RESUMO
Anti-cancer T-cell responses are often halted due to the immune-suppressive micro-environment, in part related to tumor-associated macrophages. In the current study, we assessed indigestible ß-glucans (oatßG, curdlan, grifolan, schizophyllan, lentinan, yeast whole glucan particles (yWGP), zymosan and two additional yeast-derived ß-glucans a and b) for their physicochemical properties as well as their effects on the plasticity of human monocyte-derived macrophages that were polarized with IL-4 to immune-suppressive macrophages. Beta-glucans were LPS/LTA free, and tested for solubility, molecular masses, protein and monosaccharide contents. Curdlan, yeast-b and zymosan re-polarized M(IL-4) macrophages towards an M1-like phenotype, in particular showing enhanced gene expression of CCR7, ICAM1 and CD80, and secretion of TNF-α and IL-6. Notably, differential gene expression, pathway analysis as well as protein expressions demonstrated that M(IL-4) macrophages treated with curdlan, yeast-b or zymosan demonstrated enhanced production of chemo-attractants, such as CCL3, CCL4, and CXCL8, which contribute to recruitment of monocytes and neutrophils. The secretion of chemo-attractants was confirmed when using patient-derived melanoma-infiltrating immune cells. Taken together, the bacterial-derived curdlan as well as the yeast-derived ß-glucans yeast-b and zymosan have the unique ability to preferentially skew macrophages towards a chemo-attractant-producing phenotype that may aid in anti-cancer immune responses.
Assuntos
Fatores Quimiotáticos/uso terapêutico , Macrófagos Associados a Tumor/metabolismo , Zimosan/metabolismo , beta-Glucanas/metabolismo , Fatores Quimiotáticos/farmacologia , HumanosRESUMO
BACKGROUND: Prostate cancer is recognized as a heterogeneous disease demanding appropriate preclinical models that reflect tumor complexity. Previously, we established the PSA-Cre;PtenLoxP/LoxP genetic engineered mouse model (GEMM) for prostate cancer reflecting the various stages of tumor development. Prostate tumors in this Pten KO model slowly develop, requiring more than 10 months. In order to enhance its practical utility, we established a syngeneic panel of cell lines derived from PSA-Cre targeted Pten KO tumors, designated the mouse prostate cancer (MuCap) model. METHODS: Four different MuCaP epithelial cell lines were established from three independent primary Pten KO mouse prostate tumors. Tumorigenic capacity of the MuCaP cell lines was determined by subcutaneous inoculation of these cell lines in immunocompetent mice. Response to PI3K-targeted therapy was validated in ex vivo tissue slices of the established MuCaP tumors. RESULTS: The MuCaP cell lines were all tumorigenic in immunocompetent mice after subcutaneous inoculation. Interestingly, these syngrafted tumors represented different tumor growth rates and morphologies. Treatment with the specific PI3K inhibitor GDC0941 resulted in responses very similar between syngeneic MuCaP and primary Pten KO prostate tumors. Finally, immunoprofiling of the different syngeneic MuCaP tumors demonstrated differential numbers of tumor infiltrating lymphocytes and distinct immune gene profiles with expression of CD8, INFy, and PD1 being inversely related to tumor aggressiveness. CONCLUSIONS: Collectively, we present here a well-defined MuCaP platform of in vitro and in vivo mouse prostate cancer models that may support preclinical assessment of (immune)-therapies for prostate cancer.
Assuntos
Invasividade Neoplásica/patologia , PTEN Fosfo-Hidrolase/genética , Neoplasias da Próstata/patologia , Animais , Linhagem Celular Tumoral , Modelos Animais de Doenças , Linfócitos do Interstício Tumoral/metabolismo , Linfócitos do Interstício Tumoral/patologia , Masculino , Camundongos , Camundongos Knockout , Invasividade Neoplásica/genética , PTEN Fosfo-Hidrolase/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Próstata/metabolismo , Próstata/patologia , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismoRESUMO
The discovery of genes and molecular pathways involved in the formation of brain metastasis would direct the development of therapeutic strategies to prevent this deadly complication of cancer. By comparing gene expression profiles of Estrogen Receptor negative (ER-) primary breast tumors between patients who developed metastasis to brain and to organs other than brain, we found that T lymphocytes promote the formation of brain metastases. To functionally test the ability of T cells to promote brain metastasis, we used an in vitro blood-brain barrier (BBB) model. By co-culturing T lymphocytes with breast cancer cells, we confirmed that T cells increase the ability of breast cancer cells to cross the BBB. Proteomics analysis of the tumor cells revealed Guanylate-Binding Protein 1 (GBP1) as a key T lymphocyte-induced protein that enables breast cancer cells to cross the BBB. The GBP1 gene appeared to be up-regulated in breast cancer of patients who developed brain metastasis. Silencing of GBP1 reduced the ability of breast cancer cells to cross the in vitro BBB model. In addition, the findings were confirmed in vivo in an immunocompetent syngeneic mouse model. Co-culturing of ErbB2 tumor cells with activated T cells induced a significant increase in Gbp1 expression by the cancer cells. Intracardial inoculation of the co-cultured tumor cells resulted in preferential seeding to brain. Moreover, intracerebral outgrowth of the tumor cells was demonstrated. The findings point to a role of T cells in the formation of brain metastases in ER- breast cancers, and provide potential targets for intervention to prevent the development of cerebral metastases.
