RESUMO
Immune checkpoint blockade has provided a paradigm shift in cancer therapy, but the success of this approach is very variable; therefore, biomarkers predictive of clinical efficacy are urgently required. Here, we show that the frequency of PD-1+CD8+ T cells relative to that of PD-1+ regulatory T (Treg) cells in the tumor microenvironment can predict the clinical efficacy of programmed cell death protein 1 (PD-1) blockade therapies and is superior to other predictors, including PD ligand 1 (PD-L1) expression or tumor mutational burden. PD-1 expression by CD8+ T cells and Treg cells negatively impacts effector and immunosuppressive functions, respectively. PD-1 blockade induces both recovery of dysfunctional PD-1+CD8+ T cells and enhanced PD-1+ Treg cell-mediated immunosuppression. A profound reactivation of effector PD-1+CD8+ T cells rather than PD-1+ Treg cells by PD-1 blockade is necessary for tumor regression. These findings provide a promising predictive biomarker for PD-1 blockade therapies.
Assuntos
Regulação da Expressão Gênica/efeitos dos fármacos , Inibidores de Checkpoint Imunológico/farmacologia , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Receptor de Morte Celular Programada 1/genética , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismo , Antígenos/química , Antígenos/imunologia , Biomarcadores Tumorais , Antígenos CD28/metabolismo , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Humanos , Inibidores de Checkpoint Imunológico/uso terapêutico , Imunomodulação , Ativação Linfocitária/imunologia , Linfócitos do Interstício Tumoral/imunologia , Linfócitos do Interstício Tumoral/metabolismo , Terapia de Alvo Molecular , Metástase Neoplásica , Estadiamento de Neoplasias , Neoplasias/tratamento farmacológico , Neoplasias/etiologia , Neoplasias/metabolismo , Neoplasias/mortalidade , Peptídeos/química , Peptídeos/imunologia , Prognóstico , Receptor de Morte Celular Programada 1/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Transdução de Sinais , Linfócitos T Reguladores/efeitos dos fármacos , Resultado do Tratamento , Microambiente Tumoral/imunologiaRESUMO
Regulatory T (Treg) cells are required for maintaining self-tolerance and preventing the development of autoimmune diseases. However, Treg cells are abundant in tumors and suppress antitumor immunity, contributing to tumor development and growth. Thus, the selective deletion of tumor-infiltrating Treg cells is important for successful Treg cell-targeted therapies, providing effective antitumor immunity without inducing deleterious autoimmune disorders. Advancements in sequencing technologies have exposed the diversity and heterogeneity of human Treg cells during activation and differentiation, further emphasizing the importance of understanding tumor-infiltrating Treg cells for the development of Treg cell-targeted therapies. This review provides an overview of the classification and function of Treg cells and summarizes recent knowledge on the activation and differentiation of Treg cells in the tumor microenvironment.
Assuntos
Neoplasias , Microambiente Tumoral , Humanos , Tolerância Imunológica , Linfócitos T ReguladoresRESUMO
In recent years, the dramatic increase in the number of applications for massively parallel reporter assay (MPRA) technology has produced a large body of data for various purposes. However, a computational model that can be applied to decipher regulatory codes for diverse MPRAs does not exist yet. Here, we propose a new computational method to predict the transcriptional activity of MPRAs, as well as luciferase reporter assays, based on the TRANScription FACtor database. We employed regression trees and multivariate adaptive regression splines to obtain these predictions and considered a feature redundancy-dependent formula for conventional regression trees to enable adaptation to diverse data. The developed method was applicable to various MPRAs despite the use of different types of transfected cells, sequence lengths, construct numbers and sequence types. We demonstrate that this method can predict the transcriptional activity of promoters in HEK293 cells through predictive functions that were estimated by independent assays in eight tumor cell lines. The prediction was generally good (Pearson's r = 0.68) which suggested that common active transcription factor binding sites across different cell types make greater contributions to transcriptional activity and that known promoter activity could confer transcriptional activity of unknown promoters in some instances, regardless of cell type.
