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CD44-dependent hyaluronan-binding and T-lymphocyte migration Mohseni Koochesfahani, Kasra 2002

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CD44-dependent Hyaluronan-binding and T-lymphocyte Migration by Kasra Mohseni Koochesfahani MD., Mashad University of Medical Sciences, 1996 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENT FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES Genetics Graduate Program We accept this thesis as conforming to the required standard 2002 THE UNIVERSITY OF BRITISH COLUMBIA ©Kasra Mohseni, 2002 In presenting this thesis in partial fulfillment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of . foa#&* The University of British Columbia Vancouver, Canada Date ABSTRACT CD44 has been suggested to play a role in adhesive interactions mediating migration and extravasation of T cells into the tissues. This mediation is believed to occur as a result of CD44 binding to hyaluronic acid (HA) on the surface of endothelium. Among factors suggested to regulate this binding is the pro-inflammatory cytokine TNFa, which has been suggested to upregulate HA-binding through sulfation of the CD44 molecule. One of the objectives of this research proposal was aimed at determining the role of T cell activation, TNFa and TNFa receptors in HA binding by T lymphocytes. It was found that only highly activated T cells, which are the largest in cell size and expressed the highest levels of CD44 were the most efficient HA binders. TNFa exerts its effect on HA binding by regulating T cell activation. It increases HA binding under conditions where the cells were not optimally activated. The effects of TNFR1 (p55) and TNFR2 (p75) on HA binding can also be explained by their role in T cell activation. Thus, p55, which is not required for optimal T cell activation, has no effect on HA binding by anti-CD3 activated CD8 T cells. By contrast, p75"/_ CD8 T cells, which were less optimally activated by anti-CD3 stimulation, bind much less HA than similarly activated wild type cells. The role of CD44 in migration of T lymphocytes in vivo was also determined. Various in vivo assays were used to determine whether CD44 was required for migration of polarized TH1 and TH2 cells into sites of inflammation and whether this migration correlated with HA binding. The role of CD44 in the migration of antigen-specific CD8 T cells at various times after antigen activation into lymphoid and non-lymphoid tissues was also determined. All these assays failed to demonstrate an essential role for CD44 in T cell migration. Thus, alternative mechanisms other than those mediated by CD44 are likely responsible for regulating T cell migration in vivo. ii Table of Contents Abstract ii Table of Contents iii List of Figures vi List of Abbreviations viii Acknowledgements x Chapter 1 Introduction 1 1.0 Historical perspective 1 1.1 CD44 structure 2 1.2 CD44 ligands 2 1.3 Hyaluronic acid 4 1.4 Regulation of CD44 HA-binding 4 1.5 Sulfation 6 1.6 TNFoc 6 1.7 Recirculation of naive and activated T lymphocytes 7 1.8 The role of CD44 in lymphocyte migration 8 1.9 Contact hypersensitivity 9 1.10TH1/TH2 10 1.11TC1/TC2 10 1.12 Objectives of the thesis 11 Chapter 2 Materials and Methods 12 2.0 Mice 12 2.1 Antibodies/reagents 12 2.2 Cytokines 13 2.3 Harvesting LN/spleen cells from a B6 mouce 13 2.4 Purification for CD4/CD8 T lymphocytes 14 iii 2.5 In vitro stimulation of CD4 or CD8 T cells 14 2.5.1 Stimulation by antibody 14 2.5.2 Stimulation by antigen presenting cells and antigen 14 2.6 Proliferation assay 15 2.7 Fluorescein labelling of hyaluronan 16 2.8 Flow cytometric analysis of lymphocytes 17 2.9 Polarizing CD4 cells to TH1/TH2 17 2.10 Cytokine production by the polarized cells 17 2.1.1 Cytokine ELISA 18 2.12 Induction of DTH (Delayed-type hypersensitivity) 18 2.13 Radiolabelling of cells with 5 1 Cr . 19 2.14 Labelling with CFSE (Carboxyfluorescein diacetate succinimidyl ester) 19 2.15 Perfusion of the animal 19 2.16 Separation of the lymphocytes from tissues 20 2.16.1 Blood 20 2.16.2 Liver & kidney 20 2.16.3 Peritoneal lavage cells 21 Chapter 3 Results 22 3.1 CD44-dependent HA-binding 22 3.1.1 H A binding to activated B6 and B6-CD44"'" T lymphocytes in vitro 22 3.1.2 Activated CD8 cells bind to more H A than activated CD4 cells 24 3.1.3 Influence of T N F a and TNFoc receptors on H A binding 26 3.2 DTH experiments. 33 3.2.1 B6 CD4 cells can be polarized to TH1 blasts i f exposed to a certain profile of cytokines while being activated with antiCD3 33 3.2.2 The B6 CD4 cells that were polarized to TH1 blasts demonstrated the ability to migrate to the inflammation site 35 iv 3.2.3 CD44, p55 or p75 deficiency did not affect the ability of polarized TH1 cells to migrate to the inflammation site 35 3.2.4 To determine whether there is a correlation between H A binding and lymphocyte migration 37 3.2.5 Migration of recently activated CD4 and CD8 T cells to the inflamed site is independent of CD44 37 3.3 To address the role of CD44 in the in vivo migration of CD8 T lymphocytes into various tissues 41 Chapter 4 Discussion 51 References 57 v List of Figures Figure 1.1. Schematic presentation of CD44 structure and its localization. 3 Figure 1.2. Electron micrograph of hyaluronan molecules rotary shadowed 5 with heavy metals for visualization purposes. Figure 3.1. H A binding by activated CD8 T cells is CD44-dependent. 23 Figure 3.2. Activated CD8 cells bind more H A than activated CD4 cells. 25 Figure 3.3. Effect of anti-CD3 concentration on HA-binding. 27 Figure 3.4. Correlation of H A binding with CD44 expression and cell size. 28 Figure 3.5. Enhancement of H A binding by antigen-activated CD8 T cells. 30 Figure 3.6. T N F a did not increase HA-binding by optimally activated CD8 31 T cells. Figure 3.7. Less efficient H A binding by activated p75v" CD8 T cells. 32 Figure 3.8. H A binding as a function of cell size. 34 Figure 3.9. Cytokine production by polarized TH1 and TH2 cells. 36 Figure 3.10. Preferential migration of TH1 cells to DTH site. 38 Figure 3.11. Efficient migration of polarized TH1 cells into DTH site is not 39 dependent on CD44, p55 or p75. Figure 3.12. H A binding by resting B6 TH1 and B6 TCI cells is up- 40 regulated by the IRAWB mAb. Figure 3.13. Migration of 48 hr-activated B6 CD4 cells to the inflammation 42 site. Figure 3.14. Migration of recently activated CD4 and CD8 T cells to DTH 43 site is not dependent on CD44. Figure 3.15. Optimal proliferation in response to the S Y R G L / K b ligand is 44 not CD44-dependent. Figure 3.16. Lack of CD44 did not affect expansion of activated CD8 T 45 cells in vivo. Figure 3.17. Protocol for tracking the in vivo migration of Ag-activated 47 CD8 T cells. Figure 3.18. Migration of 7-day Ag-activated 2C CD8 T cells to the 48 vi lymphoid and non-lymphoid tissues is not dependent on CD44. Figure 3.19. Increased migration of CD44-deficient CD8 T cells into 49 lymphoid and non-lymphoid tissues. Figure 3.20. Increased recovery of CD44"'" CD8+ T cells at various time 50 points. vii List of Abbreviations 2C TCR Transgenic TCR specific for p2Ca peptide presented by K b and L d class I MHC 2ME 2-Mercapthoethanole Ab Antibody Ag Antigen APC Antigen presenting cell BM Bone Marrow BSA Bovine Serum Albumin CD Cluster of differentiation CFSE Carboxyfluorescein diacetate succinimidyl ester CHS Contact Hypersensitivity ddH20 Distilled Deionized Water DNFB 2,4-dinitro-1 -fluorobenzene DMSO Dimethyl Sulfoxide DTH Delayed-type hypersensitivity ELISA Enzyme-Linked Immuno Sorbent Assay FACS Fluorescence activated cell sorter FBS Fetal Bovine Serum FCA Freund's complete adjuvant FITC Fluorescein Isothiocyanate H-2 Histocompatibility-2 HA Hyaluronic acid IFNy Interferon gamma Ig Immunoglobulin IL Interleukin I-medium Iscove's medium LN Lymph node mAb Monoclonal antibody MHC Major histocompatibility complex viii P2Ca The peptide LSPFPFDL (single letter amino acid code) PBL Peripheral Blood Lymphocyte PBS Phosphate Buffered Saline PE Phycoerythrin PMA Phorbol Myristate Acetate RBC Red Blood Cell s.c. Subcutaneous SEB Staphylococcal Enterotoxin B SYRGL The peptide SYRGL (single letter amino acid code TAP Transporters associated with antigen processing TCR T cell receptor TH1/TH2 Helper T cell 1/Helper T cell 2 TC1/TC2 Cytotoxic T cell 1 /Cytotoxic T cell 2 TNF Tumor necrosis factor TNFR Tumor necrosis factor receptor ix Acknowledgements I would like to thank Dr. Hung-Sia Teh, my supervisor, who patiently guided me in my research. This work would not have come to an end if it were not for his support and encouragement during the years. I would also like to thank Soo-Jeet. I appreciate her relentless effort to help us in our research. I would like to thank Edward Kim and Becky Diensen for their work as technicians in our lab. I would also like to thank my supervisory committee (Dr. Pauline Johnson and Dr. Fumio Takei) for their help over the years. While completing my work in Dr. Teh's lab, I am happy that I had the opportunity to work with Darryl, Edward, Jennie, John, Julia, Oliver, Payam and Salim. They were the people that helped me keep going during hard times. I would like to thank my dear friend, Farzad, whose support and help during this work has been immense and who played a critical role in my completing it. I would like to express my heartfelt thanks to my parents whom I owe this desire for intellectual pursuit, which led me here. I am grateful for their many sacrifices. x CHAPTER 1 INTRODUCTION Migration of the cells, development of the immune system, formation of the embryo and tumor progression are all processes that depend on interaction between cells or between cells and the extracellular (EC) matrix [1]. The study of cell surface molecules involved in these interactions is under intense investigation by scientists. CD44 is among the molecules characterized to play a role in these interactions. CD44 was known with different names, such as Pgp-1 (phagocytic glycoprotein-1), Hermes antigen and ECMRIII (extracellular matrix receptor type III), before its designation to CD44 [2, 3]. 1.0 HISTORICAL PERSPECTIVE Early research demonstrated that hyaluronan had the ability to bring the cells together leading to their aggregation. These studies suggested that there must be receptors for hyaluronan on the surface of the cells. The cells could be either of the same or different types, as long as they both had the receptor on the surface; for instance a low concentration of hyaluronan was able to aggregate T lymphocytes. In 1980, Underhill et al. identified a protein on the surface of SV3T3 cells with a molecular weight of about 85 kDa and suggested that it was most likely responsible for binding of hyaluronan to the cell surface [4]. Later studies demonstrated a molecule on the surface of some other tissues that had quite the same characteristics as the receptor of hyaluronan in SV3T3 cells. Underhill et al. discovered that the same antibody that can block aggregation of the cells (by hyaluronan) was able to bind to a 85-kDa leukocyte antigen called CD44 that was well-known at the time [5]. Research demonstrated later that CD44 is indeed the protein that binds to hyaluronic acid. CD44 is widely expressed on many cell types including leukocytes, endothelial cells, epithelial cells and fibroblasts. 1 1.1 CD44 STRUCTURE The CD44 family of transmembrane glycoproteins is encoded by a single gene consisting of 21 exons, of which at least 10 can be differentially spliced. Due to this great variability in splicing, at least 17 different CD44 proteins have been identified [6, 7]. CD44S (CD44H) is the most common form and has a molecular weight of 85-90 kDa. This protein is encoded by exons 1-5,16-18 and 20. The polypeptide chain synthesized has a molecular weight of only ~37kDa. The remainder of the ~85kDa mass of the CD44S molecule is contributed by N-linked and O-linked glycosylation. As displayed in figure 1.1, the CD44 molecule consists of cytoplasmic, transmembrane, membrane-proximal and extracellular parts. The distal extracellular domain is where binding to hyaluronan takes place [8]. The membrane-proximal extracellular domain is the site where most of the alternative splicing takes place. Splicing leads to CD44 variant isoforms (CD44v), which have additional sequences in their membrane-proximal domain [9]. The remarkable ability of CD44 to produce isoforms has led to the speculation that CD44, via its numerous isoforms, can have a role in the growth and metastasis of tumor cells. 