TARGETING NATURAL KILLER T CELLS TO ENHANCE IMMUNITY: A NOVEL SWINE MODEL By BIANCA LIBANORI ARTIAGA A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR TH E DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2014
Â© 2014 Bianca Libanori Artiaga
To my loving f amily and in memory of those gone
4 ACKNOWLEDGMENTS First, I would like to express my great appreciation to my professor advisor, Dr. John Driver, for giving me the opportunity and support to obtain my Master of Science degree at University of Florida. His valuable guidance and constructive suggestions duri ng the last two years were of major significance to my learning experience and the progress of this research work. I would like to extend my gratitude to the other committee members, Dr. Char les Staples and Dr. Corwin Nelson , for their useful critiques and mentoring during development and analysis of results regarding our research work. I offer my special thanks to Robert Whitener , Dr. Brian Wilson and Dr. Clayton Mathews for their guidance and ability to encourage my critical thinking about research. Also , their technical assistance was very appreciated, by offering us access to their laboratory. I wish to thank the entire faculty at the Department of Animal Sciences at University of Florida, especially Dr. JosÃ© Santos for the internship opportunity and fo llowing intervention that allowed me to be accepted into the Animal Science Graduate Program. Also, special thanks for Dr. Corwin Nelson, Dr. Stephanie Wohlgemuth, Dr. Lokenga Badinga, Dr. Timothy Hackmann, Dr. Peter Hansen, Dr. Geoffrey Dahl, Dr. William Thatcher, Dr. Charles Staples and Dr. JosÃ© Santos, for sharing their laboratories and general assistance. I would like to express my appreciation to the technical assistance prov ided by Dr. Joel Brendemuhl, Tom Crawford and the staff at UF Swine Unit for t heir patience to teach me the practical aspects of swine research and management .
5 I would like to thank the staff at the Department of Animal Sciences, particularly to Joann Fischer, Pam Krueger, Sabrina Robinson and Toyuna Grant for all their help. My sp ecial thanks are extended to all the students that helped my during the experiments, Paula Mercadante, Sarah Lewis, Qizhang Li, Megan Di Lerna, Zoe Siemienski, Mercedes Kweh, Gabriel Gomes and Vitor Mercadante. Their valuable technical assistance was essen tial for this research. I would also like to thank Dr. John Lednicky and Julia Loeb, Dr. Jeffrey Abbott and Dr. Jurgen Richt and Dr. Qinfang Liu, for enabling me to visit their laboratories, teaching and giving me access to their research protocols. I wis h to thank all my friends around the world for their help and support through bad and good times, especially Ana Monteiro, Izabella Michelon, Paula Mercadante, Maria Cortez, Ebru Karakaya, Grace Dourado, Anna Denicol, Gabriel Gomes , Alana CalaÃ§a, Erika Gan da, Fernanda Antunha, Isis de Carvalho, Mariana SimplÃcio and Rafaella Lobo . Finally, I thank all my family, particularly my parents , OsÃ³rio Artiaga and Regina Libanori , and my sister LuÃsa Libanori Artiaga for their love and encouragement throughout the last two years.
6 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ .. 4 LIST OF TABLES ................................ ................................ ................................ ............ 8 LIST OF FIGURES ................................ ................................ ................................ .......... 9 LIST OF ABBREVIATIONS ................................ ................................ ........................... 10 ABSTRACT ................................ ................................ ................................ ................... 12 CHAPTER 1 LITERATURE REVIEW ................................ ................................ .......................... 14 General Characteristics of Natural Killer T Cells ................................ ..................... 14 CD1d ................................ ................................ ................................ ................ 1 5 Thymic Development of NKT Cells ................................ ................................ ... 17 Effector Functions of NKT Cells ................................ ................................ .............. 18 NKT Cell Crosstalk with Immune Cells ................................ ............................. 19 NKT Cell Subsets ................................ ................................ ............................. 20 Modes of NKT Cell Activation ................................ ................................ ........... 21 Direct NKT cell activation ................................ ................................ ........... 22 Indirect NKT cell activation ................................ ................................ ......... 23 Role of NKT Cells in Disease ................................ ................................ ........... 24 NKT Cell Antigens ................................ ................................ ............................ 26 Therapeutic Targeting of NKT Cells ................................ ................................ ........ 28 Cancer ................................ ................................ ................................ .............. 29 Autoimmunity ................................ ................................ ................................ .... 29 Inflammation ................................ ................................ ................................ ..... 30 Infection ................................ ................................ ................................ ............ 31 Swine NKT Cells ................................ ................................ ................................ ..... 32 General Characteristics ................................ ................................ .................... 32 Effect of GC on Pig NKT Cells ................................ ................................ ....... 34 Summary ................................ ................................ ................................ ................ 35 2 ADJUVANT EFFECTS O F THERAPEUTIC GLYCOLIPIDS ADMINISTERED TO A COHORT OF NKT CELL DIVERSE PIGS ................................ .................... 41 Materials and Methods ................................ ................................ ............................ 43 Pigs ................................ ................................ ................................ .................. 43 Isolation of Leukocytes from Blood, Spleen, Thymus and Lymph Nodes ......... 43 Preparation of Glycolipids ................................ ................................ ................. 44 Ex perimental Design for Hen Egg Lysozyme Challenge ................................ .. 44 Flow Cytometry Analyses and Reagents ................................ .......................... 45
7 ELISA and IFN Enzyme Linked Immunosorbent Spot Assays ........................ 46 Statistical Analysis ................................ ................................ ............................ 47 Results ................................ ................................ ................................ .................... 48 Diversity in Pig NKT Cell Frequency, Subset Distribution and Function ........... 48 GC, OCH, and C glycoside Differentially Stimulate NKT Cell Activation and Adjuvant Responses in Pigs ................................ ................................ ... 50 Adjusting GC Dosage for Body Weight Improves the Consistency of Immune Responses Induced by NKT Cells ................................ ................... 52 Activated NKT Cells Differentially Regulate Antibody and Cellular Immune Responses ................................ ................................ ................................ .... 54 Discussion ................................ ................................ ................................ .............. 55 Conclusion ................................ ................................ ................................ .............. 60 3 GENERAL CONCLUSION AND FUTURE PERSPECTIVE ................................ .... 71 LIST OF REFERENCES ................................ ................................ ............................... 72 BIOGRAPHICAL SKETCH ................................ ................................ ............................ 95
8 LIST OF TABLES Table page 2 1 Ch aracteristics of pigs used for Experiment 1 and E xperiment 2 ....................... 61
9 LIST OF FIGURES Figure page 1 1 CD1d tetramer reagent interacting wit h a natural killer T (NKT) cell ................... 37 1 2 NKT cell crosstalk with immune cells ................................ ................................ .. 38 1 3 NKT cells subsets in mice ................................ ................................ .................. 39 1 4 Modes of NKT cell activation ................................ ................................ .............. 39 1 5 NKT cell antigens ................................ ................................ ............................... 40 2 1 Setup for hen egg lysozyme (HEL) immunization experiments .......................... 62 2 2 Comparison of pig NKT cell frequencies between different tissues .................... 62 2 3 Tissue distribution of NKT cell subsets ................................ ............................... 64 2 4 NKT cell cytokine responses to PMA/ion omycin ................................ ................. 65 2 5 Results of Experiment 1 that analyzed pig NKT cell responses to GC, OCH, and C glycoside ................................ ................................ ................................ .. 66 2 6 Results of Experiment 2 that analyzed pig responses to different doses of GC ................................ ................................ ................................ .................... 68 2 7 NKT cells differentially regulate antibody and cellular IFN production .............. 70
10 LIST OF ABBREVIATIONS GC Alpha galactosylceramide AHR Acute airway hyperreactivity APC Antigen presenting cell CCL Chemokine C C motif ligand CD Cluster of differentiation CDR Complementarity determining regions CLN Cervical lymph node CXCL Chemokine C X C motif ligand DAMP Danger associated molecular patterns DC Dendritic cell EAE Experimental autoimmune encephalomyelitis ELISA Enzyme linked immunosorbent assay ELISPOT Enzyme linked immunosorbent spot assay GM CSF Granulocyte macrophage colony stimulating factor HEL Hen egg lysozyme ICOS Inducible co stimulatory molecule IFN Interferon gamma Ig Immunoglobulin iGb3 Isoglobotrihexosylceramide IL Interleukin MAIT Mucosal associated invariant T cell MFI Mean fluorescence intensity MHC Major histocompatibility complex
11 MLN Mesenteric lymph node MNC Mononuclear cell NK Natural killer cell NKT Natural killer T cell NOD Non obese diabetic mouse strain OCH (2S,3S,4R) 1 O ( D galactopyranosyl) N tetracosanoyl 2 amino 1,3,4 nonanetriol PAMP Pathogen associated molecular patterns PB Peripheral blood PBMC Peripheral blood mononuclear cell PLZF Promyelocytic leukaemia zinc finger protein PRR Pattern Recognition Receptor RANTES Regulated upon activation normal T cell expressed and secreted ROR t Retinoid orphan receptor gamma t SLA DR Swine leukocyte antigen DR SLE Systemic lupus erythematosus TCR T cell receptor TLR Toll like receptor TNF T umor necrosis factor
12 Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for th e Degree of Master of Science TARGETING NATURAL KILLER T CELLS TO ENHANCE IMMUNITY: A NOVEL SWINE MODEL By Bianca Libanori Arti aga August 2014 Chair: John P. Driver Major: Animal Sciences CD1d restricted natural killer T (NKT) cells are a unique lymphocyte population that makes important contributions to host defense against numerous microbial pathogens. The powerful immunomodulatory effects of these cells can be exploited in mice by cognate antigens for multiple therapeutic purposes, including for protection from infectious diseases , and as adjuvants to improve vaccines against microbial organisms. These applications have potential to treat and prevent infectious diseases in livestock species that express NKT cells, including pigs. In this study, immune tissues from commercial swine of mixed genetic background were compared for NKT cell frequency, cytokine secretion an d subset ratios. We observed that pigs express NKT cells that share many characteristics with human NKT cell populations, including that they are found at low frequencies in blood and lymphoid organs, contain CD4 + and CD4 subsets and rapidly secrete large amounts of the cytokines interferon (IFN) and interleukin (IL) 4 after stimulation. Pigs were also injected with the model antigen hen egg lysozyme (HEL) in conjunction with one of three glycosphingolipids, al pha galactosylceramide ( GC), OCH , and C gly coside , that selectively activate NKT cells to
13 assess the adjuvant potential of each. There was significant variation between individual pigs for all NKT cell parameters measured. The NKT cell agonists elicited HEL specific immune responses of different qu ality, but only GC increased the systemic concentration of NKT cells. Peripheral blood NKT cell frequency measured prior to treatment was a poor predictor of how individual animals responded to NKT cell therapy. In contrast, IFN produc tion by NKT cells a nd NKT cell trans activated cell types correlated with the strength of immune responses against HEL. Our results show that although NKT cells vary considerably between pigs, there exists considerable potential to harness these cells to protect swine from i nfectious diseases.
