Regulatory T Cells – Molecular and Clinical Aspects THESIS FOR DOCTORAL DEGREE ( Ph . D . )

In the immune system’s tug of war with cancer, tolerance mechanisms by which the tumor can control anti-tumor immune responses play a central role in determining the outcome. Regulatory T cells (Treg) induced in the thymus or the periphery, represent one such tolerance mechanism that potentially can be exploited by developing tumors. In this thesis, we explore the underlying molecular mechanisms that result in Treg development, and attempt to elucidate the importance of this T lymphocyte subset in urinary bladder cancer. In humans and mice, the transcription factor FOXP3 is crucial for the function of the Treg subset and maintenance of peripheral tolerance, as illustrated by the lack of functional Treg and autoimmune disorders that result from mutations within this protein. In humans however, clinical studies of this important T lymphocyte subset are obscured by the fact that FOXP3 is transiently induced in conventional T lymphocytes upon activation. Initially, we address the epigenetic regulation of FOXP3 expression, and demonstrate that the committed Treg population has an almost completely demethylated FOXP3 promoter region, whereas conventional non-regulatory CD4+ T lymphocytes are semi-methylated in this region. Furthermore, we study the development of regulatory T cells in mice, and assess the contribution and the impact of adenosine receptor signaling on the T lymphocyte compartment. Next, the role of the Treg population in urinary bladder cancer is evaluated. First, we investigate the impact of tumor infiltrating CD3+ as well as FOXP3+ T lymphocytes on patient survival. Somewhat surprisingly, we find a positive correlation between both CD3+ and FOXP3+ lymphocytic infiltrates, suggesting that FOXP3+ lymphocytes in this case may not represent a tumor escape mechanism as initially hypothesized. In addition, we observe FOXP3 expression in a subset of tumors, and find that this expression is a negative prognostic factor for survival. To follow up these results, we characterize the T lymphocyte immune response in peripheral blood, lymph nodes and tumor tissue from patients with UBC. We demonstrate that the FOXP3+ fraction of CD4+ T lymphocytes is significantly increased compared to all other locations investigated including macroscopically healthy bladder tissue. Furthermore, these tumor infiltrating lymphocytes express high levels of activation and effector markers, but do not display a demethylated pattern in the FOXP3 promoter to match its prominent expression. Interestingly, muscle invasive tumors have a lower FOXP3+ fraction at the invasive front compared to non-invasive counterparts. In addition, we observe changes in the cellular immune response dependent on if the patients have received neo-adjuvant chemotherapy or not, both with regard to cell composition and functional reactivity to tumor antigens. Epigenetic regulation governs the commitment of T lymphocytes to the Treg lineage. The fact that FOXP3 expressing tumor infiltrating lymphocytes in UBC do not display a committed Treg phenotype could potentially explain the differences in reported clinical impact of this population in different cancers and has implications for future immunotherapy. LIST OF SCIENTIFIC PAPERS I. Winerdal ME*, Janson PC*, Marits P, Thörn M, Ohlsson R, Winqvist O. FOXP3 promoter demethylation reveals the committed Treg population in humans. PLoS One. 2008 Feb 20;3(2):e1612. *Shared first authorship II. Winerdal ME, Marits P, Winerdal M, Hasan M, Rosenblatt R, Tolf A, Selling K, Sherif A, Winqvist O. FOXP3 and survival in urinary bladder cancer. BJU Int. 2011 Nov;108(10):1672-8. III. Winerdal ME, Krantz D, Rosenblatt R, Zirakzadeh A, Ahlén Bergman E, Winerdal M, Hansson J, Holmström B, Johansson M, Marits P, Sherif A, Winqvist O. Characterization of T lymphocyte Responses in Urinary Bladder Cancer. Manuscript. IV. Winerdal ME, Winerdal M, Krantz D, Yang T, Lindmark E, Marits P, Fredholm B, Ådén U, Carlström M, Winqvist O. Adenosine receptor signaling in T cell development. Manuscript. List of publications not included in the thesis: Lofgren C, Hjortsberg L, Blennow M, Lotfi K, Paul C, Eriksson S, Albertioni F. Mechanisms of cross-resistance between nucleoside analogues and vincristine or daunorubicin in leukemic cells. Biochem Biophys Res Commun. 2004 Jul 30;320(3):825-32. Janson PC, Winerdal ME, Winqvist O. At the crossroads of T helper lineage commitment-Epigenetics points the way. Biochim Biophys Acta. 2009 Sep;1790(9):906-19. Hu J, Lou D, Carow B, Winerdal ME, Rottenberg M, Wikström AC, Norstedt G, Winqvist O. LPS Regulates SOCS2 Transcription in a Type I Interferon Dependent Autocrine-Paracrine Loop. PLoS One. 2012;7(1):e30166. Winerdal ME*, Winerdal M*, Kinn J, Urmaliya V, Winqvist O, Adén U. Long lasting local and systemic inflammation after cerebral hypoxic ischemia in newborn mice. PLoS One. 2012;7(5):e36422. *Shared first authorship