Studies of Gut-Associated Lymphoid Tissues and Other Secondary Lymphoid Tissues in 12 Week Old New Zealand White Specific Pathogen Free Rabbits

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STUDIES OF GUT-ASSOCIATED LYMPHOID TISSUES AND OTHER SECONDARY LYMPHOID TISSUES IN 12 WEEK OLD NEW ZEALAND WHITE SPECIFIC PATHOGEN FREE RABBITS

A Thesis

Presented in Partial Fulfillment of the Requirements for the Degree Master of Science in the Graduate School of The Ohio State University

Rebeccah Urbiztondo, DVM

Graduate Program in Veterinary Biosciences

The Ohio State University 2010

Master’s Examination Committee:

Professor Michael Lairmore, Advisor

Associate Professor Tracey Papenfuss

Associate Professor Mary Jo Burkhard

Rebeccah Urbiztondo

2010

ABSTRACT

Rabbits serve as valuable animal models to study the immunopathogenesis of human diseases that gain entry through the gastrointestinal (GI) tract. Rabbits possess abundant mucosa-associated lymphoid tissue (MALT), both in the GALT and at other mucosal sites. This makes them uniquely suited to studying diseases which are transmitted across these surfaces. This study looks at the gut-associated lymphoid tissues (GALT), as well as, the lymphoid populations in the spleen, mesenteric lymph nodes, and peripheral blood of twelve week old New Zealand White rabbits. Herein, we used flow cytometric and immunohistochemical methods to phenotypically characterize lymphoid populations. Results obtained via flow cytometric analysis were comparable to the distribution of leukocyte subsets in other animal species, including humans, both in the GALT inductive and effector sites and in mesenteric lymph nodes, spleen, and peripheral blood. Immunohistochemical analyses of tissues were comparable to results obtained via flow cytometry. Our data collectively indicate that New Zealand White rabbits compared to humans and mice contain a predominant CD4+ T cell population throughout their GALT and associated lymphoid tissues. Furthermore, these data will support future studies that utilize the rabbit model to study human gut-associated disease or infectious agents that gain entry via the oral route.

ACKNOWLEDGMENTS

I would like to thank Krissy and my lab mates for their help with this project. Special thanks goes to Sha who got me started on the right foot and showed me some valuable tricks regarding sample processing. Without his guidance I never would have been able to process over 100 flow samples in one day. I would also like to extend my gratitude to Robyn who has gone through this project with me. Without her help and meticulous organizational skills, things would not have run as smoothly as they did. Though I did not work directly with

Raj and Nadine, their singing in the lab provided constant amusement.

I would also like to acknowledge Sarah Leavell for her critical role in helping me ease into a research setting. She was an excellent mentor to me regarding good laboratory techniques. I am so appreciative of the patience she showed me when I was transitioning from clinical pathologist in training to graduate student.

Last but not least, I would like to thank Dr. Lairmore. His “leave no stone unturned” attitude is very refreshing in a day in age when “good enough” is the norm. I thank him for letting me be a part of his laboratory. The lessons I learned in his lab, both research and non-research related, will help me in the future regardless of my endeavors.

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VITA

March 9, 1982…………………………...... Born - San Juan, Puerto Rico

2008……………………………………………………DVM, Iowa State University College of Veterinary Medicine

2008-Present………………………………………….Graduate Research Associate/ Clinical Pathology Resident The Ohio State University Department of Veterinary Biosciences

PUBLICATION

Urbiztondo R, Chapman S, Benjamino K. Meseneteric root osteosacoma in a dog. Vet. Clin Path. In press.

FIELDS OF STUDY

Major Field: Veterinary Biosciences

Minor Field: Veterinary Clinical Pathology

TABLE OF CONTENTS

Page Abstract ………………………………………………………………………………....ii

Acknowledgments…………………………………………………………………..….iii

Vita……………………………………………………………………………..………..iv

List of Tables…………………………………………………………………………...vii

List of Figures……………………………………………………………………....….viii

1. Literature Review: Mucosal immunology with the primary emphasis on gastrointestinal-associated lymphoid tissues across all animal species and in the rabbit

Introduction…………………………………………………....….1 Mucosa-Associated Lymphoid Tissue.………………………...2 Gastrointestinal-Associated Lymphoid Tissue..…………..…..3 GALT Inductive Site………………………….. ……...... 4 GALT Effector Sites………………………………...... 7 Intraepithelial Leukocytes……………………………………….8 Lamina Propria Leukocytes…………………………...... 11 Mesenteric Lymph Nodes…………………………………...... 13 Comparative Organization of GALT in humans and other animals……………………………………………………….....14 Rabbits: A Comparative Animal Model in Investigations of Mucosal immunity And Infectious Disease Studies………...16 Conclusions……………………………………...... 19

2. Isolation and Phenotypic Characterization of New Zealand White Rabbit Gut-associated lymphoid tissues and other secondary lymphoid tissues

Introduction……………………………………………………..23 Material and Methods……………………………………….…27 Results…………………………………………………………..32 Discussion……………………………………………………....38

3. FUTURE DIRECTIONS:

Summary…………………………………………………………..56

List of References………………………………………………………………...64

LIST OF TABLES

Table Page

1.1 MALT and MALT-related abbreviation………………………………………21

1.2 Summary of Lymphocyte Phenotypes and T cell ratios in Percentages in selected lymphoid compartment across Animal species……………….22

2.1 Rabbit Monoclonal Antibody Table…………………………………………..44

2.2 White blood cell differentials of cytospin preparations from each anatomic location evaluated in percentages………………………………..45

2.3 CD4:CD8 ratios in lymphoid tissues………………………….……………...46

2.4 B:T cell ratios in lymphoid tissues……………………………………………47

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LIST OF FIGURES

Figures Pages

2.1 Gross and microscopic appearance of rabbit gastrointestinal inductive sites……………………………………………………………………………..48

2.2 H&E sections of small intestine highlighting intraepithelial and lamina propria leukocyte isolation……………………………………………………49

2.3 Cytospin preparations of isolated leukocytes from each of the nine tissues………………………………………………………………………….50

2.4 Flow cytometric scatter plots highlighting gating patterns for each of the nine tissues ……………………………………………………………………51

2.5 Flow cytometric staining patterns highlighting T cell and CD8 surface marker expression for each of the nine tissues…………………………….52

2.6 IHC images of mesenteric lymph node highlighting various T and B cell antibody markers………………………………………………………………53

2.7 IHC images of cecal tonsil highlighting various T and B cell antibody markers…………………………………………………………………………54

2.8 Bar charts for each of the nine tissues evaluated summarizing flow cytometric surface marker expression in percentages…………………….55

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CHAPTER 1

LITERATURE REVIEW

MUCOSAL IMMUNOLOGY WITH THE PRIMARY EMPHASIS ON GASTROINTESTINAL LYMPHOID TISSUES ACROSS ALL ANIMAL SPECIES AND IN THE RABBIT

Introduction

All mammals have both primitive and highly sophisticated mechanisms to keep harmful pathogens from invading the interior of the body. These mechanisms include mechanical barriers, cellular and chemical defenses.

Among these defenses is the indispensable branch of the immune system, known as, the innate immune system. The innate immune system includes physical barriers, antimicrobial peptides, and phagocytic cells that respond very quickly to invading bacteria, which are recognized as distinct from normal cells.

The second major branch of the immune system is known as the adaptive immune system. As the name implies, it “adapts” its response to the offending agent and acquires memory that will protect the body from any future attacks from the same pathogen. The adaptive immune system is characterized by specificity and memory 1, 2. There are tissue compartments throughout the body which house lymphocytes and professional antigen presenting cells that work together to create adaptive immunity. These cells originate from primary

1 lymphoid organs, mainly thymus and bone marrow, and migrate to secondary lymphoid organs 3. Secondary lymphoid tissues include the spleen, lymph nodes, and organized cellular aggregates scattered throughout mucosal surfaces 1, 2.

This latter group is commonly referred to as mucosa-associated lymphoid tissue

(MALT). Table 1.1 lists commonly used abbreviations used for describing MALT structures and functional characteristics.

MUCOSA-ASSOCIATED LYMPHOID TISSUE

Mucosa-associated lymphoid tissue comprises a large and important part of the entire immune system in mammals and contains approximately half of the lymphocytes in the entire body 4, 5. Regardless of anatomic location, MALT has similar cellular arrangements and function. This is mainly due to the comparable roles these mucosal surfaces have, which is to produce and secrete IgA across the surface epithelium, as well as, participate in cytotoxic T cell-mediated responses 4. Organized MALT compartments are present in the gastrointestinal tract, urogenital tract, conjunctiva, and upper respiratory system including the nasopharynx and upper airways. These compartments can be further subdivided into inductive and effector sites. Inductive sites are made-up of organized aggregates of lymphocytes, analogous to systemic lymph node architecture 6.

However, MALT inductive sites do not have afferent lymphatics, unlike systemic lymph nodes 5. In these inductive sites, antigen specific T-cell activation results in clonal expansion of B cells and IgA class switching 4. Antigen is sampled directly across the mucosal surface either by M cells or dendritic cells. Mucosal antigen presenting cells (APCs) are derived from monocyte precursors in blood and are

2 one of the first cells to respond to potential pathogens 7. The effector sites, on the other hand, are diffusely arranged along the entire length of the mucosa both in the surface epithelial layer, as well as, in the underlying lamina or substantia propria. The leukocyte composition of these effector sites is composed largely of

T-cells with a memory or effector phenotype and terminally differentiated IgA secreting B-cells or plasma cells. The focus of this thesis is immunophenotypic characterization of mucosa-associated lymphoid tissue in the rabbit gastrointestinal tract, commonly referred to as, gastrointestinal-associated lymphoid tissue or GALT.

GASTROINTESTINAL-ASSOCIATED LYMPHOID TISSUE

Of all the secondary lymphoid tissues, including other MALT structures,

GALT constitutes the largest collective aggregate of lymphoid tissue in the body and represents the largest interface between the outside world and the interior immune system 8. This seems appropriate since it is the major route of entry for pathogens. The gastrointestinal tract is faced with the task of allowing passage of nutrients into the body, while keeping out harmful agents. The gastrointestinal tract is strategically equipped to deal with luminal antigens due to a variety of mechanisms. These include Toll-like receptor’s (TLRs) on intestinal epithelium, production of protective peptides, and accumulations of lymphoid aggregates, also known as inductive sites 8, 9. There is a single layer of intestinal epithelium that separates the outside world from the interior of the organism connected by intercellular tight junctions, which restrict the passage of very small molecules

(<2 kDa) 10. Chemical defenses unique to the gut mucosa include defensins, secretory immunoglobulin A, mucins, and trefoil peptides 6, 11.

GALT INDUCTIVE SITES

Collections of organized lymphoid aggregates and individualized lymphocytes include inductive sites scattered along the entire length of intestine.

Depending on the species, these individualized nodules include Peyer’s patches, cryptopatches, isolated lymphoid follicles, and lymphoglandular complexes, the latter is found primarily in the large intestine. Peyer’s patches are the primary inductive sites in the gut 4. Though there are subtle variations from one inductive site to another, all inductive sites consist of the same basic compartments. These include one or more follicles with interspersed T-cell rich interfollicular areas, and an overlying subepithelial dome (SED) with a single layer of columnar epithelial cells making up the follicle-associated epithelium (FAE) 4.

