FUNCTIONS OF CD45 IN TCR SIGNALING IN

CD4+CDS+ DOUBLE-POSITIVE THYMOCYTES

Gordon W. Cheng

A thesis submitted in conformity with the requirements

for the degree of Master's of Science

Graduate Department of Immunology

University of Toronto

O Copyright by Gordon W. Cheng (1997)

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- a

Thymocytes

Master's of Science (1997)

Gordon W. Cheng

Graduate Department of Immunology University of Toronto

Abstract

T ceIl receptor (TCR) signals are essential for normal T cell developrnent, and CD45 is thought

10 be essential for coupling TCR to the intracellular signaling rnachinery. However, T cell

development is only partially compromised in CD45-deficient mice. This thesis describes a

CD45-deficien t CD4+CD8+ double-posi tive (DP) thymoma called 3T7. 1 show that in 3T7 ce1 ls

and DP thymocytes CD45 is necessary for the TCR-induced protein tyrosine phosphorylation,

as well as changes in CD5, M G - 1 and CD41CD8 expression levels. However, CO-aggregation

of TCR and either CD4 or CD8 induced signaling events in a CD45-independent rnanner,

providing a ralionale for the developmental phenotype observed in CD45-dericient mice.

Surprisingly, 1 found no disferences in the overall phosphorylation slatc of Lck or Fyn in CD45

deficient cells versus CD45-positive cells. However, 1 show that loss of CD45 is accornpaiiied

by a hyperphosphorylation of TCRS in 3T7 cells and converscly a hypophosphorylation of

TCRC in CD45-deficient thyrnocytes, providing onc possible biochemical mcchanism for ~ h c

TCR signaling dcficits dcscribed. Collectively, these observations providc a conccplual and

esperimental framcwork for understanding the role of CD45 in TCR signaling at the DP slagc of'

T ce11 dcvelopment.

Acknowledgments

1 would likc to thank al1 those without whom this work would not have been possible.

First and foremost, 1 thank my supervisor Cindy, for her guidance and support. For sharing thc

reagents rvhich allowed this work to be done: Drs. Pauline Johnson for the CD45 construct;

Michael Julius for the a-Lck antisera; Phi1 Branton for the a-CD45 antisera; Andrc Veillcttc for

the a-Fyn antisera; and Josef Penninger for the CD45-/- mice. To my cornmiitcc, Drs. Michacl

Julius and Rob Rottapel for their scientific input. Jayne Danska, for her big piclure and

conceptual input. The Danskonians (Danny, Case, Priscilla, Chns ti ne, Ildico) for their group

supporUtherapy. To my sister, Serena, for always listening & understanding - 1 thank you. To

my parents as always, your patience, lovc and confidence in me have allowcd mc to complctc

this, and mainlain somc semblance of sanity. And last and most importantly, 1 mus1 lhank my Iab

mütcs pas1 and prcscnl (a s p i a l thanks to Tim, you wcrc thcre from thc bcginning to thc end and

whüt a joumcy its becn!!; to thc rest, Dianne, Patti, Trang, BJ and thc oihers who movcd on) for

making thc daily grind bearable, and dare 1 say, enjoyabie.

Abstract

Acknowledgements

Table of Contents

List of Abbreviations

List of Figures and Tables

Chagter 1) INTRODUCTION

A) T Cell Development

i) Overview

ii) Positive Selection and Negative Seiection

iii) Markers of Positive Selection

B) T Cell Receptor (TCR) Signal Transduction Mechanisms

i) TCR Stucture

ii) Proximal/Distal Evcnts in Mature T ce1 1s

iii) TCR Signal Transduction in DP Thymocytes

C) Regulation and Function of Tyrosine Phosphatase, CD45

i) Background

ii) CD45-Deficient Cell Lines

iii) CD45 Substrates

iv) CD45 in T Cell Dcvelopment

D) Thcsis Objective

Chapter 2) MATERIALS AND METHODS

A) Ce11 lines and Cell Cullurc

B) Retroviral Gene Transfer

C) Antibodies and Second Stage Reagents

D) Flow cytomelry

Paqe I I . . . I I I

iv-v

E) Stimulation of cells for assessment of tyrosine phosphorylation

F) Stimulation of cells for assessment of surface phenotypic changcs

G) Immunoprccipitations

H) Cc11 Surfacc Biotinylation

1) SDS-PAGE and Immunoblotting

J) RNA preparation and Northern analysis

K) cDNA synthesis and RT-PCR analysis

Chaptes 3) RESULTS 3 1-59

A) Charictcnzation of a CD45-dcficienl thymoma ccll linc, 3T7

B) TCR signaling defcct in 3T7 celis

II l2illy CVG111,4 - IIIUULLIUII U1 IYlUblilC: ~ I I U h ~ I l U I ~ l i L L I I ~ I l

ii) Latc cvcnts - phcnotypic maturation,

RAG- 1 downmodulation

Differential ability of CO-rcccptors to ovcrcome

TCR signaling defect

Presence of other phosphatases in 3T7 cells

Defect in CD45 gene espression in 3T7 cclls

Re-espression of exogenous CD45 in 3T7 cells

Rescue of TCR signaling defects by CD45

i) Early events - induction of tyrosine phosphorylation

ii) iate events - phenotypic maturation,

RAG- 1 downmodulation

Biochemical basis for rescue of TCR signaling by CD45

Analysis of thymocytes from ~ ~ 4 5 - I - mice

Chapter 4) DISCUSSION

A) CD45-Dependent versus CD45-Independent

TCR Signaling Pathways

B) Molecular Targets of CD45 in TCR Signal Transduction

C) Role of CD45 in Positive Selection of DP Thymocytes

D) Future Studies

Chapter 5) REFERENCES

BSA: Bovine >ci-iirii alhiin~iri

cDN A: Coinplcniciirary cicox yrihoiliickic acid

CTL: Cytotoxic T lymphocyic

DAG: Diacylglyccnd

DEPC: Dictiiyl pyiocaihoilatc

DMF: Dinlcthyl Iormmidc

DN: Doublc ncptivc

dNTP: Dcoxyribonuclcosidc 5'-triphosphate

DP: Doublc posirivc

D R : Dilhiothrcilol

ECL: Enhanccci chcmilumincscci~sc

EDTA: Ethylcncdiaminc tctrüacictiç a d FACS: Fluorcsccncc aciivatcd cc11 sostcr

FCS: Fctial cdl ' scrum

FITC: Fluorescein isothiocyanak

GM: Growth nicdium

HBS: HEPES bufkrcd saline

MEPES: N-2-hydroxycthylpipcr~~inc-NI-2-clhancsulli~nic acid

HRP: Horsc radish peroxidasc

IP: immunoprccipilii~ion

IPj: Inositol tri-phosphate

ITAM: Iinm~inorcccptor tyrosine-hascd activation motif

LB: Lysis buflCr

mAb: Monoclunal aiilibudy

MAE: MOPS, sodiiim iicciatc, EDTA

2-ME: 7-1ncrcaptoctl1~111ol

MFI: nican 17~ioscsccncc inlcnsily

MI IC 1: Ma,ior Iiistocc,nipa~ibilily ccii~~plcx class 1

MHC II: Maior histoconipatibilily complcx class I I

mRNA: Mcsscngcr RNA

MOPS: 3-(N-niorpho1ino)-propanc sulfonic acid

PBS: Phosphate bull'ci'cd salitic

PCR: Polyincrasc chain iciictiori

PE: Phycocrytliiin

PMA: Phorbol 12-niyi-istritc 13-acclatc

L '0. ' " , L U " 'J"'.""" """"7"

PTf'iisc: Pi'0t~iil i y i l i ~ i l i ~ ~ i h o ~ ~ ) h i i l i i ~ ~

p-tyr: Phospho~yrosiiic

K AG: Rcuvnhin:isc ircriv;itirig p c

RNA: Kibonuclcic acid

RT: Rcvcrsc lranscripli~w

SDS-PAGE: Sodium dodccyl sullà~c polyacrylamidc gcl clcctrophorcsis

SHI : Src-homology 1 domain

SH3: Src-honmlogy 7 domain

SH3: Src-homology 3 domain

SP: Singlc positive

TCR: T-cc11 antigcn rcccptor

Tyr: Tyrosinc

V(D)J: Variahic, divcrsily, joining

List of Figures

F u r 1 : Surlàcc phcnotype of 317 cclls.

Figurc 2: ElTcct of CD45 dcficicncy on TCR-rilcdiatcd sisna1 ~ransduciion in 3T7 cclls.

(A) Induclion of tyrosine phosphorylation in 377 cclls aller TCRP or TCRP + co-

rcccpior crosslinking.

(B) CD5 induction in 3T7 cclls alter TCRp or TCRP + co-rcccpior crosslinking.

(C) CDS induclion in 3T7 cclls ~rcatcd with thc PTK inliibilor. Hcrhimycin A.

Figure 3: Molccular basis OC difl'crcntial signaling hctwccn CD4 and CD8 co-rcccptrirs in

3T7 cclls.

(A) Difkrcntial association of Lck wi~ti CD4 and CD8 co-rcccp~ors in 3T7 cclls.

(B) Ev;ilu;ilion o f CD8 corcccpor isoforms cxpi-csscd i n Yi7 cclls.

Figure 4: El'lcci oc thc tyrosine phosphatase inhibitor, pcrvanadatc on 3T7 cclls.

(A) Induction of lyrosinc phosphoryla~ion in 3 l7 cclls aficr pcrvanadaic &calmeni.

(B) Induction of CD5 in 3T7 cclls akcr pcrvanadalc trcatrncnt.

Figurc 5: Analysis or CD45 gctic cxprcssion in 3T7 cclls..

(A) Northcrn analysis of CD45 mRNA expression in 3T7 cclls.

(B) Surlàcc CD45 inducibly cxprcsscd in 3T7 cclls.

(C) RT-PCR anülysis of CD45 isoforms inducibly cxprcsscd in 3T7 cclls.

Figurc 6: Re-expression of cndogcnous CD45 coirclatcs with rcstorütion of TCR

rcsponsivcncss.

Figure 7: Expression of cxogcniius CD45 in 317 cclls by rctrovirnl-nicdialcd gcnc 1ransCcr.

(A) Sclicrnatic rcprcscnlation of CD45 rclroviral construcl.

(B) Wcstci-n blot anülysis of CD45 prorcin lcvcls in 3T7 inl'cçtan~s.

(C) Surface CD45 expression in (3418-rcsis~ani 3T7 infcctants.

Figure 8: El'l'cct of exogciious CD45 cxprcssion o n 'TCR-mcdiatcd signal trarisduc~ion i n

3T7 cclls.

(A) Exogcnously cxprcsscd CD45 rcstorcs TCR-mcdiatcd protcin Lyrosinc

phosphorylaiion in 317 cdls.

(B) Exogci~uusly cxprcsscd CD45 rcstorcs TCR-induced changes in cc11 surfacc

phcnolypc.

(C) Extigcnously cxprcsscd CD45 rcstores TCR-induccd RAG- 1 downmodulalion.

Figurc 9: Tyrosinc phosphorylalion indcx of potcntial CD45 suhstraics in 3T7 celIs.

(A) Aniilysis of TCR phosphorylation status.

(B) AnaIysis oSLck phosphosphorylntion status.

(C) Andysis of Fyn phosphorylation status.

Figure 10: Anülysis of'TCR< tyrosinc phosphorylation in CD45-/- thymticytcs.

List of Tables

Table 1. TCR-Mediatcd CD5 induction in Parental 3T7 cclls versus CD45 Infcctanb

CHAPTER 1

INTRODUCTION

Introduction

T cclls arc the priniriry oi-clicstrators ol' tlic imii-iunc systcni. rcspimsihlc l'or thc co-

ordinat~ rcgulation of boih c ~ l l ~ ~ l i i i and Ii~inioriil inini~inily. To ;icconiplisli this. T cclls havc

cvolvcd a complcx surlàcc rcccptor. the T cc11 aiiiigcii rcccptor (TCR) wliich rccogiiizcs antigcnic

pcptidcs hound to self niqior Iiistoçompütihility complcx (Ml-IC) molcculcs. Maturc T c d l s

mcdialc tlicir ~Sfcctor S~~nctiolis. which involvc cclliilar prolilkraiion. rclcasc ol' hici-activc

niolcculcs. and recruiinicnt of othcr cc11 types. via signals Lhrough the TCR. A criiical niolcculc

jnvolvcd in rcgiila~ing TCR signal transduction is the tyrosiiic phosphatase, CD45 Studics of

maturc T cclls have shown thüt CD45 acts to dcphosphorylütc nçgativc rcgulntory tyrosine

rcsiducs on S~-c-~iniily kinascs, thc activation of which initiates thc TCR signal tciiisduction

cüscadc. H«wcvcr, ihc rolc of CD45 in TCR signüling in immaturc T cclls has not yct hccii fiilly

dclïncd.

T cc11 devclopmcnt takes place in thc thymus, whcrc a complcx aiid inconiplctcly

undcrstod devclopmcntal pathway is Iollowcd. It is at ihe CD4+CD8+ double-positivc (DP)

s t q c of dcvclopmcnt whcrc a critical test of thc thyiiiocytc's TCR spccil'iciiy ocçurs. This

dcvclopiiicnial chcckpoiiit znsurcs that oiily thc appropriatc T cclls niütuie via die pioccss of

positivc sclcctioii, whilc riddinp thc organisrn of thymocytcs whosc spccilïcity niay hc Iiarmiùl

via thc proccss 01' ncgaiivc sclcction.

Thc work prcscntcd hcrc aticmpts to addrcss thc rcilc of CD45 i n rcgiilating TCR sigiiüls

at thc critical DP stage of dcvclopmcnt. Two mode1 systcnis arc uscd: i) a CD45-dcîïcicnt thyniic

lyniphoniii DP cc11 h i c callcd 3T7, and i i ) DP thymocytcs froiii CD4.5-1- micc. Tlic

çhanictcrizatioii of the I'uncticiiial and phcnoiypiç conscqucnccs of thc lack of CD45 i n thcsc two

sysiciiis providcs iniportmt iiisipht iiito the rcilc of this niolcculc during T cc11 dcvcloprnciit. This

iiiisoduchm will hcgin with an ovcrvicw of T ccil dcvclopmciit lixusiiig oii positivc sclccticii~,

fiillowcd hy a rcvicw oIsTCR signal traiisdoctioii nicchaiiisms, 1iiglili;litiiig dill'crcnccs hciwccii

m;iiusc and imniitturc T cclls. Thc I'inal scction will tiisc~iss CD45 linc!ion ;iiid rcg~ilation.

l'oçusiiig titi its sole dusing T cdl dcvcli~pniciit.

A) T Cell Development

i) Overview

The primary site of T cell development takes place in the thymus, wherc CD4-CDS-

(double-negative, DN) precursors bccomc DP before finally maturing in10 eithcr CD4+ or CD%+

(single-positive, SP) cells (reviewed in Guidos, 1996). While expression of CD4 and CD8 arc

widely used to definc thymocyte populations, it is important to remember that each population a n

bc further subdividcd on the ba is of other markers. For example, thc earlicst thymocytcs

exprcss low levels of CD4 and have not rearranged their TCR genes (Wu et al., 1991). Thesc

cells are the immediatc precursors of DN thymocytes, which can further bc subdivided into

CD44+CD25+, CD44-CD25+, and CD44-CD25 subsets (Godfrey et al., 1994). Signals through

a putative pre-TCR ccimplex, consisting of pre-TaiTCRplCD3 componenls, are critical at the

CD4.4-CD25+ slagc, and rcgulatc the clonal expansion during the DN to DP transition (reviewcd

in Lcvclt and Eichmann, 1995). In most mouse strains, a C D ~ - C D ~ - C D ~ ] ~ ~ dcvclopmental

intermcdiate exists, which gives rise to DP blast cells, expressing low lcvels of clonotypic

TCRup on the surface (Guidos et al., 1989). A com plex, and as yct incomplctel y undcrstood,

TCR-mediated sclection process ensues. Most devcloping thymocytes will die from ncglect

because they exprcss TCR incapable of recognizing self-MHC. Thc remainder can follow onc of

two dcvelopn~ental fatcs: i) A srnaIl subset of thymocytes bearing a TCR capable of inlcrilcting

with sclf-MHC arc rescucd from active cell death in a process known as positive sclcction. This

cnsurcs the crcation of a functicinal rcpertoirc of mature T cclls (rcvicwcd in Jamcson ct al.,

1995). i i ) Altcrnali\dy, thymocytcs with self-reactive TCRs against self-MHC arc dcstroycd or

lunctionally inactivatcd in a pi-occss known as negütive seleclion (rcvic\vcd in Nossal, 1994).

Togcthcr thcsc Iwo proccsscs, both mediated by signals delivcrcd through thc TCR, dictatc

whcthcr a givcn thymocytc dics or livcs Io continuc ihc maturation proccss. Positivcly sclcclctl

DP thymacytcs will then furlhcr dif'f'crcntistc into CD4 or CD8 SP thyinucytcs. Thc proccss of

+ L.

csprcssion of CD4, whcrcas thosc bcaring class 1 MHC-spccifrc TCR will rctain cxprcssion of

CD8, although thc csact mechanism of this proccss rcmains unresolvcd (rcvicwcd in von

Bochmer, 1996).

i i ) Positive Selection and Negntive Selection

Positive selection was discovered by thc analysis of bone marrow radiation chimeric

micc. Whcn bonc marrow from an (AxB)FI heterozygous mouse (A and B representing

diffcrent MHC haplotypes) was used to rescue a lethally irradiated homozygous A parent. T cells

responded preferentially to antigens presented by the host MHC (A) antigen preseniing cclls

(Bevan, 1977). This MHC rcstriction was shown b be mediated by H-2 antigens expresscd on

thc thymus (Fink and Bevan, 1978; Zinkernagel et al., 1978). Negative selection was

definitively demonstraled by the clonal deletion of Mls-reactive T cells in the thymus (Kappler cl

al., 1987). With the advent of TCR transgenic mice, analysis of selectivc evcnts bccame

technically easier (Teh et al., 1988). Analysis of transgenic mice expressing a TCR spccific for

the male transplanlation antigen HY revealed thit when lhe HY antigen was expressed (ic. in mdc

mice) in conjunction with the appropriate MHC restriction element, massive clonal dcletion of

TCR transgcnic thymocytes occurred, dramatically confirming thc existence of ncgalive sclcction

(Kisielow et al., 1988). In constrast, when the HY autoantigen was not expressed (ie. in fcmalc

mice), positivc selection of TCR transgcnic Lhymocyles occurred, bascd on MHC haplotypc

(Kisiclow ct al., 1988; Huesmann et al., 1991).

