RESUMO
Checkpoint blockade of CTLA-4 results in long-lasting survival benefits in metastatic cancer patients. However, patients treated with CTLA-4 blockade have suffered from immune-related adverse events, most likely due to the breadth of the induced T-cell activation. Here, we investigated the efficacy of a local low-dose anti-CTLA-4 administration for treatment of subcutaneous or orthotopic murine bladder 49 (MB49) bladder carcinoma in C57BL/6 mice. When MB49 tumors were grown s.c., peritumoral (p.t.) injection of anti-CTLA-4 treatment was equally effective as intravenous or s.c. (nontumor bearing flank) administration. Notably, p.t. injection was associated with lower circulating antibody levels and decreased IL-6 serum levels as compared to systemic treatment. Ultrasound-guided intratumoral anti-CTLA-4 antibody treatment of orthotopically growing MB49 tumors resulted in tumor regression, with more than tenfold reduction in systemic antibody levels as compared to i.v. or s.c. administration, in line with the compartmentally restrained nature of the bladder. Local anti-CTLA-4 therapy in combination with anti-PD-1 therapy resulted in complete responses, superior to each therapy alone. In addition, p.t. anti-CTLA-4 therapy was potentiated by depletion of regulatory T cells. Our results demonstrate that local anti-CTLA-4 antibody therapy is equally effective as systemic administration, but reduces systemic antibody levels and cytokine release, and enhances the response to anti-PD1 therapy.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Antineoplásicos/uso terapêutico , Antígeno CTLA-4/imunologia , Inibidores do Crescimento/uso terapêutico , Imunoterapia/métodos , Receptor de Morte Celular Programada 1/imunologia , Linfócitos T Reguladores/imunologia , Neoplasias da Bexiga Urinária/terapia , Animais , Linhagem Celular Tumoral , Citocinas/metabolismo , Quimioterapia Combinada , Depleção Linfocítica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Transplante de Neoplasias , Neoplasias Experimentais , Neoplasias da Bexiga Urinária/imunologiaRESUMO
The most important goals for the field of immuno-oncology are to improve the response rate and increase the number of tumor indications that respond to immunotherapy, without increasing adverse side effects. One approach to achieve these goals is to use tumor-directed immunotherapy, i.e., to focus the immune activation to the most relevant part of the immune system. This may improve anti-tumor efficacy as well as reduce immune-related adverse events. Tumor-directed immune activation can be achieved by local injections of immune modulators in the tumor area or by directing the immune modulator to the tumor using bispecific antibodies. In this review, we focus on therapies targeting checkpoint inhibitors and co-stimulatory receptors that can generate tumor-specific T cell responses through localized immune activation.
Assuntos
Anticorpos Biespecíficos/imunologia , Fatores Imunológicos/imunologia , Imunoterapia/métodos , Neoplasias/imunologia , Neoplasias/terapia , Linfócitos T/imunologia , Anticorpos Biespecíficos/farmacologia , Humanos , Fatores Imunológicos/farmacologiaRESUMO
BACKGROUND: Current approaches for treating metastatic malignant melanoma (MM) are not effective enough and are associated with serious adverse events. Due to its immunogenicity, melanoma is an attractive target for immunostimulating therapy. In this phase I/IIa study, local AdCD40L immunostimulatory gene therapy was evaluated in patients with MM. METHODS: AdCD40L is an adenovirus carrying the gene for CD40 ligand. Patients that failed standard treatments were enrolled. Six patients received four weekly intratumoral AdCD40L injections. Next, nine patients received low-dose cyclophosphamide conditioning before the first and fourth AdCD40L injection. The blood samples were collected at multiple time points for chemistry, haematology and immunology evaluations. Radiology was performed at enrolment and repeated twice after the treatment. RESULTS: AdCD40L was safe with mild transient reactions. No objective responses were recorded by MRI, however, local and distant responses were seen on FDG-PET. The overall survival at 6 months was significantly better when cyclophosphamide was added to AdCD40L. The patients with the best survival developed the highest levels of activated T cells and experienced a pronounced decrease of intratumoral IL8. CONCLUSIONS: AdCD40L therapy for MM was well tolerated. Local and distant responses along with better survival in the low-dose cyclophosphamide group are encouraging.
