RESUMO
Epigenetic histone modifications play critical roles in the control of gene transcription. Recently, an increasing number of histone H2A deubiquitinases have been identified and characterized. However, the physiological functions for this entire group of histone H2A deubiquitinases remain unknown. In this study, we revealed that the histone H2A deubiquitinase MYSM1 plays an essential and intrinsic role in early B cell development. MYSM1 deficiency results in a block in early B cell commitment and a defect of B cell progenitors in expression of EBF1 and other B lymphoid genes. We further demonstrated that MYSM1 derepresses EBF1 transcription in B cell progenitors by orchestrating histone modifications and transcription factor recruitment to the EBF1 locus. Thus, this study not only uncovers the essential role for MYSM1 in gene transcription during early B cell development but also underscores the biological significance of reversible epigenetic histone H2A ubiquitination.
Assuntos
Linfócitos B/citologia , Linfócitos B/enzimologia , Diferenciação Celular , Endopeptidases/metabolismo , Histonas/metabolismo , Fatores de Transcrição/metabolismo , Animais , Linfócitos B/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Linhagem da Célula/genética , Proteínas Cromossômicas não Histona/metabolismo , Regulação da Expressão Gênica , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Regiões Promotoras Genéticas , Ligação Proteica , Transativadores/genética , Fatores de Transcrição/genética , Transcrição Gênica , Proteases Específicas de UbiquitinaRESUMO
The mechanisms controlling the development of dendritic cells (DCs) remain incompletely understood. Using an Mysm1 knockout (Mysm1(-/-)) mouse model, we identified the histone H2A deubiquitinase Mysm1, as a critical regulator in DC differentiation. Mysm1(-/-) mice showed a global reduction of DCs in lymphoid organs, whereas development of granulocytes and macrophages were not severely affected. Hematopoietic progenitors and DC precursors were significantly decreased in Mysm1(-/-) mice and defective in Fms-like tyrosine kinase-3(Flt3) ligand-induced, but not in granulocyte macrophage-colony-stimulating factor (GM-CSF)-induced DC differentiation in vitro. Molecular studies demonstrated that the developmental defect of DCs from common myeloid progenitor (CMP) in Mysm1(-/-) mice is associated with decreased Flt3 expression and that Mysm1 derepresses transcription of the Flt3 gene by directing histone modifications at the Flt3 promoter region. Two molecular mechanisms were found to be responsible for the selective role of Mysm1 in lineage determination of DCs from CMPs: the selective expression of Mysm1 in a subset of CMPs and the different requirement of Mysm1 for PU.1 recruitment to the Flt3 locus vs GM-CSF-α and macrophage-colony-stimulating factor receptor loci. In conclusion, this study reveals an essential role of Mysm1 in epigenetic regulation of Flt3 transcription and DC development, and it provides a novel mechanism for lineage determination from CMP.
Assuntos
Diferenciação Celular/genética , Células Dendríticas/metabolismo , Endopeptidases/genética , Epigênese Genética , Células Progenitoras Mieloides/metabolismo , Animais , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Endopeptidases/metabolismo , Citometria de Fluxo , Expressão Gênica , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Células HEK293 , Histonas/metabolismo , Humanos , Camundongos Knockout , Regiões Promotoras Genéticas/genética , Ligação Proteica , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Receptor de Fator Estimulador de Colônias de Macrófagos/genética , Receptor de Fator Estimulador de Colônias de Macrófagos/metabolismo , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/genética , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transativadores/genética , Transativadores/metabolismo , Proteases Específicas de Ubiquitina , Tirosina Quinase 3 Semelhante a fms/genética , Tirosina Quinase 3 Semelhante a fms/metabolismoRESUMO
Breast cancer is the most common malignancy in women and the leading cause of cancer deaths in women worldwide. Breast cancers are heterogenous and exist in many different subtypes (luminal A, luminal B, triple negative, and human epidermal growth factor receptor 2 (HER2) overexpressing), and each subtype displays distinct characteristics, responses to treatment, and patient outcomes. In addition to varying immunohistochemical properties, each subtype contains a distinct gene mutation profile which has yet to be fully defined. Patient treatment is currently guided by hormone receptor status and HER2 expression, but accumulating evidence suggests that genetic mutations also influence drug responses and patient survival. Thus, identifying the unique gene mutation pattern in each breast cancer subtype will further improve personalized treatment and outcomes for breast cancer patients. In this study, we used the Ion Personal Genome Machine (PGM) and Ion Torrent AmpliSeq Cancer Panel to sequence 737 mutational hotspot regions from 45 cancer-related genes to identify genetic mutations in 80 breast cancer samples of various subtypes from Chinese patients. Analysis revealed frequent missense and combination mutations in PIK3CA and TP53, infrequent mutations in PTEN, and uncommon combination mutations in luminal-type cancers in other genes including BRAF, GNAS, IDH1, and KRAS. This study demonstrates the feasibility of using Ion Torrent sequencing technology to reliably detect gene mutations in a clinical setting in order to guide personalized drug treatments or combination therapies to ultimately target individual, breast cancer-specific mutations.
