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DNA methyltransferase 3a (DNMT3a) is an important part of the epigenetic machinery that stabilizes patterns of activated T cell responses. We hypothesized that donor T cell DNMT3a regulates alloreactivity after allogeneic blood and marrow transplantation (allo-BMT). T cell conditional Dnmt3a KO mice were used as donors in allo-BMT models. Mice receiving alloBMT from KO donors developed severe acute graft-versus-host disease (aGVHD), with increases in inflammatory cytokine levels and organ histopathology scores. KO T cells migrated and proliferated in secondary lymphoid organs earlier and demonstrated an advantage in trafficking to the small intestine. Donor T cell subsets were purified after BMT for wholegenome bisulfite sequencing (WGBS) and RNA-Seq. KO T cells had global methylation similar to that of WT cells, with distinct, localized areas of hypomethylation. Using a highly sensitive computational method, we produced a comprehensive profile of the altered epigenome landscape. Hypomethylation corresponded with changes in gene expression in several pathways of T cell signaling and differentiation. Additionally, Dnmt3a-KO T cells resulted in superior graft-versus- tumor activity. Our findings demonstrate a critical role for DNMT3a in regulating T cell alloreactivity and reveal pathways that control T cell tolerance. These results also provide a platform for deciphering clinical data that associate donor DNMT3a mutations with increased GVHD, decreased relapse, and improved survival., Introduction Allogeneic blood and marrow transplantation (allo-BMT) remains the only curative therapy for many malignant and nonmalignant disorders (1). Unfortunately, successful outcomes are limited by graft-versus-host disease (GVHD) and malignant [...]

Programmed death ligand-1 (PD-L1) is an important negative regulator of T cell immune responses via interactions with PD-1 and CD80. However, PD-L1 can also act as a positive costimulator, but the relevant counterreceptor is not known. We analyzed the role of PD-L1 in CD8-T cell responses to infection with Listeria monocytogenes (LM) or vesicular stomatitis virus (VSV). PD-L1 blockade impaired antigen-specific CD8 effector T cell expansion in response to LM, but not to VSV infection, particularly limiting short-lived effector cell differentiation. Simultaneous CD4-T cell depletion and anti-PD-L1 blockade revealed that PD-L1 provided costimulation even in the absence of CD4-T cells. Most importantly, specific blockade of PD-L1 binding to CD80 or to PD-1 did not recapitulate PDL-1 blockade. The results suggested that PD-L1 plays an important costimulatory role for antigen-specific CD8 T cells during LM infection perhaps through a distinct receptor or interaction epitope. [ABSTRACT FROM AUTHOR]

Cytotoxic T cell-induced cell death is well documented. Cytotoxic T cell releases various cytolytic proteins. The cytolytic proteins induce target cell death. T cell-induced cell death can be measured by the lytic assay. One of the well-known lytic assays uses radioactive tracer, Chromium-51 ( 51 Cr), and detects the amount of 51 Cr released from target cells. This assay can detect cell death and the efficiency of the T cell-induced cell death by coculture effector cells (T cells) and target cells. This assay can determine the kinetics of the cell lysis. The issue of this approach is the use of radioactive material. This chapter describes measuring T cell-induced cell death by determining the epigenetic remodeling and the release of cytolytic proteins. Determine the efficiency of T cell-induced cell death by using a flow cytometry-based detection method.

Tumors are heterogeneous structures that contain different cell populations. Laser capture microdissection (LCM) can be used to obtain pure cancer cells from fresh-frozen cancer tissue and the surrounded environment, thus providing an accurate snapshot of the tumor and its microenvironment in vivo. We describe a new approach to isolate pure cancer cell population and evaluate protein expression. The process includes immunocytochemistry, laser microdissection, and western blot analysis. Using this technique, we can detect proteins such as X-linked inhibitor of apoptosis protein (XIAP) and Fas ligand (FasL) with as little as 1000 cells.

Introduction: Early identification of chemoresistance in patients with ovarian cancer is of utmost importance in order to provide them with the most appropriate therapy. Recently, we described the expression of MyD88 in ovarian cancer cells that were resistant to the cytotoxic agent paclitaxel. In addition to chemoresistance, in MyD88 positive ovarian cancer cells, paclitaxel stimulates growth and production of proinflammatory cytokines. The objective of this study was to determine the correlation of MyD88 expression in primary and recurrent epithelial ovarian cancers with the response to carboplatin and paclitaxel combination chemotherapy., Methods: Tumors are heterogeneous structures that contain different cell populations, thus rendering the identification of specific tumor markers difficult. Using laser capture microdissection, pure cancer cells were isolated from ovarian malignant tumors that were obtained from 20 patients at the time of surgery. The microdissected cells were evaluated for the expression of MyD88, FasL, and XIAP by western blot analysis., Results: Protein expression was observed in samples containing as low as 500 cells. The results were correlated with the clinical course of those patients. It was evident that MyD88 expression in ovarian cancer cells accurately predicts a poor response to paclitaxel chemotherapy as shown by a short progression-free interval and overall survival., Conclusion: We describe for the first time a molecular approach to identify paclitaxel chemoresistance. Toxicity from agents without therapeutic benefit can be avoided by identifying those patients who will not respond to a specific agent. Molecular markers will enable us to design individualized treatments and improve overall survival.

Epithelial ovarian cancer is the fourth leading cause of cancer-related deaths in women and is the most lethal of the gynecological malignancies. Thle high mortality rate arises from difficulties in the early detection of the disease and the widespread development of chemoresistance. Phenoxodiol, a novel isoflavone derivative, has demonstrated antitumor activity. In addition, it has been shown to induce cell death in chemoresistant epithelial ovarian cancer cells. Moreover, suboptimal exposure of these cells to phenoxodiol lowered the IC50 value of numerous chemotherapeutic agents. In this review, the current understanding of the mechanism of action of phenoxodiol, its potential clinical application for the treatment of ovarian cancer and the concept of chemosensitization are discussed.