RESUMO
INTRODUCTION: Naked DNA vaccination could be a powerful and safe strategy to mount antigen-specific cellular immunity. We designed a phase I clinical trial to investigate the toxicity of naked DNA vaccines encoding CD8+ T-cell epitope from tumor-associated antigen MART-1 in patients with advanced melanoma. METHODS: This dose escalating phase Ia clinical trial investigates the toxicity and immunological response upon naked DNA vaccines encoding a CD8+ T-cell epitope from the tumor-associated antigen MART-1, genetically linked to the gene encoding domain 1 of subunit-tetanus toxin fragment C in patients with advanced melanoma (inoperable stage IIIC-IV, AJCC 7th edition). The vaccine was administrated via intradermal application using a permanent make-up or tattoo device. Safety was monitored according to CTCAE v.3.0 and skin biopsies and blood samples were obtained for immunologic monitoring. RESULTS: Nine pretreated, HLA-A*0201-positive patients with advanced melanoma expressing MART-1 and MHC class I, with a good performance status, and adequate organ function, were included. With a median follow-up of 5.9 months, DNA vaccination was safe, without treatment-related deaths. Common treatment-emergent adverse events of any grade were dermatologic reactions at the vaccination site (100%) and pain (56%). One patient experienced grade 4 toxicity, most likely related to tumor progression. One patient (11%) achieved stable disease, lasting 353 days. Immune analysis showed no increase in vaccine-induced T cell response in peripheral blood of 5 patients, but did show a MART-1 specific CD8+ T cell response at the tattoo administration site. The maximum dose administered was 2 mg due to lack of clinical activity. CONCLUSION: We showed that the developed DNA vaccine, applied using a novel intradermal application strategy, can be administered safely. Further research with improved vaccine formats is required to show possible clinical benefit of DNA vaccination.
Assuntos
Vacinas Anticâncer , Melanoma , Estadiamento de Neoplasias , Tatuagem , Vacinas de DNA , Humanos , Melanoma/imunologia , Melanoma/terapia , Vacinas de DNA/administração & dosagem , Vacinas de DNA/imunologia , Pessoa de Meia-Idade , Feminino , Masculino , Vacinas Anticâncer/administração & dosagem , Vacinas Anticâncer/imunologia , Vacinas Anticâncer/uso terapêutico , Injeções Intradérmicas , Idoso , Adulto , Neoplasias Cutâneas/imunologia , Antígeno MART-1/imunologia , Linfócitos T CD8-Positivos/imunologia , Vacinação/métodos , Resultado do Tratamento , Antígeno HLA-A2/imunologia , Epitopos de Linfócito T/imunologiaRESUMO
PURPOSE: Ongoing angiogenesis renders the tumor endothelium unresponsive to inflammatory cytokines and interferes with adhesion of leukocytes, resulting in escape from immunity. This process is referred to as tumor endothelial cell anergy. We aimed to investigate whether anti-angiogenic agents can overcome endothelial cell anergy and provide pro-inflammatory conditions. EXPERIMENTAL DESIGN: Tissues of renal cell carcinoma (RCC) patients treated with VEGF pathway-targeted drugs and control tissues were subject to RNAseq and immunohistochemical profiling of the leukocyte infiltrate. Analysis of adhesion molecule regulation in cultured endothelial cells, in a preclinical model and in human tissues was performed and correlated to leukocyte infiltration. RESULTS: It is shown that treatment of RCC patients with the drugs sunitinib or bevacizumab overcomes tumor endothelial cell anergy. This treatment resulted in an augmented inflammatory state of the tumor, characterized by enhanced infiltration of all major leukocyte subsets, including T cells, regulatory T cells, macrophages of both M1- and M2-like phenotypes and activated dendritic cells. In vitro, exposure of angiogenic endothelial cells to anti-angiogenic drugs normalized ICAM-1 expression. In addition, a panel of tyrosine kinase inhibitors was shown to increase transendothelial migration of both non-adherent and monocytic leukocytes. In primary tumors of RCC patients, ICAM-1 expression was found to be significantly increased in both the sunitinib and bevacizumab-treated groups. Genomic analysis confirmed the correlation between increased immune cell infiltration and ICAM-1 expression upon VEGF-targeted treatment. CONCLUSION: The results support the emerging concept that anti-angiogenic therapy can boost immunity and show how immunotherapy approaches can benefit from combination with anti-angiogenic compounds.