1.2 CD44 LIGANDS The most extensively characterized ligand for CD44 is hyaluronan, which is a major component of the extracellular matrix. CD44 isoforms bind to hyaluronan through motifs that have been identified in its extracellular part. However, binding of the T cells to the mucosal high endothelial venules can be independent of hyaluronic acid, which suggests that other molecules may serve as ligands for CD44 [10]. Examples of such ligands include serglycin, a hematopoietic cell lineage-specific proteoglycan with a possible role in lymphoid cell adherence and activation [11], osteopontin, collagen type I and fibronectin [12] . Binding to some of these ligands may be specific to certain CD44 isoforms [13]. 2 Figure 1.1. Schematic representation of CD44 structure and localization. Panel (a) a schematic representation of the extracellular, membrane-proximal, transmembrane and cytoplasmic parts of the CD44 molecule. From Toole BP. Hyaluronan in Morphogenesis and Tissue Remodeling. (vv^vvw.glycoforum.gr.jp/science/hyaluronan/HA08/HA08E.html) Panel (b) shows how addition of variant exons affects length and structure of the CD44 molecule. With changes from Knudson W, Knudson CB. The Hyaluronan Receptor, CD44. (www.glycoforum.gr.jp/science/hyaluronan/H A10/H A1 OE.html) 3 1.3 HYALURONIC ACID Hyaluronic acid was discovered by Meyer and Palmer in 1934. It is a linear polysaccharide formed by 2,000-25,000 repeating disaccharide units consisting of D-glucuronic acid and N-acetyl-D-glucosamine. HA is an important component of the extracellular matrix; it is ubiquitous in the organism, with the highest concentration found in soft connective tissue. Only one kind of hyaluronan exists. The average disaccharide has a length of ~1 nm. Thus, a hyaluronan molecule of 10,000 repeats could extend 10 um if stretched from end to end, a length approximately equal to the diameter of a human erythrocyte. Figure 1.2 shows an electron micrograph of a few intertwined hyaluronan molecules that have been deposited on a flat surface and rotary shadowed with heavy metal for contrast. 1.4 REGULATION OF CD44 HA-BINDTNG CD44 is the primary cell surface receptor for the extracellular matrix glycosaminoglycan hyaluronic acid. However, most of the normal murine hematopoietic cells cannot bind to hyaluronic acid even though they express CD44 on the surface [14]. Lesley et al. demonstrated that an anti-CD44 monoclonal antibody (IRAWB 14) can rapidly turn the non-HA binding CD44 to the binding form in T lymphocytes. These two observations suggested that the HA-binding properties of CD44 must be regulated in the cells. Different studies have shown that at least part of this regulation is through cell specific posttranslational modifications of the CD44 molecule. CD44 on the surface of naive lymphocytes is in a non-functional form known as standard (CD44S) or hematopoietic (CD44H). It has the extracellular, transmembrane and cytoplasmic domains but does not bind to hyaluronic acid; DeGrendele et al. demonstrated that binding to hyaluronic acid in T-lymphocytes can be induced in vitro by activating the cells; it does not depend on the method of activation [15]. In their experiments upregulation could be induced in vitro through stimulation by antigen, superantigen or mitogen [16]. 4 Figure 1.2. Electron micrograph of hyaluronan molecules that were rotary shadowed with heavy metals for visualization purposes. Hyaluronan is constructed from alternating monosugars of glucuronic acid and N-acetylglucosamine forming a polymer sugar. In a molecule of hyaluronic acid, the number of repeats (composed of one molecule of each monosacharide) can reach up to 10,000 or more, making a molecular weight of ~4 million daltons. The disaccharide itself has an average length of ~1 nm, that makes up about lOpm when 10,000 of them are put together. The above figure is an electron micrograph of several molecules of hyaluronan that have been rotary shadowed with heavy metals on a surface. Rotary shadowing is a technique used in electron microscopy to increase contrast in generating images. From Hascall VC, Laurent T. Hyaluronan: Structure and Physical Properties (www.glycoforum.gr.jp/science/hyaluronan/profile/biosketchE.html) Active cells express the active form of CD44, which has the ability to bind hyaluronic acid. Many factors have been implicated in regulating CD44 HA-binding. Expression of CD44v isoforms is one of these factors. Alternative splicing of variant exons is precisely regulated. Frequent association of abnormal isoform patterns with malignancy can be the result of a dysregulation in this process [17]. To increase its HA-binding, CD44 also undergoes post-translational changes like glycosylation [18], phosphorylation and addition of glycosaminoglycan chains [19] in some isoforms to convert it to the adhesive form. To discover the mechanisms involved in this modification is an area of active research. Johnson et al. have suggested that all these factors exert their role through affecting aggregation or altering the conformation of CD44 [19]. 1.5 SULFATION Johnson et al. [20] have demonstrated that TNFa can affect HA-binding ability of CD44 in the human leukemic cell line SR91 through sulfation at the post-translational level converting it from the inactive form to its active form that has the ability of binding to hyaluronan. Their results showed that incubation of the cells with TNFa (lOng/ml) for 24h resulted in increased expression of CD44 and increased binding to fluoresceinated hyaluronan. 1.6 TNFa TNFa is a member of the TNF family of cytokines postulated to have a role in migration of T-lymphocytes into tissues [21, 22]. TNFa can induce extravasation of lymphocytes from blood into tissue after local administration into skin [23]. TNFa is also suggested to play a role in upregulation of CD44-dependent HA-binding through sulfation [20]. Two distinct types of TNF receptors have been identified: a 55-kDa (p55) form designated TNFR1 and a 75-kDa (p75) form designated TNFR2. T-lymphocytes from C57BL/6 mice express both TNFRI and TNFRII on the surface [24]. 6 1.7 RECIRCULATION AND EXTRAVASATION OF NAIVE AND ACTIVATED T-LYMPHOCYTES After completing their development in the thymus, naive T lymphocytes enter the circulation; then they migrate from the bloodstream to the peripheral lymphoid tissues (lymph nodes and spleen). To maximize the chance of encountering an antigen, T-cells are continuously recirculating from the blood circulation to the lymphoid organs and back to the blood. This continuous recirculation provides the opportunity for the naive lymphocytes to make contact with a large number of antigen presenting cells in the lymphoid system everyday. To migrate to the lymphoid tissue, the naive T cells cross the walls of High Endothelial Venules (HEV). Expression of cell surface molecules such as L-selectin on the surface of naive T cells is the cause of this preferential migration to the lymph nodes [25]. Naive T cells that recognize their antigen, that is presented by an antigen presenting cell in the lymph node, undergo a series of changes leading to the generation of effector cells. Upon activation, the T cells quickly switch their pattern of trafficking to the lymphoid organs to one of sequestration in other tissues. This new pattern is believed to be a result of changes in the expression of markers on the cell surface, causing the T lymphocytes to lose their ability to enter the lymph nodes and spleen. [26]. The activated T cells express new adhesion molecules that direct them to the site of inflammation in the peripheral tissues. To migrate out of the blood vessels and into the tissues, the activated T cells undergo rolling adhesion, tight binding, diapedesis and finally migrate into the tissue. Inflammation also causes certain changes in the tissue that assist the leukocytes in iinmigrating to the inflammation site. Inflammation leads to dilatation of the blood vessels, increased permeability and slowing of the blood, which helps leukocytes get close and interact with the endothelial lining. The inflammatory mediators released in the tissue also result in changes in the expression of adhesion molecules on the endothelial lining, which results in recruiting a larger number of leukocytes to the inflammation site [27,28]. 7 1.8 THE ROLE OF CD44 IN LYMPHOCYTE MIGRATION Protin et al. [1] demonstrated that lack of CD44 in CD44-deficient mice doesn't have any effect on embryonic development. Lymphoid organs developed normally in these mice with no deviation in the number or phenotype of T cell subsets. Lack of CD44 was observed to have an effect on lymphocyte trafficking: differences were observed in trafficking of T lymphocytes to the thymus and peripheral lymph nodes. After injection of equal numbers of wild-type and CD44-deficient T lymphocytes, that had been fluorescent-labeled, into a recipient through the tail vein, homing of the injected cells to the thymus and lymph nodes was compared [1]. The study demonstrated a delay in entry of the CD44~/~ lymphocytes to the peripheral lymph nodes that was later overcompensated. Entry into the thymus was found to be 10-20 times less efficient in CD44-deficient T-lymphocytes. Zahalka et al. used LB lymphoma cells that express CD44 antigen on the surface for their experiments. Their previous results had shown that upon s.c. inoculation of LB T lymphoma cells into BALB/C mice, the cells quickly spread and invaded the peripheral lymph nodes and spleen [29]. They inoculated LB cells s.c. in the left flank of female BALB/C mice. Two hours later, some of the mice were injected s.c. with an anti-CD44 mAb containing solution near the frontal brachial and axillary lymph nodes on the same side. They did the same injection every other day for 12 days. On day 12 all the peripheral (except mesenteric) lymph nodes, in the mice with no antibody injection, were infiltrated, while the lymph nodes of the animals that received anti-CD44 mAb injection were not. To make sure the observation was specific for the type of antibody, the experiment was carried out in similar groups with the same amount of anti-CD4 or anti-CD 18 antibodies, which did not block infiltration of LB cells into the lymph nodes [30]. They also found that repeating the experiment with injection of hyaluronidase, but not the control enzyme heparinase, could block invasion of the LB cells to the lymph nodes. DeGrendele et al. injected SEB (Staphylococcal Enterotoxin B) into the peritoneal cavity of mice to induce a focus of inflammation. After injection of SEB to the peritoneal cavity, 8 they harvested the mesenteric lymph nodes from some of these animals and separated the T lymphocytes that had binding to hyaluronic acid. Then they fluorescent-labeled and injected these cells through the tail vein to another animal with similar inflammation in the peritoneal cavity [31]. On the average, 80% of the injected cells appeared in the peritoneal exudate of the recipient mice 20 hours post-injection. To address the role of CD44 in the trafficking of the cells, the same migration experiment was carried out with co-administration of KM81 (a blocking anti-CD44 antibody) and it showed reduction to 10% in migration. In a different set of experiments, Mikecz et al. demonstrated a role for CD44 in migration of leukocytes into the extravascular compartment of synovium [32] in murine arthritis. Administration of an anti-CD44 antibody prevented leukocyte infiltration and swelling in the tissue. Using an anti-CD44 antibody, Camp et al. demonstrated that CD44 is necessary for optimal contact allergic responses. To study the role of CD44, they injected the experimental animals (hapten-sensitized mice) with anti-CD44 antibodies before repainting the skin with the hapten for the challenge phase. Their assay displayed a markedly reduced inflammation at the early stages of the DTH response. A histological study of the DTH site at 24h post-challenge showed less infiltration and edema in the DTH site than the control animals with control antibody injection [33]. 