14 CHAPTER 1 LITERATURE REVIEW General C haracteristics of Natural Killer T C ells Natural killer T (NKT) cells are an immunoregulatory subset of T lymphocytes that recognize a limited range of lipid or glycolipid antigens presented by the major histocompatibility complex (MHC) class I like CD1d molecule (1 8) . They express surface molecules and receptors also found on conventional T lymph ocyte and natural killer (NK) cell lineages. These include surface markers characteristic of effector and memory T cells such as CD25, CD69 and CD122, as well as NK markers , including the activating receptor NK1.1 and receptors of the Ly49 family, which ar e mostly inhibitory (3, 9) . The distribution of NKT cells vari es between tissues. In mice, they represent approximately 30 and 15% of all liver and bone marrow T lymphocytes, respectively. In thymus, spleen, peripheral blood (PB) and pancreatic and mesenteric lymph nodes they make up between 1 and 3% of T cells, and are even rarer in other lymph nodes and lymphoid tissues (3, 10 15) . Importantly, concentrations of NKT cell s in tissues vary considera bly among different mouse strains (16, 17) . Th e frequency of NKT cells in humans is lower than in mice and differs considerably between individuals. However, within individuals , NKT cell concentrations are generally stable over time, fluctuating between 0.01 to 0.1% in PB (18 20) . In contrast to mice, human NKT cells are enr iched in the omentum rather than in the liver (21, 22) . The majority of NKT cells are classified as Type I or invariant NKT (iNKT) cells (referred to hereafter as NKT cells), and are so called because they express a limited repertoire of T cell receptors (TCR). In mice, these are composed of a V 14 J 18 chain
15 paired with a limited number of V chains (V 8.2, 7, or 2). The human NKT cell TCR repertoire is even more restricted and comp rised of a V 24 J 18 chain pairing with V 11 (1, 3). Type II or variant NKT (vNKT) cells express a more diverse variety of TCR and chains and cannot be identified using the same reagents that recognize Type I NKT cells (3) . The most common TCRs in this population are V 3 J 9 or V 8 combined with V 8.2 (3, 23) . Type I and Type II NKT cells have demonstrated opposing regulation of the immune system, which is likely due to the different cytokines they secret after activation (24) . This may explain why Type I and Type II NKT cells have contrasting effects on experimental models of infectious diseases including Trypanosoma cr uzi infection (25) , acute murine schistosomiasis (26) , autoimmune hepatitis (27) and anti tumor immunity (28) . In addition to NKT cells there exists diverse subsets of T lymphocytes that express NK cell markers but which do not depend on CD1d expression during development or to become activated. These include a fraction of CD8 + T cells that express NK1.1 upon activation and mucosal associated invariant T (MAIT) cells, which have a canonical T cell receptor (V 19 J 33 in mice and V 7.2 J 33 in humans) and appear to be stimulated by gut microbiota (3) . There is currently little known about how these cells function or contribute to immunity. CD1d The surface molecule CD1d is a conserved, non polymorphic glycoprotein, similar in structure to the MHC class I molecule (2, 29) . It is part of the CD1 family that is comprised of the following five iso forms: CD1a, CD1b, CD1c, CD1d and CD1e. All
16 CD1 family members are involved in presentation of lipid antigens, except the CD1e molecule, which has evolved to facilitate lipid processing and trafficking (30) . While CD1a c present lipid antigens to T cells with highly diverse TCR s, CD1d molecules interact with the semi invariant TCR of NKT cells, whi ch is why these lymphocytes often are referred to as CD1d restricted T cells (1, 3) . Like the MHC class I molecule, CD1d is a heterodimeric transmembrane glycoprotein comprised of a heavy chain formed by three alpha domains ( 1, 2 and 3) that non covalen tly bind to a b eta 2 microglobulin ( 2m) chain (31) . The CD1d antigen presenting site is formed by a combined 1 2 superdo main (31 33) . This is similar to the antigen presenting domain of MHC class I molecules. However, the CD1d ligand bin ding region is deeper, more hydrophobic and narrower than the MHC class I binding site (32, 34) . CD1d is anchored to the cell surface through a transmembrane segment of the immunoglobulin like 3 domain, which also includes a short cytoplasmic tail (30, 32) . CD1d is expressed on the surface of various hematopoietic cell populations including dendritic cells, B cells, macrophages and granulocytes (35) . It is also expressed on cortical thymocytes, where it pl ays a critical role in NKT cell development (36, 37) . Adipocytes have been found to express CD1d molecules that present lipids to adipose tissue resident NKT cells (38) . These NKT cells produce large amounts of th e anti inflammatory cytokines interleukin ( IL ) 4 and IL 10, and appear to protect against the development of metabolic syndrome associated with the chronic inflammation status of obesity (38 41) .
17 CD1 molecules are highly conserved between species and have been found in all m ammals studied, although there is considerable variation in the isoforms expressed. Humans produce all five CD1 molecules, but rats and mice only express CD1d, and ruminants have CD1a c and a non functional CD1d gene (2, 42) . Like humans and mice, pigs and horses possess functional CD1d molecules capable of presenting glycolipid antigens to NKT cells (42) . Mouse CD1d tetramers loaded with cognate lipid antigens can be used to distinguish NKT cells from conven tional T lymphocytes (Figure 1 1) . Because CD1d is highly conserved between species, mouse CD1d tetramer reagents also are able to identify human and pig NKT cells (11, 43, 44) . Thymic D evelopment of NKT C ells Similar to conventional T lymphocytes, NKT cells develop in the thym us and their TCR and chains undergo somatic recombination and positive selection (45) . However, their restricted TCR repertoire suggests that NKT cells are select ed for by a limited number of antigens. NKT cells develop from the same lymp hoid progenitor cells that give rise to conventional T cells (36, 45, 46) . Selection of lymphocyte precursors that eventually become NKT cells is driven by the expression of invariant TCRs that recognize glycolipids presented by CD1d. This occurs when NKT cells migrate through the thymic cortex during which double positive thymocytes present self lipid antigens to NKT cell precursors. This results in a relatively strong TCR signal that induces the expression of promyelocytic leukaemia zinc finger protein (PLZF), a transcription factor necessary to drive NKT cell development and effector function (47, 48) .
18 After initial selection, NKT cells un dergo four further stages of development in the thymus each characterized by a unique pattern of surface molecule expression. These expression patterns are as follows: St age 0: CD24 + , CD44 and NK1.1 ; Stage 1: CD24 , CD44 and NK1.1 ; S tage 2: CD24 , CD44 + and NK1.1 ; and Stage 3: CD24 , CD44 + and NK1.1 + (49, 50) . While most NKT cells remai n in the thymus until Stage 3, others appear to leave at Stage 2 to mature in the periphery (51, 52) . Different NKT cell developmental stages are also characterized by distinct cytokine expression patterns. Stages 1 and 2 produce a high quantity of IL 4 and IL 10 and a sm all amount of interferon (IFN) , wh e reas Stage 3 NKT cells produce a high quantity of IFN , little IL 4 and almost no IL 10 (53) . Effector Functions of NKT C ells NKT cells are capable of profound multiple effects on the immune system mostly through the diverse array of cytokines they secrete , which include IL 2, 3, 4, 5, 6, 9, 10, 13, 17, 21, IFN , tumor necrosis factor (TNF) and granulocyte monocyte colony stimulating factor (GM CSF). NKT cells also secrete various chemokines, such as CCL5 (RANTES) and CC L3 (MIP 1 ) (54) . In contrast to conventional T lymphocytes, which require days to develop into fully matu re effector cells, NKT cells constitutively produce cytokine encoding mRNAs that enable them to produce large quantities of cytokines within hours after activation (55, 56) . These include T helper (Th) 1, Th2 and Th17 cytokines that are capable of both stimulat ing and suppressing other immune cells through a process called transactivation (57 60) .
19 NKT cells can be activated directly through TCR interactions with lipid or glycolipid presenting CD1d molecules, or indirectly via cytokines produced by activate d antigen presenting cells (APCs) (9, 61 63) . NKT cells that are TCR activated with lipid antigens often secrete both Th1 and Th2 cytokines, whereas NKT cells activated through pro inflammatory cytokines produce only Th1 type cytokines (61, 64) . NKT Cell Crosstalk with Immune C ells Cells that respond to activated NKT cells include NK cells (65) , dendritic cells (DCs) (59, 66, 67) , B and T cells (68 70) , macrophages (71, 72) and granulocytes (73 75) . In turn, NKT cells can be stimulated to produce different mixtures of cytokines by the various immune cells with which they interact. Th us, stimulation is often bidirectional between NKT cells and the APCs that present antigen to them (33) (Figure 1 2 ) . Indeed, DC s engaged by NKT cells increase IL 12 production and upregulate the expression of various costimula tory molecules as well as lipid presenting CD1d molecules (63, 76, 77) . In response, NKT cells increase their expression of IL 12 receptor (IL 12R) and the ligands for DC costimulatory molecules. This allows NKT cells to reach a heightened state of activation upon TCR engagement by DCs o r other APCs (59, 78) . DC derived IL 12 production also leads to the sec ondary activation of NK cells that subsequently produce high levels of IFN (65) . Oth er effects of this IL 12 secretion are enhanced responses to protein antigens by MHC restricted CD4 + and CD8 + T cells (68, 69) , and increased cross presentation of antigens by DC (67) . B cells become activated by cytokines produced by activated NKT cells including IL 4, IL 5, IL 6, IL 13 and IL 21 (54) . NKT cells further stimulate B cells through CD40 CD40 ligand intera ctions (79) . In addition, NKT cells provide cognate help to B cells,
20 when the lipid antigen is linked to a specific B cell epitope (80) . Cognate help from NKT cells may have a role in enhancing early immunoglobulin production but not in genera ting memory (33, 81, 82) . Some macrophage subsets increase bacterial clearance and antigen presentation after interacting with NKT cells (71, 83) and activated NKT cells induce blood monocytes to mature into dendritic cells (72, 83, 84) . NKT cells can induce macrophage differe ntiation in both beneficial and harmful ways. During certain pulmonary infections, NKT cell derived IFN can increase phagocytosis and bacterial clearance by pulmonary macrophages (85, 86) . However, NKT cells may mature macrophages in ways that worsen common viral infections and chronic lung diseases, such as allergic asthma. It has been reported that this occurs when NKT cell derived IL 13 induces the differentiation of M2 macrophages that stimulate Th2 type immune response and large quantities of IL 10 (87) . NKT cells appear to be important for the rapid response of neutrophils to microbial infections (33) . NKT cells recruit neutrophils to inflamed tissues by secreting CXCL2 (MIP2) and/or IL 17A (73, 85, 88 90) , and by s witch ing neutrophil cytokine production from immunosuppressive IL 10 to pro inflammatory IL 12. By changing the cytokine profile of neutroph ils, NKT cells may contribute not only to microbial defense, but also to antitumor responses (74) . NKT Cell S ubsets NKT cells can be divided into different subsets based on various phenotypic characteristics (54, 91) . Human and mouse NKT cell populations can be distinguished according to the presence or absence of the surface molecule CD4. For human NKT cells, CD4 is expressed on NKT cells capable of secreting Th2 type cytokines, while
21 both CD4 + and CD4 subsets pro duce Th1 type cytokines (91, 92) . A portion of human CD4 NKT cells also express CD8 . These produce more IFN and are more cytotoxic than the CD4 + or CD4 CD8 NKT subsets (93) . Various NKT cell subsets i n mice have been identified based on the presence or absence of surface markers, including IL 17RB and NK1.1 that are analyzed often in conjunction with CD4 and CD8. Furthermore, various combinations of transcription factors , also found in some conventiona l MHC restricted T cell s, drive dis tinct modular transcriptional programs in different NKT cell subsets (94, 9 5) . Transcription factors that are differentially expressed in NKT cell subsets include PLZF, T bet, retinoic acid receptor related orphan receptor t (ROR t), and GATA binding protein 3 (GATA3) (96) (Figure 1 3 ) . PLZF and GATA3 can be detected to some extent in all NKT subsets because both transcription factors are essential for the thymic development of NKT cells (62, 9 6) . T bet that is required for Th1 responses by conventional T cell s is also expressed by Th1 cytokine producing NKT cells (95) . Similarly, ROR t ident ifies a Th17 cytokine producing NKT cell subset (95, 96) . Th2 type NKT cells are defined by the abse nce of both T bet and ROR t and the presence of GATA3, which is a transcription factor expressed by conventional CD4 T cells skewed towards the production of Th2 cytokines (33, 62, 96) . Modes of NKT Cell A ctivation Two s ignals usually contribute to the activation of NKT cells; a TCR signal provided by a lipid CD1d complex, and a cytokine signal. NKT cells can become activated with basal or weak cytokine signals when the NKT cell TCR is stimulated strongly b y high affinity lipids. NKT cell s can be indirectly activated by pro inflammatory
