RESUMO
BACKGROUND: Epirubicin/cyclophosphamide (EC) and docetaxel (D) are commonly used in a sequential regimen in the neoadjuvant treatment of early, high-risk or locally advanced breast cancer (BC). Novel approaches to increase the response rate combine this treatment with immunotherapies such as PD-1 inhibition. However, the expected stimulatory effect on lymphocytes may depend on the chemotherapy backbone. Therefore, we separately compared the immunomodulatory effects of EC and D in the setting of a randomized clinical trial. METHODS: Tumor and blood samples of 154 patients from the ABCSG-34 trial were available (76 patients received four cycles of EC followed by four cycles of D; 78 patients get the reverse treatment sequence). Tumor-infiltrating lymphocytes, circulating lymphocytes and 14 soluble immune mediators were determined at baseline and at drug change. Furthermore, six BC cell lines were treated with E, C or D and co-cultured with immune cells. RESULTS: Initial treatment with four cycles of EC reduced circulating B and T cells by 94% and 45%, respectively. In contrast, no comparable effects on lymphocytes were observed in patients treated with initial four cycles of D. Most immune mediators decreased under EC whereas D-treatment resulted in elevated levels of CXCL10, urokinase-type plasminogen activator (uPA) and its soluble receptor (suPAR). Accordingly, only the exposure of BC cell lines to D induced similar increases as compared to E. While treatment of BC cells with E was associated with cell shrinkage and apoptosis, D induced cell swelling and accumulation of cells in G2 phase. CONCLUSION: The deleterious effect of EC on lymphocytes indicates strong immunosuppressive properties of this combination therapy. D, in contrast, has no effect on lymphocytes, but triggers the secretion of stimulatory proteins in vivo and in vitro, indicating a supportive effect on the immune system. Underlying differences in the induced cell death might be causal. These divergent immunomodulatory effects of epirubicin/cyclophosphamide and docetaxel should be considered when planning future combinations with immunotherapies in breast cancer.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica , Neoplasias da Mama , Feminino , Humanos , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Ciclofosfamida/farmacologia , Docetaxel/farmacologia , Epirubicina/farmacologia , Fluoruracila , Terapia Neoadjuvante/métodos , Resultado do TratamentoRESUMO
BACKGROUND & AIMS: Surgical resection of the cancerous tissue represents one of the few curative treatment options for neoplastic liver disease. Such partial hepatectomy (PHx) induces hepatocyte hyperplasia, which restores liver function. PHx is associated with bacterial translocation, leading to an immediate immune response involving neutrophils and macrophages, which are indispensable for the priming phase of liver regeneration. Additionally, PHx induces longer-lasting intrahepatic apoptosis. Herein, we investigated the effect of apoptotic extracellular vesicles (aEVs) on neutrophil function and their role in this later phase of liver regeneration. METHODS: A total of 124 patients undergoing PHx were included in this study. Blood levels of the apoptosis marker caspase-cleaved cytokeratin-18 (M30) and circulating aEVs were analyzed preoperatively and on the first and fifth postoperative days. Additionally, the in vitro effects of aEVs on the secretome, phenotype and functions of neutrophils were investigated. RESULTS: Circulating aEVs increased at the first postoperative day and were associated with higher concentrations of M30, which was only observed in patients with complete liver recovery. Efferocytosis of aEVs by neutrophils induced an activated phenotype (CD11bhighCD16highCD66bhighCD62Llow); however, classical inflammatory responses such as NETosis, respiratory burst, degranulation, or secretion of pro-inflammatory cytokines were not observed. Instead, efferocytosing neutrophils released various growth factors including fibroblast growth factor-2 and hepatocyte growth factor (HGF). Accordingly, we observed an increase of HGF-positive neutrophils after PHx and a correlation of plasma HGF with M30 levels. CONCLUSIONS: These data suggest that the clearance of PHx-induced aEVs leads to a population of non-inflammatory but regenerative neutrophils, which may support human liver regeneration. LAY SUMMARY: In this study, we show that the surgical removal of a diseased part of the liver triggers a specific type of programmed cell death in the residual liver tissue. This results in the release of vesicles from dying cells into the blood, where they are cleared by circulating immune cells. These respond by secreting hepatocyte growth factors that could potentially support the regeneration of the liver remnant.
