The prevalent DNA modification in higher organisms is the methylation of cytosine to 5-methylcytosine (5mC), which is partially converted to 5-hydroxymethylcytosine (5hmC) by the Tet (ten eleven translocation) family of dioxygenases. Despite their importance in epigenetic regulation, it is unclear how these cytosine modifications are reversed. Here, we demonstrate that 5mC and 5hmC in DNA are oxidized to 5-carboxylcytosine (5caC) by Tet dioxygenases in vitro and in cultured cells. 5caC is specifically recognized and excised by thymine-DNA glycosylase (TDG). Depletion of TDG in mouse embyronic stem cells leads to accumulation of 5caC to a readily detectable level. These data suggest that oxidation of 5mC by Tet proteins followed by TDG-mediated base excision of 5caC constitutes a pathway for active DNA demethylation.

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The negative signal provided by interactions of programmed death-1 (PD-1) and its ligands, costimulatory molecules PD-L1 (also B7-H1) and PD-L2 (also B7-DC), is involved in the mechanisms of tumor immune evasion. In this study, we found that this negative signal was also involved in immune evasion in tumor immunotherapy. When we used different doses of a constructed eukaryotic expression plasmid, pSLC, which expresses functional murine secondary lymphoid tissue chemokine (SLC, CCL21), to treat BALB/c mice inoculated with H22 murine hepatoma cells, the inhibitory effect was enhanced along with the increase of pSLC dosage. Unexpectedly, however, the best complete inhibition rate of tumor was reached when pSLC was used at the dosage of 50 μg but not 100 or 200 μg. RT-PCR and real-time PCR revealed that both PD-L1 and PD-L2 genes were expressed in tumor and vicinal muscle tissues of tumor-bearing mice and the expression level was significantly increased if a higher dosage of pSLC was administered. We then constructed a eukaryotic expression plasmid (pPD-1A) that expresses the extracellular domain of murine PD-1 (sPD-1). sPD-1 could bind PD-1 ligands, block PD-Ls-PD-1 interactions, and enhance the cytotoxicity of tumor-specific CTL. Local gene transfer by injection of pPD-1A mediated antitumor effect and improved SLC-mediated antitumor immunity. The combined gene therapy with SLC plus sPD-1 did not induce remarkable autoimmune manifestations. Our findings provide a potent method of improving the antitumor effects of SLC and possibly other immunotherapeutic methods by local blockade of negative costimulatory molecules.

Global 5hmC and 5fC contents were decreased significantly in the very early stage of HCC. The decrease of 5hmC and 5fC was mainly due to the decrease of 5mC, and was also associated with HBV infection, decreased TET enzyme activity and uncoordinated expression of DNA methylation-related enzymes. This article is protected by copyright. All rights reserved.

Preoperative MRI is one of the most important clinical results for the diagnosis and treatment of glioma patients. The objective of this study was to construct a stable and validatable preoperative T2-weighted MRI-based radiomics model for predicting the survival of gliomas. A total of 652 glioma patients across three independent cohorts were covered in this study including their preoperative T2-weighted MRI images, RNA-seq and clinical data. Radiomic features (1731) were extracted from preoperative T2-weighted MRI images of 167 gliomas (discovery cohort) collected from Beijing Tiantan Hospital and then used to develop a radiomics prediction model through a machine learning-based method. The performance of the radiomics prediction model was validated in two independent cohorts including 261 gliomas from the The Cancer Genomae Atlas database (external validation cohort) and 224 gliomas collected in the prospective study from Beijing Tiantan Hospital (prospective validation cohort). RNA-seq data of gliomas from discovery and external validation cohorts were applied to establish the relationship between biological function and the key radiomics features, which were further validated by single-cell sequencing and immunohistochemical staining. The 14 radiomic features-based prediction model was constructed from preoperative T2-weighted MRI images in the discovery cohort, and showed highly robust predictive power for overall survival of gliomas in external and prospective validation cohorts. The radiomic features in the prediction model were associated with immune response, especially tumour macrophage infiltration. The preoperative T2-weighted MRI radiomics prediction model can stably predict the survival of glioma patients and assist in preoperatively assessing the extent of macrophage infiltration in glioma tumours.