Gcnctic studics havc dcfincd al least two "devclopiiiental chcckpoinls". Thc fïrst

chcckpoint occurs at the DN to DP transition, and is mediaied by putativc signals through thc pi-c-

TCR complex to ensurc that a funclional TCRp chain has becn made (rcvicwcd in Lcvcll and

Eichmann, 1995). Thc sccond chcckpoint occurs at the DP 10 SP transition, during which ~ h c

thymocytc undcrgocs a critical tcsl of its TCR spccificily, rcsulting in positivc and ncgativc

sclcction. In contrat to the first dcvelopmental checkpoint, positivc and ncgative sclcclion arc

2 Li u

thymocytes with the appropriate seceptors on their surface develop, both in terms of Sunction and

spcci ficity.

i i i ) Mnrkers of Positive Selectiott

The early studies described above laid the groundwork for a more detailed examination of

thc developmental events that accompany selection events. A major consequence of positive

selection is the alteration in the life span of TCRap+ thymocytes. Kinetic studies havc deduced

the average Me span of a DP thymocyte 10 be 3.5 days, after which the thymocytes cithcr dic by

apoptosis, or are positively selected for further maturation into cells that are long-lived (Egerton et

al., 1990; Huesmann et al., 1991; Kisiclow and Maizek, 1995). The upregulati«n of thc cell

survivül gene, bcl-2, during positive selection provides an attractive mechanism for this

phenomenon (Veis et al., 1993; Linette et al., 1994). Another major consequence of positive

selection is the termination of the TCR rearrangement process. TCRap DP thymocytcs still

express the recombinase-activating genes RAGl and RAGS, but expression of thcse gencs is

rapidly shut off in response to TCRap signais (Turka et al., 1991 b; Brandle et al., 1992). Thus,

rearrangement can occur at the second TCRa allele if rearrangement of the first allele was not

successful (Petrie et al., 1993), increasing the likelihood that the developing DP lhymocyte will

be positively sclccted.

Positive selection also triggers an ordered series of phenotypic changes. Adoptive

transfcr of defined subsets of thymocytes into the thymus providcd a uscful tcchniquc Ibi-

studying the developmental potential of particular thyrnocyte populations. Using this approach, i t

was Iound that DP TCRI0 blast cclls have the developmental potential for furthcr diffcrcntiation,

bccoming T C R I ~ C D ~ + C D ~ - or T C R ~ I W C D S + T cells (Guidos et al., 1989). Transitional

intcrmcdiates on thc CD4 or CD8 differentialion pathways wcrc suggcstcd to havc a

T C R " ~ ~ C D ~ + C D ~ ~ ~ or TcR1nd~D4l0c~8+ phenotypc (Guidos cl al., 1990). Howcvcr thc

csact lincagc rclationships among these transitional intcrmediales rcmains controvcrsial. Somc

stuarcs haVC SUggCSted tnat tne transirionai ceiis on rnc LUY iincagc may nor ncccssariij7 oc

comrnitted to the CD4 lincagc, and may still develop into CD8 SP thymocytcs (Lucas et al., 1995;

Lundbcrg et al., 1995; Suzuki ct al., 1995).

The quantal uprepulation of TCR espression on developing DP thymocytes likcly rclatcs

to "sub-stagcs" of diffcrentiation (Guidos et al., 1990; Ohashi ct al., 1990; Shortman ct al.,

1991), suggesting that positive selection maybe a multi-step process. Importantly, whcn puriricd

DP thymocytcs are stimulatcd through their TCR iri vitro, only a subset of maturationai proccsscs

associated with positive selection occurs (Kearse et al., 1995; Groves ct al., 1997). Whilc

increased expression of CD5, CD69, and Bcl-2 and termination of RAG- I and prc-Ta expression

were observed, clona1 deletion and CD4JCD8 Iineage cornmitment wcre not seen (Kcarse ct al.,

1995; Grovcs et al., 1997). These observations are consistent with the notion that multiple, or

sustained TCR engagements may be necessary for the complete developmental progression

associated with positive selection (Kisielow and Maizek, 1995; Wilkinson et al., 1995).

AI tcmativel y or addi tionall y, non-TCR derived signals present in the thy mic rnicrocnvironmcnt

(including growth factors, adhesion molecules, CO-stimulatory molecules, etc.) maybc rcquircd

for further developmental progression. Late events associated wi th maturation i ncludc the

downrcgulalion of heat stable antigen (HSA) and Thy- 1 (Fowi kcs ct al ., 1988; Grovcs cl al .,

7 997; Lucas et al., 1994).

Whilc thcsc markcrs provide a useful experimental tool for following positivc sclection,

somc caution must bc used in their interpretation. For cxample, CD69 is an activation markcr

uprcgulated following TCR engagement, and ihus could also be a markcr of negativc sclcciion

(Kishimoto ct al., 1995). The biologic functions of mosi of thesc moleculcs rcmain ill-dclïncd.

Onc of thcsc markcrs, CD5, appcars to bc a ncgativc rcgulator OS TCR signaling (Tarakhovsky ct

al., 1995). Thc identification of other molecules capable of impinging on TCR signaling

paihways at this stagc of dcvcloprncnt will have important çonscqucnçcs on thcsc sclcciivc

processes. Onc such molcculc, CD45, is thc focus of this thcsis. Belorc rcvicwing thc fiinction

and rcgulation of CD45, a rcvicw oSTCR signal transduction mcchanisms is prcscntcd.

B ) TCR Signal Transduction Mechanisms

i ) TCR Structure

Thc vanable domains of a and p chains of the TCR mediate recognition of antigcniç

pcptides bound to MHC molecules on the surface of antigen prcsenting cclls (APCs). Thc

cxireme diversity of TCRap specificities is mediated by somatic gene rearrangcment of n~ultiple

variable (V) , diversity (D), and joining (J) gene segments in a process known as V(D)J

recombination (reviewed in Lewis, 1994). The TCRa and TCRP subunits are non-covalently

associatcd with the invariant CD3 y$, and E chains, and either TCRS-< homodimers or <-q

hcterodimers. The cxact stoichiometry of the TCRICD3 complex is not known, but is thought to

consist of a disulfide-linked TCRa$ heterodimer, in association with a CD3ey and a CD3t6 pair,

and cither a TCRCS homo- or TCRh heterodimer. The TCRS and CD3 components perhrm two

critical functions: i) ensuring the proper assembly and surface expression of the TCRICD3

corn plex, and ii) coupling antigen recognition to the intracellular signali ng machinery (revicwcd in

Malissen and Malissen, 2996).

In addition to the TCWCM complex, the CD4and CD8 CO-receptors play important rdcs

in T ce11 recognition and signaling. CD4 is a monomeric integral mcmbranc glycoprotein, whi lc

CD8 cxists as ü disulfide-linked a-a homodimcr or a+ hetcrodirncr (reviewcd in Julius ct al.,

1993). Thc CO-rcceptors act as adhesion moleculcs by recognizing non-polymorphic rcgions on

MHC class I I (CD4) (Doylc and Strominger, 1987), and MHC class 1 molccules (CD8)

(Normcnt ct al., 1988), thus stabilizing interactions betwccn thcT ceIl and the APC. I n addition,

the co-rcccptors can transducc bi ochcmical signals during T-cc11 activation, by vi rtuc of thci r

association wi th thc tyrosine kinasc Lck (Veillette et al., 1988).

Thc TCR a and P chahs possess only short cytoplasmic domains of Iïvc arnino acids and

arc thcmsclvcs unli kcl y to bc capablc of cou pling to inlraxllular signaling pathways. I n con trast,

tlic CD3 chains and TCRt chains contain larger cytoplasmic domains, which couple

anti gcnlMHC rccogni lion to signal transduclion paihways (rcvicwed in Chan et al ., 1994a). This

L... d fi . , - . - - - . . . - -

subunits, including thc TCRICD3, BCRIIgalIgp, and FCsRIy, which rcvcaled a highly

conscrvcd motif in thc various receptor systems (Relh, 1989). This ITAM motif

(irnmunoreçeptor tyrosine-based activation motif: DIEX7DIEX2YX2LX7Y X2L) was found to bc

both necessary and sufficient for receptor signaling (Romeo et al., 1992; Irving et al., 1993).

The ability of thcse ITAM motifs to be inducibly phosphorylated on thcir two tyrosine rcsiducs

crmtes a binding site for the SH2 domain of various intracellular signaling molccules. Binding to

phosphotyrosine sites can affect SHZcontaining proteins in multiplc ways, including dircct

stimulation of enzymatic activity, cellular relocalization, and enhanccd tyrosine phosphorylation

(reviewed in Pawson, 1995).

i i ) ProxirtiallDistnl Events in Mature TCR Signai Tramdiution

Effector functions induced during T ce11 activation include lymphokine secrebon, cellular

proliferation, and cellular differentiation. The signaling events leading to these changes in genc

expression involve a complex set of biochemical events, which have been relatively wcll-

charactcrizcd (reviewed in Cantrell, 1996; Wange and Samelson, 1996). The carliesl dctcclüblc

evcnt in TCR signaling is thc induction of tyrosine kinase activity, leading to protcin lyrosinc

phosphorylalion (Samelson cl al., 1986; Hsi et al., 1989; June et al., 1990b). Two farnilics o f

PTKs have becn implicated in TCR signaling. Lck and Fyn arc lymphocytc-spccific membcrs of

thc Src-kinase fainily (Veillcttc and Davidson. 1992). The çonservcd struct.ura1 katurcs 0 1 ' this

f m i l y include: 1) an amino-terminal glycine, which is required for myrisloytation and mci-iibr~~nc

association; 2) a unique domain of roughly 60 amino acids, which allows spccific inlcractions

wilh cellular regulators; 3) the SH3 domain, a region involved in recognition of prolinc rich

sequcnccs (Rcn ct al., 1993); 4) the SH2 domain, which mcdiatcs inlcractions wi th

phosphotyrosine-containing proteins (Pawson and Gish, 1992); 5 ) the catalytic SHI domain,

coniüining an ATP-binding and an autophosphorylation site; and 6) ihc carbosy-terminal ncgativc

rcgulütory domain. Zap-70 is a member of another class of cyk)plasmic PTKs, thc SyklZap-70

.~ -" --., , - - - - ~ - - - ~ - - > -~ . -, - - ~ - - - - , i

they lack an SH3 domain and instcad havc two SH2 domains; 2) thcy lack niyristoylation si tcs;

and 3) they lack thc carboxy-tcrminal negativc rcgulatov dornain.

These three PTKs have been implicated in TCR signaling by both biochcmical and gcnctic

mcans. Experiments with T cell lines lacking Lck (Straus and Weiss, 1992) or csprcssing mutant

forms of Lck (Abraham et al., 1991; Luo and Sefton, 1992) indicatc that Lck can participatc in

TCR signaling. Furthermore, mutant mice overespressing a dominant negativc Ick transgenc

(Levin et al., 1993) or with a targeted disruption of the lck gene (Molina et al., 1992) possessed

profound defccts in thymocyte development and diminshed TCR function. A rolc for Fyn in

TCR signaling is supported by studies i n which SP thymocytes from mice overexpressing afyrr

transgene were hyperstimulable to TCR ligation (Cooke et al., 1991). Conversel y, SP

thymocytcs from mutant mice lacking Fyn displayed grcatly diminishcd TCR rcsponscs (Applcby

et al., 1992; Stcin ct al., 1992). Finally, Zap-70 has been shown to be activated by TCR ligation

and Sound to associate with the tyrosine-phosphorylated ITAMs in TCRL (Chan et al., 1992).

Futhermore, T cells from mice (Negishi et al., 1995) and human patients (Arpaia et al., 1994;

Chan et al., 1994c; Elder et al., 1994) defective in Zap-70 expression are severel y impaired in

their response to TCR ligation. A role for Syk in TCR signaling is less clear. Engagement of

TCR results in Syk activation in thyrnocytes (Chan et al., 1994). Howcver disruption of thc Syk

gene does no1 affect maturation of af3 T cells (Cheng et al., 1995; Turner el al., 1995), suggcsting

that Syk is dispensable during T cell development.

Currcnt inodcls of TCR signal transduciion posit a sequcntial activalion of PTKs

following TCR engagement, in which Lck andior Fyn are initally activated to phosphorylalc

ITAMs i n CD3 componcnts (Iwashima ct ai., 1994; van Ocrs ci al., 1996). This allows

rccrui tmcnt of Zap-70 by binding phosphorylated ITAMs (Chan ct al., 1993), and ils rapid

aciivation by tyrosine phosphoryIation (Chan et al., 1995; 1 washima ct al., 1994). Molcculcs

subsequently phosphorylated by the TCR-proximal PTK signaling cascadc includc phospholipw

Cy 1 (PLCy 1) (Mustclin ct al., 1990), p95 Vav (Gulbins et al., 1993), MAP kinasc (Ettchadich cl

al., 1992), and PI-3 kinasc (Ward et al., 1996). Following lhcsc immcdiatc carly signaling

- . -..'") . -- , . --.-.,.A-u ...- --. ."W. -. -- -.-.-- --.., , - - - - - - - O ...r-.-

(IP3), and diacylglycerol (DAG) (Weiss and Liltman, 1994). These two second mcssengers arc

i-csponsible for thc TCR-induced rise in cytoplasmic free calcium and activation of' protein kinasc

C (PKC), respective1 y.

A critical regulatory mechanism for Lck and Fyn involvcs the C-terminal negativc

rcgulatory tyrosine. I t has been demonstrated that Csk can phosphorylate this site (Bcrgrnan et

al., 1992), making Csk a negative regulator of the Src-family kinascs. When phosphorylatcd,

the C-terminal tyrosine binds to the SH2 domain of the same kinase molecule, thereby forcing i t

intoan inactivc conformation (Cooper and Howell, 1993). In contrast to Csk, CD45 is thought

to positively regulate the Src-family kinases by dephosphorylating the C-terminal tyrosine

(revicwed in Trowbridge and Thomas, 1994). The recent resolution of the crystal structures ol'c-

Src (Xu et al., 19971, Lck (Yamaguchi and Hendrickson, 1996), and anothcr membcr of thc Src-

family, Hck (Sicheri et al., 1997), have provided further insight into the structurallfunclionaI

rclationships of Srk-family PTKs. These studies have revealed thal not only is lhc

phosphorylated C-terminal tyrosine bound by the SH2 domain, but thc SH3 domain is also

involved in an intramolecular interaction with the SHNinase linker domain (Sicheri et al., 1997;

Xu cl al., 1997). Thus, the crystal structures raises the possibility that compelilivc interactions

with SH3 or SH2 ligands could also activate the molecules by displacing thc inhibitory

intrarnolecular interaction, without necessarily involving C-terminal dephosphorylation.

Following these TCR-proximal events, second messengers are thought Lo aclivatc at lcast

twoTCR-dislal signüling palhways. Onc involves thc activation of calcincurin by clcvatcd Ic \~ls

of intracellular calcium. Calcineurin, a calcium/calmodulin-depcndent serinelthrcminc

phosphahsc, stiinulritcs translocation of thc NF-AT(c) transcription factor from thc cytoplasm to

ltic nuclcus (Flanagan et al., 1991). A sccond TCR-distal pathway involvcs tlic Ras andtor PKC-

mediaicd activation of' the ERKIMAPK cytoplasmic serinelthreonine kinasc family. Thcsc

kinascs translocatc to the nuclcus and rcgulatc changcs in gcne cxprcssion by phosphorylating a

varicty of' transcription factors (Hill and Trcisman, 1995). I n this way, changcs i n gcnc

csprcssion arc incurred such that T ce11 activation occurs.

iii) TCR Signal Trartsdrcctiort in Zrltmatrlre Thy~rmcytes

In contrast to mature T cells, the elucidation of signal transduction pathways used in

immarurc thymocytes has only just begun. Many of the same signaling molecules which havc

been described in mature T cell signal transduction have been shown to participate in TCR

signajing in DP thymocytes. Most of the receptor molecules, as well as the intracellular PTKs

involved in thc proximal TCR signal transduction cascade, have been genetically disrupted, with

varying consequences on T cell development. For esample, micc dcficient in the Src-family

kinases Lck or Fyn posscss dramatically different phenotypes. Lck-deficient mice have a 10-lold

reduction in the number of thymocytas, due to a partial block at the DN to DP expansion stage,

and mature SP cells are greatly reduced (Molinaet al., 1992). In striking contrast, Fyn-deficien1

mice have no gross abnormalities in either the number or phenotype of thymocytes (Appleby ct

al., 1992; Stein et al., 1992). Yet another phenotype is observed in Zap-70 knockout micc,

where there is a block in the development of both CD4+ and CD8+ SP T cells (Negishi et al.,

1995). Finally, mice lacking expression of Itk, a cytoplasmic P f K bclonging to the BtklTec

farnily, have a modcrate reduction in the numbcr of mature T cells (Liao ct al., 1995). Howcvcr,

this cffect is morc promincnl in Itk-deficient mice expressing transgcnic TCRs, wherc positivc

sclection appcars to bc irnpaired (Liao et al., 1995). These gcnctic ablation studies suggcst thü~

the various PTKs are diffcrentially rcquircd during developmcnt, raising the possibility thüt L ~ C

inccha~iism of TCR signal lransduclion müy change over the course of T ccll dcvclopmcnl.

Several lincs of cvidence suggest that TCR signaling evenls in DP thymocytes inay dill'er

in somc respccts to lhosc characterized in mature T cells. For esamplc, aggrcgalion of TCR or

CD3 on frcsh ex vivo DP thymocytcs produces only marginal incrcascs in Lyrosinc

phosphorylation and intraccllular calcium levels (Finkel et al., 1987; Nakayama ct al., I W O ;

GilliIand el al., 1991; Turka ct al., 1991a; Sancho el al., 1992). Onc mcchanism for this bluntcd

rcsponsc has bcen proposed by Singcr in a series of studics dcmonstrüting that TCR signaling is

ncgütivcly rcgulatcd by CD4-associüted Lck (McCarthy et al., 1988; Nakayrirnü ct al., 1089;

- . - - - -. . . . - - . -. - - - - - .> - - - -, - .-a"-, -.--- - - --., - - - - 7 " - - -..> -. ", .. .--- - - -.., . r r .. , .

Iniercstingly, thc othcr co-rcccptor, CD8 docs not display this activity. While this may bc

attributable to thc intrinsic prefcrential association of Lck with CD4 over CD8 (Wiest ct al..

I993), there is also a specific developmentally controlled expression of CD8a. Through a

mechanism of alternative splicing, two polypeptide chains, a and al, that differ from onc anothcr

in the lcngths of their cytoplasmic tails, are expressed in T cells (Zamoyska and Parnes, 1988).

1 rnmaturc T cells cxprcss both CD8a and CD&rq forms on their ce11 surface, whilc maturc T cclls

cxpress on their cc11 surface predominantly the heterodimer containing CD8a (Zamoyska and

Parnes, 1988). This may have important Iuunctional implications, as the CD&' polypeptide is

unable to associate with Lck (Zamoyska et al., 1989), suggesting that DP thymocytcs arc

intrinsically morc capable of delivering CD4-mediated signals than CD8-mediaied signals.

