"Medicine, Faculty of"@en . "Medical Genetics, Department of"@en . "DSpace"@en . "UBCV"@en . "Veinotte, Linnea Lora"@en . "2011-02-10T20:32:57Z"@en . "2006"@en . "Doctor of Philosophy - PhD"@en . "University of British Columbia"@en . "Natural killer (NK) cells are the major lymphocytes of the innate immune system, but their developmental pathway is not fully defined. It is commonly assumed that all NK cells develop in the bone marrow. In this thesis, I describe a novel thymus-dependent pathway of NK cell development that is specific to those in the thymus and lymph nodes. Microarray analysis revealed TCR\u00CE\u00B3 mRNA expression in NK cells. Genomic and RT-PCR showed that some NK cells have rearranged TCR\u00CE\u00B3 genes while TCR\u00CE\u00B2 and TCR\u00CE\u00B4 genes are in germline order. NK cells with rearranged TCR\u00CE\u00B3 ( Tcr\u00CE\u00B3\u00E2\u0081\u00BA NK cells) were absent in nude mice indicating that they are thymus-dependent. Approximately half of thymus NK cells have rearranged TCR\u00CE\u00B3 genes and in vitro cultures of immature thymocytes (double negative (DN)1 and DN2 progenitors) produced Tcr\u00CE\u00B3\u00E2\u0081\u00BA NK cells, strongly suggesting that these are the thymus-dependent NK cell progenitors in vivo. Thymus NK cells are CD94/NKG2[sup hi] Ly49[sup lo] Mac-1[sup lo] IL-7R\u00CE\u00B1[sup hi] and they have normal cytotoxicity levels but reduced IFN\u00CE\u00B3 production. By using TCR\u00CE\u00B3 gene rearrangement as a marker of thymus-dependent NK cells, we showed that they are also present in lymph nodes (LNs) but in no other tissues tested. NK progenitors similar to immature thymocytes were found in LNs and LN Tcr\u00CE\u00B3\u00E2\u0081\u00BA NK cells and LN progenitors were also absent in nude mice. In vitro cultures and preliminary in vivo studies suggest that the NK progenitors in the LN give rise to mature NK cells. The results suggest that immature thymocytes migrate to LNs and differentiate into NK cells. It is likely that the thymus-dependent NK cells play a special role in the immune response since their phenotype is unique. Finally, this study suggests that multiple pathways of NK cell commitment exist in multiple tissues. The differences in tissue environment may influence the phenotype and function of NK cells, resulting in multiple subsets of NK cells throughout the body."@en . "https://circle.library.ubc.ca/rest/handle/2429/31179?expand=metadata"@en . "N O V E L P A T H W A Y O F T H Y M U S - D E P E N D E N T N K C E L L D E V E L O P M E N T by L I N N E A L O R A V E I N O T T E B .Sc .H , Acadia University, 2001 A THESIS S U B M I T T E D I N P A R T I A L F U L F I L L M E N T O F T H E R E Q U I R E M E N T S F O R T H E D E G R E E O F D O C T O R OF P H I L O S O P H Y in T H E F A C U L T Y OF G R A D U A T E S T U D I E S (Genetics) T H E U N I V E R S I T Y OF B R I T I S H C O L U M B I A December 2006 \u00C2\u00A9 Linnea Lora Veinotte, 2006 A B S T R A C T Natural killer ( N K ) cells are the major lymphocytes of the innate immune system, but their developmental pathway is not fully defined. It is commonly assumed that all N K cells develop in the bone marrow. In this thesis, I describe a novel thymus-dependent pathway of N K cell development that is specific to those in the thymus and lymph nodes. Microarray analysis revealed T C R y m R N A expression in N K cells. Genomic and R T - P C R showed that some N K cells have rearranged T C R y genes while T C R p and T C R 8 genes are in germline order. N K cells with rearranged T C R y (Tcry N K cells) were absent in nude mice indicating that they are -thymus-dependent. Approximately half of thymus N K cells have rearranged T C R y genes and in vitro cultures of immature thymocytes (double negative (DN)1 and D N 2 progenitors) produced Tcry N K cells, strongly suggesting that these are the thymus-dependent N K cell-progenitors in vivo. Thymus N K cells are C D 9 4 / N K G 2 h i L y 4 9 l 0 M a c - 1 1 0 I L - 7 R a h i and they have normal cytotoxicity levels but reduced IFNy production. B y using T C R y gene rearrangement as a marker of thymus-dependent N K cells, we showed that they are also present in lymph nodes (LNs) but in no other tissues tested. N K progenitors similar to immature thymocytes were found in L N s and L N Tcry N K cells and L N progenitors were also absent in nude mice. In vitro cultures and preliminary in vivo studies suggest that the N K progenitors in the L N give rise to mature N K cells. The results suggest that immature thymocytes migrate to L N s and differentiate into N K cells. It is likely that the thymus-dependent N K cells play a special role in the immune response since their phenotype is unique. Finally, this study suggests that multiple pathways of N K cell commitment exist in multiple tissues. The differences in tissue environment may influence the phenotype and function of N K cells, resulting in multiple subsets of N K cells throughout the body. i i T A B L E O F C O N T E N T S Abstract ii Table of contents iii List of tables vi List of figures vii List of abbreviations ix Acknowledgements xi Chapter 1. Introduction 1 1.1. Introduction to N K cells 1 1.2. Hematopoietic lineage commitment 5 1.3. Commitment to the N K cell lineage 7 1.4. N K cell development 10 1.4.1. Stages of N K cell Development: from N K P to the mature N K cell 11 1.4.2. Factors involved in N K P production: Cytokines.. . 13 1.4.3. Factors involved in N K P production: Transcription factors 14 1.5. N K cell maturation 16 1.5.1. M H C Class I Receptor acquisition 16 1.5.2. Factors involved in N K cell maturation 18 1.6. T cells and development 24 1.7. T C R rearrangement: V(D)J recombination 27 1.8. T C R y locus and rearrangement patterns 29 1.9. Thesis objectives and hypotheses 33 Chapter 2. Materials and methods 35 2.1. Mice 35 2.2. Antibodies 35 2.3. Microarray Sample Preparation and Analysis 37 2.4. Measuring D N A 37 2.5. Genomic P C R 37 2.6. Southern Blot 39 2.7. R T - P C R 40 2.8. Sequencing of P C R products 42 2.9. Tissue culture 42 2.9.1. L-cells 42 2.9.2. OP9 cells '42 2.9.3. L A K cells 42 2.9.4. Thymus or L N D N progenitor culture and B M N K P progenitor culture 42 2.10. Ce l l preparation 43 i i i 2.11. Staining and F A C S sorting or analysis of cells 44 2.12. IFNy production assay 44 2.13. Cytotoxicity assay 45 2.14. L N D N cell transplantation 45 2.14.1. Intraperitoneal injection 45 2.14.2. Intravenous inj ection 45 2.15. Statistics 46 Chapter 3: Identification of a novel pathway of N K cell development that is thymus-dependent and includes T C R gene rearrangement 47 3.1. Introduction 47 3.2. Results 48 3.2.1. Microarray analysis reveals expression of T C R y gene in N K cells 48 3.2.2. T C R y genes are rearranged and expressed in N K cells 53 3.2.3. Specificity of rearrangement 54 3.2.4. T C R y gene rearrangements in N K cells represent unselected, random recombination 55 3.2.5. Tcry+ N K cells represent a small population of total splenic N K cells 56 3.2.6. N K cells have a germline TCR(3 locus and may have initiated T C R 8 rearrangement 59 3.2.7. T C R y gene rearrangement in N K cells is not due to R A G expression in B M C L P s 60 3.2.8. The thymus is required for the development of 7 c r y + N K cells 61 3.3. Discussion 63 Chapter 4. The thymus-dependent developmental pathway generates unique subsets of N K cells in thymus and lymph node 71 4.1. Introduction 71 4.2. Results 72 4.2.1. DN1 and D N 2 thymocytes differentiate into N K cells 72 4.2.2. DN2-derived N K cells are TCRy 75 4.2.3. Thymus N K cells are phenotypically different from N K cells of other tissues 75 4.2.4. T C R y gene rearrangement in L N N K cells suggests a link with DN-derived thymus N K cells 80 4.2.5. DN1 and pre-DN2 cells in L N s give rise to N K cells in culture 81 4.2.6. The D N cells and Tcry N K cells in the L N are thymus-dependent 84 iv 4.2.7. Preliminary results suggest that L N D N progenitors give rise to N K cells in vivo 84 4.2.8. Thymus N K and L N N K cells produce lower levels of IFNy upon stimulation 86 4.2.9. Thymus N K cells have normal levels of cytotoxicity 87 4.3. Discussion 88 Chapter 5. General discussion 94 5.1. Thymus-dependent N K developmental pathway 96 5.2. Mouse N K cell subsets 98 5.3. Human N K cell subsets and L N pathway of development 99 5.4. Thymus N K cells 100 5.5. L N N K cells 102 5.6. T cell vs. N K cell lineage commitment 103 5.6.1. Notch signals 104 5.6.2. E proteins vs. Id2 105 5.7. Medical relevance 106 5.8. Final conclusions 107 Bibliography 109 L I S T O F T A B L E S Table 1.1. Genetic mutations affecting the maturation of N K cells 23 Table 2.1. List o f antibodies used in studies 36 Table 2.2. List of primers used in studies 41 Table 4.1. Global F A C S analysis of N K cells from the thymus, L N , B M , spleen, liver, and lung 77 Table 4.2. L N D N l / p r e - D N 2 cells produce small numbers of N K cells following one i.p. and one i.v. transplantation 86 vi L I S T O F F I G U R E S Figure 1.1. N K cell strategies for detecting and ki l l ing target cells 2 Figure 1.2. Progenitors with N K cell lineage potential 9 Figure 1.3. Bipotent T / N K progenitors 10 Figure 1.4. Stages of N K cell maturation 12 Figure 1.5. Stages of T cell development 26 Figure 1.6. The process o f V(D)J recombination 29 Figure 2.1. A . The murine T C R y locus 39 Figure 3.1. Microarray data analysis of differentially expressed genes between adult N K and neonatal N K cell samples 49 Figure 3.2. Detection of T C R y gene expression in N K cells by microarray analysis 52 Figure 3.3. T C R y gene rearrangement and expression in N K cells 54 Figure 3.4. Sequences of productive and non-productive T C R y gene rearrangements 56 Figure 3.5. L o w frequency of N K cells with rearranged T C R y genes 58 Figure 3.6. Tcry N K cells are not due to contamination of T cells 59 Figure 3.7. TCR(3 and T C R 5 gene rearrangements in N K cells. 60 Figure 3.8. Lack of T C R y gene rearrangement in B6 mouse B cells 61 Figure 3.9. Lack of T C R y gene rearrangement in nude mouse N K cells and high T C R y gene rearrangement in thymus N K cells 62 Figure 3.10. Thymus N K cells have high levels of T C R y gene rearrangement 63 Figure 4.1. Scheme of thymus (or L N ) D N progenitor culture 72 Figure 4.2. Thymus D N 1 and D N 2 progenitors have the potential to give rise to N K cells during in vitro cultures 74 Figure 4.3. Thymus D N 2 derived N K cells have rearranged T C R y genes 75 v i i Figure 4.4. Thymus N K cells appear 'immature' (Mac- l ' \u00C2\u00B0 IL -7Ra h l ) compared to other tissue N K cells , 78 Figure 4.5. Thymus N K cells havethe highest levels of N K G 2 A / C / E and CD94 expression and average 2B4 expression 79 Figure 4.6. Thymus N K cells have the lowest levels of L y 4 9 A , G , D , and C/I expression 79 Figure 4.7. L N N K cells have the highest percentage of Tcry N K cells other than thymus N K cells 81 Figure 4.8. D N 1 and pre-DN2 progenitors are present in the lymph node and they possess N K cell potential in vitro 82 Figure 4.9. D N progenitors from IL-15\"7\" mouse L N s still show N K cell potential in vitro 83 Figure 4.10. Nude mouse L N s have lower levels of DN1 and pre-DN2 progenitors than wi ld type L N s and nude L N N K cells do not have T C R y gene rearrangement 84 Figure 4.11. L N N K cells and thymus N K cells produce lower levels of IFNy after IL-12 and IL-18 stimulation than spleen N K cells 87 Figure 4.12. Spleen and thymus N K cells have similar levels of cytotoxicity 88 Figure 5.1. Revised model of N K cell development in the mouse 95 v i i i LIST OF ABBREVIATIONS: ADCC: antibody-dependent cell-mediated cytotoxicity BM: bone marrow CLP: common lymphoid progenitor CMP: common myeloid progenitor DC: dendritic cell DEC: dendritic epidermal T cell DMEM: Dulbecco's modified eagle's medium DN: double negative dNTP: Deoxyribonucleotide triphosphate DP: double positive ELP: early lymphoid precursor ETP: early thymus progenitor FBS: fetal bovine serum FL: fetal liver F l t 3 L : Flt-3 ligand FTOC: fetal thymic organ culture HEV: high endothelial venules HSC: hematopoietic stem cells i.p.: intraperitoneally i . v . : intravenously Id: inhibitors of D N A binding IFNy: interferon y KIR: killer inhibitory receptor LAK: lymphokine activated killer cell LN: lymph node L T P R : lymphotoxin beta receptor MCMV: mouse cytomegalovirus 2ME: 2-mercaptoethanol MEM: Min imum essential medium eagle MHC: major histocompatibility complex mNK: mature N K cell MPP: multipotent progenitor NIK: N F - K B inducing kinase NK: natural killer NKP: N K precursor PCR: polymerase chain reaction P/S: penicillin streptomycin RAET1: retinoic acid early transcripts RT-PCR: reverse transcription polymerase chain reaction RSS: recombination signal sequence ix SIPi: sphingosine 1-phosphate type 1 receptor SAGE: serial analysis of gene expression SCF: stem cell factor SP: single positive TCR: T cell receptor Tcry NK: N K cell with T C R y gene rearrangement T d T : terminal deoxyribonucleotide transferase T E C : thymic epithelial cell T-IEL: T intraepithelial lymphocyte TNF: tumour necrosis factor TNKP: bipotent T / N K cell precursor TRAF6: tumor necrosis factor receptor associated factor 6 T R A I L : TNF-related apoptosis-inducing ligand V D U P 1 : vitamin D3 upregulated protein 1 yc : common y chain A C K N O W L E D G E M E N T S Firstly, thank you to my supervisor Dr. Fumio Takei. M y studies were greatly enhanced by having you as a supervisor. I feel very lucky to have had you as my supervisor. Y o u have been extremely helpful and always interested and involved in my research. Y o u have put so much time into working with me on various projects and have helped so much with writing the thesis. Y o u have always been enthusiastic and encouraging and I have learned so much from you. Thank you! Thank you to my graduate committee members, Drs. Dixie Mager, Rob Kay, and Ke l ly McNagny, for their advice and guidance throughout my studies. Thank you to all of my lab members, past and present, for their friendship and help. Special thanks to Moto i Maeda for teaching me many laboratory techniques when I first started working in the lab. To Nooshin Tabatabaei, we have gone through classes, research, travelling to conferences together, etc. at the same time. It was nice to have you around to talk with. Thanks to Evette Haddad, Emi ly Mace, Erica Wilson, Eva Backstrom, Valeria Alcon , Lisa Dreolini, Matt MacLeod, Reza Marwali , Carmine Carpenito, for teaching me how to do certain techniques, for answering questions, helping me through problems, and for making the lab such a great place to be! Y o u are what made my grad studies so much fun! Thank you to Nastaran Mohammadi and Christine Parachoniak for doing the cloning and sequencing of the Vy-Cy R T - P C R products. Thanks to Chelsea Greenwood for helping me work on single cell R T - P C R . A n d finally thanks to T i m Hal im for working on the L N D N progenitor in vitro cultures and for working together with me on the transplantation studies, especially for getting all o f the peripheral L N s and learning how to do intravenous injections. A very big thank you to the F A C S sorting staff, Lindsey, Gayle, Jaime, Cam, and Rick. Y o u were always a big help. Thank you to K a i Lucke for help with i.v. injections and supplying mice. Thank you to the Marcel Bal ly lab for nude mice and to the J A F and A R C staff. I would like to acknowledge my funding from the Michael Smith Foundation for Health Research for both junior and senior trainee awards and from U B C U G F awards. Thank you to my family, especially my parents, for their support and encouragement. A n d finally, thank you most of all to my husband, Matt, to whom I owe very much. Thank you for giving up so much to come to Vancouver for me to do my PhD. Thank you for your constant support and understanding throughout the five years. I really needed your encouragement to keep working hard and to strive to always do my best. Y o u always make me want to reach higher. Y o u mean the world to me. Thank you! x i 1 I N T R O D U C T I O N 1.1. Introduction to N K cells Natural killer ( N K ) cells are lymphocytes that belong to the innate immune system. They have a large granular morphology and can mediate cellular cytotoxicity as well as release chemokines and inflammatory cytokines. N K cells become activated by cytokines or upon encountering target cells that express ligands for N K cell receptors. N K cells also play a role in activating adaptive immunity and participate in cross talk with dendritic cells (DCs). Upon activation, N K cells can directly lyse target cells by exocytosis of perforin and granzymes 1 . They also secrete cytokines such as interferon y (IFNy) and tumor necrosis factor (TNF) and 2 3 chemokines ' . Elimination of cells via FasL and TNF-related apoptosis-inducing ligand (TRAIL) pathways also occurs in N K cells 4 ' 5 . These ligands are mediators of caspase-dependent apoptosis in target cells expressing the corresponding receptors Fas and T R A I L - R 6 . N K cells also express C D 1 6 (FcyRIII) which binds IgG antibody coated cells and mediates antibody-dependent cell-mediated cytotoxicity ( A D C C ) . N K cells have a recognition system that is encoded by non-rearranged genes and it involves multiple types of receptors rather than one dominant receptor. These receptors can trigger N K cells individually or in combination, depending on each target cell it encounters. N K cells use various strategies to recognize target cells including recognition of pathogen-encoded molecules, induced self recognition, and the most classically described missing-self recognition (Fig. 1.1). 