Comparison of porcine CD4 and gamma-delta T cell systemic

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Comparison of porcine CD4 and gamma-delta Tcell systemic and mucosal responses to thespirochete Brachyspira hyodysenteriaeRaquel Hontecillas-MagarzoIowa State University

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ProQuest Information and Learning 300 North Zeeb Road, Ann Arbor, Ml 48106-1346 USA

800-521-0600

Comparison of porcine CD4 and gamma-delta T cell systemic and mucosal

responses to the spirochete Brachyspira hyodysenteriae

by

Raquel Hontecillas-Magarzo

A dissertation submitted to the graduate faculty

in partial fulfillment of the requirements for the degree of

DOCTOR OF PHILOSOPHY

Major: Immunobiology

Program of Study Committee: Michael J. Wannemuehler, Major Professor

Janice E. Buss Susan J. Lamont Harley W. Moon Randy E. Sacco

Iowa State University

Ames. Iowa

2002

Copyright © Raquel Hontecillas-Magarzo. All rights reserved.

UMI Number: 3073455

Copyright 2003 by

Hontecillas-Magarzo, Raquel

All rights reserved.

UMT UMI Microform 3073455

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Graduate College Iowa State University

This is to certify that the doctoral dissertation of

Raquel Hontecillas-Magarzo

has met the dissertation requirements of Iowa State University

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For the Major Program

Signature was redacted for privacy.

Signature was redacted for privacy.

iii

TABLE OF CONTENTS

ABSTRACT

CHAPTER 1. GENERAL INTRODUCTION I Background and rationale I Objectives of the dissertation 5 Dissertation organization 5 Literature review 6

Functional differences between aP and y8 T cells 6 Origin and distribution 6 Differential aspects of antigen recognition between ap and y8 T cells 7

Comparison and contrast between the (%P and yô T cell receptor 8 Structure of the T cell receptor 8 Differences between the (%P and y8 T cell receptor 12

Porcine CD4 T cells 13 Coexpression of CD8aa in mature porcine CD4T T cells 15 The CD8 coreceptor: isoforms, function and regulation of cell surface expression 16 Possible explanations for the function of mature CD4+CD8aaT cells 18

Porcine y8 T cells 19 Lymphocyte composition of the intestinal mucosa 21 Role of T cells in the development and regulation of intestinal inflammation 23

Cellular mechanisms involved in intestinal mucosa homeostasis 23 Role of CD4+ T cells in the establishment and resolution of inflammation 24 Y8 T cell involvement in the control of inflammation 27

References 29

CHAPTER 2. DIFFERENTIAL REQUIREMENTS FOR THE PROLIFERATION OF PORCINE CD4 AND yô T CELLS TO EXTRACELLULAR BACTERIAL ANTIGENS 43

Abstract 43 Introduction 44 Materials and methods 46 Results 49 Discussion 52 References 61

iv

CHAPTER 3. MODIFICATION OF THE LYMPHOCYTE COMPOSITION AND THE CYTOKINE ENVIRONMENT DURING Brachyspira hyodysenteriae-INDUCED COLITIS BY SYSTEMIC IMMUNIZATION WITH A PROTEASE DIGESTEDBACTERIN 64

Abstract 64 Introduction 65 Materials and methods 66 Results 70 Discussion 73 References 85

CHAPTER 4. GENERAL CONCLUSIONS 89

ACKNOWLEDGEMENTS 95

V

ABSTRACT

In pigs, «P CD4+ and yô T cells constitute an important fraction of the circulating

lymphocyte pool. This makes the pig an ideal animal model for functional studies of the two

cell populations. A comparative evaluation of the systemic and mucosal responses of porcine

CD4+ and yô T cells was conducted in the context of immunization and/or challenge with the

pathogen Brcichyspirci hyodysenteriae. This spirochete causes a severe colitis in pigs;

however, immunization with a pepsin-digested bacterin ameliorates clinical signs of disease.

Protection has been associated with in vitro antigen-dependent proliferation of CD41" and

yS T cells to B. hyodysenteriae antigens. A first set of experiments was conducted to evaluate

the differential functional aspects of the systemic responses to B. hyodysenteriae antigens of

CD41" and yÔ T cells. Results show that, while the proliferative responses of CD4+ were

exclusively antigen-specific, y5 T cells responded to stimuli other than B. hyodysenteriae

antigens. Proliferation of the two cell populations in vitro was dependent on the presence of

IL-2. In addition, examination of IFN-y production indicated that CD4+ T cells are the major

source of this cytokine, although stimulation with B. hyodysenteriae antigens decreased IFN-

y-producing CD4+ T cells. This effect was paralleled by an increase in yô T cells producing

IFN-y. These results suggest that, as has been demonstrated for other infectious disease

models, yô T cells may contribute to downregulate CD4+ T cell responses. A second study

describes the impact that immunization and/or challenge had on the lymphocyte composition

and cytokine environment of the colonic mucosa. Immunization decreased the colonic CD41"

T cells irrespective of the challenge status, whereas challenge with B. hyodysenteriae resulted

in depletion of epithelial yS T cells; although, vaccination ameliorated the loss of this

vi

lymphocyte subset. These cellular changes were accompanied by modulation of cytokine

expression. Particularly, TNF-a, IL-1 (3 and TGF-3i mRNA were upregulated in vaccinated

and infected pigs. The possible contribution of porcine CD4^ and yô T cells to the

development and resolution of B. hyodysenteriae-induced colitis and the mechanisms of

activation of the two subsets are discussed in this dissertation in the context of the present

findings.

1

CHAPTER 1. GENERAL INTRODUCTION

Introduction

Brachyspira hyodysenteriae is a spirochete that causes swine dysentery, a severe

mucohemorrhagic colitis (I). In addition to the etiologic agent, the presence of an appropriate

intestinal flora is required in order for the disease to develop. This was shown in experiments

involving germ-free mice or pigs, in which disease developed only after coinfection with

Bacteroides vulgatus and B. hyodysenteriae (1 ; 2).The molecular pathogenesis of swine

dysentery has not been elucidated; however, it is believed that colonization of the mucosa by

B. hyodysenteriae results in the loss of the epithelial barrier function. This is due in to the

production by the spirochete of a ^-hemolysin that is cytotoxic for enterocytes (3). This

initial phase is followed by the invasion of the mucosa by commensal bacteria from the

colon. Excessive exposure of the underlying mucosa to luminal antigens ultimately leads to a

severe, acute inflammatory response (3; 4), which was characterized in a murine model o the

disease by the upregulation of proinflammatory cytokines, such as tumor necrosis factor-

ex (TNF-a), interleukin I-(3 (IL-1(3) (5). Figure 1 depicts the most characteristic histologic

features of the disease: superficial epithelial erosion, increased thickness of the mucosa with

crypt hyperplasia, and leukocyte infiltration. The disease is basically restricted to the mucosa,

although multifocal leukocytic infiltrates may sometimes be present at the submucosa (6).

Figure 1: Photomicrographs of hematoxilin-eosin stained sections of porcine colon from a healthy (A) and a B. hyodysenteriae infected (B) pig at 160 x magnification. The most characteristic histopathologic features of the disease are increased thickness of the epithelium (indicated by arrows) with crypt hyperplasia, superficial epithelial erosions (indicated by an asterisk) and leukocytic infiltration.

Systemic immunization with a B. hyodysenteriae protease-digested bacterin provides

protection against the disease (i.e., reduced clinical signs), although it does not prevent

bacterial colonization (7; 8). The vaccine has also been used as a tool for evaluating porcine

T cell responses to extracellular bacterial antigens (8-12). A diagram showing the phenotypic

classification of porcine T cells is depicted in figure 2. Based on T cell receptor (TCR)

expression, porcine T cells can be divided into afi or yô T cells. aP T cells can be further

subdivided depending on CD4 and CD8 coreceptor expression. Besides the CD4"1" CD8" aP T

cells, three subsets of porcine CD8+ TCR aP T cells have been described: 1) CD4 CD8

(%P or cytotoxic T lymphocytes, 2) CD4+ CD8acT or memory/effector T cells, and 3) CD4"

CD8acT T cells, of unknown function (13-19). Finally, two major subsets of y5 T cells have

been characterized: 1) SWC6^ CD2", which constitute the majority of the peripheral blood

yô T cells, and 2) SWC6*CD2+ which are low in peripheral blood and preferentially migrate

to non-lymphoid organs and spleen. A fraction of the SWC6~CD2+ y5 T cells also express

CD8a (20-22).

3

Porcine T-lymphocytes

a0 T cells yS T cells

SWC6+ CD2-CD4+CD8- CD4+ CD8aa+

SWC6- CD2+CD8aa+

Figure 2: Subset classification of porcine T lymphocytes

Previous studies reported that there was an increase in the percentage of CD8cf

lymphocytes in peripheral blood, colonic lymph nodes (CLN), and colonic mucosa following

challenge of vaccinated pigs with B. hyodysenteriae (10). Also, in vitro stimulation of

peripheral blood mononuclear cells (PBMC) isolated from immunized animals resulted in

antigen-dependent proliferation of CD8cf lymphocytes, CD4+ CDSacf, CD4+ CD8aa+, and

TCR yô cell subsets. Blockade of antigen presentation with antibodies against class II major

histocompatibility complex (MHC-II) diminished in vitro proliferation of PBMC from

immunized pigs, and similar results were obtained by depleting CD4+ T cells (9). Therefore,

vaccination with a B. hyodysenteriae bacterin induces a MHC-II-dependent CD41" T cell

response. However, because significant proliferative responses of yô T cell are also induced

following in vitro antigenic stimulation of unfractionated PBMC, this immunization regime

can be utilized for the study of the differential mechanisms of activation of CD4+ and yô T

cells.

4

The identification of yô T cells is relatively recent compared to a|3 T lymphocytes

(23). Although both aP and yô T cells have the capacity to specifically recognize antigens

through their respective TCR, the type of antigens recognized and the process of antigen

recognition are different between the two subsets (23). In fact, knowledge on yS T cell

biology has greatly increased in recent years, making it evident that their function in the

immune system is different from that of ap T cells (23). The tissue distribution pattern of

aP and yô T cells is a first indicator of their differential role. In mice and humans, y5 T cells

are almost absent from peripheral blood and organized lymphoid tissues. In other species

such as poultry, cattle or pigs, yô T cells constitute a major proportion of the pool of

circulating lymphocytes, and they are present in secondary lymphoid organs although in low

numbers (24-26). In both cases, yS T cells are abundant at mucosal sites (27). On the other

hand, the T cell population within the spleen and lymph nodes are predominantly aP T cells.

These are the compartments of the immune system where naïve T cells are first exposed to

antigens processed and presented by professional antigen presenting cells (APC) and after

antigen presentation, naïve T cells undergo clonal expansion and differentiation into

effector/memory cells (28). Because yS T cells are not retained at these sites, it could be

predicted that their involvement in the immune response is different from that of ap T cells.

Questions relating to the process of antigen presentation to yÔ T cells (e.g., MHC-restriction,

requirements for APC or costimulation) and whether or not they become memory cells are

currently under investigation (27).

yô and CD41" T cells are the two T cell subsets responding following immunization

with a B. hyodysenteriae bacterin (10). During the clinical period of swine dysentery, the

bacteria invade the colonic mucosa without spreading systemically (6). Thus, by utilizing

5

different routes of antigenic exposure, this model offers the possibility of evaluating the local

response of CD4+ and y5 T cells at the colonic mucosa (i.e., following challenge),

systemically (i.e., by intramuscular immunization), or as a result of the interaction between

immunization and challenge.

Objectives of the dissertation

The objectives of the experiments conducted for this dissertation were to evaluate the

impact that immunization and /or challenge with B. hyodysenteriae had on porcine CD4~ and

yS T cells. More specifically, we investigated in vitro differential aspects of the responses

(i.e.. proliferation and cytokine production) of CD4+ and yS PBMC to B. hyodysenteriae

antigens following immunization. At the colonic mucosal level, we examined the changes

that immunization and/or challenge induced on the numbers and distribution of these subsets

at the colonic mucosa.

Dissertation organization

This dissertation is organized in four chapters: a General Introduction (chapter 1), two

journal style manuscripts (chapters 2 and 3) and a General Conclusion (chapter 4).

6

LITERATURE REVIEW

Functional differences between af) and y8 T cells

T cells derive from pluripotent precursors that follow the lymphoid lineage of

differentiation (29; 30). Their designation as "T cells" comes from their "thymus-dependent"

origin. Although this terminology has been maintained, the existence of extrathymically-

derived T lymphocytes has been known for a long time (31-33). By definition, all T cells are

lymphocytes that in mature state express a T cell receptor (TCR) (28). The TCR is a

heterodimeric molecule that results from the association of a and P (TCRaP), or y and

5 (TCRyô) polypeptide chains. Thus, two major subsets of T cells exist based on the type of

TCR expressed on the cell surface: (%P and yô T cells (34). Concepts related to T cell biology,

such as MHC restriction, immunological memory or acquired immunity resulted from studies

on «P T cells. yS T cells were identified more recently than aP T cells, and initial studies

demonstrated that some principles of aP T cell biology would not apply to them. There is

enough evidence now to support that, rather than being exceptions, y8 T cells have different

and non-overlapping roles in comparison to (%P T cells within the immune system (23; 35).

Origin and tissue distribution of C# and yS T cells

Although both cell types originate from a common lymphoid precursor, they diverge

early in ontogeny (36). During lymphocyte development, thymocytes commit to either the

aP or yô T cell lineage. Thymocytes that successfully rearrange a TCR, either aP or yS,

become naïve mature T cells (30). A difference between aP and y8 T cell development is that

y8 T cells are considered mature lymphocytes from the moment they express a functional

TCR (30; 37). At this point, they migrate to the periphery (27). Conversely, thymocytes that

have committed to the aP T cell lineage go through several checkpoints before becoming

7

mature cells. The first checkpoint occurs during the process known as |3 selection (i.e., in-

frame rearrangement and successful expression of the TCRP chain) (38), afterwards, a(3 TCR

thymocytes capable of recognizing self-MHC through the rearranged TCR are rescued from

apoptosis or are positively selected (39). Finally, the recognition of self-peptides through the

TCR generates strong signals that activate apoptosis. This phenomenon, known as negative

selection, prevents auto-reactive T cells from migrating to the periphery (40; 41).

A second aspect that differentiates a(3 and yÔ T cells is their trafficking patterns.

Naïve a(3 T cells migrate from the thymus to secondary lymphoid organs where, eventually,

they will be exposed to antigen and differentiate into effector or memory

cells. Effector/memory a(3 T cells then home to non-lymphoid tissues where they sample for

their cognate antigen. In contrast, yô T cells directly accumulate in non-lymphoid tissues,

especially at mucosae, without the need of a previous antigenic exposure (27; 42; 43). Most

challenges to the immune system of environmental origin, (e.g., infectious agents or

allergens), occur at mucosal sites. While yô T cells are ready to respond to an antigenic

challenge, aP T cells must have been previously exposed to the antigen in a secondary

lymphoid organ. Because this process requires some time for aP T cells, it has been proposed

that yô T cells constitute a first line of defense (44).

Differential aspects of antigen recognition between af) and y6 T cells

Antigen presentation to naïve aP T cells is a function of dendritic cells (DC) that

have migrated from the periphery to secondary lymphoid tissues (45). Because yô T cells are

not retained at these sites, the question then is how does antigen presentation to yô T cells

occur? aP T cells recognize peptides resulting from antigenic processing, presented in

association with MHC-class I or II molecules on the surface of target cells or APC to CD8+

8

(i.e., cytotoxic) or CD4+ (helper) T cells (28). The yô TCR is MHC-I and II-unrestricted;

instead, non-classical MHC molecules, namely T10/T22(46), its murine homologue retinoic

acid early inducible Rae (47) antigen or CD I (48) are ligands for the yô TCR. In addition,

previous research showed that inhibition of antigen processing and presentation by classical

MHC molecules did not affect y5 TCR activation (46). These results lead to the conclusion

that yô T cells recognize unprocessed antigens. However, in similar action to a(3 T cells, cell

to cell interaction and costimulation are required for yô T cell activation through the TCR

(49). Therefore, yô T cells are capable of recognizing unprocessed antigens directly on

tissues without the mediation of a professional APC (27).

MHC-I and II molecules are specialized in presenting peptidic epitopes to T cells

(50). In addition to recognizing protein-derived antigens (e.g.,glycoprotein 1 from Herpes

simplex virus) (51), yô T cells recognize non-peptidic molecules. Special attention has been

given to the capacity of phosphoantigens, low molecular mass molecules rich in phosphate

groups abundant in bacteria (e.g., Mycobacterium spp, Escherichia coli), to stimulate human

yÔ T cells (52-55). In addition, it has been shown that yÔ T cells can recognize stress-induced

self-molecules such as MHC-class I related chain A (MICA) (56), its murine homologue

retinoic acid early inducible Rae (47) antigen (56), and transformed cells (57).

