Thesis submitted in fulfillment of the requirements for the degree of Master of Science

THE EXPRESSION OF PEROXISOME PROLIFERATOR-ACTIVATED RECEPTOR 1 (PPARy1 AND PPARy.2)

J.N

.NAiVE. AND MEMORY CD4+ T

LYMPHOCYTES

by

RAFEEZUL BIN MOHAME.D

Thesis submitted in fulfillment of the requirements for the degree of Master of Science

November 2006

ACKNOWLEDGEMENTS

In the name of Allah, the most Merciful and Compassionate

All praises and gratitude is due to Allah, the lord to whom every single creature in the heavens and the earth belongs to. May

peace

and blessings be upon our prophet Muhammad SA.W, his family and companions.

A bouquet of appreciation to my supervisors, Prof. Norazmi Mohd. Nor and Assoc. Prof.

Dr. Nik Soriani Yaacob for their invaluable guidancet criticism

and

support throughout this study. I am very grateful to the Ministry of Science, Technology and Innovation (MOSTI) for awarding me the National Science Fellowship scholarship. I also wish to

thank

the Dean of the School of Health Sciences and the Director of Institute for Research

in

Molecular Medicine, Universiti Sains Malaysia for giving me the opportunity to work in their laboratories.

All the members and ex-members

of

the 'NMNINSY, Research Group especially Dr.

Zulkamain, Dr. Rapeah, Or. Rahimah, Ariffin, K. Tie, K. Halisa, K Maryam, Teo, Rozairi,

I

rna.

Asma and Kenny deserve

my

gratitude for

their

warm friendship and encouragement

Special thanks to Encik Norhissyam Yaakob, Puan Rohayu Mahamat and Encik

Jamaruddin Mat Asan for their invaluable assistance in various aspects of the labwork carried out in this study.

Finally, I am very thankful to my beloved

mother

and my family for their warm love, which

drives my spirit to keep striving in the trying times. I love you and pray for you aU.

TABLE OF CONTENTS

ACKNO~DGEMENTS

TABLE OF CONTENTS LIST OF TABLES UST OF FIGURES

LIST OF ABBREVIATIONS UST OF SYMBOLS

ABSTRAK ABSTRACT

CHAPTER 11NTRODUCTION 1.1 T cell development

1.1.1 Generation ofT cells 1.2 T cell recognition

1.2 .. 1 Antigen presenting ceHs

1.2.2 Antigen presentation by major histocompatibility complex 1.2.3 Antigen processing

1.2.4 Antigen presentation to T lymphocytes 1.2 .. 5 Second signal-co-stimulator molecules 1.2 .. 6 T cell activation

1.3 The protein tyrosine phosphatase, C045 1.3.1 Structure of CD45

1.3 .2 Biological functions

1 .. 3.3 CD45 regulates protein tyrosine kinase, Lck

1.3.4 CD45

inactivate

JAK

family kinase

1.4 Na"ive C04+ T cells

Page

ii

vii

viii

X

xiv XV

xviii

1

1

3

3

4

7 11

14

16

20

20

23

23

24

24

1.5 Memory CD4+ T cells

1.6 Peroxisome prolfferator-activated receptor {PP AR) 1.6.1 Background

1.6.2 PPAR structure 1.62.1 AlB Domain 1.6.2.2

C

Domain 1.6.2.3 D Domain 1.6.2.4 E Domain

1.6.2.5

F~cdn

1.6.3 Transcriptional activation

of

PPAR 1.6.4 PPAR isoforms

1.6.4.1 PPARa 1.6.42 PPARf3/6 1.6.4.3 PPARy

1. 7 Peroxisome proliferator-activated receptor

r

(PPARy) 1. 7.1 Genomic organization of PPARy

1. 7.2 Ugands

of

^PP

ARy 1. 7 .2.1 Natural ligands

1.7.2.2 Synthetic ligands 1. 7.3 Biological fundions of PPARy

1. 7 .3.1 Adipocyte differentiation 1.7.3.21nsuRn sensitization

1. 7 .3.3 Cancer

1.7.3.4 Inflammation

1.7.3.5 Regulation

of

immune response

iii

26 28 28 28 30

30 30 30

31 31 33

33

34 35

36

36

38

38 38 39

39 40

40

41

42

1.8 Real-TimePCR 1.9 Multiplex PCR

1.10 Rationale and research objectives CHAPTER 2 MATERIALS AND METHODS 2.1 Materials and chemicals

2~ 1.1 Chemical and reagents 2.1.2 Kits and consumabtes 2.1.3 Enzymes and antibodies

2.1.4 Laboratory apparatus and equipment

2.1.5

Computer application programmes and software 2.2 Preparation of general solutions and buffers