1 Inhibitory receptor MHC class I molecule Activating receptor Stimulatory ligand Transformation or infection Missing-self recognition Induced-self recognition or pathogen molecule recognition Figure 1.1. N K cell strategies for detecting and killing target cells. When an N K cell encounters a healthy \" s e l f cell (top), the inhibitory signals outweigh any activating signals and the N K cell does not k i l l . If an N K cell encounters a cell that has downregulated its M H C class I expression (left), inhibitory signals are absent while activating signals still occur. Therefore, the activating signals lead to target cell kil l ing. If an N K cell encounters a target cell that is expressing induced stress molecules or pathogen molecules (right), inhibitory signals w i l l still occur but more activating signals w i l l be received upon recognition of the stress or pathogens molecules and the net signal w i l l be activating, resulting in N K cell k i l l ing. Revised from Raulet 1 . As part of the well characterized missing self recognition strategy, N K cells express receptors that are specific for major histocompatibility complex ( M H C ) class I molecules (reviewed in 8 ) . M H C class I molecules are expressed on the surface of all healthy nucleated cells but are often downregulated as a consequence of infection, mutation, or transformation. Most of the M H C class I-specific receptors transduce an inhibitory signal. Ligation of these receptors delivers a dominant-negative signal to N K cells that prevents natural ki l l ing of self cells. It is 2 the balance of inhibitory and stimulatory signaling that determines the outcome of NK-target cell interactions such that i f a N K cell encounters a healthy cell, its inhibitory signals received wi l l override activating signals. If, on the other hand, an N K cell encounters an unhealthy cell ( M H C class I l o /\"), an inhibitory signal is not received and the activating signals w i l l result in cytolytic function. Adult mice express two families of M H C class I-specific receptors, Ly49 and C D 9 4 / N K G 2 , whereas fetal and neonatal N K cells express only the latter. Human N K cells express C D 9 4 / N K G 2 receptors and the killer inhibitory receptor (KIR) family. Another recognition strategy is detection of pathogen-encoded molecules. N K cells express the L y 4 9 H receptor which is stimulatory and binds to an M H C class I-like protein, m l 57, which is a mouse cytomegalovirus ( M C M V ) encoded protein 9. L y 4 9 H enables N K cells to undergo considerable proliferation and to limit early stage M C M V infection 1 0 . Other N K cell receptors that are specific for pathogens are N K p 4 6 and N K p 4 4 which bind the influenza virus hemagglutinin 1 1. N K cell receptors can also recognize induced self signals, (stress signals) that are upregulated during infection or on tumor cells. N K p 4 6 , N K p 4 4 , and N K p 3 0 receptors likely recognize ligands on tumor cells but the ligand identities are not yet known 1 2 . The best characterized receptor is N K G 2 D , which is expressed by almost all N K cells and plays a key role in immune responses, especially as an activating receptor that triggers N K cells to respond against tumors (reviewed by 6 ) . N K G 2 D ligands are stress signals encoded by the host genome. These include a diverse family of ligands, retinoic acid early transcripts ( R A E T 1 ) that are shared between mice and humans. Family members include Rae-1, H-60, M u l t l and U L B P . Although the expression patterns of N K G 2 D ligands are diverse and complex, the general 3 pattern is that they are expressed poorly on healthy cells but are upregulated on infected or tumor cells 6 . N K cells express multiple activating and inhibitory receptors that recognize M H C class I as well as many n o n - M H C class I ligands such as pathogen encoded molecules and self-induced ligands. It is ultimately the balance of signals that determines the functional outcome of the N K celhtarget cell interaction. N K cells exhibit complex repertoires of receptors because there is random coexpression of many possible combinations of N K cell receptors. This process of variable receptor gene expression results in a repertoire of N K cells that can detect multiple changes in cells. This somewhat resembles T cell and B cell lymphocytes but N K cells differ from T and B cells since they can k i l l target cells without prior sensitization, their N K cell receptor loci do not require gene rearrangement and their repertoire is much smaller. Although many of the activating/inhibitory receptors and their ligands have been discovered, the biological relevance of these molecules in host defense and the interactions between N K cells and other immune system cells is less well characterized. Through multiple pathways (cytokine secretion, direct ki l l ing, interaction with other cells), N K cells have been shown to be involved in combating viral infections (cytomegalovirus, sendai virus, influenza virus, H I V , and ebola virus), parasites (P. falciparum which causes malaria, P. berghei, and T. cruzi), and bacteria (Shingella flexneri, M. tuberculosis). These roles are reviewed by Lodoen, et al. . N K cells also interact with DCs . They can reciprocally activate one-another by unknown receptor-ligand pairs and by cytokines (reviewed in 1 4 ) . D C s secrete IL-2, IL-12, IL-18, and IFNa/p which induces cytotoxicity, proliferation, CD69 expression and IFNy production in N K cells. N K cells, on the other hand, activate or induce maturation of D C s through cell-4 contact and TNFcc 1 4 . One example of N K cel l -DC cross talk is during M C M V infection. DCs induce N K cell expansion by release of IL-12 and IL-18 and through L y 4 9 H 1 5 . Also , the presence of L y 4 9 H + N K cells results in the maintenance of D C s in the spleen during acute M C M V infection 1 5 . Since N K cells play such an important role in the immune system, it is very important to understand all aspects of N K cell biology, including their lineage commitment and maturation pathways as well as factors that affect their final phenotype and function. In this study, we have identified a new pathway o f N K cell lineage commitment that is thymus-dependent and gives rise to N K cells that reside in the thymus and L N s . This thymus-dependent pathway influences the N K cell phenotype and function. Therefore, not all N K cells develop via the B M pathway that is currently accepted in the literature as the sole location of N K cell development. We w i l l therefore review the current understanding of N K cell precursors and their development in the B M as well as their maturation steps and factors involved in this process. T cell development and V(D)J recombination of T cell receptors (TCRs) wi l l also be reviewed since this step of development occurs in the thymus-dependent pathway of N K cell development. 1.2. Hematopoietic lineage commitment A l l blood cells develop from hematopoietic stem cells (HSCs). These cells are multipotent and are capable of self renewal 1 6 ' 1 1 . Fetal hematopoiesis occurs in the liver and it switches to the B M in the adult. The first step in hematopoiesis is typically described as the division into common myeloid progenitors ( C M P s ) 1 8 and common lymphoid progenitors ( C L P s ) 1 9 . This 5 model of hematopoiesis assumes that cells consistently reach branch points in the same order and that the timing of the developmental choices is fixed such that when a cell reaches a branching point, it cannot progress further without choosing one branch or the other. It also assumes that the choice is binary so that i f a cell makes a positive choice for the one branch, it automatically makes a negative choice for the other branch. If developmental choices are considered in terms of gene regulatory mechanisms though there is no longer a need for cells to encounter binary choices but rather cells pass through overlapping 'windows of opportunity' to 20 * * give rise to certain cell types . Lineage opportunities remain open as long as the cells express essential enabling factors. B y turning on or off other subsets of transcription factors, a precursor cell can switch its cell type to more than one other choice. The exact timing and order of the choices may not always be the same and one cell fate can be blocked without commitment to another. Since there is diversity in gene expression of progenitors within the same stage, the lineage decision of each cell can occur somewhat randomly due to the fluctuations in gene expression. The end point could be reached via more than one pathway 2 0. Oligonucleotide array analysis of HSCs , M P P s (multipotent progenitors), C L P s , and C M P s revealed that H S C s express non-hematopoietic genes as well as many hematopoietic genes in a 21 lineage promiscuous manner . M P P s express both myeloid and lymphoid genes and C L P s express genes for T, B , and N K cells before they're committed to a lineage. Interestingly, C L P s express the germline transcripts of many T C R and B C R s , including germline T C R y and TCRp\ C L P s also express R A G - 1 . Lineage commitment is a result of inactivation of non-lineage specific genes and the retention or activation of enabling factors for the lineage. Therefore cells can express genes specific for a certain lineage before they are committed to that lineage and cells can express genes of other lineages even though they wi l l differentiate 6 into another lineage. This stresses the separation of the terms \"specification\" and \"commitment\" 2 0. 1.3. Commitment to the N K cell lineage It is the common assumption that all N K cells develop in the B M of adult mice. N K cells and B cells are both dependent on the B M , because B M ablation by 8 9 S R 2 2 or oestradiol 2 3 results in N K cell deficiencies. This defect is also seen in osteopetrotic animals which have a defective and much smaller B M compartment due to an osteoclast deficiency 2 4 . Commitment to the N K cell lineage requires progressive restriction of lineage potential from multipotent HSCs to oligopotent precursors to unipotent committed N K cell precursors which no longer have the ability to become any other cell type. Several oligopotent precursors have been described that retain N K cell potential (Fig. 1.2). The two main populations in the adult B M are lymphocyte precursors which posses B cell , T cell, and N K cell potential. Early lymphoid precursors (ELPs) are Lin 'c-kit F l t3L and common lymphoid progenitors (CLPs) are Lin\"c-kit '\u00C2\u00B0IL-7Ra + . It is unclear whether these are obligate intermediates for N K cells to arise. In c-kit-deficient mice, that lack all C L P s , N K P and mature N K cell numbers are normal ' . On the other hand, several studies suggest that N K cells do arise from C L P s or ELPs . Two studies using reporter genes for V(D)J recombinase 2 7 and R A G 1 2 8 show that N K cells arise from progenitors which express R A G 1 or V(D)J recombinase. One study showed that V(D)J recombinase is active at the C L P stage and its activity or evidence of its activity is detectable in B , T, N K , and D C lineages 2 7. This does not necessarily mean that N K cells or others arise only from C L P s because V(D)J recombinase could be active in other progenitors as well. The 7 recombinase expression at the C L P stage was shown to be controlled by the B cell specific enhancer, Erag and approximately 5% of N K cells do have IgH rearrangements2 7. Therefore, perhaps this I g H + portion does arise from C L P s in which the B cell specific Erag enhancer controls recombination. In the other study, R A G 1 expression was detectable in a progenitor upstream of the C L P stage, the E L P progenitors 2 8. R A G expression (or evidence of previous expression) was also detectable in mature N K cells in this study. This earlier activity in E L P s suggests that N K cells can arise from earlier progenitors as well as C L P s . Kouro, et al. examined potentials in B M precursors. N K cells can develop from early Lin\"c-kit h l cells (like ELPs) but also from more mature Lin\"c-kit'\u00C2\u00B0(Flt3L +) cells (which include CLPs) (Fig. 1.2). It seems that a significant proportion of BM-derived N K cells do arise from a Lin\"c-kit'\u00C2\u00B0 subset, because when mice are treated with estrogen, the precursors that produce N K and B cells are diminished from the Lin~c-kit'\u00C2\u00B0 fraction. When the remaining Lin\"c-kit'\u00C2\u00B0 cells are cultured, the B and N K cell numbers are reduced. Most of the progenitors for B and N K cells within the Lin\"c-kit'\u00C2\u00B0 subset are in separate populations though, because upon single cell assays most wells produced either B cells (1 of 6-10) or N K cells (1 of 19-25) but a bipotent B / N K cell precursor was rarer (1 of 64-96). The Lin\"c-kit'\u00C2\u00B0 population is heterogeneous with N K cell precursors and B cell precursors having substantial differences. For example, all B cell precursors are I L - 7 R a + while only half of N K cell precursors are. Al so , N K cell precursors are more resistant to (5-estradiol hormone treatment than B cel ls 2 9 . A committed N K cell precursor (NKP) was isolated in the adult B M 3 0 . These cells are L i n \" C D 1 2 2 + N K l . l \" D X 5 \" . CD122 is II-2 / IL- l 5RP and this is used by two cytokines, IL-2 and IL-15 , both dependent on signaling through a third subunit, known as common y chain (yc) . These cells lack mature N K cell markers and are not functionally developed. N K P s produce mature N K cells via an in vitro culture system and they lack all other cell potential. 8 l _ j n B o n e m a r r o w Sca-1hi c-kithl Lin- Lin-Figure 1.2. Progenitors with N K cell lineage potential. Multiple progenitors in the bone marrow and thymus have been shown to possess N K cell lineage potential. NK cells may share a closer developmental relationship with T cells in fetal mice and B cells in adult mice. Numerous studies have shown that fetal T cell precursors possess N K lineage potential (Fig . l .3). A bipotent T/NK cell precursor ( T N K P ) is present in the fetal liver, spleen, and blood. The earliest T N K P is B220 l o c-ki t + CD19- and is found in the fetal liver ( F L ) 3 3 . They reconstitute the T and N K cell compartments upon transplantation into mice lacking T, B and NK cells. These cells have been shown to be truly bipotent, because a single cell gives rise to both T and N K cells in a fetal thymic organ culture (FTOC) . This bipotent population represents 70% of the cells that seed the thymus 3 3 . The earliest prethymic T cell progenitors in the fetal b lood 3 4 and the first cells to colonize the fetal thymus 3 5 both have T, NK, and dendritic cell potential. O f 40 cells examined from the fetal thymic anlage, 7 cells gave rise to 9 T cells and all o f these 7 cells also gave rise to N K cells. Four o f these cells produced dendritic cells as w e l l 3 5 . Subpopulations of immature CD4\"CD8\" (DN) thymocytes in adult mice also possess N K cell potential. N K cell potential of DN1 thymocytes was also shown by Balciunaite et al?6. Adult DN1 c-kit + cells, as well as D N 2 cells, upon culturing on OP9 cells developed into functional N K cells. Limiting dilution analysis showed that 1 in 15 DN1 cells and 1 in 7 D N 2 cells developed into N K cells 3 6 . Taken together, these studies suggest a link between T cells and N K cells. This notion has further been supported by a transgenic mouse model of human C D 3 e 3 7 which displays a block in development of N K and T cells but not B 38 cells. Mice transgenic for FceRIy exhibit a similar phenotype . F e t a l l i v e r (TNKP) B22QI0 c-kit+ CD19-F e t a l b l o o d Lin-e-kit* IL-7Fr F e t a l t h y m u s CD44+ CD25-FcR-CD44* CD25-IL-2RP* Figure 1.3. Bipotent T / N K cell progenitors. T N K P cells are present in the fetal liver, blood, and thymus. Figure is revised from that designed by Tabatabaei . 