RESUMO
Langerhans cells (LCs) represent a subset of immature dendritic cells (DCs) specifically localized in the epidermis and other mucosal epithelia. As surrounding keratinocytes can produce interleukin (IL)-15, a cytokine that utilizes IL-2Rgamma chain, we analyzed whether IL-15 could skew monocyte differentiation into LCs. Monocytes cultured for 6 d with granulocyte/macrophage colony-stimulating factor (GM-CSF) and IL-15 differentiate into CD1a(+)HLA-DR(+)CD14(-)DCs (IL15-DCs). Agents such as lipopolysaccharide (LPS), tumor necrosis factor (TNF)alpha, and CD40L induce maturation of IL15-DCs to CD83(+), DC-LAMP(+) cells. IL15-DCs are potent antigen-presenting cells able to induce the primary (mixed lymphocyte reaction [MLR]) and secondary (recall responses to flu-matrix peptide) immune responses. As opposed to cultures made with GM-CSF/IL-4 (IL4-DCs), a proportion of IL15-DCs expresses LC markers: E-Cadherin, Langerin, and CC chemokine receptor (CCR)6. Accordingly, IL15-DCs, but not IL4-DCs, migrate in response to macrophage inflammatory protein (MIP)-3alpha/CCL20. However, IL15-DCs cannot be qualified as "genuine" Langerhans cells because, despite the presence of the 43-kD Langerin, they do not express bona fide Birbeck granules. Thus, our results demonstrate a novel pathway in monocyte differentiation into dendritic cells.
Assuntos
Células Dendríticas/citologia , Interleucina-15/farmacologia , Células de Langerhans/citologia , Monócitos/citologia , Monócitos/efeitos dos fármacos , Apresentação de Antígeno , Diferenciação Celular , Células Dendríticas/classificação , Células Epidérmicas , Epiderme/imunologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Humanos , Células de Langerhans/classificação , Teste de Cultura Mista de Linfócitos , FenótipoRESUMO
The goal of tumor immunotherapy is to elicit immune responses against autologous tumors. It would be highly desirable that such responses include multiple T cell clones against multiple tumor antigens. This could be obtained using the antigen presenting capacity of dendritic cells (DCs) and cross-priming. That is, one could load the DC with tumor lines of any human histocompatibility leukocyte antigen (HLA) type to elicit T cell responses against the autologous tumor. In this study, we show that human DCs derived from monocytes and loaded with killed melanoma cells prime naive CD45RA(+)CD27(+)CD8(+) T cells against the four shared melanoma antigens: MAGE-3, gp100, tyrosinase, and MART-1. HLA-A201(+) naive T cells primed by DCs loaded with HLA-A201(-) melanoma cells are able to kill several HLA-A201(+) melanoma targets. Cytotoxic T lymphocyte priming towards melanoma antigens is also obtained with cells from metastatic melanoma patients. This demonstration of cross-priming against shared tumor antigens builds the basis for using allogeneic tumor cell lines to deliver tumor antigens to DCs for vaccination protocols.
Assuntos
Antígenos de Neoplasias/imunologia , Células Dendríticas/imunologia , Melanoma/imunologia , Linfócitos T Citotóxicos/imunologia , Vacinas Anticâncer/imunologia , Diferenciação Celular , Células Cultivadas , Humanos , Células K562 , Antígeno MART-1 , Melanoma/patologia , Glicoproteínas de Membrana/imunologia , Monofenol Mono-Oxigenase/imunologia , Proteínas de Neoplasias/imunologia , Estadiamento de Neoplasias , Linfócitos T Citotóxicos/citologia , Células Tumorais Cultivadas , Antígeno gp100 de MelanomaRESUMO
Dendritic cells (DCs) are antigen-presenting cells specialized to initiate and maintain immunity and tolerance. DCs initiate immune responses in a manner that depends on signals they receive from pathogens, surrounding cells and their products. Most tumors are infiltrated by DCs. Thus, interactions between DCs and dying tumor cells may determine the balance between immunity and tolerance to tumor cells. In addition, DCs also display non-immunologic effects on tumors and the tumor microenvironment. Therefore, improved understanding of the cross talk between tumor cells and DCs may suggest new approaches to improve cancer therapy.