The inductive sites of the GALT are the location where antigen first encountered in the gut lumen, is proteolytically processed and presented to naïve

T and B-cells. Under the influence of transforming growth factor-β (TGF- β), IL-

10, and other cellular signals, B cells undergo immunoglobulin class switching from expression of sIgD+IgM+, to sIgD-IgM+ memory-effector cells, and finally IgA expression 11. Protective mechanisms in the inductive sites are complex and are characterized by several barriers, which assist in preventing entry of pathogens.

The most superficial defenses include mucus, proteases, nucleases, secreted antibodies, and the epithelial glycocalyx. Since there are no afferent lymphatics

4 in the GALT, antigen sampling occurs directly from the gastrointestinal lumen in the FAE 12. The FAE is characterized by a reduced numbers of goblet cells and enterocytes, as well as, expression of chemokine CCL20, which is responsible for recruitment of dendritic cells (DCs) 8. It is here that M-cells, which are specialized epithelial cells, and dendritic cells take-up foreign material 13-15. The

M-cells are thought to actively pinocytose and endocytose antigens and deliver them to APCs 16. In most animals, including humans, M-cells are found in the follicle-associated epithelium overlying Peyer’s patches in the small intestine.

The percentage of M cells varies between species and location in the intestinal tract (5-10% in humans and murine Peyer’s patches, and up to 50% in the rabbit and human cecal FAE 13. M-cells have a reduced glycocalyx and decreased membrane hydrolytic enzymes making these cells uniquely suited to transport antigen undisturbed to the underlying tissues 13. The region just beneath the FAE is known as sub-epithelial dome region and consists of diffusely arranged

CD11c+ DCs, HLA-DR expressing macrophages, B-cells, and CD4+ and CD8+ T cells 17. Enterocytes in the FAE secrete the chemokine macrophage inflammatory protein 3-alpha (MIP-3α, CCDL20), which recruits CD11b+ myeloid

DCs to the subepithelial dome of the inductive sites 6, 18. Dendritic cells at this location have the ability to take-up and process live bacteria and soluble antigens

7. The deepest compartment of the GALT inductive site is made up of B-cell follicles with germinal centers surrounded by T-cell rich areas, much the same as peripheral lymph nodes 7. The germinal centers of Peyer’s patches contain a large population of IgM+ B-cells that express activation-induced cytidine

5 deaminase (AID), which is essential for conversion from IgM to IgA expression 12.

Unlike spleen or systemic lymph nodes, GALT follicles are thought to be chronically active and continuously have both primary and secondary germinal centers 16. The GALT inductive sites contain large numbers of CD4+ helper T cells, which uniquely favor B-cell differentiation 19. These cells generally bear a memory phenotype and express little to no L-selectin. This population of T-cells can be further subdivided based on their profile of secreted cytokines. T helper 1

(Th1) cells produce IFN-γ, which is important for cell-mediated immunity. T- helper 2 (Th2) cells are instrumental in humoral immunity and secrete IL-4, IL-5, and IL-13 stimulating B-cell activation and differentiation 20. Prostaglandin E2

(PGE-2) and IL-10 produced in these sites (prominent in the GALT) favor a Th2 response and mucosal tolerance or anti-inflammatory responses 21, 23.

The main function of any lymphoid follicle is to process and respond to captured antigens. In the GALT inductive sites, antigen capture and MHC class

II interactions can occur via several mechanisms. The first and most common mechanism is mediated by memory B-cells in the FAE, which lack surface IgD and express HLA-DR. These cells can rapidly up-regulate the co-stimulatory B7

(CD40) molecules and acquire potent antigen-presenting properties 17. CD40 deficient mice exhibit substantial inhibition of CD8 responses in the intraepithelial and lamina propria compartments 22. Naïve B-cells capture antigen, but lack the co-stimulatory molecules present in memory B-cells and thus also promote T-cell tolerance or anergy 17. Professional antigen presenting cells (APCs) use several costimulatory molecules, including CD80 and CD86, to induce clonal expansion

6 of antigen-specific T-cells 6. Stimulation or induction of mucosal CD8+ T cells requires costimulatory molecules distinct from molecules required for stimulation of CD8+ T cells in other secondary lymphoid tissues 22.

GALT EFFECTOR SITES

The second arm of the mucosal immune system in the gut consists of the diffusely arranged gastrointestinal effector sites. As the name implies “effector” sites consist primarily of memory cells with an “effector” phenotype. The effector sites in the GI tract consist of two distinct compartments. The first compartment is made-up of lymphocytes nestled among enterocytes in the small and large intestine known as intraepithelial lymphocytes/leukocytes (IELs). The second compartment is the lamina propria, which is located beneath the surface epithelium and adjacent basement membrane. The leukocytes that reside in the intraepithelial and lamina propria compartments consist primarily of lymphocytes with a memory or effector phenotype, however, the relative proportions of

CD4:CD8 cells differ between the two compartments 24, 25. CD4+ T-cells are typically present in greater numbers than CD8+ T cells in the lamina propria, whereas the intraepithelial compartment is characterized by far greater numbers of CD8+ T cells relative to CD4+ T cells. Despite the differences in cellular composition in these two compartments, there is considerable cross-talk between the two areas mediated by cytokines, professional APCs, lymphocytes, and granulocytes 5,11.

INTRAEPITHELIAL LEUKOCYTES

There are approximately 10 to 20 IELs per 100 villous enterocytes in the small intestine of humans 20. These lymphocytes are quite unique and consist primarily of T-cells with a strong bias toward CD8+ expression (>70%), whereas, lymph nodes, peripheral blood and spleen have a much smaller percentage of

CD8+ T cells with CD4+ T cells predominating 26. Furthermore, the CD8+ T cells in the intraepithelial compartment are a heterogeneous population. The most recent classification scheme groups IELs into thymus-dependent and thymus- independent categories in mice. The thymus dependent IELs consist of the traditional MHC class Ia restricted TCRαβ+CD8αβ+ cells. The thymus- independent cells represent a unique population of lymphocytes 27,28. These cells consist of TCRαβ+CD8αα+, TCRγδ+CD8αα+, and TCRγδ+CD4-CD8- T-cells. This second population of cells makes the IEL compartment exceedingly unique compared to systemic lymphoid tissues, which house primarily TCRαβ+CD8αβ+

20. Cells that express the γδ TCR are reportedly the first T-cell receptor bearing cells to be detected early in development 29. A recent study found that both murine and human gut effector sites have increased numbers of CD34+CD45+ hematopoietic stem cells (HSCs) relative to bone marrow and peripheral blood.

This population of cells also co-expresses the T-cell progenitor antigen, CD7, which is important for lymphopoiesis 30. A small percentage of lamina propria

HSCs (<10%) also co-expressed CD56, a natural killer (NK) T cell marker. In the intraepithelial compartment almost 50% of HSCs co-expressed CD56 in the human and murine intestine 30. In addition, there are increased levels of IL-7 and

IL-15 cytokines in murine small intestine that are essential for TCR-γδ development and TCRγδ+CD8αα+ and NKT cell development, respectively 30.

These T-cells subsets are not restricted by MHC antigen presentation, and have the ability to recognize a mixed array of bacterial heat shock proteins, classical and non-classical MHC antigens, ligands, and various self-antigens 26,31. Animals are generally born with undeveloped IEL compartments that subsequently develop with enteral nutrition and exposure to environmental antigens 29.

Interestingly, it is the TCRαβ+CD8αβ+ cells that expand, while the unconventional T-cells, mainly TCRγδ+CD4-CD8- cells remain static despite exposure or lack of exposure to environmental antigens 29. The proportion of

TCRαβ vs. TCRγδ is variable and depends on the species and location in the gut. For example, T-cell subsets in the IEL of the large intestine differ from those in the small intestine, which itself is highly variable for T-cell subsets between proximal and distal regions 32. These differences are believed to be due, in part, to differences in the intestinal flora between regions of the intestine. The prototypic TCRαβ+CD8αβ+ T-cells in the small intestine are MHC I restricted 31.

These cells are usually cytolytic and express both granzymes and FAS ligand.

Human IEL, that are phenotypically CD8+TCRαβ T-cells, may have the most cytotoxic potential of any of the various IEL subtypes and express the highest levels of mRNA for five cytotoxic proteins 33. Thymus-independent cytolytic T- cells are granzyme positive and secrete cytokines and chemokines 29. In mice, however, the more traditional, TCRαβ+CD8αβ+ exert more potent virus-specific cytotoxicity, than the thymus-independent T cells. In general, TCRαβ+CD8αα+ are

9 less effective at neutralizing antigen primed target cells, than TCRαβ+CD8αβ+ 26.

Based on previous studies it appears that TCRαβ+CD8αα+ are responsible for maintaining local immune homeostasis and TCRαβ+CD8αβ+ are more important in cytotoxic responses across many species.

Homing of intraepithelial lymphocytes requires expression of specific integrins and chemokine receptors, which mediate adhesion, rolling, and diapedesis of lymphocytes in gut epithelium and adjacent lamina propria 25. The

α4β7 and αεβ7 integrins bind to the mucosal adhesion ligand MadCAM and the epithelial adhesion molecule E-cadherin, respectively 24. Lamina propria leukocytes express high levels of the integrin α4β7, whereas, IELs express high levels of αεβ7 integrin. The integrin α1β1 mediates IEL adhesion to the collagen IV component of the basement membrane and is thought to be important in IEL retention 24. In addition to the integrins, enterocytes secrete various chemokines that attract appropriate lymphocytes subsets to the intraepithelial compartment.

For example, gut enterocytes secrete interferon-inducible protein (IP-10,

CXCL10) and mucosa-associated epithelial chemokine (MEC) induced by INF-γ.

Intraepithelial lymphocytes in turn, express chemokine receptors that bind to the cytokines. For example, CCR9 receptor binds to MEC and CXCR3 binds to IP-10

20.

LAMINA PROPRIA LEUKOCYTES

The lamina propria compartment is more similar to systemic lymphoid tissues than the intraepithelial compartment. However, like the intraepithelial

10 compartment, the lamina propria has an abundant population of T-cells with a memory immunophenotype, but these tend to be CD4+ T-cells, rather than CD8+

T-cells 3, 19, 34. CD4+ T-cells in the lamina propria are generally unresponsive or hyporesponsive to TCR mediated signals 21. Interestingly, the unresponsiveness of these cells can be reversed by decreasing local concentrations of IL-10 and

TGF-β. It is likely CD4+ T-cells have several roles in the lamina propria including helping local B-cells produce IgA, as well as, performing regulatory functions important for maintaining gut homeostasis and tolerance. Although less numerous than CD8+ T-cells in the intraepithelial compartment, lamina propria

CD8+ cells still have potent cytotoxic activity 21. Other leukocytes present in this compartment include terminally differentiated B-cells, plasma cells, dendritic cells, macrophages and scattered granulocytes. Plasma cell numbers in different species typically range between 10 to 30% 35. It is estimated that 80% of all Ig producing B-cell blasts and plasma cells reside in the intestinal lamina propria 17.

Terminal differentiation of plasma cells is enhanced by “IgA-enhancing” cytokines such as IL-5, IL-6, and IL-10 12. Of these plasma cells, approximately 75 to -90% secrete polymeric or dimeric IgA. Efficient secretory immunity depends on cooperation between immunoglobulin secreting B-cells and secretory component or polymeric Ig receptor (pIgR), also known as membrane secretory component.