Assuntos
Neoplasias da Mama/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Mutação/genética , Receptor ErbB-2/genética , Adulto , Idoso , Neoplasias da Mama/patologia , Análise Mutacional de DNA , Feminino , Humanos , Pessoa de Meia-Idade , Proteínas de Neoplasias/genética , Medicina de PrecisãoRESUMO
Histone modifications play critical roles in regulating immunity; however, little is known about the epigenetic control of natural killer (NK) cell development. Here, we found that NK cell development is severely impaired in mice deficient in the histone H2A deubiquitinase MYSM1. We demonstrated that MYSM1 is required for NK cell maturation but not for NK lineage specification and commitment. We also found that MYSM1 intrinsically controls this NK cell maturation. Mechanistic studies revealed that the expression of transcription factor, inhibitor of DNA-binding protein (ID2), a critical factor for NK cell development, is impaired in Mysm1(-/-) NK cells. MYSM1 interacts with nuclear factor IL-3 (NFIL3, also known as E4BP4), a critical factor for mouse NK cell development, and the recruitment of nuclear factor Il-3 to the ID2 locus is dependent on MYSM1. Further, we observed that MYSM1 is involved in maintaining an active chromatin at the ID2 locus to promote NK cell development. Hence this study demonstrates the critical epigenetic regulation of NK cell development by the histone H2A deubiquitinase MYSM1 through the transcriptional control of transcription factors important for NK cell development.
Assuntos
Imunidade Adaptativa/imunologia , Endopeptidases/imunologia , Epigênese Genética/imunologia , Células Matadoras Naturais/imunologia , Animais , Imunoprecipitação da Cromatina , Endopeptidases/genética , Endopeptidases/metabolismo , Citometria de Fluxo , Células HEK293 , Humanos , Immunoblotting , Proteína 2 Inibidora de Diferenciação/metabolismo , Interleucina-3/metabolismo , Células Matadoras Naturais/citologia , Camundongos , Camundongos Knockout , Reação em Cadeia da Polimerase em Tempo Real , Transativadores , Transdução Genética , Proteases Específicas de UbiquitinaRESUMO
Colorectal cancer (CRC) is widespread with significant mortality. Both inherited and sporadic mutations in various signaling pathways influence the development and progression of the cancer. Identifying genetic mutations in CRC is important for optimal patient treatment and many approaches currently exist to uncover these mutations, including next-generation sequencing (NGS) and commercially available kits. In the present study, we used a semiconductor-based targeted DNA-sequencing approach to sequence and identify genetic mutations in 91 human rectal cancer samples. Analysis revealed frequent mutations in KRAS (58.2%), TP53 (28.6%), APC (16.5%), FBXW7 (9.9%) and PIK3CA (9.9%), and additional mutations in BRAF, CTNNB1, ERBB2 and SMAD4 were also detected at lesser frequencies. Thirty-eight samples (41.8%) also contained two or more mutations, with common combination mutations occurring between KRAS and TP53 (42.1%), and KRAS and APC (31.6%). DNA sequencing for individual cancers is of clinical importance for targeted drug therapy and the advantages of such targeted gene sequencing over other NGS platforms or commercially available kits in sensitivity, cost and time effectiveness may aid clinicians in treating CRC patients in the near future.
Assuntos
Sequenciamento de Nucleotídeos em Larga Escala , Proteínas de Neoplasias/genética , Neoplasias Retais/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , MutaçãoRESUMO
Epigenetic histone modifications play critical roles in the control of self-renewal and differentiation of hematopoietic stem cells (HSCs). Mysm1 is a recently identified histone H2A deubiquitinase with essential and intrinsic roles for maintaining functional HSCs. In this study, in addition to confirming this function of Mysm1, by using Mysm1-deficient (Mysm1(-/-)) mice, we provide more evidence for how Mysm1 controls HSC homeostasis. Mysm1 deletion drives HSCs from quiescence into rapid cycling and increases their apoptotic rate, resulting in an exhaustion of the stem cell pool, which leads to an impaired self-renewal and lineage reconstituting abilities in the Mysm1-deficient mice. Our study identified Gfi1 as one of the candidate genes responsible for the HSC defect in Mysm1-deficient mice. Mechanistic studies revealed that Mysm1 modulates histone modifications and directs the recruitment of key transcriptional factors such as Gata2 and Runx1 to the Gfi1 locus in HSCs. We found that Mysm1 directly associates with the Gfi1 enhancer element and promotes its transcription through Gata2 and Runx1 transactivation. Thus, our study not only elaborates on the initial reports of Mysm1 association with HSC homeostasis but also delineates a possible epigenetic mechanism through which Mysm1 carries out this function in the HSCs.