Another difference inTCR signaling between DP thymocytes and mature T cells involves

their requirements for co-stimulation. In the classic mode1 of T ceIl activation, two signals arc

required: the first via the TCRICD3 complex and the second provided by CD281B7 ligand-

receptor system (Robey and Allison, 1995). In striking contrast, DP thymocytes do not appear to

rcquirc co-stimulation. Mice deficient in CD38 conlain normal numbers of T cclls, and

apparcntly normal developmental profiles (Shahinian et al., 1993). Furthcrmore, whcn CD78-

deficient mice were made to cxpress a$ transgenic TCR, no obvious deficiencics in cilher positivc

or ncgütivc sclection were observed (Walunas et al., 1996), suggesting that alternative co-

stimuiülory pathways exist, os that the two-signal hypothesis does not apply to DP thymocytcs.

Thus several differences cxist in the TCR signal response of DP thymocytes as comparcd

to maturc T cells. To complicate malters further, several groups have suggcstcd ihat

biochcmically distinct signal-transduction palhways may distinguish positive and ncgüiivc

selection. Various groups have looked for qualitative differenccs in TCR signaling Icading to

cilher positivc or ncgativc selection. Two groups found positivc sclcction to bc prcfcrcnhlly

inhibiled by the calcincurin inhibitors, cyclosporin A or FK506, and not ncgcilivc sclcclion

(Anderson cl al., 1995; Wang ct al., 1995), suggcsting that ncgativc selcclicin acts via a

calcineunn-indcpcndcnl pathway. Similarily, ovcrcxprcssion OS doininant-ncgativc Ras or MEK-

-

selection intact (AI berola-IIa cl al., 1995; Swan et al., 1995). Howcvcr, thc lailure to addrcss

whethcr there was a complctc or partial block in the signaling pathways, leavcs opcn Lhc

interprclation that quantitalive, ralhcr than qualitative, differences in signaling may distinguish

positivc from negative selection. As studies îurther define, by both genctic and biochcmical

means, the signaling molecules invohed in the TCR signal transduction pathway in DP

thymocytes, a greater understanding of how a thymocyte undergoes selective processes will bc

achieved. One critical molecule, CD45 has been shown to be important in regulating TCR

signals in DP thymocytes as well as in mature T cells. The final section will review the regulaiion

and function of this molecuie.

C) Regulation and Function of the Tyrosine Phosphatase, CD45

CD45 (also known as leukocyte common antigcn, Ly5, T200 in T cells and B330 In B

cclls) is a transmembrane glycoprolein, highly expressed on al1 nucleated cells of hematc~poieiic

origin (reviewed in Trowbridge and Thomas, 1994; Okumura and Thomas, 1995; Frcarson and

Alcxander, 1996). The function of CD45 rcmained unidcntificd until in 1989, ivhcn significiinl

homology bctwecn the cytoplasmic domain of CD45 and a tyrosinc phosphatasc, mP-1B was

identificd, suggcsting that CD45 is a receptor phosphtase (PTPasc; Charbonneau ct al., 1989).

CD45 has an cxtcnsivcl y gl ycosylated amino-terminal extcrnal domain, a single mcmbranc-

spanning rcgion, and a largc cytoplasmic domain containing two 300 amino acid tandcinly

rcpcatcd PTPase domains, with the proximal domain containing lhc calalytic aclivity (Johnstin cl

al., 1993).

CD45 is csprcsscd as multiple isoSorn~s ranging in molcculür mass from 180-335 kDa,

which scsult from allcrnativc splicrng of four variable csons (csons 4, 5, Ci and 7) in tlic

cstraccllular N-terminal domai n of thc moleculc. CD45 isoSorms conlaining cson 4-, cson 5, or

, , , u u , ' . L , 7 " , T A , C L 7 . . U,\,21CLI.I1\III . I I

diffcrcnl CD45 isoforms is ce11 type-specific and depends on the diffèrenliation and aclivalion

statc of the lymphocyte (Lefrancois and Goodman, 1987; Hathcock et al., 1992). The majority

of developing thymocytes and activated T cells esprcss the CD45RO 180 kDa isoform (lacking

csons 4, 5 and G), whcreas CD4+ and CD8+ SP thymocytes and naive pcriphcral T cclls can

cspress various 1 eson forms. In contrast, B lymphocytes espress the CD45ABC 330 kDa

isoform (Cot'fman and Weissman, 1981). The finding thal diffcrent isoforms dif1'cr in their

ability to participate in antigcn-rnediatcd slimulation in a mode1 cc11 line systcm (Novük ct al.,

1994) has Icd to the hypothesis that changes in isoform expression can direçtly alter the signaling

characteristics of the T cell, although the physiologie significance of this remains to bc

determined.

i i ) CD45-Deficieat Cell Lirzes

Dircct cvidence for the involvement of CD45 in regulating TCR signal transduction \vas

first providcd by studics of CD45-deficient T cclls. By mutagcnizing an antigcn-spccifïc CD4+

murinc T ccll linc and selccting for variants which lost surfacc CD45 cxprcssion, Pingel &

Thomas corrclalcd loss of CD45 with an inability to prolifcrate in responsc to antigen or lo CD3

l igation. A s poniancous CD45 rcvcrtant rcgaincd KR-mcdiated ac~ivation responscs (Pi ngcl and

Thomas, 1989). Thc rcquirement for CD45 was soon estcnded by analysis of olhcr CD45

dcficicntccll linc systcms: a CD8f cytolyticT-ccll clonc (Weavcr ct al., 1991), a human DP T ccll

lcukcmic linc (Koretzky ct ai., 1990), a DN T lymphoma Iinc (Volarcvic ct al., 1993), hunirin

C M + furkat Icukcinic T cclls (Korctzky et al., 199 l ) , a B ce11 plasniacytoma cc11 linc (Juslcnicnl

cl al., 1991) and a nalural killcr ceIl linc (Bell et al., 1993). In al1 of thcsc syslcins, antigcn

rcccplors wcre uncouplcd from signaling pathways and thcir downstrcam outcomcs. Thus, thc

CD45 molcculc plays an obligalory rolc in rcgulaling anligcn rcccplor signaling in a varicly 01'

1 ymphocytc lincagcs.

" .-- ..---.- -.. -m.- -.. -. O"-'-."C --.-- ..-- -7 .- --..--- .-- . ---. ....-- ..-" ..---.. -,.--...,. . -. .

charactcrixcd. CD45-deficicnt T cells arc unablc to prolifcratc or to produce cytokines, such as

IL-?. i n rcsponsc to antigcn or to TCR ligation (Pineel and Thomas. 1989). Thc signaling

c ~ ~ c a d c initiated by TCK ligation is interrupted at the earliest stagc i n CD45-dcficicnt cells. Most

CD45-dcficicnt cclls show an inability to induce tyrosine phosphorylation of sevcral protcins

(Koretzky et al., 1991), including TCRC chah (Volarevicel al., 1992) and PLC-y 1 (Koretzky et

al., 1992) in response to TCR ligation. In addition, they fail to increase intracellular calcium

(Koretzky et al., 1990; Volarevic et al., 1992), generate inosilol phosphates (Koretzky et al.,

1990; Volarevic et al., 1992), and activate PKC (Shiroo et al., 1992) in response to TCR

ligation. However, not al1 studies have found the same manifestations on TCR signaling in thc

absencc of CD45 One study found markedly elevated levels of basal tyrosine phosphorylation in

a CD45-deficient leukemic ccll line compared to the CD45+ parental cell line (Volarcvic et al.,

1992), suggesting that CD45 might be responsible for maintaining low basal Ievels of Lyrosinc

phosphorylation in parental cclls. Other groups Sound that the block in TCR signaling was nnot

absolutc. For cxample, the inability Io signal through the TCR could bc overcornc whcn TCR

and CD4 or CD8 were CO-aggregated (Deans et al., 1992; Shiroo ct al., 1992). Yct anothcr siudy

described a CD45deficient Jurkat T cell line which was fully capable of responding to TCR

stimulation despite the absence of CD45 (Peyron et al., 19911, an cffcct that was later shown to

be inediated by the Syk P ï K (Chu et al., 1996). Taken togethcr, thcsc studics rcveal a critical

rolc for CD45 in regulating TCR signais. However, its role may diffcr dcpcnding on thc

particular T cell studied, and the types of stimulatory conditions uscd.

Various groups furthcr dissected the regulation and funclion of CD45 by rc-csprcssing

various mutant forms of CD45 in the CD45-dcficicnt cc11 line systems by gcnc trünsfcr

tcchniqucs. Ashwcll's group demonstratcd that the TCR signaling dcl'cct could bc complcmcntcd

using thc enzymalically activc inlncellular portion of CD45 (Volarcvic ct al., 1993), so long as il

was teihercd to the plasma membrane and catalytically active (Niklinska et al., 1994). Wciss's

group made chimcric molecules in which the eslracellular and transmcmbrane domains of CD45

wcrc rcplaccd with those of thc EGF rcccptor (EGFR) (Dcsai ct al., 1993). Once morc, thc

c~Lraçc~~u~ür uomarn oi ~ u 4 3 W ~ S UlSpenSilDlC ICII I~CSCUL: CIL I LK slgnallng. A n InLcrcsung

finding was that thc addition of EGFR ligands during TCR signaling resulted in a rapid and

dramntic inactivation of TCR-mediated signals (Desai et al., 1993: Desai cl al ., 1994)' siiggcsting

lhat ligand binding could regulate CD45 activity, although the exacl rnechanism 1-cmains

undctïncd. Finally, a heterologous phosphatase from yeast espressed as a chimcric protcin wilh

estracellular and transmembrane domains of a MHC clas 1 molecule could restore TCR-medialcd

signal transduction, albcit lcss cfficicntly than thc analagous CD45 chimcric protein (Desai ct al.,

1993; Desai et al., 1994). Together, these studies helped define the striictural rcquiremenis ol'

CD45 activity in TCR signaling: the intracellular active catalytic domain is absolutely nccessary,

whilc the estracellular domain is dispensable.

The above results do not rule out a role for the estracellular domain, however, as

physiologic TCR signaling is likely to be more subtle than the highly artificial stimulatory

conditions used in in vitro signaling assays. Indeed, a comparison of the ability of various CD45

isoforms to promote IL-2 secretion in a thymoma ceIl line stimulated with antigenlMHC rcvealed

thal subclones expressing CD45RO were most effeclive while those expressing CD45RABC wcrc

Icrist cffectivc (Novak et al., 1994). Notably, this diffcrence was not obscrvcd whcn TCR-

spccific mAb was used as the stimulus. Further studies revealed that thc CD45RO isof'orm

prefercntially caps with TCRICD4 when compared with CD45RABC, and that ihis interaction

does not rcquirc the CD45 cytoplasmic tail (Dianzani et al., 1992; Leitenbcrg et al., 1996).

Rcccnlly, CD45-I- micc have bcen generated that express CD45RO or CD45RABC transgcncs

(Kozicradzki cl al., 1997). While both isoforms rcstorcd thc developmcnt of CD4+ and CD8+

SP thyrnocylcs, only CD45RO micc wcre able 10 gencrate cytotoxic T cc11 rcspnscs açüinst viral

infeciion (Kozieradzki ct al., 1997). Thus, the CD45 ectodomain can, i n soinc circurnstianccs,

play a role in modulating TCR signaling.

iii) CD45 Srrbstrntes

* & A - "A..,-..- . . m . ---a .A.-- .....m.. -- .... -- -- .- . - O - - - - . . . - . . . - - - - . --c----

transduction have been extensively studied. CD45 positivcly rcgulates mcrnbcrs of the Src family

of kinases by dcphosphorylation of their C-terminal negative regulatory tyrosine. This was

infcrrcd frorn studies of CM5-deficicnt ce11 lines, which were found to posscss dccrcascd IcvcIs

of both Lck and Fyn kinase activity due to a hyperphosphorylation of their C-terminal tyrosines

(Mustclin ct al., 1989; Ostergaard et al., 1989; Mustelin ct al., 1992; Gervais and Vcilletlc,

1995). Therefore, i t is likely that Lck and Fyn are physiological substrates of CD45 Othcr

studies indicate a more cornplex role for CD45 i n its relationship with Src family kinases. I n

three different CD45- T-cell lines (YAC-1, SARKTLS, and HPB-ALL), Lck was found 10 be

hyperphosphorylated at the C-terminai tyrosine site, consistent with previous studics (Burns ct

al., 1994). Surprisingl y, however, the kinases were found to be hyperactive. Further studies

demonstrcited that in addition to Tyr-505, CD45 can dephosphorylate Tyr-394 iri virro (D'Oro ct

al., 1996). In the absence of CD45, the hyperphosphorylütion of Tyr-394 can cause an increase

in kinase activity, despite the inhibitory hyperphosphorylation of Tyr-505 (D'Oro ct al., 1996).

Thus, while the exact nature of the inability to signal through the TCR in CD45-deficicnt cclls

rcrnains controversial, it seems to strongly correlate with the dysregulation of Lck andlor Fyn.

In most of the studies alluded to above, CD45 appeared to differentiall y regulaie thc two

Src-family kinases, Lck and Fyn. In one study, the degree of C-terminal phosphorylation of Lck

and FynT in thrcc diffcrenl CD45-deficient ceIl lines (SAKRTLS 12.1, BW5147, NZB. 1 ) was

assessed (Hurley et al., 1993). In each cell line, the C-terminal hyperphosphorylation of Lck

was more pronounccd than for Fyn (Hurley ct al., 1993). Similarly, in anothcr CD45-deficicnt

ccll line (L3M-93), an 8-fold increase in C-terminal phosphorylation for Lck and a 2-fold incrcasc

for Fyn was rcported, dong with decreased kinase activi ty for cach (McFarland cl al ., 1993). 1 n

contrat, anolher group studying a diffcrcnl CD45-dcficicnt cc11 linc (HPB-ALL), found thri t Fyn

had decreased kinase activity but not Lck (Shiroo et al., 1993). Howcvcr, thc statc of C-tcrminal

phosphorylation was no1 examincd in that study. Instcad, data was prcscnted suggcsting thxt

CD45 could regulate spcçii'ic pools of the Src-family kinases, spccifically thosc pools which wcrc

rcceptor-associatcd, ic. TCR-associatcd h r Fyn and CD4 associatcd I'or Lck (Bil'I.cn cl al.,

cspcrimcntal proccdurcs remains to be rcsolved.

A numbcr of other potential CD45 substntcs have reccntly been dcfincd. CD45 has bccn

shown to associatc with both TCRS (Furukawa et al., 1994) and Zap-70 (Mustelin et al., 1995)

in i mrnunoprccipitation experiments, and is able to dephosphorylatc them i ~ i vitro. Final 1 y, onc

more potential rcgulator of CD45 activi ty may involve a novel protein called CD45 AP. This

protein of approximately 30 kDa has been shown to immunoprecipitatc with CD45 and Lck,

suggesting that it may be an adaptor protein linking the two enzymes (Schravcn et al., 1994).

Clearly much remains to be learned about the nature of CD45 substrates and how CD45 acts to

regulate them.

i v ) CD45 irc T Ce11 Developmerrt

Definitive evidence that CD45 is involved in thymocyte development camc from thc

gcneration of CD45 exon 6-deficient mice (Kishihara et al., 1993). Disruption of cvon 6 might

havc been cxpectcd to result in mice deficient on1 y in expression the CD45RC isoform, howevcr

cxprcssion of al1 isoforms of CD45 was compromised. Pan-CD45 staining of thymocytcs [rom

~ ~ 4 5 - 1 - mice showed that the DN and DP populations lacked dctectable cxprcssion of CD45,

howcvcr, a fraction of SP thymocytes and peripheral lymph nodc T cclls (10-30%) did cxprcss

CD45 (Kishihara et al., 1993). The total nurnber of thyrnocytes in ~ ~ 4 5 - 1 - micc wüs only

slightly reduccd as compared with wildtype mice (Kishihara cl al., 1993). CD45-'- micc had

slightly incrcüscd frequencics of DN, normal frequencics of DP, and signifiçantly rcduccd

I'rcqucncics of SP thymocytcs as cornparcd to littcrmatc control micc (Kishihan ct al., 1993).

Pcriphcral T cclls wcrc markcdly reduced in iiurnbcr and wcrc rcfractory to CDS-induccd

activation (Kishihara ct al., 1993). Morc recently, CD45-nul1 micc wcrc gcncratcd by gcnctiç

disrupiion of cson 9, rcsulting in complcte abrogation of CD45 cxprcssion (Byth ct al., 1996).

I n thcsc micc, thc CD41CD8 dcvelopmental profilc obscrvcd was vcry similar to that sccn in the

cson 6-knockout micc, but thc frcquency of T cells in thc peri pherd coinpartincnt wüs morc

frcquency of CD#-CD35+ DN cells (Byth et al., 1996). Thus, thc phcnotype of the two

indcpcndcntly-dcrivcd CD45 knockout mice suggcsts a minor rolc for CD45 in the DN 10 DP

transition rcgulritcd by putativc signals through thc pre-TCR cornplex, and a more important rolc

in the DP to SP transition regulated by signals through thc maturc TCRnfliCD3 cornplcs.

Howcvcr, the nccd for CD45 at cither of these checkpoints is not absolutc, as somc DP and SP

thymocytes are still observed.

Another line of evidence suggesting a role for CD45 in T cell development came from thc

gcneration of mice expressing a CD45RO transgene (Ong et al., 1994). The augmcntcd

expression of the phosphatase led to a marked reduction in numbers of DP thyrnocytcs, which

correlated with increased Lck kinase activily and enhanced calcium influx in responsc to TCR

ligation (Ong et al., 1994). By examining the effect of CD45RO overexpression on a ncgativc

sclccting background, investigators found that there was an enhanced MHC-rcstriclcd ncgativc

selcction ol'anti-HY TCR-bcaring DP thymocytes (Ong el al., 1994), suggesting that CD45 could

rnodulatc TCR signals to cffcct ncgativc sclcction proccsses.

