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In the original publication of the article the following abstract and keywords were inadvertently omitted.
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Background: In this study the toxicity and efficacy of an irradiated autologous tumor cell vaccine (ATV) co-injected with a class-B CpG oligodeoxynucleotide (CpG-B) and GM-CSF, followed by systemic CpG-B and IFN-α administration, were examined in patients with metastatic renal cell carcinoma (mRCC). Methods: A single-arm Phase II trial was conducted, in which patients with mRCC were intradermally injected with a minimum of three whole-cell vaccines containing 0.71.3 × 107 irradiated autologous tumor cells (ATC), admixed with 1 mg CpG-B and 100 µg GM-CSF, followed by bi-weekly s.c. injections with 8 mg CpG-B and s.c. injections with 6 MU IFN-α three times per week. Results: Fifteen patients were treated according to the protocol. Treatment was well tolerated. Objective clinical responses occurred in three patients, including one long-term complete response. Disease stabilization occurred in another three patients. Positive delayed type hypersensitivity (DTH) responses to ATC were absent before treatment but present in 13 out of 15 patients during treatment. Immune monitoring revealed activation of plasmacytoid dendritic cells, non-classical monocytes and up-regulation of both PD-1 and CTLA4 on effector T cells upon treatment. Moreover, a pre-existing ex vivo IFN-γ response to ATC was associated with clinical response. Conclusions: ATV combined with systemic CpG-B and IFN-α is tolerable, safe, immunogenic and able to elicit anti-tumor responses in patients with mRCC. Immune activation and treatment-induced up-regulation of PD-1 and CTLA4 on circulating T cells further suggest an added benefit of combining this approach with immune checkpoint blockade [added]
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Vacinas Anticâncer/uso terapêutico , Carcinoma de Células Renais/terapia , Neoplasias Renais/terapia , Oligodesoxirribonucleotídeos/farmacologia , Idoso , Carcinoma de Células Renais/imunologia , Diferenciação Celular , Feminino , Humanos , Imunoterapia/métodos , Interferon-alfa/farmacologia , Neoplasias Renais/imunologia , Subpopulações de Linfócitos , Masculino , Pessoa de Meia-Idade , Metástase Neoplásica , Nefrectomia , Resultado do TratamentoRESUMO
In elderly acute myeloid leukemia (AML) patients post-remission treatment options are associated with high comorbidity rates and poor survival. Dendritic cell (DC)-based immunotherapy is a promising alternative treatment strategy. A novel allogeneic DC vaccine, DCP-001, was developed from an AML-derived cell line that uniquely combines the positive features of allogeneic DC vaccines and expression of multi-leukemia-associated antigens. Here, we present data from a phase I study conducted with DCP-001 in 12 advanced-stage elderly AML patients. Patients enrolled were in complete remission (CR1/CR2) (n = 5) or had smoldering disease (n = 7). All patients were at high risk of relapse and ineligible for post-remission intensification therapies. A standard 3 + 3 dose escalation design with extension to six patients in the highest dose was performed. Patients received four biweekly intradermal DCP-001 injections at different dose levels (10, 25, and 50 million cells DCP-001) and were monitored for clinical and immunological responses. Primary objectives of the study (feasibility and safety) were achieved with 10/12 patients completing the vaccination program. Treatment was well tolerated. A clear-cut distinction between patients with and without detectable circulating leukemic blasts during the vaccination period was noted. Patients with no circulating blasts showed an unusually prolonged survival [median overall survival 36 months (range 7-63) from the start of vaccination] whereas patients with circulating blasts, died within 6 months. Long-term survival was correlated with maintained T cell levels and induction of multi-functional immune responses. It is concluded that DCP-001 in elderly AML patients is safe, feasible and generates both cellular and humoral immune responses.
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Vacinas Anticâncer/imunologia , Células Dendríticas/imunologia , Imunoterapia , Leucemia Mieloide Aguda/prevenção & controle , Linfócitos T/imunologia , Idoso , Vacinas Anticâncer/administração & dosagem , Feminino , Humanos , Leucemia Mieloide Aguda/imunologia , Masculino , Pessoa de Meia-Idade , Indução de Remissão , Resultado do TratamentoRESUMO
Impaired immune effector functions in the melanoma sentinel lymph node (SLN) may allow for early metastatic events. In an effort to determine the optimal way to strengthen immune defenses, 28 clinical stage I-II melanoma patients were randomized in a 3-arm Phase II study to receive, prior to excision and sampling of the SLN, i.d. injections of saline or low-dose CpG-B (CpG), alone or combined with GM-CSF (GM), around the melanoma excision site. We previously described the combined administration of these DC-targeting agents to result in activation and recruitment of potentially cross-presenting BDCA3(+) DCs to the SLN. In this report we describe the effects on effector and regulatory T and NK cell subsets. Local low-dose CpG administration resulted in lower CD4/CD8 ratios, Th1 skewing, increased frequencies of melanoma-specific CD8(+) T cells and possible recruitment of effector NK cells, irrespective of GM co-administration. These immune-potentiating effects were counterbalanced by increased IL-10 production by T cells and significantly higher levels of FoxP3 and CTLA4 in regulatory T cells (Tregs) with correspondingly higher suppressive activity in the SLN. Notably, CpG ± GM-administered patients showed significantly lower numbers of SLN metastases (saline: 4/9, CpG + GM: 1/9, CpG: 0/10, p = 0.04). These findings indicate that i.d. delivery of low-dose CpG ± GM potentially arms the SLN of early-stage melanoma patients against metastatic spread, but that antitumor efficacy may be further boosted by counteracting the collateral activation of Tregs.
