ELUCIDATING THE ROLE OF BCL6 IN HELPER T CELL ACTIVATION, PROLIFERATION, AND DIFFERENTIATION Kristin N. Hollister Submitted to the faculty of the University Graduate School in partial fulfillment of the requirements for the degree Doctor of Philosophy in the Department of Microbiology and Immunology, Indiana University June 2014 Accepted by the Graduate Faculty of Indiana University, in partial fulfillment of the requirements for the degree of Doctor of Philosophy. ________________________________ Alexander L. Dent, Ph.D., Chairman Doctoral Committee ________________________________ Randy R. Brutkiewicz, Ph.D. March 7, 2014 ________________________________ Maureen A. Harrington, Ph.D. ________________________________ Mark H. Kaplan, Ph.D. ii ACKNOWLEDGEMENTS I would like to thank my mentor, Dr. Alexander Dent. Without his guidance and support this work would not have been possible. He has been a phenomenal mentor, and any student would be lucky to have him as such. Also, I would like to thank my committee members, Dr. Randy Brutkiewicz, Dr. Mark Kaplan, and Dr. Maureen Harrington. I always looked forward to our meetings and the constructive criticism and helpful encouragement they provided. Several Dent lab members, past and present, contributed greatly to this work and enabled me to complete complex experiments resulting in excellent data. These people include Hao Wu, Arpita Mondal, and Ninah Clegg. Members of the Kaplan lab were also instrumental in assisting with the work presented here. In particular, I would like to acknowledge the contributions of Dr. Duy Pham, who provided excellent technical assistance with some of the assays presented in this thesis. Our collaborators at the University of Massachusetts Medical School played a pivotal role in ascertaining the data for our HIV-1 vaccine model. Dr. Shan Lu’s group included Dr. Shixia Wang and Yuxin Chen. I have been fortunate enough to be funded by multiple sources during my graduate studies, and I would like to thank Dr. Janice Blum, Dr. Hal Broxmeyer, and Dr. Stanley Spinola for providing me with financial support during my studies. Finally, I would like to thank my family for providing me with the support and encouragement needed to make it through graduate school. iii Kristin N. Hollister ELUCIDATING THE ROLE OF BCL6 IN HELPER T CELL ACTIVATION, PROLIFERATION, AND DIFFERENTIATION The transcriptional repressor BCL6 has been shown to be essential for the differentiation of germinal center (GC) B cells and follicular T helper (TFH) cells. The interaction of TFH and GC B cells is necessary for the development of high affinity antibodies specific for an invading pathogen. Germline BCL6-deficient mouse models limit our ability to study BCL6 function in T cells due to the strong inflammatory responses seen in these mice. To overcome this, our lab has developed a new BCL6 conditional knockout (cKO) mouse using the cre/lox system, wherein the zinc finger region of the BCL6 gene is flanked by loxP sites. Mating to a CD4-Cre mouse allowed us to study the effects of BCL6 loss specifically in T cells, without the confounding effects seen in germline knockout models. Using this cKO model, we have reaffirmed the necessity of BCL6 for TFH differentiation, including its role in sustained CXCR5 surface expression, a signature marker for TFH cells. This model also allowed us to recognize the role of BCL6 in promoting the expression of PD-1, another key surface marker for TFH cells. Without BCL6, CD4+ T cells cannot express PD-1 at the high levels seen on TFH cells. Our discovery of DNMT3b as a target for BCL6 suggests BCL6-deficient T cells have increased DNA methyltransferase activity at the PD-1 promoter. This data establishes a novel pathway for explaining how BCL6, a transcriptional repressor, can activate genes. Experiments with the BCL6 cKO model have also established a role for BCL6 in naïve CD4+ T cell activation. Furthermore, we did not observe increased differentiation of other helper T cell subsets, in contrast to what has been reported elsewhere with germline BCL6-deficient models. Unexpectedly, we found decreased T helper type 2 (Th2) cells, whereas mouse models with a germline mutation of BCL6 have increased Th2 cells. These results indicate that BCL6 activity in non-T cells is critical for controlling T cell differentiation. Finally, using an HIV-1 gp120 immunization model, we have, for the first time, shown BCL6-dependent GCs to be limiting for antibody development and affinity maturation in a prime-boost vaccine scheme. Alexander L. Dent, Ph.D., Chairman iv TABLE OF CONTENTS List of Tables .................................................................................................................. ix List of Figures ................................................................................................................. x List of Abbreviations .................................................................................................... xiii CHAPTER 1 – A GENERAL INTRODUCTION TO THE IMMUNE RESPONSE ............. 1 Immune System Origins ........................................................................................ 1 The Innate Immune Response .............................................................................. 1 Granulocytes.............................................................................................. 2 Monocytes, macrophages, dendritic cells .................................................. 3 Major histocompatibility complex class II molecules .................................. 3 Natural killer cells....................................................................................... 3 The Adaptive Immune Response .......................................................................... 4 T helper type 1, type 2, type 17, and type 9 cells ...................................... 4 Th-B cell interactions ................................................................................. 6 Regulatory Th cells .................................................................................... 8 Follicular T helper cells .............................................................................. 8 Germinal Centers .................................................................................................. 9 Germinal center development .................................................................... 9 Somatic hypermutation and isotype class switching of B cells ................ 11 Germinal center structure ........................................................................ 12 Regulating the germinal center response ................................................ 12 Goals of Research ............................................................................................... 14 CHAPTER 2 – DEVELOPMENT OF A NEW BCL6 CONDITIONAL KNOCKOUT MOUSE MODEL ............................................................................................................. 15 Introduction .......................................................................................................... 15 BCL6 structure and activity ...................................................................... 15 BCL6 germline knockout mouse model ................................................... 16 Materials and Methods ........................................................................................ 17 Results ................................................................................................................ 20 Generation of BCL6Neofl/Neofl mice ............................................................. 20 Functional testing of BCL6Neofl/Neofl mice ................................................... 21 Removal of the neomycin resistance gene .............................................. 28 New germline knockout mice – BCL6∆ZF/∆ZF ............................................ 29 v BCL6fl/fl mice ............................................................................................ 33 Discussion ........................................................................................................... 33 CHAPTER 3 – BCL6 IS NECESSARY FOR PROPER NAÏVE CD4+ T CELL ACTIVATION .................................................................................................................. 37 Introduction .......................................................................................................... 37 T cell development ................................................................................... 37 Th cell activation signaling ....................................................................... 37 Materials and Methods ........................................................................................ 40 Results ................................................................................................................ 43 Efficient and complete deletion of BCL6 zinc finger exons in BCL6+/fl CreCD4 mice ................................................................................. 43 Skewed naïve and effector memory CD4+ T cell populations in BCL6fl/fl CreCD4 mice ................................................................................. 43 Skewed naive and effector CD4+ T cell populations confirmed in mixed bone marrow chimera model ......................................................... 44 Naïve/effector memory CD4+ T cell phenotype seen early after immunization and only exacerbates over time ......................................... 48 Differences in T