A third linc of evidence implicating CD45 i n T ceIl development involved neonatal

injection of a pan-reactive anti-CD45 antibody. This treatment was found to inhibit diffcrentiation

of DP thymocytcs in10 mature SP thymocytcs (Benveniste ct al., 1994). To examine the role 01'

CD45 in positive selcction, the same antibody treatment was perfomed in fernale mice espressing

trdnsgcnic TCR spccific for the male HY antigen. The development OC CD&+ SP was inhibitcd in

a ktal thyinic organ culture system, suggesling thai lhc CD45 engageincnt inhibitcd positivc

sclcction (Bcnvcnistc ct al., 1994). IntcrestingIy, anli-CD45 prc-trcalmcnt of primary lyinph

ncxic CD4+ T cclls inhibits many TCR-induced activation priramctcrs, such ris Ca2+ mobilixation

and DNA synthesis (Maroun and Julius, 1994). In marked contrat, the samc lrcatmcnt had littlc

cffcct in CD8+ T cclls (Maroun and Julius, 1994). This implies thal thc two lincagcs havc a

diffcrcntial rcquircmcnt for CD45 inT ce11 activation. Thc mechanism bchind lhcsc obscrvaiions

has no1 bccn dcfined. Conccivably, thc addition ol'anti-CD45 could inducc a rcdistribution ol'

CD45 such that i t no longcr funclions to rcgutate TCR signals. Altcrnativcly, ligat~on ol' CD45

- -. -. - - -. . - - . - . -. - - - - -. - - -. . - - - - - . - - - a - . -. - . - - - - - - . - - , , - ---..-.- - - . ..-- ~. r . - -.-- -~ - - - - - - ~ - - - -

support of thc l'ormcr hypothcsis. By disriipting thc physical association bctwccn CD4 and

CD45 in CD4+ T cells (Bonnard et al., 1997). anti-CD45 treatmcnt may spccif'ically prcvcnt thc

appropriatc participation of CD45 in theTCRlCD3 complcs.

Clearly, regulation of CD45 function at the DP stage of T-ce11 deveiopmcnt has many

potcntial implications on the outcomes of positive and negative sclection. Intriguingly, a rcccnt

report suggests thal CD45 may be involved in setting thresholds mediating B ccll selcction cvcnts

(Cystcr et al., 1996). Investigators assessed positive and negative seleclion in CD45-del'icicnt

micc esprcssi ng immunoglobulin transgenes specific for hen egg lysozyme (HEL) in the prescncc

or absence of thc autoantipen. Their data showed that the absence of CD45 rendcred B cclls

hypo-responsive 10 BCR stimulation iti vitro, thus resulting in the positive, rather than thc normal

ncgative sclcction of HEL-spccific B cells in vivo (Cyster ct al., 1996). Thcsc results support a

signal-threshold mode1 for B-ceIl selection, and provide a framework for investigating the role ol'

CD45 in T-cell sclection. as wcll.

D ) Thesis Objective

The goal of my project involves the characterization of signaling proccsses rcgulating

positivc selection. To address the rolc of CD45 in TCR signaling in DP thyrnocyes, 1 havc

characierized a CD45-dericicnt DP thymomaccll linc called 3T7. Using this modcl systcm, 1

havc esamincd ihc requirements and rcgulatory rolc of this molcculc. By rclating thcsc

obscrvations io the itt vivo counterpart, thc ~D45-1- mousc, 1 proposc that thcsc signalrng rcsults

providc a salional basis for undcrstanding thc dcvclopmcnhl phcnotypc of CD45-/- niicc. Thcsc

~cslrlts furthcr dcfinc thc rch of CD45 in rcgulating TCR signals at thc DP siagc of dcvclopnicnt,

whilc providing a rational fianicwork I r our basic undcrstanding 01' antigcn rcccptor signaling

pi*,ccsscs.

CHAPTER 2

MATERIALS AND METHODS

3T7 derivatives are dl subclones of AKR 3T, ü thyrriic DP lymphoma ceIl linc (Grovcs ci

al., 1995). 3T7 was derived from a spontaneous thymoma in AKR mice. The AKR strain of

mice was bred for high incidence of thymornas, associated wi th endogenous retroviruses (Rowc

and Pincus, 1973). Ce11 lines were maintained in growth medium (GM) consisting of RPMI

1640 supplemented with 5% FCS, 5sl0-5 M 2-ME, 10 mM glutamine, and 10 mM HEPES,

pH7.4. 3T7.GC 14 was derived by sorting 3T7 cells that had becorne C D S ~ ~ aster overnight

cross1 inking wi th H57-5971GK 1.5 heteroconjugate antibody (see Anti bodies). C D S ~ ~ cells werc

defined as those cells with a mean fluoresence intensity (Mm) greater than 100. Unstimulatcd

cells contained less than 1% CD* cells and had a MFI of 30. Pervanadate was freshly made by

mixing 10 mM sodium orthovanadate (Sigma Chernical Co., %Louis, MO) with 10 mM Hz%.

The 10 mM stock pervanadate solution was incubated at RT for 10 min , prior to addition to thc

the ce11 culture. Herbimycin A, generously provided from Dr. P Benveniste (Hospital for Sick

Childrcn, Ont), was added to ce11 culture at a finai concentration of O. 1 PM.

B ) Retroviral Gene Transfer

Retroviral-mcdiatcd genc transfer was used to express CD45RO in 3T7.GC 14. Thc

plasmid T200/0 provided by Dr. P. Johnson (University of British Columbia, BC) (Johnson ct

al., 1989b) contains Su11 lcngth murine CD45RO cDNA cloncd into thc rctroviral vcctor pARV 1

(McLachlan ct al., 1987). pARV 1 also contains the neom ycin phophotransfcrasc genc ( M I )

which confcrs rcsiscüncc to G418. Al tcrnative splicing of transcripts drivcn by thc 5' long

tcrminal repcat (LTR) gcncratcs CD45RO and neo messengcr RNA (mRNA). DNA conslruçts

wcrc transfcctcd by calcium phosphate prccipitation into thc fibroblast ccotropic viral packaging

l inc, GP+E (Markowi tz ct al., 1988). A pol yclonal retrovirus-producing cc1 l linc, GP+EIT30010

was cstablishcd by growth in the prcscnce of 500 pglrnl G418 (Gibco, Grand Island, NY). To

infcct 3T7.GC 14 cells, 5- 10 s 106 cells were CO-incubated ovcrnight with irradiakd (7000 riid)

U l T L 1 1 -UUIU. L l U l l U I I I I I L C I L U I I L ~ I Y b L I , U U L U I I I b U Ur 1 1 1 1 1 1 1 1 1 1 5 U l I U l l U l l 111 U l V l L \ J 1 1 L L I 1 1 I I I 1 ~ t l J U tL$llll

G418. Surviving cells wcrc cvaluatcd for expression of' CD45 by tlow cytornclric analysis

(FACS) analysis using a fluorcsccin isothiocyanate (F1TC)-conjugated pan-CD45 spccil'ic

antibody (ALI-4A2). Posilive cells were expanded and maintained in GM containing 300 pg/rnl

(3418. As a negative control, paraIIel infections were done as above, espect using thc q2-ncci

packaging line, provided by Dr. D. Vaux (WEHI, Melbourne) to generate 3T7.nco subcloncs.

The $-ne0 packaging line contains the pLXSN retroviral vector containing the rieo gene dri\..cn

by thc SV40 early promoler (Vaux et al., 1988).

C ) Antibodies and Second Stage Reagents

Thc following monoclonal antibodies (MAb) were used in this study: anti-CD4 (GK1.5)

(Dialynas et al., 1983) and (YTS-191.1); anti-CD& (YTS-169.4) (Cobbold et al., 1984) and

(53-6.7); anti-CD5 (53-7.3) (Ledbetter and Herzenberg, 1979); anti-CD69 (H1.2F3) (Yokoyama

et al., 1988); anti-TCRp (H57-597) (Kubo et al., 1989); and pan-anti-CD45 (ALI-4A3)

(Spangrude and Scollay, 1990). Antibodies were purified from tissuc culture supcrnatanls

coniaining 5% NuScrum (Collabontivc Rcsearch Inc., Bcdford, MA) instead of' FCS, by protcin

G- or protcin A-Scpharose chromatography, and conjugated to FITC or biotin using siandard

tcchniqucs. Avidin-phycoerythrin was purchased from Caltag (San Franscico, CA). All

antibodics wcrc pi-c-titratcd and uscd at saturating conccntrations. Rat IgGb, rat IgG2.,, and

hainstcr IgG isotypc control antibodies conjugated to fiuoresccin, biotin, or phycocrylhrin wcrc

purchascd Srom Pharmingcn (San Dicgo, CA), and used at saturating concentrations OS 1-5

pg/1111.

The l'ollowing antisera wcrc used Ior weslern blol detcction andlor immunprccipiilitions:

anti-lck polyclonal rabbit antiscra, kindly providcd by Dr. M. Julius (University 01' Toroiilo,

Toronto, Ont); anti-Fyn polyclonal rabbi1 anliscra for wcslern blotting (Dr. A. Vcillcltc; McGill

University, Montreal, Quc), or immunoprecipi(ations (Santa-Cruz Biotechnology, Santa Cruz,

CA); anti-CD45 polyclonal rabbit anliscra, kindly providcd by Dr. P. Brrinton (McGill

d . . - , . , . , %

1994); and anti-phosphotyrosinc MAb 4G 10 (Upstate Biotechnology, Lake Placid, NY).

Sccondary reagents used in western blot detection were: horseradish perosidase (HRP)-

conjugated protein A and HRP-conjugated strepavidin, purchased from Amersham Corp.

(Arlington Heights, IL); and HRP-conjugated goat anti-mouse IgG, purchased from Bio-Rad

Laboratones (Richmond, CA).

To generate heteroconjugate anti bodies (H57-5971GK1.5 and H57-597lYTS 169.4) by

chemical coupling, H57-597 was modified using 15 pg N-gamma-

maleimidobutyryloxysuccinimide (GMBS; Calbiochem, San Diego, CA) pcr mg of antibody,

while GK1.5 and YTS- 169.4 were modified using 250 pg 2-iminothiolane HCI (3-IT; Picrce,

Rockford, IL) per mg of antibody (Lcdbctter ct al., 1989). GMBS is a hcierobifunctional

crosslinking reagent which introduces thiol-reactive maleimides into the prolein. ModiIïed

antibodies were purified through PD-IO columns (Pharmacia, Baie d'Urfe, Que) to removc

unreacted GMBS and 2-IT. The GMBS modified antibody then reacts with the second thiol-

conlaining 2-IT modified antibody to form a stable thioester crosslink (Fujiwara et al., 188 1).

This reaction was performed by mixing the GMBS-modified H57-5!37 and 3-IT-modificd GK 1.5

or YTS 169.4 together for 1 h at RT in coupling buffer ( 100 mM Na2HP04, pH6.8; 50 mM

NaCI). The reaction was quenched by adding 5x10-2 M 2-ME for 15 min and ihen O. 1 M N-

cthylmaleimide in DMF a further 15 min. Heteroconjugates were purified through PD-IO

columns to exchangc coupling buffer with PBS + 10 mM NaN3.

CNBr coupling of antibodies was pcrformcd üccording 10 manufacturer's instructions.

CNBr beads (Pharrnacia, Baie d'Urfc, Quc) were swelled foi- 30 min in 1 mM HC1 and rhen

washed thrcc tiincs in coupling buffer (0.1 M Na2HC03, pH 8; 0.5 M NaCI). 1 nig of pui-ificd

antibody was added per 79.12 mg ol' CNBr beads (dry wcight). The coupling rcaclion wns

pcrl'orined for 3 houis al room Lcmperature with constant mixiiig. Thc proccdurc crcütcs a

covülcnt bond bclwccn CNBr-aclivated bcads and ligands conlaining primary amincs. Thc

rcaction was qucnched by addilion of 0.1 M Tris, pH 8 for 2 hours al room lemperaturc undct-

consianl shaking. Couplcd bcads wcrc washcd three limes in coupling bul'f'cr, Lwicc in PBS, and

. - .

antibodies to protein-G-sepharosc beads, 10 pg of purified antibody was addcd pcr 50 ol' a

25% protein-G slurry. The beads and antibody wcrc rniscd ovcrnight at 4 0 ~ , washed thrcc

Limes in PBS and rcsuspcndcd in PBS + O. 1% NaN3 al a final mtibody conccniralion ol 1 p$p1.

D) Flow Cytometry

For llow cytornetric anal .yses, exponentidly growing cells or freshl .y isolated thymocytcs

wcre harvested in staining media (SM), consisting of HBSS plus 3% calf scrum and 10 mM

HEPES, pH 7.4. Cells werc stained by incubation of 0.5 - 1.0 x 106 cclls with satunting

amounts of FiTC- or biotin-conjugated antibodies at a concentration of 1 x IO7 ccllslml on icc for

20 min. The cells were washed in SM and resuspended at a concentration of 0.5 - 1 .O s 10"

cclls/ml in SM plus 1 &ml propidium iodide. Flow cytornetric analyscs werc pcrl'orrned on a

FACScan (Becton Dickinson & Co., Mountain View, CA). Data was acquircd on al lcast

10,000 cells/sample and anal yzed using an HP 340 computer and Lysis II software. Dead cclls,

idcntificd by thcir low forward scatter and high propidium iodidc fluorcscencc, wcrc escludcd

from analysis. Note that x axis labels on al1 histograms have becn converted by the dab analysis

software from thc fluorcscencc channel number (nonlinear scalc) to mcan lincar fluorcsccncc

intcnsi ty. Because unstained cul tured ceIls exhibi t varying degrces of autolluorcscencc that can

intcrfcre wi th discrimination of fluorcscencc due to antibody binding, fluorcscencc coin pensalion

was uscd to subtract ccllular autofluorescence (Alberti et al., 1987). This tcchnique I'acilitatcd

dctcction of low Icvcls of surfacc staining on highly autofluorcscent culturcd cclls.

E) Stirriuiation of cells for assessment of tyrosine phosphorylation

Cells were harvested in early lo mid-log phase, counted and aliquoted at 30 s 1061tnI

prior to staining for 20 min at OC with saturating concentrations of biotinylatcd anli-TCRp

(H57-597), biotinylatcd anli-CD4 (GKIS) , biotinylatcd anti-CDXtr (53-6.7), donc or in

b \ I I I I V I I I U L I V I I . I I I L d b C 1 I 1 J V V b 1 L i I I L W I I b U UllU L b I I L I O ~ b I I U L d U U L -.J i\ 1" 11111, YlLd I > U L I I I b U L I , J I b 1111

3 min, and cross-linking was pcrformcd for 1 min by adding 5 &ml avidin (Molccular Probcs,

Eugcnc, OR). Cc11 stimulation was tcrminated by adding 10 volumcs of icc-cold phosphatc-

buffcrcd salinc (PBS) containing 400 pM Na3VO4. Cell pcllets wcrc thcn rcsuspcndcd at 5 s

10~1ml in lysis buffcr (LB) containing 1% NP-40 (Fluka), 50 mM Tris, pH 8.0, 20 m M EDTA,

30 pM Na3VO4, 50 mM NaF, and 20 pglml leupeptin and aprotinin. Nuclci wcre pellctcd by

centrifugation (12,000 x g at 4 * ~ for 10 min) and the postnuclear proteins werc scparatcd by

SDS-PAGE gcls (8% non-reducing) (Laemmli, 1970) and then transferred to nitrocellulose, and

immunoblotted with anti-phosphotyrosine mAb (4G10).

F) Stimulation of cells for assessment of surface phenotypic changes

Cclls wcrc harvcskd i n carly to mid-log phase, and cultured at 3 7 O ~ overnight in 6-well

tissue culture plates (1 x 10~/well; Coming, New York, NY), pre-coated with PBS or various

protein A- or protein G-purified antibodies at 5 pglml, the optimal conccntration determincd by

titration cxperimcnls, for 2 hours at 37O~. The antibodies used for stimulation werc: anti-TCRp

(H57-597), anti-TCRVanti-CD4 heteroconjugate (H57-597/GK1.5), anti-TCRplanti-CD&

heteroconjugate (H57-597lYTS-169.4), GK1.5, and YTS- 169.4. To bypass stimulation

through the TCR, PMA (Sigma Chemical Co., St.Louis, MO) and ionomycin (Calbiochem, La

Jolla, CA) werc added at 10 nglml and 250 ngiml, rcspcctively. The cclls were hawestcd aftcr 1

or 2 days in culture and stained to quanlitate expression of surfacc CD5 (53-7.3), CD4 (YTS-

191. l ) , or CD8 (53-6.7) using FITC-conjugated anlibodics.

G ) Iinmunoprecipitations

Cclls wcrc lyscd at 5 x lo7/ml in LB for 20 min at OC, csccpl for cclls to be subjcct LO

anti-TCRp iinmunoprccipiiations (IP), which were lysed in 1% digitonin (Wako; Richmon, VA)

LB for 1 hr at OC. Lysatcs (10 x 106 cc11 cquivalents) wcrc ccntrifugcd (13,000 s g Ior 10 min

". . -, '- .-*..-. - ...L,Y.-Y.V . . . I L 1I.l. ...a- ...W.. r.--.--. -- " J ".--"L'.\'.. " L * . . d.' p..L \,A U - 2 ' L

protcin G-Sepharosc slurry overnight at 40C undcr constant mixing. Precleared matcrial n'as

rcmoved by centrifugation (13.000 s ç for 10 min al OC), and lhc rcmaining supernatant was

spli t into two equal volumes (5 x 106 cell equivalents) for IP. IPs wcrc performed for 1 hr at

4 0 ~ with constant mixing using 50 pg of antibody pre-couplcd 10 either protein G-scpharosc

bcads (Pharmacia, Uppsala, Sweden) or CNBr-activated-sepharosc beads (Pharmacia, Uppsala,

Swcdcn). Control IPs were performed identically but with uncouplcd IP bcads, unlcss

othcrwise indicated. Sequential IPs with either anti-CD4 (GK1.5) or anti-CD8 (53-6.7) an11 body

ivcrc performcd as abovc esccpt that following the intial IP, the supernatant w u transfcrred to a

frcsh tube and re-immunoprecipitated with an equivalent amount of antibody for 1 hr at 4 0 ~ with

constant mixing. The procedure was repeated a toial of five times. Immune complexes wcrc

washed 3 times in LE More k i n g resuspended in non-reducing SDS-PAGE loading bulTcr and

boilcd for 5 min. The beads were removed by centrifugation (13,000 x g for 2 min) and protcins

were resolved by SDS-PAGE (Laemmli, 1970) and then lranslcrrcd to nitroccllulosc mcmbrancs

for Western blotting (Towbin et al., 1979).

H ) Cell Surface Biotinylation

Cells werc washed three times in ice-cold biotinylation buffer (HEPES buffcred sülinc

(HBS), pH8.8; 1 m M MgCl-; and O. 1 mM CaCI-) and incubaled at 4OC for 20 min at 20 x 106

ccllslml in bioiinylation buffer containing 0.5 mglm1 sulfo-NHS-biolin (Piercc; Rocklord, IL).

The NHS ester of biotin reacts wi th the deprolonated form of primary amincs (Iysinc rcsidues) to

crcate a peptidc bond. The rcaction wüs qucnchcd by addition of biotinylation buffèr cciniaining

35 mM NH4CI. Cclls wcrc ivashed twice and then counted. A small aliquot of biotinylatcd cclls

was stained with avidin-PE to assess the degrcc of biotinylation by flow cyloinelry (biotinylatcd

cclls typically possesscd 1000-fold higher MF1 over unbiotinylated cclls). Thc rcrnaindcr oi'cclls

wcrc lyscd al 5 s lo7 ccllslml in LB with 20 &ml lcupeptin and aprolinin rcx- 20 min at 4 0 ~ .

immunoprccipitation.

