The Role of MHC Antigens in Heterotopic Murine

Small Bowel Transplant Rejection

BY

Catherine Cagiannos

Department of Pathology

Submitted in partial fulfillment

of the requirements for the degree of

Master of Science

Faculty of Graduate Studies

The University of Western Ontario

London. Ontario, Canada

January, 1 997

0 C. Cagiannos, 1997

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Major histocompatibility complex antigens (MHC ags) are

targets against which a rejection response is mounted. Using a

heterotopic murine intestinal transplantation model, this thesis

examines the consequence(s) of MHC ag deficiency on graft

survival- Grafts from MHC class I-deficient and MHC class II-

deficient animals were compared against MHC-expressing grafts to

assess if there was: a) an altered rejection histology, b) a

difference in mechanisms contributing to early graft damage and c)

a difference in the phenotype of cells or cytolytic proteins in cells

(assessed by immunoperoxidase) comprising the graft infiltrate.

Regardless of MHC expression, increased crypt cell apoptosis, as

detected by in situ hybridization, indicated intestinal rejection.

MHC class l-deficient grafts had a survival advantage and

decreased frequency of crypt cell apoptosis. Apoptosis. perforin

and CD8a positivity of infiltrating cells were good markers that

correlated with the severity of rejection and stratified the groups of

allografts. CD4 and IL-2Ra expression by infiltrating cells were

unable to distinguish between groups of allografts and did not

correlate with severity of rejection.

Dedication

To my father, from whom I learned the meaning of hard work

and perseverance.

Acknowledgments

I would like to thank, Or. David Grant for his financial and

emotional support.

I would like to thank, Dr. Robert Zhong. Dr. Zheng Zhang and

the entire microsurgical lab for the meticulous and efficient work

they performed.

I would like to thank, Dr. Bertha Garcia for reading the

histology and immunohistology.

I would like to thank, Dr. Luisa Garcia for doing the

morphometric analysis.

I would like to thank, Dr. Braham Shahi for his cooperation

and willingness to accept a difficult "on - call" schedule while

working with me during the completion of this thesis.

I would like to thank, Dr. Candace Gibson for helping me

finish this project; without her encouragement I doubt that I would

have persisted.

I would like to thank, Dr. Subrata Chakrabarti for introducing

me to the concept of apoptosis and for his infectious enthusiasm.

I would like to thank, the technologists at University Hospital

for teaching me the techniques that were used in this project.

Table of Contents

CERTIFICATE OF EXAM INATION .............................................................................. 11

ABSTRACT .......................................................................................................................lll ........................................................................................... DEDICATION ............ ... ....... ., IV

ACKNOWLEDGMENTS ................................................................................................. V TABLE OF CONTENTS ................................................................................................. Vl

....................................................................................... LIST OF PHOTOGRAPHS Vlll ............................................................................................................ LIST OF TABLES IX

LIST OF FIGURES ........................................................................................................... X

CHAPTER 1 . LITERATURE REVIEW: INTESTINAL ......................................................... TRANSPLANTATION .. 1

........................................................................ . 1 1 INTESTINAL TRANSPLANTATION 1 . ..................................................... 1 . 1 I History and Indications for SBTx 1

........................................................................... . 1 7.2 Results and Outcomes 3 1 . 1.3 Propensity for Rejection in Intestinal Grafts ............................... 6

........................ ........ . 1 2 CURRENT CONCEPTS OF GRAFT REJECTION.. ....... 7 ........................... 1.2. 1 The Major Histocompatibility Complex (MHC) 7

....... ................... 1.2.2 Mechanisms of Rejection : Afferent Loop ... 8 .................................................. Antigen Processing and Presentation 8

Allorestricted "Direct" Recognition ................................................ 10 ............ S elf-Restricted "Indirect" Recognition ................... ... 1 0

.......................................................................................... 1.2.3 Co-stimulation 11 ...................................... 1.2.4 Mechanisms of Rejection: Efferent loop 13

................................................................................................. Effector Cel ls 13 .............................................................. .................... Effector Proteins ... 15

1 . 3 APO PTOS IS ............................................................................................................ 16 1.3.1 Definition and Features which Distinguish Apoptosis

from Necrosis .................................................................................................... 16 .................................................... 7.3.2 Triggers / Induction of Apoptosis 17

............................................................ 1.3.3 Genes Regulating Apoptosis 19 1.3.4 Gut Physiology and Mucosal Regeneration .............................. 20 1.3.5 Apoptosis Plays a Role in the Regulation of

Cell Number in Intestinal Mucosa ........................................................... 21 1.3.6 Apoptosis in Transplantation and GVHD .................................... 22

....................... . 1 4 USE OF GENE KIO ANIMALS TO INVESTIGATE REJECTION 23 1.4.1 Generation and Reasoning for the Use of

......................................................................................................... k/o Animals 23 .......................................... 1.4.2 MHC I (p2-microglo buIin) k/o Animals 28

................................................................ 1.4.3 MHC I 1 k/o Animals .............. .. 29 1.4.4 Perforin k/o Animals ..... ......................................................................... 30

......................................................... CHAPTER 2 - AIMS AND OBJECTIVES 31

................................................ . CHAPTER 3 MATERIALS AND METHODS 33 3.1 ANIMALS ................................................................................................................. 33 3 -2 S URGICAL MODEL, SAMPLING POINTS ......................................................... 33

........................................................ 3 -3 H ISTO PATH0 LOGICAL ASSESSMENT..... 36 .................................................................................. 3 -4 IMMUNOHISTOCHEMISTRY 36

3 -5 IN SlTU DETECTION OF APOPTOSlS ................................................................ 41 ..................................................................................... 3 -6 MoRPHOMETRY .............. ,. 43 ..................................................................................... 3 -7 s TATISTICAL ANALYSIS 45

........................................................................................ CHAPTER 4 - RESULTS 46

.......................... ................... 4.1 TIME TO ONSET OF CLINICAL REJECTION .. 46 4.2 f EMPORAL ASSESSMENT O F HISTOLOGY .................................................... 52 4.3 TEMPORAL IMMUNOHISTOCHEMISTRY: C03, CD4, CD8a

MOMA-2, IL-2Ra, PERFORIN ........... -60

4.4 MORPHOMETRIC IMMUNOSTAINING: APOPTOS IS. PERFORIN

CD8a. CD4. IL-PRa ........................... 65 4.4.1 Apoptosis in Intestinal Grafts ........................................................... 65 . *

4.4.2 Perforin lmmunosfarnrng ..................................................................... 73 4- 4.3 CD8a .......................................................................................................... 85 4.4.4 CD4 ............................................................................................................. 100 4 - 4 5 IL-2Ra ........................................................................................................ 0 8

CHAPTER 5 = DISCUSSION .............................................................................. 113

CHAPTER 6 = SUMMARY AND CONCLUSIONS ................................... 124

REFERENCES .......................................................................................................... 126

VITA ............................................................................................................................... 145

List of Photographs

................................................................... . Photograph 1 Terminal histology 50

......................................... . Photograph 2 Comparative histology - POD S 56

. ...................................... Photograph 3 Comparative histology - POD 10 59

.......................................... . Photograph 4 POD 5 Comparative apoptosis 69

........................................ . Photograph 5 POD 1 0 Comparative apoptosis 72

.......................... . Photograph 6 POD 5 perforin immunohistochemistry 76

....................... . Photograph 7 POD 10 perforin immunohistochemistry 79

.............................. Photograph 8- POD 5 CD8a immunohistochemistry 89

............................ . Photograph 9 POD 1 0 CD8a immunohistochemistry 92

Table 1 . Heterotopic cardiac allograft survival ........................................ 26

.............. . Table 2 Summary of antibodies, specificities and controls 37

................... Table 3 . Differences in time to onset of clinical rejection 46

............... . Table 4 Temporal comparison of median histology scores 52

...................................................... . Table 5 Summary of histologic grading 53

......................................................... Table 6 . POD 5 immunohistochemistry 62

...................................................... . Table 7 POD 10 immunohistochemistry 64

Table 8 . Comparison of number of crypt apoptotic nuclei

...................................................................................................... counted 66

Table 9 . Comparison of number of perforin positive cells

.............................................................. counted in intestinal grafts 73

Table 10 . Number of CD8a cells counted in various classes

............................................................. of pre-transplant intestine 85

Table 11 . Comparison of number of CD8a cells counted in

........................................................................... intestinal allografts 86

Table 12. Number of CD4 cells counted in various classes

..................................... ................... of pre-transplant intestine ... 100

Table 13 . Comparison of number of C04 cells counted in

......................................................................... intestinal allografts 101

Table 14 . Comparison of number of IL-2Ra cells counted in

.............-..... ............................................... intestinal allografts .. 108

List of Figures

..................................................... . Figure 1 Morphometry diagram 44

................................... . Figure 2 Time to onset of clinical rejection 47

. ................................................ Figure 3 Rejection criteria seen on POD 5 54

. ............................................. Figure 4 Rejection criteria seen on POD 10 57

. ........................... Figure 5 Presence of apoptosis in POD 5 crypts 67

................................. . Figure 6 Presence of apoptosis in POD 10 crypts 70

.............................. . Figure 7 Presence of perforin in POD 5 crypts 74

. ..................................... Figure 8 Presence of perforin in POD 10 crypts 77

............. ........ . Figure 9 Presence of perforin in POD 5 intercrypts ... 80

. ......................... Figure 1 0 Presence of perforin in POD 10 intercrypts 81

Figure 1 1 . Comparison of intracrypt perforin and

crypt cell apoptosis . POD 5 ....................................................... 83

Figure 12 . Comparison of intercrypt perforin and

crypt cell apoptosis . POD 5 .................................................... 84

. . ........................... Figure 13 Presence of CDBa cells in crypts POD 5 87

. . ........................ Figure 14 Presence of CD8a cells in crypts POD 1 0 90

. ................. Figure 15 . Presence of CD8a cells in intercrypts POD 5 93

. . ............... Figure 16 Presence of CD8a cells in intercrypts POD 10 94

Figure 17- Comparison of intracrypt CD8a cells and

crypt cell apoptosis . POD 5 ........................................................ 96

Figure 18 . Comparison of intercrypt CD8a cells and

........................... ........................ . crypt cell apoptosis POD 5 .. 97

Figure 19 . Comparison of intracrypt CD8a cells and

perforin expression = POD 5 ................................................ 98

Figure 20 . Comparison of intercrypt CQ8u cells and

. ........................................................ perforin expression POD 5 99

Figure 21 . Presence of intracrypt CD4 cells - POD 5 .......................... 102

. . ....................... Figure 22 Presence of intracrypt CD4 cells POD 10 103

Figure 23- Presence of intercrypt CD4 cells - PO0 5 .......................... 104

. Figure 24 Presence of intercrypt CD4 cells . POD 10 ....................... 105

Figure 25 . Comparison of intracrypt CD4 cells and

crypt cell apoptosis - POD 5 .................................................... 106

Figure 26 . Comparison of intercrypt CD4 cells and

crypt cell apo ptosis - POD 5 ...................................................... 107

. Figure 27 Presence of IL-2Ra cells in crypts . POD 5 .................... ...1 09

. ................... Figure 28 Presence of IL-2Ra cells in crypts . POD 10 -1 10

. . ........*.... Figure 29 Presence of IL-2Ra cells in intercrypts POD 5 111

. ..........- Figure 30 Presence of IL-2Ru cells in intercrypts - POD 10 112

A A

ABC

allos

ALS

ANOVA

APC

B6

BSA

CTL

CYA

DAB

d ATP

DC

DNA

DTH

amino acid

avidin-biotin complex

antigen(s)

class I major histocompatibility deficient grafts

class II major histocompatibility deficient grafts

major histocompatibility antigen expressing grafts

antilymphocyte serum

analysis of variance

antigen presenting cell

C57BL/6 mouse strain

bovine serum albumin

base pair

cry Pt

calcium

cluster of differentiation

radioactive chromium

cytotoxic T lymphocyte

cyclosporine A

diaminobenzidine

deoxyadenosine triphosphate

dendritic cell

degrees of freedom

deoxyri bonucleic acid

delayed -type hypersensitivity

deoxyuracil triphosphate

EBV

EC

epi

ER

F1

FasL

FK 506

FLS D

Go

G1

GALT

GFR

gld

GVHD

H -2

H & E

HLA

1251

tC

ICAM

ICE

IEL

IFN-y

IL

IL-2Ra

isos

Epstein-Barr virus

en terocyte

epithelium

endoplasmic reticul um

the offspring of two different parental strains

Fas ligand

tacrolimus

Fisher's least significant difference

zero growth stage of cell division cycle

first growth stage of cell division cycle

gut-associated lymphoid tissue

glomerular filtration rate

generalized lymphoproliferative disease mutation

graft-versus-host disease

histocompatibility - 2

haematoxylin and eosin

human leukocyte antigen

radioactive iodine

in tercrypt

intercellular adhesion molecule

interleukin 1 $ converting enzyme

intraepithelial lymphocyte

interferon gamma

interleukin

interleukin 2 receptor - the alpha subunit

i sog rafts

kDa

k/o

KIW

LAK

LFA

LN

LP

w Mo mAb

Mg2+

MHC

MLN

mM

pM

mRNA

NK

P

PARP

PBS

PI

PLC

PMN

POD

PP

preen

kilodalton

knock-out

Kruskal-Wallis

lymphokine-activated killer

lymphocyte function-associated antigen

lymph node

lamina propria

Iymphoproliferation mutation

macrophage

monoclonal an tibody

magnesium

major histocompatibility complex

mesenteric lymph node

millimole

micrometer

messenger ribonucleic acid

natural killer

parent

po I y-(ADP-ribose)-polymerase

phosphate buffered saline

polyphosphoinositide

phospholipase Cyl

polymorphonuclear

post-operative day

Peyer's patch

pre-transplant MHC-expressing B6 intestine

XIV

pre61 K

pre62K

RPF

S BTx

S EM

SM

TAP

TCR

TGF-$

TN F - a

TPN

TUNEL

VCAM

VLA

WT

pre-transplant M HC I-deficient intestine

pre-transplant MHC 11-deficient intestine

renal plasma flow

small bowel transplantation

standard error of the mean

submucosa

transporter associated with antigen processing

T cell receptor

transforming growth factor beta

tumor necrosis factor alpha

total parenteral nutrition

terminal deoxynucleotid yl transferase (TdT)-

mediated dUTP-biotin nick end labeling

vascular adhesion molecule

very late antigen

wild type

Chapter 1

Literature Review

I I Intestinal Transplantation 7 History and Indications for Smell Bowel

Transplantation (SBTx)

Intestinal failure results in diarrhea, fluid and electrolyte

losses and ma1 n utrition which impose many lifestyle restrictions

and result in serious complications for patients. Although the

introduction of total parenteral nutrition (TPN) in 1968 resulted in a

remarkable improvement in patient survival (Jeejeebhoy et al.,

1976), the accompanying expenses (Wateska et al., 1980), catheter

infections and TPN-induced liver disease have prompted the

development of alternative solutions such as S BTx.

Lillehei described a technique for orthotopic canine SBTx in

1959 (Lillehei et al., 1959). Since then, several animal models

have been used to study SBTx. Monchik and Russell developed a

heterotopic, caval outflow, semiallogeneic model of SBTx in rats

(Monchik et al., 1971). The great availability of reagents to examine

rodents has allowed detailed yet cost effective immunologic and

immunohistochemical studies to be done which may be

extrapolated to human transplantation. These studies help to piece

together the mechanisms causing rejection by examining the

repopulation of grafts by recipient cells, the expression of intra-

graft cytokines, and the role played by cytotoxic proteins.

By using rats from inbred parent strains (P) and their offspring

(F1), it i s possible to distinguish between rejection and graft-

versus-host disease (GVHD). Transplantation of P into F1 results in

isolated GVHD, whereas, transplantation of F1 into P results in

isolated rejection. Clinical transplantation involves a fully

allogeneic model thus there is opportunity for both GVHD and

rejection to occur.

The intestinal graft is placed either in a heterotopic or

orthotopic position. In the heterotopic model, the native bowel

remains in situ, and the graft ends are brought out as stomas

through the abdominal wall. In the orthotopic model, the native

bowel is resected and the graft is placed in continuity with the

recipient's intestine.