2.2.1 ACK

2.2.2 PBS

2.2.3 DEPC

2.2.4 Ethanol (70%) 2.2.5 NaCI {5 M}

2.2.6 NaOH (3 M) 2.2 7 HCI (1M)

2.3 Agarose gel electrophoresis 2.3.1 Buffers and reagents

2.4 DNA agarose gel electrophoresis 2.4.1 Preparation of agarose gel

2.4.2 Agarose gel electrophoresis

2.5 Cell

culture

2.5.1 Reagents

43 44

45

48

48 48 48

48 48

54 54

54

54

54 55 55

55

55 55 57

57

57

57

57

2.5.2 Culture condition 2.6 Peripheral blood

2.71solation of naive and memory CD4+ T cells 2. 7.1 Isolation of PBMC

2. 7.2 Isolation of CD4+ T cells

2.7.2.1 Magnetic labeling 2.7.2.2 Magnetic separation

2. 7.3 Isolation

of naive

and memory CD4+ T cells 2.7.3.1

Magnetic labeling

2.7 .3.2

Magnetic

separation 2.8 Flow cytometric analyses

2.9 Stimulation

of naive

and memory CD4+ T cells 2.9.1 Preparation

of

ciglitazone (100 mM) 2.9.2 Preparation

of rHJ thymidine

⁽¹

p.Ci}

2.9.3 In vitro stimulation of

naive

and memory CD4+ T cells 2.9.4 Proliferation

assay

2.10 Isolation of Total RNA

2.1 0.1 Total RNA extraction

2.1 0.2

Electrophoresis

of total RNA

2~

10.3 Measurement of RNA purity and concentration

2.11 First strand eDNA synthesis

2.11.1 Verification of eDNA synthesis

2.12 Real-Time PCR

2.12.1 Reagents for Real-Time PCR 2.12.2 Real-Time PCR setup

2.13 Multiplex PCR

v

58 58

59 59 59 59 60 60 60 60

61 61

·61 63 63

63

64

64

65

65

65 66

66

66

70

71

2.13.1 Preparation

of reaction mixture

for MPCR 2.13.2 MPCR thermal profile

2.13.3 Densitometric analysis of MPCR products 2.14 Statistical analysis

CHAPTER 3 RESULTS

71

72 72 74

3.1 Efficiency of naive and memory CD4+ T cell isolation 75 3.2 Proliferative response of CD3/CD28-stimulated

naive

and memory C04+ T 75

cells

3.31solati0n of total RNA 79

3.4 First strand eDNA analysis 79

3.5 Quantification of PPARy1 and PPARy2 expression 82

3.6 The expression of selected cytokines in unstimulated and stimulated naive 88

and memory C04+ T cells ·

3. 7

Summary of results

95

CHAPTER 4 GENERAL DISCUSSIONS AND CONCLUSIONS

4.1

General

discussions

4.2 Conclusions 4.3 Future studies REFERENCES APPENDICES

97

118

119

121

147

LIST OF TABLES

Table

Idle

^Page

Table 2.1 List of general chemicals and reagents 49

Table2.2 List of commercial kits and consumables 50

Table 2.3 List of enzymes and antibodies

51

Table 2.4 Ust of laboratory apparatus and equipment

52

Table2.5 List

of

computer application programmes and software 53 Tab1e2.6 Fluorescence-labeled antibodies for dual-labeled flow cytometry

62

Table 2..7 Primer and

probe

sequences_ of human PPARy1 and PPARy2

68

Table2 .. 8 Composition of Real-Time PCR mixture 69

Table2.9A Optimum thennal profiles for MPCR analysis of cytokine genes

73

using the MPCR kit for Human Inflammatory Cytokine Genes Set-1

Table 2.98

Optimum thermal profiles for MPCR analysis of cytokine genes 73

using the MPCR kit for Human Th1ITh2 Cytokine Genes Set-1

vii

UST OF

FIGURES

Figure Title

Page

Figure 1.1 Pathways ofT cell development 2

Figure 1.2 Antigen presenting cells (APCs)

5

Figure 1.3 The genetic organization of MHC class J and II molecules 6

Ftgure 1.4 MHC molecules ⁸

Figure 1.5 Antigens processing pathways

10

Figure 1 .. 6 TCR complex

13

Figure 1.7 Critical molecules involved in antigen presentation

15

Figure 1.8 The IT AM complex 17

Figure 1.9 Intracellular signaling in T cell activation 19

Figure 1.10 The CD45 strudure.

21

Figure 1.11 Genomic organization of CD45ABC

22

Figure 1 ..

12

Schematic representation of the structure of PPARs

29

Figure 1.13 Transcriptional adivation of PPARy 32

Figure

1.14 Genomic organization of the human PPARy gene 37

Figure 1.15 Workflow for the current study

47

Figure 3.1 A representative dot plot after separation

of

non-CD4+ T

cells 76

Figure 3.2 A representative dot plot of the (a) unseparated na"ive and memory ⁷⁷

CD4+ T cells (b) memory

CD4+

T celt (c) naive CD4+ T cell

Figure 3.3 Profiles of [~]thymidine incorporation

of naive

and memory CD4+ T

78

cells after in vitro stimulation

Figure 3.4 Veriftcation

of

the integrity

of

total RNA extracted from unstimulated

80 and stimulated naive and memory CD4+ T cells with or

without

ciglitazone

Figure 3.5 Verifscation of the success of the first strand .. ~NA synthesis

81

Figure 3.6 A) The amplifiCation plot and B) the standard curve for measurement

83

of PPARy1 gene expression in unstimulated and stimulated

na·ive

and memory CD4+ T cells

Figure 3.7 A) The amplification plot and B) the standard curve for measurement 84 of PPARr2 gene expression in unstimulated and stimulated naive

and memory CD4+ T cells

Figure 3 .. 8 PPARy1 gene expression levels in unstimulated and stimulated

86

na"ive and memory CD4+ T cells with or without the presence

of

ciglitazone

Figure 3.9 PPARy2 gene expression levels

in

unstimulated and stimulated 87 na"ive and memory CD4+ T cells with or without ciglitazone

treatment

Figure 3.10 An example of MPCR products for the analysis of selected gene

89

expression using MPCR kit for Human Inflammatory Cytokine Set-1

Figure 3.11 An example of MPCR products for the analysis of selected gene

90

expression using MPCR kit for Human Th 1/Th2 Cytokine Set-2

Figure 3.12 Relative mRNA expression Jevels

of

Human Inflammatory cytokine

92

genes in naive and memory CD4+ T cells

Figure 3.13 Relative mRNA expression levels of Human Th1rrh2 cytokine genes 94 in

naive

and memory C04+ T cells

Figure 3.14 Summary of results

96

ix

13-ME

15d-PGJ₂

AF-1 AF-2

AP-1

APC

bp

CaCb

eDNA

C02

Co A CoR Ct

CDR CTLA-4

DAG

DBO

DC

DMSO

ON DNA

DP

UST OF ABBREVIATIONS

(3-Mercaptoethanol

15-deoxyi-A ¹2. ¹⁴-prostaglandin J2

activation function-1 activation function-2 Activated protein·1

Antigen presenting cells

Base pair

Calcium chloride Complementary DNA Carbon dioxide Co-activator Co-repressor Threshold cycle

Complementary determining region

Cytotoxic T lymphocyte

associated

antigen-4 Diacylglycerol

DNA-binding domain Dendritic cell

Dimethyl

sulphaxide

Double negative

Deoxylribonucleic acid

Double positive

EDTA

EtBr FAM FBS g

GM-CSF h

HCI IKB

IT AM

kDa L

LBO

LDH

M mg

MgCI2

min

mAb MHC

NaCt NaOH

Ethylenediamine-tetra acetic acid Ethidium bromide

6-carboxyfluorescein Foetal bovine albumin gram

Granulocyte monocyte colony-stimulating factor hour

Hydrochloric acid

Inhibitor

of

KB

lmunoreceptor tyrosine adivation motif kiloDalton

litre

Ugand binding domain Lactate dehydrogenase

molar

miligram

Magnesium chloride

minute

Monoclonal antibody

Major histocompatibility complex Sodium chloride

Sodium hydroxide

xi

NFAT Nuclear factor activated T ceJls

NF-KB Nuclear factor tcB

NHR

Nuclear hormone receptor

ng

Nanogram

NTC No template control

PBS Phosphate-buffered saline

PCR Polymerase chain reaction

PKC Protein kinase C

PTK Protein tyrosine kinase

PTP

Protein tyrosine phosphatase

PTPRC

Protein tyrosine phosphatase receptor type

C

PPAR Peroxisome proliferator-activated receptor PPRE Peroxisome proliferator response element