22 cytokines from pat tern recognition receptor (PRR) stimulated APCs that combine with weak TCR signals from low affinity microbial or self lipid antigens (9, 33, 77) (Figure 1 4 ) . ). NKT cells that are TCR activated with lipid antigens often secrete both Th1 and Th2 cytokines, whereas NKT cells activated principally through pro inflammatory cytokines produce only Th1 type cytokines (61, 64) . The mechanisms of direct and indirect NKT cell action are fully described below. Direct NKT c ell a ctivation NKT cells have a distinct mode of antigen recognition (34) . Two hydrophobic channels within the CD1d antigen hydrophobic regions of glycolipid antigens leaving the hydrophilic portions exposed for interaction with the NKT cell TCR (97, 98) . Presentation of peptides by MHC class I molecules to conventi onal T cells requires that both and chains of the TCR contact the MHC helices. This allows the two hypervariable complementarity determining region 3 (CDR3) loops of the TCR to interact with antigen (33) . To facilitate this, conventional TCRs bind MHC molecules diagonally (34) . In contrast, the TCR chains of NKT cells are orientated in parallel to the antigen binding cleft of CD1d. This allows the semi invariant TCR chain to bind the lipid antigen as well as the CD1d antigen binding cleft (34) . The TCR chain stabilizes the i nteraction of NKT cells with CD1d, and in so doing modulates the affinity of the NKT cells for individual antigens (34, 99, 100) . Structural studies suggest that instead of using a diverse repertoire of TCR s to recognize different lipid antigens, NKT ce ll s employ a limited combination of TCR and chains with variable degrees of induced fit for binding different lipid antigen structures
23 (99, 101) . Th is allows the TCR of NKT cells to recognize a wide range of glycolipid antigen s (33, 101) . Microbial lipid antigens with an linked sugar head group require a minimal induce fit , and bind CD1d in a similar orientation to the synthetic NKT cell superantigen galactosylceramide ( GC) (102, 103) . Self lipid antigens that contain a linked sugar head group have been suggested to require a mu ch greater degree of structural alteration to bind the CD1d molecule for recognition by the NKT cell TCR (104, 105) . Therefore, the structure of the lipid antigen largely regulates the strength of the interaction between TCR and lipid CD1d and consequently the activation of the NKT cell (101) . (101) . Indeed, it has been postulated that different glycolipid antigens control the mixture of Th1 and Th2 cytokines produced by NKT cells through the streng th of their interactions with the NKT cell TCR (33, 101) . Indirect NKT cell a ctivation NKT cells express particularly high baseline levels of the IL 12 receptor, which suggests that IL 12 is particularly important for NKT cell activation. Some bacterial and viral infections stimulate sufficient IL 12 production by APCs to activate NKT cells even in the absence of TCR engagement (61, 106, 107) . Also, weakly binding endogenous ligands are sufficient to trigger NKT cells w hen they are presented by APCs in combination with enough APC derived IL 12 (108) . NKT cells express NK receptors that provide both activation and regulation signals in response to stress induced ligands (1, 109) . Activation of these NK receptors provides another pathway for NKT cells to respond to inflammation in the absence of lipid antigen stimulation (110) . The above mechanisms demonstrate that NKT cells are capable of responding to almost any microbial infection or non infectious inflammatory state in which APCs are
24 triggered by pathogen associated molecular patterns (PAMPs) or danger associated molecular patterns (DA MPs) (33) . Role of NKT Cells in D isease As discussed above, the effector functions of NKT cells can polarize the immune response to be both pro inflammatory and/or anti inflammatory, depending on their subsets and cytokine environment, and their mode, strength and duration of activation (54, 111) . Because different organs contain distinct combinations of these factors, the location where NKT cells a re activated can influence how these cells contribute to an immune response (112) . For this reason, the outcome of NKT cell stimulation can be difficult to predict. It has been reported that NKT cells can play either a protective o r a pathogenic role during infection. A protective role has been described for infections caused by several types of bacteria, viruses, fungi, protozoa and helminthes. The level of cognate NKT cell lipid antigen present during an infection appears to contr ibute to disease outcome (113, 114) . For example, during Novos phingobium infection NKT cells may suppress or exacerbate disease. This bacteria contains a lipid antigen capable of activating NKT cells responses that protect animals during low level infections with this organism. However, lethal sepsis results when mic e are inoculated with a large dose of the bacteria (113 115) . Patholo gical effects sometimes induced by NKT cells appear to be a consequence of their rapid release of high quantities of cytokines, which may contribute to the development of sepsis syndrome (116) . Pathogenicity of NKT cell responses often varies between mouse strains. For example, NKT cells protect BALB/c mice against syncytial virus infection but aggravate disease in C57BL/6 mice (117) .
25 NKT cells have been harnessed to treat experimental models of various autoimmune diso rders (118, 119) . It is thought that activated NKT cells suppress the development of auto reactive cells through the large quantities of anti inflammatory Th2 cytokines they produce and by their ability to recruit immune regulatory cell types such as FoxP3 + regulatory T cells and tolerogenic DC s that in turn inhibit autoimmune responses (120 122) . Animal models of autoimmunity have demonstrated that NKT cells suppress the following diseases: type I diabetes in the non obese diabetic (NOD) mouse (123 125) , the experimental autoimmune encephalomyeliti s (EAE) model of multiple sclerosis (126 128) , multiple models of systemic lupus ery thematosus (SLE) (129, 130) , and an age induced, acute model of rheumatoi d arthritis (131) . NKT cells have been shown to contribute to the maintenance of immune tolerance in a number of animal models. Indeed, NKT cells are necessary for tolerance induced by injection of antigens in to the anterior chamber of the eye, spontaneous tolerance to hepatic allografts, transplant tolerance induced by antibodies against CD4 + T cells or co stimulatory molecules, oral tolerance, and fetal tolerance (132) . NKT cells also prevent the development of graft ve rsus host disease after bone marrow transplantation (133) . The pro inflammatory properties of NKT cel ls impact numerous inflammatory diseases (7, 111) . NKT cell activation heightens the severity of airway hyper responsiveness in asthma (9) and exacerbates acute and chronic viral hepatitis (14) , contact hypersensitivity (134) , colitis (135) , acute tissue in jury induced to liver or kidney in ischemia reperfusion models (73) and atherosclerosis (136, 137) . The capacity of NKT cells to worsen these disorders is l ikely linked to the large amounts of pro -
26 inflammatory IFN they themselves produce and which they stimulate from NK cells and cytotoxic T lymphocytes. This ability to induce high levels of IFN from the immune system may underlie why NKT cells are reported to enhance tumor immunity (138, 139) . Loss of NKT cell s through defects in their development and/or function have been linked with a more severe disease for some pathologies (8, 111, 118) . The mechanisms underlying this association are still not well defined. However, pathogenic immune responses may develop when the immunoregulatory functions usually performed by NKT cells are absent. These types of defects have been linked to increased susceptibility to autoimmunity, cancer and infections (1, 138, 140) . NKT Cell A ntigens NKT cells can recognize endogenous and exogenous lipid antigens presented by the CD1d molecule (141) . NKT cells likely recognize multiple endogenous self antigens, which are necessary for their thymic development (36, 45) . Some s elf antigens may also mediate autoreactivity when overexpressed by injured or stressed tissues (142) . In humans, lysophospholipids have been reported to be important self antigens that are often expressed by inflamed tissues where they trigger NKT cells (143) . Exogenous antigens are synthetic glycolipids or antigens derived from microbial organisms (141) . Bacterial antigens are recognized by both human and mouse NKT cells. Examples include glycosphingolipids derived from the cell wall of Novosphingobium and Ehrlichia (115, 144) . Synthetic antigens have been designed that strongly stimulate NKT cells. Most of these are structural analogs of GC, a glycosphingolipid originally isolated from a marine sponge (79) . GC is a ceramide -
27 based glycolipid composed of a carbohydrate head group attached to two saturated long chain fatty acids through an or ientated glycosidic linkage ( 33, 79) . Most NKT cell therapy studies use the synthetic GC an alog KRN7000 to activate NKT cells (8, 145) . GC binds TCRs with high affinity and induces NKT cells to produce large quantities of both Th1 and Th2 cytokines (54, 56) . However, structural analogs of GC have been synthesized that stimulate NKT cells to produce either Th1 or Th2 cytokines (57, 146 150) (Figure 1 5 ) . Glycolipid analogs with short fatty acid chains skew NKT cells towards producin g Th2 cytokines. One often used as a therapeutic agent is OCH which, compared to GC, consists of a sphingosine base reduced from 18 to 9 c arbons and an acyl chain shorter by two carbon residues (146, 150) . Another analog, C20:2 contains acyl chains with double bonds (145, 148) . The shortened sphingosine base of OCH may reduce its binding to the CD1d molecule and decrease the TCR affinity of the OCH CD1d complex (146, 151) . Other properties that may contribute to the effects of Th2 biasing analogs include that they: (1) are preferentially presented by specific APC subsets (60, 148) , (2) intracellularly load on to CD1d (152, 153) , (3) enhance recycling of the CD1d glycolipid complexes to lysosomes (152) and (4) exhibit a shorter half life in vivo (145, 154) . Other GC analogs have been synthesized that preferentially activate NKT cells to produce Th1 cytokines (8) . These include the C glycoside analog C GC that contains a CH 2 group in place of the glycosidic oxygen present in GC (57) , or the car ba GC analog that contains an oxygen residue in the galactose sugar ring (154) . Additional GC variants 7DW8 5 and C34 respectively possess methylene and aromatic residues inserted into their fatty acid chains (155, 156) . The reason why the
28 above glycolipids induce Th1 cytokines remains uncertain. However, studies in mice have shown that C glycoside causes a great er downstream activation of NK cells to produce IFN . This appears to be because the C glycoside CD1d complex is highly stable in vivo (145) . Therapeutic T argeting of NKT C ells Therapeutically targeting NKT cells holds promise for the treatment of a host of diseases (157, 158) and for the enhancement of vaccines (140, 159, 160) . This has been demonstrated in animal studies using the prototypical NKT cell antigen GC (KRN7000) or the structurally related analogs OCH and C glycoside (9, 32) . Treatment of mice with GC induces NKT cells to proliferate , up to 10 fold in the spleen, five fold in blood, bo ne marrow and lymph nodes, and two to three fold in the liver. NKT cells ex pand to maximum levels between three and four days after injection and return to pre treatment concentr ation s one to two weeks later (9, 12, 161) . However, it has been reported that NKT cells become anergic after initial activation and cannot be restimulated for at least one month after encountering GC (162, 163) . The same phenomenon has been report ed in mice challenged with bacterial pathogens (164 1 66) , toxins (167) , and TLR agonists (164) that activate NKT cells. It is important to note that NKT cell popula tions vary significantly among tissues. This presents a challenge when trying to understand the effects of NKT cell therapeutic s using PB NKT cells alone, which is the only viable alternative for human studies. Therefore, there is much interest is understanding the relationship between NKT cells in PB compared to other tissues (118) .