Prior to the introduction of cyclosporine (CyA), results with

immunosuppression were not very encouraging. Despits use of

azathioprine, prednisone, antilymphocyte serum (ALS) there was

only marginal prolongation of graft survival (Russell et al., 1967).

The discouraging results and the availability of TPN diminished

interest in SBTx in humans in the late 1960s. By the 1980s, the

complications of long term TPN and the success enjoyed by

researchers using CyA for cardiac and hepatic transplants

prompted a return to clinical SBTx.

Tacrolimus (FK 506) is a macrolide antibiotic derivative from

the Streptornyces fungus which has potent immunosuppressive

properties. FK 506 was initially used as a rescue agent in

recipients who were rejecting grafts despite CyA

immunosuppression. Subsequent clinical trials have shown the

efficacy of this agent in both animal and human transplantation

models (Tzakis et al., 1994). FK 506 has resulted in 40% two year

human intestinal graft survival in contrast to 11% seen with CyA

(Brousse et al., 1996).

There are two major generic indications for SBTx: a) loss of

the native intestine's absorptive surface (short gut syndrome) or b)

loss of the native intestine's function. In adults, Crohn's disease.

vascular accidents and trauma are frequent causes of short gut

syndrome. Radiation enteritis and pseudoobstruction are common

causes of lost function despite normal length. In children.

necrotizing enterocolitis, gastroschisis, midgut volvulus and

intestinal atresias are the major indications for SBTx. The intestine

is transplanted in isolation, unless there is associated cholestatic

liver disease caused by long term TPN. Corn bining intestinal

transplantation with liver grafting is technically more difficult and

makes the resultant postoperative course more dangerous and

complicated (Todo et al., 1992). Patients who receive an isolated

SBTx tolerate enteral feeding and leave the hospital sooner than

patients with combined transplants (Tzakis et al., 1994).

Lj .2 Results and Outcomes Despite immunosuppression, SBTx has not enjoyed the

success of kidney and liver transplantation. Rejection is the most

common complication of intestinal transplantation. There are high

rates of graft failure with 30% risk of graft loss after each rejection

episode (Todo et al., 1992). In unmodified murine SBTx, graft

survival i s between 7-10 days (Zhong et al., 1993). After

heterotopic S BTx, the rejecting graft wi l l either undergo necrosis

and perforate or will encapsulate and become fibrotic. If the graft

becomes fibrotic. the recipient survives despite graft loss thus

making the end point of rejection more difficult to define. With

orthotopic transplantation, recipient survival is dependent on graft

function thus making it easier to define rejection end points.

Clinically, rejection in heterotopic transplants presents as: a)

increased mucus per stoma, b) stoma1 necrosis I stenosis or c)

development of a palpable intra-abdominal mass. Unfortunately by

the time that there are clinical signs of rejection, there are

irreversible changes in intestinal graft histology. Both histological

and immunohistochemical changes precede clin ical changes.

Histologically, rejection presents as loss of goblet cells, loss and

blunting of villi, lymphocytic infiltration of the lamina propria and

mucosal ulceration. Terminal rejection manifests as mucosal

destruction, transmural cellular infif tration and microvascuIar

thrombosis (Garcia et al.. 1 990). lmmunohistochemical changes

precede histological changes and are early markers of rejection

that occur at a time when the process is still reversible.

Upregulation of MHC II ags on crypt enterocytes (EC) and

enterocytes of the villous epithelium is an early indicator of

rejection (Garcia et al., 1990; Schmid et al., 1990). Appearance of

pericrypt interleukin-2 receptor positive (IL-2Ra) cells and

infiltration by CD3CD8 positive lymphocytes are other early

immu nopathologic features of rejection which precede irreversible

changes in graft architecture (Cerf-Bensussan et al., 1990). There

is also a significant accumulation of macrophages and CD4 positive

T helper cell subset suggesting that a delayed-type hypersensitivity

response may be occuring (Lowry et al.. 1983; Kim et al., 1990).

Infection is the second most common complication of

intestinal transplantation. Use of potent immunosuppression to

prevent rejection, makes recipients susceptible to bacterial. fungal

and viral infections. Mucosal injury secondary to improper graft

preservation, ischemia. rejection or viral infection disrupts the

barrier function of the intestine thus allowing translocation of

enteric organisms and predisposing the recipient to sepsis.

Development of lymphoproliferative malignancies is another

complication resulting from potent immunosuppression. These

malignancies are associated with use of intense, non-specific

immunosuppression, use of antilymphocyte products and Epstein-

Barr virus (EBV) infection (Tzakis et al., 1992). There is an 80%

mortality associated with development of 6 cell lymphoma.

Although graft-versus-host disease (GVH D) is theoretically

possible because intestinal grafts possess a large, migrating

lymphoid compartment, it has not been a major feature in large

animal or fully allogeneic human SBTx (Goulet at al., 1994). Use of

immunosuppression, allows the survival of immunocompetent graft

cells in recipient lymphoid tissue.

At present, more than 170 patients have received small bowel

transplants. Children comprise two thirds of the recipients. In the

1960s (the preCyA era), none of the seven recipients survived for

more than two to three months. Introduction of CyA improved graft

survival but only two of twenty recipients between 1985 to 1990

were able to resume oral feeds. The introduction of FK 506 has

allowed 65% one year intestinal graft survival and 29% three year

survival (Brousse et al., 1996).

1.1.3 Propensity for Rejection in intestinal Grafts

At present, the propensity for rejection in intestinal grafts has

not been explained; there is no specific mechanism against which

anti-rejection therapy can be directed. Factors that may conti bute to

the high rate of intestinal allograft rejection include:

1) The intestine contains a large population of MHC class I and II-

expressing cells e.g. intraepithelial lymphocytes (IEL) (Poussier et

al., 1994), Peyer's patch lymphocytes, lamina propria lymphocytes,

mesenteric node lymphocytes. Many studies have shown

bidirectional lymphocyte traffic following S BTx. Graft "passenger

leukocytes" can leave the graft to atlosensitize the recipient

(Gundlach et al., 1990; 1992). Attempts at purging intestinal grafts

with antilymphocyte preparations have not reduced rejection rates

(Stangl et al., 1990; Shaffer et al., 1991; Saat et al., 1991).

2) Enterocytes (EC) constitutively express MHC I1 ags and

upregulate expression with rejection (Quan et al., 1993; Schmid et

al., 1990). EC can present antigens and release cytokines;

therefore, they can act as a trigger as well as a target for rejection

(Bland et al., l986a; 1986b; 1987; Brornander et al., 1993; Hoyne

et al., 1993; Zhang et al., 1990).

3) The intestinal graft is the only solid organ graft that is heavily

colonized with micro-organisms. Translocating luminal aerobic and

anaerobic bacteria have the potential to release endotoxin which in

turn can stimulate graft-infiltrating cells to produce proinflammatory

cytokines. Cytokines play many roles in rejection by: a) direct

toxicity to epithelium (Madara et al., 1989; Maessen et al., 1989;

Sun et al., 1988); b) induction of MHC and adhesion molecules

which have been shown to augment the rejection response (Milton

et al., 1986); c) promotion of diapedesis and activation of other

inflammatory I alloreactive cells (S teinman et al., 1988).

1.2 Current Concepts of Graft Rejection

1.2.1 The Major Histocompatibility Complex (MHC)

The MHC molecules were first recognized because of an

ability to induce rejection of tumours and skin grafts. The MHC

encodes a region of polymorphic genes which help to distinguish

an organism's own cells from foreign. In humans, the HLA region is

found on chromosome 6. HLA-A. 6, and C are designated class I

molecules. HLA-DR. DQ and DP are class I1 molecules. In mice, the

H-2 region is encoded on chromosome 17. H-2 K, D, and L are

designated class I molecules. H-2 I-E and I-A are class I1 molecules

(Zaleski et al., 1983).

The polymorphic residues on MHC molecules form a peptide

binding groove. The MHC molecules interact with both peptide and

the T cell receptor : CD3 complex (TCR:CD3). Monomorphic

determinants on MHC I and I1 molecules are ligands for COB and

CD4 respectively (Julius et al., 1993). MHC ags on transplanted

organs differ from those expressed on the recipient. MHC ags may

serve both as presenting molecules and as foreign peptides

against which a response i s mounted (Sayegh et al., 1994).

The MHC I molecule is composed of a 45kDa u chain that is

non-covalently associated with f12-microglobulin at the a3 domain.

The a1 and the a2 domains form the peptide binding groove. The aa

domain is the site for interaction with CD8 positive cytotoxic T cells

(Abbas et al., 1991). MHC I molecules are present on most

nucleated cells of the body. MHC I molecules present peptides

derived from endogenous cytosolic protein sources (Germain and

Marguilies, 1993; Germain, 1994).

The MHC II molecule is composed of a 33 kDA a and a 29

kDa fi chain. The a1 and P I domains form the peptide binding

groove. The p2 domain in the site of interaction with CD4 positive T

lymphocytes. MHC II molecules are expressed on dendritic cells

(DC), macrophages (Mws), monocytes, 6 cells and on endothelial

cells, however, under the influence of cytokines, these molecules

can be induced on a wide variety of cells. MHC II molecules

present molecules from exogenously derived sources (Germain and

Marguilies, 1993; Germain. 1994).

1.2.2 Mechanisms of Rejection :Afferent Loop

ANTlGEN PROCESSING AND PRESENTA TlON

Peptides are derived from either the cytosol or the endosornal

compartment. MHC I and II molecules have developed different

abilities to present peptides from these two intracellular locations.

Newly synthesized MHC I heavy chains are folded and

noncovalently associated with $2-microglobulin in the endoplasmic

reticulum (ER). In the absence of peptide, MHC I is not transported

to the cell surface but is retained in the ER. Calnexin is a resident

ER protein which inhibits movement out of the ER into the Golgi

complex (Degen et al.. 1992). Cytosolic proteins are digested into

8-10 amino acid (AA) peptides and transported by the transporter

associated with antigen processing (TAP) into the ER (Shepherd at

al., 1993; Neefjes et al., 1993). The binding of peptide makes the

MHC I stable for transport to the cell surface via the Golgi complex.

MHC II molecules assemble with invariant chain in the ER.

MHC I1 molecules have an open binding site which permits

interaction with protein segments ranging from 13-25 AA in length

(Chicz et al., 1992). MHC II molecules acquire peptide in acidic

proteolytic intracellular compartments called endosomes. Without

the invariant chain. MHC II molecules are deficient in their ability to

reach endosomes and bind peptide (Viville et al., 1993). The

invariant chain prevents the binding of MHC II with proteins in the

ER. The invariant chain i s cleaved in the acidic environment of the

endosome thus exposing the MHC II peptide binding site. The

binding of peptide makes the MHC II stable for transport to the cell

su rface.

The specific TCR alp heterodimers usually recognize

antigenic peptide fragments bound to self MHC I or II molecules.

Transplantation rejection is an exception to this rule. In

alloreactivity, T cells recognize foreign MHC molecules as the ags.

The frequency of T cells able to recognize allogeneic MHC

molecules is 10-100 times greater than the frequency of T cells

able to recognize other ags (Shoskes and Wood, 1994).

ALLORESTRlCTED PIRECT" RECOGNITION

A main reason for allograft rejection is the presence on graft

tissue of incompatible class I and II MHC ags. Class I and I1 ags are

major targets which are recognized by alloreactive cytotoxic T

lymphocytes (CTLs). Initiation of the cell-mediated immune

reactions traditionally requires donor antigen presenting cells

(APCs) such as macrophages or dendritic cells (Halloran et al..

1989). These professional APCs take up extracellular antigen,

degrade it and present it to effector cells. The allo-MHC ags are

presented to recipient CD4 positive T cells which become

activated, elaborate cytokines and induce clonal expansion of

activated ag-specific CD4 and CD8 cells. Damage of the allograft is

mediated by several cell populations including CD3CD8 expressing

CTLs, CD3CD4 expressing T helper cells (Steinmuller. 1985) and

CD56 positive NK cells (Hall, 1991 ; Kummer et al., 1995). NK cells

are non-MHC restricted (Nemlander et al., 1983).

The precursor frequency of T cells able to respond to foreign

MHC is high. Estimates range between 1 - 1 0 1 (Eckels et al.. 1988;

Chandler et al., 1993). The dependence of direct allorecognition on

donor DCs may limit its effect to the early stages of rejection. Graft

DCs are migratory and are replaced by recipient DCs within two

weeks of transplantation (Milton et al., 1986).

SELF-RESTRICTED "INDIRECT" RECOGNITION

S elf-restricted ag presentation resu Its when host APCs

activate host T cells by presenting graft proteins in the form of

peptides associated with self-MHC molecules (S hoskes and Wood.

1994). Alloantigens are treated as exogenous ags. The indirect

pathway is dominated by CD4 positive T cells which recognize ags

shed from the graft as allopeptides bound to MHC II on recipient

APCs. The precursor frequency of T cells involved in indirect

recognition is relatively low as for any other nominal ag. Indirect

recognition may play a role in chronic rejection.

There are several important differences between direct and

indirect ag presentation:

a) indirect recognition involves the presentation of peptides in the

context of self MHC II to CD4 positive T cells; CD8 expressing CTLs

activated by self-restricted recognition may attack host cells

b) CD8 expressing CTLs directly activated by al lo restricted

mechanisms lyse graft cells without attacking host cells

C) direct recognition involves the provision of competent APCs by

the graft, whereas indirect recognition involves the provision of

APCs by the host.

1.2.3 Co-stimulation The peptides that load onto class II MHC molecules are

derived from proteins that have been endocytosed from the

extracellular medium (Germain, 1994). The peptides that load onto

class I MHC come from the cytosolic proteins that have been

processed by proteasornes and transported into the endoplasmic

reticulum via the specialized transporter associated with antigen

processing (TAP) (Shepherd st al.. 1993). T cell receptor

occupancy alone is insufficient to cause clonal expansion and

signal transduction in effector cells. Although TCR occupancy

facilitates progression of T cells from Go to GI (i.e. stages of the

cell cycle), it is insufficient to allow progression from GI to the

synthesis phase of cell division. Interleukin-2 (IL-2) messenger

ribonucleic acid (mRNA) transcription and IL-2 receptor occupancy

is required for clonal expansion. IL-1 is a cytokine released by

APCs which acts on the IL-2 mRNA to enhance stability (Paetkau et

al.. 1989).

Most encounters with antigen involve low affinity interactions

and low frequency of receptor occupancy. Lymphocytes

compensate for this problem by the use of coreceptors that act in

conjunction with the TCR and ag to amplify signal transduction

(Weiss and Littman, 1994). CD4 and CD8 are two important

coreceptors which increase the avidity of TCR - MHC ag

interactions. Ag presented in association with MHC on APCs results

in co-aggregation of CD4 I CD8 and the TCR : CD3 complex. CD4

binds to the 82 segment of the class I1 MHC (Cammarota et al.,

1992). CD8 binds to the a3 segment of class I MHC (Konig et al.,

1992). CD4 and CD8 bind to p56lck, a cytoplasmic tyrosine kinase

which phosphorylates the TCR (Veillette et al., 1991) and activates

phospholipase C (PLC) to result in the hydrolysis of

polyphosphoinositide (PI).

Release of proinflammatory cytokines and engagement of the

TCR augment the adhesion of other T cell accessory molecules to

their ligands on targets. TCR occupancy activates binding of

lymphocyte function-associated antigen4 (LFA-1) to intercellular

adhesion molecule (ICAM), very late antigens (VLAs) to

extracellular matrix proteins and CD2 to LFAd (O'Rourke et al.,

1993). APCs release IL-I and IL-6 and are a source of T cell

surface molecule ligands. The interactions between various

molecules serve to strengthen contact and provide costimulation to

amplify signalling via the TCR after ag-specific activation.

1.2.4 Mechanisms of Rejection : Efferent loop

Effector Cells

Both CD4 and CD8 phenotypes are found in the cellular

infiltrate of grafts in the process of rejection. Studies have

attempted to determine the relative roles of CD4 and CD8 cells in

allograft rejection (Hall, 1991 ). Both subsets participate, although

their functions differ. In general. CD4 cells interact with MHC II ags

and secrete cytokines which mediate delayed-type hypersensitivity

(DTH), whereas, CD8 cel Is mediate cytotoxicity after sensitization

by MHC I ags. However, there is no distinct separation of function

between CD4 and CD8 cells. Both CD4 and CD8 cells can be

cytotoxic, provide helper function or induce DTH (Golding et al.,

1987).