Rn

Normalized reporter

RNA Ribonucleic acid

RPM I Roselle's Park Memorial Institute Medium

RT-PCR Reverse transcriptase PCR

RXR

Retinoid

acid

receptor

TAE Tris-acetate-EDTA

TAMRA

6-alrboxyl-tetramethyl-rhodamine

TCR T ceU receptor

TBE

Tris-borate-EOTA

TNF TZD

tlv

Melting temperature Tumour necrosis factor Thiazolidinedione Ultra violet

xiii

UST OF SYMBOLS

j.l micro

< less than

oc

degree Celcius

a alpha

(3 beta

a

^delta

r gamma

K kappa

TM trademark

® registered l;

zeta

s

epselon

PENGEKSPRESAN RESEPTOR TERAKTIF PEMPROLIFERASI PEROKSISOM

y (PPARy1- DAN PP.ARy2) DALAM LIMFOSIT T CD4+ NAIF DAN MEMORI

ABSTRAK

Set T CD4+ periferi boleh dibahagikan kepada dua kumpulan berfungsi berdasarkan ekspresi isofom di permukaan motekut yang mengandungi dua

domain

fosfat intrasellular dikenali sebagai CD45. Sel T memori mengekspres isoform yang mempunyai berat molekul

terendah

iaitu CD45RO manakala

set

T

na·Jl mengekspres

isofom CD45RA (manusia) atau

CD45RB

(tikus). CD45 iatah protein tirosina fosfatase yang memainkan peranan penting sebagai pengantara isyarat TCR dengan mengaktifkan lck melalui defosforilasi pengawalatur

Tyros.

Sel T CD4+ naif dan memori manusia

berbeza

dari segi keperluan untuk pengaktifan dan magnitud tindakbalas sel. Sejenis reseptor nukleus, reseptor teraktif pemptoriferasi peroksisom y (PPARy), dilaporkan terlibat di dalam pengawalaturan aktiviti sel imun seperti makrofaj atau monosit dan limfosit

T.

Memandangkan peranannya di dalam pengawalaturan imun~ kajian terkini

dijalankan

untuk menentukan tahap pengekspresan PPARy di dalam set T CD4+ naif dan memori kerana tahap ekspresi

PPARy

berkemungkinan berbeza di dalam isofom CD45 yang berlainan. Tambahan

lagi,

perbezaan oorak pengisyaratan dan rembesan sitokin bagi se1 T subset tersebut mungkin disebabkan oleh gabungan dengan isofom PPARy- kemungkinan yang masih be1um dieksplorasi setakat ini. Bagi mengenalpasti peranan PPARr di dalam pengawalaturan pengaktifan sel T CD4+ nalf dan memori, ciglitazone

yang merupakan agonis bagi PPARy

digunakan

untuk memodulasi status pengaktifan se1 T CD4+

nail dan

memori

selain pengekspresan

PPARy

~ndiri dan sitokin terpilih. Denga~

menggunakan Real-Time PCR,

sel

T CD4+ nm1 dan m~.

talc

teraktif tidak

mengekspres

PPARy1

dan

PPARy2

manakala sel T CD4+ naif dan memori teraktif mengekspres reseptor tersebut pada paras yang tinggi dengan pengekspresan PPARy2 lebih tinggi berbanding PPARr1 di dalam kedua-dua jenis sel (p<0.01). Tambahan lagi, ekspresi PPARy1 lebih tinggi di

dalam se1

memori

ter.aktif

berbanding

sel

T CD4+

naif teraktif

{p<O .05}

manakala tiada perbezaan

bagi

pengekspresan .PPARr2 di dalam kedua-

dua sel yang teraktif. Penambahan agonis bagi PPARy iaitu cigtitazone meningkatkan pengekspresan PPARy1 kira-kira 61 kali dan 175

kaJi

masing-masing di dalam seJ T CD4+

naif dan memori yang teraktif (p<0 .. 01). 8erbeza dengan PPARy1, penambahan

ciglitazone mengurangkan ekspresi PPARy2 kira-kira 650 kali dan 140 kali di dalam sel T

CD4+

naif

dan memori teraktif {p<0.01 ) .. Tambahan lagi,

tahap ekspresi

gen TGF-Ji dan IL- 1

f3

adalah tinggi di dalam sel T C04+

naif

dan

memori

yang tak teraktif

tetapi

berkurangan di dalam keadaan teraktif (p<0.01). Gen IL-8 mengekspres

pacta

tahap yang rendah di dalam

set

T CD4+ naif dan memori yang tak teraktif tetapi meningkat di dalam keadaan

sel

tersebut yang teraktif (p<0.01). Wafaubagaimanapun tiada perbezaan bagi ekspresi

sitokin

tersebut di antara sel T CD4+ naif dan memori di dalam kedua-dua keadaan. IL-2, IFN-y, IL-5, ll-13,

TNF-a.,

GM-CSF dan IL-6 hanya mengekspres

di

dalarn

set

T CD4+ naif dan memori yang teraktif tetapi tidak dalam keadaan yang tak tetaktif. Tahap pengekspresan

IL-2 dan IL-13 adalah tinggi di dalam sef naif yang teraktif berbanding

se1

T CD4+

memori teraktif (p<0.01 ). Berbeza dengan IL-2 dan IL-13. tahap pengekspJesan IFN-ty adatah tinggi di dalam sel

memori

teraktif berbanding sel nail yang

teraktif

(p<0.05).

Walaubagaimanapun, tiada perbezaan di dalam pengekspresan

ll-5,

IL-6, TNF-a dan GM-CSF di antara kedua-dua jenis sel

yang teraktif.

Penambahan ciglitazone

mengurangkan tahap pengekspresan TGF-fl, IL-1[i,IL-8, IL-2. IFN-y, IL-5, TNF-a. dan GM-

CSF di dalam sel T CD4+

nail

dan memori yang teraktif. Peningkatan PPARy1 dan perencatan ekspresi PPARy2 di dalam sel T CD4+ nail dan memori di dalam kehadiran

ciglitazone mencadangkan bahawa isoform PPARy mungkin mempunyai fungsi yang berbeza dalam pengawalaturan

sel

T. Pengekspresan gen sitokin yang terpilih di dalam sel T CD4+ naif dan memori yang teraktif adaJah konsisten dengan kajian terdahulu.

