RESUMO
Tumor cells always exhibit differences to normal cells. These differences can be recognized by the immune system, enabling the destruction of tumor cells by T cells, as was impressively demonstrated by the success of immune checkpoint inhibition, e.g., in malignant melanoma. Many cancers, however, do not respond to this kind of therapy. In these cases, vaccination against tumor antigens could be very helpful. Nevertheless, all of the efforts made in this respect during the past 30 years have been virtually futile. With current knowledge and technology there is new hope.
Assuntos
Antígenos de Neoplasias/imunologia , Vacinas Anticâncer/uso terapêutico , Melanoma/imunologia , Neoplasias/imunologia , Vacinas Anticâncer/imunologia , Humanos , Melanoma/prevenção & controle , Neoplasias/prevenção & controle , Neoplasias/terapia , Linfócitos T/imunologia , VacinaçãoRESUMO
Immune therapy and tumor cell vaccination is a challenging option in prostate cancer therapy, especially as side effects rarely occur. This review highlights recent developments in vaccination therapy of prostate cancer. The FDA approved antigen presenting cell vaccine Sipuleucel-T is described and new strategies of immune therapy like RNA and peptide vaccination are discussed in detail. Currently the effect of prostate cancer vaccination has still limitations, at least partially due to the immune suppressive effects of the tumor microenvironment and regulatory T cells, which suppress the immune effector function. To overcome these hurdles the concept of immune checkpoint modulation, which has the aim to break tolerance mechanisms, is discussed. Potential clinical therapies of checkpoint modulation are outlined.
Assuntos
Vacinas Anticâncer/uso terapêutico , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/prevenção & controle , Vacinação/tendências , Humanos , MasculinoRESUMO
No consensus has been reached on how to determine if an immune response has been detected based on raw data from an ELISPOT assay. The goal of this paper is to enable investigators to understand and readily implement currently available methods for response determination. We describe empirical and statistical approaches, identifying the strengths and limitations of each approach to allow readers to rationally select and apply a scientifically sound method appropriate to their specific laboratory setting. Five representative approaches were applied to data sets from the CIMT Immunoguiding Program and the response detection and false positive rates were compared. Simulation studies were also performed to compare empirical and statistical approaches. Based on these, we recommend the use of a non-parametric statistical test. Further, we recommend that six medium control wells or four wells each for both medium control and experimental conditions be performed to increase the sensitivity in detecting a response, that replicates with large variation in spot counts be filtered out, and that positive responses arising from experimental spot counts below the estimated limit of detection be interpreted with caution. Moreover, a web-based user interface was developed to allow easy access to the recommended statistical methods. This interface allows the user to upload data from an ELISPOT assay and obtain an output file of the binary responses.
Assuntos
Técnicas Imunoenzimáticas , Reações Falso-Positivas , Humanos , Técnicas Imunoenzimáticas/métodos , Técnicas Imunoenzimáticas/estatística & dados numéricos , Padrões de Referência , Sensibilidade e EspecificidadeRESUMO
The Cancer Immunotherapy Immunoguiding Program has conducted an IFN-gamma ELISPOT proficiency panel to examine the influence of serum supplementation of test media on assay performance. Sixteen European laboratories analyzed the same PBMC samples using different locally established protocols. Participants generated two simultaneous data sets-one using medium supplemented with serum and one without serum. Performances of the two test conditions were compared by quantifying: (1) the number of viable cells, (2) background spot formation induced in the medium only control and (3) the ability to detect antigen-specific T cell responses. The study demonstrated that the number of viable cells recovered and the overall background spot production were not significantly different between the two conditions. Furthermore, overall laboratory performance was equivalent for the two test conditions; 11 out of 16 laboratories reported equal or greater detection rates using serum-free medium, while 5 laboratories reported decreased detections rates under serum-free conditions. These results show that good performance of the IFN-gamma ELISPOT assay can be achieved under serum-free conditions. Optimization of the protocol for serum-free conditions should result in excellent detection rates and eliminate the requirement of serum batch and stability testing, allowing further harmonization of the assay.