T cell-mediated cytotoxicity is dependent on two mechanisms.

Both mechanisms are ag-specific (Kagi et al.. 1994a). Despite the

mechanism used, the target cell is induced to undergo apoptosis.

By contrast, when death is induced by antibodies, the cells undergo

necrosis. Cycling cells are susceptible to CTL-induced DNA

fragmentation, whereas quiescent cells are relatively resistent

(Nishio ka and Welsh, 1 994).

One mechanism is based on granule exocytosis (Heusel et

al., 1994). The granule exocytosis model proposes that CTLs

release the content of granules upon binding ag on target cells.

Ligation of the CTL TCR stimulates a Ca++-dependent

degran ulation. Cooperation between various constituents of the

granules is needed for optimal CTL-induced target cell lysis.

The second independent mechanism is mediated through the

interaction of Fas I Apo-1 on target cells and Fas ligand (Fas L) on

effector cells. This pathway may account for cytotoxicity in perforin

klo mice and for lysis by CD4 expressing CTLs (Ju et al., 1994).

Similar to CD8 expressing CTLs, CD4 expressing CTLs induce

apoptosis in their targets (Grogg et al., 1992), however, unlike C08

expressing C f Ls which kill preferentially by exocytosis of granules

(Kagi et a1.J 994b), CD4 expressing CTLs don't appear to contain

cytoplasmic granules and induce target cell death via engagement

with Fas (Stalder et al.. 1994). Disruption of the target cell genome

and target cell lysis are more rapid when induced by CD8

expressing CTLs (Hahn et al.. 1995).

Support that these two mechanisms account for the majority

of T cell cytotoxicity is provided by experiments showing that

perforin-deficient effector cells can lyse wild-type thymocytes but

are unable to lyse ipr (i.e.Fas mutation) thymocytes (Kagi et al.,

1994a). The authors interpreted the absence of in vitro cytotoxicity

when effectors were unable to exert perforin-based lysis and

targets were unable to be lysed through the Fas pathway to mean

that no other cytotoxic mechanisms were operable. However, in a

later in vivo study using perforin knock-out (klo) recipients and

cardiac grafts from lpr donors, prolongation of graft survival could

not be shown (Schulz et al., 1995). These authors hypothesized

that the failure of the Fas pathway to compensate for perforin

deficiency in causing rejection may be due to low expression of Fas

on target cardiac graft cells. They proposed that rejection could be

mediated by secretion of soluble factors such as tumor necrosis

factor (TNF-a) or interferon gamma (IFN-y) (Apasov et al., 1993).

TNF-a i s expressed on the surface of CTLs.

Effector Proteins

Granzymes are effector molecules which are stored in the

granules of CTL and NK cells. Granzymes are serine proteases

which cleave substrates at aspartate residues. It has been shown

that purified granzyme A or B can induce deoxyribonucleic acid

(DNA) fragmentation in cells permeabilized with sub-lytic levels of

perforin (Shi el al.. 1992; reviewed in Henkart, 1994; Heusel et al.,

1994). Entrance of granzymes into target cells is hypothesized to

occur through pores made in the target cell membrane by perforin

(Henkart, 1994). Activated CTLs from granzyme 8 klo mice are

deficient in the induction of DNA fragmentation and apoptosis in

allogeneic target cells (Heusel et al., 1994).

Perforin i s also found in the granules of CTLs and NK cells. In

the presence of Ca++ (Young et al.. 1987), perforin polymerizes to

form pores. Perforin has functional and structural homology to C9

in the complement cascade (Young et al.. 1986). Rat basophilic

leukemia cells transfected with perforin can lyse both nucleated

cells and ABCs but can't cause DNA fragmentation in the nucleated

cells (Shiver and Henkart, 1991). Rat basophilic leukemia cells

transfected with both perforin and granzyme lyse target cells and

also trigger DNA fragmentation (Shiver et al., 1992).

FasL is a 40 kDa type II transmembrane protein of the TNF

family that is expressed on activated T cells. The gld mutation

represents a loss of function mutation at the FasL gene locus on

chromosome one (Nagata and Suda, 1995b). FasL is upregulated

in lymphocytes upon activation and can induce apoptosis in Fas-

expressing cells.

1.3 Apoptosio

Parenchymal cell apoptosis has been recognized in several

transplant models as a marker of impending rejection and CTL - mediated injury. Apoptosis has been noted in crypts of rejecting

intestinal allografts (White et al., 1 995).

1.3. I Definition and Features which Distinguish

Apoptosk from Necrosis Apoptosis is derived from the ancient Greek for "falling off of

tree leaves". Necrosis and apoptosis are two types of cell death

that display different morphological and biochemical features.

During apoptosis, chromatin condenses, the cell membrane forms

blebs, DNA fragments into oligonucleosomal length segments, the

cytoplasm shrinks, the endoplasmic reticulum dilates and

eventually the entire cell fragments. The condensation of chromatin

seen in apoptotic cells precedes both 51Cr release and 12Wabeled

DNA release (Russell et al., 1982).

On haematoxylin and eosin (H & E) sections, apoptotic cells

are characterized by having fragmented nuclei with condensed

chromatin and condensed clear cytoplasm. Apoptotic bodies are

defined as round or ovoid structures bounded by a discrete

membrane and enclosing aggregates of pyknotic intensely

basophilic nuclear chromatin often surrounded by a thin mantle of

cytoplasm (Lee. 1993)- These nuclear or cytoplasmic fragments are

sloughed off into the lumen or phagocytosed by adjacent epithelial

cells or rnacrophages. The hallmark of apoptosis is the

endonucleolytic cleavage of DNA into oligonucleosomal fragments

of 180 base pairs (bp) which result in the formation of the

characteristic ladder seen on agarose gel (Wyllie. 1980a).

The triggered cell responds to activation of surface receptors,

or to the receipt of a signal by undergoing internal changes which

regulate the production of enzymes I proteins to cause the

metabolic and morphologic changes representative of apoptosis.

The apoptotic process serves many purposes: a) removes excess

cells in embryological development; b) removes cells containing

genetic defects either secondary to errors in DNA replication or

exposure to cytotoxic materials c) homeostasis d) thymic clonal

selection.

7.3.2 Triggers / Induction of Apoptosis

Apoptosis results from physiologic and non-ph ysiologic

stimuli. Biologic agents such as Mullerian inhibiting factor, TGF-p

(Lsmo et al., 1995) or INF-a (Opipari et al., 1992) can interact with

cell surface receptors to initiate apoptosis. Similarly, lipophilic

molecules such as glucocorticoids or thyroxine bind to nuclear

receptors and activate transcription of genes that induce apoptosis

(Fesus et al.. 1991). Loss of trophic signals such as cytokines or

hormones can also induce apoptosis e-g. loss of testosterone

causes atrophy of the prostatic epithelium (Rouleau et al., 1990).

Non-physiologic causes of apoptosis such as

chemotherapeutic agents, ionizing or ultraviolet radiation, induce

DNA strand breakage. Apoptosis resulting from these stimuli is

dependent on p53 - a tumor suppressor gene (Clarke et al., 1993).

Following irradiation, murine intestinal crypt cells undergo

apoptosis, whereas crypt cells from irradiated p53-deficient mice

fail to do so (Merritt et al., 1994). P53 does not appear to play a

role in TCR receptor-mediated apoptosis (Howie et al., 1994).

Cytotoxic T lymphocytes are induced to express Fas ligand

upon TCR activation (Nagata and Golstein. 1995). Fas I APO-1 is

expressed on cells such as hepatocytes and enterocytes (ECs).

Signalling through Fas I APO-1 causes cells to undergo apoptosis

upon ligation with cells expressing FasL (Nagata and Golstein.

1995). It i s possible that the Fas I FasL pathway is a physiological

regulator of apoptosis in cells both inside and outside the lymphoid

system (Wyllie, 1995).

Cytotoxic T lymphocytes (CTLs) contain perforin and

granzyme in their granules that may induce apoptosis in target

cells (Ando et al., 1994). The CTL granules can induce DNA

fragmentation in target cells. The cytoplasmic granules are

released into the intercellular space between the target cell and

the CTL. Perforin forms pores in the target cell membrane (Young

et al., 1987). Granzymes enter the cytoplasm of the target and are

transported into the nucleus where they mediate DNA

fragmentation (Heusel et al., 1994).

1 J.8 Genes Regulating Apoptosis

Apoptosis is controlled by a variety of growth factors e.g.

TGF-p1 (Lsmo et al., 1995), cytokines e.g. IL-2, oncogenes e.g. myc

or bcl-2 and tumour suppressor genes e.g. p53. Much of our

understanding of apoptosis comes from studying the nematode

Caenorhabditis elegans. During nematode development, cedd

and ced-4 are required for programmed cell death. In mammals,

there are members of a family of cysteine protease(s) which are

homologous to ced-3, i-e. interleukin-l p-converting enzyme (ICE)

(Yuan et al, 1993); Nedd-2llCH-1 (Kumar et al., 1994); and the

proenzyme CPP-32 (Nicholson et al., 1995). One substrate for the

ICE I CED-3 proteases is poly-(ADP-ribose)-polymerase (PARP).

PARP is involved in DNA repair and genome surveillance during

periods of stress (Whyte and Evan, 1995). PARP inhibits the

Ca2+/Mg2+-dependent endonuclease implicated in internucleosomal

DNA cleavage. PAAP is cleaved at the onset of apoptosis thus

losing its genomic reparative function. In mammals, CPP-32

(apopain) is responsible for the cleavage of PARP (Nicholson et al.,

1995). The ability to manipulate CPP-32 activity may have

therapeutic implications in conditions such as rejection where

excess apoptosis is present.

Ced-9 is a nematode gene which suppresses apoptosis. In

mammals, the proto-oncogene bcl-2 serves a similar function

(Hengartner et al., 1994). Expression of the bcl-2 gene results in a

26kDa protein which inhibits apoptosis that is induced by the

withdrawal of growth factors or the exposure to cytotoxic stimuli

(Reed, 1994). Inhibition of apoptosis by bcl-2 i s thought to occur

through reciprocal interaction with Bax, a pro-apoptotic protein

(Oltvai et af ., 1 993). Coprecipitation experiments show that the

apoptosis inhibitory ability of bcl-2 requires heterodimerization with

bax. Bax I bax homodimerization promotes apoptosis (Yin et al.,

1994).

Although bcl-2 can prevent apoptosis caused by: noxious

agents e.g. radiation, antibodies to CD3, steroids (Hawkins and

Vaux, 1994), ICE (Nicholson et al-, 1995) and p53 (Chiou et al.,

1994); i t does not confer protection against all forms of CTL-

mediated injury (Vaux et al., 1992; Chiu et al., 1995). Bcl-2 may

block apoptotic lysis induced by perforin plus granzymes, but not

apoptotic lysis induced via the Fas pathway (Chiu et al., 1995).

1.3.4 Gut Physiology and Mucosal Regeneration

The small intestine represents one of the most dynamic body

tissues. Cell division in murine crypts produces approximately 109

cells every 5 days (Potten, 1992). Murine crypts have 4-1 6 actual

stem cells which produce the various lineages of cells making up

the intestinal epithelium and up to 30-40 potential clonogenic cells

which can take over stem cell function following perturbations of

the former (Potten and Loeffler, 1990). Stem cells in the crypts of

small intestine are observed about four cell positions from the base

of crypts - above the Paneth cells (Potten et al., 1982). A low but

constant level of spontaneous cell death occurs in the crypt but this

frequency increases with exposure to radiation (Merritt et al.,

1994), chemotherapeutic or other cytotoxic agents (Potten et al.,

1992; 1994).

1.3.5 Apoptosis Plays a Role in the Regulation of

Cell Number in intestinal Mucosa Gut epithelial cells which originate in crypts, move toward the

tips of villi and are extruded into the lumen (Leblond, 1981). The

lining of the gastrointestinal tract is replaced every two to three

days in rodents (Wright, 1984). After leaving crypts. intestinal

epithelial cells differentiate, mature and acquire barrier, absorptive

or secretory functions. Apoptosis accounts for the bulk of epithelial

cell loss in the gut and is a central feature of the regulation of cell

number (Hall. 1994). Apoptosis occurs within the crypts of the small

intestine at a low spontaneous rate- The apoptotic deletion of cells

is greatest at position 4 i-e. where the stem cells reside (Merritt et

al., 1995). The propensity for cell death in this location is

maintained regardless if the process is spontaneous or cytotoxic-

induced. Apoptotic cells I fragments which occur along the length of

the villus originate from cells undergoing cell death in the crypt

(Potten and Allen. 1977). These fragments are phagocytosed and

digested by macrophages or healthy neighbouring epithelial cells

which migrate out of the crypt with maturation (Potten, 1992). On

average there are 1200 apoptotic cells lost per villus in a 24 hour

period (Hall et al., 1994). This is equal to the number of cells

migrating onto a villus per day (Potten et al., 1990).

There is a great deal of speculation about the mechanism(s)

regulating intestinal cell apoptosis. Differential expression of

adhesion molecules or survival factors which influence interactions

between cells and the substratum may play a role in the decreased

survival of cells along the crypt-villus axis (Beaulieu. 1992).

8cl-2 is expressed in the intestine. The distribution is non-

uniform with maximum expression in the base of colonic crypts

where stem cells are found. By comparison, expression of bcl-2 is

low in the small intestine especially in the positions occupied by

stem cells. The low expression of bcl-2 may account for the

increased apoptotic activity seen in the small intestine and the

removal of cells which have incurred genomic damage. This may

explain the relatively low propensity of the small intestine to

develop neoplasms while the colonic epithelium, where stem cells

are protected by expressing bcl-2, is prone to carcinogenesis

(Merritt et al.. 1 995).

1 J .6 Apoptosls in Transplantation and GVHD

Apoptosis was recognized using the light microscope in

hepatocytes from rejecting porcine hepatic allografts (Battersby et

al., 1974).

Acute rejection and cyclosporine nephropathy are associated

with the occurrence of apoptosis in renal tubular epithelia leading

to tubular atrophy and loss (Ito et al., 1995). The incidence of

apoptotic hepatocytes is increased in rejecting rat hepatic

allografts when compared to isografts. This increase parallels the

development of histologic and biochemical criteria of rejection

(Krams et al., 1995).

The presence of crypt apoptosis is not physiologic and is

considered one of the criteria indicative of histologic rejection.

Apoptotic crypt epithelial cells are rarely seen in mucosal biopsies

from nonintestinal transplant patients. One study found that under

normal conditions there is < 1 apoptotic body per 100 human

colonic crypts, however, there are no studies to show what the

frequency of crypt cell apoptosis is in normal human small intestine

(Lee, 1993). One group made the histologic diagnosis of rejection

when finding at least two apoptotic figures in one crypt or one

apoptotic figure in each of two crypts in the presence of a lymphoid

infiltrate (White et al., 1995). In the intestine, the influx of host

lymphocytes does not necessarily indicate rejection and occurs

even in immunosuppressed recipients (Grover et al., 1993).

Apoptotic crypt cells were seen by POD 3 in a heterotopic intestinal

transplant rat model (Krarns et al.. 1996). The authors commented

that the incidence of apoptosis increased with the severity of

rejection.

Apoptosis in individual crypt epithelial cells with the presence

of intra-epithelial lymphocytes is also a morphologic change that

appears in intestinal GVHD. Apoptosis is found in a high frequency

of patients with intestinal symptoms and GVHD in other organs

such as skin or liver (Bornbi et al.. 1995).

1.4 Use of Gene k/o Animals to investigate Rejection

1.4.1 Generation and Reasoning for the Use of

Wo Animals

Gene disruption techniques permit selective elimination of

particular antigens or cells thus allowing researchers the

opportunity to see the influence that they exert on allograft survival.

Researchers proposed that cells within the graft are

responsible for initiating rejection (Snell, 1957). Dendritic cells are

strong stimulators of the alloimmune response (Lafferty et al.,

1984). They are able to process and present ag in the context of

MHC I and II, as well as provide costimulatory signals which

facilitate activation of effector cells. Depletion of functional DCs

has resulted in long-term graft survival (Lechler et al., 1982).