Assuntos
Vesículas Extracelulares , Hiperplasia Nodular Focal do Fígado , Humanos , Hepatectomia , Neutrófilos , Transporte Biológico , Regeneração HepáticaRESUMO
Fibroblasts are the most abundant stromal constituents of the tumour microenvironment in primary as well as metastatic colorectal cancer (CRC). Their supportive effect on tumour cells is well established. There is growing evidence that stromal fibroblasts also modulate the immune microenvironment in tumours. Here, we demonstrate a difference in fibroblast-mediated immune modulation between primary CRC and peritoneal metastasis. Cancer-associated fibroblasts (CAFs) were isolated from primary cancer and from peritoneal metastases (MAFs) from a total of 17 patients. The ectoenzyme CD38 was consistently expressed on the surface of all MAFs, while it was absent from CAFs. Furthermore, MAFs secreted higher levels of IGFBP2, CXCL2, CXCL6, CXCL12, PDGF-AA, FGFb, and IL-6. This was associated with a decreased activation of macrophages and a suppression of CD25 expression and proliferation of co-cultivated T-cells. Downregulation of IGFBP2 abolished these immunosuppressive effects of MAFs. Taken together, these results show that MAFs contribute to an immunosuppressive tumour microenvironment in CRC metastases by modulating the phenotype of immune cells through an IGFBP2-dependent mechanism.
Assuntos
Fibroblastos Associados a Câncer , Neoplasias Colorretais , Fibroblastos Associados a Câncer/metabolismo , Movimento Celular/genética , Proliferação de Células/genética , Neoplasias Colorretais/patologia , Fibroblastos/metabolismo , Humanos , Microambiente Tumoral/genéticaRESUMO
Numerous disease states are associated with cell death. For many decades, apoptosis and accidental necrosis have been assumed to be the two ways how a cell can die. The recent discovery of additional cell death processes such as necroptosis, ferroptosis or pyroptosis revealed a complex interplay between cell death mechanisms and diseases. Depending on the particular cell death pathway, cells secrete distinct molecular patterns, which differ between cell death types. This review focusses on released molecules, detectable in the blood flow, and their potential role as circulating biomarkers of cell death. We elucidate the molecular background of different biomarkers and give an overview on their correlation with disease stage, therapy response and prognosis in patients.
Assuntos
Biomarcadores/sangue , Morte Celular , Animais , Apoptose , Humanos , Necrose/sangueAssuntos
Anticorpos/metabolismo , Misturas Complexas/metabolismo , Inflamação/imunologia , Monócitos/imunologia , Sepse/tratamento farmacológico , Adulto , Idoso , Antígeno CD11b/metabolismo , Células Cultivadas , Feminino , Voluntários Saudáveis , Humanos , Imunoglobulina A/sangue , Imunoglobulina G/sangue , Imunoglobulina M/sangue , Imunomodulação , Lipopolissacarídeos/imunologia , Masculino , Pessoa de Meia-Idade , Receptores de IgG/metabolismo , Ácidos Teicoicos/imunologia , Receptor 2 Toll-Like/metabolismo , Receptor 4 Toll-Like/metabolismo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
When apoptotic cells are not cleared in an efficient and timely manner, they progress to secondary necrosis and lose their membrane integrity. This results in a leakage of immunostimulatory, danger associated molecular patterns (DAMPs), similar to accidental (or primary) necrosis. However, primary necrosis is a sudden event with an inadvertent release of almost unmodified DAMPs. Secondary necrotic cells, in contrast, have gone through various modifications during the process of apoptosis. Recent research revealed that the molecules released from the cytoplasm or exposed on the cell surface differ between primary necrosis, secondary necrosis, and regulated necrosis such as necroptosis. This review gives an overview of these differences and focusses their effects on the immune response. The implications to human physiology and diseases are manifold and will be discussed in the context of cancer, neurodegenerative disorders and autoimmunity.
Assuntos
Alarminas/imunologia , Apoptose/imunologia , Morte Celular/imunologia , Necrose/imunologia , Alarminas/metabolismo , Animais , Apoptose/fisiologia , Morte Celular/fisiologia , Humanos , Imunoterapia/tendências , Inflamação/imunologia , Necrose/patologia , Necrose/fisiopatologia , Fagocitose/imunologia , Transdução de SinaisRESUMO
BACKGROUND: Interleukin-15 has become a promising molecule in the context of eliciting an effective, antitumor immune response because it is able to stimulate cells of the innate and adaptive immune system. METHODS: We generated an interleukin-15-expressing oncolytic influenza A virus for the treatment of an established murine tumor model. RESULTS: Our oncolytic influenza A virus produced large amounts of interleukin-15 and induced proliferation and activation of human T cells in vitro. Intraperitoneal administration increased the amount of mouse natural killer cells and effector memory T cells, as well as T cell reactivity in vivo. Moreover, intratumoral injection induced a profound decrease in growth of established tumors in mice and increased the amount of tumor-infiltrating T cells and natural killer cells. CONCLUSION: We established a stable, IL-15-producing oncolytic influenza A virus with promising immunostimulatory and antitumor attributes.
