Washington University in St. Louis Washington University Open Scholarship Arts & Sciences Electronic Theses and Dissertations Arts & Sciences Spring 5-15-2020 Dendritic Cell Development and Function Vivek Durai Washington University in St. Louis Follow this and additional works at: https://openscholarship.wustl.edu/art_sci_etds Part of the Allergy and Immunology Commons, Genetics Commons, Immunology and Infectious Disease Commons, Medical Immunology Commons, and the Molecular Biology Commons Recommended Citation Durai, Vivek, "Dendritic Cell Development and Function" (2020). Arts & Sciences Electronic Theses and Dissertations. 2179. https://openscholarship.wustl.edu/art_sci_etds/2179 This Dissertation is brought to you for free and open access by the Arts & Sciences at Washington University Open Scholarship. It has been accepted for inclusion in Arts & Sciences Electronic Theses and Dissertations by an authorized administrator of Washington University Open Scholarship. For more information, please contact [email protected]. WASHINGTON UNIVERSITY IN ST. LOUIS Division of Biology and Biomedical Sciences Immunology Dissertation Examination Committee: Kenneth M. Murphy, Chair Brian Edelson Takeshi Egawa Daved Fremont Chyi-Song Hsieh Dendritic Cell Development and Function by Vivek Durai A dissertation presented to The Graduate School of Washington University in partial fulfillment of the requirements for the degree of Doctor of Philosophy May 2020 St. Louis, Missouri © 2020, Vivek Durai Table of Contents List of Figures ................................................................................................................................ iv List of Tables ................................................................................................................................ vii Acknowledgments ........................................................................................................................ viii Abstract .......................................................................................................................................... ix Chapter 1: Introduction to Dendritic Cells ...................................................................................... 1 1.1 Abstract ............................................................................................................................ 2 1.2 Dendritic cells and the immune response ......................................................................... 3 1.3 Mouse models for studying dendritic cells ...................................................................... 5 1.4 Functions of dendritic cells ............................................................................................ 17 1.5 Conclusions .................................................................................................................... 31 1.6 Author contributions ...................................................................................................... 32 1.7 References ...................................................................................................................... 32 Chapter 2: Altered compensatory cytokine signaling underlies the discrepancy between Flt3–/– and Flt3l–/– mice ............................................................................................................................ 59 2.1 Abstract .......................................................................................................................... 60 2.2 Introduction .................................................................................................................... 60 2.3 Results ............................................................................................................................ 63 2.4 Discussion ...................................................................................................................... 72 2.5 Materials and Methods ................................................................................................... 74 2.6 Acknowledgements ........................................................................................................ 82 2.7 Author Contributions ..................................................................................................... 82 2.8 References ...................................................................................................................... 83 Chapter 3: Cryptic activation of an Irf8 enhancer governs cDC1 fate specification .................. 110 3.1 Abstract ........................................................................................................................ 111 3.2 Introduction .................................................................................................................. 112 3.3 Results .......................................................................................................................... 114 3.4 Discussion .................................................................................................................... 123 3.5 Materials and Methods ................................................................................................. 126 3.6 Acknowledgements ...................................................................................................... 140 ii 3.7 Author Contributions ................................................................................................... 141 3.8 References .................................................................................................................... 142 Chapter 4: Discussion and Future Directions ............................................................................. 176 4.1 Abstract ........................................................................................................................ 177 4.2 The role of supportive cytokines in hematopoiesis ...................................................... 178 4.3 Evolving enhancer landscapes in hematopoiesis ......................................................... 179 4.4 Future Directions .......................................................................................................... 180 4.5 References .................................................................................................................... 181 iii List of Figures Figure 1.1: Functions of dendritic cell subsets in the immune response……………………...…58 Figure 2.1: Flt3l–/– mice have a more severe DC deficiency than Flt3–/– mice at two weeks of age ……………………………………………………………………………………………………88 Figure 2.2: DC deficiency in skin-draining lymph nodes and lungs is more severe in Flt3l–/– mice than in Flt3–/– mice…………………………………………………………………………….…89 Figure 2.3: Flt3l–/– mice continue to have a more severe DC deficiency than Flt3–/– mice at eight weeks of age……………………………………………………………………………………...91 Figure 2.4: Flt3L fails to generate DCs in Flt3–/– mice………………………………………….93 Figure 2.5: Flt3–/– DCs are transcriptionally and functionally similar to WT DCs…………...…95 Figure 2.6: Flt3–/– bone marrow generates mature DCs in response to M-CSF or SCF and contains committed DC progenitors……………………………………………………………..97 Figure 2.7: cDCs from M-CSF and SCF cultures are phenotypically similar to cDCs from Flt3L cultures…………………………………………………………………………………………...99 Figure 2.8: M-CSF and SCF support pDC development from Flt3–/– bone marrow…………...100 Figure 2.9: Committed DC progenitors can mature in response to M-CSF and SCF………….101 Figure 2.10: cDC development in Flt3–/– mice is dependent upon CSF1R…………………….103 Figure 2.11: Flt3–/– and Flt3l–/– BM progenitors and cDCs express normal levels of CSF1R and c-Kit.… ………………………………………………………………………………………...105 Figure 2.12: Committed DC progenitors arise in Flt3l–/– bone marrow and do not display increased sensitivity to M-CSF relative to WT progenitors…….……………………………...106 Figure 2.13: Flt3–/– DC progenitors display increased sensitivity to M-CSF..............................107 Figure 2.14: Deletion of Flt3 rescues the severe DC defect in Flt3l–/– mice…………………...108 Figure 2.15: Deletion of Flt3 in Flt3l–/– mice reverses the severe DC defect in skin-draining lymph nodes and lungs………………………………………………………………………….109 Figure 3.1: The +32 kb Irf8 enhancer is required for cDC1 development..................................148 Figure 3.2: AICEs in the +32 kb Irf8 enhancer drive cDC1 development……………………..150 Figure 3.3: Loss of the +32 kb Irf8 enhancer does not impact non-cDC1 immune lineages…..152 iv Figure 3.4: The +32 kb Irf8 enhancer is required for compensatory cDC1 development but not for cDC1 specification………………………………………………………………………….154 Figure 3.5: The +32 kb Irf8 enhancer is required for all compensatory cDC1 development…..156 Figure 3.6: CD226 uniquely identifies pre-cDC1s in the bone marrow………………………..158 Figure 3.7: The -50 kb Irf8 enhancer is not required for dendritic cell development…………..160 Figure 3.8: Loss of the -50 kb Irf8 enhancer does not impact non-monocyte/macrophage immune lineages…………………………………………………………………………………………162 Figure 3.9: The -50 kb Irf8 enhancer controls IRF8 expression in monocytes and macrophages……………………………………………………………………………………164 Figure 3.10: ATAC-seq identifies the +41 kb Irf8 enhancer as transiently active in cDC1 progenitors……………………………………………………………………………………...166 Figure 3.11: The +41 kb Irf8 enhancer is required for cDC1 specification and development…168 Figure 3.12: Loss of the +41 kb Irf8 enhancer does not impact non-DC immune lineages……170 Figure