Although simple, non-vascularized grafts can be depleted of cells

by in vitro culture, gamma irradiation, ultraviolet irradiation or

antibody and complement fixation, the purging of complex

vascularired grafts by similar methods has not been as successful

(Campos et al., 1995). Attempts to render intestinal allografts less

immunogenic have failed because there is a large quantity of

lymphoid tissue in the intra-epithel ial compartment, the lamina

propria, the Peyer's patches and the MLNs. Even if a small number

of dendritic cells survive the treatment, they are sufficient to initiate

rejection.

The inability to eliminate cel Is, prompted researchers to

explore other avenues of immunomodulation. The MHC I ags are

constitutively expressed on almost all nucleated cells. The MHC II

ags are expressed on DCs, Mq, B cells, thymic stromal cells and on

epithelial cells. MHC I1 ags play a major role in: a) recognition and

presentation of at loantigen, b) T lymphocyte and M ~ I interactions, c)

anti body production and d) thymocyte education. The rationale

behind the use of MHC depleted organs is an attempt to prolong

graft survival.

Prior to the development of gene transfaction I targetting

technology, researchers attempted to assess the role of

histoincompatibility between donor and recipient by the use of

congenic strains. Through repeated backcrossing, strains were

generated that share the same non-MHC background and which

differ only in their MHC genotype. These co-isogeneic (congenic)

strains are widely available and represent a useful tool for

understanding the role played by MHC in allograft rejection.

However, use of congenic animals may result in poor localization of

recombination sites thus causing incompatibility from differences in

minor antigens or variation in unknown gene products close to the

selected gene locus. Use of transgenic mice generated by

homologous recombination using embryonic stem cells results in

germline transmission I deletion of a modified gene. This

methodology introduces a planned alteration at a specific locus

with minimal involvement of the surrounding genome. Mice lacking

MHC I or II provide a novel experimental system for investigating

MHC-directed immune responses in vivo. Elimination of these ags

should, in theory, preclude direct ag presentation and reduce

allorecognition.

Unfortunately, there appear to be organ-specific mechanisms

contributing to graft rejection because use of MHC depleted skin

(Auchincloss et al., 1993; Grusby et al., 1993), pancreas

(Markmann et al.. 1 S92), kidney (Coffman et al.. 1993) and heart

(Campos et al., 1995) allografts has resulted in tremendous

variation of survival benefit. There are organs which are difficult to

engraft such as skin and intestine and organs which are accepted

with relative ease such as liver. It is not possible to extrapolate

results from one transplant model to all models especially if grafts

are free versus vascularized -

MHC I klo pancreatic islets survive indefinitely in allogeneic

hosts (Markmann et al., 1992; Osorio et al., 1993). MHC I klo renal

allografts also demonstrate improved renal function i.e. glomerular

filtration rate (GFR) and renal plasma flow (RPF) when compared

with allograft controls (Coffman et al.. 1993). By comparison, skin

grafts from MHC I klo (Zijlstra et al.. 1990). MHC II klo (Auchincloss

et al., 1993) and MHC-deficient mice are rejected without delay

(Grusby et al., 1993) suggesting that tissue from these animals is

still highly antigenic. When the recipient is in vivo depleted of CD8

cells. the tempo of rejection is still unaffected, however, when the

recipient is in vivo depleted of CD4 cells there is prolonged

survival (Auchincloss et al.. 1993). The rejection of MHC II k/o skin

grafts requires the presence of CD4 positive helper T cells primed

to donor ags by indirect presentation. In comparison, murine

cardiac allografts require interaction between recipient CD4

positive T cells and donor class II ags to reject; results argue

against indirect sensitization occurring in this model (Campos et

al., 1995).

ral - Median Survival (days)

Table 1. Heterotopic cardiac allograft survi

**=p 4.05 vs. 66 I klo (Campos et al.. 1 995)

Donor (H-2b) B6 control B6 I k/o B6 II k/o 86 1+11 k/o DBN2 DBN2

MHC ags are a major barrier against the transplantation of

organs between individuals. MHC ags may play a particularly

important role in SBTx because the intestine has a large

component of MHC I expressing lymphocytes and MHC II

expressing ECs which are tranplanted with the graft. These MHC-

expressing cells are potent inducers of the alloimmune response

Recipient (H-2% DBAI2

u

I 8

u

B6 I klo 66 II Wo

*=pc0.05 vs. B6 control

against the intestinal graft. The release of pro-inflammatory

cytokines induces the expression of adhesion molecules and MHC

ags (Halloran et al., 1992; Rothlein et al., 1988; Thornhill et al.,

1991). There is upregulation of MHC ags in the mucosa and in

crypts with rejection (Schmid et al., 1990). The increased

expression of MHC ags may augment the rejection response

because it occurs prior to the onset of histologic damage.

The effect of MHC matching on intestinal graft survival was

examined in a rat heterotopic SBTx model using congenic and

recombinant strains (Gundlach et al., 1990). The researchers

showed that the A (MHC I) sublocus represented a stronger genetic

barrier than the 6 (MHC 11) sublocus. Generation of MHC I and I or

II klo animals by homologous recombination has given researchers

an opportunity to assess the consequences of tissue

transplantation in the absence of these molecules. Success with

the small animal model for MHC deficiency may serve as the

impetus for the genetic engineering of larger animals that can

donate organs to humans which can be accepted with reduced

immunosuppression requirements and risk of rejection.

1.4.2 MHC I (p2-microglobulin) Wo Animals Cells that fail to synthesize f%2-microglobulin, have incomplete

folding and accelerated breakdown of class I heavy chains in the

endoplasmic reticulum thus reducing the expression of MHC I ags

on the cell surface (Williams et al., 1989). The assembly of MHC I

ags is a multi-step process which results in formation of a

heterodimer. Calnexin anchors free heavy chain in the ER until it

can complex with pa-microglobulin (Rajagopalan et al., 1979).

Proteasomes digest cytosolic proteins into peptides which are

transported by TAP into the ER to stabilize the p2-microglobulin and

heavy chain complex. Upon peptide binding the MHC I ag is

exocytosed to the cell surface (Zeff, 1995).

MHC I k/o mice have a marked reduction in CD8 alp T cells.

Class I ags on thymic epithelium are required for the positive

selection of CD4-CD8+ thymocytes from CD4+CD8+ precursors

(Kisielow et al., 1988). By comparison, the absolute number of y/6 T

cells and CD4 a@ T cells is unaffected, however, the precursor

frequency of CD4 expressing MHC Il-specific CTLs is higher

(Marusic-Galesic et al., 1993).

MHC class I heavy chains (H-2Db) are expressed on the cell

surface in the absence of f32-rnicroglobulin. S urface expression of

H-2Kb is absent from MHC I klo mice. Free heavy chain H-2Db is

present but is reduced by twenty-fold (Zijlstra et al., 1990). Recent

evidence suggests that class I negative CD8 T cells in MHC I ltlo

animals may be the result of positive thymic selection using the

free class I heavy chains (Apasov et al., 1993b; Glas et a!., 1994;

Lamouse-Smith et al., 1993).

1.4.3 MHC I1 Wo Anima Is

To produce MHC II klo mice, one must disrupt expression of

both I-A and I-E. To accomplish this, a mutated Ap gene was

introduced into the 03 embryonic stem cell line. The 03 l ine is

derived from a strain that is b-haplotype at the H-2 locus. Strains of

b-haplotype carry a mutated E, gene promoter and fail to express

the I-E complex on the cell surface (Mathis et al., 1983). A

neomycin resistance gene is cloned into the second exon of a b-

haplotype AB clone. Then, a copy of herpes simplex virus thymidine

kinase is added to each end of the construct. The construct is

electroporated into D3 cells where it is incorporated into the

genome by homologous recombination. D3 cell clones which have

appropriately integrated the mutation are selected by exposure to

G418 and gancyclovir. Transfected clones are injected into

C57BL16 (66) blastocysts and the embryos implanted into foster

mothers. Chimeric males are mated with 66 females and the

resultant heterozygous offspring are mated to yield hornozygous

MHC II k/o animals (Grusby et al., 1991; Cosgrove et al., 1991).

MHC If klo animals have a reduced number of CD4 a/f3 TCR

cells in the thymus (0.6% versus 9.9% in control animals) and in

the periphery (approximately 5% of the level seen in control mice).

This reduction results because there is a lack of MHC II ags on

thymic stromal cells for progression from the CD4+CD8+ stage to

the single positive CD4+CD8- stage (Grusby et al.. 1991). The CD4

cells in MHC II klo mice are large cells which express low levels of

a@ TCR and high levels of (3044. These T cells are located in B

cell follicles rather than in the usual T cell zones of spleen and

LNs. The decrease in the number of CD4 cells in MHC II k/o

animals is accompanied by an increased number of CD8 cells

(Grusby et al., 1991). 6 cell function and development is

unaffected.

1.4.4 Perforin Wo Animals

Perforin klo mice were generated by homologous

recombination to test the role played by CTLs in graft rejection,

antiviral responses and carcinogenesis. The mice generated have

normal numbers of CD8 and NK cells and appropriately expand

these populations to antigenic stimulation.

CTLs are involved in the recognition of tumour antigens and

tumour rejection. Perforin klo mice have a considerably weaker

ability to eliminate fibrosarcoma tumour cells and develop tumours

when inoculated with low number of fibrosarcoma cells (Kagi et al.,

1994a; 1994b). Perforin klo fail to clear lymphocytic

choriomeningitis virus (LCMV) infection. However, lysis of LCMV

peptide-presenting EL4 target cells which express Fas by perforin

klo effectors occurs readily (Clark et al., in press). Thus, the

inability of perforin klo cells to lyse LCMV-infected cells may be

due to the lack of Fas expression by those cells.

Chapter 2

Aims and Objectives

Rationale: There have been many transplant models

which have looked at improving survival by reducing the

immunogenicity of the graft. Recent gene disruption techniques

have resulted in mice lacking MHC I or MHC II antigens. These

"knock-out" mice have been used with variable success in skin,

pancreatic, kidney and heart transplants. There is a large quantity

of lymphoid tissue contained in the epithelium. Peyer's patches,

lamina propria and mesenteric lymph nodes of intestinal grafts

which expresses MHC I alloantigens. Also, enterocytes express

MHC II constitutively and upregulate expression of MHC II antigens

in crypts and in the mucosa with impending rejection (Garcia et al.,

1990). The question of whether MHC elimination is beneficial in

SBTx remains unanswered. This thesis attempts to address the

issue.

Hypothesis: MHC antigens are a potent stimulus which

are recognized by the recipient as being foreign and against which

a rejection response is mounted. Elimination of these

immunogenetic stimuli of allorecognition should decrease the

tempo and I or intensity of rejection in heterotopic murine intestinal

allografts.

Objectives: This thesis investigates whether depletion of

MHC I or II confers a survival advantage to intestinal allografts. The

specific objectives for this study were:

1 . To determine if elimination of either MHC I or MHC II antigens is

protective in mice receiving heterotopic intestinal allografts.

2. To determine if MHC I ags play a more critical role than MHC II

ags.

3. To identify early indicators of rejection Le. determination of the

phenotype of cells or effector proteins associated with or predating

the histologic damage seen.

Chapter 3

Materials and Methods

3.1 Animals Intestinal transplantation was performed using male mice

weighing 25-30 grams. The following strains were used:

1) Balblc (H-26) [Harlan Sprague Dawley, Indianapolis. IN].

2) B6WT ("wild typew C57BL16 x 1291Sv; sixth backcross onto

C57BL16 pedigree; H-2b) [Gen Pharm lnternational, Mountain View.

CAI,

3) B6 all061 K (homozygous MHC I -deficient; B6WT pedigree; t i -2b)

[Gen Pharm International].

4) 66 allo62K (homozygous MHC II -deficient; B 6 M pedigree; H-

2 b ) [Gen Pharm International].

MHC-deficient animals were housed in the pathogen-free J.P.

Robarts Research Institute's Transgenic Facility (London, Ontario).

Non-transgenic animals were housed in the Animal Quarters at the

University of Western Ontario. A l l animals were treated in

accordance with guidelines established by the Canadian Council

on Animal Care (1 984).

3.2 Surgical Model, Sampling Points Heterotopic small bowel transplants with proximal and distal

stomas and portosystemic drainage were performed as previously

described (Zhong et al.. 1993). Donors and recipients were MHC I

and MHC II disparate. The surgical model was fully allogeneic

allowing for both rejection and GVHD to occur. Balblc mice served

as the universal recipients. Donor grafts (n=3-6 per sacrifice

period) were harvested from Balblc, B 6 M , 86 MHC l-deficient

(all061 K) and 66 MHC 11-deficent (a11062K). Balblc grafts

transplanted into Balblc recipients served as the isograft control

group.

Time Course of Clinical Rejection

A pilot study was performed to assess whether MHC class I or

I1 depletion exerted an effect on the latency to onset of clinical

rejection :

Bal blc (H-2d)--->~al blc (n=5) i so s**

~ 5 7 ~ ~ / 6 ( ~ - 2 b ) - - - z ~ a l b / c (n =6) allos

M H C-l klo (H -2b)--->8al blc (n=5) all061 K

M HC- Il k lo (~-2b) - - ->~a l blc (n=5) allo62K **lsos did not show signs of clinical rejection; they were sacrificed on POD 28 which was set as the end-point of the study.

Phenotypically, MHC-expressing and MHC-deficient animals

looked identical, but in addition. Dr. R. Zhong and Dr. 2. Zhang, the

two surgeons performing the transplants were blinded as to the

MHC expression of donors . Graft recipients were observed daily

for the development of any physical signs of rejection:

a) stoma1 necrosis I stenosis or b) palpable intra-abdominal mass

or C) increase in mucus production.

Animals were sacrificed at the development of the first sign of

clinical rejection or after a maximum of 28 days. At necropsy

tissues were collected for histological verification of graft rejection

and further immunohistochemical analyses.

Time Course of H istopathological Rejection

A temporal study was also performed comparing graft

histology, immunohistology and apoptotic activity between

isografts, MHC-expressing, MHC I-deficient and MHC il-deficient

allografts. Animals were sacrificed on POD 5 and 10:

POD 5 Bal blc (H-2d)--->~al blc (17-3) isos

~ 5 7 ~ ~ / 6 ( ~ - 2 b ) - - - > ~ a l blc (n-3) allos

M H C-l klo (H -2b)--->6al blc (n=3) all061 K

MHC-II k l o ( ~ -2b)--->~al blc (n=3) allo62K

POD 1 0 Bal blc (H-2d)--->6al blc (n=3) isos

~ 5 7 6 ~ 1 6 ( ~ - 2 b ) - - - > ~ a l blc (n =8) allos

MHC-I klo (H-2b)--->6al blc (n=4) all061 K

MHC-If klo(~-2b)--->6alblc (n-3) allo62K

To facilitate histologic and immunohistologic examination, at

necropsy, portions of the graft were fixed in 4% paraformaldehyde

(Marivac Limited, Halifax, NS) or "snap-frozen'' in a 5 0 5 0 mixture

of isopentane (Fisher Scientific, Fair Lawn. NJ): liquid nitrogen.

3.3 Histopathological Assessment Intestinal samples were fixed in 4% paraformaldehyde,

embedded in paraffin, cut 3-5 pm thick and stained with

hematoxylin-eosin (H & E) on a Linistain GLX automatic stainer

(Lerner Labs, Pittsburg, PA).

Sections were examined by Or. 6. Garcia under light

microscopy and graded from 0-2 (0-no change, I -minimal change,

2-marked change) for each of the following mucosal features:

mitosis, cryptitis, loss of goblet cells, lymphocytic infiltration,

intraepithelial PMNs, shortening of villus height and sloughing of

villus tips (as previously described by Garcia et al., 1990).

Increased mitosis and cryptitis are nonspecific histologic criteria

which are features of other bowel pathology. The remaining criteria

are specific to rejection.

3.4 lmmunohistochemistry Sections of intestinal grafts were embedded in 0.C.f. (Miles

Inc., Elkhart, IN). frozen in a 50:50 mixture of isopentane: liquid

nitrogen and stored at -70°C until processed for

immunohistochemistry. A panel of monoclonal antibodies was

examined in each graft harvested.