Assuntos
Citocinas/metabolismo , Células Dendríticas/imunologia , Células Dendríticas/fisiologia , Neoplasias/imunologia , Neoplasias/fisiopatologia , Receptores de Superfície Celular/metabolismo , Receptores Toll-Like/metabolismo , Animais , Morte Celular , Citocinas/imunologia , Humanos , Tolerância Imunológica , Imunidade Celular , Imunoterapia , Neoplasias/patologia , Neoplasias/terapia , Receptores de Superfície Celular/imunologia , Transdução de Sinais , Linfócitos T Reguladores/imunologia , Receptores Toll-Like/imunologiaRESUMO
Dendritic cells (DCs) are important in regulating both immunity and tolerance. Hence, we hypothesized that systemic lupus erythematosus (SLE), an autoimmune disease characterized by autoreactive B and T cells, may be caused by alterations in the functions of DCs. Consistent with this, monocytes from SLE patients' blood were found to function as antigen-presenting cells, in vitro. Furthermore, serum from SLE patients induced normal monocytes to differentiate into DCs. These DCs could capture antigens from dying cells and present them to CD4-positive T cells. The capacity of SLE patients' serum to induce DC differentiation correlated with disease activity and depended on the actions of interferon-alpha (IFN-alpha). Thus, unabated induction of DCs by IFN-alpha may drive the autoimmune response in SLE.
Assuntos
Células Dendríticas/citologia , Células Dendríticas/imunologia , Interferon-alfa/fisiologia , Lúpus Eritematoso Sistêmico/imunologia , Monócitos/imunologia , Adolescente , Apresentação de Antígeno , Antígenos CD/análise , Contagem de Células Sanguíneas , Linfócitos T CD4-Positivos/imunologia , Diferenciação Celular , Células Cultivadas , Criança , Homeostase , Humanos , Interferon-alfa/sangue , Interferon-alfa/farmacologia , Lúpus Eritematoso Sistêmico/sangue , Teste de Cultura Mista de Linfócitos , Monócitos/citologiaRESUMO
Immunization to multiple defined tumor antigens for specific immune therapy of human cancer has thus far proven difficult. Eighteen HLA A*0201(+) patients with metastatic melanoma received injections s.c. of CD34(+)progenitor-derived autologous dendritic cells (DCs), which included Langerhans cells. DCs were pulsed with peptides derived from four melanoma antigens [(MelAgs) MelanA/MART-1, tyrosinase, MAGE-3, and gp100], as well as influenza matrix peptide (Flu-MP) and keyhole limpet hemocyanin (KLH) as control antigens. Overall immunological effects were assessed by comparing response profiles using marginal likelihood scores. DC injections were well tolerated except for progressive vitiligo in two patients. DCs induced an immune response to control antigens (KLH, Flu-MP) in 16 of 18 patients. An enhanced immune response to one or more MelAgs was seen in these same 16 patients, including 10 patients who responded to >2 MelAgs. The two patients failing to respond to both control and tumor antigens experienced rapid tumor progression. Of 17 patients with evaluable disease, 6 of 7 patients with immunity to two or less MelAgs had progressive disease 10 weeks after study entry, in contrast to tumor progression in only 1 of 10 patients with immunity to >2 MelAgs. Regression of >1 tumor metastases were observed in seven of these patients. The overall immunity to MelAgs after DC vaccination is associated with clinical outcome (P = 0.015).
Assuntos
Antígenos CD34/imunologia , Vacinas Anticâncer/imunologia , Vacinas Anticâncer/uso terapêutico , Células Dendríticas/imunologia , Melanoma/imunologia , Melanoma/terapia , Adulto , Idoso , Antígenos de Neoplasias/imunologia , Linfócitos T CD8-Positivos/imunologia , Vacinas Anticâncer/efeitos adversos , Feminino , Antígeno HLA-A2/imunologia , Hemocianinas/imunologia , Humanos , Hipersensibilidade Tardia/imunologia , Imunoterapia Adotiva/métodos , Células de Langerhans/imunologia , Masculino , Pessoa de Meia-Idade , Resultado do TratamentoRESUMO
It is generally believed that relapse of acute leukemia heralds progression of the disease into a more aggressive stage. The biological behavior of leukemic cells collected from four patients with adult acute lymphoblastic leukemia (ALL) prior to treatment and at relapse was studied after engraftment into 28 unconditioned mice with severe combined immunodeficiency (SCID). Leukemic cells engrafted in all but one mouse, with major differences observed in the growth and aggressiveness of the leukemias. Recipient mice of cells derived from all patients at relapse died more rapidly in overt leukemia than those which were injected with cells obtained prior to induction treatment (P=0.0002). SCID mice that received cells from one patient at the time of diagnosis also died in terminal leukemia. Other SCID mice however, that received cells from the remaining three patients prior to treatment developed occult leukemia that was detectable in the blood or bone marrow with the use of polymerase chain reaction (PCR) or flow cytometry only. Leukemic cells recovered from mice with terminal leukemia exhibited a larger proliferating fraction than cells originally injected (P=0.004). Our results demonstrate, that during the evolution from initial presentation to relapse, ALL cells may acquire biological properties which render them more aggressive in SCID mice.