Assuntos
Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/secundário , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Proteínas de Ligação ao GTP/metabolismo , Linfócitos T/metabolismo , Adulto , Idoso , Animais , Barreira Hematoencefálica/metabolismo , Barreira Hematoencefálica/patologia , Células Cultivadas , Técnicas de Cocultura , Feminino , Proteínas de Ligação ao GTP/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos , Pessoa de Meia-Idade , Metástase Neoplásica/fisiopatologia , Transplante de Neoplasias , Proteoma , RNA Mensageiro/metabolismoRESUMO
Adoptive transfer of T cells that are gene engineered to express a defined TCR represents a feasible and promising therapy for patients with tumors. However, TCR gene therapy is hindered by the transient presence and effectiveness of transferred T cells, which are anticipated to be improved by adequate T cell costimulation. In this article, we report the identification and characterization of a novel two-chain TCR linked to CD28 and CD3ε (i.e., TCR:28ε). This modified TCR demonstrates enhanced binding of peptide-MHC and mediates enhanced T cell function following stimulation with peptide compared with wild-type TCR. Surface expression of TCR:28ε depends on the transmembrane domain of CD28, whereas T cell functions depend on the intracellular domains of both CD28 and CD3ε, with IL-2 production showing dependency on CD28:LCK binding. TCR:28ε, but not wild-type TCR, induces detectable immune synapses in primary human T cells, and such immune synapses show significantly enhanced accumulation of TCR transgenes and markers of early TCR signaling, such as phosphorylated LCK and ERK. Importantly, TCR:28ε does not show signs of off-target recognition, as evidenced by lack of TCR mispairing, as well as preserved specificity. Notably, when testing TCR:28ε in immune-competent mice, we observed a drastic increase in T cell survival, which was accompanied by regression of large melanomas with limited recurrence. Our data argue that TCR transgenes that contain CD28, and, thereby, may provide T cell costimulation in an immune-suppressive environment, represent candidate receptors to treat patients with tumors.
Assuntos
Antígenos CD28/imunologia , Complexo CD3/imunologia , Melanoma/terapia , Receptores de Antígenos de Linfócitos T/imunologia , Neoplasias Cutâneas/terapia , Linfócitos T/imunologia , Animais , Antígenos CD28/química , Antígenos CD28/genética , Complexo CD3/química , Complexo CD3/genética , Linhagem Celular Tumoral , MAP Quinases Reguladas por Sinal Extracelular/genética , MAP Quinases Reguladas por Sinal Extracelular/imunologia , Expressão Gênica , Regulação da Expressão Gênica , Humanos , Sinapses Imunológicas , Interleucina-2/genética , Interleucina-2/imunologia , Ativação Linfocitária , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/genética , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/imunologia , Melanoma/genética , Melanoma/imunologia , Melanoma/mortalidade , Camundongos , Recidiva Local de Neoplasia/prevenção & controle , Transplante de Neoplasias , Ligação Proteica , Engenharia de Proteínas , Receptores de Antígenos de Linfócitos T/química , Receptores de Antígenos de Linfócitos T/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Transdução de Sinais , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/imunologia , Neoplasias Cutâneas/mortalidade , Análise de Sobrevida , Linfócitos T/metabolismo , Linfócitos T/transplante , Carga TumoralRESUMO
Clinical therapy with T cells shows promise for cancer patients, but is currently challenged by incomplete responses and tumor relapse. The exact mechanisms that contribute to tumor relapse remain largely unclear. Here, we treated mouse melanomas with T cell receptor-engineered T cells directed against a human peptide-major histocompatibility complex antigen in immune-competent mice. T cells resulted in significant tumor regression, which was followed by relapse in about 80-90% of mice. Molecular analysis revealed that relapsed tumors harbored nonmutated antigen genes, not silenced by promoter methylation, and functionally expressed surface antigen at levels equal to nontreated tumors. Relapsed tumors resisted a second in vivo T cell treatment, but regained sensitivity to T cell treatment upon retransplantation in mice. Notably, relapsed tumors demonstrated decreased levels of CD8 T cells and monocytes, which were substantiated by downregulated expression of chemoattractants and adhesion molecules. These observations were confirmed when using T cells specific for a less immunogenic, endogenous mouse melanoma antigen. We conclude that tumors, when exposed to T cell treatment, can relapse without loss of antigen and develop a milieu that evades recruitment of effector CD8 T cells. Our findings support the concept to target the tumor milieu to aid T cell therapy in limiting tumor relapse.