Comparison and contrast between the a0 and the y8 TCR

Structure of the TCR

Studies on the structure of the TCR have provided a basis for some of the differences

between a(3 and yô of T cells. As already mentioned, the TCR is a heterodimer formed by

two disulphide-linked polypeptide chains; a-fi or y-ô . It is always expressed along with the

9

CDS complex, thus phenotypically, T cells are designated as CD3+. The CD3 complex is the

association of three different polypeptide chains (i.e., CD3y, CD3S and CD3e), with an

extracellular immunoglobulin-like (Ig-like) domain and a short cytoplasmic tail. Also, most

of the TCR-CD3 complexes are associated with a homodimer formed by two Ç chains, or a

heterodimer formed by Ç and its alternative spliced form, x\. The latter proteins have a very

short extracellular domain and a long cytoplasmic tail that very actively participate in TCR

signal transduction (58). The composition of the TCR complex is not constant and varies

depending on developmental or activation state of the cell (28; 59; 60).

Based on genetic features and protein structure, the TCR has been classified within

the immunoglobulin superfamily. Figure 3 depicts the structure of a TCR. Each polypeptide

chain results from the combination of one invariant, or constant (C) (i.e., Ca and CP in the

TCRaP, or Cy and CÔ in the TCRyô), and one variable (V) region (i.e., Va and VP or Vy

and Vô) (28; 30; 61-63). The C portion of the protein has an Ig-like domain followed by a

hinge region, a transmembrane region and a short cytoplasmic tail. Cystein residues at the

hinge region are involved in dimer formation between a and P or y and Ô chains. The

transmembrane domain facilitates the association between the TCR and the CD3 signaling

complex. In the case of antibodies, different isoforms exist for the C region (i.e., isotypes). In

contrast, isotypic variation at the TCR C region is lower (28). Two isoforms of the Cp genes

have been described in the mouse with apparently no significant functional differences (64)

and three isoforms of the Cy have been identified in the pig (65).

The variable domain of the TCR results from the somatic recombination of germline

encoded gene segments (29). The great advantage that this unique system shared by the TCR,

B cell receptor (BCR) and antibodies, is the generation of receptor diversity. This allows for

10

the specific recognition of the great variety of antigens to which the immune system is

exposed. TCR diversity is achieved by recombination of different segments that will form the

V chain. The a and y V chains result from the recombination of V and joining (J) segments,

(i.e., VJ). In the P and S chains, an additional diversity (D) segment is incorporated into the V

chain (i.e., VDJ). The existence of several V, D and J genes that can be recombined in the V

chain is what brings about receptor diversity (66).

Amino acid sequence comparisons of V domains of each of the polypeptide chains

that form the TCR indicated that within the V chain there are regions of hypervariability

(HV) (67). The HV regions of the a-P or y-S chains form the complementary determining

regions (COR) on the TCR. The CDR1 and CDR2 domains interact with MHC-I or II for aP

T cells, or a non-classical MHC molecule in the case of yô T cells. The CDR3 is the region of

the TCR that is directly implicated in antigen recognition (68; 69). This feature is indicated in

figure 3.

Il

Structural differences between the C regions of the aP and yô TCR suggest that the two types of

receptors form different signaling complexes.

CDR3 has the same length in Va and Vp, but it is longer in Vô than in Vy.

This feature is also present in antibody molecules. From this finding it was interpreted that yô T cells recognize

epitopes in a similar fashion to immunoglobulins.

Figure 3. Crystal structure of an aP T cell receptor (TCR) interacting with a major histocompatibility complex (MHC-l) molecule loaded with a peptide (P1-P8). The complementary determining regions (CDR) are the loops represented in colors at the are area of contact between the TCR and the MHC-peptide complex. The major structural differences between the ap and the yô TCR, and the functional implications that they have are indicated in boxes (with permission from LA, Wilson) (70).

12

Differences Between the (*P and y5 TCR

Comparative studies examining the structure and function of the a(3 and yô TCR

showed that the CDR3 of VS is longer than the CDR3 of Vy (70). This structural feature

results from the rearrangement of up to three D segments and the addition of N-nucleotides

in VÔ (71). In contrast to this, the CDR3 of the a and p chains are of the same length (70).

Besides, the number of amino acid residues that form the CDR3 is larger in the yô than in the

aP TCR. These structural differences have important functional implications (70). T cells

recognize peptides of 6 to 12 aminoacids in length that result from antigen processing and are

presented by MHC molecules on APC (28). In turn, yô T cells can recognize antigens of

different chemical structure other than proteins (e.g., phosphoantigens or lipoproteins) (52-

55; 72; 73). In fact, based on the basis of the CDR3, the yÔ TCR closely resembles

antibodies, which have longer CDR3 in the heavy than in the light chain (70). These

structural similarities between immunoglobulins and the yôTCR suggest that yÔ T cells can

recognize unprocessed antigens (46; 74-76). However, in contrast to antibodies, antigen

recognition by yô TCR requires cell to cell contact (49).

The first crystallographic model of a complete human yô TCR (i.e., Vy9 VÔ2) was

recently published (63). Vy9 VÔ2 T cells constitute the majority of the circulating y5 T cells

in humans (77-79). In this specific case, the CDR3 regions are of the same length in the y and

Ô chains. The most significant differences with the aP TCR are found in the C chain. The

comparison of both the structure and shape of the «P and yô TCR reveals that they form

different signaling complexes. First, within the C chain Ig-like domain, the polypeptide

domain that in the «P TCR apparently associates with the CD3E (i.e., FG loop), is longer

than the corresponding region in the yô TCR. In addition, both receptors differ in their hinge

13

regions, with a maximum of 7 versus 23 residues between the end of the Ig-like domain and

the interchain disulphide bond in the ap and yÔ TCR, respectively. These particularities

indicate that a different type of association exists between the CD3 complex and the a(3 or

yS TCR and that the receptor on yS T cells has higher flexibility (63). As already mentioned,

three Cy isoforms have been described in the pig (65; 80). It is interesting that the three

isoforms are different from one another in the hinge region and in the cytoplasmic tail.

Again, these differences suggest that yô T cells form different signaling complexes and,

therefore, distinct signal transduction pathways may regulate activation of the different yô T

cell subsets.

In summary, both the a(3 and the yô TCR belong to the immunoglobulin superfamily,

however, modifications of the C and V domains account for their differential biological

properties. Mainly, the type of antigen that they recognize, the mechanisms of antigen

recognition, and the association with signaling complexes are characteristic for each receptor.

The overall result is that the two cell types have different roles within the immune system.

Porcine aP CD4 T cells

An important role of the CD4+ T cells is to coordinate acquired immune responses.

They are involved in both cellular and humoral responses by serving as intermediates

between antigen presenting cells (APC) and B cells, for the production of antibodies, or

cytotoxic T cells (28). T helper 1 (Thl) and Th2 cells regulate immune responses involved in

the elimination of foreign antigens of microbial origin (81). CD4+ T cells are implicated in

the discrimination of self from non-self are equally important. In this regard, CD4+

14

regulatory T cells control other lymphocytes capable of recognizing self-antigens preventing

autoimmune diseases (82).

CD4 is a glycoprotein that possesses 4 Ig-like domains. It interacts with a non-

polymorphic region on the (32 domain of the MHC-II molecule stabilizing antigen

recognition by the TCR. In addition, its cytoplasmic tail associates with Lck, a tyrosine

kinase of the Src family that participates in proximal signal transduction events (83-85).

Although no monoclonal antibodies that recognize the a or p chains of the porcine TCR are

available, CD4+ T cells are CD3+ and are considered TCRaP cells (14; 86; 87). The Thl/Th2

paradigm has not been demonstrated for porcine T cells. However, it is assumed that porcine

CD4+ T cells have similar roles to CD4T T cells from other species, and that they use the

similar effector mechanisms (87; 88). CD4 T cells provide help for the generation of

alloantigen-specific responses (89). The expression of MHC-II antigens is unique to a subset

of porcine CD4+ T cells (i.e., CD44" CD8acO- Although the significance is unknown, it

seems that it does not confer them with the ability to present antigens, because the absence of

other accessory APC abrogated alloantigen-specific responses (89). It has also been

suggested that CD4"1* T cells interact with B-cells for the generation of humoral responses

(90).

Most of the information currently available with regards to the biology of porcine

CD4+ T cells has been obtained in the context of the cellular immune responses to infectious

agents, mainly of viral origin. As it can be expected, infections of bacterial, viral or parasitic

etiology induce detectable in vivo changes in the CD4+ subset (91-96). In vitro, CD4+T cells

proliferate in response to viral or bacterial antigens and these antigen-specific responses are

15

MHC-II restricted (9; 88; 97). In addition, CD4+ T cells become CD8aa+ during antigen-

specific recall responses (18).

Coexpression of CD8aa in mature porcine CD4 T cells

The simultaneous expression of the CD8aa coreceptor on peripheral mature CD4+T

cells has been emphasized as one of the distinctive characteristics of the porcine immune

system. However, other species have also detectable numbers of CD4+ CD8aa+ T cells (e.g.,

monkeys and chickens) (17). The CD8 molecule is coexpressed on mature CD4+ T cells in

low levels and always as a CD8aa homodimer, which differentiates it from the CD8«P

heterodimer on CD8+ cytotoxic T cells (9; 14; 98). CD4+CD8aa+T cells are normal

components of the gut mucosa (99). In healthy individuals conditions, a small percentage of

peripheral lymphocytes are CD4+CD8aa+T cells. However, the presence of this subset is

associated with certain diseases such as neoplasia (100) or rheumathoid arthritis (99).

It is has been accepted that porcine CD4^CD8acTT cells represent a fraction of

memory/activated cells within the CD4"1" pool (18). This assumption is based on the lack of

expression of CD1 (101), a molecule present on immature T cells, and the fact that the

numbers of CD4 CD8aa T cells increase with age, suggesting that this is the result of

antigenic exposure (17; 18). Ontogenetically CD4+CD8aa+T lymphocytes are undetectable

until day 90 of gestation. The peripheral blood of newborn piglets posses approximately I %

of CD4+CD8aa+T cells (102). This number increases to 30-55 % of the total PBMC in 3

year old pigs (18).

In vitro experiments showed that sorted CD4+ T cells from pigs immunized against

pseudorabies virus (PRV) became CD8acT after ex vivo stimulation with PRV antigens (18).

Moreover, most of the CD4 CDScxct T cells also coexpressed CD29, a phenotypic marker of

16

memory T cells (18). Neither the mechanisms regulating CD8aa expression on porcine T

cells, nor its functional role on these cells have been investigated to date.

The CDS coreceptor: isoforms, function and regulation of cell surface expression

CD8 is a glycoprotein of the immunoglobulin superfamily. There are two isoforms:

CD8a and CD8P; the association of two a chains or an a and a P chain form the

homodimeric or heterodimeric forms of the coreceptor, respectively (103; 104). The CD8aP

form is characteristically expressed on cytotoxic T cells (104; 105), while CDSaa can be

expressed on dendritic cells (106), natural killer cells, yô T cells and on mature CD4T T cells

(107). The CD8 coreceptor facilitates the interaction with APC. Both forms of the coreceptor

recognize non-variable regions on the MHC-l molecule (108) and, in addition, CDSaa

recognizes non-classical MHC antigens as has been reported for thymus leukemia (TL)

antigens expressed by enterocytes ( 109).

There has been some controversy regarding which form of CDS is more efficient as a

coreceptor. Soluble CDSaa and CDSaP molecules have equal binding affinity for MHC-I

(110), however, cell-associated CDSaP is more efficient coreceptor than CDSaa (111). The

clearest difference between the coreceptors is in their contribution to T cell activation. CDSa

has a long cytoplasmic tail that associates with Lck (85). As it has been indicated for the CD4

coreceptor, this interaction is important in early signal transduction events mediated by the

TCR. CD8P has a short cytoplasmic tail and does not associate with Lck (112); however, it

has a domain that is necessary for palmitoylation and for the recruitment of CDSaP into lipid

rafts, where most of the cellular Lck is found (113). Deletion of the palmitoylation residue on

CDSP abrogated its ability to partition into lipid rafts, and chemical inhibition of

palmitoylation decreased T cell activation (114; 115). Thus, CDSP is crucial role for the

17

formation of efficient signaling complexes and activation of cytotoxic lymphocytes through

the TCR. On the other hand, although CD8aa can associate with Lck, the lack of a

palmitoylation residue suggests that it cannot come in contact with the TCR, and therefore, it

does not participate in T cell activation in the same way that CD8«P would (115).

In the mouse, the genes that code for CD8a and CD8(3 are in close proximity on

chromosome 6(116). Both transcriptional and postranscriptional mechanisms for the control

of the expression of these genes have been described. Within the transcriptional mechanisms,

there are four enhancers (E8t-E8iv) that are tissue-, lineage-, and developmentally-specific

(117; 118). For instance, E8, regulates CD8a expression in T cells and mature single positive

(SP) thymocytes, but it has no effect in double positive (DP) thymocytes. Also, it controls the

expression of CD8a in NK cells, intestinal TCRyô CD8aa and TCRaP CD8aa T cells but

not in TCRaP CD8a|3 intraepithelial lymphocytes (IEL) (118-120). Neither CD8a nor

CD8P genes of the pig have been sequenced, and no information is available on how its

expression is regulated. It could be anticipated that an element similar to E8, exists which

regulates the expression of porcine CD8a gene, because the homodimeric form of CD8a is

expressed in equivalent cell subsets. With regards to postranscriptional regulation, mature

human CD4+ T cells transcribe CD8a, although the mRNA is very unstable and the protein is

never produced (121). However, it was reported that phorbol myristate acetate (PMA)-

activated human CD4+ T cell clones were able to synthesize and coexpress CD8a in the

presence of interleukin-4 (IL-4). The synthesis of CD8a was transient and downregulated by

IL-2 (122). Although these are in vitro studies, certain human diseases such as neoplasia

(100) or rheumathoid arthritis (99) are associated with an increase in recirculating mature

18

CD4+CD8TT cells. Therefore, molecular mechanisms that stabilize CD8a mRNA could

enhance the expression of CD8a on the surface of CD4+ T cell.

Possible explanations for the function of mature CD4+CD8aa+T cells

A question that remains to be answered is what are the functional differences between

CD4""T cells and CD4+CD8acf T cells. It has been hypothesized that human CD4+CD8acT

are regulatory cells. Consistent with this hypothesis, cloned CD4+CD8+aaT cells from

lepromatous leprosy patients inhibited activated T cells (124). In line with the findings in

human lymphocytes, porcine CD4TCD8+aaT cells isolated from peripheral blood were

shown to express high levels of IL-10 mRNA (92). IL-IO is a cytokine associated with

regulatory responses (125; 126). However, a different study showed that peripheral blood

CD4^CD8aa*T cells produced high levels of IFN-y when stimulated with PMA and

ionomycin (127).

A second theory proposes that circulating human CD4TCD81aaT cells have

cytotoxic activity. In support to this theory, addition of anti-CD8a monoclonal antibodies

blocked anti-CD3-induced cytotoxicity of human CD4+CD8+aaclones (123). Porcine CD4T

CD8+aaT cells isolated from PRV-immunized pigs contributed to MHC-class [-restricted,

antigen-specific cytotoxic activity of CTLs (128). It has also been suggested that porcine

CD4+CD8+aaT cells could themselves be cytotoxic (129). It is improbable that the

mechanism of cytotoxicity depends on dual MHC-I/MHC-II restriction of CD4+CD8aoTT

cells, because the CD4+ T cell precursors express a TCR specific for a MHC-II-dependent

epitope. Although this rules out the dual MHC-I/MHC-II-restriction of porcine CD41"

CD8aa+T cells, they could still induce cytotoxicity of target cells by alternative mechanisms

(e.g., Fas-FasL interaction). This hypothesis, however, has not been tested. Studies on the

19

localization of CD8aa on the cell membrane of CD4+CD8aa+T cells and the determination

of the molecules that associate with the cytoplasmic tail would help in elucidating if

CD8aa contributes to TCR-mediated activation of CD4+T cells. In addition, the

identification of the factors that stimulate CD8aa expression would provide some insights

into the possible role of porcine CD4+CD8aa+T cells as memory/effector T cells.

Porcine yfi T cells

yô T cells were discovered during the identification and sequencing of the

genes for the a and (3 chains of the TCR (130-132). One of the principal characteristics of y5

T cells is that they tend to accumulate in non-organized lymphoid tissues, especially the skin

and intestinal mucosa (27). However, species-specific differences have been reported, with

mice and humans having very low numbers of circulating yô T cells while in pigs, ruminants

and chickens they constitute an important fraction of the PBMC (24-26). When monoclonal

antibodies became available for some of the cell surface markers of porcine lymphocytes, it

was observed that a high proportion of PBMC were negative for either B or T cell surface-

expressed proteins (i.e., CD4, CD8 or slgM). These "non-B, non-T cells" were subsequently

phenotypically characterized as y8 T lymphocytes (133).

