RESUMO
Optimal ex vivo expansion protocols for adoptive cell therapy (ACT) must yield T cells able to effectively home to tumors and survive the inhospitable conditions of the tumor microenvironment (TME), while simultaneously exerting persistent anti-tumor effector functions. Our previous work has shown that ex vivo activation in the presence of IL-12 can induce optimal expansion of murine CD8+ T cells, thus resulting in significant tumor regression after ACT mostly via sustained secretion of IFN-γ. In this report, we further elucidate the mechanism of this potency, showing that IL-12 additionally counteracts the negative regulatory effects of autocrine IFN-γ. IL-12 not only downregulates PD-1 expression by T cells, thus minimizing the effects of IFN-γ-induced PD-L1 upregulation by tumor stromal cells, but also inhibits IFNγR2 expression, thereby protecting T cells from IFN-γ-induced cell death. Thus, the enhanced anti-tumor activity of CD8+ T cells expanded ex vivo in the presence of IL-12 is due not only to the ability of IL-12-stimulated cells to secrete sustained levels of IFN-γ, but also to the additional capacity of IL-12 to counter the negative regulatory effects of autocrine IFN-γ.
Assuntos
Linfócitos T CD8-Positivos/efeitos dos fármacos , Citotoxicidade Imunológica/efeitos dos fármacos , Interferon gama/fisiologia , Interleucina-12/farmacologia , Linfócitos do Interstício Tumoral/efeitos dos fármacos , Transferência Adotiva , Animais , Linfócitos T CD8-Positivos/imunologia , Linhagem Celular Tumoral , Humanos , Ativação Linfocitária , Linfócitos do Interstício Tumoral/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Receptores de Interferon/análise , Receptores de Interferon/fisiologia , Receptor de Interferon gamaRESUMO
Shifting the balance away from tumor-mediated immune suppression toward tumor immune rejection is the conceptual foundation for a variety of immunotherapy efforts currently being tested. These efforts largely focus on activating antitumor immune responses but are confounded by multiple immune cell populations, including myeloid-derived suppressor cells (MDSCs), which serve to suppress immune system function. We have identified immune-suppressive MDSCs in the brains of GBM patients and found that they were in close proximity to self-renewing cancer stem cells (CSCs). MDSCs were selectively depleted using 5-flurouracil (5-FU) in a low-dose administration paradigm, which resulted in prolonged survival in a syngeneic mouse model of glioma. In coculture studies, patient-derived CSCs but not nonstem tumor cells selectively drove MDSC-mediated immune suppression. A cytokine screen revealed that CSCs secreted multiple factors that promoted this activity, including macrophage migration inhibitory factor (MIF), which was produced at high levels by CSCs. Addition of MIF increased production of the immune-suppressive enzyme arginase-1 in MDSCs in a CXCR2-dependent manner, whereas blocking MIF reduced arginase-1 production. Similarly to 5-FU, targeting tumor-derived MIF conferred a survival advantage to tumor-bearing animals and increased the cytotoxic T cell response within the tumor. Importantly, tumor cell proliferation, survival, and self-renewal were not impacted by MIF reduction, demonstrating that MIF is primarily an indirect promoter of GBM progression, working to suppress immune rejection by activating and protecting immune suppressive MDSCs within the GBM tumor microenvironment. Stem Cells 2016;34:2026-2039.
Assuntos
Neoplasias Encefálicas/imunologia , Glioblastoma/imunologia , Evasão da Resposta Imune , Fatores Inibidores da Migração de Macrófagos/metabolismo , Células Supressoras Mieloides/metabolismo , Células-Tronco Neoplásicas/metabolismo , Animais , Arginase/metabolismo , Neoplasias Encefálicas/patologia , Carcinogênese/metabolismo , Carcinogênese/patologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Meios de Cultivo Condicionados/farmacologia , Feminino , Glioblastoma/patologia , Humanos , Evasão da Resposta Imune/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Camundongos Nus , Células Supressoras Mieloides/efeitos dos fármacos , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/patologia , Microambiente Tumoral/efeitos dos fármacosRESUMO
BACKGROUND: A subset of patients with metastatic renal-cell carcinoma show indolent growth of metastases. Because of the toxicity and non-curative nature of systemic therapy, some of these patients could benefit from initial active surveillance. We aimed to characterise the time to initiation of systemic therapy in patients with metastatic renal-cell carcinoma under active surveillance. METHODS: In this prospective phase 2 trial, we enrolled patients with treatment-naive, asymptomatic, metastatic renal-cell carcinoma from five hospitals in the USA, Spain, and the UK. Patients were radiographically assessed at baseline, every 3 months for year 1, every 4 months for year 2, then every 6 months thereafter. Patients continued on observation until initiation of systemic therapy for metastatic renal-cell carcinoma; a decision that was made at the discretion of the treating physician and patient. The primary endpoint of the study was time to initiation of systemic therapy in the per-protocol population. The follow-up of patients is ongoing. FINDINGS: Between Aug 21, 2008, and June 7, 2013, we enrolled 52 patients. Median follow-up of patients in the study was 38·1 months (IQR 29·4-48·9). In the 48 patients included in analysis, median time on surveillance from registration on study until initiation of systemic therapy was 14·9 months (95% CI 10·6-25·0). Multivariate analysis showed that higher numbers of International Metastatic Database Consortium (IMDC) adverse risk factors (p=0·0403) and higher numbers of metastatic disease sites (p=0·0414) were associated with a shorter surveillance period. 22 (46%) patients died during the study period, all from metastatic renal-cell carcinoma. INTERPRETATION: A subset of patients with metastatic renal-cell carcinoma can safely undergo surveillance before starting systemic therapy. Additional investigation is required to further define the benefits and risks of this approach. FUNDING: None.