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Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Células Dendríticas/efeitos dos fármacos , Fator Estimulador de Colônias de Granulócitos e Macrófagos/uso terapêutico , Melanoma/tratamento farmacológico , Oligodesoxirribonucleotídeos/uso terapêutico , Neoplasias Cutâneas/tratamento farmacológico , Imunidade Adaptativa/efeitos dos fármacos , Imunidade Adaptativa/imunologia , Adulto , Idoso , Relação CD4-CD8 , Células Dendríticas/imunologia , Relação Dose-Resposta a Droga , Feminino , Fator Estimulador de Colônias de Granulócitos e Macrófagos/administração & dosagem , Humanos , Células Matadoras Naturais/efeitos dos fármacos , Células Matadoras Naturais/imunologia , Linfonodos/efeitos dos fármacos , Linfonodos/imunologia , Linfonodos/patologia , Masculino , Melanoma/imunologia , Melanoma/patologia , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Oligodesoxirribonucleotídeos/administração & dosagem , Biópsia de Linfonodo Sentinela , Método Simples-Cego , Neoplasias Cutâneas/imunologia , Neoplasias Cutâneas/patologia , Subpopulações de Linfócitos T/efeitos dos fármacos , Subpopulações de Linfócitos T/imunologia , Linfócitos T Reguladores/efeitos dos fármacos , Linfócitos T Reguladores/imunologia , Resultado do TratamentoRESUMO
Cancer immunotherapy induces a variety of autoinflammatory responses, including those against the thyroid gland, which can be exploited to predict clinical outcomes. Considering the paucity of information about thyroid autoimmunity in patients receiving cancer vaccines, we designed our study to assess the development of thyroglobulin antibodies (TgAbs) in patients treated with GVAX (vaccine made of a tumor cell type transfected with GM-CSF) and/or ipilimumab and correlated seroconversion with survival. Using both in house and commercial ELISA assays, we measured TgAbs in patients with pancreatic (No. = 53), prostate (No. = 35) or colon (No. = 8) cancer, before and after treatment with GVAX only (No. = 34), GVAX plus ipilimumab (No. = 42) or ipilimumab (No. = 20), and correlated their levels with patient's survival, disease status and T-cell surface markers. Antibodies to thyroperoxidase, myeloperoxidase, proteinase 3, insulin and actin were also measured. TgAbs specifically developed after GVAX, independent of the underlying cancer (81% in prostate, 75% colon cancer and 76% pancreatic cancer) and co-administration of ipilimumab (75% in GVAX only and 78% in GVAX plus ipilimumab). This TgAbs seroconversion could be detected mainly by the in house assay, suggesting that the thyroglobulin epitopes recognized by the antibodies induced by GVAX are different from the epitopes seen in the classic form of Hashimoto thyroiditis. Notably, TgAbs seroconversion was associated with significantly prolonged survival (p = 0.01 for pancreas and p = 0.005 for prostate cancer). In conclusion, GVAX immunotherapy induces the appearance of TgAbs that recognize a unique antigenic repertoire and associate with prolonged survival.
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Vacinas Anticâncer/administração & dosagem , Neoplasias do Colo/terapia , Neoplasias Pancreáticas/terapia , Neoplasias da Próstata/terapia , Tireoglobulina/imunologia , Anticorpos Monoclonais/administração & dosagem , Anticorpos Antineoplásicos/sangue , Antineoplásicos/administração & dosagem , Autoanticorpos/sangue , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Vacinas Anticâncer/imunologia , Linhagem Celular Tumoral , Estudos de Coortes , Neoplasias do Colo/sangue , Neoplasias do Colo/imunologia , Neoplasias do Colo/mortalidade , Terapia Combinada , Humanos , Ipilimumab , Masculino , Neoplasias Pancreáticas/sangue , Neoplasias Pancreáticas/imunologia , Neoplasias Pancreáticas/mortalidade , Neoplasias da Próstata/sangue , Neoplasias da Próstata/imunologia , Neoplasias da Próstata/mortalidade , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Análise de Sobrevida , Tireoglobulina/genética , Tireoglobulina/metabolismo , Tireotropina/sangue , VacinaçãoRESUMO
Regulatory T cell (Treg)-mediated immunosuppression is considered a major obstacle for successful cancer immunotherapy. The association between clinical outcome and Tregs is being studied extensively in clinical trials, but unfortunately, no consensus has been reached about (a) the markers and (b) the gating strategy required to define human Tregs in this context, making it difficult to draw final conclusions. Therefore, we have organized an international workshop on the detection and functional testing of Tregs with leading experts in the field, and 40 participants discussing different analyses and the importance of different markers and context in which Tregs were analyzed. This resulted in a rationally composed ranking list of "Treg markers". Subsequently, the proposed Treg markers were tested to get insight into the overlap/differences between the most frequently used Treg definitions and their utility for Treg detection in various human tissues. Here, we conclude that the CD3, CD4, CD25, CD127, and FoxP3 markers are the minimally required markers to define human Treg cells. Staining for Ki67 and CD45RA showed to provide additional information on the activation status of Tregs. The use of markers was validated in a series of PBMC from healthy donors and cancer patients, as well as in tumor-draining lymph nodes and freshly isolated tumors. In conclusion, we propose an essential marker set comprising antibodies to CD3, CD4, CD25, CD127, Foxp3, Ki67, and CD45RA and a corresponding robust gating strategy for the context-dependent analysis of Tregs by flow cytometry.
