The goal of this project is to test the hypothesis that the tumor microenvironment plays a critical role in influencing T cell responses to tumor antigens. This microenvironment is comprised of a complex interaction between tumor cells, lymphocytes, myeloid cells, and stromal cells. This project has historically used TRAMP mice which express the transforming SV40 T antigen (TAg) under the transcriptional control of a prostate-specific promoter, which causes the development of murine prostate cancer. Naive TAg-specific T cells are transferred into TRAMP mice. We previously reported that CD8+ T cells become tolerized when they enter the tumor microenvironment, acquire suppressive activity, and the induction of tolerance and suppressor activity is dependent on infiltration into the tumor. These regulatory cells have the capacity to suppress proliferation of other T cells. We also reported that transfer of TAg-specific CD4+ T cells also undergo transient activation before deletion and trafficking to the prostate. Taking advantage of this transient activation, we further demonstrated that co-transfer of both CD4+ and CD8+ T cells delays tolerization of the CD8+ T cells. Continuous administration of the tumor-specific CD4+ T cells prevented T cell tolerance and reduced tumor growth. More recently, we reported on the mechanism by which T cells become tolerized in the tumor microenvironment. We identified a population of plasmacytoid dendritic cells (pDCs) which exist in both normal and transformed prostate tissues. Interestingly, the dendritic cells isolated from the TRAMP prostate are incapable of stimulating proliferation of nave T cell proliferation in vitro, whereas dendritic cells from normal prostate tissues are stimulatory. A similar population was identified in murine B16 melanoma tumors, as well. Further examination of the T cells primed by these tumor-associated DC (TADC) revealed that they were tolerized and had suppressor function. Depletion of the TADC resulted in efficient T cell priming and reduced tumor growth. A similar population of pDCs was identified in human prostate cancer specimens, and these cells had similar functional attributes. Most striking was our observation that the transcription factor FOXO3 was over-expressed in TADC from human and murine prostatic tumors (relative to non-cancerous prostatic tissues), and silencing expression of Foxo3 resulted in a loss of TADC tolerogenicity. These findings implicate FOXO3 as a novel target for prostate cancer immunotherapy. Based on these findings, a patent application was filed in January, 2011 and was fully executed in June 2012.Our on-going and future studies will identify the mechanism(s) by which FOXO3 controls TADC function. Specifically, we will study its interaction with other intracellular signaling molecules and transcription factors. In addition, we will identify transcriptional targets of FOXO3 using profiling approaches such as microarray and ChIP-Seq. In addition, we will study FOXO3 expression by other tumor-associated myeloid cells.A newer project is focused on studying the role of mast cells in immune regulation in the tumor microenvironment. Mast cells play an important role in allergic responses, but have also been implicated in tumor progression. We have identified a population of tumor-derived mast cells that accumulate in TRAMP tumors as they develop. These mast cells inhibit T cell responses in vitro. More importantly, we have identified two mast cell-derived factors (TGF-beta and IL-13) that are capable of inducing 'normal'DC into cells which have phenotypic and functional similarities to TADC. Our on-going studies will characterize the mechanisms by which these mast cells suppress T cells and identify ways to inhibit this suppression to enhance immunity to prostatic tumors. Our long-term studies will identify the mechanisms by which tumors induce these suppressive properties in mast cells.

National Institute of Health (NIH)
National Cancer Institute (NCI)
Investigator-Initiated Intramural Research Projects (ZIA)
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National Cancer Institute Division of Basic Sciences
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Zhu, Ziqiang; Cuss, Steven M; Singh, Vinod et al. (2015) CD4+ T Cell Help Selectively Enhances High-Avidity Tumor Antigen-Specific CD8+ T Cells. J Immunol 195:3482-9
Shurin, Michael R; Umansky, Viktor; Malyguine, Anatoli et al. (2014) Cellular and molecular pathways in the tumor immunoenvironment: 3rd Cancer Immunotherapy and Immunomonitoring (CITIM) meeting, 22-25 April 2013, Krakow, Poland. Cancer Immunol Immunother 63:73-80
Hudson, R S; Yi, M; Esposito, D et al. (2013) MicroRNA-106b-25 cluster expression is associated with early disease recurrence and targets caspase-7 and focal adhesion in human prostate cancer. Oncogene 32:4139-47
Hurwitz, Arthur A; Watkins, Stephanie K (2012) Immune suppression in the tumor microenvironment: a role for dendritic cell-mediated tolerization of T cells. Cancer Immunol Immunother 61:289-93
Watkins, Stephanie K; Hurwitz, Arthur A (2012) FOXO3: A master switch for regulating tolerance and immunity in dendritic cells. Oncoimmunology 1:252-254
Watkins, Stephanie K; Zhu, Ziqiang; Watkins, Keith E et al. (2012) Isolation of immune cells from primary tumors. J Vis Exp :e3952
Watkins, Stephanie K; Zhu, Ziqiang; Riboldi, Elena et al. (2011) FOXO3 programs tumor-associated DCs to become tolerogenic in human and murine prostate cancer. J Clin Invest 121:1361-72
Chen, Xin; Hamano, Ryoko; Subleski, Jeffrey J et al. (2010) Expression of costimulatory TNFR2 induces resistance of CD4+FoxP3- conventional T cells to suppression by CD4+FoxP3+ regulatory T cells. J Immunol 185:174-82
Shafer-Weaver, Kimberly A; Watkins, Stephanie K; Anderson, Michael J et al. (2009) Immunity to murine prostatic tumors: continuous provision of T-cell help prevents CD8 T-cell tolerance and activates tumor-infiltrating dendritic cells. Cancer Res 69:6256-64
Shafer-Weaver, Kimberly A; Anderson, Michael J; Stagliano, Katherine et al. (2009) Cutting Edge: Tumor-specific CD8+ T cells infiltrating prostatic tumors are induced to become suppressor cells. J Immunol 183:4848-52

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