RESUMO
Clinical outcomes from cancer vaccine trials in patients with advanced melanoma have so far been disappointing. This appears at least partially due to a state of immunosuppression in these patients induced by an expansion of regulatory cell populations including regulatory T cells (Tregs). We have previously demonstrated potent immunogenicity of the NY-ESO-1/ISCOMATRIX™ vaccine in patients with resected melanoma (study LUD99-08); however, the same vaccine induced only a few vaccine antigen-specific immune responses in patients with advanced disease (study LUD2002-013). Pre-clinical models suggest that the alkylating agent cyclophosphamide can enhance immune responses by depleting Tregs. Therefore, we have enrolled a second cohort of patients with advanced melanoma in the clinical trial LUD2002-013 to investigate whether pre-treatment with cyclophosphamide could improve the immunogenicity of the NY-ESO-1/ISCOMATRIX™ vaccine. The combination treatment led to a significant increase in vaccine-induced NY-ESO-1-specific CD4(+) T cell responses compared with the first trial cohort treated with vaccine alone. We could not detect a significant decline in regulatory T cells in peripheral blood of patients 14 days after cyclophosphamide administration, although a decline at an earlier time point cannot be excluded. Our observations support the inclusion of cyclophosphamide in combination trials with vaccines and other immune-modulatory agents.
Assuntos
Antígenos de Neoplasias/imunologia , Antineoplásicos Alquilantes/administração & dosagem , Linfócitos T CD4-Positivos/imunologia , Vacinas Anticâncer/administração & dosagem , Colesterol/imunologia , Ciclofosfamida/administração & dosagem , Melanoma/terapia , Proteínas de Membrana/imunologia , Fosfolipídeos/imunologia , Saponinas/imunologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Vacinas Anticâncer/imunologia , Estudos de Coortes , Terapia Combinada , Relação Dose-Resposta a Droga , Combinação de Medicamentos , Feminino , Seguimentos , Humanos , Metástase Linfática , Masculino , Melanoma/imunologia , Melanoma/secundário , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Prognóstico , Linfócitos T Reguladores/imunologiaRESUMO
BACKGROUND: NY-ESO-1 protein formulated in ISCOMATRIX™ results in CD4+, CD8+ T cell and antibody-mediated immunity. We evaluated persistence of immunity, relapse-free survival and tumour antigen expression upon relapse in patients vaccinated in an earlier trial. METHODS: Immunity was measured in 28 patients with resected NY-ESO-1-expressing tumours (melanoma 25, breast 3) 252-1,155 days (median = 681) after vaccination. In the earlier vaccination, trial patients received NY-ESO-1 with ISCOMATRIX™ adjuvant at three protein doses 10 µg, 30 µg or 100 µg (n = 14); 100 µg NY-ESO-1 protein (n = 8) or placebo (n = 6), together with 1 µg of intradermal (ID) NY-ESO-1 protein twice for DTH skin testing. Immune responses assessed in the current study included antibody titres, circulating NY-ESO-1-specific T cells and DTH reactivity 2 days after DTH skin testing with NY-ESO-1 protein (1 µg) or peptides (10 µg). Relapse-free survival was determined for 42 melanoma patients. On relapse NY-ESO-1 and HLA, class I was assessed by immunohistochemistry in 17. RESULTS: Persisting anti-NY-ESO-1 immunity was detected in 10/14 recipients who had previously received vaccine with ISCOMATRIX™ adjuvant. In contrast, immunity only persisted in 3/14 who received 100 µg un-adjuvanted NY-ESO-1 protein (3/8) or 2 µg DTH protein (0/6) P = 0.02. Hence, persisting NY-ESO-1 immunity was associated with prior adjuvant. Tumour NY-ESO-1 or HLA class I was downregulated in participants who relapsed suggesting immunoediting had occurred. CONCLUSION: Immunoediting suggests that a signal of anti-tumour activity was observed in high-risk resected melanoma patients vaccinated with NY-ESO-1/ISCOMATRIX™. This was associated with measurable persisting immunity in the majority of vaccinated subjects tested. A prospective randomised trial has been undertaken to confirm these results.