Membrane secretory component is a 100 kDa transmembrane epithelial glycoprotein 11. This structure mediates the active external transport of dimers and polymers of IgA (pIgA) and pentameric IgM 16.

To maximize secretory immunity MALT must promote expression of B- cells with prominent J (joining) chain expression 17. The germinal centers of human GALT structures produce relatively more IgA immunocytes with J-chain expression, than tonsillar germinal centers 36. The j-chain is believed to protect the secretory immunoglobulin component from proteolytic cleavage in the gut 38.

The j-chain gene is evolutionary highly conserved which suggests it was once part of the innate immune response. IgA function includes excluding antigen from crossing the epithelium and excreting IgA bound antigen out of the lamina propria via the polymeric immunoglobulin receptor 6, 16. Small numbers of DCs, macrophages, granulocytes, and mast cells are also scattered amongst the lymphocytes.

A unique subset of CD11b+ F4/80+ CD11c- lamina propria macrophages has recently been described. This macrophage population constitutively produces higher levels of the immunoregulatory cytokines IL-10 and TGF-β, as well as, higher levels of retinoic-acid converting enzymes than splenic macrophages. These lamina propria macrophages are also hyporesponsive to toll-like receptor stimulation 7. Interestingly, the IL-10 and TGF- β produced by these lamina propria macrophages seems to favor generation of FoxP3+ T regulatory cells 34. This subset of cells adds further support to the significant anti- inflammatory nature of GALT necessary to prevent excessive inflammation and tissue trauma following antigenic stimulation.

MESENTERIC LYMPH NODES

Although not officially considered part of the GALT, mesenteric lymph nodes (MLNs) are a critical site of leukocyte trafficking from GALT inductive to

GALT effector sites. Not only are MLNs a checkpoint for lymphocytes, they are also important for complete and specific IgA antibody responses. Mice lacking

MLNs lack robust IgA responses in the GALT effector sites 21. It is estimated that the percentage of B cells with secretory IgA is approximately 2% in human

Peyer’s patches, increases to 50% in mesenteric lymph node, and fully expands to 90% in the lamina propria 11. The development of MLNs differs from peripheral lymph node development. Peripheral lymph node ontogeny depends on the presence of tumor necrosis factor (TNF), TNF receptor, LTα1β2, LTβ receptor, whereas MLN development is not dependent upon these cytokines 11.

Lymphocytes in MLN require expression of both L-selectin and α4β7 integrin, peripheral and mucosal tissue homing molecules, respectively. Lymphocytes that were primed to antigen in the GALT inductive sites lose L-selectin expression and up regulate α4β7 integrins. In contrast, to lymphocytes primed in peripheral lymph nodes which acquire the α4β1 integrin preventing migration of these cells to gut mucosa. Over time this homing mechanism may be impaired as aging has been associated with decreased IgA antibody titers in the intestinal lumen of older rats due to impaired migrations of immunoblasts from the Peyer’s patches to the lamina propria 37.

COMPARATIVE ORGANIZATION OF GALT IN HUMANS AND OTHER

ANIMALS

An in depth understanding of mucosal immunity has great potential for improving vaccine development and immunotherapy. However, the mucosal immune system is more complex than the systemic immune system both in anatomy and cellular interplay. There is some information regarding GALT in healthy and ill individuals. However, due to obvious limitations with doing in depth longitudinal studies in humans, several animal models have been studied to elucidate cellular and chemical properties of GALT with respect to disease pathogenesis. Mucosal lymphoid tissues have been studied in many animals including mice, rats, rhesus macaques, and cats 40, 41, 49. The basic organizational structure of the mucosal tissues are similar between mammals, however key differences exist. For example, in humans, ruminants, horses, dogs, and cats,

Peyer’s patches occur primarily in the distal ileum 33. In rabbits and some rodents, on the other hand, Peyer’s patches are randomly distributed throughout the small intestine. In all species Peyer’s patches typically contain between 5 and 200 lymphoid follicle aggregates 36. In most animals, GALT inductive sites do not develop until shortly after birth when there is direct antigenic stimulation via enteral nutrition. In humans, Peyer’s patch numbers start out at around 50 in the last trimester, increase to 100 at birth, expand to ~250 in adolescence, and diminish to less than 100 in late adulthood 33. Human Peyer’s patches are made- up of 40% B-cells, 45% T-cells, and CD4:CD8 ratios of 3:1 45. In cats, B cell percentages (~40%) and T cells percentages (~48%) are quite similar to humans

14 with CD4:CD8 ratios of 1.2 to 1.9 38. Table 1.2 summarizes the lymphoid phenotypes for Peyer’s patches in humans, rabbits, and cats.

Human small intestinal epithelial lymphocytes on average consist of 1% B- cells and 85% T-cells, with CD4:CD8 Ratios of 0.2:1. The T cells in the intraepithelial compartment consist primarily of CD8+TCRαβ, followed by γδ T- cells (3-24%), and small numbers of CD4+αβ T cells 30. The proportion of T-cells in the intraepithelial compartment in rhesus macaques, cats, and rabbits is also similar to humans 40. In mice the intraepithelial compartment, on the other hand, has a predominance γδ T-cells 42. Cats have large percentages of lymphocytes in the intraepithelial compartment (>85%), however, unlike humans or mice these animals have 40% CD8+αα T-cells and CD4:CD8 ratios of 0.25 38, 41. Table 1.2 summarizes the intraepithelial lymphoid phenotypes in humans, rabbits, and cats. Some variability in T-cell populations in all species occurs depending on the segment of intestine being evaluated, i.e., small vs. large intestine.

Human lamina propria lymphocytes consist of approximately 30% B-cells,

70% T-cells, and a CD4:CD8 ratio of 2:1 42. The αβ T -cells predominate in the lamina propria of humans 31. The CD4:CD8 ratios in mice, rhesus macaques, and cats are closer to 1:1, with the exception of cows and pigs, which reportedly have

CD4:CD8 ratios of less than one 22, 38, 40, 43. All human lamina propria CD4+ T cells proliferate poorly and constitutively express glucocorticoid-induced TNFR family related protein, CTLA-4, and FoxP3 44.

A large percentage (80 to 90%) of the small intestinal plasma cells in humans produce IgA, followed by 6 to 17% IgM and approximately 4% IgG

15 production 45. Plasma cells in the LPL of Rhesus macaques also produce predominantly IgA immunoglobulin 43. In human jejunum the majority of the T- cells are CD8+ αβ T cells, with lesser numbers of γδ T-cells 47. Murine small intestinal lamina propria, like human lamina propria houses primarily CD4+ T- cells (60 to 70%), the majority of which express the conventional αβ TCR. There are also large numbers (20 to 40%) of plasma cells in the murine lamina propria

19.

Human MLN contain approximately 60% naïve T cells, 25% naïve B cells,

10% memory T and B cells combined, and 2% B-cell blasts 48. Furthermore, many of the memory B and T-cells display high expression of α4β7, and low expression of L-selectin, which is not an uncommon phenotype for mucosal effector cells. Rat MLN lymphocytes are composed of 20% B cells, 64% T cells which predominantly express TCR αβ, and CD4:CD8 ratios of approximately 3:1

49. Table 1.2 summarizes the MLN lymphoid phenotypes in humans and rabbits.

RABBITS: A COMPARATIVE ANIMAL MODEL IN INVESTIGATIONS OF MUCOSAL IMMUNITY AND INFECTIOUS DISEASE STUDIES

Recently there has been renewed interest in using the rabbit as an animal model, over some of the other more common animal models. Rabbits have been used as models to study a wide range of mucosally transmitted diseases, including human T-cell leukemia virus (HTLV-1), human immunodeficiency virus

(HIV), Herpes simplex virus 1 (HSV-1), pathogenic Neisseria gonorrhoeae infections, etc 50-53. This is due in large part to similarities that exist between rabbit mucosa-associated lymphoid tissue and human MALT 52, 54, 55. In addition,

16 rabbits represent a good compromise between using non-human primates and small rodents, such as, mice and rats. Although non-human primates are most ideal, these animals are expensive to house and are often difficult to handle.

Small rodents tend to be cheaper to house, however, given their small size it is often difficult to collect large enough sample volumes for evaluation.

Rabbits are true non-ruminant herbivores and are considered hind gut fermenters. They have a large cecum that can hold up to 40% of the intestinal contents and enables them to eat a primarily fibrous diet 56. Rabbits have an abundance of lymphoid tissue scattered throughout their small and large intestine

(Fig. 2.1). Peyer’s patches are numerous and are randomly dispersed throughout the small intestine including duodenum (personal observation). At the distal end of the cecum is the appendix, which is a long tubular structure 56. Lymphoid follicles comprise approximately 70% of the entire thickness of the intestinal wall.

These follicles are lined by a single layer of low columnar epithelium 57.

Two lymphoid structures unique to rabbits are the cecal tonsil, at the most distal end of the ileum and the ileocecal plaque at the most proximal end of the cecal lumen. The cecal tonsil is spherically-shaped and approximately 1 to 1.5 inches in diameter depending on the size and age of the rabbit (personal observation). The cecal tonsil like the appendix consists primarily of lymphoid follicles of variable thickness 57. The ileocecal plaque is a small structure, approximately 2-3 cm in diameter. Unlike the cecal tonsil and appendix, the ICP lymphoid follicles are in direct contact with the lumen. Rabbits have up to 35% M cells in the tonsil crypt epithelium. These cells are scattered throughout the crypt

17 epithelium and are most abundant at the projections of the crypt surfaces. These areas closely resemble the dome areas of Peyer’s patches 58. It appears the rabbit M cells in FAE originate from a separate pre-programmed lineage within the crypt that is different from cells that give rise to enterocytes 59. Intestinal M cells are consistently positive for vimentin (an immunohistochemical mesenchymal cell marker), and only weakly positive for the epithelial cell marker cytokeratin 60.

Rabbits, like humans have Peyer’s patches, a cecum and an appendix.

The rabbit appendix serves as a primary lymphoid structure, which serves as a site of primary antibody development 61-63. T-cells in the human appendix range from 7 to 40% with CD4+ T helper cells having a key role in germinal center formation with CD8+ T cells that secrete IL-4 to enhance B cell proliferation 62.

Although the appendix is similar in the rabbit and the human, the rabbit has relatively more lymphoid tissue than a human appendix.

In addition to similarities in GALT between humans and rabbits, these animals also have MALT comparable to humans in other locations. For example, rabbits have ocular conjunctiva-associated lymphoid tissue (O-CALT) and nasal- cavity associated lymphoid tissue (NALT). The relative number of lymphoid follicles and immunophenotypic make-up of the lymphoid populations in rabbits is similar to human nasal and conjunctiva-associated lymphoid tissues 52, 54, 55.

Normal rabbit conjunctiva has a higher frequency of CD4+CD25+ (bright), T cells relative to spleen or peripheral blood mononuclear cells (PBMCs). These conjuctival cells also express high levels of FoxP3, glucocorticoid-induced tumor

18 necrosis factor related peptide (GITR), and cytotoxic T-lymphocyte antigen 4

(CTLA-4) 52. This cell population appears to have a suppressive function critical for immune tolerance and homeostasis at mucosal surfaces. This population of cells efficiently suppressed Herpes simplex virus specific CD4+ and CD8+ effector cells 52. Furthermore, there was a selective increase in the frequency and selective capacity of Foxp3+CD4+CD25+ (bright) regulatory T cells in conjunctiva, but not in spleen or peripheral blood mononuclear cells 52.

