RESUMO
Cellular immune responses against the oncoantigen anaplastic lymphoma kinase (ALK) in patients with ALK-positive anaplastic large cell lymphoma (ALCL) have been detected using peptide-based approaches in individuals preselected for human leucocyte antigen (HLA)-A*02:01. In this study, we aimed to evaluate nucleophosmin (NPM)-ALK-specific CD8(+) T cell responses in ALCL patients ensuring endogenous peptide processing of ALK antigens and avoiding HLA preselection. We also examined the HLA class I restriction of ALK-specific CD8(+) T cells. Autologous dendritic cells (DCs) transfected with in-vitro-transcribed RNA (IVT-RNA) encoding NPM-ALK were used as antigen-presenting cells for T cell stimulation. Responder T lymphocytes were tested in interferon-gamma enzyme-linked immunospot (ELISPOT) assays with NPM-ALK-transfected autologous DCs as well as CV-1 in Origin with SV40 genes (COS-7) cells co-transfected with genes encoding the patients' HLA class I alleles and with NPM-ALK encoding cDNA to verify responses and define the HLA restrictions of specific T cell responses. NPM-ALK-specific CD8(+) T cell responses were detected in three of five ALK-positive ALCL patients tested between 1 and 13 years after diagnosis. The three patients had also maintained anti-ALK antibody responses. No reactivity was detected in samples from five healthy donors. The NPM-ALK-specific CD8(+) T cell responses were restricted by HLA-C-alleles (C*06:02 and C*12:02) in all three cases. This approach allowed for the detection of NPM-ALK-reactive T cells, irrespective of the individual HLA status, up to 9 years after ALCL diagnosis.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Linfoma Anaplásico de Células Grandes/imunologia , Proteínas Tirosina Quinases/imunologia , Adolescente , Alelos , Animais , Anticorpos/sangue , Anticorpos/imunologia , Antígenos de Neoplasias/imunologia , Linfócitos T CD8-Positivos/metabolismo , Linhagem Celular , Criança , Pré-Escolar , Antígenos de Histocompatibilidade Classe I/genética , Antígenos de Histocompatibilidade Classe I/imunologia , Humanos , Lactente , Ativação Linfocitária/imunologia , Linfoma Anaplásico de Células Grandes/diagnóstico , Linfoma Anaplásico de Células Grandes/tratamento farmacológico , Linfoma Anaplásico de Células Grandes/metabolismo , Proteínas Tirosina Quinases/metabolismo , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismoRESUMO
Lymphocytes of melanoma patients can be restimulated in vitro with autologous tumor cells to generate antitumor cytolytic T lymphocytes (CTL). Previous reports have indicated that, when such CTL are obtained from HLA-A2 melanoma patients, they often display broad reactivity on A2 melanoma cell lines. Such antitumor CTL clones, which appeared to recognize the same antigen, were isolated from two patients. We report here the cloning of a cDNA that directs the expression of the antigen recognized by these CTL. This cDNA corresponds to the transcript of the tyrosinase gene. The gene was found to be active in all tested melanoma samples and in most melanoma cell lines. Among normal cells, only melanocytes appear to express the gene. The tyrosinase antigen presented by HLA-A2 may therefore constitute a useful target for specific immunotherapy of melanoma. But possible adverse effects of antityrosinase immunization, such as the destruction of normal melanocytes and its consequences, will have to be examined before clinical pilot studies can be undertaken.