Assuntos
Antígenos de Neoplasias/administração & dosagem , Neoplasias da Mama/terapia , Vacinas Anticâncer/administração & dosagem , Colesterol/administração & dosagem , Melanoma/terapia , Proteínas de Membrana/administração & dosagem , Fosfolipídeos/administração & dosagem , Saponinas/administração & dosagem , Adjuvantes Imunológicos/administração & dosagem , Adulto , Idoso , Sequência de Aminoácidos , Antígenos de Neoplasias/biossíntese , Antígenos de Neoplasias/imunologia , Neoplasias da Mama/imunologia , Vacinas Anticâncer/imunologia , Colesterol/imunologia , Intervalo Livre de Doença , Regulação para Baixo , Combinação de Medicamentos , Hipersensibilidade a Drogas/etiologia , Hipersensibilidade a Drogas/imunologia , Feminino , Humanos , Imuno-Histoquímica , Masculino , Melanoma/imunologia , Proteínas de Membrana/biossíntese , Proteínas de Membrana/imunologia , Pessoa de Meia-Idade , Dados de Sequência Molecular , Fosfolipídeos/imunologia , Estudos Prospectivos , Saponinas/imunologia , Pele/imunologiaRESUMO
PURPOSE: NY-ESO-1 is a highly immunogenic antigen expressed in a variety of malignancies, making it an excellent target for cancer vaccination. We recently developed a vaccine consisting of full-length recombinant NY-ESO-1 protein formulated with ISCOMATRIX adjuvant, which generated strong humoral and T-cell-mediated immune responses and seemed to reduce the risk of disease relapse in patients with fully resected melanoma. This study examines the clinical and immunologic efficacy of the same vaccine in patients with advanced metastatic melanoma. EXPERIMENTAL DESIGN: Delayed-type hypersensitivity responses, circulating NY-ESO-1-specific CD4(+) and CD8(+) T cells, and proportions of regulatory T cells (Treg) were assessed in patients. RESULTS: In contrast to patients with minimal residual disease, advanced melanoma patients showed no clinical responses to vaccination. Although strong antibody responses were mounted, the generation of delayed-type hypersensitivity responses was significantly impaired. The proportion of patients with circulating NY-ESO-1-specific CD4(+) T cells was also reduced, and although many patients had CD8(+) T cells specific to a broad range of NY-ESO-1 epitopes, the majority of these responses were preexisting. Tregs were enumerated in the blood by flow cytometric detection of cells with a CD4(+)CD25(+)FoxP3(+) and CD4(+)CD25(+)CD127(-) phenotype. Patients with advanced melanoma had a significantly higher proportion of circulating Treg compared with those with minimal residual disease. CONCLUSIONS: Our results point to a tumor-induced systemic immune suppression, showing a clear association between the stage of melanoma progression, the number of Treg in the blood, and the clinical and immunologic efficacy of the NY-ESO-1 ISCOMATRIX cancer vaccine.
Assuntos
Adjuvantes Imunológicos/administração & dosagem , Antígenos de Neoplasias/imunologia , Vacinas Anticâncer/imunologia , Colesterol/administração & dosagem , Melanoma/terapia , Proteínas de Membrana/imunologia , Fosfolipídeos/administração & dosagem , Saponinas/administração & dosagem , Linfócitos T Reguladores/fisiologia , Linfócitos T/imunologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Antígenos de Neoplasias/administração & dosagem , Vacinas Anticâncer/administração & dosagem , Combinação de Medicamentos , Feminino , Humanos , Hipersensibilidade Tardia/etiologia , Masculino , Proteínas de Membrana/administração & dosagem , Pessoa de Meia-Idade , VacinaçãoRESUMO
PURPOSE: Ipilimumab is a monoclonal antibody that blocks the immune-inhibitory interaction between CTL antigen 4 (CTLA-4) and its ligands on T cells. Clinical trials in cancer patients with ipilimumab have shown promising antitumor activity, particularly in patients with advanced melanoma. Often, tumor regressions in these patients are correlated with immune-related side effects such as dermatitis, enterocolitis, and hypophysitis. Although these reactions are believed to be immune-mediated, the antigenic targets for the cellular or humoral immune response are not known. EXPERIMENTAL DESIGN: We enrolled patients with advanced melanoma in a phase II study with ipilimumab. One of these patients experienced a complete remission of his tumor. The specificity and functional properties of CD8-positive T cells in his peripheral blood, in regressing tumor tissue, and at the site of an immune-mediated skin rash were investigated. RESULTS: Regressing tumor tissue was infiltrated with CD8-positive T cells, a high proportion of which were specific for Melan-A. The skin rash was similarly infiltrated with Melan-A-specific CD8-positive T cells, and a dramatic (>30-fold) increase in Melan-A-specific CD8-positive T cells was apparent in peripheral blood. These cells had an effector phenotype and lysed Melan-A-expressing tumor cells. CONCLUSIONS: Our results show that Melan-A may be a major target for both the autoimmune and antitumor reactions in patients treated with anti-CTLA-4, and describe for the first time the antigen specificity of CD8-positive T cells that mediate tumor rejection in a patient undergoing treatment with an anti-CTLA-4 antibody. These findings may allow a better integration of ipilimumab into other forms of immunotherapy.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Antígenos de Neoplasias/imunologia , Antineoplásicos/uso terapêutico , Melanoma/tratamento farmacológico , Proteínas de Neoplasias/imunologia , Neoplasias Cutâneas/tratamento farmacológico , Linfócitos T Citotóxicos/imunologia , Autoimunidade , Citotoxicidade Imunológica , Método Duplo-Cego , Exantema/induzido quimicamente , Exantema/imunologia , Humanos , Ipilimumab , Linfócitos do Interstício Tumoral/imunologia , Antígeno MART-1 , Melanoma/diagnóstico por imagem , Melanoma/imunologia , Neoplasias Cutâneas/diagnóstico por imagem , Neoplasias Cutâneas/imunologia , Tomografia Computadorizada por Raios XRESUMO