Assuntos
Imunoterapia Adotiva , Melanoma/imunologia , Linfócitos T/imunologia , Animais , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/imunologia , Antígenos de Neoplasias/metabolismo , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Quimiotaxia de Leucócito/imunologia , Metilação de DNA , Modelos Animais de Doenças , Epitopos de Linfócito T , Expressão Gênica , Ativação Linfocitária/imunologia , Contagem de Linfócitos , Linfócitos do Interstício Tumoral/imunologia , Linfócitos do Interstício Tumoral/metabolismo , Melanoma/genética , Melanoma/metabolismo , Melanoma/patologia , Melanoma/terapia , Melanoma Experimental , Camundongos , Monócitos/imunologia , Monócitos/metabolismo , Recidiva Local de Neoplasia , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/metabolismo , Recidiva , Linfócitos T/metabolismoRESUMO
CD8(+) CTLs are essential for effective immune defense against intracellular microbes and neoplasia. CTLs recognize short peptide fragments presented in association with MHC class I (MHCI) molecules on the surface of infected or dysregulated cells. Ag recognition involves the binding of both TCR and CD8 coreceptor to a single ligand (peptide MHCI [pMHCI]). The TCR/pMHCI interaction confers Ag specificity, whereas the pMHCI/CD8 interaction mediates enhanced sensitivity to Ag. Striking biophysical differences exist between the TCR/pMHCI and pMHCI/CD8 interactions; indeed, the pMHCI/CD8 interaction can be >100-fold weaker than the cognate TCR/pMHCI interaction. In this study, we show that increasing the strength of the pMHCI/CD8 interaction by approximately 15-fold results in nonspecific, cognate Ag-independent pMHCI tetramer binding at the cell surface. Furthermore, pMHCI molecules with superenhanced affinity for CD8 activate CTLs in the absence of a specific TCR/pMHCI interaction to elicit a full range of effector functions, including cytokine/chemokine release, degranulation and proliferation. Thus, the low solution binding affinity of the pMHCI/CD8 interaction is essential for the maintenance of CTL Ag specificity.
Assuntos
Apresentação de Antígeno/imunologia , Linfócitos T CD8-Positivos/imunologia , Citotoxicidade Imunológica/imunologia , Antígenos de Histocompatibilidade Classe I/imunologia , Ativação Linfocitária/imunologia , Linfócitos T Citotóxicos/imunologia , Antígenos CD8/imunologia , Antígenos CD8/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Separação Celular , Células Cultivadas , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Humanos , Receptores de Antígenos de Linfócitos T/imunologia , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T Citotóxicos/metabolismoRESUMO
BACKGROUND: The tumor-selective human adenovirus Delta24-RGD is currently under investigation in phase II clinical trials for patients with recurrent glioblastoma (GBM). To improve treatments for patients with GBM, we explored the potential of combining Delta24-RGD with antibodies targeting immune checkpoints. METHODS: C57BL/6 mice were intracranially injected with GL261 cells and treated with a low dose of Delta24-RGD virus. The expression dynamics of 10 co-signaling molecules known to affect immune activity was assessed in tumor-infiltrating immune cells by flow cytometry after viral injection. The antitumor activity was measured by tumor cell killing and IFNγ production in co-cultures. Efficacy of the combination viro-immunotherapy was tested in vitro and in the GL261 and CT2A orthotopic mouse GBM models. Patient-derived GBM cell cultures were treated with Delta24-RGD to assess changes in PD-L1 expression induced by virus infection. RESULTS: Delta24-RGD therapy increased intratumoral CD8+ T cells expressing Inducible T-cell co-stimulator (ICOS) and PD-1. Functionality assays confirmed a significant positive correlation between tumor cell lysis and IFNγ production in ex vivo cultures (Spearman r = 0.9524; P < .01). Co-cultures significantly increased IFNγ production upon treatment with PD-1 blocking antibodies. In vivo, combination therapy with low-dose Delta24-RGD and anti-PD-1 antibodies significantly improved outcome compared to single-agent therapy in both syngeneic mouse glioma models and increased PD-1+ tumor-infiltrating CD8+ T cells. Delta24-RGD infection induced tumor-specific changes in PD-L1 expression in primary GBM cell cultures. CONCLUSIONS: This study demonstrates the potential of using low-dose Delta24-RGD therapy to sensitize glioma for combination with anti-PD-1 antibody therapy.