Assuntos
Antígenos de Neoplasias/genética , Antígeno HLA-A2/imunologia , Melanoma/imunologia , Monofenol Mono-Oxigenase/genética , Neoplasias Cutâneas/imunologia , Linfócitos T Citotóxicos/imunologia , Animais , Antígenos de Neoplasias/imunologia , Linhagem Celular , Células Clonais , Clonagem Molecular , DNA , Feminino , Humanos , Melanoma/patologia , Monofenol Mono-Oxigenase/imunologia , Neoplasias Cutâneas/patologia , Células Tumorais CultivadasRESUMO
From the peripheral blood of the melanoma patient (AV), we derived cytolytic T lymphocyte (CTL) clones that lysed the autologous tumor line SK-MEL-29, but not autologous EBV-B cells, K562, and other tumor targets. By immunoselection experiments it was shown that the CTL clones recognized at least three different antigens on the autologous tumor cells. We demonstrate here that these melanoma antigens are presented to the CTL in association with HLA-A2. First, HLA-A2-reactive pregnancy sera as well as an mAb against HLA-A2 inhibited the CTL lysis. Second, immunoselected melanoma subclones that were resistant to lysis by CTL clones against the three antigens described were found to lack expression of HLA-A2. By sensitizing the patient's lymphocytes against an HLA-A2- melanoma clone, we established a new series of CTL clones recognizing autologous AV melanoma cells. However, efficient lysis was only seen when target cells were pretreated with IFN-gamma. The lytic activity of these CTL was selectively inhibited by an mAb against a common HLA-B determinant. These results indicate that in addition to HLA-A2, other class I antigens are involved in the recognition of AV melanoma cells by autologous CTL.
Assuntos
Antígenos de Neoplasias/análise , Citotoxicidade Imunológica , Antígenos HLA-A/fisiologia , Melanoma/imunologia , Linfócitos T Citotóxicos/imunologia , Adulto , Anticorpos Monoclonais/imunologia , Células Clonais , Antígenos HLA-A/imunologia , Antígeno HLA-A2 , Humanos , Soros Imunes/imunologia , MasculinoRESUMO
It has been reported previously that antitumor cytolytic T lymphocyte (CTL) clones can be isolated from blood lymphocytes of HLA-A2 melanoma patients, after stimulation in vitro with autologous tumor cells, and that some of these CTL clones lyse most HLA-A2 melanomas. A first antigen recognized by such CTL clones was previously shown to be encoded by the tyrosinase gene. We report here the identification of another gene that also directs the expression of an antigen recognized on most melanomas by CTL clones that are restricted by HLA-A2. The gene, designated Melan-A, is unrelated to any known gene. It is 18 kb long and comprises five exons. Like the tyrosinase gene, it is expressed in most melanoma tumor samples and, among normal cells, only in melanocytes.
Assuntos
Antígenos de Diferenciação/genética , Antígenos de Neoplasias/genética , Antígeno HLA-A2/imunologia , Melanoma/imunologia , Linfócitos T Citotóxicos/imunologia , Sequência de Aminoácidos , Sequência de Bases , DNA Complementar/análise , Expressão Gênica , Humanos , Melanoma/genética , Dados de Sequência Molecular , Monofenol Mono-Oxigenase/genética , Células Tumorais CultivadasRESUMO
T lymphocytes recognize antigens consisting of peptides presented by class I and II major histocompatibility complex (MHC) molecules. The peptides identified so far have been predictable from the amino acid sequences of proteins. We have identified the natural peptide target of a CTL clone that recognizes the tyrosinase gene product on melanoma cells. The peptide results from posttranslational conversion of asparagine to aspartic acid. This change is of central importance for peptide recognition by melanoma-specific T cells, but has no impact on peptide binding to the MHC molecule. This posttranslational modification has not been previously described for any MHC-associated peptide and represents the first demonstration of posttranslational modification of a naturally processed class I-associated peptide. This observation is relevant to the identification and prediction of potential peptide antigens. The most likely mechanism for production of this peptide leads to the suggestion that antigenic peptides can be derived from proteins that are translated into the endoplasmic reticulum.
Assuntos
Antígeno HLA-A2 , Melanoma/imunologia , Proteínas de Membrana/metabolismo , Monofenol Mono-Oxigenase/imunologia , Processamento de Proteína Pós-Traducional , Sequência de Aminoácidos , Antígenos de Neoplasias/imunologia , Asparagina/metabolismo , Ácido Aspártico/biossíntese , Células Clonais , Epitopos , Humanos , Melanoma/enzimologia , Modelos Biológicos , Dados de Sequência Molecular , Fragmentos de Peptídeos/imunologia , Células Tumorais CultivadasRESUMO
A mutated cyclin-dependent kinase 4 (CDK4) was identified as a tumor-specific antigen recognized by HLA-A2. 1-restricted autologous cytolytic T lymphocytes (CTLs) in a human melanoma. The mutated CDK4 allele was present in autologous cultured melanoma cells and metastasis tissue, but not in the patient's lymphocytes. The mutation, an arginine-to-cysteine exchange at residue 24, was part of the CDK4 peptide recognized by CTLs and prevented binding of the CDK4 inhibitor p16INK4a, but not of p21 or of p27KIP1. The same mutation was found in one additional melanoma among 28 melanomas analyzed. These results suggest that mutation of CDK4 can create a tumor-specific antigen and can disrupt the cell-cycle regulation exerted by the tumor suppressor p16INK4a.
