TRANSCRIPTIONAL CONTROL OF INTERFERON GAMMA SYNTHESIS BY NATURAL KILLER CELLS DISSERTATION Presented in Partial Fulfillment of the Requirements for The Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Michael Brian Becknell, B.A. ***** The Ohio State University 2006 Dissertation Committee: Michael A. Caligiuri, M.D., Adviser Denis Guttridge, Ph.D. Approved by Danilo Perrotti, M.D., Ph.D. _________________________________________ Saïd Sif, Ph.D. Adviser Integrated Biomedical Sciences Graduate Program ABSTRACT Natural killer (NK) cells fill an critical niche in the mammalian immune system. In addition to deleting compromised host cells through a variety of cytolytic mechanisms, NK cells orchestrate the innate and adaptive immune response via the production of a set of protein factors called cytokines. In particular, NK cells elaborate interferon gamma (IFN-γ) in response to a variety of pathologic stimuli, both extrinsic and intrinsic to the host. The importance of NK-derived IFN-γ is tragically illustrated by rare cases of pediatric immune deficiency, in which children lacking NK cells and/or a functional IFN- γ signaling pathway succumb to microbial infection early in life. Despite the clear importance of IFN-γ in host defense against intracellular pathogens and tumorigenesis, it is equally apparent that excessive IFN-γ production contributes to the etiology of pro- inflammatory disease states, including but not limited to the generalized Shwartzman reaction, inflammatory bowel disease, and graft versus host disease. A better understanding of the regulation of IFN-γ by NK cells may facilitate the manipulation of this process in patients whose disease is due to too much or too little IFN-γ production. With this goal in mind, this study has focused on the regulation of IFN-γ messenger RNA (mRNA) synthesis in NK cells. In the work summarized herein, we developed the methodology to retrovirally transduce primary NK cells and NK-derived cell lines with -ii - transcription factors and determined their role in IFN-γ transcription. We applied this methodology to the study of two candidate regulators of IFN-γ transcription, T-bet and Hlx. Whereas T-bet is required for optimal IFN-γ synthesis by NK cells, we found that Hlx is a novel inhibitor of IFN-γ transcription in NK cells, and that this regulatory role is achieved through both direct and indirect mechanisms. These studies – when integrated with parallel work conducted in our laboratory and by many other talented laboratories worldwide – enable us to begin to comprehend the network of positive and negative regulatory events that ultimately dictate the outcome of transcriptional activity at the IFN-γ promoter. -iii - To Debbie and Sophia. -iv - ACKNOWLEDGMENTS I wish to especially thank my wife, Debbie: for her unflagging love and support; for her encouragement; and for her honesty. For all these things, I am forever grateful. Thank you next to my parents, Jerry and Eileen Becknell, for believing in me and for your unconditional love. I want to express my most sincere gratitude to my mentor, Dr. Michael Caligiuri, who has always been a source of wisdom, both about NK cell biology and about life. Mike, I am constantly in awe of the way in which you inspire me and the people around you. You truly bring out the best in a person. I am deeply honored to have worked with you for this long. To my colleagues in the Caligiuri Lab: you are my dear friends. Thank you for your support, your criticism (scientific and otherwise), and the comic relief you have provided (consciously or otherwise) over the years. In particular, I wish to acknowledge the tremendous effort of Tiffany Hughes in contributing to this work. I want to thank my thesis committee, Dr. Denis Guttridge, Dr. Danilo Perrotti, and Dr. Saïd Sif. Each of you has provided valuable scientific instruction over the years. I respect you deeply and thank you for the time you have spent on my behalf. - v - I also thank Dr. Sif and Sharmistha Pal, his talented graduate student, who taught me a great deal during my time in the Sif laboratory in 2004. I thank Dr. Allan Yates for his enormous investment of time and talent in creating the Integrated Biomedical Science Program at OSU, as well as his resuscitation of the Medical Scientist Program. I wish to acknowledge the following sources of financial support over the course of my tenure as a graduate student: the NSF Predoctoral Fellowship, the Dean's Distinguished University Fellowship, the Bennett Fellowship, and the Medical Scientist Program. - vi - VITA July 16, 1974 ……………………………………………Born, Clarksville, Tennessee 1997 ……………………………………………………..B.A., Kenyon College, Magna cum Laude, with Distinction and Highest Honors in Molecular Biology 1997-1999 ……………………………………………..Ph.D. student, Molecular and Cellular Biology Program, Washington University in St. Louis 1999-present…………………………………………….M.D. Student, The Ohio State University PUBLICATIONS Peer-Reviewed Research Articles 1. Yu J, Wei M, Becknell B, Trotta R, Liu S, Boyd Z, Jaung MS, Blaser BW, Sun J, Benson DM Jr, Mao H, Yokohama A, Bhatt D, Shen L, Davuluri R, Weinstein M, Marcucci G, Caligiuri MA. Pro- and Antiinflammatory Cytokine Signaling: Reciprocal Antagonism Regulates Interferon-gamma Production by Human Natural Killer Cells. Immunity 2006 May; 24(5): 575-90. 