The classification of mouse and human yô T cells is based on their variable TCR gene

usage (35) while the pig it is based on their phenotypic characteristics. There are two main

subsets of porcine y5 T cells. One is the analogue of bovine WCl^yô T cells and is

phenoty pically defined by the expression of SWC6 and CC101 antigens. Bovine WC1 is a

member of the scavenger receptor cysteine-rich family and, antibodies against CC101

recognize a related molecule expressed by pig yô T cells. This subset is also CD2~ and CD8a.

20

SWC61" TCRyô cells constitute the majority of the yô T cells in PBMC (26; 134). The second

subset can be phenotypically defined as SWC6" CD2+, of which a small fraction expresses

CD8a. Cells from this subset have been detected in the spleen and lymph nodes and as

recirculating lymphocytes (134; 135).

Protein analyses of the yS TCR yield a unique form of the 8 chain of 38 kDa, and 3

isoforms of the y chain of 37, 38 and 46 kDa. The 37 and 38 kDa isoforms are expressed in

CD2CD8" (i.e., SWC6*) y5 T cells. These two y chains are subset-specific, as the 38 kDa

iso form is only present in circulating SWC6+ cells that also express the 86D molecule, while

the 37 kDa form is characteristic of circulating but also tissue-resident cells. Finally, the 46

kDa iso form was attributed to CD21" CD8*(SWC6") cells present in secondary lymphoid

tissues (81). Cloning of the porcine Cy chain revealed the existence of 3 iso forms different in

their hinge regions. Two of them (G10 and G15) are similar to each other with longer hinge

regions that have 2 additional cysteine residues in comparison with the G4 isoform. The third

iso form (04) differs from G10 and G15 in its shorter hinge region and in the cytoplasmic

tail. G10 and G15 were identified as the 37 and 38 kDa isoforms of the y chain. The 46 kDa

isoform, however, has not been matched to any Cy sequence (65). A possible explanation for

this is that PBMC were used as a source of T cells for cloning the Cy, when cells bearing the

46 kDa chain are found in lymphoid tissue.

Very little information is available regarding functional aspects of porcine y5 T cells.

There are reports describing yô T cells from peripheral blood proliferating in vitro to viral or

bacterial antigens (9-11; 89; 98). However, they appear to have very low capacity of IFN-y

production in vitro (127). Porcine CD6* lymphocytes are functionally characterized by having

non-specific cytotoxic activity (128). The SWC6+ subset of porcine yS T cell is CD6

21

and has NK-like activity (127; 135). In addition, it was shown that this cell type

preferentially migrated to sites of cutaneous inflammation induced by administration of

phytohemagglutinin (136; 137). There are contradictory results with regard to the SWC6"

yÔ T cells. They have been described as both CD6" (I28)and CD6+ (133) by different authors.

Thus, their function remains unclear.

Lymphocyte composition of the intestinal mucosa

The intestinal mucosa is the most inner layer of the gut wall. It is divided in two

functional and structurally different compartments: lamina propria and epithelium. The

intestinal epithelium is a single cell layer of polarized cells exposed to the lumen, thus, in

direct contact with the intestinal contents (138). Lymphocytes constitute an important portion

of both the lamina propria (i.e., lamina propria lymphocytes, LPL) and the epithelium (i.e.,

intraepithelial lymphocyes, IEL) (139; 140). However, the lymphocyte composition of the

mucosa is not random, but characteristic of the different anatomical regions of the intestine

(e.g., small versus large intestine), and of each of the two compartments (i.e., epithelium

versus lamina propria) (141; 142). In addition, due to the specific functions that the immune

system has at this site, lymphocytes from the intestine are different in several aspects (138).

In regards to the phenotype, they have an atypical pattern of expression of cell surface

molecules compared to conventional lymphocytes at the systemic/central lymphoid system.

First of all, the gut associated lymphoid tissue (GALT) is rich in y5 T cells (143) and, within

the aP T cell fraction, cells expressing CD4 (TCRaP CD4+CD8"), CD8 (TCRaP CD4*

CD84), or neither of the coreceptors (TCRaP CD4~CD8~) have been described (139; 144). In

addition, the expression of the CD8aa homodimer is common among cells of both the aP

and yô T cell lineage (145; 146). In the mouse, it has been proven that the CD8aa I EL

develop extrathymically while the rest are of thymic origin and migrate from the periphery to

colonize the intestine (147; 148). This separation in origin has not been elucidated in other

species

Lymphocytes of the epithelium and lamina propria constitute the diffuse lymphoid

tissue of the GALT as opposed to isolated follicles, Peyer's patches, lymphocyte-filled villi

and cryptopatches, which constitute the organized lymphoid tissue (140; 149). Functionally,

diffuse and oganized lymphoid structures are effector and inductor sites, respectively (140).

In the porcine small intestine, the crypt areas of the lamina propria are rich in plasma cells

while T cells, most them ofCD4+ phenotype although there are also some CD8T, localize

within the villi. The CD4+ cells are in close association with the epithelium, as they are

located right underneath the basement membrane (150). The IEL constitute approximately 20

percent of the epithelial cells and are distributed mainly in the villus. In a recent study, it was

reported that in adult pigs approximately 50 % of the IEL were CD2+ CD4" CD8+ and were

localized in the proximity of the basement membrane. The remaining, CD2+ CD4" CD8" IEL,

were aligned with the enterocyte nucleus except for a small percentage located towards the

apical side (151). This study did not include additional markers, therefore, it cannot be

concluded to which T cell lineage (i.e.,oc(3 oryÔ) these cells belong. When the lymphocyte

composition of the intestinal mucosa was first evaluated, monoclonal antibodies to the

yd TCR were not available yet. For this reason the classification has been incomplete until

recently. yS T cells have been found in both epithelium and lamina propria (152). Currently,

there are no publications describing the lymphocyte composition of the large intestine of the

pig. Immunohistochemical analyses done in our laboratory have shown similar distribution of

23

CD4\ CDS and y§ cells in the colon: CD4+ cells are only present as LPL and, CD8T and

TCRyS localize to both compartments (unpublished data). However, the exact percentages of

and numerical evaluation of each cell type has not been completed yet.

Role of T cells in the development and regulation of intestinal inflammation

Cellular mechanisms involved in intestinal mucosa homeostasis

Cells of the systemic/central immune system are constrained within a sterile

environment and will react to any antigen recognized as non-self. However, the G ALT has

evolved to be in a non-sterile environment, under constant antigenic challenge. Several

mechanisms operate for maintaining tolerance towards antigens from food origin and from

the enteric normal flora. At the same time, the G ALT has to keep its capacity to react to

pathogenic microorganisms (144). Thus, establishment of equilibrium between active

responses and tolerance ensures the maintenance of the barrier function across the mucosa

(153).

Anergy, or lack of reactivity, is one of the mechanisms by which tolerance towards

food antigens or intestine-associated commensal flora is maintained (144). Several theories

have been proposed to explain how anergy is established. It has been hypothesized that

antigen presentation in the absence "danger signals" leads to anergy (154; 155). Such signals

originate from bacterial products referred to as pathogen-associated molecular patterns

(PAMPs) (e.g., lipopolysaccharide or peptidoglycan) which are specifically recognized by

pattern recognition receptors (PRR), such as toll-like receptors (TLR) on the surface of APC

(156; 157). Signaling through TLR results in the surface upregulation of costimulatory

24

molecules thus, in the absence of PAMPs, APC will not provide sufficient costimulation,

which will result in incomplete T cell activation, or even apoptosis (i.e., clonal deletion).

The route of antigenic exposure is an important parameter that contributes to the

maintenance of tolerance. In normal conditions, antigens from the intestinal lumen are

sampled through M-cells (i.e., at the small intestine), or through the apical side of the

absorptive epithelium (138; 140). Enterocytes are polarized cells (i.e., their plasma

membrane is divided into domains with different properties). TLR are expressed by

absorptive enterocytes, but only at the basolateral side (158). Tight junctions prevent

paracellular transport across the epithelium (159-161), therefore, this physical separation

between luminal contents and the basolateral side of enterocytes blocks recognition of

bacterial products by TLR or other PRR. However, whenever the integrity of the intestinal

epitheluim is compromised, the basolateral side of the enterocyte will be exposed to luminal

bacteria, which will ultimately lead to an inflammatory response (162; 163).

Several experimental models have been developed for the study of intestinal

inflammation (153; 164). Irrespective of the etiopathogenesis for each model, a common

denominator is that inflammation results from a break in tolerance and/or an imbalance in the

production of molecules that maintain intestinal homeostasis (153). Cellular interactions at

the intestinal mucosa are extremely complex, however, because of the focus of this

dissertation, only CD4+and yÔT cell involvement in the development and regulation of

intestinal inflammation will be discussed below.

Role of CD4 in the establishment and resolution of inflammation

CD4+T cells have been implicated both in the development of colitis and in the

maintenance of tolerance. Following antigen presentation to naïve CD4+T cells, they

25

differentiate into either Thl or Th2 effector cells (165; 166). The Thl phenotype is

associated with cellular responses modulated by proinflammatory cytokines, mainly IFN-y

and DC-derived IL-12 (167; 168). On the other hand, Th2 polarized responses are

characterized by the predominance of cytokines that favor humoral responses. Increased

production of IL-4 or IL-5 is an indicator of an ongoing Th2 response (166). The Thl/Th2

paradigm was first investigated in vitro using differentiated T cell clones. Usually, in vivo

both Thl and Th2 cells are present and there is a reciprocal regulation between cells of each

type (169). However, genetic and environmental factors may alter this equilibrium resulting

in a biased Thl or Th2 effector response (170).

Even though both polarized Thl and Th2 responses can be involved in the induction

of intestinal inflammation, this pathology is more commonly associated with Thl than with

Th2 responses (171). It has been speculated that the reason for this is that bacteria of the

normal flora, which play a critical role in the pathogenesis of inflammation, trigger the

production of IL-12 by DC, and therefore, the differentiation of naïve T cells into effector

Thl cells (153; 172-174). In line with this mechanism of pathogenesis, the cellular

mechanism underlying B. hyodysenteriae-induced colitis would be activation of intestinal

resident DC by B. hyodysenteriae and members of the normal flora. These activated DC

migrate to colonic lymph nodes, where they present antigens to naïve T cells in a

microenvironment influenced by IL-12 that favors the differentiation of naïve T cells into

Thl cells. Subsequently, bacterial-sensitized T cells would home to the colonic mucosa,

establishing a predominant Thl profile.

Cytokines produced in the context of a polarized Thl response, mainly IL-12 and.

IFN-y, inhibit the development of Th2 cells (175; 176). It has been proposed that the absence

26

of an active Thl response favors the differentiation of Th2 cells (168), while IL-10 and TGF-

P, produced by T reg cells, inhibit the production of IL-12, and therefore the development of

Thl cells (177; 178). During an inflammatory response driven by Thl cells, Th2 cells

migrating into the mucosa could block the effects of activated Thl effector cells responding

to members of the normal flora. This model could explain why vaccination with a B.

hyodysenteriae bacterin protects against swine dysentery. Basically, whereas challenge

would induce a Thl-type response, parenteral immunization would favor the development of

Th2 effector cells. Following infection, Th2 cells could migrate either to the colonic lymph

nodes and block the differentiation of naïve T cells into Thl cells, or directly to the mucosa

and inhibit effector Thl cells. Studies in mice have shown that infection with B.

hyodysenteriae increased IFN-y-producing cells in mesenteric lymph nodes and spleen, and

favored immunoglobulin isotype switching to IgG2a, which is characteristic of a Thl

response. However, cytokine analysis on vaccinated mice did not show increased production

of IL-4, which would indicate a predominant Th2 response. The lack of IL-4 and increased

production of IL-10 and TGF-(3 suggest that, alternatively, vaccination could stimulate

regulatory T cells (T reg), rather than inducing a Th-2-type response (179).

T reg cells have been identified in mice as memory CD4+ CD45RB10 cells that

provided protection against colitis induced by naïve CD4* CD45RBhl cells, when both were

transferred into a T cell-deficient SCID mouse (180). Functionally, T reg cells are

characterized by the production of TGF-P and IL-10, but not IL-4 (181). Additionally, T reg

cells have been shown to inhibit auto-reactive T cells, therefore, preventing autoimmunity

(82; 182). It could be hypothesized that immunization with B. hyodysenteriae stimulates T

27

reg cells, which would block the effect of Thl cells reacting to antigens of the normal flora

by producing TGF-P and IL-10.

yS T cell involvement in the control of inflammation

In regards to the involvement of yô T cells in intestinal inflammatory disorders, it

appears that they do not have a crucial role in the development of inflammation (153).

However, it has been suggested that they contribute to the process of epithelial restitution

that follows severe inflammation (72; 183; 184) and to downregulation of inflammatory

responses. Several models of infectious disease in which inflammation is present indicate

that a(3 T cells are increased initially, whereas y5 T cells are elevated later in the course of

the disease (185-188). The use of mice deficient in yô T cells indicated that they are

specifically implicated in the resolution of inflammatory damage. For instance, lesions

associated with infection with Listeria monocytogenes were more persistent in mice depleted

ofyô T cells (189; 190). It has been reported that during L. monocytogenes infection, yÔ T

cells shut down inflammation by inducing apoptosis of activated macrophages (191).

Another observation of these studies is that yô T cell responses are not specific for the

microorganism causing the disease (185). The fact that a subset of murine intraepithelial,

skin resident, yô T cells, (i.e., dendritic epidermal T cells (DETC)), were activated in vitro by

transformed or injured, but not healthy keratinocytes, suggested that stress-induced antigens,

rather that bacterial products might activate yô T cells (192; 193).

Although yS T cells are present both in the intestinal lamina propria and epithelium,

special emphasis has been given to the contribution of yô IEL to the resolution of

inflammation (194). yô IEL produce chemokines (e.g., RANTES or lymphotactin) for the

recruitment of lymphocytes and macrophages (e.g., MCP-1) (195). Also, neutrophil

28

recruitment into bacterial-induced pulmonary lesions was shown to be dependent on the

presence ofyô T cells, and correlated with protection from pneumonia (196; 197).

Additionally, yÔ IEL produce growth factors that promote proliferation and differentiation of

epithelial cells (198). The possibility of keratinocyte growth factor (KGF) being produced by

yS T cells was initially investigated in the context of the close interaction between epidermal

keratinocytes and DETC (198; 199). KGF belongs to the fibroblast growth factor family

(200). KGF was initially believed to be exclusively produced by stromal cells, mainly

fibroblasts, and act in a paracrine fashion on epithelial cells (201). Expression of KGF has

been detected in epithelial cells in vivo, and in DETC stimulated with anti-CD3 in vitro (202-

204). Also, murine DETC isolated from wounded skin expressed KGF mRNA, whereas

DETC obtained from healthy skin did not. In addition, skin lesions in mice lacking yô T cells

healed two to three days later than in wild-type mice (205).

A direct correlation between the production of KGF during intestinal epithelium

restitution by intraepithelial yô T cells had not been demonstrated until very recently. In a

murine model of dextran sodium sulfate (DSS) -induced colitis the number of yô IEL were

increased at the colonic mucosa during the process of tissue repair. These cells were

associated with damaged areas and were shown to produce KGF by reverse-transcriptase

polymerase chain reaction. In addition, both KGF- and TCRô-deficient mice had a delay in

recovery from DSS-induced colitis (206).

In summary, three basic mechanisms have been reported in regard to the participation

of yô T cells in mucosal protection. First, by recruiting cells which contribute to control of

infection. Second, by eliminating activated cells that otherwise would perpetuate

inflammation, such as activated macrophages or CD4+ T cells; and third, by producing

29

growth factors that contribute to regeneration of the intestinal epithelium and to restoration of

the epithelial cell barrier (27).