Assuntos
Vírus da Influenza A , Interleucina-15/fisiologia , Melanoma Experimental/patologia , Terapia Viral Oncolítica , Vírus Oncolíticos , Neoplasias Cutâneas/patologia , Animais , Técnicas de Cultura de Células , Proliferação de Células/efeitos dos fármacos , Feminino , Humanos , Células Matadoras Naturais/efeitos dos fármacos , Melanoma Experimental/terapia , Camundongos , Camundongos Endogâmicos C57BL , Neoplasias Cutâneas/terapia , Linfócitos T/efeitos dos fármacosRESUMO
Toll-like receptors (TLR) are crucial sensors of microbial agents such as bacterial or viral compounds. These receptors constitute key players in the induction of inflammation, e.g. in septic or chronic inflammatory diseases. Colony-stimulating factors (CSFs) such as granulocyte-macrophage-CSF (GM-CSF) or granulocyte-CSF (G-CSF) have been extensively investigated in their capacity to promote myelopoiesis in febrile neutropenia or to overcome immunosuppression in patients suffering from sepsis-associated neutropenia or from monocytic immunoincompetence. We report here that GM-CSF, downregulates TLR1, TLR2 and TLR4 in a time- and dose-dependent fashion in human monocytes. Diminished pathogen recognition receptor expression was accompanied by reduced downstream p38 and extracellular-signal-regulated kinase (ERK) signaling upon lipoteichoic acid (LTA) and lipopolysaccharide (LPS) binding-and accordingly led to impaired proinflammatory cytokine production. Knockdown experiments of the transcription factors PU.1 and VentX showed that GM-CSF driven effects on TLR regulation is entirely PU.1 but not VentX dependent. We further analysed monocyte TLR and CD14 expression upon exposure to the IMID® immunomodulatory drug Pomalidomide (CC-4047), a Thalidomide analogue known to downregulate PU.1. Indeed, Pomalidomide in part reversed the GM-CSF-mediated effects. Our data indicate a critical role of PU.1 in the regulation of TLR1, 2, 4 and of CD14, thus targeting PU.1 ultimately results in TLR modulation. The PU.1 mediated immunomodulatory properties of GM-CSF should be taken into consideration upon usage of GM-CSF in inflammatory or infection-related conditions.
Assuntos
Fator Estimulador de Colônias de Granulócitos e Macrófagos/fisiologia , Monócitos/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Receptores Toll-Like/biossíntese , Transativadores/metabolismo , Citocinas/biossíntese , Regulação para Baixo , Citometria de Fluxo , Humanos , Receptores de Lipopolissacarídeos/biossíntese , Monócitos/fisiologia , Proteínas Proto-Oncogênicas/fisiologia , Interferência de RNA , Reação em Cadeia da Polimerase em Tempo Real , Receptor 1 Toll-Like/biossíntese , Receptor 2 Toll-Like/biossíntese , Receptor 4 Toll-Like/biossíntese , Transativadores/fisiologiaRESUMO
The response to neoadjuvant chemotherapy in breast cancer patients is usually assessed by pCR and RCB score. However, the prognostic value of these parameters is still in discussion. We showed recently that an epirubicin/docetaxel therapy is associated with an increase in the cell death marker high-mobility group box 1 protein (HMGB1) in the circulation. Here, we investigate whether this increase correlates with the long-term outcome. Thirty-six early breast cancer patients under neoadjuvant epirubicin/docetaxel combination chemotherapy were included in this study. To determine the immediate effect of this treatment on HMGB1, we collected blood samples before and 24-96 h after the initial dose. This time course was then compared to the 5-year follow-up of the patients. HMGB1 levels varied before chemotherapy between 4.1 and 11.3 ng/mL and reacted differently in response to therapy. Some patients showed an increase while others did not show any changes. Therefore, we subdivided the patient collective into two groups: patients with an at least 1.1 ng/mL increase in HMGB1 and patients with smaller changes. The disease-free survival was longer in the HMGB1 increase group (56.2 months vs. 46.6 months), but this difference did not reach significance. The overall survival (OS) was significantly better in patients with an increase in HMGB1 (log rank P = 0.021). These data suggest that an immediate increase in HMGB1 levels correlates with improved outcome in early breast cancer patients receiving neoadjuvant chemotherapy, and may be a valuable complementary biomarker for early estimation of prognosis.