1.9 CONTACT HYPERSENSITIVITY Contact hypersensitivity (CHS) is a type of T cell reaction characterized by delayed reaction to antigen. It can be induced by exposure to poison ivy or industrial and household chemicals. Experimentally, it can be induced by applying a hapten on the skin [34]. A contact hypersensitivity reaction consists of two stages: sensitization and challenge. In the sensitization phase, the subject is exposed to the hapten for the first tune. This exposure does not usually result in any symptoms. The hapten makes covalent bonds to cell-associated or extracellular proteins. The Langerhans' cells will engulf the modified protein and move 9 toward the lymph nodes draining the region. Once in the regional lymph node, the Langerhans' cell will present the antigen to T cells. As a result, the reactive T cells will expand, differentiate into memory cells and finally take residence in different tissues. In the challenge phase, re-application of the same hapten on the skin will lead to hapten-protein complexes being presented to the memory T cells that were produced after the first exposure [35] Recognition of the antigen by the memory cells will lead to rapid development of an inflammatory response at the site of challenge. 1.10 TH1/TH2 Once activated, naive CD4 cells can differentiate into TH1 or TH2 cells. TH1 and TH2 cells produce different profiles of cytokines. TH1 cells produce IL2, IFN-y and lymphotoxin (LT), while only TH2 cells secrete IL4, IL5 and probably IL6. The difference in cytokine production also manifests itself in functional differences [36]. TH1 cells, secrete IFN-y, which activates macrophages; they are capable of migrating to the site of inflammation and activating pro-inflammatory mechanisms. TH1 cells are considered inducers of contact hypersensitivity response. TH2 cells, conversely, are believed to suppress inflammatory reactions. TH1 and TH2 immune response seem to be mutually exclusive. Polarization of CD4 lymphocytes to TH1 cells is induced by secretion of IL12 from antigen presenting cells. Alternatively, secretion of IL10 from the APCs can turn CD4 polarization to TH2 cell type. Experimentally, purified CD4 cells can be polarized to TH1 or TH2 cells by stimulating them with anti-CD3 in the presence of IL12 and IFN-y or IL4 and IL2 respectively [37]. 1.11 TC1/TC2 CD8 blasts with cytokine secretion similar to TH1 and TH2 cells have also been recognized and are known as TCI and TC2 respectively. Unlike CD4 cells that can easily differentiate to TH1 or TH2, CD8 cells have a strong preference to develop to TCI blasts. IL-10 12 and IFNycan induce CD8 cell differentiation to TCI blasts. For CD8 cells to differentiate to TC2 cells there is a requirement for IL-4 to be added in the medium and IFNy to be blocked for example by using blocking antibodies in the medium [38]. 1.12 OBJECTIVES OF THE THESIS It has been demonstrated that TNFa and other inflammatory cytokines can upregulate HA-binding in monocytes. One of the objectives of this project is to investigate the role of TNFa in regulating HA-binding of T-lymphocytes upon activation and determine if this effect is mediated by TNFR1 (p55) or TNFR2 (p75). The question was addressed by developing an in vitro assay that included staining for CD44 expression and HA-binding of the T-lymphocytes. We used the assay to measure CD44-dependent HA-binding in T-lymphocytes under different conditions. By comparing CD44-dependent HA-binding in B6, B6-p55"A and B6-p75v~ T lymphocytes, the role of TNFa and its receptors in regulation of HA-binding was addressed. CD44 is expressed both on the endothelial cell and the lymphocyte surface. It has been suggested that the CD44 molecules on the endothelial surface hold a layer of hyaluronic acid molecules on the surface generating a layer for the CD44 on the activated T lymphocytes to bind to [39]. The other objective of this thesis is to assess the role of CD44 in the migration of activated and memory T cells in vivo. 11 CHAPTER 2 MATERIALS & METHODS 2.0 M I C E Breeders for C57BL/6, B6-p55_/" and B6-p75"A mice were obtained from The Jackson Laboratory (Bar Harbor, ME). The breeders for B6-CD44"'" mice were kindly provided by Dr. C. Eaves of the Terry Fox Laboratory, U.B.C. All the animal breeding was carried out in the animal unit of the Department of Microbiology and Immunology of the University of British Columbia. 2.1 ANTIBODIES/REAGENTS Anti-CD44 antibody IM7 was generated using IM7.8.1 clone. K M 114 was another anti-CD44 antibody that was obtained from PharMingen, San Diego, CA (Catalogue no. 09410D). Anti-CD44 antibody IRAWB14.4 was kindly provided by Dr. Pauline Johnson, University of British Columbia and used at 10|^ g/ml. Anti-CD4 (GK1.5), anti-CD8 (53.67), anti-CD3 (145-2C11), anti-CD25 (PC61) were obtained from American Type Culture Collection (Manassas, VA). Anti-CD4, anti-CD8, anti-CD44 (IM7) and anti-CD25 antibodies were precipitated by (NH4)2S04. In case of anti-CD3 antibody, Protein A columns were used for purification. Depending on the experiment, the above antibodies were used either in the FITC-labeled form (FITC - Fluorescein Isothiocyanate Isomere 1 - Sigma F-7250) or were biotinylated. Anti-CD69 antibody was purchased from PharMingen, San Diego, CA (Catalogue no. 01502D). Anti 2C TCR antibody was generated using 1B2 hybridoma, a courtesy of Dr. Eisen (Cambridge, MA). Anti-IFNy antibody R4-6A2 was purified by Protein G in Dr. Teh's laboratory and used as capture antibody for ELISA. For detection, buitinylated anti-IFNy Ab (SMG1.2) was used. 12 Anti-IL2 capture antibody (Clone JES6-1A12) and Anti IL2 detection antibody (Clone JES6-5H4) (biotinylated) were purchased from PharMingen, San Diego, CA (Catalogue no 18161D and 18172D, respectively). We used anti-IL4 antibody (11B11) for capture. For detection anti-IL4 detection antibody (BVD6-24G2) was purchased in biotinylated form from PharMingen, San Diego, CA (Catalogue no 18042D). Streptavidin Phycoerythrin (PE) (catalogue number 7100-09L) and Streptavidin tricolor (catalogue number CLCSA 1006) were obtained from Southern Biotechnology Associates, Birmingham, AL. and Cederlane Laboratories, Hornby, ON respectively. 2.2 CYTOKINES Mouse recombinant IL-4, IFNy and IL-12 were purchased from Sigma (Catalogue numbers 1-1020,1-4777,1-8523, respectively). Recombinant TNFa was obtained from R&D systems, Minneapolis, MN (Catalogue number 410-TRNC). 2.3 HARVESTING LN/SPLEEN CELLS FROM A B6 MOUSE Mice were sacrificed by cervical dislocation. Lymph nodes harvested and single cell suspension was prepared in RPMI 1640 medium (Catalogue no 31800-071 GibcoBRL, Burlington, Ontario) supplemented with 2% FBS (Fetal Bovine Serum Catalogue no F-2442 Sigma). The resulting cell suspension was spun down and subsequently the pellet resuspended in RPMI. This procedure was repeated twice to remove the clumps. Finally the suspension was resuspended in Iscove's (I) medium (Life Technologies, Burlington, Ontario, Canada) supplemented with 10% FBS (Life Technologies), 5xl0"5 uJvl 2-ME, and antibiotics. In case of spleen, the procedure is the same as lymph nodes, with the only difference that the RBCs are removed from the suspension by hypotonic lysis using the RBC 13 Lysis Buffer (155 jxM Ammonium Chloride, 10 |J.M Tris Base, pH 7.3). For this purpose cell suspensions were centrifuged, resuspended in the lysis buffer for 5 minutes in room temperature and then washed twice. The remaining cells were resuspended in I medium for further study. 2.4 PURIFICATION FOR CD4/CD8 T LYMPHOCYTES To isolate CD4+ CD8" (CD4) T lymphocytes from the mouse lymph nodes, the lymph node cell suspension was incubated with biotinylated anti-CD4 mAb (GK1.5 biot) for 15 minutes, washed once with FACS-medium followed by incubation with positive selection using a MACS MS + Separation Column and MiniMACS magnet following the procedure provided by the manufacturer (Miltenyi Biotech, Auburn, CA). The purified CD4 population was >95% pure. To isolate CD8+ CD4" (CD8) cells the procedure was the same as above except that the antibody used was biotinylated anti-CD8p mAb. 2.5 IN VITRO STIMULATION OF CD4 OR CD8 T CELLS 2.5.1 STIMULATION BY ANTIBODY For this purpose, flat bottom tissue culture plates (Falcon Microtest™ plates, Becton Dickinson) were used. Anti-CD3e antibody was added to the wells in a flat-bottom culture plate with varying concentrations of 1 to 10 |ig/ml PBS depending on the experiment. The wells were very gently washed twice with PBS after the plate was left in the 37°C incubator at least for an hour. The responding cells were added to the wells in I media and incubated in 37°C (5% C02). 2.5.2 STIMULATION BY ANTIGEN PRESENTING CELLS AND ANTIGEN For this purpose, irradiated splenocytes or mitomycin C-treated T2-Ld cells were employed. T2-Ld cell line (which is deficient in peptide transporter protein) [40] was kindly provided by Dr. Peter Cresswell, Yale University School of Medicine. The cells were treated 14 with mitomycin C (Sigma Catalogue no M0503) before being used. For this purpose the cells were incubated in the number of 5x106 cell/ml serum free I-media with mitomycin-C added to a final concentration of 50 |ig/ml. The cells were left in the 37°C incubator for an hour and washed three times with normal I-media before use. To stimulate 2C TCR transgenic CD8 T lymphocytes, 5x104 mitomycin-treated T2-Ld cells were added along with 105 purified CD8 T lymphocytes in a volume of 200ul in round-bottom wells. The p2Ca peptide, which is presented by L d and is recognized by the 2C TCR [40] was added to make the final concentration of 0, 0.10 and 1.00 )iM in the culture medium. To use splenocytes for stimulation, B6-TAP-T'" splenocytes were resuspended at 107 cells/ml in cold PBS and irradiated with 2000 rads using Gamma irradiation. The cells were centrifuged and resuspended in I-media before use. To stimulate 2C cells, irradiated B6 splenocytes were incubated with the 2C cells in round-bottom wells with the SIYRYYGL (SYRGL) peptide added to the media in concentrations of 0.001, 0.01, 0.1 and 1 |JM in different wells. The SYRGL peptide binds to K b and is recognized with high affinity by the 2C TCR [41]. The SYRGL peptide was synthesized by the NAPS Unit at UBC. 2.6 PROLIFERATION ASSAY The proliferation assay was carried out by incubating 5xl05 B6-TAP"" splenocytes, added to the wells as antigen presenting cells with 104 purified CD8 T cells from lymph nodes of 2C and 2C-CD44"'" mice as responders and varying concentrations of SYRGL peptide. The cells were cultured in a volume of 0.20 ml in 96-well round-bottom plates in triplicate. For the last 6h of the 72h culture, 1 |xCi [3H]thymidine was added to each well. After the 6h incubation with [3H]thymidine, the contents of the wells were passed through Whatman Glass Microfibre Filters (Catalogue no. 1820866 Whatman Nucleopore Canada, Toronto, ON) and washed extensively with distilled water. The process lyses the cells, allows the soluble components and unincorporated [3H]thymidine to be washed away. The DNA that stuck to the Whatman filter was fixed with methanol. The samples were then transferred to 15 Mini Poly-Q Vials (Beckman Coulter, Mississauga, ON) and left in room temperature to dry. After the samples were dried, 1 ml of scintillation liquid was added to each vial. To measure the amount of [3H]thymidine existing in each sample, a Beckman Liquid Scintillation Counting System (LS 6000TA) was used. The reading for each sample corresponds to the amount of [3H]thymidine that was incorporated in the last 6h of culture. A blank vial was counted along with the sample vials to give a measure of the background reading. The background was deducted from the reading of the samples. 