Assuntos
Inibidores da Angiogênese , Carcinoma de Células Renais , Células Endoteliais , Neoplasias Renais , Neovascularização Patológica , Humanos , Bevacizumab/imunologia , Bevacizumab/farmacologia , Bevacizumab/uso terapêutico , Carcinoma de Células Renais/tratamento farmacológico , Carcinoma de Células Renais/imunologia , Carcinoma de Células Renais/patologia , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/imunologia , Células Endoteliais/patologia , Endotélio/efeitos dos fármacos , Endotélio/imunologia , Endotélio/patologia , Molécula 1 de Adesão Intercelular/imunologia , Neoplasias Renais/tratamento farmacológico , Neoplasias Renais/imunologia , Neoplasias Renais/patologia , Neovascularização Patológica/tratamento farmacológico , Neovascularização Patológica/imunologia , Neovascularização Patológica/patologia , Sunitinibe/imunologia , Sunitinibe/farmacologia , Sunitinibe/uso terapêutico , Fator A de Crescimento do Endotélio Vascular/imunologia , Tolerância Imunológica/efeitos dos fármacos , Tolerância Imunológica/imunologia , Invasividade Neoplásica/imunologia , Inflamação/tratamento farmacológico , Inflamação/imunologia , Inflamação/patologia , Inibidores da Angiogênese/imunologia , Inibidores da Angiogênese/farmacologia , Inibidores da Angiogênese/uso terapêuticoRESUMO
In this study, we investigated a large series of immune (escape) markers, relevant to T-cell function, as potential biomarkers for clinical outcome following immunotherapy. We retrospectively studied the expression of immune (escape) markers in metastatic melanoma tissues of 27 patients before autologous tumor cell vaccination, and 16 patients who were intended to treat but were not vaccinated because of rapid disease progression. Immunohistochemical data of infiltrating (suppressive) cells, such as T cells, regulatory T cells, myeloid-derived suppressor cells, and mast cells, or the expression of T-cell inhibitory factors (PD-1/PD-L1, IDO, and galectins), cytotoxic molecules (granzyme-B), melanocyte differentiation antigens, HLA class-I and tolerogenic cytokines [interleukin (IL)-1, IL-6, IL-10, TNF-α, and TGF-ß] were correlated statistically to clinical outcome and overall survival (OS). Significantly more tumor-infiltrating CD4(+) and CD8(+) T cells (both P < 0.05) were found in nonprogressors to vaccination (n = 9; median OS, 56 months), compared with progressors (n = 18; median OS, 9.5 months). Moreover, granzyme-B expression was elevated in the tumors of nonprogressors, suggesting activated cytotoxic T cells or natural killer cells. T-cell infiltration and granzyme-B expression significantly correlated with overall OS. T-cell inhibitory factors and suppressive cells did not correlate with OS, suggesting minor influence of these immune-escape markers on clinical outcome. The data of progressors were comparable with those from patients with rapid progression (not vaccinated; n = 16; median OS, 3 months). Our study shows that high numbers of intratumoral activated CD4(+) or CD8(+) T cells, before autologous tumor cell vaccination, are associated with favorable clinical outcome. Analyses of these markers in the patients' tumor tissues before immunotherapy may therefore be a valuable tool to select patients for whom the treatment may result in potential clinical benefit.
Assuntos
Biomarcadores Tumorais/metabolismo , Imunoterapia/métodos , Melanoma/terapia , Neoplasias Cutâneas/terapia , Evasão Tumoral/imunologia , Adulto , Idoso , Vacinas Anticâncer/uso terapêutico , Feminino , GTP Fosfo-Hidrolases/genética , Fator Estimulador de Colônias de Granulócitos e Macrófagos/uso terapêutico , Humanos , Masculino , Melanoma/genética , Melanoma/imunologia , Proteínas de Membrana/genética , Pessoa de Meia-Idade , Mutação/genética , Proteínas Proto-Oncogênicas B-raf/genética , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/imunologia , Linfócitos T/imunologia , Resultado do TratamentoRESUMO
PURPOSE: To investigate the angiogenic changes in primary tumor tissue of renal cell carcinoma (RCC) patients treated with VEGF-targeted therapy. EXPERIMENTAL DESIGN: Phase II trials of VEGF pathway-targeted therapy given before cytoreductive surgery were carried out with metastatic RCC patients with the primary tumor in situ to investigate the necessity of nephrectomy. Primary tumor tissues were obtained and assessed for angiogenesis parameters. Results were compared with similar analyses on untreated tumors. RESULTS: Sunitinib or bevacizumab pretreatment resulted in a significant reduction of microvessel density in the primary tumor. Also, an increase in vascular pericyte coverage was found in sunitinib-pretreated tumors, consistent with efficient angiogenesis inhibition. Expression of several key regulators of angiogenesis was found to be suppressed in pretreated tissues, among which VEGFR-1 and VEGFR-2, angiopoietin-1 and angiopoietin-2 and platelet-derived growth factor-B. In addition, apoptosis in tumor and endothelial cells was induced. Interestingly, in sunitinib-pretreated tissues a dramatic increase of the number of proliferating endothelial cells was observed, which was not the case in bevacizumab-pretreated tumors. A positive correlation with the interval between halting the therapy and surgery was found, suggesting a compensatory angiogenic response caused by the discontinuation of sunitinib treatment. CONCLUSION: This study describes, for the first time, the angiostatic response in human primary renal cancers at the tissue level upon treatment with VEGF-targeted therapy. Discontinuation of treatment with tyrosine kinase inhibitors leads to accelerated endothelial cell proliferation. The results of this study contribute important data to the ongoing discussion on the discontinuation of treatment with kinase inhibitors.
