RESUMO
HIV- and SIV-envelope (Env) trimers are both extensively glycosylated, and antibodies identified to date have been unable to fully neutralize SIVmac239. Here, we report the isolation, structure, and glycan interactions of antibody ITS90.03, a monoclonal antibody that completely neutralized the highly neutralization-resistant isolate, SIVmac239. The co-crystal structure of a fully glycosylated SIVmac239-gp120 core in complex with rhesus CD4 and the antigen-binding fragment of ITS90.03 at 2.5-Å resolution revealed that ITS90 recognized an epitope comprised of 45% glycan. SIV-gp120 core, rhesus CD4, and their complex could each be aligned structurally to their human counterparts. The structure revealed that glycans masked most of the SIV Env protein surface, with ITS90 targeting a glycan hole, which is occupied in â¼83% of SIV strains by glycan N238. Overall, the SIV glycan shield appears to functionally resemble its HIV counterpart in coverage of spike, shielding from antibody, and modulation of receptor accessibility.
Assuntos
Anticorpos Monoclonais/química , Anticorpos Neutralizantes/química , Infecções por HIV/imunologia , HIV/fisiologia , Polissacarídeos/química , Síndrome de Imunodeficiência Adquirida dos Símios/imunologia , Vírus da Imunodeficiência Símia/fisiologia , Animais , Anticorpos Monoclonais/isolamento & purificação , Anticorpos Monoclonais/metabolismo , Anticorpos Neutralizantes/isolamento & purificação , Anticorpos Neutralizantes/metabolismo , Antígenos CD4/metabolismo , Células Cultivadas , Cristalização , Cristalografia por Raios X , Modelos Animais de Doenças , Glicosilação , Anticorpos Anti-HIV/imunologia , Anticorpos Anti-HIV/metabolismo , Proteína gp120 do Envelope de HIV/metabolismo , Humanos , Macaca mulatta , Glicoproteínas de Membrana/metabolismo , Polissacarídeos/metabolismo , Ligação Proteica , Relação Estrutura-Atividade , Proteínas do Envelope Viral/metabolismoRESUMO
A central goal of HIV-1 vaccine research is the elicitation of antibodies capable of neutralizing diverse primary isolates of HIV-1. Here we show that focusing the immune response to exposed N-terminal residues of the fusion peptide, a critical component of the viral entry machinery and the epitope of antibodies elicited by HIV-1 infection, through immunization with fusion peptide-coupled carriers and prefusion stabilized envelope trimers, induces cross-clade neutralizing responses. In mice, these immunogens elicited monoclonal antibodies capable of neutralizing up to 31% of a cross-clade panel of 208 HIV-1 strains. Crystal and cryoelectron microscopy structures of these antibodies revealed fusion peptide conformational diversity as a molecular explanation for the cross-clade neutralization. Immunization of guinea pigs and rhesus macaques induced similarly broad fusion peptide-directed neutralizing responses, suggesting translatability. The N terminus of the HIV-1 fusion peptide is thus a promising target of vaccine efforts aimed at eliciting broadly neutralizing antibodies.
Assuntos
Vacinas contra a AIDS/imunologia , Anticorpos Neutralizantes/imunologia , Epitopos/imunologia , Anticorpos Anti-HIV/imunologia , HIV-1/imunologia , Peptídeos/farmacologia , Proteínas Recombinantes de Fusão/farmacologia , Sequência de Aminoácidos , Animais , Feminino , Cobaias , HIV-1/efeitos dos fármacos , Imunização , Macaca mulatta , Camundongos Endogâmicos C57BL , Modelos Moleculares , Testes de Neutralização , Produtos do Gene env do Vírus da Imunodeficiência Humana/química , Produtos do Gene env do Vírus da Imunodeficiência Humana/metabolismoRESUMO
Th22 cells traffic to and retain in the colon cancer microenvironment, and target core stem cell genes and promote colon cancer stemness via STAT3 and H3K79me2 signaling pathway and contribute to colon carcinogenesis. However, whether Th22 cells affect colon cancer cell proliferation and apoptosis remains unknown. We studied the interaction between Th22 cells and colon cancer cells in the colon cancer microenvironment. Colon cancer proliferation was examined by flow cytometry analysis and H(3) thymidine incorporation. Cell cycle related genes were quantified by real-time PCR and Western blotting. We transfected colon cancer cells with lentiviral vector encoding specific gene shRNAs and used chromatin immunoprecipitation (ChIP) assay to determine the genetic signaling involved in interleukin (IL)-22-mediated colon cancer cell proliferation. We showed that Th22 cells released IL-22 and stimulated colon cancer proliferation. Mechanistically, IL-22 activated STAT3, and subsequently STAT3 bound to the promoter areas of the Polycomb Repression complex 2 (PRC2) components SUZ12 and EED, and stimulated the expression of PRC2. Consequently, the activated PRC2 catalyzed the promoters of the cell cycle check-point genes p16 and p21, and inhibited their expression through H3K27me3-mediated histone methylation, and ultimately caused colon cancer cell proliferation. Bioinformatics analysis revealed that the levels of IL-22 expression positively correlated with the levels of genes controlling cancer proliferation and cell cycling in colon cancer. In addition to controlling colon cancer stemness, Th22 cells support colon carcinogenesis via affecting colon cancer cell proliferation through a distinct histone modification.