RESUMO
A novel mutation F826L located within the ligand binding domain (LBD) of the human androgen receptor (AR) was investigated. This mutation was found in a boy with severe penoscrotal hypospadias (classified as 46,XY DSD). The AR mutant F826L appeared to be indistinguishable from the wild-type AR, with respect to ligand binding affinity, transcriptional activation of MMTV-luciferase and ARE2-TATA-luciferase reporter genes, protein level in genital skin fibroblasts (GSFs), and sub-cellular distribution in transfected cells. However, an at least two-fold higher NH2-/COOH-terminal domain interaction was found in luciferase and GST pull-down assays. A two-fold increase was also observed for TIF2 (transcription intermediary factor 2) co-activation of the AR F826L COOH-terminal domain. This increase could not be explained by a higher stability of the mutant protein, which was within wild-type range. Repression of transactivation by the nuclear receptor co-repressor (N-CoR) was not affected by the AR F826L mutation. The observed properties of AR F826L would be in agreement with an increased activity rather than with a partial defective AR transcriptional activation. It is concluded that the penoscrotal hypospadias in the present case is caused by an as yet unknown mechanism, which still may involve the mutant AR.
Assuntos
Substituição de Aminoácidos , Síndrome de Resistência a Andrógenos/genética , Mutação/genética , Coativador 2 de Receptor Nuclear/metabolismo , Receptores Androgênicos/química , Receptores Androgênicos/genética , Linhagem Celular , Pré-Escolar , Feminino , Fibroblastos/citologia , Fibroblastos/metabolismo , Prepúcio do Pênis/citologia , Humanos , Imunoprecipitação , Lactente , Ligantes , Masculino , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Proteínas Nucleares/metabolismo , Correpressor 1 de Receptor Nuclear , Ligação Proteica , Estrutura Terciária de Proteína , Transporte Proteico , Receptores Androgênicos/metabolismo , Proteínas Repressoras/metabolismo , Frações Subcelulares/metabolismo , Ativação Transcricional/genéticaRESUMO
Androgen insensitivity syndrome (AIS) is caused by defects in the androgen receptor (AR) that render the AR partially or completely inactive. As a result, embryonic sex differentiation is impaired. Here, we describe a novel mutation in the AR found in a patient with partial AIS. The mutation results in a substitution of a glutamine (Q) by a lysine (K) residue at position 902, Q902K. The AR Q902K mutation was investigated in vitro with respect to its functional properties. The equilibrium dissociation constants (K(d)s) of AR Q902K in the presence of either the synthetic androgen R1881 or the natural ligand DHT were slightly elevated. The R1881 dissociation rate (t(1/2)) was increased 3-fold for AR Q902K compared with wild type. Transcriptional activity was decreased to 85% of wild type, and the dose-response curve revealed that the sensitivity to hormone was decreased due to the mutation. Furthermore, the 114-kDa androgen-induced phosphorylated AR protein band was not detectable in genital skin fibroblasts. However, it could be detected in transfected CHO cells expressing the mutant receptor in the presence of 10 and 100 nm R1881. Functional interaction assays and a GST pull-down assay showed that the interaction between the NH2 and COOH terminus of AR Q902K was reduced to 50% of wild type. Furthermore, the transactivation by the coactivator TIF2 (transcriptional intermediary factor 2) was decreased 2- to 3-fold. The half-maximal response in both assays was shifted to a higher hormone concentration compared with wild type. These results indicate that residue Q902 is involved in TIF2 and NH2/COOH interaction and that the Q to K mutation results in a mild impairment of AR function, which can explain the partial AIS phenotype of the patient.
Assuntos
Síndrome de Resistência a Andrógenos/genética , Mutação , Receptores Androgênicos/genética , Animais , Células CHO , Pré-Escolar , Cricetinae , Humanos , Masculino , Receptores Androgênicos/química , Receptores Androgênicos/fisiologia , Ativação TranscricionalRESUMO
Among nuclear receptors, the androgen receptor (AR) is unique in that its ligand-binding domain (LBD) interacts with the FXXLF motif in the N-terminal domain, resembling coactivator LXXLL motifs. We compared AR- and estrogen receptor alpha-LBD interactions of the wild-type AR FXXLF motif and coactivator transcriptional intermediary factor 2 LXXLL motifs and variants of these motifs. Random mutagenesis revealed a key role for the F residues in FXXLF motifs in high-affinity and selective AR LBD interaction. The FXXLF motif in full-length AR and transcriptional intermediary factor 2 LXXLL motifs competed for an overlapping binding site. A computer model of the AR LBD/AR FXXLF complex showed that the bulky F residues are buried in a deep coactivator-binding groove. The corresponding groove in estrogen receptor alpha LBD is considerably shallower, explaining lack of binding of any of the FXXLF motifs tested. FXXLF and LXXLL motif interaction depended on different charged amino acid residues in the AR LBD present at opposite ends of the coactivator groove. In conclusion, our data demonstrate the importance of a deep hydrophobic groove and alternative usage of charged amino acids in specifying peptide binding to the AR LBD.