Assuntos
Sobrevivência de Enxerto , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Adolescente , Adulto , Animais , Ciclo Celular , Humanos , Camundongos , Camundongos SCID , Pessoa de Meia-Idade , Transplante de Neoplasias , Reação em Cadeia da Polimerase , Leucemia-Linfoma Linfoblástico de Células Precursoras/diagnóstico , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , RNA Mensageiro/análise , RecidivaRESUMO
Conjugate formation by AML blasts with fresh peripheral blood lymphocytes (PBL) and lymphokine activated killer (LAK) effectors was studied by flow cytometry. Leukemic blasts formed very low numbers of conjugates with fresh PBL and were resistant to natural killer (NK) cytotoxicity. When LAK effectors were used a significant increase in conjugate formation was observed, which in the majority of cases was followed by an increased killing. There was a positive correlation between the percentages of conjugates formed by AML blasts with LAK effectors and the susceptibility to lysis. No significant difference in binding activity between the CD3+ and CD56+ LAK subpopulations was found. There was no correlation between the expression of ICAM-1, LFA-3 and Transferrin receptor (CD71) and the conjugate formation. The blocking of CD71 on the control K562 cell line reduced the conjugate formation with LAK effectors but no such effect could be observed with CD71+ AML blasts.
Assuntos
Citotoxicidade Imunológica , Células Matadoras Ativadas por Linfocina/imunologia , Células Matadoras Naturais/imunologia , Leucemia Mieloide Aguda/patologia , Antígenos CD/análise , Antígenos CD/fisiologia , Antígenos de Diferenciação de Linfócitos B/análise , Antígenos de Diferenciação de Linfócitos B/fisiologia , Moléculas de Adesão Celular/análise , Humanos , Molécula 1 de Adesão Intercelular , Interleucina-2/farmacologia , Leucemia Mieloide Aguda/imunologia , Receptores da TransferrinaRESUMO
The expression of adhesion molecules on blasts from 14 patients with acute myeloid leukemia (AML) was investigated by immunofluorescence and flow cytofluorometry. All tested blast populations expressed CD18/CD11a complex [leukocyte function antigen-1 (LFA-1)] and CD29 (very-late antigen (VLA)) and the majority were positive for CD54 [intercellular adhesion molecule-1 (ICAM-1), 78.6%] and CD56 [neural cell adhesion molecule (NCAM), 64.3%]. The expression of two other alpha chains of CD18/CD11b and CD11c varied considerably (64.3% and 42.8% of positive cases, respectively). Only one case (AML-M4) showed a weak expression of the activated platelet antigen CD41b. None of the tested blasts expressed the vitronectin receptor (CD61/CD51). No significant correlation between the expression of adhesion molecules and the FAB type of leukemia could be found. All tested blast populations were completely resistant to NK-mediated cytotoxicity and relatively resistant to LAK-mediated cytotoxicity in the standard 51Cr release assay. While no statistically significant correlation of the results in cytotoxicity assays with the expression of adhesion molecules or the expression of HLA-DR antigen could be observed, 2 out of 3 completely resistant cases lacked ICAM-1. These results show that even leukemic blasts which express all of the tested adhesion molecules can still be resistant to LAK-mediated cytotoxicity.
Assuntos
Moléculas de Adesão Celular/biossíntese , Citotoxicidade Imunológica , Células Matadoras Ativadas por Linfocina/fisiologia , Leucemia Mieloide/imunologia , Linfócitos/imunologia , Adulto , Idoso , Antígenos CD/biossíntese , Antígenos CD11 , Antígenos CD18 , Feminino , Citometria de Fluxo , Imunofluorescência , Antígenos HLA-DR/biossíntese , Humanos , Imunofenotipagem , Técnicas In Vitro , Integrina beta1 , Molécula 1 de Adesão Intercelular , Células Matadoras Naturais/fisiologia , Antígeno-1 Associado à Função Linfocitária/biossíntese , Masculino , Pessoa de Meia-IdadeRESUMO
The kinetics of conjugate formation between leukemic cell lines (K562 and Daudi) and lymphokine-activated killer (LAK) cells was studied. A flow cytofluorometry method using double immunofluorescence staining was applied. During the first 15 min of incubation of LAK effectors with leukemic targets, a rapid binding occurred, followed by a plateau phase lasting until 30 min of observation. A considerable, yet not statistically significant, between-donor variability was noticed. A mathematical model of conjugate formation kinetics, based on the analogy to enzyme kinetics, was formulated and validated. Parameters of the model were related to the binding capacity of effector and target cells, and to the lifetime of conjugates and free cells. The concordance of theoretical curves with experimental data proved that the described model can be considered as a useful tool for the evaluation of kinetic and dynamic characterization of conjugate formation between leukemic targets and LAK effectors.