Assuntos
Endopeptidases/fisiologia , Epigênese Genética , Células-Tronco Hematopoéticas/citologia , Animais , Apoptose , Ciclo Celular , Diferenciação Celular , Proliferação de Células , Sobrevivência Celular , Subunidade alfa 2 de Fator de Ligação ao Core/metabolismo , Proteínas de Ligação a DNA/metabolismo , Endopeptidases/genética , Fator de Transcrição GATA2/metabolismo , Deleção de Genes , Histonas/metabolismo , Homeostase , Camundongos , Camundongos Transgênicos , Transativadores , Fatores de Transcrição/metabolismo , Proteases Específicas de UbiquitinaRESUMO
Stem cells are normally maintained in a quiescent state and proliferate only under certain conditions; however, little is known about the biological stimuli that initiate the proliferation and differentiation of stem cells. In this study, we found that functional Toll-like receptors (TLRs) are expressed on mouse embryonic stem (ES) cells and that TLR ligands stimulate ES cell proliferation and promote their hematopoietic differentiation. TLR ligands activate TLR-mediated signaling pathways, leading to the altered expression of numerous genes in ES cells. Moreover, TLR ligands efficiently stimulate the proliferation and expansion of adult stem cells and progenitors of nonhematopoietic tissues, such as mammary glands and intestine as well. We further found that mammary luminal progenitor cells (Lin(-)CD29(+)CD61(+)) express TLR4-MD2 complex and actively proliferate, resulting in the enhanced growth of mammospheres in response to TLR ligands. Thus, mouse ES cells and adult tissue-specific stem cells/progenitors directly sense and respond to microbial products, which function as a class of foreign, but biological stimuli for stem cell/progenitor proliferation. This finding expands the biological role of TLRs and has implications in understanding stem cell biology, tissue repair/homeostasis, and the role of infection and inflammation in malignant transformation.
Assuntos
Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Glândulas Mamárias Animais/citologia , Células-Tronco/citologia , Células-Tronco/metabolismo , Receptores Toll-Like/metabolismo , Animais , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Proliferação de Células , Células Cultivadas , Células-Tronco Embrionárias/efeitos dos fármacos , Citometria de Fluxo , Imuno-Histoquímica , Lipopolissacarídeos/farmacologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Camundongos SCID , Análise de Sequência com Séries de Oligonucleotídeos , Poli I-C/farmacologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/efeitos dos fármacos , Células-Tronco/efeitos dos fármacosRESUMO
Tumor cells harbor unique genetic mutations, which lead to the generation of immunologically foreign antigenic peptide repertoire with the potential to induce individual tumor-specific immune responses. Here, we developed an in situ tumor vaccine with the ability to elicit antitumor immunity. This vaccine comprised an E1B-deleted oncolytic adenovirus expressing beta-defensin-2 (Ad-BD2-E1A) for releasing tumor antigens, recruiting and activating plasmacytoid dendritic cells (pDCs). Intratumoral injections of Ad-BD2-E1A vaccine inhibited primary breast tumor growth and blocked naturally occurring metastasis in mice. Ad-BD2-E1A vaccination induced potent tumor-specific T-cell responses. Splenic and intratumoral DCs isolated from Ad-BD2-E1A-immunized mice were able to stimulate or promote the differentiation of naive T cells into tumor-specific cytotoxic T cells. We further found that the increased numbers of mature CD45RA(+)CD8alpha(+)CD40(+) pDCs infiltrated into Ad-BD2-E1A-treated tumors. The antitumor effect of Ad-BD2-E1A vaccination was abrogated in toll-like receptor 4 (TLR4) deficient mice, suggesting the critical role of TLR4 in the induction of antitumor immunity by Ad-BD2-E1A. The results of this study indicate that in situ vaccination with the oncolytic BD2-expressing adenovirus preferentially attracts pDCs and promotes their maturation, and thus elicits potent tumor-specific immunity. This vaccine represents an attractive therapeutic strategy for the induction of individualized antitumor immunity.