Assuntos
Antígenos Virais/imunologia , Técnicas de Laboratório Clínico/normas , Meios de Cultura Livres de Soro/farmacologia , Ensaio de Imunoadsorção Enzimática/métodos , Imunoensaio/métodos , Interferon gama/imunologia , Leucócitos Mononucleares/imunologia , Sobrevivência Celular , Células Cultivadas , Técnicas de Laboratório Clínico/estatística & dados numéricos , Europa (Continente) , Humanos , Imunoensaio/normas , Fragmentos de Peptídeos/imunologia , Padrões de ReferênciaRESUMO
The choice of serum for supplementation of media for T cell assays and in particular, Elispot has been a major challenge for assay performance, standardization, optimization, and reproducibility. The Assay Working Group of the Cancer Vaccine Consortium (CVC-CRI) has recently identified the choice of serum to be the leading cause for variability and suboptimal performance in large international Elispot proficiency panels. Therefore, a serum task force was initiated to compare the performance of commercially available serum-free media to laboratories' own medium/serum combinations. The objective of this project was to investigate whether a serum-free medium exists that performs as well as lab-own serum/media combinations with regard to antigen-specific responses and background reactivity in Elispot. In this way, a straightforward solution could be provided to address the serum challenge. Eleven laboratories tested peripheral blood mononuclear cells (PBMC) from four donors for their reactivity against two peptide pools, following their own Standard Operating Procedure (SOP). Each laboratory performed five simultaneous experiments with the same SOP, the only difference between the experiments was the medium used. The five media were lab-own serum-supplemented medium, AIM-V, CTL, Optmizer, and X-Vivo. The serum task force results demonstrate compellingly that serum-free media perform as well as qualified medium/serum combinations, independent of the applied SOP. Recovery and viability of cells are largely unaffected by serum-free conditions even after overnight resting. Furthermore, one serum-free medium was identified that appears to enhance antigen-specific IFNgamma-secretion.
Assuntos
Técnicas de Laboratório Clínico/normas , Meios de Cultura Livres de Soro/farmacologia , Ensaio de Imunoadsorção Enzimática/métodos , Imunoensaio/métodos , Interferon gama/imunologia , Leucócitos Mononucleares/imunologia , Linfócitos T/imunologia , Técnicas de Laboratório Clínico/estatística & dados numéricos , Humanos , Imunoensaio/normas , Padrões de ReferênciaRESUMO
A total of 93 frozen primary renal cell carcinoma (RCC) samples and 31 frozen samples of corresponding normal renal tissue were analyzed for human leukocyte antigen (HLA) class I and HLA-DR expression. Unexpectedly, HLA class I expression was much higher on RCC cells than on normal renal tubular cells. Immunohistochemistry analysis of frozen and paraffin-embedded tissue samples, applying an extended panel of specific anti-HLA monoclonal antibodies, showed elevated HLA class I antigen expression in 95.6% of the tumors vs only 12.9% of normal renal tissues. These findings were confirmed by molecular analysis of HLA heavy chain and beta2-microglobulin (beta2m) transcription levels using quantitative real-time polymerase chain reaction (PCR) on microdissected tissue samples (isolated tumor nests and autologous normal renal tubules) from four patients. These results might help to explain the relatively high success rate of immunotherapy in patients with RCC. The molecular mechanism underlying the increased HLA class I expression in RCC has yet to be elucidated.
Assuntos
Carcinoma de Células Renais/imunologia , Carcinoma de Células Renais/patologia , Antígenos HLA-DR/genética , Antígenos de Histocompatibilidade Classe I/análise , Neoplasias Renais/imunologia , Neoplasias Renais/patologia , Carcinoma de Células Renais/genética , Antígenos HLA-DR/biossíntese , Antígenos HLA-DR/imunologia , Antígenos de Histocompatibilidade Classe I/genética , Antígenos de Histocompatibilidade Classe I/imunologia , Humanos , Imuno-Histoquímica , Rim/química , Rim/imunologia , Neoplasias Renais/genética , Leucócitos/química , Leucócitos/imunologia , Leucócitos/patologia , Inclusão em ParafinaRESUMO
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
Cancer Immunotherapy 2005 was the third international meeting organized by the Association for Immunotherapy of Cancer (AIC). About 200 participants were attracted by the excellent scientific program that consisted of overview lectures from 25 international speakers in the plenary auditorium and four guided poster sessions during both days of the meeting. The first day of the symposium mainly focused on experience with, and new perspectives in, antibody therapy. On the second day of the meeting, organized as a joint conference together with the Combined Research Grant "Mechanisms of Tumor Defense and Therapeutic Intervention" funded by the German Research Council, the participants had the chance to gain deeper insights into the principles of antigen processing and the regulation of immune responses. Further topics that were discussed mainly in the poster sessions and in the special lecture given by M. Nishimura (Chicago, USA), were "cellular therapies" and "vaccination against cancer". The lectures selected for this report aim to provide an overview of the complete scientific program and give an impression of the lively atmosphere that could be felt from the first until the last session of CIMT 2005.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Imunoterapia/métodos , Antígenos de Neoplasias/imunologia , Ensaios Clínicos como Assunto , Humanos , Neoplasias/imunologia , Neoplasias/terapiaRESUMO