Rabbits can be consistently infected with HTLV-1 both intravenously and orally 64-69. Rabbits were first infected in the in the mid 1980’s via intravenous inoculation with a human T cell leukemia cell line from a patient with ATL 70. A recent study from our laboratory demonstrated that as early as two weeks following intravenous infection with HTLV-1 transfected cells rabbits develop viral reservoirs in spleen, mesenteric lymph, and intraepithelial compartments 69. In this study, infected rabbits developed lymphocytosis one week after intravenous inoculation and accumulated infected leukocytes in the MLN, spleen, and IELs of the small intestine 69. Data from this thesis supports further development of the rabbit model of HTLV-1 infection following oral transmission.

CONCLUSIONS

It is clear that MALT plays a large role in immune surveillance, immunoregulation, and is a primary interface for infectious agents that enter the body via epithelial surfaces supported by MALT components. The rabbit has served as an excellent model for studying human disease, because of the

19 similarities that exist between the two species with respect to the MALT.

Unfortunately, only limited information exists regarding immunocompetent T and

B-cell areas in the GALT of rabbits. Furthermore, there is a large gap in the literature regarding normal reference ranges with regard to immunophenotypic make-up of resident mononuclear cells in the lymphoid tissue associated with the intestinal tract of rabbits. The data presented in this thesis, provide foundational information to further define rabbit GALT and support studies designed to use the rabbit to understand the pathogenesis of human infectious agents such as HTLV-

Table 1.1: List of MALT and MALT-related abbreviations

Abbreviations Complete Word/Phrase

MALT Mucosa-associated lymphoid tissue GALT Gut-associated lymphoid tissue IEL Intraepithelial leukocytes LPL Lamina propria leukocytes FAE Follicle-associated epithelium SED Sub-epithelial dome PP Peyer’s patch MLN Mesenteric lymph node ICP Ileocecal plaque IgA Immunoglobulin alpha TLR Toll-like receptor APC Antigen-presenting cell DC Dendritic cell CD Cluster designation NK Natural killer MHC Major-histocompatibility complex TGF-β Transforming growth factor beta J-Jain Joining chain CTLA-4 Cytotoxic T-lymphocyte antigen 4 FoxP3 Forkhead helix transcription factor

Th1 T-helper 1

Th2 T-helper 2 MIP-3α Macrophage inflammatory protein-three alpha AID Activation induced cytidine deaminase MEC Mucosa-associated epithelial chemokine IP-10 Interferon-inducible protein 10

Table 1.2: Summary of lymphocyte phenotypes and T cell ratios in percentages in selected lymphoid compartments across various animal species

Peyer’s Patch Human1,2 Rabbit Cat3

B cells 40 33 40

T cells 45 34 48

CD4:CD8 Ratio 3:1 7:1 2:1

Intraepithelial Lymphocytes

B cells 1 5 6

T cells 85 69 75

CD4:CD8 Ratio 0.2 0.8 0.3

Mesenteric Lymph Node

B cells 27 37 39

T cells 68 56 60

CD4:CD8 Ratio 2:1 4.5:1 2:1

1Farstad et al. Gastroenterology (1997); 112:163-173. 2Westermann et al. Clin Investig (1992); 70:539-544. 3Howard et al. J.Immunol.Methods (2005); 302: 36-53

Chapter 2

ISOLATION AND PHENOTYPIC CHARACTERIZATION OF NEW ZEALAND WHITE RABBIT GUT-ASSOCIATED LYMPHOID TISSUES AND OTHER SECONDARY LYMPHOID TISSUES

2.1 INTRODUCTION

The rabbit has been used as an animal model to study a variety of human pathogens transmitted across mucosal surfaces. These diseases include

Herpes simplex virus 1 (HSV-1) 52, human T-cell leukemia/lymphoma virus 1 51, human immunodeficiency virus (HIV) 63, pathogenic Neisseria gonorrhoeae infections 53, tuberculosis 64 and syphilis 65. In addition to serving as effective animal models for the study of a myriad of human diseases, the rabbit is advantageous compared to other animal models such as small rodents (e.g., mice and rats) and non-human primates. Rabbits are less expensive to house and easier to handle than non-human primates, but are larger than the traditionally used smaller laboratory animals offering larger sample volumes for collection e.g., blood and gut-associated lymphoid tissues (GALT). Few studies have evaluated the structure of certain mucosa-associated lymphoid tissues in the rabbit which include nasal and ocular-associated lymphoid tissues (NALT and

OALT) 52, 54-55. The relative number of lymphoid follicles and immunophenotypic characteristics of the lymphoid populations in rabbit nasal-associated lymphoid

23 tissue (NALT) and ocular-associated lymphoid tissues (OALT) are similar to those tissues in humans 52, 54. In addition to structural similarities, there are age related changes in rabbit OALT, which parallel changes described in humans 55.

Thus, rabbits provide a unique model system to study the pathogenesis of mucosally transmitted human infectious agents in these MALT tissues. Despite these advantages, limited information is available regarding the phenotypic characterization of major rabbit leukocyte populations in both MALT and other secondary lymphoid tissues.

In all mammalian species, extensive mucosal surfaces interface with the external environment and include the upper respiratory tract, conjunctiva, urogenital tract, and the gastrointestinal tract extending from the oral cavity to the rectum 4. These surfaces are typically covered by a single layer of epithelium that allows entry of vital nutrients and molecules necessary for maintenance of life, but must act as a barrier, preventing entrance of harmful pathogens 4, 8, 14, 17.

Mucosal surfaces and the associated lymphoid tissues are of particular interest in the study of the immunopathogenesis of infectious organisms that gain entry across these surfaces. Knowledge of cellular alterations during innate and adaptive immune responses at mucosal surfaces is critical to understanding immune surveillance to pathogens. The gastrointestinal tract is estimated to contain the greatest amount of MALT lymphocytes 4, 23. These aggregates of organized lymphoid tissues are collectively known as the gastrointestinal- associated lymphoid tissue (GALT).

Similar to other MALT structures, GALT it is made up of inductive sites and effector sites 5, 14. The most commonly identified inductive GALT sites are known as Peyer’s patches, but depending on the species may also include, isolated lymphoid follicles, lymphoglandular complexes in the large intestine, and specialized inductive sites, such as the appendix 6, 11. Inductive sites within the

GALT across all animal species consist of one or more follicles with interspersed

T-cell rich interfollicular areas, an overlying subepithelial dome (SED), with a single layer of columnar epithelial cells making up the follicle-associated epithelium (FAE) 4-6. Inductive sites of the GALT are sites where antigens from the gut lumen are processed and presented to naïve T and B cells 11. The effector sites of GALT are dispersed along the entire length of the gut epithelium and consist primarily of lymphocytes with a memory phenotype 24, 25. These cells are located between individual enterocytes, known as intraepithelial leukocytes, and just beneath the intestinal epithelium, in the lamina propria. The latter population of cells is referred to as lamina propria leukocytes.

Rabbits are true non-ruminant herbivores and are considered hind gut fermenters. They have a large cecum, which can hold up to 40% of the intestinal contents enabling them to eat a high fiber diet 56. Rabbits have an abundance of lymphoid tissue scattered throughout their small and large intestine (Figure 2.1).

Peyer’s patches are numerous and are randomly dispersed throughout the small intestine including duodenum (personal observation). At the distal end of the cecum is the appendix, which is a long tubular structure 56. In addition to a large appendix, rabbits have a large, round, blind-ended pouch at the proximal end of

25 the cecum called the sacculus rotundus or cecal tonsil (CT). A plaque-like structure known as the ileocecal plaque (ICP) is located adjacent to the CT at the proximal entrance of the cecal lumen. Both these structures, CT and ICP, consist of lymphoid follicles and are unique to rabbits 57.

Rabbits serve as valuable animal models for the study of pathogens that enter the body via the GALT 51, 63, 65. Thus, it is important to completely characterize phenotypes of major leukocyte populations in rabbit GALT compartments. Unfortunately, there are limited reports of phenotypic characterization of rabbit GALT tissues. Most scientific literature has focused on rabbit lymphoid microanatomy, M cell structure and function, and development of the primary antibody repertoire 59-62. Limited reports have evaluated the T cell and B cell surface markers in rabbit mesenteric lymph nodes (MLN), spleen, and peripheral blood 67-69, 80. No studies to date have evaluated the phenotypic make-up of the leukocytes in the GALT of rabbits.

The objective of the study presented herein was to comprehensively evaluate the immunophenotypic make-up of major leukocyte populations in rabbit

GALT inductive and effector sites, and other secondary lymphoid tissues. A second objective was to develop improved methods for isolation of intraepithelial and lamina propria leukocytes in the rabbit small intestine. Although this technique has been described previously for other animal species, it has not been described for rabbits 38, 46, 49. Our data indicate that New Zealand White rabbits compared to humans and mice contain a predominant CD4+ T cell population throughout their GALT and associated lymphoid tissues.

2. MATERIALS AND METHODS

2.1 Animals Ten, specific pathogen free (SPF), 12-week old New Zealand white rabbits (Harlan, Indianapolis, IN) were used for the study. All rabbits were maintained and treated according to approved animal care and use protocols in accordance with the guidelines established by the Ohio State University

Institutional Animal Care and Use Committee. All rabbits were pre-medicated with intramuscular injections of 10mg/ml acepromazine (Vedco,Inc. St. Joseph,

MO), 100mg/ml ketamine (Ft. Dodge Animal Health, Ft. Dodge, IA) and 10mg/ml butorphanol (Vedco Inc., St. Joseph, MO) prior to injection of sodium thiopental

(Abbott Laboratories, Chicago, IL) given via the auricular vein to accomplish complete anesthesia. Median thoracotomy was performed and the pericardial sac was exposed and excised. Perfusion with 500ml of heparanized saline was performed in the left ventricle via an 18 gauge needle. Following saline perfusion the ventral midline incision was extended to the pubis and internal abdominal organs were exposed for sample collection.

2.2 Sample Collection and Processing Entire mesenteric lymph nodes (MLNs) and Peyer’s patches, and partial sections of spleen, CT, appendix, and ICP were collected. Approximately 15 cm of duodenum beginning just distal to the pylorus was collected, as well. All

Peyer’s patches and grossly visible lymphoid follicles were excised from the small intestine prior to intraepithelial leukocyte (IEL) and lamina propria leukocyte

(LPL) processing. All tissue samples were placed in 20 ml of lymphocyte media consisting of 1 x RPMI medium (Invitrogen, Auckland, NZ), supplemented with

5% fetal bovine serum (Sigma, St. Louis, MO), 1% Penicillin/Streptomycin

(Invitrogen, Auckland, NZ), and 1% Na-pyruvate (Sigma)., 10% neutral buffered formalin, or were snap frozen and placed in OCT media for flow cytometric, histologic, and immunohistochemical evaluation, respectively.