1) SDS-PAGE and Irnrnunoblotting

Samples werc boiled for 5 min in SDS sample buffer and elcctrophoresed on 8% or

10.5% SDS-PAGE gels under non-reducing conditions unless othenvise noted. The gcls werc

thcn equilibrated in Towbin's transfer buffer (25 mM Tris; 193 mM glycine; 30% methanol) and

trmferred to nitroccllulose membranes at 72 V for 1.5 h in a Transblor apparalus (Bio-Rad

Lüboratories, Mississauga, Ont). After transfer, blots were blocked for 1 h at room icmpcraturc

in TBS-T (O. 1% Tween-30, 10 mM Tris, 2.5 mM EDTA, 50 mM NaCI) with 5% wlv carnation

milk and then probed for 1 h wilh primary antibody diluted in 2.5% milk protein (1:5000 I.or

anti-lck, 1:800 for anti-Fyn, 1: 1000 for anti-CD45, and 1 &ml foranti-TCR1;). The blois wcrc

washed for 10 min with TBS-T and then incubated 1 h with protein A-HRP (15000, Amcrsham

Life Science, Oakville, Ont), after which they were washed five times for a total of 1 h.

Antiphosphotyrosine blots were performed as above exccpt that blocking and rintibody

incubations were perforrned in 2.5% bovine serum albumin (BSA, I'raction V, l t t y acid-frec;

Bochringcr Mannhcirn, Indianapolis, IN) in TBS-T. The blots were subsequently developcd

with the cnhanced chemiluminescence detection assay (ECL kit, Amersham Lifc Scicncc). Blets

10 bc rcprobcd wcrc strippcd by vigorous washing in 10 mM Tris, pH2.3; 150 mM NaCl for 20

min, f'ollowed by two washes in 10 mM Tris, pH8; 150 mM NaCl for I O min. Dcnsitomctry

was performed to asscss relative amounis of protein using a Protcin Databases Inc. Discovciy

Scrics DNA 35 Dcnsitomcter.

J ) RNA preparation and Northern analysis

Total RNA was crtractcd Sroin cells by thc Trizol acid guanidinium singlc stcp rnclhod

(Molccular Rcscarch Ccntcr, Cincinnati, OH), and quantitatcd by UV spcctropholomctry. RNA

1 \ ' C . , .. 3.2M tormaldehydc, 1s MAE) and clcctrophoresed in 1s MAE in a denaturing 1.3%) aagrosc gel

conlaining 2.2M formaldehyde. Thc gel was blotted by capillary transfer in 30s SSC onto Zcla-

probc GT membrane (Biorad, Mississauga, Ont). Membranes wcre UV cross-linked (1300 J,

Stmtalinkcr, Stratagcne, La Jolla, CA) and prehybndized overnight at 4 2 0 ~ in of 50% dFA; 5X

SSPE; 3X Denhardt's; 0.5% SDS; 100 &ml salmon sperrn DNA. BIots werc ihcn hybridizcd

with 3 2 ~ - d ~ ~ ~ - l a b e l l e d DNA fragments (106 cpmlml) for 1 hr at 42OC with constant shaking,

washed twice with 1s SSC/O.l% SDS at RT, and twice with 0.2X SSC/O.I% SDS al G°C.

DNA probes wcre labelled using T7QuickPrime Kit, according to manufacturer's instructions

(Pharmacia, Baie d'Urfe, Que). Unincorporated nucleotides were removed by passing the probc

through NICK-Columns (Pharmacia). The hybridization probes used in this study werc: human

0-actin cDNA (0.6 kb Pst-1 fragment); murine RAG-1 cDNA (1.0 kb Bgl-II fragment) (Schatz el

al., 1989), and murine CD45 cDNA (1.35 kb XbaI fragment), kindly provided by Dr. H.

Ostergaard (U. of Alberta, Canada). BIots were exposed to the Molecular Dynamics

Phosphorimager (Sunnyvale, CA) system and then analyzed using Imagequant 3.0 software

(Molccular Dynamics).

K) cDNA synthesis and RT-PCR analysis

cDNA wüs synthcsizcd iri a 25 pl reaction with 5 pg of totül RNA, Promcga (Madison,

WI) RT buffer (final concentration: 50 mM Tris (pH8.3), 75 mM KCI, 3 m M MgCI2, 10 mM

DTT), 0.5 dNTPs (Pharmacia, Baie d'Urfc, Quc), O. 1 mglml BSA (Bochringcr Mannheim,

Indianapolis, IN), 40 U RNAsin (Proii~cga), 0.5 FM random hcsamcrs (Gi bco BRL, Grand

Island, NY), 2.5 U Promcga AMV RT. For the RT-PCR analysis of CD45 isolorms,

oligonuclcotides corrcsponding to thc mutine CW5 cDNA Genbank scqucncc (Johnson cl al.,

1989a) wcrc synthcsized: thc scnsc primcr (ATG ACA GCT GAT CTC CAG ATA TGA CCA

TG) corrcsponding 10 positions 110-134, and lhc anti-scnsc primcr (ATG AGT CGA CAA TCC

TCA TTT CCA CAC TTA GC) corrcsponding to psilions 821-2344, wcrc uscd. For thc murinc

L b

positions 330-340, and thc anti-sense primer (CAC GCA GCT CAT AGC TCT TCT)

corrcsponding to positions 790-8 10, were used.

RT-PCR reactions, camed out in 35 PI , containcd Taq buffcr (final concentration: 10 m M

Tris (pH9.0), 50 mM KCI, 1.5 mM MgCI2, 0.1% (WIV) Triton-X 100), 200 pM dNTPs

(Pharmacia, Baie d'Urfe, Que), 0.5 pM 3' and 5' primers, 1.35 u of 'Cïrertnirs nqzinticra ( k q )

DNA polymerase (purchased from Dr. J. Friesen, Hospital for Sick Childrcn, Ont). Sarnplcs

wcre overlaid with 50 pl mineral oil (Sigma Chemical Co., St.Louis, MO) to prcvent

condensation and subjected to 37 cycles of amplication using a programmed thermal cycler

(Perkin Elmer Cetus, Nonvalk, CT). For the CD45 PCR, each cycle consisted of denaturation at

9 4 0 ~ for 1 min, annealing and extending at 72% for 2.75 min. For p-actin PCR, samples rvcrc

subjected to 30 cycles consisting of denaturation at 94O~ for 1 min, anncaling at 55OC for I min,

extending at 7 2 0 ~ for 1 min. A fter amplication, PCR products were anal yzed by electrophoresis

in 7.5% agarosc (1.5% NuSicve and 1% Multipurpose) gcl i n a TrislAcetate buffer systcm and

DNA prcducts wcre visualized by ethidium bromide staining.

CHAPTER 3

RESULTS

Previous work in our laboratory characterized a panel of DP thymic Iymphomas. which

rcspond to TCR cngagcrncnt in vitro by undergoing multiplc maturation cvcnts associatcd with

positive selection in vivo, including increased expression of CD5 and Bcl-2, and dccrcased

cspression of RAG-1 and RAG-2 (Groves et al., 1995). These ceIl lines offcr a uscful rnodcl

systcm to elucidatc molecular mechanisms involved at this cntical stagc of T ce11 developmcnt.

Onc of thcse ccll lincs, 3T7, was identified as king TCR-non-responsive, failing to undcrgo

any of the phenotypic changes observed in TCR-responsive ce11 lines (Groves et al., 1995).

Thc defect appeared to be proximal, in that addition of PMA and ionomycin, pharinocologic

agents which bypass carly events in TCR signaling, upregulated expression of CD5 and CD69

(Grovcs ct al., 1995). To attcrnpt to idcntify thc TCR signaling defect in 3T7 cells, the surlàcc

expression levcls of various cell markers was assessed. Figure I shows that 3T7 cclls express

both of the co-receptors CD4 and CDS, and the great majority of cells (90-95%) stably cxprcss

medium levels of surface TCR (MF1 = 11 1), while a small fraction (5-10%) express low levcls

of surface TCR (MF1 = 5) by flow cytometry. In constrast, 3T7 cells lack dctcctablc surfacc

expression of CD45 by flow cytometry (Fig. 1). Western blot, Northern blot, and RT-PCR

analysis also revealed no dctcctablc CD45 expression (see Iater). Thus, 3T7 is a CD45-

dcfïcient DP lymphoma ce11 linc.

B) TCR signaling defect in 3T7 cells

i) Early events - induction of tyrosine phosphorylation

Thc carlicst detcctablc cvcnl in TCR signaling is Lhc induction of tyrosinc kinase

xiivity, as rcvcalcd by induciblc protein tyrosine phosphorylation (Samclson ct al., 1986; Hsi

cl al., 1989; Junc et al., 1990b). Givcn the critical rolc of CD45 in rcgulating Src-family PTKs,

thc rcquircmcnts Ior CD45 in TCR signaling with or without Lhc ddibcralc CO-aggrcgalion 01'

Figure 1: Surface phenotype of 3T7 cells.

Ccils wcrc stained wilh FITC-conjugatcd antibodics spccific for CD4 (GK1 S), CD8tr (53-A.7),

TCRP (H57-597), or pan-CD45 (ALI-4A2). Shadcd hislograms show lhc lcvcl of staining with

isotypc-matched control antibodies.

stin~ulated for onc minute and tyrosinc phosphorylatcd protcins dclcclcd by SDS-PAGE and

Wcstcrn blotting with the anti-phosphotyrcisinc antibody (4G 10). I n rcsponse to TCR

aggrcgation, thcrc was a marginal increase in the tyrosinc phosphorylation of two protcins 01'

around 80 kDa and 90 kDa (Fig. 2A). However, when eithcr CD4 or CD8 was delibcratcly co-

aggregated with TCR, a striking induction of phosphoproteins, notably 40 kDa, 70 kDa, 80

kDa, and 120 kDa was obsenled. Crosslinking of either CO-receptor alone rcsulled in only a

marginal induction of phosphoproteins, simiIar to that observed in response to TCR stimulation

alone. Furthermore, TCRICD4 co-aggregation was far more robust in inducing Lyrosinc

phosphorylation than TCRICD8 CO-aggregation. These data suggest that the need for CD45 in

early TCR signaling is obviated when the TCR and either CO-receptor are CO-aggregated.

ii) Late events - CD5 induction, RAG-1 downmodulation

To detcrmine whether the proximal TCR defects observed extended to Iater cvcnts in

TCR signaling, 1 assessed the ability of 3T7 cel!s to undergo phenotypic maturilion cvcnts.

Prcvious work has indicated that both fresh ex vivo DP thymocytes and various DP thymoma

cc11 lines undergo a number of phenotypic changes, including upregulation of CD5, CD69 and

Bcl-2, as well as dccreased expression of RAG-1 and RAG-2, in rcsponse to TCR ligation irr

vitro (Turka et al., 1991 b; Brandle et al., 1992; Groves et al., 1995; Kearse et al., 1995; Grovcs

ct al., 1997). Furthermore, these events have been s h o w to occur during posilive selcction itr

vivo (reviewed in Guidos, 1996). Therefore, 1 determined whcther 3T7 cells could incrcasc

CD5 and decrcasc RAG-1 expression in response to TCR signais. Following overnight culturc

of 3T7 cclls alonc, or i n wclls that had bcen precoated with purified antibodics, flow cyloinctry

was uscd to quantitatc surfacc cxprcssion of' CD5 as a markcr of maturationlac~iva~iun.

1-Iclcroconjugate aniibodies, generaled by chernical crosslinking of anti-TCRp and cithcr anti-

CD4 or anli-CD8 anlibodies, were used for stimulations involving thc co-cngagenicnt of TCR

with cither co-rcçeptor. Whilc TCR engagement donc fàiled to induce CD5 csprcssion i n 3T7

a 2i 6 Y P : d c 4 Stimulation: j ÿ y n u Z k U E - c U

Figure 2: Effect of CD45 deficicncy on TCR-mediatcd signal transduction in 3T7 cclls.

(A) Induction of tyrosine phosphorylation in 3T7 cells aftcr TCRp or TCRp + CO-rcccptor

crosslinking. Cclls were cultured for 1 min at 37OC with or without antibody-incdiatcd cross-

linking of thc indicatcd surface molecules. Postnuclear lysates rrom cqual cc11 numbcrs

(0.5~106) wcrc scpaiated by 8% SDS-PAGE (non-reducing), traiisferrcd to nitroccllulosc, and

probcd with a monoclonal anli-phospholyrosine antibody (4G 10) followcd by goat anti-mousc

HRP, and dctectcd by ECL. Arrows indicate sevcral prolcins that undcrgo TCR-induciblc

tyrosinc phosphorylation. Numbcrs on the left indicate the migration of MW standards.

, - - -.- --- . . ---. - - - - - . - - . . - - - - - - --. -..- - .... -. -- . -. --., ,.-.- dclibcrately co-aggrcgated (Fig. 2B). The ligation of eilher CD4 or CD8 co-rcceptors without

TCR lailed to induce CDS expression i n 3T7 cells (Fig. 2B). SimiIarly, a decrease in RAG-1

espression was observed when TCR and CD4 were co-aggregated, but not in rcsponse to TCR

cngagemenl alone (see later, Fig. 8C). As was observed for protein lyrosine phosphorylation,

TCRICD4 co-aggregation was more efficient at inducing CD5 (70-90% became C D S ~ ~ ) than

was TCRICD8 co-aggregation (20-50s became CD*; Fig. 2B). Two populations of cclls

were consislently observed: lhose that were responsive (~~518, MFI>100), and those that

remained CDJO, MFI<100. This suggests that 3T7 cells may harbour additional delècts

bcsidcs CD45, accounting for the inability of some cells to respond.

The signaling capaci ty of the CO-receptors CD4 and CD8 is large1 y attribukblc to thcir

association with Lck (Veiliette et al., 1988). Thus, 1 hypthesized that thc ability to inducc

TCR signaling events by coaggregation of TCR with CD4 or CD8 could bc attributcd to the

activation of Lck when brought into close proximity to the TCR complex. To asscss whclher

TCR plus co-receplor signaling was associated with E K activity, 1 used the tyrosinc kinase

inhibitor hcrbimycin A, which has becn shown to markedly diininish TCR signaling (June ci

al., 1990a). The induction of CD5 after ovemight stimulation by TCRICD4 was almost

completely abolished when cells were cultured with O. 1 PM Herbimycin A (Fig. 2C). This

effccl was dose-dependent, with no obscrvablc cffcct when the dnig was added al 0.01 (Fig.

2C). Thus as expected, the generation of TCRlco-reccptor signals not only corrclates wilh KTK

activity, but requires it.

C) Differential ability of CD4 versus CD8 CO-receptors to overcome TCR signaling

defect

I t has becn rcp~rted that CD4 associatcs rnorc strongly than CD8 wiih Lck in wild-typc

DP lhymocytcs (Wicst cl al., 1993). Thus, differential Lck association might csplain thc

grcalcr abilily of CD4 to signal than CD8 when coaggregatcd wi th Lhc TCR. To dctcrminc if

Stimulation: Stimulation: Stimulation:

TCRPICD4 .'l,:;: !y:.-,. .+.:$.. -...,

IO* 10' 10' id 10' io* 10' IO' id 10'

Figure 2B: CDS induction in 3T7 cells arter TCRp or TCRp + CO-receptor crosslinking.

Cells were cultured overnight alone or in culture wells coated with the indicated antibodies: anti-TCRp (H57-597), anti-

TCRPanti-CD4 (H57-597lGK 1.5 heteroconjugate), anti-TCRf3Ianti-CD8a (H57-597lYTS- 169 heteroconj ugate), anti-CD4

(GK 1 S), or anti-CD8a (YTS- 169). Cells were stained with RTC-conjugated anti-CDS (53-7.3). Shaded histograms represent

staining with an isotypc-matched control antibody.

Stimulation:

Herbhycin A:

TCRfYCD4

Nooe

Figure SC: CD5 induction in 3T7 cells treated with the PTK inhibitor? Herbimycin A.

Cells were cultured overnight alone or in culture wells c o a a with anti-TCRP/anti-CD4 (H57-597lGK1.5 heteroconjugate)

and the indicated arnount of Herbirnycin A. Cells were stained with FITC-conjugated anti-CD5 (53-7.3). Shaded

histograrns represent staining with an isotype-matched control antibody.

tliis was the case, I performed sequential immunoprecipitations OS CD4 and CDS. I n

aggreernent wi th studies of DP thymocytes, CD4 immunoprecipi tates Srom 3T7 cells containcd

signilïcantly greater amounts of Lck than the corresponding CD8 immunoprecipitatcs (Fig.

3A). Lck associates with the cytoplasmic tail of CD& (Veillette et al., 1988). An altematively

spliced version of CD8a, CD8a' contains a truncation in the cytoplasmic domain such that i t no

longer associates with Lck (Zamoyska and Parnes, 1988; Zamoyska et al., 1989). Therefore, 1

assessed whether the low stoichiometry of the association between Lck and CD8 might rellect a

low ratio of CD8cJCD&tt in 3T7 cells. Cells were biotinylated to label surface protçins prior to

CD8a immunoprecipitation. Immunoprecipitated proteins were then sepanted under reducing

conditions on SDS-PAGE to dissociate the disulfide linked CD8 dimers. Two bands of 40 and

35 kDa were observed, corresponding to CD8a and CD&' polypeptides, respective1 y (Fig. 3B).

The "smearing" appearance of these bands is likely due to the presence of diffcrentially

glycosylated forms of the CD8a polypeptide. Also, the CD8p labels poorly in these procedurcs,

and is not readily observed in CD8 immunoprecipitates. 3T7 cells expressed significantly

greater amounts of CD&' on their surface than CD8a (Fig. 3B). In contrast,

immunoprccipitatcs from unfractionated thymocytes revealed roughly equivalcnt surlacc

expression of the CD& and CD8a' polypeptides (Fig. 3B), in agreement with previous studies

(Zamoyska and Parnes, 1988). Thus, the lower association of Lck wi th CD8 than CD4 in 3T7

cells can be explained, in part, by the preferential expression of CD8a' which can not associatc

with Lck. However, the low stoichiometry of the association between Lck and CD8 in 3T7

cclls could also bc explained by the intrinsically poor association between CD8n and Lck. This

is demonstrated in CDS+ T cells from the periphery (cg. lymph nodes), which prcdominantly

express the full-length CD8a polypeptide, yel slill have 10-lold lcss associütcd Lck than Sound

in CD4+ T cells (Zamoyska and Parnes, 1988).