Mekanisma sebenar bagaimana

PPARr

merencat pengekspresan sitokin di dafam sel T CD4+

na"1T

dan memori teraktif atau isoform PPARy yang mana bertanggung jawab terhadap kesan ini masih belum dapat di pastikan. PPARy berkemungkinan merencat pengekspresan gen sitokin di dalam sel subset yang teraktif melalui interaksi dengan NF- KB. AP-1 dan STATs yang merupakan faktor transkripsi yang penting bagi sitokin tersebut sebagaimana kajian terdahulu di dalam

set

yang lain ..

xvil

THE EXPRESSION OF PEROXISOME PROLIFERA TOR-ACTIVATED RECEPTOR

y

(PPARy1 AND PPARy2) IN NAi.VE AND MEMORY CD4+ T LYMPHOCYTES

ABSTRACT

Peripheral CD4+ T cells can be divided into two functional groups based on the expression of distinct isoforms of the surface molecule that contains an intracellular two- domain phosphatase portion, known as CD45. Memory T cells express the lowest molecular weight CD45RO isofonn. whereas

naive

T cells express CD45RA (human) or CD45RB (mouse) isoforms. CD45 is a protein tyrosine phosphatase which plays an important role in TCR-mediated signaling through its activation of Lck by de- phosphorylating the regulatory

Tyros.

Human

naive

and memory CD4+ T cells differ in the.

requirements for activation and magnitude

of

the cellular responses. The nuclear receptor,

peroxisome proliferator-activated receptor 1 {PPARy) has been reported to be involved in

regufatinQ

the activities

of

immune cells such as macrophages or monocytes and T lymphocytes. Given their roles in immune regulation, the current study was carried out to determine the expression

of

PPARy in human

naive

and

memory

CD4+ T cells since it is possible that

PPARy

may be differentially

expressed

in the

different

isoforms of CD45. In addition. the differential signaling patterns and cytokine secretion

of

these subsets of T cells may require engagement with PPARy isoforms ... a possibility that has not been explored thus far. To further dissect

the

rote of PPARy in the regulation of naive. and

memory

CD4+ T cell

activation,

the PP AR.y

agonist, ciglitazone, was used to modulate the

activation status

of

naive and memory CD4+ T cells as wetl as the expression

of

PPARy

itself and selected cytokines. Using Real .. Time

PCR.

unstimulated

naive

and memory CD4+ T cells were found not to express PPARy1 and

PPARy2,

whereas stimulated naive

and memory CD4+ T cells express high levels of these receptors with PPARy2 expression being higher than PPARy1 in both cell types (p<0.01). In addition, the PPARy1 expression was higher in stimulated memory as compared to stimulated

naive

C04+ T cells {p<0.05) whereas there was no significant difference between PPARy2 expression in both types of stimulated cells. The addition- of the PPARy agonist, ciglitazone signifiCantly increased the expression of PPARy1 by about 61-fold and 175-fold

in

stimulated naive and memory CD4+ T cells respectively (p<0.01). In contrast to PPARy1, the addition of ciglitazone significantly decreased the expression of PPARy2 by about 650-fokl and 140-fold in stimulated na"ive and memory CD4+ T

cells

respectively {p<0.01). In addition, the expression levels of TGF-)3 and· IL-1 J3 gene were higher in unstimulated naive and memory CD4+ T cells but decreased in their stimulated state (p<0.01). lL-8 gene was expressed at low levels in unstimulated but elevated in stimulated naive and memory CD4+ T cells (p<0.01}. However, there were no significant differences in the levels of these cytokines between naive and

memory

CD4+ T celts between both states. IL-4 lFNy, IL-5, IL-13, TNFa, GM-CSF and IL-6 were only expressed in stimulated naive and memory CD4+ T ceUs but not. in their unstimulated state. The expression levels of IL-2 and IL-13 were significantly higher in stimulated

naive

as compared to stimulated memory C04+ T cells (p<0 .. 01 ). In contrast, the expression levels

of

IFNy were significantly higher in stimulated memory as compared

to

stimulated naive CD4+ T cells (p<0.05). However, there were no significant differences in the expression of IL-5, IL-6, TNFa and GM..CSF between both stimulated cell types. The addition

of

ciglitazone. decreased the expression levels of TGF-

p,

IL-1p, IL-8, IL-2, IFNy, IL-5, TNFa. and GM-CSF in stimulated memory and na"ive CD4+

T cell& The induction of PPARr1, and suppression of PPARy2 expression in naTve and

memory CD4+ T cells in the p~sence of ciglitazone suggest

that

the PPARy isoforms may have different functions in T cell regulation. The expreqi~n of $elected cytokine genes

in

I : ' • '

xix

activated naive and memory CD4+ T cells is consistent

with

previous studies. The exact mechanism of how PPARy inhibit cytokine expression in stimulated

naive

and memory CD4+ T cells or which PPARy isoforms is responsible for this effect remain uncertain. It is possible that PPARy inhibit the expression of cytokine genes in these stimulated cell subsets. via interacting with NF-KB, AP-1 and STATs. which is important transcription factors for these cytokines as shown by previous studies in other cells.

1.1 T cell development 1.1.1 Generation ofT cells

CHAPTER ONE

INTRODUCTION

T cell development occurs in the thymus (Surh and Sprent, 2005). The thymus is a multi- lobed organ consisting of cortical and medullary areas surrounded by a capsule (Figure 1.1). T cell precursors enter the subcapsular cortical areas, where they encounter networks

of

cortical epithelial cells (the thymic stroma) and undergo a period of proliferation. After differentiation. they migrate from

the

cortex towards

the

meduUa of

the

thymus.

In the thymic cortex, progenitor

cells

derived from

the

bone marrow differentiate into T cell lineage by rearranging the TCRJ3 chain and expression of the

pre-

TCR complex which lead to multiple changes including, massive expansion of 'double negative• (ON)

cD4- a-

precursors

by

IL-7, upregulation of CD4 and CDS on the 'double-positive' (DP)

c04• a•

thymocytes, and rearrangement

of

TCRa chain for expression of the TCR (Michie

et

^al.,

2002; von Boehmer, 2004) (Figure 1.1).