2.3 Tissue Processing Tissue samples from MLNs, Peyer’s patches (1-3 per animal), cecal tonsil, appendix, ileocecal plaque, and spleen were mechanically dissociated using sterile forceps and scissors. Dissociated tissue and media mixes were then passed through 40 micron mesh sieves to obtain cell suspensions. Samples were centrifuged at 1400 RPM for 5 minutes at 37°C. Cell pellets were resuspended and filtered through another 40 micron nylon basket style sieve (BD

Falcon, Franklin Lakes, NJ) and washed twice with 1x PBS to obtain highly purified single cell suspensions. A red blood cell lysis step using a hemolytic solution containing NH4Cl, KHCO3, EDTA, and HCl was added to the cell pellet for 5 minutes to obtain a pure sample of splenic leukocytes free of RBCs.

Following the lysis step, cells were washed twice to remove RBC debris.

Remaining pellets were re-suspended in 5 ml of lymphocyte media and counted with a hemacytometer.

2.4 Peripheral Blood Leukocyte Isolation

Blood samples (10 to15ml) were collected from the lateral auricular artery following sedation with acepromazine. Whole blood was placed into 10 ml

EDTA tubes (BD, Franklin Lakes, NJ) and stored at room temperature for less than 60 minutes prior to further processing. Blood samples were centrifuged at

1400 RPM for 10 minutes followed by collection of leukocytes (Buffy coat), which were then suspended in 5ml of lymphocyte media. Red blood cells (RBCs) were lysed using a hemolytic solution containing NH4Cl, KHCO3, EDTA, and HCl and cells were then washed twice to remove RBC debris (same protocol as for spleen). The final cell pellet was then resuspended in 5ml of lymphocyte media and cells were counted using a hemacytometer.

2.5 Intraepithelial Leukocyte Isolation To isolate intraepithelial leukocytes (IELs) a protocol was adapted based on a method to isolate IELs from cats 38, 41. Approximately 15cm of duodenum were collected starting just distal to the pyloric antrum. All grossly visible lymphoid follicles were excised prior to processing. Intestinal sections were flushed twice with lymphocyte media. Intestinal sections were then opened longitudinally and cut into 0.5 to 1 cm strips. The strips were then placed into a

250 ml Erlenmeyer flask containing 40 ml of lymphocyte media. Sections were stirred at 900-1200 RPM for 45 minutes at 37 °C on a hot plate. Following stirring, the remaining tissues were strained from the supernatant using a 40 micron sieve. The supernatant was centrifuged at 1200 RPM for 10 minutes at room temperature. Following centrifugation, the supernatant was carefully discarded and the pellet was resuspended in 40 ml of lymphocyte media,

29 strained through a 40 micron mesh sieve and centrifuged a second time.

Following the second centrifugation step, the supernatant was discarded and the pellet was resuspended in 5 ml of lymphocyte media and cells were counted.

Figure 2.2 demonstrates the histological appearance of duodenal sections throughout the various stages of leukocyte collection

2.6 Lamina Propria Leukocyte Isolation The remaining intestinal tissue strips from the IEL isolation, following initial stirring steps, were used for isolation of lamina propria leukocytes (LPLs)

(Fig. 2.2). Tissue strips were re-suspended in 40 ml of lymphocyte media containing 1 ml of Dispase II-neutral protease (1.9 units/ml,Roche Diagnostics,

Indianapolis, IN). Tissues were then stirred at 700 to 800 RPM for 45 minutes at

37° C. Following the first digestion step, the supernatant was collected by filtering through 40 micron mesh sieve. Remaining tissue strips were re-suspended in 40 ml of lymphocyte media containing 1 ml Dispase II-neutral protease (1.9 units/ml) and stirred at 700 RPM for 30 minutes at 37° C. It is this second supernatant sample that was used for antibody staining (Fig. 2.2)

2.7 Cytospin preparation and cell counting An aliquot of 1 x 103 freshly isolated cells from each of the nine tissues evaluated was placed in lymphocyte media and spun in a cytospin cassette using a Cytospin 3 machine (Shandon Inc., Pittsburg, PA) at 10,000 RPM for 5 minutes at room temperature. Samples were subsequently air dried and stained with

Wright Giemsa (Astral Diagnostics, West Depord, New Jersey, USA). A manual

30 differential count of 100 cells was performed using a Nikon Eclipse 50i Y-THR-L light microscope (Nikon, Japan) at the 100x objective for every tissue site sampled for six of the ten rabbits used in this study. Cells were classified as small lymphocytes, medium/large lymphocytes, macrophages/monocytes, heterophils, eosinophils, basophils, or plasma cells.

2.8 Antibodies Commercially available rabbit specific monoclonal antibodies (mAb) were used for cell surface staining for flow cytometric and immunohistochemical evaluation. The following anti-rabbit mAbs were used as primary immunoreagents for antibody staining: anti-CD5 (Pan T cell), anti-CD4 (helper T cell costimulatory molecule), anti-CD8 (cytotoxic T cell costimulatory molecule), anti-IgM (Present on naïve B cells), anti-CD45 (common leukocyte antigen), and

CD11b (cellular adhesion molecule, expressed by monocytes/macrophages and heterophils) 81. Monoclonal antibodies were either directly conjugated to fluorescein isothiocyanate (FITC), phycoerythrin (PE), spectral red (R-Pe), or were detected using directly labeled secondary antibodies. Secondary antibody

F(ab’)2 fragment goat anti-mouse IgG PE and FITC was used to detect CD45 and anti-IgM (B cell marker). Isotype control mouse IgG1 PE/FITC, IgG2α PE, and IgG2β were used to test for non-specific binding of antibodies. Antibodies, dilutions, and manufacturer information are summarized in table 2.1.

2.9 Flow Cytometric Staining

Cell surface phenotypic characterization of blood and tissue-derived

leukocytes was accomplished using one and two color flow cytometry. Briefly, 1x

105 cells suspended in 100 µl of lymphocyte media were incubated in 96 well

plates with one unconjugated/purified mAb for 30 minutes at room temperature,

gently agitated, and protected from light. Cells were subsequently washed twice

with room temperature lymphocyte media and centrifuged at 1000 RPM for 5

minutes at room temperature. Following the first staining procedure, cells were

then incubated with a fluorochrome conjugated secondary antibody, as well as, a

directly conjugated antibody if two color flow cytometric analysis was to be

performed. Cells were subsequently washed twice with room temperature

lymphocyte media and centrifuged at 1000 RPM for 5 minutes at room

temperature. Samples were fixed using 2% paraformaldehyde and analyzed

(FACS Calibur, Immunocytometry Systems, BD). Gating was based on

expression of the common leukocyte antigen (CD45). Ten thousand events were

collected from the gated region. Representative gating schemes and

immunofluorescence staining patterns are illustrated in Figures 2.3 and 2.4,

respectively.

3. RESULTS

3.1 Cell Sample Purity Sample purity for all nine tissues evaluated was established using the CD45,

common leukocyte antigen. Only the CD45+ cells were gated on and analyzed

via flow cytometry. Mean purity ranged from 99%+/- 1-2% in the mesenteric

32 lymph node, cecal tonsil, appendix, Peyer’s patch, ileocecal plaque, and peripheral blood leukocytes, to 97%+/- 3 in the spleen and 95%+/- 1 and 91%+/-

1 for IELS and LPLs, respectively. These data confirmed that our methods were effective in isolation of pure populations of intestinal leukocytes. As a further assessment of sample purity, cytospin preparations of each tissue were made for

6 of the 10 rabbits included in the study (Fig. 2.5). Mean percentages for peripheral blood leukocytes consisted of 72% small lymphocytes, 19% heterophils, 5% monocytes, 2% basophils, and 2% medium/large lymphocytes.

Peyer’s patches, appendix, CT, ICP, and MLN consisted of 87-89% small lymphocytes, 8-10% medium/large lymphocytes and scattered macrophages and heterophils. Mean percentages for splenic samples consisted of 68% small lymphocytes, 16% macrophages, 13% medium/large lymphocytes, and 3% heterophils. Intraepithelial lymphocytes consisted primarily of small lymphocytes

(83%), with scattered heterophils (6%), macrophages (6%), plasma cells (3%), and medium/large lymphocytes (2%). Lamina propria leukocytes consisted of

55% small lymphocytes, scattered heterophils (5%), macrophages (5%), medium/large lymphocytes (2%), and a large percentage of plasma cells (30%)

(Table 2.2).

3.2 Peripheral Blood Leukocytes

Cell yield averaged 1.1 x 10 3 leukocytes/µl. Consistent with other reports 66-67, our rabbits had a predominance of lymphocytes in their peripheral blood with percentages ranging from 62 to 82%. Granulocytes comprised

33 approximately 10 to 29% of blood leukocytes and were predominantly heterophils, with lesser numbers of basophils and rarely eosinophils, and less than 5% of the leukocytes were monocytes based on blood smear and cytospin evaluation. Among circulating lymphocytes the mean of T cells was 45% +/-7 and the mean of B cells was 44% +/- 13. The proportion of B to T cells was approximately 1:1. The mean CD4:CD8 T cell ratio was approximately 2:1. The average percentage of lymphocytes gated for flow cytometry generally correlated with the average percentage of lymphocytes seen on the leukocyte differential counts performed manually.

3.3 Mesenteric Lymph Node

Two to three MLNs, approximately 2 cm X 1.5 cm in diameter were collected from each rabbit. Cell yield averaged 4 x 10 7 leukocytes per gram of tissue. Lymph node samples contained increased numbers of T cells, mean 56%

+/- 9 relative to B cells, mean 37% +/- 8. Mean B to T cell ratios were 0.7:1.

Mesenteric lymph nodes contained approximately 4 fold more CD4+ T cells versus CD8+ T cells with mean CD4:CD8 ratios of 4.5. Mean CD11b expression was 14% +/- 9. Immunohistochemical (IHC) staining patterns for anti-T cell, anti-

B cell, anti-CD4, and anti-CD8 antibody markers performed in a parallel study supported our flow cytometric analysis and had similar percentages of these leukocyte markers. Lymphocytes in tissue sections are substantially more positive for CD4, than CD8 in the interfollicular T cell rich areas of the lymph node. B cells are largely found in the follicles of the nodal cortex, subjectively

34 correlated well with the B to T cell ratios from our flow cytometric analysis (1.3:1).

Figure 2.6 illustrates typical immunohistochemical staining patterns for MLN.

3.4 Spleen

Standardized spleen samples (2 cm x 1 cm) from each rabbit were processed for leukocyte analysis. Cell yields averaged 2 x 108 leukocytes per gram of tissue.

In contrast to the other secondary lymphoid tissues evaluated, splenic leukocytes consisted of a more heterogeneous population of cells. Cytospin samples revealed a large percentage of large mononuclear cells, approximately 16%, which likely represented macrophages. Among lymphocyte populations, B cells predominated in splenic samples, mean 49% +/- 10, which were reflected in an elevated B to T cell ratio of 1.3:1. Among T cells, mean 37% +/- 10, CD4+ T cell predominated over CD8+ T cells, with an average CD4:CD8 ratio of 1.6:1.