Thymocytcs Parental 3T7

Figure 3: Molecular basis of differential signaling between CD4and CD8 CO-receptors in 3T7 cells.

(A) Differential association of Lck wi th CD4 and CD8 coreceptors in 3T7 cells. Cells were lysed in LB and sequentiall y

immunoprecipitated 5 times with protein G-coupled anti-CD4 (GKlS), protein G-coupled anti-CD8 (53-6.7) or with protein G-

coupled total rat IgG (control IP). 2x106 ce11 equivalents were used for the sequential immunoprecipitations, and h l 0 6 cell

equivarents were used in the lysate control. Proteins were separated by 8% SDS-PAGE (non-reducing), transferred to

nitrocellulose, and probed with rabbit anti-Lck antisera followed by protein A-HRP, and detected by ECL.

( B ) Evaluation of CD8 coreceptor isoforms expressed in 3T7 cells. Parental 3T7 cells or B6 thymocytes were surface labeled

with bioiin and then lysed in LB. Lysates from 5 x 106 (3T7) or 5 x 107 (B6 thymocytes) cells were immunoprecipitated with

protein G-coupled anti-CD8a (53-6.7) or with protein G beads alone (control IP). Proteins were separated by 10.5% SDS-

PAGE (reducing), transferred to nitrocellulose, and probed with strepavidin-HRP followed by ECL detection.

&. T% -- 4- CDScx

4- CDSa'

Thc ability of 3T7 cclls to signal when TCR and CO-rcccptor wcrc co-aggrcgatcd

suggests that CD45 is dispensable under these stimulatory conditions. To addrcss whcthcr

altcrnative phosphatases (PTPases) might be espressed in 3T7 cells, the effèct 01' lhc

phosphatase inhibitor, pervanadate was assessed. Pervanadate treatment has been shown to

mimic T-cell activation via inhibition of PTPases and activation of some FTKs (Secrist ct al.,

1993; Imbert et al., 1994). Accordingly, pervanadate treatment led to a dose-dependent

induction of several phosphoproteins, notably 40 kDa, 46 kDa, 60 kDa, 70 kDa, 120 kDa (Fig.

4A). 1 proceeded to addrcss whether the induction of phosphoproteins extended to later cvenls,

such as CD5 induction. Following overnight culture of 3T7 cells alone, or with pervanadatc,

Slow cytometq was used to quantitate surface expression or CD5 While highcr concentrütions

of thc drug were toxic after overnight culture with 3T7 cells (data not shown), a 5-fold

induction of CD5 was observed after treatment with 10 pM pervanadate (Fig. 4B). This

induction was less than the 10-fold induction of CD5 observed when surface receptors werc

ligatcd (Fig. 2B) and may rclatc 10 thc absence OC a nucleating physical structurc ont0 which ihc

induced phosphoproteins can interact. Nonethcless, thesc results suggcst that altcrnativc

FTPases are indecd expressed in 3T7 cclls, and can function to activatc TCR signal

Lrünsduction palhways when perturbed.

E) Defect in CD45 gene expression in 3T7 cells

Having charactcrizcd somc SunctionaI consequences of lhc lack of CD45 on TCR

signaling in 3T7 ccIls, 1 next wished to idcntify lhc naturc of thc dcfcct in CD45 gcnc

csprcssion in 3T7 cclls. As prcviously staled, Northcrn analysis revcaled that rcsting 3T7 cells

do no1 contain dctcctablc lcvels of stcady-stale CD45 mcssagc (Fig. 5A). Thus, the csprcssion

is no1 constiiulivc. Howevcr, ovcrnighl slimulalion with TCRICD4 or TCRICD8

hetcroconjugütc antibodics induced CD45 mRNA and suri'acc CD45 protein expression (Fig.

Pervanadate

(B) Pervanadate: None 10 pM

k,w, $" .a , ,,w , , ,k B"$ t<.. ,:,.c:,. q p.;::, &.; ,.-..,* "Ad'.

"Fw: \ "':::.,.

.-'+'-..+(ib&

p:,: :.. .: 1. :j 4 .::- 4 . .+L Ar..; ... l *$, ,,,-,...

1 q'lB ' 1 1W "'î

Figure 4: Effect of the tyrosine phosphatase inhibitor, pervanadate on 3T7 cells.

(A) Induction of tyrosine phosphorylation in 3T7 cells after pervanadate treatment. CeIls wert

cultured for 10 min at 3 7 ' ~ after addition of the indicated amount of pervanadate. Postnuclear lysate!

froni equal ce11 numbers ( 0 . 5 ~ 109 were sepamted by 8% SDS-PAGE and probed for phosphotyrosini

as described in Fig. 2A.

(B) Induction of CD5 in 3T7 cells after pervanadate keatment. Cells were cultured ovemight dont

or with the indicated amount of pervanadate. Cells were stained with FITC-conjugated &-CD:

(53-7.3). Shaded histograms represent staining with an isotype-matched control antibody.

VL3-3M2 Parental 3T7

Figure 5: Ailalysis of CD45 gene expression i n 3T7 cells.

(A) Noi-thei-n andysis of CD45 niRNA expression in 3T7 cells cultureci ovcrnight

alone or with indicakd iitimobilized anlibodies. Tolnl RNA was separated on

formaldehyde-agarose gels, blottcd onto nylon riieiiibrane, probed witli 33-P-

Iribelled RAG-1 and p-actin cDNA fragiiients, and exposed to a phosphosima~es

scieeii. VL3-3M2 (positive control) is a DP ttiyinonin ce11 linc whicli expicsses

high lcvcls of suiflice CD4.5.

. , ~~ ~ - - - C - - - - -

esprcssion of CD45 than was TCRICD8.

The pattcrn of CD45 alternative splicing and its changes during lhymacyte development

suggcst that i t is an important mcchanism for controlling CD45 function. To idcntify which

CD45 isoform is re-expressed, 1 performed RT-PCR analysis on cDNA from rcsting and

stimulakd 3T7 cells. Primers specific for exon 2 and cson 9 were designed such that mRNA

containing differcnt combinations of variable exons 4, 5, 6 and 7 would generatc RT-PCR

products of different sizes, as indicated in Fig. 5C (top panel). While resling 3T7 cells

contained no detectable CD45 mRNA products, stimulated 3T7 cells contained RT-PCR

products whose size corresponded to the expected products of thc CD45R(O) and CD45R(-1)

isoforms (331 and 257 bp, respectively, Fig. 5C). These bands co-migrated with the PCR

products generated frcim the positive control, wild-type thymocytes, which pre-dominanily

expressed CD45R(O) and CD45R(-1) isoforms (Chang et al., 1991). Thus, 3T7 cells cxpress

littlc or no steady state CD45 message, but can be stimulated to express isoforms of CD45

cxpressed in normal thymocytes. Together these data are consistent with a defect at thc

transcriptional level of CD45 However, it is not possible to formally cxcludc a dcfcct in CD45

mRNA stability.

I next sought to determine whether CD45 exprcssion in slimulated 3T7 cells was

functional. 3T7 cclls wcrc cultured overnight alone or wiih PMA -t ionomycin to inducc CD45,

and thcn subjccted to f'unctional assays of TCR signaling. 1 chose 10 use PMA + ionomycin to

inducc CD45 because these agents bypass surfacc ligation events, and thus avoid the problem

of reccpior internalizalinnlblocking that would occur if anti -TCRlanti-CD4 heteroconjugatcs

wcrc L ISC~ . Thc PMA + ionomycin-trcated ceils relaincd surfacc TCRF csprcssion (data not

shown) and bccümc lransienlly CD45-positivc (Fig. 6, top panel) bcîorc gradually losing CD45

cxprcssion over the course of 34 hours (data not shown). PMA + ionomycin-lrcatcd 3T7 cclls

wcrc now able to rcspond to TCR ligation with the induction or protcin tyrosinc

phusphorylalion (data not shown), and also incrcascd CD5 esprcssion in rcsponsc to TCR

cngagcmcnt (Fig. 6, bottorn panel). Notc that the PMA + ionomycin trcatcd cclls csprcsscd

Stimulation:

1

Stimulation:

Figure 5B: Surfacc CD45 inducibly cxprcsscd in 3T7 cells.

Cclls wcrc culturcd ovcrnighr donc or in cullurc wclls coatcd wilh thc indicalcd antibodics:

anti-TCRp (H57-597), anti-TCRpianti-CD4 (H57-5971GK 1.5 hcteroconjugalc), or anti -

TCRplanti-CD8a (H57-597iYTS-169 helcroconjugaie). Cells wcre staincd wilh biolinylatcd

pan-anli-CD45 (ALI-4A2) followcd by avidin-PE. Shaded histograms reprcscnt staining with

an isolypc-matchcd conlrol anli body.

Isoiomi Namc SpUced Producl PCK Product (bp) RAl3C 2-3-45-6-7-8-9 743 €3 2-3-47-8-9 460 RB 2-3-17-8-9 478 RC 2-3-6-7-8-9 472

RAB 2-3-4-5-7-8-9 607 R4C 2-3-4-6-7-8-9 601 RBC 2-3-16-7-8-9 619 R(O) 2-3-7-8-9 33 1

Figure 5C: RT-PCR analysis of CD45 isoforms inducibly exprcssed in 3T7 cells.

Thc schematic represenis the exonlintron structurc of' thc unspliced CD45 prc-mRNA (not t(

scalc). The # of nuclcotidcs in cach cson is indicated insidc the boxes. Location ol' lhc prlmcc

used for thc RT-PCR analysis arc dcpictcd by thc arrows. Thc cspectcd sizcs of RT-PCI.

product from cach isoform arc s h o w in thc table. Cells werc cultured overnight donc or witl

anti-TCRpIanli-CD4 (H57-597/GK1.5) hcteroconjugatc antibody. cDNA was rcvcrsl

transcri bcd îrom tolaI RNA and PCR-ampli ficd wi th primcrs spccific for CD45 and pactii

(contra! -RT sampIcs wcrc subject to the samc trcatmcnt cxccpt rcvcrsc transcriptase was no

added). RT-PCR products wcrc scpamtcd by agarosc gcl and stüincd with clhidium bromidc.

Parental 3T7 "CD45-Positive"3T7

Stimulation:

Figure 6: Rc-expression of endogcnous CD45 comelatcs with restoralion of TCR

rcsponsivcncss.

Cclls wcrc culturcd ovcrnighl alonc or with PMA + Ionomycin, harvcslcd, and a small aliqunt

was slaincd with FITC-conjugatcd pan-CD45 antibody (ALI-4A2) or an isolypc niatchcd

control antibody and anaIyLed by fl ow cytomelry. Thc rcmaining cclls wcrc rc-çul~urcd

ovcinigtit alonc, or wiih immobilizcd anti-TCRP (H57-597). Cclls wcrc haivcslcd and siaincd

wilh FITC-conjugatcd anti-CD5 (53-7.3) (open histograms) or an isolypc inalchcd conLr.01

antibody (shadcd histogsains).

b d L .

= 100), but this was furthcr incrcascd by anti-TCR stimulation i n a subsct of cclls (MF1 =

2000). The rcsults suggest that thc cndogcnous CD45 1s functional, and ablc to rcstorc, at lcast

in a subset of cclls, thc ability to rcspond to TCR ligation.

F) Re-expression of exogenous CD45 in 3T7 cells

The above resulls demonstrated that effective coupling of the TCR to TCR signal

transduction machinery correlates with the expression of CD45 Horvever, i t remained possiblc

that other changes induced by the PMA + ionomycin treatmenl could be indirectly responsiblc

for restored TCR responsiveness, as opposed to a direct effect of CD45 re-expression. To

determine if the primary signaling defect in 3T7 cells was due to Lack of CD45 1 expressed

exogenous CD45 and assessed i ts functional consequences on TCR signal transduction. 3T7

celis were infected with a retroviral construcl containing the cDNA encoding the 180-kDa

CD45RO isoform of murine CD45 (Fig. 7A), the isoform normally expresscd in DP thymocytcs

(Chang cl al., 1991). Aftcr G418 selection, clonal infcctrints (obiaincd by limiting dilution)

expressing CD45 were identified by llow cytometry. Western analysis showcd that the two

infectanls, 3T7.CD45 C1.l and 3T7.CD45 C1.3 cxprcssed a single specics of CD45 prolcin

corrcspanding to thc cxpcctcd 180-kDa band, while parcntal 3T7 cclls and 3T7.nco cclls

contained no detectable CD45 protein (Fig. 7B). The siaining characteristics of parcntal 3T7

cells and two clona1 infectants, 3T7.CD45 C1.l and 3T7.CD45 C1.2, are presentcd in Fig. 7C.

Ench infectant cxprcsscd surlacc CD45 protein, while parental 3T7 cells and a negativc conlrol

infectant, 3T7.nco did not slain übove background. Clona1 infcctants wcrc also staincd for

CD4, CD8, and TCRP and wcrc found to express similar levcls of tliesc surl'acc markcrs, whcn

coinparcd to parental 3T7 cclls (data not shown).

Figure 7: Expression of cxogcnous CD45 in 3T7 cclls by relroviral-mediated gcne transfer.

(A) Schcinatic representation of the CD45R(O) retroviral construct iised in this study.

(B) Western blot aiialysis of CD45 protcin levcls in 3T7 inrectants. Post-nuclear supernatants

were prcpared from 2.5~106 cells lysed in LB. Lysriies were separated by 8% SDS-PAGE (non-

rcducing), transferred to nitrocellulose, and probed with CD45-specific antiscra (#788/9-4)

followcd by protein A-HRP, and developcd by ECL deiection.

(C) Surface CD45 expression in G41X-sesistrint 3T7 infectants. Parental 3T7 cclls, 3T7.nco

(3T7 infèctcd with neo-control constsrrct), 3T7.CD45 Cl. I and C1.2 werc stainccl with FITC-

corljugatcd pan-CD45 (ALI-4A2). Sliaded histogra~i-is show ttic lcvcl of stnining with isotype-

niatclicd coiitrol nntibody.

i) Early events - induction of tyrosine phosphorylation

To esaminc the lunctional consequences of CD45 re-expression on TCR signaling in

3T7 cells, 1 analyzed CD45 infectants for tyrosine phosphorylation upon TCR cngagcmcnt.

Comparing the basal phosphorylation state of proteins in parental 3T7 cells to 3T7.CD45 Cl. 1,1

observed that the overall level of phosphoproteins was similar, except for a 32 kDa protein,

which is hyperphosphorylated in parental 3T7 cells (Fig. 8A) . This suggests that CD45

csprcssion is not required for the maintenance of the overall tyrosine phosphorylation

homeostasis of most phosphoproteins in 3T7 cells. In response to TCR crosslinking, the

3T7.CD45 Cl. 1 infectant regained i ts abili ty to induce protein tyrosine phosphorylation (Fig.

8A). The degree and pattern of phosphoprotein induction was similar to that obscrvcd in

parental 3T7 cclls when stimulated with TCRICD4. Similar results were obtaincd in another

i ndependentl y derived infectant, 3T7.CD45 Cl .2 (data not show n). These results indicatc that

parental 3T7 cells require expression of the CD45 glycoprotein for the induction of protcin

tyrosine phosphorylation in response to K R ligation alone.

ii) Late events - phenotypic maturation, RAG-1 downmodulation

To asscss whether the rescue in proximal TCR signaling evcnts observed in 3T7.CD45

infcctants extended to downstream events, 1 evaluated whether TCR engagement coiild indiicc

csprcssion of CD5, as well as othcr maluration cvents associatcd with TCR signaling in DP

ihymocytcs. As prcviously obscrvcd, TCR engagement of parcnial 3T7 cclls did not causc

CD5 induction. Howcvcr, a largc fraction of the 3T7. CD45 Cl. 1 infcctünts (75%) rcspondcd to

TCRp ligation wiih a IO-Sold induction of CD5 (Fig. 8B). Similarly, a significant proportion of'

ihc 3T7.CD45 Cl. 1 infcciants (40%) responded to TCR ligaîion by downmodulating CD4 and

CD8 csprcssion (Fig. 8B), an effect not observed in the parental cell linc. Downrcgulation »I'

Parental 3ïS 3i7.CD45 CL1

Figure 8: Effect of exogenous CD45 expression on TCR-rnediated signal transduction

in 3T7 cclls.

(A) Esogcnously expressed CD45 rcstores TCR-induccd protcin tyrosine phosphoylaricin i n 3T'

cclls. Parcntal 3T7 cells or 3T7.CD45 C1.l cells werc subject to antibody-mediatcd cross-linkin;

of thc indrcatcd surface inolecules as describcd in Fig. 2A. Arrows indicatc scvcral prolcins tha

undcrgo TCR-induciblc tyrosine phosphoylation. Astcrisk (*) indicales phosphoprokin which i

dil'fcrcntially phosphorylatcd in parental 3T7 cclls vcrsus 3T7.CD45 Cl. 1 cclls.

Stimulation:

i o V o ' IO' 10' 10' ioO 10' io'""io3 lo\oO 10' 10' 10' 10'

, $ .

10" IO' 10' 10' 10' 1 0 9 0 ' io2 10' 10' 10' IO' IO* IO' IO* -CDBa.-+ - - CD5 -b

Figure SB: Esogcnously espressed CD45 rcsiorcs KR-induccd changcs in ccll sui-làcc

phenotype.

Cells wese culluscd overnighl alone or with irn~nobilized anli-TCR13 (1-157-597), and ~licri

s~ained wi th FITC-conjugatcd antibodics specif'ic for: CD5 (53-7.3), CD4 (YTS-191.1), os

CD8a (53-6.7). Shadcd liisrogsams rcprescnt staining wiih isotypc-malchcd conlrol anlibodics.

co-rcccptor cspression also occurs wiicn trcshly isolated Dl-' tthytnocytcs arc sliniulalcci

ovcrnight with anti-TCR (Grovcs et al., 1995; Kcarsc cl al., 1995; Groves cl al., 1997). Table

1 summarizcs thc levcl of TCR-induccd CD5 expression in several 3T7.CD45 infcctants.

Whilc in al1 cascs, some rescue in TCR-induced signaling was observed, thc ability of CD45 to

complemcnt the signaling defect varied considerably between clones. This may rclatc to thc

additional signaling defect(s) present in 3T7 cells, alluded to before.

1 extended my functional analysis of the CD45 infectants to expression of RAG- 1. To

assess whether CD45 infectants also regained their ability to downregulate RAG-1 mRNA i n

response to TCR ligation, Northern analysis was performed. Parental 3T7 cel!s did no1

downmodulatc RAG-1 message in responsc to TCR ligation alone (Fig. 8C). In contrast,

3T7.CD45 (21.1 cells responded to TCR Iigation with a 3-fold reduction in RAG-1 mcssagc.

However, this was no1 as çomplete as thal obsctved in parental 3T7 cells following TCRICD4

CO-stimulation, whcrc a 30-foId reduciion in the amount of RAG-1 message wüs observcd (Fig.