RESUMO
Effector T cells and fibroblasts are major components in the tumor microenvironment. The means through which these cellular interactions affect chemoresistance is unclear. Here, we show that fibroblasts diminish nuclear accumulation of platinum in ovarian cancer cells, resulting in resistance to platinum-based chemotherapy. We demonstrate that glutathione and cysteine released by fibroblasts contribute to this resistance. CD8(+) T cells abolish the resistance by altering glutathione and cystine metabolism in fibroblasts. CD8(+) T-cell-derived interferon (IFN)γ controls fibroblast glutathione and cysteine through upregulation of gamma-glutamyltransferases and transcriptional repression of system xc(-) cystine and glutamate antiporter via the JAK/STAT1 pathway. The presence of stromal fibroblasts and CD8(+) T cells is negatively and positively associated with ovarian cancer patient survival, respectively. Thus, our work uncovers a mode of action for effector T cells: they abrogate stromal-mediated chemoresistance. Capitalizing upon the interplay between chemotherapy and immunotherapy holds high potential for cancer treatment.
Assuntos
Linfócitos T CD8-Positivos/metabolismo , Resistencia a Medicamentos Antineoplásicos , Neoplasias Ovarianas/tratamento farmacológico , Animais , Antineoplásicos/uso terapêutico , Técnicas de Cultura de Células , Linhagem Celular Tumoral , Cisplatino/uso terapêutico , Feminino , Fibroblastos/metabolismo , Glutationa/metabolismo , Humanos , Interferon gama/metabolismo , Camundongos , Camundongos Endogâmicos NOD , Camundongos NusRESUMO
Myeloid-derived suppressor cells (MDSC) contribute to immune suppression in cancer, but the mechanisms through which they drive metastatic progression are not fully understood. In this study, we show how MDSC convey stem-like qualities to breast cancer cells that coordinately help enable immune suppression and escape. We found that MDSC promoted tumor formation by enhancing breast cancer cell stem-like properties as well as by suppressing T-cell activation. Mechanistic investigations indicated that these effects relied upon cross-talk between the STAT3 and NOTCH pathways in cancer cells, with MDSC inducing IL6-dependent phosphorylation of STAT3 and activating NOTCH through nitric oxide leading to prolonged STAT3 activation. In clinical specimens of breast cancer, the presence of MDSC correlated with the presence of cancer stem-like cells (CSC) and independently predicted poor survival outcomes. Collectively, our work revealed an immune-associated mechanism that extrinsically confers cancer cell stemness properties and affects patient outcome. We suggest that targeting STAT3-NOTCH cross-talk between MDSC and CSC could offer a unique locus to improve cancer treatment, by coordinately targeting a coupled mechanism that enables cancer stemness and immune escape. Cancer Res; 76(11); 3156-65. ©2016 AACR.