Assuntos
Proteínas de Transporte/farmacologia , Proteínas de Ciclo Celular , Quinases Ciclina-Dependentes , Melanoma/imunologia , Proteínas Serina-Treonina Quinases/imunologia , Proteínas Proto-Oncogênicas , Linfócitos T Citotóxicos/imunologia , Proteínas Supressoras de Tumor , Sequência de Aminoácidos , Animais , Sequência de Bases , Proteínas de Transporte/metabolismo , Linhagem Celular , Clonagem Molecular , Quinase 4 Dependente de Ciclina , Inibidor de Quinase Dependente de Ciclina p15 , Inibidor p16 de Quinase Dependente de Ciclina , Inibidor de Quinase Dependente de Ciclina p21 , Inibidor de Quinase Dependente de Ciclina p27 , Ciclinas/metabolismo , Ciclinas/farmacologia , Antígeno HLA-A2/imunologia , Humanos , Melanoma/enzimologia , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Associadas aos Microtúbulos/farmacologia , Dados de Sequência Molecular , Mutação Puntual , Reação em Cadeia da Polimerase , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Transfecção , Células Tumorais CultivadasRESUMO
Acute graft-versus-host disease (aGVHD) is a life-threatening complication after solid-organ transplantation, which is mediated by host-reactive donor T cells emigrating from the allograft. We report on two liver transplant recipients who developed an almost complete donor chimerism in peripheral blood and bone marrow-infiltrating T cells during aGVHD. By analyzing these T cells directly ex vivo, we found that they died by apoptosis over time without evidence of rejection by host T cells. The host-versus-donor reactivity was selectively impaired, as anti-third-party and antiviral T cells were still detectable in the host repertoire. These findings support the acquired donor-specific allotolerance concept previously established in animal transplantation studies. We also observed that the resolution of aGVHD was not accompanied by an expansion of circulating immunosuppressive CD4/CD25/FoxP3-positive T cells. In fact, graft-versus-host-reactive T cells were controlled by an alternative negative regulatory pathway, executed by the programmed death (PD)-1 receptor and its ligand PD-L1. We found high PD-1 expression on donor CD4 and CD8 T cells. In addition, blocking PD-L1 on host-derived cells significantly enhanced alloreactivity by CD8 T cells in vitro. We suggest the interference with the PD-1/PD-L1 pathway as a therapeutic strategy to control graft-versus-host-reactive T cells in allograft recipients.
Assuntos
Antígenos CD/metabolismo , Antígenos de Superfície/metabolismo , Proteínas Reguladoras de Apoptose/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Regulação da Expressão Gênica , Doença Enxerto-Hospedeiro/sangue , Transplante de Fígado/métodos , Animais , Linfócitos T CD4-Positivos/metabolismo , Transplante de Células , Fatores de Transcrição Forkhead/biossíntese , Doença Enxerto-Hospedeiro/diagnóstico , Humanos , Imunossupressores/uso terapêutico , Subunidade alfa de Receptor de Interleucina-2/biossíntese , Masculino , Camundongos , Camundongos Knockout , Pessoa de Meia-Idade , Receptor de Morte Celular Programada 1RESUMO
The key issue in tumor immunology is to identify antigens as target structures for a cancer-selective immunological attack in the tumor-bearing host, resulting in tumor rejection. There is a growing detailed understanding of structural and regulatory gene alterations giving rise to candidate rejection antigens and peptides in tumor cells. As well as reviewing the development of new adjuvant and recombinant vector systems, new approaches are suggested for the construction of cancer vaccines.