Assuntos
Adjuvantes Imunológicos/administração & dosagem , Ligante de CD40/administração & dosagem , Ciclofosfamida/administração & dosagem , Melanoma/tratamento farmacológico , Melanoma/terapia , Adenoviridae/genética , Adulto , Idoso , Feminino , Terapia Genética/métodos , Vetores Genéticos/genética , Humanos , Masculino , Pessoa de Meia-Idade , Adulto JovemRESUMO
Autologous haematopoietic stem cell transplantation (HSCT) for relapsing-remitting multiple sclerosis is a potentially curative treatment, which can give rise to long-term disease remission. However, the mode of action is not yet fully understood. The aim of the study was to evaluate similarities and differences of the CD4(+) T-cell populations between HSCT-treated patients (n = 12) and healthy controls (n = 9). Phenotyping of memory T cells, regulatory T (Treg) cells and T helper type 1 (Th1) and type 17 (Th17) cells was performed. Further, T-cell reactivity to a tentative antigen, myelin oligodendrocyte glycoprotein, was investigated in these patient populations. Patients treated with natalizumab (n = 15) were included as a comparative group. White blood cells were analysed with flow cytometry and T-cell culture supernatants were analysed with magnetic bead panel immunoassays. HSCT-treated patients had similar levels of Treg cells and of Th1 and Th17 cells as healthy subjects, whereas natalizumab-treated patients had lower frequencies of Treg cells, and higher frequencies of Th1 and Th17 cells. Cells from HSCT-treated patients cultured with overlapping peptides from myelin oligodendrocyte glycoprotein produced more transforming growth factor-ß1 than natalizumab-treated patients, which suggests a suppressive response. Conversely, T cells from natalizumab-treated patients cultured with those peptides produced more interleukin-17 (IL-17), IL-1 and IL-10, indicating a Th17 response. In conclusion, we demonstrate circumstantial evidence for the removal of autoreactive T-cell clones as well as development of tolerance after HSCT. These results parallel the long-term disease remission seen after HSCT.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Transplante de Células-Tronco Hematopoéticas , Esclerose Múltipla Recidivante-Remitente/imunologia , Esclerose Múltipla Recidivante-Remitente/cirurgia , Adolescente , Adulto , Idoso , Anticorpos Monoclonais Humanizados/uso terapêutico , Separação Celular , Feminino , Citometria de Fluxo , Humanos , Masculino , Pessoa de Meia-Idade , Esclerose Múltipla Recidivante-Remitente/tratamento farmacológico , Natalizumab , Adulto JovemRESUMO
BACKGROUND: Successful cell therapy relies on the identification and mass expansion of functional cells for infusion. Cryopreservation of cells is an inevitable step in most cell therapies which also entails consequences for the frozen cells. MATERIAL AND METHODS: This study assessed the impact of cryopreservation and the widely used protocol for rapid expansion of T lymphocytes. The effects on cell viability, immunocompetence and the impact on apoptotic and immunosuppressive marker expression were analyzed using validated assays. RESULTS AND CONCLUSION: Cryopreservation of lymphocytes during the rapid expansion protocol did not affect cell viability. Lymphocytes that underwent mass expansion or culture in high dose IL-2 were unable to respond to PHA stimulation by intracellular ATP production immediately after thawing (ATP = 16 ± 11 ng/ml). However, their reactivity to PHA was regained within 48 hours of recovery (ATP = 356 ± 61 ng/ml). Analysis of mRNA levels revealed downregulation of TGF-ß and IL-10 at all time points. Culture in high dose IL-2 led to upregulation of p73 and BCL-2 mRNA levels while FoxP3 expression was elevated after culture in IL-2 and artificial TCR stimuli. FoxP3 levels decreased after short-term recovery without IL-2 or stimulation. Antigen specificity, as determined by IFNγ secretion, was unaffected by cryopreservation but was completely lost after addition of high dose IL-2 and artificial TCR stimuli. In conclusion, allowing short-time recovery of mass expanded and cryopreserved cells before reinfusion could enhance the outcome of adoptive cell therapy as the cells regain immune competence and specificity.
Assuntos
Técnicas de Cultura de Células/métodos , Criopreservação/métodos , Imunoterapia Adotiva/métodos , Linfócitos T , Trifosfato de Adenosina/metabolismo , Apoptose/fisiologia , Sobrevivência Celular/fisiologia , Humanos , Tolerância Imunológica/fisiologia , Imunocompetência/fisiologia , Melanoma , Mitógenos/farmacologia , Fito-Hemaglutininas/farmacologiaRESUMO
Adoptive cell therapy with tumor-infiltrating lymphocytes (TIL) can mediate objective responses in up to 50% of malignant melanoma patients with a good performance status refractory to standard treatments. Current protocols for generation of TILs rely on open surgery for access to tumor tissue. We obtained tumor material by ultrasound-guided core needle biopsy or surgery from melanoma patients with progressive disease and were able to isolate >5 × 10(6) TILs from 23 of 24 patients who were subsequently treated with these cells. One-third of the individual TIL-positive cultures displayed interferon gamma activity after stimulation with relevant melanoma cell lines. When expanded TILs were used for treatment in combination with daily low dose s.c. IL-2 after prior lymphodepleting chemotherapy, we observed objective clinical responses in one patient treated with TILs obtained from surgery and 4 patients treated with TILs from core biopsies. The results of this study demonstrate for the first time the potential of core biopsies for generation of relevant numbers of TILs that can mediate objective responses in patients with metastatic malignant melanoma. Ultrasound-guided core needle biopsy is a robust, safe and inexpensive approach to obtain tumor tissue for TIL generation, and is especially valuable in instances where surgery is contraindicated.