Assuntos
Células Dendríticas/citologia , Células Dendríticas/imunologia , Neoplasias/imunologia , Adenoviridae/genética , Proteínas E1B de Adenovirus/genética , Animais , Western Blotting , Células COS , Vacinas Anticâncer/genética , Vacinas Anticâncer/imunologia , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Linhagem Celular , Linhagem Celular Tumoral , Proliferação de Células , Chlorocebus aethiops , Células Dendríticas/metabolismo , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Vetores Genéticos/genética , Humanos , Imuno-Histoquímica , Camundongos , Vírus Oncolíticos/genética , beta-Defensinas/genética , beta-Defensinas/fisiologiaRESUMO
Oncolytic viruses offer an in situ vaccination approach to activate tumor-specific T cell responses. However, the upregulation of PD-L1 expression on tumor cells and immune cells leads to tumor resistance to oncolytic immunotherapy. In this study, we generate an engineered oncolytic virus that coexpresses a PD-L1 inhibitor and GM-CSF. We find that the oncolytic virus is able to secrete the PD-L1 inhibitor that systemically binds and inhibits PD-L1 on tumor cells and immune cells. Importantly, the intratumoral injection with the oncolytic virus overcomes PD-L1-mediated immunosuppression during both the priming and effector phases, provokes systemic T cell responses against dominant and subdominant neoantigen epitopes derived from mutations, and leads to an effective rejection of both virus-injected and distant tumors. In summary, this engineered oncolytic virus is able to activate tumor neoantigen-specific T cell responses, providing a potent, individual tumor-specific oncolytic immunotherapy for cancer patients, especially those resistant to PD-1/PD-L1 blockade therapy.
Assuntos
Antígeno B7-H1/antagonistas & inibidores , Terapia Viral Oncolítica , Vírus Oncolíticos/genética , Vírus Oncolíticos/metabolismo , Animais , Antígenos de Neoplasias/metabolismo , Antineoplásicos , Antígeno B7-H1/genética , Antígeno B7-H1/metabolismo , Linfócitos T CD8-Positivos/imunologia , Linhagem Celular Tumoral , Modelos Animais de Doenças , Engenharia Genética , Fator Estimulador de Colônias de Granulócitos e Macrófagos/imunologia , Células HEK293 , Humanos , Terapia de Imunossupressão , Imunoterapia , Camundongos , Camundongos Endogâmicos C57BL , Vírus Oncolíticos/imunologia , Proteínas RecombinantesRESUMO
DC-based tumor vaccine research has largely focused on enhancing DC maturation/costimulation and antigen presentation in order to break tolerance against self tumor-associated antigens. DC immunization can activate autoreactive T cells but rarely causes autoimmune pathologies, indicating that self tolerance at the host level is still maintained in the vaccinated hosts. This study in mice reveals a novel regulatory mechanism for the control of self tolerance at the host level by DCs through the restriction of positive cytokine feedback loops by cytokine signaling inhibitor SOCS1. The study further finds the requirement of persistent antigen presentation by DCs for inducing pathological autoimmune responses against normal tissues and tumor, which can be achieved by silencing SOCS1 to unleash the unbridled signaling of IL-12 and the downstream cytokine cascade. However, the use of higher-affinity self peptides, enhancement of DC maturation, and persistent stimulation with cytokines or TLR agonists fail to break tolerance and induce pathological antitumor immunity. Thus, this study indicates the necessity of inhibiting SOCS1, an antigen presentation attenuator, to break self tolerance and induce effective antitumor responses.
Assuntos
Proteínas de Transporte/fisiologia , Células Dendríticas/imunologia , Interleucina-12/genética , Melanoma Experimental/imunologia , Proteínas Repressoras/fisiologia , Tolerância a Antígenos Próprios/fisiologia , Proteínas Supressoras da Sinalização de Citocina/fisiologia , Animais , Linhagem Celular Tumoral , Feminino , Regulação da Expressão Gênica , Interleucina-12/deficiência , Interleucina-12/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Transdução de Sinais/imunologia , Proteína 1 Supressora da Sinalização de CitocinaRESUMO
Dendritic cells (DCs) are potent antigen-presenting cells that play a critical role in activating cellular and humoral immune responses. DC-based tumor vaccines targeting tumor-associated antigens (TAAs) have been extensively tested and demonstrated to be safe and potent in inducing anti-TAA immune responses in cancer patients. Sipuleucel-T (Provenge), a cancer vaccine of autologous DCs loaded with TAA, was approved by the United States Food and Drug Administration (FDA) for the treatment of castration-resistant prostate cancer. Sipuleucel-T prolongs patient survival, but has little or no effect on clinical disease progression or biomarker kinetics. Due to the overall limited clinical efficacy of tumor vaccines, there is a need to enhance their potency. PD-L1 is a key immune checkpoint molecule and is frequently overexpressed on tumor cells to evade antitumor immune destruction. Repeated administrations of PD-L1 or PD-1 antibodies have induced sustained tumor regression in a fraction of cancer patients. In this study, we tested whether vaccinations with DCs, loaded with a PD-L1 immunogen (PDL1-Vax), are able to induce anti-PD-L1 immune responses. We found that DCs loaded with PDL1-Vax induced anti-PD-L1 antibody and T cell responses in immunized mice and that PD-L1-specific CTLs had cytolytic activities against PD-L1+ tumor cells. We demonstrated that vaccination with PDL1-Vax DCs potently inhibited the growth of PD-L1+ tumor cells. In summary, this study demonstrates for the first time the principle and feasibility of DC vaccination (PDL1-Vax) to actively induce anti-PD-L1 antibody and T cell responses capable of inhibiting PD-L1+ tumor growth. This novel anti-PD-L1 vaccination strategy could be used for cancer treatment and prevention.