References

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CHAPTER 2. DEFERENTIAL REQUIREMENTS FOR THE PROLIFERATION OF

PORCINE CD4+ AND yS T CELLS TO EXTRACELLULAR BACTERIAL

ANTIGENS

A manuscript to be submitted to Immunology

Raquel Hontecillas, Josep Bassaganya-Riera, and Michael J. Wannemuehler

Abstract

dp and yô T cells have different mechanisms of epitope recognition and are

stimulated by antigens of different chemical nature. Humans and mice have very low

numbers of circulating yô T cells, whereas in ruminants, chickens, and pigs this T cell subset

constitutes a significant fraction of the peripheral blood mononuclear cells (PBMC). Both

peripheral blood CD4+ and yô T cells from pigs immunized with a pepsin-digested bacterin of

the spirochete Brachyspira hyodysenteriae proliferate in vitro to stimulation with spirochetal

antigens. This antigenic preparation was used to assess the requirements for the proliferation

of each of the two subsets in mixed lymphocyte cultures. While CD4+ T cells only responded

to stimulation with B. hyodysenteriae antigens, but not to exogenous recombinant porcine

1L-2 whereas yô T cells proliferated when cultures were stimulated with either bacterial

antigens or exogenous recombinant IL-2. PBMC that had proliferated after 5 days in culture

expressed high levels of IL-2Ra and neutralization of IL-2 at the beginning of the culture

period was more efficient in blocking yô T cell than CD4+ T cell proliferation. After 5 days in

culture, CD4+ cells were the major source of IFN-y, while only a small number of yô T cells

had the capacity to produce this cytokine. Together, these results indicate that porcine CD4+

and yô T cells differ in the mechanisms that drive their proliferation, and in their effector

functions.

44

Introduction

Porcine lymphocytes, and more specifically T cells, are characterized by the

heterogeneous expression of the CD8aa coreceptor and by their diffuse distribution into the

different compartments of the immune system (I; 2). Although some murine or human T

cells also express CD8aa, numerically, the CD8aa+ subsets are not as abundant as in pigs.

In addition, whereas in humans and mice certain cell types are only present at specific

anatomic locations (e.g., murine yÔ CD8GCGC+ T cells within the intestinal epithelium), this

clear association between phenotype and tissue distribution has not been demonstrated in the

pig. Particularly, porcine peripheral blood mononuclear cells (PBMC) are characterized by

the presence of mature CD41" CD8aa" T cells (3) and by having very high numbers of yÔ T

cells (2).

yô T cells differ from afi T cells in several aspects. Through the TCR, a(5 T cells

recognize peptides presented in association with MHC-class I or II molecules on the surface

of target cells (i.e., CD8+ cytotoxic T cells) or APC (i.e., CD4+). yô T cells react to antigens

with chemical composition other than proteins (e.g., phosphoantigens) (4-6) and, although

costimulation is necessary for cell activation (7), antigen recognition does not require

processing and presentation by professional APC (8). In addition, the yô TCR is MHC-I and

II-unrestricted; instead, non-classical MHC molecules, such as T10 and T22 (8), or CD1 (9)

are ligands for the yô TCR.

Functional studies of porcine CD4+ CD8acT T cells have indicated that this cell type

represents memory/effector T helper cells. Moreover, in vitro experiments showed that

45

antigen-experienced CD4+ CD8aa~T cells from peripheral blood upregulate

CDSaa following a subsequent antigenic exposure (10; 11). Thus, it is currently accepted

that CD4^ CDSaa-T cells are the precursors of CD4+ CDSaa* T cells. With regard to yô T

cells, two major subsets have been identified based on the expression of the molecule SWC6

(12). Porcine SWC6+ cells constitute the majority of the circulating yô T cells and, although

their function is currently unknown, they are capable of inducing nonspecific cytotoxicity in

vitro (13; 14). The SWC6' yÔ T cells are phenotypically characterized as CD2+ CD6+ (15),

and a fraction of this subpopulation additionally expresses CDSaa (16). SWC6" yô T cells

are found in spleen, secondary lymphoid organs and they are also detectable as circulating

lymphocytes, although in lower numbers than SWC6+ yô T cells (15)

Porcine CD4+ and yÔ T cells are thought to contribute to cell-mediated immune

responses to extracellular bacterial antigens (17). Previous experiments demonstrated that

immunization with a pepsin-digested bacterin of Brachyspira hyodysenteriae, a gram-

negative spirochete, increases the pool of circulating CD8aa+ PBMC (18), which also

proliferate to B. hyodysenteriae specific antigen in vitro. Further analysis, indicated that

CD4+ and yÔ subsets constitute the majority of the T cells proliferating in vitro to antigenic

stimulation. In addition, CD4+ T cell depletion or blockade of antigen presentation through

MHC-II greatly diminished proliferative responses of the PBMC (17). Based on these results,

immunization with this bacterin induces a MHC-II-dependent CD41" T cell response.

However, because the vaccine affects both CD4 and yô" T cells, additional mechanisms of

activation might exist besides antigen presentation through MHC-II. We chose this

immunization regime as a model to investigate differential functional aspects of the two cell

types in the pig. In the present study, it was determined that the requirements for the

46

proliferation of porcine CD4+ and yô T cells are different: while CD4+ T cell responses were

strictly antigen-dependent, yô T cells proliferated, in addition to specific antigens, to

stimulation with exogenous IL-2. It is also shown that most of the IFN-y produced in culture

originates from CD4+ T cells, whereas, in spite of being one of the most numerous cell type

among PBMC, yô T cells have very reduced capacity of IFN-y production in culture.

Materials and methods

Animals and immunization

Four-week-old cross-bred pigs were immunized with either a squalene-adj uvanted

protease-digested preparation of Brachyspira hyodysenteriae, or adjuvant alone as previously

described (18-21). B. hyodysenteriae strain B204 was grown in tripticase soy broth (Becton

Dickinson; Cockesville, Maryland) supplemented with 5% horse serum (Hyclone, Logan,

Utah), 0.5% yeast extract (Difco, Detroit, Michigan) and VPI salts under anaerobic

conditions. Lyophilized bacterial cells were digested by incubation with pepsin (10"3 g, per g

of lyophilized B. hyodysenteriae) at pH 1.9-2.2 for 25 h at 37° C. The vaccine was prepared

by mixing at 1:1 (v:v) the pepsin-digested B. hyodysenteriae with the adjuvant (10% of

squalene/pluronic acid (80:20 v:v) in 2% Tween 80-Saline. Pigs received 3 intramuscular

doses of either vaccine or adjuvant alone at 15 day intervals. All animal experiments were

approved by the Iowa State University Committee on Animal Care.

Isolation of peripheral blood mononuclear cells

One week after the last immunization PBMC were obtained from whole blood by

gradient centrifugation. Briefly, a 1:4 dilution of blood in PBS was overlaid onto 4 mL of

1.077 lymphocyte separation media (Mediate, VA), and centrifuged at 800 x g for 40

47

minutes. PBMC from the plasma/ media interface were aspirated with a pasteur pipete,

washed 3 times in PBS, and resuspended in complete RPMI 1640 medium (10 % fetal calf

serum (Hyclone, UT), 25 mM HEPES buffer (Sigma., St. louis, MO), 100 units/ml penicillin

(Sigma), 0.1 m/ml streptomycin (Sigma), 5 x 10*5 2-mercaptoethanol (Sigma), ImM sodium

pyruvate (Sigma), 1 mM nonessential aminoacids (Sigma), and 2 mM essential amino acids

(Mediatech). Cells were enumerated with a Coulter Z1 Single Particle Counter (Beckman

Coulter, Miami, Florida).

Proliferation assay

A total of 20 x 106 PBMC isolated following the procedure described above were

labeled with the green fluorescent dye PKH67 (Sigma) following manufacturer instructions.

Briefly, PBMC were aliquoted into 15 mL polystyrene conical tubes and centrifuged at 400 x

g for 5 min. After eliminating the supernatant, cells were resuspended in 1 mL of Diluent C

(Sigma). The cell suspension was immediately transferred to a 15 mL polystyrene conical

tube containing 1 mL of a 2 mM solution of PKH67 dye in Diluent C (Sigma). Cells were

incubated with the dye for 5 min and the reaction was stopped by the addition of 2 mL of

Fetal Bovine Serum (Hyclone) and, after 1 min, 4 mL of RPMI 1640. To eliminate excess

fluorescent dye, cells were washed 3 times in RPMI 1640 and resuspended in complete

RPMI to a final 2xl06 cells/mL.

PBMC at 106 cells/ mL were seeded in 96 well flat-bottom microliter plates and

incubated with complete RPMI only, the pepsin digested preparation of B. hyodysenteriae

B204 antigen at 5 gg/mL, Concanavalin A at 5 ng/mL (Sigma) or 5 ng/mL of recombinant

porcine IL-2 (Biosource, MA), in a 200 p.L final volume. After 5 d in culture, PBMC were

harvested and labeled for the analysis cell surface marker expression by flow cytometry as

48

previously described ( 17). For IL-2 neutralization experiments, polyclonal anti-porcine IL-2

(R&D Systems, MN) was added at the beginning of the culture period at 3 gg/mL.

The optimal dose of recombinant IL-2 and the inhibitory concentration of polyclonal

anti-porcine IL-2 were determined in a titration experiment in which PBMC were incubated

with increasing amounts of recombinant IL-2, ranging from 0 to 20 ng/mL. Neutralizing IL-

2 antibodies were added at 0, 0.75, 1.5 or 3 mg/mL to triplicate cultures of the various

amounts of recombinant IL-2. Proliferation was assessed by titriated thymidine

incorporation. Results showed that addition of more than 5 ng/mL of recombinant IL-2 did

not result in stronger stimulation and that 3|ig/mL of polyclonal anti-IL-2 completely

inhibited exogenous IL-2-induced proliferation.

Antibodies used in flow cytometry

The following antibodies were used in the proliferation assay for the detection of cell

surface marker expression: mouse IgG2b anti-porcine CD4 (74-12-1), biotinylated mouse

IgG2a anti-porcine CD8a (76-2-11), mouse IgGlanti-porcine TCR5 chain (PGBL22A,

VMRD, Pullman, WA), mouse IgGl anti-porcine CD3 (8E6, VMRD), mouse IgG2a anti-

porcine CD8(3 (PG145A, VMRD), and mouse IgGl anti-porcine CD25 (PGBL25A, VMRD).

Intracellular IFN-y detection

PBMC were incubated in 96 well flat-bottom microliter plates at 106 cells/ mL with

either medium alone, or with the pepsin digested preparation of B. hyodysenteriae B204

antigen 5 |ig/mL, in a final volume of 200 |AL. On d 5, cultures were stimulated with 50

ng/mL of phorbol myristate acetate (PMA) (Sigma) and 100 ng/mL ionomycin (Sigma).

Golgi transport was blocked by the addition of 4 |xL of Golgi Stop (Pharmingen, San Diego,

CA) for every 6 mL of cell culture. After CD4/CD8a and TCRyô/CDSa cell surface staining,

49

cells were fixed and permeablized with the Cytofix-cytoperm kit (Pharmingen) following

manufacturer's instructions. PE-labeled mouse IgGl anti-porcine IFN-y (P2G10,

Pharmingen) was used for intracellular cytokine detection. Equally stained non-PMA and

ionomycin-stimulated cultures were used as negative controls, and mouse IgGl-PE (Sigma)

was used as isotype control.

Statistical analysis

Data were analyzed as a randomized complete block design. Analysis of variance (ANOVA)

was used to investigate the main effects of vaccination. ANOVA was performed using the

general linear model procedure (GLM) of SAS. Differences with P < 0.05 were considered

significant.

Results

Lymphocyte activation and proliferation in vitro

Previous studies demonstrated that immunization with a B. hyodysenteriae bacterin

induced antigen-specific T cell proliferation (21). In order to further investigate these

responses, PBMC from control and immunized animals were cultured with medium alone, or

with pepsin-digested whole cell B. hyodysenteriae antigens. As it had been reported before, T

cells from immunized animals proliferated in vitro in the presence of the pepsin digested B.

hyodysenteriae antigens as determined by the decrease in PKH67 mean fluorescence

intensity of CD3+ PBMC (figure 1 A). The same results were obtained by 3H-thymidine

incorporation (data not shown). In addition, a fraction of CD3" cells from immunized pigs

(5.6 ± 0.04 vs. 1.3 ± 0.34 % in non-immunized) also proliferated to antigenic stimulation,

although we did not phenotypically characterize the responding CD3~ lymphocytes.

50

Anti-CD25 staining of PBMC from immunized pigs cultured with B. hyodysenteriae

antigens (figure IB) indicates that antigenic stimulation upregulated cell-surface IL-2Ra .

Based on the expression of CD25 on cells from immunized pigs that had proliferated, IL-

2Ra upregulation correlated with cell division. However, by d 5, a small proportion of non-

stimulated PBMC from immunized pigs (6.09 ± 0.9), or PBMC from control pigs cultured

with either medium (7.5 ± 2.7) or B. hyodysenteriae antigens (8.9 ± 0.02) stained positive for

IL-2Ra, although these cells failed to proliferate.

Subset analysis of T cells responding to B. hyodysenteriae

In order to dissect the pool of T cells that had proliferated, CD4T(i.e., helper a(3 T

cells), CD8afT (i.e., cytotoxic a(3 T cells), and yô T cells were individually analyzed. An

additional T cell subset (i.e., TCR CD4~ CD8aaT) has been identified in porcine PBMC;

however, due to the lack of monoclonal antibodies specific for either the porcine TCRa or (3

chains, it was not possible to analyze this subset individually. Proliferation results show that

6.4 ± 2.3 % of CD4+ and 8.3 ± 3.97 % ofy5 T cells isolated from immunized pigs

proliferated in vitro to B. hyodysenteriae antigens. Thus, these two subsets account for the

vast majority of the T cells responding to antigenic stimulation (figure 2). No proliferative

responses were detected in CD8a|3+ T cells. Additionally, when non-specific T cell responses

were detected (i.e., PBMC from immunized pigs cultured with medium or PBMC from

controls cultured with B. hyodysenteriae antigens), it was due to proliferation of yô T cells.

Both CD4+ and yô T cells can be further subdivided based on the expression of

CD8a. In order to determine differential responses within CD4+ or y8 T cell subsets, cells

were stained with both anti-CD4 and anti-CD8a, or anti-TCRS chain and anti- CD8a for the

phenotypic analysis of cells that had proliferated after 5 d in culture. Results show that when

51

PBMC from immunized pigs were stimulated with B. hyodysenteriae antigens, 9.9 ± 3.9 % of

CD4* cells were CD4+ CDSaa" PKH67 10 and 12.4 ±6 % were CD4+ CDSaa*

PKH67 lo. With regard to the yô T cell subset, 17.13 ± 8.0 of the cells that proliferated were

yÔ CDSaa", wheras only a small fraction of TCRyS CDSaa* (1.4 ± 0.5 %) proliferated in the

presence of B. hyodysenteriae antigens (Table I). Also, non-specific background

proliferation (7.4 ± 2.4) was observed for TCRyô CDSaa" T cells isolated from non-

immunized animals in cultures where bacterial antigens were added.

Differential requirements for proliferation of CD4* and y& T cells

Lymphocytes that had proliferated during the 5 d in culture upregulated and

maintained cell surface CD25 expression (figure I). In order to determine the IL-2

requirements for activation and proliferation of the different cell types responding to

antigenic stimulation, PBMC were cultured with either B. hyodysenteriae pepsin-digested

antigen or medium supplemented with recombinant porcine IL-2. This cytokine alone was an

insufficient stimulus for a(3 T cells (i.e., CD4+ and CD8a0*) to proliferate (figure 3A).

However, in some instances, CD4+ T cells weakly responded (data not shown). Cell

phenotype based on CDSaa expression indicated that only CD4*CD8aa~, but not

CD4*CD8aa* T cells could undergo cell division following stimulation with exogenous IL-

2. In contrast to a(3 T cells, a significant proportion of yô T cells (13.21 ±5.7) responded to

the addition of this cytokine (figure 3 A). The majority of the yô T cells that proliferated were

CDSaa" as well. No significant differences were found between PBMC isolated from control

or B. hyodysenteriae-immunized pigs in their proliferation to the addition of exogenous IL-2.

Treatment of cultures with neutralizing anti-IL-2 antibodies at the beginning of the

culture period, blocked proliferation of yô CDSaa" T cells to exogenous IL-2, and resulted in

52

50 % reduction of the proliferative response of this cell type when PBMC from immunized

pigs where stimulated with antigen (figure 3 B). With regards to the CD4+ pool, proliferation

of CD4+CD8aa+ T cells to B. hyodysenteriae diminished from 21.01 ± 4.58 to 11.9 ± 3.6 %

by the addition of lL-2-neutralizing antibodies (figure 3B).

CD4* T cells are the major source of IFN-y

Previously, we reported the presence of IFN-y-producing cells in B. hyodysenteriae-

stimulated PBMC from vaccinated pigs. Additionally, we showed that the IFN-y response

was CD4+T cell-dependent because in vitro CD4T cell depletion decreased the numbers of

IFN-y-producing cells (18). In the present study, intracellular cytokine staining results

indicate that CD4+T cells are the major source of IFN-y in PBMC (figure 4). In addition,

when CD4* T cells were stimulated with B. hyodsyenteriae, the percent IFN-y+-producing

cells decreased (2.53±1.09 to l.07±0.1) along with the mean fluorescence intensity of the

cytokine staining (1758.7±114 to 436±18.). This effect was observed in all pigs, irrespective

of the in vivo treatment, although it was more marked in CD4+ cells from immunized pigs.

