RESUMO
Although remission rates for metastatic melanoma are generally very poor, some patients can survive for prolonged periods following metastasis. We used gene expression profiling, mitotic index (MI), and quantification of tumor infiltrating leukocytes (TILs) and CD3+ cells in metastatic lesions to search for a molecular basis for this observation and to develop improved methods for predicting patient survival. We identified a group of 266 genes associated with postrecurrence survival. Genes positively associated with survival were predominantly immune response related (e.g., ICOS, CD3d, ZAP70, TRAT1, TARP, GZMK, LCK, CD2, CXCL13, CCL19, CCR7, VCAM1) while genes negatively associated with survival were cell proliferation related (e.g., PDE4D, CDK2, GREF1, NUSAP1, SPC24). Furthermore, any of the 4 parameters (prevalidated gene expression signature, TILs, CD3, and in particular MI) improved the ability of Tumor, Node, Metastasis (TNM) staging to predict postrecurrence survival; MI was the most significant contributor (HR = 2.13, P = 0.0008). An immune response gene expression signature and presence of TILs and CD3+ cells signify immune surveillance as a mechanism for prolonged survival in these patients and indicate improved patient subcategorization beyond current TNM staging.
Assuntos
Regulação Neoplásica da Expressão Gênica/imunologia , Melanoma/diagnóstico , Melanoma/genética , Estadiamento de Neoplasias/métodos , Perfilação da Expressão Gênica/métodos , Humanos , Imuno-Histoquímica , Linfócitos do Interstício Tumoral/patologia , Melanoma/imunologia , Melanoma/secundário , Índice Mitótico/métodos , Análise de Sequência com Séries de Oligonucleotídeos , Prognóstico , Análise de SobrevidaRESUMO
Autologous cellular immunotherapies have been used experimentally in humans to treat many types of cancer. These therapies are divided into two principal types: active cellular immunotherapies that rely on autologous dendritic cells or other antigen presenting cells; and adoptive T-cell therapies, in which large numbers of antigen-specific T lymphocytes are propagated ex vivo and then infused back into the patient. With the FDA approval of the antigen presenting cell vaccine sipuleucel-T for prostate cancer, active immunization has become an accepted approach for the treatment of established cancer.
Assuntos
Vacinas Anticâncer/uso terapêutico , Células Dendríticas/imunologia , Imunoterapia , Linfócitos T/imunologia , Extratos de Tecidos/uso terapêutico , Transferência Adotiva , Animais , Células Apresentadoras de Antígenos/imunologia , Vacinas Anticâncer/imunologia , Humanos , Masculino , Neoplasias da Próstata/terapia , Extratos de Tecidos/imunologiaRESUMO
Dendritic cells (DCs) are important antigen-presenting cells (APCs) that can prime naive T cells and control adaptive immune responses with respect to magnitude, memory and self-tolerance. Understanding the biology of these cells is central to the development of new generation immunotherapies for cancer and chronic infections. This review presents a brief overview of DC biology and of the preparation and use of DC-based vaccines.
Assuntos
Vacinas Anticâncer/uso terapêutico , Células Dendríticas/imunologia , Imunoterapia Ativa/métodos , Infecções/terapia , Neoplasias/terapia , Vacinas/uso terapêutico , Apresentação de Antígeno , Vacinas Anticâncer/imunologia , Doença Crônica , Humanos , Vacinas/imunologiaRESUMO
The purpose of this study was to determine the lineage progression of human and murine very small embryonic-like (HuVSEL or MuVSEL) cells in vitro and in vivo. In vitro, HuVSEL and MuVSEL cells differentiated into cells of all three embryonic germ layers. HuVSEL cells produced robust mineralized tissue of human origin compared with controls in calvarial defects. Immunohistochemistry demonstrated that the HuVSEL cells gave rise to neurons, adipocytes, chondrocytes, and osteoblasts within the calvarial defects. MuVSEL cells were also able to differentiate into similar lineages. First round serial transplants of MuVSEL cells into irradiated osseous sites demonstrated that â¼60% of the cells maintained their VSEL cell phenotype while other cells differentiated into multiple tissues at 3 months. Secondary transplants did not identify donor VSEL cells, suggesting limited self renewal but did demonstrate VSEL cell derivatives in situ for up to 1 year. At no point were teratomas identified. These studies show that VSEL cells produce multiple cellular structures in vivo and in vitro and lay the foundation for future cell-based regenerative therapies for osseous, neural, and connective tissue disorders.