Assuntos
Neoplasias da Mama/patologia , Interleucina-6/metabolismo , Células Supressoras Mieloides/patologia , Células-Tronco Neoplásicas/patologia , Óxido Nítrico/metabolismo , Receptores Notch/metabolismo , Fator de Transcrição STAT3/metabolismo , Animais , Apoptose , Western Blotting , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Proliferação de Células , Estudos de Coortes , Feminino , Citometria de Fluxo , Seguimentos , Humanos , Técnicas Imunoenzimáticas , Interleucina-6/genética , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Células Supressoras Mieloides/metabolismo , Estadiamento de Neoplasias , Células-Tronco Neoplásicas/metabolismo , Fosforilação , Prognóstico , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Receptor Cross-Talk , Receptores Notch/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Transcrição STAT3/genética , Transdução de Sinais , Taxa de Sobrevida , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Infiltration of tumors with effector T cells is positively associated with therapeutic efficacy and patient survival. However, the mechanisms underlying effector T-cell trafficking to the tumor microenvironment remain poorly understood in patients with colon cancer. The polycomb repressive complex 2 (PRC2) is involved in cancer progression, but the regulation of tumor immunity by epigenetic mechanisms has yet to be investigated. In this study, we examined the relationship between the repressive PRC2 machinery and effector T-cell trafficking. We found that PRC2 components and demethylase JMJD3-mediated histone H3 lysine 27 trimethylation (H3K27me3) repress the expression and subsequent production of Th1-type chemokines CXCL9 and CXCL10, mediators of effector T-cell trafficking. Moreover, the expression levels of PRC2 components, including EZH2, SUZ12, and EED, were inversely associated with those of CD4, CD8, and Th1-type chemokines in human colon cancer tissue, and this expression pattern was significantly associated with patient survival. Collectively, our findings reveal that PRC2-mediated epigenetic silencing is not only a crucial oncogenic mechanism, but also a key circuit controlling tumor immunosuppression. Therefore, targeting epigenetic programs may have significant implications for improving the efficacy of current cancer immunotherapies relying on effective T-cell-mediated immunity at the tumor site.
Assuntos
Quimiocinas/imunologia , Neoplasias do Colo/genética , Complexo Repressor Polycomb 2/genética , Linfócitos T/imunologia , Células Th1/imunologia , Movimento Celular/genética , Movimento Celular/imunologia , Proliferação de Células/genética , Neoplasias do Colo/imunologia , Neoplasias do Colo/patologia , Epigênese Genética , Humanos , Complexo Repressor Polycomb 2/imunologia , Linfócitos T/metabolismo , Células Th1/metabolismo , TransfecçãoRESUMO
Epigenetic silencing including histone modifications and DNA methylation is an important tumorigenic mechanism. However, its role in cancer immunopathology and immunotherapy is poorly understood. Using human ovarian cancers as our model, here we show that enhancer of zeste homologue 2 (EZH2)-mediated histone H3 lysine 27 trimethylation (H3K27me3) and DNA methyltransferase 1 (DNMT1)-mediated DNA methylation repress the tumour production of T helper 1 (TH1)-type chemokines CXCL9 and CXCL10, and subsequently determine effector T-cell trafficking to the tumour microenvironment. Treatment with epigenetic modulators removes the repression and increases effector T-cell tumour infiltration, slows down tumour progression, and improves the therapeutic efficacy of programmed death-ligand 1 (PD-L1; also known as B7-H1) checkpoint blockade and adoptive T-cell transfusion in tumour-bearing mice. Moreover, tumour EZH2 and DNMT1 are negatively associated with tumour-infiltrating CD8(+) T cells and patient outcome. Thus, epigenetic silencing of TH1-type chemokines is a novel immune-evasion mechanism of tumours. Selective epigenetic reprogramming alters the T-cell landscape in cancer and may enhance the clinical efficacy of cancer therapy.
Assuntos