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Citometria de Fluxo , Neoplasias Ovarianas/imunologia , Linfócitos T Reguladores/imunologia , Animais , Antígenos CD/metabolismo , Biomarcadores/metabolismo , Separação Celular , Células Cultivadas , Consenso , Feminino , Fatores de Transcrição Forkhead/metabolismo , Humanos , Cooperação Internacional , Antígeno Ki-67/metabolismo , Ativação Linfocitária , Monitorização Imunológica , Padrões de Referência , Evasão TumoralRESUMO
Immune checkpoint blockade enhances antitumor responses, but can also lead to severe immune-related adverse events (IRAE). To avoid unnecessary exposure to these potentially hazardous agents, it is important to identify biomarkers that correlate with clinical activity and can be used to select patients that will benefit from immune checkpoint blockade. To understand the consequences of CTLA-4 blockade and identify biomarkers for clinical efficacy and/or survival, an exploratory T cell monitoring study was performed in a phase I/II dose escalation/expansion trial (n = 28) of combined Prostate GVAX/ipilimumab immunotherapy. Phenotypic T cell monitoring in peripheral blood before and after Prostate GVAX/ipilimumab treatment revealed striking differences between patients who benefited from therapy and patients that did not. Treatment-induced rises in absolute lymphocyte counts, CD4(+) T cell differentiation, and CD4(+) and CD8(+) T cell activation were all associated with clinical benefit. Moreover, significantly prolonged overall survival (OS) was observed for patients with high pre-treatment frequencies of CD4(+)CTLA-4(+), CD4(+)PD-1(+), or differentiated (i.e., non-naive) CD8(+) T cells or low pre-treatment frequencies of differentiated CD4(+) or regulatory T cells. Unsupervised clustering of these immune biomarkers revealed cancer-related expression of CTLA-4(+) in CD4(+) T cells to be a dominant predictor for survival after Prostate GVAX/ipilimumab therapy and to thus provide a putative and much-needed biomarker for patient selection prior to therapeutic CTLA4 blockade.
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Anticorpos Monoclonais/uso terapêutico , Antineoplásicos/uso terapêutico , Linfócitos T CD4-Positivos/efeitos dos fármacos , Antígeno CTLA-4/imunologia , Vacinas Anticâncer/uso terapêutico , Neoplasias da Próstata/terapia , Idoso , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/imunologia , Antígeno CTLA-4/análise , Vacinas Anticâncer/imunologia , Linhagem Celular Tumoral , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Humanos , Ipilimumab , Ativação Linfocitária/efeitos dos fármacos , Ativação Linfocitária/imunologia , Masculino , Neoplasias da Próstata/imunologia , Neoplasias da Próstata/mortalidadeRESUMO
BACKGROUND: New treatment modalities are needed for the treatment of cancers of the head and neck region (HNSCC). Survivin is important for the survival and proliferation of tumor cells and may therefore provide a target for immunotherapy. Here we focused on the ex vivo presence and in vitro induction of survivin specific T cells. METHODS: Tetramer staining and ELIspot assays were used to document the presence of survivin specific T cells in patient derived material, and to monitor the presence and persistence of survivin specific T cells after repeated in vitro stimulation with autologous dendritic cells. RESULTS: Ex vivo analysis showed the presence of survivin-specific T cells in the peripheral blood (by tetramer analysis) and in the draining lymph node (by ELIspot analysis) in a HNSCC and a locally advanced breast cancer patient respectively. However, we were unable to maintain isolated survivin specific T cells for prolonged periods of time. For the in vitro generation of survivin specific T cells, monocyte derived DC were electroporated with mRNA encoding full length survivin or a survivin mini-gene together with either IL21 or IL12 mRNA. Western blotting and immunohistochemical staining of dendritic cell cytospin preparations confirmed translation of the full length survivin protein. After repeated stimulation we observed an increase, followed by a decrease, of the number of survivin specific T cells. FACS sorted or limiting dilution cloned survivin specific T cells could not be maintained on feeder mix for prolonged periods of time. Protein expression analysis subsequently showed that activated, but not resting T cells contain survivin protein. CONCLUSIONS: Here we have shown that survivin specific T cells can be detected ex vivo in patient derived material. Furthermore, survivin specific T cells can be induced in vitro using autologous dendritic cells with enforced expression of survivin and cytokines. However, we were unable to maintain enriched or cloned survivin specific T cells for prolonged periods of time. Endogenous expression of survivin in activated T cells and subsequent fratricide killing might explain our in vitro observations. We therefore conclude that survivin, although it is a universal tumor antigen, might not be the ideal target for immunotherapeutic strategies for the treatment of cancer of the head and neck.
