells (iRSCs) that are cells of origin for the periepithelial intestinal mesenchymal sheath. Grem1 expression identifies distinct connective tissue stem cells in both the bone (OCR stem cells) and the intestine (iRSCs).


Assuntos
Osso e Ossos/citologia , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Intestino Delgado/citologia , Células-Tronco Mesenquimais/citologia , Animais , Cartilagem/metabolismo , Intestino Delgado/metabolismo , Células-Tronco Mesenquimais/metabolismo , Camundongos , Camundongos Endogâmicos C57BL
2.
Immunology ; 172(2): 279-294, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38444199

RESUMO

In 2015, the oncolytic herpes simplex virus 1 (HSV-1) T-VEC (talimogene laherparepvec) was approved for intratumoral injection in non-resectable malignant melanoma. To determine whether viral replication is required for oncolytic activity, we compared replication-deficient HSV-1 d106S with replication-competent T-VEC. High infectious doses of HSV-1 d106S killed melanoma (n = 10), head-and-neck squamous cell carcinoma (n = 11), and chondrosarcoma cell lines (n = 2) significantly faster than T-VEC as measured by MTT metabolic activity, while low doses of T-VEC were more effective over time. HSV-1 d106S and, to a lesser extent T-VEC, triggered caspase-dependent early apoptosis as shown by pan-caspase inhibition and specific induction of caspases 3/7, 8, and 9. HSV-1 d106S induced a higher ratio of apoptosis-inducing infected cell protein (ICP) 0 to apoptosis-blocking ICP6 than T-VEC. T-VEC was oncolytic for an extended period of time as viral replication continued, which could be partially blocked by the antiviral drug aciclovir. High doses of T-VEC, but not HSV-1 d106S, increased interferon-ß mRNA as part of the intrinsic immune response. When markers of immunogenic cell death were assessed, ATP was released more efficiently in the context of T-VEC than HSV-1 d106S infection, whereas HMGB1 was induced comparatively well. Overall, the early oncolytic effect on three different tumour entities was stronger with the non-replicative strain, while the replication-competent virus elicited a stronger innate immune response and more pronounced immunogenic cell death.


Assuntos
Apoptose , Herpesvirus Humano 1 , Terapia Viral Oncolítica , Vírus Oncolíticos , Replicação Viral , Herpesvirus Humano 1/fisiologia , Humanos , Terapia Viral Oncolítica/métodos , Linhagem Celular Tumoral , Vírus Oncolíticos/genética , Vírus Oncolíticos/fisiologia , Caspases/metabolismo , Animais , Melanoma/terapia , Melanoma/imunologia
3.
Mol Cell ; 49(4): 668-79, 2013 Feb 21.
Artigo em Inglês | MEDLINE | ID: mdl-23317503

RESUMO

The HIV Nef protein recruits the polycomb protein Eed and mimics an integrin receptor signal for reasons that are not entirely clear. Here we demonstrate that Nef and Eed complex with the integrin effector paxillin to recruit and activate TNFα converting enzyme (TACE alias ADAM 17) and its close relative ADAM10. The activated proteases cleaved proTNFα and were shuttled into extracellular vesicles (EVs). Peripheral blood mononuclear cells that ingested these EVs released TNFα. Analyzing the mechanism, we found that Pak2, an established host cell effector of Nef, phosphorylated paxillin on Ser272/274 to induce TACE-paxillin association and shuttling into EVs via lipid rafts. Conversely, Pak1 phosphorylated paxillin on Ser258, which inhibited TACE association and lipid raft transfer. Interestingly, melanoma cells used an identical mechanism to shuttle predominantly ADAM10 into EVs. We conclude that HIV-1 and cancer cells exploit a paxillin/integrin-controlled mechanism to release TACE/ADAM10-containing vesicles, ensuring better proliferation/growth conditions in their microenvironment.


Assuntos
Proteínas ADAM/metabolismo , Secretases da Proteína Precursora do Amiloide/metabolismo , Proteínas de Membrana/metabolismo , Paxilina/fisiologia , Produtos do Gene nef do Vírus da Imunodeficiência Humana/fisiologia , Quinases Ativadas por p21/fisiologia , Proteínas ADAM/sangue , Proteína ADAM10 , Proteína ADAM17 , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Substituição de Aminoácidos , Secretases da Proteína Precursora do Amiloide/sangue , Estudos de Casos e Controles , Ativação Enzimática , Células HEK293 , Infecções por HIV/sangue , Infecções por HIV/enzimologia , Ribonucleoproteínas Nucleares Heterogêneas Grupo K , Humanos , Melanoma/sangue , Melanoma/enzimologia , Microdomínios da Membrana/enzimologia , Proteínas de Membrana/sangue , Mutagênese Sítio-Dirigida , Paxilina/genética , Paxilina/metabolismo , Fosforilação , Complexo Repressor Polycomb 2/metabolismo , Ligação Proteica , Proteína Quinase C-delta/metabolismo , Precursores de Proteínas/metabolismo , Processamento de Proteína Pós-Traducional , Transporte Proteico , Ribonucleoproteínas/metabolismo , Vesículas Secretórias/metabolismo , Transdução de Sinais , Células Tumorais Cultivadas , Fator de Necrose Tumoral alfa/metabolismo , Produtos do Gene nef do Vírus da Imunodeficiência Humana/metabolismo , Quinases Ativadas por p21/metabolismo
4.
Int J Cancer ; 144(5): 1147-1150, 2019 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-30151962