DP thymocytes undergo positive selection

to

remove cells that have significant TCR reactivity for self~HC/peptide complexes. After positive selection, DP cells migrate from the cortex to the medulla and differentiate into single-positive CD4 +

a-

^{and CD4-}

a+

^cells.

During this differentiation process, negative selection takes over to destroy autoreactive T

cells

with

high

avidity

for self components (Sprent

et

^{at .. , 1995;}

Starr

^~tat., 2003) .. After

positive and negative selection,

depending

on the affinity and the conwxt

of

such binding,

1

Progenitor cells derived from bone marrow

L ~ ~

y&+, C03+, C04-, COB-

Positive selection

ap+, C03+, C04+, COB+, TCR+

(Ooublel positive)

• >95%

Apoptosis

Thymus

Figure 1.1 Pathways ofT cell development (modified from Mehr eta/., 1995)

95% of developing DP cells die via apoptosis while

only

a small

fraction (

<5%) of DP ceUs,

move in1o the periphery to form the T cell pool (Surh and Sprent.

2005).

In

addition to T ceUs expressing o:J3 TCR, which constitute the majority

of

T cells in

adultst there

is a lineage ofT cells expressing the yo

TCR. These

cells are

abundant

in various

epithelial tissues, such as the epidermis {in mice but not humans), intestinal epithelium, uterus and tongue .. The y5 T ceUs recognize both exogenous antigens such as viral and protozoal peptides. and autoantigen such as heat shock proteins_ Moreover, the peptides can be presented by either class I or class II MHC molecules, and there are several studies which suggest the y8 T cells do not require antigen to

be

presented by MHC molecules

at

all (Adams et al., 2005).

1.2 T cell recognition

Approximately 90-95% of peripheral blood T cells are

a.J3

T cells.

aJJ

T cells are mainly divided into CD4+ helper T cells and COS+ cytotoxic T cells. CD4+ T cells recognize their specific

antigens

in association

with

the major

histocompatibility complex

(MHC) dass II molecules, whereas COS+ T cells recognizes antigen in association with MHC class I

molecules.

1.2.1 Antigen presenting cells

Antigens must be processed by antigen presenting cells (APCs) before presentation toT lymphocytes. The APCs which are essential forT cell activation includes dendritic cells (DCs}, mature B cells and macrophages (Sille eta/., 2005).

Des

are found in lymphoid

tissues, connective tissues and epithelial cells (Bell et

al.,

1999). OCs capture antigens in

peripheral tissues and then move to lymph nodes, where they express high levels

of

3

adhesion and ·co-stimulatory molecules, as well as MHC class II antigens (Bell

et

a7.,

1999)~ DCs stop synthesis of these molecules when they migrate but express high levels of MHC class II molecules containing peptides from antigens produced

by

tissues where the DCs originated (Bell eta/., 1.999).

8 ceUs can bind to a specific antigen,

internalize it

and then degrade

it

into

peptides

whiCh

associate with MHC class II . molecules~ B cells which have high affinity antigen receptors {lgM or lg.O)

are

^{the most} potent APC at low concentrations of antigens because other

APCs cannot capture enough antigens (MeUman et al., 1998). B celis

do

nOt eXpress eo-

stimulatory molecules such

as

87 but · can

be

induced · to produce

87

by bacteria1 constituents.

Macrophages ingest microbes and particulate antigens by ·phagocytosis for -processing

and presentation

by

MHC class II molecules (Kalish, 1995). High levels

of MHC and co-

stimulatory molecules such as 87 are induced by the uptake of bacteria which is enhanced by receptors specific to certain surface components of bacteria (Mellman et

at.,

^1998).

These APCs are illustrated in Figure 1.2..

1.2.2

Antigen presentation by major

histocompatability complex molecules

The MHC is involved in presentation of antigens to T cells. The human MHC class I molecules includes the HLA-A, B and C whereas the human MHC class II molecules comprise

of

the DR, OP and DQ

(van

den Elsen

et at.,

2004). The

genetic

locus of MHC class

1

and II is presented in Figure 1.3. MHC class I and II molecules are members of the

immunoglobulin supergene family which consist of multiple "immunoglobulin domains" that

Dendritic cells

http://www.dukecancervaccines.org/images/

dend.jpg

B cells

http://www.4-antibody .comlimages/cells.jpg

Macrophage

http :1/www. healingdaily. com/detoxification- diet/macrophages.jpg

Figure 1.2 Antigen presenting cells (APCs)

5

Class Ill

DP

DQDR

~ B C

^A

• D []:[] I I I I I []:[] 0=

HLA dass II

HLA

class

I

HLA-DR} HLA-A}

HLA-DQ Human

HLA-B Human

HLA-DP HLA-C

I-A

} Mouse

H·2K}

1-E

H-20 Mouse

H-2L Figure 1.3 The genetic organization of MHC class I and II molecules

have similar structure and amino acid homology to the constant and variable domains of immunoglobulins (Kalish, 1995}.

i)

MHC class I molecules

MHC class l molecule consists of a heavy chain with three immunoglobuUn domains, in noncovalent association

with

Pz-microglobutin (van den E1sen et

a/.,

2004) and are expressed on all nucleated

ceus

(Kalish, 1995) (Figure 1.4) .. The first two domains of the alpha chain,

a1

and a2 form a "groovelf that binds a peptide and together

with J3:r

microglobufin, the three member complex allows for its stable expression on the cell surface {Anderson eta/., 1993). MHC class I molecules bind peptides consisting of 8 to 10 amino acids (Falk et a/., 1993). The peptide-binding groove contains pockets with amino acid residues which formed specific interactions with the amino and carboxylic acid terminals of the peptide (Wilson and Fremont, 1993) ..

il) MHC

class

II molecules

MHC class II molecules are comPQSed of two a. chains and two f} chains (Kalish, 1995)

(Figure 1.4). The peptide-binding groove

of

MHC class II molecules is open at the ends

and enables binding of longer peptides compared to the MHC class I molecules {Rudensky

et

a/., 1991 ). In the absence of stimulation, MHC dass II molecules are only expressed on professional APCs such as macrophages, mature B ceiJs, Langerhans cells and dendritic cells (Kalish,

1995).

The expression

of

MHC class II molecules can be

induced

by

interferon gamma on kerattnocytes and

endothelial

cells (Kalish, 1995).