The primary monoclonal antibodies used in this study

included: biotin anti-mouse I-Ab, biotin anti-mouse I-Ad, purified

anti-mouse CD4 (L3T4), purified anti-mouse CD8a (all from

PharMingen. San Diego, CA); rat anti-mouse CD3, rat anti-mouse

macrophages (MOMA-2), rat anti-mouse VCAM-1 (MIK-2) (all from

Serotec Ltd. Toronto. ONT); rat anti-mouse perforin (KM585)

(Kamiya Biomedical, Thousand Oaks, C A ) and rat anti-mouse anti-

interleukind receptor (AMT-13) (Boehringer Mannheim Canada.

Montreal. QUE). The secondary and tertiary antibodies included:

mouse adsorbed affinity purified biotinylated anti-rat (H+L) IgG

(Dimension Laboratories. Mississauga, ONT) and tertiary avidin-

biotin complexes (ABC Elite) (Vector Laboratories, Burlingame,

CA). Al l antibodies used produced the expected staining of positive

control tissues and no staining in negative controls.

i a ble 2. Summary of antibodies, specificities and controls.

I

' Antibodies I

(all are rat anti-mouse)

CD3

L3T4

Ly-2

MOMA-2

anti-l L-2 receotor

Antigenlcells

stained

all CD3

all CD4

all CD8 (a-chain)

perforin

VCAM-1

macrophages

IL-2Ra; p55

Biotinylated I-Ab

Biotinylated I-Ad

Positive Controls

spleen

spleen

spleen

perforin

VCAM (CD106)

Negative Controls

1

no lo

no lo

no lo

spleen

CTLL-2

I-Ab (MHC II)

I-Ad (MHC I I)

no lo

no lo

CTLL-2

mRUl~r kidnev

no lo

no lo

spleen (H-2b)

spleen (H -2d)

spleen (H-26)

spleen (H-26)

Three pm cryostat (Kryostat 1720. Leitz MGW Lauda,

GERMANY) sections were cut and mounted on Superfrost Plus

positively charged slides (Fisher Scientific. Fair Lawn, NJ).

Sections were air-dried overnight at 4OC. Sections were fixed with

either a) 4OC acetone (BDH lnc.. Toronto, ON) for 10 minutes or b)

4°C acetone for 3 minutes immediately followed by 4%

paraformaldehyde for 1 minute. After fixation, sections were

equilibrated with phosphate buffered saline (PBS) (Sigma

Chemical Co., St. Louis, MO) or 50 mM Tris-HCI (pH 7.6) (Sigma

Chemical Co., St. Louis, MO). Indirect immunoperoxidase was

performed using the avidin-biotin complex (ABC) technique.

A standardized protocol developed by the University Hospital

Department of Pathology was used. Acetone-fixed frozen sections

were washed and equilibrated with PBS for 6 minutes on a Clinical

Rotator (Fisher Scientific Co.. Pittsburg, PA). A humidity chamber

was used to prevent drying of sections during incubation with

reagents. To diminish nonspecific binding of immunoglobulins to

negatively charged protein sites, sections were blocked for 15

minutes with 10% human AB serum supplied by the London

Regional Blood Bank. At the end of the serum block, excess

reagant was blotted off and primary monoclonal antibody was

applied. The primary antibody was applied for one hour at room

temperature. Sections were subsequently washed with PBS for 5

minutes on the rotary shaker. Endogenous peroxidase activity was

blocked for 5 minutes using 3% H202 (Fisher Scientific Co., Fair

Lawn, NJ). A 9:l mixture of methanol:30% H202 was used. The

sections were again washed with PBS and incubated with

biotinylated secondary antibody for 30 minutes at room

temperature. Sections were again washed with PBS for 5 minutes

and subsequently incubated with the tertiary ABC reagent for 30

minutes at room temperature. A 15 minute final wash was

performed on the rotary shaker. 3'.3' diaminobenzidine (DAB)

(Sigma Chemical Co.. St. Louis. MO) was used as the chromogen.

Ten mg of DAB was mixed with 10 rnl PBS and 4 drops of 3Oh H202

were added. This was applied for 5-10 minutes on sections

resulting in a brown reaction product when the antigen in question

was present. The chrornogen reaction was terminated by quenching

sections with distilled water. Sections were counterstained with

Harris haematoxylin, dehydrated in graded alcohols and

coverslipped with Permount mounting medium (Surgipath Canada

Inc., Winnipeg. MAN).

Primary antibodies were made in the following dilutions of

PBS:

a) CD3 @ 1:100

b) CD4 8 150

C) CD8a @ 1 :SO

d) MOMA-2 @ 1 :I60

e) IL-2Ra Q neat; 30 PI applied undiluted directly on the section

f) perforin 9 1 :200

g) VCAM @ 1 :I00

h) biotinylated I-Ab @ 1 :40

i) biotinylated I-Ad @ 1:40

Secondary reagents were made in dilutions of PBS : a) mouse

adsorbed affinity purified biotinylated rabbit anti-rat IgG and normal

rabbit serum (Dimension Laboratories, Mississauga, ONT) @ (1 + 3):200. The Vectastain ABC Elite kit (Vector Laboratories.

Burlingame, CA) was used as the tertiary reagent according to the

manufacturer's instructions.

Modified staining protocols were used for the IL-2Ra and

perforin antibodies. The IL-2Ra monoclonal antibody was applied

undiluted (30 pl I section) for 30 minutes at room temperature after

the serum blocking step. Sections were washed with PBS and

incubated with biotinylated secondary as previously described.

After washing with PBS, endogenous peroxidase activity was

blocked for 15 minutes with 0.3% H202 in double distilled water.

The remainder of the staining protocol was the same as previously

described.

The staining protocol for perforin was kindly provided by Dr.

A. Kawasaki (personal communication) from the Kamiya Biomedical

Company. Slides for perforin immunostaining were equilibrated

with 50 mM Tris-HCI (pH 7.6) for 6 minutes. Slides were incubated

for 10 minutes with 0.5% periodic acid (Fisher Scientific Co., Fair

Lawn, NJ) in double distilled water. After a 5 minute wash with Tris

HCI, sections were blocked for 30 minutes with normal rabbit serum

(1 :50 dilution in 1% BSA (Sigma Chemical Co., St. Louis, MO) I

PBS). The primary antibody was used at a 1 :200 dilution in 1 % BSA

I PBS. Incubation was one hour at room temperature. Slides were

washed with Tris and incubated with mouse adsorbed biotinylated

rabbit anti-rat IgG for one hour at room temperature. Sections were

washed with Tris, blocked for 10 minutes with 0.3% H202 in double

distilled water and washed again with Tris. The tertiary ABC

reagent was used as previously described. A 15 minute final wash

was performed in Tris. Again, DAB was the chrornogen used. Ten

mg of DAB was mixed with 5 pl of 30% H202 and 50 ml of 50mM

Tris-HCI (pH 7.6) and applied to sections for 5 min to develop the

reaction product.

In the initial latency to onset of clinical rejection study, the

expression of CD3, CD4, CD8a, MOMA-2, I-Ab and I-Ad was graded

in a semi-quantitative fashion by Dr. 6. Garcia.

When doing the POD 5 and POD 10 temporal studies.

expression of CD3. CD4. CD8a. MOMA-2, IL-2Ra, perforin and

VCAM was evaluated in sections of transplanted intestine in a

semi-quantitative fashion. In addition, the expression of CD4,

CDBa, IL-2Ra, perforin and apoptosis was quantified

morphometrically by Br. 6. Garcia.

3.5 In situ detection of apoptosis

Terminal deoxynucleotidyl transferase (TdT)-mediated d UTP-

biotin nick end labeling (TUNEL) is a method which detects the 3'-

OH ends of DNA characteristic of cells undergoing apoptosis. This

method can detect apoptotic cells in formalin-fixed, paraffin-

embedded tissue samples (Gavrieli et al., 1992). The TdT

technique has been used to examine apoptosis in murine small

intestine and colon (Merritt et al., 1995), as well as human kidney

(Ito et al., 1995). When using the TUNEL technique. it is important

to correlate staining with H & E routine microscopic criteria

because stringency of the labeling procedure can result in

nonspecific signals in nuclei of necrotic cells (Que and Gores,

1996).

Apoptotic nuclei were detected in 4% paraformaldehyde-fixed

paraffin-em bedded intestinal sections using indirect

immunoperoxidase to detect digoxigenin-labelled genomic 3'-OH

DNA ends. Paraffin embedded sections were processed on

Superfrost Plus positively charged slides (Fisher Scientific, Fair

Lawn, NJ) using the protocol suggested by the ApopTagn in situ

apoptosis peroxidase detection kit (ONCOR, Gaithersburg. MD).

Briefly, sections were deparaffinized in xylene, absolute

ethanol, 95% and 70% ethanols. Proteinase K (20 pglml) (Sigma

Chemical Co., St. Louis, MO) was applied for 15 minutes at room

temperature to digest protein in tissue sections. Slides were

washed in distilled water and endogenous peroxidase activity was

quenched for 5 minutes in 2% hydrogen peroxide:PBS (Sigma

Chemical Co., St. Louis, MO). Sections were subsequently

incubated for 15 seconds under plastic coverslips with

Equilibration Buffer containing digoxigenin-nucleotides. The

manufacturer's solution of terminal deoxynucleotidyl transferase

(TdT), the enzyme which catalyzes template independent addition

of deoxyribonucleotide triphosphate to the 3'-OH ends of double or

single-stranded DNA, was applied and sections were incubated in

a humidity chamber at 37OC for 1 hour. The reaction was terminated

by submerging slides in 37OC pre-warmed Stopmash Buffer for 30

minutes. Specimens were washed in PBS and the incorporated

digoxigenin-1 1-dUTP and dATP were tagged with peroxidase-

congugated anti-digoxigenin antibody. 1 his step was performed in

a humidity chamber for 30 minutes at room temperature. The color

was developed over 3-6 minutes using DAB. Sections were

counterstained for 10 minutes at room temperature with 0.5%

methyl green in 0.1 M sodium acetate, pH 4.0. Subsequently, the

slides were washed in distilled water followed by 100% butanol

(BDH lnc.. Toronto, ONT), dehydrated in 3 changes of xylene (BDH

Inc., Toronto, ONT) and coverslipped with Permount (S urgipath

CANADA Inc., Winnipeg, MA). Positive control slides were

purchased from the manufacturer. Negative control slides were run

for every section processed. For the negative controls, the TdT was

replaced with distilled water. This was done in an attempt to see if

endogenous peroxidase activity was responsible for any of the

staining seen on sections.

3.6 Morphometry

Morphometric analysis was performed on slides

imrnunostained for the CD4, CD8a. IL-2Ra and perforin markers.

Morphornetric analysis was also performed on sections which were

used to detect apoptosis.

The morphometric analysis was performed by Luisa Garcia

using techniques established in Dr. Colin Anderson's laboratory. Or

S. Chakrabarti assisted with the analysis of apoptosis. Slides were

counted under the 4 0 X objective of a Karl Zeiss microscope. A 1 Ox

gridlined counting ocular (Intergrattionsplatte II, Karl Zeiss. West

Germany) was used to standardize results into number of positively

stained cells per mm2.

So as to be consistent, sections were analyzed with villi in

longitudinal cross section. In this orientation. data was obtained for

the following four locations of intestinal grafts: a) villous

epithelium, b) lamina propria, c) crypts and d) intercrypts (see

figure 1).

For each marker, ten separate visual fields were counted for

each location. A minimum of two different grafts were analyzed for

each transplant group at each selected time point.

An inherent bias was incorporated into the counting scheme

in that for all sections counted, the entire slide was scanned and

subsequently the regions with the greatest staining were selected.

This was done in an attempt to focus on the most intense areas of

cellular infiltration, which like the rejection process are patchy and

non-continuous (White et al., 1995).

Figure 1 . Locations for which morphometric data was obtained: Epi = villous epithelium; LP = lamina propria; IC = intercrypt; C = crypt. The bowel is shown in cross-section.

3.7 Statistical Analysis

Survival and morphometric data are presented as mean t

standard error of the mean (S EM). Statistical analysis between

groups was performed using analysis of variance (ANOVA). The

statistical package STATVIEW (Abacusn, Berkeley, CA) was used

on a PowerBook 5300 (Apple Computer Inc., Cupertino, CA). Post

hoc comparisons were done using Fisher's least significant

difference (FLSD) with p < 0.05 considered as being significant.

Kruskal-Wal l is tests (non-parametric ANOVA eq uivalents) were

used to assess differences in histologic and immunohistologic

grades between transplant groups (p < 0.05 considered

significant). Correlation coefficients (r) were calculated using

STATVIEW and were considered significant if Fisher's r to z

transformation had a p-value < 0.05.

Chapter 4

Results

4.1 Time to Onset of Clinical Rejection Animals were sacrificed at the first sign of clinical rejection

determined by Dr. R. Zhong and Dr. 2. Zhang ("blinded"

investigators) as the presence of a) a palpable abdominal mass or

b) stoma1 necrosis or c) increased mucus production. Animals

which did not manifest any of the above criteria were sacrificed on

POD 28.

Al l mice receiving isografts survived to POD 28. Al l groups of

mice receiving allografts showed significantly different survival

times compared to isograft controls (F = 16.2; df = 20; p < 0.0001).

Table 3. Differences in time required to manifest initial signs of clinical rejection .

Graft . Sacrifice (da y s) Mean * SEM

I s o s ( ~ = ~ ) 28, 28, 28, 28, 28 2 h O C AUos(n=6) 6, 7, 8, 8, 9, 16 9 * 2 All06 1 K(n=S) 13, 14, 16, 27, 28 20*3a All062K(n=~) 9, 11, 13, 14, 22 1 4 k 2 b

a Mean time to sacrifice for all061 K>allos p0.002 (ANOVA).

b No statistical difference between allo62K and allos.

All isos survived without clinical signs of rejection to the end point of the study.

Balblc mice receiving MHC-expressing allografts began to

show signs of rejection at POD 6 with a mean onset of rejection of 9

* 2 days. The two groups of Balblc mice receiving allografts from

MHC gene knock-outs had a delayed time to onset of clinical

rejection. Recipients of all06 1 K grafts demonstrated a significant

increase in the time required to manifest the initial signs of clinical

rejection (p c 0.002) (Table 3 and Figure 2). Rejection was also

delayed in the group receiving allo62K grafts, but, this was not

significantly different from allograft controls (p = 0.1 1 ).

all os all061 k allo62k isos Group

Figure 2. Time to onset of clinical rejection. Anova analysis supported a significant increase in the time to onset of clinical rejection for the MHC-I deficient allografts. (df = 20; F = 16.2; p < 0.0001)

* p0.05 isos>>all types of allografts * * p<O.O02 all06 1 K>allos PO. 1 1 all06 2K=allos

lsografts did not develop any features of histologic rejection.

All allografts which had clinical signs of rejection also had

corresponding histologic changes compatible with rejection.

Despite differences in latency, all a1 log rafts eventually developed

equally severe features of histologic rejection (Photograph 1 ).

Photograph 1. Terminal histology using H & E staining: lsografts

sacrificed on POD 28 (A; 80x magnification) had preserved

histology, whereas, a l lo6lK sacrificed on POD 27 (B; 1OOx

magnification), allo62K sacrificed on POD 14 (C; 1OOx

magnification) and allos sacrificed on POD 9 (D; 1OOx

magnification) had erosions. short villi and cellular infiltration.

Despite using MHC-expressing and MHC-deficient allografts

there were no differences in the terminal staining patterns seen for

CD3, CD4, CD8a or MOMA-2. Frozen sections were graded from 0-

2 (0 = no staining; 1 = mild to moderate staining; 2 = marked

staining). Median scores were converted to symbols to facilitate

presentation in tabular format and a Kruskal-Wall is (KMI) analysis

was performed:

( 0 ) - (no positive staining)

(0.1 - 0.74) +I- (few positively staining cells)

(0.75 - 1.49) + (moderate positive staining)

(1 -50 - 2.00) ++ (marked positive staining)

Allo62K grafts demonstrated significantly less I-Ab staining

than either allos or all061 K (KMI with p < 0.004, df = 2. H = 1 1.3 for

crypts; KAN with p c 0.005. df = 2, H = 10.5 for epithelium),

nevertheless, they showed all other features of severe histologic

rejection.