1.4. N K cell development Much about the N K cell commitment pathway was first determined from in vitro cultures. Nearly a decade ago, an in vitro culture system was used to demonstrate that there are two major stages in N K cell development. The first stage involves acquisition of C D 122, which marks commitment to the N K cell lineage and allows the cells to become responsive to IL-15 which is crucial for the second stage of N K cell maturation. When Will iams et al.40 cultured multipotent progenitors with IL-15 alone there was no cell expansion whereas when the cells 10 were cultured in IL-6, IL-7, S C F , and flt3L substantial expansion occurred but little to no N K cells were produced. But i f these cells were first cultured in the cytokine cocktail (where they acquired C D 122 and became IL-15-responsive) and then switched to a culture with IL-15 alone, cells expanded well and almost all became N K 1 . 1 + and had lytic capabilities. There is also a critical requirement for stromal cells in N K cell development. Cells cannot express Ly49 receptors without stroma cells in the culture 4 1. 1.4.1. Stages of N K cell Development: from NKP to the mature N K cell Recently K i m et al.10 defined a developmental model of N K cell maturation that divides their in vivo development in the B M into five stages (Fig. 1.4). These stages are defined by the cells' functional characteristics and expression of molecules which may not provide specific functional significance but work for defining specific stages of maturity in N K cells. Developmental stage I represent the N K P s which are defined as CDlZZ^TSfKl.rDXS\". Vossenrich et al.42 have further characterized this population and have shown that a subset of N K P s express C D 127 (IL-7Ra), IL-21R, CD117(c-Kit), and C D 135 (Flk2), but are CD25 (IL-2Ra) negative. These markers are characteristic of early hematopoietic precursors but C D 122 is not. In addition, they found that N K P s express N K G 2 D , while earlier studies have seen this at stage II. Stage II of development involves acquisition of the pan-NK cell markers; high NK1.1 expression and low D X 5 expression. The cells also express their first functional receptors, C D 9 4 / N K G 2 and, as reported by K i m et al.10, N K G 2 D . This stage of development resembles mature fetal/neonatal N K cells which do not express Ly49 receptors. Stage II cells also express the integrin a v + and are Mac- l l o CD43 l o c-k i t \" . The profile of Stage III N K cells is similar to that of the previous stage except that they are now c-kit + and they begin to acquire 11 the Ly49 molecules. D X 5 is upregulated in stage IV and the N K cells undergo a substantial proliferation in the bone marrow. A t stage V , N K cells complete their maturation by highly expressing Mac-1 and CD43 , and proliferation is slowed until N K cells are stimulated by pathogens1 0. These mature N K cells have full functional capabilities as well . The N K cell maturation status varies in the peripheral tissues. Mature N K cells in the periphery express DX5 and CD 1 l b and lack CD51 and CD117. Expression of K L R G 1 , a killer cell lectin-like receptor, is restricted to these most mature cells. Therefore, D X 5 + C D 1 l b + K L R G l + C D 5 1 \" CD 117\" NK cell population is the most mature phenotype in the periphery . S t a g e 1 S t a g e I I S t a g e I I I S t a g e I V S t a g e V N K P M H C c l a s s I r e c e p t o r a c q u i s i t i o n E x p a n s i o n F u n c t i o n a l l y m a t u r e CD94/ NKG2 CD94/ NKG2 Ly49 CD94/ CD94/ NKG2 Ly49 \u00C2\u00AE & IFNy ( v C # ) IFNy C D 1 2 2 + N K 1 . 1 -D X 5 -N K G 2 D C D 1 2 2 + N K 1 . 1 * D X 5 ' \u00C2\u00B0 C D 9 4 / N K G 2 + M a c - 1 i 0 a v h i C D 4 3 ' 0 C D 1 2 2 + N K 1 . 1 * D X 5 ' \u00C2\u00B0 C D 9 4 / N K G 2 + M a c - 1 1 0 a v h i C D 4 3 1 0 c - k i t + L y 4 9 + C D 1 2 2 + N K 1 . 1 * D X 5 h i C D 9 4 / N K G 2 + M a c - 1 1 0 a v ' \u00C2\u00B0 C D 4 3 1 0 c - k i t + L y 4 9 + C y t o t o x i c i t y 1 0 I F N y 1 0 C D 1 2 2 + N K 1 . 1 + D X 5 h i C D 9 4 / N K G 2 + M a c - 1 h l C D 4 3 h l c - k i t + A L y 4 9 + C y t o t o x i c i t y h i I F N y h i ' Figure 1.4. Stages of N K cell maturation. N K cells proceed through multiple stages following commitment to the N K cell lineage until they are fully mature. Each stage is marked by changes in receptor expression, proliferation and functional capabilities. Figure is from Veinotte et a l 8 . Mature N K cells express a full repertoire of receptors and have the functional capacity to k i l l target cells and produce cytokines. N K cells are functionally mature when they can distinguish between se l f -MHC class I + and self class I\" (expressing allogeneic class I or no class I) cells, rather than just when the cells acquire cytotoxicity potential. Mature N K cells are relatively static in terms of self renewal and proliferative capacity, except after pathogen infection. 1.4.2. Factors involved in NKP production: Cytokines To delineate the crucial cytokines for N K cell commitment, Wil l iams et al.41 cultured multipotent progenitors with various combinations of IL-7, S C F , and Fl t3L. TL-7 and Flt3L combination yielded the highest proportion of N K P s . Although addition of S C F did not change N K P numbers, it did increase N K cell yield. Therefore, IL-7 and Fl t3L expand or induce N K P s , and S C F has an additive effect to the overall N K cell numbers. The IL-7Ra chain is expressed by some N K P s 3 0 and splenic N K cells are reduced 3-fold in IL-7 7 \" mice 4 4 , whereas normal numbers of N K cells are produced in IL-7Ra\" A mice 4 5 . Since IL-7 only minimally affects N K cell development, Will iams et al.41 further concentrated on Flt3L and its role in N K cell development. F l t 3 L + progenitors produce more N K cells than Flt3L\" progenitors. It seems that Fl t3L either induces N K P s or more likely expands the N K P population, because N K P cell numbers are only slightly reduced in Fl t3L\" A mice 4 6 . F l t3L v \" mice have a 5.3-fold reduction of mature N K cells and a loss of cytotoxicity against Y A C - 1 4 7 . A s for a role for S C F , when c-kit\" /_ (receptor for SCF) progenitors were transplanted into RAG2/y c \" / \" mice, they produced N K cells at lower levels; absolute cell numbers were 40% of AO that of wild type progenitors. Their lytic capacity was greatly reduced . N K P cell numbers are not affected in c-kit_ /\" mice though so it appears that it is important for survival and 13 proliferation of committed N K c e l l s 2 5 ' 2 6 . Individually, these pathways are not essential for the first step of N K cell lineage commitment but there may be redundancy in the system and analysis of the double-mutant mice would be informative. Even though early studies suggested that signaling via the common y chain (yc) is essential for N K cell commitment since defects in the gene coding for yc (U2rg) cause an X-l inked severe combined immune deficiency characterized by several immune defects, including an absence of mature N K cells and IL15 _ /\" mice have reduced N K cell numbers 4 9 \" 5 2 , further investigations showed that the commitment to the N K lineage does not occur through yc-dependent cytokine stimulation 4 2, indicating that factors other than early^acting cytokines or yc-cytokines lead to N K cell lineage commitment. Recent studies indicates the expression N K G 2 D at the N K P cell stage4 2 but it has yet to be clarified whether it plays a role in restriction of the progenitors to the N K cell lineage. 1.4.3. Factors involved in N K P production: Transcription factors There are some factors that indirectly affect the production of N K P cells probably by affecting earlier upstream progenitors. One example is Ikaros, a zinc-finger D N A binding protein that is present in H S C s and is up-regulated during lymphocyte differentiation. A n Ikaros null 53 mutation in mice causes a severe defect in the lymphoid compartment including N K cells . Analyses of B M hematopoietic progenitors from Ikaros mutant mice revealed very low expression of Flt3 and c-Kit receptors in these progenitors 5 4, which suggested that Ikaros might regulate E L P / C L P homeostasis through growth factor receptors, thereby affecting the development of all lymphoid lineages including N K P s . Another factor is P U . l , an Ets family 14 transcription factor, which is highly expressed in most hematopoietic cells and regulates the development of myeloid and lymphoid lineages 5 5. P U . l and G A T A 1 have a mutually antagonistic relationship because they bind each other in the cytoplasm. This causes them to lose their translocation and binding ability respectively. The levels of these two transcription factors mediate the decision between lymphoid and myeloid lineages. High P U . l induces myeloid genes and high G A T A - 1 promotes erythroid and megakaryocyte genes. A t low P U . l levels, lymphocyte lineages are permitted. This is likely because only at low levels can P U . 1 induce IL-7Roc expression which promotes survival and proliferation of T and B cel ls 5 6 . P U . l knockouts do show defects in N K cells, but they are less severe than T and B cell defects, and this is likely due to N K cells' reduced dependency on I L - 7 5 7 . When transferred to Rag2\"A yc'7\" mice, P U . 1-deficient fetal liver cells generate normal numbers of H S C s , but produce reduced numbers of N K P s and N K cel ls 5 7 . Although the N K P cell numbers were affected, the phenotype of mature N K cells was not substantially different but the deficiency does affect cell expansion and homeostasis. 58 Ets - l , a winged helix-turn-helix transcription factor is essential for development of N K cells . In Ets-l\" 7\" mice, T and B cells develop normally but the number of D X 5 + C D 3 \" splenic N K cells and their cytolytic activity is remarkably reduced. The defects in Ets-l\" 7\" N K cells are intrinsic to N K precursors. One mechanism by which Ets- l controls N K cell development might be through survival, as the increased apoptosis has been reported in B - and T-cell populations from Ets-1\"/\" mice 5 9 . A class of b H L H proteins known as inhibitors of D N A binding (Id) proteins are key molecules for N K cell development. They negatively regulate E proteins that are crucial for B and T cell 15 lineages. Overexpression of Id3 in human C D 3 4 + H S C s has been shown to block T cell development and promotes N K cell development in fetal thymic organ cultures 6 0. Development of N K cells is also accelerated when both Id2 and Id3 are constitutively expressed in C D 3 4 + HSCs, whereas the generation of T, B , and lymphoid-DCs is completely abrogated 6 1. These observations were further complemented by the finding that targeted disruption of Id2 gene results in a selective block in N K cell development 6 2, accompanied by a lack of N K P s 6 3 . These data present a model in which Id proteins promote commitment of common lymphoid precursors to N K P s while inhibiting their option to develop to committed T- and B -progenitors. 1.5. N K cell maturation 1.5.1. M H C Class I Receptor acquisition The MHC-class I receptor acquisition on N K cells is complex. Although the numbers of Ly49 and C D 9 4 / N K G 2 receptors is quite low in comparison to T and B cell receptor diversity, the repertoire of the N K cell population, as a whole, is very diverse. Each Ly49 or C D 9 4 / N K G 2 receptor family member is expressed on a subset o f N K cel ls 6 4 . Therefore, their expression partially overlaps with the expression of the other receptors. N K cells express at least one M H C I-specific receptor and individual N K cells can coexpress up to five different Ly49 receptors in addition to C D 9 4 / N K G 2 receptors 6 4. Expression o f the receptors is not co-regulated and it appears that they are expressed with a great degree of independence from each other8. 16 The receptor expression on developing N K cells differs between the fetal and neonatal mouse and the adult mouse. Most fetal and neonatal N K cells express C D 9 4 / N K G 2 but not Ly49 receptors, with the exception of Ly49E, which is expressed in the fetal cells but not the adult cells 6 5 . A s the mouse matures, N K cells begin to acquire Ly49 receptors, while C D 9 4 / N K G 2 + N K cells decline. The frequency of N K cells expressing high levels of C D 9 4 / N K G 2 decreases from - 9 0 % on neonatal N K cells to - 5 0 % on adult N K cel ls 6 6 . This decease in C D 9 4 / N K G 2 expression coincides with the increase in the frequencies of N K cells expressing Ly49. Ly49 receptor expression begins very gradually, starting at approximately 1 week after birth and reaching adult levels at 6 to 8 weeks of l i fe 6 7 . The decease of C D 9 4 / N K G 2 is not due to a downregulation in cells but rather due to a generation o f C D 9 4 / N K G 2 X y 4 9 + N K cells in young mice 6 7 . N K cells appear to undergo a process of ensuring self tolerance during development. Since many normal self cells express ligands for N K cells, it is important that N K cells express self M H C class I inhibitory receptors to ensure that autoreactivity does not occur. Evidence shows that N K cells that lack inhibitory receptors for self M H C class I are hyporesponsive. Two main theories exist on how this self tolerance is acquired by N K cells during development (reviewed in 7 ) . The first is the arming model. In this model, N K cells receive positive signals i f their Ly49 receptors bind self M H C class I. This positive signal 'arms' the N K cells and allows them to mature into functional N K cells while those that do not express a self Ly49 remain unarmed and unresponsive. The second model is the disarming model. In this model N K cells express a variety of stimulatory and inhibitory signals and interact with self cells. If the N K cell binds both activating ligands and inhibitory ligands (the self M H C class I) no net stimulation occurs and the responsiveness of the N K cell is maintained. Conversely, i f an N K 17 cell without a self Ly49 binds a self cell, it w i l l only receive activating signals and hyporesponsiveness w i l l be induced in this cell. In other words, the cell is 'disarmed'. 