8C). This likely relates to the presence of a TCR-non-responsive subset of cells, as show in

Fig. 4.

Collectively, these data formally demonstrate that expression of exogenous CD45 in

3T7 cells rcstores TCR coupling Lo the proximal PTK signal ing pathway and to downslrcam

maturation cvents at least in a subset of cells. These data dernonstrate a critical role for CD45

in rcgulating specific TCR-induced maturation events known to occur at the DP stagc of T cc11

dcvelopment.

H) Biochemical basis for rescue of TCR signaling by CD45

To identify thc sitc of action of CD45 in rcgulation of thc TCR signal transduclion

pathway, 1 invcstigatcd thc phosphorylalion slatc of various known CD45 substrütcs. Iiilporlant

targcts 01' CD45 arc thc Src-l'aniily kinases, Lck and Fyn (Ostcrgaard ct al., 1989; Muslclin ct

al., 1993; McFarland cl al., 1993), as ivcll as TCRT, (Furukawa cl al., 1994). Thcrclbrc, I

asscsscd thc inllucncc of CD45 cspression on Lhc tyrosinc phosphorylation of Lck, Fyn, and

S t i m u l a t i o n :

k B

7.5 -

Parental 3T7 3T7.CD4 CL1

u u f! a

Figure 8C: Expression of exogenous CD45 restores TCR-induced RAG-1 downmodulation.

Northcrn analysis of RAG-1 and p-actin lranscripts in cclls cultured ovcrnighl alonc or with thc

indicatcd immobilizcd antibadies was performcd as dcscribcd in Fig. 5A. Nuinbers on lcft

indicatc migration of RNA MW standards. Dcnsitomctric analysis was perlormcd, and thc

rcsults wcrc normalizcd for cach cc11 linc by triking thc ralio of RAG- 1 signal to p-actin signal

and arbitrarily dcsignating this ratio in unstimulatcd cells as 1.0.

Table 1.

TCR-mediated CD5 induction in

Parental 3T7 Cells versus CD45 Infectants

Mi3 is rcportcd as a mcasurc of surfacc CD45 cxprcssion on thc indicatcd 3T7 subcloncs, as

dclected by staining with FITC-conjugated pan-CD45 antibody (ALI-4A2). Control stains wilh

an isotype-matched antibody had an average MF1 of 3.0. To rncasurc thc various subclone ccll's

funclional response to TCR cross-linking, they were cultured ovcrnight in wells coatcd wilh

antihdics specific l'or: TCRp (H57-597) or TCRplCD4 hetcroconjugritc (H57-5971GK1 .S), and

rvcrc thcn staincd with FITC-conjugated CD5 (53-7.3) and analyzcd by llow cytomctry. C D S ~ ~

cclls wei-c dcl'incd as those cells with a MF1 gseater than 100. Unstimiilated cclls coniaincd lcss

ihan 1% ~ ~ 5 ~ h l l s .

- - - - . - - . - - - - - - _. . _ _ _ _. _ _ _ . . _ _ _ - - - - - - - - - - . . - - - - - -- - - , . , - - . - - - -. .

SDS-PAGE, and transl'crred to nitroccllulosc. The blots wcrc prcibcd f'irst witli unti-

phosphotyrcisinc, and lhcn strippcd and re-probcd iising antibodies specific for the midcculc 01'

inicrcst. To quantitate the degree of' phosphorylation, densitometric scanning of

autoradiographs was performed and the results werc normalized by dividing thc

phosphotyrosinc signal of a particular band by the amount of protein in that same band. This

ratio was designated as 1.0 in parental 3T7 cells.

A sljght variation on the immunoprecipitation procedure was used 10 asscss thc

phosphorylation status of TCRT. Mild lysis conditions (digitonin), which presewe TCRICD3

associations were used, and TCRq was immunoprecipitated indircctly by using antibody

specific for TCRp. Thus, TCRp-associated 5 was measured, as oppscd to the total ccllular

pool ol' TCRT. A s can been seen in Fig. 9A, TCR-associated < chain is 3.5 timcs morc

phosphorylated in parental 3T7 cells than in 3T7.CD45 CI. 1. Thus, an inverse relülionship

csists between expression of CD45 and TCRS phosphorylation, suggesting that CD45 can

regulate, either directly or indirectly, the phosphorylation state of TCRS. This is in agrccment

with data from other groups who have found hypcrphosphorylation of TCRC in T-cell Iines

deficient i n CD45 (Volarevic et al., 1992; Niklinska et al., 19%). In contrast, no apprcciablc

differencc was observed in the overall phosphorylation state of either Lck or Fyn in parental

3T7 cclls versus 3T7.CD45 CI. 1 (Fig. 9B, C).

1) Analysis of thymocytes from ~ ~ 4 5 - 1 - mice

Whilc lhe results from the ce11 line syslem off'cr a uscful, inanipulablc tool 101- asking

questions iibout CD45 l'unction and rcgulation, il was impoi-lant 10 coi-1-clak and cossoborak ttic

rcsulls in a niore physiologic setling. Thcrcforc, other members of thc laboratoi-y analyzcd

TCR signaling in DP Ihymocytes [rom CD45 eson 6-1- niicc (Smilcy P., Grovcs T. and Guidos

C., unpublished rcsults), provided by Josel' Pcnningcr (Kishihara ct al., 1903). Thc i-csulrs

closcly parallclcd thosc rcporlcd in 3T7 cells, i n that thyniocytcs froni C~4.5-/- inicc rcspciridcd

I';ireril;~l 3T7 31'7.C'I)JS CI.] II' II' - - II' - Ip ,

"

Illi~ltinp Aiilibiid? :

.Iti111 rr (p-tjrl

-C * - .;

.

înliscrï Lc k

r e 4- Lch

45

Figure 9: Tyrosine phosphorylation index of potential CD45 substrates in 3T7 cells.

Phosphorylation status of TCRC (Fig. 9A), Lck (Fig. 9B), and Fyn (Fig. 9C) in parental 3T7 cells and 3T7.CD45 C1.l. Cells were

lysecl in LB, except in Fig. 9A in which cells were lysed in digitonin LB. Lysates from 5s106 cells were immunoprecipitated with the

indicated antibody: anti-TCRp (H57-597), anti-Lck, or anti-Fyn, coupled to CNBr sepharose beads. Control IPs were performed

identically using inactivated CNSr beads. Proteins were separated by 12.5% (Fig. 9A) or 10.5% (Fig. 9B, C) SDS-PAGE (non-

reducing), and analyzed by sequential 4G10 and TCRS, Lck or Fyn immunoblotting. Densitometnc analysis was performed, and the

rcsults wcre normalizcd by taking the ratio of p-tyr signal : TCRC, Lck, or Fyn signal and assigning the value from parental 3T7 cclls as

1 .o.

I - - - - - J - - U

receptors were CO-engaged, both in tcrms of induction of protcin tyrosine phosphorylation and

CD51CD69 induction (Smilcy, Grovcs and Guidos, unpublished rcsults). To asscss thc

biochcmical basis of the impaircd TCR signaling obsenred in DP thymocytes from the ~ ~ 4 5 - I -

mouse, 1 analyzed the phosphorylation state of various CD45 substrates as described Sor 3T7

cclls. No differences in the overall phosphoryIation state of Lck or Fyn werc obscn~ed (dala not

shown). However, TCRp-associated TCR 5 was round to be 5 times less phosphorylated in

~ ~ 4 5 - 1 - thymocytes when compared to ~ ~ 4 5 + / - littermate controls (Fig. 10). Additionally, a

protcin of approximately 40 kDa (*) was hyperphosphorylated in ~ ~ 4 5 - 1 - thymocytcs whcn

compared to cD45+jb littermate controls (Fig. 10). Thus, CD45 acts to regulaic lhc

phosphorylation state of several phosphoproteins eithcr dircctly or indirectly. The profound

hypophosphorylation of TCRI; i n ~~45-1- thyrnocytcs is contrary to thc rcsults oblaincd in

CD45- 3T7 cells, where a hyperphosphorylation of TCRS was obscrvcd (Fig. 9A). The bwis of

this discrepancy remains to bc detcrmined, but müy rcflect differcnccs belwecn studying

Lransformcd culturcd cells grown i r i vilru, versus fresh ex vivo thymocytcs. Nonethclcss, ihc

disrcgulation of TCRt phosphorylation that accompanies loss of CD45 provides a biochcmicnl

corrclate with the TCR signaling defecis observed.

Figure 10: Analysis of TCRS tyrosine phosphorylation in ~ ~ 4 5 - / - thyniocytes.

Fresliy isolated thyniocytes froni ~ ~ 4 5 - 1 - and CD&+/- littermatecontrols (4 months of

age) were lyed in digitonin LB and ir~~m~iiioprecipitated (7x 106 cells/lrim) as describcd

in Fig. 9. Astcriiik (":) indiciites phosphoprotein whicli is difrercniiully phospl-iorylatecl

iii CD4S-1- vetsus CD45+/- thyniocyies

CHAPTER 4

DISCUSSION

L

thc well-documcntcd inübility to signal through the TCR i n CD4S-deficient cell lincs, thc

immature DPcell line, 3T7, described here is also TCR non-responsive. 1 have shown that the

re-expression of endogenous CD45 or an exogenous CD45 construct largcly rcstorcs TCR

signaling capabilities in 3T7 cells. CD45 was found to bc necessary not only for thc TCR-

triggcred protein tyrosine phosphorylation, but also for several other TCR-induced downstrcarn

events, including CD5 upregulation, RAG-1, and CD4lCD8 downregulation. This reprcscnts thc

first demonstration of CD45 regulating these specific developmental events, known 10 occur

during positive selection in vivo. Intriguingly, 3T7 cells could respond when TCR and co-

rcceplor were deliberately co-aggregated, an effect presumably mediated by the co-rcceptar

associated kinase, Lck. This CD45independent signaling pathway has also bccn demonstrütcd in

DP thymocytes [rom ~ ~ 4 5 - 1 - micc (Smiley P., Groves T., and Guidos C., unpublished results).

Thus, 1 propose that the partial developmental block observed irr vivo in ~ ~ 4 5 - 1 - rnice can bc

rationalized by the existence of CD45-i ndependen t but CD4lCD8-dependent TCR signaling,

allowing some DP thymocytes to be positively selected. Finally, 1 demonstrated that lack of

CD45 in 3T7 cells results in hyperphosphorylation of TCRC chain. In constrast, the lack of

CD45 in thyrnocytcs from the ~ ~ 4 5 - / - micc resulls in a profound hypophosphorylation of TCRS

chain. Thus, the disrcgulation of TCRC phosphorylation that accompanies the loss of CD45

providcs a biochcmical correlate of the profound TCR signaling deficits obscrved.

A ) CD45-Dependent versus CD45-Independent TCR Signaling Pathways

This analysis of TCR signal transduction i n a CD45-dcficicnt DP thymoma, 3T7,

supports a critical rolc for CD45 in coupling the TCR to the intraccllulas signaling machincry.

Howcvcr, Lhc icquirement for CD45 is not absolute, in that both proximal (PTK activation, Fig.

3A) and distal (phcnotypic maturation, Fig. 2B; RAG-1 downrcgulation, Fig. 8C) outcomcs ol'

TCR ligation can be induced by the co-aggrcgation of TCR with cithcr co-rcccptor, CD4 or CDS.

Similar findings havc bccn reportcd by two groups (Dcans ct al., 1992; Shiroo ct al., 1993). I n

onc study, a subclone 01 the human L)Y leukemic Cell Iine HYB.ALL was isolaled thal IücKcd

surl'ace expression of CD45 (possessing a ~ranslationallpost-translational defeçt). In agrcemeni

with my findings, thcy found that while CD3 ligation alone failed to inducc protcin tyrosine

phosphorylation, calcium influs, or PLC-y 1 activation, CO-ligation of CD3 with CD4 was ablc lo

induce thesc cvents (Deans et al., 1992). This was correlated with increased activation of CD4

associated Lck i n CD45- cells (Deans et al., 1992). In another study, an indepcndently dcrivcd

CD45-negative (possessing a CD45 transcriptional/post-transcriptional defect) subclone of

HPB.ALL was isolated and compared to a CD45RAB transfectant. Similar iïndings wcrc

reported, in that CD3 ligation in the CD45 cells failed to induce protein tyrosine phosphorylation,

inositol phosphate production, calcium influx and PKC activation, while CO-aggregation of CD3

with CD4 or CD8 restored these events (Shiroo et al., 1992). The lack of ability to signal when

TCR was ligatcd alone was correlated with lower basal Fyn kinase activities in CD4S- cclls,

whereas Lck kinase activies were comparable in CD45 versus CD45f cells (Shiroo et al., 1997).

Howcver in one report this signaling phenotype was not observed (Biffen cl al., 1994).

Invcstigators found that CD45- cclls (isolated from a human DP T-ce11 linc callcd CB 1) lailcd tri

inducc protcin tyrosinc phosphorylation and calcium influx in rcsponse to CD3 as wcll as CD3 s

CD41CD8 stimulation (Biffen et al., 1994). The reason for this contradictory result remains

undefined, but may relate to differences in the activationldifferentiative state of the various T ccll

lincs utilizcd.

A potential cavent of the restored TCR signaling observed when TCR and co-reccptors

wcre CO-ligated is the ability of 3T7 cells to inducibly re-express endogenous CD45 (Fig. 5A, B).

I I coiild bc argucd that the rescue in TCR signaling observed was simply due to the rc-esprcssion

of CD45 Two lines of cvidcnce however argue against this. First, thc CD45 rc-cxpi'cssion as

dctcçtcd by FACS occurs after 24-48 hours. Yct, the ability to inducc an carly responsc

(phosphotyrosinc induction) is dctectcd alkr 1 minute of TCRICD4 aggregation. Thus, i l would

bc unlikely that CD45 could bc signiricanlly re-cxpressed. Second, sludics of CD45-'-

thymocytcs, which do no1 inducibly rc-cxprcss CD45 (Smilcy P., Grovcs T., and Guidos C.,

unpublishcd results), show similar funclional rcsponscs lo TCRIco-rcccptor stimulation . Thus,

- - -". , - - - - . . - - - - - - - - - - - - - -- -~ -~ - . - ..- - - - - . - r - - " . . - - .- , L' - - - - - U - ~ - - -~ 7 - -.-

rathcr is truly CD45-independent. If this is lhe casc, it suggests that the CO-receptor assnciatcd

Lck can be activated in thc abscnce of CD45, whilc thc TCR-associated F'TKs (ie. Fyn) can not.

Notably, whcn cithcr CO-rcceptor was ligated without TCR, liltlc or no signaling was obscrvcd

(Fig. ?A, B). This suggests that elements in the TCR comples, namely the CD3 and TCRC

chains, are rcquired for the entire signal transduction pathway to occur, perhaps by providing a

physical structure ont0 which other molecules can be recruited, such as Zap-70.

Several potcntial mechanisms could explain why TCR signaling absolutely requircs

CD45 while TCRko-receptor signaling does not. One possible esplanation is ihat co-receptor

associated Lck can be activated without C-terminal tyrosine dephosphorylation, in effcct

bypassing the requirement for CD45 I t is interesthg to note that Moarefi et al. reccntly

dcmonstratcd that addition of a SH3 ligand stimulated the activity of purified Hck (a Src-rclated

PTK) that is phosphorylated at Tyr 527 (Moarefi et al., 1997). These results suggest that whilc

dephosphorylation of C-terminal tyrosine may be a key regulatory switch in Src-làmily kinasc

activation, cornpetition for binding of Src-family kinase SH3 domains by cxogcnous ligands may

also result in kinasc activation by releasing inhibitory intramolecular conformations (sec later).

Thus, it is possiblc thal C-terminal phosphorylated Lck andfor Fyn in 3T7 cells are nonetheless

aclivated when TCR and CO-receptor are CO-aggregated duc to thc presence of other proteins

whicli rclicvc thcir inhibitory conformation. Onc molecule that may act in this capacity is Syk,

which has been shown to bc constitutively bound to the TCRICD3 complcx and bccome activatcd

in a Lck-independent and CD45-indcpendent mannes (Couture e l al., 1994; Pao and Cambicr,

1997), although this remains contentious. I t has bcen propsed ihat tyrosine phosphoiylatcd Syk

may mcdiate interactions with Lck SH2 domains and lhus recruit Lck io ihc TCRICD3 complcs

(Thomc cl al., 1995). Thus, i t is possible that Syk can become autophosphorylatcd in 3T7 cclls,

allowing i t to compcte for Lck SHZbinding when TCRlco-reccptor are co-aggrcgatcd. This

would rclease Lck Srom its inlramolccular inhibition and allow efficicnt signal amplificalion 10

occur. Intercstingly, Syk is cxpsesscd highly in immaturc DP thymocytcs bcforc bcing down-

L , . W" .. cclls.

Thc diffcrcntial requirement for CD45 in TCR CO-reccptor indcpendcnt signaling vcrsus

TCWco-receptor signaling could then be explained by quantitative differenccs in the signaling

capacity of thc two stimulatory conditions. Only 1-3% of cellular Fyn is thoughl to bc associalcd

with TCRICD3 compIes (Samelson et al., 1990), while greater than 50% of cclluiar Lck

associates with the CO-receptors, CD4 and CD8, in DP thymocytes (Wiest et al., 1993). Perhaps

CD45 is a prerequisite when TCR is ligated alone, needed for the efficient initial activation of

srnall amounts of Fyn. In contrast, when TCR and CO-rcccptor are CO-ligated, CD45 becomes

dispcnsablc, owing 10 the higher arnaunts of Lck recruited to the signaling cornplex and becoming

activated by the mechanism proposed above. The observation that TCRICD4 co-aggregation gave

consislcntly stronger signals than TCRICD8 CO-aggrcgation (Fig. ?A, B), an effect that direclly

correlated with the greater association of Lck with CD4 than CD8 in 3T7 cells (Fig. 3A), lcnds

support to this quantitative argument.

A second explanation for the differential requirement for CD45 in CO-receptor-dependent

ircrsus co-rcceptor-independent TCR signaling could involve qualitative diffcrcnccs in ~ h c

aclivation requirements of CO-receptor associated PTKs versus TCR-associatcd PTKs. For

cxamplc, thc CO-receplor associated PTK activity cauld bc "prc-activated" perhaps by being

inücccssiblc to Csk, which itself is predarninantly localized in thc cytoplasm (Nada et al., 1991;

Okada et al., 1991; Bergman et al., 1992). In this scenario, the CO-receptor-bound Lck would not

be cngaged in an intramolccular C-tcrminal phosphotyrosinelSH2 inkraction bccausc thc C-

Lcrminal tyrosine is not phosphorylated, and could thus be aclivatcd upon TCRICD4 co-

aggrcgation. Intriguingly, a inuhnt rncinbrane-targeted form of' Csk more slrongly inhi bilcd TCR

signaling than the WT cytoplasmic form (Chow et al., 1993), suggcsling that thc ccllular

localimtions of thc molcculcs can have a critical influcncc on lhcir activiiy. Also, 1 can not rulc

out thc possibility that olhcr unidenlilïed PTKs, not rcquiring CD45 for their activation, could

bccomc aclivatcd whcn TCiUCD4 arc co-aggrcgatcd.