Assuntos
Células-Tronco Embrionárias/citologia , Células-Tronco Multipotentes/citologia , Osteoblastos/citologia , Adipócitos/citologia , Adipócitos/fisiologia , Animais , Biomarcadores/metabolismo , Regeneração Óssea/fisiologia , Diferenciação Celular , Linhagem da Célula/fisiologia , Separação Celular , Tamanho Celular , Células Cultivadas , Condrócitos/citologia , Condrócitos/fisiologia , Células-Tronco Embrionárias/fisiologia , Células-Tronco Embrionárias/transplante , Feminino , Humanos , Masculino , Camundongos , Camundongos SCID , Células-Tronco Multipotentes/fisiologia , Neurônios/citologia , Neurônios/fisiologia , Osteoblastos/fisiologia , Crânio/citologia , Crânio/lesões , Crânio/fisiologiaRESUMO
The method of isolation of bone marrow (BM) mesenchymal stem/stromal cells (MSCs) is a limiting factor in their study and therapeutic use. MSCs are typically expanded from BM cells selected on the basis of their adherence to plastic, which results in a heterogeneous population of cells. Prospective identification of the antigenic profile of the MSC population(s) in BM that gives rise to cells with MSC activity in vitro would allow the preparation of very pure populations of MSCs for research or clinical use. To address this issue, we used polychromatic flow cytometry and counterflow centrifugal elutriation to identify a phenotypically distinct population of mesenchymal stem/progenitor cells (MSPCs) within human BM. The MSPC activity resided within a population of rare, small CD45â»CD73âºCD90âºCD105⺠cells that lack CD44, an antigen that is highly expressed on culture-expanded MSCs. In culture, these MSPCs adhere to plastic, rapidly proliferate, and acquire CD44 expression. They form colony forming units-fibroblast and are able to differentiate into osteoblasts, chondrocytes, and adipocytes under defined in vitro conditions. Their acquired expression of CD44 can be partially downregulated by treatment with recombinant human granulocyte-colony stimulating factor, a response not found in BM-MSCs derived from conventional plastic adherence methods. These observations indicate that MSPCs within human BM are rare, small CD45â»CD73âºCD90âºCD105⺠cells that lack expression of CD44. These MSPCs give rise to MSCs that have phenotypic and functional properties that are distinct from those of BM-MSCs purified by plastic adherence.
Assuntos
Células da Medula Óssea/citologia , Separação Celular/métodos , Tamanho Celular , Citometria de Fluxo/métodos , Receptores de Hialuronatos/metabolismo , Células-Tronco Mesenquimais/citologia , Biomarcadores/metabolismo , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/metabolismo , Diferenciação Celular/efeitos dos fármacos , Fracionamento Celular , Linhagem da Célula/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Centrifugação , Fator Estimulador de Colônias de Granulócitos/farmacologia , Humanos , Imunofenotipagem , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/metabolismo , Fenótipo , Reprodutibilidade dos TestesRESUMO
Progressive multifocal leukoencephalopathy (PML) is a demyelinating disease of the brain caused by JC virus (JCV) for which there is no cure. PML patients who have JCV-specific CD8(+) cytotoxic T lymphocytes (CTL) in their blood have a better clinical outcome. We compared JCV-specific CTL responses in vitro elicited either by JCV peptide-loaded dendritic cells (DC) or by direct peptide stimulation of lymphocytes from 20 HLA-A0201(+) healthy controls, HIV(+) and PML patients. JCV peptide-loaded DC elicited a stronger CTL expansion in 13/15 responders. DC can induce a potent JCV-specific CTL response in vitro, and may constitute a promising approach for PML immunotherapy.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Células Dendríticas/imunologia , Vírus JC/imunologia , Leucoencefalopatia Multifocal Progressiva/imunologia , Adulto , Idoso , Proliferação de Células , Infecções por HIV/complicações , Humanos , Técnicas In Vitro , Pessoa de Meia-IdadeRESUMO