4.2 Temporal Assessment of Histology

On POD 5, allos had definite signs of histologic rejection. The

allo61K had preservation of histology on POD 5 (Photograph 2) and

POD 10 (Photograph 3). By comparison, although the allo62K had

preserved histology on POD 5, they developed definite signs of

histologic rejection by POD 10 (Tables 4 and 5; Figures 3 and 4).

lsografts did not develop any histologic criteria which could be

attributed to rejection.

All analyses were Kruskal-Wallis with df = 2. (Shaded areas have significant differences with p e 0 0 5 )

53

To ble 5. Summary of histologic grading.

POD 5 *infiltration of lyrnphs(WW) *sloughing of villi(K/W) .erosion epithelium areduction villus height(K/W) *cryptitis( W W) mitosis(WW) .goblet cell loss(K/W) *PMN intraepith(K/W)

* At POD 5, MHCexpressing allografts showed definite histologic features of mild to moderate rejection while both allo61K and allo62K lacked specific rejection criteria.

POD 10 .infiltration of lymphs(W W) .sloughing of villi(W W) .erosion epithelium(WW) areduction villus height( WW) *cryptitis(K/W) *mitosis(WW) .goblet cell loss(WW) .PMN intraepith(K/W)

" By POD 10. MHC-expressing allografts showed features of severe I terminal rejection. Allo62K began to show mild I moderate rejection. All061 K still only showed nonspecific histologic criteria wlh preservation of villi and lack of heavy cellular infiltration.

Increased mitosis and ayptitis are nonspedfic rejedm criteria i.e. they are also features of other bowel pathology. Goblet cell loss, infiltration by lymphocytes, sloughing villi, reduction of villus height and intraepithelial PMNs are specific rejedkm criteria Kruskal-Wallis (K/ W) analysis was performed with df = 2.

Intraepi. PMN

Goblet loss

Cryptitis I Allos

Figure 3. Rejection criteria seen on POD 5. At POD 5, there was an absence of specific histologic criteria indicating rejection in all061 K and allo62K.

Sloughing tips - 0 Y

0

5 d~ Height loss - h

Erosion -

Infiltration -

0 lsos

All061 K

Mitosis -

0 0.5 1 1.5 2 2.5 Severity (median grade)

Photograph 2. POD 5 comparative histology using H & E staining:

Allografts (D; 125x magnification) had short, sloughing villi with few

goblet cells, while isos (A; lOOx magnification), all061 K (6; lOOx

rnagification), allo62K (C; 125x magnification) had long villi with

goblet cells clearly evident.

Intraepi. PMN I Goblet loss I-,

Cryptitis Allos 0 lsos

Sloughing tips-- I 0 C

rn All061 K - r cr d~ Height loss e

Infiltration

Severity (median grade)

Figure 4. Rejection criteria seen on POD 10. A t POD 10, the allo62K were showing specific rejection criteria, while the all061 K were showing nonspecific changes.

Photograph 3. POD 10 comparative histology using H & E

staining: Allo62K (C; 1 OOx magnification) had rejection parameters

although not as severe as allos (D; lOOx magnification). lsos (A;

125x magnification) and all061 K (B; 1 OOx magnification) did not

show rejection histology.

4.3 Temporal lmmunohistochemistry: CD3, CD4, CD8a,

MOMA-2, IL-2Ra, Perforin Semi-quantitative grading at POD 5 for CD3. CD4, CDBa, 11-

2Ra and MOMA-2 immunostaining did not show differences

between allograft groups (Table 6). Perforin expression was

greater in MHC-expressing allografts (KMI with p c 0.02 in crypts;

df = 2; H = 8.0) . There was a tendency, also, for greater perforin

staining by allos in the lamina propria and submucosa (LP + SM),

but, statistical significance was not reached (KMI with p = 0.06).

This was addressed more closely using morphometric analysis in a

later section-

Semi-quantitative grading at POD 10 for CD3. CD4. CDBa, IL-

2Ra and MOMA-2 immunostaining also did not show differences

between allograft groups (Table 7 ) . There were no statistically

significant differences in perforin expression between groups of

allografts at this time. Allo62K sections tended to have more

perforin staining in crypts at this point in time. This was addressed

more closely using morphometric analysis.

Table 6 . POD 5 lmmunohistochemistry. (shaded areas have significant differences with p < 0.05)

Is0 s . Pertorin Allos

, All061 K

Allo62K i

Table 7 . POD 10 lmmunohistochemistry.

MAb

CD3

C 0 4

C D 8 a

MOMA-2

IL-2Ra I

I

Graft

I

Perforfn

I

_LP + S M Crypt

lsos (n=2)

Allos (8)

All06 1 K (4)

Allo62K (3)

Is0 s

AIIOS

All061 K

Allo62K

Is0 s

Allos

All061 K

Allo62K

Is0 s

Allos

All061 K

Allo62K

lsos

Allos

All061 K

All062 K

Is0 s

Allos

All0 6 1 K

Allo62K

Muscle

.

+ + + - g

- - 0

+/- + + - - - -

+/-

- +I-

- *

+ + ++

+I-

+ + + +I-

+/- + +

+/-

+ + + +I-

++ ++ ++ +/- +/- +

+/- t

+/-

+ +

- + + + - +I-

+ +I-

9

+ +

++ -

++ ++ ++

N /A

N /A

N /A

N /A

- - +I-

-

4.4 Morphometric Immunostaining: Apoptosis, Perforin,

CDBa, C04 and lL-2Ra

Using semi-quantitative analyses it was difficult to

demonstrate specific differences between the groups of allografts.

During preliminary investigations, it was noted that apoptosis was

occurring in intestinal grafts after transplantation. S imilar to

histologic criteria of rejection, the occurrence of apoptotic ceils was

non-uniformally distributed throughout intestinal sections. The

degree of apoptosis in different intestinal zones was assessed by

morphometry. Correlations between markers specific for activated

a) cytotoxic (CDBa. perforin, IL-2Ru) or b) helper lymphocytes

(C04, IL-2Ra) and apoptosis were examined. Values are presented

as the mean number of cells / mm2 k S EM-

4.4.1 Apoptosis in Intestinal Grafts

There is controversy regarding the role played by apoptosis

in intestinal epithelial turnover, however, crypt cell apoptosis as

assessed by the TUNEL technique was a positive marker for

allograft rejection. Occurrence of apoptosis was rare in the crypts

of nontransplanted bowel and isografts. Apoptosis occurred in the

crypts of MHC-expressing and MHC-deficient allografts. Like

histologic changes seen in rejection, the occurrence of apoptosis

was non-uniformally distributed.

A t POD 5, there was increased apoptosis in the crypts of

MHC-expressing allografts (ANOVA; df = 11; F = 28.6; p < 0.0001).

Allografts had more apoptosis when compared to allo61K (p = 0.02)

and allo62K (p < 0.01). By POD 10. allo62K had more apoptosis

(ANOVA; df = 14; F = 7.2; p < 0.006) than both allos (p c 0.01 ) and

all061 K (p s 0.05). In general, the relative delay to onset of clinical

and histologic rejection seen in MHC-deficient animals was

associated with a decreased rate of crypt cell apoptosis at POD 5.

Table 8. Comparison of number of crypt apoptotic nuclei counted.

(Numbers in parentheses represent the number of animals used per group)

Group

l so s Al los A l l061 K Allo62K

# Counted in Crypts a t POD 5 /mm2 0.1 f 0.1 ( 3 )

15.6 + 1.7 (3) 10.6 f 1.2 ( 3 ) 5.0 f 1.4 (3)

# Counted in Crypts a t POD 10 /mm2 0.7 2 0.2 ( 3 ) 4.0 f 0.9 (5) 6.4 f 1.6 (4)

11.3 f 2.8 (3 )

isos all061 k allo62k allos Group

Figure 5. Presence of apoptosis in POD 5 crypts. The ANOVA statistic (df = 11; F = 28.6; p c 0.0001) supported the occurrence of increased apoptosis in the crypts of MHC-expressing allografts at POD 5.

* p<0.05 isos< all allografts * * ~ 0 . 0 2 allos>allo6 1 K * * pa01 allos>allo62K

Photograph 4. POD 5 comparative apoptosis using the TUNEL

method: lsos (A; 250x magnification) did not show apoptotic crypt

cells. Allo61K (6; 313x magnification) and allo62K (C; 250x

magnification) had few positive cells in their crypts. Allos (D; 313x

magnification) had a high frequency of crypt cell apoptosis.

1 6 . 1 4 - - . 12- .,

C) = 10 - Z L

s o 8 - )C

- a # .

L

isos all061 k allo62k allos Group

Figure 6. Presence of apoptosis in POD 10 crypts. The ANOVA statistic supported the occurrence of increased apoptosis in the crypts of allo62K. (df = 14; F = 7.2; p < 0.006)

Photograph 5 . POD 10 comparative apoptosis using the TUNEL

method: lsos (A; 250x magnification) did not have crypt cell

apoptosis. All061 K (B; 3 13x magnification) and allo62K (C; 400x

magnification) had increasing frequency of crypt cell apoptosis.

POD 10 allos (D; 313x magnification) had few positive cells when

compared to POD 5.

4.4.2 Perforin immunostaining After observing that there was variability in crypt cell

apoptosis between transplant groups. we decided to stain for CD8

positive cells and perforin. C08 positive phenotype and perforin

are markers of activated CTLs which have been considered as

effectors of cellular rejection. CTLs kill targets via an apoptotic

mechanism.

Although it is controversial whether CD4 expressing CTLs

contain perforin, there is strong support that CD8 expressing CTLs

need perforin-induced pore formation in target cell membranes

before inducing the nuclear changes representative of apoptosis.

The generation of perforin klo mice has provided proof for the

crucial role played by perforin in CD8 expressing CTL or NK-

mediated cytotoxicity. Although the expression of perforin by itself

does not induce apoptosis, i t was felt that perforin was a better

indicator of cytotoxic CD8 activity (i-e. granule exocytosis - mediated injury) than of CD4 activity (i.e. Fas I FasL - mediated

killing).

Table 9. Comparison of number of perforin positive in intestinal grafts.

cells counted

Group

Isos

I # Perforin positive cells in POD 10 IC /

- - --

(IC 2 intercrypt; numbers in parentheses represent the number of animals used per

group)

# Perforin positive cells in POD 5 crypts / mm*

0.0f 0.0 (3)

crypts / mm2 1 mmz

0.4 f 0.2 ( 2 ) 10.3 k0.3

# Perforin positive celts in POD 10

# Perforin positive cells in POD S IC /

isos all06 1 C allo62k allos Group

Figure 7. Presence of perforin in POD 5 crypts. The ANOVA statistic supported the presence of increased perforin expression in the crypts of MHC-expressing allografts. (df = 11; F = 14.7; p =0.001)

* ~ 0 . 0 1 isos<all allografts * * p0.03 allos>allo61 K * * ~ 0 . 0 2 allos>allo62K

Photograph 6. POD 5 comparative perforin

immunohistochemistry: Allos (D; 400x magnification) had more

perforin staining than isos (A; 250x magnification), allo6lK (B;

31 3x magnification) or allo62K (C; 3 13x magnification).

isos all061 k allo62k allos Group

Figure 8. Presence of perforin in POD 10 crypts. There was increased expression of perforin in the crypts of POD 10

allo62K grafts. (ANOVA analysis; df = 15; F = 7.1 ; p = 0.005)

* p0.06 all06 1 K=allos not significant * * p0.04 allo62K>allos

Photograph 7. POD 10 comparative perforin

immunohistochemistry (A-D ; all 250x magnification): Allo62K (C)

and allo61K (B) had increased perforin staining in crypts. lsos (A)

had little perforin staining. Allos (0) had decreased numbers of

crypts and maintained positive staining with perforin.

isos all061 k allo62k allos Group

Figure 9. Presence of perforin in POD 5 intercrypts. There was a significant difference in intercrypt perforin expression between isografts and all allografts (ANOVA; df = 11 ; F = 37.9; p < 0.0001'). There was no significant difference in staining for intercrypt perforin between POD 5 MHC-expressing and MHC-I deficient grafts (p = 0 1 however, there was a significant difference between MHC-expressing and MHC-I1 deficient grafts (p < 0.001 ").

isos all061 k allo62k allos Group

Figure 1 0 . Presence of perforin in POD 10 intercrypts. There was a significant difference in POD 10 intercrypt perforin expression between isografts and all allografts (ANOVA; df = 15; F = 6.2; p c 0.01 '). There was no significant difference in POD 10

intercrypt perforin expression between groups of allografts.

When comparing pictures of perforin imrnunostaining with

pictures of TUNEL crypt cell apoptosis there was a marked

similarity to the pattern of positivity obtained. Similarities were also

noted when comparing CD8a staining with crypt cell apoptosis and

when comparing CD8a staining with perforin expression. Data from

the latter two correlations will be presented in the CD8a section.

Using bivariate plotting, the groups of transplants appeared

to be stratified with respect to perforin expression within crypts and

intercrypts. lsografts which had little crypt cell apoptosis also had

little perforin staining. whereas. MHC-expressing allografts which

had a great deal of crypt cell apoptosis also had the most perforin

staining. Although causality could not be assumed, there was a

significant correlation between POD 5 crypt cell apoptosis and POD

5 perforin staining within crypts (r = 0.87; Fisher's r to z

transformation p-val ue < 0.0001 ). Although expression of perforin

in the intercrypt location was not as predictive as extent of perforin

staining within crypts at gauging the severity of histologic rejection

between allografts, there was still a positive correlation (r = 0.86;

Fisher's r to z transformation p-value = 0.0001) between POD 5

crypt cell apoptosis and POD 5 intercrypt perforin staining.

. isos

- . allo6lk . - A allo62k

1 O allos . - L

-.5 0 -5 1 1.5 2 2.5 3 3.5 4 4.5 Number of Perforin Positive Cells in Crypts / mm2

Figure 11. Comparison of intracrypt perforin staining and crypt cell apoptosis - POD 5. The coefficient of determination was R2 = 0.75 meaning that 75% of the variation seen in crypt cell apoptosis (y; the dependent variable) could be explained by POD 5 perforin expression within crypts (x; the independent variable).

isos

all06 1 k

allo62k

allos

- 1 0 1 2 3 4 5 6 7 Number of Perforin Positive Cells in Intercrypts / mm2

Figure 12. Comparison of intercrypt perforin staining and crypt cell apoptosis - POD 5. The coefficient of determination was R2 = 0.73 thus 73% of the variation seen in crypt cell apoptosis (y; the dependent variable) could be explained by POD 5 perforin expression in intercrypts (x; the independent variable).

There were no statistical differences in the number of CD8a

positive cells between MHC-I deficient and other classes of

intestine. MHC-I deficient animals have less CD8 positive a/p TCR

positive cells in the thymus and lymph nodes, but, they have normal

numbers of CD8 positive y/8 TCR positive cells (Ziljstra et al.,

1990). The marker used recognizes the a-chain of the CD8

differentiation antigen. Normally CD8a and CD8p chains form

heterodimers on the surface of T cytotoxic I suppressor cells,

however, intestinal epithelial lymphocytes (IELs) can express CD8a

without CD8p and are of the y/8 TCR positive variety thus

developing independently of the a/p TCR positive cells which are

deficient in animals with MHC-I deficiency. This may explain why

there were no differences between classes of intestine used in this

study.

Table 10. Comparison of number of CD8a positive cells in various classes of pre-transplanted intestine.

Group # CD8a positive cells in epithdium

I t CD8a positive cells in lamina propria /mm2

4.8 f 2.5

in crypts in IC /mm2

There was infiltration by CD8a positive cells in and around

crypts post-transplant. By POD 5. MHC - expressing allografts had

the most infiltration with 2.8 t 0.5 positively stained intracrypt cells

per mm2 post-transplant, in comparison to 0.3 * 0.3 cells per mm2

pre-transplant. MHC I-deficient allografts had a relatively minor

infiltration with 1.5 t 0.3 positively stained intracrypt cells per mrn2

post-transplant, in comparison to 1.1 + 0.3 cells per mm2 pre-

transplant. MHC 11-deficient grafts had 1.4 * 0.2 positively stained

intracrypt cells per mm2 post-transplant, in comparison to 0.4 t 0.3

cells per mm2 pre-transplant. MHC - expressing allografts also had

the most CD8a positive cellular infiltration in the intercrypt

location.