29 Another concern is whether glycolipid antigens will be tolerated safely if used to treat humans and other large mammals. Under some conditions GC has been observed to induce liver toxicity in mice (168, 169) . However, the analog was well tolerated when used to treat human cancer patients in a Phase I clinical trial (169 173) . Cancer GC was first discovered as part of a drug screen to identify molecules with antimetastatic properties (79, 174) . This le d to studies showing that NKT cells often play a role in cancer immunit y (118) . Innate NKT cell defects predispose mice to cancer while their adoptive transfer or stimulation in vivo can protect animals from disease (138, 175, 176) . NKT cells do not directly destroy tumors as NKT mediated protection does not require CD1d expression by cancer cells (177) . Instead, it i s likely that NKT cell derived pro inflammatory cytokines activate Th1 cytokine producing cytotoxic CD8 + T lymphocytes and NK cells that in turn destroy cancer cells (59, 139) . NKT cells activated by glycolipids also condition DCs to produce cytokines and costimulatory molec ules that enhance the activation of cytotoxic CD8 + T cells, NK cells, and conventional CD4 + T cells (59, 138) . Apart from stimulating immune responses, the increased amounts of IFN produced after NKT cell activation has anti a ngiogenic effects that restrict blood flow to tumor cells (178, 179) . Auto immunity Activated NKT cells are capable of producing a wide range of anti inflammatory cytokines that contribute to suppress ing different autoimmune disorders (111, 119) . The advantage of harnessing NKT cells to treat autoimmune disease is that a single cognate
30 antigen can be used to target all NKT cells. In contrast, CD4 + FoxP3 + regulatory T cells that also suppress autoimmunity expres s a vast range of TCR specificities each with its own cognate antigen making these cells very difficult to target (180) . Using NKT cell activating GC to treat autoimmunity does not always lead to disease suppression and the effectiveness of treatment often depends on the mouse model used (119, 181) , the timing of treatment related to disease onset (130, 182) , dose of GC injected and the genetic background of the treated mice (183) . For example, it has been observed in a case of pristine induced lupus like disease that GC protected BALB/c mice but aggravated the disease in SJL/J mice (184) . Also, in an adoptive transfer model of type I diabetes with diabetogenic CD8 T cells, GC protected NOD mice but promoted disease in a stock of C57BL/6 mice congenially expressing the diabetogenic NOD MHC haplotype (183) . In general, amelioration of autoimmune disease has been associated with conditions where GC treatment promoted the release of more Th2 cytokines and reduced Th17 cytokines by NKT cells. In contrast, disease was wors ened if NKT cell s did not produce more Th2 cytokines after GC stimulation (127, 128, 185) . Inflammation Stimulating NKT cells to produce Th2 cytokines, including IL 4 and IL 10, has been used as a strategy to treat a variety of IFN mediated inflammatory conditions, including experimental models of induced colitis, allergic asthma and allogeneic skin transplantation ( 135, 186, 187) . However, GC treatment in some studies actually exacerbated disease, probably for the same reasons that this agent sometimes worsens certain autoimmune dis orders. Two pathologies reported to be both suppressed and
31 exacerbated by GC treatment are graft versus host disease and graft rejection (187 191) . However, under conditions where GC induced NKT cells to increase secretion of the Th2 cytokine IL 10 (187) or reduce secretion of the Th1 cytokine IFN (188, 189) , graft survival was prolonged and graft versus host disease was suppressed (190, 191) . Some studies have shown that glycolipid analogs that favor Th2 cytokine secretion by NKT cells inhibit inflammatory diseases normally exacerbated by GC. For example, protection was achieved when mice were treated with OCH in a model of induced colitis (135) , and with DPPE in a model of contact hypersensitivity (134) and allergen induced airway hyper reactivity (192) . The same d iseases were worsened by GC treatment (135, 193) . Infection NKT cells have been harnessed successfully for the treatment and prevention of numerous infectious diseases in mice (8) . Treatment with GC protected against diseases caused by Pseudomonas aeruginosa (85, 194) , Streptococcus pneumoniae (195) , Mycobacterium tuberculosis (196, 197) , Listeria monocytogenes (198) , Novosphingobium capsulat a (156) , Staphylococcus aureus (156) , Plasmodium falciparum (199) , Trypanosoma cruzi (200) , Cryptococcus neoformans (201) , influenza virus (202, 203) , respiratory syncytial virus (117) , murine cytomegalovirus (204) , diabetogenic encephalomyocarditis virus (205) , Japanese encephalitis virus (156) and hepatitis B virus (206) . The mechanisms of a ction through which NKT cell activation eliminates the above infectious agents have yet to be fully determined. However, they are likely to include stimulating pro inflammatory effector functions in DCs, CD8 + cytotoxic T
32 lymphocytes, and NK cells (8) . This is likely the reason that GC analogs that induce Th1 biased responses from NKT cells are often more potent than GC at treating infectious diseases (57, 156) . The effectiveness of NKT cell agonists may be enhanced when these agents are used in combination with other antimicrobial treatments. For instance, the combination of GC and the antimycobacterial agent isoniazid was more effective than either compound alo ne for protecting mice from infection by M. tuberculosis (197) . Animal models have shown that NKT cell agonists can be used with great effect as adjuvants to enhance vaccines against many infectious diseases (159, 160) . For example, GC treatment increased the effect iveness of an experimental malaria vaccine by increasing the frequency and activity of parasite specific CD8 + T cells (207) . Intranasal administration of an influenza vacc ine formulated with GC induced the development of memory CD8 + T cells and mucosal and systemic humoral responses that protected mice from subsequent influenza infection (208 211) . Mice treated wi th the GC vaccine therapy also developed IgA antibodies that provided cross protection against multiple other influenza virus strains (212) . The adjuvant effects of GC also enhanced experimental vaccines against HIV 1 (155, 213) , Haemophilus influenzae (214) , herpes simplex virus 2 (215) , Mycobacterium tuberculosis (216) and the toxin of Bacillus anthracis (217) . Swine NKT C ells General C h aracteristics NKT cells have been identified in lungs, spleen and PB of pigs (43, 44) . Like ot her species, pig NKT cells rapidly activate and secrete cytokines after in vitro
33 stimulation with PMA/ionomycin or GC (44) . They also undergo activation in vivo after exposure to GC as demonstrated in a model of induced acute airway hy perreactivity (AHR) (43) . Pig NKT cells in previous studies were identified with a cross reactive mouse CD1d tetramer reagent (43, 44) . This cross reactivity is because genes encoding CD1 molecules are highly conserved between mammalian species (218) . Thus, murine glycolipid conjugated CD1d can be recognized b y porcine, human and monkey NKT cells (11) . Porcine NKT cells were identified by flow cytometry using a combination of anti porcine CD3 and mouse CD1d tetramer conjugated with the glycolipid antigen PBS 57 (43) or GC (44) . Consistent with findings in humans, porcine PB NKT cells range in frequency between 0.01 and 1% of total lymphocytes (91) . Pigs express four main NKT cell subsets that can be identified based on the surface expression of CD4 and CD8 molecules. The CD4 CD8 + and CD4 CD8 populations are present at similar concentrations compared to humans; 45.5% versus 42.14% for CD4 CD8 + and 35.9% versus 36.57% for CD4 CD8 in humans and pigs, respectively. Another subset identified as CD4 + CD8 is reduced in pigs compared to humans (18.4 % versus 31.1%) (44) . Pigs also produce a PB NKT cell subset that expresses both CD4 and CD8 (219) . Pig NKT cells possess a similar memory activation phenotype to human and mouse NKT cells, characterized by high expression of MHC class II SLA DR and CD5, and low expression of CD45RA in comparison to conve ntional T cells (44, 92, 220) . The activation/memory phenotype of mouse NKT cells has been associated with their expression of the transcriptional factor PLZF (221) . PLZF is also expressed in mouse and human T cells and in a large percentage of human CD8 + T cells (222) . A recent
34 analysis of pig PB lymphocyte populations found that porcine NKT cells also express high levels of PLZF relative to conventional T cells (44) . Other surface markers have been examined for their ability to differentiate NKT cells from conventional T cells in porcine PB. These include CD16, a NK cell surface marker often used to identify NKT cells in mice and humans. CD16 was found at higher levels but not exclusively on NKT cells compared to conventional T cells (20.8% versu s 12.5%) (44) . However, the alpha M integrin CD11b is expressed by a significant proportion of pig NKT cells and is almost absent from conventional T cells (44) . Another way NKT cells can be distinguished from conventional T cells is through their heightened ability to rapidly produce large quantities of cytokines after PMA/ionomycin stimulation. It was recently shown that a greater proportion of pig NKT cells prod uce IFN (19.3%) compared to conventional CD3 + T cells after stimulation (5.7%) (44) . Effect of GC on Pig NKT C ells It has been observed in mice, humans and monkeys that GC specifically stimulates NKT cells (1, 43) . GC also induced the activation and proliferation of pig NKT cells in vitro . As in other species, the expansion of NKT cells by GC was augmented by addition of cytokines IL 2 and IL 15 or IL 33 (44) . This is in line with a report that IL 33 specifically enhances NKT cell expansion without affecting other T cells (223) . A recent report describing a pig model of airway hyperreactivity (AHR) showed that pig NKT cells in the lung became activated after intratracheally dosing animals with GC (43) . Symptoms induced by this treatment included an increase in respiratory rate,
35 mucus secretion and body temperature. Ex vivo it was observed that lungs contained petechial hemorrhages, infiltration of CD4 + cells and high amounts of Th2 cytokines (43) . There was no increase in the frequency of NKT cells after treatment, but ex vivo restimulation of lung mononuclear cells (MNC) and PBMC with GC induced NKT cell proliferation and secretion of Th1 and Th2 cytokines, but only for the GC treated animals (43) . The mixture of cytokines secreted by the NKT cells was significantly altered if different C glycoside analogs were used in place of GC for ex vivo restimulation (43) . This indicates that pig NKT cells may be manipulated in vivo with different glycolipid analogs for different therapeutic purposes (43) . Summary Natural killer T cells are a regulatory lymphocyte popula tion capable of bridging the adaptive and innate immune systems through their rapid and potent immunomodulatory effects. NKT cells have been greatly studied in mice and humans, with particular emphasis on how they may be harnessed to treat various infectio us and inflammatory diseases, autoimmune disorders and cancer. Various safety concerns related to the poor translatability of mouse studies to humans have slowed the use NKT cells therapeutics for clinical applications. Thus, a better animal model is neede d. One model with considerable potential is swine. This is because pigs have similar body size, physiology, and genetic heterogeneity compared to humans. Importantly, they are also susceptible to almost identical contagion. This likely makes pigs a good m odel with which to study how manipulating NKT cells affects a wide variety of common zoonotic diseases and the vaccines against them. These discoveries
36 may also lay the groundwork for using NKT cell therapeutics to improve the health of commercial swineher ds and possibly other domestic animal species.