Assuntos
Receptores Androgênicos/química , Receptores Androgênicos/metabolismo , Fatores de Transcrição/química , Motivos de Aminoácidos/genética , Sequência de Aminoácidos , Substituição de Aminoácidos/genética , Sítios de Ligação , Ligação Competitiva , Análise Mutacional de DNA , Receptor alfa de Estrogênio/química , Receptor alfa de Estrogênio/genética , Receptor alfa de Estrogênio/metabolismo , Humanos , Ligantes , Modelos Moleculares , Dados de Sequência Molecular , Coativador 2 de Receptor Nuclear , Peptídeos/química , Conformação Proteica , Receptores Androgênicos/genética , Fatores de Transcrição/metabolismo , Técnicas do Sistema de Duplo-HíbridoRESUMO
Antiandrogens are widely used agents in the treatment of prostate cancer, as inhibitors of AR (androgen receptor) action. Although the precise mechanism of antiandrogen action is not yet elucidated, recent studies indicate the involvement of nuclear receptor co-repressors. In the present study, the regulation of AR transcriptional activity by N-CoR (nuclear receptor co-repressor), in the presence of different ligands, has been investigated. Increasing levels of N-CoR differentially affected the transcriptional activity of AR occupied with either agonistic or antagonistic ligands. Small amounts of co-transfected N-CoR repressed CPA (cyproterone acetate)- and mifepristone (RU486)-mediated AR activity, but did not affect agonist (R1881)-induced AR activity. Larger amounts of co-transfected N-CoR repressed AR activity for all ligands, and converted the partial agonists CPA and RU486 into strong AR antagonists. In the presence of the agonist R1881, co-expression of the p160 co-activator TIF2 (transcriptional intermediary factor 2) relieved N-CoR repression up to control levels. However, in the presence of RU486 and CPA, TIF2 did not functionally compete with N-CoR, suggesting that antagonist-bound AR has a preference for N-CoR. The AR mutation T877A (Thr877-->Ala), which is frequently found in prostate cancer and affects the ligand-induced conformational change of the AR, considerably reduced the repressive action of N-CoR. The agonistic activities of CPA- and hydroxyflutamide-occupied T877A-AR were hardly affected by N-CoR, whereas TIF2 strongly enhanced their activities. These results indicate that lack of N-CoR action allows these antiandrogens to act as strong agonists on the mutant AR.
Assuntos
Antagonistas de Androgênios/farmacologia , Antagonistas de Receptores de Andrógenos , Regulação da Expressão Gênica , Proteínas Nucleares/metabolismo , Receptores Androgênicos/metabolismo , Proteínas Repressoras/metabolismo , Transcrição Gênica , Animais , Ligação Competitiva , Células CHO , Linhagem Celular Tumoral , Cricetinae , Acetato de Ciproterona/farmacologia , Regulação para Baixo , Ligantes , Masculino , Metribolona/antagonistas & inibidores , Metribolona/farmacologia , Camundongos , Mifepristona/farmacologia , Mutação/genética , Correpressor 1 de Receptor Nuclear , Coativador 2 de Receptor Nuclear , Regiões Promotoras Genéticas/genética , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo , Receptores Androgênicos/genética , Fatores de Transcrição/metabolismo , TransfecçãoRESUMO
During sex differentiation, androgens are essential for development of the male genital tract. The Wolffian duct is an androgen-sensitive target tissue that develops into the epididymis, vas deferens, and seminal vesicle. The present study aimed to identify androgen-regulated proteins that are involved in development of Wolffian duct-derived structures. We have used male mouse embryos transgenic for temperature-sensitive simian virus 40 large tumor antigen at 18 d of gestation, to generate immortalized mouse fetal vas deferens (MFVD) parental and clonal cell lines. The MFVD parental and clonal cell lines express androgen receptor protein and show features of Wolffian duct mesenchymal cells. Clonal cell line MFVD A6 was selected for proteomic analysis and cultured in the absence or presence of androgens. Subsequently, two-dimensional gel electrophoresis was performed on total cell lysates. Differentially expressed proteins were analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and two androgen-regulated proteins were identified as mElfin and CArG-binding factor-A (CBF-A). CBF-A and mElfin are known to bind to cytoskeletal F-actin. Both proteins appeared to be regulated by androgens at the posttranslational level, possibly involving phosphorylation. Posttranslational modification of mElfin and CBF-A by androgens may be associated with a cytoskeletal change that is involved in androgen-regulated gene expression.
Assuntos
Androgênios/farmacologia , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B , Proteômica , Receptores Androgênicos/análise , Ducto Deferente/embriologia , Ducto Deferente/fisiologia , Animais , Linhagem Celular Transformada/química , Linhagem Celular Transformada/fisiologia , Linhagem da Célula/fisiologia , Proteínas de Ligação a DNA/análise , Proteínas de Ligação a DNA/genética , Feminino , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Proteínas com Domínio LIM , Masculino , Camundongos , Camundongos Endogâmicos CBA , Camundongos Transgênicos , Gravidez , Receptores Androgênicos/genética , Proteínas Repressoras/análise , Proteínas Repressoras/genética , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Células Estromais/citologia , Fatores de Transcrição/análise , Fatores de Transcrição/genética , Ducto Deferente/citologiaRESUMO
Antiandrogens can efficiently block androgen receptor (AR) mediated gene expression, and are therefore useful tools in the treatment of androgen dependent prostate cancer. Antiandrogens are either complete or partial inhibitors of AR activity, depending on the nature of the compound. As compared to androgens, antiandrogens induce a different AR conformation, thereby influencing the recruitment of co-regulators (coactivators and corepressors). This ligand-selective modulation of AR activity is affected by an AR mutation (Thr877Ala substitution) found in prostate cancer. In contrast to the wild-type AR, the mutant AR conformation induced by cyproterone acetate (CPA) and hydroxyflutamide (OHF) is comparable to that induced by androgens. As a consequence, this might affect recruitment of co-regulators, thereby allowing CPA and OHF to act as strong agonists on the mutant AR.