Assuntos
DNA/genética , DNA/metabolismo , Genes Reporter , Transcrição Gênica , Sítios de Ligação/genética , Biologia Computacional , Bases de Dados de Ácidos Nucleicos/estatística & dados numéricos , Células HEK293 , Sequenciamento de Nucleotídeos em Larga Escala/estatística & dados numéricos , Humanos , Regiões Promotoras Genéticas , Análise de Regressão , Análise de Sequência de DNA/estatística & dados numéricos , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
BACKGROUND: Histone epigenome data determined by chromatin immunoprecipitation sequencing (ChIP-seq) is used in identifying transcript regions and estimating expression levels. However, this estimation does not always correlate with eventual RNA expression levels measured by RNA sequencing (RNA-seq). Part of the inconsistency may arise from the variance in RNA stability, where the transcripts that are more or less abundant than predicted RNA expression from histone epigenome data are inferred to be more or less stable. However, there is little systematic analysis to validate this assumption. Here, we used stability data of whole transcriptome measured by 5'-bromouridine immunoprecipitation chase sequencing (BRIC-seq), which enabled us to determine the half-lives of whole transcripts including lincRNAs, and we integrated BRIC-seq with ChIP-seq to achieve better estimation of the eventual transcript levels and to understand the importance of post-transcriptional regulation that determine the eventual transcript levels. RESULTS: We identified discrepancies between the RNA abundance estimated by ChIP-seq and measured RNA expression from RNA-seq; for number of genes and estimated that the expression level of 865 genes was controlled at the level of RNA stability in HeLa cells. ENCODE data analysis supported the idea that RNA stability control aids to determine transcript levels in multiple cell types. We identified UPF1, EXOSC5 and STAU1, well-studied RNA degradation factors, as controlling factors for 8% of cases. Computational simulations reasonably explained the changes of eventual mRNA levels attributable to the changes in the rates of mRNA half-lives. In addition, we propose a feedback circuit that includes the regulated degradation of mRNAs encoding transcription factors to maintain the steady state level of RNA abundance. Intriguingly, these regulatory mechanisms were distinct between mRNAs and lincRNAs. CONCLUSIONS: Integrative analysis of ChIP-seq, RNA-seq and our BRIC-seq showed that transcriptional regulation and RNA degradation are independently regulated. In addition, RNA stability is an important determinant of eventual transcript levels. RNA binding proteins, such as UPF1, STAU1 and EXOSC5 may play active roles in such controls.
Assuntos
Estabilidade de RNA , RNA/metabolismo , Antígenos de Neoplasias/metabolismo , Imunoprecipitação da Cromatina , Proteínas do Citoesqueleto/metabolismo , Complexo Multienzimático de Ribonucleases do Exossomo/metabolismo , Regulação da Expressão Gênica , Meia-Vida , Células HeLa , Sequenciamento de Nucleotídeos em Larga Escala , Histonas/metabolismo , Humanos , RNA/química , RNA Helicases , RNA Longo não Codificante/química , RNA Longo não Codificante/metabolismo , RNA Mensageiro/química , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Análise de Sequência de RNA , Transativadores/metabolismoRESUMO
We recently developed a novel transcriptome analysis method, termed 5'-bromo-uridine (BrU) immunoprecipitation chase-deep sequencing analysis (BRIC-seq). BRIC-seq enables the determination of genome-wide RNA stability by chasing chronological decreases of BrU-labeled RNAs under physiologically undisturbed conditions. The RNA half-life of each transcript is calculated from the decreasing number of BrU-labeled RNA sequence tags measured by deep sequencing of BrU-labeled RNAs. Here, we describe a detailed protocol and provide tips for BRIC-seq, followed by computational analysis.