1.5.2. Factors involved in N K cell maturation N K cell maturation requires cell-to-cell interactions and soluble factors derived from B M l stromal cells. In vitro culture systems that are designed to give rise to mature N K cells, as described earlier, are not complete without stromal cells in the culture 4 1. Multipotent HSCs can be cultured with certain cytokines to produce C D 1 2 2 + N K P s but without addition of IL-15 and stromal cells in a second stage of N K cell culture, the cells w i l l not fully mature. If IL-15 is added without stromal cells, the cells become functionally mature and express C D 9 4 / N K G 2 but not L y - 4 9 4 1 . This shows the importance of cell-cell interactions between developing N K cells and stromal cells. IL-15 is the critical cytokine, or the \"essential fuel\" for N K cell development. IL-15 works by binding the IL-15R complex, which is made up of IL-2/IL-15RP (CD 122), IL-15Ra, and the yc 68 \u00E2\u0080\u00A2 but it appears that there may be other methods in which IL-15 signaling occurs . There is an N K cell deficiency, as well as deficiencies in N K T cells, intestinal epithelial cells, and memory C D 8 + T cells, in IL-15 and IL-15Ra-deficient m i c e 4 9 ' 5 2 . Disrupting yc , the third subunit of IL-15R, generates a similar defect in N K cell production, suggesting an essential role for IL-15, overall, in N K cell development 6 9 ' 1 0 . In an effort to identify the developmental stage at which IL-15 and other y c cytokines are important for N K cell development, Vossehenrich et al.42 produced various yc deficient mice (IL-2, IL-4, IL-7, IL-15) including multiple y c deficiencies per mouse (i.e. I L - 2 v l L - 7 \" / \" , IL-2\" /\"IL-4\" /\"IL-7\" /\", and IL-4\" /\"IL-7\" /\"IL-15\" /\") on a Rag2-deficient 18 background. They were able to show that N K P s do not require yc cytokines because they are present in normal numbers and IL-2, IL-4, and IL-7 have no role in the generation of mature N K cells. Conversely, there is an essential singular role for IL-15 in the generation of immature and mature N K cells in the B M and mature N K cells in the spleen. The N K cells that do remain in IL-15-Rag2 deficient mice have an immature phenotype of Mac-1 l o CD43\". The cells were also CD94 , N K G 2 A / C / E and Ly49D + bu t Ly49G2 and Ly49C/I N K cell numbers were reduced. The cells could k i l l Y A C - 1 and produce IFNy upon stimulation but at reduced levels 4 2 . One clue to IL-15 signaling function is the antiapoptotic factor Bcl -2 . Transgenic 71 overexpression of Bcl -2 in IL-2RP-deficient mice leads to normal N K cell numbers . It also allows peripheral N K cells to persist in IL-15 deficient hosts after adoptive transfer ' . A recent study has isolated a downstream target of IL-15 in human N K cells and therefore provides another new clue to its function . Because IL-2 and IL-15 signal through common receptor subunits, it is likely that they regulate a shared set of downstream target genes. They showed that IL-2 and IL-15 stimulation results in the post-transcriptional increase in Ets-1 protein. The exact role for Ets-1 in N K cell function and the particular target genes controlled by Ets-1 are still unknown . These studies still have not revealed the exact role of IL-15 in N K cell development and many possibilities exist. To summarize, IL-15 may promote N K cell development by indirectly controlling the responsiveness of maturing N K cells to other growth and survival factors. It may also act as a survival factor for N K cells at certain developmental stages or it may act to support proliferation of developing N K cells. Also , IL-15 may have an indirect effect on N K cells by regulating their susceptibility to the IL-21 maturation factor 4 2. Factors that regulate the IL-15 signaling pathway and its effects on N K cells have been identified. These include lymphotoxin and IRF-1. The lymphotoxin L T a ^ i s expressed on N K 19 cells and it binds the L T p receptor on non-lymphoid cells. A reduction of N K cell numbers is seen in mice lacking the L T a on N K cells or L T p R on stromal c e l l s 7 4 ' 7 5 . The impaired N K cell development is due to the absence of LT|3R triggered signals in the stromal cells. F L cells from wild type mice cannot develop into N K cells when grown in vitro on L T p R deficient stromal cells. If the reverse culture is performed, LTa-deficient F L cells can differentiate into N K cells when grown on wi ld type stroma 7 6. Lian et al?1 recently suggested that this impaired N K cell development is a result of impaired IL-15 production in the stromal cells. The signals derived from L T and L T p R interaction result in the up-regulation of IL-15 production by the B M stroma 7 7. IRF-1 deficient mice also have severely reduced numbers of N K cells due to a 78 79 defect in the production of IL-15 by the bone marrow microenvironment ' . A very recent study has made a breakthrough in determining factors required for Ly49 receptor expression and N K cell maturation 8 0. Tyro3 receptors' ( A x l , Tyro3, Mertk) expression on committed N K cell precursors is crucial for both the acquisition of nearly all inhibitory and activating receptors on N K precursors in the B M and for the functional maturation of these cells in the spleen. N K cells express all three receptors and B M stroma expresses their two ligands (Gas6 and protein S ) 8 0 . They seem to be required for progression from the immature N K cell stage because N K cells in Tyro3-deficient mice have high N K G 2 and CD94 expression and low Ly49, D X 5 , N K G 2 D , Mac-1, CD43 expression. Their cytotoxicity is substantially decreased and the cells cannot produce IFNy either. The Tyro3 receptors seem to act through an uncharacterized pathway because they do not alter expression of known factors SO that play roles in N K cell development . 20 Other factors, besides those that play a role in the B M stroma signals, are important for N K cell maturation and/or effector function. For example, V D U P 1 (Vitamin D3 upregulated protein 1)\"A mice show a profound reduction in the number of mature N K cells in the spleen and B M . Expression of C D 122 (the marker of N K P s ) was reduced and V D U P 1 expression is known to be induced from the N K P stage. Therefore V D U P 1 may be important for the differentiation of N K P s or for their maturation because upregulated V D U P 1 may induce cell cycle arrest, which is necessary for the onset of differentiation 8 1. The G A T A - 3 transcription factor may also play an important role in the transition from the immature M a c - l ' \u00C2\u00B0 C D 4 3 l 0 stage to the M a c - l h l C D 4 3 h l stage. Peripheral N K cells that develop in GATA-3\" 7 \" mice resemble immature M a c - l ' \u00C2\u00B0 C D 4 3 l 0 N K cel ls 8 2 . A third factor that controls N K cell maturation is N F - K B . The N F - K B family members normally are kept in an inactive state by inhibitors but i f these inhibitors are degraded the N F - K B transcription factors become ac t ive 8 3 , 8 4 . When the N F - K B transcription factor family members are hyperactivated, N K cell maturation is arrested and the residual N K cells in the B M , spleen, and liver are immature (Mac-1 l o C D 4 3 l 0 ) . N K cell cytotoxicity is not affected but there is reduced capacity for IFN-y production. The defect in maturation is l ikely a result o f N K cells being unable to provide adequate proliferative signals. The cells express normal levels of IL-2RP and y c but that they could not proliferate in the presence of IL-2 or IL-15. The molecular mechanism for this defect is unclear . T-bet is a transcription factor that regulates N K cell maturation and effector functions (cytokine production and to a lesser extent, cytotoxicity). In T-bet deficient mice there is a reduction in N K cell number, specifically in cells with late maturation markers. The perforin and granzyme B genes are T-bet target genes even though T-bet only plays a minor role in controlling cytotoxicity 8 6 . There may be a compensatory relationship with T-bet. E O M E S , 21 which is also a T-box family transcription factor, is highly expressed in N K cells (even in T-bet\"7\" mice) and it regulates IFNy, perforin, and granzyme expression 8 7. It w i l l be informative to study these transcription factors' roles in EOMES^T-bet\" 7 \" mice. G A T A 3 participates with T-bet in N K cell function as well . There is an intrinsic defect in IFN-y production in G A T A - 3 _ / \" g o N K cells 0 0 . In these mice, T-bet expression was 4-fold reduced while H l x (another transcription factor that controls IFNy production) expression was 10-fold reduced. T-bet establishes an active chromatin configuration of the IFN-y locus and it cooperates with Hlx , which is broadly expressed in multiple hematopoietic cell lineages, including N K cells, to upregulate IFN-y expression 8 9 ' 9 0 . The combined deficiency in T-bet and H l x expression may explain the poor inducible IFN-y production in G A T A 3 \" \" mice . A few other factors that are critical for N K cell effector function rather than maturation are M E F - 1 , C /EBPy , M I T F and N E M O . A member of the ETS-family, M E F - 1 , is required for N K and N K T cell development as a profound reduction of these cells is observed in MEF-deficient mice. The few N K cells found in these mice are functionally impaired because M E F regulates transcription of the perforin gene 8 2. MITF-deficient mice have a cytotoxicity defect due to decreased perforin expression 9 1 while NEMO-deficient N K cells have a defect in their N F - K B signaling pathway which is important in the regulation of perforin and other cytotoxic factors 9 2. The basic leucine zipper transcription factor, C / E B P y , is also important for proper N K cell cytotoxicity and IFNy production but not for earlier N K cell development. C /EBPy expression is ubiquitous and constitutive and while it does not have a transcription activating domain, it can interact with other transcription factors and augment their D N A binding. In C/EBPy\"\" mice, N K cell cytotoxic activity and IFNy production are impaired . 22 A cytokine that plays a late role in N K maturation is IL-21. IL-21 induces further differentiation of activated, mature N K cells and is an initiator of IFN-y production as well . The terminal differentiation of mouse N K cells leads to an increase in cell size and granularity, loss of NK1.1 expression, and upregulation of the N K G 2 - C D 9 4 complex. It also leads to a significant increase in cytotoxicity and a massive induction of cytokine secretion. This late function of IL-21 is important for activated N K cells because it enhances IFN-y production more than IL-15 alone 9 4 . In conclusion, the precise pathway of N K cell lineage commitment and subsequent maturation is still unclear. The stages of N K cell development are not yet fully characterized but we are reaching a point now where an accepted model of N K cell development is present and being built upon. Future studies w i l l contribute to this model by characterizing more cell surface markers and molecular pathways which contribute to either the commitment or the maturation of N K cells. Table 1.1. Genetic mutations affecting the maturation of N K cells. Revised from DiSanto . Transcription factor Phenotype Repertoire Stage Cytotoxicity Cytokines Ref. PU.1 Normal Decreased Ly49 NKP Nomal Normal bl Ets-1 ? ? NKP Reduced Reduced ba Id2 ? ? NKP Reduced Reduced Gata-3 Mac-110, CD43 1 0 Decreased Ly49 Immature NK Normal Reduced 88 T-bet Mac-110, CD43 1 0 Normal Mature NK Normal Normal Bb MEF, MITF, C/EBPy Normal Normal Late mature NK Reduced Normal 82, 91, 93 23 1.6. T cells and development The T cell population is able to mount a response to virtually any foreign antigen because each T cell expresses a unique variant of heterodimeric receptors (a.p or y8) 9 5 . These T cell receptors (TCRs) are formed from rearrangement and co-expression of either a and P genes or y and 8 genes. aP T cells are part of the adaptive immune system. They localize primarily in secondary lymphoid organs and respond to infection by facilitating the production of antibodies and by lysing target cells. aP T cells recognize peptide ligands presented by class I and II M H C molecules. y8 T cells participate in the early immune response, similar to the innate immune system 9 6 . They comprise only a minor population in the blood (1-5%) but in epithelial tissues, they represent 50% of the T cells. They recognize a much wider variety of antigens such as nonclassical M H C molecules, heat shock proteins, and l ip ids 9 6 . The structure and signaling potential of the y8TCR complex differs slightly from the ocpTCR complex. Both complexes include invariant accessory chains such as the C D 3 proteins. While in the a P T C R complex there is one heterodimer of CD3e and 8 and another heterodimer of CD3e and y, the ySTCR complex lacks CD38 and has two heterodimers of CD3s and y . The signal transduction by the y 8 T C R complex is superior to that of the a P T C R complex . T cell development occurs in the thymus and T cell precursors migrate from the fetal liver and the adult B M to continuously seed the thymus. The first half of T cell development is independent of the T C R while the second half is T C R dependent (Fig. 1.5). T cell development can be defined by a series of stages (reviewed by Janeway 9 5). The initial stages are traditionally termed double negative since they do not express C D 4 or C D 8 but they are also referred to as triple negative to include CD3 as well . The initial precursor stage is termed 24 double negative 1 (DN1) and the cells are CD44 + CD25\". The D N 2 stage is marked by gain of C D 2 5 + and the initiation of T C R gene rearrangement. This rearrangement continues in CD44\" C D 2 5 + cells at the D N 3 stage. T cell development then becomes T C R dependent and cells die i f they fail to generate a productive in-frame T C R P chain or a pair of T C R y and T C R 8 chains. Because the joining events of V(D)J recombination are imprecise, two out of three attempts are non-productive and fail to maintain the translational reading frame of the T C R subunit. Cells that do successfully rearrange and express a productive T C R chain(s) on their surface wi l l then continue differentiation to either the y8 lineage or the otp lineage. H o w the T cell progenitors decide to follow the y8 T cell lineage versus the aP T cell lineage is not yet known. One OR model suggests that it depends on the strength of the signal . If a weak signal is received from either a pre-TCR (the T C R p chain is expressed on the surface at this stage with an invariant p T a chain) or a y8 T C R that has not encountered ligand, the cells w i l l become ocP T cells. Conversely, i f a stronger signal is received from a yS T C R that has encountered a ligand of intermediate affinity, cells w i l l become y8 T cells. Finally, i f a very strong signal is received OR from a y8 T C R and ligand, the cells w i l l die . aP T cells continue their development by undergoing proliferation and differentiate into C D 4 + C D 8 + double positive (DP) aP T cells 9 5 . These od3 T cells then rearrange their T C R a genes and undergo positive and negative selection for self/nonself discrimination. T C R s must be able to recognize M H C class I or II on cortical thymic epithelial cells to pass positive selection and become C D 8 SP cells or C D 4 SP cell respectively. If SP cells recognize a self antigen presented by B M D C s or macrophages in the thymus, they are kil led by negative selection to ensure that self-reactive cells do not leave the thymus. After selection and maturation, T cells finally exit the thymus to take up their roles in the immune system 9 5 . 25 Natural killer T ( N K T ) cells are a conserved T cell sublineage with unique properties (reviewed by Kronenberg\"). The main subpopulation of N K T cells are invariant N K T cells, which likely arise from D P T cells during development in the thymus (Fig. 1.5). These cells express invariant T C R s and they recognize a synthetic glycolipid presented by an M H C class I-like molecule, C D Id. Upon stimulation, N K T cells rapidly produce many cytokines and can influence diverse immune responses. Another subset of N K T cells is C D l d independent and their developmental pathway is not defined. Figure 1 . 5 . Stages of T cell development. The initial stages are T C R independent and involve V(D)J recombination of T C R y , 5, and p genes as well as loss o f alternate cell potential and final commitment to the T cell lineage at the D N 3 stage. The remainder of the stages are T C R dependent and result in mature single positive CD4 and C D 8 T cells. Two groups characterized the kinetics and timing of T C R recombination during T cell development 1 0 0 ' 1 0 1 . TCRy, 5, and P recombination occurs before T C R a . T C R rearrangements 26 at the DN1 stage are negligible and rearrangements that were detected are likely contamination. A t the D N 2 stage, both T C R y and T C R 5 rearrangements are present while T C R p rearrangements are almost absent. Specifically for TCRy , Vy2-Jy l rearrangements were more abundant than Vy5-Jy l and by the DN3 stage, the level of Vy2-Jy l rearrangements were within range of mature y8 T cells while Vy5-Jyl levels were still lower. For T C R 5 , Capone et al.100 detected V55-J51 and V54-J81 rearrangements at D N 2 stage but V55 rearrangements are more prominent at this stage. Livak et al.101 saw that D N 2 cells have substantial amounts of partial D51 and D82 to J81 rearrangements but not V8-DJ8 and that by D N 3 75-100% have completed V - D J 8 rearrangements where only a minority of cells have full V-DJP rearrangements. A l l major y and 8 genes are rearranged at D N 3 or D N 4 at levels similar to total thymocytes. Therefore, the recombination of the majority of T C R y and T C R 8 loci is completed by D N 3 but completion of V-DJP rearrangement to maximal levels is not seen until more advanced stages. Also , rearrangement of the T C R y and T C R 8 loci occurs in most ap T ce l l s 1 0 2 \" 1 0 7 and productive T C R p rearrangements can be found in y8 T ce l l s 1 0 4 ' 1 0 8 \" m . 1.7. TCR rearrangement: V ( D ) J recombination The ability of T cells to respond to a vast array of antigens is dependent on the generation of unique surface receptors with diverse binding specificities. The T C R s are assembled during lymphocyte development from germline variable (V), diversity (D), and joining (J) gene 1 1 7 segments by the process of V(D)J recombination (Fig. 1. 