There are good arguments for suggesting that two seminal papers published 50 years ago can be taken as the beginning of modern tumour immunology. These papers by R. Baldwin, "Immunity to transplanted tumour: the effect of tumour extracts on the growth of homologous tumours in rats" and "Immunity to methylcholanthrene-induced tumours in inbred rats following atrophy and regression of the implanted tumours" (Br J Cancer 9:646-51 and 652-657, 1955) showed that once tumours are established, they and their products can be recognised by the adaptive immune system and rejected. However, the tumour normally co-evolves with immunity, like a parasite, rather than being suddenly introduced as in these, and many other, experimental models. Dynamics of this co-evolution are illustrated by findings that inflammation enhances tumorigenicity, yet is important to enable T cells to respond properly to tumour antigen and exert anti-tumour effects. The important thing is to maintain the balance between effective anti-tumour immunity and tumour escape and/or stimulatory mechanisms. Tumours almost always co-exist with immune defence systems over extended periods and interact chronically with T cells. The effect of this is potentially similar to other situations of chronic antigenic stress, particularly lifelong persistent virus infection, most strikingly, CMV infection. The questions briefly explored in this symposium paper are what happens when T lymphocyte clones are chronically stimulated by antigen which is not or cannot be eliminated? What are the similarities and differences between chronic antigenic stimulation by tumour antigen versus CMV antigen? What can we learn in one system which may illuminate the other?
Assuntos
Citomegalovirus/imunologia , Vigilância Imunológica , Modelos Imunológicos , Neoplasias/imunologia , Evasão Tumoral/imunologia , Idoso , Animais , Células Clonais , Progressão da Doença , Humanos , Linfócitos T/imunologiaRESUMO
The liver is the second only to lymph nodes as the most common site of metastatic disease irrespective of the primary tumor. Up to 50% of all patients with malignant diseases will develop liver metastases with a significant morbidity and mortality. Although the surgical resection leads to an improvement of the survival time, only approximately 20% of the patients are eligible for surgical intervention. Radiofrequency (RF) ablation represents one of the most important alternatives as well as complementary methods for the therapy of liver metastases. RF ablation can lead in a selected patient group to a palliation or to an increased life expectancy. RF ablation appears either safer (vs. cryotherapy) or easier (vs. laser) or more effective (percutaneous ethanol instillation [PEI], transarterial chemoembolisation [TACE]) in comparison with other minimal invasive procedures. RF ablation can be performed percutaneously, laparoscopically or intraoperatively and may be combined with chemotherapy as well as with surgical resection. Permanent technical improvements of RF systems, a better understanding of the underlying electrophysiological principles and an interdisciplinary approach will lead to a prognosis improvement in patients with liver metastases.
Assuntos
Ablação por Cateter/instrumentação , Ablação por Cateter/métodos , Neoplasias Hepáticas/secundário , Neoplasias Hepáticas/cirurgia , Ablação por Cateter/tendências , Humanos , Seleção de Pacientes , Padrões de Prática Médica , Resultado do TratamentoRESUMO
Vacuum-assisted closure involving the implantation of polyvinyl alcohol foam is a technique recently developed for the treatment of patients suffering from either wound infection or chronic wounds. This method has been shown to improve and accelerate wound healing. However, little is known about the cell populations that infiltrate the foam, and their potential role in resolving the infection and promoting granulation tissue formation. Our study demonstrates that wound-implanted foams are mainly infiltrated with granulocytes, but that mononuclear cells, including macrophages and minor populations of T, B and natural killer lymphocytes, are also present. We show that foam-infiltrating T cells, especially CD4(+) T cells, constitute a phenotypically and functionally heterogeneous population influenced by wound-infecting bacteria. Thus, T lymphocytes could play a role in wound cleansing. In addition, our data indicate that implanted polyvinyl alcohol foams might be suitable microenvironments for manipulating T cell-mediated immune responses in patients.