2.7 FLUORESCEIN LABELLING OF HYALURONAN Labelling of hyaluronan with Fluorescein was carried out following the isocyanide reaction method described by De Belder et al. [42] Hyaluronan (HA) from Rooster comb was obtained from Sigma-Aldrich Oakville, ON, Canada. To make a solution of hyaluronan in water 10 mg of rooster comb HA was dissolved in 8 ml of ddH20 over 15h at room temperature. After HA was completely dissolved, 4 ml of dimethylsulfoxide (DMSO; Sigma-Aldrich Oakville, ON, Canada) was added to the solution. Then 5 pj of acetaldehyde (Sigma-Aldrich, Oakville, ON, Canada), 5 ul of cyclohexyl isocyanide (Sigma-Aldrich, Oakville, ON, Canada), and 5 mg of fluoresceinamine isomer I (Sigma-Aldrich, Oakville, ON, Canada), was dissolved in a small amount of DMSO. The resulting solution was then added to the HA solution (pH was kept between 5.0 and 7.0). The mixture was left stirring in the dark for 5 hours at ambient temperature. Finally to precipitate the Fluoresceinated hyaluronan (HA-FL1) the mixture was added to about 800ml of ethanol. The precipitated HA was collected and precipitated again from water in ethanol (with a few drops of saturated NaCl in water). After removing the free Fluoresceinamine (the supernatant was not orange anymore), the pellet was dried in vacuum at 50°C for 15h. The resulting Fluoresceinated HA was diluted to a concentration of 15 p:g/ml and used at a dilution of 1/100 to stain the cells for flow cytometry. 16 2.8 FLOW CYTOMETRIC ANALYSIS OF LYMPHOCYTES Cells (lxlO5) were stained with mAb for 15 min on ice in lOOul of FACS buffer (PBS with 2% FCS). Cells were washed and then incubated with secondary antibody for 15 min on ice in 100 |il of FACS buffer. Cells were then washed and resuspended in FACS buffer for analysis on a FACScan flow cytometer using Cell Quest software (Becton Dickinson, Mountain View, CA). 2.9 POLARIZING CD4 CELLS TO TH1/TH2 A flat-bottomed well plate was coated with anti-CD3 mAb (2C11) at 10p:g/ml in PBS, using 0.5ml per well. The plate was incubated for more than an hour at 37°C temperature. Then the 2C11 was removed, and the wells were washed twice at room temperature with PBS. Lymphocytes were then cultured in the 2C11-coated wells in I media for 2 days at 37°C. To polarize the cells to TH1 or TH2, the cultures were started with the following supplements: POLARIZING TO TH1 106 CD4+ T lymphocytes in 1.5 ml IL12 lOOU/ml IL2 50 U/ml Anti-IL4(11B11 mAb) 10 p:g/ml POLARIZING TO TH2 106 CD4+ T lymphocytes in 1.5 ml IL4 300 U/ml IL2 50 U/ml Anti-IFN-y (XMG1.2) 10 pig/ml After two days, the cells were transferred to an uncoated well. The cells were allowed to grow for 5 days on the uncoated plate to return to resting state before use. The cultures were split and cultured in additional wells in I media supplemented with 50 U/ml of IL-2 as required to prevent overgrowth. 2.10 CYTOKINE PRODUCTION BY THE POLARIZED CELLS Cytokine production by the polarized cells was determined as follows: The polarized cells 17 were washed with fresh medium 3 times, resuspended in I media at 2.5x1 OVml and stimulated with PMA (20ng/ml) and Ionomycin (500ng/ml). The supernatants were collected after 6-8 hrs stimulation at 37°C temperature for cytokine assay. The concentration of individual cytokine in the supernatants was determined using ELISA assays. 2.11 CYTOKINE ELISA The ELISA plate was coated with the capture antibody by adding 50|il solution of the antibody at 4 jig/ml in carbonate buffer and leaving it in the 4°C fridge overnight. Then the wells were washed three times with PBS-Tween 20. Then the plate was blocked by adding 100p:l of 1% BSA in 0.1% Sodium azide in PBS into the wells and leaving the plate in the incubator at 37°C. After an hour in the incubator, the wells were washed three times with PBS-Tween 20. Then the samples were added to the wells in three different dilutions. Serial dilutions of the standard antibody and three dilutions of the test samples in 1% BSA/0.1% Sodium azide in PBS was added to the wells and incubated for 1 hour at 37°C. The wells were washed three times with PBS-Tween 20 (with 0.1% Sodium azide). Then the detection antibody was added to the wells at 1 |Xg/ml in 1% BSA/0.1% Sodium azide in PBS and incubated for an hour at 37°C. After another wash with PBS-Tween 20 with azide, strepavidin-alkaline phosphatase (BD PharMingen) was added (1/2000 in 1% BSA/0.1% azide in PBS) and left at room temperature for an hour. Finally the wells were washed and the substrate solution (no 104, Sigma, St. Louis, MO) (1 tablet per 5 ml of Diemanolamine buffer) added. The yellow color that developed in the wells was quantified with an ELISA plate reader at 405 nm. 2.12 INDUCTION OF DTH (DELAYED-TYPE HYPERSENSITIVITY) The mice were sensitized to the hapten - DNFB (2,4-dinitro-l-fluorobenzene)- by painting their shaved abdomen with 20jxl 0.5% DNFB in acetone-olive oil (4:1) for two consecutive days. DTH responses to DNFB were measured 20 days later by painting the 18 right ear with 20|il 0.3% DNFB solution. The peak inflammation appeared 24 hours post-challenge on the ear. The unpainted left ear served as an internal control for the DTH reaction. 2.13 RADIOLABELLING OF CELLS WITH s l C r Cells were labeled with 5 1 Cr prior to injection into live animals to track their migration. The cells to be injected were centrifuged and 0.1 ml of 51Cr-sodium chromate in PBS was added to the cell pellet. The cell pellet was then gently resuspended and incubated at 37°C for 1 to 1.5 hour. The cells were then washed three times with Iscove's medium to remove the extra radioactivity that was not taken up by the cells. The cells were finally resuspended in PBS at a density of 4xl06/ml. The amount of 5 I Cr incorporated into the injected cells was determined by gamma counting prior to injection. 2.14 CFSE LABELLING Single cell suspensions of lymph node cells were resuspended in PBS at a concentration of lxlO 7 cells/ml in PBS. To label cells with carboxyfluorecein diacetate succinimidyl ester (CFSE), CFSE was added at a final concentration of 1 |jM and incubated for 10 minutes at room temperature. To stop the labeling reaction, an equal volume of fetal calf serum (FCS) was added and the cells were subsequently washed three times with Iscove's Media. 2.15 PERFUSION OF THE ANIMAL Perfusion of the animals was carried out to remove PBL from the tissues. The mouse was euthanized by C02 narcosis. The thorax was rapidly opened and a 25-G needle connected to a catheter was inserted into the left ventricle. The catheter was connected to a source of heparinized PBS positioned 50cm higher. An incision was made in the right atrium to let blood out of the circulation. The animal was perfused with 25-30 ml of heparinized PBS. 19 2.16 SEPARATION OF THE LYMPHOCYTES FROM TISSUES Lymph nodes and spleen were collected as described in 2.3. Bone marrow cells were collected from the femur of the mice as follows. After removing the soft tissue and muscles from the surface, using surgical instruments, the bone excised. The bone marrow was then extracted by flushing with 3ml of PBS from one end. The BM washout was collected, centrifuged, RBCs were lysed by resuspending the pellet in hypotonic lysis buffer. The remaining cells were resuspended in Imedia for further study. 2.16.1 BLOOD To isolate mononuclear cells from whole blood, Histopaque was used. It has a density of 1.077. 2 ml of Histopaque was introduced to the bottom of the tube with the cell suspension lying on top. The tube was spun at 1500 rpm for 15 minutes. This process allowed erythrocytes and granulocytes to pellet. The mononuclear cells appear as a band at the interface of plasma and Histopaque and can be separated easily. The separated cells were washed three times with I-medium to remove the remaining Histopaque from the suspension. 2.16.2 LIVER & KDDNEY The liver and kidney were dissected out and cut into little pieces and then put over a sieve soaked in I-medium. The tissue was then mashed with the end of a syringe. This procedure, mashing followed by rinsing was repeated three times for kidney or liver. The cell suspensions derived from these washings were combined into a 50 ml tube. The cell suspension was spun at 2000 rpm for 20 minutes. Using a suction tube the supernatant was removed very carefully. Then 14ml of 35% percoll/Heparin solution was added to the tube. The cells were resuspended in the 35% percoll/Heparin solution, vortexed to break the cell aggregates and spun again at 2000 rpm for 20 minutes. The centrifuge was stopped very slowly to avoid resuspension of the loose pellet into the supernatant (brakes off). The 20 supernatant was removed very carefully. The cells were resuspended in RPMI and transferred to 10ml tubes and centrifuged again to form a pellet. The RBCs were removed from the suspension by hypotonic lysis. The remaining cells were resuspended in Iscove's medium for further study. 100% Percoll Solution 35% Percoll/Heparin Solution 92.5 ml Percoll (Sigma) 10.5 ml of 100% Percoll Solution 8.5mll0xPBS 19.5 ml PBS pH should be 7.2-7.5. 3000 U of Heparin 2.16.3 PERITONEAL L A V A G E CELLS For this purpose, 3 ml of PBS solution was injected into the peritoneal cavity of a mouse that was recently sacrificed by cervical dislocation. A small incision was made in the upper part of the peritoneal cavity and the cavity was thorough rinsed with the injected PBS with the help of a pasture pipet that was inserted into the incision. The cells collected from this procedure is referred to as peritoneal lavage cells. 21 CHAPTER 3 Results 3.1 CD44-DEPENDENT HA-BINDING GENERAL OBJECTIVE To determine the role of TNFa and TNF receptors in regulating CD44-dependent HA-binding in T lymphocytes. RATIONALE CD44 is the recognized receptor for hyaluronic acid on the surface of T-lymphocytes. Naive T-lymphocytes express CD44S or CD44H isoform of CD44 that does not bind to hyaluronic acid. Once the lymphocytes are activated, they upregulate their CD44 expression and express new isoforms of CD44 that bind to hyaluronic acid. Based on this knowledge, in vitro assays of CD44-HA binding utilized anti-CD3-activated T cells. Binding of HA was quantified by flow cytometric analysis of binding of fluoresceinated HA to CD44. Based on the Uterature, HA-binding of T lymphocytes is highest at 48h time point and then remains high for about 48h before diminishing [15]. Our preliminary experiments of binding of HA to CD44 on activated T cells were therefore carried out at the 48h time point. 3.1.1 HA binding to activated B6 and B6-CD44"'" T lymphocytes in vitro The binding of fluoresceinated HA to anti-CD3-activated CD8 T cells from B6 and B6-CD44"'" mice are shown in Fig. 3.1. It is clear from this figure that only CD8 blasts from B6 mice bind HA efficiently. Very little HA binding by activated CD8 T cells from B6-CD44 mice could be detected using the flow cytometric assay. Co-staining the activated CD8 T cells with an anti-CD44 mAb (IM7-PE) demonstrated that HA binding was mediated primarily by those that expressed high levels of CD44. This assay also confirms the lack of expression of CD44 by CD44 null cells. 22 a) B6 CD8 Blasts b) B6-CD44' CD8 Blasts c) B6 CD8 Blasts incubated with KM114 before staining Figure 3.1 HA-binding by activated CD8 T cells is CD44-dependent. Panel (a) demonstrates the results of the assay when B6 CD8 blasts activated with anti-CD3 were used. Panel (b) is the same assay using B6-CD44"" CD8 blasts. In panel (c), B6 CD8 blasts were incubated with KM114 (antiCD44) that blocks HA-binding of CD44 by adhering to its HA-binding site. 23 KM114 is an anti-CD44 antibody that binds to the HA-binding site of the CD44 molecule. Incubating the activated cells with KM114 should block the HA-binding sites of CD44. As shown in Fig. 3.1 the binding of HA to B6 blasts was almost completely inhibited by the KM114 mAb. This observation further supports our conclusion that binding of HA to B6 blasts was indeed CD44-specific. It is noted that a small percentage (1.18%) of CD44"'" CD8 blasts bound HA. Furthermore, there is a small amount of residual HA binding by B6 CDS blasts after blocking with the KM114 mAb. These data indicate that a small amount of HA binding to CD8 blasts could occur via CD44-independent mechanisms. It is unclear whether this binding is due to impurities in the HA preparation and/or other cell surface molecules which can potentially bind HA. Nevertheless, the results clearly indicate that the vast majority of HA binding by CD8 blasts in CD44-mediated. 