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Vacinas Anticâncer/metabolismo , Carcinoma/metabolismo , Células Dendríticas/citologia , Neoplasias de Cabeça e Pescoço/metabolismo , Proteínas Inibidoras de Apoptose/metabolismo , Linfócitos T/metabolismo , Antígenos de Neoplasias/metabolismo , Carcinoma/terapia , Morte Celular , Proliferação de Células , Separação Celular , Células Cultivadas , Regulação Neoplásica da Expressão Gênica , Neoplasias de Cabeça e Pescoço/terapia , Humanos , Imunoterapia/métodos , Fenótipo , RNA Mensageiro/metabolismo , Survivina , Linfócitos T/imunologiaRESUMO
BACKGROUND: Adoptive cell transfer of tumor infiltrating lymphocytes has shown clinical efficacy in the treatment of melanoma and is now also being explored in other tumor types. Generation of sufficient numbers of effector T cells requires extensive ex vivo expansion, often at the cost of T cell differentiation and potency. For the past 20 years, IL-2 has been the key cytokine applied in the expansion of TIL for ACT. However, the use of IL-2 has also led to collateral expansion of regulatory T cells (Tregs) and progressive T cell differentiation, factors known to limit in vivo persistence and activity of transferred TIL. The use of alternative T cell growth factors is therefore warranted. Here, we have compared the effects of IL-2, -15 and -21 cytokines on the expansion and activation of TIL from single-cell suspensions of non-small cell lung cancer, ovarian cancer and melanoma. METHODS: We applied the K562-based artificial APC (aAPC) platform for the direct and rapid expansion of tumor infiltrating lymphocytes isolated from primary cancer specimens. These aAPC were engineered to express the Fc-γ receptor CD32 (for anti-CD3 antibody binding), the co-stimulatory molecule 4-1BBL, and to secrete either IL-2, IL-15 or IL-21 cytokine. RESULTS: Although IL-2 aAPC induced the greatest overall TIL expansion, IL-21 aAPC induced superior expansion of CD8+ T cells with a CD27+ CD28+ "young" phenotype and superior functional cytotoxic effector characteristics, without collateral expansion of Tregs. CONCLUSION: Our data rationalize the clinical application of IL-21-secreting aAPC as a standardized cell-based platform in the expansion of "young" effector TIL for ACT.
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Antígenos CD28/metabolismo , Interleucinas/metabolismo , Linfócitos do Interstício Tumoral/citologia , Linfócitos T Reguladores/citologia , Membro 7 da Superfamília de Receptores de Fatores de Necrose Tumoral/metabolismo , Biópsia , Linfócitos T CD8-Positivos/citologia , Proliferação de Células , Citocinas/metabolismo , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Humanos , Células K562 , Neoplasias/metabolismo , Fenótipo , Receptores de IgG/metabolismoRESUMO
BACKGROUND: The granulocyte-macrophage colony-stimulating factor-transduced allogeneic prostate cancer cells vaccine (GVAX) has antitumour activity against prostate cancer; preclinical studies have shown potent synergy when combined with ipilimumab, an antibody that blocks cytotoxic T-lymphocyte antigen 4. We aimed to assess the safety of combined treatment with GVAX and ipilimumab in patients with metastatic castration-resistant prostate cancer (mCRPC). METHODS: We did an open-labelled, single-centre, dose-escalation study of ipilimumab concurrent with a fixed dose of GVAX, with a subsequent expansion phase, both at the VU University Medical Centre (Amsterdam, Netherlands). Eligible patients had documented mCRPC and had not been previously treated with chemotherapy. All patients received a 5×10(8) cell priming dose of GVAX intradermally on day 1 with subsequent intradermal injections of 3×10(8) cells every 2 weeks for 24 weeks. The vaccinations were combined with intravenous ipilimumab every 4 weeks. We enrolled patients in cohorts of three; each cohort received an escalating dose of ipilimumab at 0·3, 1·0, 3·0, or 5·0 mg/kg. Our primary endpoint was safety. This study is registered with ClinicalTrials.gov, number NCT01510288. FINDINGS: We enrolled 12 patients into our dose-escalation cohort. We did not record any severe immune-related adverse events at the first two dose levels. At the 3·0 mg/kg dose level, one patient had grade 2 and two patients grade 3 hypophysitis; at the 5·0 mg/kg dose level, two patients had grade 3 hypophysitis and one patient developed grade 4 sarcoid alveolitis (a dose-limiting toxic effect). Due to observed clinical activity and toxic events, we decided to expand the 3·0 mg/kg dose level, rather than enrol a further three patients at the 5·0 mg/kg level. 16 patients were enrolled in the expansion cohort, two of whom developed grade 2 hypophysitis, three colitis (one grade 1 and two grade 2), and one grade 3 hepatitis--all immune-related adverse events. The most common adverse events noted in all 28 patients were injection-site reactions (grade 1-2 events seen in all patients), fatigue (grade 1-2 in 20 patients, grade 3 in two), and pyrexia (grade 1-2 in 15 patients, grade 3 in one). 50% or greater declines in prostate-specific antigen from baseline was recorded in seven patients (25%); all had received 3·0 mg/kg or 5·0 mg/kg ipilimumab. INTERPRETATION: GVAX combined with 3·0 mg/kg ipilimumab is tolerable and safe for patients with mCRPC. Further research on the combined treatment of patients with mCRPC with vaccination and ipilimumab is warranted. FUNDING: Cell Genesys Inc, Prostate Cancer Foundation, Dutch Cancer Society (KWF-VU 2006-3697), and Foundation Stichting VUmc Cancer Center Amsterdam.