Assuntos
Estabilidade de RNA , RNA Mensageiro/genética , Animais , Bromouracila/análogos & derivados , Mapeamento Cromossômico , Biblioteca Gênica , Ontologia Genética , Genoma , Células HEK293 , Meia-Vida , Células HeLa , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , RNA Mensageiro/metabolismo , Análise de Sequência de RNA , Coloração e Rotulagem , Uridina/análogos & derivados , Uridina/químicaRESUMO
We developed a computer program that can predict the intrinsic promoter activities of primary human DNA sequences. We observed promoter activity using a quantitative luciferase assay and generated a prediction model using multiple linear regression. Our program achieved a prediction accuracy correlation coefficient of 0.87 between the predicted and observed promoter activities. We evaluated the prediction accuracy of the program using massive sequencing analysis of transcriptional start sites in vivo. We found that it is still difficult to predict transcript levels in a strictly quantitative manner in vivo; however, it was possible to select active promoters in a given cell from the other silent promoters. Using this program, we analyzed the transcriptional landscape of the entire human genome. We demonstrate that many human genomic regions have potential promoter activity, and the expression of some previously uncharacterized putatively non-protein-coding transcripts can be explained by our prediction model. Furthermore, we found that nucleosomes occasionally formed open chromatin structures with RNA polymerase II recruitment where the program predicted significant promoter activities, although no transcripts were observed.
Assuntos
Regiões Promotoras Genéticas , Software , Ativação Transcricional , Sequência de Bases , Sítios de Ligação , DNA/química , Genes Reporter , Genoma Humano , Células HEK293 , Humanos , Modelos Lineares , Luciferases/análise , Luciferases/genética , Fatores de Transcrição/metabolismo , Sítio de Iniciação de TranscriçãoRESUMO
UPF1 eliminates aberrant mRNAs harboring premature termination codons, and regulates the steady-state levels of normal physiological mRNAs. Although genome-wide studies of UPF1 targets performed, previous studies did not distinguish indirect UPF1 targets because they could not determine UPF1-dependent altered RNA stabilities. Here, we measured the decay rates of the whole transcriptome in UPF1-depleted HeLa cells using BRIC-seq, an inhibitor-free method for directly measuring RNA stability. We determined the half-lives and expression levels of 9,229 transcripts. An amount of 785 transcripts were stabilized in UPF1-depleted cells. Among these, the expression levels of 76 transcripts were increased, but those of the other 709 transcripts were not altered. RNA immunoprecipitation showed UPF1 bound to the stabilized transcripts, suggesting that UPF1 directly degrades the 709 transcripts. Many UPF1 targets in this study were newly identified. This study clearly demonstrates that direct determination of RNA stability is a powerful approach for identifying targets of RNA degradation factors.
Assuntos
Códon sem Sentido , Estabilidade de RNA , RNA Mensageiro/genética , Transativadores/genética , Transativadores/metabolismo , Transcriptoma , Linhagem Celular Tumoral , Células HeLa , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , RNA Helicases , Interferência de RNA , RNA Interferente Pequeno , Análise de Sequência de RNARESUMO
Regulatory T (Treg) cells suppress effective antitumor immunity in tumor-bearing hosts, thereby becoming promising targets in cancer immunotherapy. Despite the importance of Treg cells in tumor immunity, little is known about their differentiation process and epigenetic profiles in the tumor microenvironment (TME). Here, we showed that Treg cells in the TME of human lung cancers harbored a completely different open chromatin profile compared with CD8+ T cells, conventional CD4+ T cells in the TME, and peripheral Treg cells. The integrative sequencing analyses including ATAC, single-cell RNA, and single-cell ATAC sequencing revealed that BATF, IRF4, NF-κB, and NR4A were important transcription factors for Treg cell differentiation in the TME. In particular, BATF was identified as a key regulator, which leveraged Treg cell differentiation through epigenetically controlling activation-associated gene expression, resulting in the robustness of Treg cells in the TME. The single-cell sequencing approaches also revealed that tissue-resident and tumor-infiltrating Treg cells followed a common pathway for differentiation and activation in a BATF-dependent manner heading toward Treg cells with the most differentiated and activated phenotypes in tissues and tumors. BATF deficiency in Treg cells remarkably inhibited tumor growth, and high BATF expression was associated with poor prognosis in lung cancer, kidney cancer, and melanoma. These findings indicate one of the specific chromatin remodeling and differentiation programs of Treg cells in the TME, which can be applied in the development of Treg cell-targeted therapies.