6) (reviewed by ). The segments are flanked by recombination signal sequences (RSSs). These conserved noncoding sequences guide the D N A rearrangements. The RSS includes a heptamer sequence which is always 27 contiguous with the coding sequence. Next is a nonconserved spacer region that is either 12 or 23 bases long, which is followed by a second conserved nonamer sequence. Therefore the heptamer-spacer-nonamer sequence motif makes up the RSS and it is always directly adjacent to the coding sequence of the V , D , or J gene segment. Normally, a 12-RSS is joined with a 23-RSS. The complex of enzymes that carries out recombination is collectively called V(D)J recombinase. This contains lymphocyte specific enzymes, R A G - 1 and R A G - 2 , and ubiquitously expressed DNA-modify ing proteins. Two R A G complexes recognize and bind two RSS sequences. The R A G complexes then bind each other and therefore bring together the two gene segments to be joined. The R A G complexes each introduce a single strand nick precisely between the R S S and the coding sequence. This leaves a free 3' O H group which attacks the phosphodiester bond on the other strand, creating a hairpin at the end of the gene coding segment. This process simultaneously creates a double-stranded break at the ends of the heptamer sequences. The two non-coding RSSs are joined in a precise head to head linkage to form a signal joint. The coding joint formation involves extra steps. The R A G complex remains bound to the hairpin structures and proteins in the complex cleave the hairpins at random. The D N A repair enzymes in the complex may remove some nucleotides while the terminal deoxyribonuclease transferase (TdT) enzyme adds nucleotides randomly. Finally, D N A ligase IV joins the ends together and repair enzymes trim off non-matching bases and synthesize complementary bases to f i l l in the remaining single stranded D N A . Since the number of nucleotides added by TdT is random, the added nucleotides often disrupt the reading frame of the coding sequence. This produces a nonproductive rearrangement, which occurs in 2 /3 r d s o f rearrangements, as mentioned above. 28 _J_J I Coding joint Signal joint 3 A J xJ) Proteins Figure 1.6. The process of V(D)J recombination. 1. Two R A G complexes recognize and bind two RSS sequences, one on the V segment and one on the J segment. 2. The R A G complexes then bind each other and therefore bring together the two gene segments to be joined. 3. The R A G complexes cleave the D N A to create hairpin ends on the V and J segments. Other proteins bind the hairpins and the cleaved RSS ends of the gene coding segment. 4. The D N A hairpins are cleaved at random. Additional bases are added (TdT) or subtracted (exonuclease) to create imprecise ends. 5. D N A ligase IV joins the ends of the segments to form the coding joint. It also joins the RSSs to form a signal joint. The process of V(D)J recombination is regulated by the availability of the recombination machinery and also by accessibility of the target gene segments . Multiple epigenetic mechanisms are involved in regulating rearrangements including: histone acetylation, D N A methylation, allelic exclusion, nuclear location, and cis- and trans- acting factors 1 1 3. Specifically for T C R y recombination, IL-7 regulates chromatin accessibility for germline expression and R A G enzymes via histone acetylation which opens the chromatin structure1 1 4\" 116 1 . 8 . TCRy locus and rearrangement patterns The T C R y locus in mice features four clusters of Vy , Jy, and Cy regions containing 7 V y segments, 4 Jy segments and Cy regions (Fig. 2 .1) 1 0 3 ' 1 1 7 . Each cluster contains one C region, 29 one J segment and one to four V segments. The V segments rearrange to the J segment in the same cluster. Cluster 3 has been deleted in most T C R y haplotypes and is believed to be non-functional in some s t r a i n s 1 0 2 ' m . The C y l cluster contains four closely linked but distantly related V region genes. The utilization of V y genes is developmentally regulated such that Vy3 and Vy4 gene rearrangements are prominent in the fetal thymus but are very rare in the adult while Vy2 and Vy5 rearrangements have the opposite pa t t e rn 1 0 3 ' 1 1 9 ' 1 2 0 . This rearrangement pattern is reflected in the y5 T cells present in the mouse. V y 3 + y8 T cells appear first at approximately embryonic day 13 and disappear from the thymus by embryonic day 18 1 2 1 . V y 4 + y8 T cells are also seen in the fetal mouse 1 2 2 . Interestingly, the cells expressing different V y rearrangements have specific functions as well . V y 3 + y8 T cells home to epidermal epithelial tissues and are called dendritic epidermal T cells ( D E C s ) 1 2 3 . These cells are unique in that they secrete keratinocyte growth factor 1 2 4 . V y 4 + yS T cells, on the other hand, concentrate in the female reproductive tract and tongue. These cells also have a fixed T C R 8 chain and therefore, the population only has one T C R specificity. These cells likely recognize common stress-induced self antigens and eliminate damaged cells as well as promote epithelial growth and differentiation 1 2 3. In the adult environment, y8 T cells rearrange Vy2 and Vy5 chains but they are not as limited with T C R specificity since they can pair to different T C R 8 chains, to some extent, and they also have V(D)J junctional diversity, which is lacking in the fetal cells. 125 Therefore, these cells l ikely recognize foreign antigens . 30 It appears that developmentally regulated V y gene recombination is an intrinsic genetically programmed process. In models where the T C R y locus contains frame shift mutations that prevent functional expression, the V y genes are still rearranged at the appropriate times even though they cannot influence the fate of the cells . For the fetal T C R genes (Vy3 and Vy4), the location of the V segment influences the rearrangement pattern. In a transgenic model where Vy2 and Vy3 are switched, fetal cells had higher Vy2 rearrangement levels than Vy3, the 12 opposite of what normally occurs . V segment location does not affect the rearrangement pattern in adult thymocytes though and they are mostly influenced by promoters. The sequences upstream of Vy2 , 3, and 4 are quite divergent, suggesting that each promoter could be regulated by distinct trans-acting factors. Baker et al.126 swapped the promoter regions of Vy2 and Vy3 and the pattern was reversed in adult with Vy3 being rearranged. Since germline transcription is present for both Vy2 and Vy3 in the fetal stage, it seems that both genes are accessible in the early stages, with an advantage to the more proximal gene, followed by a stage when Vy3 is repressed and/or Vy2 is activated, leading to a strong preference for Vy2 rearrangement. Therefore, the \"developmental switch\" (i.e. Vy3,4 to Vy2,5) in V y gene usage is imposed by two mechanisms, one sensitive to gene location and the other dependent on differential V-promoter activity. During development, each precursor can try multiple rearrangements at the T C R y locus . For example when y5 T cells were sorted for Vy2, Vy5 or V y l . l + y8 T C R s on their cell surface (these three represent 90% of the y8 thymocytes in adult B6 mice), all clones had multiple rearrangements at the y locus. Most cells had 2 to 6 rearrangements and V y 2 + cells normally 31 had a maximum of 4 rearrangements. Rearrangements involving Jy l were found in all cells and about half had Jy l rearrangements in both chromosomes. Since multiple V - J rearrangements occur in a single cell there are several factors that ensure that y8 T cells w i l l not express more than one T C R specificity on the cell surface. These include: 1. the frequencies at which each V y and V 8 gene segments participate in recombination, 2. the frequencies at which the rearranged products produce a functional chain, and 3. whether the functionally rearranged T C R y and T C R 8 chains can pair to form a y 8 T C R 1 2 7 . The end result is the formation of a pool of y8 T cells expressing a diverse repertoire o f V y and V 8 chains in which the majority o f the cells bear a single T C R specificity at the cell surface. This works in these cells without specific checkpoints to test and control for the functionality of each of the rearranged chains, unlike a.p T cells. This is only possible because the three mechanisms work differently on each V gene segment. For example, Vy2 and V y l . 2 rearrange at similar frequencies but they are approximately -10-12 times more frequent than V y l . l rearrangements and 16-20 times more common than Vy5 rearrangements. V S segments also rearrange at different frequencies . The frequencies at which different V y and V 8 segments participate in recombination are inversely correlated with the apparent ability of the resulting T C R y or T C R 8 chain to participate in the formation of a functional ySTCR. For example, V y l . 2 is one of the most frequent rearrangements but it shows the highest level o f restriction for pairing with a T C R 5 chain. In contrast, V y l . 1 and Vy5 , which are rearranged at low frequencies, lack restriction in pairing with V 8 chains. 32 Also, there is a stop codon at the 3'end of the very frequently rearranged germline Vy2 gene 100 128 segment ' . This substantially lowers the frequency at which rearrangement wi l l produce a functional chain. This therefore lowers the frequency of a functional Vy2 being expressed on the cell surface and therefore increases the chances that y8 T cells can express T C R y chains on their surface that are from low frequency rearrangements (i.e. Vy5). 1.9. Thesis objectives and hypotheses The original objective of my thesis was to determine i f adult and neonatal N K cells follow separate pathways of lineage commitment. Since adult and neonatal N K cells differ in their phenotype and function, I hypothesized that the differences are due to their developmental pathways. In Chapter 3, this hypothesis was tested by examining differences in gene expression patterns between the two types of N K cells. This study resulted in the detection of TCRy gene rearrangement in a subset of N K cells. Based on this interesting and unexpected finding, my subsequent thesis research was redirected to further characterize N K cells that have rearranged T C R y genes. The objectives of my subsequent study were to determine the developmental pathway responsible for the generation of N K cells with T C R y gene rearrangement and to further characterize these N K cells. M y hypotheses were: 1) there are at least two separate developmental pathways for N K cells, one in the bone marrow and the other in the thymus, 2) a subset of N K cells arises from immature T cell progenitors that have begun TCRy gene rearrangement but still retain N K cell potential, and 3) N K cells with rearranged T C R y genes are different from bone marrow-derived conventional N K cells in phenotype and function. To test these hypotheses, the following objectives were set: 1. to determine i f the T C R y gene-rearranged N K cells are thymus-dependent, 2. to identify the progenitors that give 33 rise to the N K cells with T C R y gene rearrangement, 3. to determine i f N K cells generated by the developmental pathway involving T C R y gene rearrangement differ from conventional B M -derived N K cells in phenotype, function, and tissue distribution. 34 2 M A T E R I A L S A N D M E T H O D S 2.1. Mice . C 5 7 B L / 6 (B6) mice were bred in our animal facility. Adult mice used in this study were 6 to 10 weeks old and neonatal mice were 1 to 3 days old. R A G 2 \" / \" H Y T C R transgenic mice were also bred from breeders purchased from Taconic Farms (Germantown, N Y ) . Adult TCRp7TCR6-double knockout (B6.\29P2-Tcrb\"\"IMomTcraimlMc\"n/J) mice were purchased from The Jackson Laboratories (Bar Harbour, M E ) . Heterozygous nude (B6.Cg-Foxnlnu) mice were purchased from The Jackson Laboratories and were mated, and athymic nude neonatal mice (3 days old) were used. IL-15\"7\" mice (C57BL/6NTac- / I i5\"\" 7 / T O C ) were purchased from Taconic Farms. ~NOD.Cg-PrkdcsciclIl2rg\"\"IWjl/SzS mice were from The Jackson Laboratories. 2.2. Antibodies. Antibodies used in this thesis are listed in Table 2.1. 35 Table 2.1. L i s t of antibodies: Antibody Clone Animal Isotype Conjugate Company NK1.1 PK136 mouse Ms I g G 2 a , K APC PM NK1.1 PK136 mouse Ms I g G 2 a , K PE PM NK1.1 PK136 mouse Ms I g G 2 a , K FITC PM NK1.1 PK136 mouse Ms I g G 2 a , K biotin PM CD3e 145-2C11 mouse A r H a m I g G l , K FITC PM CD3e 145-2C11 mouse A r H a m I g G l , K biotin PM CD3e 145-2C11 mouse Ar H a m I g G l , K PE PM CD3e 145-2C11 mouse A r H a m I g G l , K PerCP-Cy5.5 PM NKG2A/C/E 20d5 mouse \u00E2\u0080\u00A2 Rat I g G 2 a , K biotin PM NKG2A/C/E 20d5 mouse Rat I g G 2 a , K FITC PM CD94 18d3 mouse Rat I g G 2 a , K biotin PM Ly-49A YE/148 mouse FITC homemade Ly49H 1F8 mouse FITC homemade Ly-49 C and Ly-49 I 5E6 mouse Ms I g G 2 a , K FITC PM Ly-49 C and Ly-49 I 5E6 mouse Ms I g G 2 a , K biotin PM Ly-49 D 4E5 mouse Rat I g G 2 a , K FITC PM Ly-49 G2 4D11 mouse Rat I g G 2 a , K FITC PM Ly49 G2 4D11 mouse biotin homemade NKG2D C7 mouse A r H a m IqG biotin Biolegend CD127 A7R34 mouse Rat I g G 2 a , K biotin eBioscience KLRG1 2F1 mouse S y r H a m IgG2, K biotin PM Mac-1 mouse biotin homemade CD8a (LY-2) 53-6.7 mouse Rat I g G 2 a , K biotin PM CD8b.2 (Ly-3.2) 53-5.8 mouse Rat I g G w K biotin PM CD8 FITC Boehringer CD3e 145-2C11 mouse A r H a m I g G l , K FITC PM CD3e 145-2C11 mouse A r H a m I g G l , K biotin PM gamma-delta TCR GL3 mouse Ar H a m IgG2, K biotin PM gamma-delta TCR GL3 mouse Ar H a m IgG2, K FITC PM TCR beta chain H57-597 mouse A r H a m IgG2, XI biotin PM NK1.1 PK136 mouse Ms I g G 2 a , K biotin PM NK1.1 PK136 mouse Ms I g G 2 a , K FITC PM Mac-1 FITC homemade Mac-1 biotin homemade TER-119 Ter-119 mouse Rat I g G 2 b , K biotin PM Gr-1 biotin homemade B220 RA3-6B2 mouse Rat lgG 2 a , K FITC PM B220 biotin homemade CD19 1D3 mouse Rat I g G 2 a , K FITC PM CD19 1D3 mouse Rat I g G 2 a , K biotin PM CD44 1M7 mouse Rat I g G 2 b , K biotin PM CD25 3C7 mouse Rat I g G 2 b , K PE PM CD122 TM-31 mouse Rat I g G 2 b , K PE PM Pan-NK cells DX5 mouse R a t I g M , K FITC PM Pan-NK cells DX5 mouse R a t I g M , K PE PM IFN-gamma XMG1.2 mouse Ra t I g d , K APC PM 36 2.3. Microarray Sample Preparation and Analysis. Total R N A was isolated by using the RNeasy M i n i K i t ( Q I A G E N Inc., Mississauga, ON) . Double stranded c D N A was synthesized from total R N A with the Superscript double stranded c D N A kit (Invitrogen, Carlsbad, C A ) . The Enzo BioArray high yield R N A transcript labeling kit (Affymetrix Inc., Santa Clara, C A ) produced biotin labeled c R N A which was fragmented and hybridized to Affymetrix GeneChip Mouse Genome U 7 4 A v 2 arrays. The first two microarray experiments were performed at the D N A Array Laboratory, Wine Research Centre, University of British Columbia and the third experiment was performed at the Affymetrix GeneChip Facility at the Michael Smith Genome Sciences Centre, British Columbia Cancer Agency. A l l data analysis was performed with Genespring version 7 (Silicon Genetics, Redwood City, C A ) . Expression values were background corrected, normalized, and summarized by using the default settings of the program package. 2.4. Measuring DNA. For determining the percentage of N K cells that had T C R y gene rearrangements, D N A templates were first quantitated using Pico Green\u00C2\u00AE d s D N A quantitation kit (Molecular Probe, Invitrogen, Carlsbad, C A ) , and the fluorescence was measured with a CytoFluor\u00E2\u0084\u00A2 2300 Fluorescence Measurement System (Millipore, Bil lerica, M A ) . 2.5. Genomic PCR. To isolate genomic D N A , cells were lysed with 50 ul of dF^O and vigorous pipetting, placed at 98\u00C2\u00B0C for 10 minutes, and then 5 ul of 1 mg/ml proteinase K was added and incubated at 55\u00C2\u00B0C for 2 hours followed by incubation at 98\u00C2\u00B0C for 10 minutes. D N A thus isolated was used as template for P C R . Primers for Vy-Jy and N K G 2 A genomic P C R are listed in Table 2.2. The genomic P C R scheme is shown in Fig . 