37 Figure 1 1. CD1d tetramer reagent interacting with a natural killer T ( NKT ) cell. The CD1d tetramer is composed of four biotin labeled GC loaded CD1d molecules conjugated to a streptavidin molecule. Each CD1d molecules is capable of interacting with the NKT cell TCR. Tetramers are also conjugated to a fluorochrome molecule for their identification by flow cytometry. Figure adapted from MB L International website ( http://www.mblintl.com/tetramer/Detection_of_antigen specific_T_cells_by_MHC_tetramers.aspx ).
38 Figure 1 2 .NKT cell crosstalk with immune cells. Activation of NKT cell after interaction with antigen presenting cells (APC) such as dendritic cells (DC), B lympho cytes and macrophages. NKT cell cytokine secretion and presentation of costimulatory molecules immunomodula tes the responses by other cells. (33) .
39 Figure 1 3 . NKT cells subsets in mice. Three subsets of NKT cells in mice are classified according to the expression of transcription factors that define subsets of conventional T lymphocytes. Th1 , Th2 or Th17 like NKT cells are represented with characteristic transcription f actors, surface molecules and cytokines production. review (33) . Figure 1 4 . Modes of NKT cell activation. Direct activation of NKT cell is characterized by a strong TCR signal and a weak cytokine signal. Indirect activation of NKT cell is described by a strong cytokine signal and a weak TCR signal. Figure adopted from Brennan, Bri (33) .
40 Figure 1 5 . NKT cell antigens. Structure of three glycolipid antigens recognized by NKT cells, a lpha galactosylceramide ( GalCer or GC) and two GC analogs, OCH and C glycoside. Figure adopted from Sullivan et al (145) .
41 CHAPTER 2 ADJUVANT EFFECTS OF THERAPEUTIC GLYCOLIPIDS ADMINISTERED TO A COHORT OF NKT CELL DIVERSE PIGS Invariant natural killer T (NKT) cells are a regulatory lymphocyte subset characterized by their unique ability to recognize glycolipid antigens presented by the major histocompatibility complex (MHC) class I like CD1d molecule (9) . NKT cells recognize endogenous glycolipids as well as various microbial ligands through semi invariant T cell r eceptors (141, 224, 225) . Once activated, these cells induce profound multiple effects o n the innate and adaptive immune systems, primarily through the rapid secretion of various cytokines (9) . Mouse studies have revealed that NKT cells m ake important contributions to immunity against a wide variety of pathogens including bacteria, viruses, fungi, protozoa and parasites (1, 113, 114, 140) . NKT cells have also been therapeutically targeted by synthetic glycolipid superago nists to treat a range of disord ers in mice including cancer, graft rejection, autoimmunity and various infectious diseases (8) . Although these ligands hold great promise for treating the ab ove maladies, safety concerns have slowed their translation to humans for fears they may actually exacerbate rather than ameliorate some diseases (8, 111, 119) . The significance of these studies is difficult to assess because considerable differences in frequency, subsets, cytokine secretion patterns and tissue localization exist between mouse and human NKT cel ls (1, 36) . Although it may require several more years to fully exploit NKT superagonists for humans, it could be easier to harness these agents for livestock, which typically do not develop diseases that risk exacerbation by acute NKT cell activation. One species with significant promise is pig s. This is because it is known already that swine NKT cells can be detected using a mouse CD1d tetramer reagent (43, 44) . Also, intratracheal
42 inoculation with the NKT superagonist alpha galactosy lceramide ( GC) stimulates NKT cell activation in pig lungs (43) . Previous reports have demonst rated that NKT cell superagonis ts can protect mice from several pathogens that affect swine, including influenza (202) , Pseudomonas aeruginosa (85, 194) Streptococcus pneumonia (195) , Mycobacterium tuberculosis (196, 197) , Listeria monocytogenes (198) , Staphylococcus aureus (156) , cytomegalovirus (204) , Trypanosoma cruzi (200) , Japanese encephalitis virus (156) and Bacillus anthrax (21 7) . In addition, the adjuvant effects of NKT cell superagonists have been exploited in mice to improve vaccines against microorganisms that also infect pigs, including influenza (208 211, 226) , M. tuberculosis (216) and B. anthrax (217) . Thus, the tools and incentive exist to characterize pig NKT cells and ascertain whether these cells may be harnessed to mitigate important swine pathogens in the future. An added benefit may be establishing safety and optimal dosage ranges for NKT cell therapeutics for clinical application given that both pigs and humans are genetically outbred, of similar size and express the full repertoire of CD1 genes (42, 218) . Thus, our hypothesis is that pigs express NKT cells that may be therapeutically targeted to enhance immunity against foreign antigens. To advance these goals, the current work characterizes the frequency, function, sub sets, and localization of pig NKT cells. It also evaluates how three well known NKT cell superagonist s , GC, OCH and C glycoside, perform as adjuvants in swine when co injected with a foreign antigen.
43 Materials and Methods Pigs Yorkshire, Chester White, Du roc, and Landrace crossbred pigs are maintained at the Department of Animal Science s swine unit at the University of Florida. Breeding of sows is performed by artificial insemination using sem en collected on site or from Swine Genetics International (SGI) (Cambridge , IA), Top Cut Genetics (Farmland, IN) or In vivo experiments were performed according to the recommendations of the United States Department of Agriculture regulations, the he Care and Use of Laboratory Animals, as well as all relevant state and federal regulations and policies. The animal care and use committee at the University of Florida approved the protocol. In addition to experimental animals, tissue and blood samples w ere collected from swine unit raised pigs Isolation of Leukocytes from Blood, Spleen, Thymus and Lymph N odes Blood from live and euthanized animals was collected in hepariniz ed vacutainers (BD Biosciences, San Jose, CA). Spleen, thymus and lymph node tissues from euthanized pigs were collected in Hanks balanced salt solution, washed, and dispersed to single cell suspension using 40 ml glass homogenizers. Erythrocytes in whole blood and spleen were l ysed using an ammonium chloride based lysis buffer. Isolation of peripheral blood mononuclear cells (PBMCs) was performed using Ficoll PREMIUM (GE Healthcare Bio Sciences Corp., Uppsala, Sweden). Up to 17 ml of blood was mixed with the same volume of PBS and then layered on top of 15 ml of Ficoll Paque Premium in a 50 ml conical tube. Samples were centrifuged at 1200 x g for 25 minutes at 25Â°C. The PBMCs were removed and washed twice with PBS after which
44 residual red blood cell s were lysed. PBMCs were re suspended in culture media (RPMI 1640 containing 10% fetal bovine serum and 1% Penicillin/Streptomycin solution) at the appropriate concentration. Preparation of G lycolipids Alpha galactosylceramide was purchased from Avanti Po lar Lipids, Inc. (Alabaster, AL) and Toronto Research Chemicals (Toronto, ON, Canada). The National glycoside and OCH. GC and C glycoside were dissolved in DMSO at 2 and 1 mg/ml , respec tively. OCH was reconstituted in distilled water and 0.5% Tween 20, 56 mg sucrose and 7.5 mg histidine at 0.2 mg/ml. All mixtures were sonicated for 30 minutes or until glycolipids were fully dissolved. Stock solutions were stored at 20 o C. For in vitro s tudies, glycolipid stock solutions were added directly to RPMI 1640 culture media. For in vivo use, glycolipid stock solutions were further dissolved in PBS at the indicated concentrations. Experimental Design for Hen Egg Lysozyme C hallenge Fifty nine two week old female piglets were bled from the jugular vein to determine their NKT cell frequencies. From those, thirty seven piglets were assigned to individual treatments so that each treatment group contained animals with the same range of NKT cell frequenc ies (Table 2 1). For the first experiment, hen egg lysozyme (HEL) purchased from Sigma Aldrich (St. Louis, MO) was dissolved in sterile PBS only or mixed with GC, C glycoside or OCH and was injected i. m. into the neck of piglets at three and five weeks of age ( Figure 2 1). Dosages of HEL were adjusted accord ing to body weight at both the three and five week injection times so as to receive 200 g/kg
45 of body weight . Glycolipids were injected at a rate of 50 g/kg of body weight (5.84 x 10 8 mol/kg) at three weeks of age and the same amount of each compoun d was used for inoculations at five weeks of age without adjusting for increased body weight. For the second experiment, piglets were injected with 200 g/kg of HEL dissolved in PBS only or with GC ad justed for body weight at both three and five weeks of age at a rate of 50 g/kg (5.84 x 10 8 mol/kg) or 100 g/kg (1.16 x 10 7 mol/kg). For both experiments, blood samples were taken from the jugular vein at the indicated time points (Fig ure 2 1) for analysis o f NKT cell frequencies, HEL specific IgG and T lymphocyte responses and plasma cytokine concentrations. Rectal temperatures were recorded before each blood sample was taken (Table 2 1). All pigs were euthanized by exsanguination at 28 days after the start of each experiment . Sp leen, cervical lymph nodes (CLN) and mesentery lymph nodes (MLN) were collected to quantify the frequency of NKT cells and HEL reactive T lymphocytes in each tissue. Flow Cytometry A nalyses an d R eagents NKT cells were characterized b y flow cytometry using an Accuri C6 flow cytometer. Cell suspensions were Fc receptor blocked with rat IgG from Sigma Aldrich and stained with the indicated fluorochrome conjugated antibodies at 4 o C. NKT cells from spleen, thymus, CLN and MLN were identif ied using anti CD3 FITC BB23 8E6 8C8) and PE conjugated GC analog PBS57 loaded mouse CD1d (mCD1d) tetramer reagent respectively provided by BD Biosciences and the National Institutes of Health Tetramer Core Facility. NKT cell subsets were distinguished using anti CD4 (74 12 4) from SouthernBiotech (Birmingham, AL) that was conjugated to Alexa Fluor 647 (A647) fluorescent dye (Invitrogen, Carlsbad, CA) according to directions. For