Table 11. Comparison of number of CD8a positive cells counted in intestinal allografts.

There were more CD8a positive cells infiltrating the crypts of

POD 5 MHC-expressing allografts (ANOVA; df = 11; F = 5.054; p =

0.03). MHC-I and MHC-II deficient allograft groups demonstrated

delayed infiltration by CD8a positive cells with rising values on

POD 10, while MHC-expressing allografts had peaked on POD 5.

Group

L

lsos A l l 061 K A l l o 6 2 K

I A I I o s 12.8&O.5(311lm9k0.3(7) 11Z.Ok2.8 17.Zkl.O I

# CD8a positive cells in POD 5 crypts / mm2

0.6 k 0.5 (3) 1.5 k 0.3 (3) 1.4f 0.2(3)

# CD8a positive cells in POD 10 crypts / rnm2

0.1 & 0.1 (2) 2.4 f 0.6 (4)

2.6f 0.6 (3)

# CD8a positive cells in POD 5 1C / mm2

2.5 f 1.4 5.7 f 1 . I 7.9f 2.8

# CD8a positive cells in POD 10 IC /

mm 2

2.8 f 1.4 8.9f 1.1 10.2 f 3.3

3.5 cv 7 . f 3 - - m = 2.5 - 41

U rc 0 1 - L

isos all061 k allo6Zk allos Group

Figure 1 3. Presence of CD8a positive cells in crypts - POD 5. MHC-expressing allografts had significantly more CD8a positive cells in their crypts than other allograft groups on POD 5. (ANOVA; df = 11; F = 5.1; p = 0.03')

Photograph 8. POD S comparative CD8a imrnunohistochernistry:

lsos (A; 250x magnification) had few CD8a positive cells around

crypts. Allos (0 ; 31 3 x magnification) had heavy infiltration by CD8a

positive cells in and around crypts. All061 K (6; 313x magnification)

and allo62K (C; 313x magnification) had low positive CD8a

staining.

isos all061 k allo62k allos Group

Figure 1 4. Presence of CD8a positive cells in crypts - POD 10.

There were no significant differences between groups of allografts in the number of CD8a positive cells infiltrating crypts on POD 10. lsografts had less infiltration of crypts by CD8a positive cells on POD 10. (ANOVA; df = 15; F = 3.7; p < 0.05 ')

Photograph 9. POD 10 comparative CD8a immunohi~tocherni~try:

Allo62K (C; 313x magnification) and allo61K (6; 313x

magnification) had increased CD8a infiltration. Allos (D; 313x

magnification) had decreased staining when compared to POD 5.

lsos (A; 250x magnification) had few CD8a positive cells.

isos all061 k allo62k allos Group

Figure 15. Presence of CD8a positive cells in intercrypts - POD 5. There were no significant differences in CD8o levels between groups of allografts on POD 5. (ANOVA; df = 11; F = 3.36; p = 0.08)

isos all061 k allo62k allos Group

Figure 16. Presence of CD8a positive cells in intercrypts - POD

10. There were no significant differences in CD8a infiltrate between

groups of allografts on POD 10. (ANOVA; df = 15; F = 2.4; p = 0.1 2)

Similarities in staining between CD8a immunohistochemistry

and the pattern of crypt cell apoptosis at POD 5 prompted us to look

for further correlations. There was a significant correlation between

POD 5 crypt cell apoptosis and POD 5 CD8a staining within crypts

(r = 0.66; Fisher's r to z transformation p-value = 0.02). The

correlation was not significant for the intercrypt location (r = 0.50;

Fisher's r to z transformation p-value = 0.1 0). MHC-expressing

allografts tended to have more CD8a staining in crypts and

intercrypts than MHC-deficient allografts, but these correlations

were weaker than the correlation between apoptosis and perforin in

crypts on POD 5 (r = 0.87; Fisher's r to z transformation p-value <

0.0001 ).

isos

all061 k

allo62k

ailos

-. 5 0 -5 1 1.5 2 2.5 3 3.5 4 Number of CD8 Cells in Crypts / mm2

Figure 17. Comparison of intracrypt CD8a positive cells and crypt cell apoptosis - POD 5. The coefficient of determination for the effect of intracrypt CD8a

staining on crypt cell apoptosis was R2 = 0.44.

-

isos

I all061 k

A allo62k

0 allos

- 2 0 2 4 6 8 10 12 14 16 18 Number of CD8 Celk in Intercrypts / mm2

Figure 18. Comparison of intercrypt CD8a positive cells and crypt cell apoptosis - POD 5. The coefficient of determination for the effect of intercrypt CD8a

staining on crypt cell apoptosis was R2 = 0 . 2 5 . This correlation was not significant.

98

Cytoplasmic granules of CD8 expressing CTLs contain

perforin and granzyrnes. Perforin expression is indicative of

cytotoxic potential in CD8 positive cells (Garcia-Sanz et al., 1988;

Liu et al., 1995).There was a positive correlation between perforin

expression and CD8a immunostaining (see figure 19).

- - -

-. 5 0 -5 1 1.5 2 2.5 3 3.5 4 Number of C08 Positive Cells in Crypts / mm2

isos

all061 k

allo62k

allos

Figure 19. Comparison of intracrypt CDBU positive cells and perforin expression - POD 5. The coefficient of determination for the effect of intracrypt CD8a

positive cells on perforin expression was R* = 0.39 (r = 0.62; Fisher's r to z transformation p-value = 0.03').

isos

all061 k

allo62k

allos

-2 0 2 4 6 8 10 12 14 16 18 Number of CD8 Positive Cells in Intercrypts / mm2

Figure 2 0 . Comparison of intercrypt CD8a positive cells and perforin expression - POD 5. The coefficient of determination for the effect of intercrypt CD8a

positive cells on perforin expression was R2 = 0.39 (r = 0.62; Fisher's r to z transformation p-value = 0.03').

4.4.4 C04

An antibody that recognizes the L3T4 differentiation antigen,

expressed on mature T helper cells, showed minor differences

between sections from pre-transplant MHC-II deficient intestines

and other classes (pre62K c preen in IC location with p c 0.05') .

Nevertheless, MHC-II deficient intestines tended to have less CD4

positive cells. In the literature, MHC-I1 deficient animals are quoted

as having only 3.5% peripheral CD4 positive cells. These cells are

hypothesized to be either MHC-I-restricted or of y/8 TCR variety

(Cosgrove et al., 1991 ).

Table 12. Comparison of number of CD4 cells in various classes of pre-transplant Intestine.

(The shaded area has a significant difference with p < 0.05; pre62K < preen')

Group

Pre6n

# CD4 positive cells in epithelium / mm2

0.2 f 0.2

# CD4 positive cells in lamina propria /mm2

5.1 2 2.3

# CD4 positive cells in crypts rnm2

0.0 k 0.0

# CD4 positive cells in IC / rnm2

3.9 k 0.8

Regardless of class of allograft, there was a doubling of CD4

cells in the intercrypt (IC) location by POD 5. These CD4 cells were

likely of recipient origin because the MHC-I1 deficient allografts

showed increases which were comparable to other groups.

There was infiltration by CD4 positive cells in and around

crypts post-transplant. By POD 5 MHC - expressing and MHC I-

deficient allografts had the most intracrypt infiltration with 1.4 a 0.5

and 1.3 0.6 positively stained cells per mm2 respectively. In

comparison, isografts and MHC 11-deficient allografts had 0.0 * 0.0

and 0.1 * 0.1 intracrypt cells per mm* respectively. MHC l-deficient

allografts maintained a relatively high infiltration with 1.0 t 0.2

positively stained intracrypt cells per mm2 at POD 10. In

comparison, MHC - expressing and MHC 11-deficient allografts both

had 0.1 0.1 intracrypt cells per rnm2 at POD 10. Similarities in

CD4 positive staining were seen for the intercrypt location (see

Table 13). Again. MHC I-deficient grafts had more CD4 positive

intercrypt cells per mm2.

Table 13. Comparison of CD4 cells counted in intestinal allografts. Group

lsos

(The numbers in parentheses represent pre-transplant values for different locations)

A l los

f CD4 positive cells in POD 5 crypts / mm2

0.0 k 0.0

(0.0) 1.4 & 0.2

# CD4 positive cells in POD 10 crypts / mm2

0.5 f 0.5

0.1 f 0.1

# C04 positive cells in PO0 5 IC / rnm 2

1.7 & 0.2

# CD4 positive cells in POD 10 IC / m m2

3.3 A 1.3

(1.7) 8.0 & 1.8 1.9 f 0.4

The presence of CD4 cells in crypts was not an indicator of

impending rejection i.e. this marker was found in high numbers in

MHC-I deficient allografts which were not undergoing rejection. In

fact, MHC-I deficient grafts which had higher numbers of CD4 cells

through to POD 10 demonstrated features of less severe histologic

rejection relative to MHC-expressing allografts which did not. CD4

cells secreting a Th2 cytokine profile (Mosmann et al., 1986) could

be suppressing cytotoxic T cell activity.

isos all061 k allo62k all os Group

Figure 21. Presence of intracrypt CD4 cells - POD 5. There were fewer CD4 cells in the crypts of allo62K compared with allo61K on POD 5.

(ANOVA; df = 11; F = 5.3; p c 0.03; FLSD: all061 K > allo62K p c 0.05'; FLSD: allos > allo62K p c 0.02")

isos all061 k allo62k allos Group

Figure 22. Presence of intracrypt CD4 cells - POD 10. There were more CD4 cells in the crypts of allo61K on POD 10.

(AN0VA;df = 15; F = 8.7; p < 0.003; FLSD: allo62K c allo61K p c 0.01 *; FLSD: allos < all061 K p < 0.001 ")

isos all061 k allo62k allos Grwp

Figure 23. Presence of intercrypt CD4 cells - POD 5. There were fewer CD4 cells in the intercrypt location of allo62K on POD 5. (ANOVA; df = 11; F = 7.8; p < 0.01; FLSD: allo61K > allo62K p c 0.01 *; FLSD: allos > allo62K p < 0.05")

i sos all061 k allo62k allos Group

Figure 24. Presence of intercrypt CD4 cells - POD 10. There were more CD4 cells in the intercrypt location of allo61K on POD 10.

(ANOVA; df 4 5 ; F = 8.1; p c 0.01; FLSD: allo61K > allos p < 0.001'; FLSD: allo62K > allos p < 0.03")

l . l . l . l . l . l . l , , , l I .

. 0 L

w

m

- m

1 # . , . 1 . 1 ' 1 . 1 ' 1 ' 1 . 1 .

-.2 0 .2 .4 .6 .8 1 1.2 1.4 1.6 1.8 2 Number of CD4 cells in crypts/mmZ

isos

all061 k

allo62k

alios

Figure 25. Comparison of intracrypt CD4 cells and crypt apoptosis - POD 5. The coefficient of determination for the effect of CD4 cells on crypt cell apoptosis was R* t 0.41. (r = 0.64; Fisher's r to z transformation p - value = 0.02')

isos

all061 k

allo62k

allos

1 2 3 4 5 6 7 8 9 10 t 1 Number of CD4 Cells in Intercryptd mm2

Figure 26. Comparison of intercrypt CD4 cells and crypt apoptosis - POD 5. The coefficient of determination for the effect of intercrypt CD4 cells on crypt cell apoptosis was R2 = 0.43. (r = 0.66; Fisher's r to z transformation p - value = 0.02')

4m4m5 IL-2Ra The IL-2Ra (CD25) antibody used. detected the low affinity a chain

(p55 subunit) which is expressed on activated murine CD4 and

CD8 lymphocytes as well as NK cells. Staining the distribution of

IL-2Ra cells was not useful at predicting impending rejection or

distinguishing between allografts with respect to the severity of

rejection.

Table 14. Comparison of number of IL-2Ra positive cells in intestinal allografts.

Group

r

Isos A l l 0 6 1 K A I l o 6 2 K A l l o s

# IL-2Ra positive cells in POD S crypts / mmz

0.9 +_ 0.9 1.3 k 0.2 0.7 & 0.3 2.0 & 0.5

# IL-2Ra positive cells in POD 10 crypts / mm2

0.8 & 0.4 0.8 f 0.3 0.5 f 0.5 0.0 2 0.0

# IL-2Ra positive cells in POD 5 IC / mm2

1.5 +- 0.8 8.0 & 1.8 9.2 & 3.7 14.0 f 0.3

- -

# IL-2Ra positive cells in POD 10 IC / mm2

1.3 + 0.7 6.8 f 1.7 5.0 f 2.9 2.5 + 0.5

isos all061 k allo62k allos Group

Figure 27. Presence of IL-2Ra cells in crypts - POD 5. IL-2Ra positive staining was present in the crypts of isografts and allografts. There were no significant differences between classes of intestinal allografts with respect to the infiltration of POD 5 crypts by IL-2Ra positive cells. (ANOVA; df = 11 ; F = I .2; p = 0.37)

isos all061 k allo62k all os Group

Figure 28. Presence of IL-2Ra cells in crypts - POD 10. Statistical analysis did not show significant differences in IL-2Ra

positive cells between groups of transplanted intestine. (ANOVA for crypts; df = 15; F = 3.3; p = 0.06)

isos all06 t k allo62k allos Group

Figure 29. Presence of IL-2Ra cells in intercrypts - POD 5. There were no significant differences in IL-2Ra infiltration between groups of allografts, however, there were less IL-2Ra positive cells infiltrating isografts. (ANOVA for intercrypts; df = 11 ; F = 5.9; p = 0.02')

isos all061 k allo62k allos Group

Figure 30. Presence of IL-2Ra cells in intercrypts - POD 10.

Statistical analysis did not show a significant difference between groups of transplants for the presence of lL-2Ra cells in the

intercrypt location. (ANOVA for intercrypts; df = 1 5 ; F = 2.7; p = 0.1 0 ; Post hoc analysis using FLSD supported more IL-2Ra staining in all061 K than allos p = 0.03')

Chapter 5

Discussion 1) MHC PLAYS A ROLE IN INTESTINAL GRAFT REJECTION

WITH MHC I AGS PLAYING A MORE CRUCIAL ROLE THAN MHC II

AGS

One basic principle of immunology states that the severity of

allograft rejection is influenced by the degree of histocompatibility

between donor and recipient. MHC typing has proven effective in

experimental as well as clinical transplantation by minimizing graft

antigenicity without altering the recipient's immune responsiveness

(Takemoto et al., 1992). MHC matching has been examined in

canine and (Westbroek et al., 1971) and rat intestinal allografts

(Lee et al., 1986b). Using various rat strain combinations Lee

showed that MHC differences between donor - recipient resulted in

earlier rejection than non-MHC differences (Lee et al., l986b).

Using MHC matching and cyclosporine immunosuppression it was

possible to achieve long-term survival in canine orthotopic

segmental ileal allografts (Meijssen et al., 1993).

The small bowel has a large amount of MHC-expessing

lymphoid cells collectively known as gut-associated lymphoid

tissue (GALT). In man, every gram of small bowel mucosa contains

6 x 106 lymphoid cells (Goodacre et al., 1979). It has been

suggested that it is the absolute amount of MHC I or II Ags which

influence the graft's immunogenicity rather than one population of

cells such as dendritic cells (Gundlach et al., 1990). The

alloantigen load from immunocompetent intragraft lymphocytes and

mucosal enterocytes may account for the immunogenicity of

intestinal allografts. The relative importance of MHC I versus II was

assessed in a heterotopic intestinal model using congeneic rat

strains (Gundlach et al., 1990). The authors showed that grafts with

isolated class I1 disparity (i.e. MHC I matching) had 11 days mean

survival, whereas, isolated class I disparity (i-e. MHC II matching)

resulted in a mean survival of 6.7 days. The difference was

attributed to the high degree of MHC I expressed on intestinal

lymphocytes. Our results agreed with the above study thus

supporting the more crucial role played by MHC I in SBTx. MHC I-

deficient animals in our study are comparable to animals with an

isolated MHC II disparity. Increased latency to onset of clinical

rejection and relative absence of histologic parameters of rejection

favour MHC I ags as playing a greater role.