Assuntos
Imunoterapia Adotiva/métodos , Linfócitos do Interstício Tumoral/imunologia , Melanoma/imunologia , Melanoma/terapia , Linfócitos T/imunologia , Adolescente , Adulto , Idoso , Biópsia por Agulha/métodos , Feminino , Humanos , Interferon gama/imunologia , Interleucina-2/imunologia , Masculino , Melanoma/diagnóstico por imagem , Melanoma/patologia , Pessoa de Meia-Idade , UltrassonografiaRESUMO
Cytotoxic CD4(+) T cells have been found in patients with chronic lymphocytic leukaemia (CLL) and seem to be involved in the regulation of malignant B cells. The CD4(+) T regulatory cells (Tregs) can regulate various immune cells, including B cells, by inducing their apoptosis. Hence, different subgroups of CD4(+) T cells may be involved in the regulation of malignant B cells. In this study, the cytotoxic phenotype and function of various CD4(+) T-cell subgroups were investigated in patients with B-cell malignancies. Peripheral blood was collected from patients with CLL, various B-cell lymphomas, healthy adult donors, children with precursor B-cell acute lymphoblastic leukaemia (pre-B ALL) and from healthy children. CD4(+) T cells (CD3(+) CD4(+) FoxP3(-)), Tregs (CD3(+) CD4(+) CD127(low) FoxP3(+)) and CD127(high) FoxP3(+) T cells (CD3(+) CD4(+) CD127(high) FoxP3(+)) were analysed for their expression of the cytolytic markers CD107a and Fas ligand. Patients with CLL had increased CD107a expression on all tested T-cell subgroups compared with healthy donors. Similar results were found in patients with B-cell lymphomas whereas the CD107a expression in children with pre-B ALL was no different from that in healthy controls. Fas ligand expression was similar between patient cells and cells of healthy donors. CD4(+) T cells and Tregs from patients with CLL and healthy donors were subsequently purified and cultured in vitro with autologous B cells. Both subgroups lysed B cells and killing was confirmed by granzyme ELISAs. In conclusion, cytotoxic populations of CD4(+) T cells, including Tregs, are present in patients with B-cell malignancy and may be an important factor in immune-related disease control.
Assuntos
Linfócitos B/citologia , Linfócitos T CD4-Positivos/imunologia , Fatores de Transcrição Forkhead/imunologia , Leucemia Linfocítica Crônica de Células B/imunologia , Morte Celular , Células Cultivadas , Ensaio de Imunoadsorção Enzimática , Humanos , Linfócitos T Reguladores/fisiologiaRESUMO
Phosphorothioate oligodeoxynucleotides can activate complement, and experimental murine studies have revealed differential effects upon simultaneous TLR stimulation and complement activation compared with either event alone. We set out to investigate the immune stimulatory effects of CpG 2006 in fresh non-anticoagulated human blood with or without presence of active complement. We also sought to elucidate the mechanism behind complement activation upon stimulation with phosphorothioate CpG 2006. In a human blood loop system, both backbone and sequence-specific effects by CpG were counteracted by selective inhibition of C3. Furthermore, DNA backbone-mediated CD40 and CD83 expression on monocytes and sequence-specific IL-6 and TNF production were reduced by complement inhibition. CpG-induced complement activation occurred via either the classical or the alternative pathway and deposits of both IgM and properdin, two activators of complement, were detected on CpG after incubation with EDTA plasma. Quartz crystal microbalance with dissipation monitoring demonstrated alternative pathway convertase build-up onto CpG as a likely pathway to initiate and sustain complement activation. Specific inhibition of C3 suppressed CpG 2006 uptake into monocytes indicating that C3 fragments are involved in CpG internalization. The interplay between complement and TLR9 signaling demonstrated herein warrants further investigation.