- --.-.-- ,, -~ - - , - ~ - - - - -

lcast one study, thc dcfect in TCR signaling caused by lack 01' CD45 \vas partially conipcnsatcd

by csprcssing a hetcrologous PTPase from yeast (Mntto et al.. 1994). suggesting that othcr

PTPascs can indced perform somc of CD45 functions. With the identification of ovcr 75

diffcrcnt receptor and cytoplasmic PTPases (Tonks and Neel, 1996), many of which arc

cspressed in T cells, the possibility of overlapping funclion becomcs more and morc likcly. In

support of this contention, treatment of 3T7 cells wi th the FïPase inhibitor penlanadate lcads to

an accumulation of tyrosine phosphoproteins and CD5 upregulation in a dose-dependcnt Sashion

(Fig. 4A, B). This indicates that other PTPases are expressed in 3T7 cclls, acting to maintain thc

ovcrall phosphotyrosine homcostasis in the cell. When this balance is perturbcd, TCR signal

transduction pathways can become activated without dclibente aggregalion of lhc TCR.

B ) Molecular Targets of CD45 in TCR Signal Transduction

In this study, no significant differences were observed in the overall tyrosinc

phosphorylation statc of Lck or Fyn in 3T7 cells compared to thc 3T7.CD45 inkctants (Fig. 9B,

C). These were unexpected findings, considering several studies have Sound a corrclation

bctwcen a lack of CD45 expression and a hyperphosphorylation ol' thc C-terminal tyrosinc of total

ccllular LcklFyn (Ostergaard et al., 1989; McFarland ct al., 1993; Sich cl al., 1993). Thc

rcsul tant dccrcase in kinase activi ty providcd an allraclivc mcchanism for thc impaircd TCR

signaling obscrvcd in CD45-deficient cells. However, in my study neither LckIFyn kinasc

activily nor their C-terminal phosphorylalion shlus wcrc assessed. Givcn that Src-l'amily kinascs

conlain thrce potcntial rcgulatory tyrosine sites, i t is entircly possible that whilc thc ovcrall

phosphoiylation slatc of Lck and Fyn may not bc diffcrcnt in lhc prcscncc or absencc of CD45,

thc sites of phosphorylation may be diffcrcnt, cither qualitativcly or quantitativcly. Thus, a

disrcgulation of LcklFyn may still exist in 3T7 cells. Thereforc, thc asscssmcnt of LcklFyn

kinasc activitics in 3T7 cclls vcrsus 3T7.CD45 cells will clarify this mattcr. A n altcrnatc

hypothcsis is Lhat CD45 may no1 bc invalvcd i n rcgulating thc basal LcUFyn kinasc activitics, but

ra~ncr may cscrr ils Iuncuon concurrcnr. w1r.n or jusr ai Lcr r LK iigarion. 1 t may wcii oc inc Daiance

of inhibitory vcrsus stimulatory activities of CD45 that determincs its net cffcct during TCR

signaling. Nonethelcss, thc results prcsentcd here clearly indicatc that the funciion OS CD45 i n

3T7 cclls is more complex: than simply kecping Lck/Fyn in an "on" conf~guration. This is

dcmonstratcd by the finding that TCRlco-receptor c m signal independent of CD45, impl ying that

thc activation of CO-receptor Lck may occur indepndently of C-terminal dephosphorylation.

Thc comples rolc of CD45 in regulating Lck/Fyn is dernonstrated by scvcral studics

whose results wcre incompafible wi th the mode1 of CD45 aclivating LcklFyn simply by

dcphosphorylation of C-terminal tyrosine. Two studies reportcd that thc kinase activity of

LcWFyn \vas higher in CD45 cells than in CD45+ cells, despite the hyperphosphorylation of thc

C-terminal tyrosinc (Dcans et al., 1992; Burns et al., 1994). Also, some investigators have

suggested a negative regdatory role Tor CD45 For example, the CO-ligation of CD45 with the

TCR cornplex suppressed T ce11 activation (Turka et al., 1997), while the co-ligation of CD45

with CD4 inhibi ted the anti-CD4-induced phosphorylation of Lck and the concomitant incrcasc in

Lck kinase activi ty (Ostergaard and Trowbridge, 1990). These antibody-mediated ncgatik~c

cllCcts may bc duc to the inappropriatc antibody-rncdiatcd coaggrcgation of CD45 with thc

TCRICD3 complcx, lcadi ng to an increased PTPase activi ty which cCfcctivcly prcvcn ts thc

accumulation of tyrosine phosphoproteins. Alternatively, ihc inappropriatc dcphosphorylation of

LcHFyn auto-phosphorylation site, which is rcquired for activation, could occur.

T hc considerablc con troversy regardi ng the ef f ec ts of CD45 dc fiçicncy on LçklFy n

rcgulation may bc cxplained by the complex activation mcchanisms of Src-family kinases. Thc

rcccnt resolution of the crystal struciurcs of c-Src and another Src-family PTK, Hck, havc

providcd insights into thc activation requirements of Src-family PTKs (Siclicri ct al., 1997; Xu ct

al., 1997). I n thesc studics, both the SH2 and SH3 domains arc involvcd in intrimolccular

iiitcractions rcsulting in conformational constraints on thc kinase active site (Sichcri ct al., 1997;

Xu et al., 1997). Moiarcfi et nl. rccently proposcd a hypothetical modcl of Hck activation i n

which SH3 doinain displacement, SH3 domain displaccmcnt, autophosphorylation and C-

~erininal tyrosinc dcphosphorylation may al1 activatc Hck io difl'cring dcgrccs (MoarclÏ ct al.,

. , , . ,. ' """, ...- . ' ...--....i "' "'- '-".."., '., D-.- "" "'., "' " .- ". t! ", ", """

rclalc to thc complcment of othcr molcculcs wiLh SH2 and SH3 ligands (incluciing other PTPascs,

PTKs and "adapter" moleciilcs) c~pressed in the varioiis T cc11 lines, which mriy var); clcpcnding

on the particular developmental and differentiative state of the ccll being studied.

In contrat to the lack of difference in the overall tyrosine phosphorylation of LckIFyn, 1

obscrvcd a hyperphosphorylation of TCRS in 3T7 cells (Fig. 9A). Similar Sindings wcrc rcportcd

by lrvo other groups studying CD45-deficient ce11 lines (Volarevic ct al., 1993; Niklinska ct al.,

1994). The hyperphosphorylation of TCRC is somewhat surprising considering that Lck and

Fyn, which are thoughl to be rcsponsible for TCRI; phosphorylation, would be cspectcd to bc

inactivc in 3T7 cclls. Hencc, TCRS should bc largcly unphosphorylatcd in the absencc OS CD45

I n Sacl lhis is prcciscly what was observed in thymocytes from C D 4 9 mice, in which TCRC was

hypophosphosphorylatcd (Fig. 10). Several potentiül cxplanations cxist for this parados. Thc

phosphorylation state of TCRg depends on multiple factors, a number of which may difl'cr

bctween 3T7 cells versus fresh ex vivo thymocytes. First, the constitutive CD4 engagement on

DP thymocytes by MHC II on thymic stroma in situ has been shown tu result i n thc hypcr-

phosphorylation of TCRI; (Nakayama et al., 1990). 3T7 cells cul turedin suspension in thc

abscnce of MHC II+ cells obviously lack this interaction. Second, 3T7 cells k i n g a transforincd

cc11 line, may possess other defects which could affect TCRI; phosphorylation. For esamplc,

many transformed ce11 lines possess high PTK activity, and this could resull i n a

hypcrphosphorylation of TCRI;. The decrease in TCRC phosphorylation thal accompanics CD45

rc-expression in 3T7 cells could thcn bc explained as a dircct cffccl of CD45 which has bccn

shown to dephosphorylatc TCRC (Furukawa ct al., 1994).

C) Role of CD45 in Positive Selection of DP Thyniocytes

Thc data prcscntcd in this thcsis support a critical rolc for CD45 in rcgulaling TCR sigiials

rit thc DP stagc of dcvclopmcnt. The importance of this molcculc is cvidcnt in thc dcvclopmcnlal

phcnolypc of ~ ~ 4 5 - 1 - micc, in ivhich thymocytc dcvclopmcnl is scvcrely impaircd in thc DP to

3r L I ~ I I S I L I C I I I (h1srlinürü el ai., 1 rr3; ~y ln c i ai., I Y Y ~ ) . r el, givcn Lnc cviacncc cnar ~ ~ 4 3 -

indcpcndcnt TCR signaling can occur when TCR and co-rcceptor arc co-ligatcd, thc scvcrc

devclopmcntal block is sornewhat surprising. Physiologically, i t has bcen proposcd lhat mosl T

cells are activated only when TCR and CO-receptor are brought in10 close prosimity via

recognition of the same MHC molecule (Weiss and Littman, 1994). I f this TCRIco-rcccptor

signaling is CD45-independent, then one might expect that most DP thymocytes could gcneraic

the necessary signals in the absence of CD45 to be positively selected. Howcver, thc TCR

signals involved in positive selection are likely more subtle than the artil'ical stimulatory

conditions used in signaling assays, where antibodies are used to crosslink surface moleculcs in

saturating amounts. Thus, while many of the outcomcs of TCR ligation can be induced in the

absence of CD45 iti vitro by driving TCR signals using strong stimuli, i i ~ vivo such

ovcrwhclming stimulatory conditions are unlikely to exist. In fact, cven in the presence of ihesc

artifical stimulatory conditions, most, but not al1 DP thymocytes from ~ ~ 4 5 - 1 - mice induced CD5

upon TCRIco-receptor crosslinking (Smiley P., Groves T., and Guidos C., unpublishcd rcsults),

suggesting that some cells are unable to bypass the requirement for CD45 Similarly, not al1 3T7

cells undergo CD5 induction Iollowing TCRlco-receptor crosslinking (Fig. 2B). I t semains

unclcar whether this reflects secondary defects in a subset of cells or a modulation in the signaling

characteristics of somc cclls, rendering them non-responsive.

In 3T7 cells, 1 consistenily found the CD4 CO-receptor possessed a grcatcr ability to signal

than thc CD8 CO-rcceplor when CO-aggregated with TCR (Fig. 2A, B, 5A,B). Thc diffcrcntial

signaling capacity of the two CO-reccplors correlated with thcir diffcrcntial association wilh Lck

(Fig. 3A). This resull prcdicls thai CD4+ T cells should bc morc cfficicntly positivcly sclcçicd

than CD8+ 'ï cclls in CD45-1- mice, owing to the strongcr signals dclivcrcd by thc CD4 co-

reccptor. This is indeed the case, as the ralio of CD4' to CD8f T cclls in the periphcry of CD45

cson A-/- micc was skewed forn 3: I in WT animaIs to 4: 1 in CD45 cxon 6-1- rnicc (Kishihara ci

al., 19931, suggesting a more profound impairment in ihc dcvclopmcnt of thc CD8 lincagc ihan

CD4 lincagc in thc absencc of CD45. In contrast, CD45 cxon 9'- micc appcaicd to have a nioic

scvcre impairn~ent in thc devclopment of CD4+ than CDS+ SP T cclls, both in thc thymus and in

1 1 d \ , ~ ~ , ~ ~, - , -

- - - - - - - ~

C - - - - - C ~ l ~-

the CD8+ SP thyrnocytes cells were clearly C D ~ ~ ~ and thus likely immature. I t is intcresting lo

notc that in both CD45 eson 6-1- and exon 9-1- knockout mice, there is ri marked iiprquloticm of

CD4 and CD8 CO-receptor molecules on DP thymocytes (Kishihara et al., 1993; Bytli cl al.,

1996). Considcring iny observation that TCRko-receptor signaling is largely CD45-indepcndcnt,

i t could be inferred that upregulation of CD4/CD8 expression on DP thymocytes increases thc

ability of DP thymocytes to generate TCR signals. Furthemore, CD5, a negative regulator o f

TCR signals (Tarakhovsky et al., 1995), was shown to be downregulüted on DP thymocylcs

l'rom CD45 exon 6-/- rnice (Smiley P., Groves T., and Guidos C., unpublished results).

Collectively, the downregulation of CD5 and upregulation of CO-receptors may thus rcprcsent

adaptive responses made to overcome the CD45 deficiency, increasing the likelihood that DP

thymocytes will be positive1 y selected.

The lack of expression of CD45 in 3T7 cells represents an extremc cxamplc of the

consequences of CD45 expression on TCR signaling in DP thymocytes. Howcver, under

physiologie circumstances more subtle adaptations are likely employed to modulüte thc signaling

charactcrislics of thc ccll. Mcchanisticall y, this could involvc changcs in thc surfacc cxprcssion

levcl of CD45 The recent finding that CD45 is upregulatcd during positivc seleciion

concomitantly with the TCRICD3 complex supports the idea that the thymocyte adapts CD45

lcvels to incrcasing antigcn receptor levels during development (Ong et al., 1994; Kirbcrg and

Brockcr, 1996). This increüsc in CD45 expression would be espectcd to lowcr thc signaling

thrcshold in thc maturing thymocyte, making i t more competent to generate TCR signals.

Altcrnativcly, changcs in CD45 isoform expression could altcr the signaling characteristics 01' the

cc11 (Novak ct al., 1994), possibl y by intcracting wilh distinct ligands andlor substratcs (Dimmni

ct al., 1992; Leitenberg et al ., 1996). Intcrcstingly, studies of TCR transgcnic micc on sclccling

backgrounds showcd that CD45RA and ~ ~ 4 5 R B t " d ' isofornls specifically werc uprcgulatcd

during positivc and ncgalive selection (Wallace et al,, 1992), suggesting that isolOrm patterns do

indccd change during thymic sclection cvents. Furthcrmorc, thymocytcs fi-on1 CD45R1113C

lrünsgenic micc rcspondcd morc robustly toTCR stimulalion ihan did thymocytcs froni CD45RO

""""b'""' """" ,""' -. W.' " " 9 , ' * a'-"- .-"-"" "- ""' -.. ' -.-"' ..," "'...." ," ,& " '.+,,

difl'cr in Lhcir abilitics Lo participalc in TCR signaling, and thus allcr thc signaling chanictcrislics

of dcveloping thymocy tes.

ln summary, this study has estended our undcrstanding of thc rolc of CD45 in TCR

signaling i n DP thymocytes. My results suggest that TCR signals can bc gcncratcd in a CD45

independent manner, providing a rational basis for understanding the devclopmentai phcnotype 01'

CD45-'- mice.

D) Future Studies

Tlic results described herc indicate that TCRICD4 and TCRICD8 signaling in DP

thymocyics is largcly CD45independent. 1 hypothesize that this signaling is inilatcd via co-

rcccptor associatcd Lck. To formally dcmonstrate that Lhis is the case, the following expcrimcnt

could be performed. Using the CD4 lineage as an example, I would gcncratc CD~~- / - ;CM- ; -

double-deficient mice and then reconstitute them with a tmncated form of CD4 that can no longcr

associate with Lck. If the ability to generate CD4f SP in CD4.5-1- micc is via the postulatcd

mcchanism, then in CD4w~t&CD45-~-; CD4-I- mice, no CD4+ SP should be observcd, as thc

ncccssary signal can ncit bc transduccd.

Toaltempt 10 further dctïnc signaling pathways, lhe cffccts of ovcrcsprcssicin 01' acli\'atcd

forms of molcculcs lhought 10 bc rcgulatcd by CD45 could bc assesscd. Thc complcmcntalion (il'

CD45 defccts by such gain-of-function molecules would provide gcnclic evidcncc of a signaling

pathway. Such an approach might involvc csprcssing LckF505 andlos FynT528 transgcncs o n

thc ~ ~ 4 5 - 1 - background, Lo scc if thymocytc dcvclopmcnt rcvcrts to wild-typc. This approach

has been attcinptcd In the 3T7 cclls, whcrc LckF505 or FynT528 wcrc ovcrcxprcsscd, and

assesscd for a rescuc in TCR signaling. Whilc a partial rcstoration of TCR function was

somclimcs observed in FynT528 ovcrespressors, the resulls wcrc no1 rcproduciblc (data not

shown), suggcsting that othcr defects cxist in 3T7 cclls or that CD45 dcficicncy can not bc Sully

complcincntcd by constitulivcly activc Fyn. To rcsalvc somc OS Lhc contradiclory rcsults ivith

--Q-..- .- - - - - - -.--r--.-,--.-. - - - , - . . . -. . - - - - - - - - - - - - . - - - - -- - - . . -. - - - - . -. . . - . . - - - - . . .-J --.- -

tcrminal tyrosine phosphorylation status of Lck and Fyn in 3T7 cclls versus CD45 inlcctants.

A h , atternpts could be made to mimic itt sitic effects by ligaiinç CD4 on 3T7 cclls irr vitro, and

asscssing if' this restored the TCRS phosphorylation patterns observed in thymocytcs from CD45-

1- micc. Finally, thc expression and activity of Syk i n 3T7 cells should be assessed as a first stcp

in determining if this PTK could be responsible for the CD45-independent signaling obscrvcd.

The defect in CD45 gcne expression in 3T7 ceIIs appears to bc ai the levcl of transcription.

This could involve a defcct in trnns, i n which resting 3T7 cells lack expression of a positive-

acting transcription factor, or the defect couId be in cis, in which mutation(s) in thc regulatory

sequcnces of the CD45 locus make it non-transcribablc. Allcrnritivcly, svabilizing factorslsplicing

factors neccssary to maintain steady statc message of CD45 could be missing in resting 3T7 cclls,

but induced in stimulated cells. The inducible re-expression of CD45 in 3T7 cells suggcsts that

differences exist i n CD45 expression betwccn resling stale cclls and activated cells. Somc

possible future experiments to address the defect in CD45 expression in 3T7 cells might includc

ceIl-cell fusion procedures, to see if the defect in CD45 espression can be cornplemcntcd. Thc

sclcction of propcr fusion partncrs is critical; idcally i t would bc a cc11 which csprcsscs al1 thc

transcription factors necessary for CD45 expression, but itself has a disrupted CD45 gcne. I f

aftcr such a fusion, CD45 was re-expressed, then this would indicatc a dcfect in a transcriptional

factor in 3T7 cells In summary, the cxperimcnts outlined i n this section would estcnd our

undcrstanding of how CD45 acts 10 regulate TCR signal tranduction, and furtlicr our

understanding of how TCR signals control T cc11 dcvclopmcnt.

CHAPTER 5

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