3.1.2 Activated CD8 cells bind to more HA than activated CD4 cells It is not clear if activated CD4 and CD8 T cells bind HA equally. To determine the relative ability of these activated cells to bind HA, CD4 and CD8 T cells from B6 mice were purified from B6 lymph node cells by positive selection using a MACS MS + Separation Column and MiniMACS magnet. The purified cells were activated with identical concentrations of anti-CD3 mAb and IL-2 and the ability of these cells to bind HA was determined after 48 h of activation. As shown in Fig. 3.2 CD4 cells demonstrated a much lower percent of HA-binding compared to CD8 cells. Interestingly, the lower HA-binding by activated CD4 T cells correlates with the lower proportion of CD44hi cells in this population. This raised the issue of whether the CD4 T cells were not activated to the same extent as the CD 8 T cells. Staining of the activated CD4 and CD8 T cells indicates that they express the same amount of CD25 (IL-2R.O) but lower amounts of CD69. Thus, the lower HA-binding by activated CD4 T cells may be due to fewer activated T cells as evidenced by lower expression of CD69 and a lower proportion of CD44hi cells. Alternatively, this result suggests that activated CD4 T cells 24 Figure 3.2. Activated CD8 cells bind more HA than activated CD4 cells. Purified B6 CD4 and CD8 lymphocytes were activated with antiCD3 (10p:g/ml) coated on the plate and IL2 concentration of 20U/ml added to the medium. At 48h time point CD8 blasts had a higher HA-binding than CD4 cells. Staining the cells for CD25, CD44 and CD69 showed that B6 CD8 cells had higher expression of the activation markers. 25 expressed less HA-binding CD44 isoforms than activated CD8 T cells. Since activated CD8 T cells bind HA much more efficiently than activated CD4 T cells further experiments on HA binding were focused on activated CD8 T cells. We next detemiined the concentration of anti-CD3 stimulation that will lead to maximal binding of HA by activated B6-CD8 T cells. The results in Fig. 3.3 indicate that maximum binding of HA was achieved with cells that were activated with 3 to 20 fig/ml of immobilized anti-CD3 mAb. A concentration of 10 u.g/ml was used in further studies to maximally activate CD8 T cells. The preceding studies are consistent with the conclusion that the higher percent of HA binding in activated CD8 T cells is associated with a higher activation of the CD8 cells compared to the CD4 T cells. To further investigate this phenomenon we determined whether HA binding by CD8 T cells also correlates with their degree of activation. Day 2 activated CD8 T cells were divided into large, medium and small cells based on their FSC/SSC values (Fig. 3.4). The expression level of CD44 corresponded with cell size with the large cells expressing the highest level of CD44. Analysis of HA binding by these cells revealed that the large cells are also the most efficient binders of HA (78%). About 38% of the medium size cells also bind HA whereas no binding of HA was detected in the small cell population. 3.1.3 Influence of TNFa and TNFa receptors on HA binding Previous studies have demonstrated that TNFa can affect HA binding to CD44 in a human leukemic cell line [20]. Furthermore, studies from the Teh lab have indicated that TNFR2 (p75) plays an important costimulatory role in the activation of CD8 T cells [43]. Previous studies have shown that T cell development is not affected by the p75"/_ mutation [44]. We have also shown that the hypo-responsiveness of p75_/" T cells to TCR stimulation can be largely rescued by the addition of an exogenous source of IL-2 [43]. These studies indicate that the p75v" mutation affects TCR signalling and provide a system for correlating the level of T cell activation to HA binding. We used the in vitro HA-binding assay to address 26 B6CD8 60 i § 10 • PL, 0 I i i i — — — • 1 3 10 20 Concentration of 2C11 (M-g/ml) Figure 3.3. Effect of anti-CD3 concentration on HA-binding. Purified B6 CDS lymphocytes were activated with different concentrations of anti-CD3 coated on the plate for 48 hours (IL2=20U/ml). The figure demonstrates the % H A binding in the live population for every concentration of anti-CD3 used based on one experiment. 27 a) b) 400 600 FSC-H 1000 Population 1 (Large Blasts)— Population 2 (Medium-sized cells) Population 3 (Small cells) 10° 101 102 to- toH FL2+I Large Cells Medium Cells Small Cells CD44-PE" c) 90 SO fcO . § 5 0 *40 30 ^20 10 0 % HA-binding in B6 CDS cells of different size i l l Large Cells Medium Cells Small Cells Figure 3.4. Correlation of H A binding with CD44 expression and cell size. Panel (a) shows how we can differentiate three populations in the CD8 + live T cells on the basis of FSC/SSC values. Panel (b) illustrates CD44 expression by the gated cells. Panel (c) illustrates the average of HA binding by gated cells in three experiments. The cells were activated on the plate coated with anti-CD3=1 Opg/ml, IL2=20U/ml. 28 the following questions: (i) Does the addition of exogenous TNFa increase HA binding by activated T cells? (ii) Which TNF receptor (p55 or p75) is responsible for the effects of TNFa? and (iii) Does the binding of HA correlate with cell size in CD8 T cells that are deficient in either p55 or p75? The 2C TCR transgenic receptor is positively selected by D b and is specific for the L d alloantigen [45]. The 2C TCR can also recognize the SYRGL peptide that is presented by K b . To determine the effect of TNFa on T cell activation and HA binding, 2C CD 8 T cells were activated for 48 hours with the SYRGL peptide presented by B6-TAP-1"'" splenocytes with and without exogenous TNFa added during culture. The results in Fig. 3.5 indicate that the addition of exogenous TNFa led to an increase in HA binding by Ag-activated 2C CD8 T cells. This increase in HA binding correlated with a higher proportion of large cells that expressed higher levels of CD44. We also determined whether TNFa had an effect on HA binding by maximally activated CD8 T cell. CD8 T cells from B6 mice were maximally activated with 2C11 + IL-2. Exogenous TNFa was added to half the cultures. HA binding was determined 48 h after activation. As indicated in Fig. 3.6 CD8 T cells that were activated in the presence of TNFa did not bind HA better than those that were activated in its absence. This is likely due to the fact that maximally activated CD8 T cells can produce TNFa and the amount of TNFa produced under these conditions is sufficient for optimal activation and HA binding. We next determined HA binding by 2C11-activated CD 8 T cells from B6, B6-p55'A and B6-p75"A mice. The CD8 cells were activated in the absence or presence of exogenous IL-2. The results in Fig. 3.7 indicate that activated p75'/_ CD8 cells had the least percentage of HA binding cells. This result is consistent with the conclusion that p75 is an important costimulatory receptor for CD8 T cells and the lower percent HA binding in these cells is due to the lower level of activation of the cells. In the presence of exogenous IL-2 the percentage of HA binders was significantiy increased in cells with the p75_/" mutation. This is consistent with the conclusion that p75 deficiency can be rescued by exogenous IL-2. Interestingly, 29 a) b) c) CD44-PE Without TNF 13.83% With TNF 25.19% o-1 n i . .n . | ' •• ••••^ <nu tn' i n ' irv* ^Qi HA-FL1 10" io' 10' 10^  ion FL1-H 10° 10' 10' 10J 10n FL1-H HA-binding with/without TNF BTNF=0 a TNF=30ng/ml Large cells Medium cells Cells size Small cells CD44 Expression with/without TNF 700 600 fl 500 8 '8 <00 § W 200 700 0 mTNF=0 D TNF=30ng/mI mJB Large cells Medium cells Cells size Small cells Figure 3.5. Enhancement of H A binding by antigen-activated CD8 T cells. (a) 2C CD8 cells were activated with the SYRGL peptide presented by B6 splenocytes with or without exogenously added T N F a (30ng/ml). H A binding by activated 2C CD8 T cells was determined at 48 hr. after activation, (b) Illustrates %HA-binding as a function of cell size, (c) Illustrates expression of CD44 as a function of cell size. The plots reflect the results of one experiment. 30 Q without TNF • With TNF Large cells Medium Small cells Cells Cell type Figure 3.6. TNFa did not increase HA-binding by optimally activated CD8 T cells. Purified B6 CD8 cells were activated with anti-CD3 (lOug/ml) coated on the plate (IL-2 20U/ml). The cells were stained with HA-FL1 and IM7-PE (anti-CD44PE) after 48h. The figure compares the average of the HA-binding of the large, medium and small cells with/without TNFa added to the medium in three experiments (concentration of TNF=30ng/ml). 31 50 45 40 35 30 25 20 15 10 5 0 • Without IL2 • with IL2 iBfli • iilliil H I •Bis B<5 p55 Cell type p75 Figure 3.7. Less efficient HA binding by activated p75_/" CD8 T cells. Purified CD8 T cells from B6, B6-P55"'" or B6-p75"'" mice were activated with anti-CD3 (lOjxg/ml) +/- IL-2 (20U/ml). HA binding was determined 48 hr. after activation. The plot reflects the results of one experiment. 32 when activated CD8 cells from B6, p55"'" and p75"/_ mice were gated into large, medium small cells only the large and medium cells from these mice were efficient HA binders.' result indicates that the effect of TNFa on HA binding is likely a direct consequence o: effect of this cytokine on T cell activation (Figure 3.8). 3.2 DTH EXPERIMENTS GENERAL OBJECTIVE The purpose of this series of experiments was to address the role of CD44 in migration of T lymphocytes to sites of mflammation. Based on the literature CD4+ and CD8+ cells can differentiate to TH1/TH2 (T-helper 1/T-helper 2) and TC1/TC2 (T-cytotoxic 1/T-cytotoxic 2) cells, respectively. They have demonstrated that this polarization can also take place in vitro if the T-lymphocytes are exposed to (certain cytokines in the culture medium while being activated. The method commonly employed entails activation of T lymphocytes with anti-CD3 antibody for 48 hours (with the cytokines in the medium) and then transferring the cells with the media to a blank plate for 4-5 days to expand and then they will be polarized. Austrup et al. demonstrated that injection of these resulting blasts to recipients through the tail vein results in accumulation of the TH1 but not TH2 cells in the inflammation site [37]. Our assay involved labeling polarized TH1 and TH2 cells with 5 1 Cr and injecting the labeled cells into recipient mice via the tail vein. Active DTH is induced in the right ear at the time of injection. The left ear (without inflammation) can act as control for the right (inflamed) ear. The amount of radioactivity that accumulated in the experimental (right) and control (left) ears was determined at 24h post-injection. By knowing the total radioactivity of the injected cells, percent accumulation of radioactivity in the ears can be used as a measure of the percent migration to either the inflamed or control sites. 3.2.1 B6 CD4 cells can be polarized to TH1 blasts if exposed to a certain profile of cytokines while being activated with anti-CD3 33 0 B6 B6 B6 p 5 5 p 5 5 p 5 5 p 7 5 p 7 5 p 7 5 (1) (2) (3) (1) (2) (3) (1) (2) (3) Cell type Figure 3.8. HA binding as a function of cell size. Purified CD8 T cells from B6, B6-p55_/" and B6-P75"'" mice were activated on anti-CD3 (10|xg/ml) coated plate and IL-2 (20U/ml). HA binding by blasts (1), medium-sized cells (2) and small cells (3) were detenriined 48 hr. after activation. The plot shows the average of two experiments. 34 The first part of this experiment was to ensure that the cells used for injection were correctly polarized. Purified CD4 T cells were polarized to TH1 and TH2 cells as described in Chapter 2. The cytokine profiles of the polarized cells were determined by stimulating polarized cells with PMA and ionomycin. The data in Fig. 3.9 indicate that polarized TH1 cells were highly efficient in producing IFN-y but not IL-4 whereas the converse was observed for polarized TH2 cells. These data indicate that the CD4 T cells were indeed correctly polarized into TH1 and TH2 cells. 