Assuntos
Anticorpos Monoclonais Humanizados/administração & dosagem , Carcinoma de Células Renais/tratamento farmacológico , Indóis/administração & dosagem , Neovascularização Patológica/tratamento farmacológico , Pirróis/administração & dosagem , Adulto , Idoso , Apoptose/efeitos dos fármacos , Bevacizumab , Carcinoma de Células Renais/complicações , Carcinoma de Células Renais/patologia , Carcinoma de Células Renais/secundário , Ensaios Clínicos Fase II como Assunto , Feminino , Humanos , Masculino , Microvasos/efeitos dos fármacos , Pessoa de Meia-Idade , Neovascularização Patológica/complicações , Estudos Retrospectivos , SunitinibeRESUMO
The introduction of targeted agents has substantially improved treatment of metastatic clear-cell renal cell carcinoma (RCC). However, complete responses are rare and therapy is not curative. Moreover, information on the latest generation of potent and selective vascular endothelial growth factor receptor (VEGFR) tyrosine kinase inhibitors (TKI) suggests that a plateau has been reached in terms of efficacy. Recent data reveal that targeted agents are involved in modulating immune responses in RCC. In addition, current research adds to our understanding of how RCC escapes an effective anti-tumor response with the potential to modulate these processes by drug development. This review provides specific insight into targeted therapy induced changes in the immunological microenvironment of RCC, summarizes the available evidence, and discusses potential therapeutic implications.
Assuntos
Inibidores da Angiogênese/uso terapêutico , Antineoplásicos/uso terapêutico , Carcinoma de Células Renais/tratamento farmacológico , Fatores Imunológicos/uso terapêutico , Neoplasias Renais/tratamento farmacológico , Terapia de Alvo Molecular/métodos , Microambiente Tumoral/efeitos dos fármacos , Carcinoma de Células Renais/imunologia , Humanos , Imunidade Celular , Neoplasias Renais/imunologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Microambiente Tumoral/imunologiaRESUMO
HY-specific T cells are presumed to play a role in acute graft-versus-host disease (aGVHD) after female-to-male stem cell transplantation (SCT). However, infiltrates of these T cells in aGVHD-affected tissues have not yet been reported. We evaluated the application of HLA-A2/HY dextramers for the in situ detection of HY-specific T cells in cryopreserved skin biopsy specimens. We applied the HLA-A2/HY dextramers on cryopreserved skin biopsy specimens from seven male HLA-A2(+) pediatric patients who underwent stem cell transplantation with confirmed aGVHD involving the skin. The dextramers demonstrated the presence of HY-specific T cells. In skin biopsy specimens of three male recipients of female grafts, 68% to 78% of all skin-infiltrating CD8(+) T cells were HY-specific, whereas these cells were absent in biopsy specimens collected from sex-matched patient-donor pairs. Although this study involved a small and heterogeneous patient group, our results strongly support the hypothesis that HY-specific T cells are actively involved in the pathophysiology of aGVHD after sex-mismatched stem cell transplantation.
Assuntos
Doença Enxerto-Hospedeiro/imunologia , Transplante de Células-Tronco Hematopoéticas/métodos , Pele/imunologia , Linfócitos T/imunologia , Adolescente , Criança , Pré-Escolar , Criopreservação , Imunofluorescência , Doença Enxerto-Hospedeiro/patologia , Humanos , Masculino , Microscopia Confocal , Pele/patologia , Linfócitos T/patologiaRESUMO
PURPOSE: To predict the potential antitumor effect of antigen-specific T cells in melanoma patients, we investigated T-cell effector function in relation to tumor-escape mechanisms. EXPERIMENTAL DESIGN: CD8(+) T cells isolated from tumor, adjacent normal skin, and peripheral blood of 17 HLA-A2(+) patients with advanced-stage melanoma were analyzed for their antigen specificity and effector function against melanocyte differentiation antigens MART-1, gp100, and tyrosinase by using HLA-A2/peptide tetramers and functional assays. In addition, the presence of tumor-escape mechanisms PD-L1/PD-1 pathway, FoxP3 and loss of HLA or melanocyte differentiation antigens, both required for tumor cell recognition and killing, were studied. RESULTS: Higher percentages of melanocyte antigen-specific CD8(+) T cells were found in the melanoma tissues as compared with adjacent normal skin and peripheral blood. Functional analysis revealed 2 important findings: (i) in 5 of 17 patients, we found cytokine production after specific peptide stimulation by tumor-infiltrating lymphocytes (TIL), not by autologous peripheral blood lymphocytes (PBL); (ii) CD8(+) T cells from 7 of 17 patients did not produce cytokines after specific stimulation, which corresponded with significant loss of tumor HLA-A2 expression. The presence of other tumor-escape mechanisms did not correlate to T-cell function. CONCLUSIONS: Our data show that functional T-cell responses could be missed when only PBL and not TIL are evaluated, emphasizing the importance of TIL analysis for immunomonitoring. Furthermore, loss of tumor HLA-A2 may explain the lack of T-cell functionality. These findings have important implications for selecting melanoma patients who may benefit from immunotherapy.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Imunoterapia , Linfócitos do Interstício Tumoral/imunologia , Melanoma/imunologia , Pele/imunologia , Idoso , Idoso de 80 Anos ou mais , Antígeno B7-H1/biossíntese , Linfócitos T CD4-Positivos , Células Cultivadas , Citocinas/biossíntese , Citotoxicidade Imunológica , Feminino , Fatores de Transcrição Forkhead/biossíntese , Antígeno HLA-A2/biossíntese , Antígeno HLA-A2/imunologia , Humanos , Ativação Linfocitária , Antígeno MART-1/imunologia , Masculino , Melanoma/sangue , Melanoma/patologia , Melanoma/terapia , Pessoa de Meia-Idade , Monofenol Mono-Oxigenase/imunologia , Evasão Tumoral , Antígeno gp100 de Melanoma/imunologiaRESUMO