RESUMO
Anti-CD19 chimeric antigen receptor (CAR) T cell therapies can cause severe cytokine-release syndrome (CRS) and neurotoxicity, impeding their therapeutic application. Here we generated a new anti-CD19 CAR molecule (CD19-BBz(86)) derived from the CD19-BBz prototype bearing co-stimulatory 4-1BB and CD3ζ domains. We found that CD19-BBz(86) CAR T cells produced lower levels of cytokines, expressed higher levels of antiapoptotic molecules and proliferated more slowly than the prototype CD19-BBz CAR T cells, although they retained potent cytolytic activity. We performed a phase 1 trial of CD19-BBz(86) CAR T cell therapy in patients with B cell lymphoma (ClinicalTrials.gov identifier NCT02842138 ). Complete remission occurred in 6 of 11 patients (54.5%) who each received a dose of 2 × 108-4 × 108 CD19-BBz(86) CAR T cells. Notably, no neurological toxicity or CRS (greater than grade 1) occurred in any of the 25 patients treated. No significant elevation in serum cytokine levels after CAR T cell infusion was detected in the patients treated, including in those who achieved complete remission. CD19-BBz(86) CAR T cells persistently proliferated and differentiated into memory cells in vivo. Thus, therapy with the new CD19-BBz(86) CAR T cells produces a potent and durable antilymphoma response without causing neurotoxicity or severe CRS, representing a safe and potent anti-CD19 CAR T cell therapy.
Assuntos
Antígenos CD19/imunologia , Imunoterapia Adotiva/métodos , Linfoma de Células B/imunologia , Linfoma de Células B/terapia , Receptores de Antígenos Quiméricos/imunologia , Adulto , Idoso , Antígenos CD19/genética , Citocinas/sangue , Feminino , Humanos , Imunoterapia Adotiva/efeitos adversos , Linfoma de Células B/diagnóstico por imagem , Masculino , Pessoa de Meia-Idade , Tomografia por Emissão de Pósitrons combinada à Tomografia Computadorizada , Indução de Remissão , Linfócitos T/imunologia , Resultado do Tratamento , Adulto JovemRESUMO
BACKGROUND: DC-based tumor vaccines have had limited clinical success thus far. SOCS1, a key inhibitor of inflammatory cytokine signaling, is an immune checkpoint regulator that limits DC immunopotency. METHODS: We generated a genetically modified DC (gmDC) vaccine to perform immunotherapy. The adenovirus (Ad-siSSF) delivers two tumor-associated antigens (TAAs), survivin and MUC1; secretory bacterial flagellin for DC maturation; and an RNA interference moiety to suppress SOCS1. A 2-stage phase I trial was performed for patients with relapsed acute leukemia after allogenic hematopoietic stem cell transplantation: in stage 1, we compared the safety and efficacy between gmDC treatment (23 patients) and standard donor lymphocyte infusion (25 patients); in stage 2, we tested the efficacy of the gmDC vaccine for 12 acute myeloid leukemia (AML) patients with early molecular relapse. RESULTS: gmDCs elicited potent TAA-specific CTL responses in vitro, and the immunostimulatory activity of gmDC vaccination was demonstrated in rhesus monkeys. A stage 1 study established that this combinatory gmDC vaccine is safe in acute leukemia patients and yielded improved survival rate. In stage 2, we observed a complete remission rate of 83% in 12 relapsed AML patients. Overall, no grade 3 or grade 4 graft-versus-host disease incidence was detected in any of the 35 patients enrolled. CONCLUSIONS: This study, with combinatory modifications in DCs, demonstrates the safety and efficacy of SOCS1-silenced DCs in treating relapsed acute leukemia. TRIAL REGISTRATION: ClinicalTrials.gov NCT01956630. FUNDING: National Institute of Health (R01CA90427); the Key New Drug Development and Manufacturing Program of the "Twelfth Five-Year Plan" of China (2011ZX09102-001-29); and Clinical Application Research of Beijing (Z131107002213148).