Assuntos
Antagonistas de Androgênios/metabolismo , Receptores Androgênicos/metabolismo , Androgênios/metabolismo , Animais , Linhagem Celular , Regulação da Expressão Gênica , Humanos , Masculino , Metribolona/metabolismo , Mifepristona/metabolismo , Ligação Proteica , Receptores Androgênicos/genéticaRESUMO
T cell-sorting technologies with peptide-MHC multimers or antibodies against gene markers enable enrichment of antigen-specific T cells and are expected to enhance the therapeutic efficacy of clinical T cell therapy. However, a direct comparison between sorting reagents for their ability to enrich T cells is lacking. Here, we compared the in vitro properties of primary human T cells gene-engineered with gp100(280-288)/HLA-A2-specific T cell receptor-αß (TCRαß) on magnetic-activated cell sorting (MACS) with various peptide-MHC multimers or an antibody against truncated CD34 (tCD34). With respect to peptide-MHC multimers, we observed that Streptamer(®), when compared with pentamers and tetramers, improved T cell yield as well as level and stability of enrichment, of TCR-engineered T cells (>65% of peptide-MHC-binding T cells, stable for at least 6 weeks). In agreement with these findings, Streptamer, the only detachable reagent, revealed significant T cell expansion in the first week after MACS. Sorting TCR and tCD34 gene-engineered T cells with CD34 monoclonal antibody (mAb) resulted in the most significant T cell yield and enrichment of T cells (>95% of tCD34 T cells, stable for at least 6 weeks). Notably, T cells sorted with CD34 mAb, when compared with Streptamer, bound about 2- to 3-fold less peptide-MHC but showed superior antigen-specific upregulated expression of CD107a and production of interferon (IFN)-γ. Multiparametric flow cytometry revealed that CD4(+) T cells, uniquely present in CD34 mAb-sorted T cells, contributed to enhanced IFN-γ production. Taken together, we postulate that CD34 mAb-based sorting of gene-marked T cells has benefits toward applications of T cell therapy, especially those that require CD4(+) T cells.
Assuntos
Linfócitos T CD4-Positivos/citologia , Linfócitos T CD8-Positivos/citologia , Separação Celular/métodos , Engenharia Genética/métodos , Magnetismo/métodos , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Anticorpos Monoclonais , Antígenos CD34/genética , Antígenos CD34/imunologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Marcadores Genéticos/genética , Antígeno HLA-A2/genética , Humanos , Técnicas In Vitro , Interferon gama/metabolismo , Proteína 1 de Membrana Associada ao Lisossomo/metabolismo , Fragmentos de Peptídeos , Peptídeos/genética , Antígeno gp100 de Melanoma/genéticaRESUMO
Adoptive cell therapy using T-cell receptor (TCR)-engineered T cells is a clinically feasible and promising approach to target tumors, but is currently faced with compromised antitumor efficacies in patients. Here, we extensively validated immune-deficient mice to facilitate further development of the therapeutic potential of TCR-engineered T cells. Treatment of human melanoma-bearing SCID or NSG mice with high doses of human T cells transduced with an hgp100/HLA-A2-specific TCR did not result in antitumor responses irrespective of chemotherapeutic preconditioning. Imaging of human green fluorescent protein-labeled T cells demonstrated significant T-cell accumulation in intratumoral vasculature directly upon T-cell transfer, which was followed by loss of T cells within 72 hr. Peripheral persistence of human T cells was highly compromised and appeared related to T-cell differentiation. On the contrary, adoptive transfer (AT) of relatively low numbers of hgp100/HLA-A2 TCR-transduced mouse T cells resulted in rapid clearance of large established human melanomas. Unexpectedly and in contrast to reported studies with chimeric antibody receptor-engineered T cells, antitumor activity and homeostatic expansion of T cells were independent of TCR transgene as evidenced in two SCID strains and using two different human melanoma cell lines. Interestingly, the xeno-reactive melanoma response of mouse T cells appeared to be dictated by CD4(+) tumor-infiltrating lymphocytes and did not require in vitro T-cell activation, retroviral gene transfer, or subcutaneous interleukin-2 support. Taken together, AT of human but not mouse T cells in human melanoma-bearing immune-deficient mice is in close accordance with clinical studies.