T cells specific for hematopoietic system restricted minor Histocompatibility (H) antigens target normal and malignant hematopoietic cells. Thus, cellular immune responses against the latter miHAS eradicate the recipient's hematopoiesis including residual leukemic cells after HLA-matched minor H antigen-mismatched stem-cell transplantation (SCT). However, there are controversial reports on the role of HA-1 in the development of graft-versus-host-disease (GVHD) as well. Here, we address the behavior of HA-1-specific cytotoxic T cells (CTLs) in an ex vivo in situ skin explant model wherein HA-1-expressing dendritic cells (DCs) were added as antigen-presenting cells (APCs). Infiltration and activation of HA-1 CTLs occurred only in those cases where both HLA-A2 and HA-1 were expressed, either by the skin or by the DCs, or by the combination of HLA-A2(+) skin and HA-1(+) DCs. These results point toward the role of recipient's HA-1(+) DCs in the chimeric patient suffering from GVHD after HA-1-mismatched SCT. Although in our model the infiltrated and activated CTLs did not cause skin tissue destruction, our results provide a first step in understanding the reported association of HA-1 mismatching with clinical GVHD.
Assuntos
Células Dendríticas/transplante , Doença Enxerto-Hospedeiro/terapia , Imunoterapia/métodos , Antígenos de Histocompatibilidade Menor , Oligopeptídeos , Linfócitos T Citotóxicos/imunologia , Movimento Celular , Células Dendríticas/imunologia , Humanos , Técnicas In Vitro , Ativação Linfocitária , Modelos Biológicos , Pele/citologia , Pele/imunologia , Dermatopatias/terapiaRESUMO
Tumours express tumour-associated antigens that are recognised as self-antigens precluding the induction of effective anti-tumour immune responses. Inflammatory conditions which facilitate appropriate antigen presentation and reduce the immuno-suppressive micro-milieu may break tolerance. However, tumours have evolved mechanisms to escape cytotoxic T-cell attack by expressing inhibitory molecules on their surface, secreting suppressive factors, attracting regulatory T cells to the tumour environment or downregulating MHC molecules. Induction of angiogenesis by tumours may represent another mechanism by which tumours escape from immune attack. It provides an anti-inflammatory milieu that will prevent appropriate activation and maturation of antigen presenting cells, allow tumours to secrete suppressive factors and inhibit expression of tumour endothelial adhesion receptors, such as intercellular adhesion molecule-1, vascular cell adhesion molecule-1 and E-selectin, needed for appropriate interactions with immune cells. Inhibition of angiogenesis may, apart from its direct detrimental effects on the tumour, reverse these processes and contribute to anti-tumour immune reactivity. Without trying to give a complete overview of the field, this paper reviews insights on angiogenesis inhibition in relation to tumour immune responsiveness, mainly based on the Maastricht-Amsterdam experience. This review adds to the hypothesis of improvement of immuno-directed therapies for cancer by angiostasis.
Assuntos
Inibidores da Angiogênese/uso terapêutico , Moléculas de Adesão Celular/metabolismo , Imunoterapia , Neoplasias/irrigação sanguínea , Neoplasias/imunologia , Neoplasias/terapia , Neovascularização Patológica/tratamento farmacológico , Animais , Antígenos de Neoplasias/imunologia , Adesão Celular/imunologia , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/imunologia , Quimioterapia Combinada , Humanos , Tolerância Imunológica , Imunidade Celular , Vigilância Imunológica , Neoplasias/patologia , Países Baixos , Evasão TumoralRESUMO
In vitiligo, cytotoxic T cells infiltrating the perilesional margin are suspected to be involved in the pathogenesis of the disease. However, it remains to be elucidated whether these T cells are a cause or a consequence of the depigmentation process. T cells we obtained from perilesional skin biopsies, were significantly enriched for melanocyte antigen recognition, compared with healthy skin-infiltrating T cells, and were reactive to melanocyte antigen-specific stimulation. Using a skin explant model, we were able to dissect the in situ activities of perilesional T cells in the effector phase of depigmentation. We show that these T cells could infiltrate autologous normally pigmented skin explants and efficiently kill melanocytes within this microenvironment. Interestingly, melanocyte apoptosis was accompanied by suprabasal keratinocyte apoptosis. Perilesional T cells did, however, not induce apoptosis in lesional skin, which is devoid of melanocytes, indicating the melanocyte-specific cytotoxic activity of these cells. Melanocyte killing correlated to local infiltration of perilesional T cells. Our data show that perilesional cytotoxic T cells eradicate pigment cells, the characteristic hallmark of vitiligo, thereby providing evidence of T cells being able to mediate targeted autoimmune tissue destruction.