2. Freud AG, Yokohama A, Becknell B, Lee MT, Mao HC, Ferketich AK, Caligiuri MA. Evidence for discrete stages of human natural killer cell differentiation in vivo. Journal of Experimental Medicine 2006 April 17; 203(4): 1033-43. 3. Vandeusen JB, Shah MH, Becknell B, Blaser BW, Ferketich AK, Nuovo GJ, Ahmer BM, Durbin J, Caligiuri MA. STAT-1-mediated repression of monocyte - vii - interleukin-10 gene expression in vivo. European Journal of Immunology 2006 Feb 15; 36(3): 623-30. 4. Mikhalkevich N, Becknell B, Caligiuri MA, Bates MD, Harvey R, Zheng WP. Responsiveness of naive CD4 T cells to polarizing cytokine determines the ratio of Th1 and Th2 cell differentiation. Journal of Immunology 2006 Feb 1; 176(3): 1553-60. 5. Freud AG, Becknell B, Roychowdhury S, Mao HC, Ferketich AK, Nuovo GJ, Hughes TL, Marburger TB, Sung J, Baiocchi RA, Guimond M, Caligiuri MA. A human CD34(+) subset resides in lymph nodes and differentiates into CD56bright natural killer cells. Immunity 2005 March; 22(3): 295-304. 6. Whitman SP, Liu S, Vukosavljevic T, Rush LJ, Yu L, Liu C, Klisovic MI, Maharry K, Guimond M, Strout MP, Becknell B, Dorrance A, Klisovic RB, Plass C, Bloomfield CD, Marcucci G, Caligiuri MA. The MLL partial tandem duplication: evidence for recessive gain-of-function in acute myeloid leukemia identifies a novel patient subgroup for molecular-targeted therapy. Blood 2005 July 1; 106(1): 345-52. 7. Liu S, Shen T, Huynh L, Klisovic MI, Rush LJ, Ford JL, Yu J, Becknell B, Li Y, Liu C, Vukosavljevic T, Whitman SP, Chang KS, Byrd JC, Perrotti D, Plass C, Marcucci G. Interplay of RUNX1/MTG8 and DNA methyltransferase 1 in acute myeloid leukemia. Cancer Research 2005 February 15; 65(4): 1277-84. 8. Yu L, Liu C, Vandeusen J, Becknell B, Dai Z, Wu YZ, Raval A, Liu TH, Ding W, Mao C, Liu S, Smith LT, Lee S, Rassenti L, Marcucci G, Byrd J, Caligiuri MA, Plass C. Global assessment of promoter methylation in a mouse model of cancer identifies ID4 as a putative tumor-suppressor gene in human leukemia. Nature Genetics 2005 March; 37(3): 265-74. 9. Becknell B, Trotta R, Yu J, Ding W, Mao HC, Hughes T, Marburger T, Caligiuri MA. Efficient infection of human natural killer cells with an EBV/retroviral hybrid vector. Journal of Immunological Methods 2005 January; 296(1-2): 115-23. 10. Trotta R, Parihar R, Yu J, Becknell B, Allard J 2nd, Wen J, Ding W, Mao H, Tridandapani S, Carson WE, Caligiuri MA. Differential expression of SHIP1 in CD56bright and CD56dim NK cells provides a molecular basis for distinct functional responses to monokine costimulation. Blood 2005 April 15; 105(8): 3011-8. 11. Yu L, Liu C, Bennett K, Wu YZ, Dai Z, Vandeusen J, Opavsky R, Raval A, Trikha P, Rodriguez B, Becknell B, Mao C, Lee S, Davuluri RV, Leone G, Van den Veyver IB, Caligiuri MA, Plass C. A NotI-EcoRV promoter library for studies of genetic and epigenetic alterations in mouse models of human malignancies. Genomics 2004 October; 84(4):647-60. - viii - 12. Igarashi T, Wynberg J, Srinivasan R, Becknell B, McCoy JP Jr, Takahashi Y, Suffredini DA, Linehan WM, Caligiuri MA, Childs RW. Enhanced cytotoxicity of allogeneic NK cells with killer immunoglobulin-like receptor ligand incompatibility against melanoma and renal cell carcinoma cells. Blood 2004 July 1; 104(1):170-7. 13. Becknell B, Shen T, Maghraby E, Taya S, Kaibuchi K, Caligiuri MA, Marcucci G. Characterization of leukemia-associated Rho guanine nucleotide exchange factor (LARG) expression during murine development. Cell and Tissue Research 2003 December; 314(3): 361-6. 14. Lesinski GB, Anghelina M, Zimmerer J, Bakalakos T, Badgwell B, Parihar R, Hu Y, Becknell B, Abood G, Chaudhury AR, Magro C, Durbin J, Carson WE 3rd. The antitumor effects of IFN-alpha are abrogated in a STAT1-deficient mouse. Journal of Clinical Investigation 2003 July; 112(2): 170-80. 15. Farag SS, George SL, Lee EJ, Baer M, Dodge RK, Becknell B, Fehniger TA, Silverman LR, Crawford J, Bloomfield CD, Larson RA, Schiffer CA, Caligiuri MA. Postremission therapy with low-dose interleukin 2 with or without intermediate pulse dose interleukin 2 therapy is well tolerated in elderly patients with acute myeloid leukemia: Cancer and Leukemia Group B study 9420. Clinical Cancer Research 2002 September; 8(9): 2812-9. 16. Whitman SP, Archer KJ, Feng L, Baldus C, Becknell B, Carlson BD, Carroll AJ, Mrozek K, Vardiman JW, George SL, Kolitz JE, Larson RA, Bloomfield CD, Caligiuri MA.