46 intracellular NKT cell staining, PBMCs w ere incubated in duplicate for five h ours at 37 o C with or without 2 l/ml Cell Stimulation Cocktail from eBiosciences (San Diego, CA), containing PMA (40.5 M), ionomycin (670 M), and 1 l/ml of the protein transport inhibitor BD Golgi Plug. Following the incubation period, PBMCs were surface labeled with anti CD3 and mCD1d tetrame r and fixated and permeabilized with solutions from the BD Cytofix/Cytoperm Plus fixation/permeabilization kit (BD Biosciences). Cells were then restained with anti IFN PerCP P2G10; BD Biosciences) and A647 conjugated IL 4 (A155B 16F2; Invitrogen). Data were analyzed using FlowJo software (Treestar, Palo Alto, CA). ELISA and IFN Enzyme Linked Immunosorbent Spot A ssays HEL specific IgG responses were measured in plasma by ELISA. Wells of ELISA plates were coated with 100 l of 10 g/ml HEL and incubated overnight at 4 o C. HEL solution was removed and plates were incubated with blocking buffer (5% skim mi lk powder in PBS) for one h our at 37 o C. Plates were washed and incubated with plasma diluted at indicated concentrations with blocking buffer. Plates were washed and incubated with 100 l of blocking buffer containing a 1:1000 dilution of alkaline phosphatase linked secondary antibody specific for porcine IgG (SouthernBiote ch) and incubated for one h our at 37 o C. Plates were washed and incubated with 10 0 l of 1 mg/ml p nitrophenol phosphate substrate (Sigma) for 20 minutes at room temperature and read at 405 nm using an ELISA plate reader. The concentration of HEL reactive T lymphocytes in PBMC, spleen, CLN and MLN was assessed using IFN enzyme linked immunos orbent spot (ELISPOT) assays. MultiScreen HTS plates (Milipore, Billerica, MA) as well as BD Bioscience supplied capture (P2G10) and detection antibodies
47 (P2C11) and colorimetric reagents from the BD Cytokine ELISPOT pair kit were used for the analyses. Si ngle cells were suspended in culture medium and plated in triplicate at between 0.125 and 2 x 10 6 cells per well with increasing concentrations of HEL ranging from 0 to 10 M. Plate s were incubated for 72 h at 37 o C. After development, spots were read using an automated ELISPOT reader (AID EliSpot High Resolution Reader System ELHR03). Data are presented as mean number of spots Â± SEM/10 4 viable cells in each of the HEL stimulated triplicates after subtracting spots counted in unstimulated wells. The same ass ay was used to determine the concentrations of GC reactive T lymphocytes in PBMCs with the exception that cells were plated per well at concentrations between 0.25 and 1 x 10 6 with increasing concentrations of GC ranging from 0 to 1 g/ml. IFN and IL 4 in plasma samples was measured by ELISA using a NovexÂ® Antibody pa irs kits for swine (Invitrogen). Statistical A nalysis The data were analyzed using PROC MIXED of SAS , v ersion 9.3 (SAS Institute Inc., Cary, NC) when assessed to be normally distrib uted using the PROC UNIVERIATE and QQPLOT procedures. Data that were not normally distributed were analyzed using a nonparametric Wilcoxon U test from JMPÂ®, version 11 (SAS Institute Inc.) . To evaluate the effects of the treatments on changes in NKT cell f requency and HEL specific antibodies with time in the HEL vaccination studies, pretrial NKT cell frequency was used as a covariate, pigs were nested within treatment and a repeated measures statement was used. Where appropriate, the SAS slice command was u sed to examine treatment differences at each time poin t for each dependent variable. All
48 linear regression analyses were performed using GraphPad Prism, version 6.0d for Macintosh (GraphPad Software, Inc., La Jolla, CA). Results Diversity in Pig NKT Cell F requency, Subset Distribution and F unction Peripheral blood (PB), spleen , thymus, CLN and MLN were sampled from 79 crossbred male and female pigs to characterize swine NKT cell frequencies, distribution, subset ratios and cytokine secretion patterns using a PE conjugated CD1d tetramer reagent (Fig ure 2 2A). The frequency of splenic NKT cells was greater and more variable among animals compared to the other tissues analyzed (Fig ure 2 2B). Within individual pigs, we detected weak to moderate correlations betw een PB, spleen, and CLN NKT cell concentrations (Fig ure 2 2C). NKT cells in spleen and MLN were also weakly correlated but no association was found between PB and MLN or CLN and MLN. To investigate the likelihood that genetic effects contribute to the de velopment of NKT cells in pigs, we compared their frequency in PB of between two to nine two week old female piglets from 15 genetically heterogeneous sows that were farrowed in four separate groups. There was wide diversity in frequency among piglets born of different sows, with average NKT cell concentrations ranging from 0.06 (sow 15) to 1.19% (sow 1) of CD3 + T cells (Fig ure 2 2 D). More variation among piglets was encountered in litters with greater average NKT cell frequencies. We examined whether the breed c omposition of sows and boars influenced the NKT frequency of piglets they produced, but no significant relationships were detected. NKT cell subsets that are functionally distinct were identified by their expression of membrane bound CD 4. CD4 + and CD4 NKT cell populations are not clearly
49 distinguishable in pigs. Therefore, in each sample CD4 + and CD4 subsets were identified according to transposed gates that separated total CD4 + T cells from global CD3 + lymphocytes (Fig ure 2 3A). This strategy was used to determine the proportion of NKT cells that express CD4 within each tissue sampled (Fig ure 2 3B). We observed greater mean but more variable proportions of CD4 + NKT cells in lymph nodes, but particularly in CLN. On average, l ess than 10 percent of the NKT cells in PB, thymus and spleen expressed CD4, which is consistent with a previous report describing NKT cells in pig PB (44) . Our results confirm that NKT cell subset distribution is tissue specific. However, further characterization of pig NKT cell populations is required to discover the implications of this observation. To evaluate NKT cell functional varia tion among pigs, PBMCs were stimulated with or without PMA/ionomycin for five hours at 37Â°C and subsequently analyzed for intracellular IFN and IL 4 expression. The proportion of PBMC resident NKT cells stimulated to produce one or both cytokines was quantified according to gates from the unstimulated samples (Fig ure 2 4A). Overall, a similar proportion of activated NKT cells expressed IFN and IL 4 or IL 4 only, while comparatively few produced IFN alone (Fig ure 2 4A). These different cytokine producing populations may represent distinct NKT cell subsets. Larg e variation was detected among individual pigs for mean fluorescence intensity of IFN and IL 4 antibody staining in stimulated NKT cells ( 2 4B). Interestingly, some animals responded very little or not at all to stimulation. The wide range of cytokine responses we observed is sig nificant because the ability of pigs to react to microbial or therapeutic glycolipids likely depends on both their number of NKT
50 cells as well as the amount and variety of cytokines these cells produce when stimulated. GC, OCH, and C glycoside Differentially S t imulate NKT Cell A ctivation and Adjuvant Responses in P igs To investigate how different glycolipid antigens impact NKT cells an d adjuvant responses in swine, three week old newly weaned female piglets were injected i.m. with 200 g/kg of h en egg lysozyme (HEL) in combination with 5.83 x 10 8 mol/kg of GC, C glycoside, OCH or vehicle (PBS) . Immunizations were boosted at five weeks of age with 200 g/kg of HEL with vehicle or the same quantity of glycolipid that was used for inoculati ons at three weeks of age without adjusting for increased body weight (Fig ure 2 1). Piglets from amongst seven litters were assigned to individual treatments so that each group contained animals with a similar distribution of NKT cell frequencies (Fig ure 2 2D and Table 2 1). Blood samples were collected at the indicated time points to measure the frequency of NKT cells and HEL reactive PBMCs as well as cytokine concentrations in the plasma. Spleen, CLNs and MLNs also were analyzed for frequency of NKT cells and HE L reactive cells after pigs were sacrificed at seven weeks of age. Peripheral blood NKT cell conc entrations respectively peaked nine and four d ays after the first and second GC/HEL injections (Fig ure 2 5A). As reported previously for other species, NKT c ells down regulated TCR expression within hours after GC administration (9) . No increase in PB NKT cells was detected for pigs immunized with C glycoside or OCH. GC, but not the other glycolipids, increased the frequency of NKT cells in spleen and CLNs. No change in NKT cell frequency was detected in MLNs for any treatments, pos sibly because of their distance from the injection site (Fig ure 2 5B).