1.2.3 Antigen processing

Antigen processing involves antigen degradation into peptide fragments which

are

recognized by T

cells

via the TCR. A minority of peptide·

fragments

from protein antigens is 7

HLA-Aw68 (class I)

HLA·DR1 l~ U)

Figure 1.4 MHC molecules (adapted from Roitt eta/., 2001)

able to bind to particular MHC molecules. Furthermore, different MHC molecules engage with different sets of peptides.

i) Endogenous antigens

Protein antigens arising from inside the cell (endogenous antigens) are processed by

the

endogenous pathway for presentation to T cells. Examples of endogenous antigens are viral antigens, transplantation antigens and tumor-associated antigens (Kalish, 1995).

Endogenous antigens derived from cytoplasmic proteins (Moore

et

a/., 1988) are targeted

by

conjugation

with

ubiquitin (Michalek

et

a/.,

1993;

Ciechanover and Schwartz.

1994)

facilitating them to be degraded into peptides by the proteosome complex (Yang

et

aL, 1992). Peptides derived from the degradation of cytoplasmic proteins are transported from the cytoplasm to endoplasmic reticulum (ER) by the transporter-associated antigen

processing (TAP1 and TAP2) molecules {Spies eta/., 1990; Attaya

et

^al., 1992; Colonna

et

al.,

1992;

Spies eta/., 1992; Suh eta/.,

1994).

Within the ER, a series of chaperone proteins, induding calnexin, calreticulin, ERp57, and immunoglobulin binding protein (Bip) facilitates the proper folding of MHC class 1 and its association with J32 microglobulin. The partially folded MHC class I molecules then interact

with

TAP via tapasin. Newly synthesized MHC class I molecules cornplexed with peptides are then transported from the ER through the Golgi complex to the cell surface for recognition by CDS+ T cells (Cox

eta/.,

1990) (Figure 1.5).

li) Exogenous antigen

Protein antigens from outside the ceH (exogenous antigens) are processed

by the

exogenous pathway for presentation to

T

cells. Examples of exo~nous antigens are extracellular bacteria, bacterial toxins, vaccines and allergens which include pollen and

9

Exogenous

antigen Endocyrtc Class I MliC

I

compartments

- - - t - - - , . r - - . .

End,>gcnc>cts pathway

(cbs~ J :\U!C)

Exogenous p;Hh wa) (cbs!'\ II ~·1!HC'J

Figure 1.5 Antigens processing pathways (cited from http://www.anzies.com.au)

dust mites (Kalish, 1995). Exogenous antigens are internalized in endosomes by phagocytosis or pinocytosis. The endosomal vacuoles fused with lysosome to form lysosome/endosome compartment. This compartment has an acidic pH and contains multiple degradative enzymes such as acid proteases and cathepsins (Rodriguez and Diment, 1992) to degrade the exogenous antigen into peptides. MHC class II molecules are transported into this lysosome/endosome compartment to bind to the processed peptides (Neefhes and Ploegh, 1992).

MHC class II molecules are

firstly

synthesized in the ER (Kafish, 1995). The invariant chain facilitates MHC class II export from ERin a vesicle (Teyton

eta/.,

1990). This vesicle

will fuse with endosome containing degraded proteins and then broken down to leave only a small fragment called CLIP which responsible to inhibit the binding of peptides to the MHC class II molecules before arrival in the endosome/lysosomal compartment {Bodmer

et

a/., 1994). Peptides generated in the lysosome/endosome compartment are presented by MHC

class

II

molecules on the cell surface for recognition by

C04+ T

cells (Fabbri

et a/., 2003; van den Elsen

et at.,

2004) (Figure 1.5).

1.2A

Antigen presentation

to T lymphocytes

Antigen presentation is the process by

which

T cells recognize antigen on the surface of an APC (Kalish, 1995). The peptide-MHC (pMHC) complex is recognized by af3 T cells through their TCR (Garcia and Adams, 2005). The TCR is a genetically recombined receptor and analogous to an immunoglobulin molecule (Garcia and Adams, 2005). It is a

heterodimer consisting

of

a and p chain held together

by

disulfide bonds,

.each

with one variable and one constant domain. However, 5% Of the T cells, especially those found in the skin and mucous layers, have TCR consisting of the

1

and o chain. Va. and vp are the

hypervariabte regions of TCR, that form three complementary detetmining regions (CDRs}

11

on each chain. CDR1 and CDR2 usually bind to MHC while CDR3 usually binds to the peptide (Garcia and Adams, 2005). TCR has a very short cytoplasmic tails, Which is not appropriate in signal transduction. So it is expressed on the membrane together with the signal transduction complex, CD3. as TCR complex (Figure

1.6).

CD3 is composed of four invariant polypeptides called y, e.

o

^and t;. The CD3 chains are organized as heterodimers of either

rs

or 8e and a homodimer of ~~- The

y, o

and s chains have negatively charged transmembrane region which forms salt bridges with positively charged transmembrane regions of

TCR.

The l; chain comprises a small extracellular domain of only nine amino acids which includes the disulphide bond, a transmembrane segment including a negatively charged residue and a large cytoplasmic tail. All CD3 chains contain one immunoreceptor tyrosine activation motif (IT AM), while three copies of ITAMs are present on each of the l; chain•s cytoplasmic tail (Chan eta/., 1994). FoDowing antigen binding, the ITAMs will associate with tyrosine kinase to initiate intracellular signaling cascade. CD4 and COS are co-receptor molecules that bind

to

the non-polymorphic regions

of

MHC molecules and enhance the binding avidity of T cells to the APC. CD4 is a monomeric protein with four domains where two distal domains that bind to the f32 domain of MHC class II. COB is an

a

and

p

chain heterodimer, each with one domain and a long extended region. COB binds to the a3 domain of MHC

dass I.

Both CD4 and C08 have Lck associate with its cytoplasmic tail to initiate signal transduction (Figure 1.6). The Lck or p561ck is a lymphocyte-specific tyrosine kinase of 56 kDa that is attached

to

the intracellular

portions of CD4 or CDS.