Vitiligo, a skin disorder characterized by the spontaneous destruction of melanocytes, is believed to be of autoimmune origin. We investigated the presence and functionality of CD8(+) T-cells specific for the melanocyte-associated antigens Melan-A, gp100, tyrosinase, and TRP-2 in the blood of HLA-A2(+) vitiligo patients. We enumerated antigen-specific CD8(+) T cells by major histocompatibility complex multimer staining directly ex vivo, as well as after 9 days of in vitro stimulation and assessed IFN-gamma secretion by enzyme-linked immunospot (Elispot) assay. Tyrosinase-, gp100-, or TRP-2-specific CD8(+) T cells could not be identified in the peripheral blood of individuals with vitiligo. Although Melan-A-specific T cells were detectable at levels comparable to Flu-MP-specific T cells by multimer staining, these lymphocytes did not express the skin-homing receptor cutaneous lymphocyte antigen, were phenotypically naïve (CD45RA(+)), and were unresponsive in the IFN-gamma Elispot assay, suggesting that they are unlikely to be involved in the etiopathogenesis of vitiligo.
Assuntos
Antígenos de Neoplasias/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Antígeno HLA-A2/metabolismo , Proteínas de Neoplasias/metabolismo , Vitiligo/sangue , Antígenos de Neoplasias/sangue , Linfócitos T CD8-Positivos/patologia , Estudos de Casos e Controles , Humanos , Interferon gama/metabolismo , Antígeno MART-1 , Glicoproteínas de Membrana/sangue , Glicoproteínas de Membrana/metabolismo , Proteínas de Membrana/metabolismo , Monofenol Mono-Oxigenase/metabolismo , Proteínas de Neoplasias/sangue , Fragmentos de Peptídeos/metabolismo , Vitiligo/etiologia , Vitiligo/patologia , Antígeno gp100 de MelanomaRESUMO
T cell-mediated immunity to microbes and to cancer can be enhanced by the activation of dendritic cells (DCs) via TLRs. In this study, we evaluated the safety and feasibility of topical imiquimod, a TLR7 agonist, in a series of vaccinations against the cancer/testis Ag NY-ESO-1 in patients with malignant melanoma. Recombinant, full-length NY-ESO-1 protein was administered intradermally into imiquimod preconditioned sites followed by additional topical applications of imiquimod. The regimen was very well tolerated with only mild and transient local reactions and constitutional symptoms. Secondarily, we examined the systemic immune response induced by the imiquimod/NY-ESO-1 combination, and show that it elicited both humoral and cellular responses in a significant fraction of patients. Skin biopsies were assessed for imiquimod's in situ immunomodulatory effects. Compared with untreated skin, topical imiquimod induced dermal mononuclear cell infiltrates in all patients composed primarily of T cells, monocytes, macrophages, myeloid DCs, NK cells, and, to a lesser extent, plasmacytoid DCs. DC activation was evident. This study demonstrates the feasibility and excellent safety profile of a topically applied TLR7 agonist used as a vaccine adjuvant in cancer patients. Imiquimod's adjuvant effects require further evaluation and likely need optimization of parameters such as formulation, dose, and timing relative to Ag exposure for maximal immunogenicity.