51 All glycolipids induced HEL specific antibody production (Fig ure 2 5C). However, the response s stimulated by GC and C glycoside were more variable compared to OCH. Indeed, the relati ve HEL specific IgG concentration was great er in OCH treated pigs compared to the GC group between days 18 and 28 and compared to the C glycoside group between days 23 and 28. There was no difference in HEL specific mean IgG concentrations between GC and C glycoside treated pigs. The concentration of HEL reactive cells resident within PB and lymphoid organs was analyzed by IFN ELISPOT assay, which involved restimulating single cell suspensions with increasing concentrations of HEL protein (Fig ure 2 5D). A dose dependent HEL response was detected in PBMCs from GC treated pigs 12 days after the booster injection (26d), but not after the primary immunization (12d). Low numbers of HEL reactive cells were identified in PBMCs from OCH treated pigs. However, t his glycolipid was the most efficient at generating HEL reactive splenocytes. Only CLNs from GC treated pigs res ponded to HEL restimulation wh e reas no IFN production was detected from any MLNs. In addition, no HEL responsive cells were detected in PBMCs or lymphoid organs of any C glycoside treated pigs. Cytokine levels in plasma were measured by ELISA (Fig ure 2 5E). Four of the six GC treated pigs produced IFN between 12 and 24 h after the primary immunization. However, no IFN was found after the sec ond GC administration or at any time point for pigs treated with the other glycolipids. No IL 4 was detected in any of the samples collected. Peripheral blood NKT cell concentrations analyzed from piglets one week before the start of the experiment was used as a covariate for the statistical analysis of the
52 different immune parameters measured. This approach showe d initial NKT cell frequency had a significant effect on how glycolipid treatment changed PB NKT cell frequencies and HEL specific IgG concentrations over time. However, this parameter did not influence lymphoid organ NKT cell frequencies or the concentration of HEL reactive cells in PB and lymphoid organs. Body weights and temperatures were recorded regularly to determine whether pig growth and health were affected by any of the glycolipids. Neither parameter was affected by the glycolipid treatments (Table 2 1). Adjusting GC D osage f o r Body Weight Improves the Consistency of Immune Responses Induced by NKT C ells Our second study (E xperiment 2) was designed to determine whether changi ng the dosing strategy used in E xperiment 1 could improve the adjuvant effects of GC. Instead of treat ing animals with the same quantity of GC for both primary and booster immunizations against HEL, piglets were injected with GC ad justed for body weight at both three and five weeks of age at a rate of 50 g/kg (5.83 x 10 8 mol/kg) or 100 g/kg (1.16 x 10 7 mol/kg). Control pigs received HEL with vehicle. Blood and tissue samples were collected as for E xperiment 1 (Fig ure 2 1), to measure the frequency of NKT cells and HEL and GC reactive cells as well as plasma cytokine concentrations. Peripheral blood NKT cell concentrations increased similarly in pigs injected with either dose of GC (Fig ure 2 6A). Only at peak expansion, nine days after the initial immunization (9d) were NKT cell levels greater in the group treated with 100 than 50 g /kg of GC. The timing of PB NKT cell expansion was similar to E xperiment 1. However, responses were more consistent and sustained. The improved consistency was likely, in p art, because pigs selected for E xperiment 2 possessed more similar pre -
53 trial NKT ce ll concentrations, compared to pigs in E xperiment 1 (Table 2 1). I nterestingly, although pigs in E xperiment 2 began with ~2.5 fold fewer PB NKT cells, their average peak (9d) NKT cell concen tration was similar to pigs in E xperiment 1. This difference in ex pansion was not entirely due to variation in treatment method as some animals in both studies received the same initial dose of GC (50 g/kg). Within lymphoid organs, only the 100 g/kg dose of GC significantly increased NKT cell concentrations compared to controls, and only in spleen (Fig ure 2 6B). In addition, NKT cell concentrations were considerably greater in lymphoid organs compared to PB (Fig ure 2 6A and B), including the vehicle treated pigs. The opposite distribution pa ttern was observed for pigs in E xperiment 1 (Fig ure 2 5A and B). Pigs treated with either dose of GC generated comparable levels of anti HEL IgG antibody (Fig ure 2 6C) and HEL reactive PBMCs (Fig ure 2 6D). Antibody responses in E xperiment 2 were more consiste nt between animals comp ared to E xperiment 1. However, HEL reactive PBMC responses remained highly variable and HEL reactive cells within spleen, CLN and MLN were undetectable (data not shown). We also measured in vitro IFN production by PBMCs in response to increasing concent rations of GC using ELISPOT assays (Fig ure 2 6E). This was performed to quantify the frequency GC responsive NKT cells and NKT trans activated leukocyte s resident within PBMCs. A dose dependent response in IFN production was detected w hen GC concentrations were increased, at both 12 d and 26d, although only the 50 g/kg dose was different from controls at the 12d time point. PBMCs from GC injected pigs generated considerably more IFN compared to vehicle treated animals,
54 presumably because of the large increase in PB NKT cells that the glycolipid treatments induced. PMA/ionomycin induced IFN and IL 4 production by pig NKT cells was used as an additional measure of NKT cell function (Fig ure 2 6F). S timulated NKT cells from both vehicle and GC treated pigs produced comparable levels of IFN and IL 4 at 12d. However, at 26d pigs that received the 50 g/kg dose of GC produced more IFN and pigs that received the 50 or 100 g/kg doses of GC produced more IL 4 compared to control animals. Thes e results suggest that treating pigs with multiple doses of GC increases the capacity of their N KT cells to produce cytokines. Plasma cytokines were also measured (Fig ure 2 6G). Within 24 h after the first vaccination, IFN was found in two of five pigs i n both the 50 and 100 g/kg GC treatment groups. However, only one of these pigs in the 50 g/kg group produced IFN after the booster vaccination. No IL 4 was found in any of the plasma samples. Pre trial PB NKT cell concentration was not a significant c ovariate for any of the immune parameters measured in E xperiment 2. Furthermore, GC treatment did not affect body weight gain or body temperatures (Table 2 1). Activated NKT C ells Differentially Regulate Antibody and Cellular Immune R esponses To identify factors that are associated with how individual pigs respond to the adjuvant effects of GC, we correlated various NKT cell responses to anti HEL IgG production and HEL reactive PBMC concentrations at 26d in E xperiment 2. The concentration of GC reactive PBMCs was compared with the relative concentration of anti HEL IgG or IFN produced by HEL reactive PBMCs (Fig ure 2 7A).
55 No relationship was found between GC reactive PBMCs and antibody production. However, PBMCs that responded strongly to G C also possessed more HEL reactive cells. We investigated whether NKT cell production of IFN or IL 4 after PMA/ionomycin activation correlated with how pigs responded to GC. IFN but not IL 4 production was positively associated with IgG production (Fig u re 2 7B). No link was found between cytokine production and the concentration of HEL reactive PBMCs (data not shown). Thus, production of HEL specific IgG and HEL reactive PBMCs was respectively correlated with the level of IFN synthesized by NKT cells a nd by IFN produced by all PBMCs that respond to GC stimulation in vitro , including trans activated cell types. Discussion Others have used a cross reactive mouse CD1d tetramer to demonstrate that pig NKT cells share many phenotypic characteristics with h uman and mouse NKT cells, including the expression of surface molecules associated with T cell activation and memory and the transcription factor PLZF (43, 44) . We employed the same strategy to survey NKT cell populations in a variety of tissues for a large cohort of genetically sciences laboratory . Although the range o f NKT cell frequencies was wide for all tissues tested, they were also similar to what has been reported previously for pigs and other species (43, 44, 227, 228) . Furthermore, our finding that NKT cell concentrations in spleen are higher and more variable compared to PB and lymph nodes is co nsistent with the distribution of NKT cells in mice (96, 228) . Also similar to mice, we found a
56 moderate correlation in the frequency of NKT cells between PB and spleen within individual pigs (228) . However, both blood and spleen were respectively weakly and not significantly correlated to CLN and MLN for NKT cells. Taken together, these results suggest that PB only partially reflects the NKT cell status in pigs. The same is likely to be true for humans. The range of PB NKT cell frequencies between litters was large, with two out of 15 sows farrowing piglets with particularly high and variable concentrations of these lymphocytes. Although, high and low NKT cell producing sows farr owed at the same time and in adjacent crates, piglets were born in a conventional farrowing house where environmental factors may have influenced NKT cell development during the first two weeks of life. However, it is likely much of the variation was due t o genetic effects, because it is known that NKT cell development and homeostasis is influenced strongly by multiple loci in mice (228 230) . Differences in NKT cell frequency between lit ters may impact the health of piglets given that these immunoregulatory cells make important contributions to both innate and adaptive immunity against multiple pathogens, and appear to develop soon after birth when animals are most susceptible to infectio ns. Further studies are needed to establish the importance of NKT cells for neonatal immunity as it may be possible to improve pig health by selectively breeding for greater numbers of these cells. Besides frequency, immune responses modulated by NKT cells are influenced heavily by their ability to produce cytokines after stimulation. Significant variation in the capacity of NKT cells to produce different cytokines has been reported for both humans and inbred mouse strains (54, 227) . This phenomenon may in part explain why specific
57 NKT cell activation protocols a ctually exacerbate rather than ameliorate some mouse models of disease (8, 111, 119) . Our study confirms tha t a large spectrum of IFN and IL 4 responses exists between pigs for PB NKT cells. Some of this diversity may have been due to the pig to pig variation in NKT cell subset ratios we observed, as each subset produces different mixtures of cytokines. Particu larly large variation in subset ratios was detected in lymph nodes. This may be significant because lymph nodes are likely where NKT cells first encounter therapeutic glycolipids injected i.m., s.c. or i.p. For the first time, adjuvant effects of NKT cell agonists have been demonstrated in pigs. The three glycolipids selected for testing are reported to promote different patterns of cytokine production in mice (8 ) . Multiple model systems have shown that GC has exceptionally high stimulatory activity and induces both Th1 and Th2 cytokines (8) . In contrast, C glycos ide and OCH respectively promote Th1 and Th2 immune responses (8) , but both analogs stimulate less secretion of a smaller variety of cytokines compared to GC (145, 231 233) . These differences in cytokine expression are likely related to the di stinct pharma c okinetic and TCR stimulatory properties of each glycolipid in vivo (145) . The same factors probably explain why GC but not OCH or C glycoside expanded NKT cells in our study. A comparison of HEL specific immune responses induced by the different NKT cell agonists has provided interesting results. Although OCH did not increase measurably NKT cell proliferation, this glycolipid stimulated pigs to produce hig her and more consistent HEL specific IgG responses compared to the other agonists. This may indicate that in pigs OCH is especially effective at stimulating cytokines that drive antibody responses. OCH was also able to stimulate HEL specific IFN ELISPOT
58 r eactions from blood and spleen cells while C glycoside only induced antibodies. This is inconsistent wi th mouse studies that have reported that C glycoside triggers a Th1 biasing cytokine response with superior adjuvant activities on NK and CD8 T cells aga inst malaria and tumor metastases (57, 154 156, 234) . However, swine reactions to NKT cell agonists may not mimic necessarily other species because the invariant TCR of NKT cells rather than the CD1d molecule determines the differential response of NKT cells to structurally different glycolipids (235) . This probably underlies why OCH and C glycoside are not potent activators of human invariant NKT cells. Our second immunization experiment was designed to determine whether changi ng the dosing strategy used in E xperiment 1 could improve the adju vant efficiency GC. We found that adjusting the booster immunization concentration of GC for body weight led to more sustained NKT cell expansion and more consistent HEL specific IgG reactions. The improved consistency of IgG production suggests our modi fied approach better stimulates pigs that are inherently insensitive to NKT cell therapy. However, doubling the concentration of GC had no significant impact on the adjuvant effects measured, which indicates that 50 g/kg is sufficient to maximize NKT c ell responses in most pigs. NKT cells in the pigs treated with GC did not appear to become anergic at the second boosting injection, unlike in mice where a single administration of GC induced long term NKT cell unresponsiveness (162) . HEL specific antibody levels in individual pigs were correlated positively with the amount of IFN produced by their NKT cells after PMA/ionomycin stimulation . Instead, HEL reactive PMBC concentrations were correlated positively with the number of all IFN producing cells induced by GC stimulation. These likely included NK cells,
59 conventional T cells and neutrophils that are trans activated to produce IFN by stimulated NKT cells (9) . One interpretation of these results is that IgG production relies more on direct interactions between NKT cells with B cells, and less on other immune cells. This is consistent with reports that B cells directly present G C to NKT cells that in turn drive IgG production through a variety of T helper type signals, including CD40L, IL 21 and ICOS (236) . On the other hand, T cells that do not directly interact with NKT cells must rely more on trans activation effects. It is important to note that although our assays used IFN as a read out for the activation status of NKT cells and other leukocytes, it is likely the antibody and T cell responses observed resulted from multiple additional stimulatory signals up regulated in conjunction with this cytokine. Another factor associated with how different humans and inbred mouse strains respond to glycolipid antigens is their frequency of circulating NKT cells prior to treatment (17, 171, 183, 230) . This parameter only correlated with how individual pigs responded to NKT cell therapy in E xperiment 1, presumably because th is study included animals expressing a much wider range of NKT cells compared to the second trial. This indicates that PB NKT cell frequency is a relatively insensitive predictor of how individual pigs will respond to the adjuvant effects of glycolipid ant igens. Finally , we observed that all pigs in E xperiment 2 developed a mild allergic reaction after receiving their second injection of GC and HEL. Pigs that received HEL alone were not affected. Symptoms manifested included whole body skin rash, excessiv e salivation, and lethargy for a period or 30 to 60 minutes after vaccination. It is likely that the pigs experienced an allergic reaction to HEL and/or GC. This was surprising as none of the GC treated pigs in E xperiment 1 developed reactions after
60 their booster immunization, although some were initially primed with the same dose of GC and HEL. Variables that may have contributed to the different reactions between studies include that GC was supplied by different manufacturers and boosted at differ ent concentrations for each experiment. Further experiments are needed to evaluate the importance of these factors. However, the adverse reactions we encountered serve to highlight that dangers remain to be overcome before NKT cell antigens can be safely u sed as adjuvants for humans and swine. Conclusion C onsiderable variability in tissue frequency, subset distribution and cytokine secretion patterns were detected for NKT cells analyzed from a large cohort of crossbred commerc ial pigs. Importantly, NKT cel l mediated adjuvant effects were demonstrated. Furthermore, useful information was generated about how pigs respond to different varieties and dosage levels of therapeutic glycolipids, which will provide a better understanding of how NKT cells could be har nessed to protect swineherds and possibly humans from dangerous pathogens.