Assuntos
Neoplasias/imunologia , Vacinas/imunologia , Animais , Anticorpos Antineoplásicos/imunologia , Formação de Anticorpos , Antígenos de Neoplasias/imunologia , Vacina BCG , Citocinas/biossíntese , Gangliosídeos/imunologia , Humanos , Imunidade Celular , Linfocinas/biossíntese , Camundongos , Neoplasias/prevenção & controle , Neoplasias Experimentais/imunologia , Linfócitos T Citotóxicos/imunologia , Vacinas SintéticasRESUMO
Vaccination with tumor-associated antigens is a promising approach for cancer immunotherapy. Because the majority of these antigens are normal self antigens, they may require suitable delivery systems to promote their immunogenicity. A recombinant vector based on the modified vaccinia virus Ankara (MVA) was used for expression of human tyrosinase, a melanoma-specific differentiation antigen, and evaluated for its efficacy as an antitumor vaccine. Stable recombinant viruses (MVA-hTyr) were constructed that have deleted the selection marker lacZ and efficiently expressed human tyrosinase in primary human cells and cell lines. Tyrosinase-specific human CTLs were activated in vitro by MVA-hTyr-infected, HLA-A*0201-positive human dendritic cells. Importantly, an efficient tyrosinase- and melanoma-specific CTL response was induced in vitro using MVA-hTyr-infected autologous dendritic cells as activators for peripheral blood mononuclear cells derived from HLA-A*0201-positive melanoma patients despite prior vaccination against smallpox. Immunization of HLA-A*0201/Kb transgenic mice with MVA-hTyr induced A*0201-restricted CTLs specific for the human tyrosinase-derived peptide epitope 369-377. These in vivo primed CTLs were of sufficiently high avidity to recognize and lyse human melanoma cells, which present the endogenously processed tyrosinase peptide in the context of A*0201. Tyrosinase-specific CTL responses were significantly augmented by repeated vaccination with MVA-hTyr. These findings demonstrate that HLA-restricted CTLs specific for human tumor-associated antigens can be efficiently generated by immunization with recombinant MVA vaccines. The results are an essential basis for MVA-based vaccination trials in cancer patients.
Assuntos
Vetores Genéticos , Antígenos HLA-A/imunologia , Melanoma/imunologia , Monofenol Mono-Oxigenase/biossíntese , Monofenol Mono-Oxigenase/genética , Linfócitos T Citotóxicos/imunologia , Vaccinia virus , Animais , Linhagem Celular , Células Dendríticas/imunologia , Células Dendríticas/fisiologia , Indução Enzimática , Marcadores Genéticos , Humanos , Ativação Linfocitária , Camundongos , Camundongos Transgênicos , Proteínas Recombinantes de Fusão/biossíntese , Deleção de Sequência , Vacina Antivariólica , TransfecçãoRESUMO
A bispecific antibody construct (bAb) recognising CD3 and epidermal growth factor receptor (EGFR) was studied in vitro. Human peripheral blood lymphocytes (PBL), pre-activated with monoclonal antibody OKT-3 or with irradiated tumour cells, were armed with the bAb construct and targeted to autologous and allogeneic tumour target cells in culture. bAb EGFR x CD3 promoted significant cytolysis even at a concentration of 1 ng/ml. The specificity of target cell lysis was provided by the EGFR specificity of the bAb, as tumour cells negative for EGFR were not lysed. However, not only EGFR-positive tumour cells but also EGFR-positive normal cells were killed. Human renal cancer cell lines and the normal autologous kidney cell cultures expressing the same level of EGFR molecules were lysed to a similar extent. These results may contribute toward the planning of future clinical trials with such bAb.