As more tumor antigens are discovered and as computer-guided T cell epitope prediction programs become more sophisticated, many potential T cell epitopes are synthesized and demonstrated to be antigenic in vitro. However, it is estimated that about 50% of such tumor antigen-specific T cells have not been demonstrated to recognize the naturally presented epitopes due to either technical difficulties, such as T cell cloning which is still challenging for many laboratories; or the predicted T cell epitopes are not generated or not generated in sufficient amounts by the antigen processing machinery. However, to potentially identify clinically relevant vaccine candidate epitopes, it is essential to demonstrate natural antigen presentation. Here we combine the advantages of MHC tetramer and intracellular cytokine staining to sensitively detect natural antigen presentation by tumor cells for epitopes of interest. The novel method does not require T cell cloning or long-term T cell culture. Because the antigen-specific T cells are positively identified, this method is much less influenced by IFNgamma producing cells with unknown specificities and should be widely applicable.
Assuntos
Antígenos de Neoplasias/análise , Linfócitos T CD8-Positivos/imunologia , Epitopos de Linfócito T/análise , Antígenos de Histocompatibilidade/imunologia , Melanoma/imunologia , Antígenos de Neoplasias/imunologia , Linhagem Celular Tumoral , Citocinas/análise , Citocinas/imunologia , Epitopos de Linfócito T/imunologia , Citometria de Fluxo , HumanosRESUMO
The amino acids that confer aldosterone binding specificity to the mineralocorticoid receptor (MR) remain to be determined. We had previously analyzed a panel of chimeras created between the MR and the glucocorticoid receptor and determined that amino acids 804-874 of the MR ligand binding domain are critical for aldosterone binding. In the present study a further series of chimeras was created within this region. The chimeras were analyzed by a transactivation assay and [(3)H]aldosterone binding, and the critical region was narrowed down to amino acids 820-844. Site-directed mutagenesis was used to create single and multiple amino acid substitutions in this region. These studies identified 12 of the 16 amino acids that differ in the MR and the glucocorticoid receptor in this region as being critical to conferring aldosterone responsivity. The amino acids that differ in the region 820-844 lie on the surface of the molecule and, therefore, it appears that MR ligand binding selectivity is conferred by residues that do not form part of the ligand binding pocket. Other studies have found that the corresponding regions of the androgen and glucocorticoid receptors are critical for the binding of natural and synthetic ligands, suggesting a common mechanism governing ligand binding specificity. The new chimeras also displayed, as previously reported, a dissociation between cortisol binding and transactivation and, intriguingly, only those that bound aldosterone with high affinity were activated by cortisol, suggesting a common mechanism that underlies specificity of aldosterone binding and the ability of cortisol to activate the MR.
Assuntos
Aldosterona/metabolismo , Hidrocortisona/metabolismo , Receptores de Mineralocorticoides/agonistas , Receptores de Mineralocorticoides/metabolismo , Sequência de Aminoácidos , Animais , Células Cultivadas , Hidrocortisona/farmacologia , Ligantes , Dados de Sequência Molecular , Mutação , Conformação Proteica , Receptores de Mineralocorticoides/genética , Receptores de Esteroides/agonistas , Receptores de Esteroides/genética , Receptores de Esteroides/metabolismo , Proteínas Recombinantes de Fusão , Ativação TranscricionalRESUMO
Cancer cell lines can be useful to model cancer stem cells. Infection with Mycoplasma species is an insidious problem in mammalian cell culture. While investigating stem-like properties in early passage melanoma cell lines, we noted poorly reproducible results from an aliquot of a cell line that was later found to be infected with Mycoplasma hyorhinis. Deliberate infection of other early passage melanoma cell lines aliquots induced variable and unpredictable effects on expression of putative cancer stem cell markers, clonogenicity, proliferation and global gene expression. Cell lines established in stem cell media (SCM) were equally susceptible. Mycoplasma status is rarely reported in publications using cultured cells to study the cancer stem cell hypothesis. Our work highlights the importance of surveillance for Mycoplasma infection while using any cultured cells to interrogate tumor heterogeneity.