MHC I klo grafts are rejected eventually, because although

gene targetted disruption results in less immunogenic grafts, it may

not completely abolish the antigen in question. For example, it has

been suggested that expression of functional MHC I may be

possible even in klo animals because of reconstitution of donor

MHC I heavy chain by recipient pa-microglobulin (Bix et al., 1992).

Also, MHC I H-20'3 heavy chains can reach the cell surface in the

absence of p2-microglobulin (Bix et al., 1992; Allen et al., 1986).

These heavy chains are functional and can present peptides to

alloreactive CTLs which are induced to attack them (Bix et al..

1992; Glas et al., 1992). Although cells from MHC I klo animals are

less susceptible to C 0 8 expressing CTL-mediated lysis, low levels

of killing occur in vitro and may occur in vivo (Glas et al.. 1992).

The strength of in vitro alloimmune responses may not be

representative of in vivo responses. The graft microenvironment

may provide extra-cellular matrix constituents, co-stimulatory

molecules and immunoregulatory cytokines (i-e. from C04 positive

cells) that serve to regulate activation, proliferation, migration or

tolerization of alloreactive cells in a manner that i s independent of

CD8 positive precursor frequency (Mannon et al., 1995).

Another reason that may account for the eventual rejection of

MHC I klo grafts may be increased vulnerability to NK cell-mediated

lysis (Liao et al., 1991). NK cells express inhibitory receptors for

specific MHC class I molecules. Ligation of MHC class I molecules

on targets inhibits NK cell lytic machinery. Fetal liver and bone

marrow cells from MHC l-deficient donors were vigorously rejected.

This susceptibiity to rejection by NK cells is predominantly a

feature of hematopoietic cell transplants and may not apply to

grafts of non hematopoietic tissues i.e. pancreatic islet grafts

(reviewed in Raulet, 1994). However, i f NK cells were playing a

major role in SBTx rejection, then, MHC l-deficient grafts should

have been rejected more quickly. This was not the case. MHC I-

deficient grafts were rejected at a relatively slow tempo. Despite

several attempts, it was impossible to stain NK cells in our tissue

sections. It would have been useful to stain these cells because

they contain perforin. The correlation between CD8 positive cells

(which express perforin) and apoptosis was not as strong as the

correlation between perforin staining and apoptosis. Perforin found

in NK cells may have accounted for this difference. The role of NK

cells in graft rejection, however, is in question.

lmmunohistochemical analysis of cellular infiltrates from rejected

renal grafts showed minimal NK staining (Okabayashi et al., 1985).

S imifarly, myocardial biopsies placed under the kidney capsule

had granzyme expressed in Thy-1 positive (i.e. lymphocyte origin)

cells only. There was no infiltration by or granzyme expression in

Thy-1 negative (i.e. NK origin) cells in this model (Mueller et al.,

1988).

2) CRYPT CELLS ARE THE INITIAL TARGET

Intestinal crypts have cycling pluripotential progenitor cells

from which the intestinal mucosa differentiates and regenerates

(Potten et al., 1992). It is not surprising that if these cells were

targetted during rejection that there would be widespread

detrimental effects. Examination of slides revealed rejection to be

associated with apoptosis in crypts. MHC-expressing grafts had the

earliest increases and the greatest frequency of crypt cell

apoptosis. The MHC I and MHC 11-deficient grafts had delayed

increases and severity of crypt cell apoptosis.

There is a constant level of spontaneous apoptosis in the

small intestine. Usually one apoptotic event is detected in every

fifth to tenth crypt in transverse section (Potten et al., 1994).

Normally, there is a 10% stem cell apoptotic index to remove cells

with DNA damage or cells in excess of homeostatic levels (Potten

et al., 1994). The turnour suppressor gene p53 and the oncogene

bcl-2 have opposite effects on apoptosis. p53 induces apoptosis in

DNA damaged cells (Haffner and Oren, 1995). There is expression

of p53 in the small intestine. Bcl-2 suppresses apoptosis (Hawkins

and Vaux, 1994). There is little to no expression of bcl-2 in the

crypts of the small intestine (Merritt et al., 1995). In particular, there

is no bcl-2 expression in the stem cell region of crypts (Merritt et

al.. 1995).

The level of apoptosis seen in sections of intestinal allografts

was much greater than that which could be attributed to physiologic

spontaneous crypt cell apoptosis. We did not attempt to determine

if apoptosis was maximal in the stem cell zone, as is the case post

exposure to cytotoxic chemicals, temperature or ionizing radiation.

One would expect stem cells or adjacent dividing transit

(clonogenic) cells to be at greatest risk because these cells lack

bcl-2 and are cycling thus making them susceptible to CTL-

mediated injury (Lu et al.. 1996; Nishioka and Welsh, 1994). Bcl-2

expression can block CTL-induced apoptosis via the degranulation

(perforin and granzyme) pathway but has no effect on necrotic

target cell death or fas-based apoptosis (Chiu et a!.. 1995). Fas-

based cytotoxicity plays a limited role in transplantation rejection

(Larsen et al., 1995). Researchers found that similar levels of Fas

and FasL transcripts were present in both intestinal isografts and

allografts, whereas, transcripts of granzyme 6 were up-regulated in

allografts only (Krams et al., 1996). The apoptosis-suppressive or

promoting effect of bcl-2 in an experimental system may be

modified by the presence or absence of other proteins with which

bcl-2 interacts. The distribution of these proteins in the

gastrointestinal tract is still under investigation.

3) CD8 EXPRESS ING CTL-MEDIATED APOPTOS IS

CONTRIBUTES TO EARLY GRAFT DAMAGE VIA THE

DEGRANULATION PATH WAY

Using morphometry to evaluate immunoperoxidase sections

we found correlation(s) between severity of rejection, apoptosis,

intracrypt infiltration by CD8a positive cells and expression of

perforin. Although crypt cell apoptosis, presence of intracrypt

perforin and CD8a positive cells were good indicators of the

severity of histologic or clinical rejection, the presence of intracrypt

CD4 positive cells and IL-2Ra positive cells was not.

Histologic parameters of rejection, cellular infiltrates and the

presence of apoptosis are patchy in distribution. To examine the

role played by MHC depletion, apoptosis and cellular markers in

rejection it was necessary to bias morphometry by only counting

areas which displayed the greatest positivity. When using standard

morphometric techniques, differences between groups of allografts

were less obvious.

Presence of staining inside crypts was a better marker of

severity of rejection than staining in between crypts i-e. in

intercrypts (IC). Although MHC-expressing grafts tended to have

more cells in their IC relative to MHC-deficient grafts, statistical

significance was not achieved, whereas, presence of more

intracrypt cells and perforin correlated well with impending

rejection and increased frequency of apoptosis.

We observed that the incidence of crypt cell apoptosis

increased with severity of rejection. The concept of cell mediated

damage has been accepted in transplantation although the

phenotype reponsible has been in question. Activated, perforin-

expressing CD8 positive cells recognize and kill cells displaying

foreign antigen bound to MHC I molecules. Knowing that CTLs

mediate target cell damage via apoptosis, we attempted to

correlate apoptosis with CD8a infiltration. lntracrypt infiltrates of

CD8a positive cells correlated with severity of rejection in this

study.

Apoptosis was seen in the crypts of both MHC-expressing and

MHC I or 11-deficient grafts. Despite a delay, all allografts had

similarities in the phenotype of infiltrating cells and expression of

cytolytic proteins. This suggests that although full presentation of

MHC is best for alloaggtession, either MHC class incompatibility

can compensate. Although the apoptotic process can be mediated

by growth factor withdrawal (Fesus et al., 1991) or pro-inflammatory

cytokines i.e. TNF-a (Steller, 1995) and TGF-p (Oberhammer et al.,

1992). the similarities in the relative density of CD8a positive

infiltrating cells and the presence of perforin bearing cells support

CTL-induced apoptosis as playing a role in rejection for all types of

allografts.

The intestine has a tremendous MHC load which can

sensitize effector cells, however, it also has a large population of

resident lymphocytes which complicates the diagnosis of rejection

because the presence of these cells does not necessarily mean

that they are alloreactive. There has been a great deal of debate

regarding which cell phenotype predominates in the infiltrate of

rejecting grafts. Most authors favour C08 positive cells (Kataoka et

al., 1992; Rosenberg et al., 1991; 1993; Mueller et al., 1993) or

CD4 positive cells (Campos et al., 1995); some favour Mcp

(Andersen et al.. 1994); a few favour NK cells (Gundlach et al.,

1990). The relative role played by CD4 and CD8 co-receptors is

determined by the dependence of CD8 expressing cells on CD4

positive cel Is for help. Generation of cytotoxic effector cells

generally requires cytokines released from CD4 positive cells.

Recently, it was shown that in C57BL16 strains of mice. MHC class

I-reactive CD8 positive cells could be activated independent of

MHC class 11-reactive CD4 positive cells or MHC Il-expressing

dendritic cells. The CD8 positive cells from this strain could

produce IL-2 and theoretically promote activation, clonal expansion

and proliferation of CTLs (reviewed in Hall, 1991).

Perforin and granzymes are cytolytic proteins which are found

in granules of CTLs and NK cells (Kawasaki et al., 1992; S h i et al..

1992). The presence of perforin and granzymes correlates with

activation and cytotoxic function of alloaggressive effector cells.

Cytotoxic T lymphocytes are triggered to release perforin and

granzymes upon recognition of MHC I alloantigens. T cell receptor

ligation results in reorientation and vectorial release of granules

toward the target (Liu et al.. 1995; Ortaldo et al.. 1992). Perforin

and granzymes cooperate to induce apoptosis in target cells

(Heusel et al., 1994). Perforin is unable to induce the DNA changes

characteristic of apoptosis on its own (Duke et al., 1989).

Granzymes alone are not cytolytic. The combination of perforin and

granzymes is necessary to lyse nucleated cells and cause DNA

fragmentation (Shiver et al., 1992). Perforin may promote entry of

granzymes into the target cell via the formation of membrane pores

which can act as conduits or secondary to uptake during endocytic

repair of the damaged cell membrane (Liu et al., 1995). The

importance of perforin I granzymes as markers of rejection has

been shown in heart (Clement et al.. 1991 ; Mueller et al., 1993),

kidney (Kataoka et al., 1992) and intestinal (McDiarmid st al..

1995) transplantation models. In this study we showed that

increased perforin staining was associated with increased severity

of rejection. MHC-expressing allografts had the most intracrypt

perforin staining and the most vigorous rejection, whereas, MHC I

or 11-deficient grafts which had a slower tempo of rejection also had

decreased intracrypt perforin staining.

In this study we also showed CD4 positive cell infiltration

correlated with apoptosis but not with severity of rejection. MHC I-

deficient and MHC-expressing allografts had relatively large

infiltrates of CD4 positive cells likely, because they both have

foreign MHC II ags which can allosensitize recipient CD4 positive

cells. These activated CD4 positive cells can provide co-stirnulatory

signals for C08 expressing CTLs which recognize MHC I ags as

targets; MHC I ags were present on MHC-expressing allografts. but.

they were missing from the MHC I klo allografts. Although the

expression of C04 positive cells was similar, the resultant intensity

of the immune attack, as manifested by apoptosis, was greater in

grafts which had a large quantity of MHC I than in grafts which were

MHC I-deficient. The eventual rejection process, however, was

apoptotic even in these MHC depleted grafts suggesting that

although MHC expressing crypts are better targets, there are other

alloantigens which can compensate to drive the immune response.

These ags may be minor ags which are characterized by a weaker

immunological reaction when compared to MHC ags (Bevan, 1976).

The minor ags can stimulate both CTLs and CD4 positive MHC II-

restricted T helper cells to independently mount effector responses

against them (Roopenian, 1 992). The infiltration of MHC 11-deficient

grafts by CD4 positive cells may be mediated by: a) multiple minor

ag disparities between donor and recipient or b) graft MHC I

al loan tigen which has been processed by recipient APCs (i-e.

indirect presentation) and presented in the context of self MHC II

ags.

The intragraft cytokine milieu has an impact on induction of

MHC ags, expression of adhesion molecules, cellular chemotaxis

and promotion of cytotoxicity versus tolerance (reviewed in Colvin,

1990). Mosrnann and Coffman described two populations of murine

CD4 positive cells (designated Thl and Th2) which released two

distinct cytokine profiles upon activation (Mosmann and Coffman,

1989). Thl CD4 positive cells can secrete IL-2, IFN-y and TNFg

which induce DTH responses, or, provide help for CTLs to kill

alloantigens by apoptosis (Powrie and Coffman, 1993). In

comparison, the release of IL-4 and IL-10 from Th2 CD4 positive

cells may mediate specific unresponsiveness in organ

transplantation (reviewed in Lowry. 1993).

CD4 expressing CTLs exist, but, but utilize Fas I FasL to kill

rather than perforin (granule exocytosis) (Hahn and Erb, 1995). The

Fas I FasL pathway may function as an imrnunoregulatory balance

to regulate clonal expansion of alloreactive cells, but, it plays a

limited role in the induction of allogeneic cell apoptosis (Larsen et

al., 1995).

Activation ags such as IL-2Ra are mainly expressed on CD4

positive subsets and NK cells whereas CD8 positive T lymphocytes

and macrophages stain to a lesser degree (Andersen et al., 1994).

The staining of activated CD4 positive Thl cells, CD4 positive Th2

cells and CD8 positive cells by the IL-2Ra monoclonal antibody

used, made it difficult to separate suppressor I tolerizing from

cytotoxic activity. Although activated CD4 positive cells may

account for the majority of IL-2Ra expression, since we did not

know the relative contribution provided from each set, we could not

determine whether the presence of cells staining positive was

protective or destructive.

Chapter 6

Summary and Conclusions In summary, this study supports that MHC ags play a strong

role in intestinal rejection. f he MHC expressing allografts showed

clinical signs of rejection on average at 9 days. whereas MHC I-

deficient and MHC ll-deficient allografts did so at 20 days and 14

days respectively. MHC I ags appeared to play a more crucial role

than MHC II ags. MHC I-deficient allografts maintained a

comparatively normal histology on POD 5 and POD 10, whereas,

MHC 11-deficient allografts had preserved histology on POD 5 but

demonstrated definite rejection histology by POD 10. The

protection conferred by MHC I and MHC II depletion was temporary.

Despite differences in latency, all allografts were eventually

rejected clinically and histologically.

The frequency of apoptosis in crypts appeared to parallel the

intensity of rejection. Grafts with full MHC expression underwent

the most vigorous rejection and also demonstrated the most crypt

cell apoptosis. Crypt cell apoptosis predated the development of

severe histologic rejection. The elimination of MHC I or II from

grafts resulted in reduced crypt cell apoptosis and delayed onset of

clinical rejection thus suggesting that cytotoxic attack was directed

against these ags.

Apoptosis in crypts may destroy the stem cells responsible for

intestinal mucosal regeneration thus resulting in sloughing and

erosion. The expression of allogeneic MHC ags on crypt stem cells

may provide a target for the host's immune response. Why crypt

cells are the dominant cells undergoing apoptosis may be

explained by the relative resistance of other noncycling intestinal

graft cells to CTL-induced apoptosis (Nishioka et al., 1994). Stem

cells in crypts are cyciing cells which may make them more

susceptible to CTL-induced apoptosis.

Expression of perforin and Coda phenotype correlated with

the intensity and distribution of apoptosis. POD 5 MHC-expressing

allografts with earlier high rates of crypt cell apoptosis also had

earlier infiltration of crypts by more perforin and CD8a positive

cells when compared to MHC I or It-deficient grafts. Although IL-

2 R a positivity i s indicative of T lymphocyte activation, it did not

help in distinguishing between classes of allografts and severity of

rejection. This is likely because the antibody recognizes CD4

positive cells (in addition to CDBa cells) which may not contribute

to crypt cell apoptosis. Thus, crypt cell apoptosis and presence of

perforin and CD8a positive cells in crypts were good markers of

rejection, whereas the presence of intracrypt CD4 positive cells

and IL-2Ra positive cells was not. The presence of CD4 positive

cells in crypts and intercrypts could actually be tolerizing recipient

cells i.e. CD4 positive cells secreting a Th2 cytokine profile

(Mosmann et al., 1986) could be suppressing cytotoxic T cell

activity.

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