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Anticorpos Monoclonais/uso terapêutico , Antineoplásicos/uso terapêutico , Vacinas Anticâncer/uso terapêutico , Fator Estimulador de Colônias de Granulócitos e Macrófagos/uso terapêutico , Neoplasias da Próstata/terapia , Adulto , Idoso , Terapia Combinada , Fator Estimulador de Colônias de Granulócitos e Macrófagos/imunologia , Humanos , Imunoterapia , Ipilimumab , Masculino , Pessoa de Meia-Idade , Orquiectomia , Neoplasias da Próstata/imunologia , Neoplasias da Próstata/secundário , Transplante Homólogo , Células Tumorais CultivadasRESUMO
Vγ9Vδ2-T cells constitute a proinflammatory lymphocyte subpopulation with established antitumor activity. Phosphoantigens activate Vγ9Vδ2-T cells in vivo and in vitro. We studied whether the antitumor activity of Vγ9Vδ2-T cells can be potentiated by invariant NKT cells (iNKT), an important immunoregulatory T cell subset. When activated by the glycolipid α-galactosylceramide (α-GalCer), iNKT produce large amounts of cytokines involved in antitumor immune responses. Monocyte-derived dendritic cells were loaded with both phosphoantigens (using aminobisphosphonates) and α-GalCer during maturation and subsequently co-cultured with Vγ9Vδ2-T and iNKT cells. Aminobisphosphonates dose-dependently enhanced Vγ9Vδ2-T cell activation, and this was potentiated by α-GalCer-induced iNKT co-activation. iNKT co-activation also enhanced the IFN-γ production and cytolytic potential of Vγ9Vδ2-T cells against tumor cells. Using transwell experiments and neutralizing antibodies cross-talk between iNKT and Vγ9Vδ2-T cells was found to be mediated by TNF-α. Our data provide a rationale for combining both activating ligands to improve Vγ9Vδ2-T cell based approaches in cancer-immunotherapy.
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Ativação Linfocitária/imunologia , Células T Matadoras Naturais , Neoplasias , Linfócitos T Citotóxicos , Fator de Necrose Tumoral alfa/biossíntese , Antígenos de Neoplasias/imunologia , Células Cultivadas , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Difosfonatos/farmacologia , Galactosilceramidas/imunologia , Galactosilceramidas/farmacologia , Hemiterpenos/metabolismo , Humanos , Fatores Imunológicos/imunologia , Imunoterapia/métodos , Interferon gama/biossíntese , Interferon gama/imunologia , Células T Matadoras Naturais/imunologia , Células T Matadoras Naturais/metabolismo , Neoplasias/imunologia , Neoplasias/terapia , Compostos Organofosforados/metabolismo , Linfócitos T Citotóxicos/imunologia , Linfócitos T Citotóxicos/metabolismo , Fator de Necrose Tumoral alfa/imunologiaRESUMO
Varicella-zoster virus (VZV) causes varicella and herpes zoster, diseases characterized by distinct cutaneous rashes. Dendritic cells (DC) are essential for inducing antiviral immune responses; however, the contribution of DC subsets to immune control during natural cutaneous VZV infection has not been investigated. Immunostaining showed that compared to normal skin, the proportion of cells expressing DC-SIGN (a dermal DC marker) or DC-LAMP and CD83 (mature DC markers) were not significantly altered in infected skin. In contrast, the frequency of Langerhans cells was significantly decreased in VZV-infected skin, whereas there was an influx of plasmacytoid DC, a potent secretor of type I interferon (IFN). Langerhans cells and plasmacytoid DC in infected skin were closely associated with VZV antigen-positive cells, and some Langerhans cells and plasmacytoid DC were VZV antigen positive. To extend these in vivo observations, both plasmacytoid DC (PDC) isolated from human blood and Langerhans cells derived from MUTZ-3 cells were shown to be permissive to VZV infection. In VZV-infected PDC cultures, significant induction of alpha IFN (IFN-alpha) did not occur, indicating the VZV inhibits the capacity of PDC to induce expression of this host defense cytokine. This study defines changes in the response of DC which occur during cutaneous VZV infection and implicates infection of DC subtypes in VZV pathogenesis.