3.2.2 The B6 CD4 cells that were polarized to TH1 blasts demonstrated the ability to migrate to the inflammation site The ability of polarized TH1 and TH2 cells to migrate to the inflammation site was determined (Fig. 3.10). In agreement with a previous report [37] we found that polarized TH1 cells are much more efficient in migrating to the inflamed ear when compared to polarized TH2 cells. That this migration is inflammation-dependent is supported by the observation that only background radioactivity accumulated in the control ear. 3.2.3 CD44, p55 or p75 deficiency did not affect the ability of polarized TH1 cells to migrate to the inflammation site After making sure the B6 TH1 cells had the ability to migrate to the site of delayed type hypersensitivity, we addressed the role of CD44, p55 and p75 in this migration. For this purpose, we used CD4 lymphocytes from either wild type B6 mice or B6 mice that have targeted null mutations for CD44, p55 or p75. The CD4 cells from these mice were polarized into TH1 cells and the ability of these polarized cells to migrate to the inflamed site determined in the same manner. The results in Fig. 3.11 Indicate that polarized TH1 cells from CD44"A, p55_/" or p75v" mice were just as efficient as the cells from wild type mice in migrating to the inflamed site. This result indicates that migration of polarized TH1 cells to the inflamed site is not dependent on CD44 or TNFa receptors. The experiment in Fig. 3.11 35 J 20000 s 15000 ,<s> '« § 10000 i e 5000 a 0 IFN-Gamma Concentration (U/ml) f i t * VttA THl TH2 a IFN-Gamma Concentration (U/ml) I C « I s a 2000 1500 1000 500 0 IL-2 Concentration (U/ml) THl TH2 a IL-2 Concentration (U/ml) I C •I 550 300 250 200 150 100 50 0 IL-4 Concentration (U/ml) THl TH2 UIL4 Concentration (UM) Figure 3.9. Cytokine production by polarized T H l and TH2 cells. Purified CD4 T cells from B6 mice were polarized into THl and TH2 cells as described in chapter 2. The polarized cells were restimulated with PMA and ionomycin on day 7. The amount of cytokines produced by the polarized cells is determined using ELISA assays. 36 was a separate set up from Fig. 3.10, explaining the difference in percent recovery of B6 CD4 cells in the two experiments that is being used as the baseline for comparing migration of the B6-CD44"'-, B6-p55_/- and B6-p75'A cells. In Fig. 3.11 the B6-CD44"'" group shows a high variation within the group, but considering that even the lowest percent recovery in the group is higher than the B6 group proves that lack of CD44 is indeed not playing any role in the process. 3.2.4 To determine whether there is a correlation between HA binding and lymphocyte migration. In the DTH experiments described above, the polarized THl cells were in a resting state. In vitro staining of resting THl cells demonstrated a low percent of HA-binding. Consequently, these studies do not address the question of whether migration of the polarized THl cells is dependent on binding of CD44 to HA. Previous studies have shown that the IRAWB 14.4 mAb binds to a CD44 site that is distinct from the HA binding site. Furthermore, binding of CD44 on resting cells by the IRAWB mAb induces a conformational change that leads to increased percent binding to hyaluronic acid [14]. We therefore used the IRAWB mAb to upregulate HA-binding of B6 THl blasts before injecting the cells (Figure 3.12). 3.2.5 Migration of recently activated CD4 and CD8 T cells to the inflamed site is independent of CD44. We have shown that activated CD4 and CD8 T cells bind HA maximally at 48 h after activation. In the first part of this experiment we determined whether activated CD4 T cells were able to migrate to the DTH site. For this purpose CD4 T cells from B6 mice were activated under THl polarizing conditions for 48 h, then labeled with 5 1Cr and injected into B6 mice with their right ear sensitized by DNFB. The amount of radioactivity that accumulated in the sensitized and control ears was then determined 24 hours later. 37 a) B6 CD4 T-lymphocytes, Polarized to TH1 s lCr-labeled Inject to B6 mouse (DTH on the Rt. ear) b) B6 CD4 T-lymphocytes, Polarized to TH2 5 1 Cr -labeled Inject to B6 mouse (DTH on the Rt. ear) Measure the % Radioactivity in the inflamed/non-inflamed ear ELB6 mi cells UB6 TH2 cells DNFB Control Figure 3.10. Preferential migration of TH1 cells to DTH site. The experimental protocol is illustrated in panel (a). 2xl0 6 radiolabeled B6 TH1 and TH2 cells were injected (i.v. route) into B6 recipients in the number of. Percent radioactivity recovered in the inflamed (DNFB) and control ears are indicated in panel (b). 3 8 Figure 3.11. Efficient migration of polarized TH1 cells into DTH site is not dependent on CD44, p55 or p75. Purified CD4 T lymphocytes from B6, B6-CD44'", B6-p55v' and B6-p75v- mice were polarized into TH1 cells. The polarized cells were then 51Cr-labeled and injected into B6 recipients and analyzed as described in fig. 3.10. The results in the figure are from the inflamed ears. The results demonstrate that polarized TH1 cells from CD44"'", p55"'", p75v" mice migrate just as efficiently as wild type B6 TH1 cells into the DTH site. For each group receiving a different cell type, three B6 recipient mice were used. 39 B6TH1 B6TC1 Figure 3.12. HA binding by resting B6 TH1 and B6 TCI cells is upregulated by the IRAWB mAb. CD4 and CD8 T cells from B6 mice were polarized to TH1 and TCI cells respectively. The resting TH1 and TCI cells were incubated with IRAWB mAb (10mg/ml) for 30 minutes and then stained with HA-FL1 and anh-CD44PE. The thin and bold lines indicate HA binding before and after IRAWB treatment 40 As shown in Fig. 3.13, recovery of radioactivity from the inflamed ear was 3 times the non-inflamed ear. Therefore, this experiment demonstrates that activated CD4 T cells can migrate to sites of inflammation, although the efficiency of migration was less than for THl cells that have returned to the resting state. We then compared the rate of migration of CD4 and CD8 T cells from B6 and B6-CD44"'" mice that were activated under polarizing conditions for TH1/TC1 or TH2/TC2 conditions, respectively to the DTH ear. The results in Fig. 3.14 indicate that there is no dependence on CD44 for the migration of activated CD4 and CD8 T cells to the DTH ear. 3.3 To address the role of CD44 in the in vivo migration of CD8 T lymphocytes into various tissues The availability of TCR transgenic mice allows us to track antigen-specific T cells in vivo with mAbs that are specific for the TCR idiotype. The 2C TCR transgenic receptor is positively selected by D b and is specific for the L d alloantigen [45]. The 2C TCR is expressed by CD8 T cells and is detected by the 1B2 mAb [45, 46]. The 2C TCR can also recognize the SYRGL peptide that is presented by K b [41]. This latter observation allows the activation of CD8 2C TCR+ cells in B6 mice as a result of immunization with the SYRGL peptide. We used this system to determine whether the migration of antigen-activated CD8 T cells into various tissues is affected by CD44 deficiency. The first control was to determine whether the antigen responsiveness of CD8 2C TCR+ cells to the SYRGL peptide is affected by CD44 deficiency. The results in Fig. 3.15 clearly indicate that the CD44 deficiency did not affect the proliferative responses of CD8 2C TCR+ cells to the SYRGL/Kb ligand. We also determined whether the in vivo expansion of anti-CD3-activated CD8 2C TCR+ cells was affected by CD44 deficiency. This was done by activating CD8 2C TCR+ cells with anti-CD3 mAb for 48 h, labeling the activated cells with CFSE and tracking the division of the CFSE labeled cells in TCRa " mice. The results in Fig. 3.16 indicate that CD44"7" CD8 2C TCR+ cells expanded normally after injection into TCRa"'" mice. 41 a) B6 CD4 T-lymphocytes, Activate in vitro with anti-CD3 for 48h, 51Cr-labeled Inject to DTH-induced B6 mouse (Rt. Ear Inflamed) After 24 h the % Radioactivity in the inflamed/non-inflamed ear was determined. b) "8 0.1 « 0.09 § 0.08 & °-07 £ 0.06 •£ 0. OS « 0.04 § 0.03 '•V 0.02 S, 0.01 5? 0 DNFB ear (A verage of 2 Control ear (A verage of 2 mice) mice) Figure 3.13. Migration of 48 hr-activated B6 CD4 cells to the inflammation site. The experimental protocol is illustrated in (a). Panel (b) shows the result at 24h post-injection. %Radioactivity recovered was used as a measure of percent migration to the inflammation site. The left ear (non-inflamed) was used as control. The cells were activated with anti-CD3 (l^ig/ml) and IL-2 (20 U/ml). 42 0.16 0.14 t 0.12 > o o Pi 0.1 0.08 § 0.06 • * — ( <2 0.04" 0.02 BB6CD4 Blast Recipients (Average of 2 mice) DB6CD8 Blast Recipients (Average of 3 mice) QB6-CD44-/-CD4 Blast Recipients (Average of 2 mice) BB6-CD44-/-CD8 Blast Recipients (Average of 3 mice) DNFB ear Control ear Figure 3.14. Migration of recently activated CD4 and CD8 T cells to DTH site is not dependent on CD44. Purified CD4 and CD8 T cells from B6 and B6-CD44"'" mice were cultured under polarizing condition for THl and TCI for 48 hr. The cells were then labeled with 5 1Cr and injected and analyzed as in fig. 3.13. 43 8000 7000 6000 5000 -] § 4000 U 3000 2000 1000 0 —o—2C cells + SYRGL Peptide -Q-2C-CD44-/ cells + SYRGL Peptide 0 0.001 0.01 0.1 Concentration of SYRGL peptide ( pM) Figure 3.15. Optimal proliferation in response to the SYRGL/K b ligand is not CD44-dependent. Purified CD8 T cells (lxl04) from 2C and 2C-CD44'" mice were stimulated with 5x10s B6-TAP-1"'" splenocytes plus the indicated concentration of SYRGL peptide. Incorporation of [3H]-thymidine for the last 6 hours of a 72 hr. culture period was used as a measure of proliferation of the cells. 44 CFSE Staining after 48h activation with 2C11 CFSE profiles after 3 days expansion in vivo Figure 3.16 Lack of CD44 did not affect expansion of activated CD8 T cells in vivo. CD8 cells from 2C and 2C-CD44/" mice were activated in vitro by anti-CD3 (lOug/ml) and 11-2 (20U/ml). After 48h activation, the CD8 cells were labeled with CFSE and injected into B6-TCRoc"' mice. After 3 days the CFSE profiles of gated CD8+ 1B2+ spleen cells from recipient mice were determined by flow cytometry. The spleen cells were depleted of CD4+ and Ig+ cells prior to staining with anti-CD8 and 1B2 mAbs. 45 To track the migration of Ag-activated CD8 T cells in vivo we adoptively transferred C D 8 2C TCR+ cells into B6-TCRa"A mice. B6-TCRa"' mice were chosen since they do not possess any 0 $ TCR+ cells, express K b, and all responses to the SYRGL peptide can be attributed to the adoptively transferred C D 8 cells. The adoptively transferred cells were activated 24 h later by s.c. immunization with the SYRGL peptide. At various times after SYRGL immunization the mice were sacrificed. Lymphoid (spleen, lymph node, bone marrow, peritoneal cells) and non-lymphoid (liver and kidney) tissues were harvested and the frequency and absolute numbers of CD8 +1B2 + cells in these tissues determined. To determine the role of CD44 in the migration of Ag activated C D 8 cells into these various tissues the responses of CD8 +1B2 + cells were compared to those of CD44"/"CD8+1B2+ cells in parallel experiments. A graphic summary of this protocol is illustrated in Fig. 3.17. The harvested tissues were enriched for lymphocytes and were stained with lB2-FitC, aCD44 -PE and 0CCD8-TC. Fig. 3.18 illustrates the results 7 days post SYRGL peptide injection. It is clear from this analysis that CD44 is not required for the migration of CD8 +1B2 + cells into various tissues. There was an increase in the frequency of CD44"/"CD8+1B2+ cells in the spleen, blood, bone marrow, liver, kidney and peritoneum. This increase in frequency also translated into an increase in the absolute numbers of CD8 +1B2 + cells (Fig. 3.19) since roughly equal number of lymphocytes were isolated from the various tissues of recipient mice that were injected with wild type or CD44-deficient CD8 +1B2 + cells. A more detailed kinetic study revealed that the increased accumulation of CD44"/"CD8+1B2+ cells in liver, spleen and kidney was observed over a 45 day period (Fig. 3.20). These results indicate that there is no requirement for CD44 for the circulation of antigen-activated and presumably memory C D 8 T cells into lymphoid and non-lymphoid tissues. 46 Inject 2x106 naive 2C CD8 T cells Inject 2x l06 naive 2C-CD447" CD8 T cells B6-TCRoc mouse B6-TCRa mouse After 24h, inject S Y R G L , s.c. -Harvest the lymphoid & non-lymphoid tissues after various times -Compare the two groups Figure 3.17. Protocol for tracking the in vivo migration of Ag-activated CD8 T cells. 