Assuntos
Anticorpos Biespecíficos/imunologia , Complexo CD3/imunologia , Citotoxicidade Imunológica , Receptores ErbB/imunologia , Neoplasias/terapia , Anticorpos Biespecíficos/uso terapêutico , Humanos , Células Matadoras Naturais/imunologia , Neoplasias/imunologia , Células Tumorais CultivadasRESUMO
Enzyme-linked immunospot (ELISPOT) analysis is a sensitive technique for the detection and quantification of single T lymphocytes forming cytokine spots after antigen contact in vitro. Herein computer-assisted video image analysis (CVIA) was applied to automatically determine the number and size of tumor necrosis factor alpha (TNF-alpha) spots formed by single blood-derived CD8+ T cells after contact with peptide-loaded target cells. With CVIA and TNF-alpha ELISPOT analysis we quantified CD8+ T cells responsive to HLA-A2.1-binding tyrosinase and influenza matrix peptides in healthy donors. We followed the course of the virus-specific T cell response in two HLA-A2-positive patients with reactivation of latent cytomegalovirus (CMV) infection during immunosuppressive therapy. The test proved sufficiently sensitive to detect in the blood of both patients a temporary expansion of CD8+ T lymphocytes reactive with a known immunogenic HLA-A2.1-binding peptide from glycoprotein B of CMV. Reactivity to peptide antigens was not only reflected by numeric increases of spot formation, but also by the appearance of larger spot areas, presumably formed by strongly peptide-reactive CD8+ T cells. We conclude that the combined use of the TNF-alpha ELISPOT assay and CVIA allows reliable monitoring of the T cell responsiveness to peptide antigens in peripheral blood.
Assuntos
Linfócitos T CD8-Positivos/metabolismo , Processamento de Imagem Assistida por Computador/métodos , Peptídeos/imunologia , Fator de Necrose Tumoral alfa/química , Adulto , Linfócitos T CD8-Positivos/imunologia , Linhagem Celular , Citomegalovirus/imunologia , Infecções por Citomegalovirus/imunologia , Ensaio de Imunoadsorção Enzimática/métodos , Antígeno HLA-A2/imunologia , Humanos , Contagem de Linfócitos/métodos , Microscopia de Vídeo/métodos , Pessoa de Meia-Idade , Proteínas do Envelope Viral/imunologiaRESUMO
We applied an enzyme-linked immunospot (ELISPOT) assay for the detection and quantification of blood-derived CD8+ T cells recognizing peptide antigens presented by HLA-A2.1. CD8+ T lymphocytes were isolated from peripheral blood and were stimulated for 40 h with peptide-loaded A2.1-positive 0.174 x CEM.T2 cells. Tumor necrosis factor alpha (TNF-alpha) secreted by single T cells in response to antigen contact was trapped on nitrocellulose membranes precoated with anti-TNF-alpha antibodies and was then immunochemically visualized as spots. With this assay, up to 25% of cloned cytolytic T lymphocytes (CTL) were detected during the test period that recognized defined melanoma antigens in association with HLA-A2.1. CD8+ lymphocytes responsive to a known immunogenic HLA-A2.1-binding peptide from reverse transcriptase of the human immunodeficiency virus (HIV) were only detectable in HIV-infected patients, but not in anti-HIV-negative donors. T cells reacting with a peptide derived from a mutated cyclin-dependent kinase 4 (CDK4-R24C) were exclusively detected among CD8+ lymphocytes isolated from blood of the patient, whose melanoma had previously been found to carry the CDK4-R24C allele. T cells responding to HLA-A2.1-associated peptides of normal melanocyte differentiation antigens tyrosinase and Melan-A/MART-1 were found at low frequencies in almost all donors tested, which might reflect a natural autoimmunity to these antigens. However, in a melanoma patient we found a few days after surgery of melanoma metastases high frequencies of T cells against Melan-A/MART-1 and tyrosinase peptides (up to 38 per 10(5) CD8+ T cells), which gradually decreased during the following months. In an HIV-infected patient with progressive disease we observed a loss of T cells reactive with the HIV reverse transcriptase peptide. These observations provide evidence that peptide-dependent TNF-alpha spot formation in vitro resulted from previous antigen exposure in vivo. Therefore, the TNF-alpha ELISPOT assay might be useful in monitoring antigen-specific T lymphocyte responses during the natural course of diseases as well as during therapeutic interventions aiming at the induction of protective T cell immunity. In addition, it might help to identify immunodominant T cell epitopes.