Assuntos
Adjuvantes Imunológicos/farmacologia , Ativação do Complemento/efeitos dos fármacos , Citocinas/imunologia , Monócitos/imunologia , Oligodesoxirribonucleotídeos/farmacologia , Oligonucleotídeos Fosforotioatos/farmacologia , Antígenos CD/imunologia , Antígenos CD/metabolismo , Antígenos CD40/imunologia , Antígenos CD40/metabolismo , Ativação do Complemento/imunologia , Via Alternativa do Complemento/efeitos dos fármacos , Via Alternativa do Complemento/imunologia , Proteínas do Sistema Complemento/efeitos dos fármacos , Proteínas do Sistema Complemento/imunologia , Proteínas do Sistema Complemento/metabolismo , Citocinas/efeitos dos fármacos , Citocinas/metabolismo , Humanos , Imunoglobulina M/imunologia , Imunoglobulina M/metabolismo , Imunoglobulinas/imunologia , Imunoglobulinas/metabolismo , Interleucina-6/imunologia , Interleucina-6/metabolismo , Glicoproteínas de Membrana/imunologia , Glicoproteínas de Membrana/metabolismo , Monócitos/citologia , Monócitos/efeitos dos fármacos , Monócitos/metabolismo , Peptídeos Cíclicos/farmacologia , Properdina/imunologia , Properdina/metabolismo , Fator de Necrose Tumoral alfa/imunologia , Fator de Necrose Tumoral alfa/metabolismo , Antígeno CD83RESUMO
Malignant melanoma is a serious disease in both humans and dogs, and the high metastatic potential results in poor prognosis for many patients. Its similarities with human melanoma make spontaneous canine melanoma an excellent model for comparative studies of novel therapies and tumor biology. Gene therapy using adenoviruses encoding the immunostimulatory gene CD40L (AdCD40L) has shown promise in initial clinical trials enrolling human patients with various malignancies including melanoma. We report a study of local AdCD40L treatment in 32 cases of canine melanoma (23 oral, 5 cutaneous, 3 ungual and 1 conjunctival). Eight patients were World Health Organization (WHO) stage I, 9 were stage II, 12 stage III, and 3 stage IV. One to six intratumoral injections of AdCD40L were given every seven days, combined with cytoreductive surgery in 20 cases and only immunotherapy in 12 cases. Tumor tissue was infiltrated with T and B lymphocytes after treatment, suggesting immune stimulation. The best overall response based on result of immunotherapy included 7 complete responses, 5 partial responses, 5 stable and 2 progressive disease statuses according to the World Health Organization response criteria. Median survival was 285 days (range 20-3435 d). Our results suggest that local AdCD40L therapy is safe and could have beneficial effects in dogs, supporting further treatment development. Clinical translation to human patients is ongoing.
RESUMO
AdCD40L is a replication-deficient virus carrying the gene for CD40 ligand which has previously been evaluated in patients with urothelial cancer and malignant melanoma. Herein, we present the results of repeated intratumoral injections of AdCD40L in seven patients with metastatic solid cancer. One patient who developed urothelial cancer derived from a renal transplant was treated with repeated injections of AdCD40L alone. The remaining patients suffered from cholangiocarcinoma, kidney, breast, rectal, or ovarian cancer and received AdCD40L repeatedly (4x) in combination with cyclophosphamide. The treatment was safe and generally well-tolerated. Two patients had clinical benefit of the treatment and one of them was accepted for re-treatment. Circulating proinflammatory cytokines were commonly increased after treatment, but save for TNFα, significances were not reached which could be due to the low number of patients. Similar to earlier findings in AdCD40L-treated melanoma patients, IL8 plasma levels were high in the present study. In conclusion, gene therapy by repeated intratumoral AdCD40L injections alone, or in combination with cyclophosphamide, is feasible and safe in patients with solid cancers. The potential of intratumoral CD40L gene transfer as treatment of cancer was illustrated by the clinical improvement in two out of seven patients.
Assuntos
Terapia Genética/métodos , Imunização/métodos , Neoplasias/genética , Animais , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Neoplasias/mortalidade , Análise de SobrevidaRESUMO
BACKGROUND: Cytomegalovirus (CMV) infection is still the leading opportunistic infection following solid organ transplantation. The aim of this prospective study of renal transplant recipients was to evaluate the dynamics of CMV-specific T-cells, viral load, and clinical symptoms of CMV infection. METHODS: Levels of tetramer-selected CD8(+) T-cells (TetraCD8), CMV-specific interferon-gamma producing CD8(+) T-cells (IFNgammaCD8), and CD4(+) T-cells (IFNgammaCD4), measured using major histocompatibility complex-tetramer and cytokine flow cytometry techniques, and CMV DNA were monitored monthly in 17 CMV-seropositive patients up to one yr (median 12 months, range 3-12) after transplantation and correlated to clinical outcome. RESULTS: CMV DNAemia was detected in 94% of the patients, but only one patient developed CMV disease. CMV DNAemia >1 million copies/mL was seen in asymptomatic patients. CMV-specific T-cells decreased rapidly after transplantation. TetraCD8 and IFNgammaCD8 regenerated within three months, whereas IFNgammaCD4 recovery was impaired up to one yr after transplantation. The proportion of IFNgammaCD4 at two months post-transplantation as compared with baseline, correlated strongly with the magnitude of the CMV DNAemia. CONCLUSIONS: Monitoring the reduction of IFNgammaCD4 compared with baseline during the first months after transplantation could be considered in predicting risk for high-grade CMV DNAemia and in deciding strategic approaches for pre-emptive and prophylactic therapy.