Assuntos
Carcinoma de Células Renais/secundário , Neoplasias Renais/patologia , Tomografia Computadorizada por Raios X/métodos , Idoso , Carcinoma de Células Renais/diagnóstico por imagem , Carcinoma de Células Renais/epidemiologia , Carcinoma de Células Renais/cirurgia , Feminino , Seguimentos , Humanos , Neoplasias Renais/diagnóstico por imagem , Neoplasias Renais/epidemiologia , Neoplasias Renais/cirurgia , Masculino , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Nefrectomia , Vigilância da População , Prognóstico , Estudos Prospectivos , Espanha/epidemiologia , Taxa de Sobrevida , Reino Unido/epidemiologia , Estados Unidos/epidemiologiaRESUMO
BACKGROUND: Renal cell carcinoma (RCC) patients treated with tyrosine kinase inhibitors (TKI) typically respond initially, but usually develop resistance to therapy. We utilised transcriptome analysis to identify gene expression changes during development of sunitinib resistance in a RCC patient-derived xenograft (PDX) model. METHODS: RCC tumours were harvested during pre-treatment, response and escape phases. Direct anti-proliferative effects of sunitinib plus MEK inhibitor were assessed. Activation status (phosphorylation) of MEK1/2 and ERK1/2 was determined, myeloid-derived suppressor cells (MDSC) sub-fractions were quantitated and G-CSF was measured by ELISA. RESULTS: During the response phase, tumours exhibited 91% reduction in volume, characterised by decreased expression of cell survival genes. After 4-week treatment, tumours developed resistance to sunitinib, associated with increased expression of pro-angiogenic and cell survival genes. During tumour escape, cellular movement, inflammatory response and immune cell trafficking genes were induced, along with intra-tumoural accumulation of MDSC. In this PDX model, either continuous treatment with sunitinib plus MEK inhibitor PD-0325901, or switching from sunitinib to PD-0325901 was effective. The combination of PD-0325901 with TKI suppressed intra-tumoural phospho-MEK1/2, phospho-ERK1/2 and MDSC. CONCLUSIONS: Continuous treatment with sunitinib alone did not maintain anti-tumour response; addition of MEK inhibitor abrogated resistance, leading to improved anti-tumour efficacy.
Assuntos
Benzamidas/uso terapêutico , Carcinoma de Células Renais/tratamento farmacológico , Difenilamina/análogos & derivados , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Indóis/farmacologia , Neoplasias Renais/tratamento farmacológico , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Proteínas de Neoplasias/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Pirróis/farmacologia , Adulto , Animais , Benzamidas/farmacologia , Carcinoma de Células Renais/enzimologia , Carcinoma de Células Renais/genética , Carcinoma de Células Renais/imunologia , Linhagem Celular Tumoral , Difenilamina/farmacologia , Difenilamina/uso terapêutico , Sinergismo Farmacológico , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Indóis/uso terapêutico , Neoplasias Renais/enzimologia , Neoplasias Renais/genética , Neoplasias Renais/imunologia , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Células Supressoras Mieloides/efeitos dos fármacos , Células Supressoras Mieloides/imunologia , Neovascularização Patológica/tratamento farmacológico , Neovascularização Patológica/genética , Fosforilação/efeitos dos fármacos , Inibidores de Proteínas Quinases/uso terapêutico , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Pirróis/uso terapêutico , Receptores de Interleucina-2/deficiência , Sunitinibe , Carga Tumoral/efeitos dos fármacos , Evasão Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
IL-17 is a proinflammatory cytokine implicated in the pathogenesis of autoimmune diseases including psoriasis. ACT1 is an essential adaptor molecule in the IL-17 signaling pathway. A missense single nucleotide polymorphism (rs33980500; SNP-D10N) that resulted in the substitution of an asparagine for an aspartic acid at position 10 of ACT1 (ACT1-D10N) is associated with psoriasis susceptibility. Due to alternative splicing in humans, SNP-D10N encodes two mutated ACT1 proteins, ACT1-D10N and ACT1-D19N. Although both ACT1 isoforms are Hsp90 client proteins, the nine additional amino acids in ACT1-D19N provide an additional Hsp90 binding site that is absent in ACT1-D10N. Therefore, whereas ACT1-D10N is a dead protein that is unable to transduce IL-17 signals for gene expression, ACT1-D19N is fully responsive to IL-17. Intriguingly, the two ACT1 isoforms are differentially expressed in ACT1(D10N/D10N) fibroblasts and T cells. Fibroblasts express both isoforms equally, enabling ACT1-D19N to compensate for the loss of ACT1-D10N function. ACT1(D10N/D10N) T cells, however, express predominantly ACT1-D10N. Lacking this compensatory mechanism, ACT1(D10N/D10N) T cells behave like ACT1-deficient T cells, exhibiting a dysregulated and hyperactive Th17 phenotype with overproduction of IL-22 and IL-17. The hyperactive Th17 response combined with fully responsive fibroblasts likely synergized to contribute to psoriasis susceptibility in SNP-D10N patients.