RESUMO

Recent studies suggest that the age-related remodeling of the immune system, known as immunosenescence, could impact the efficacy of immune checkpoint inhibitors in leukemia or nonsmall cell lung cancer. We investigated whether senescence markers can predict response to checkpoint inhibitor therapy in melanoma patients. The peripheral blood of patients with newly diagnosed, untreated metastatic melanoma was analyzed by flow cytometry to correlate the frequency of senescence markers with clinical response as measured by RECIST after 12 weeks of treatment with immune checkpoint inhibitors. The loss of surface markers CD27 and CD28 or the expression of Tim-3 and CD57 on T cells was associated with resistance to checkpoint inhibitor blockade, presenting these phenotypes as possible predictive biomarkers for checkpoint inhibitor therapy. Immunosenescence seems to impact on the response to checkpoint inhibitor therapy in melanoma patients. Thus, lymphocyte phenotyping for senescence markers, with the introduction of immunosenescence panels, could be predictive for checkpoint inhibitor response.


Assuntos
Imunossenescência/imunologia , Melanoma/imunologia , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais , Antígenos CD28/imunologia , Antígenos CD57/imunologia , Feminino , Citometria de Fluxo/métodos , Receptor Celular 2 do Vírus da Hepatite A/imunologia , Humanos , Linfócitos/imunologia , Masculino , Pessoa de Meia-Idade , Membro 7 da Superfamília de Receptores de Fatores de Necrose Tumoral/imunologia
5.
Development ; 143(14): 2616-28, 2016 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-27287799

RESUMO

Intestinal hormone-producing cells represent the largest endocrine system in the body, but remarkably little is known about enteroendocrine cell type specification in the embryo and adult. We analyzed stage- and cell type-specific deletions of Nkx2.2 and its functional domains in order to characterize its role in the development and maintenance of enteroendocrine cell lineages in the mouse duodenum and colon. Although Nkx2.2 regulates enteroendocrine cell specification in the duodenum at all stages examined, it controls the differentiation of progressively fewer enteroendocrine cell populations when deleted from Ngn3(+) progenitor cells or in the adult duodenum. During embryonic development Nkx2.2 regulates all enteroendocrine cell types, except gastrin and preproglucagon. In developing Ngn3(+) enteroendocrine progenitor cells, Nkx2.2 is not required for the specification of neuropeptide Y and vasoactive intestinal polypeptide, indicating that a subset of these cell populations derive from an Nkx2.2-independent lineage. In adult duodenum, Nkx2.2 becomes dispensable for cholecystokinin and secretin production. In all stages and Nkx2.2 mutant conditions, serotonin-producing enterochromaffin cells were the most severely reduced enteroendocrine lineage in the duodenum and colon. We determined that the transcription factor Lmx1a is expressed in enterochromaffin cells and functions downstream of Nkx2.2. Lmx1a-deficient mice have reduced expression of Tph1, the rate-limiting enzyme for serotonin biosynthesis. These data clarify the function of Nkx2.2 in the specification and homeostatic maintenance of enteroendocrine populations, and identify Lmx1a as a novel enterochromaffin cell marker that is also essential for the production of the serotonin biosynthetic enzyme Tph1.


Assuntos
Linhagem da Célula , Células Enterocromafins/citologia , Células Enteroendócrinas/citologia , Proteínas de Homeodomínio/metabolismo , Proteínas com Homeodomínio LIM/metabolismo , Serotonina/biossíntese , Fatores de Transcrição/metabolismo , Envelhecimento/metabolismo , Animais , Biomarcadores/metabolismo , Linhagem da Célula/genética , Colo/metabolismo , Duodeno/metabolismo , Deleção de Genes , Regulação da Expressão Gênica , Proteína Homeobox Nkx-2.2 , Proteínas de Homeodomínio/química , Camundongos Endogâmicos C57BL , Modelos Biológicos , Mutação/genética , Reação em Cadeia da Polimerase , Domínios Proteicos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Análise de Sequência de RNA , Células-Tronco/citologia , Fatores de Transcrição/química , Proteínas de Peixe-Zebra
6.
Apoptosis ; 23(3-4): 201-209, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29435687

