The ontogeny and regulation of human natural killer cells

Natural Killer (NK) cells are members of the innate lymphoid cell (ILC) family and take part in the detection and eradication of virus-infected and transformed cells. In this thesis, together with my colleagues I have investigated how NK cells and other ILCs develop and function during human fetal development, how NK cells are functionally regulated (educated) via the activating receptor KIR2DS1, and how NK cells in our body are affected during the early phase of an acute viral infection. Little is known about the ontogeny of NK cells and other ILCs in fetal development. The characterization of ILCs has been hampered by their overlapping surface phenotypes. In contrast, ILC transcription factor expression is more specific, and by combining multicolor flow cytometry analysis of transcription factors and surface markers expressed by different fetal ILC subsets, we were able to study and model their development and differentiation. All ILC subsets were detected as early as gestational week six, and their distribution varied depending on both tissue and gestational age. Moreover, putative precursors of NK cells were identified as cells that sequentially lost CD34 and acquired CD122, Eomes, CD94/NKG2A, T-bet, and CD16. In addition putative CD34 progenitors of RORγt ILCs were identified. In the second trimester of fetal development, analysis of mature fetal NK cell subsets revealed that stage 4 and stage 5 NK cells differed in frequency in fetal organs, and the highest NK cell frequency was found in the fetal liver and lung. The vast majority of fetal NK cells were NKG2A, and fetal lung NK cells also frequently expressed killercell immunoglobulin-like receptors (KIR). Interestingly, while NKG2A educated fetal NK cells similar to adult NK cells, KIR expression on fetal NK cells was linked to hyporesponsiveness, thus contrasting education of NK cells after birth. Nevertheless, fetal NK cells were highly responsive to cytokines, as well as to antibody-coated target cells, suggesting they may take part in fetal immune responses against in utero infections, while remaining tolerant to maternal cells crossing the placenta. While it is established that NK cells in adults are educated via inhibitory KIRs, it is not known how activating KIRs such as KIR2DS1 affects NK cells. By combining antibodies against four inhibitory KIRs, NKG2A, and KIR2DS1, we were able to interrogate the regulation of NK cells by KIR2DS1. We found that KIR2DS1 singlepositive NK cells exist in vivo, and that the presence of the ligand for KIR2DS1, HLAC2, resulted in hyporesponsiveness of such NK cells, thus ensuring self-tolerance. Our findings represent the first identification of NK cell education via activating KIR. The human NK cell response to viral infections is not well understood. To this end we employed the live attenuated yellow fever vaccine 17D as an in vivo model of an acute viral infection. Our results show that the vaccine primed NK cells, and that less differentiated CD57 NK cells dominated the response, which peaked at day 6-10 post vaccination. Moreover, KIR expression on NK cells did not affect their response to the vaccine, indicating that NK cells expressing self-KIR and non-self KIR contributed equally to the NK cell response to the vaccination. LIST OF PUBLICATIONS This thesis is based on the following publications and manuscripts, which are referred to in the text by their Roman numerals: I. High-resolution mapping of transcription factor expression reveals distinct developmental pathways of human fetal innate lymphoid cells. Martin A. Ivarsson, Nicole Marquardt, Liyen Loh, Jeff E. Mold, Magnus Westgren, Erik Sundström, Elisabet Åkesson, Åke Seiger, Jenny Mjösberg, Niklas K. Björkström, Douglas F. Nixon, and Jakob Michaëlsson Manuscript II. Differentiation and functional regulation of human fetal NK cells. Martin A. Ivarsson, Liyen Loh, Nicole Marquardt, Eliisa Kekäläinen, Lena Berglin, Niklas K. Björkström, Magnus Westgren, Douglas F. Nixon, and Jakob Michaëlsson Journal of Clinical Investigation 2013;123(9):3889–3901 III. Education of human natural killer cells by activating killer cell immunoglobulin-like receptors. Cyril Fauriat, Martin A. Ivarsson, Hans-Gustaf Ljunggren, Karl-Johan Malmberg, and Jakob Michaëlsson Blood 2010 115: 1166-1174 IV. The human NK cell response to yellow fever virus is primarily governed by NK cell differentiation independently of NK cell education. Nicole Marquardt*, Martin A. Ivarsson*, Kim Blom, Veronica D. Gonzalez, Monika Braun, Karolin Falconer, Rasmus Gustafsson, Anna Fogdell-Hahn, Johan K. Sandberg, and Jakob Michaëlsson Manuscript * Authors contributed equally Paper II: © 2013 Journal of Clinical Investigation Paper III: © 2010 The American Society of Hematology LIST OF ADDITIONAL RELEVANT PUBLICATIONS SI. Invariant natural killer T cells developing in the human fetus accumulate and mature in the small intestine Liyen Loh, Martin A. Ivarsson, Jakob Michaëlsson, Johan K. Sandberg, and Douglas F Nixon Mucosal Immunology 2014 (Accepted for publication Feb 4th 2014) SII. Expression patterns of NKG2A, KIR, and CD57 define a process of CD56 NK-cell differentiation uncoupled from NK-cell education. Niklas K. Björkström, Peggy Riese, Frank Heuts, Sandra Andersson, Cyril Fauriat, Martin A. Ivarsson, Andreas T. Björklund, Malin FlodströmTullberg, Jakob Michaëlsson, Martin E. Rottenberg, Carlos A. Guzmán, Hans-Gustaf Ljunggren, and Karl-Johan Malmberg Blood 2010 116: 3853-3864 SIII. Temporal dynamics of the primary human T cell response to yellow fever virus 17D as it matures from an effectorto a memory-type response. Kim Blom, Monika Braun, Martin A. Ivarsson, Veronica D. Gonzalez, Karolin Falconer, Markus Moll, Hans-Gustaf Ljunggren, Jakob Michaëlsson, and Johan K. Sandberg Journal of Immunology 2013; 190:2150-2158 SIV. NK cell responses to cytomegalovirus infection lead to stable imprints in the human KIR repertoire and involve activating KIRs. Vivien Béziat, Lisa L. Liu, Jenny-Ann Malmberg, Martin A. Ivarsson, Ebba Sohlberg, Andreas T. Björklund, Christelle Retière, Eva SverremarkEkström, James Traherne, Per Ljungman, Marie Schaffer, David A. Price, John Trowsdale, Jakob Michaëlsson, Hans-Gustaf Ljunggren, and Karl-Johan Malmberg Blood 2013 121: 2678-2688 SV. Activating killer immunoglobulin-like receptors in health and disease. Martin A. Ivarsson, Jakob Michaëlsson, and Cyril Fauriat Invited review. Submitted to Frontiers in Immunology TABLE OF CONTENTS 1! Introduction . 1! 1.1! Flow Cytometry.1! 1.2! NK cell biology . 2! 1.2. 1! NK cell receptors.3! 1.2. 2! NK cell education.7! 1.2. 3! Models of NK cell development . 8! 1.2. 4! Innate lymphoid cells . 9! 1.2. 5! NK cell differentiation . 12! 1.2. 6! NK cells in virus infections.14! 1.3! Methodological development.17! 2! Aims of this thesis . 18! 3! Results and discussion.19! 3.1! Development of fetal NK cells and other ILCs . 19! 3.2! Fetal NK cell differentiation and functional regulation.24! 3.3! Education of NK cells via activating KIR . 29! 3.4! NK cell responses in a model of acute viral infection . 33! 4! Concluding remarks . 37! 5! Acknowledgements . 39! 6! References . 42! LIST OF ABBREVIATIONS ADCC Antibody-dependent cellular cytotoxicity