Quimiocinas/genética , Epigênese Genética , Inativação Gênica , Imunoterapia , Neoplasias Ovarianas/imunologia , Neoplasias Ovarianas/terapia , Células Th1/metabolismo , Animais , Antígeno B7-H1/metabolismo , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/imunologia , Quimiocina CXCL10/biossíntese , Quimiocina CXCL10/genética , Quimiocina CXCL10/imunologia , Quimiocina CXCL9/biossíntese , Quimiocina CXCL9/genética , Quimiocina CXCL9/imunologia , Quimiocinas/biossíntese , Quimiocinas/imunologia , DNA (Citosina-5-)-Metiltransferase 1 , DNA (Citosina-5-)-Metiltransferases/antagonistas & inibidores , DNA (Citosina-5-)-Metiltransferases/metabolismo , Metilação de DNA/efeitos dos fármacos , Proteína Potenciadora do Homólogo 2 de Zeste , Epigênese Genética/efeitos dos fármacos , Feminino , Histonas/química , Histonas/metabolismo , Humanos , Imunoterapia/métodos , Linfócitos do Interstício Tumoral/imunologia , Lisina/metabolismo , Camundongos , Neoplasias Ovarianas/enzimologia , Neoplasias Ovarianas/patologia , Complexo Repressor Polycomb 2/antagonistas & inibidores , Complexo Repressor Polycomb 2/metabolismo , Prognóstico , Células Th1/imunologia , Células Tumorais Cultivadas , Evasão Tumoral/imunologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Little is known about how the immune system impacts human colorectal cancer invasiveness and stemness. Here we detected interleukin-22 (IL-22) in patient colorectal cancer tissues that was produced predominantly by CD4(+) T cells. In a mouse model, migration of these cells into the colon cancer microenvironment required the chemokine receptor CCR6 and its ligand CCL20. IL-22 acted on cancer cells to promote activation of the transcription factor STAT3 and expression of the histone 3 lysine 79 (H3K79) methytransferase DOT1L. The DOT1L complex induced the core stem cell genes NANOG, SOX2, and Pou5F1, resulting in increased cancer stemness and tumorigenic potential. Furthermore, high DOT1L expression and H3K79me2 in colorectal cancer tissues was a predictor of poor patient survival. Thus, IL-22(+) cells promote colon cancer stemness via regulation of stemness genes that negatively affects patient outcome. Efforts to target this network might be a strategy in treating colorectal cancer patients.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Neoplasias Colorretais/imunologia , Interleucinas/imunologia , Metiltransferases/imunologia , Células-Tronco Neoplásicas/imunologia , Fator de Transcrição STAT3/imunologia , Animais , Linhagem Celular Tumoral , Proliferação de Células , Quimiocina CCL20/imunologia , Quimiocina CCL20/metabolismo , Neoplasias Colorretais/mortalidade , Neoplasias Colorretais/patologia , Ativação Enzimática/imunologia , Células HT29 , Histona-Lisina N-Metiltransferase , Proteínas de Homeodomínio/imunologia , Proteínas de Homeodomínio/metabolismo , Humanos , Metiltransferases/metabolismo , Camundongos , Proteína Homeobox Nanog , Transplante de Neoplasias , Células-Tronco Neoplásicas/patologia , Fator 3 de Transcrição de Octâmero/imunologia , Fator 3 de Transcrição de Octâmero/metabolismo , Receptores CCR6/imunologia , Receptores CCR6/metabolismo , Fatores de Transcrição SOXB1/imunologia , Fatores de Transcrição SOXB1/metabolismo , Fator de Transcrição STAT3/metabolismo , Interleucina 22RESUMO
Multiple layers of suppressive components including regulatory T (T(Reg)) cells, suppressive antigen-presenting cells, and inhibitory cytokines form suppressive networks in the ovarian cancer microenvironment. It has been demonstrated that as a major suppressive element, T(Reg) cells infiltrate tumor, interact with several types of immune cells, and mediate immune suppression through different molecular and cellular mechanisms. In this paper, we focus on human ovarian cancer and will discuss the nature of T(Reg) cells including their subsets, trafficking, expansion, and function. We will briefly review the development of manipulation of T(Reg) cells in preclinical and clinical settings.
RESUMO
Earlier, we demonstrated the essential role of Kruppel-like transcription factor, TIEG1, in TGF-ß-induced regulatory T cell (Treg) development. In this article, we demonstrate that IL-6, which promotes Th17 development, abrogated TIEG1 nuclear translocation and inhibited TGF-ß-induced Treg development. Tyrosine kinase Tyk2-mediated phosphorylation of TIEG1 at Tyr179 promoted noncanonical K-27-linked polyubiquitination, which inhibited TIEG1 nuclear translocation. To test the role of TIEG1-regulated Treg/Th17 development in antitumor immunity, we analyzed TRAMP-C2 tumor growth in TIEG1(-/-) mice. The defective Treg development and elevated Th17 response resulted in enhanced immune reactivity in the tumor and inhibition of TRAMP-C2 tumor growth in TIEG1(-/-) mice. Thus, our results uncovered a novel regulatory mechanism that modulates Tregs and may regulate tumor progression.