Assuntos
Vacinas Anticâncer/administração & dosagem , Células Dendríticas/imunologia , Leucemia Mieloide Aguda/terapia , Recidiva Local de Neoplasia/terapia , Leucemia-Linfoma Linfoblástico de Células Precursoras/terapia , Adenoviridae/genética , Adolescente , Adulto , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/imunologia , Vacinas Anticâncer/efeitos adversos , Vacinas Anticâncer/genética , Vacinas Anticâncer/imunologia , Engenharia Celular/métodos , Criança , Células Dendríticas/transplante , Feminino , Seguimentos , Vetores Genéticos/genética , Doença Enxerto-Hospedeiro/epidemiologia , Doença Enxerto-Hospedeiro/imunologia , Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Humanos , Imunoterapia Adotiva/métodos , Leucemia Mieloide Aguda/imunologia , Leucemia Mieloide Aguda/mortalidade , Transfusão de Linfócitos , Masculino , Pessoa de Meia-Idade , Recidiva Local de Neoplasia/imunologia , Recidiva Local de Neoplasia/mortalidade , Leucemia-Linfoma Linfoblástico de Células Precursoras/imunologia , Leucemia-Linfoma Linfoblástico de Células Precursoras/mortalidade , Análise de Sobrevida , Transplante Autólogo , Resultado do Tratamento , Adulto JovemRESUMO
BACKGROUND: Current efforts to develop HIV vaccines that seek to stimulate immune responses have been disappointing, underscoring the inability of natural immune responses to control HIV-1 infection. Here we tested an alternative strategy to induce anti-HIV immune responses by inhibiting a host's natural immune inhibitor. METHODS AND FINDINGS: We used small interfering RNA (siRNA) to inhibit suppressor of cytokine signaling (SOCS) 1, a key negative regulator of the JAK/STAT pathway, and investigated the effect of this silencing on the ability of dendritic cells (DCs) to induce anti-HIV-1 immunity. We found that SOCS1-silenced DCs broadly induced enhanced HIV-1 envelope (Env)-specific CD8+ cytotoxic T lymphocytes and CD4+ T helper cells, as well as antibody responses, in mice. Importantly, SOCS1-silenced DCs were more resistant to HIV Env-mediated suppression and were capable of inducing memory HIV Env-specific antibody and T cell responses. SOCS1-restricted signaling, as well as production of proinflammatory cytokines such as interleukin-12 by DCs, play a critical role in regulating the anti-HIV immune response. Furthermore, the potency of HIV DNA vaccination is significantly enhanced by coimmunization with SOCS1 siRNA expressor DNA. CONCLUSIONS: This study demonstrates that SOCS1 functions as an antigen presentation attenuator to control both HIV-1-specific humoral and cellular responses. This study represents the first, to our knowledge, attempt to elicit HIV-specific T cell and antibody responses by inhibiting a host's antigen presentation attenuator, which may open a new and alternative avenue to develop effective therapeutic and prophylactic HIV vaccines.
Assuntos
Vacinas contra a AIDS/imunologia , Adjuvantes Imunológicos , Apresentação de Antígeno , Células Dendríticas/imunologia , Proteína gp120 do Envelope de HIV/imunologia , Infecções por HIV/imunologia , HIV-1/imunologia , Vacinação/métodos , Transferência Adotiva , Animais , Formação de Anticorpos , Linfócitos B/imunologia , Células Cultivadas , Células Dendríticas/metabolismo , Células Dendríticas/transplante , Infecções por HIV/prevenção & controle , Interleucina-12/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Interferência de RNA , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/imunologia , RNA Interferente Pequeno/metabolismo , Receptores de Interleucina-12/deficiência , Receptores de Interleucina-12/genética , Proteína 1 Supressora da Sinalização de Citocina , Proteínas Supressoras da Sinalização de Citocina/genética , Proteínas Supressoras da Sinalização de Citocina/imunologia , Proteínas Supressoras da Sinalização de Citocina/metabolismo , Linfócitos T/imunologiaRESUMO
Tumor cells harbor a repertoire of unique, mutated antigens and shared self-antigens but generally are incapable of provoking an effective immune response, likely because of inadequate antigen presentation by professional antigen-presenting cells. Heat shock proteins (HSPs) play important roles in eliciting innate and adaptive immunity by chaperoning peptides for antigen presentation and providing endogenous danger signaling. Although effective in inducing tumor-specific immunity in mice and in some clinical trials, tumor-derived HSPs have many limitations like vaccines, such as the technical difficulty of ex vivo preparation of adequate quantities of HSPs from the resected tumors of individual patients. Here we have developed an in vivo HSP-suicide gene tumor vaccine by generating a recombinant replication-defective adenovirus (Ad-HT) that coexpresses HSP70 and a herpes simplex virus thymidine kinase suicide gene. The combination of HSP70 overexpression in situ and tumor killing by thymidine kinase/ganciclovir treatment, but neither strategy alone, provoked potent systemic antitumor activities after intratumor injection of Ad-HT. Tumor-specific CD4+ and CD8+ T-cell responses were induced by Ad-HT intratumor injection. CD11c+ dendritic cells (DCs) isolated from mice treated with Ad-HT were able to prime tumor-specific CTLs. Collectively, these results indicate that the combination of tumor killing by activation of a suicide gene to release tumor antigens and in situ HSP70 overexpression to enhance DC antigen presentation overcomes host immune tolerance to tumor antigens, leading to the induction of potent antitumor immunity. Our findings may have broad relevance to the use of the in vivo HSP/suicide gene tumor vaccine in therapy for human solid tumors.