3.5 GALT Inductive Sites

Cell yield averaged 7 x 107, 3 x 108, 1 x 108, 9 x 107 leukocytes per gram of tissue for Peyer’s patches, ileocecal plaque, cecal tonsil and appendix, respectively. T cells examined from these sites exhibited a predominance of

CD4+ T cells relative to CD8+ T cells in these compartments. CD4:CD8 ratios in

GALT inductive sites ranged from 9.0 in the ileocecal plaque, 8.0 in the cecal tonsil, 7.0 in Peyer’s patches, and 3.5 in the appendix (Table 2.3). Interestingly, these ratios were significantly higher than ratios reported in cats, rhesus macaques, rats, and humans. 38,40,46-47,49. Mean T cell percentages were 33% +/-

4 in Peyer’s patch, 27% +/- 6 in cecal tonsil, 32% +/- 4 in the ilecoecal plaque, and 13% +/- 2 in the appendix. Mean B cell percentages were 34% +/- 4 in

Peyer’s patch, 36% +/- 6 in cecal tonsil, 25% +/- 7 in the ilecoecal plaque, and

50% +/- 9 in the appendix. Immunohistochemical evaluation of these same tissues supported our flow cytometric findings, demonstrating large areas of

CD4+ T cells in inductive sites and only small, scattered areas of CD8+ cells (Fig.

2.7). Similarly immunohistochemical staining for anti- B cell and the pan T-cell marker CD5 paralleled our B to T cell ratios obtained using flow cytometry. B to

T cell ratios across the four inductive tissue compartments were variable and shifted from primarily B cells in the cecal tonsil and appendix, to equal ratios in

Peyer’s patches, and a predominance of T cells in the ileocecal plaque (Table

2.4) . All four compartments evaluated had relatively small numbers of CD11b expression, ranging from 4% to 7%. This was supported by cytospin examinations performed on these tissues, which yielded a minor population of large mononuclear cells (<5%).

3.6 GALT Effector Sites: Intraepithelial and Lamina Propria Leukocytes

To examine leukocytes associated with GALT effector sites, we adapted a method to isolate these unique subsets of cells from intestinal tissues.

Histologic evaluation of small intestinal sections following isolation of IELs and

LPLs confirmed preferential digestion of intestinal layers containing these cell populations (Figure 2.2). Intraepithelial leukocyte (IEL) cell yield averaged 4.7 x

106 leukocytes per gram of duodenum. The IELs were predominantly T cells

(mean of 69% +/- 12) and had a greater percentage of mean CD8+ T cells (37%

+/- 7), compared to CD4+ T cells (mean 31% +/- 6), resulting in a mean CD4 to

CD8 ratio of 0.8:1. Total percentages of B cells in the IEL compartments was low

(mean 5% +/- 4.). IEL leukocytes contained a mean of 3% +/- 2 CD11b expressing cells, which correlated with the cytopsin analysis for large mononuclear cells/macrophages (mean of 5-6%).

Lamina propria leukocyte cell yield averaged 1.4 x 10 6 leukocytes per gram of duodenum. Leukocytes isolated following the second digestion step were used and leukocytes isolated following the first digestion step were discarded since these cells had intraepithelial leukocyte contaminants. The mean CD4 expression in the lamina propria compartment was 27%+/- 12 and mean CD8 expression was 17% +/- 6. As a result, the LPL compartment had on average a higher CD4 to CD8 ratio (1.6:1) compared to the IEL compartment.

Approximately 1% +/- 1 of the lamina propria leukocytes expressed the B cell surface marker. Cytospin analysis consistently demonstrated approximately 32% plasma cells in the LPL compartment. Thus, with plasma cells included in total B cell percentages the average is B to T cell ratio in the LPL was significantly larger than the IEL compartment. The mean number of T cells in the lamina propria compartment was 56% +/- 11, while CD11b+ cells comprised a mean of 6% +/- 6.

3.7 Statistical Analysis

Figure 2.6 shows bar graphs summarizing means and standard deviations of six of the monoclonal antibodies evaluated for each of the nine

37 tissues evaluated. Calculations were performed using Microsoft Excel software

2003 and are based on evaluation of 6 New Zealand White rabbits.

4. Discussion

To fully utilize the rabbit as a predictive animal model to study human diseases an understanding of the rabbit mucosal immune system is required.

Our data presented herein, defines key leukocyte populations in specific gastrointestinal lymphoid tissues, as well as, spleen, mesenteric lymph node, and peripheral blood. We combined flow cytometric analyses of specific leukocyte subsets and compared these data to parallel immunohistochemical studies of these same tissues. We also developed methods for isolation of intraepithelial and lamina propria leukocytes in the rabbit small intestine and found unique differences in leukocyte populations in these effector sites. Our data indicate that

New Zealand White rabbits compared to humans and mice contain a predominant CD4+ T cell population throughout their GALT and associated lymphoid tissues.

We utilized the rabbit specific monoclonal anti-CD45 antibody to test the purity of our samples. This parameter was particularly important in our methods to distinguish intestinal epithelial cells from intestinal leukocytes, since intestinal epithelial cells do not express CD45 77. This method has been previously established to be a reliable way to assess leukocyte purity from mixed cell populations which include leukocytes 78. Overall our data indicated that our

38 methods achieved mean purity ranges from 95 to 99%, with the exception of lamina propria leukocytes (mean 91%) and verified that our methods were effective in isolation of pure populations of intestinal leukocytes.

For the non-gut associated lymphoid tissues, mesenteric lymph node and peripheral blood, our data indicated that the percentages of B cells, T cells, and CD11b+ cells typically added up to 100%. This implied that the markers used in the study effectively captured the majority of leukocyte populations present in these tissues. In other words, the mean percentages of lymphocytes and large mononuclear cells (macrophages and dendritic cells) added up to 100%. Spleen was unique in that lymphocyte markers and CD11b expression was in excess of

100%. A possible explanation for this is that CD11b expression is not limited to macrophages and a subset of dendritic cells, but is also upregulated on heterophils 8. The data obtained in this study was comparable to data obtained in other rabbit studies evaluating phenotypic characteristics of lymphocytes in mesenteric lymph node, spleen, and peripheral blood compartments 67-69, 80.

Phenotypic evaluation of GALT inductive and effector sites was not as straight forward as evaluation of peripheral blood leukocytes, mesenteric lymph node and spleen. The percentages of B cells, T cells, and CD11b expressing cells in these compartments never added up to 100% and comprised 63-77% of the total populations. However, cytologic analysis of these tissues demonstrated a predominance of small lymphocytes, which suggests that the population of cells not being identified likely represented other subsets of lymphocytes. This is not surprising, since it is well known there are increased numbers of atypical T

39 cells present in the gut in many species, including mice and human 29-31. Both these populations were morphologically small lymphocytes and most likely represent T cells with an atypical phenotype. Most T cells in systemic secondary lymphoid compartments consist of TCRαβ+CD8αβ+ or TCRαβ+CD4αβ+ cells 42. In the gastrointestinal tract, on the other hand, there are increased numbers of

TCRαβ+CD8αα+, TCRγδ+CD8αα+, and TCRγδ+CD4-CD8- expressing cells 29-31.

The proportion of these cells is variable among species. For example, cattle have a large percentage of TCRγδ+CD4-CD8- cells in all secondary lymphoid tissues, whereas, humans and mice have smaller percentages of these cells 1. Another possibility is that these lymphoid cells represent a population of natural killer cells. These may represent the more commonly described CD56+CD16+ and variably positive CD8+ cells or a population of invariant NK T cells 78. The only caveat is that natural killer cells often contain intracytoplasmic granules and are also known as large granular lymphocytes 1, 2. We rarely identified large granular lymphocytes in our cytospin samples. A third possibility is that the percentage of

B cells is actually higher than what is reported here. Since the IgM marker used in this study is most commonly expressed on naïve B cells it is possible that we under estimated B cell percentages 11.

We tested a variety of antibodies to identify B cell populations in the various tissue compartments evaluated. The B cell markers tested consisted of both rabbit specific and human specific monoclonal antibodies. Ideally, a more widely used B cell specific surface antibody would have been used, such as,

CD79α (B cell antigen receptor, present on B-cell lineage and plasma cells),

CD20 (important for B-cell activation and present on activated B cells), or CD21

(important in complement activation and present on mature resting B cells) 81.

Unfortunately, these failed to bind to surface antigens on rabbit leukocytes (data not shown). Only the anti-rabbit IgM monoclonal antibody was found to consistently identify B cell populations in all the tissue compartments evaluated.

Studies in mice have shown that resting B cells express surface IgM 82, 83. It is only after circulating antigen binds the BCR in the presence of helper T cells and various cytokines (i.e. IL-2, IL-4, etc.) that expression of other B cells surface markers is upregulated. This upregulation of surface markers signifies the beginning of B cell affinity maturation and division and isotype switching. These markers include MHC class II, IL-2, IL-4, IL-5, IL-6, TNF-α, and TGF-β receptors

82-84. Once the process of isotype switching is underway, B cells generally lose

IgM expression and upregulate expression of IgA, IgG, IgE, etc., depending on the response needed to neutralize the offending antigen 82-85. This being said it is quite plausible that a population of of B cells undergoing isotype switching were not identified. However, comparative evaluation of flow cytometric data with immunohistochemical evaluation of B cell populations was subjectively similar.

With respect to GALT tissues it is widely recognized that effector sites house a predominance of IgA secreting plasma cells 7, 11, 17. Although a terminally differentiated B cell or plasma cell marker would have been ideal, it was not available. This problem was easily circumvented by cytologic evaluation of these tissues as plasma cells have a unique and easily recognizable cytologic appearance. Our data indicated that a monoclonal anti-rabbit IgM did consistently

41 bind a minority subset (~1%) of B cells from GALT. Although this provided a good starting point for evaluating B cell populations in the rabbit, there are likely other

B cell subsets or differentiated B cells that were not identified, as discussed previously. Cytologic examination of lamina propria leukocytes revealed a large number of plasma cells in parallel samples. This illustrates that plasma cells were not identified by the B cell marker used in this study, but were a prominent population of leukocytes in this compartment.

A second marker that was of interest in this study, specifically for identification of regulatory T-cells in conjuction with CD4 expression, was the lymphocyte activation marker CD25 (IL-2 receptor alpha chain) 93, 94. Several anti-human CD25 monoclonal antibodies were used with no success. Only one anti-rabbit CD25 antibody clone was available for evaluation. After multiple attempts, this antibody non-specifically bound to leukocytes leading spurious fluorescence patterns. Possible explanations for this non-specific binding include damage to epitopes during processing, steric hindrance caused by use of large fluorochromes, or alternate isoform expression within mucosal sites.

Previous studies of disease pathogenesis in the rabbit have looked at lymphocyte and other leukocyte responses in lymphoid compartments following inoculation with certain pathogens 51-53, 63. Most recently, spatial and temporal events were evaluated following intravenous inoculation of 12 week old New

Zealand White rabbits with an HTLV-1 infected rabbit CD4+ T cell line (ACH- transformed R49 cells) 75. Significant findings included a transient lymphocytosis that correlated with peak virus load one week following infection and homing of

HTLV-1 infected cells to splenic and mesenteric lymph node reservoirs, in addition to the intraepithelial compartment of the GALT 2-4 weeks following intravenous inoculation. This data in conjunction with data regarding normal leukocyte populations in lymphoid tissues of uninfected rabbit can lead the way for evaluation of significant shifts in these populations. A better understanding of the immune response prior to and shortly after virus inoculation may be elucidated. Therapeutic and prophylactic measures can be tested and optimized once a thorough understanding of the immunopathogenesis of the HTLV-1 virus and other infectious agents is more completely understood.