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Varicela/imunologia , Células Dendríticas/imunologia , Herpes Zoster/imunologia , Herpesvirus Humano 3/imunologia , Pele/imunologia , Antígenos CD/análise , Moléculas de Adesão Celular/análise , Células Cultivadas , Varicela/patologia , Células Dendríticas/virologia , Citometria de Fluxo , Herpes Zoster/patologia , Humanos , Imunoglobulinas/análise , Imuno-Histoquímica , Interferon-alfa/metabolismo , Lectinas Tipo C/análise , Proteínas de Membrana Lisossomal/análise , Glicoproteínas de Membrana/análise , Microscopia , Receptores de Superfície Celular/análise , Pele/patologia , Antígeno CD83RESUMO
PURPOSE: Impaired immune effector functions in the melanoma sentinel lymph node (SLN) may allow for early metastatic events. Local administration of PF-3512676 (formerly known as CpG 7909) has shown immunostimulatory effects of both dendritic cell and T-cell subsets in the melanoma SLN. Here, we set out to ascertain whether these PF-3512676-induced immunostimulatory effects translate into higher frequencies of melanoma-specific CD8(+) T cells. EXPERIMENTAL DESIGN: Twenty-four stage I to III melanoma patients were randomized to preoperative local administration of either PF-3512676 or saline. CD8(+) T cells from SLN and peripheral blood were tested for reactivity by IFN-gamma ELISPOT assay against several HLA-A1/A2/A3-restricted epitopes derived from various melanoma-associated antigens (MAA) in 21 of 24 enrolled patients. Frequencies of natural killer (NK) cells and frequencies and maturation state of dendritic cell subsets in the SLN were determined by flow cytometry. RESULTS: Melanoma-specific CD8(+) T-cell response rates against >1 MAA epitope in the SLN were 0 of 11 for the saline group versus 5 of 10 for the PF-3512676-administered group (P = 0.012). Of these 5 responding patients, 4 also had a measurable response to >1 MAA epitope in the blood. Increased frequencies in the SLN of both MAA-specific CD8(+) T cells and NK cells correlated to CpG-induced plasmacytoid dendritic cell maturation. CONCLUSIONS: These data show an increase in melanoma-specific CD8(+) T-cell frequencies as well as an increased effector NK cell rate after a single dose of PF-3512676 and thus support the utility of local PF-3512676 administration as adjuvant treatment in early-stage melanoma to try and halt metastatic spread.
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Linfócitos T CD8-Positivos/imunologia , Imunoterapia Ativa/métodos , Melanoma/tratamento farmacológico , Oligodesoxirribonucleotídeos/administração & dosagem , Neoplasias Cutâneas/tratamento farmacológico , Antígenos de Neoplasias/imunologia , Quimioterapia Adjuvante , Células Dendríticas/citologia , Células Dendríticas/imunologia , Citometria de Fluxo , Humanos , Injeções Intralesionais , Células Matadoras Naturais/citologia , Células Matadoras Naturais/imunologia , Melanoma/imunologia , Pessoa de Meia-Idade , Terapia Neoadjuvante , Biópsia de Linfonodo Sentinela , Neoplasias Cutâneas/imunologiaRESUMO
In human skin, two main populations of dendritic cells (DC) can be discriminated: dermal DC (DDC) and epidermal Langerhans cells (LC). Although extensively studied, most of the knowledge about DDC and LC phenotype and function is obtained from studying DDC and LC cultured in vitro or DDC and LC migrated from skin explants. These studies have left the exact relationship between steady-state human LC and DDC unclear: in particular, whether CD1a+ DDC represent migrated LC or whether they constitute a separate subset. To gain further insight in the kinship between skin-resident CD1a+ DDC and LC, we analyzed CD1a+ DDC and LC, isolated from steady-state skin samples, by high-density microarray analysis. Results show that the CD1a+ DDC specifically express markers associated with DDC phenotype, such as the macrophage mannose receptor, DC-specific ICAM-grabbing nonintegrin, the scavenger receptor CD36, coagulation factor XIIIa, and chemokine receptor CCR5, whereas LC specifically express Langerin, membrane ATPase (CD39), and CCR6, all hallmarks of the LC lineage. In addition, under steady-state conditions, both DC subsets display a strikingly different activation status, indicative of distinct functional properties. CD1a+ DDC exhibit a more activated, proinflammatory, migratory, and T cell-stimulatory profile, as compared with LC, whereas LC mainly express molecules involved in cell adhesion and DC retention in the epidermis. In conclusion, transcriptional profiling is consistent with the notion that CD1a+ DDC and LC represent two distinct DC subsets but also that under steady-state conditions, CD1a+ DDC and epidermal LC represent opposites of the DC activation spectrum.