2.1. The reaction volume for these PCRs was 50 ul, containing 5 ul of 10* P C R buffer, 1.5 ul of 50 m M M g C l 2 , 1 ul of 10 37 m M dNTPs, 1.25 ul each of 10 u M primers, and 0.5 ul of 5 U / u l Taq D N A polymerase. Thermocycling conditions were as follows: 3 minutes at 94\u00C2\u00B0C followed by 30 cycles of 45 seconds at 94\u00C2\u00B0C, 2 minutes at 55\u00C2\u00B0C, 1 minute at 72\u00C2\u00B0C and finally 7 minutes at 72\u00C2\u00B0C. 10 ul of P C R products mixed with 1 ul 10* loading buffer were analyzed on a 1% agarose gel. T C R P and T C R 8 P C R primers are listed in Table 2.2. P C R for T C R P gene rearrangement was as described by Ikawa, et al63. The reaction volume was 20 ul, containing 1.5 ul of 10x P C R buffer (with M g C l 2 ) , 0.16 ^1 of 25 m M dNTPs, 0.4 ul each of 10 u M primers, and 0.2 ul of 5U/ul Taq D N A polymerase. Thermocycling conditions were as follows: 5 minutes at 94\u00C2\u00B0C followed by 35 cycles of 1 minute at 94\u00C2\u00B0C, 1 minute at 60\u00C2\u00B0C, 2 minutes at 72\u00C2\u00B0C and finally 10 minutes at 72\u00C2\u00B0C. Primers for T C R 5 P C R were described by Capone et al. 1 0 \u00C2\u00B0 . Thermocycling conditions were as follows: 5 minutes at 94\u00C2\u00B0C followed by 32 cycles of 1 minute at 94\u00C2\u00B0C, 30 seconds at 55\u00C2\u00B0C, 2 minutes at 72\u00C2\u00B0C and finally 7 minutes at 72\u00C2\u00B0C. 38 a Cluster y1 y2 y4 Vy 5 2 43 f - H f Cy1 Jy1 y1E i rnn cpJy3 Vy1.3 ^ cpCy3 y3E I i n n Cy2 y2E ^ Jy2 r~i n i Vy1.1 JY 4 jj II I G e n o m i c P C R s c h e m e Vy5 + Cy1 JY1 _L \ y1E Cy1 OR Vy5 Jy1 \ y1E _o_ Germline order NO PCR product Rearranged PCR product Figure 2.1. A . The murine TCRy locus. There are 4 clusters in the T C R y locus. Each cluster contains one J segment and one C segment and variable numbers of V segments. The nomenclature is that of Garman et a l . 1 0 3 . B. Schematic of genomic P C R design. The genomic P C R design was based on that by Itohara et a l . 1 2 9 . If the locus is in germline order, the primers specific to the V and J segment w i l l be too far apart to produce a P C R product. On the other hand, i f the V and J segment have been rearranged, the primers w i l l be close enough together and a P C R product w i l l result. 2.6. Southern blot. 10 ul of P C R products mixed with 1 ul 10* loading buffer were analyzed on a 1% agarose gel. The gel was alkaline blotted to BioRad's (Hercules, C A ) Zeta-Probe\u00C2\u00AE membrane. The southern blot was probed with a biotin-labeled oligonucleotide and visualized by Pierce's (Rockford, IL) North2South\u00C2\u00AE Chemiluminescent Nucleic A c i d Hybridization and Detection kit. The oligonucleotide probes were labeled with 3' end labeling D N A with biotin-14-dATP Invitrogen protocol. 39 2 . 7 . R T - P C R . R N A was isolated from cells with Q I A G E N ' s RNeasy\u00C2\u00AE M i n i K i t and reversed transcribed into c D N A with Q I A G E N ' s Omniscript\u00C2\u00AE Reverse Transcription kit. The c D N A samples for R T - P C R templates were equal to lOOng of R N A . Forward primers for Vy2 , 3, 4, and 5 were paired with a reverse primer for a constant region sequence that is shared by all T C R y gene clusters ( C y l , 2, and 4). The primer sequences are listed in Table 2.2. The reaction volume was 50 pi , containing 5ul of 10x P C R buffer, 1.5 u.1 of 50 m M M g C l 2 , 1 pi of 10 m M dNTPs, 1.25 ui each of 10 u M forward and reverse primers, and 0.5 pi of 5U/ul Taq D N A polymerase. Thermocycling conditions were as follows: 5 minutes at 96\u00C2\u00B0C followed by 32 cycles of 15 seconds at 96\u00C2\u00B0C, 40 seconds at 50\u00C2\u00B0C, 1 minutes at 72\u00C2\u00B0C and finally 10 minutes at 72\u00C2\u00B0C. 1 pi of P C R product was analyzed on a 1% agarose gel. 40 Table 2.2. List of primers Genomic P C R : Vy2: T G G A C A T G G G A A G T T G G A G Vy3: G A T C A G C T C T C C T T T A C C C Vy4: C T G G G G T C A T A T G T C A T C A A Vy5: G C T A A C C T A C C A T T C T C T G T J y l : C A G A G G G A A T T A C T A T G A G C V y l . l , Vyl.2: C T T C C A T A T T T C T C C A A C A C A G C Jy4: (PAIRS W I T H V y l . l ) : A C T A C G A G C T T T G T C C C T T T G G Jy2: (PAIRS W I T H V y l . 2 ) : A C T A T G A G C T T T G T T C C T T C T G C A A V54: C C G C T T C T C T G T G A A C T T C C V55: C A G A T C C T T C C A G T T C A T C C J51: C A G T C A C T T G G G T T C C T T G T C C V84: C C G C T T C T C T G T G A A C T T C C Dp2: G C A C C T G T G G G G A A G A A A C T Jp2.6: T G A G A G C T G T C T C C T A C T A T C G A T T N K G 2 A ( 5 ' ) : C C T T C T C A G G A G C A T C C C T G G A T N K G 2 A ( 3 ' ) : G A C A A A A C A G A T G A G G C C C A G G G Oligonucleotide probes: Jy2: C A A A T A C C T T G T G A A A G C C C G A G C Jy4: C A A A T A T C T T G A C C C A T G A T G T G C J y l : T G C A A A T A C C T T G T G A A A A C C T G A G J81: G T T C C T T G T C C A A A G A C G A G T T R T - P C R : Vy2: C C T T G G A G G A A G A A G A C G A Vy3: C A T C G G A T G A A G C C A C G T A Vy4: A G T G A C A G A A G A G G A C A C G Vy5: C G A T T C T G C T C T G T A C T A C T V y l . l : A A C T T C T A C C T C A A C C T T G A V y l . 2 : A A G T T C T A C C T C A A C C T T G G C-region: C T T A T G G A G A T T T G T T T C A G C 41 2.8. Sequencing of P C R products. R T - P C R products from thymocytes, IL-2-activated adult N K and newborn N K cells were purified using Wizard P C R preps D N A purification from Promega (Madison, WI). The P C R products were ligated into the p G E M - T easy vector (Promega). The plasmid clones were sequenced at the N A P S Sequencing Service (University of British Columbia, Vancouver, Canada). 2.9. Tissue culture: 2.9.1. L cells. The murine fibroblast L-cells were cultured in Dulbecco's modified eagle's medium (with 4500 mg D-glucose/L) ( D M E M ) plus 10% F B S , L-glutamine, penicillin, streptomycin, and 5x10\" 5 M 2-mercaptoethanol. 2.9.2. OP9 cells. OP9 stroma cells were cultured in Min imum essential medium eagle, alpha modification with nucleosides ( M E M ) with 10% F B S , penicillin, and streptomycin. 2.9.3. L A K cells. Single cell suspensions of bulk cells from spleen, lymph node, B M , thymus, liver or lung were cultured with 1000 U / m l IL-2 (PeproTech, Rocky H i l l , NJ) to expand the population. Cells were incubated in tissue culture dishes for 3 hours at 37\u00C2\u00B0C and the non-adherent cells were cultured for 7-10 days in RPMI1640 media containing 10% F B S , L -glutamine, penicillin, streptomycin, and 5x10\"5 M 2-mercaptoethanol. Media was changed during the middle of the culture. 2.9.4. Thymus or L N DN progenitor culture and B M NKP progenitor culture. Thymocytes, L N cells, or B M cells were blocked with 2.4G2 hybridoma culture supernantant and then stained with lineage marker mAbs (CD3, C D 8 , T C R p \ TCRy5 , CD19 , B220, Mac-1, G R - 1 , NK1.1 (and possibly N K G 2 A / C / E , Ly49G, and Ly49D), and Ter l 19). Lineage marker positive cells were removed from the sample with EasySep F ITC Positive Selection kit (StemCell Technologies). Thymus and L N cells were then stained with CD44 and CD25 42 mAbs and DN1 (Lin\"CD44 + CD25\") and D N 2 (Lin\"CD44 + CD25 + ) or pre-DN2 (Lin\" C D 4 4 + C D 2 5 l 0 ) cells were sorted. For B M N K P cells, cells were stained with NK1.1 and C D 122 and N K P cells were sorted ( L i n \" N K l . l \" C D 1 2 2 + ) . Cells were then seeded onto 0 P 9 stroma at 20,000-40,000 cells per 500 ul well of a 24-well plate. If less cells were sorted, all cells were seeded into one well . OP9 stroma was grown 2 days in advance in Min imum essential medium eagle, alpha modification with nucleosides ( M E M ) with 10% F B S and P/S to ensure that the stroma was confluent before D N or N K P progenitors were added. The OP9 media was removed and the progenitor cells were grown in M E M with 10% F B S and P/S as well as 150 u M monothioglycerol, 30 ng/ml stem cell factor (SCF), 100 ng/ml recombinant human Flt-3 ligand (Flt3L), 1 ng/ml IL-7, and 25 ng/ml IL-15. Ha l f o f the media was replaced on day 4 and i f necessary, cells were transferred to new OP9 with new media at a later stage. Cultures were grown for 10-12 days. 2.10. C e l l preparation. Cel l suspensions were prepared from spleen, thymus, or L N tissue and passed through a 70 um filter. Cells were washed, red blood cells were lysed with ammonium chloride and washed again. To prepare single cell suspensions of B M , muscle was removed from femur and tibia and B M was plunged from the bone with a syringe and 28 gauge needle. B M was then made to single cell suspension by passing through a 21 gauge needle repeatedly. Cells were washed, red blood cells were lysed and cells were washed again. For lung and liver, the tissues were perfused with 2% Phosphate buffered saline (PBS). The tissue was cut into small sections and digested with DNase and collagenase. Liver was digested in R P M I with 5% F B S , P/S, 2 M E . Lung was digested in D M E M with 5% F B S , P/S. Liver tissue was digested with 25 U / m l Dnase I and 250 U / m l collagenase IV while rotating at room temperature for 45 minutes. Lung tissue was digested with 50 U / m l Dnase and 250U/ml 43 collagenase IV while rotating at 37\u00C2\u00B0C for 1 hour. Tissues were then passed through a 70 urn filter and washed. The liver pellet was resuspended in 40% percoll (dilutions made with PBS) and layered on top of 70% percoll. For the lung, the percoll was first diluted to 90% percoll with 1 OX P B S . The lung pellet was resuspended in 44% percoll and layered on 67% percoll (dilutions made with D M E M ) . Gradients were spun at 2100 rpm for 20 minutes. The interface was collected, cells were washed, and red blood cells were lysed. To isolate B cells, bulk splenocytes from adult and newborn mice were stained with anti-CD19-biotin plus streptavidin-PE and anti-CD3-FITC, and C D 1 9 + CD3\" cells were purified by cell sorting. 2.11. Staining and F A C S sorting or analysis of cells. Cells were washed in 2% P B S , counted and pellets were incubated on ice for 15 minutes in 50 u.1 2.4G2 hybridoma supernantant per 4x 10 6 cells to block Fc receptors. m A b was then added at appropriate concentration for 30 minutes at 4\u00C2\u00B0C. Finally, propidium iodide was added to 5 u,g/ml. Cells were purified by cell sorting by a F A C S Caliber\u00C2\u00AE (BD, Mountain V i e w , C A ) . Sorted cells were checked for purity. For analysis of F A C S data CellQuestPro and W i n M D I were used. 2.12. IFNy production assay. Bulk cells (2 x 106) from L N , thymus or spleen were resuspended in 2 ml of R P M I media with 10% F B S , P/S, and 2 - M E with 1 ng of IL-12 and 0.5 ng of IL-18. These cells were incubated at 37\u00C2\u00B0C for 24 hours. Approximately 6 hours before the end of the culture, 1 ul of Golgi Plug (BD Biosciences) was added to each well . This inhibits the secretion of the IFNy produced and makes it accumulate in the cells. Intracellular 44 staining of IFNy was performed with the B D Biosciences B D Cytofix/Cytoperm\u00E2\u0084\u00A2 Plus kit. Cells were then analyzed by F A C S . 2.13. Cytotoxicity assay. R M A - S cells were used as target cells. These cells were fluorescence labeled using Invitrogen Vybrant C F D A SE Cel l Tracer kit. A stock C F D A SE solution was made at 100 u M and a working dilution of 1 u M was used to stain the R M A - S cells. The effector cells were either splenic or thymus N K cells that were cultured with IL-2 in a L A K culture. Thymus N K cells were prepared from B6 thymocytes depleted of C D 3 + cells or from TCRp\"7\"8\"7\" mice. 10,000 C F D A SE labeled R M A - S cells were mixed with either thymus N K or splenic N K cells at ratios of 1:1, 1:2, 1:5, 1:10, and 1:20 in 500 ul of R P M I in 24-well plates. Following a four-hour incubation, the cells were collected, washed and resuspended in PI buffer. The cells were then analyzed by F A C S and the percentage of C F D A S E + cells that were positive for PI buffer was recorded. 2.14. L N DN cell transplantation. 2.14.1. Intraperitoneal injection. DN1 and pre-DN2 cells were sorted from IL-15\"7\" mice as above. 20,000 cells were injected intraperitoneally in 500 ul P B S into three Nod Scid IL-2Ry\"7\" mice. Three weeks later, spleens, thymuses, B M , and L N were removed and examined by F A C S analysis for N K cells. 2.14.2. Intravenous injection. DN1 and pre-DN2 cells were sorted from Pep3b mice. 20,000 cells were injected intravenously in 500 ul P B S into two N o d Scid IL-2Ry\"7\" mice. Four weeks later, spleens and B M were removed and examined by F A C S analysis for N K cells. 45 2.15. Statistics. Data was analyzed statistically using the Student's T-test (Microsoft Excel). Differences of p<.05 were considered statistically significant. 46 3 IDENTIFICATION O F A N O V E L P A T H W A Y O F N K C E L L D E V E L O P M E N T T H A T IS T H Y M U S - D E P E N D E N T AND INCLUDES T C R G E N E R E A R R A N G E M E N T 1 3.1. Introduction Currently, the relationship between BM-derived N K cells and the N K cell potential demonstrated by T cell progenitors is unclear. Although adult D N thymocytes have N K cell potential, it is believed that this pathway is not realized in steady state N K cell development in normal mice since N K cell numbers are normal in nude mice, which lack a thymus. While it is well demonstrated that N K cells arise from bipotent T / N K cell progenitors found in the fetal liver, blood, spleen and thymus, it is currently thought that this pathway is restricted to the fetal environment. A s hematopoiesis switches from fetal liver to adult B M , it is assumed that the N K cell developmental pathway switches exclusively to the B M as well . In this regard, it is of interest that N K cells in fetal and neonatal mice are different from those in adult mice. The former express C D 9 4 / N K G 2 , which recognizes non- classical M H C class I Q a - l b , and Ly49E, but not other Ly49 receptors that recognize classical M H C class I, whereas the latter express a full repertoire of N K cell receptors. What causes the differences between fetal/neonatal and adult N K cells is still unknown, especially the control of Ly49 expression. We began this study by comparing gene expression patterns between adult and neonatal N K cells to look for differences that may suggest different pathways o f N K lineage commitment in neonatal and adult mice. Unexpectedly, we found that a subpopulation of N K cells expresses 1 A version of this chapter has been published. Veinotte L L , Greenwood CP, Mohammadi N , Parachoniak C A , Takei F. Expression of rearranged TCRy genes in natural killer cells suggests a minor thymus-dependent pathway of lineage commitment. Blood, 107, 2673-2679 (2006). 47 T C R y genes and that expression is higher in neonatal N K cells than adult N K cells. These studies suggest that a subset of N K cells develop in the thymus from T cell precursors that have rearranged T C R y genes. Therefore, since both adult and neonatal N K cells express T C R y genes, the bipotent T / N K cell pathway still contributes in the adult environment. 3.2. Results 3.2.1. Microarray analysis reveals expression of TCRy gene in N K cells To examine whether adult and neonatal N K cells follow different pathways of lineage commitment, global gene expression of IL-2 activated N K cells, which were purified by two rounds of cell sorting (over 99% N K 1 . 1 + CD3\"), were compared by performing triplicate microarray experiments on Affymetrix M G - U 7 4 A v 2 chips. The raw expression values of the microarray experiments were submitted to an online microarray database and they are available, at EBI ArrayExpress (http://www.ebi.ac.uk/arrayexpress/) with accession # E - M E X P - 3 5 4 . The genes were normalized and then filtered on expression, confidence, and fold change. A parametric student's t-test with a p-value cut off of 0.05 and a Benjamini and Hochberg false discovery rate multiple testing correction was applied. Out of 12,488 genes on the chip, 12 had statistically significant differences in expression between the two samples (Fig. 3.1). A s expected, 7 of the 12 differences in gene expression were from Ly49 genes with high expression in adult N K cells and low or absent in neonatal N K cells. 48 a Affymetrix probe ID p-value: : 6 e n e name 100325_at 0.0499 glycoprotein48 B 102744_at 0.0475 M.muscutus T cell receptor V gamma 2 arid T cell receptor J gamma 2 mRHA 102424_at .0.C0777 chemokine(C-C motJt)ligand3 93430/at; 0.00771 . chemokine (C-Cmotif) l igand5 94146_at 0.00267: : M.muscufus MIP-1b gene tot macrophage inflammatory protein 1b.; 10038?_f_at 0.00287 Ml us musculus natural killer cell receptor (L/491) gene, exon 7, partial cds L y 4 9 B 100326_f_at 0.00267 killer cell ledri-like receptor, subfamily A , members L y 4 9 H 94779_f_at 0.00287 killer cell lectin-like receptor, subfamily A . member3 L y 4 9 C 93893_f_at 0.000633 killer cell lectm-like receptor subfamily A . member 12 L y 4 9 C 93894_f_at ' 0.000333 killer'cell lectm-like receptor, subfamily A . member3 L y 4 9 C 97781J_at I 0.000443 killer cell lectin-like receptor, subfamily A , member? L y 4 9 G 2 97782_f_at 0.000381 killer cell lectsn-like receptor, subfamily A , member? L y 4 9 G 2 b II ;: \u00E2\u0080\u00A2 - \u00E2\u0080\u00A2 \u00E2\u0080\u00A2 - < \u00E2\u0080\u00A2 Adult .. . \u00E2\u0080\u00A2\u00E2\u0080\u00A2\u00E2\u0080\u00A2 ^Neonatal NK NK Figure 3 . 