Assuntos
Biomarcadores Tumorais , Neoplasias da Mama/sangue , Neoplasias da Mama/mortalidade , Proteína HMGB1/sangue , Adulto , Idoso , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Feminino , Humanos , Estimativa de Kaplan-Meier , Lactato Desidrogenases/metabolismo , Pessoa de Meia-Idade , Terapia Neoadjuvante , Estadiamento de Neoplasias , Prognóstico , Resultado do TratamentoRESUMO
New anti-viral agents and strategies are urgently needed to fight rapidly mutating viruses, as vaccine programs cannot react fast enough to prevent pandemics. Recently, we have shown that interleukin-24 (IL-24) sensitizes tumor cells to toll-like receptor 3 (TLR3) mediated apoptosis. As influenza A virus stimulates the TLR3 receptor, we hypothesized that IL-24 might also exert an anti-viral effect. This study demonstrates that IL-24 reduces the titer of different influenza A virus subtypes independently of type I interferon in an apoptosis dependent manner. The anti-viral effect of IL-24 correlated with caspase-3 activation and could be blocked by a pan-caspase inhibitor and by small interfering RNA (siRNA) directed towards TLR3. Surprisingly, caspase-3 activation in influenza A virus/IL-24-stimulated cells correlated with the down-regulation of the B-cell lymphoma 2 (Bcl-2) family member myeloid cell leukemia 1 (Mcl-1). Correspondingly, knockdown of Mcl-1 by siRNA enhanced caspase activation in influenza A virus infected cells and was furthermore linked to a reduction of viral titers. We conclude that IL-24 exerts an anti-viral role selectively purging virally infected cells by leading to a down-regulation of Mcl-1. Our findings might therefore represent the first step towards a new rational concept in the development of anti-viral strategies based on the induction of apoptosis.
Assuntos
Apoptose , Vírus da Influenza A/imunologia , Vírus da Influenza A/fisiologia , Interleucinas/metabolismo , Receptor 3 Toll-Like/metabolismo , Replicação Viral , Animais , Linhagem Celular , Humanos , Camundongos Endogâmicos BALB CRESUMO
BACKGROUND: Monoclonal antibodies (mAb), such as trastuzumab are a valuable addition to breast cancer therapy. Data obtained from neoadjuvant settings revealed that antibody-dependent cell-mediated cytotoxicity (ADCC) is a major mechanism of action for the mAb trastuzumab. Conflicting results still call into question whether disease progression, prolonged treatment or concomitant chemotherapy influences ADCC and related immunological phenomena. METHODS: We analyzed the activity of ADCC and antibody-dependent cell-mediated phagocytosis (ADCP) of peripheral blood mononuclear cells (PBMCs) from human epidermal growth factor receptor 2 (HER2/neu) positive breast cancer patients receiving trastuzumab therapy either in an adjuvant (n = 13) or metastatic (n = 15) setting as well as from trastuzumab treatment-naive (t-naive) HER2/neu negative patients (n = 15). PBMCs from healthy volunteers (n = 24) were used as controls. ADCC and ADCP activity was correlated with the expression of antibody binding Fc-gamma receptor (FcγR)I (CD64), FcγRII (CD32) and FcγRIII (CD16) on CD14+ (monocytes) and CD56+ (NK) cells, as well as the expression of CD107a+ (LAMP-1) on CD56+ cells and the total amount of CD4+CD25+FOXP3+ (Treg) cells. In metastatic patients, markers were correlated with progression-free survival (PFS). RESULTS: ADCC activity was significantly down regulated in metastatic, adjuvant and t-naive patient cohorts as compared to healthy controls. Reduced ADCC activity was inversely correlated with the expression of CD107a on CD56+ cells in adjuvant patients. ADCC and ADCP activity of the patient cohorts were similar, regardless of treatment duration or additional chemotherapy. PFS in metastatic patients inversely correlated with the number of peripheral Treg cells. CONCLUSION: The reduction of ADCC in patients as compared to healthy controls calls for adjuvant strategies, such as immune-enhancing agents, to improve the activity of trastuzumab. However, efficacy of trastuzumab-specific ADCC and ADCP appears not to be affected by treatment duration, disease progression or concomitant chemotherapy. This finding supports the application of trastuzumab at any stage of the disease.