Assuntos
Álcool de Polivinil , Linfócitos T/imunologia , Cicatrização/imunologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Linfócitos B/imunologia , Feminino , Humanos , Imuno-Histoquímica/métodos , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/imunologia , Ativação Linfocitária , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Masculino , Pessoa de Meia-Idade , Neutrófilos/efeitos dos fármacos , Neutrófilos/imunologia , Álcool de Polivinil/administração & dosagem , Álcool de Polivinil/farmacologia , Linfócitos T/efeitos dos fármacos , Vácuo , Cicatrização/efeitos dos fármacosRESUMO
Specific immunotherapy of cancer utilizes tumor-directed cytotoxic T lymphocytes (CTL) that lyse tumor cells presenting MHC class I-associated peptides derived from tumor-associated proteins. Many tumor-associated gene products are known, but corresponding T cell epitopes are only known for relatively few of these. The most commonly used approaches to identify such antigens require pre-existing CTL lines or clones. By using a CTL-independent high performance liquid chromatography mass spectrometry (HPLC MS)-based approach we identified HLA-A2-presented peptides from carcinoembryonic antigen and wild-type p53 with a copy number as low as eight molecules per cell. Potential epitopes were predicted from the sequences of known tumor antigens and the corresponding synthetic peptides were analyzed by nanocapillary HPLC MS. In parallel, peptides were extracted from fresh, solid tumor tissue or tumor cell lines and analyzed in the same way. Upon co-elution of a natural peptide with a predicted peptide of the same mass, the peptide sequence was confirmed by on-line tandem MS. This approach allows rapid screening of large numbers of tumor-associated gene products for naturally processed peptides presented by different MHC class I molecules as a prerequisite for efficient epitope identification and rapid transfer to therapeutic vaccine trials.
Assuntos
Antígenos de Neoplasias/análise , Antígeno HLA-A2/metabolismo , Linfócitos T Citotóxicos/imunologia , Calibragem , Cromatografia Líquida de Alta Pressão , Epitopos de Linfócito T , Neoplasias Gastrointestinais/imunologia , Humanos , Ligantes , Espectrometria de Massas , Sensibilidade e Especificidade , Células Tumorais CultivadasAssuntos
Neoplasias Experimentais/terapia , Linfócitos T Citotóxicos/imunologia , Membro 2 da Subfamília B de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/uso terapêutico , Animais , Apresentação de Antígeno , Antígenos de Neoplasias/uso terapêutico , Vacinas Anticâncer/uso terapêutico , Cisteína Endopeptidases/genética , Cisteína Endopeptidases/uso terapêutico , Citotoxicidade Imunológica , DNA/uso terapêutico , Imunoterapia , Camundongos , Complexos Multienzimáticos/genética , Complexos Multienzimáticos/uso terapêutico , Complexo de Endopeptidases do Proteassoma , Evasão Tumoral/imunologiaRESUMO
The origin and the function of HLA class I molecules present on the surface of human platelets are still unclear. In particular, it is controversial which fraction of these class I molecules represents integral membrane components derived from the megakaryocyte-platelet lineage versus soluble plasma HLA molecules acquired by adsorption. Results of the present study show that HLA-A2 ligands isolated from platelets possess the same peptide motif as described for HLA-A2-associated peptides obtained from nucleated cells. Sequencing of these platelet-derived peptides reveals that they originate mainly from ubiquitously expressed proteins also present in the megakaryocyte-platelet lineage. Moreover, one of these peptides derives from the GPIX protein, which is specifically expressed by platelets and their precursors. Platelet HLA molecules are unstable in vitro at 37 degrees C, but can be partially stabilized by addition of exogenous beta(2)-microglobulin and HLA class I binding peptide, suggesting that platelets cannot load HLA molecules with endogenous peptides. In in vitro experiments platelets were used to stimulate peripheral blood mononuclear cells. No allospecific cytotoxicity was observed after primary stimulation, or secondary restimulation, with allogenic resting or activated platelets, even in the presence of additional third-party helper activity. These data indicate that HLA class I molecules from platelets cannot directly induce allogenic CD8(+) cytotoxic T-cell response in vitro.