RESUMO

The tumor environment critically influences responsiveness of cancer cells to chemotherapies, most of which activate the mitochondria-regulated (intrinsic) apoptotic cascade to kill malignant cells. Especially skin tumors encounter an environment with remarkable biophysical properties. Cutaneous accumulation of Na+ locally establishes osmotic pressure gradients in vivo (hypertonicity or hyperosmotic stress), but whether cutaneous hypertonicity is a factor that modulates the responsiveness of skin cancers to therapeutic apoptosis-induction has thus far not been investigated. Here, we show that hyperosmotic stress lowers the threshold for apoptosis induction in malignant melanoma, the deadliest form of skin cancer. Hypertonic conditions enforce addiction to BCL-2-like proteins to prevent initiation of the mitochondria-regulated (intrinsic) apoptotic pathway. Essentially, hyperosmotic stress primes mitochondria for death. Our work identifies osmotic pressure in the tumor microenvironment as a cell extrinsic factor that modulates responsiveness of malignant melanoma cells to therapy.


Assuntos
Apoptose , Melanoma/fisiopatologia , Pressão Osmótica , Humanos , Melanoma/genética , Melanoma/metabolismo , Proteína de Sequência 1 de Leucemia de Células Mieloides/genética , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo
7.
Immunology ; 146(2): 327-38, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26194553

RESUMO

Malignant melanoma is an aggressive tumour of the skin with increasing incidence, frequent metastasis and poor prognosis. At the same time, it is an immunogenic type of cancer with spontaneous regressions. Most recently, the tumoricidal effect of plasmacytoid dendritic cells (pDC) and their capacity to overcome the immunosuppressive tumour microenvironment are being investigated. In this respect, we studied the effect of the infectious, but replication-deficient, herpes simplex virus 1 (HSV-1) d106S vaccine strain, which lacks essential immediate early genes, in pDC co-cultures with 11 melanoma cell lines. We observed a strong cytotoxic activity, inducing apoptotic and necrotic cell death in most melanoma cell lines. The cytotoxic activity of HSV-1 d106S plus pDC was comparable to the levels of cytotoxicity induced by natural killer cells, but required only a fraction of cells with effector : target ratios of 1 : 20 (P < 0·05). The suppressive activity of cell-free supernatants derived from virus-stimulated pDC was significantly neutralized using antibodies against the interferon-α receptor (P < 0·05). In addition to type I interferons, TRAIL and granzyme B contributed to the inhibitory effect of HSV-1 d106S plus pDC to a minor extent. UV-irradiated viral stocks were significantly less active than infectious particles, both in the absence and presence of pDC (P < 0·05), indicating that residual activity of HSV-1 d106S is a major component and sensitizes the tumour cells to interferon-producing pDC. Three leukaemic cell lines were also susceptible to this treatment, suggesting a general anti-tumour effect. In conclusion, the potential of HSV-1 d106S for therapeutic vaccination should be further evaluated in patients suffering from different malignancies.


Assuntos
Vacinas Anticâncer/imunologia , Células Dendríticas/transplante , Herpesvirus Humano 1/imunologia , Imunoterapia Adotiva/métodos , Leucemia/terapia , Melanoma/terapia , Neoplasias Cutâneas/terapia , Apoptose , Linhagem Celular Tumoral , Proliferação de Células , Sobrevivência Celular , Técnicas de Cocultura , Citotoxicidade Imunológica , Células Dendríticas/imunologia , Células Dendríticas/virologia , Herpesvirus Humano 1/genética , Herpesvirus Humano 1/patogenicidade , Humanos , Interferon Tipo I/imunologia , Interferon Tipo I/metabolismo , Leucemia/imunologia , Leucemia/metabolismo , Leucemia/patologia , Leucemia/virologia , Melanoma/imunologia , Melanoma/metabolismo , Melanoma/patologia , Melanoma/virologia , Necrose , Neoplasias Cutâneas/imunologia , Neoplasias Cutâneas/metabolismo , Neoplasias Cutâneas/patologia , Neoplasias Cutâneas/virologia , Fatores de Tempo , Transfecção , Microambiente Tumoral
8.
Am J Physiol Gastrointest Liver Physiol ; 309(12): G975-87, 2015 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-26492922

RESUMO

There are two major stem cell populations in the intestinal crypt region that express either Bmi1 or Lgr5; however, it has been shown that other populations in the crypt can regain stemness. In this study, we demonstrate that the transcription factor NK2 homeobox 2 (Nkx2.2) is expressed in enteroendocrine cells located in the villus and crypt of the intestinal epithelium and is coexpressed with the stem cell markers Bmi1 and Lgr5 in a subset of crypt cells. To determine whether Nkx2.2-expressing enteroendocrine cells display cellular plasticity and stem cell potential, we performed genetic lineage tracing of the Nkx2.2-expressing population using Nkx2.2(Cre/+);R26RTomato mice. These studies demonstrated that Nkx2.2+ cells are able to give rise to all intestinal epithelial cell types in basal conditions. The proliferative capacity of Nkx2.2-expressing cells was also demonstrated in vitro using crypt organoid cultures. Injuring the intestine with irradiation, systemic inflammation, and colitis did not enhance the lineage potential of Nkx2.2-expressing cells. These findings demonstrate that a rare mature enteroendocrine cell subpopulation that is demarcated by Nkx2.2 expression display stem cell properties during normal intestinal epithelial homeostasis, but is not easily activated upon injury.