Assuntos
Anticorpos Monoclonais Humanizados/farmacologia , Citotoxicidade Celular Dependente de Anticorpos/efeitos dos fármacos , Antineoplásicos/farmacologia , Neoplasias da Mama/imunologia , Genes erbB-2 , Fagocitose/efeitos dos fármacos , Adulto , Idoso , Antígenos CD/imunologia , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Estudos de Casos e Controles , Linhagem Celular Tumoral , Feminino , Citometria de Fluxo , Humanos , Imunofenotipagem , Pessoa de Meia-Idade , Metástase Neoplásica , Trastuzumab , Adulto JovemRESUMO
Previously we have developed a prototype for conditionally replicating oncolytic influenza A virus which is based on deletions in the non-structural (NS1) protein. Multi-cycle replication of influenza A virus in malignant tissue is critically dependent on a protease which cleaves the viral entry protein. Here we demonstrate that the malignant colon cancer cell lines Caco-2, HT-29 and SW-620 can endogenously provide a virus-activating protease, which allows lytic multi-cycle replication of NS1 deletion viruses in those cancer cells in vitro. The oncolytic potency of an influenza NS1 deletion virus (NS1-80) was further tested in SCID mice bearing HT-29 derived tumors. The intra-tumoral injection of live, but not of heat inactivated NS1-80 virus significantly inhibited progression of established tumors. We conclude that a selected set of human cancer expressing virus activating- proteases will be a preferred target for oncolytic tumor therapy using influenza A virus mutants.
Assuntos
Neoplasias do Colo/enzimologia , Vírus da Influenza A/metabolismo , Vírus Oncolíticos/metabolismo , Peptídeo Hidrolases/metabolismo , Proteínas não Estruturais Virais/metabolismo , Animais , Células CACO-2 , Linhagem Celular Tumoral , Chlorocebus aethiops , Neoplasias do Colo/patologia , Neoplasias do Colo/virologia , Feminino , Células HCT116 , Células HT29 , Interações Hospedeiro-Patógeno , Humanos , Vírus da Influenza A/genética , Vírus da Influenza A/fisiologia , Camundongos , Camundongos SCID , Mutação , Neoplasias Experimentais/enzimologia , Neoplasias Experimentais/patologia , Neoplasias Experimentais/virologia , Vírus Oncolíticos/genética , Vírus Oncolíticos/fisiologia , Infecções por Orthomyxoviridae/enzimologia , Infecções por Orthomyxoviridae/patologia , Infecções por Orthomyxoviridae/virologia , Peptídeo Hidrolases/genética , Transplante Heterólogo , Células Vero , Proteínas não Estruturais Virais/genéticaRESUMO
Dendritic cells (DCs) use pattern recognition receptors to sense invading viruses and triggering of these receptors induces a maturation program. Human rhinoviruses (HRVs) belong to the family of Picornaviridae, which have a single-stranded, coding RNA genome. Because HRV does not replicate in DCs, we used genomic RNA from HRV in this study to analyze the impact of natural occurring viral ssRNA on DC function. We found that transfection of human monocyte-derived DCs with viral ssRNA induced type I IFN production but failed to activate the NF-kappaB pathway in DCs. In line with this observation, the up-regulation of typical maturation markers such as CD83 or the production of the proinflammatory cytokines IL-12p40, IL-6, and TNF-alpha was not detectable. Most importantly, the T cell stimulatory capacity of viral ssRNA-treated DCs was not enhanced and remained at the level of immature DCs. Taken together, our results demonstrate that viral ssRNA efficiently activates the innate defense arm of DCs, whereas it is insufficient to activate the stimulatory capacity of DCs for the adaptive defense responses.