Assuntos
Testes Imunológicos de Citotoxicidade , Citometria de Fluxo/métodos , Células Matadoras Ativadas por Linfocina/imunologia , Adesão Celular , Humanos , Cinética , Leucemia Eritroblástica Aguda , Linfoma de Células B , Modelos Biológicos , Reprodutibilidade dos Testes , Células Tumorais CultivadasRESUMO
Conjugate formation by natural killer (NK)-resistant and NK-sensitive leukaemic cell lines with fresh and IL-2-stimulated (lymphokine-activated killer, LAK) peripheral blood lymphocytes (PBL) was studied by a flow cytofluorometry method with double staining. A significant difference in binding of NK-resistant T-cell lymphoma (HuT 78) and NK-sensitive myeloid (K562) blasts to fresh PBL was observed (P < 0.01). Activation of lymphocytes with IL-2 resulted in a significant increase of binding and killing of both K562 and HuT 78. However, in the case of blasts from NK-resistant cell line Daudi a similar conjugate formation with fresh PBL and LAK effectors was observed, despite a significant increase in killing. Various subpopulations of LAK effectors (CD3, CD16 and CD56 positive) displayed similar binding activity towards myeloid (K562) and lymphoid (Raji) blasts, which shows that conjugate formation occurs not only with NK-cell-derived, but also with T-cell-derived LAK cells. The blocking of CD71 antigen (transferrin receptor) on K562 blasts inhibited binding of cytotoxic lymphocytes, which was mostly due to the blocking of binding of CD56+ subpopulation. Our results indicate that the resistance of leukaemic blasts to cell-mediated cytotoxicity may depend on different (and probably several) steps of this process.
Assuntos
Antígenos CD/metabolismo , Antígenos de Diferenciação de Linfócitos B/metabolismo , Células Matadoras Ativadas por Linfocina/metabolismo , Leucemia/patologia , Subpopulações de Linfócitos/metabolismo , Anticorpos/metabolismo , Antígenos CD/imunologia , Antígenos de Diferenciação de Linfócitos B/imunologia , Antígenos de Diferenciação de Linfócitos T/metabolismo , Ligação Competitiva , Complexo CD3/metabolismo , Antígeno CD56 , Humanos , Células Matadoras Ativadas por Linfocina/imunologia , Leucemia/metabolismo , Receptores da TransferrinaRESUMO
The expression and function of neural cell adhesion molecule (NCAM, CD56, Leu19) on leukaemic blasts was investigated. The expression of NCAM was frequent (64%) in 14 studied cases of acute myeloid leukaemia (AML) but not in chronic lymphocytic leukaemia (CLL: 1/3 cases positive) or immunocytoma (IC: no positivity). No correlation with the expression of other AM (intercellular adhesion molecule-1 (ICAM-1), leucocyte function antigen-1 (LFA-1). VLA beta chain) or with AML type, according to FAB classification, was observed. Blocking of NCAM with anti-Leu 19 MoAb on AML targets resulted in a significant decrease of their susceptibility to LAK killing and in inhibition of conjugate formation. In the case of B prolymphocytic leukaemia (B-PLL) which did not express ICAM-1 or LFA-1 but was NCAM+, a complete resistance to LAK activity and lack of conjugate formation was observed. Blocking of NCAM on LAK effectors did not decrease their cytotoxic activity. Our results suggest that NCAM, in the presence of other AM, may have a supportive role in adhesion of leukaemic targets to LAK effectors.