Assuntos
Diferenciação Celular , Linhagem da Célula , Proliferação de Células , Células Enteroendócrinas/metabolismo , Proteínas de Homeodomínio/metabolismo , Mucosa Intestinal/metabolismo , Células-Tronco Pluripotentes/metabolismo , Fatores de Transcrição/metabolismo , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Biomarcadores/metabolismo , Células Cultivadas , Células Enteroendócrinas/patologia , Células Enteroendócrinas/efeitos da radiação , Genótipo , Proteína Homeobox Nkx-2.2 , Proteínas de Homeodomínio/genética , Mucosa Intestinal/patologia , Mucosa Intestinal/efeitos da radiação , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas do Tecido Nervoso/metabolismo , Fenótipo , Complexo Repressor Polycomb 1/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Fatores de Transcrição/genética , Irradiação Corporal Total , Proteínas de Peixe-Zebra , Proteína Vermelha Fluorescente
9.
Blood ; 122(13): 2185-94, 2013 Sep 26.
Artigo em Inglês | MEDLINE | ID: mdl-23958949

RESUMO

Denileukin diftitox (DD), a diphtheria toxin fragment IL-2 fusion protein, is thought to target and kill CD25(+) cells. It is approved for the treatment of cutaneous T-cell lymphoma and is used experimentally for the depletion of regulatory T cells (Treg) in cancer trials. Curiously enough, clinical effects of DD did not strictly correlate with CD25 expression, and Treg depletion was not confirmed unambiguously. Here, we report that patients with melanoma receiving DD immediately before a dendritic cell (DC) vaccine failed to develop a tumor-antigen-specific CD4 and CD8 T-cell immune response even after repeated vaccinations. Analyzing the underlying mechanism, so far we found unknown effects of DD. First, DD modulated DCs toward tolerance by downregulating costimulatory receptors such as CD83 and CD25 while upregulating tolerance-associated proteins/pathways including Stat-3, ß-catenin, and class II transactivator-dependent antigen presentation. Second, DD blocked Stat3 phosphorylation in maturing DCs. Third, only activated, but not resting, Treg internalized DD and were killed. Conversely, resting Treg showed increased survival because of DD-mediated antiapoptotic IL-2 signaling. We conclude that DD exerts functions beyond CD25(+) cell killing that may affect their clinical use and could be tested for novel indications.


Assuntos
Antineoplásicos/uso terapêutico , Células Dendríticas/efeitos dos fármacos , Toxina Diftérica/uso terapêutico , Interleucina-2/uso terapêutico , Melanoma/terapia , Neoplasias Cutâneas/terapia , Linfócitos T Reguladores/efeitos dos fármacos , Vacinas Anticâncer , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/imunologia , Terapia Combinada , Células Dendríticas/imunologia , Células Dendríticas/transplante , Citometria de Fluxo , Humanos , Tolerância Imunológica , Teste de Cultura Mista de Linfócitos , Melanoma/imunologia , Microscopia Confocal , Análise de Sequência com Séries de Oligonucleotídeos , Fenótipo , Proteínas Recombinantes de Fusão/uso terapêutico , Neoplasias Cutâneas/imunologia , Linfócitos T Reguladores/imunologia
10.
Cancer Immunol Immunother ; 63(4): 381-94, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24487961

RESUMO

PURPOSE: Survivin is a member of the inhibitor-of-apoptosis family. Essential for tumor cell survival and overexpressed in most cancers, survivin is a promising target for anti-cancer immunotherapy. Immunogenicity has been demonstrated in multiple cancers. Nonetheless, few clinical trials have demonstrated survivin-vaccine-induced immune responses. EXPERIMENTAL DESIGN: This phase I trial was conducted to test whether vaccine EMD640744, a cocktail of five HLA class I-binding survivin peptides in Montanide(®) ISA 51 VG, promotes anti-survivin T-cell responses in patients with solid cancers. The primary objective was to compare immunologic efficacy of EMD640744 at doses of 30, 100, and 300 µg. Secondary objectives included safety, tolerability, and clinical efficacy. RESULTS: In total, 49 patients who received ≥2 EMD640744 injections with available baseline- and ≥1 post-vaccination samples [immunologic-diagnostic (ID)-intention-to-treat] were analyzed by ELISpot- and peptide/MHC-multimer staining, revealing vaccine-activated peptide-specific T-cell responses in 31 patients (63 %). This cohort included the per study protocol relevant ID population for the primary objective, i.e., T-cell responses by ELISpot in 17 weeks following first vaccination, as well as subjects who discontinued the study before week 17 but showed responses to the treatment. No dose-dependent effects were observed. In the majority of patients (61 %), anti-survivin responses were detected only after vaccination, providing evidence for de novo induction. Best overall tumor response was stable disease (28 %). EMD640744 was well tolerated; local injection-site reactions constituted the most frequent adverse event. CONCLUSIONS: Vaccination with EMD640744 elicited T-cell responses against survivin peptides in the majority of patients, demonstrating the immunologic efficacy of EMD640744.