Assuntos
Regulação Neoplásica da Expressão Gênica , Mycoplasma hyorhinis/isolamento & purificação , Proteínas de Neoplasias/genética , Células-Tronco Neoplásicas/microbiologia , RNA Ribossômico 16S/isolamento & purificação , Técnicas de Tipagem Bacteriana , Técnicas de Cultura de Células/normas , Linhagem Celular Tumoral , Interações Hospedeiro-Patógeno , Humanos , Melanoma/genética , Melanoma/metabolismo , Melanoma/microbiologia , Melanoma/patologia , Mycoplasma hyorhinis/genética , Proteínas de Neoplasias/metabolismo , Células-Tronco Neoplásicas/metabolismo , Reação em Cadeia da Polimerase , Cultura Primária de Células , Controle de Qualidade , RNA Ribossômico 16S/genética , Reprodutibilidade dos Testes , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/metabolismo , Neoplasias Cutâneas/microbiologia , Neoplasias Cutâneas/patologiaRESUMO
Immune responses to cancer vaccines are commonly tested by measuring cutaneous reactions to intradermal (i.d.) antigen. When well-characterized peptide epitopes are injected i.d., infiltrates of CD4+ and CD8+ T lymphocytes are frequently seen. In this study, we have further characterized T cells derived from vaccine-infiltrating lymphocyte (VIL) responses. We found that the infiltrates capable of producing IFN-gamma and cytolytic activity could recognize vaccine peptide, as well as antigen-positive melanoma cells. We studied antigen-specific T cell responses from VILs and peripheral blood in 10 patients who participated in a clinical trial. All patients received systemic Flt3 ligand (20 microg/kg/d) and i.d. peptides: Three NY-ESO-1 peptides, SLLMWITQCFL (157-167), SLLMWITQC (157-165), QLSLLMWIT (155-163); tyrosinase internal peptide YMDGTMSQV (368-376); Melan-A/MART-1 analogue peptide ELAGIGILTV (26-35, E27L substitution); and influenza matrix peptide GILGFVFTL (58-66). In 54 paired VIL and peripheral blood analyses, a good correlation was found between responses in skin and in blood. These cells could be rapidly expanded in a short-term assay and thus appear to be memory T cells. The demonstrated presence of antigen-specific T cells at vaccination sites validates this method of assessing the immune response to i.d. vaccines.
Assuntos
Antígenos de Neoplasias/imunologia , Linfócitos T CD8-Positivos/imunologia , Vacinas Anticâncer/imunologia , Hipersensibilidade Tardia/imunologia , Linfócitos do Interstício Tumoral/imunologia , Células Sanguíneas/imunologia , Linfócitos T CD4-Positivos/imunologia , Vacinas Anticâncer/administração & dosagem , Linhagem Celular Tumoral , Testes Imunológicos de Citotoxicidade , Humanos , Injeções Intradérmicas , Interferon gama/biossíntese , Melanoma/imunologia , Pele/imunologia , Neoplasias Cutâneas/imunologia , Vacinas de Subunidades Antigênicas/administração & dosagem , Vacinas de Subunidades Antigênicas/imunologiaRESUMO
Many tumor antigenic determinants have been identified and included in cancer clinical trials. Due to low T-cell frequencies even after vaccination, few T-cell responses can be revealed ex vivo without in vitro stimulation. Various expansion protocols have been employed for this purpose and the outcomes tend to be quite variable, partly due to the high complexity involved in the protocols. Here we systematically studied various common culture conditions including sera, cytokines and feeders and describe a reliable "bulk" culture method that is robust, simpler and more economical. We demonstrated that fetal calf serum (FCS) supported T-cell proliferation better than multiple commercially available pooled human AB sera. IL-2 is critical in our cultures, but IL-7, IL-15 and anti-CTLA-4 in combination with IL-2 did not further enhance T-cell expansion. We typically achieve more than a 40-fold expansion within a 10-day culture period for antigen-specific T cells measured by HLA-peptide tetramer before and after culture. This method was not only validated by multiple operators as a standard operating procedure for monitoring T-cell responses but was also successfully used for discovering novel CD8+ and CD4+ T cells specific to previously unknown epitopes from the NY-ESO-1 tumor antigen.