On the other hand, very few yô T were able to produce IFN-y in vitro. However, the percent

of cytokine-positive yÔ T cells increased in both vaccinated (from 0.687± 0.158 to l.52±0.82)

and controls (from 0.69±0.19 to 1.157 ±0.827) after stimulation with B. hyodysenteriae

antigens.

Discussion

Our results show that both a(3 and yô T cells respond in vitro following antigenic

stimulation, however, the requirements for activation are different for the two T cell subsets.

The fact that cells that had divided after 5 d in culture (i.e., lower intensity of PKH67

53

staining) expressed high levels of CD25, and that IL-2 neutralization blocked proliferation,

suggest that IL-2 is necessary for porcine T cell proliferation. However, based on the

responses to exogenous IL-2, the addition of this cytokine alone is insufficient to induce

CD4+ and CD8aP T cell division. The antigen used in vitro in these experiments was the

pepsin-digested preparation of B. hyodysenteriae. As it would be expected for an

extracellular antigen. CD4+ (i.e, T helper) but not CD8aP T cells (i.e., cytotoxic T cells)

responded to antigenic stimulation. Thus, both IL-2Ra and TCR-derived signals were

necessary for «P T cell activation as measured by proliferation assays.

In contrast to aP T cells, yô T cells proliferated in response to specific antigen as well

as to addition of exogenous IL-2. Earlier studies have shown that blockade of antigen

presentation through MHC-II. or culture of PBMC depleted from CD4+ T cells diminished

yô T cell proliferation to B. hyodysentariae antigens (17). It could be interpreted that yô T

cell proliferation is secondary to CD41"T cell activation. In this regard, it could be proposed

that B. hyodysenteriae-antig&n specific cells are within the CD4™ pool, and yô T cell

proliferation is bystander to the activation of antigen-specific CD4+ T cells. A similar

interaction between CD4+and yô T cells was described for in vitro pseudorabies virus-

stimulated PBMC from immunized pigs (22). It remains unknown whether, in addition to IL-

2, TCR signals are needed for proliferation of porcine yô T cells.

Yang et al. reported that, in contrast to (%P T cells, porcine yô T cells failed to

proliferate in vitro following stimulation with a panel of anti-CD3 monoclonal antibodies.

These results suggest that signal transduction events that lead to TCR activation are distinct

for porcine aP and yô T cells (23). Comparative studies of the structure of the human aP and

yô TCR have shown differences that constitute the basis for some of the biological properties

54

of each the two cell populations. Among these, the fact that the hinge region of the yô TCR

has additional amino-acid residues, and that the domain that has been proposed to interact

with CD3E is shorter in the <*P TCR, suggest that aP and yô TCR form different signaling

complexes (24). In fact, the molecular composition of the TCR-CD3 complex in human and

murine yô T cells differs from aP T cells in that the first lacks CD3S subunits. However, at

the functional level, the threshold for TCR activation appears to be lower in yS than in (%P T

cells based on calcium mobilization, extracellular signal-regulated kinase (ERK.) activation,

and proliferation following stimulation with anti-CD3 (25).

Our results do not provide sufficient evidence to support the concept that all yô T

cells that proliferated in B. hyodysenteriae-stimulated cultures responded solely to IL-2-

induced stimulation. A portion of yô T cells could have responded independently from CD4T

T cells and, equally to ctp T cells, also require TCR activation for proliferation. This could be

explained by non-MHC-II-restricted specific recognition of B. hyodysenteriae epitopes by yô

T cells. This hypothesis is supported by the fact that yô T cell proliferation at background

levels was observed when PBMC from non-immunized pigs were cultured with bacterial

antigens. Also, because yô T cells proliferated in the absence of B. hyodysenteriae antigens

but in the presence of exogenous IL-2, an additional CD4* and B. hyodysenteriae antigen-

independent mechanism must exist for the activation of yô T cells. A CD41"-independent

pathway of yô T cell activation in vitro by an unknown ligand present on monocytes and

granulocytes has been described in cattle (26). Porcine and bovine yô T cells have similar

phenotypic characteristics. It is also probable that they also have functional aspects in

common. A similar ligand might also exist in porcine cells, however, while stimulation with

55

this ligand induced IFN-y production by bovine cells, in our experiments yô T cells failed to

produce this cytokine.

Several in vivo models have shown an increase in yô T cells following activation of

a(3 T cells (27-29). Such changes in the yô T cell population initially depend on the

production of IL-2 (30; 31). It has been suggested that a negative feed back loop is

established between yô and a(3 T cells by which, antigen-specific a(3 T cell responses are

inhibited by the expanded yô T cells (28). Activated yô T cells express high levels of Fas

ligand and can induce apoptosis of Fas* CD4+ T cells (32). Porcine yô T cells have non

specific cytotoxic activity in vitro, although the molecular basis has not been investigated.

Our results show that B. hyodysenteriae antigen stimulates CD4* CDSaa™ and CD4*

CDSaa* from immunized pigs to proliferate. In addition, antigenic stimulation decreased the

number of CD4+ cells producing IFN-y. It is possible that the activated yô T cells

proliferating in B. hyodysenteriae-stimulated cultures downregulated CD4+

CDSaa" and CD4+ CDSaa* responses (i.e., less IFN-y production).

The magnitude of in vitro T cell proliferation in antigen recall responses is a

functional parameter frequently used to evaluate the efficacy of a cellular immune response.

T cell proliferative responses to an antigen indicate the presence of antigen-specific cells, and

therefore the generation of immunological memory. We have shown in a mixed lymphocyte

culture of porcine PBMC that vaccination with a gram-negative spirochete bacterin generated

antigen-dependent CD4 T cell responses. Additionally, yô CDSaa" T cells proliferated in

vitro. We analyzed yô CDSaa* T cells separately because we had reported that CDSaa*

PBMC from B. hyodysenteriae-immunized pigs were increased in vivo, and proliferated in

antigen recall responses. In these experiments, yô CDSaa* T cells failed to respond to either

56

antigen or IL-2 stimulation. Future studies should address the subset composition (i.e.,

SWC6+ or SWC6") of the responding yô T cells.

Immunization with this bacterin protects pigs against B. hyodysenteriae-indxiced

colitis. We are currently investigating the function of CD4T and yô T cells at the colonic

mucosa during infection. Very few studies have addressed the role of porcine yÔ T cells in

vivo. SWC6+ yS T cells (i.e., CD2" CDSaa") were recruited to skin inflammatory lesions

induced phytohemagglutining injection (33; 34). In several murine models, yô T cells have

been reported to downregulate inflammation (28). If these responses were beneficial, it

would be of interest to elucidate the basis of the CD4T-yô T cell interactions and whether this

mechanism should be exploited for the treatment or prevention of inflammatory diseases.

Table 1. Cell surface phenotype of proliferating peripheral blood T cell subsets from control or immunized pigs '

Medium2 B. hyodysenteriae antigen

Phenotype3 Control Immunized Control Immunized

CD4* CDSaa" PKH671"4

CD4* CDSaa1- PKH67'0

yô CDSaa" PKH67loS

yô CDSaa' PKH6710

2.228 ± 0.998 1.263 ±0.709 3.804 ±2.061 0.438 ±0.279

2.244 ±0.826 0.961 ±0.261 7.226 ± 4.226 0.326 ±0.161

3.345 ± 1.422 1.831 ±0.891 7.366 ±2.414 0.689 ±0.401

9.894 ±3.971*6

12.394 ±6* 17.131 ±8.063* 1.383 ±0.487

1 Results are expressed as mean (n=5) percentage of CD4*or TCR5* cells that had proliferated (i.e., PKH6710) after 5 d in culture. " Peripheral blood mononuclear cells isolated from control and B. hyodysenteriae-immumzsd pigs were labeled with PK.H67 and cultured with either complete RPMI medium or 5 gg/mL of B. hyodysenteriae pepsin-digested antigenic preparation. 3 Cells were harvested on day 5 and double stained with monoclonal antibodies specific for CD4 and CD8a or TCRS and CD8a, and analyzed by 3 color flow cytometry. 10,000 events within the viable cell gate were acquired. 4 CD4* cells within the mononuclear viable gate were analyzed by plotting CD8a versus RK.H67. 5 TCR5* cells within the monocyte viable gate were analyzed by plotting CD8a versus PK.H67. 6 *, f-value < 0.05 when compared to the non-immunized control group.

57

Medium Medium

B

mn 0.7

1 i :-•» ft.

PKH67

Immunized Control

Figure 1. Proliferation (A) and CD25 expression (B) of PBMC from an immunized (left panel) and a non-immunized control pig (right panel) after 5 d in culture with either RPMI medium or B. hyodysenteriae antigens. PBMC were isolated one week after three intramuscular immunizations with a pepsin-inactivated B. hyodysenteriae bacterin. CD3 staining (top row) indicates that T cells from immunized pigs specifically responded to stimulation with spirochetal antigens. The forward versus side scatter plot shows the cells included in the analysis (i.e., live mononuclear cells). Plots are representative from 2 experiments with a total of 5 pigs per group.

58

Medium

O

ÎÏS16

PKH67

Medium B. hyo

I 0.2 26.6

a: 0.3 25.5

|*r

1.63 ' 0.6 - • £*:

•* rfl

0.3

Immunized Control

Figure 2. Proliferation of CD41", CD8afT and yô T cells from an immunized (left panel) and a non-immunizad control pig (right panel) after 5d in culture with either RPMI medium or B. hyodysenteriae antigens. PBMC were isolated one week after three immunizations with a pepsin-inactivated B. hyodysenteriae bacterin. CD4+ and y5 T cells from vaccinated pigs account for the majority of the CD3+ proliferating cells. The forward versus side scatter plot shows the cells included in the analysis (i.e., live mononuclear cells). Plots are representative from 2 experiments with a total of 5 pigs per group.

59

PKH67

B. hyo rpIL-2

Anti-lL-2: • + •

TCRS

PKH67

Figure 3. IL-2 requirement for the proliferation of porcine T cells.

A) y§ T cells proliferated to the addition of 5 ng/mL of exogenous porcine recombinant IL-2 while neither CD4 nor CD8«P+ T cells responded to stimulation with this cytokine alone. Plots correspond to 5 d cultured PBMC from a representative non-immunized control pig. Two experiments were conducted with a total of 4 pigs per group. Cells included in the analysis correspond to live mononuclear cells gated as indicated in the forward versus side scatter plot.

B) IL-2 neutralization blocked proliferation of y8 CD8~ T cells stimulated with exogenous porcine IL-2 (lower row in right panel). In PBMC cultures from immunized pigs stimulated with B. hyodysenteriae antigen, the addition of anti-porcine IL-2 antibodies at the beginning of the culture period diminished proliferation of both CD4+CD8aot+ (top row in left panel) and yô CDS" T cells (bottom row in left panel), although proliferative responses of the latter were more affected by IL-2 neutralization. CD4+ or TCR§+ T cells within the viable gate were analyzed by plotting CD8a versus PKH67 as indicated in the CD4 versus CDSa or TCR5 versus CDSa plots, respectively. Plots are representative of one experiment with 3 pigs per group.

60

I Medium B. hyo FSC ] 2:3

i 1 :

Z ! be. ;

0.3 2 1-1

4 0:5 < i

4

Medium B. hyo

1 0.6

•32.2 Iliiêf'

3.4 1.7

Immunized

O.R. 0.5 -

0.6 . . 0.5 Control

w : u CD4 TCR-Ô

Figure 4. IFN-y production by CD4+ and yS T cells from an immunized (top row) and a non-immunized control pig (bottom row). PBMC were isolated from controls and B. hyodysenteriae pepsin-digested-immunized pigs one week after the third immunization. After 5d in culture with either medium alone or B. hyodysenteriae antigens, cells were stimulated with PMA and ionomycin for 4 hours for the detection of intracellular IFN-y by flow cytometry. CD41" T cells are the major source of IFN-y, and stimulation with B. hyodysenteriae antigens modulates IFN-y production by CD41" and yô T cells. Initial gate was set on the mononuclear cell population as indicated in the forward versus side scatter plot. Plots are representative from one experiment of 3 pigs per treatment.

61

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8. Schild, H., Mavaddat, N., Litzenberger, C., Ehrich, E. W., Davis, M. M., Bluestone, J. A., Matis, L., Draper, R. K., & Chien, Y. H. (1994). The nature of major histocompatibility complex recognition by yô T cells. Cell 76: 29-37. 9. Spada, F. M., Grant, E. P., Peters, P. J., Sugita, M., Melian, A., Leslie, D. S., Lee, H. K., van Donselaar, E., Hanson, D. A., Krensky, A. M., Majdic, O., Porcelli, S. A., Mori ta, C. T., & Brenner, M. B. (2000). Self-recognition of CD1 by y/ô T cells: implications for innate immunity. J. Exp. Med. 191:937-948. 10. Zuckermann, F. A., & Husmann, R. J. (1996). Functional and phenotypic analysis of porcine peripheral blood CD4/CD8 double-positive T cells. Immunology 87: 500-512. 11. Zuckermann, F. A. (1999). Extrathymic CD4/CD8 double positive T cells. Vet. Immunol. Immunopathol. 72: 55-66. 12. Carr, M. M., Howard, C. J., Sopp, P., Manser, J. M., & Parsons, K. R. (1994). Expression on porcine yô lymphocytes of a phylogenetically conserved surface antigen previously restricted in expression to ruminant yÔ T lymphocytes. Immunology 81: 36-40. 13. Pauly, T., Weiland, E., Hirt, W., Dreyer-Bux, C., Maurer, S., Summerfield, A., & Saalmuller, A. (1996). Differentiation between MHC-restricted and non-MHC-restricted porcine cytolytic T lymphocytes. Immunology 88:238-46. 14. de Bruin, T. G., van Rooij, E. M., de Visser, Y. E., Voermans, J. J., Samsom, J. N., Kimman, T. G., & Bianchi, A. T. (2000). Discrimination of different subsets of cytolytic cells in pseudorabies virus-immune and naive pigs. J. Gen. Virol. 81: 1529-1537.

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15. Yang, H., & Parkhouse, R. M. (2000). Characterization of the porcine yô T-cell receptor structure and cellular distribution by monoclonal antibody PPT27. Immunology 99: 504-9 16. Yang, H., & Parkhouse, R. M. (1997). Differential expression of CDS epitopes amongst porcine CDS-positive functional lymphocyte subsets. Immunology 92: 45-52. 17. Waters, W. R., Hontecillas, R., Sacco, R. E., Zuckermann, F. A., Harkins, K.. R., Bassaganya-Riera, J., & Wannemuehler, M. J. (2000). Antigen-specific proliferation of porcine CDSaa cells to an extracellular bacterial pathogen. Immunology 101: 333-341. 18. Waters, W. R., Sacco, R. E., Dorn, A. D., Hontecillas, R., Zuckermann, F. A., & Wannemuehler, M. J. (1999). Systemic and mucosal immune responses of pigs to parenteral immunization with a pepsin-digested Serpulina hyodysenteriae bacterin. Vet. Immunol. Immunopathol. 69: 75-87. 19. Bassaganya-Riera, J., Hontecillas, R., Zimmerman, D. R., & Wannemuehler, M. J. (2002). Long-term influence of lipid nutrition on the induction of CDS* responses to viral or bacterial antigens. Vaccine 20: 1435-1444. 20. Bassaganya-Riera, J., Hontecillas, R., Zimmerman, D. R., & Wannemuehler, M. J. (2001) Dietary conjugated linoleic acid modulates phenotype and effector functions of porcine CDS* lymphocytes. J. Nutr. 131:2370-2377. 21. Waters, W. R., Pesch, B. A., Hontecillas, R., Sacco, R. E., Zuckermann, F. A., & Wannemuehler, M. J. (1999). Cellular immune responses of pigs induced by vaccination with either a whole cell sonicate or pepsin-digested Brachyspira (Serpulina) hyodysenteriae bacterin. Vaccine 18: 711-719. 22. Summerfield, A.. & Saalmuller, A. (1998). Interleukin-2 dependent selective activation of porcine yô T lymphocytes by an extract from the leaves of Acanthospermum hispidum. Int. J. Immunopharmacol. 20: 85-98. 23. Yang, H., & Parkhouse, R. M. (1998). Differential activation requirements associated with stimulation of T cells via different epitopes of CD3. Immunology 93:26-32. 24. Allison, T. J., Winter, C. C„ Fournie, J. J., Bonneville, M., & Garboczi, D. N. (2001). Structure of a human yô T-cell antigen receptor. Nature 411: 820-824. 25. Hayes, S. M., & Love, P. E. (2002). Distinct structure and signaling potential of the yô TCR complex. Immunity 16: 827-838. 26. Sathiyaseelan, T., Naiman, B., Welte, S., Machugh, N., Black, S. J., & Baldwin, C. L. (2002). Immunological characterization of a yô T-cell stimulatory ligand on autologous monocytes. Immunology 105: 181-189. 27. Carding, S. R., Allan, W., Kyes, S., Hayday, A., Bottomly, K., & Doherty, P. C. (1990). Late dominance of the inflammatory process in murine influenza by y/ô * T cells. J. Exp. Med. 172: 1225-1231. 28. Born, W., Cady, C., Jones-Carson, J., Mukasa, A., Lahn, M., & O'Brien, R. (1999). Immunoregulatory functions ofyô T cells. Adv. Immunol. 71:77-144. 29. Doherty, P. C., Allan, W., Eichelberger, M., & Carding, S. R. (1992). Roles of a(3 and yô T cell subsets in viral immunity. Annu. Rev. Immunol. 10:123-151. 30. Haque, A., Echchannaoui, H., Seguin, R., Schwartzman, J., Kasper, L. H., & Haque, S. (2001). Cerebral malaria in mice: interleukin-2 treatment induces accumulation of yô T cells

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in the brain and alters resistant mice to susceptible-like phenotype. Am. J. Pathol. 158: 163-172. 31. Choi, K. D., & Lillehoj, H. S. (2000). Role of chicken IL-2 on yS T-cells and Eimeria acervulina-induced changes in intestinal IL-2 mRNA expression and yô T-cells. Vet. Immunol. Immunopathol. 73: 309-321. 32. Vincent, M. S., Roessner, K., Lynch, D., Wilson, D., Cooper, S. M., Tschopp, J., Sigal, L. H., & Budd, R. C. (1996). Apoptosis of Fashlgh CD4T synovial T cells by borrelia-reactive Fas-ligandhlgh yô T cells in Lyme arthritis. J. Exp. Med. 184: 2109-2117. 33. Whyte, A., Haskard, D. O., & Binns, R. M. (1994). Infiltrating yô T-cells and selectin endothelial ligands in the cutaneous phytohaemagglutinin-induced inflammatory reaction.PG. Vet. Immunol. Immunopathol. 41: 31-40. 34. Whyte, A., Licence, S. T., Robinson, M. K.., & van der Lienden, K. (1996). Lymphocyte subsets and adhesion molecules in cutaneous inflammation induced by inflammatory agonists: correlation between E-selectin and yÔ TcR* lymphocytes. Lab. Invest. 75: 439-449.