Assuntos
Melanoma , Linfócitos T Reguladores , Fatores de Transcrição de Zíper de Leucina Básica/genética , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Linfócitos T CD8-Positivos , Cromatina/metabolismo , Humanos , NF-kappa B/genética , NF-kappa B/metabolismo , RNA , Microambiente TumoralRESUMO
Bronchoalveolar lavage is commonly performed to assess inflammation and identify responsible pathogens in lung diseases. Findings from bronchoalveolar lavage might be used to evaluate the immune profile of the lung tumor microenvironment (TME). To investigate whether bronchoalveolar lavage fluid (BALF) analysis can help identify patients with non-small cell lung cancer (NSCLC) who respond to immune checkpoint inhibitors (ICIs), BALF and blood were prospectively collected before initiating nivolumab. The secreted molecules, microbiome, and cellular profiles based on BALF and blood analysis of 12 patients were compared with regard to therapeutic effect. Compared with ICI nonresponders, responders showed significantly higher CXCL9 levels and a greater diversity of the lung microbiome profile in BALF, along with a greater frequency of the CD56+ subset in blood T cells, whereas no significant difference in PD-L1 expression was found in tumor cells. Antibiotic treatment in a preclinical lung cancer model significantly decreased CXCL9 in the lung TME, resulting in reduced sensitivity to anti-PD-1 antibody, which was reversed by CXCL9 induction in tumor cells. Thus, CXCL9 might be associated with the lung TME microbiome, and the balance of CXCL9 and lung TME microbiome could contribute to nivolumab sensitivity in patients with NSCLC. BALF analysis can help predict the efficacy of ICIs when performed along with currently approved examinations.
Assuntos
Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Líquido da Lavagem Broncoalveolar , Carcinoma Pulmonar de Células não Pequenas/patologia , Humanos , Neoplasias Pulmonares/patologia , Nivolumabe/farmacologia , Nivolumabe/uso terapêutico , Receptor de Morte Celular Programada 1/metabolismo , Microambiente TumoralRESUMO
PD-1 blockade exerts clinical efficacy against various types of cancer by reinvigorating T cells that directly attack tumor cells (tumor-specific T cells) in the tumor microenvironment (TME), and tumor-infiltrating lymphocytes (TILs) also comprise nonspecific bystander T cells. Here, using single-cell sequencing, we show that TILs include skewed T cell clonotypes, which are characterized by exhaustion (Tex) or nonexhaustion signatures (Tnon-ex). Among skewed clonotypes, those in the Tex, but not those in the Tnon-ex, cluster respond to autologous tumor cell lines. After PD-1 blockade, non-preexisting tumor-specific clonotypes in the Tex cluster appear in the TME. Tumor-draining lymph nodes (TDLNs) without metastasis harbor a considerable number of such clonotypes, whereas these clonotypes are rarely detected in peripheral blood. We propose that tumor-infiltrating skewed T cell clonotypes with an exhausted phenotype directly attack tumor cells and that PD-1 blockade can promote infiltration of such Tex clonotypes, mainly from TDLNs.
Assuntos
Linfócitos T CD8-Positivos/efeitos dos fármacos , Inibidores de Checkpoint Imunológico/farmacologia , Linfócitos do Interstício Tumoral/efeitos dos fármacos , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Microambiente Tumoral/imunologia , Animais , Linfócitos T CD8-Positivos/metabolismo , Linhagem Celular Tumoral/efeitos dos fármacos , Humanos , Linfócitos do Interstício Tumoral/metabolismo , Camundongos , Receptor de Morte Celular Programada 1/metabolismo , Microambiente Tumoral/efeitos dos fármacosRESUMO
The balance of programmed death-1 (PD-1)-expressing CD8+ T cells and regulatory T (Treg) cells in the tumor microenvironment (TME) determines the clinical efficacy of PD-1 blockade therapy through the competition of their reactivation. However, factors that determine this balance remain unknown. Here, we show that Treg cells gain higher PD-1 expression than effector T cells in highly glycolytic tumors, including MYC-amplified tumors and liver tumors. Under low-glucose environments via glucose consumption by tumor cells, Treg cells actively absorbed lactic acid (LA) through monocarboxylate transporter 1 (MCT1), promoting NFAT1 translocation into the nucleus, thereby enhancing the expression of PD-1, whereas PD-1 expression by effector T cells was dampened. PD-1 blockade invigorated the PD-1-expressing Treg cells, resulting in treatment failure. We propose that LA in the highly glycolytic TME is an active checkpoint for the function of Treg cells in the TME via upregulation of PD-1 expression.
