Analysis of the role of the transcription factor C/EBPβ in controlling uterine functions during early pregnancy

Embryo implantation into the endometrium is a complex biological process involving the integration of steroid hormone signaling, endometrial tissue remodeling and maternal- fetal communications. A successful pregnancy is the outcome of the timely integration of these events during the early stages of implantation. The involvement of ovarian steroid hormones, estrogen (E) and progesterone (P), acting through their cognate receptors, is essential for uterine functions during pregnancy. The molecular mechanisms that control the process of implantation are undergoing active exploration. Through our recent efforts, we identified the transcription factor, CCAAT Enhancer Binding Protein Beta (C/EBPb) as a prominent target of estrogen and progesterone signaling in the uterus. The development of a C/EBPb-null mouse model, which is infertile, presented us with an opportunity to analyze the role of this molecule in uterine function. We discovered that C/EBPb functions in two distinct manners: (i) by acting as a mediator of E-induced proliferation of the uterine epithelium and (ii) by controlling uterine stromal cell differentiation, a process known as decidualization, during pregnancy. My studies have delineated important mechanisms by which E regulates C/EBPb expression to induce DNA replication and prevent apoptosis of uterine epithelial cells during E-induced epithelial growth. In subsequent studies, I analyzed the role of C/EBPb in decidualization and uncovered a unique mechanism by which C/EBPb regulates the synthesis of a unique laminin-containing extracellular matrix (ECM) that supports stromal cell differentiation and embryo invasion. In order to better define the role of laminin in implantation, we developed a laminin gamma 1-conditional knockout mouse model. This is currently an area of ongoing investigation. The information gained from our analysis of C/EBPb function in the uterus provides new insights into the mechanisms of steroid hormone action during early pregnancy. Ultimately, our findings may aid in the understanding of dysregulation of hormone-controlled pathways that underlie early pregnancy loss and infertility in women.

Evaluation of Adoptive T Cell Therapy and Oncolytic Virotherapy for Treatment of Brain Tumors

Adoptive immunotherapy and oncolytic virotherapy are two promising strategies for treating primary and metastatic malignant brain tumors. We demonstrate the ability of adoptively transferred tumor-specific T cells to rapidly mediate the clearance of established brain tumors in several mouse models. Similar to the clinical situation, tumor recurrences are frequent and result from immune editing of tumors. T cells can eliminate antigen-expressing tumor cells but are not effective against antigen loss variant (ALV) cancer cells that multiply and repopulate a tumor. We show that the level of tumor antigen present affects the success of adoptive T cell therapy. When high levels of antigen are present, tumor stromal cells such as microglia and macrophages present tumor peptide on their surface. As a result, T cells directly eliminate cancer cells and cross-presenting stromal cells and indirectly eliminate ALV cells. We were able to show the first direct evidence of tumor antigen cross-presentation by CD11b+ stromal cells in the brain using soluble, high-affinity T cell receptor monomers. Strategies that target brain tumor stroma or increase antigen shedding from tumor cells leading to increased crosspresentation by stromal cells may improve the clinical success of T cell adoptive therapies. We evaluated one potential strategy to complement adoptive T cell therapy by characterizing the oncolytic effects of myxoma virus (MYXV) in a syngeneic mouse brain tumor model of metastatic melanoma. MYXV is a rabbit poxvirus with strict species tropism for European rabbits. MYXV can also infect mouse and human cancer cell lines due to signaling defects in innate antiviral mechanisms and hyperphosphorylation of Akt. MYXV kills B16.SIY melanoma cells in vitro, and intratumoral injection of virus leads to robust, selective and transient infection of the tumor. We observed that virus treatment recruits innate immune cells iii to the tumor, induces TNFα and IFNβ production in the brain, and results in limited oncolytic effects in vivo. To overcome this, we evaluated the safety and efficacy of co-administering 2C T cells, MYXV, and neutralizing antibodies against IFNβ. Mice that received the triple combination therapy survived significantly longer with no apparent side effects, but eventually relapsed. Based on these findings, methods to enhance viral replication in the tumor and limit immune clearance of the virus will be pursued. We conclude that myxoma virus should be further explored as a vector for transient delivery of therapeutic genes to a tumor to enhance T cell responses.