CDS

CD4

~C03~

Figure 1.6 TCR complex (cited from http://www.immuno.path.cam.ac.uk)

13

1.2.5 Second signal-co-stimulator molecules

Co-stimulatory molecules do not bind to antigen directly but act when TCR binds to antigen. The main functions of co-stimulatory and other accessory molecules are to transduce signal for the complete activation of T cell, ads as adhesion molecules to stabilize the interaction between T cells and APCs and facilitate the migration of T cells. T

cells

that do not receive co-stimulation become anergic (not functioning) and this is important to ensure that self-antigen will not activate self-specific T cells that escaped negative selection (Tan eta/., 1993). There are perhaps many co-stimulator molecules that are involved in the T cell activation process. Some of the important ones are briefly described

below.

i) B7-CD28

87 molecules are expressed mainly by professional APCs such as dendritic

cells,

macrophages and mature B lymphocytes. 87-1 and 87-2 are structurally similar, single chain glycoprotein,

each

with

extracellular lg-like domain, a transmembrane segment and

cytoplasmic tail. Co-stimulation ofT cells mainly comes from the binding of 87-1 (COSO) and 87-2 (CD86)

on

APC to the CD28 molecule on T cell membrane {Turka

et at.,

1990) (Figure 1 .. 7). The binding between these molecules brings upon the expression of anti ..

apoptosis proteins such as Bcl-x, production of cytokines such as IL-2 and proliferation and differentiation ofT cells.

II) Cytotoxic T lymphocyte associated antigen 4 (CTLA-4) (CD152)

Activated T cells express CTLA-4 instead of CD28 .. CTLA-4 is also a receptor for

87

molecules. Its binding to 87 inhibits T cell function

by

interrupting signals transduced

by

CD28. Mutant T cell that lack CTLA-4

cannot be deactivated, resulting in the increase

incidence of autoimmune reactions.

C02

LFA-3 (CD58)

LFA-1

ICAM-1

Tcell

APC TCR

MHC II

CD28

87-1/87-2 (CD80/CD86)

Figure 1.7 Critical molecules involved in antigen presentation (adapted from Roitt eta/.

2001)

15

iii) Other accessory molecules

CD2 act as an adhesion molecule by binding to LFA-3 (CD58) molecules found on a wide range of cells (Damle

et at.,

1992; Perlmutter, 1993) {Figure 1.7). lt is involved in the interaction between T cells and APCs. CD11 b/CD18 is also an adhesion molecule Hke CD2. It binds to the intracellular adhesion molecule-1 (ICAM-1) on APCs or target cells (Damle et a/., 1992~ Perlmutter, 1993) (Figure· 1 .. 7). Activated CD4 cells express.

CD401igands (CD40L) that binds

to

the CD40 molecules on. B cells and macrophages .. The-

binding induces the activation of B cell and macrophages accompanied

by

the induction of

87 molecule expression on APC and enhancement of proliferation and differentiation of T

cells.

1.2.6 T cell activation

T cell activation is initiated

by

the interaction of the TCR with peptide-MHC complexes.

TCR engagement triggers the tyrosine phosphorylation

of

the IT AM present on the TCR- associated CD3-l; subunit by the protein tyrosine kinase (PTK) Lck (Chan et al., 1994;

Myung et a/., 2000). All ITAMs contain two sequence elements (Tyr-X-X-Leu) with potential tyrosine phosphorylation sites which is separated by seven or eight variable

amino acids (Hegedus et at., 1999) (Figure 1.8). When two tyrosines in a single

motif are phosphorylated, IT AM forms a binding site for a cytoplasmic PTK, known as ZAP-70. This recruitment initiates

ZAP-70

activation and downstream signaling cascade such as the activation of phospholipase ~1 and protein kinase C (PKC).

Engagement of TCR induces activation of phospholipase Cy1 (PLCy1),

which

catalyzes

the hydrolysis of inositol phospholipids (PIP2) to produce inositol 1, 4, 5-triphosphate (IP3)

and diacylglycerol (DAG)

which

activate two distinct signaling

pathways

in T cells (van

Leeuwen and Samelson, 1999}. IP3 migrate from the cytosol to the endoplasmic reticulum,

r-X-X-Leu-X-X-X-X-X-X-X-Leu-X-X-Tyr

Figure 1.8 The IT AM complex

The ITAMs contain two sequence elements (Tyr-X-X-Leu) separated by 7 or 8 variable amino acids. When the tyrosine residues are phosphorylated, IT AM acts as a docking site for the tyrosine kinase, ZAP-70.

17

where it binds' to its receptor and initiates the release of internal Ca²+ stores which then cause a rapid increase in the cytosolic free Ca²+ ion concentration (Lewis, 2001). Cytosolic free Ca²⁺ acts as a signaling molecule by binding to calmodulin (Ca^{2 ....} dependent regulatory protein) (Quintana et al.,

2005).

This calcium-calmodulin complex activate several

enzymes especially calcineurint

a

Ca

²+

-calmodulin dependent

protein phosphatase which subsequently dephosphorylates the nuclear factor of activated T cells (NFAT) (Rao et al., 1997). This transcription factor is an inducible regulatory complex critical for transcriptional induction of IL-2 in activated T cells, but also regulate the transcription of various genes such as cytokines, cell surface receptors and regulatory enzymes (Rao et al., 1997;

Martinez-martinez

et

a/.t 2004). The increase in free cytosotic C<f+' results in the translocation of inactive cytosolic PKC to the cell membrane.. DAG activates PKC by inducing a conformational change that becomes the catalytic site of the kinase accessible to ~ubstrate (Lewis, 2001 ). Although DAG activates multiple isofonns of PKC, only PKCa is

required forT cell activation in vivo (Sun et al., 2000; Pfeifhofer eta/., 2003). The GTP

bound Ras (GTP·Ras) functions as adivator of a cascade of enzymes namely mitogen·

activated protein (MAP) kinases. The MAP kinase cascade consist of three different kinases, the extracellular signal-regulated kinases (ERKs) (Schaeffer and Weber, 1999), p38

Map-kinases (Han

and

Ueitch,

1999)

and

c-Jun NH2-tenninal kinases (JNKs) (Davis, 2000). The activated ERK phosphorylates Elk1, which then stimulates transcription of Fos, the first component of the activation protein-1 (AP-1) (Genot eta/., 1996). Parallel to this pathway, the adapter protein also activates a GTP exchange protein namely Vav that

acts

on Rae. This Rac-GTP initiates the activation of the c-Jun

by

JNK

at

Ser-63 and Ser-73 within the transactivation domain (Whitmarsh and Davis; 1996). c-Jun heterodimerlzes

with c-Fos to form AP-1, the regulatory element in the IL-2 promoter

that

is important for

early transcription

of

the IL-2 gene (Durand et

al.,

1988). The brief overall intracellular signaling involved in T cell activation as described herein is summarized in

Figure

1.9.