Assuntos
Antígenos CD1/metabolismo , Diferenciação Celular , Perfilação da Expressão Gênica , Células de Langerhans/citologia , Células de Langerhans/metabolismo , Pele/citologia , Transcrição Gênica , Movimento Celular , Quimiocinas/genética , Quimiocinas/metabolismo , Análise por Conglomerados , Citometria de Fluxo , Regulação da Expressão Gênica , Humanos , Tolerância Imunológica , Fenótipo , Receptores Imunológicos/genética , Receptores Imunológicos/metabolismo , Pele/metabolismoRESUMO
Dendritic cells (DC) are increasingly applied in the immunotherapy of cancer. As the development of a standardized DC vaccine product is often hampered by the limited availability of DC precursors and inter- and intra-donor variability, and the preparation of individual vaccines is labor-intensive, it would be preferable to use DC from a readily available and unlimited source, such as cell lines can provide. It has been described that leukemia-derived cell lines are able to differentiate into functional DC, creating possibilities for the development of highly reproducible DC vaccines and providing in vitro model systems for in-depth studies about DC physiology. This review discusses the different human DC cell line differentiation models described so far. Based on the available data, characteristics that determine the ability of leukemia cells to differentiate along the different precursor stages into functional DC will be formulated. In addition, evidence will be provided that the human CD34+ acute myeloid leukemia cell line MUTZ-3 provides DC that exhibit the functional properties that are crucial for the in vivo generation of CTL-mediated immunity and thus, currently, represents the most valuable, sustainable model system for myeloid DC differentiation and clinical DC vaccination studies.
Assuntos
Vacinas Anticâncer/uso terapêutico , Diferenciação Celular/fisiologia , Células Dendríticas/fisiologia , Modelos Biológicos , Linhagem Celular , Humanos , Leucemia/imunologia , Leucemia/terapiaRESUMO
Resistance to antitumor immunity can be promoted by the oncogenic pathways operational in human cancers, including the epidermal growth factor receptor (EGFR) pathway. Here we studied if and how EGFR downstream signaling in head and neck squamous cell carcinoma (HNSCC) can affect the attraction of immune cells. HPV-negative and HPV-positive HNSCC cell lines were analyzed in vitro for CCL2, CCL5, CXCL9, CXCL10, IL-6 and IL-1ß expression and the attraction of T cells under different conditions, including cetuximab treatment and stimulation with IFNγ and TNFα using qPCR, ELISA and migration experiments. Biochemical analyses with chemical inhibitors and siRNA transfection were used to pinpoint the underlying mechanisms. Stimulation of HNSCC cells with IFNγ and TNFα triggered the production of T-cell attracting chemokines and required c-RAF activation. Blocking of the EGFR with cetuximab during this stimulation increased chemokine production in vitro, and augmented the attraction of T cells. Mechanistically, cetuximab decreased the phosphorylation of MEK1, ERK1/2, AKT, mTOR, JNK, p38 and ERK5. Chemical inhibition of EGFR signaling showed a consistent and pronounced chemokine production with MEK1/2 inhibitor PD98059 and JNK inhibitor SP600125, but not with inhibitors of p38, PI3K or mTOR. Combination treatment with cetuximab and a MEK1/2 or JNK inhibitor induced the highest chemokine expression. In conclusion, overexpression of EGFR results in the activation of multiple downstream signaling pathways that act simultaneously to suppress type 1 cytokine stimulated production of chemokines required to amplify the attraction of T cells.
Assuntos
Carcinoma de Células Escamosas/tratamento farmacológico , Cetuximab/uso terapêutico , Citocinas/metabolismo , Neoplasias de Cabeça e Pescoço/tratamento farmacológico , Linfócitos T/metabolismo , Idoso , Idoso de 80 Anos ou mais , Antineoplásicos Imunológicos/uso terapêutico , Carcinoma de Células Escamosas/patologia , Carcinoma de Células Escamosas/cirurgia , Linhagem Celular Tumoral , Citocinas/sangue , Citocinas/genética , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/metabolismo , Feminino , Neoplasias de Cabeça e Pescoço/patologia , Neoplasias de Cabeça e Pescoço/cirurgia , Humanos , Masculino , Pessoa de Meia-Idade , Inibidores de Proteínas Quinases/uso terapêutico , Transdução de Sinais/efeitos dos fármacosRESUMO
Purpose: Human papillomavirus (HPV)-associated oropharyngeal squamous cell cancer (OPSCC) has a much better prognosis than HPV-negative OPSCC, and this is linked to dense tumor immune infiltration. As the viral antigens may trigger potent immunity, we studied the relationship between the presence of intratumoral HPV-specific T-cell responses, the immune contexture in the tumor microenvironment, and clinical outcome.Experimental Design: To this purpose, an in-depth analysis of tumor-infiltrating immune cells in a prospective cohort of 97 patients with HPV16-positive and HPV16-negative OPSCC was performed using functional T-cell assays, mass cytometry (CyTOF), flow cytometry, and fluorescent immunostaining of tumor tissues. Key findings were validated in a cohort of 75 patients with HPV16-positive OPSCC present in the publicly available The Cancer Genome Atlas database.Results: In 64% of the HPV16-positive tumors, type I HPV16-specific T cells were present. Their presence was not only strongly related to a better overall survival, a smaller tumor size, and less lymph node metastases but also to a type I-oriented tumor microenvironment, including high numbers of activated CD161+ T cells, CD103+ tissue-resident T cells, dendritic cells (DC), and DC-like macrophages.Conclusions: The viral antigens trigger a tumor-specific T-cell response that shapes a favorable immune contexture for the response to standard therapy. Hence, reinforcement of HPV16-specific T-cell reactivity is expected to boost this process. Clin Cancer Res; 24(3); 634-47. ©2017 AACRSee related commentary by Laban and Hoffmann, p. 505.