Assuntos
Vacinas Anticâncer/imunologia , Epitopos de Linfócito T/imunologia , Proteínas Inibidoras de Apoptose/imunologia , Neoplasias/terapia , Linfócitos T/imunologia , Vacinação , Vacinas Anticâncer/administração & dosagem , Vacinas Anticâncer/efeitos adversos , Vacinas Anticâncer/uso terapêutico , Relação Dose-Resposta Imunológica , Feminino , Antígenos HLA-A/imunologia , Antígeno HLA-B7/imunologia , Humanos , Testes de Liberação de Interferon-gama , Masculino , Pessoa de Meia-Idade , Proteínas de Neoplasias/imunologia , Neoplasias/imunologia , Fragmentos de Peptídeos/imunologia , Survivina , Especificidade do Receptor de Antígeno de Linfócitos T
11.
J Clin Med ; 12(15)2023 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-37568553

RESUMO

Open spina bifida (OSB) is a congenital, non-lethal malformation with multifactorial etiology. Fetal therapy can be offered under certain conditions to parents after accurate prenatal diagnostic and interdisciplinary counseling. Since the advent of prenatal OSB surgery, various modifications of the original surgical techniques have evolved, including laparotomy-assisted fetoscopic repair. After a two-year preparation time, the team at the University of Giessen and Marburg (UKGM) became the first center to provide a three-port, three-layer fetoscopic repair of OSB via a laparotomy-assisted approach in the German-speaking area. We point out that under the guidance of experienced centers and by intensive multidisciplinary preparation and training, a previously described and applied technique could be transferred to a different setting.

12.
Blood ; 116(13): 2277-85, 2010 Sep 30.
Artigo em Inglês | MEDLINE | ID: mdl-20566893

RESUMO

The use of dendritic cells (DCs) in therapeutic cancer vaccination requires their loading with tumor-specific antigen(s). DEC-205, a phagocytosis receptor mediating antigen uptake, is associated with CD8(+) T-cell responses in mice. Here we fused an anti-DEC-205scFv to an HLA-DP4-restricted epitope from the tumor antigen MAGE-A3, and examined the suitability and efficacy of DEC-205 to deliver a helper epitope to human monocyte-derived DCs (moDCs). The construct specifically bound DEC-205 on human moDCs without negative impact on DC phenotype and function. We measured antigen presentation with specific autologous CD4(+) T cells, generated by TCR-RNA transfection. DEC-205 targeting resulted in significant major histocompatibility complex class II-restricted antigen presentation, and was superior to loading DCs by electroporation of mRNA encoding endosome-targeted MAGE-A3-DCLAMP or by direct peptide pulsing. Anti-DEC-205scFv-MAGE-A3 was presented 100 times more efficiently than the control constructs. DC maturation before or during incubation with anti-DEC-205scFv-MAGE-A3 reduced the interleukin-10/interleukin-2 ratio. Moreover, we successfully applied the DEC-205 targeting strategy to moDCs from malignant melanoma patients. Again, DEC-205-targeted mature DCs (mDCs) presented the antigen more efficiently than peptide-pulsed DCs and maintained their stimulatory capacity after cryoconservation. Thus, DEC-205 targeting represents a feasible and effective method to deliver helper epitopes to DCs in anticancer vaccine strategies, which may also be suitable for DC targeting in vivo.


Assuntos
Apresentação de Antígeno/imunologia , Antígenos CD/imunologia , Células Dendríticas/imunologia , Antígenos de Histocompatibilidade Classe II/metabolismo , Lectinas Tipo C/imunologia , Receptores de Superfície Celular/imunologia , Sequência de Aminoácidos , Anticorpos/administração & dosagem , Anticorpos/genética , Antígenos CD/metabolismo , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/imunologia , Linfócitos T CD4-Positivos/imunologia , Diferenciação Celular/imunologia , Citocinas/biossíntese , Células Dendríticas/citologia , Epitopos/administração & dosagem , Epitopos/genética , Antígenos HLA-DP/metabolismo , Cadeias beta de HLA-DP , Humanos , Imunoterapia Ativa , Técnicas In Vitro , Lectinas Tipo C/metabolismo , Melanoma/imunologia , Melanoma/terapia , Antígenos de Histocompatibilidade Menor , Dados de Sequência Molecular , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/imunologia , Receptores de Superfície Celular/metabolismo , Proteínas Recombinantes de Fusão/administração & dosagem , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia
13.
Cancers (Basel) ; 13(12)2021 Jun 19.
Artigo em Inglês | MEDLINE | ID: mdl-34205379