In summary, our data indicate that New Zealand White rabbits compared to humans and mice contain a predominant CD4+ T cell population throughout their GALT and associated lymphoid tissues. We also established methods to isolate and identify IEL and LPL populations from gut effector sites in rabbits.

Collectively, our findings provide new focus on this important laboratory animal species for studies seeking information on the immunopathogenesis of orally transmitted human pathogens, such as HTLV-1 infection.

TABLE 2.1: Antibodies used in flow cytometric and immunohistochemical evaluation of rabbit intestinal leukocyte

Antigen Conjugation Clone Specificity Source Dilution CD4 FITC KEN-4 Mouse Anti-rabbit IgG2a Serotec 1:100

CD4 FITC KEN-4 Mouse Anti-rabbit IgG2a Antigenix 1:100

CD8 FITC 12.C7 Mouse Anti-Rabbit IgG1 Serotec

CD8 FITC C7 Mouse Anti-Rabbit IgG1 Antigenix 1:50 Tcell (CD5) FITC KEN-5 Mouse Anti-Rabbit IgG1 Serotec 1:100 T cell (CD5) Purified KEN-5 Mouse Anti-Rabbit Antigenix 1:100 CD25 Unconjugated Kei-Alpha 1 Mouse anti-rabbit IgG2b Antigenix CD25 Unconjugated Kei-Alpha Mouse anti-rabbit Serotec 1 CD45 Unconjugated L12 Mouse anti-rabbit IgG1 Antigenix 1:100

CD21 PE B-Iy4 Mouse anti-human IgG1 BD Biosciences CD79α RPE HM57 Mouse anti-human IgG1 Dako CD79α FITC HM57 Mouse anti-human IgG1 Serotec CD19 SPRD SJ25-C1 Mouse anti-human IgG1 Southern Biotechnolog y Bcell (IgM) Unconjugated NRBM Mouse anti-rabbit IgG1 Antigenix 1:25

CD18 PE 6.7 Mouse anti-human IgG1 BD Biosciences CD18 RPE 68-5A5 Mouse anti-human IgG2a Southern Biotechnolog y CD14 FITC K4 Mouse anti-rabbit IgG2a Antigenix 1:20 CD11a RPE 38 Mouse anti-human IgG2a Souther Biotechnolog y CD11b Unconjugated 198 Mouse anti-rabbit IgG1 Antigenix 1:100 CD56 FITC MEM-188 Mouse anti-human IgG2a Serotec CD16 FITC LNK16 Mouse anti-human IgG1 Seroec Anti-mouse FITC/PE Goat anti-mouse IgG Antigenix 1:100 IgG (Heavy & Light chain specific)

TABL 2.2: Cytospin analysis from each anatomic location evaluated (%)

Tissue Small Medium/Larg Macrophages/ Plasma Heterop Basophils Lymphocytes e Monocytes cells hils Lymphocytes Periph 72 2 5 0 1 2 eral 9 Blood MLN 89 8 3 0 0 0 Spleen 68 13 16 3 Peyer’ 87 9 4 0 0 0 s Patch Cecal 88 9 3 0 0 0 tonsil Ileocec 88 10 2 0 0 0 al Plaque Appen 88 9 3 0 0 0 dix IEL 83 2 6 3 6 0 LPL 56 2 5 3 5 0 2

One hundred cell differential counts of cytospin preparations are summarized for 6 rabbits in the study. Results shown here represent mean percentages.

Table 2.3: Distribution of CD4+ AND CD8+ T cells in MALT and systemic lymphoid tissues Percentage of Positive Cells (+/- SD)

Tissues CD4 CD8 Ratio of CD4/CD8 Mucosa-associated Lymphoid tissues Ileocecal plaque 28+/-5 3+/-1 9.0 Cecal tonsil 24+/-3 3+/- 8.0 Peyer’s Patch 29+/-4 4+/-2 7.0 Appendix 11+/-2 3+/-1 3.5 Lamina Propria Leukocytes 27+/-12 17+/-6 1.6 Intraepithelial Leukocytes 31+/-6 37+/-7 0.8 Systemic Tissues Mesenteric Lymph Node 50+/-6 11+/-3 4.5 Peripheral Blood 29+/-7 15+/-4 2.0 Spleen 28+/-7 17+/-9 1.6

Table 2.4: Distribution of B and T cell subsets in MALT and systemic lymphoid tissues

Percentage of Positive Cells (+/- SD)

Tissues B cell T cell Ratio of B to T cells Mucosa-associated Lymphoid tissues Appendix 50+/- 9 13+/- 2 4.0 Cecal tonsil 36+/- 6 27+/- 3 1.3 Peyer’s Patch 34+/- 4 33+/- 4 1.0 Ileocecal plaque 25+/- 7 32+/- 4 0.8 Intraepithelial Leukocytes 5+/- 4 69+/- 12 0.1 Lamina Propria Leukocytes 1+/- 1 56+/- 11 0.01 Systemic Tissues Spleen 49+/- 10 37+/- 10 1.3 Peripheral Blood 44+/- 13 45+/- 7 1.0 Mesenteric Lymph Node 37+/- 8 56+/- 9 0.7

Figure 2.1: Illustration of rabbit gastrointestinal tract drawn to scale highlighting grossly visible lymphoid follicles. There are approximately 4-6 round to oval-shaped Peyer’s patches in the small intestine starting at the duodenum and ending at the ileum. There is a 1cm x 1cm round, plaque like structure at the proximal end of the cecum which is direct contact with the cecal lumen known as the ileocecal plaque (ICP). Adjacent to the ICP is the sacculus rotundus, also known as, cecal tonsil. This is a 4cm X 4cm round pouch. At the terminal end of the cecum is a long, approximately 10 cm in length tubular structure known as the appendix vermiformis. Mesenteric lymph nodes can be found at the root of the mesentery and drain the lymphoid follicles previously described. Representative H&E sections for all five lymphoid structures are also shown at 4x magnification.

A (4X) B (4X)

50x 50x

C (4X) D (4X)

50x 50x

Figure 2.2: Isolation of intraepithelial and lamina propria leukocytes from the proximal small intestine, H&E tissue secions, 4x and 50x magnification (A) Normal rabbit duodenum prior to processing. (B) Image demonstrating microscopic appearance of small intestine following first centrifugation step which removes surface epithelium and releases intraepithelial leukocytes. (C) Microscopic appearance of duodenum following first digestion step. (D) Microscopic appearance of duodenum following second digestion step and release of lamina propria leukocytes. Results are representative of multiple tissue sections.

MLN Spleen PBMC

Peyer’s Patch Cecal Tonsil Appendix

Ileocecal Plaque IEL LPL

Figure 2.5: 100x, Wright Giemsa Stain. Cytospin preparations from the 9 tissue compartments evaluated. Images reflect typical leukocyte populations isolated following tissue processing steps. There is more heterogeneity of leukocytes in the IEL, LPL, and spleen in comparison to Peyer’s patches, ileocecal plaque, appendix, cecal tonsil, mesenteric lymph node, and peripheral blood.

Peripheral blood Spleen Mesenteric lymph node

Appendix Cecal tonsil Ileocecal plaque

Peyer’s patch Intraepithelial Leukocytes Lamina propria leukocytes Side Scatter Side

Forward Scatter

Figure 2.4: Representative scatter plots for the nine tissues evaluated. Gated regions represent CD45+ cells with the exception of peripheral blood which illustrates gating on lymphocytes only. Results are representative of multiple cell suspensions for each of the specified tissues.

18% 17% 51% 15% 32% 8%

PBMC MLN Spleen

25% 27% 6% 13% 2%

4% T Cell PE T

Cecal Tonsil Peyer’s Patch Appendix

31% 3% 35% 40% 37% 13%

Ileocecal Plaque Intraepithelium Lamina Propria

CD8 FITC

Figure 2.5: Flow cytometric fluorescence patterns highlighting phenotypic analysis for the 9 tissues evaluated in the rabbit.Each plot is representative of dual staining for monoclonal antibodies anti-rabbit pan T cell conjugated to a secondary antibody PE and anti-rabbit CD8-FITC markers. Percentages in the plots are from the data presented in each plot, but are similar to mean values from tables 2.3 and 2.4. Only CD45 positive cells were gated on for phenotypic evaluation.

A B

C D

Figure 2.6: Mesenteric lymph node tissue sections illustrating immunohistochemical staining patterns with various markers, 4x. (A) CD4 IHC marker highlighting marked diffuse CD4 positivity in the T cell rich areas of the lymph node. (B) CD8 IHC marker highlighting scant multifocal CD8 positivity in the T cell rich areas. (C) Pan T cell IHC demonstrating similar staining pattern to CD4, suggesting that the majority of the T cells are CD4 positive. (D) CD79a IHC demonstrating diffuse positivity in the B cell rich areas of the lymph node. Note: IHC results paralleled flow cytometric results. Pan T cell and CD79a positive staining are subjectively similar (Flow cytometry: B:T cell ratios of 0.7:1). IHC subjectively demonstrated a marked predominance of CD4 T cells relative to CD8 T cells with also correlates well with flow cytometry results CD4:CD8 ratios of 4.5:1. IHC sections courtesy of Robyn Haines, Lairmore laboratory.

A B

C D

Figure 2.7: Cecal tonsil tissue sections illustrating immunohistochemical staining patterns with various markers, 20x. (A) CD4 IHC marker highlighting marked diffuse CD4 positivity in the T cell rich areas of lymphoid tissue (B) CD8 IHC marker highlighting scant multifocal CD8 positivity in the T cell rich areas. (C) Pan T cell IHC demonstrating similar staining pattern to CD4, suggesting that the majority of the T cells are CD4 positive. (D) CD79a IHC demonstrating diffuse positivity in the B cell rich areas of the lymph node. Note: IHC results subjectively correlate hightly with flow cytometric results. Pan T cell and CD79a positive staining are subjectively similar (Flow cytometry: B:T cell ratios of 1.3:1). IHC subjectively demonstrates a marked predominance of CD4 T cells relative to CD8 T cells with also correlates well with flow cytometry results CD4:CD8 ratios of 8:1.

Peripheral Blood Mononuclear Cells Mesenteric Lymph Node SPLEEN 100 100 100 90 90 90 80 80 80 70 70 70 60 60 60 50 50 50 40 40 40 30 30 30 20 20 20 10 10 10

0 0 Percent Positive Leukocytes Positive Percent

0 Leukocytes Positive Percent Percent Positive Leukocytes Positive Percent Series1 CD45 Tcell CD4 CD8 Bcell CD11b CD45 Tcell CD4 CD8 Bcell CD11b CD45 Tcell CD4 CD8 Bcell CD11b Series1 Series1

Peyer's Patch CECAL TONSIL Ileocecal Plaque 100 100 100 90 90 90 80 80 80 70 70 70 60 60 60 50 50 50 40 40 40 30 30 30 20 20 20 10 10

Percent Positive Leukocytes 10 Percent Positive Leukocytes Positive Percent 0 0 0 Cells Mononuclear Positive Percent Series1 CD45 Tcell CD4 CD8 Bcell CD11b CD45 Tcell CD4 CD8 Bcell CD11b Series1 CD45 Tcell CD4 CD8 Bcell CD11b

Appendix Intraepithelial Leukocytes Lamina Propria Leukocytes

100 100 100 90 90 90 80 80 80 70 70 70 60 60 60 50 50 50 40 40 40 30 30 30 20 20 20

10 10 10 Percent Positive Leukocytes Positive Percent 0 Leukocytes Positive Percent 0 Percent Positive Mononuclear Cells Mononuclear Positive Percent 0 CD45 Tcell CD4 CD8 Bcell CD11b Series1 CD45 Tcell CD4 CD8 Bcell CD11b Series1 CD45 Tcell CD4 CD8 Bcell CD11b

Figure 2.8: Bar graphs for each of the 9 tissues evaluated highlighting the six main surface antibody markers used in the study. Means and standard deviations were calculated based on results obtained from ten, twelve week old New Zealand White rabbits.