Assuntos
Regulação Neoplásica da Expressão Gênica , Ácido Láctico/metabolismo , Receptor de Morte Celular Programada 1/genética , Linfócitos T Reguladores/metabolismo , Microambiente Tumoral/genética , Animais , Biomarcadores Tumorais , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Linfócitos T CD8-Positivos/patologia , Linhagem Celular Tumoral , Modelos Animais de Doenças , Imunofluorescência , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Glicólise , Humanos , Inibidores de Checkpoint Imunológico/farmacologia , Inibidores de Checkpoint Imunológico/uso terapêutico , Proteínas de Checkpoint Imunológico/metabolismo , Imunofenotipagem , Ácido Láctico/farmacologia , Ativação Linfocitária , Contagem de Linfócitos , Linfócitos do Interstício Tumoral/imunologia , Linfócitos do Interstício Tumoral/metabolismo , Linfócitos do Interstício Tumoral/patologia , Camundongos , Terapia de Alvo Molecular , Prognóstico , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Receptor de Morte Celular Programada 1/metabolismo , Linfócitos T Reguladores/efeitos dos fármacos , Linfócitos T Reguladores/imunologia , Resultado do Tratamento , Microambiente Tumoral/efeitos dos fármacosRESUMO
We developed linear regression models which predict strength of transcriptional activity of promoters from their sequences. Intrinsic transcriptional strength data of 451 human promoter sequences in three cell lines (HEK293, MCF7 and 3T3), which were measured by systematic luciferase reporter gene assays, were used to build the models. The models sum up contributions of CG dinucleotide content and transcription factor binding sites (TFBSs) to transcriptional strength. We evaluated prediction accuracies of the models by cross validation tests and found that they have adequate ability for predicting transcriptional strength of promoters in spite of their simple formalization. We also evaluated statistical significance of the contributions and proposed a picture of regulatory code hidden in promoter sequences. That is, CG dinucleotide content and TFBSs mainly determine strength of transcriptional activity under ubiquitous and specific environments, respectively.
Assuntos
Modelos Genéticos , Regiões Promotoras Genéticas , Transcrição Gênica , Células 3T3 , Animais , Composição de Bases , Sítios de Ligação , Células HEK293 , Humanos , Modelos Lineares , Células MCF-7 , Camundongos , Fatores de Transcrição/metabolismoRESUMO
Combining our full-length cDNA method and the massively parallel sequencing technology, we developed a simple method to collect precise positional information of transcriptional start sites (TSSs) together with digital information of the gene-expression levels in a high throughput manner. We applied this method to observe gene-expression changes in a colon cancer cell line cultured in normoxic and hypoxic conditions. We generated more than 100 million 36-base TSS-tag sequences and revealed comprehensive features of hypoxia responsive alterations in the transcriptional landscape of the human genome. The features include presence of inducible 'hot regions' in 54 genomic regions, 220 novel hypoxia inducible promoters that may drive non-protein-coding transcripts, 191 hypoxia responsive alternative promoters and detailed views of 120 novel as well as known hypoxia responsive genes. We further analyzed hypoxic response of different cells using additional 60 million TSS-tags and found that the degree of the gene-expression changes were different among cell lines, possibly reflecting cellular robustness against hypoxia. The novel dynamic figure of the human gene transcriptome will deepen our understanding of the transcriptional program of the human genome as well as bringing new insights into the biology of cancer cells in hypoxia.