Assuntos
Papillomavirus Humano 16 , Linfócitos do Interstício Tumoral/imunologia , Neoplasias Orofaríngeas/etiologia , Neoplasias Orofaríngeas/patologia , Infecções por Papillomavirus/complicações , Linfócitos T/imunologia , Microambiente Tumoral , Células Apresentadoras de Antígenos/imunologia , Células Apresentadoras de Antígenos/metabolismo , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Cisplatino/farmacologia , Citocinas/biossíntese , Resistencia a Medicamentos Antineoplásicos , Feminino , Papillomavirus Humano 16/genética , Humanos , Linfócitos do Interstício Tumoral/metabolismo , Linfócitos do Interstício Tumoral/patologia , Masculino , Neoplasias Orofaríngeas/tratamento farmacológico , Neoplasias Orofaríngeas/mortalidade , Infecções por Papillomavirus/virologia , Prognóstico , Especificidade do Receptor de Antígeno de Linfócitos T , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Linfócitos T/metabolismo , Linfócitos T/patologia , Microambiente Tumoral/imunologiaRESUMO
The study of early events in dendritic cell (DC) differentiation is hampered by the lack of homogeneous primary cell systems that allow the study of cytokine-driven, transitional DC differentiation steps. The CD34(+) acute myeloid leukemia cell line MUTZ-3 displays a unique ability to differentiate into interstitial DC (IDC) and Langerhans cells (LC) in a cytokine-dependent manner. Phenotypic characterization revealed MUTZ-3 to consist of three distinct subpopulations. Small CD34(+)CD14(-)CD11b(-) progenitors constitute the proliferative compartment of the cell line with the ability to differentiate through a CD34(-)CD14(-)CD11b(+) stage to ultimately give rise to a morphologically large, nonproliferating CD14(+)CD11b(hi) progeny. These CD14(+)CD11b(hi) cells were identified as common, immediate myeloid DC precursors with the ability to differentiate into LC and IDC, exhibiting characteristic and mutually exclusive expression of Langerin and DC-specific ICAM-grabbing nonintegrin, respectively. The identity of the MUTZ-3-derived LC subset was confirmed further by the presence of Birbeck granules. We conclude that the MUTZ-3 cell line provides a ready and continuous supply of common myeloid precursors, which should facilitate further study of the ontogeny of myeloid DC lineages.
Assuntos
Antígenos CD34/imunologia , Antígeno CD11b/imunologia , Diferenciação Celular/imunologia , Células de Langerhans/imunologia , Receptores de Lipopolissacarídeos/imunologia , Modelos Imunológicos , Antígenos CD/biossíntese , Antígenos CD/imunologia , Antígenos CD34/metabolismo , Antígeno CD11b/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Grânulos Citoplasmáticos/imunologia , Grânulos Citoplasmáticos/metabolismo , Regulação da Expressão Gênica/imunologia , Humanos , Células de Langerhans/metabolismo , Lectinas Tipo C/biossíntese , Lectinas Tipo C/imunologia , Receptores de Lipopolissacarídeos/metabolismo , Lectinas de Ligação a Manose/biossíntese , Lectinas de Ligação a Manose/imunologia , Células Progenitoras Mieloides/imunologia , Células Progenitoras Mieloides/metabolismoRESUMO
Immunotherapy shows promising clinical results in patients with different types of cancer, but its full potential is not reached due to immune dysfunction as a result of several suppressive mechanisms that play a role in cancer development and progression. Monitoring of immune dysfunction is a prerequisite for the development of strategies aiming to alleviate cancer-induced immune suppression. At this point, the level at which immune dysfunction occurs has to be established, the underlying mechanism(s) need to be known, as well as the techniques to assess this. While it is relatively easy to measure general signs of immune suppression, it turns out that accurate monitoring of the frequency and function of immune-suppressive cells is still difficult. A lack of truly specific markers, the phenotypic complexity among suppressive cells of the same lineage, but potentially with different functions and functional assays that may not cover every mechanistic aspect of immune suppression are among the reasons complicating proper assessments. Technical innovations in flow and mass cytometry will allow for more complete sets of markers to precisely determine phenotype and associated function. There is, however, a clear need for functional assays that recapitulate more of the mechanisms employed to suppress the immune system.