Assuntos
Proteínas de Choque Térmico HSP90/imunologia , Interleucina-17/imunologia , Psoríase/imunologia , Transdução de Sinais/imunologia , Peptídeos e Proteínas Associados a Receptores de Fatores de Necrose Tumoral/genética , Proteínas Adaptadoras de Transdução de Sinal , Processamento Alternativo/genética , Sequência de Bases , Sítios de Ligação , Predisposição Genética para Doença , Células HEK293 , Células HeLa , Humanos , Interleucina-17/biossíntese , Interleucinas/biossíntese , Dados de Sequência Molecular , Mutação de Sentido Incorreto , Polimorfismo de Nucleotídeo Único , Isoformas de Proteínas/biossíntese , Isoformas de Proteínas/genética , Psoríase/patologia , Interferência de RNA , Pele/imunologia , Pele/patologia , Células Th17/imunologia , Peptídeos e Proteínas Associados a Receptores de Fatores de Necrose Tumoral/biossíntese , Interleucina 22RESUMO
Myeloid derived suppressor cells (MDSCs) are bone marrow derived cells with immunosuppressive properties. We have shown previously that MDSCs numbers are elevated in the circulation of GBM patients and that they produce reversible T cell dysfunction. Here, we evaluated whether MDSCs infiltrate human GBM tissues, and whether a commonly used mouse model of GBM reproduces the biology of MDSCs that is observed in patients. We evaluated tumor specimens from patients with newly diagnosed GBM. We harvested and evaluated normal brain, tumors and hematopoietic tissues from control, vehicle and sunitinib-treated mice. In human GBM tumors, MDSCs represented 5.4 ± 1.8 % of total cells. The majority of MDSCs (CD33+HLADR-) were lineage negative (CD14-CD15-), followed by granulocytic (CD15+CD14-) and monocytic (CD15-CD14+) subtypes. In murine GBM tumors, MDSCs were 8.06 ± 0.78 % of total cells, of which more were monocytic (M-MDSC, CD11b+ Gr1-low) than granulocytic (G-MDSC, CD11b+ Gr1-high). Treatment with the tyrosine kinase inhibitor sunitinib decreased the infiltration of both granulocytic and monocytic MDSCs in murine GBM tumors. In the hematopoietic tissues, circulating G-MDSC blood levels were reduced after sunitinib treatment. In tumors, both CD3(+) and CD4(+) T cell counts increased following sunitinib treatment (p ≤ 0.001). Total T cell proliferation (p < 0.001) and interferon gamma production (p = 0.004) were increased in the spleens of sunitinib treated mice. Sunitinib-treated mice survived longer than vehicle-treated mice (p = 0.002). MDSCs are present in both human and mouse GBM tumors. Sunitinib may have an immunostimulatory effect, as its use is associated with a reduction in G-MDSCs and improvement in anti-tumor immune function.
Assuntos
Neoplasias Encefálicas/fisiopatologia , Encéfalo/fisiopatologia , Glioma/fisiopatologia , Linfócitos do Interstício Tumoral/fisiologia , Células Mieloides/fisiologia , Animais , Antineoplásicos/farmacologia , Medula Óssea/efeitos dos fármacos , Medula Óssea/fisiopatologia , Encéfalo/efeitos dos fármacos , Encéfalo/patologia , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/patologia , Linhagem Celular , Galinhas , Glioma/tratamento farmacológico , Glioma/patologia , Humanos , Indóis/farmacologia , Linfócitos do Interstício Tumoral/efeitos dos fármacos , Linfócitos do Interstício Tumoral/patologia , Camundongos Transgênicos , Células Mieloides/efeitos dos fármacos , Células Mieloides/patologia , Transplante de Neoplasias , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/metabolismo , Pirróis/farmacologia , Baço/efeitos dos fármacos , Baço/fisiopatologia , Sunitinibe , Análise de Sobrevida , Linfócitos T/efeitos dos fármacos , Linfócitos T/patologia , Linfócitos T/fisiologia , Resultado do TratamentoRESUMO
Sunitinib, a protein tyrosine kinase inhibitor is the frontline therapy for renal and gastrointestinal cancers. We hypothesized that by virtue of its well documented tumor apoptosis and immune adjuvant properties, combination of Sunitinib with anti-tumor immunotherapeutics will provide synergistic inhibition of tumor growth. Our study was designed to evaluate the impact of Sunitinib on immunotherapy mediated anti-tumor immune responses and evaluate its efficacy as a combinatorial therapy with tumor targeted immunotherapeutic vaccination. Mice immunized with recombinant α-lactalbumin, a lactation protein expressed on majority of breast tumors were treated with 1 mg of Sunitinib for seven consecutive days beginning (1) concurrently, on the day of α-lactalbumin immunization or (2) sequentially, on day 9 after immunization. Ten-day lymph nodes or 21 day spleens were tested by ELISPOT assays and flow cytometry to evaluate responsiveness to α-lactalbumin immunization in presence of Sunitinib and distribution of cells involved in T cell antigen priming and proliferation in different lymphoid compartments. In addition, therapeutic efficacy of the α-lactalbumin/ Sunitinib combination was evaluated by monitoring tumor growth in the 4T1 transplanted tumor model. Our studies reveal that concurrent administration of Sunitinib with active vaccination against a targeted tumor antigen inhibits priming to the immunogen due to a drastic decrease in CD11b+CD11c+ antigen presenting cells, leading to failure of vaccination. However, sequential delivery of Sunitinib timed to avoid the priming phase of vaccination results in the desired vaccination mediated boost in immune responses.