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Infecções por Citomegalovirus/imunologia , Citomegalovirus/fisiologia , Transplante de Rim/imunologia , Imunologia de Transplantes/fisiologia , Carga Viral , Adulto , Idoso , Estudos de Casos e Controles , DNA Viral/genética , Feminino , Citometria de Fluxo , Seguimentos , Sobrevivência de Enxerto , Humanos , Imunidade Celular , Masculino , Pessoa de Meia-Idade , Projetos Piloto , Prognóstico , Estudos Prospectivos , Estudos SoroepidemiológicosRESUMO
Relapsing-remitting multiple sclerosis (RRMS) is a complex autoimmune disease of the central nervous system with oscillating phases of relapse and remission. RRMS has been considered to be driven by T helper type 1 (Th1) lymphocytes but new data indicate the involvement of Th17 responses. In the present study, blood samples from patients (n=48) and healthy individuals (n=44) were evaluated for their immunological status. T cells from patients with RRMS expressed high levels of the activation marker CD28 (P<0.05) and secreted both interferon-gamma (CD8: P<0.05) and interleukin-17 upon polyclonal mitogen or myelin oligodendrocyte glycoprotein antigen stimulation. However, T cells from patients with RRMS in remission, in contrast to relapse, had poor proliferative capacity (P<0.05) suggesting that they are controlled and kept in anergy. This anergy could be broken with CD28 stimulation that restored the T-cell replication. Furthermore, the patients with RRMS had normal levels of CD4(+) Foxp3(+) T regulatory cells but the frequency of Foxp3(+) cells lacking CD127 (interleukin-7 receptor) was lower in patients with MS (mean 12%) compared to healthy controls (mean 29%). Still, regulatory cells (CD25(+) sorted cells) from patients with RRMS displayed no difference in suppressive capacity. In conclusion, patients in relapse/remission demonstrate in vitro T-cell responses that are both Th1 and Th17 that, while in remission, appear to be controlled by tolerogenic mechanisms yet to be investigated.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Anergia Clonal/imunologia , Esclerose Múltipla Recidivante-Remitente/imunologia , Subpopulações de Linfócitos T/imunologia , Adolescente , Adulto , Proliferação de Células , Células Cultivadas , Feminino , Humanos , Interferon gama/biossíntese , Interleucina-17/biossíntese , Subunidade alfa de Receptor de Interleucina-7/análise , Ativação Linfocitária/imunologia , Masculino , Pessoa de Meia-Idade , Proteínas da Mielina , Glicoproteína Associada a Mielina/imunologia , Glicoproteína Mielina-Oligodendrócito , Linfócitos T Reguladores/imunologia , Células Th1/imunologiaRESUMO
BACKGROUND: In order to obtain a sustained cytotoxic T lymphocyte (CTL) response against cancer cells it is preferable to have CTLs directed against multiple peptide epitopes from numerous tumor-associated antigens. METHODS: We used a Flow Cytometry-based interferon (IFN)-gamma secretion assay with peptide-pulsed C1R-A2 as antigen-presenting cells to analyze whether CD8+ T cells directed against any of 24 HLA-A*0201-binding peptides from 15 prostate-associated proteins can be found in the peripheral blood of patients with localized prostate cancer. We also investigated whether multiple prostate antigen-specific CD8+ T cells can be generated simultaneously, from a naïve T cell repertoire. In that case, dendritic cells (DCs) from peripheral blood of healthy donors were divided in six portions and separately pulsed with six peptides. The peptide-pulsed DCs were then pooled and used to stimulate autologous T cells. The T cells were re-stimulated with peptide-pulsed monocytes. RESULTS: We found prostate antigen-restricted CD8+ T cells in the peripheral blood in 48 out of 184 (26.1%) analyzed samples from 25 cancer patients. This is significantly higher than 17 out of 214 analyzed samples (7.9%) from 10 healthy age-matched male individuals (P = 0.0249). In the cases when antigen-specific T cells could not be detected, we were able to generate IFN-gamma-producing CD8+ T cells specific for up to three prostate antigens simultaneously from a naïve T cell repertoire. CONCLUSIONS: CD8+ T cells directed against prostate antigen peptides can be found in, or generated from, peripheral blood. This indicates that such T cells could be expanded ex vivo for adoptive transfer to prostate cancer patients.