47 a) 1B2-TC b) O ~\ I I H ' « | • M | I I | I • III ^10° 101 102 103 10 FL1-H CD8-FitC %1B2+CD8+/CD8+ in 2C- injected animal %1B2+CD8+/CD8+ in 2C-CD44"'" -injected animal LN 19.6 21 Spleen 26.7 40 Blood 36.5 78 BM 8.4 22 Liver 28 81.5 Kidney 64.5 90.5 PEC 46.6 59 Figure 3.18. Migration of 7-day Ag-activated 2C CD8 T cells to the lymphoid and non-lymphoid tissues is not dependent on CD44. The protocol is as illustrated in Fig. 3.17. At day 7 post SYRGL peptide injection lymphocytes were isolated from lymphoid and non-lymphoid tissues as described in Chapter 2. The cells were then stained with anti-CD8FitC, anti-CD44PE and 1B2-Cy5 and analyzed by flow cytometry. The numbers reflect the results of one experiment that was carried out at this time point. 48 eg /-v S3 O U O o o l+<N s * 7 6 5 5 2 1 0 0 2C • 2C-CD44-/-liver Spleen Time Kidney Figure 3.19. Increased migration of CD44-deficient CD8 T cells into lymphoid and non-lymphoid tissues. The protocol is the same as in Figure 3.18. In this figure the absolute number of 2C cells recovered from liver, spleen and kidney from one experiment are compared. 49 a) Kinetics of migration of 2C/2C-CD44-/- cells into tissues 100 4 days 10 days Time 45 days • LN(2C) @LN(2C-CD44-/-) • Spleen(2C) B Spleen(2C-CD44-/-) • Liver(2C) ® Liver(2C-CD44-/-) • Kidney(2C) 0 Kidney(2C-CD44-/-) ffl BM(2C) S BM(2G-CD44-/-) M PEC(2C) E3 PEC(2C-CD44-/-) b) Kinetics of migration of 2C/2C-CD44-/- cells into tissues 2500000 2000000 + oo Q 1500000 + 3 1000000 3fc 500000 0 • Liver(2C) m Liver(2C-CD44-/-) • Spleen(2C) • Spleen(2C-CD44-/-) • Kidney(2C) B Kidney(2C-CD44-/-) 4 days 45 days Figure 3.20. Increased recovery of CD44-deficient CD8+ T cells at various time points. The experimental protocol is as illustrated in Fig. 3.17. In (a) the %1B2+CD8+ of all CD8 + in different tissues at different time points were compared . In (b) the absolute number of 1B2+CD8+ cells harvested were compared. The results show that more CD44"'" 1B2+CD8+ cells were recovered from these tissues at the indicated time points. The plots reflect the results of one experiment. 50 CHAPTER 4 DISCUSSION CD44 has been suggested to play a role in adhesive interactions that are important for the migration and extravasation of T cells into lymphoid and non-lymphoid tissues. Furthermore, it is believed that this function of CD44 is mediated as a result of CD44 binding to HA on endothelial surfaces. In the first part of this study we demonstrated that binding of HA to activated T cells is absolutely dependent on CD44. The cells demonstrated HA binding above a certain threshold of CD44 expression with higher expression of CD44 by the activated T cells leading to higher HA binding. We also came to the conclusion that blast cells expressed the highest level of CD44 and bound the highest amount of HA. We also investigated the role of TNFa and its receptors in the binding of HA to activated T cells. These studies are also consistent with the conclusion that TNFa is involved in T cell activation and regulates HA binding through its effect on T cell activation. In the second part of this thesis the role of CD44 and TNF receptors in the migration of T lymphocytes into inflamed tissues and in the circulation of activated and memory T cells in lymphoid and non-lymphoid tissues were investigated. These results clearly indicate that CD44 and TNF receptors are not required for these processes. Our in vitro binding assay for HA clearly indicates that the binding of HA by activated T cells is CD44 dependent. Two approaches were used. In the first approach we compared HA binding by CD8 T cells from B6 and B6-CD44"'" mice that were activated under identical conditions. We found that activated CD44-deficient CD8 T cells were unable to bind HA. In the second approach, activated CD8 T lymphocytes were incubated with KM114 before staining with HA-FL1. KM114 is an anti-CD44 antibody that binds to the HA-binding site of the CD44 molecule, hence blocking HA binding [47]. Our results showed that HA binding by activated CD8 T cells was in fact completely abrogated by pre-incubation with the KM114 51 mAb. These two approaches indicate that HA binding by activated CD 8 T cells is in fact absolutely CD44-dependent. We also observed that a lower percent of CD4 T lymphocytes that were activated under the same conditions as CD8 T cells bind to hyaluronic acid. We found that the less efficient binding of HA by activated CD4 T cells correlated with a lesser degree of activation of the CD4 T cells relative to CD8 T cells. This was evidenced by a higher expression of activation markers (CD25, CD44, CD69) in CD8 blasts. Interestingly, we also found that blast cells, which expressed the highest level of CD44, are the most efficient binders of HA. This observation indicates that there is a direct relationship between activation, CD44 expression and HA binding. TNFa is known to be required for optimal T cell activation [43]. It also affected HA binding by a human leukemic cell line [20]. This is thought to occur through sulfation at the post-translational level, converting it from the inactive (non-HA-binding) to its active form (HA-binding) [20]. We found that TNFa can affect HA binding by activated CD8 T cells under some conditions. It appears to increase percent HA binding if the CD8 T cells were not optimally activated and does not increase percent HA binding if the cells were already maximally activated. This conclusion can be extended to CD8 T cells with the p55"A and p75''' mutation. Our laboratory has recently shown that p75, but not p55, functions as an important costimulatory molecule for T cells [43]. Our observation that anti-CD3 activated p75~/_ CD8 T cells have a lower percent HA binding than those from wild type or p55"A mice is consistent with the conclusion that there are fewer activated T cells in the p75''~ population. We also found that the percent HA binding by p75"'~ cells can be further increased by the addition of exogenous IL-2. Based on these observations we propose the following explanation for our observations: TNFa is required for optimal T cell activation. This effect of TNFa is likely mediated via the p75 receptor. Optimal activation of T cells leads to expression of very high levels of CD44. It is clear that the CD44 expressed by the most activated cells contains CD44 isoforms that are most effective in HA binding. However, it is not clear if the conversion of 52 inactive to active form is mediated by TNFa. It is possible that the largest amount of TNFa is produced by the most activated T cells and this would facilitate the conversion of inactive to active forms of CD44 through sulfation. However, it is also clear that this putative action of TNFa is not dependent on the p75 or the p55 receptor since p757" and p55_/" blasts are also the most efficient HA binders. Thus, we favor the view that T cell activation is accompanied by high expression of CD44 and HA binding CD44 isoforms. However, the conversion from the inactive to active isoform may occur via TNFa-independent mechanisms. In 1997 DeGrendele et al. [31] used an experimental model involving injection of Staphylococcal Enterotoxin B (SEB) into the peritoneal cavity of B6 mice. The injection led to inflammation in the peritoneal cavity and finally proliferation of Vp8+ lymphocytes in vivo and their relocation in the mesenteric lymph nodes. The basic experimental plan consisted of harvesting HA-binding lymphocytes from the mesenteric lymph nodes of one mouse, fluorescent-labeling the cells and injecting them through the tail vein to another mouse with a similar inflammation in the peritoneal cavity. Some of the recipient mice received an injection of the blocking KM114 mAb along with the cells. To address the role of CD44 in this assay, migration of the labeled T cells with/without the blocking antibody was compared. Their result demonstrated a reduction of migration in the mouse with blocking antibody. However, the conclusion of this study is compromised by the fact that binding of anti-CD44 mAb may also have an effect on the CD44-bearing endothelial cells, which introduces another variable to the study. Therefore, this study does not conclusively demonstrate a role for CD44/HA-mediated migration of lymphocytes in vivo. In 1999 Protin et al. [1] addressed the role of CD44 in vivo by producing mice lacking CD44. Their study showed that CD44"'" mice developed normally and possess a normal immune system. These mice were used to study the role of CD44 in the migration of naive T-lymphocytes in vivo. Their results showed a delay in migration of the cells into the peripheral lymph nodes at the 2 h but not the 24 h time point. They also showed that the migration of B6-CD44"'" cells to the thymus was 10-20 times less than their wild-type counterparts at the 2 53 h and 24 h time points. Naive T lymphocytes were used for these studies. However, it is well known that naive T lymphocytes do not bind HA [14]. Furthermore, CD44-mediated HA binding has been proposed as a common mechanism for explaining the role of CD44 in the attachment/diapedesis of lymphocytes. Since resting and naive T cells do not bind HA, the role of HA in these processes need to be re-evaluated. We used three different approaches to evaluate the potential role of CD44 and TNF receptors in affecting the migration of lymphocytes in vivo. In the first approach we polarized CD4 T cells into THl andTH2 cells and determined the ability of the polarized T cells to migrate to a DTH site. The results from this series of measurement did not show any reduction in migration of B6-CD44" ~ THl cells compared to B6 cells. We also found that migration of THl cells was not dependent on either the p55 or p75 TNF receptors. These results are consistent with an earlier study that concluded that migration of polarized THl cells to DTH site is more dependent on other adhesion molecules such as LFA-1 and VLA-4 existing on the surface of the activated T cell [37]. We found that although polarized THl cells from B6 mice after 5 days of expansion express fairly high levels of CD44 these cells do not bind HA. Therefore these experiments do not provide a critical evaluation of whether CD44/HA interactions can affect the migration of T cells into DTH site. We were successful in inducing HA-binding activity in resting THl cells by incubating with the IRAWB mAb. However, we found that this HA binding activity was rapidly lost upon culture at 37° and therefore this approach was not suitable for in vivo studies. We therefore used an alternative approach to address this question. This alternative approach involved the use of recently activated T cells in our studies. HA binding was maximal in 48 h activated cells and these cells were used to evaluate the role of CD44 and HA in the migration of lymphocytes to DTH sites. The primary concern of this approach was whether recently activated cells have the ability to recirculate. Our preliminary experiments showed that although activated T cells do not recirculate as efficiently as resting T cells sufficient migration of activated T cells to the DTH site was observed to allow these 54 experiments to proceed. Using this approach it was clear that the migration of 48 h activated CD4 and CD8 T cells to DTH sites was independent of CD44. This result indicates that the binding of CD44 to HA likely has no role in the migration of activated T cells to sites of inflammation. The third approach involved the adoptive transfer of antigen-specific CD8 T cells into TCRcc"/_ mice. The adoptively transferred cells were then activated by its specific antigen and the distribution of antigen-activated CD8 T cells in lymphoid and non-lymphoid tissues was determined at various times after antigen injection. The role of CD44 in the distribution of antigen-specific CD8 T cells in these tissues was evaluated by comparing the pattern of distribution in wild type and CD44"'" antigen-specific CD8 T cells. Previous studies have shown that activated T lymphocytes were distributed to different extent in different lymphoid and non-lymphoid tissues [48]. Thus, by following the distribution of antigen-specific CD44+/+ and CD44"'' CD8 T cells at various times after antigen administration it was possible to evaluate the role of CD44 in the distribution of activated and memory T cells to lymphoid and non-lymphoid tissues. The use of CD8 T cells from the 2C TCR transgenic mice allows us to track the distribution of these