61 T able 2 1. Ch aracteristics of pigs used for Experiment 1 and E xperiment 2 Treatment Groups Number of animals Initial NKT cell frequency 1 (% o f CD3 + cells) Body temperature 2 (Â°C) Average daily gain (kg/day) Final body weight (kg) Experiment 1 PBS + HEL 6 0.60 Â± 0.16 39.58 Â± 0.08 0.51 Â± 0.03 18.29 Â± 1.10 GC + HEL 6 0.83 Â± 0.29 39.45 Â± 0.07 0.58 Â± 0.03 20.26 Â± 0.92 C glycoside + HEL 6 0.79 Â± 0.23 39.35 Â± 0.07 0.52 Â± 0.04 18.07 Â± 1.56 OCH + HEL 6 0.77 Â± 0.27 39.53 Â± 0.05 0.53 Â± 0.04 18.67 Â± 1.29 Experiment 2 PBS + HEL 3 0.29 Â± 0.11 39.32 Â± 0.05 0.53 Â± 0.04 18.75 Â± 1.60 GC 50 + HEL 5 0.31 Â± 0.12 39.28 Â± 0.11 0.56 Â± 0.02 20.50 Â± 0.89 GC 100 + HEL 5 0.33 Â± 0.13 39.33 Â± 0.10 0.53 Â± 0.03 18.87 Â± 1.26 Values represent least squares mean s Â± SEM. No d ifferences were detected among treatment groups for any of the above measurements. NKT: natural killer T; HEL: hen egg lysozyme; GC: alpha galactosylceramide. 1 Analyzed from peripheral blood at 2 weeks of age 2 Average of 11 rectal temperature measurements taken during the course of each experiment
62 Figure 2 1. Setup for hen egg lysozyme ( HEL ) immunization experiments. For E xperime nt 1, piglets were injected at three (0d) and five (14d) weeks of age with 200 g/kg of HEL alone or in combination with alpha galactosylceramide ( GC ) , OCH, or C glycoside at concentrations indicated in Materials and Methods. For E xperime nt 2, piglets were injected at three and five weeks of age with 200 g/kg of HEL alone or in combination with 50 or 100 g/kg of GC. For both experiments, blood sampl es were taken from the jugular vein at all indicated time point s. All pigs were euthanized at seven weeks of age (28d) to collect peripheral blood ( PB ) , spl een, cervical lymph nodes ( CLN ) and mesenteric lymph nodes ( MLN ) for analysis. Figure 2 2. Comparis on of pig NKT cell frequencies between different tissues. ( A ) Gating strategy to identify NKT cells in pigs. Single cell suspensions from different tissues were membrane labeled with anti CD3 mAb and PBS57 loaded CD1d tetramer. Some samples were also label ed with an empty CD1d tetramer to validate the staining of PBS57 loaded CD1d tetramer. One representative case is shown. ( B ) NKT cell frequency expressed as a proportion of CD3 + cells for PB (n=79), spleen (n=79), thymus (n=3), CLN (n=78) and MLN (n=35) wh ere n represents the number pigs sampled for each tissue from the total of 79 animals. Each symbol corresponds to an individual. Tissues were compared by Wilcoxon U test. ( C ) Correlation of NKT cell frequencies as a proportion of CD3 + cells between PB and lymphoid organs (top panel) and between lymphoid organs (bottom panel). Correlations were determined using linear regression analysis and the line in each plot represents the best linear fit. ( D ) Frequency of PB NKT cells in 2 week old female piglets from 15 different sows. The labels E xperim ent 1 and E xperiment 2 indicate the piglets tested for enrollment into glycolipid immunization studies. Bars represent the mean SEM. *P < 0.05 .
64 Figure 2 3. Tissue distribution of NKT cell subsets. ( A ) Gating strategy to distinguish NKT cell subsets in pig tissues. NKT cells identified using anti CD3 mAb and CD1d tetramer were co stained with anti CD4. Gating to discriminate CD4 + and CD4 NKT cell subsets was based on CD4 staini ng of the total CD3 + lymphocytes population in each sample. One representative case is shown for each tissue. ( B ) NKT cell subset frequency expressed as a proportion of CD1d tetramer + CD3 + cells for PB (n=65), spleen (n=69), thymus (n=3), CLN (n=70) and ML N (n=37) where n represents the number pigs sampled for each tissue from the total of 79 animals . Each symbol corresponds to an individual. Tissues with no common superscripts differ significantly ( P<0.05 ) when compared by Wilcoxon U test. Bars represent t he mean SEM.
65 Figure 2 4. NKT cell cytokine responses to PMA/ionomycin. Pig PBMCs were stimulated wit h or without PMA/ionomycin for five h ours , membrane labeled with anti CD3 and CD1d tetramer, fixed and permeabilized, and intracellularly stained with anti IFN and anti IL 4. ( A ) The proportion of stimulated NKT cells producing one or both cytokines was quantified using gates from unstimulated samples. One representative case is shown (left panel) as well as the average prop ortion of activated p ig NKT cells that expressed IFN and IL 4, IL 4 only, or IFN only (right panel). Data represent ed as the mean SEM. ( B ) Mean fluorescence intensity of IFN (left panel) and IL 4 (right panel) antibody staining for unstimulated versus stimulated NKT cells for individual pigs. Averages were compared by Wilcoxon U test. * P < 0.05 .
66 Figure 2 5. Results of E xperiment 1 that anal yzed pig NKT cell responses to GC, OCH, and C glycoside. Pigs were immunized with HEL and glycolipids as described in Materials and Methods. ( A & B ) The frequency of NKT cells as a proportion of CD3 + cells was analyzed in PB over the course of the experiment represented in days (d) and hours (h) after the first injection ( *P<0.05 , GC versus other glycolipids ) ( A ) and in various lymphoid organs after pigs were euthanized ( B ). ( C ) Plasma was assessed by ELISA for relative levels of HEL specific IgG at a 1/50 dilution over the course of the experiment represented in days (d) after the first injection . Data are r epresented as mean OD405 SEM. IgG levels were significantly higher in OCH pigs between 23d and 28d compared to the other treatments. No significant differences were found between C glycoside and GC treated pigs. ( D ) IFN production by HEL reactive cells in PBMCs, spleen and CLN. Results represent mean IFN spots per 1 x 10 4 PBMCs, splenocytes or CLN cells. ( E ) Plasma IFN as measured by ELISA over the course of the experiment represented in days (d) and hours (h) after the first injection . Cumulative dat a from two experiments using 6 pigs per group are shown. Changes in PB NKT cells, HEL specific IgG concentrations and plasma IFN were analyzed using the SAS PROC MIXED procedure and the slice command was used to examine treatment differences at each time point for each dependent variable. All other means were compared using the Wilcoxon U test. Bars represent the mean SEM. * P < 0.05 .
68 Figure 2 6. Results of E xperiment 2 that analyzed pig responses to different doses of GC. Pigs were immunized with HEL and vehicle, 50 or 100 g/kg of GC as described in Materials and Methods. ( A & B ) The frequency of NKT cells as a proportion of CD3 + cells was analyzed in PB over the course of the experiment represented in days (d) and hours (h) after the first injection (* P<0.05, GC versus other glycolipids) ( A ) and in various lymphoid organs at 28d ( B ). ( C ) Plasma was assessed by ELISA for relative levels of HEL specific IgG at a 1/50 dilution over the course of the experiment represented in days (d) after the first injection . There was no significant difference between the 50 or 100 g/kg treatment groups. ( D & E ) IFN production by PBMCs was measured in response to HEL ( D ) and GC ( E ) restimulation. Results represent mean IFN spots per 1 x 10 4 PBMCs. ( F ) Fold change in mean fluorescence intensity (MFI) of antibody staining of IFN and IL 4 within PBMC resident NKT cells incubated with or without PMA/ionomycin ( G ) Plasma IFN as measured by ELISA over the course of the experiment represented in days (d) and hours (h) after the first injection . Data are represented as mean SEM. PB NKT cells, IgG concentrations and plasma IFN were analyzed using the SAS PROC MIXED procedure and the slice command was used to examine treatment differences at each time point for each dependent variable. All other means were compared using the Wilcoxon U test. Data represent ed as the mean SEM. *P < 0.05.
70 Figure 2 7. NKT cells differentially regulate antibody and cellular IFN production. ( A ) The frequency of GC reactive PBMCs in pig blood correlates with HEL reactive PBMCs, but not anti HEL IgG concentrati on. ( B ) IFN but not IL 4 levels in PMA/ionomycin stimulated NKT cells is correlated with anti HEL IgG production. Comparisons were made using blood collected at 26d and analyzed by ELISPOT, ELISA and intracellular cytokine staining. Correlations were determined using linear regression analysis and the l ine in each plot represents the best linear fit. Analyses did not include PBS + HEL treated pigs.
71 CHAPTER 3 GENERAL CONCLUSION AND FUTURE PERSPECTIVE Extensive study of NKT cells over the last 15 years has revealed these cells have great potential to be h arnessed to treat a wide range of diseases. NKT cells possess properties of both the innate and adaptive immune systems and are capable of stimulating other immune cell types in ways that augment overall immunity. NKT cells and their immunoregulatory funct ions can be targeted by synthetic glycolipids such as GC and related analogs. These have potential as disease treatments and vaccine adjuvants. Various obstacles have prevented the rapid translation of NKT cell therapeutics to humans, including the lack o f an appropriate animal model with which to test the safety and efficacy of these agents for protection ag ainst human relevant pathogens. Our study has identified swine as a promising animal model to address this deficit. The current work shows that pig NK T cells share many common characteristics with human NKT cells. Importantly, we also show that pig NKT cells can be targeted with synthetic glycolipids to generate immune responses against exogenous antigens. In addition, valuable information was generated about how pigs tolerate and respond to different varieties and dosage levels of glycolipid antigens. Taken together, our results demonstrate the promise of using a pig model to study and understand NKT cell biology and the therapeutics that activate these cells. We expect our work to underpin future studies testing whether NKT cell can be used to treat and protect pigs from live pathogens, which will provide a better understanding of how NKT cells could be employed to protect against important diseases tha t threate n humans and swineherd health.
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95 BIOGRAPHICAL SKETCH Bianca Libanori Artiaga was born in GoiÃ¢nia, GoiÃ¡s, Brazil, in 1988. In March 2007 she began her studies in the School of Veterinary Medicine and Animal Sciences at the Federal Univ ersity of GoiÃ¡s (EVZ/UFG), and she graduated as a Bachelor of Veterinary Medicine in March 2012. In August 2012 she moved to Gainesville, Florida, USA to join the Graduate Program in the Department of Animal Sciences at University of Florida as a Master of Science student under the supervision of Dr. John Driver. She obtained her Master of Science degree in August 2014.