Assuntos
Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/imunologia , Técnicas de Cultura de Células/métodos , Ensaios Clínicos como Assunto , Neoplasias/imunologia , Animais , Anticorpos/imunologia , Anticorpos/farmacologia , Células Apresentadoras de Antígenos/citologia , Células Apresentadoras de Antígenos/imunologia , Antígenos CD , Antígenos de Diferenciação/imunologia , Antígeno CTLA-4 , Bovinos , Divisão Celular/efeitos dos fármacos , Células Cultivadas , Epitopos/química , Epitopos/imunologia , Humanos , Interleucina-15/farmacologia , Interleucina-7/farmacologia , Cinética , Neoplasias/sangue , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/imunologia , Reprodutibilidade dos Testes , SoroRESUMO
Dendritic cells (DCs) show promise as adjuvants in anticancer immunotherapeutic strategies. Flt3 ligand (FL) is a hematopoietic growth factor that increases the number of immature DCs in the blood and other tissues. We treated 27 patients with metastatic or high-risk resected melanoma with s.c. FL daily for 14 d in three 28 d cycles. Eighteen of these patients also received vaccination with influenza (Flu), Melan-A (Mel), tyrosinase (Tyr), and NY-ESO-1 peptides. To induce local DC maturation, 8 of the vaccinated patients had imiquimod, a Toll-like receptor-7 ligand (TLR7L), applied topically to their vaccine sites. Patients were monitored for clinical and hematological effects. Immune responses were assessed by cutaneous reactivity to vaccination and by the induction of peptide-specific CD8+ T-cells. Eight patients did not complete the protocol due to adverse events related to their cancer. The treatment was generally safe and well tolerated, although some patients developed clinically significant toxicities related to FL. FL induced increases in immature CD11c+ and CD123+ peripheral blood (PB) DCs. Other hematological effects included monocytosis, granulocytosis, and thrombocytosis, which were marked in some patients. Cutaneous reactions to peptide vaccination and circulating peptide-specific CD8+ T-cells were more frequent in imiquimod-treated patients. FL treatment of melanoma patients has pleiotropic clinical and hematological effects. In vivo maturation of FL-generated DCs using imiquimod may increase immune responses to tumor antigens.
Assuntos
Aminoquinolinas/administração & dosagem , Vacinas Anticâncer/administração & dosagem , Imunoterapia Ativa/métodos , Melanoma/imunologia , Melanoma/terapia , Proteínas de Membrana/administração & dosagem , Fragmentos de Peptídeos/imunologia , Adjuvantes Imunológicos/administração & dosagem , Adjuvantes Imunológicos/efeitos adversos , Adulto , Idoso , Aminoquinolinas/efeitos adversos , Aminoquinolinas/imunologia , Antígenos de Neoplasias/imunologia , Antineoplásicos/administração & dosagem , Antineoplásicos/efeitos adversos , Linfócitos T CD8-Positivos/imunologia , Vacinas Anticâncer/efeitos adversos , Vacinas Anticâncer/imunologia , Citocinas/sangue , Citocinas/imunologia , Células Dendríticas/imunologia , Feminino , Humanos , Imiquimode , Antígeno MART-1 , Masculino , Proteínas de Membrana/efeitos adversos , Proteínas de Membrana/imunologia , Pessoa de Meia-Idade , Monofenol Mono-Oxigenase/imunologia , Proteínas de Neoplasias/imunologia , Fragmentos de Peptídeos/administração & dosagem , Fragmentos de Peptídeos/efeitos adversosRESUMO
Combination therapy with BRAF and MEK inhibition is currently in clinical development for the treatment of BRAF-mutated malignant melanoma. BRAF inhibitors are associated with enhanced antigen-specific T-lymphocyte recognition in vivo. Consequently, BRAF inhibition has been proposed as proimmunogenic and there has been considerable enthusiasm for combining BRAF inhibition with immunotherapy. MEK inhibitors inhibit ERK phosphorylation regardless of BRAF mutational status and have been reported to impair T-lymphocyte and modulate dendritic cell function. In this study, we investigate the effects on isolated T lymphocytes and monocyte-derived dendritic cells (moDC) of a MEK (trametinib) and BRAF (dabrafenib) inhibitor combination currently being evaluated in a randomized controlled clinical trial. The effects of dabrafenib and trametinib, alone and in combination, were studied on isolated normal T lymphocytes and moDCs. Lymphocyte viability, together with functional assays including proliferation, cytokine production, and antigen-specific expansion, were assessed. MoDC phenotype in response to lipopolysaccharide stimulation was evaluated by flow cytometry, as were effects on antigen cross-presentation. Dabrafenib did not have an impact on T lymphocytes or moDCs, whereas trametinib alone or in combination with dabrafenib suppressed T-lymphocyte proliferation, cytokine production, and antigen-specific expansion. However, no significant decrease in CD4(+) or CD8(+) T-lymphocyte viability was observed following kinase inhibition. MoDC cross-presentation was suppressed in association with enhanced maturation following combined inhibition of MEK and BRAF. The results of this study demonstrate that MEK inhibition, alone or in combination with BRAF inhibition, can modulate immune cell function, and further studies in vivo will be required to evaluate the potential clinical impact of these findings.