cells in vivo with the use of the 1B2 mAb, which detects the TCR idiotype of the 2C TCR [46]. The use of 2C CD8 T cells from CD44+/+ and CD44'" mice allowed us to determine the role of CD44 in the distribution of lymphocytes at various stages of their developmental pathway to lymphoid and non-lymphoid tissues. The adoptive transfer of 2C CD8 T cells into TCRct7" mice allowed us to do these mice in the absence of any contribution of ap T cells from the recipient mice to immune responses induced by the injection of the SYRGL peptide. Furthermore, these experiments were all performed on a B6 background and therefore any difference in the results can be directly attributed to CD44. The injected 2C CD8 T cells were activated by the SYRGL/Kb ligand [41]. The population of 1B2+CD8+ cells that is marked in Fig. 3.18(a) was not detected in the animals that had received an injection of 2C CD8 T cells but not of SYRGL peptide afterwards. The results 55 from this series of experiments demonstrate quite conclusively that the distribution of 2C CD8 T cells in lymphoid and non-lymphoid tissues at various times after antigen administration (4 to 45 days) is not dependent on CD44 expression. The surprising finding is that there is better accumulation of 2C CD44"'" CD8 T cells in these tissues at various times after antigen administration. This result challenges a widely held notion that CD44-mediated adhesive interactions is required for the extravasation of T cells into lymphoid and non-lymphoid tissues. The distribution of antigen-specific CD8 T cells in a particular tissue at any given time is a function of the rate of migration to and from the tissues and the extent of cell proliferation and death in these tissues. We have shown that antigen-activated CD44"'" CD8 cells proliferated to the same extent as CD44+/+ cells in vitro. Furthermore, anti-CD3 activated CD44"'" CD8 T cells also proliferated to the same extent in vivo as CD44+/+ cells. Consequently, it is unlikely that the higher recovery of CD44"A CD8 T cells is due to increased proliferation by these cells in response to antigen stimulation. The increased accumulation of antigen-specific CD44"7" CD8 T cells in lymphoid and non-lymphoid tissues may be due to an increase in migration and/or decrease in the rate of cell death. It is interesting to speculate that adhesive interactions between activated CD8 T cells and HA may serve to inhibit the migration of activated CD 8 T cells out of the lymphoid organs where the immune response is initiated. The increased accumulation of CD44"'" CD 8 T cells in various tissues can then be attributed to increased migration of these cells into these tissues. A more detailed analysis of the kinetics of migration of antigen-activated CD8 T cells into non-lymphoid tissues is required for testing this hypothesis. This study can be coupled with an analysis of the cell-surface molecules that are implicated in the migration of activated/memory T cells into non-lymphoid tissues to determine whether the expression of these molecules is affected by the CD44"'" mutation. 56 REFERENCES 1. Protin, U., T. Schweighoffer, W. Jochum, and F. Hilberg, CD44-deficient mice develop normally with changes in subpopulations and recirculation of lymphocyte subsets. J Immunol, 1999.163(9): p. 4917-23. 2. de los Toyos, J., S. Jalkanen, and E.C. Butcher, Flow cytometric analysis of the Hermes homing-associated antigen on human lymphocyte subsets. Blood, 1989. 74(2): p. 751-60. 3. Lynch, F. and R. Ceredig, Ly-24 (Pgp-1) expression by thymocytes and peripheral T cells. Immunol Today, 1988. 9(1): p. 7-10. 4. Underhill, C.B. and B.P. Toole, Physical characteristics ofhyaluronate binding to the surface of simian virus 40-transformed 3T3 cells. J Biol Chem, 1980. 255(10): p. 4544-9. 5. Underhill, C.B., S.J. Green, P.M. Comoglio, and G. Tarone, The hyaluronate receptor is identical to a glycoprotein of Mr 85,000 (gp85) as shown by a monoclonal antibody that interferes with binding activity. J Biol Chem, 1987. 262(27): p. 13142-6. 6. Hirano, H., G.R. Screaton, M.V. Bell, D.G. Jackson, J.I. Bell, and R.J. Hodes, CD44 isoform expression mediated by alternative splicing: tissue-specific regulation in mice. Int Immunol, 1994. 6(1): p. 49-59. 7. Tolg, C , M. Hofmann, P. Herrlich, and H. Ponta, Splicing choice from ten variant exons establishes CD44 variability. Nucleic Acids Res, 1993. 21(5): p. 1225-9. 8. Peach, R.J., D. Hollenbaugh, I. Stamenkovic, and A. Aruffo, Identification of hyaluronic acid binding sites in the extracellular domain of CD44. J Cell Biol, 1993. 122(1): p. 257-64. 9. Lesley, J. and R. Hyman, CD44 structure and function. Front Biosci, 1998. 3: p. D616-30. 10. Culty, M., K. Miyake, P.W. Kincade, E. Sikorski, E.C. Butcher, C. Underhill, and E. Silorski, The hyaluronate receptor is a member of the CD44 (H-CAM) family of cell surface glycoproteins. J Cell Biol, 1990.111(6 Pt 1): p. 2765-74. 11. Toyama-Sorimachi, N., H. Sorimachi, Y. Tobita, F. Kitamura, H. Yagita, K. Suzuki, and M. Miyasaka, A novel ligand for CD44 is serglycin, a hematopoietic cell lineage-specific proteoglycan. Possible involvement in lymphoid cell adherence and activation. J Biol Chem, 1995. 270(13): p. 7437-44. 12. Jalkanen, S. and M. Jalkanen, Lymphocyte CD44 binds the COOH-terminal heparin-binding domain offibronectin. J Cell Biol, 1992. 116(3): p. 817-25. 57 13. Sleeman, J.P., K. Kondo, J. Moll, H. Ponta, and P. Herrlich, Variant exons v6 and v7 together expand the repertoire of glycosaminoglycans bound by CD44. J Biol Chem, 1997. 272(50): p. 31837-44. 14. Lesley, J. and R. Hyman, CD44 can be activated to function as an hyaluronic acid receptor in normal murine T cells. Eur J Immunol, 1992. 22(10): p. 2719-23. 15. DeGrendele, H.C., M. Kosfiszer, P. Estess, and M.H. Siegelman, CD44 activation and associated primary adhesion is inducible via T cell receptor stimulation. J Immunol, 1997.159(6): p. 2549-53. 16. Lesley, J., N. Howes, A. Perschl, and R. Hyman, Hyaluronan binding function of CD44 is transiently activated on T cells during an in vivo immune response. J Exp Med, 1994.180(1): p. 383-7. 17. Lesley, J., R. Hyman, N. English, J.B. Catterall, and G.A. Turner, CD44 in inflammation and metastasis. Glycoconj J, 1997.14(5): p. 611-22. 18. Kincade, P.W., Z. Zheng, S. Katoh, and L. Hanson, The importance of cellular environment to function of the CD44 matrix receptor. Curr Opin Cell Biol, 1997. 9(5): p. 635-42. 19. Johnson, P., A. Maiti, K.L. Brown, and R. Li, A role for the cell adhesion molecule CD44 and sulfation in leukocyte-endothelial cell adhesion during an inflammatory response? Biochem Pharmacol, 2000. 59(5): p. 455-65. 20. Maiti, A., G. Maki, and P. Johnson, TNF-alpha induction of CD44-mediated leukocyte adhesion by sulfation. Science, 1998. 282(5390): p. 941-3. 21. Green, D.M., J. Trial, and H.H. Birdsall, TNF-alpha released by comigrating monocytes promotes transendothelial migration of activated lymphocytes. J Immunol, 1998.161(5): p. 2481-9. 22. de Jong, A.L., D.M. Green, J.A. Trial, and H.H. Birdsall, Focal effects of mononuclear leukocyte transendothelial migration: TNF-alpha production by migrating monocytes promotes subsequent migration of lymphocytes. J Leukoc Biol, 1996. 60(1): p. 129-36. 23. Hay, J.B., N.J. Abemethy, A.N. Kalaaji, G.F. Teare, and P. Borron, The relevance of lymphoid cell migration to immunodeficiency syndromes. Lymphology, 1990. 23(2): p. 64-72. 24. Peschon, J.J., D.S. Torrance, K.L. Stocking, M.B. Glaccum, C. Otten, C.R. Willis, K. Charrier, P.J. Morrissey, C.B. Ware, and K.M. Mohler, TNF receptor-deficient mice reveal divergent roles for p55 and p75 in several models of inflammation. J Immunol, 1998.160(2): p. 943-52. 58 25. Arbones, M.L., D.C. Ord, K. Ley, H. Ratech, C. Maynard-Curry, G. Otten, D.J. Capon, and T.F. Tedder, Lymphocyte homing and leukocyte rolling and migration are impaired in L-selectin-deficient mice. Immunity, 1994.1(4): p. 247-60. 26. Hamann, A., K. Klugewitz, F. Austrup, and D. Jablonski-Westrich, Activation induces rapid and profound alterations in the trafficking of T cells. Eur J Immunol, 2000. 30(11): p. 3207-18. 27. Ebnet, K., E.P. Kaldjian, A.O. Anderson, and S. Shaw, Orchestrated information transfer underlying leukocyte endothelial interactions. Annu Rev Immunol, 1996. 14: p. 155-77. 28. Springer, T.A., Traffic signals for lymphocyte recirculation and leukocyte emigration: the multistepparadigm. Cell, 1994. 76(2): p. 301-14. 29. Zahalka, M.A., E. Okon, and D. Naor, Blocking lymphoma invasiveness with a monoclonal antibody directed against the beta-chain of the leukocyte adhesion molecule (CD18). J Immunol, 1993.150(10): p. 4466-77. 30. Zahalka, M.A., E. Okon, U. Gosslar, B. Holzmann, and D. Naor, Lymph node (but not spleen) invasion by murine lymphoma is both CD44- and hyaluronate-dependent. J Immunol, 1995.154(10): p. 5345-55. 31. DeGrendele, H.C., P. Estess, and M.H. Siegelman, Requirement for CD44 in activated Tcell extravasation into an inflammatory site. Science, 1997. 278(5338): p. 672-5. 32. Mikecz, K., F.R. Brennan, J.H. Kim, and T.T. Giant, Anti-CD44 treatment abrogates tissue oedema and leukocyte infiltration in murine arthritis. Nat Med, 1995.1(6): p. 558-63. 33. Camp, R.L., A. Scheynius, C. Johansson, and E. Pure, CD44 is necessary for optimal contact allergic responses but is not required for normal leukocyte extravasation. J Exp Med, 1993.178(2): p. 497-507. 34. Black, C.A., Delayed type hypersensitivity: current theories with an historic perspective. Dermatol Online J, 1999. 5(1): p. 7. 35. Shelley, W.B. and L. Juhlin, Selective uptake of contact allergens by the Langerhans cell. Arch Dermatol, 1977.113(2): p. 187-92. 36. Mosmann, T.R. and R.L. Coffman, TH1 and TH2 cells: different patterns of lymphokine secretion lead to different functional properties. Annu Rev Immunol, 1989. 7: p. 145-73. 37. Austrup, F., D. Vesrweber, E. Borges, M. Lohning, R. Brauer, U. Herz, H. Renz, R. Hallmann, A. Scheffold, A. Radbruch, and A. Hamann, P- and E-selectin mediate 59 recruitment ofT-helper-1 but not T-helper-2 cells into inflammed tissues. Nature, 1997. 385(6611): p. 81-3. 38. Mosmann, T.R., L. Li, and S. Sad, Functions of CD8 T-cell subsets secreting different cytokine patterns. Semin Immunol, 1997. 9(2): p. 87-92. 39. Knudson, W., D.J. Aguiar, Q. Hua, and C.B. Knudson, CD44-anchored hyaluronan-rich pericellular matrices: an ultrastructural and biochemical analysis. Exp Cell Res, 1996. 228(2): p. 216-28. 40. Alexander, J., J.A. Payne, R. Murray, J.A. Frelinger, and P. Cresswell, Differential transport requirements ofHLA and H-2 class I glycoproteins. Immunogenetics, 1989. 29(6): p. 380-8. 41. Cho, B.K., D. Palliser, E. Guillen, J. Wisniewski, R.A. Young, J. Chen, and H.N. Eisen, A proposed mechanism for the induction of cytotoxic T lymphocyte production by heat shockfusion proteins. Immunity, 2000.12(3): p. 263-72. 42. de Belder, A.N. and K.O. Wik, Preparation and properties offluorescein-labelled hyaluronate. Carbohydr Res, 1975. 44(2): p. 251-7. 43. Kim, E.Y. and H.S. Teh, TNF type 2 receptor (p75) lowers the threshold of Tcell activation. J Immunol, 2001. 167(12): p. 6812-20. 44. Erickson, S.L., F.J. de Sauvage, K. Kikly, K. Carver-Moore, S. Pitts-Meek, N. Gillett, K.C. Sheehan, R.D. Schreiber, D.V. Goeddel, and M.W. Moore, Decreased sensitivity to tumour-necrosis factor but normal T-cell development in TNF receptor-2-deficient mice. Nature, 1994. 372(6506): p. 560-3. 45. Motyka, B. and H.S. Teh, Naturally occurring low affinity peptide/MHC class I ligands can mediate negative selection and Tcell activation. J Immunol, 1998. 160(1): p. 77-86. 46. Kranz, D.M., D.H. Sherman, M.V. Sitkovsky, M.S. Pastemack, and H.N. Eisen, Immunoprecipitation of cell surface structures of cloned cytotoxic T lymphocytes by clone-specific antisera. Proc Natl Acad Sci U S A , 1984. 81(2): p. 573-7. 47. Miyake, K , C.B. Underhill, J. Lesley, and P.W. Kincade, Hyaluronate can function as a cell adhesion molecule and CD44 participates in hyaluronate recognition. J Exp Med, 1990.172(1): p. 69-75. 48. Masopust, D., V. Vezys, A.L. Marzo, and L. Lefrancois, Preferential localization of effector memory cells in nonlymphoid tissue. Science, 2001. 291(5512): p. 2413-7. 60 

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