Assuntos
Regulação Neoplásica da Expressão Gênica , Sítio de Iniciação de Transcrição , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Hipóxia Celular , Linhagem Celular , Linhagem Celular Tumoral , Neoplasias do Colo/genética , Biblioteca Gênica , Redes Reguladoras de Genes , Genoma Humano , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Regiões Promotoras Genéticas , Análise de Sequência de DNA , Transcrição GênicaRESUMO
Patients with colorectal cancers (CRCs) generally exhibit improved survival through intensive lymph node (LN) dissection. However, recent progress in cancer immunotherapy revisits the potential importance of regional LNs, where T cells are primed to attack tumor cells. To elucidate the role of regional LN, we investigated the immunological status of nonmetastatic regional LN lymphocytes (LNLs) in comparison with those of the tumor microenvironment (tumor-infiltrating lymphocytes; TILs) using flow cytometry and next-generation sequencing. LNLs comprised an intermediate level of the effector T cell population between peripheral blood lymphocytes (PBLs) and TILs. Significant overlap of the T cell receptor (TCR) repertoire was observed in microsatellite instability-high/mismatch repair-deficient (MSI-H/dMMR) CRCs with high tumor mutation burden (TMB), although limited TCRs were shared between nonmetastatic LNs and primary tumors in microsatellite stable/MMR proficient (MSS/pMMR) CRC patients with low TMB. In line with the overlap of the TCR repertoire, an excessive LN dissection did not provide a positive impact on long-term prognosis in our MSI-H/dMMR CRC cohort (n = 130). We propose that regional LNs play an important role in antitumor immunity, particularly in MSI-H/dMMR CRCs with high TMB, requiring care to be taken regarding excessive nonmetastatic LN dissection in MSI-H/dMMR CRC patients.
Assuntos
Neoplasias Colorretais/imunologia , Linfonodos/imunologia , Linfócitos do Interstício Tumoral/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Subpopulações de Linfócitos T/imunologia , Linfócitos T/imunologia , Microambiente Tumoral/imunologia , Idoso , Linfócitos T CD4-Positivos/imunologia , Estudos de Coortes , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Neoplasias Colorretais/cirurgia , Reparo de Erro de Pareamento de DNA/genética , Feminino , Citometria de Fluxo , Fatores de Transcrição Forkhead/imunologia , Humanos , Excisão de Linfonodo , Linfonodos/patologia , Linfonodos/cirurgia , Masculino , Células T de Memória/imunologia , Instabilidade de Microssatélites , Pessoa de Meia-Idade , Receptor de Morte Celular Programada 1/imunologiaRESUMO
Regulatory T (Treg) cells are important negative regulators of immune homeostasis, but in cancers they tone down the anti-tumor immune response. They are distinguished by high expression levels of the chemokine receptor CCR4, hence their targeting by the anti-CCR4 monoclonal antibody mogamulizumab holds therapeutic promise. Here we show that despite a significant reduction in peripheral effector Treg cells, clinical responses are minimal in a cohort of patients with advanced CCR4-negative solid cancer in a phase Ib study (NCT01929486). Comprehensive immune-monitoring reveals that the abundance of CCR4-expressing central memory CD8+ T cells that are known to play roles in the antitumor immune response is reduced. In long survivors, characterised by lower CCR4 expression in their central memory CD8+ T cells possessed and/or NK cells with an exhausted phenotype, cell numbers are eventually maintained. Our study thus shows that mogamulizumab doses that are currently administered to patients in clinical studies may not differentiate between targeting effector Treg cells and central memory CD8+ T cells, and dosage refinement might be necessary to avoid depletion of effector components during immune therapy.