Assuntos
Antígenos de Neoplasias/imunologia , Linfócitos T CD8-Positivos/metabolismo , Antígenos HIV/imunologia , Antígeno HLA-A2/química , Melanoma/imunologia , Fragmentos de Peptídeos/imunologia , Fator de Necrose Tumoral alfa/metabolismo , Sequência de Aminoácidos , Antígenos de Neoplasias/química , Linfócitos T CD8-Positivos/química , Linfócitos T CD8-Positivos/imunologia , Ensaio de Imunoadsorção Enzimática/métodos , Antígenos HIV/química , Antígeno HLA-A2/imunologia , Humanos , Cinética , Ativação Linfocitária , Contagem de Linfócitos , Dados de Sequência Molecular , Ligação Proteica/imunologia , Células Tumorais CultivadasRESUMO
CD8-positive cytotoxic T cells (CTL) recognize antigenic peptides in combination with major histocompatibility complex (MHC) class I molecules on the surface of syngeneic antigen presenting cells (APC). In the present paper we show that cells from tumor antigen-specific CTL clones present their cognate antigenic peptide to other CTL from the same clone. Inter-CTL peptide presentation resulted in activation of the cells of one CTL clone to MHC-unrestricted lysis of bystander cells. In contrast to the behaviour of this clone, another CTL clone did not lyse bystander cells after incubation with the cognate peptide, but was activated to self-destruction. The human herpes virus Epstein-Barr virus is involved in the pathogenesis of a broad spectrum of human neoplasias. Using freshly established non-clonal T cells with specificity for a peptide derived from an Epstein-Barr virus encoded antigen we found again lysis of MHC mismatched bystander cells as a consequence of inter-CTL peptide presentation, indicating that bystander lysis following antigen self-presentation is not a phenomenon restricted to long-term in vitro cultured T cell clones. The potential implications for immunosurveillance against cancer and for tumor escape mechanisms are discussed.
Assuntos
Apresentação de Antígeno/imunologia , Linfócitos T Citotóxicos/imunologia , Linhagem Celular Transformada , Citotoxicidade Imunológica , Antígeno HLA-A2/imunologia , Humanos , Células Tumorais CultivadasRESUMO
Therapeutic effects of haematopoietic stem cell transplantation are not limited to maximal chemoradiotherapy and subsequent bone marrow regeneration, but include specific as well as unspecific immune reactions known as graft-versus-leukaemia (GvL) effects. Specific immune reactions are likely to be particularly relevant to the long-term treatment of diseases, such as chronic myeloid leukaemia (CML), in which residual cells may remain quiescent and unresponsive to cytotoxic and molecular therapies for long periods of time. Specific GvL effects result from the expression on leukaemic cells of specific tumour-associated antigens (TAAs) in the context of HLA proteins. As human leukocyte antigen (HLA) types vary widely, the development of broadly applicable tumour vaccines will require the identification of multiple TAAs active in different HLA backgrounds. Here, we describe the identification of NM23-H2 as a novel HLA-A32-restricted TAA of CML cells and demonstrate the presence of specifically reactive T cells in a patient 5 years after transplantation. As the NM23 proteins are aberrantly expressed in a range of different tumours, our findings suggest potential applications beyond CML and provide a new avenue of investigation into the molecular mechanisms underlying CML.