Assuntos
Células Dendríticas/efeitos dos fármacos , Células Dendríticas/metabolismo , Linfócitos/efeitos dos fármacos , Linfócitos/metabolismo , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Antígenos de Neoplasias/imunologia , Diferenciação Celular , Proliferação de Células/efeitos dos fármacos , Apresentação Cruzada/imunologia , Citocinas/biossíntese , Células Dendríticas/citologia , Epitopos de Linfócito T/imunologia , Humanos , Imidazóis/farmacologia , Lipopolissacarídeos/imunologia , Ativação Linfocitária/efeitos dos fármacos , Subpopulações de Linfócitos/efeitos dos fármacos , Subpopulações de Linfócitos/imunologia , Subpopulações de Linfócitos/metabolismo , Oximas/farmacologia , Piridonas/farmacologia , Pirimidinonas/farmacologiaRESUMO
NY-ESO-1 has been a major target of many immunotherapy trials because it is expressed by various cancers and is highly immunogenic. In this study, we have identified a novel HLA-B*1801-restricted CD8(+) T cell epitope, NY-ESO-1(88-96) (LEFYLAMPF) and compared its direct- and cross-presentation to that of the reported NY-ESO-1(157-165) epitope restricted to HLA-A*0201. Although both epitopes were readily cross-presented by DCs exposed to various forms of full-length NY-ESO-1 antigen, remarkably NY-ESO-1(88-96) is much more efficiently cross-presented from the soluble form, than NY-ESO-1(157-165). On the other hand, NY-ESO-1(157-165) is efficiently presented by NY-ESO-1-expressing tumor cells and its presentation was not enhanced by IFN-γ treatment, which induced immunoproteasome as demonstrated by Western blots and functionally a decreased presentation of Melan A(26-35); whereas NY-ESO-1(88-96) was very inefficiently presented by the same tumor cell lines, except for one that expressed high level of immunoproteasome. It was only presented when the tumor cells were first IFN-γ treated, followed by infection with recombinant vaccinia virus encoding NY-ESO-1, which dramatically increased NY-ESO-1 expression. These data indicate that the presentation of NY-ESO-1(88-96) is immunoproteasome dependent. Furthermore, a survey was conducted on multiple samples collected from HLA-B18(+) melanoma patients. Surprisingly, all the detectable responses to NY-ESO-1(88-96) from patients, including those who received NY-ESO-1 ISCOMATRIX™ vaccine were induced spontaneously. Taken together, these results imply that some epitopes can be inefficiently presented by tumor cells although the corresponding CD8(+) T cell responses are efficiently primed in vivo by DCs cross-presenting these epitopes. The potential implications for cancer vaccine strategies are further discussed.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Células Dendríticas/imunologia , Epitopos/imunologia , Antígeno HLA-B18/imunologia , Western Blotting , Linhagem Celular Tumoral , Humanos , Melanoma/imunologia , Melanoma/patologiaRESUMO
The tumor antigen NY-ESO-1 is a promising cancer vaccine target. We describe here a novel HLA-B7-restricted NY-ESO-1 epitope, encompassing amino acids 60-72 (APRGPHGGAASGL), which is naturally presented by melanoma cells. The tumor epitope bound to HLA-B7 by bulging outward from the peptide-binding cleft. This bulged epitope was not an impediment to T-cell recognition, however, because four of six HLA-B7(+) melanoma patients vaccinated with NY-ESO-1 ISCOMATRIX vaccine generated a potent T-cell response to this determinant. Moreover, the response to this epitope was immunodominant in three of these patients and, unlike the T-cell responses to bulged HLA class I viral epitopes, the responding T cells possessed a remarkably broad TCR repertoire. Interestingly, HLA-B7(+) melanoma patients who did not receive the NY-ESO-1 ISCOMATRIX vaccine rarely generated a spontaneous T-cell response to this cryptic epitope, suggesting a lack of priming of such T cells in the natural anti-NY-ESO-1 response, which may be corrected by vaccination. Together, our results reveal several surprising aspects of antitumor immunity and have implications for cancer vaccine design.