RESUMO

Talimogene laherparepvec (T-VEC), an oncolytic herpes simplex virus, is approved for intralesional injection of unresectable stage IIIB/IVM1a melanoma. However, it is still unclear which parameter(s) predict treatment response or failure. Our study aimed at characterizing surface receptors Nectin-1 and the herpes virus entry mediator (HVEM) in addition to intracellular molecules cyclic GMP-AMP synthase (cGAS) and stimulator of interferon genes (STING) as potential bio-markers for oncolytic virus treatment. In 20 melanoma cell lines, oncolytic activity of T-VEC was correlated with the expression of Nectin-1 but not HVEM, as evaluated via flow cytometry and immunohistochemistry. Knockout using CRISPR/Cas9 technology confirmed the superior role of Nectin-1 over HVEM for entry and oncolytic activity of T-VEC. Neither cGAS nor STING as evaluated by Western Blot and immunohistochemistry correlated with T-VEC induced oncolysis. The role of these biomarkers was retrospectively analyzed for the response of 35 cutaneous melanoma metastases of 21 patients to intralesional T-VEC injection, with 21 (60.0%) of these lesions responding with complete (n = 16) or partial regression (n = 5). Nectin-1 expression in pretreatment biopsies significantly predicted treatment outcome, while the expression of HVEM, cGAS, and STING was not prognostic. Altogether, Nectin-1 served as biomarker for T-VEC-induced melanoma regression in vitro and in vivo.

14.
Ther Adv Med Oncol ; 11: 1758835919891622, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31853267

RESUMO

BACKGROUND: In therapeutic cancer vaccination, monocyte-derived dendritic cells (moDCs) efficiently activate specific T-cell responses; however, optimizing the activation of innate immune cells could support and improve the antitumor effects. A major disadvantage of moDCs matured with the standard cytokine cocktail (consisting of IL-1ß, IL-6, TNFα, and PGE2) is their inability to secrete IL-12p70. IL-12 prominently activates natural killer (NK) cells, which are crucial in innate antitumor immunity, as they act as helper cells for the induction of a cytotoxic T lymphocyte (CTL) response and are also able to directly kill the tumor. METHODS: Previously we have shown that triggering the NF-κB pathway in moDCs by transfection of mRNA encoding constitutively active IKKß (caIKKß) led to IL-12p70 secretion and improved the dendritic cells' capability to activate and expand CTLs with a memory-like phenotype. In this study, we examined whether such dendritic cells could activate autologous NK cells. RESULTS: moDCs matured with the standard cytokine cocktail followed by transfection with the caIKKß-RNA were able to activate autologous NK cells, detected by the upregulation of CD54, CD69, and CD25 on the NK cells, their ability to secrete IFNγ, and their high lytic activity. Moreover, the ability of NK-cell activation was not diminished by simultaneous T-cell activation. CONCLUSION: The capacity of caIKKß-DCs to activate both the adaptive and innate immune response indicates an enhanced potential for clinical efficacy.

15.
Front Immunol ; 10: 2, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30723467

RESUMO

Robust anti-tumor immunity requires innate as well as adaptive immune responses. We have shown that plasmacytoid dendritic cells develop killer cell-like activity in melanoma cell cocultures after exposure to the infectious but replication-deficient herpes simplex virus 1 (HSV-1) d106S. To combine this innate effect with an enhanced adaptive immune response, the gene encoding human MelanA/MART-1 was inserted into HSV-1 d106S via homologous recombination to increase direct expression of this tumor antigen. Infection of Vero cells using this recombinant virus confirmed MelanA expression by Western blotting, flow cytometry, and immunofluorescence. HSV-1 d106S-MelanA induced expression of the transgene in fibroblast and melanoma cell lines not naturally expressing MelanA. Infection of a melanoma cell line with CRISPR-Cas9-mediated knockout of MelanA confirmed de novo expression of the transgene in the viral context. Dependent on MelanA expression, infected fibroblast and melanoma cell lines induced degranulation of HLA-matched MelanA-specific CD8+ T cells, followed by killing of infected cells. To study infection of immune cells, we exposed peripheral blood mononuclear cells and in vitro-differentiated macrophages to the parental HSV-1 d106S, resulting in expression of the transgene GFP in CD11c+ cells and macrophages. These data provide evidence that the application of MelanA-encoding HSV-1 d106S could enhance adaptive immune responses and re-direct MelanA-specific CD8+ T cells to tumor lesions, which have escaped adaptive immune responses via downregulation of their tumor antigen. Hence, HSV-1 d106S-MelanA harbors the potential to induce innate immune responses in conjunction with adaptive anti-tumor responses by CD8+ T cells, which should be evaluated in further studies.