Assuntos
Diferenciação Celular/imunologia , Células Dendríticas/imunologia , Regulação para Baixo/imunologia , Genoma Viral/imunologia , Interferon Tipo I/biossíntese , Monócitos/imunologia , RNA Viral/imunologia , Rhinovirus/imunologia , Biomarcadores/metabolismo , Diferenciação Celular/genética , Linhagem Celular , Células Dendríticas/patologia , Células Dendríticas/virologia , Regulação para Baixo/genética , Inibidores do Crescimento/genética , Inibidores do Crescimento/fisiologia , Células HeLa , Humanos , Imunidade Inata/genética , Imunofenotipagem , Mediadores da Inflamação/metabolismo , Interferon Tipo I/genética , Ativação Linfocitária/genética , Monócitos/patologia , Monócitos/virologia , RNA Viral/antagonistas & inibidores , RNA Viral/metabolismo , Rhinovirus/genética , Replicação Viral/genética , Replicação Viral/imunologiaRESUMO
BACKGROUND: We developed a novel intranasal influenza vaccine approach that is based on the construction of replication-deficient vaccine viruses that lack the entire NS1 gene (DeltaNS1 virus). We previously showed that these viruses undergo abortive replication in the respiratory tract of animals. The local release of type I interferons and other cytokines and chemokines in the upper respiratory tract may have a "self-adjuvant effect", in turn increasing vaccine immunogenicity. As a result, DeltaNS1 viruses elicit strong B- and T- cell mediated immune responses. METHODOLOGY/PRINCIPAL FINDINGS: We applied this technology to the development of a pandemic H5N1 vaccine candidate. The vaccine virus was constructed by reverse genetics in Vero cells, as a 5:3 reassortant, encoding four proteins HA, NA, M1, and M2 of the A/Vietnam/1203/04 virus while the remaining genes were derived from IVR-116. The HA cleavage site was modified in a trypsin dependent manner, serving as the second attenuation factor in addition to the deleted NS1 gene. The vaccine candidate was able to grow in the Vero cells that were cultivated in a serum free medium to titers exceeding 8 log(10) TCID(50)/ml. The vaccine virus was replication deficient in interferon competent cells and did not lead to viral shedding in the vaccinated animals. The studies performed in three animal models confirmed the safety and immunogenicity of the vaccine. Intranasal immunization protected ferrets and mice from being infected with influenza H5 viruses of different clades. In a primate model (Macaca mulatta), one dose of vaccine delivered intranasally was sufficient for the induction of antibodies against homologous A/Vietnam/1203/04 and heterologous A/Indonesia/5/05 H5N1 strains. CONCLUSION/SIGNIFICANCE: Our findings show that intranasal immunization with the replication deficient H5N1 DeltaNS1 vaccine candidate is sufficient to induce a protective immune response against H5N1 viruses. This approach might be attractive as an alternative to conventional influenza vaccines. Clinical evaluation of DeltaNS1 pandemic and seasonal influenza vaccine candidates are currently in progress.
Assuntos
Virus da Influenza A Subtipo H5N1/genética , Vacinas contra Influenza/uso terapêutico , Administração Intranasal , Animais , Brônquios/citologia , Linhagem Celular , Galinhas , Chlorocebus aethiops , Cães , Avaliação Pré-Clínica de Medicamentos , Células Epiteliais/citologia , Furões , Glicoproteínas de Hemaglutininação de Vírus da Influenza/química , Humanos , Vacinas contra Influenza/metabolismo , Macrófagos/metabolismo , Macrófagos/virologia , Camundongos , Células Vero , Replicação ViralRESUMO
Cryopreservation of immature or mature dendritic cells (DC) has been proposed as a suitable method to gain large numbers of DC for immunotherapeutic trials against cancer. However, clinical studies using cryopreserved DC have demonstrated only limited success so far. The aim of this study was to investigate whether cryopreservation of monocytes elicits more potent DC and whether these DC are comparable to freshly generated DC preparations. Monocytes, either separated immunomagnetically or by means of leukapheresis and elutriation, were differentiated into DC and cryopreserved at various developmental stages. DC preparations were analyzed regarding recovery, viability, phenotype, and functional properties. In contrast to DC frozen at their immature or semi-mature state, generation of DC from cryopreserved monocytes elicited viability values comparable with freshly generated DC. Furthermore, using frozen monocytes for DC differentiation revealed improved expression of DC surface markers and interleukin-12p70 secretion as compared with DC generated from frozen immature or frozen semi-mature DC. Impaired phenotypical appearance of the latter DC variants was further substantiated by functional analysis. T-cells cocultured with these DC showed decreased expression of interferon-gamma and granzyme B, and lowered proliferation when compared with T-cells cocultured with DC generated from frozen monocytes or DC generated from freshly isolated monocytes. Induction of regulatory T-cell populations was negligible among all investigated DC preparations. These findings may further improve DC-based immunotherapeutical protocols. Cryopreservation of unchallenged monocytes enables targeted therapy by loading DC with varying antigenic compositions in case of tumor escape during treatment.