1 . Microarray data analysis of differentially expressed genes between adult N K and neonatal N K cell samples. Genes with statistically significant differences when grouped by 'Sample Source' (adult vs. neonatal samples); parametric test, variances assumed equal (Student's t-test). p-value cutoff 0.05, multiple testing correction: Benjamini and Hochberg False Discovery Rate. Genes from adult and neonatal were normalized and were filtered on expression, confidence, and fold change. These remaining genes were then tested by 1 - w a y A N O V A . (a) List of the genes with statistically significant differences after 1 - w a y A N O V A test, (b) Graph representation of the same group of genes as in (a). 49 Gp49 T cell receptor gamma chain Ccl3 Ccl5 Ly49H Ly49C Ccl4 Ly49B Ly49C Ly49C Ly49G2 Ly49G2 S a m p l e S a m p t e source s o u r c e P-valUft n a o n a t a l a d u l t O . M M 0.0475 0.00777 0.00771 0.00267 0.00267 0.00287 0.00287 0.000633 0 .000833 0000443 0.000381 O I S S3 BS Figure 3 . 1 . Microarray data analysis of differentially expressed genes between adult N K and neonatal N K cell samples. (c) Graph representation of the same group of genes as in (a). 50 The most striking and unexpected result was the expression of T C R y genes in both adult and neonatal N K cells. A n analysis of various T C R gene expression in N K cells showed that only TCRy genes were consistently detected in both neonatal and adult N K cells (Fig. 3.2a). T C R 8 gene expression was detectable at a lower level and the probe identification describes it as germline T C R 5 expression. T C R P gene expression was undetectable. Only one of three T C R a gene probes detected positive expression. The other T C R a and the p re -TCRa probes were negative and T C R a rearrangement only begins after full T cell lineage commitment and therefore, it is likely that this probe detected non-specific gene expression. Not only was T C R y gene m R N A expression in N K cells detected, its expression was also shown to be significantly higher in neonatal N K cells than in adult N K cells with a student's t-test p-value of 0.0475 (Fig. 3.2b). To confirm that T C R y is not expressed on the N K cell surface, T cells and N K cells were stained with TCRy8 m A b and F A C S analysis showed no expression on N K cells (Fig. 3.2c). 51 Figure 3.2. Detection of TCRy gene expression in N K cells by microarray analysis. (a) Gene expression patterns of purified IL-2 activated adult and neonatal N K cells were analyzed in triplicate using Affymetrix GeneChip Mouse Genome U 7 4 A v 2 arrays. Expression of a, P, y, and 8 T C R genes \u00C2\u00B1 s.d. are shown. The black bars represent neonatal N K cells and the white bars represent adult N K cells. The expression values (0-700 on the graph) are based on raw values after default normalization of the 6 chips, with the 3 adult samples grouped together and the 3 neonatal samples grouped together. For Affymetrix gene chips, each gene is represented by a probe set of 10-25 oligonucleotide pairs, each pair consisting of a perfectly matching probe and a probe with one nucleotide mismatch in the middle of the sequence. The detection call of whether a gene is present (expressed) or absent (not expressed) is based on binding to the perfect match and mismatch pairs. Therefore, an expression value can be assigned but it does not necessarily mean the gene w i l l be called present. Those with a * show that they are present and the expression values are valid. The affymetrix probe numbers for these genes are: p i : 101311, P 2 : 94202, P3: 99798, a l : 101823, a 2 : 97944, a3: 97945, pa: (preTa): 98354, 8 : 92328, y l : 102745, y2: 102685, y3: 102744. (b) Significant difference in expression levels of T C R y gene (probe 102744) in IL-2 activated adult N K cells (white bar) and neonatal N K cells (black bar) determined by a 1-way A N O V A (p-value: 0.0475). The values represent the log ratio which is the intensity ratio (adult N K cell sample gene divided by the neonatal N K cell sample gene) log transformed (log2). (c) Surface staining for TCRSy on neonatal N K cells and T cells from a splenic L A K culture show that N K cells do not express T C R on their cell surface. 52 3.2.2. TCRy genes are rearranged and expressed in N K cells The microarray data revealed T C R y gene expression in N K cells. However, the probes for the T C R y genes on the microarrays were specific for the 3' end of the transcripts and the results did not reveal whether the microarray data were detecting germline expression or expression of rearranged T C R y gene segments that resulted from V J recombination. In addition, i f it was detecting rearrangement, the microarray data did not show the extent of possible rearrangement combinations that were expressed. There are four clusters in the murine T C R y locus, each containing variable (V) , joining (J), and constant (C) regions. Cluster 1, which is the most commonly studied, consists of four V segments (Vy2, 3, 4, and 5), one J segment (Jyl) and one constant region (Cy l ) . To further characterize the T C R y gene expression in N K cells, genomic P C R was performed to determine whether T C R y genes are rearranged in N K cells. Forward primers specific to the V segments in the locus (Vy2, 3, 4, 5) and the reverse primers specific to their respective J segment (Jyl) were used. The genomic P C R was designed so that i f the locus was in germline configuration, the V and J primers would bind to segments too far apart from each other to produce a P C R product. On the other hand i f the V and J segments were rearranged, the primers would be close enough to each other to produce a P C R product of about 350 bp. IL-2-activated N K cells from adult and neonatal mouse spleen were purified as above and analyzed by genomic P C R . Southern blot analysis of the P C R products hybridized to Jy-specific oligonucleotide probes determined that neonatal N K cells exhibited rearrangement of all possible V - J combinations that were examined while adult N K cells had some of the possible rearrangements (Fig. 3.3a, left panel). The identity of the larger band seen for the adult N K cell Vy3- Jy l P C R is unknown. These results showed that T C R y gene rearrangement with multiple V - J combinations does occur in N K cells. To determine whether the rearranged T C R y genes are expressed in N K cells, R T - P C R was performed using the 53 primers specific to the V-segments and their corresponding C-region. Consistent with the genomic P C R results, neonatal N K cells expressed all of the possible combinations while adult N K cells expressed only some (Fig. 3.3a, right panel). The genomic and R T - P C R experiments were also performed with freshly isolated N K cells without culturing with IL-2, and the results were the same to those with IL-2 activated N K cells (data not shown). o 2j z as E o s c c c 3 a r - U _ < Z Control I i Z 1 l e g s \u00C2\u00A3 A t) r - U _ < 0) Vy2 p V J Vy3 . -Vy4 - 4m Vy5 Mil ! Genomic PCR RT-PCR Z JS . o e 2 < i? ^ DZ I- u_ Vy2-Jy1 Control Figure 3.3. T C R y gene rearrangement and expression in N K cells. (a) Southern hybridization with Jyl-specific oligonucleotide probe of genomic P C R (left panel) or R T - P C R (right panel) of IL-2 activated adult and neonatal N K cells to test for rearrangement of T C R y locus and expression of rearranged T C R y genes. Thymocytes are the positive control and fibroblasts (L cells) are the negative control. N K G 2 A P C R and Glyseraldehyde-3-phosphate dehydrogenase ( G A P D H ) R T - P C R were used as control for genomic and R T - P C R , respectively, (b) N K cell D N A from adult Rag2\"A was tested by genomic P C R for Vy2- Jy l rearrangement as in (a). 3.2.3. Specificity of rearrangement To confirm that the detected T C R y gene rearrangement occurs as a result of normal V(D)J recombination that requires R A G enzymes, N K cells from adult R A G 2 7 \" mice were sorted and tested by genomic P C R . A s expected, no T C R y gene rearrangement was detected in these N K cells (Fig. 3.3b). The specificity of the R T - P C R was also confirmed by cloning and 54 sequencing the P C R products from thymocytes and from purified adult and neonatal N K cells. The sequences showed that P C R cross-amplified non-specific T C R y genes. However, Southern hybridization to Jy-specific oligonucleotide probes detected only the specific sequences (data not shown). 3 .2 .4 . TCRy gene rearrangements in N K cells represent unselected, random recombination Since Vy2-Jy l recombination was most prominent among N K cells, R T - P C R sequencing results for this rearrangement were further analyzed. Vy2 has an in-frame stop codon at the 3' end, which can be removed during the V J gene recombination process. Out of the Vy2-Jy l rearrangements that were sequenced, 4 out of 9 (44%) were in-frame, productive rearrangements in adult N K cells and 3 out of 8 (44%) were in-frame, productive rearrangements in neonatal N K cells (Fig. 3.4). These are similar to the expected frequencies (33%) of random unselected rearrangements. 55 Vy2 Jy1 Germline TCC TAC GGC TAA T A G C T C A GGT TCC TAC GGC TAA TAG CTC AGG T > o TCC TAC GGC TAA CTG AGC TAT ATA T3 O i _ TCC TAC GGC T TT TAG CTC AGG T CL t o TCC TAC GGC TAA A G C T TA GCT CAG GT z TCC TAC GGC ATT A TA GCT CAG GT tive TCC TAC GGC TA T AGC TCA GGT )duc TCC TAC GGC CA T AGC TCA GGT i\u00E2\u0080\u0094 o_ TCC TAC GGC TA T AGC TCA GGT Figure 3.4. Sequences of productive and non-productive T C R y gene rearrangements. Sequences of neonatal N K cell R T - P C R products for V y 2 - C y l transcripts. Only the sequences at the junction of V y 2 - J y l are shown. The in-frame stop codon in Vy2 is underlined. 3.2.5. Tcrf N K cells represent a small population of total splenic N K cells To determine the percentage of splenic N K cells with T C R y gene rearrangement (termed Tcry N K cells hereafter), fresh and IL-2 activated N K cells from adult and neonatal mice were purified by two rounds of cell sorting. The levels of purity of the N K cell samples used in this experiment were always over 99% with 0-0.05% C D 3 + or T C R y 8 + (Fig. 3.5a). D N A was isolated from the purified N K cells and subjected to genomic P C R analysis for T C R Vy2-Jyl gene rearrangement. To determine the frequency of Vy2-Jy l rearrangements among N K cells, D N A was also isolated from purified y8T cells and mixed with fibroblast (L-cell) D N A at various ratios, and genomic P C R was performed in the same way. The intensity of the bands for adult and neonatal N K cells was compared to the various control percentages. To ensure that the starting amount of D N A was identical for each sample, the D N A was first measured with PICO green staining. Results consistently showed that about 5% of neonatal splenic N K 56 cells and about 1% of adult splenic N K cells had Vy2-Jy l gene rearrangements (Fig. 3.5b, c). The frequency was the same with freshly isolated N K cells and IL-2-activated N K cells, as the P C R bands were of similar intensity (Fig. 3.5b, d). Since these percentages are low, it was important to rule out the possibility of T cell contamination in the N K cell samples. The same experiment was also performed with N K cells from TCRP\"'^\" 7 \" mice which have no T cells in the spleen. T C R y gene rearrangement was still observed in these N K cell samples, thus ruling out the possibility of T cell contamination (Fig. 3.6). 57 a A _ J . .11 Adult Neonatal t 99.3 \u00E2\u0080\u00A2 CD3e 99.3 m id' ' ' w CWf f l C \u00E2\u0080\u00A2 * * 5 Z Z d Z = (0 (0 * i \u00C2\u00B1i C C D D O O 20% 10% 5% 2.5% 1% 0% < < z z %yoTcell DNA CM \u00E2\u0080\u0094 d 1 10% 5% 1% 0% -g \u00C2\u00A7 < z % y5T cell DNA 7 1 % v5T cell DNA t c ~5 o T3 0) 10% 5% 1% 0% < z Figure 3 . 5 . L o w frequency of N K cells wi th rearranged T C R y genes. (a) Purity of IL-2 activated N K cell samples from adult and neonatal mice after two rounds of cell sorting. The numbers show the percentages of N K cells (NK1.1 + CD3\") . (b) Genomic P C R (Vy2-Jyl) performed with 8yT cell D N A and fibroblast D N A mixed at various ratios and with fresh and IL-2 activated adult and neonatal N K cells. The P C R products were analyzed by agarose gel electrophoresis and stained with ethidium bromide, (c) Southern hybridization to Jy l probe of the genomic P C R products generated from IL-2-activated N K cells from adult and neonatal mice in (b). The top panel shows a short exposure of the southern blot whereas the bottom panel shows a long exposure to visualize rearranged Vy2- Jy l in adult N K cells, (d) Southern blot of genomic P C R , as in (c), but with freshly isolated adult and neonatal cell D N A . Gels divided by lines are groupings of images from different parts of the same gel. 58 % y8T cell DNA 10% 5% 2.5% 1% 0% or \u00C2\u00AB o * I\u00E2\u0080\u0094 z Figure 3.6. 7c#y+ N K cells are not due to contamination of T cells. The frequency of IL-2 activated N K cells from TCRp'^TCRS\" 7 \" mice with Vy2-Jy l rearrangement was estimated by genomic P C R and ethidium bromide staining of agarose gel. Genomic P C R (Vy2-Jyl) was performed with 8yT cell D N A and fibroblast D N A mixed at various ratios and with N K cell D N A . 3.2.6. N K cells have a germline TCRP locus and may have initiated TCR8 rearrangement Rearrangement of T C R p and T C R 8 genes in N K cells was also tested by genomic P C R . Rearrangement of T C R p genes was not detectable in neonatal and adult N K cells with the genomic P C R scheme used (Fig. 3.7a). On the other hand, a very small fraction (less than 1%) of neonatal N K cells had V84-J81 rearrangement (Fig. 3.7b). 59 a Germline TCRp H Rearranged TCRp % yST cell DNA W 1 a) \u00C2\u00B1; c 10% 2.5% 1% 0% -a S < z V54 -J51 V55 -J51 Figure 3.7. T C R p and T C R 8 gene rearrangements in N K cells. D N A from purified IL-2 activated adult and neonatal N K cells was tested by genomic P C R for T C R P (a) or T C R 8 (b) rearrangement, (a) Genomic P C R using primers specific to Dp2 and JB2.6 genes was analyzed by agarose gel electrophoresis and stained with ethidium bromide. The largest band represents non-rearranged germline T C R p locus whereas multiple smaller bands represent T C R p gene rearrangements. Thymocyte D N A was used as positive control and fibroblast D N A was used as negative control, (b) Genomic P C R (V84-J81 or V85-J81) performed with 8yT cell D N A and fibroblast D N A mixed at various ratios and with fresh and IL-2 activated adult and neonatal N K cells. The P C R products were analyzed by agarose gel electrophoresis, blotted and hybridized to J81 -specific oligonucleotide probe. Gels divided by lines are groupings of images from different parts of the same gel. 3.2.7 TCRy gene rearrangement found in N K cells does not occur in CLPs which generate mature B cells 7 7 7 8 R A G genes have been shown to be activated in C L P s in the B M ' , and about 5% of adult N K cells have been shown to have rearranged immunoglobulin heavy chain gene 2 7. Therefore, whether T C R y genes are also rearranged in B cells in neonatal and adult mice was tested. Genomic P C R analysis of purified B cells detected no Vy2-Jyl rearrangement in B cells (Fig. 3.8). These results suggest that a subpopulation of N K cells develop from thymic T / N K bipotential progenitors that have rearranged T C R y genes and lost B cell potential. 60 Neonatal Adult o CO 0 ) S 1 IT 5 t\u00E2\u0080\u0094 u-Vy2-Jy1 control Vy2-Jy1 control Figure 3.8. L a c k of T C R y gene rearrangement in B6 mouse B cells. Southern blot with J y l probe of genomic P C R (Vy2-Jyl) of B6 adult and neonatal splenic B (CD19 + CD3\"). Genomic P C R for a part of N K G 2 A gene confirms the presence of B cell D N A . 