Assuntos
Expressão Gênica , Vetores Genéticos/genética , Herpesvirus Humano 1 , Antígeno MART-1/genética , Vírus Oncolíticos , Transgenes , Células Apresentadoras de Antígenos/imunologia , Células Apresentadoras de Antígenos/metabolismo , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Linhagem Celular Tumoral , Fibroblastos/metabolismo , Marcação de Genes , Engenharia Genética , Herpesvirus Humano 1/genética , Humanos , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismo , Melanoma , Vírus Oncolíticos/genética , Linfócitos T/imunologia , Linfócitos T/metabolismo
16.
Immunol Lett ; 116(1): 7-14, 2008 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-18164076

RESUMO

Tumor cells often evoke specific immune responses that, however, fail to eliminate all the tumor cells. The development of cancer immunotherapies is, therefore, mostly focused on the generation of large numbers of activated anti-tumor effector cells by vaccination or adoptive T cell transfer. These developments are built on an ever-extended list of identified tumor-associated antigens and corresponding T cell epitopes, and a steady flow of reports from proof-of-principle animal model experiments demonstrating cure from disease by immune interventions. However, the promises have not translated into clinical successes for cancer patients. Even where tumor regression or complete responses were achieved there is usually relapse of the disease. Increasing numbers of reports over recent years highlight potential immunosuppressive mechanisms that act in tumors and systemically in cancer patients to block effective anti-tumor immune responses. They account in large parts for the failures of cancer immunotherapy and need to be overcome before progress can be expected. We review here the current state of the research on immunosuppressive networks in human cancer.


Assuntos
Imunoterapia , Neoplasias/imunologia , Neoplasias/terapia , Evasão Tumoral , Animais , Apresentação de Antígeno , Antígenos de Neoplasias/imunologia , Apresentação Cruzada , Humanos , Tolerância Imunológica , Imunoterapia/tendências , Camundongos , Neoplasias/irrigação sanguínea , Neoplasias/patologia , Neovascularização Patológica/imunologia
17.
Oncogene ; 37(30): 4122-4136, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29706657

RESUMO

Attempts to exploit the cytotoxic activity of death receptors (DR) for treating cancer have thus far been disappointing. DR activation in most malignant cells fails to trigger cell death and may even promote tumor growth by activating cell death-independent DR-associated signaling pathways. Overcoming apoptosis resistance is consequently a prerequisite for successful clinical exploitation of DR stimulation. Here we show that hyperosmotic stress in the tumor microenvironment unleashes the deadly potential of DRs by enforcing BCL-2 addiction of cancer cells. Hypertonicity robustly enhanced cytotoxicity of tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) and other DR ligands in various cancer entities. Initial events in TRAIL DR signaling remained unaffected, but hypertonic conditions unlocked activation of the mitochondrial death pathway and thus amplified the apoptotic signal. Mechanistically, we demonstrate that hyperosmotic stress imposed a BCL-2-addiction on cancer cells to safeguard the integrity of the outer mitochondrial membrane (OMM), essentially exhausting the protective capacity of BCL-2-like pro-survival proteins. Deprivation of these mitochondrial safeguards licensed DR-generated truncated BH3-interacting domain death agonist (tBID) to activate BCL-2-associated X protein (BAX) and initiated mitochondrial outer membrane permeabilization (MOMP). Our work highlights that hyperosmotic stress in the tumor environment primes mitochondria for death and lowers the threshold for DR-induced apoptosis. Beyond TRAIL-based therapies, our findings could help to strengthen the efficacy of other apoptosis-inducing cancer treatment regimens.


Assuntos
Morte Celular/fisiologia , Pressão Osmótica/fisiologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Receptores de Morte Celular/metabolismo , Apoptose/fisiologia , Proteínas Reguladoras de Apoptose/metabolismo , Linhagem Celular Tumoral , Células HCT116 , Células HEK293 , Células HT29 , Humanos , Mitocôndrias/metabolismo , Membranas Mitocondriais/metabolismo , Proteínas Mitocondriais/metabolismo , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Transdução de Sinais/fisiologia , Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Microambiente Tumoral/fisiologia , Proteína X Associada a bcl-2/metabolismo
18.
Oncol Lett ; 14(5): 5443-5451, 2017 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-29098034