Assuntos
Vacinas Anticâncer/farmacologia , Indóis/farmacologia , Lactalbumina/imunologia , Pirróis/farmacologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Adjuvantes Imunológicos/farmacologia , Animais , Células Apresentadoras de Antígenos/efeitos dos fármacos , Células Apresentadoras de Antígenos/imunologia , Vacinas Anticâncer/imunologia , Proliferação de Células/efeitos dos fármacos , Progressão da Doença , Feminino , Imunoterapia/métodos , Linfonodos/efeitos dos fármacos , Linfonodos/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Baço/efeitos dos fármacos , Baço/imunologia , Sunitinibe , Vacinação/métodosRESUMO
Renal cell carcinoma (RCC) constitutes more than 90 % of primary kidney tumors with the development of metastatic disease in the lung, bone, liver, and brain. Clear-cell RCC (CCRCC) is the most common histologic form of sporadic kidney cancer where the majority of tumors have inactivation of the von Hippel-Lindau (VHL) tumor-suppressor gene resulting in the accumulation of hypoxia-inducible factor (HIF) leading to dysregulation of cell growth and angiogenesis. Understanding of the genetic changes in RCC and the downstream events have led to the development of tyrosine kinase inhibitors (TKI) that target HIF-regulated proteins which currently represents front-line therapy for metastatic disease although resistance develops in most patients overtime. Despite the fact that RCC is an immunogenic tumor, there is mounting evidence that immune cells and inflammatory pathways can enhance tumor growth and immune escape. However, recent studies are beginning to uncover the mechanisms of immune escape in RCC, and the role inflammatory immune cells and cytokines play is this process. These new findings have led to renewed interest in the use of immunotherapy for the treatment of this disease that includes strategies to regulate inflammatory responses. Here, we will discuss the different inflammatory signaling pathways (e.g., VHL, hypoxia, TNF-α, STAT, and TGF-ß) and the downstream transcription factors, cytokines, and chemokines involved in tumor development, and disease progression. This will include assessment of the role inflammatory molecules (e.g., pVHL, TGFb, IL6, select chemokines/chemokine receptors) play in promoting cell transformation, survival, proliferation of tumor cells, and metastasis derived from in vitro and in vivo studies. Included is a section on how select inflammatory cells (TAM, MDSC, and neutrophils) promote tumor evasion of immune cells. We also provide examples of molecules/cells that correlate negatively (CXCL12, CXCR4, and MMP, neutrophils, and MDSC) and positively (TH1 cells, IP-10, and MIG) with tumor progression and survival. Finally, there is a discussion of different inhibitors of inflammation that may be useful in the treatment of RCC.
Assuntos
Carcinoma de Células Renais/etiologia , Inflamação/complicações , Neoplasias Renais/etiologia , Animais , Anti-Inflamatórios/uso terapêutico , Carcinoma de Células Renais/patologia , Carcinoma de Células Renais/terapia , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/imunologia , Humanos , Inflamação/tratamento farmacológico , Inflamação/genética , Mediadores da Inflamação/fisiologia , Neoplasias Renais/patologia , Neoplasias Renais/terapia , Transdução de Sinais/genética , Transdução de Sinais/imunologiaRESUMO
The receptor tyrosine kinase inhibitor, sunitinib, is astonishingly effective in its capacity to reduce MDSCs in peripheral tissues such as blood (human) and spleen (mouse), restoring responsiveness of bystander T lymphocytes to TcR stimulation. Sunitinib blocks proliferation of undifferentiated MDSCs and decreases survival of more differentiated neutrophilic MDSC (n-MDSC) progeny. Ironically, sunitinib's profound effects are observed even in a total absence of detectable anti-tumor therapeutic response. This is best explained by the presence of disparate MDSC-conditioning stimuli within individual body compartments, allowing sensitivity and resistance to sunitinib to coexist within the same mouse or patient. The presence or absence of GM-CSF is likely the major determinant in each compartment, given that GM-CSF's capacity to preempt STAT3-dependent with dominant STAT5-dependent hematopoietic programming confers sunitinib resistance and redirects differentiation from the n-MDSC lineage to the more versatile monocytoid (m-MDSC) lineage. The clinical sunitinib experience underscores that strategies for MDSC and Treg depletions must be mindful of disparities among body compartments to avoid sanctuary effects. Ironically, m-MDSCs manifesting resistance to sunitinib also have the greatest potential to differentiate into tumoricidal accessory cells, by virtue of their capacity to respond to T cell-secreted IFN-γ or to TLR agonists with nitric oxide and peroxynitrate production.
Assuntos
Indóis/uso terapêutico , Células Progenitoras Mieloides/efeitos dos fármacos , Neoplasias/patologia , Neoplasias/terapia , Inibidores de Proteínas Quinases/uso terapêutico , Pirróis/uso terapêutico , Linfócitos T/efeitos dos fármacos , Evasão Tumoral , Animais , Fator Estimulador de Colônias de Granulócitos e Macrófagos/genética , Fator Estimulador de Colônias de Granulócitos e Macrófagos/imunologia , Humanos , Tolerância Imunológica , Indóis/farmacologia , Camundongos , Células Progenitoras Mieloides/imunologia , Células Progenitoras Mieloides/patologia , Neoplasias/imunologia , Neoplasias/metabolismo , Especificidade de Órgãos , Inibidores de Proteínas Quinases/farmacologia , Pirróis/farmacologia , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/imunologia , Fator de Transcrição STAT5/genética , Fator de Transcrição STAT5/imunologia , Transdução de Sinais , Sunitinibe , Linfócitos T/imunologia , Linfócitos T/patologiaRESUMO
Study of human monocytic Myeloid-Derived Suppressor cells Mo-MDSC (CD14+ HLA-DRneg/low) has been hampered by the lack of positive cell-surface markers. In order to identify positive markers for Mo-MDSC, we performed microarray analysis comparing Mo-MDSC cells from healthy subjects versus CD14+ HLA-DRhigh monocytes. We have identified the surface ectoenzyme Vanin-2(VNN2) protein as a novel biomarker highly-enriched in healthy subjects Mo-MDSC. Indeed, healthy subjects Mo-MDSC cells expressed 68 % VNN2, whereas only 9% VNN2 expression was observed on CD14+ HLA-DRhigh cells (n = 4 p < 0.01). The top 10 percent positive VNN2 monocytes expressed CD33 and CD11b while being negative for HLA-DR, CD3, CD15, CD19 and CD56, consistent with a Mo-MDSC phenotype. CD14+VNN2high monocytes were able to inhibit CD8 T cell proliferation comparably to traditional Mo-MDSC at 51 % and 48 % respectively. However, VNN2 expression on CD14+ monocytes from glioma patients was inversely correlated to their grade. CD14+VNN2high monocytes thus appear to mark a monocytic population similar to Mo-MDSC only in healthy subjects, which may be useful for tumor diagnoses.