Assuntos
Autoimunidade , Melanócitos/patologia , Pele/patologia , Vitiligo/imunologia , Apoptose , Citotoxicidade Imunológica , Humanos , Interleucina-17/fisiologia , Ativação Linfocitária , Melanócitos/imunologia , Pele/imunologia , Linfócitos T/imunologia , Vitiligo/etiologia , Vitiligo/patologiaRESUMO
AIMS: To assess the strategy of using an absence of progression at metastatic sites following initial cytokine therapy outcome as a selection criterion for nephrectomy in patients with synchronous metastatic renal carcinoma and an intermediate prognosis according to the Memorial Sloan Kettering prognostic index classification. MATERIALS AND METHODS: A combined retrospective analysis of patients with clear-cell subtype from studies of initial cytokine treatment response to assist with selection of patients for nephrectomy. We analyzed survival times, UCLA integrated staging system scores, number of nephrectomies and risk of progression to unresectability of the primary tumor during treatment. RESULTS: There were 33 patients in total. Nephrectomies were not performed in 10 (30%) patients whose cancers had progressed at metastatic sites. Median survival time was 4 months with none of the patients dying of local tumor progression. The median survival time of the 21 patients with nonprogressive cancer and the primary removed was 17 months. Of those, 8 had a survival time < or =1 year (median 8.5 months) and a progression-free survival time of 4 months and 13 had a survival time >1 year (median 25 months). The median progression-free survival time was 7 months (4-57 months). Four of the 5 objective responses at metastatic sites (5/33, 14%) occurred in those surviving >1 year. CONCLUSIONS: We propose that progression at metastatic sites during initial immunotherapy may be used to identify patients with a short survival time and who are unlikely to benefit from nephrectomy.
Assuntos
Carcinoma de Células Renais/tratamento farmacológico , Carcinoma de Células Renais/cirurgia , Interferon-alfa/uso terapêutico , Neoplasias Renais/tratamento farmacológico , Neoplasias Renais/cirurgia , Adulto , Idoso , Carcinoma de Células Renais/mortalidade , Carcinoma de Células Renais/secundário , Terapia Combinada , Intervalos de Confiança , Feminino , Humanos , Imuno-Histoquímica , Injeções Subcutâneas , Interferon alfa-2 , Neoplasias Renais/mortalidade , Neoplasias Renais/patologia , Metástase Linfática , Masculino , Pessoa de Meia-Idade , Invasividade Neoplásica/patologia , Estadiamento de Neoplasias , Nefrectomia/métodos , Seleção de Pacientes , Probabilidade , Prognóstico , Ensaios Clínicos Controlados Aleatórios como Assunto , Proteínas Recombinantes , Estudos Retrospectivos , Medição de Risco , Análise de Sobrevida , Fatores de Tempo , Resultado do TratamentoRESUMO
Dendritic cells (DCs) are key cells in innate and adaptive immune responses that determine the pathophysiology of Crohn's disease. Intestinal DCs migrate from the mucosa into mesenteric lymph nodes (MLNs). A number of different markers are described to define the DC populations. In this study we have identified the phenotype and localization of intestinal and MLN DCs in patients with Crohn's disease and non-IBD patients based on these markers. We used immunohistochemistry to demonstrate that all markers (S-100, CD83, DC-SIGN, BDCA1-4, and CD1a) showed a different staining pattern varying from localization in T-cell areas of lymph follicles around blood vessels or single cells in the lamina propria and in the MLN in the medullary cords and in the subcapsular sinuses around blood vessels and in the T-cell areas. In conclusion, all different DC markers give variable staining patterns so there is no marker for the DC.