Assuntos
Vacinas Anticâncer/imunologia , Terapia Genética/métodos , Proteínas de Choque Térmico HSP70/genética , Proteínas de Choque Térmico HSP70/imunologia , Adenoviridae/genética , Adenoviridae/fisiologia , Animais , Vacinas Anticâncer/genética , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/imunologia , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/terapia , Linhagem Celular Tumoral , Terapia Combinada , Células Dendríticas/imunologia , Feminino , Ganciclovir/farmacocinética , Ganciclovir/farmacologia , Proteínas de Choque Térmico HSP70/biossíntese , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/imunologia , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/terapia , Ativação Linfocitária/imunologia , Neoplasias Mamárias Experimentais/genética , Neoplasias Mamárias Experimentais/imunologia , Neoplasias Mamárias Experimentais/metabolismo , Neoplasias Mamárias Experimentais/terapia , Camundongos , Camundongos Endogâmicos BALB C , Simplexvirus/enzimologia , Simplexvirus/genética , Linfócitos T/imunologia , Linfócitos T Citotóxicos/imunologia , Timidina Quinase/biossíntese , Timidina Quinase/genética , Timidina Quinase/metabolismo , Replicação ViralRESUMO
CD4+ T cells play critical roles in initiating, regulating, and maintaining antitumor immune responses. One way to improve current tumor vaccines that mainly induce CTLs would be to activate antigen-specific CD4+ T cells that recognize MHC class II restricted tumor associated antigens. Human telomerase reverse transcriptase (hTRT) is preferentially expressed by various tumors and, therefore, could be a universal tumor antigen. In this study, we used a combined approach of using the prediction software TEPITOPE to select class II epitope candidates and in vitro T-cell biological analysis to identify class II-restricted epitope(s) in hTRT. We first identified several HLA-DR7-restricted class-II epitope candidates in hTRT by examining human T-cell responses to synthetic peptides. We then characterized these HLA-DR7-restricted hTRT epitope candidates by establishing and analyzing peptide-specific T-cell clones. It was demonstrated that CD4+ T cells specific for the HLA-DR7-restricted hTRT(672) epitope (RPGLLGASVLGLDDI) can respond to naturally processed hTRT proteins. Furthermore, the hTRT(672)-specific T cells recognized hTRT antigen from various tumors, including prostate cancer, breast cancer, melanoma, and leukemia. Thus, the identification of the naturally processed HLA-DR7-restricted hTRT epitope, together with the previous finding of class I-restricted hTRT epitopes, provide a basis for the combined application of class I- and II-restricted hTRT epitopes to induce potent, long-term CD4+ and CD8+ T-cell responses against a broad spectrum of tumors.
Assuntos
Epitopos de Linfócito T/imunologia , Antígeno HLA-DR7/imunologia , Linfócitos T Auxiliares-Indutores/imunologia , Telomerase/imunologia , Sequência de Aminoácidos , Afinidade de Anticorpos , Linfócitos T CD4-Positivos/imunologia , Proteínas de Ligação a DNA , Mapeamento de Epitopos , Humanos , Ativação Linfocitária/imunologia , Dados de Sequência Molecular , Neoplasias/imunologia , Fragmentos de Peptídeos/imunologia , Células Tumorais CultivadasRESUMO
Each tumor harbors unique repertoire of mutated antigenic peptides that are immunogenic and potentially can induce tumor-specific immune responses. Because heat shock proteins (HSPs) have the promiscuous ability to chaperone and present a broad repertoire of tumor antigens to antigen presenting cells, HSP tumor vaccine has been tested in clinical trials. However, this vaccine has many limitations, including individual preparation of HSP vaccines from each tumor ex vivo, and quantity of HSPs for therapy strictly limited by the size of the resected tumor mass. Hence, we developed a novel HSP-mediated oncolytic tumor vaccine, referred to as HOT vaccine, by combining the versatile ability of overexpressed HSPs to chaperone antigenic peptides and induce immune responses against a broad array of mutated tumor antigens, with the oncolytic activity of viruses. The results of this study demonstrate that intratumor vaccination with a recombinant oncolytic adenovirus overexpressing the HSP70 protein can eradicate primary tumors, as well as inhibit the growth of established metastatic tumor in mice. Because of its capacity to induce individual tumor-specific immune responses, this HSP-mediated oncolytic tumor vaccine might become a universally applicable, personalized vaccine against any type of solid tumor.