Many tumor immunotherapy efforts are focused on the generation of strong T-cell response against tumor antigens. However, strong T-cell response does not always coincide with tumor rejection, for which upregulated expression of immunoinhibitory molecules may be responsible. In this study, the treatment with heat shock protein 70 (HSP70) vaccine induced an infiltration of T cells into the tumor site as well as the expression of IFN-c and IL-2, and delayed lung metastases of tumor, but the tumor progression nonetheless occur finally. We demonstrated that B7-H1 expressed by residual tumor cells was responsible for the resistance of tumor to the therapy with HSP70 vaccine. Blockade of B7-H1 by i.v. injection pPD-1A, a plasmid encoding the extracellular domain of PD-1 (sPD-1), could reverse this resistance and enhance the therapeutic efficacy. To complement these findings, we investigated the gene expression of tumor-infiltrating lymphocytes (TILs) by Realtime PCR analysis, which revealed that the expression of T H 1 cytokines IFN-c and IL-2 by TIL in the mice treated with HSP70 vaccine in combination with sPD-1 was increased and the expression of negative regulatory molecules IL-10, TGF-b and foxp3 was decreased, demonstrating that multifunctional properties afforded by the combination therapy can effectively overcome tumor resistance and promote effective antitumor immunity. The in vivo transfection with pPD-1A could be performed as infrequently as once a week and still produce a significant antitumor effect. These findings suggest that the treatment with HSP70 vaccine followed by blockade of tumor-B7-H1 with sPD-1 may provide a promising approach for tumor immunotherapy. ' 2006 Wiley-Liss, Inc.Key words: tumor immunotherapy; tumor immunity; immune escape; gene therapy The molecular hallmark of antigens preferentially expressed by tumor cells has attracted numerous interests in the development of tumor antigen based vaccinations. The ability of heat shock proteins (HSPs) to bind cellular peptides makes them the attractive candidates for cancer vaccines. 1-5 HSPs obtained from tumors or virus-infected cells have been shown to induce CTL responses in vitro and in vivo against a variety of antigens expressed in the cells from which HSPs were purified. The immunogenicity of HSP preparations has been attributed to peptides bound to HSPs. 1 HSPs are able to chaperon antigenic peptides into antigen-presenting cells (APCs) by binding to specific receptor on APCs. 6-9 The binding of HSP-peptide complex to APCs triggers a cascade of events, including re-presentation of the chaperoned peptides by the major histocompatibility complex, secretion of proinflammatory cytokines and maturation of DCs. 10-12 These properties make HSPs-based vaccination a powerful therapeutic approach to produce tumor antigen specific T cells.Besides HSPs-based vaccination, many current attempts at harnessing the immune system are capable of eliciting strong T-cell responses against tumor antigens, as high frequencies of tumor antigen specific T cell...

Although the proinflammatory cytokine interferon-gamma (IFN-gamma) has been generally thought to enhance antitumor immune responses and be involved in antitumor mechanisms of many other immunotherapy molecules, it has also been reported that IFN-gamma could promote tumor immune evasion. In this report, by using an ideal mouse model that expresses IFN-gamma locally in muscle, we demonstrate that sustained low-level expression of IFN-gamma promotes the development of several types of tumor including H22 hepatoma, MA782/5S mammary adenocarcinoma and B16 melanoma. However, transitory expression of IFN-gamma does not have such an effect. On the other hand, sustained high-level expression of IFN-gamma mediates significant antitumor effect on H22 hepatoma. Low level of IFN-gamma upregulates expression of PD-L1, PD-L2, CTLA-4 and Foxp3, which may partly account for the tumor immune evasion promoted by IFN-gamma. Furthermore, blockade of PD-L inhibits IFN-gamma's tumor-promoting effect. Our findings provide a mechanistic link between chronic inflammation and cancer and would have potential implications for cancer prevention and also for the design of cytokine-based cancer immunotherapy.

Toll-like receptor 4 (TLR4) specifically recognizes lipopolysaccharide (LPS) to initiate signal transduction events that modulate host inflammatory responses. Although increasing numbers of genes have been characterized individually for their involvement in TLR4 signaling, the LPS-induced TLR4-mediated signaling pathway and connected networks are incompletely delineated. Given that most components of signaling pathways are activated at an early phase of the LPS-induced response, we have employed a subcellular, SILAC-based quantitative proteomics approach to identify proteins in LPS-stimulated macrophages showing either cytosolic- or nuclear-specific changes in abundance. Subcellular fractionation not only reduces the spectral complexity for identifying maximum numbers of proteins but also enriches for low-abundance proteins within the compartment in which they function. Following 10 min LPS stimulation, the abundances of 508 proteins were found elevated in the cytosol, while the elevated levels of 678 proteins together with the decreased abundances of another 80 proteins were quantified in nuclei. Coincident with observations that many key proteins involved in signal relays in the MAPK and NF-kappaB cascades were found simultaneously regulated in the cytosol, various transcriptional factors (TFs) such as IRFs were found activated in the nuclei. We also extended links between these intracellular pathways and various biological processes by identifying multiple pathway modules. For the first time, our combined data sets from quantitative proteomics and bioinformatics analyses provide a direct, system-wide insight into how cross-talk between upstream signaling pathways modulates the activities of particular TFs for regulating sets of pro-inflammatory genes.