Assuntos
Linfócitos T CD8-Positivos/patologia , Próstata/imunologia , Neoplasias da Próstata/imunologia , Linfócitos T Citotóxicos/patologia , Transferência Adotiva , Fatores Etários , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Divisão Celular/imunologia , Linhagem Celular Transformada , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Células Dendríticas/patologia , Epitopos , Epitopos de Linfócito T/imunologia , Feminino , Citometria de Fluxo , Antígeno HLA-A2/imunologia , Humanos , Interferon gama/metabolismo , Masculino , Próstata/patologia , Neoplasias da Próstata/patologia , Linfócitos T Citotóxicos/imunologia , Linfócitos T Citotóxicos/metabolismoRESUMO
The gene expression profile of metastasizing serotonin-producing neuroendocrine carcinomas, which arise from enterochromaffin cells in the jejunum and ileum, is still largely unknown. The aim of this study was to identify genes and proteins, which are preferentially expressed by neuroendocrine carcinoma and enterochromaffin cells and therefore potential novel biomarkers and/or therapeutic targets. Six carcinoma specimens and six normal ileal mucosas were profiled by Affymetrix microarrays. Advanced bioinformatics identified differentially and specifically expressed genes, which were validated by quantitative real-time-PCR on tumor cells extracted by laser capture microdissection and normal enterochromaffin cells extracted by immunolaser capture microdissection. We identified six novel marker genes for neuroendocrine carcinoma cells: paraneoplastic antigen Ma2 (PNMA2), testican-1 precursor (SPOCK1), serpin A10 (SERPINA10), glutamate receptor ionotropic AMPA 2 (GRIA2), G protein-coupled receptor 112 (GPR112) and olfactory receptor family 51 subfamily E member 1 (OR51E1). GRIA2 is specifically expressed by neuroendocrine carcinoma cells whereas the others are also expressed by normal enterochromaffin cells. GPR112 and OR51E1 encode proteins associated with the plasma membrane and may therefore become targets for antibody-based diagnosis and therapy. Hierarchical clustering shows high similarity between primary lesions and liver metastases. However, chemokine C-X-C motif ligand 14 (CXCL14) and NK2 transcription factor related locus 3 Drosophila (NKX2-3) are expressed to a lower level in liver metastases than in primary tumors and normal enterochromaffin cells, which implies a role in neuroendocrine carcinoma differentiation. In conclusion, this study provides a list of genes, which possess relatively specific expression to enterochromaffin and neuroendocrine carcinoma cells and genes with differential expression between primary tumors and metastases. We verified six novel marker genes that may be developed as biomarkers and/or therapeutic targets.
Assuntos
Biomarcadores Tumorais/genética , Carcinoma Neuroendócrino/genética , Células Enterocromafins/química , Regulação Neoplásica da Expressão Gênica , Neoplasias do Íleo/genética , Íleo/química , RNA Mensageiro/análise , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/análise , Carcinoma Neuroendócrino/química , Carcinoma Neuroendócrino/patologia , Análise por Conglomerados , Bases de Dados Genéticas , Feminino , Perfilação da Expressão Gênica/métodos , Humanos , Neoplasias do Íleo/química , Neoplasias do Íleo/patologia , Imuno-Histoquímica , Mucosa Intestinal/química , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/secundário , Masculino , Microdissecção , Pessoa de Meia-Idade , Neoplasias Mesoteliais/genética , Neoplasias Mesoteliais/secundário , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase , Reprodutibilidade dos TestesRESUMO
INTRODUCTION: Our aim was to investigate the immune status of midgut carcinoid patients. Cancer patients generally display suppressed Th1-type immunity that disables mounting of an efficient anti-tumor response. However, little is known about patients with neuroendocrine midgut carcinoids. MATERIAL AND METHODS: Circulating regulatory T cells were determined in patient blood by staining for CD4, CD25 and FoxP3 in flow cytometric analysis. T cell proliferation was measured by Alamar Blue in response to polyclonal activation and the regulatory phenotype of patient CD25+ cells was validated by allogeneic stimulation of CFSE labelled responders. Cytokine levels in patient peripheral blood were measured by ELISA and CBA. Tumor infiltrating T cells were analyzed by immunohistochemistry and immunofluorescence. RESULTS: The results demonstrate that midgut carcinoid patients exhibit increased frequencies of circulating Tregs and patient T cells have a decreased proliferative capacity compared to healthy donors. Systemic Th1-promoting cytokines are reduced. Midgut carcinoid tumors display CD4+ and CD8+ T cell infiltration, always in the presence of regulatory CD4+FoxP3+ cells. DISCUSSION: Midgut carcinoid patients display elevated T regulatory cell numbers and T cell dysfunction. Therapeutic strategies to overcome tumor-induced Th1 immunosuppression are required in combination with anti-tumor vaccinations.
Assuntos
Tumor Carcinoide/sangue , Neoplasias Intestinais/sangue , Neoplasias Hepáticas/sangue , Linfócitos do Interstício Tumoral/imunologia , Linfócitos T Reguladores/imunologia , Antígenos CD4/metabolismo , Tumor Carcinoide/secundário , Estudos de Casos e Controles , Citocinas/metabolismo , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Imunofluorescência , Humanos , Técnicas Imunoenzimáticas , Neoplasias Intestinais/patologia , Neoplasias Hepáticas/secundário , Ativação Linfocitária , Linfócitos do Interstício Tumoral/patologiaRESUMO
Cytomegalovirus (CMV) reactivation can cause severe complications for transplant patients. Such patients can be protected against CMV-associated diseases through reconstitution of donor-derived CMV-reactive cytolytic and helper T cells. We have developed a strategic protocol for efficient simultaneous generation of CMV-reactive CD8+ and CD4+ T cells ex vivo. The protocol uses peripheral blood lymphocytes (PBLs), antigen-modified mature dendritic cells (DCs) generated in only 3 d and an adenoviral vector encoding the CMV pp65 antigen (Adpp65) both as an endogenous and exogenous source of antigen. PBLs stimulated once with Adpp65-transduced DCs (endogenously expressed pp65) resulted in preferential activation and expansion of pp65-specific CD8+ T cells while PBLs stimulated with DCs pulsed with cell lysate from Adpp65-transduced autologous monocytes (exogenously expressed pp65) yielded pp65-specific CD4+ T cells. Stimulation with double-modified DCs efficiently activated and expanded cytolytic and helper T cells simultaneously. The frequency of T cells producing interferon-gamma in response to pp65 increased after one stimulation on average 9.6-fold to 4.3% for CD8+ T cells and 25.8-fold to 6.5% for CD4+ T cells. This implies that sufficient number of pp65-specific cytolytic and helper T cells for adoptive transfer may be obtained in only 2 weeks.