Assuntos
Células Dendríticas/imunologia , Imunoterapia Adotiva , Monócitos/imunologia , Neoplasias/terapia , Linfócitos T Reguladores/imunologia , Antígenos CD/imunologia , Antígenos CD/metabolismo , Antígenos de Diferenciação de Linfócitos T/imunologia , Antígenos de Diferenciação de Linfócitos T/metabolismo , Vacinas Anticâncer/imunologia , Linhagem Celular Tumoral , Criopreservação , Células Dendríticas/transplante , Humanos , Separação Imunomagnética , Interferon gama/imunologia , Interferon gama/metabolismo , Interleucina-10/imunologia , Interleucina-10/metabolismo , Interleucina-12/imunologia , Interleucina-12/metabolismo , Interleucina-4/imunologia , Interleucina-4/metabolismo , Lectinas Tipo C , Monócitos/transplante , Linfócitos T Reguladores/metabolismoRESUMO
Immunotherapy with autologous dendritic cells (DCs) loaded with tumor lysate(s) from allogeneic tumor cell lines is a novel strategy to induce immune responses in cancer patients. We report on a pilot trial of autologous DCs pulsed with tumor cell lysate derived from allogeneic medullary thyroid carcinoma (MTC) cell lines in patients with metastatic MTC. The purpose of this study was to assess the safety, resulting immune responses and clinical activity of the DCs. DCs were injected into a groin lymph node at 3-week intervals. Monitoring included serial calcitonin tumor marker measurements, radiological imaging and immunological in vitro tests (T-cell interferon-gamma detection assay, T-cell cytotoxicity assay). Ten patients (median age 47 years, range 29-77) were enrolled. DC vaccinations were well-tolerated and safe. After a median follow-up of 11 months, (range 7-26), 3 (30%) of 10 patients had stable disease, while 7 (70%) of the patients progressed during treatment. In 2 patients with stable disease, calcitonin decreased below treatment levels, paralleled by a T-cell-mediated immune response. Notably, treatment with DCs pulsed with a combination of different tumor cell lysates was followed by a calcitonin decrease in 4 patients who had previously experienced a calcitonin increase during monotherapy with DCs pulsed with a single lysate. Allogeneic tumor cell lysate-based DC immunotherapy is well-tolerated and safe. Combined treatment with different tumor cell lysate-pulsed DCs increases the likelihood of a calcitonin tumor marker response and should therefore be preferred over monotherapy with DCs pulsed with a single lysate.
Assuntos
Carcinoma Medular/terapia , Células Dendríticas/transplante , Imunoterapia Adotiva , Neoplasias da Glândula Tireoide/terapia , Adulto , Idoso , Biomarcadores Tumorais/sangue , Calcitonina/sangue , Vacinas Anticâncer , Carcinoma Medular/diagnóstico , Carcinoma Medular/imunologia , Carcinoma Medular/secundário , Linhagem Celular Tumoral , Células Dendríticas/imunologia , Progressão da Doença , Feminino , Humanos , Imunoterapia Adotiva/efeitos adversos , Interferon gama/metabolismo , Masculino , Pessoa de Meia-Idade , Projetos Piloto , Linfócitos T Citotóxicos/imunologia , Neoplasias da Glândula Tireoide/diagnóstico , Neoplasias da Glândula Tireoide/imunologia , Neoplasias da Glândula Tireoide/secundário , Fatores de Tempo , Transplante Autólogo , Resultado do TratamentoRESUMO
Prolonged or repeated stimulation of Toll-like receptor (TLR) 4 leads to hyporesponsiveness of monocyte-derived macrophages, which seems to be a hallmark of immunosuppression related to sepsis and cancer. Two negative regulators of TLR-4 signaling are IL-1 receptor-associated kinase M and B-cell leukemia 3. Here, we demonstrate that the expression of both proteins is inhibited when the TLR-7/TLR-8 agonist CL097 is added to monocyte cultures despite costimulation with the TLR-4 agonist LPS or hyaluronic acid. Reduction of IL-1 receptor-associated kinase M and B-cell leukemia 3 was paralleled by a significant increased cytokine induction of TNF-alpha, IL-10, and IL-12 observed after intracellular and extracellular TLR stimulation. In ex vivo stimulated whole blood of patients who have prolonged sepsis or metastatic cancer, TLR-7/TLR-8 agonists retained their ability of increased stimulation of TNF-alpha. These data might add to the understanding of sepsis and cancer-associated immune suppression in men.