Assuntos
Terapia Genética , Antígeno HLA-A2/genética , Imunoterapia Adotiva , Melanoma/terapia , Receptores de Antígenos de Linfócitos T/genética , Antígeno gp100 de Melanoma/genética , Animais , Diferenciação Celular , Genes Reporter , Vetores Genéticos , Proteínas de Fluorescência Verde , Antígeno HLA-A2/imunologia , Humanos , Melanoma/imunologia , Melanoma/patologia , Camundongos , Camundongos SCID , Receptores de Antígenos de Linfócitos T/imunologia , Retroviridae/genética , Especificidade da Espécie , Linfócitos T/imunologia , Linfócitos T/metabolismo , Transdução Genética , Transgenes , Antígeno gp100 de Melanoma/imunologiaRESUMO
The Chimeric Antigen Receptor (CAR) consists of an antibody-derived targeting domain fused with T-cell signaling domains that, when expressed by a T-cell, endows the T-cell with antigen specificity determined by the targeting domain of the CAR. CARs can potentially redirect the effector functions of a T-cell towards any protein and nonprotein target expressed on the cell surface as long as an antibody or similar targeting domain is available. This strategy thereby avoids the requirement of antigen processing and presentation by the target cell and is applicable to nonclassical T-cell targets like carbohydrates. This circumvention of HLA-restriction means that the CAR T-cell approach can be used as a generic tool broadening the potential of applicability of adoptive T-cell therapy. Proof-of-principle studies focusing upon the investigation of the potency of CAR T-cells have primarily focused upon the genetic modification of human and mouse T-cells for therapy. This chapter focuses upon methods to modify T-cells from both species to generate CAR T-cells for functional testing.
Assuntos
Imunoterapia Adotiva/métodos , Receptores de Antígenos/imunologia , Proteínas Recombinantes/imunologia , Linfócitos T/imunologia , Animais , Anticorpos/imunologia , Proliferação de Células , Separação Celular , Eletroporação , Humanos , Linfonodos/citologia , Ativação Linfocitária/imunologia , Subpopulações de Linfócitos/imunologia , Camundongos , Microesferas , RNA/metabolismo , Retroviridae/fisiologia , Baço/citologia , Linfócitos T/citologia , Linfócitos T/virologia , Transcrição Gênica , Transdução Genética , TransfecçãoRESUMO
Gene-engineered T cell therapy represents a promising strategy to treat cancers. To enable pre-selection of patients sensitive to this type of treatment we have setup and validated a T cell activation assay to test antigen expression on patient-derived tumor tissues. Chimeric antibody-based receptor (CAR) directed against CAIX, currently used in a clinical trial to treat RCC patients, was used as a model receptor. Primary human T cells expressing CAIX CAR were able to respond to CAIX-positive but not CAIX-negative tumor tissue and showed an increased production of IFNgamma, TNFalpha, IL-10 and IL-4, but not IL-2 or IL-5. Tumor tissue driven responses of primary T cells were paralleled by NFAT activation measured in CAR-transduced Jurkat T cells, which was shown to be triggered in a CAR and antigen-specific manner. Next, the reporter gene assay was applied to two independent PSMA CARs, which both mediated NFAT activation in response to tumor tissue. Taken together, a sensitive and donor-independent assay was established to measure T cell activation upon exposure to patient-derived tumor tissue, which may facilitate pre-selection of patients for clinical adoptive T cell therapy.
Assuntos
Transferência Adotiva/métodos , Carcinoma de Células Renais/imunologia , Carcinoma de Células Renais/terapia , Neoplasias Renais/imunologia , Neoplasias Renais/terapia , Ativação Linfocitária/imunologia , Linfócitos T/imunologia , Linfócitos T/transplante , Reações Antígeno-Anticorpo , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/imunologia , Anidrase Carbônica IX , Anidrases Carbônicas/genética , Anidrases Carbônicas/imunologia , Carcinoma de Células Renais/patologia , Engenharia Genética , Humanos , Células Jurkat , Neoplasias Renais/patologia , Seleção de Pacientes , Receptores de Antígenos de Linfócitos T/imunologia , Células Tumorais CultivadasRESUMO
Therapeutic success of TCR gene transfer to treat tumors depends on the ability of redirected T cells to become activated upon tumor recognition in vivo. Help provided by tumor-specific Th1 cells is reported to relieve T cells from an anergized state and to induce tumor regression. We recently demonstrated the ability to generate melanoma-specific Th1 cells by genetic introduction of both a CD8-dependent TCR and the CD8alpha coreceptor into CD4+ T cells. In this study, we analyzed a TCR that binds Ag independently of CD8, a property generally preferred to induce tumor-specific T cell responses, and addressed the contribution of CD8alpha following introduction into TCR-transduced CD4+ T cells. To this end, primary human CD4+ T cells were gene transferred with a high-avidity TCR, and were shown not only to bind peptide/MHC class I, but also to effectively kill Ag-positive tumor cells in the absence of CD8alpha. The introduction of CD8alpha up-regulates the tumor-specific production of TNF-alpha and IL-2 to some extent, but significantly down-regulates production of IL-4, IL-5, and IL-10 in CD4+ T cells. The introduction of a mutated cysteine motif in CD8alpha, which prevents its binding to LCK and linker for activation of T cells, did not adversely affect expression and T cell cytotoxicity, but counteracted the CD8alpha-mediated down-regulation of IL-4 and IL-5, but not IL-10. In conclusion, CD8alpha down-regulates the production of major Th2-type cytokines, in part mediated by LCK and/or linker for activation of T cells, and may induce differentiation of tumor-specific Th1 cells, which makes this coreceptor an interesting candidate to improve the clinical potential of TCR gene transfer to treat cancer.