Assuntos
Leucemia Mielogênica Crônica BCR-ABL Positiva/imunologia , Nucleosídeo NM23 Difosfato Quinases/imunologia , Adulto , Células Apresentadoras de Antígenos/imunologia , DNA Complementar , Ensaio de Imunoadsorção Enzimática , Humanos , Hibridização in Situ Fluorescente , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Leucemia Mielogênica Crônica BCR-ABL Positiva/cirurgia , Masculino , Nucleosídeo NM23 Difosfato Quinases/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Linfócitos T/imunologiaRESUMO
The interpretation of the results obtained from immunomonitoring of clinical trials is a difficult task due to the variety of methods and protocols available to detect vaccine-specific T-cell responses. This heterogeneity as well as the lack of standards has led to significant scepticism towards published results. In February 2005, a working group was therefore founded under the aegis of the Association for Immunotherapy of Cancer ("CIMT") in order to compare techniques and protocols applied for the enumeration of antigen-specific T-cell responses. Here we present the results from two consecutive phases of an international inter-laboratory testing project referred to as the "CIMT monitoring panel". A total of 13 centers from six European countries participated in the study in which pre-tested PBMC samples, synthetic peptides and PE-conjugated HLA-tetramers were prepared centrally and distributed to participants. All were asked to determine the number of antigen-specific T-cells in each sample using tetramer staining and one functional assay. The results of the first testing round revealed that the total number of cells analyzed was the most important determinant for the sensitive detection of antigen-specific CD8(+) T-cells by tetramer staining. Analysis by ELISPOT was influenced by a combination of cell number and a resting phase after thawing of peripheral blood mononuclear cells. Therefore, the experiments were repeated in a second phase but now the participants were asked to change their protocols according to the new guidelines distilled from the results of the first phase. The recommendations improved the number of antigen-specific T-cell responses that were detected and decreased the variability between the laboratories. We conclude that a two-step approach in inter-laboratory testing allows the identification of distinct variables that influence the sensitivity of different T-cell assays and to formally show that a defined correction to the protocols successfully increases the sensitivity and reduces the inter-center variability. Such "two-step" inter-laboratory projects could define rational bases for accepted international guidelines and thereby lead to the harmonization of the techniques used for immune monitoring.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Epitopos/imunologia , Antígenos HLA-A/imunologia , Monitorização Imunológica/métodos , Monitorização Imunológica/normas , Linfócitos T CD8-Positivos/química , Ensaio de Imunoadsorção Enzimática/métodos , Ensaio de Imunoadsorção Enzimática/normas , Europa (Continente) , Citometria de Fluxo/métodos , Citometria de Fluxo/normas , Antígenos HLA-A/química , Humanos , Imunoterapia , Leucócitos Mononucleares/imunologia , Peptídeos/síntese química , Peptídeos/química , Peptídeos/imunologia , Comitê de Profissionais , Valores de Referência , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Coloração e RotulagemRESUMO
The immunological and molecular mechanisms that govern T cell responses to human malignant tumours are just starting to be understood within a more complex framework of humoral, cellular and molecular interactions. The definition of multiple antigens simultaneously expressed on human melanoma, as detected with cytolytic T cells in immunoselection experiments, is a first step towards the molecular characterization of these antigens. Observations on the influence of expression of restriction elements of the major histocompatibility complex on the recognition of these tumour associated antigens have advanced our understanding of how the immune system responds to cancer cells in vivo. It is specificity that is tuning the immune system, not only in cancer. The molecular characterization of the first human cancer antigen recognized by CTL is now under way as outlined by Boon et al in this issue.
Assuntos
Neoplasias/imunologia , Linfócitos T/imunologia , Antígenos de Neoplasias/imunologia , Células Clonais , Humanos , Imunoterapia , Melanoma/imunologia , Neoplasias/terapia , Sensibilidade e EspecificidadeRESUMO
Stage IV melanoma is still a disease with poor prognosis. Although modern chemo- or chemoimmunotherapies give high response rates in stage IV patients, remissions are usually followed by fast relapses. In order to avoid early relapses after chemotherapy, patients with stage IV disease and either stable disease or partial or complete remission following therapy were treated with 9 million IU IFN alpha subcutaneously 5 times weekly and 6 million IU IL-2 subcutaneously twice weekly. Compared with untreated controls, the rate of progression in the treatment group was reduced from 95% to 35%. Also, time to progression was significantly prolonged. Median survival times in the control group were 25 weeks, whereas median survival time in the treatment group has not yet been reached. Furthermore, TNF-ELISPOT assays showed a significant increase in MAGE-3 reactive cytotoxic T-cells in the treatment, but not in the control group. Thus, immunotherapy in stage IV disease seems to prolong survival in melanoma patients.