Assuntos
Amidoidrolases/metabolismo , Moléculas de Adesão Celular/metabolismo , Glioma/diagnóstico , Receptores de Lipopolissacarídeos/metabolismo , Monócitos/imunologia , Células Supressoras Mieloides/imunologia , Biomarcadores/análise , Linfócitos T CD8-Positivos/imunologia , Proliferação de Células/fisiologia , Proteínas Ligadas por GPI/metabolismo , Glioma/patologia , Antígenos HLA-DR/metabolismo , Humanos , Ativação Linfocitária/imunologia , Proteínas de Membrana/metabolismo , Gradação de Tumores , Análise Serial de Proteínas , Lectina 3 Semelhante a Ig de Ligação ao Ácido Siálico/metabolismoRESUMO
The multikinase inhibitor sunitinib malate (SUT) has been reported to reduce levels of myeloid suppressor cells and Treg cells in cancer patients, hypothetically diminishing intrinsic impediments for active immunization against tumor-associated antigens in such individuals. The goal of this study was to identify longitudinal immune molecular and cellular changes associated with tumor regression and disease-free status after the treatment of established day 7 s.c. MO5 (B16.OVA) melanomas with SUT alone (1 mg/day via oral gavage for 7 days), vaccination using ovalbumin (OVA) peptide-pulsed dendritic cell [vaccine (VAC)] alone, or the combination of SUT and VAC (SUT/VAC). We observed superior anti-tumor efficacy for SUT/VAC combination approaches, particularly when SUT was applied at the time of the initial vaccination or the VAC boost. Treatment effectiveness was associated with the acute loss of (and/or failure to recruit) cells bearing myeloid-derived suppressor cells or Treg phenotypes within the tumor microenvironment (TME) and the corollary, prolonged enhancement of Type-1 anti-OVA CD8(+) T cell responses in the tumor-draining lymph node and the TME. Enhanced Type-1 T cell infiltration of tumors was associated with treatment-induced expression of vascular cell adhesion molecule-1 (VCAM-1) and CXCR3 ligand chemokines in vascular/peri-vascular cells within the TME, with SUT/VAC therapy benefits conditionally negated upon adminsitration of CXCR3 or VCAM-1 blocking antibodies. These data support the ability of a short 7 day course of SUT to (re)condition the TME to become more receptive to the recruitment and prolonged therapeutic action of (VAC-induced) anti-tumor Tc1 cells.
Assuntos
Vacinas Anticâncer/imunologia , Indóis/farmacologia , Melanoma Experimental/imunologia , Pirróis/farmacologia , Animais , Anticorpos Bloqueadores/administração & dosagem , Anticorpos Bloqueadores/imunologia , Linhagem Celular Tumoral , Terapia Combinada , Células Dendríticas/imunologia , Epitopos de Linfócito T/imunologia , Feminino , Imunoterapia Adotiva , Indóis/administração & dosagem , Linfonodos/imunologia , Melanoma Experimental/terapia , Camundongos , Camundongos Endogâmicos C57BL , Pirróis/administração & dosagem , Receptores CXCR3/imunologia , Sunitinibe , Linfócitos T/imunologia , Microambiente Tumoral/imunologia , Molécula 1 de Adesão de Célula Vascular/imunologiaRESUMO
INTRODUCTION: Cycloxygenase-2 (COX-2) is an enzyme involved in prostaglandin E2 (PGE(2)) synthesis associated with higher renal cell carcinoma stage. COX-2 inhibition enhances interferon (IFN-α) anti-tumor immune effects in pre-clinical models. A phase II trial of celecoxib and IFN-α in a targeted population of metastatic renal cell carcinoma patients with maximal COX-2 expression was conducted. METHODS: Cytokine-naive metastatic renal cell carcinoma patients with tumors expressing ≥10% maximal COX-2 staining by immunohistochemistry received IFN-α 5 million units daily and celecoxib 400 mg orally twice daily in an open-label, single-arm phase II trial. RESULTS: There were 3 partial responses among 17 patients (objective response rate 18%; 95% confidence interval, 4-43%). Time to progression was 5.6 months. Increased tumor staining 3+ for COX-2 was associated with increased baseline peripheral blood PGE(2) levels, and these patients demonstrated less PGE(2) decrease with therapy. Patients with more 3+ COX-2 staining had significantly more CD3(+) (p = 0.004) and CD4(+) (p = 0.002) IFN-γ T cells at baseline and a significantly greater decrease in these cells with therapy. DISCUSSION: Celecoxib plus IFN-α in renal cell carcinoma (RCC) patients with maximally staining COX-2 tumors does not significantly enhance overall response rates over IFN monotherapy. CONCLUSION: COX-2-expressing RCC demonstrates inherent immunosuppression. COX-2 inhibition with IFN results in minimal immunomodulation and no augmented clinical activity in RCC.