Assuntos
Antígenos CD8/genética , Regulação para Baixo/imunologia , Técnicas de Transferência de Genes , Interleucina-10/antagonistas & inibidores , Interleucina-4/antagonistas & inibidores , Interleucina-5/antagonistas & inibidores , Melanoma/terapia , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Motivos de Aminoácidos/genética , Antígenos CD8/fisiologia , Antígenos CD8/uso terapêutico , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Linhagem Celular , Linhagem Celular Tumoral , Cisteína/genética , Citotoxicidade Imunológica/genética , Regulação para Baixo/genética , Epitopos de Linfócito T/imunologia , Epitopos de Linfócito T/metabolismo , Antígeno HLA-A2/imunologia , Antígeno HLA-A2/metabolismo , Humanos , Interleucina-10/biossíntese , Interleucina-4/biossíntese , Interleucina-5/biossíntese , Melanoma/genética , Melanoma/imunologia , Glicoproteínas de Membrana/imunologia , Glicoproteínas de Membrana/metabolismo , Fragmentos de Peptídeos/imunologia , Fragmentos de Peptídeos/metabolismo , Ligação Proteica/genética , Ligação Proteica/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Receptores de Antígenos de Linfócitos T alfa-beta/uso terapêutico , Células Th1/citologia , Células Th1/imunologia , Células Th1/metabolismo , Transdução Genética/métodos , Antígeno gp100 de MelanomaRESUMO
EBV is associated with a broad range of malignancies. Adoptive immunotherapy of these tumors with EBV-specific CTL proved useful. We generated a panel of primary human T cells specific to various EBV antigens (i.e. Epstein-Barr nuclear antigen 3A, 3B and BamHI-M leftward reading frame) via transfer of modified TCR genes that are either coupled to CD3zeta or Fc(epsilon)RIgamma. TCR-transduced T cells from 20-60% of donors (total number of 25) demonstrated specific lysis of EBV peptide-loaded target cells, whereas lymphoblastoid cell lines expressing native EBV antigens were not killed by any of the EBV-specific T cell populations. This non-responsiveness, confirmed at the level of nuclear factor of activated T cells activation, is not due to receptor configuration since identical receptor formats specific for melanoma antigens successfully re-targeted T cells to native melanoma cells. In an effort to generate a more potent receptor, we introduced a CD28 domain into one of the EBV-specific TCR. This TCR did not affect the cytotoxic response of re-targeted T cells, but dramatically enhanced antigen-specific IFNgamma production. We therefore conclude that these novel CD28-containing EBV-specific TCRs provide a basis for further development of TCR gene transfer to treat EBV-induced diseases.
Assuntos
Antígenos Nucleares do Vírus Epstein-Barr/imunologia , Técnicas de Transferência de Genes , Genes Codificadores dos Receptores de Linfócitos T , Interferon gama/biossíntese , Linfócitos T/imunologia , Antígenos CD28/imunologia , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Terapia Genética/métodos , Humanos , Células Jurkat , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/imunologia , Transdução GenéticaRESUMO
The N-terminal domain (NTD) and the ligand-binding domain (LBD) of the androgen receptor (AR) exhibit a ligand-dependent interaction (N/C interaction). Amino acids 3-36 in the NTD (AR3-36) play a dominant role in this interaction. Previously, it has been shown that a PhixxPhiPhi motif in AR3-36, 23FxxLF27, is essential for LBD interaction. We demonstrate in the current study that AR3-36 can be subdivided into two functionally distinct fragments: AR3-13 and AR16-36. AR3-13 does not directly interact with the AR LBD, but rather contributes to the transactivation function of the AR.NTD-AR.LBD complex. AR16-36, encompassing the 23FxxLF27 motif, is predicted to fold into a long amphipathic alpha-helix. A second PhixxPhiPhi candidate protein interaction motif within the helical structure, 30VREVI34, shows no affinity to the LBD. Within AR16-36, amino acid residues in and flanking the 23FxxLF27 motif are demonstrated to modulate N/C interaction. Substitution of Q24 and N25 by alanine residues enhances N/C interaction. Substitution of amino acids flanking the 23FxxLF27 motif by alanines are inhibitory to LBD interaction.