Assuntos
Colo/patologia , Doença de Crohn/patologia , Células Dendríticas/patologia , Mucosa Intestinal/patologia , Linfonodos/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Antígenos CD/metabolismo , Antígenos CD1/metabolismo , Antígenos de Superfície/metabolismo , Biomarcadores/metabolismo , Moléculas de Adesão Celular/metabolismo , Colo/metabolismo , Doença de Crohn/metabolismo , Células Dendríticas/metabolismo , Glicoproteínas , Humanos , Imunoglobulinas/metabolismo , Imuno-Histoquímica , Mucosa Intestinal/metabolismo , Lectinas Tipo C/metabolismo , Linfonodos/metabolismo , Glicoproteínas de Membrana/metabolismo , Mesentério , Pessoa de Meia-Idade , Receptores de Superfície Celular/metabolismo , Proteínas S100/metabolismo , Trombomodulina , Antígeno CD83RESUMO
Successful induction of functional tumor-specific T cells by peptide vaccination in animal models has resulted in many clinical trials to test this approach in advanced-stage melanoma patients. In this phase I clinical trial, 11 end-stage melanoma patients were vaccinated intradermally with 3 peptides: MART-1(26-35) E27L (ELAGIGILTV), tyrosinase(368-376) N375Q (YMDGTMSQV), and gp100(209-217) T210M (IMQVPFSV), admixed with tetanus toxoid and granulocyte-monocyte colony stimulating factor. The peptide vaccine was well tolerated at all tested doses, and led to grade 1-2 toxicity only. Although all patients did show a rise in antitetanus IgG titers, in only 3 patients peptide-specific CD8 T-cells were induced. In 2 cases, the response was directed against MART-1(26-35) and consisted of 0.2% and 3.3% of the CD8 population; however, in both instances these cells did not produce interferon-gamma on stimulation with the unmodified peptide. The third patient mounted a small (0.1%) response against gp100. In a fourth patient, a nonfunctional tyrosinase-specific response (0.6%) was found that was present before vaccination, but was not affected in size nor in function by the vaccine. None of the 11 patients responded clinically according to response evaluation criteria in solid tumors criteria. Although this study is a small scale phase I clinical trial, the efficacy that was observed was disappointingly low. In accordance with previously published peptide vaccination studies, these results add to the increasing evidence that peptide vaccination in itself is not potent enough as an effective melanoma immunotherapy in advanced-stage patients.
Assuntos
Vacinas Anticâncer/uso terapêutico , Neoplasias Oculares/tratamento farmacológico , Fator Estimulador de Colônias de Granulócitos e Macrófagos/uso terapêutico , Melanoma/tratamento farmacológico , Peptídeos/uso terapêutico , Neoplasias Cutâneas/tratamento farmacológico , Toxoide Tetânico/uso terapêutico , Adulto , Sequência de Aminoácidos , Neoplasias Oculares/química , Feminino , Antígenos HLA-A/análise , Antígeno HLA-A2 , Humanos , Masculino , Melanoma/química , Pessoa de Meia-Idade , Dados de Sequência Molecular , Peptídeos/química , Neoplasias Cutâneas/química , Resultado do Tratamento , Vacinação , Vacinas de Subunidades Antigênicas/química , Vacinas de Subunidades Antigênicas/uso terapêuticoRESUMO
Tetrameric MHC/peptide complexes are important tools for analyzing antigen-specific T cells. The in situ use of tetrameric MHC/peptide complexes in viable tissue sections has several shortcomings: it does not allow the execution of multiple analyses on one single biopsy, the storage of the biopsies, and the co-staining of the tetramer-positive cells for various intracellular molecules. We have developed a novel approach using overnight pre-labeling of viable human tissues with MHC/peptide tetramers, followed by cryopreservation and labeling of the cryosections. The visualization of antigen-specific T cells, combined with detection of other membrane, cytoplasmic, or nuclear markers is now feasible.
Assuntos
Pele/imunologia , Linfócitos T/imunologia , Biópsia , Criopreservação , Feminino , Corantes Fluorescentes/química , Antígeno H-Y/análise , Antígeno H-Y/imunologia , Antígeno HLA-A2/análise , Antígeno HLA-A2/imunologia , Humanos , Imuno-Histoquímica , Técnicas In Vitro , Masculino , Pele/citologia , Linfócitos T/citologiaRESUMO
OBJECTIVES: In a phase I study the feasibility, toxicity and immunological effects of peri-operative cytokine immunotherapy of renal cell carcinoma were studied. Main goals were to determine the maximal tolerable dose and detailed in situ analysis of tumor infiltrates. METHODS: Fifteen patients with renal cell carcinoma, undergoing nephrectomy, received subcutaneous immunotherapy, consisting of low-dose IL-2, IFNalpha and GM-CSF, from day -3 prior, until day +5 following surgery in a dose escalation study. Infiltrates from resected tumor tissues from patients undergoing immunotherapy or control patients that underwent nephrectomy only, were examined using quantitative immunohistological analysis and 3-color immunofluorescence staining and confocal laser scanning microscope analysis. RESULTS: Toxicity was limited and the maximal tolerable dose was established. In peripheral blood an increase was found in total lymphocytes, (activated) T cells, NK cells and monocytes. Quantitative immunohistological analysis of tumor infiltrates showed enhanced numbers of CD3+ T cells, S100+ DC, CD83+ DC and IL-2 receptor positive cells (4-fold, 2-fold, 10-fold and 20-fold, respectively, compared to controls). In treated patients preferential invasion was observed of TNFalpha positive CD8+ T cells and DC, positive for DC-SIGN (CD209), CD83, CD80, IL-12 and the DC specific chemokine, DC-CK1 (CCL18). CONCLUSIONS: These findings show increased infiltration of activated, mature DC and functionally active CD8+ T cells in renal tumors, which may suggest clinical potential of cytokine immunotherapy.