Assuntos
Carcinoma de Células Renais/tratamento farmacológico , Ciclo-Oxigenase 2/biossíntese , Interferon-alfa/uso terapêutico , Neoplasias Renais/tratamento farmacológico , Pirazóis/uso terapêutico , Sulfonamidas/uso terapêutico , Adulto , Idoso , Carcinoma de Células Renais/imunologia , Celecoxib , Células Dendríticas/efeitos dos fármacos , Dinoprostona/biossíntese , Dinoprostona/metabolismo , Quimioterapia Combinada/efeitos adversos , Feminino , Humanos , Fatores Imunológicos/uso terapêutico , Interferon-alfa/administração & dosagem , Interferon-alfa/efeitos adversos , Neoplasias Renais/patologia , Masculino , Pessoa de Meia-Idade , Pirazóis/administração & dosagem , Pirazóis/efeitos adversos , Sulfonamidas/administração & dosagem , Sulfonamidas/efeitos adversos , Células Th1/imunologia , Equilíbrio Th1-Th2 , Células Th2/imunologia , Resultado do TratamentoRESUMO
Scientific discoveries that provide strong evidence of antitumor effects in preclinical models often encounter significant delays before being tested in patients with cancer. While some of these delays have a scientific basis, others do not. We need to do better. Innovative strategies need to move into early stage clinical trials as quickly as it is safe, and if successful, these therapies should efficiently obtain regulatory approval and widespread clinical application. In late 2009 and 2010 the Society for Immunotherapy of Cancer (SITC), convened an "Immunotherapy Summit" with representatives from immunotherapy organizations representing Europe, Japan, China and North America to discuss collaborations to improve development and delivery of cancer immunotherapy. One of the concepts raised by SITC and defined as critical by all parties was the need to identify hurdles that impede effective translation of cancer immunotherapy. With consensus on these hurdles, international working groups could be developed to make recommendations vetted by the participating organizations. These recommendations could then be considered by regulatory bodies, governmental and private funding agencies, pharmaceutical companies and academic institutions to facilitate changes necessary to accelerate clinical translation of novel immune-based cancer therapies. The critical hurdles identified by representatives of the collaborating organizations, now organized as the World Immunotherapy Council, are presented and discussed in this report. Some of the identified hurdles impede all investigators; others hinder investigators only in certain regions or institutions or are more relevant to specific types of immunotherapy or first-in-humans studies. Each of these hurdles can significantly delay clinical translation of promising advances in immunotherapy yet if overcome, have the potential to improve outcomes of patients with cancer.
Assuntos
Imunoterapia , Neoplasias/terapia , Humanos , Cooperação Internacional , Pesquisa Translacional BiomédicaRESUMO
Increased expression of gangliosides by different tumor types including renal cell carcinoma (RCC) is thought to contribute to the immune suppression observed in cancer patients. In this study, we report an increase in apoptotic T cells from RCC patients compared with T cells from normal donors that coincided with the detection of T cells staining positive for GM2 and that the apoptosis was predominantly observed in the GM2(+) but not the GM2(-) T cell population. Ganglioside shedding from tumor rather than endogenous production accounts for GM2(+) T cells since there was no detectable level of mRNA for GM2 synthase in RCC patient T cells and in T cells from normal healthy donors after incubation with either purified GM2 or supernatant from RCC cell lines despite their staining positive for GM2. Moreover, reactive oxygen species as well as activated caspase 3, 8, and 9 were predominantly elevated in GM2(+) but not GM2(-) T cells. Similarly, increased staining for GD2 and GD3 but not GD1a was detected with patient T cells with elevated levels of apoptosis in the GD2(+) and GD3(+) cells. These findings suggest that GM2, GD2, and GD3 play a significant role in immune dysfunction observed in RCC patient T cells.
Assuntos
Carcinoma de Células Renais/imunologia , Gangliosídeos/imunologia , Neoplasias Renais/imunologia , Linfócitos T/imunologia , Apoptose/imunologia , Carcinoma de Células Renais/metabolismo , Caspases/imunologia , Caspases/metabolismo , Linhagem Celular Tumoral , Gangliosídeos/metabolismo , Gangliosidoses GM2/imunologia , Gangliosidoses GM2/metabolismo , Humanos , Neoplasias Renais/metabolismo , Espécies Reativas de Oxigênio/imunologia , Espécies Reativas de Oxigênio/metabolismo , Linfócitos T/metabolismoRESUMO
Myeloid-derived suppressor cells (MDSC) and tumor-associated macrophages (TAM) contribute to cancer-related inflammation and tumor progression. While several myeloid molecules have been ascribed a regulatory function in these processes, the triggering receptors expressed on myeloid cells (TREMs) have emerged as potent modulators of the innate immune response. While various TREMs amplify inflammation, others dampen it and are emerging as important players in modulating tumor progression-for instance, soluble TREM-1 (sTREM-1), which is detected during inflammation, associates with disease progression, while TREM-2 expression is associated with tumor-promoting macrophages. We hypothesized that TREM-1 and TREM-2 might be co-expressed on tumor-infiltrating myeloid cells and that elevated sTREM-1 associates with disease outcomes, thus representing a possibility for mutual modulation in cancer. Using the 4T1 breast cancer model, we found TREM-1 and TREM-2 expression on MDSC and TAM and that sTREM-1 was elevated in tumor-bearing mice in multiple models and correlated with tumor volume. While TREM-1 engagement enhanced TNF, a TREM-2 ligand was detected on MDSC and TAM, suggesting that both TREM could be functional in the tumor setting. Similarly, we detected TREM-1 and Trem2 expression in myeloid cells in the RENCA model of renal cell carcinoma (RCC). We confirmed these findings in human disease by demonstrating the expression of TREM-1 on tumor-infiltrating myeloid cells from patients with RCC and finding that sTREM-1 was increased in patients with RCC. Finally, The Cancer Genome Atlas analysis shows that TREM1 expression in tumors correlates with poor outcomes in RCC. Taken together, our data suggest that manipulation of the TREM-1/TREM-2 balance in tumors may be a novel means to modulate tumor-infiltrating myeloid cell phenotype and function.