Assuntos
Plaquetas/imunologia , Antígeno HLA-A2/imunologia , Plaquetas/citologia , Linhagem da Célula/imunologia , Citotoxicidade Imunológica , Antígenos de Histocompatibilidade Classe I/imunologia , Humanos , Isoantígenos/imunologia , Megacariócitos/citologia , Megacariócitos/imunologia , Ativação PlaquetáriaRESUMO
RAS oncogenic proteins are frequently found mutated in human cancers, where they are known to be implicated in the tumoral process. Mutations occur preferentially at positions 12, 13 or 61. Identification of potential T cell epitopes is the first step to determine it RAS mutated proteins can generate tumor specific antigens which could be further used as targets for cancer immunotherapy protocols. We have investigated the capacity of synthetic wild-type and mutant RAS derived peptides encompassing positions 12 and 13 to bind to three frequent HLA-A alleles: HLA-A*0201, HLA-A*0301 and HLA-A*1101. Binding was evaluated by two methods using TAP-defective cell lines: a cytometric assay based on HLA molecules stabilization at the cell surface, and an assembly assay detecting interactions between solubilized HLA molecules and peptides. Positive HLA binding was observed for two sets of synthetic peptides, one specific for HLA-A*0201 allele (RAS 5-14), and the other one specific for HLA-A*0301 and HLA-A*1101 alleles (RAS 8-16). Interestingly, the different substitutions at positions 12 and 13 were not equivalent for HLA binding. These observations will be useful for the in vitro generation of restricted CD8+ T lymphocytes specific for mutated RAS proteins and recognizing tumoral cells expressing such RAS mutations.
Assuntos
Alelos , Antígenos HLA-A/genética , Fragmentos de Peptídeos/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Linhagem Celular , Humanos , Mutação , Relação Estrutura-Atividade , Linfócitos T Citotóxicos/imunologiaRESUMO
Lymphocyte activation induces or increases the expression of several surface structures, none of which is characteristic of an activated cell subset. In particular, structures such as CD45RO, CD25, CD26, CD49b, CD54, CD71 are expressed by the vast majority of lymphocytes at various times following in vitro activation. CD39 molecules were originally identified on activated B lymphocytes and have recently been described on activated T cell clones. In the present report, we have characterized phenotypically and functionally defined cell subsets generated during an in vitro allostimulation. Results indicated that the percentage of CD39+ cells reached a maximum at day 6 and remained stable thereafter. We demonstrate that CD39 expression allows the identification within the allosensitized CD8+ cytotoxic cells of distinct subsets of cells mediating allo cytotoxic T lymphocyte or natural killer (NK)-like reactivity. More precisely, CD8+CD39+ alloactivated cells mainly mediate specific killer activity, whereas CD8+CD39- alloactivated cells predominantly exhibit NK-like reactivity. Further, we show a high functional correlation associated with the lack of CD39 expression on NK-like alloactivated CD8+ cells, while there is no association with CD56 or CD57 NK-associated structures.
Assuntos
Adenosina Trifosfatases , Antígenos CD/análise , Antígenos CD8/análise , Ativação Linfocitária , Subpopulações de Linfócitos T/imunologia , Linfócitos T Citotóxicos/imunologia , Animais , Apirase , Células Cultivadas , Humanos , Células Matadoras Naturais/imunologia , Camundongos , Camundongos Endogâmicos BALB CRESUMO
By using several monoclonal antibodies (mAb) reacting either with the constant or variable regions of the T cell receptors (TcR) αß and γδ or various CD molecules, differences between two clinically related entities e.g. T cell acute lymphoblastic leukemia (T-ALL) and lymphoblastic lymphoma (T-LL) have been demonstrated. We studied a panel of fifteen T-ALL and fifteen T-LL because of their cell surface expression of the CD3-TcR molecules. The results indicated that TcR γδ is more frequently expressed in T-ALL (10 out of the 15 patients tested) than TcR αß. This is in contrast to the results obtained with T-LL where the vast majority showed TcR αß (13 out of the 15 patients). We discuss the significance of these findings which may imply that the leukemic cells are of a different origin in these two diseases. In addition analysis of TcR variable regions expressed by the leukemic blasts showed that in most cases they had rearranged functional Vδ1 gene to Jδ1 or Jδ2 segments (8 out of 11 patients) whereas in a unique case Vδ2 gene segment was evident. Taken together these results and those showing that T-ALL cells coexpress the CD1a, b and c molecules strengthen the possibility that despite the fact that these leukemic cells express the CD3-TcR complex at their surface their normal counterparts are not found in peripheral blood.