Assuntos
Antígenos CD/metabolismo , Antígenos de Diferenciação de Linfócitos T/metabolismo , Moléculas de Adesão Celular Neuronais/metabolismo , Células Matadoras Ativadas por Linfocina/imunologia , Leucemia Mieloide Aguda/imunologia , Adulto , Idoso , Antígeno CD56 , Adesão Celular , Citotoxicidade Imunológica , Humanos , Imunidade Celular , Células Matadoras Ativadas por Linfocina/citologia , Leucemia Mieloide Aguda/patologia , Pessoa de Meia-IdadeRESUMO
Monocytes can give rise to either antigen presenting dendritic cells (DCs) or scavenging macrophages. This differentiation is initiated when monocytes cross the endothelium. But the regulation of DC and macrophage differentiation in tissues remains elusive. When stimulated with granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4), monocytes yield DCs. However, we show here that the addition of fibroblasts switches differentiation to macrophages. On contact with monocytes, fibroblasts release IL-6, which up-regulates the expression of functional M-CSF receptors on monocytes. This allows the monocytes to consume their autocrine M-CSF. Thus, the interplay between IL-6 and M-CSF switches monocyte differentiation to macrophages rather than DCs, and IL-6 is an essential factor in the molecular control of antigen presenting cell development.
Assuntos
Células Dendríticas/citologia , Células Dendríticas/imunologia , Interleucina-6/metabolismo , Macrófagos/citologia , Macrófagos/imunologia , Monócitos/citologia , Monócitos/imunologia , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Técnicas de Cocultura , Células Dendríticas/efeitos dos fármacos , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Humanos , Interleucina-4/farmacologia , Receptores de Lipopolissacarídeos/metabolismo , Fator Estimulador de Colônias de Macrófagos/metabolismo , Macrófagos/efeitos dos fármacos , Monócitos/efeitos dos fármacos , Receptor de Fator Estimulador de Colônias de Macrófagos/metabolismoRESUMO
Strength of binding (Sb), a new parameter defined by the ratio of binding capacity and dissociation rate constants, was estimated and used for the characterization of conjugate formation between leukemic blasts and cytotoxic lymphocytes. Lymphokine activated killer (LAK) cells, i.e., interleukin-2 activated peripheral blood lymphocytes (PBL) displayed significantly (P < 0.001) higher Sb with leukemic blasts when compared to fresh, nonactivated PBL. Interaction of both fresh PBL and LAK effector cells with acute myeloid leukemia (AML) blasts was characterized by a significantly (P < 0.05) lower Sb when compared to the K562 cell line. Interaction of LAK effector cells with leukemic cell-lines of lymphoid origin (Daudi, Raji, and HuT78) was characterized by a significantly (P < 0.005) lower Sb than with K562 myeloid cell line. Sb for the interaction of natural killer (NK)-derived CD16+/CD56+ LAK with leukemic blasts was significantly (P < 0.001) higher than that of T-cell-derived CD3+LAK. We conclude that calculation of Sb provides a relatively simple and independent way to evaluate systems of interacting cells at a single time and may be used to compare results between different cell systems and laboratories.
Assuntos
Células Matadoras Naturais/imunologia , Leucemia Monocítica Aguda/imunologia , Leucemia Mieloide Aguda/imunologia , Leucemia Mielomonocítica Aguda/imunologia , Computação Matemática , Humanos , Modelos Imunológicos , Células Tumorais CultivadasRESUMO
Previous studies have analyzed the lymphoid and myeloid foci within the gingival mucosa in health and chronic periodontitis (CP); however, the principal APCs responsible for the formation and organizational structure of these foci in CP have not been defined. We show that in human CP tissues, CD1a(+) immature Langerhans cells predominantly infiltrate the gingival epithelium, whereas CD83(+) mature dendritic cells (DCs) specifically infiltrate the CD4(+) lymphoid-rich lamina propria. In vivo evidence shows that exacerbation of CP results in increased levels of proinflammatory cytokines that mediate DC activation/maturation, but also of counterregulatory cytokines that may prevent a Th-polarized response. Consistently, in vitro-generated monocyte-derived DCs pulsed with Porphyromonas gingivalis strain 381 or its LPS undergo maturation, up-regulate accessory molecules, and release proinflammatory (IL-1beta, PGE(2)) and Th (IL-10, IL-12) cytokines. Interestingly, the IL-10:IL-12 ratio elicited from P. gingivalis-pulsed DCs was 3-fold higher than that from Escherichia coli-pulsed DCs. This may account for the significantly (p < 0.05) lower proliferation of autologous CD4(+) T cells and reduced release of IFN-gamma elicited by P. gingivalis-pulsed DCs. Taken together, these findings suggest a previously unreported mechanism for the pathophysiology of CP, involving the activation and in situ maturation of DCs by the oral pathogen P. gingivalis, leading to release of counterregulatory cytokines and the formation of T cell-DC foci.