Assuntos
Carcinoma de Células Renais/terapia , Células Dendríticas/imunologia , Imunoterapia/métodos , Neoplasias Renais/terapia , Linfócitos T/imunologia , Adulto , Carcinoma de Células Renais/cirurgia , Terapia Combinada , Feminino , Fator Estimulador de Colônias de Granulócitos e Macrófagos/uso terapêutico , Humanos , Técnicas Imunoenzimáticas , Interferon-alfa/uso terapêutico , Interleucina-2/uso terapêutico , Neoplasias Renais/cirurgia , Masculino , Microscopia Confocal , Pessoa de Meia-Idade , Nefrectomia , Coloração e Rotulagem , Resultado do TratamentoRESUMO
It is unknown how B cells that mature during a germinal center reaction 'decide' between plasma or memory cell fate. Here we describe a previously unknown subpopulation of B cells in the human germinal center that is characterized by tyrosine phosphorylated transcriptional activator STAT5. These cells had an activated centrocyte phenotype and had abundant expression of BCL6 but low expression of PRDM1, both encoding transcriptional repression proteins. Using RNA interference and ectopic expression of constitutively activated forms of STAT5, we demonstrate here a function for STAT5 in the self-renewal of B cells in vitro. STAT5b isoform seemed to directly upregulate Bcl-6, and ectopic expression of Bcl-6 in B cells resulted in self-renewal and inhibition of plasma cell differentiation. These data indicate that activation of STAT5 is involved in regulation of memory B cell differentiation.
Assuntos
Linfócitos B/imunologia , Proteínas de Ligação a DNA/metabolismo , Memória Imunológica , Proteínas do Leite/metabolismo , Transativadores/metabolismo , Linfócitos B/citologia , Diferenciação Celular , Proteínas de Ligação a DNA/efeitos dos fármacos , Humanos , Proteínas do Leite/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-bcl-6 , Fator de Transcrição STAT5 , Tamoxifeno/farmacologia , Transativadores/efeitos dos fármacos , Regulação para CimaRESUMO
The importance of CD45RB expression on T cells was already shown in mice where CD45RB(high) expression determines pathogenic potential. In this study, we analyzed the expression of CD45RA, CD45RB, and CD45RO on CD4(+) T lymphocytes in the intestinal mucosa and in the circulation of patients with inflammatory bowel disease (IBD). In addition, we studied the cytokine profile of these cells. In the circulation, virtually all CD4(+)CD45RB(high) T cells expressed the naive marker CD45RA, and circulating CD4(+)CD45RB(low) cells expressed the memory marker CD45RO in IBD patients and a control patient population. In contrast, the intestinal CD4(+) CD45RB(high) T cells are in normal controls for 90% CD45RO(+). However, in IBD, 27.7% [Crohn's disease (CD)] and 49% [ulcerative colitis (UC)] of the intestinal CD4(+) CD45RB(high) T cells are CD45RA(+). This special CD4CD45RA(+) T cell in IBD can be found in the lamina propria as well as in lymphoid follicles (confocal laser-scanning microscopy). The CD4(+)CD45RB(high) T lymphocytes produce significantly less interleukin (IL)-10 and IL-4 and produce more tumor necrosis factor alpha than CD45RB(low) T lymphocytes in control patients. CD4(+)CD45RB(low) T cells from IBD patients produced less IL-10 than CD4(+)CD45RB(low) T lymphocytes of controls, and interferon-gamma production by both T lymphocyte subsets was decreased in IBD. These data indicate that CD and UC are characterized by an influx of CD4(+)CD45RB(high) T lymphocytes. These CD4(+)CD45RB(high) T lymphocytes seem to be important in the pathogenesis of IBD, as they produce more proinflammatory cytokines and less anti-inflammatory cytokines compared with CD4(+)CD45RB(low) T lymphocytes.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Colite Ulcerativa/imunologia , Doença de Crohn/imunologia , Regulação da Expressão Gênica/imunologia , Mucosa Intestinal/metabolismo , Antígenos Comuns de Leucócito/metabolismo , Adulto , Idoso , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/ultraestrutura , Estudos de Casos e Controles , Colite Ulcerativa/sangue , Doença de Crohn/sangue , Feminino , Humanos , Interleucina-10/metabolismo , Interleucina-4/metabolismo , Mucosa Intestinal/imunologia , Masculino , Microscopia Confocal , Pessoa de Meia-Idade , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis, is primarily mediated by CD4 T cells specific for Ags in the CNS. Using MHC class II tetramers, we assessed expansion and phenotypic differentiation of polyclonal self-reactive CD4 T cells during EAE after primary and secondary challenge with the specific Ag. After EAE induction in SJL mice with proteolipid protein 139-151, CNS-specific T cells up-regulated activation markers and expanded in the draining lymph nodes and in the spleen. Less than 20% of total autoreactive T cells entered the CNS simultaneously with Th cells of other specificities. Almost all tetramer-positive cells in the CNS were activated and phenotypically distinct from the large peripheral pool. When EAE was induced in Ag-experienced mice, disease symptoms developed earlier and persisted longer; autoreactive T cells were more rapidly activated and invaded the CNS earlier. In striking contrast to specific CTLs that respond after secondary viral challenge, the absolute numbers of autoreactive CD4 T cells were not increased, indicating that the accelerated autoreactivity in Ag-experienced mice is not related to higher frequencies of autoreactive CD4 T cells.