Assuntos
Interferon-alfa/administração & dosagem , Interleucina-2/administração & dosagem , Melanoma/terapia , Neoplasias Cutâneas/terapia , Adulto , Idoso , Terapia Combinada , Feminino , Humanos , Injeções Subcutâneas , Interferon alfa-2 , Interferon-alfa/efeitos adversos , Interleucina-2/efeitos adversos , Masculino , Melanoma/imunologia , Melanoma/patologia , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Proteínas Recombinantes , Neoplasias Cutâneas/imunologia , Neoplasias Cutâneas/patologia , Resultado do TratamentoRESUMO
Epoxide hydrolase (epoxide hydratase, epoxide hydrase, E.C. 3.3.2.3) activity so far has only been measured in subcellular preparations. We show here that, with the highly lipophilic substrate (3H)-benzo(a)pyrene 4,5-oxide, the activity can be determined in intact cells. Whole human lymphocytes hydrolyze it at a similar rate to that in lymphocyte homogenate. We have previously reported that cultivation of lymphocytes in a medium containing 5,6-benzoflavone leads to an increase in epoxide hydrolase activity. We now demonstrate that this stimulation is due to enzyme activation and that enzyme induction does not contribute to this increase to any measurable extent. Moreover, both 5,6-benzoflavone and 7,8-benzoflavone activate epoxide hydrolase. This activation occurs not only in cell homogenate, but also - with a similar concentration-response relationship - in whole lymphocytes. Hence measurement of epoxide hydrolase activity in subcellular preparations reflects the activity in these intact cells. Furthermore, insofar as a concentration of 1 microM of the benzoflavones is sufficient to cause a measurable (10 to 20%) activation, it appears likely that foreign compounds can activate epoxide hydrolase in man.
Assuntos
Benzoflavonas/farmacologia , Epóxido Hidrolases/metabolismo , Flavonoides/farmacologia , Linfócitos/enzimologia , Ativação Enzimática/efeitos dos fármacos , Humanos , Técnicas In Vitro , beta-NaftoflavonaRESUMO
A limiting-dilution assay was developed and used to determine the frequency of autologous tumor-reactive cytotoxic T lymphocytes (CTL) in peripheral blood of a melanoma patient MZ2, who has been free of detectable disease since several years. In this patient, the frequencies of tumor-reactive CTL spontaneously varied only by a factor of 1.5. After vaccinations with autologous mutagenized and lethally irradiated tumor cells a two- to tenfold increase in frequencies of tumor-reactive CTL was found within the first 2 weeks. Thereafter, CTL frequencies returned to values measured prior to vaccinations. We conclude, that the limiting-dilution assay applied in this study can detect changes in the T cell response to autologous tumor cells. The frequency of tumor-reactive CTL determined with this approach can serve as an immunological parameter for monitoring the T cell response to autologous tumor cells in individual cancer patients receiving tumor cell vaccinations.
Assuntos
Imunoterapia , Melanoma/sangue , Melanoma/imunologia , Linfócitos T Citotóxicos/citologia , Linfócitos T Citotóxicos/imunologia , Vacinação , Células Cultivadas , Citotoxicidade Imunológica , Humanos , Contagem de Leucócitos , Teste de Cultura Mista de Linfócitos , Melanoma/terapia , Reprodutibilidade dos Testes , Células Tumorais CultivadasRESUMO
Cytolytic T-lymphocyte (CTL) clones against an autologous melanoma (SK-MEL-29) were generated by mixed lymphocyte tumor culture and subsequent cloning of responder lymphocytes at limiting dilutions. These CTL clones lysed autologous melanoma but not autologous Epstein-Barr virus-transformed B cells and none of the allogeneic tumor targets included in the specificity analysis. The lysis of autologous melanoma targets could be inhibited by monoclonal antibodies against monomorphic HLA class I determinants. For proliferation of CTLs, the stimulation with the relevant target antigen on autologous tumor cells was essential. Immunoselection experiments carried out with two CTL clones revealed the existence of melanoma subclones that were resistant to lysis by the CTL clones used for immunoselection but were still lysed by other autologous CTL clones. This analysis allowed us to identify three stable simultaneously expressed antigens on the melanoma cells defined by autologous CTLs.