Assuntos
Antígenos de Neoplasias/imunologia , Vacinas Anticâncer/imunologia , Epitopos Imunodominantes/imunologia , Melanoma/imunologia , Proteínas de Membrana/imunologia , Fragmentos de Peptídeos/imunologia , Alanina/genética , Sequência de Aminoácidos , Substituição de Aminoácidos , Apresentação de Antígeno , Linfócitos T CD8-Positivos/imunologia , Vacinas Anticâncer/uso terapêutico , Linhagem Celular Tumoral , Antígenos HLA-B/imunologia , Antígeno HLA-B7 , Humanos , Ativação Linfocitária , Melanoma/terapia , Modelos Moleculares , Dados de Sequência Molecular , Conformação ProteicaRESUMO
Immunodominance has been well-demonstrated in many antiviral and antibacterial systems, but much less so in the setting of immune responses against cancer. Tumor Ag-specific CD8+ T cells keep cancer cells in check via immunosurveillance and shape tumor development through immunoediting. Because most tumor Ags are self Ags, the breadth and depth of antitumor immune responses have not been well-appreciated. To design and develop antitumor vaccines, it is important to understand the immunodominance hierarchy and its underlying mechanisms, and to identify the most immunodominant tumor Ag-specific T cells. We have comprehensively analyzed spontaneous cellular immune responses of one individual and show that multiple tumor Ags are targeted by the patient's immune system, especially the "cancer-testis" tumor Ag NY-ESO-1. The pattern of anti-NY-ESO-1 T cell responses in this patient closely resembles the classical broad yet hierarchical antiviral immunity and was confirmed in a second subject.
Assuntos
Antígenos de Neoplasias/fisiologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Epitopos Imunodominantes/imunologia , Proteínas de Membrana/fisiologia , Animais , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Células CHO , Linhagem Celular Transformada , Linhagem Celular Tumoral , Cricetinae , Cricetulus , Epitopos de Linfócito T/imunologia , Epitopos de Linfócito T/metabolismo , Humanos , Imunidade Inata , Epitopos Imunodominantes/metabolismo , Masculino , Pessoa de Meia-IdadeRESUMO
Flt3 ligand mobilizes dendritic cells (DCs) into blood, allowing generation in vivo of large numbers of DCs for immunotherapy. These immature DCs can be rapidly activated by soluble CD40 ligand (CD40L). We developed a novel overnight method using these cytokines to produce DCs for cancer immunotherapy. Flt3 ligand-mobilized DCs (FLDCs) were isolated, activated with CD40L, loaded with antigenic peptides from influenza matrix protein, hepatitis B core antigen, NY-ESO-1, MAGE-A4, and MAGE-A10, and injected into patients with resected melanoma. Three injections were given at 4-week intervals. Study end points included antigen-specific immune responses (skin reactions to peptides alone or peptide-pulsed FLDCs; circulating T-cell responses), safety, and toxicity. No patient had a measurable tumor. Six patients were entered. FLDCs were obtained, enriched, and cultured under Good Manufacturing Practice grade conditions. Overnight culture with soluble CD40L caused marked up-regulation of activation markers (CD83 and HLA-DR). These FLDCs were functional and able to stimulate antigen-specific T cells in vitro. No significant adverse events were attributable to FLDCs. Peptide-pulsed FLDCs caused strong local skin reactions up to 60 mm diameter with intense perivascular infiltration of T cells, exceeding those seen in our previous peptide-based protocols. Antigen-specific blood T-cell responses were induced, including responses to an antigen for which the patients were naive (hepatitis B core antigen) and MAGE-A10. MAGE-A10-specific T cells with a skewed T-cell receptor repertoire were detected in 1 patient in blood ex vivo and from tumor biopsies. Vaccination with FLDCs pulsed with peptides is safe and primes immune responses to cancer antigens.
Assuntos
Ligante de CD40/imunologia , Linfócitos T CD8-Positivos/imunologia , Vacinas Anticâncer/uso terapêutico , Células Dendríticas/imunologia , Melanoma/terapia , Proteínas de Membrana/imunologia , Neoplasias Cutâneas/terapia , Adulto , Apresentação de Antígeno , Antígenos CD8/imunologia , Vacinas Anticâncer/efeitos adversos , Vacinas Anticâncer/imunologia , Células Cultivadas , Citotoxicidade Imunológica , Feminino , Humanos , Imunoterapia Adotiva , Leucaférese , Ativação Linfocitária , Masculino , Melanoma/imunologia , Pessoa de Meia-Idade , Peptídeos/imunologia , Peptídeos/uso terapêutico , Projetos Piloto , Receptores de Antígenos de Linfócitos T/metabolismo , Neoplasias Cutâneas/imunologiaRESUMO
"Cross-priming" refers to the activation of naive CD8+ T cells by antigen-presenting cells that have acquired nominal antigens from another cell. The biological relevance of cross-priming of CD8+ T cells has recently been challenged (Zinkernagel, R. M., Eur. J. Immunol. 2002. 32: 2385-2392), on the basis that responses are weak or poorly quantitated, and the determinants recognized are undefined. Here we show that cross-priming is a robust process that elicits vigorous primary responses to multiple peptides in two well-defined systems. Our findings support the relevance of cross-priming in CD8+ T cell responses to viruses and tumor cells, and demonstrate that cross-priming elicits CD8+ T cells to determinants generated by the endogenous processing pathway.