Assuntos
Ativação Enzimática/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Imidazóis/farmacologia , Quinases Associadas a Receptores de Interleucina-1/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Quinolinas/farmacologia , Receptores Toll-Like/agonistas , Fatores de Transcrição/metabolismo , Adulto , Idoso , Proteína 3 do Linfoma de Células B , Células Cultivadas , Humanos , Ácido Hialurônico/farmacologia , Imidazóis/química , Interleucina-10/metabolismo , Interleucina-12/metabolismo , Ligantes , Lipopolissacarídeos/farmacologia , Pessoa de Meia-Idade , Quinolinas/química , Receptor 4 Toll-Like/metabolismo , Receptor 7 Toll-Like/agonistas , Receptor 8 Toll-Like/agonistas , Receptores Toll-Like/metabolismo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
BACKGROUND: A replication defective influenza A vaccine virus (delNS1 virus) was developed. Its attenuation is due to potent stimulation of the innate immune system by the virus. Since the innate immune system can also target cancer cells, we reasoned that delNS1 virus induced immune-stimulation should also lead to the induction of innate cytotoxic effects towards cancer cells. METHODOLOGY/PRINCIPAL FINDINGS: Peripheral blood mononuclear cells (PBMCs), isolated CD56+, CD3+, CD14+ and CD19+ subsets and different combinations of the above subsets were stimulated by delNS1, wild type (wt) virus or heat inactivated virus and co-cultured with tumor cell lines in the presence or absence of antibodies against the interferon system. Stimulation of PBMCs by the delNS1 virus effectively induced cytotoxicity against different cancer cell lines. Surprisingly, virus induced cytotoxicity was exerted by all major subtypes of PBMCs including CD56+, CD3+, CD14+ and CD19+ cells. Virus induced cytotoxicity in CD3+, CD14+ and CD19+ cells was dependent on virus replication, whereas virus induced cytotoxicity in CD56+ cells was only dependent on the binding of the virus. Virus induced cytotoxicity of isolated cell cultures of CD14+, CD19+ or CD56+ cells could be partially blocked by antibodies against type I and type II (IFN) interferon. In contrast, virus induced cytotoxicity in the complete PBMC preparation could not be inhibited by blocking type I or type II IFN, indicating a redundant system of activation in whole blood. CONCLUSIONS/SIGNIFICANCE: Our data suggest that apart from their well known specialized functions all main subsets of peripheral blood cells also initially exert a cytotoxic effect upon virus stimulation. This closely links the innate immune system to the adaptive immune response and renders delNS1 virus a potential therapeutic tool for viro-immunotherapy of cancer.
Assuntos
Citotoxinas/imunologia , Imunidade Inata/imunologia , Vírus da Influenza A/imunologia , Vírus da Influenza A/patogenicidade , Leucócitos Mononucleares/imunologia , Animais , Antígenos CD/imunologia , Biomarcadores/metabolismo , Linhagem Celular Tumoral , Técnicas de Cocultura , Citocinas/imunologia , Feminino , Humanos , Vírus da Influenza A/genética , Vírus da Influenza A/fisiologia , Camundongos , Camundongos Endogâmicos BALB C , Oligodesoxirribonucleotídeos/genética , Oligodesoxirribonucleotídeos/imunologia , Baço/citologia , Replicação ViralRESUMO
BACKGROUND: Recent preclinical and clinical evidence suggests the use of allogeneic tumor as a source of antigen for DC-based immunotherapy against cancer. We hypothesized that addition of allogeneic tumor lysate to monocyte-derived DC culture could serve a dual purpose: (1) antigen source and (2) protein supplementation of DC culture media. Protein supplementation whether of known origin (human serum/plasma, fetal bovine serum, human serum albumin) or undeclared origin ("serum-free" media) is a source of variability and bias. We addressed the question whether protein supplementation can be omitted in the presence of allogeneic tumor lysate. MATERIALS AND METHODS: Human DC cultured in the presence of lysate from medullary thyroid carcinoma (MTC) cell line SHER-I (TuLy-DC) and DC pulsed with the same lysate but cultured in the presence of FBS (FBS-DC) were assessed for morphology, phenotype, maturation and functional properties. RESULTS: In comparison of FBS-DC/TuLy-DC no significant differences in morphology, phenotype and maturation could be detected. Both culture conditions produced CD1a(high), CD14(low) DC with high expression of costimulatory molecules and CD83 upon stimulation. TuLy-DC gave significantly better yields and produced more IL12p70. DC showed high (allo)stimulatory capacity toward T-cells. TuLy-DC induced more intracellular IFNgamma in CD8+T-cells of vaccinated MTC patients. Both types of DC induced killing of SHER-I after short in vitro restimulation. Tumor lysate from SHER-I can substitute for further protein supplementation in DC culture. Allogeneic tumor lysates should be taken into consideration as both source of antigen and protein supplementation in monocyte-derived DC culture.