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CHAPTER 3. MODIFICATION OF THE LYMPHOCYTE COMPOSITION AND

CYTOKINE ENVIRONMENT DURING Brachyspira hyodysenteriae-INDUCED

COLITIS BY SYSTEMIC IMMUNIZATION WITH A PROTEINASE DIGESTED

BACTERIN

A manuscript to be submitted to Immunology

Raquel Hontecillas, Josep Bassaganya-Riera, Jennifer Wilson. David L. Hutto, and Michael

J. Wannemuehler.

Abstract

Systemic immunization has proven to be of limited efficacy in preventing bacterial

mucosal infections. Here, we have examined the mechanism by which an enteric disease can

be ameliorated by parenteral immunization with a protease-digested bacterin of the

spirochete Brachyspira hyodysenteriae. We have previously shown that systemic

immunization with B. hyodysenteriae induced proliferation of CDSaa*, CD4* and yÔ* T cells

to specific B. hyodysenteriae antigens. Here, we have examined how the colonic profiles of

immunoregulatory and pro-inflammatory cytokines as well as the lymphocyte subset

distribution were influenced by immunization and/or challenge with B. hyodysenteriae.

Following immunization with the protease-digested B. hyodysenteriae bacterin, colonic

inflammation was triggered by challenging the pigs with B. hyodysentariae. Colonic mucosal

lesions and lymphocyte subset distribution were evaluated by histology and

immunohistochemistry. Immunoregulatory and pro-inflammatory cytokines were assayed in

colonic tissue. Immunization up-regulated TNF-a and IL-1 (5 mRNA expression. In addition,

TGF-(3l mRNA was increased in infected pigs previously immunized. Finally, vaccination

diminished the loss of yô T cells observed following the bacterial challenge and favored a

65

decrease in numbers of lamina proprial CD4+ cells. Therefore, we report a unique case of

parenteral immunization that regulates colonic lymphocyte subset distribution and cytokine

expression. This model of immunoregulation of colitis may have important applications in

the treatment of inflammatory bowel disease in humans.

Introduction

Enteric inflammatory processes associated with inflammatory bowel disease (IBD)

have been studied using a number of murine models of coltis (IBD) (I). Antigens expressed

by the intestinal normal flora contribute to the pathogenesis of IBD by triggering abnormal

immune and/or inflammatory responses (1; 2). Excessive activity of T helper (Th) cells (i.e.,

CD4*), especially polarized Thl, induces a cytokine imbalance often observed in Crohn's

disease patients (3-6). On the other hand, Th cells are controlled by regulatory T cells (i.e.,

Th3. Trl or CD4+ CD25+), mainly through the production of suppressor cytokines such as

transforming growth factor-^ (TGF-P) or IL-10 (7-10). Thus, defects in the regulation of T

cell responses contribute to the pathogenesis of inflammation by negatively affecting

mucosal homeostasis (1).

Brachyspira hyodysenteriae is a bacterial enteric pathogen that causes swine

dysentery, a severe hemorrhagic colitis (11). The bacteria invade the colonic mucosa without

spreading systemically (12). Similarly to IBD, the normal intestinal flora is involved in the

disease pathogenesis (13). One of the virulence factors of B. hyodysenteriae is a low

molecular mass P-hemolysin that was shown to induce apoptosis of intestinal epithelial cells

(14). Following loss of the epithelial cell barrier, excessive exposure of the underlying

mucosa to luminal antigens triggers a severe inflammatory response.

66

Parenetral immunization of pigs with a pepsin-inactivated bacterin of B.

hyodysenteriae ameliorates clinical and histopathological signs associated with swine

dysentery (15). CD8act\ CD4+ and yô T cells isolated from peripheral blood of immunized

pigs respond in vitro to stimulation with B. hyodysenteriae antigens (16-19); however, the

effect that vaccination has on these cell subsets in the colonic mucosa during infection has

not been investigated. In the present study it was shown that both vaccination and infection

modulated lymphocyte composition of the colonic mucosa. Particularly, it is shown that the

protection against disease induced by immunization correlated with a decrease in lamina

proprial CD4+ cells. Furthermore, although infected pigs had lower yÔ intraepithelial

lymphocytes (IEL), those that were previously immunized maintained higher numbers of this

lymphocyte subset. These phenotypic changes were accompanied by an increase in TGF-

P mRNA in colonic tissue recovered from immunized pigs. Immunization also increased the

expression of IL-1(3 and TNF-a. Thus, on the basis of these results, systemic immunization

modified the outcome of colonic inflammatory and immune responses.

Materials and methods

Experimental design

Immunization: a total of 12 four-week old pigs were immunized with either a squalene-

adjuvanted, pepsin-digested preparation of Brachyspira hyodysenteriae (n=6) or adjuvant

alone (n=6). The vaccine was prepared as previously described (20). Pigs received 3

intramuscular doses at 15 days intervals.

Challenge: two weeks after the third immunization, half of the control or immunized pigs

were orally challenged with two doses of 1010 CFU of B. hyodysenteriae strain B204

given on consecutive days. All cultures used for infection studies were >90% motile and

67

had been passed in vitro 23 to 25 times. After challenge, pigs were monitored daily for

clinical signs of disease (e.g., diarrhea, depression, anorexia, abdominal pain, and

dehydration). Stools were observed and scores were based on consistency: (0), no diarrhea,

stools formed: (1) mild, (2) moderate, and (3) severe diarrhea. Stools were also observed for

the presence of blood and/or mucus. Clinical response scores were calculated daily following

challenge by adding the diarrhea score (i.e., 0 to 3) plus a 1 for the presence of blood and/or a

1 for the presence of mucus. All animal related procedures were approved by the Iowa State

University Animal Care and Use Committee.

Necropsy: 15 days post-challenge pigs were anesthetized by administration of Rompun

(Bayer; Shawnee, KS)/Telazol (Fort Dodge Laboratories; Fort Dodge, IA) intramuscularly

and euthanized via electrocution. At necropsy, swabs of cecal and spiral colon contents were

streaked onto modified BJ blood agar plates containing antibiotics for isolation of B.

hyodysenteriae (21). Sections of spiral colon and cecum were obtained, fixed in 10%

buffered formalin, later embedded in paraffin, and then sectioned for histological

examination. Samples of colon and mesenteric lymph nodes were embedded in RNA Later

(Ambion Inc., Austin, TX) for isolation of total RNA and analysis of cytokine m RNA

expression. For immunohistochemistry, samples of colonic tissue were placed in tissue

freezing medium (Triangle Biomedical Sciences, Durham, NC) and snap frozen in

ethanol/dry ice bath.

Histopathological evaluation of colonic samples.

Hematoxilin-eosin (H & E)-stained sections from colon and cecum were labeled with

accession numbers lacking any reference to immunization or infection status (i.e., blinded to

evaluator). Histological evaluation was conducted on the basis of gland depth (i.e., expressed

68

as crypt depth as a function of crypt width), metaplasia/hyperplasia (i.e., increased cell

turnover and basal cell basofilia, or goblet cell metaplasia), lamina propria vascular change

(i.e., capillary dilation and lamina propria hemorrhage), inflammation and submucosal

change (i.e.. inflammatory cells infiltrates at the lamina propria or submucosa), and epithelial

erosions (i.e., 1 ) no erosion, 2) mild erosion and 3) severe erosion).

Determination of the lymphocyte subset composition of the mucosa by

immunohistology.

Frozen colonic tissue embeded in OCT tissue freezing medium were cut at 5 to 7 |im-

thick sections on a cryostat at - 18 °C. Sections were placed on poly-L-lysine coated slides,

fixed in 95% methanol for 2 minutes and soaked in cryopreservative solution (0.5 M sucrose,

0.006 M MgCh, 50 % glycerol) for 10 minutes. Slides were stored at - 20 °C until processed

for immunohistchemistry. Prior to staining tissues with monoclonal antibodies, slides were

warmed to room temperature and rehydrated in 0.5 M Tris solution. Endogenous peroxidase

activity was blocked by adding 0.3 % hydrogen peroxide for 10 minutes. Non-specific

binding was blocked by adding immunohistochemistry buffer containing 5 % normal goat

Serum/ 3 % bovine serum albumin/tris buffer (NGS/BSA/Tris) solution at room temperature

for 2 hours. Slides were incubated with the primary antibody solution overnight at 4 °C.

Primary antibodies were diluted in NGS/BSA/Tris. Sections were stained with anti-porcine

CD4 (74-12-1 hybridoma supernatant), anti-porcine-CD8a (76-2-11 hybridoma supernatant),

anti- porcine TCRÔ (PGBL22A, VMRD, Pullman, WA), or anti-porcine CD3 (8E6, VMRD).

Prior to the incubation with the secondary antibodies, slides were rinsed with Tris solution.

Peroxidase-conjugated goat anti-mouse IgG (H+L) (Jackson ImmunoResearch, West Grove,

PA) diluted at 1:300 in NGS/BSA/Tris was used for the detection of anti-porcine CD4, anti-

69

porcine CD3, and anti-porcine TCR8. Biotinylated goat anti-mouse IgG2a F(ab')i fragments

(Southern Biotechnologies Associates Inc. Birmingham, AL) were used for the detection of

anti-porcine CD8a primary antibody diluted in NGS/BSA/Tris at 1:250. All secondary

antibodies were incubated for 2 hours at RT. Slides were then treated with peroxidase-

conjugated streptavidin, diluted in Tris solution (1:500) and incubated for 1 hour (RT).

Positive antibody binding was visualized with diaminobenzediene (Biomedia Corporation,

Foster City. CA). Tissue sections were counterstained with Instant Hematoxylin (Shandon,

Pittsburg, PA) and coversliped with Immu-mount (Shandon). The numbers of CD4+, CD8a+,

CD3T, and TCRyfT cells in either epithelium or lamina propria were enumerated for each pig

and antibody staining. Data are presented as the sum of positive-staining cells in 5 randomly

chosen fields of 0.375 mm2 area, observed at 400 x magnification.

Isolation of total RNA.

Total RNA from full-thickness colonic tissue recovered with a 3 mm diameter punch

biopsy 15 days after challenge was isolated using the total RNA isolation MiniKit (Qiagen,

Valencia. CA) following manufacturer's instructions. RNA was diluted in 0.02% diethyl

pyrocarbonate (DEPC)-treated water (Qiagen). RNA concentration and purity were

determined using a spectrophotometer at an optical density (OD)2eo and OD26o/OD2so ratios,

respectively. All samples had OD260/OD280 ratios above 1.80 corresponding to 90-100% pure

nucleic acid. Samples were stored at —70°C until processed.

Ribonuclease protection assay

A custom template set (Pharmingen, San Diego, CA) was used for the synthesis of

antisense-RNA probes for the detection of porcine IL-4, IL-12p40, TNFa, IL-lfî, TGF-

Pt, IL-6, IFN-y, L32 and GAPDH mRNAs. [32P]UTP (Amersham Pharmacia)-labeled probes

70

were synthesized with an in vitro transcription kit (Pharmingen) following manufacturer's

instructions. Target RNA was hybridized overnight to the antisense RNA probes. Single-

stranded unbound RNA and free probe were subjected to RNases A and Tl digestion.

Protected double-stranded RNA duplexes were purified and resolved on a urea-

polyacrylamide denaturing gel. The gel was dried for I h at 80 C and bands were visualized

and quantified by phosphorimaging.

Statistical analysis

Post-challenge, data were analyzed as a 2 X2 factorial arrangement of treatments

within a split-plot design. Analysis of variance (ANOVA) was used to investigate the main

effects of vaccination and infection as well as the interaction between infective status and

vaccination. ANOVA was performed using the general linear model procedure (GLM) of

SAS. Differences with P < 0.05 were considered significant.

Results

Vaccination ameliorates the severity of B. hyodysenteriae-induced colitis

Following challenge, pigs were monitored daily for the development of clinical signs

associated with B. hyodysenteriae-induced colitis. No signs were observed until 4 days post

infection (figure 1). Disease progression from this point was similar for both of the groups

that were challenged although severity was milder in the group previously immunized with

the B. hyodysenteriae bacterin, as shown by the lower daily average score. Examination of

colonic sections recovered on day 15 post-infection revealed the typical microscopic lesions

associated with B. hyodysenteriae;-induced colitis. A total lesion score was generated based

on grading changes in gland depth, hyperplasia/metaplasia, lamina proprial vascular change,

71

submucosal change, inflammation, and superficial epithelial erosions (Table 1), as described

in the materials and methods section. No differences in total average cecal scores were found

between the different treatment groups (data not shown). In the colon, the average total score

of immunized and infected pigs was lower (15.6± 5.7) than that of the corresponding

challenged pigs not receiving immunization (19.6± 4.8). All infected pigs were culture

positive for B. hyodysenteriae the day they were necropsied. The histopathological score in

the non-infected groups was equal (11.3± 2.4and 11.33± 1.8) for the non-vaccinated and non-

infected and vaccinated and non-infected pigs.

Severe inflammation was manifested by increased mucosal thickness due to a

pronounced hyperplasia of crypt epithelial cells, which was accompanied by increased

cellular basophilia, and increased mitotic figures. This lesion was associated with multifocal

inflammatory cell infiltrates across the lamina propria muscularis (figure 2H). On the other

hand, pigs recovering from disease (as determined by clinical evaluation) showed goblet cell

metaplasia with excess mucus production (figure 2G). Non-vaccinated and infected pigs had

a 2.33 ± 0.44 hyperplasia/metaplasia average score, with 13 ± 2.67 gland depth whereas the

score for infected and vaccinated pigs were 1.66 ± 1.11 for hyperplasia/metaplasia, and

10.31 ±3.1 for gland depth. Thus, vaccinated and non-vaccinated groups differed in the

intensity rather than in the type of lesions developed following challenge.

Lymphocyte distribution in the colonic mucosa

Studies of the lymphocyte composition of the intestinal mucosa have revealed

regional specialization between the small and large intestine, and between lamina propria and

epithelium. Also, species-specific differences have been reported. Phenotypic

characterization of the small intestine of pigs revealed the absence of CD4+ cells within the

72

epithelium. Immunohistochemical analysis of colonic sections showed the same distribution

of CD4+ cells (figure 3B). yô T cells are present in both compartments of the colonic mucosa,

as lamina propria lymphocytes (LPL) and as intraepithelial lymphocytes (IEL) (figure 3C).

CDScT cells follow the same distribution as y8 T cells (not shown).