Assuntos
Autoantígenos/imunologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Encefalomielite Autoimune Experimental/imunologia , Antígenos de Histocompatibilidade Classe II/metabolismo , Proteína Proteolipídica de Mielina/administração & dosagem , Glicoproteína Associada a Mielina/administração & dosagem , Fragmentos de Peptídeos/administração & dosagem , Animais , Autoantígenos/metabolismo , Linfócitos T CD4-Positivos/citologia , Diferenciação Celular/imunologia , Divisão Celular/imunologia , Sistema Nervoso Central/imunologia , Sistema Nervoso Central/patologia , Encefalomielite Autoimune Experimental/patologia , Epitopos de Linfócito T/imunologia , Feminino , Adjuvante de Freund/administração & dosagem , Adjuvante de Freund/imunologia , Antígenos de Histocompatibilidade Classe II/imunologia , Imunização , Imunização Secundária , Imunofenotipagem , Ativação Linfocitária , Contagem de Linfócitos , Camundongos , Proteínas da Mielina , Proteína Proteolipídica de Mielina/imunologia , Proteína Proteolipídica de Mielina/metabolismo , Glicoproteína Associada a Mielina/imunologia , Glicoproteína Associada a Mielina/metabolismo , Glicoproteína Mielina-Oligodendrócito , Fragmentos de Peptídeos/imunologia , Fragmentos de Peptídeos/metabolismoRESUMO
Many tumor-associated antigens are derived from nonmutated "self" proteins. T cells infiltrating tumor deposits recognize self-antigens presented by tumor cells and can be expanded in vivo with vaccination. These T cells exist in a functionally tolerant state, as they rarely result in tumor eradication. We found that tumor growth and lethality were unchanged in mice even after adoptive transfer of large numbers of T cells specific for an MHC class I-restricted epitope of the self/tumor antigen gp100. We sought to develop new strategies that would reverse the functionally tolerant state of self/tumor antigen-reactive T cells and enable the destruction of large (with products of perpendicular diameters of >50 mm2), subcutaneous, unmanipulated, poorly immunogenic B16 tumors that were established for up to 14 d before the start of treatment. We have defined three elements that are all strictly necessary to induce tumor regression in this model: (a) adoptive transfer of tumor-specific T cells; (b) T cell stimulation through antigen-specific vaccination with an altered peptide ligand, rather than the native self-peptide; and (c) coadministration of a T cell growth and activation factor. Cells, vaccination, or cyto-kine given alone or any two in combination were insufficient to induce tumor destruction. Autoimmune vitiligo was observed in mice cured of their disease. These findings illustrate that adoptive transfer of T cells and IL-2 can augment the function of a cancer vaccine. Furthermore, these data represent the first demonstration of complete cures of large, established, poorly immunogenic, unmanipulated solid tumors using T cells specific for a true self/tumor antigen and form the basis for a new approach to the treatment of patients with cancer.
Assuntos
Autoimunidade , Linfócitos T CD8-Positivos/imunologia , Tolerância Imunológica , Melanoma Experimental/imunologia , Melanoma Experimental/patologia , Glicoproteínas de Membrana/imunologia , Proteínas de Neoplasias/imunologia , Transferência Adotiva , Animais , Antígenos de Histocompatibilidade Classe I , Humanos , Interferon gama/imunologia , Interferon gama/metabolismo , Interleucina-2/metabolismo , Complexo Principal de Histocompatibilidade , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/metabolismo , Tolerância a Antígenos Próprios , Taxa de Sobrevida , Vacinação , Antígeno gp100 de MelanomaRESUMO
Dentritic cells (DC) as antigen-presenting cells are most likely responsible for regulation of abnormal T cell activation in Crohn's disease (CD), a chronic inflammatory bowel disease. We have analyzed the expression of activation and maturation markers on DC in the colon mucosa from patients with CD compared with normal colon, using immunohistochemical techniques. We found two distinct populations of DC present in CD patients: a DC-specific ICAM-3 grabbing non-integrin (DC-SIGN)(+) population that was present scattered throughout the mucosa, and a CD83(+) population that was present in aggregated lymphoid nodules and as single cells in the lamina propria. In normal colon the number of DC-SIGN(+) DC was lower and CD83(+) DC were detected only in very few solitary lymphoid nodules. Co-expression of activation markers and cytokine synthesis was analyzed with three-color confocal laser scanning microscopy analysis. CD80 expression was enhanced on the majority of DC-SIGN(+) DC in CD patients, whereas only a proportion of the CD83(+) DC co-expressed CD80 in CD as well as in normal tissue. Surprisingly, IL-12 and IL-18 were only detected in DC-SIGN(+) DC and not in CD83(+) DC. A similar pattern of cytokine production was observed in normal colon albeit to a much lesser extent. The characteristics of these in-situ-differentiated DC markedly differ from the in-vitro-generated DC that simultaneously express DC-SIGN, CD83 and cytokines.