Assuntos
Citomegalovirus/imunologia , Fosfoproteínas/imunologia , Linfócitos T Citotóxicos/imunologia , Linfócitos T Auxiliares-Indutores/imunologia , Proteínas da Matriz Viral/imunologia , Adenoviridae/genética , Antígenos Virais/imunologia , Células Cultivadas , Células Dendríticas/imunologia , Vetores Genéticos , Humanos , Imunofenotipagem , Ativação Linfocitária/imunologia , Transdução GenéticaRESUMO
BACKGROUND: AdCD40L is an immunostimulatory gene therapy under evaluation for advanced melanoma, including ocular melanoma. Herein, we present the final data of a Phase I/IIa trial using AdCD40L alone or in combination with low dose cyclophosphamide +/- radiation therapy. METHODS: AdCD40L is a replication-deficient adenovirus carrying the gene for CD40 ligand (CD40L). Twenty-four patients with advanced melanoma were enrolled and treated with AdCD40L monotherapy, or combined with cyclophosphamide +/- single fraction radiotherapy. The patients were monitored for 10 weeks using immunological and radiological evaluations and thereafter for survival. RESULTS: AdCD40L treatment was safe and well tolerated both alone and in combination with cyclophosphamide as well as local radiotherapy. Four out of twenty-four patients had >1 year survival. Addition of cyclophosphamide was beneficial but adding radiotherapy did not further extend survival. High initial plasma levels of IL12 and MIP3b correlated to overall survival, whereas IL8 responses post-treatment correlated negatively with survival. Interestingly, antibody reactions to the virus correlated negatively with post IL6 and pre IL1b levels in blood. CONCLUSIONS: AdCD40L was safely administered to patients and effect was improved by cyclophosphamide but not by radiotherapy. Immune activation profile at baseline may predict responders better than shortly after treatment.
RESUMO
PURPOSE: Local administration of immune-activating antibodies may increase the efficacy and reduce the immune-related adverse events associated with systemic immunotherapy of cancer. Here, we report the development and affinity maturation of a fully human agonistic CD40 antibody (IgG1), ADC-1013. EXPERIMENTAL DESIGN: We have used molecular engineering to generate an agonistic antibody with high affinity for CD40. The functional activity of ADC-1013 was investigated in human and murine in vitro models. The in vivo effect was investigated in two separate bladder cancer models, both using human xenograft tumors in immune deficient NSG mice and using a syngeneic bladder cancer model in a novel human CD40 transgenic mouse. RESULTS: Activation of dendritic cells (DC) by ADC-1013 results in upregulation of the costimulatory molecules CD80 and CD86, and secretion of IL12. ADC-1013 also activates DCs from human CD40 transgenic mice, and peptide-pulsed and ADC-1013-stimulated DCs induce antigen-specific T-cell proliferation in vitro. In vivo, treatment with ADC-1013 in a syngeneic bladder cancer model, negative for hCD40, induces significant antitumor effects and long-term tumor-specific immunity. Furthermore, ADC-1013 demonstrates significant antitumor effects in a human bladder cancer transplanted into immunodeficient NSG mice. CONCLUSIONS: Our data demonstrate that ADC-1013 induces long-lasting antitumor responses and immunologic memory mediated by CD40 stimulation. To the best of our knowledge, ADC-1013 represents the first immunomodulatory antibody developed for local immunotherapy of cancer.