RESUMO

Malignant brain tumors, including gliomas, brain metastases and anaplastic meningiomas, are associated with poor prognosis, and represent an unmet medical need. ASA404 (DMXAA), a vascular disrupting agent, has demonstrated promising results in several preclinical tumor models and early phase clinical trials. However, two phase III trials in non-small cell lung cancer reported insufficient results. The aim of the present study was to determine the effects of ASA404 on brain tumors. The effects of ASA404 were evaluated in vitro and in vivo using subcutaneous, and orthotopical models for malignant glioma (U-87, LN-229, U-251, LN-308 and Tu-2449), brain metastasis (HT-29) and malignant meningioma (IOMM-Lee). The acute effects of ASA404 on tumor tissue were analyzed using conventional and immunohistochemical staining techniques [hematoxylin and eosin, MIB-1 antibody/proliferation maker protein Ki-67, cleaved caspase-8, stimulator of interferon genes (STING), ionized calcium-binding adapter molecule 1]. Furthermore, the sizes of subcutaneous tumors were measured and the symptom-free survival rates of animals with intracranial tumors receiving ASA404 treatment were analyzed. ASA404 demonstrated low toxicity in vitro, but exhibited strong effects on subcutaneous tumors 24 h following a single dose of ASA404 (25 mg/kg). ASA404 induced necrosis, hemorrhages and inhibited the proliferation, and growth of tumors in the subcutaneous glioma models. However, ASA404 failed to demonstrate comparable effects in any of the intracranial tumor models examined and did not result in a prolongation of survival. Expression of STING, the molecular target of ASA404, and infiltration of macrophages, the cells mediating ASA404 activity, did not differ between subcutaneous and intracranial tumors. In conclusion, ASA404 demonstrates clear efficacy in subcutaneous tumor models, but has no relevant activity in orthotopic brain tumor models. The expression of STING and infiltration with macrophages were not determined to be involved in the differential activity observed among tumor models. It is possible that the low penetration of ASA-404 into the brain prevents concentrations sufficient enough reaching the tumor in order to exhibit acute effects in vivo.

19.
JCI Insight ; 2(8)2017 Apr 20.
Artigo em Inglês | MEDLINE | ID: mdl-28422751

RESUMO

BACKGROUND: Reports on long-term (≥10 years) effects of cancer vaccines are missing. Therefore, in 2002, we initiated a phase I/II trial in cutaneous melanoma patients to further explore the immunogenicity of our DC vaccine and to establish its long-term toxicity and clinical benefit after a planned 10-year followup. METHODS: Monocyte-derived DCs matured by TNFα, IL-1ß, IL-6, and PGE2 and then loaded with 4 HLA class I and 6 class II-restricted tumor peptides were injected intradermally in high doses over 2 years. We performed serial immunomonitoring in all 53 evaluable patients. RESULTS: Vaccine-specific immune responses including high-affinity, IFNγ-producing CD4+ and lytic polyfunctional CD8+ T cells were de novo induced or boosted in most patients. Exposure of mature DCs to trimeric soluble CD40 ligand, unexpectedly, did not further enhance such immune responses, while keyhole limpet hemocyanin (KLH) pulsing to provide unspecific CD4+ help promoted CD8+ T cell responses - notably, their longevity. An unexpected 19% of nonresectable metastatic melanoma patients are still alive after 11 years, a survival rate similar to that observed in ipilimumab-treated patients and achieved without any major (>grade 2) toxicity. Survival correlated significantly with the development of intense vaccine injection site reactions, and with blood eosinophilia after the first series of vaccinations, suggesting that prolonged survival was a consequence of DC vaccination. CONCLUSIONS: Long-term survival in advanced melanoma patients undergoing DC vaccination is similar to ipilimumab-treated patients and occurs upon induction of tumor-specific T cells, blood eosinophilia, and strong vaccine injection site reactions occurring after the initial vaccinations. TRIAL REGISTRATION: ClinicalTrials.gov NCT00053391. FUNDING: European Community, Sixth Framework Programme (Cancerimmunotherapy LSHC-CT-2006-518234; DC-THERA LSHB-CT-2004-512074), and German Research Foundation (CRC 643, C1, Z2).

20.
Oncotarget ; 8(40): 67439-67456, 2017 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-28978044

RESUMO

Immunotherapy for metastatic melanoma offers great promise but, to date, only a subset of patients have responded. There is an urgent need to identify ways of allocating patients to the most beneficial therapy, to increase survival and decrease therapy-associated morbidity and costs. Blood-based biomarkers are of particular interest because of their straightforward implementation in routine clinical care. We sought to identify markers for dendritic cell (DC) vaccine-based immunotherapy against metastatic melanoma through gene expression analysis of peripheral blood mononuclear cells. A large-scale microarray analysis of 74 samples from two treatment centers, taken directly after the first round of DC vaccination, was performed. We found that phosphatidylethanolamine binding protein 1 (PEBP1)/Raf Kinase inhibitory protein (RKIP) expression can be used to identify a significant proportion of patients who performed poorly after DC vaccination. This result was validated by q-PCR analysis on blood samples from a second cohort of 95 patients treated with DC vaccination in four different centers. We conclude that low PEBP1 expression correlates with poor overall survival after DC vaccination. Intriguingly, this was only the case for expression of PEBP1 after, but not prior to, DC vaccination. Moreover, the change in PEBP1 expression upon vaccination correlated well with survival. Further analyses revealed that PEBP1 expression positively correlated with genes involved in T cell responses but inversely correlated with genes associated with myeloid cells and aberrant inflammation including STAT3, NOTCH1, and MAPK1. Concordantly, PEBP1 inversely correlated with the myeloid/lymphoid-ratio and was suppressed in patients suffering from chronic inflammatory disease.

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