RESUMO
T helper type 1 (Th1)-type CD4(+) antitumor T cell help appears critical to the induction and maintenance of antitumor cytotoxic T lymphocyte (CTL) responses in vivo. In contrast, Th2- or Th3/Tr-type CD4(+) T cell responses may subvert Th1-type cell-mediated immunity, providing a microenvironment conducive to disease progression. We have recently identified helper T cell epitopes derived from the MAGE-6 gene product; a tumor-associated antigen expressed by most melanomas and renal cell carcinomas. In this study, we have assessed whether peripheral blood CD4(+) T cells from human histocompatibility leukocyte antigens (HLA)-DRbeta1*0401(+) patients are Th1- or Th2-biased to MAGE-6 epitopes using interferon (IFN)-gamma and interleukin (IL)-5 enzyme-linked immunospot assays, respectively. Strikingly, the vast majority of patients with active disease were highly-skewed toward Th2-type responses against MAGE-6-derived epitopes, regardless of their stage (stage I versus IV) of disease, but retained Th1-type responses against Epstein-Barr virus- or influenza-derived epitopes. In marked contrast, normal donors and cancer patients with no current evidence of disease tended to exhibit either mixed Th1/Th2 or strongly Th1-polarized responses to MAGE-6 peptides, respectively. CD4(+) T cell secretion of IL-10 and transforming growth factor (TGF)-beta1 against MAGE-6 peptides was not observed, suggesting that specific Th3/Tr-type CD4(+) subsets were not common events in these patients. Our data suggest that immunotherapeutic approaches will likely have to overcome or complement systemic Th2-dominated, tumor-reactive CD4(+) T cell responses to provide optimal clinical benefit.
Assuntos
Carcinoma de Células Renais/imunologia , Neoplasias Renais/imunologia , Melanoma/imunologia , Proteínas de Neoplasias/imunologia , Células Th1/imunologia , Células Th2/imunologia , Adulto , Idoso , Apresentação de Antígeno , Antígenos de Neoplasias/genética , Sequência de Bases , Carcinoma de Células Renais/genética , Carcinoma de Células Renais/terapia , Epitopos/genética , Feminino , Antígenos HLA-DR/genética , Antígenos HLA-DR/metabolismo , Cadeias HLA-DRB1 , Humanos , Imunoterapia , Técnicas In Vitro , Neoplasias Renais/genética , Neoplasias Renais/terapia , Masculino , Melanoma/genética , Melanoma/terapia , Pessoa de Meia-Idade , Proteínas de Neoplasias/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Neoplásico/genética , RNA Neoplásico/metabolismoRESUMO
PURPOSE: The immunological consequences of cryoablation for renal cell carcinoma are largely unknown. Cryoablation is an attractive therapeutic option for tumors due to its minimally invasive nature. Cryoablation is also potentially immunogenic. We describe the development of an animal model to deliver in vivo renal cryotherapy to orthotopically implanted renal cell carcinoma and the results of multiple immunological interrogations after cryoablation. MATERIALS AND METHODS: Four to 6-week-old female Balb/c mice (Jackson Laboratories, Bar Harbor, Maine) underwent renal subcapsular implantation of the syngeneic murine renal cell carcinoma Renca. Two weeks later contact cryoablation was done in tumor bearing kidneys. Another group of animals underwent cryoablation of normal kidneys. Animals were sacrificed 2 weeks after tumor injection or 1 and 2 weeks after cryoablation, respectively. Kidneys, spleens and draining lymph nodes were harvested. Evaluation consisted of immunohistochemistry, immunofluorescence and gene expression profiling using reverse-transcriptase polymerase chain reaction. RESULTS: Subcapsular tumor implantation was successful in all cases and confirmed histologically. No significant lymphocytic infiltrate was seen in tumor only animals but those treated with cryoablation (tumor and nontumor bearing) had a significant inflammatory response primarily in sublethal tissue injury and perivascular areas. After cryoablation most infiltrating cells were neutrophils, macrophages and T cells. Polymerase chain reaction showed increased interferon-gamma production in kidneys after cryoablation. CONCLUSIONS: This study shows the potential feasibility of this animal model for studying cryo-immunology. We confirm the absence of any significant immune cell infiltration in tumor bearing kidneys and report a significant inflammatory infiltrate after cryoablation, consisting primarily of neutrophils, macrophages, and CD4+ and CD8+ T cells with an increase in the T helper type 1/2 ratio. This orthotopic murine model can form the basis of future studies of additional immunological aspects of renal cryoablation.