Assuntos
Anticorpos Monoclonais Humanizados/uso terapêutico , Antineoplásicos/uso terapêutico , Linfócitos T CD8-Positivos/efeitos dos fármacos , Células T de Memória/efeitos dos fármacos , Idoso , Idoso de 80 Anos ou mais , Linfócitos T CD8-Positivos/metabolismo , Relação Dose-Resposta a Droga , Feminino , Humanos , Imunoterapia , Células Matadoras Naturais/efeitos dos fármacos , Células Matadoras Naturais/metabolismo , Masculino , Pessoa de Meia-Idade , Neoplasias/tratamento farmacológico , Neoplasias/imunologia , Receptores CCR4/antagonistas & inibidores , Receptores CCR4/metabolismo , Linfócitos T Reguladores/efeitos dos fármacos , Linfócitos T Reguladores/metabolismo , Resultado do TratamentoRESUMO
PD-1 blockade exerts antitumor effects by reinvigorating tumor antigenspecific CD8+ T cells. Whereas neoantigens arising from gene alterations in cancer cells comprise critical tumor antigens in antitumor immunity, a subset of nonsmall cell lung cancers (NSCLCs) harboring substantial tumor mutation burden (TMB) lack CD8+ T cells in the tumor microenvironment (TME), which results in resistance to PD-1 blockade therapy. To overcome this resistance, clarifying the mechanism(s) impairing antitumor immunity in highly mutated NSCLCs is an urgent issue. Here, we showed that activation of the WNT/ß-catenin signaling pathway contributed to the development of a noninflamed TME in tumors with high TMB. NSCLCs that lacked immune cell infiltration into the TME despite high TMB preferentially up-regulated the WNT/ß-catenin pathway. Immunologic assays revealed that those patients harbored neoantigen-specific CD8+ T cells in the peripheral blood but not in the TME, suggesting impaired T cell infiltration into the TME due to the activation of WNT/ß-catenin signaling. In our animal models, the accumulation of gene mutations in cancer cells increased CD8+ T cell infiltration into the TME, thus slowing tumor growth. However, further accumulation of gene mutations blunted antitumor immunity by excluding CD8+ T cells from tumors in a WNT/ß-catenin signaling-dependent manner. Combined treatment with PD-1 blockade and WNT/ß-catenin signaling inhibitors induced better antitumor immunity than either treatment alone. Thus, we propose a mechanism-oriented combination therapy whereby immune checkpoint inhibitors can be combined with drugs that target cell-intrinsic oncogenic signaling pathways involved in tumor immune escape.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/imunologia , Neoplasias Pulmonares/imunologia , Evasão Tumoral/imunologia , Animais , Linfócitos T CD8-Positivos/imunologia , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Feminino , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Microambiente Tumoral/imunologia , Via de Sinalização Wnt/imunologiaRESUMO
Single-cell level analysis is powerful tool to assess the heterogeneity of cellular components in tumor microenvironments (TME). In this study, we investigated immune-profiles using the single-cell analyses of endoscopically- or surgically-resected tumors, and peripheral blood mononuclear cells from gastric cancer patients. Furthermore, we technically characterized two distinct platforms of the single-cell analysis; RNA-seq-based analysis (scRNA-seq), and mass cytometry-based analysis (CyTOF), both of which are broadly embraced technologies. Our study revealed that the scRNA-seq analysis could cover a broader range of immune cells of TME in the biopsy-resected small samples of tumors, detecting even small subgroups of B cells or Treg cells in the tumors, although CyTOF could distinguish the specific populations in more depth. These findings demonstrate that scRNA-seq analysis is a highly-feasible platform for elucidating the complexity of TME in small biopsy tumors, which would provide a novel strategies to overcome a therapeutic difficulties against cancer heterogeneity in TME.
Assuntos
Análise de Célula Única , Neoplasias Gástricas/patologia , Microambiente Tumoral , Adulto , Biópsia , Feminino , Humanos , Leucócitos Mononucleares/metabolismo , Masculino , Pessoa de Meia-Idade , RNA-Seq , Neoplasias Gástricas/genéticaRESUMO
Using full-length cDNA sequences, we compared alternative splicing (AS) in humans and mice. The alignment of the human and mouse genomes showed that 86% of 199 426 total exons in human AS variants were conserved in the mouse genome. Of the 20 392 total human AS variants, however, 59% consisted of all conserved exons. Comparing AS patterns between human and mouse transcripts revealed that only 431 transcripts from 189 loci were perfectly conserved AS variants. To exclude the possibility that the full-length human cDNAs used in the present study, especially those with retained introns, were cloning artefacts or prematurely spliced transcripts, we experimentally validated 34 such cases. Our results indicate that even retained-intron type transcripts are typically expressed in a highly controlled manner and interact with translating ribosomes. We found non-conserved AS exons to be predominantly outside the coding sequences (CDSs). This suggests that non-conserved exons in the CDSs of transcripts cause functional constraint. These findings should enhance our understanding of the relationship between AS and species specificity of human genes.