3.2.8. The thymus is required for the development of Tcry+ N K cells The T C R gene rearrangement in N K cell subsets suggested that they develop in the thymus, since this is the location where the majority of T cells undergo T C R V ( D ) J recombination. To examine whether the Tcry+ N K cells develop in the thymus, N K cells were isolated from nude mice, which lack a proper thymic environment and lack conventional T cells. Only extrathymic T cells accumulate in the spleen of older nude mice (up to 5.4% of splenocytes) (Fig. 3.9a). It was known that N K cells were present at normal or elevated levels in adult nude mice, but the N K cell status of neonatal nude mice was not known. Normal numbers o f N K cells were found in the spleen of neonatal (3 day old) nude mice. Genomic P C R analysis of freshly isolated N K cells and IL-2-activated N K cells from nude mice showed no Vy2-Jy l rearrangement in N K cells from nude mice (Fig. 3.9b). It should be noted that extrathymic T cells, which accumulate in the spleen of old nude mice, had rearranged T C R y genes, but N K cells isolated from the same mice did not (Fig. 3.9b) demonstrating that the T C R rearrangement can still occur in these mice and the absence of it in N K cells is due to the lack of the thymus. 61 Furthermore, genomic P C R analysis of highly purified fresh and IL-2 activated N K cells from the B M and thymus showed that T C R y gene rearrangement is present in at least half of thymic N K cells while very low (-5% or less) rearrangement was detected in B M N K cells (Fig. 3.10). Therefore, the thymus is required for the development of Tcry+ N K cells. Adult nude CD3 Neonatal nude 2 . 3 0 . 0 5 m 0 . 1 5 0' w w CD3 b Figure 3 . 9 . Lack of TCRy gene rearrangement in nude mouse N K cells and high TCRy gene rearrangement in thymus N K cells. ( a ) Percentages of N K (NK1.1 + CD3\") cells and T (NK1.1\"CD3 + ) cells in the spleen of 1 year old (left) and three day old (right) nude mice, ( b ) Agarose gel electrophoresis and ethidium bromide staining of genomic P C R (Vy2-Jyl) of IL-2 activated and fresh N K cells from adult and neonatal nude mice. T cells that accumulate in aged nude mice were also isolated from spleen of the same adult mouse by cell sorting. Thymocytes were used as positive control and fibroblasts (L cells) were used as negative control. Genomic P C R for a part of N K G 2 A gene confirms that comparative amounts of template D N A was used for all the genomic P C R . 62 I\"* \u00E2\u0080\u00A2'\u00E2\u0080\u00A2Jv-\"- o o o> 6 \u00E2\u0080\u00A2->% o o o 1 0 ' o CD \u00C2\u00B0 10\u00C2\u00B0 101 102 10\u00C2\u00B0 104 CD3 87 c CD -O) IM o 4 10\u00C2\u00B0 4 o crioj 10 3 10 4 0.2 ft 0.1 J i g 101 1 0 2 103 104 10\u00C2\u00B0 101 102 103 10* NK1.1 NK1.1 NK1.1 120 110 100 90 80 u D l 70 RJ C 60 S y so 6 D_ 40 -H 30 20 10 0 \u00C2\u00B1 o f gP a* 4* / \u00E2\u0080\u00A2 DN1 \u00E2\u0080\u00A2 D M 2 \u00E2\u0080\u00A2 NKP 4T Figure 4.2. Thymus D N 1 and D N 2 progenitors have the potential to give rise to N K cells during in vi t ro cultures. B M N K P s and DN1 and D N 2 progenitors were cultured on OP9 in N K cell differentiation conditions. (a,b) Profiles of cells at the end of cultures, (a) values are representative of one culture set. (b) Averages of receptor expression each from at least 4 cultures (except 2B4). 74 4.2.2. DN2-derived N K cells are Ter/ The D N l - and DN2-derived N K cells were sorted and T C R y gene rearrangements were examined. Semi-quantitative genomic P C R showed that 25-50% of DN2-derived N K cells had Vy2-Jyl rearrangements while DNl-der ived N K cells had low T C R y gene rearrangement with about 5% of cells being positive (Fig. 4.3a,b). This is to be expected since T C R gene rearrangement only begins at the D N 2 cell stage. Therefore, Tcry N K cells likely arise from DN2 cells in vivo while other thymus N K cells likely arise from the earlier D N l progenitors which have not begun T C R y gene rearrangement yet. %y8T cell DNA 100 50 25 10 5 0 d) o o Z Z TJ TJ aj 0) > > \u00E2\u0080\u00A2c \u00E2\u0080\u00A2c 0) 0) T> TJ T\u00E2\u0080\u0094 (N z z Q Q = j * % a> s z z \" 2 - CM k - a Z Z % rr Q Q %y5T cell DNA 0) 0) 0 o ^ z z \u00E2\u0080\u00A2 a T J 1 s c c D a) XJ TJ T - CN 100 50 25 10 5 0 Q \u00C2\u00B0 tot H Genomic PCR control Figure 4.3. Thymus DN2 derived N K cells have rearranged TCRy genes. N K cells produced during in vitro cultures of D N l and D N 2 thymocytes were tested by genomic P C R for T C R y gene rearrangements. Genomic P C R (Vy2-Jyl ) was performed with 8yT cell D N A and fibroblast D N A mixed at various ratios and with N K cell D N A . (a) Ethidium bromide stained gel of genomic P C R and control N K G 2 A P C R . (b) Southern blot of genomic P C R with Jy l probe. Two exposures of the same membrane are shown. 4.2.3. Thymus N K cells are phenotypically different from N K cells in other tissues In the previous chapter, it was shown that almost 50% of thymus N K cells have rearranged TCRy genes, suggesting that the majority of thymus N K cells develop in the thymus. To determine i f the thymus-dependent pathway of N K cell development produces N K cells that 75 differ from those in other tissues, we performed F A C S analysis of N K cells from the thymus and compared them to N K cells from the spleen, liver, lung, B M , and mesenteric lymph nodes. The N K cell profile is difficult to visualize in the wild type thymus with such a large percentage of C D 3 + cells, and depletion of T cells seems to skew the N K cell profile as well . Therefore, thymuses from T C R P \" 7 ^ 7 \" mice were used. N o T cells or N K T cells are present in these mice and the N K cell population is easily visualized. A s expected, N K cell receptor expression on splenic N K cells from these mice is the same as B6 splenic N K cells (Table4.1b). There are many significant differences in receptor expression on thymus N K cells compared to those of other tissues. Thymus N K cells exhibit both extremes of expression when compared to other tissues (Table 4.1a). 76 Table 4 . 1 . Global F A C S analysis of N K cells from the thymus, L N , B M , spleen, liver and lung. (a) Averages of percentages of receptor expression on N K cells from various tissues determined by F A C S analysis. Values are from triplicate (or more) experiments. Values marked with * are significantly different than spleen N K cells with a p-value <0.05. (b) Percentages of receptor expression on N K cells from the spleen of T C R p ^ 7 \" mice. a Thymus LN B M Spleen Liver Lung NKG2ATC/E 74.3 \u00C2\u00B1 10.4* 55.7 \u00C2\u00B1 1.5 47.7 \u00C2\u00B1 5.7 45.0 \u00C2\u00B1 5.6 56.8 \u00C2\u00B1 5.0 59.0 \u00C2\u00B14 .6 CD94 83.7 \u00C2\u00B1 1.2* 56.3 \u00C2\u00B10 .6 52.3 \u00C2\u00B13 .5 47 .7+4 .0 57.0 \u00C2\u00B1 5.6 59.7 \u00C2\u00B16 .0 Ly49A 4.0 \u00C2\u00B1 1.0* 16.5 \u00C2\u00B1 2.3 17.3 \u00C2\u00B1 2.9 14.5 \u00C2\u00B1 0.7 13.3 \u00C2\u00B1 2.5 11.0+1.0 Ly49G 19 \u00C2\u00B1 3.6* 43.8 \u00C2\u00B1 2.5 51.0 \u00C2\u00B1 2.8 47.7 \u00C2\u00B1 6.8 41.0 \u00C2\u00B1 0.0 35.0 \u00C2\u00B1 5.2 Ly49C/l 8.3 \u00C2\u00B1 1.5* 25.8 \u00C2\u00B1 2.2 29.0 \u00C2\u00B15 .3 46.0 \u00C2\u00B1 2.6 33.0 \u00C2\u00B1 9.5 23.0 \u00C2\u00B1 3.0 Ly49D . 11.0 \u00C2\u00B1 3.5* 34.6 \u00C2\u00B1 6.6 41.5 \u00C2\u00B1 0.7 43.5 \u00C2\u00B1 3.3 n.d n.d. Ly49H 21.0 \u00C2\u00B1 1.7 12.3 \u00C2\u00B1 3.5 17.7 \u00C2\u00B12 .1 27.3 \u00C2\u00B1 5.9 n.d. n.d. 2B4 94.0 \u00C2\u00B1 2.0 81.0 \u00C2\u00B1 6.1 81.3 \u00C2\u00B1 12.9 87.3 + 9.7 94.7 \u00C2\u00B1 2.9 82.0 \u00C2\u00B1 26.0 KLRG1 n.d. 6.0 + 2.9 2.4 \u00C2\u00B1 2.2 5.0 \u00C2\u00B1 3.6 4.3 \u00C2\u00B1 2.5 14.0 \u00C2\u00B1 1.7 Mac-1 12.0 \u00C2\u00B1 5.0* 52.5 \u00C2\u00B1 8.8 59.8 \u00C2\u00B1 8.2 76.3 \u00C2\u00B1 9.0 78.0 + 5.7 96.7 \u00C2\u00B1 0.6 IL7Ra 65.0 + 7.9* 25.5 \u00C2\u00B1 1.7 12.8 \u00C2\u00B1 3.7 7.3 \u00C2\u00B1 3.2 4.3 \u00C2\u00B12 .2 5.7 \u00C2\u00B12 .3 b TCRS'-<-6-'- Spleen N K G 2 A / C / E 49.0 Ly49G 48.0 Ly49C/ l 50.0 Mac-1 76.0 IL7Ra 5.0 Cells expressing N K G 2 A , CD94, 2B4, and I L - 7 R a are most abundant among thymus N K cells while those expressing Mac-1, L y 4 9 A , C/I, G , and D are very rare (Fig. 4.4, 4.5, 4.6). More thymus N K cells express L y 4 9 H than lymph node and B M N K cells but less than splenic N K cells. For the L y 4 9 H values, double staining with 5E6 (Ly49C/I) and 1F8 (Ly49C/I/H) mAbs was performed. The L y 4 9 H value represents L y 4 9 H single positive cells, so the value is lower 77 than reported values since double positive (Ly49H + Ly49C/ I + ) cells were excluded. Interestingly, the percentage o f I L - 7 R a + cells is very high in thymus N K cells and L N N K cells are the only other population with a moderate percentage o f I L - 7 R a + cells (Fig. 4.4). Also, thymus N K cells have the lowest Mac-1 expression, with L N N K cells a distant second. A l l thymus N K cell receptors have significantly different expression pattern from splenic N K cells except L y 4 9 H and 2B4. The intensity of receptor staining did not differ between N K cell populations. These results suggest an immature N K cell phenotype ( C D 9 4 / N K G 2 h l L y 4 9 l o M a c 1 \u00C2\u00B0) of thymus N K cells and also a most closely shared phenotype with L N N K cells. 120 Thymus Lymph BM Spleen Liver Lung node Figure 4 . 4 . Thymus N K cells appear 'immature' ( M a c - l l o I L - 7 R a h l ) compared to other tissue N K cells. F A C S analysis of Mac-1 and I L 7 R a expression on N K cells from various tissues. The percentages of I L - 7 R a and Mac-1 expression in the thymus N K cells are statistically significant from spleen N K cells with a p-value >0.05. 78 Figure 4.5. Thymus N K cells have the highest percentages of N K G 2 A / C / E and CD94 expression and average 2B4 expression. F A C S analysis of N K G 2 A / C / E , CD94, and 2B4 expression on N K cells from various tissues. The percentages of N K G 2 A / C / E and CD94 on thymus N K cells are statistically significant from spleen N K cells with a p-value >0.05. 60 L y 4 9 A L y 4 9 G L y 4 9 C / l L y 4 9 D L y 4 9 H Figure 4.6. Thymus N K cells have the lowest percentages of Ly49A, G , D, and C/I expression. F A C S analysis of Ly49 expression on N K cells from various tissues. The expression of Ly49A, Ly49G, Ly49C/I and Ly49D are all significantly different between thymus and spleen N K cells. 4.2.4. TCRy gene rearrangement in L N N K cells suggests a link with DN-derived thymus N K cells To determine whether the N K cells produced via the thymus-dependent developmental pathway migrate to other tissues, we purified N K cells from lung, liver, and both mesenteric and peripheral L N s and checked for presence of T C R y gene rearrangement by genomic P C R since this is a marker of N K cells that originated from the thymus pathway rather than the B M pathway. Tissues with a significant Tcry N K cell population have N K cells that originate via the thymus-dependent pathway and selectively migrate to the tissue. A s shown in chapter 3, 50% of thymus N K cells have T C R y gene rearrangement while less than 5% of splenic and B M N K cells do (Fig. 4.7c). Lung N K cells have negligible levels of T C R y gene rearrangement as do liver N K cells, except on one occasion where a visible band was detected. Both peripheral and mesenteric L N N K cells, on the other hand, consistently had T C R y gene rearrangement (Fig. 4.7a). Semi-quantitative genomic P C R showed that approximately 20-25% of L N N K cells are positive for T C R y gene rearrangement (Fig. 4.7b). 80 a b 0) 0) CL V) CO. 0) T3 o CL r-n i _ o E C &> CN >i 3 .> X _ l _ l _ l T J %yoT cell DNA 100 50 25 1 0 Thymus: 50% LN: 15-20% BM: 5% Spleen: 1% Lung: 1% Liver: 1% %y8T cell DNA 100 50 25 10 z z Figure 4.7. L N N K cells have the highest percentage of Tcry N K cells other than thymus N K cells. (a) Equivalent amounts of D N A from N K cells from each tissue were examined by genomic P C R for the presence of T C R y gene rearrangement. Agarose gel electrophoresis and ethidium bromide staining of genomic P C R is shown, (b) Southern blots o f genomic P C R with Jy l probe was carried out as in Figure 3.5 to estimate the percentage o f T C R y gene rearrangement in mesenteric ( M - L N ) and peripheral (P-LN) L N N K cells. Genomic P C R (Vy2-Jyl) was performed with 8yT cell D N A and fibroblast D N A mixed at various ratios and with N K cell D N A . (c) A comparison of the percentage of T C R y gene rearrangement in N K cells of multiple tissues. 4.2.5. D N l and pre-DN2 cells in LNs give rise to N K cells in culture The above results suggested thymic origin of L N N K cells. However, it was unknown whether they develop in the thymus and migrate to the L N or whether immature thymocytes migrate to the L N and differentiate into N K cells within the L N . Terra et al.141 previously showed that L N s have D N l and pre-DN2 (CD44 + CD25 l 0 ) progenitors present. The D N l cells differ from the E T P profile of the thymus, and no C D 2 5 h l D N 2 or D N 3 cells are present in the L N . Furthermore, L N D N cells in normal mice are unable to differentiate into T cells whereas their a 56 12 L.I ,. , Lin cocktail C D 2 5 b ! - | :\u00E2\u0080\u009E\u00E2\u0084\u00A2..\u00E2\u0084\u00A2\u00E2\u0080\u009E\u00E2\u0080\u009E_\u00E2\u0080\u009E,\u00E2\u0080\u009E. - O NK1.1 NK1.1 Z z I %y8T cell DNA | z 100 50 25 10 0 g CL I (WPWHr I Figure 4.8. D N 1 and pre-DN2 progenitors are present in the lymph node and they possess N K cell potential in vitro. (a) Lin\" cells in the L N were gated and DN1 and pre-DN2 profiles are shown, (b) DN1 and pre-DN2 cells were sorted and cultured in N K cell differentiation conditions as in Figure 4.1. F A C S profile of cells at the end of one representative culture is shown, (c) Southern blot of genomic P C R with Jy l probe of N K cells that differentiate during the culture was used to estimate the percentage of N K cells with T C R y gene rearrangement. Genomic P C R (Vy2-Jyl) was performed with 5yT cell D N A and fibroblast D N A mixed at various ratios and with N K cell D N A . N K cell potential has not been addressed. To test the possibility of thymus-derived progenitors migrating to the L N and producing thymus-dependent N K cells, the N K cell potential of the L N DN1 and pre-DN2 cells was tested. The D N cells were purified and cultured for N K cell differentiation as described for D N thymocytes. The majority of cells recovered from the 82 cultures were N K cells very similar to those from D N thymocyte cultures. These N K cells expressed a detectable level of Ly49 receptors as well (Fig. 4.8a,b). L N DN-derived N K cells were purified by cell sorting and T C R y gene rearrangement was checked by genomic P C R . A s seen in the thymus cultures, DNl-der ived N K cells had negligible levels of rearrangement while pre-DN2-derived N K cells had significant levels of rearrangement (Fig. 4.8c). To confirm that the N K cells were not from contaminating N K cells that expanded during the culture, the same in vitro culture was performed with D N l and pre-DN2 cells from IL-15\"7\" mice which have greatly reduced N K cell numbers. The end result of these cultures was the same as the wi ld type cultures. Once again the majority of cells were N K cells and they expressed detectable levels of Ly49 receptors (Fig. 4.9). IL15' DN1 92 i9 -:io! ' : - W .10* i .\u00E2\u0080\u00A2.: fl.2H - \u00E2\u0080\u00A2 IL15-/- Pre-DN2 91 R2 3 CD3 12 \u00E2\u0080\u00A2if* *> 19? \u00E2\u0080\u00A2 ' \u00E2\u0080\u00A2:1D 18V. 10' \u00E2\u0080\u00A2 . :5SC-W.: .. Ly49G Figure 4.9. DN progenitors from IL-15\"7\" mouse LNs still show N K cell potential in vitro. D N progenitors from IL-15\"7\" mice, which lack N K cells, were cultured in N K cell differentiation conditions as in Figure 4.1. F A C S profiles of cells recovered from a representative culture. 83 4.2.6. The DN cells and Ter/ N K cells in the L N are thymus-dependent To confirm that the Tcry N K cells in the L N were thymus-dependent rather than being derived through an alternate B M developmental pathway, we checked for Tcry N K cells in the LNs of nude mice, which lack a thymus. The N K cells in nude L N s lack T C R y gene rearrangement. Therefore, the population from the thymus-dependent pathway is absent in these mice (Fig. 4.10b). In addition, we found that D N l and pre-DN2 cells are greatly reduced in nude mouse L N . In wi ld type mice, approximately 50% of Lin\" cells are D N l whereas in the nude mouse, only 3-9% of Lin\" cells are D N l progenitors. Pre-DN2 cells were 7 fold fewer in nude LNs (Fig. 4.10a). These results indicate that L N D N cells derive from the thymus. a UJ R2 \u00E2\u0080\u00A2xt \"xt Q O 10\u00C2\u00B0 10' 10* 10\" 10* Lin cocktail 2 1 2 2 0) >1 o o E "Thesis/Dissertation"@en . "10.14288/1.0100554"@en . "eng"@en . "Genetics"@en . "Vancouver : University of British Columbia Library"@en . "University of British Columbia"@en . "For non-commercial purposes only, such as research, private study and education. Additional conditions apply, see Terms of Use https://open.library.ubc.ca/terms_of_use."@en . "Graduate"@en . "Novel pathway of thymus-dependent NK cell development"@en . "Text"@en . "http://hdl.handle.net/2429/31179"@en .