Assuntos
Antígenos de Neoplasias/imunologia , Antígenos Virais/imunologia , Linfócitos T CD8-Positivos/imunologia , Apresentação Cruzada/imunologia , Animais , Antígenos Transformantes de Poliomavirus/imunologia , Vírus da Influenza A/imunologia , CamundongosRESUMO
Immunodominance is a central feature of CD8+ T cell (TCD8+) responses to pathogens, transplants, and tumors. Determinants occupy a stable position in an immunodominance hierarchy (alpha-, beta-, etc.) defined by the frequencies of responding TCD8+. In this paper, we study the mechanistic basis for place-swapping between alpha- (acid polymerase (PA)(224-233)) and beta-determinants (nuclear protein 366-374) in primary vs secondary anti-influenza A virus (IAV) responses in mice. This phenomena was recently correlated with the inability of IAV-infected nondendritic cells (DCs) to generate PA(224-233), and it was proposed that secondary TCD8+ are principally activated by IAV-infected epithelial cells, while primary TCD8+ are activated by IAV-infected DCs. In this study, we show that the inability of non-DCs to generate PA(224-232) is relative rather than absolute, and that the preferential use of cross-priming in secondary anti-IAV responses can also account for the revised hierarchy. We further show that immunodomination of PA(224-233)-specific TCD8+ by nucleoprotein 366-374-specific TCD8+ plays a critical role in the phenomena, and that this is unlikely to be mediated by TCD8+ lysis of APCs or other cells.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Apresentação Cruzada , Citotoxicidade Imunológica , Vírus da Influenza A/imunologia , Transferência Adotiva , Animais , Linhagem Celular Tumoral , Memória Imunológica , Camundongos , Camundongos Endogâmicos C57BLRESUMO
There is increasing evidence showing the involvement of CD4(+) T cells in initiating and maintaining antitumor immune responses. NY-ESO-1 is expressed by various tumors but not normal tissues except testis. We conducted a cancer clinical trial by using full-length NY-ESO-1 protein formulated with ISCOMATRIX adjuvant and injected into patients intramuscularly. Autologous dendritic cells pulsed with NY-ESO-1 ISCOMATRIX in combination with overlapping synthetic peptides were used to identify immunodominant T cells from a vaccinated patient. We show here the identification and characterization of two novel CD4(+) T cell epitopes. T cells specific to these epitopes not only recognized autologous dendritic cells loaded with NY-ESO-1 but also NY-ESO-1-expressing tumor cell lines treated with IFN-gamma. One of the two responses identified was greater than the previously identified immunodominant HLA-DP4-restricted response and correlated with NY-ESO-1-specific CD8(+) T cell induction after vaccination. This T cell response was vaccinated in most patients who expressed HLA-DR2. This study has systematically surveyed patients vaccinated with full-length tumor antigen for a vaccinated CD4 helper T cell response.
Assuntos
Antígenos de Neoplasias/imunologia , Linfócitos T CD4-Positivos/imunologia , Proteínas de Membrana/imunologia , Vacinas Sintéticas/imunologia , Sequência de Aminoácidos , Linfócitos T CD8-Positivos/imunologia , Linhagem Celular Tumoral , Células Cultivadas , Epitopos/química , Epitopos/imunologia , Humanos , Ativação Linfocitária , Complexo Principal de Histocompatibilidade/imunologia , Masculino , Dados de Sequência Molecular , Testículo/imunologiaRESUMO
NY-ESO-1 is a "cancer-testis" antigen expressed in many cancers. ISCOMATRIX is a saponin-based adjuvant that induces antibody and T cell responses. We performed a placebo-controlled clinical trial evaluating the safety and immunogenicity of recombinant NY-ESO-1 protein with ISCOMATRIX adjuvant. Forty-six evaluable patients with resected NY-ESO-1-positive tumors received three doses of vaccine intramuscularly at monthly intervals. The vaccine was well tolerated. We observed high-titer antibody responses, strong delayed-type hypersensitivity reactions, and circulating CD8(+) and CD4(+) T cells specific for a broad range of NY-ESO-1 epitopes, including known and previously unknown epitopes. In an unplanned analysis, vaccinated patients appeared to have superior clinical outcomes to those treated with placebo or protein alone. The vaccine is safe and highly potent immunologically.