Assuntos
Anticorpos Monoclonais/farmacologia , Antineoplásicos/farmacologia , Antígenos CD40/agonistas , Linfócitos T/imunologia , Neoplasias da Bexiga Urinária/imunologia , Animais , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais/química , Anticorpos Monoclonais Humanizados , Citotoxicidade Celular Dependente de Anticorpos/imunologia , Antineoplásicos/administração & dosagem , Antineoplásicos/química , Antígenos CD40/química , Antígenos CD40/genética , Antígenos CD40/metabolismo , Linhagem Celular Tumoral , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Modelos Animais de Doenças , Feminino , Humanos , Ativação Linfocitária/imunologia , Masculino , Camundongos , Camundongos Knockout , Modelos Moleculares , Conformação Molecular , Ligação Proteica , Linfócitos T/metabolismo , Carga Tumoral/efeitos dos fármacos , Carga Tumoral/imunologia , Neoplasias da Bexiga Urinária/tratamento farmacológico , Neoplasias da Bexiga Urinária/genética , Neoplasias da Bexiga Urinária/metabolismo , Neoplasias da Bexiga Urinária/patologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
PURPOSE: Triggering of CD40 on antigen-presenting cells via its ligand CD154 is an important event in the initial phase of an immune response against cancer cells. In this study, we investigated the effects of adenoviral CD154 immunomodulatory gene therapy on the activation of human dendritic cells (DCs) in a well-defined in vitro system. EXPERIMENTAL DESIGN: Human bladder cancer cell lines and tumor cells from patients with renal cell carcinoma (RCC) were transduced with Ad-CD154 vectors or control vectors. Activation of human in vitro generated DCs after coculture with transduced tumor cells was analyzed. Therapeutic efficacy and cytotoxic T-lymphocyte (CTL) activity were assessed in a subcutaneous (s.c.) murine bladder cancer model. RESULTS: Human bladder cancer cell lines expressing CD154 showed a decreased growth rate, increased apoptosis, and modulated expression of molecules important for recognition by cytotoxic lymphocytes. Further, CD154-expressing allogeneic bladder tumor cell lines and autologous tumor cells from patients with renal cell cancer induced maturation of DCs and stimulated IFN-gamma production from lymphocytes cocultured with mature DCs. In vivo studies showed that CD154 gene therapy was highly effective in wild-type mice but only minimally effective in nude mice. Consequently, strong tumor-specific CTL activity was detected in mice vaccinated with tumor cells expressing CD154. CONCLUSIONS: Using tumor cell lines as well as patient-derived material, we could show that tumor cells expressing CD154 efficiently induce maturation and activation of DCs as well as activation of lymphocytes. Our murine in vivo studies demonstrate that lymphocytes contribute to the observed antitumor effect in a s.c. bladder tumor model. These studies should stimulate CD154 gene therapy approaches for the treatment of urologic malignancies.
Assuntos
Adenoviridae/genética , Ligante de CD40/genética , Carcinoma de Células Renais/genética , Carcinoma de Células de Transição/genética , Células Dendríticas/imunologia , Terapia Genética/métodos , Neoplasias Renais/genética , Neoplasias da Bexiga Urinária/genética , Animais , Apoptose , Carcinoma de Células Renais/imunologia , Carcinoma de Células de Transição/imunologia , Divisão Celular , Citometria de Fluxo , Técnicas de Transferência de Genes , Engenharia Genética , Vetores Genéticos , Humanos , Técnicas In Vitro , Interferon gama/metabolismo , Neoplasias Renais/imunologia , Ativação Linfocitária/imunologia , Transfusão de Linfócitos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Nus , Linfócitos T Citotóxicos/imunologia , Células Tumorais Cultivadas , Neoplasias da Bexiga Urinária/imunologiaRESUMO
BACKGROUND: HLA class I peptide tetramers represent powerful diagnostic tools for detection and monitoring of antigen-specific CD8(+) T cells. The impetus for the current multicenter study is the critical need to standardize tetramer flow cytometry if it is to be implemented as a routine diagnostic assay. Hence, the European Working Group on Clinical Cell Analysis set out to develop and evaluate a single-platform tetramer-based method that used cytomegalovirus (CMV) as the antigenic model. METHODS: Absolute numbers of CMV-specific CD8(+) T cells were obtained by combining the percentage of tetramer-binding cells with the absolute CD8(+) T-cell count. Six send-outs of stabilized blood from healthy individuals or CMV-carrying donors with CMV-specific CD8(+) T-cell counts of 3 to 10 cells/microl were distributed to 7 to 16 clinical sites. These sites were requested to enumerate CD8(+) T cells and, in the case of CMV-positive donors, CMV-specific subsets on three separate occasions using the standard method. RESULTS: Between-site coefficients of variation of less than 10% (absolute CD8(+) T-cell counts) and approximately 30% (percentage and absolute numbers of CMV-specific CD8(+) T cells) were achieved. Within-site coefficients of variation were approximately 5% (absolute CD8(+) T-cell counts), approximately 9% (percentage CMV-specific CD8(+) T cells), and approximately 17% (absolute CMV-specific CD8(+) T-cell counts). The degree of variation tended to correlate inversely with the proportion of CMV-specific CD8(+) T-cell subsets. CONCLUSIONS: The single-platform MHC tetramer-based method for antigen-specific CD8(+) T-cell counting has been evaluated by a European group of laboratories and can be considered a reproducible assay for routine enumeration of antigen-specific CD8(+) T cells.