CD45 CD4

phosphatase

domains PLC-y

•

PIP~~-

IP

f:1? ~ DAG

C{J' +

Ca²•

+ ®

+ + + +

+ + +

Figure 1.91ntracellular signaling in T cell activation (adapted from Roitt eta/., 2001)

19

1.3 The protein tyrosine phosphatase, CD45

The protein tyrosine phosphatase (PTPase) CD45 is a transmembrane glycoprotein expressed on the surface of all nucleated haematopoietic cells except mature erythrocytes and platelets (Trowbridge and Thomas, 1994). CD45 consists

of

its splice variants namely CD45RA, CD45RB, CD45RC and CD45RO. CD45 comprises up to 10% of the T and 8 cell surface molecules (Sasaki et al., 2001 ). This high level of expression coupled with the differential expression of selected variants on naive and memory T cells and their role in immune regulation makes the CD45 molecules are important molecule to be investigated.

1.3.1 Structure of CD45

CD45 is expressed in muHiple isoforms as a result of alternative RNA splicing of exon 4 to

s

(Trowbridge and Thomas, 1994; Alexander

et

at~, 1997) with a molecular weight range

'

from 180-240 kDa. The high molecular weight isoforms contain exon 4/A, 518 and 6/C (CD45ABC) and the low molecular weight is the CD45RO isoform (Figure 1.10). The genetic organization

of

CD45 is shown in Figure 1.11. The CD45 ectodomain is characterized by the three alternatively sp1iced exons A, 8 and C that are rich in serine, threonine and proline residues, a cysteine rich domain followed

by

fibronectin (FN)

type Ill

repeats at the N-tenninus (McNeill eta/., 2004). The ectodomain is heavily glycosylated,

mainly

N-linked

in the FN-111 and cysteine

rich

regions and O..Jinked in the A, B and C exon

encoded regions (McNeill et al .• 2004). The CD45RO lacks the A, 8 and C exon encoded region (McNeill

et

^a/., 2004). The intracytoplasmic tail of CD45 is highly conserved between all mammalian species and contains the so-called 01-domain which has PTPase activity, whereas the 02-domain has no signifiCant PTPase activity due to changes in critical

amino acids required for catalytic

activity (Sasaki

eta/.,

2001).

Figure 1.10 The CD45 structure (adapted from McNeHI eta/., 2004)

21

CD45Exons

4

I 5 I 6

3

4

5

6 7

CD45RABC

CD45RAB

CD45RAC

CD45BC

CD45RA

CD45RB

CD45RC

CD45RO

Figure 1.11 Genomic organization of CD45ABC (adapted from Fukuhara

et

al.., 2002)

1.3.2 Biological functions

CD45 is an important positive regulator of TCR and BCR mediated signaling required for the activation and development of lymphocytes (Kishihara et al., 1993; Byth et al., 1996).

CD45 PTPase activity is required for histamine degranulation following lgE receptor cross- linking in mast cells (Berger et al., 1994). CD45 expression is also invOlved in antigen receptor-driven thymocyte maturation and B cel1 selection (Kishihara eta/., 1993; Byth et

at.,

1996). Mutation in the protein tyrosine phosphatase receptor type C (PTPRC} gene encoding CD45 and abnormalities in the expression of CD45 splice variants increased the incidence of severe combined immunodeficiency disease (SCID) in humans (Kung et al.,

2000).

1.3.3 CD45 regulates protein tyrosine kinase, Lck

An important early step in TCR signaling is the phosphorylation of ITAMs by the Src-like protein tyrosine kinase Lck (lrles

et

a/., 2002) .. This kinase is located in glyosphingoUpid- enriched membrane {GEMs) and its activity is regulated by the opposing actions of the CD45 PTPase and the carboxyl-terminal Src kinase (Csk) (Bergman

et

al., 1992; Thomas and Brown, 1999). Phosphorylation of

Ty,SOS

in Lck

by

Csk leads

to

^an Src homology domain 2 (SH2)-mediated intramolecular interaction

which

inhibits activation of Lck by trans-autophosphorylation of Ty~ in its catalytic domain (Sicheri and Kuriyan, 1997).

CD45 dephosphocytates the

Ty,SOS

to relieve

this

inhibition (Yamaguchi and Hendrickson.

1996; Sicheri and Kuriyanr 1997) allowing the Lck to promote full phophorylation

of

the IT AM motifs of the CD3/' components and faalitate the recruitment and activation of the ZAP-70 tyrosine kinase (Leitenberg

et at .•

1999). In addition,

C045 may

maintain Lck in an

"open" configuration

which

enhances

Lck.

interaction and recruitment of various adapter proteins and other signaling molecules into large macromolecular complex that facilitates T

23

cell activation and IL-2 secretion independently of Lck kinase activity (Collin and Burakoff, 1993; Sieh eta/., 1993; Xu and

Littman,

1993).

1.3.4 CD45 inactivate Jak

family

kinase

The Janus .. kinase/signal transducers and activators of transcription (Jak/STA

T) pathway

are involved in the signaling of many cytokines (Scott eta/., 2002). Following stimulation of cytokine receptors, Jaks (Jak1, Jak2, Jak3 and Tyk2) are phosphorylated on their tyrosine residues and are activated to phosphorylate STATs. STATs phosphorylation by Jaks

results in dissociation and homo- or hetero.dimerization and ultimately translocation to the nucleus where they can rapidly and specifically activate target genes (lvaskhiv, 1995).

CD45 dephosphorylates all Jaks and inhibit the secretion of cytokines (Sasaki et al., 2001). CD45 also negatively regulates JL .. 13 mediated cellular

proliferation.

erythropoetin- dependent hematopoiesis and antiviral responses in vitro and in vivo (Sasaki et at., 2001 ).

1.4 Naive CD4+ T cells

Naive CD4+ T cells are thymic emigrants which had already undergone various maturation and selection processes in the thymus and populate the peripheral blood and secondary lymphoid organs such as spleen,

lymph

nodes and the mucosal-associated lymphoid tissue.

They

have not been activated

by

specific antigen in

vivo

{Van Uer and Baars,

1999). These cells can

be

characterized by the expression

of

the high molecular weight isoform

of

CD45, CD45RA, and low levels of 131 and P2 integrins (Sanders et

a/.,

1988;

Prince et al., 1992; Masopust

et

al., 2004). Naive CD4+ T cells express CCR7 and the

peripheral lymph node homing receptor CD62L

that

facilitate the migration

of

these cells to

the T cell areas

of

the secondary lymphoid organs (SaOusto

et a/., 1999; Masopust et a/.,

2004; Sallusto et

^al., 2004). Naive

CD4+ T cells require the engagement not

only

of TCR,

but also the

costimulatory

receptor, CD28 for complete

activation which

leads to