While yô LPL were dispersed in the tissue, clusters of CD4+ cells were present across

the lamina propria. These aggregates were more abundant in tissues recovered from B.

hyodysenteriae infected pigs (figure 4A-4C). In addition, immunohistological analysis of

longitudinal sections revealed that the multifocal cellular infiltrates found in the lamina

propria muscularis (figure 2H) were mainly composed of CD4+ T cells. yÔ T cells were also

detected in these lesions although in much smaller numbers (figures 4D-4F).

Changes in the lymphocyte subset composition of the colonic mucosa induced by

immunization and/or challenge.

Peripheral blood CD4^and yô T cells from immunized pigs respond in vitro to B.

hyodysenteriae antigens. In order to investigate whether immunization affects the

lymphocyte composition of the mucosa, CD3+, CD4+, yÔ T cells and CD8a* from the colonic

epithelium or lamina propria were enumerated. Immunization decreased the numbers of

CD4+ cells in the lamina propria (figure 5A). This effect was found irrespective of challenge

status. Changes were also detected at the epithelium, with lower numbers of epithelial T cells

(i.e., CD3+), consistent with a decrease in yô IEL. Vaccination, however, attenuated the loss

of this T cell subset (figure 5B).

Proinflammatory cytokine expression

A ribonuclease protection assay was utilized to investigate how infection and/or

immunization modulated the expression of cytokines in vivo (Figure 6). Transcriptional

73

activity of TNF-a and IL-1(3 was increased in the group of vaccinated and infected pigs when

compared to those pigs infected only and non-infected pigs. Immunization also upregulated

the expression of IL-1(3. In addition, vaccinated and infected pigs had higher expression of

TGF-(3, mRNA. Infection also stimulated the expression of IFN-y and IL-6 without any

significant differences between immunized and non-immunized pigs (data not shown).

Finally, transcriptional activity of IL-4 or IL-12p40 was undetectable (not shown).

Discussion

Protection induced by systemic immunization with a proteinase-digested bacterin of

B. hyodysenteriae correlates with changes in the lymphocytic composition of the colonic

mucosa and in the proinflammatory cytokine expression. B. hyodysenteriae is a non-invasive

bacterium which disrupts the colonic epithelium (14). Lymphocytes are part of the

population of leukocytes infiltrating the lamina propria of inflammatory lesions induced by

B. hyodysenteriae. Our results show that immunization decreased the numbers of CD4+ cells

in both infected and non-infected pigs. In the former group, these changes were accompanied

with a milder disease manifestation and less severe microscopic lesions. Interestingly, the

expression of the proinflammatory cytokines TNF-a and IL-1(3 m RNA was higher in

vaccinated pigs.

The cytokine profile of colonic tissue recovered from infected pigs is suggestive of a

predominant Thl response. In the majority of murine models of colitis, lesions result from

aberrant Thl responses, irrespective of the factor triggering the process (1). These pathogenic

CD4+ T cells are specific for antigens expressed by members of the normal flora (2; 4). The

presence of members of the normal colonic flora is also necessary for the development of B.

74

hyodysenteriae-induced colitis because disease was not detected in germ-free/gnotobiotic

pigs or when mice were selectively depleted of gram negative bacteria via antibiotics prior to

challenge (22-25). Antibiotic treatment has also proven to be effective in decreasing

intestinal inflammation in murine models of IBD (26; 27). Thus, part of the LP CD4^ cells

might be responding to antigens from the colonic flora.

An interesting finding is that vaccination alone modified the expression of

proinflammatory cytokines at the colonic mucosa. B. hyodysenteriae produces a

lipopolysaccharide (LPS) that stimulates the production of IL-1(3 and TNF-a by

macrophages (28; 29). However, because the vaccine was administered parenterally (i.e.,

intramuscular), the increase in proinflammatory cytokine expression is secondary to the

cellular immune response induced by vaccination. Peripheral blood CD8aar, CD4+, and yôT

cells from immunized pigs respond in vitro to B. hyodysenteriae antigens (17-19; 30). Also,

the production of IFN-y in antigen-recall responses to B. hyodysenteriae was shown to be

CD4-dependent (20). Thus, it is possible that immunization favors differentiation of naïve

cells into Thl cells that home to the colonic mucosa where they activate macrophages to

synthesize IL-1(3 and TNF-a. However, cytokine upregulation induced by previous

vaccination may contribute to epithelial regeneration. Specifically, IL-IJ3 is considered a

restitution-promoting cytokine (31). Skin keratinocytes-derived IL-1(3 stimulates the

production of keratinocyte growth factor (K.GF) by fibroblasts which, in turn favor

differentiation and proliferation of epithelial cells (32). This mechanism is dependent on the

transcription factors c-fos and c-jun, which are transcribed as early response genes during

epithelial restitution and modulate the expression of genes involves in cell migration (33; 34).

75

TGF-P, mRNA expression was increased in immunized and infected pigs. The

production of this cytokine by T reg cells has been associated with the downregulation of Th

I responses (7; 35). It has been shown that the same antigens that activate effector cells, can

also prime regulatory T cells (36; 37). Thus, it is possible that both Thl effector cells and T

reg cells are present at the colonic mucosa following immunization and challenge.

Additionally, yô T cells have been implicated in the downregulation of inflammatory

responses (38-40). Based on immunohistochemical analysis, the numbers of yô T cells were

not increased in vaccinated pigs, although the loss of yô IEL was less severe than in the non-

immunized and challenged group. It has been shown that yÔ T cells downregulate

inflammation by inducing apoptosis of activated aP T cells and activated macrophages (41;

42). Also yô IEL are important in maintaining epithelial homeostasis (43; 44). This subset in

addition contributes to the process of epithelial wound healing (45). The possible

contribution of yô T cells in protection against B. hyodysenteriae-induced colitis should be

addressed in future experiments. Finally, it has been shown that NK and NK-T cells provided

protection in murine models of colitis (46; 47). NK cells have been characterized in the pig

as CD3" CD8aa+. In addition, a subset of TCRotP CD8aa* has been identified as part of the

circulating lymphocyte pool (48; 49). Although CD8aa+ cells were analyzed during the

course of this study, several cell types within the mucosa express this coreceptor; thus, a

more specific phenotypic analysis should be performed in order to determine whether the

composition of NK cells or CD3" CD8aa+ subsets is modulated by infection and/or

vaccination.

Mechanisms involved in downregulation of inflammation are impaired in IBD. The

present study describes an immunization treatment that modulates the course of an

76

inflammatory response. The elucidation of the cellular and molecular mechanisms of action

of this vaccine could reveal alternative therapies for the treatment of IBD.

Tabic. I: Histopathological evaluation of colonic lesions of vaccinated and/or infected pigs following challenge with Brachyspira hyodysenteriae'

Category

Non-infected Infected*

Non-vaccinated Vaccinated Non-vaccinated Vaccinated Gland depth

HyperVMetapl.5

Inflammation6

LP Vasc.change7

Subm. change8

Epithelial erosion9

Total score 10

8.33 ± 1.11 0.66 ± 0.44

1.0 ± 0.0 0.0 ± 0.0

0.66 ± 0.44 0.66 ± 0.44

11.33 ± 1.85

7.66 ± 0.88 1.0 ±0.0

1.33 ±0.44 0.0 ± 0.0

0.66 ± 0.44 0.66 ±0.44

11.33 ± 1.33

13.0 ±2.66* 2.33 ±0.44* 1.33 ±0.88

1.0 ±0.6 0.66 ± 0.44 1.33 ±0.88

19.66 ±4.88

10.66 ±3.11 1.66 ± 1.11 1.33 ±0.44 0.66 ± 0.8

0±0.0 1.33 ± 0.44 15.66 ±5.7

' Values represent mean ± SEM (n=3) 2 Pigs were orally challenged with two doses of 1010 CPU of B. hyodysenteriae strain B204 given on consecutive days 3 Pigs were immunized with three doses of a pepsin-digested preparation of Brachyspira hyodysenteriae at 15 d intervals 4 Gland depth is expressed as a function of crypt width (i.e., a score of 5 indicates that crypts are 5 times as deep as they are wide) 5 Hyperplasia/metaplasia was evaluated as increased cell turnover and basal cell basofilia, or goblet cell metaplasia 6 inflammation was scored based on the presence of cellular infiltrates at the lamina propria 7 Lamina propria vascular change was evaluated as capillary dilation and/or lamina propria hemorrhage 8 Submucosal change score is based on the presence of inflammatory foci at the submucosa 9 Epithelial erosion scores are based on a subjective scale of : 0 = no erosion, 1 = mild erosion, or 2 = severe erosion 10 Total score is the addition of the scores obtained from the previous parameters

P< 0.05 compared to non-infected groups

77

e ti C«

4-

3-

2 -

1-

TAT

o-

jA rti111

7 12 Days post-infection

Non-vacc., non-inf ... Vacc., non-infected - Vacc., inf. — Non-vacc., inf.

Figure 1. Clinical scores of non-vaccinated and non-infected, vaccinated and non-infected, vaccinated and infected, and non-vaccinated and infected pigs. Vaccinated pigs received 3 doses of a pepsin-digested bacterin of B. hyodysenteriae. Pigs were challenged with 2 doses of 1010 CPU of B. hyodysenteriae given intragastrically on consecutive days. Diarrhea associated with the development of swine dysentery was monitored daily after challenge. Scores are based on stool consistency: (0), no diarrhea, stools formed; (1) mild, (2) moderate, and (3) severe diarrhea. Clinical scores were calculated by adding diarrhea score (i.e., 0 to 3) plus a 1 for the presence of blood and/or a 1 for the presence of mucus represents significant differences (P<0.05) between infected groups attributed to the effect of vaccination. Values are means ± SEM (n=3), from day 1 to day 13 post infection.

Figure 2. Photomicrographs of colonic mucosa stained with hematoxylin and eosin. Tissues were recovered from non-vaccinated and non-infected (A and E), vaccinated and non-infected (B and F), vaccinated and infected (C and G), and non-vaccinated and infected pigs (D and H) on day 15 post-challenge with B. hyodysenteriae. Inflammation was more severe in non-vaccinated and infected pigs (D and H). Protection induced by vaccination resulted in lesser mucosal thickening when compared to non-immunized pigs (C and G). Original magnification, top panel 63 X, bottom panel 160 X.

Non-vaccinated, non-infected

80

•S Figure 3. Photomicrographs of colonic sections from a representative non-vaccinated and non-infected pig stained for CD3, CD4 and TCR8 chain by immunohistochemistry. T cells are present, both in the lamina proprial and epithelial compartments (A). However, while CD4+ cells exist only as lamina propria lymphocytes (B), yô T cells are present in both epithelium and lamina propria (C).

Figure 4. Photomicrographs of colonic sections recovered on day 15 post-challege to depict CD4+ T cell distribution in the mucosa. Top panel: CD4+ T cell clusters in the lamina propria of non-vaccinated and infected pigs (C) were more abundant than in non-vaccinated and non-infected (A) or vaccinated and infected pigs (B) (indicated with arrowheads). Original magnification, 200 X. Bottom panel: hematoxylixn and eosin staining (D) and immunohistochemical staining (E and F) of the basal lamina propria revealed multifocal leukocytic infiltrates composed mainly of CD4+ T cells (E). Very few yô T cells were present at these inflammatory foci (F). Original magnification, 400 X.

83

Lamina propria

ë s a

I !

rr in c

« U

B

200

150-

100-

50-

Non-vacc., non-inf.

Vacc., non-inf. Vacc, inf . Non-vacc, inf „

i i r CD3 CD4 TCRyS CD8a

Epithelium

Non-vacc., non-inf.

Vacc., non-inf. Vacc, inf. Non-vacc, inf.

i r CD3 CD4 TCRyS CD8a

Phenotype

Figure 5. Changes in lymphocyte composition induced by vaccination and/or infection with Brcichyspira hyodysenteriae. Colonic sections recovered from non-vaccinated and non-infected, vaccinated and non-infected, vaccinated and infected, or non-vaccinated and infected pigs were analyzed by immunohistochemistry for the presence of CD3+, CD4+, TCR5+, and CD8a+cells in the lamina propria (A) and epithelium (B). Vaccination reduced the numbers of CD4+ lamina propria lymphocytes (*P<0.05). Infected pigs had lower numbers of y5 intraepithelial lymphocytes (5 P<0.05), although previous immunization ameliorated the loss of this subset. Values are means + SEM (n=3).

84

1 2 3 4 S 6 7 8 9 10 II 12 13 14 IS 16

******

« * * «* •

* 8 î = * | l #

TNF-a

IL-1P

TGF-P

L32

GAPDH

Figure 6. Detection of cytokine mRNA from colonic tissue using a ribonuclease protection assay. Full thickness colonic samples were obtained with a 3 mm-diameter punch biopsy from non-vaccinated and infected (1-4), vaccinated and infected (5-8), vaccinated and non-infected (9-12) and non-vaccinated and non-infected pigs (13-16) at day 15 post-challenge.

85

References

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15. Hontecillas, R., Wannemuelher, M. J., Zimmerman, D. R., Hutto, D. L., Wilson, J. H., Ahn, D. U., & Bassaganya-Riera, J. (2002) Nutritional regulation of porcine bacterial-induced colitis by conjugated linoleic acid. J. Nutr. 132: 2019-2027. 16. Bassaganya-Riera, J., Hontecillas-Magarzo, R., Bregendahl, K., Wannemuehler, M. J., & Zimmerman, D. R. (2001) Effects of dietary conjugated linoleic acid in nursery pigs of dirty and clean environments on growth, empty body composition, and immune competence. J. Anim. Sci. 79: 714-721. 17. Bassaganya-Riera, J., Hontecillas, R., Zimmerman, D. R., & Wannemuehler, M. J. (2002) Long-term influence of lipid nutrition on the induction of CD8+ responses to viral or bacterial antigens. Vaccine 20: 1435-1444. 18. Waters, W. R., Pesch, B. A., Hontecillas, R„ Sacco, R. E., Zuckermann, F. A., & Wannemuehler, M. J. (1999) Cellular immune responses of pigs induced by vaccination with either a whole cell sonicate or pepsin-digested Brachyspira (Serpulina) hyodysenteriae bacterin. Vaccine 18: 711-719. 19. Waters, W. R., Hontecillas, R., Sacco, R. E„ Zuckermann, F. A., Harkins, K. R., Bassaganya-Riera, J., & Wannemuehler, M. J. (2000) Antigen-specific proliferation of porcine CD8aa cells to an extracellular bacterial pathogen. Immunology 101: 333-341. 20. Waters, W. R., Sacco, R. E., Dorn, A. D., Hontecillas, R., Zuckermann, F. A., & Wannemuehler, M. J. (1999) Systemic and mucosal immune responses of pigs to parenteral immunization with a pepsin-digested Serpulina hyodysenteriae bacterin. Vet. Immunol. Immunopathol. 69: 75-87. 21. Kunkle, R. A., & Kinyon, J. M. (1988) Improved selective medium for the isolation of Treponema hyodysenteriae. J Clin Microbiol 26: 2357-2360. 22. Hayashi, T., Suenaga, I., Komeda. T., & Yamazaki, T. (1990) Role of Bacteroides uniformis in susceptibility of Ta:CF# 1 mice to infection by Treponema hyodysenteriae. Zentralbl. Bakteriol. 274: 118-125. 23. Joens, L. A., Robinson, I. M., Glock, R. D., & Matthews, P. J. (1981) Production of lesions in gnotobiotic mice by inoculation with Treponema hyodysenteriae. Infect. Immun. 31:504-506. 24. Li, X. (1998) Characterization of acute proinflammatory cytokine responses within the cecal mucosafollowing infection with Serpulina hyodysenteriae. MSc Thesis. Iowa State University. 25. Nibbelink, S. K. (1992) Abrogation of cecal lesions, but not colonization, in conventional mice experimentally challenged with Serpulina hyodysenteriae and maintained on oral antibotics. Ph.D. dissertation. Iowa State University. 26. Steinhart, A. H., Feagan, B. G., Wong, C. J.. Vandervoort, M., Mikolainis, S., Croitoru, K., Seidman, E., Leddin, D. J., Bitton, A., Drouin, E., Cohen, A., & Greenberg, G. R. (2002) Combined budesonide and antibiotic therapy for active Crohn's disease: a randomized controlled trial. Gastroenterology 123: 33-40. 27. Farrell, R. J., & LaMont, J. T. (2002) Microbial factors in inflammatory bowel disease. Gastroenterol. Clin. North. Am. 31:41-62. 28. Sacco, R. E., Nibbelink, S. K., Baarsch, M. J., Murtaugh, M. P., & Wannemuehler, M. J. (1996) Induction of interleukin (IL)-ip and IL-8 mRNA expression in porcine macrophages by lipopolysaccharide from Serpulina hyodysenteriae. Infect. Immun. 64:4369-4372.