Assuntos
Adjuvantes Imunológicos/farmacologia , Aminoquinolinas/farmacologia , Antígenos de Neoplasias/imunologia , Vacinas Anticâncer/imunologia , Imunização , Melanoma/imunologia , Proteínas de Membrana/imunologia , Receptor 7 Toll-Like/agonistas , Adjuvantes Imunológicos/efeitos adversos , Adulto , Idoso , Aminoquinolinas/efeitos adversos , Formação de Anticorpos/imunologia , Biópsia , Vacinas Anticâncer/efeitos adversos , Mapeamento de Epitopos , Eritema/induzido quimicamente , Eritema/imunologia , Eritema/patologia , Feminino , Humanos , Imiquimode , Masculino , Melanoma/metabolismo , Melanoma/patologia , Melanoma/terapia , Pessoa de Meia-Idade , Projetos Piloto , Receptor 7 Toll-Like/metabolismoRESUMO
Ikaros is a critical transcriptional regulator of hematopoietic cell differentiation. In addition to its effects on the lymphoid system and hematopoietic stem-cell compartment, we have previously shown that Ikaros is also required for normal erythroid development. In this report, we compare Ikaros-dependent gene expression in erythroid cells of mice lacking the Ikaros protein with that of normal mice in purified adult bone-marrow erythroid cells (BMRC). Gene expression, measured by Affymetrix microarray analysis, indicates that in the BMRC of Ikaros-null mice, there is significant up-regulation of SMADs 6 and 7, serine protease inhibitor 3, and immediate-early protein 3 (IER3), all proteins that play a modulating role in apoptosis. We investigate the role of Ikaros in oxidative stress-induced apoptosis using Annexin-V staining and FACS analysis. We find a decrease in apoptosis in the BMRC of Ikaros-null mice compared to normal mice. This effect is also seen in nonerythroid cells but is stronger in BMRC. We conclude that normal Ikaros function increases normal apoptosis in erythroid cells. The data also suggest that Ikaros plays a role in apoptosis-mediated events in other normal hematopoietic cell lineages.
Assuntos
Apoptose/fisiologia , Células Precursoras Eritroides/fisiologia , Fator de Transcrição Ikaros/biossíntese , Regulação para Cima/fisiologia , Animais , Células Precursoras Eritroides/citologia , Eritropoese , Citometria de Fluxo , Perfilação da Expressão Gênica/métodos , Fator de Transcrição Ikaros/deficiência , Camundongos , Camundongos Knockout , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Estresse Oxidativo/fisiologiaRESUMO
Dendritic cells (DCs) are potent antigen-presenting cells (APC) that are important in the initiation and control of cellular immune responses. Commonly used in T cell-stimulation experiments, DCs are typically "matured" in vitro with microbial products or proinflammatory cytokines, and then loaded with antigens from any number of sources, including peptides, whole proteins, cell lysates, RNA, microbes, or killed tumor cells. This unit presents a simple and commonly used method for the generation of mature human dendritic cells--differentiating them from peripheral blood monocytes.
Assuntos
Células Dendríticas/citologia , Monócitos/citologia , Diferenciação Celular , Células Cultivadas , HumanosRESUMO
Dendritic cells are professional antigen presenting cells that are central to the induction and regulation of immunity. This review discusses recent advances in the understanding of dendritic cell biology.
Assuntos
Células Dendríticas/fisiologia , Animais , Apresentação de Antígeno , Comunicação Celular , Diferenciação Celular , Células Dendríticas/citologia , Células Dendríticas/imunologia , Humanos , Linfócitos/imunologiaRESUMO
Dendritic cells are professional antigen presenting cells that are central to the induction and regulation of immunity. This review discusses recent advances in the understanding of dendritic cell biology.
Assuntos
Células Dendríticas/citologia , Células Dendríticas/imunologia , Animais , Apresentação de Antígeno/imunologia , Antígenos/imunologia , Antígenos/metabolismo , Diferenciação Celular , Células Dendríticas/classificação , Células Dendríticas/metabolismo , Humanos , Linfócitos T/imunologiaRESUMO
Dendritic cells (DCs) are specialized antigen-presenting cells (APCs) that have an unequaled capacity to initiate primary immune responses, including tolerogenic responses. Because of the importance of DCs in the induction and control of immunity, an understanding of their biology is central to the development of potent immunotherapies for cancer, chronic infections, autoimmune disease, and induction of transplantation tolerance. This review discusses recent advances in DC research and the application of this knowledge toward new strategies for the clinical manipulation of DCs for cancer immunotherapy.