Assuntos
Carcinoma de Células Renais/imunologia , Carcinoma de Células Renais/cirurgia , Criocirurgia , Modelos Animais de Doenças , Neoplasias Renais/imunologia , Neoplasias Renais/cirurgia , Nefrectomia/métodos , Animais , Estudos de Viabilidade , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Transplante de NeoplasiasRESUMO
The EphA2 receptor tyrosine kinase is an attractive therapeutic target that is commonly overexpressed on solid tumors, with the degree of overexpression associated with disease progression, metastatic potential, and poor prognosis. Agonistic mAbs or ligand (ephrinA1)-Fc fusion protein are capable of inducing EphA2 internalization and degradation, thereby (at least transiently) eliminating the influence of this oncoprotein. We and others have also shown that EphA2 contains multiple peptide epitopes that can be recognized by effector CD4(+) and CD8(+) T cells isolated from tumor-bearing patients. Herein, we show that "agonist" reagents that trigger the proteasome-dependent degradation of tumor cell EphA2 result in the improved presentation of peptides derived from (both the extracellular and intracellular domains of) EphA2 in MHC class I complexes expressed on the tumor cell membrane for at least 48 h, as manifested by increased recognition by EphA2-specific CD8(+) T cells in vitro. We also observed that while delivery of ephrinA1-Fc fusion protein or agonist mAb into EphA2(+) tumor lesions promotes EphA2 degradation in situ, this single administration of agent does not dramatically alter tumor progression in a humanized SCID model. However, when combined with the adoptive transfer of normally nontherapeutic (human) anti-EphA2 CD8(+) CTL, this dual-agent regimen results in complete tumor eradication. These results suggest that strategies targeting the conditional proteasome-mediated destruction of tumor cell EphA2 may enable EphA2-specific CD8(+) T cells (of modest functional avidity) to realize improved therapeutic potential.
Assuntos
Apresentação de Antígeno/imunologia , Linfócitos T CD8-Positivos/imunologia , Epitopos de Linfócito T/imunologia , Proteínas de Neoplasias/imunologia , Peptídeos/imunologia , Neoplasias da Próstata/imunologia , Receptor EphA2/imunologia , Transferência Adotiva , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacologia , Anticorpos Antineoplásicos/farmacologia , Apresentação de Antígeno/efeitos dos fármacos , Linhagem Celular Tumoral , Modelos Animais de Doenças , Feminino , Humanos , Masculino , Camundongos , Camundongos SCID , Metástase Neoplásica , Transplante de Neoplasias , Neoplasias da Próstata/terapia , Complexo de Endopeptidases do Proteassoma/imunologia , Estrutura Terciária de Proteína , Receptor EphA2/antagonistas & inibidoresRESUMO
PURPOSE: Immune dysfunction reported in renal cell carcinoma (RCC) patients may contribute to tumor progression. Myeloid-derived suppressor cells (MDSC) represent one mechanism by which tumors induce T-cell suppression. Several factors pivotal to the accumulation of MDSC are targeted by the tyrosine kinase inhibitor, sunitinib. The effect of sunitinib on MDSC-mediated immunosuppression in RCC patients has been investigated. EXPERIMENTAL DESIGN: Patient peripheral blood levels of MDSC and regulatory T-cell (Treg) and T-cell production of IFN-gamma were evaluated before and after sunitinib treatment. Correlations between MDSC and Treg normalization as well as T-cell production of IFN-gamma were examined. The in vitro effect of sunitinib on patient MDSC was evaluated. RESULTS: Metastatic RCC patients had elevated levels of CD33(+)HLA-DR(-) and CD15(+)CD14(-) MDSC, and these were partially overlapping populations. Treatment with sunitinib resulted in significant reduction in MDSC measured by several criteria. Sunitinib-mediated reduction in MDSC was correlated with reversal of type 1 T-cell suppression, an effect that could be reproduced by the depletion of MDSC in vitro. MDSC reduction in response to sunitinib correlated with a reversal of CD3(+)CD4(+)CD25(hi)Foxp3(+) Treg cell elevation. No correlation existed between a change in tumor burden and a change in MDSC, Treg, or T-cell production of IFN-gamma. In vitro addition of sunitinib reduced MDSC viability and suppressive effect when used at >/=1.0 microg/mL. Sunitinib did not induce MDSC maturation in vitro. CONCLUSIONS: Sunitinib-based therapy has the potential to modulate antitumor immunity by reversing MDSC-mediated tumor-induced immunosuppression.
Assuntos
Antineoplásicos/farmacologia , Carcinoma de Células Renais/tratamento farmacológico , Indóis/farmacologia , Neoplasias Renais/tratamento farmacológico , Células Mieloides/imunologia , Pirróis/farmacologia , Fatores Supressores Imunológicos/efeitos dos fármacos , Adulto , Idoso , Idoso de 80 Anos ou mais , Carcinoma de Células Renais/imunologia , Feminino , Humanos , Indóis/uso terapêutico , Interferon gama/biossíntese , Neoplasias Renais/imunologia , Masculino , Pessoa de Meia-Idade , Pirróis/uso terapêutico , Sunitinibe , Fatores Supressores Imunológicos/fisiologia , Linfócitos T Reguladores/efeitos dos fármacos , Linfócitos T Reguladores/fisiologiaRESUMO
Renal cell carcinoma (RCC) is the most common type of kidney cancer and comprises several subtypes with unique characteristics. The most common subtype (~70% of cases) is clear-cell RCC. RCC is considered to be an immunogenic tumour but is known to mediate immune dysfunction in large part by eliciting the infiltration of immune-inhibitory cells, such as regulatory T cells and myeloid-derived suppressor cells, into the tumour microenvironment. Several possible mechanisms have been proposed to explain how these multiple tumour-infiltrating cell types block the development of an effective anti-tumour immune response, including inhibition of the activity of effector T cells and of antigen presenting cells via upregulation of suppressive factors such as checkpoint molecules. Targeting immune suppression using checkpoint inhibition has resulted in clinical responses in some patients with RCC and combinatorial approaches involving checkpoint blockade are now standard of care in patients with advanced RCC. However, a substantial proportion of patients do not benefit from checkpoint blockade. The identification of reliable biomarkers of response to checkpoint blockade is crucial to facilitate improvements in the clinical efficacy of these therapies. In addition, there is a need for the development of other immune-based strategies that address the shortcomings of checkpoint blockade, such as adoptive cell therapies.