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29. Greer, J. M., & Wannemuehler, M. J. (1989) Pathogenesis of Treponema hyodysenteriae: induction of interleukin-1 and tumor necrosis factor by a treponemal butanol/water extract (endotoxin). Microb. Pathog. 7: 279-288. 30. Bassaganya-Riera, J., Hontecillas, R., Zimmerman, D. R., & Wannemuehler, M. J. (2001) Dietary conjugated linoleic acid modulates phenotype and effector functions of porcine CD8+

lymphocytes. J. Nutr.131: 2370-2377. 31. Podolsky, D. K. (1997) Healing the epithelium: solving the problem from two sides. J. Gastroenterol. 32: 122-126. 32. Maas-Szabowski, N., Szabowski, A., Stark, H. J., Andrecht, S., fColbus, A., Schorpp-Kistner, M., Angel, P., & Fusenig, N. E. (2001) Organotypic cocultures with genetically modified mouse fibroblasts as a tool to dissect molecular mechanisms regulating keratinocyte growth and differentiation. J. Invest. Dermatol. 116: 816-820. 33. Angel, P., Szabowski, A., & Schorpp-Kistner, M. (2001) Function and regulation of AP-1 subunits in skin physiology and pathology. Oncogene 20: 2413-2423. 34. Angel, P., & Szabowski, A. (2002) Function of AP-1 target genes in mesenchymal-epithelial cross-talk in skin. Biochem Pharmacol 64:949-956. 35. Letterio, J. J., & Roberts, A. B. (1998) Regulation of immune responses by TGF-|3. Annu. Rev. Immunol. 16: 137-161. 36. McGuirk, P., & Mills, K.. (2002) Pathogen-specific regulatory T cells provoke a shift in the Thl/Th2 paradigm in immunity to infectious diseases. Trends. Immunol. 23: 450. 37. McGuirk, P., McCann, C., & Mills, K. H. (2002) Pathogen-specific T regulatory 1 cells induced in the respiratory tract by a bacterial molecule that stimulates interleukin 10 production by dendritic cells: a novel strategy for evasion of protective T helper type 1 responses by Bordetella pertussis. J. Exp. Med. 195:221-231. 38. Bagot, M., Echchakir, H., Mami-Chouaib, F., Delfau-Larue, M. H., Charue, D., Bernheim, A., Chouaib, S., Boumsell, L„ & Bensussan, A. (1998) Isolation of tumor-specific cytotoxic CD41" and CD4+CD8dim+ T-cell clones infiltrating a cutaneous T-cell lymphoma. Blood 91:4331-4341. 39. Born, W., Cady, C., Jones-Carson, J., Mukasa, A., Lahn, M., & O'Brien, R. (1999) Immunoregulatory functions of yô T cells. Adv. Immunol. 71: 77-144. 40. Hayday, A. C. (2000) yÔ T cells: a right time and a right place for a conserved third way of protection. Annu. Rev. Immunol. 18: 975-1026. 41. Vincent, M. S., Roessner, K., Lynch, D., Wilson, 0., Cooper, S. M., Tschopp, J., Sigal, L. H., & Budd, R. C. (1996) Apoptosis of Fash'8h CD4+ synovial T cells by borrelia-reactive Fas-ligandhlgh yô T cells in Lyme arthritis. J. Exp. Med. 184: 2109-2117. 42. Egan, P. J., & Carding, S. R. (2000) Downmodulation of the inflammatory response to bacterial infection by yô T cells cytotoxic for activated macrophages. J. Exp. Med. 191 : 2145-2158. 43. Boismenu, R., & Havran, W. L. (1994) Modulation of epithelial cell growth by intraepithelial yô T cells. Science 266: 1253-1255. 44. Boismenu, R., & Havran, W. L. (1998) yô T cells in host defense and epithelial cell biology. Clin. Immunol. Immunopathol. 86: 121-133. 45. Chen, Y., Chou, K., Fuchs, E., Havran, W. L., & Boismenu, R. (2002) Protection of the intestinal mucosa by intraepithelial yô T cells. Proc. Natl. Acad. Sci. U S A 99: 14338-14343.

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46. Fort, M. M., Leach, M. W., & Rennick, D. M. (1998) A role for NK cells as regulators of CD4* T cells in a transfer model of colitis. J. Immunol. 161: 3256-3261. 47. Saubermann, L. J„ Beck, P., De Jong, Y. P., Pitman, R. S., Ryan, M. S., Kim, H. S„ Exley, M., Snapper, S., Balk, S. P., Hagen, S. J., Kanauchi, O., Motoki, K., Sakai, T., Terhorst, C., Koezuka, Y., Podolsky, D. K., & Blumberg, R. S. (2000) Activation of natural killer T cells by a-galactosylceramide in the presence of CD Id provides protection against colitis in mice. Gastroenterology 119: 119-128. 48. Yang, H., & Parkhouse, R. M. (1997) Differential expression of CD8 epitopes amongst porcine CD8-positive functional lymphocyte subsets.PG - 45-52. Immunology 92. 49. de Bruin, M. G., van Rooij, E. M., Voermans, J. J., de Visser, Y. E., Bianchi, A. T., & Kimman, T. G. (1997) Establishment and characterization of porcine cytolytic cell lines and clones. Vet. Immunol. Immunopathol. 59: 337-347.

89

CHAPTER 4. GENERAL CONCLUSIONS

The major focus of this dissertation has been the comparison between porcine CD4+

and yô T cells. However, the experiments conducted for this aim should be considered within

the long-term objective of achieving a better understanding of the immune regulation of

mucosal inflammation. The major strength of utilizing Brachyspira hyodysenteriae-induced

colitis is the possibility of evaluating systemic and mucosal responses resulting from

vaccination or infection, respectively. In addition, the fact that swine is among the species

with large numbers of circulating yô T cells, made it an ideal animal model for conducting

these studies.

Intramuscular administration of the B. hyodysenteriae bacterin does not prevent

infection and, in most of the cases it does not prevent dysentery. However, immunization

results in a milder disease manifestation as reflected by the clinical signs and microscopic

lesions described in chapter 3. Due to the functional compartmentalization of the mucosal

immune system, oral immunization is a requirement in order to mount a protective immune

response against mucosal pathogens (e.g., production of neutralizing IgA) (1; 2). Of course

this is not the case here: the route of immunization is not the appropriate to induce this type

of response, and colonization by the spirochete is not blocked by vaccination. Then, is

vaccination inducing an anti-inflammatory response rather than an anti-fi. hyodysenteriae

response? The major effects of vaccination reported here are I) a decrease in CD4+ T cells at

the colonic mucosa, 2) the 5. hyodysenteriae-induced proliferation of CD4+ and yô T cells in

vitro, and 3) the upregulation of IL-ip and, TNF-a and TGF-P mRNA expression following

90

infection, at the colonic mucosa. It is therefore expected that these cellular and molecular

events contribute to the mechanisms of protection.

Results presented in chapter 2 suggest that a cross-talk between CD4+ and yô T cells

occurs following stimulation with B. hyodysenteriae antigens, in which IL-2 appears to have

a synergistic effect. This type of interaction was described in vivo in murine models of

influenza and listeriosis. In both cases, yô T cells were necessary for downregulating

inflammation (3-6). Thus it appears that several infectious diseases with an inflammatory

component trigger this response. Very interestingly, in Lyme disease, which is caused by

another spirochete, Borrelia burgdorferi, responses of CD41* T cells isolated from synovial

fluid were downregulated by yô T cells. Lipoproteins from B. burgdorferi were later shown

to activate yÔ T cells (7; 8). It is evident that CD4+ T cell proliferation described in chapter 2

is B. hyodysenteriae-spccific. The specificity of the yô T cells is unclear, although it is

possible that yÔ T cells are responding to B. hyodysenteriae antigens recognized through a

different mechanism than CD4+. Thus, the capacity of B. hodysenteriae antigens to stimulate

yô T cells in the absence of CD4+ T cells should be investigated in the future.

yô T cells induce apoptosis of activated CD4+T cells and macrophages. The

selectivity for activated cells has been explained on the basis of the high expression level of

Fas on target cells, whereas yô T cells constitutively express FasL (7; 9). Because

immunohistochemical analysis did not reveal a significant increase of yô T cells at the

colonic mucosa of B. hyodysenteriae vaccinated pigs, initially this does not appear to be the

mechanism that explains the decrease of CD4+ LPL. Alternatively it could be possible that

91

this response took place soon after challenge. A time course study would be necessary to test

this hypothesis.

The decrease of CD4^ T cells in protected animals was a preliminary observation

from previous experiments, which was confirmed here. As it was discussed in chapters 1 and

2, CD4+ T cells specific for intestinal flora antigens are involved in the pathogenesis of IBD.

Therefore vaccination had the beneficial effect of lowering a cell type putatively involved in

the pathogenesis of swine dysentery. Intestinal colonization by B. hyodysenteriae alone is

insufficient for the development of swine dysentery as it was demonstrated in experiments

with gnotobiotic pigs, or when mice were treated with antibiotics that eliminated specific

bacteria from the colonic commensal flora (10-12). Besides the possible interaction described

above, two additional hypotheses can explain the effect that immunization has on CD4+ LPL.

First stimulation of regulatory T cells by vaccination. Second, vaccination could partially

block inflammation (i.e., lower epithelial damage), or accelerate the process of epithelial

restitution. The upregulation of TGF-Pi mRNA expression is consistent with the stimulation

of regulatory T cells (13; 14). These results are in agreement with those reported in a murine

model in which vaccination and infection with B. hyodysenteriae increased antigen-specific

TGF-P protein levels in mesenteric lymph nodes. Measurement of bioactive TGF-P protein

in the colon would further support this hypothesis (15).

Vaccination also upregulated the expression of IL-ip and TNF-a mRNA. The

implications of these results are intriguing. These cytokines are a hallmark of

proinflammatory responses, yet they also have a role in epithelial wound healing (16; 17). It

was previously reported that mice challenged with B. hyodysenteriae upregulated colonic

TNF-a m RNA 24 hours post-infection. The increased expression of IL-ip did not occurr

92

until 5 days post-infection (12). This is the first study that evaluates the interaction between

vaccination and infection in regard to the in vivo modulation of cytokine expression. Again, a

time-course study seems to be necessary in order to determine whether these changes are

beneficial or, in the contrary, this is a negative effect of the vaccine. The increased

transcriptional activity of these cytokines, especially of IL-1(3, is most likely due to B.

hyociysenteriae lipopolysaccharide. Stimulation of murine peritoneal exudate cells or porcine

alveolar macrophage macrophage with B. hyociysenteriae LPS increased the expression of

IL-1(3 (18). Evaluation of protection against B. hyociysetitericie-'induced colitis in pigs

immunizd with a preparation of B. hyociysenteriae LPS would clarify whether the

upregulation of IL-1(3 mRNA has a beneficial effect.

Because of their complexity, the use of porcine models for immunological studies has

the disadvantage of the difficulty in analyzing the role of specific factors within a cellular or

molecular mechanism. In the case of immunization and or vaccination with B.

hyociysenteriae, the evaluation of immune responses at the single cell level would aid in

elucidating the mechanism of action of the vaccine, or the molecular pathogenesis of with B.

hyociysenteriae-induced colitis. Specifically, in vitro experiments with purified

subpopulations of yô or CD4+T cells will be necessary for the elucidation of the exact

mechanism of activation by B. hyociysenteriae antigens or by other stimuli (i.e., IL-2). In

regards to the regulation of inflammation associated with swine dysentery, there are two

aspects of major priority: the identification of the cellular source of cytokines involved in

immune regulation and the expression of molecules that participate in cell-cell interactions at

the mucosa.

93

References

1. Czerkinsky, C., Anjuere, F., McGhee, J. R., George-Chandy, A., Holmgren, J., Kieny, M. P., Fujiyashi, K., Mestecky, J. F., Pierrefite-Carle, V., Rask, C., & Sun, J. B. (1999). Mucosal immunity and tolerance: relevance to vaccine development. Immunol. Rev. 170: 197-222. 2. Kraehenbuhl, J. P., & Neutra, M. R. (1992). Molecular and cellular basis of immune protection of mucosal surfaces. Physiol. Rev. 72: 853-879. 3. Doherty, P. C., Allan, W., Eichelberger, M., & Carding, S. R. (1992). Roles of a(3 and yô T cell subsets in viral immunity. Ann. Rev. Immunol. 10: 123-151. 4. S keen, M. J., & Ziegler, H. EC (1993). Intercellular interactions and cytokine responsiveness of peritoneal a(3 and yô T cells from Listeria-infected mice: synergistic effects of interleukin I and 7 on yô T cells. J. Exp. Med. 178: 985-996. 5. Skeen, M. J., & Ziegler, H. K. (1993). Induction of murine peritoneal yô T cells and their role in resistance to bacterial infection. J. Exp. Med. 178: 971-984. 6. Carding, S. R., Allan, W., McMickle, A., & Doherty, P. C. (1993). Activation of cytokine genes in T cells during primary and secondary murine influenza pneumonia. J. Exp. Med. 177: 475-482. 7. Vincent, M. S., Roessner, K., Lynch, D., Wilson, D., Cooper, S. M., Tschopp, J., Sigal, L. H„ & Budd, R. C. (1996). Apoptosis of Fash'8h CD4+ synovial T cells by borrelia-reactive Fas-ligandh'eh yô T cells in Lyme arthritis. J. Exp. Med. 184: 2109-2117. 8. Vincent, M. S., Roessner, K.., Sellati, T., Huston, C. D., Sigal, L. H., Behar, S. M., Radolf, J. D., & Budd, R. C. (1998). Lyme arthritis synovial yô T cells respond to Borrelia burgdorferi lipoproteins and lipidated hexapeptides. J. Immunol. 161: 5762-5771. 9. Egan, P. J., & Carding, S. R. (2000). Downmodulation of the inflammatory response to bacterial infection by yô T cells cytotoxic for activated macrophages. J. Exp. Med. 191 : 2145-2158. 10. Hayashi, T.. Suenaga, L, Komeda. T., & Yamazaki, T. (1990). Role of Bacteroides uniformis in susceptibility of Ta:CF# 1 mice to infection by Treponema hyodysenteriae. Zentralb.l Bakteriol. 274: 118-125. 11. Nibbelink, S. K. (1992). Abrogation of cecal lesions, but not colonization, in conventional mice experimentally challenged with Serpulina hyodysenteriae and maintained on oral antibotics. Ph.D. dissertation. Iowa State University. 12. Li, X. (1998). Characterization of acute proinflammatory cytokine responses within the cecal mucosafollowing infection with Serpulina hyodysenteriae. MSc thesis. Iowa State University. 13. Singh, B., Read, S., Asseman, C., Malmstrom, V., Mottet, C., Stephens, L. A., Stepankova, R., Tlaskalova, H., & Powrie, F. (2001). Control of intestinal inflammation by regulatory T cells. Immunol. Rev. 182: 190-200. 14. Letterio, J. J., & Roberts, A. B. (1998). Regulation of immune responses by TGF-0. Ann. Rev. Immunol. 16: 137-161. 15. Khalifeh, M. S. (1999). The role of T cell mediated immune response in the development and resolution of colitis induced by Serpulina hyodysenteriae. MSc Thesis. Iowa State University. Iowa State University.

94

16. Strober, W., Fuss, I. J., & Blumberg, R. S. (2002). The immunology of mucosal models of inflammation. Ann. Rev. Immunol. 20:495-549. 17. Podolsky, D. K. (1997). Healing the epithelium: solving the problem from two sides. J. Gastroenterol. 32: 122-126. 18. Sacco, R. E., Nibbelink, S. K., Baarsch, M. J., Murtaugh, M. P., & Wannemuehler, M. J. (1996). Induction of interleukin (IL)-l (3 and IL-8 mRNA expression in porcine macrophages by lipopolysaccharide from Serpulina hyodysenteriae. Infect. Immun. 64: 4369-4372.

95

ACKNOWLEDGEMENTS

The author wishes to express her most sincere appreciation to Dr. Michael J.

Wannemuehler, her major professor for sharing his ideas, talent and mentorship with the

author throughout this doctoral dissertation.

Acknowledgements are also extended to Dr. Jan Buss, Dr. Susan Lamont, Dr. Harley

W. Moon and Dr. Randy E. Sacco for their assistance and advice while serving as members of

this Ph.D. committee at Iowa State University.

The author is very grateful to Dr. Josep Bassaganya-Riera, whose friendship,

constructive criticisms, positive intellectual interactions and assistance in experimental work

have converted immunology research into an enjoyable task and the author's graduate

program into a highly valuable learning experience.

The author deepest and kindest appreciation is expressed to her family for their

support and encouragement.

Documents

Comparison of porcine CD4 and gamma-delta T cell systemic