Assuntos
Proliferação de Células , Proteínas de Ligação a DNA/metabolismo , Fatores de Transcrição Forkhead/genética , Neoplasias Experimentais/metabolismo , Neoplasias Experimentais/patologia , Fatores de Transcrição/metabolismo , Transporte Ativo do Núcleo Celular , Transferência Adotiva , Animais , Western Blotting , Núcleo Celular/metabolismo , Citometria de Fluxo , Fatores de Transcrição Forkhead/metabolismo , Imunoprecipitação , Interleucina-6/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fosforilação , Reação em Cadeia da Polimerase , Receptores de Antígenos de Linfócitos T/imunologia , Transdução de Sinais , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismo , TYK2 Quinase/metabolismo , Células Th17/metabolismo , Fator de Crescimento Transformador beta/metabolismo , UbiquitinaçãoRESUMO
MKP-2 is a member of the dual-specificity phosphatase family that can dephosphorylate and inactivate mitogen-activated protein kinases (MAPKs). Although MKP-2 can be induced by ERK signaling, little is known about the regulation of MKP-2 at the post-translational level. Here we show that MKP-2 is phosphorylated by ERK and that such phosphorylation leads to stabilization of MKP-2 protein. Importantly, we find that MKP-2 can be phosphorylated on Ser386 and Ser391 at its C-terminus. Blockage of ERK activation results in enhanced proteasomal degradation of MKP-2 protein. Moreover, we find that phosphorylation has no effect on MKP-2 phosphatase activity. Taken together, these results illustrate an important post-translational regulation of MKP-2 protein as a feedback mechanism to control ERK activity.
Assuntos
Fosfatases de Especificidade Dupla/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Fosfatases da Proteína Quinase Ativada por Mitógeno/metabolismo , Domínio Catalítico , Linhagem Celular Tumoral , Fosfatases de Especificidade Dupla/genética , Humanos , Fosfatases da Proteína Quinase Ativada por Mitógeno/genética , Mitógenos/farmacologia , Fosforilação , Complexo de Endopeptidases do Proteassoma/metabolismo , Transdução de SinaisRESUMO
The mechanism of cisplatin resistance in cancer cells is not fully understood. Here, we show that the Akt/mTOR survival pathway plays an important role in cisplatin resistance in human ovarian cancer cells. Specifically, we found that cisplatin treatment activates the Akt/mTOR survival pathway and that inhibition of this pathway by the PI3K inhibitor LY294002 or knockdown of Akt sensitizes ovarian cancer cells to cisplatin. Furthermore, we generated cisplatin-resistant cells and found that resistant cells express a higher level of activated Akt as compared to their cisplatin sensitive counterparts. Importantly, inhibition of Akt or mTOR sensitized resistant cells to cisplatin-induced apoptosis. Taken together, our data indicate that activation of the Akt/mTOR pathway prevents cisplatin-induced apoptosis, leading to cisplatin resistance. Therefore, our study suggests that cisplatin resistance can be overcome by targeting the Akt/mTOR survival pathway in human ovarian cancer cells.
Assuntos
Antineoplásicos/farmacologia , Cisplatino/farmacologia , Resistencia a Medicamentos Antineoplásicos , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Proteínas Serina-Treonina Quinases/fisiologia , Proteínas Proto-Oncogênicas c-akt/fisiologia , Apoptose , Linhagem Celular Tumoral , Sobrevivência Celular , Cromonas/farmacologia , Técnicas de Silenciamento de Genes , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Peptídeos e Proteínas de Sinalização Intracelular/genética , Morfolinas/farmacologia , Inibidores de Fosfoinositídeo-3 Quinase , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/genética , Serina-Treonina Quinases TORRESUMO
Apoptin protein harbors tumor-selective cell death activity, which makes it a potential anticancer therapy candidate. This study reports an apoptin therapy approach based on protein transduction domain 4 (PTD4)-mediated transduction of recombinant apoptin protein. In vitro, the PTD4-apoptin fusion protein is located in the nucleus and induces cell death in, e.g., human hepatocarcinoma HepG2 cells. In normal human L-02 hepatocytes, PTD4-apoptin protein retained mainly cytoplasmic and did not induce detectable levels of cell death, illustrating that the PTD4 domain does not affect apoptin's tumor-selective characteristics. In vivo, liver, cervix and gastric carcinoma xenografts treated with PTD4-apoptin protein for 6 days via the tumor epidermis exhibited a significant tumor growth inhibition because of apoptin-mediated cell death. In addition, treatment of human hepatocarcinoma xenografts during 3 weeks showed that PTD4-apoptin protein has significant anticancer activity, whereas control treatment with PTD4-enhanced green fluorescence protein or saline did not. Cell death and disruption of the tumor integrity were apparent in the PTD4-apoptin transduced xenografted tumors. As important, although PTD4-apoptin protein could be detected in the epidermal tissue covering the subcutaneous tumor tissue and in several organs, such as liver and brain, of the treated mice, no tissue disruption or signs of cell death could be detected. Our in vivo data reveal that apoptin protein delivery constitutes a novel powerful and safe anticancer therapy.