Assuntos
Processamento Alternativo , DNA Complementar/química , Evolução Molecular , Aminoacil-tRNA Sintetases/genética , Animais , Sequência de Bases , Sequência Conservada , Interpretação Estatística de Dados , Éxons , Genômica , Humanos , Íntrons , Camundongos , Fosfatidilinositol 3-Quinases/genética , RNA Mensageiro/química , Especificidade da EspécieRESUMO
PURPOSE: To evaluate the detailed immunosuppressive role(s) of PD-L2 given that its detailed role(s) remains unclear in PD-1 signal blockade therapy in animal models and humans. EXPERIMENTAL DESIGN: We generated mouse cell lines harboring various status of PD-L1/PD-L2 and evaluated the tumor growth and phenotypes of tumor-infiltrated lymphocytes using several PD-1 signal blockades in animal models. In humans, the correlation between immune-related gene expression and CD274 (encoding PD-L1) or PDCD1LG2 (encoding PD-L2) was investigated using The Cancer Genome Atlas (TCGA) datasets. In addition, PD-L1 or PD-L2 expression in tumor cells and CD8+ T-cell infiltration were assessed by IHC. RESULTS: In animal models, we showed that PD-L2 expression alone or simultaneously expressed with PD-L1 in tumor cells significantly suppressed antitumor immune responses, such as tumor antigen-specific CD8+ T cells, and was involved in the resistance to treatment with anti-PD-L1 mAb alone. This resistance was overcome by anti-PD-1 mAb or combined treatment with anti-PD-L2 mAb. In clinical settings, antitumor immune responses were significantly correlated with PD-L2 expression in the tumor microenvironment in renal cell carcinoma (RCC) and lung squamous cell carcinoma (LUSC). CONCLUSIONS: We propose that PD-L2 as well as PD-L1 play important roles in evading antitumor immunity, suggesting that PD-1/PD-L2 blockade must be considered for optimal immunotherapy in PD-L2-expressing cancers, such as RCC and LUSC.
Assuntos
Antineoplásicos Imunológicos/uso terapêutico , Antígeno B7-H1/antagonistas & inibidores , Terapia de Imunossupressão , Neoplasias Renais/imunologia , Neoplasias Experimentais/imunologia , Proteína 2 Ligante de Morte Celular Programada 1/antagonistas & inibidores , Microambiente Tumoral/imunologia , Animais , Antígeno B7-H1/imunologia , Linfócitos T CD8-Positivos/imunologia , Carcinoma de Células Renais/tratamento farmacológico , Carcinoma de Células Renais/imunologia , Carcinoma de Células Renais/patologia , Carcinoma de Células Escamosas/tratamento farmacológico , Carcinoma de Células Escamosas/imunologia , Carcinoma de Células Escamosas/patologia , Linhagem Celular Tumoral , Feminino , Humanos , Neoplasias Renais/tratamento farmacológico , Neoplasias Renais/patologia , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/patologia , Linfócitos do Interstício Tumoral/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Nus , Neoplasias Experimentais/tratamento farmacológico , Neoplasias Experimentais/patologia , Proteína 2 Ligante de Morte Celular Programada 1/imunologia , Microambiente Tumoral/efeitos dos fármacosRESUMO
In order to understand an overview of promoter activities intrinsic to primary DNA sequences in the human genome within a particular cell type, we carried out systematic quantitative luciferase assays of DNA fragments corresponding to putative promoters for 472 human genes which are expressed in HEK (human embryonic kidney epithelial) 293 cells. We observed the promoter activities of them were distributed in a bimodal manner; putative promoters belonging to the first group (with strong promoter activities) were designated as P1 and the latter (with weak promoter activities) as P2. The frequencies of the TATA-boxes, the CpG islands, and the overall G + C-contents were significantly different between these two populations, indicating there are two separate groups of promoters. Interestingly, similar analysis using 251 randomly isolated genomic DNA fragments showed that P2-type promoter occasionally occurs within the human genome. Furthermore, 35 DNA fragments corresponding to putative promoters of non-protein-coding transcripts (ncRNAs) shared similar features with the P2 in both promoter activities and sequence compositions. At least, a part of ncRNAs, which have been massively identified by full-length cDNA projects with no functional relevance inferred, may have originated from those sporadic promoter activities of primary DNA sequences inherent to the human genome.