CHAPTER 3: FUTURE DIRECTIONS

FURTHER CHARACTERIZATION OF RABBIT GUT- ASSOCIATED LYMPHOID TISSUES

In this thesis, the phenotypic characterization of multiple lymphoid compartments in the rabbit GALT and other secondary lymphoid tissues were described; however, significant gaps remain in knowledge of leukocyte subsets in rabbit secondary lymphoid tissues. The rabbit is a useful animal model of human diseases, in part, due to their low cost to house compared to non-human primates and their larger size compared to mice and other small laboratory animals. Another potential advantage for the use of rabbit models is their similar mucosal histologic and functional properties compared to humans 55. Thus, rabbit models provide the opportunity to study the pathogenesis of mucosally transmitted human infectious agents.

We used CD45, a pan-leukocyte marker to assess sample purity in tissues analyzed via flow cytometry. This proved to be effective as we demonstrated a high purity in tissues examined. We demonstrate that the mesenteric lymph nodes, spleen, and peripheral blood tissue compartments contained typical populations of B-cells, T-cells, and macrophages, comparable to other animal species, including cats, humans, mice, and rhesus macaques 38,40,46,48. Our data indicate that New Zealand White rabbits compared to humans and mice contain a predominant CD4+ T cell population throughout their GALT and associated

56 lymphoid tissues. Interestingly, our data indicates that the rabbit contains significantly larger proportions of CD4+ cells relative to CD8+ T cells in the inductive sites of the gut-associated lymphoid tissues. While these data confirmed that our methods were effective in isolation of pure populations of intestinal leukocytes, as well as, phenotypic characterization of major leukocyte subsets in the rabbit GALT and associated lymphoid tissues, there are important caveats to our data.

Our data from non-gut associated lymphoid tissues indicated that the percentages of B cells, T cells, and CD11b+ cells typically added up to 100%.

This implied that the markers used in the study effectively captured the majority of leukocyte populations present in these tissues. In contrast, the percentages of

B cells, T cells, and CD11b expressing cells in GALT comprised 63-77% of the total populations. We suspect that populations of cells representing other subsets of leukocytes were not detected by our antibody panels. This is not an unexpected finding since GALT inductive and effector compartments are known for being comprised of unique subsets of T-cells in a multitude of species 29-31.

These subsets include T-cells expressing the alpha homodimer

(TCRαβ+CD8αα+) and T cells expressing the gamma/delta TCR (TCRγδ+CD8αα+ and TCRγδ+CD4-CD8- 31. Between 63 and 77% of the total leukocytes in each rabbit GALT compartment were found to express the more common lymphoid markers. However there were a significant number of lymphoid cells, roughly 23-

37% depending on the GALT compartment, which did not express the T and B cell markers used in this study. This unidentified population of cells likely

57 represents an atypical population of T cells. Another population of lymphoid cells likely present in the rabbit GI tract, but not classified in this study, includes natural killer (NK) cells. Recent studies have made significant strides in elucidating the phenotype and maturation of NK cells. Originally, NK cells were thought to be cytolytic, but not restricted to activation by major histocompatibility

(MHC) complex expression on target cells 96-98. This theory has since been abandoned since it appears the NK cells do in fact possess a wide array of inhibitory and activating surface receptors, which engage MHC class I molecules and other similar molecules. 99,100. NK cell function is broad and includes production of interferon gamma (IFN-γ), a key cytokine mediator of Th1 responses 101, induction and up regulation of MHC class 1 molecules 102 on

APCs, and potent cytotoxic potential on viral and other transformed malignant cells 103. The most widely accepted NK cell surface phenotype in humans includes CD56 (a neural adhesion molecule) and a lack of CD3 (Pan T cell marker) expression 79. In murine species, on the other hand, CD56 is not expressed by NK cells, Instead, the natural cytotoxicity receptor NKp46 is used as a marker for NK cell identification 100. Identification of this subset of cells in rabbits would be highly advantageous, specifically for evaluation of responses to virus induced malignant transformation of cells. (e.g., HTLV-1 adult T cell leukemia/lymphoma cells or “flower” cells). Although several atypical T cell markers, including mouse anti-γδ T cell, and NK markers (human anti-CD16 and human anti-CD56) were tested, none proved to cross-react with rabbit

58 leukocytes. Future studies are needed to more completely characterize these cells in the rabbit GALT.

A more robust panel of B cell markers, in addition to the rabbit anti-IgM marker used in this study, would also be ideal to completely characterize this population and should include CD79α (B cell antigen receptor, present on B-cell lineage and plasma cells), CD20 (important for B-cell activation and present on activated B cells), CD21 (important in complement activation and present on mature resting B cells), or CD19 (important for signal transduction in B cell maturation, present on B lineage cells) 81. Studies in mice have shown that resting B cells express surface IgM 82, 83. It is only after circulating antigen binds the B cell receptor in the presence of helper T cells and various cytokines (i.e. IL-

2, IL-4, etc.) that expression of other B cells surface markers is up regulated.

This expression of surface markers signifies the beginning of B-cell affinity maturation and division and isotype switching. Once the process of isotype switching is underway, B cells generally lose IgM expression and produce IgA,

IgG, IgE, etc depending on the response needed to neutralize the offending antigen 82-85. It is quite plausible that a population of B cells undergoing isotype switching were not identified in our study. Since the IgM marker used in our study likely identified only naïve B cells it is possible that we under estimated B cell populations. Thus, future studies need to be directed at development of reagents to identify mature or fully differentiated B cells in rabbits.

In addition to complete characterization of leukocyte subsets in the various secondary lymphoid compartments in the rabbit, it would also be advantageous

59 to establish the activation state, specifically of the lymphocytes. Activation markers that could be evaluated include CD80 (B7.1) and CD25 (IL-2 alpha receptor). The CD80 surface marker is expressed by APCs and activated T-cells in the periphery 6, 7. Previous studies in cats 38 have found that the activation marker CD80, which can be up regulated on activated T-cells, is expressed by more IEL and LPL T-cells (40 to 70%), than T cells in the periphery. CD25 represents the IL-2 alpha receptor which binds to the IL-2 cytokine 81.

Expression of CD25, like expression of CD80 on T-cells, is associated with an activated state 86. Expression of CD25, like expression of CD80, also appears to be increased in LPL lymphocytes. Human and Rhesus macaque LPL can approach up to 25% CD25 expression 87, 88. Several rabbit specific CD25 monoclonal antibodies were evaluated in this study. However, all antibodies tested consistently demonstrated non-specific binding. Possible explanations for lack of reactivity or non-specific reactivity between the rabbit specific CD25 marker are decreased density of surface receptor sites, damage to epitopes during processing, steric hindrance caused by use of large fluorochromes, alternate isoform expression within mucosal sites, or potential lack of mucosal expression within mucosal sites. Future studies, should focus on identification of a CD25 antibody which effectively identifies this surface marker in the rabbit.

Regulatory T-cells are key regulators of the immune response to many viral pathogens including HIV and human T-cell leukemia/lymphoma virus 1

(HTLV-1).89, 90. To date, the regulatory T-cells have been associated with a

CD4+Foxp3+ phenotype 90-91, 93. It is well-documented that there is massive CD4+

T-cell depletion acutely following HIV infection in humans 89. This is followed by an increase in T regulatory cells, which has been characterized as pre-mature, allowing virus infected cells to replicate un-checked 89, 91. This phenomenon has also been documented with HTLV-1 infection in humans, where the frequency of regulatory T-cells is inversely correlated with the rate of cytotoxic T-lymphocyte

(CTL) mediated lysis 90, 92. In other words, regulatory T cells have a potent inhibitory effect on the rate of CD8+ cytotoxic T cell mediated lysis of virus infected cells. Presumably, an overly robust regulatory T cell response would be advantageous to a virus trying to replicate in as many cells as possible.

HTLV-1, like HIV, has an intimate association with CD4+ T cells. HTLV-1 is a member of the delta retroviridae and infects 10-20 million people worldwide. A small percentage of infected individuales, ~4% develop leukemia/lymphoma or adult T cell leukemia/lymphoma (ATLL) following a latency period of 20-30 years.

72-74. For years it was thought that the phenotype of HTLV-1 transformed cells or

ATLL cells and regulatory T cells were one and the same. However, recent literature has found that ATLL cells which are characteristically CD4+CD25+ and regulatory T cells, phenotypically CD4+FoxP3+ are in fact two distinct populations

90-92. Characterization of regulatory T cells in rabbits would be highly advantageous to future studies focused on mucosally transmitted retroviruses, such as, HTLV-1, since current literature suggests this subset of T cells is a key determinant of the outcome of viral infections.

Collectively data presented herein, indicate that New Zealand White rabbits compared to humans and mice contain a predominant CD4+ T cell

61 population throughout their GALT and associated lymphoid tissues. These findings suggest directions to more fully characterize this important laboratory animal species. Once complete characterization of all lymphocyte subsets and specialized lymphoid subsets has been established, further studies should focus on evaluation of cell proliferation and the cytokine profile of these cells with the aim of assessing immune competence of leukocytes in each lymphoid compartment. Cytokines and chemokines represent a family of low molecular weight proteins that mediate leukocyte chemotaxis and extravasation in tissues

24. Thus, these proteins are critical mediators of inflammation. It is well- established that the production of cytokines in various T cell subsets is not random and varies depending on microenvironment 95. For example, it appears that GALT inductive sites tend to favor anti-inflammatory cytokine production (i.e.

PGE-2α and IL-10), rather than pro-inflammatory cytokines 21. Other cytokines that would need to be evaluated in rabbits include, but are not limited to IL-2,

TNF-α, IFN-γ, TGF-β1, IL-6, and IL-8. Future studies are needed to focus on isolation of lymphoid populations in the GALT inductive and effector sites followed by antibody and mitogenic stimulation (e.g., Concavalin A), and evaluation of cytokine mRNA expression with reverse-transcriptase polymerace chain reaction (PCR) technology, flow cytometry and immunohistochemcial analysis if applicable.

In summary, while our data provides a foundation of description of rabbit

GALT and associated lymphoid tissues, future studies will be required to fully realize the potential of the rabbit as a model of human disease. Our data

62 indicate that New Zealand White rabbits compared to humans and mice contain a predominant CD4+ T cell population throughout their GALT and associated lymphoid tissues. Collectively, our findings provide new focus on this important laboratory animal species for studies seeking information on the immunopathogenesis of orally transmitted human pathogens, such as HTLV-1 infection.

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