Assuntos
Vacinas Anticâncer/imunologia , Proteínas de Choque Térmico/imunologia , Adenovírus Humanos/genética , Animais , Vacinas Anticâncer/genética , Linhagem Celular Tumoral , Feminino , Proteínas de Choque Térmico/genética , Masculino , Melanoma Experimental/imunologia , Melanoma Experimental/terapia , Camundongos , Camundongos Endogâmicos C57BL , Linfócitos T Citotóxicos/imunologia , Replicação ViralRESUMO
Circulating tumor DNA (ctDNA) isolated from peripheral blood has recently been shown to be an alternative source to detect gene mutations in primary tumors; however, most previous studies have focused on advanced stage cancers, and few have evaluated ctDNA detection in early-stage lung cancer. In the present study, blood and tumor samples were collected prospectively from 58 early-stage non-small lung cancer (NSCLC) patients (stages IA, IB, and IIA) and a targeted sequencing approach was used to detect somatic driver mutations in matched tumor DNA (tDNA) and plasma ctDNA. We identified frequent driver mutations in plasma ctDNA and tDNA in EGFR, KRAS, PIK3CA, and TP53, and less frequent mutations in other genes, with an overall study concordance of 50.4% and sensitivity and specificity of 53.8% and 47.3%, respectively. Cell-free (cfDNA) concentrations were found to be significantly associated with some clinical features, including tumor stage and subtype. Importantly, the presence of cfDNA had a higher positive predictive value than that of currently used protein tumor biomarkers. This study demonstrates the feasibility of identifying plasma ctDNA mutations in the earliest stage lung cancer patients via targeted sequencing, demonstrating a potential utility of targeted sequencing of ctDNA in the clinical management of NSCLC.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/patologia , DNA Tumoral Circulante/sangue , Neoplasias Pulmonares/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Antígenos Glicosídicos Associados a Tumores/sangue , Biomarcadores Tumorais/sangue , Antígeno Ca-125/sangue , Carcinoma Pulmonar de Células não Pequenas/genética , DNA Tumoral Circulante/química , Classe I de Fosfatidilinositol 3-Quinases/genética , DNA de Neoplasias/sangue , DNA de Neoplasias/química , Receptores ErbB/genética , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Neoplasias Pulmonares/genética , Masculino , Proteínas de Membrana/sangue , Pessoa de Meia-Idade , Mutação , Estadiamento de Neoplasias , Proteínas Proto-Oncogênicas p21(ras)/genética , Análise de Sequência de DNA , Proteína Supressora de Tumor p53/genéticaRESUMO
Non-small cell lung cancers (NSCLC) have unique mutation patterns, and some of these mutations may be used to predict prognosis or guide patient treatment. Mutation profiling before and during treatment often requires repeated tumor biopsies, which is not always possible. Recently, cell-free, circulating tumor DNA (ctDNA) isolated from blood plasma has been shown to contain genetic mutations representative of those found in the primary tumor tissue DNA (tDNA), and these samples can readily be obtained using non-invasive techniques. However, there are still no standardized methods to identify mutations in ctDNA. In the current study, we used a targeted sequencing approach with a semi-conductor based next-generation sequencing (NGS) platform to identify gene mutations in matched tDNA and ctDNA samples from 42 advanced-stage NSCLC patients from China. We identified driver mutations in matched tDNA and ctDNA in EGFR, KRAS, PIK3CA, and TP53, with an overall concordance of 76%. In conclusion, targeted sequencing of plasma ctDNA may be a feasible option for clinical monitoring of NSCLC in the near future.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/genética , DNA de Neoplasias/sangue , Neoplasias Pulmonares/genética , Adulto , Idoso , Carcinoma Pulmonar de Células não Pequenas/patologia , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Neoplasias Pulmonares/patologia , Masculino , Pessoa de Meia-Idade , Mutação , Estadiamento de NeoplasiasRESUMO
Circulating tumor DNA (ctDNA) in peripheral blood is a "liquid biopsy" that contains representative tumor information including gene mutations. Additionally, repeated ctDNA samples can be easily obtained to monitor response to treatment and disease progression, which may be especially valuable to lung cancer patients with tumors that cannot be easily biopsied or removed. To investigate the changes in ctDNA after surgical tumor resection, tumor and blood samples obtained before and after surgery were collected prospectively from 41 non-small lung cancer (NSCLC) patients. Somatic driver mutations in tumor DNA (tDNA) and pre- and post-op plasma ctDNA sample pairs were identified by targeted sequencing in several genes including EGFR, KRAS, and TP53 with an overall study concordance of 78.1% and sensitivity and specificity of 69.2% and 93.3%, respectively. Importantly, the frequency of 91.7% of ctDNA mutations decreased after surgery and these changes were observed as little as 2 days post-op. Moreover, the presence of ctDNA had a higher positive predictive value than that of six tumor biomarkers in current clinical use. This study demonstrates the use of targeted sequencing to reliably identify ctDNA changes in response to treatment, indicating a potential utility of this approach in the clinical management of NSCLC.