FASCIN EXPRESSION AND FUNCTION IN MOUSE DENDRITIC CELLS

Monther bi- ru-r'wan

A thesis submitted in pattïal hl fament of the requirements for the degree of

Mas ter O K Science

Dalhousie Cniversitv

O Copyright by Monther M. AL-iU\vm, 1999

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TABLE OF CONTENTS

....................................................................................... Table of Contents

List of Figures ............................................................................................

Abstract ......................................................................................................

Introduction ................................................... ...................................

1.1 Dendritic Ceils ..................................................................................... MvcIoid Derived IDC ...................................................................

Origin and Maturation O f DC ...................................................... Chemokines and Chemokine Receptors on DC ........................ Cytokines and Cvtolcine Receptors on DC ................................. hiarkers of DC ...............................................................................

1.2 Fascin as a Potmuai Marker of DC blaturitv ...................................

Fascin E~pression in Humm DC ................................................

Objective of the Study ..................................................................

h. iaterids and Methods ..............................................................................

.............................................................................................. Ani mal s

............................................................................................. Rcagcnts .......................................................................................... An tibodies

Growth Factors .................................................................................

Epidermal S b Sheet Preparation ...................................................

IgG Coated-Petri Dish Preparation .................................................

2.8 Prepantion of DC from Bone Marrow . . . . . . . . . . . . .. . . . . . -. - .-. . .-. . . . - - -. . -. -. . . -

2.9 Immunostauimg .......................... .. ...... ...... ........................................ 3.10 Flow Crtometric Analvsis .... ... . . .. .... . ... . . . .... ...-.--...--. -.-.-..--... ....--........ 3.1 1 T CeIl Isolation .................................................................................. 3.1 2 kf~xed Lvmphocyte Reaction P L R ) . . . . . . . . . . . . . . . . . . . . . . -. . . - .-. -. . . . . . -. . . - -. - -. 3.13 Cultivation of Bone Marrow-Precursors with ht isense

Oligonucleo tides . . . . . . . . . . . .. . . . . . . . -. . -. . . . . . . . -. . . . . -. . . . -. -. . . .. . . . . -. . . . -. . - -. . . . - -. . . . . -. .

Rcsuits ..... . .. . . . . . . .... ..... . ............--.... ..-. ..-- .-.-.. .....-... . ..... .. . . . .-... .....-.. ... .... . .

3.1 Fascin Expression in Human DC ..................................... ... .............. 3.2 Characterization of Fascin in Mouse DC In vivo ............................

(a) Lvmph Node .................................................................................. (b) Epidermal S h Sheet . .. .. . ... ..................... ..... . .. ... .... . . . . . . . . . .. . . -. ... .. .. .

3.3 Characterization of Fascin in Mouse DC In vitro ............................

(a) Generation oFDC from Mouse Bone Marrow DC-precursors

(b) Immunocytochemical Esamination of Fascin Espression in

Bhf-DC ....... ............... ............ .... ,. .................................. .. .....-....-... .

(c) Esamination of Fascin and b H C class II Espression on BM-DC during Maturation ..........................................................................

(d) Double Color Flow Cvtometnc Analysis of BM-DC for Fascin

and hhEIC class II Coespression .................................................

(c) Investigation of the Correlation between Fascin and hEHC Class I I

Expression on BM-DC during Maturation ................................

(f) Esamination of B7-2 Expression on BM-DC du&g Maturation

Cg) Effect of TGF-B and TNF-a on Fascin and bEIC class II

Expression on BM-DC during Maturation ................................

3.3 Investigation of the Effect of Fascin Level on BM-DC - .

AlIo-s tirnulaton- Activity . . . . . . . . . . . . . . . . . . . . . . . -. . . . . . . . . . . . . . . . . . . ... -. . . . . . . . . . . . . . -. . . -. . (a) Cornparison of the No-stimulatory Activitv benveen Day 6- and

Day 9-DC ....................................... - ............................................... (13) The E ffect of Fascin Levcls on BiM-DC No-stimulatory r-cti\*ity

3.4 Investigation of the Rolc of Fascin in DC-T Cell Interactions .......

(a) Esamination of the Effect of Fascin Inhibition in bfahlre

................................. B M-DC on their Mo-stllnulaton- Activitv 77

(b) Confirmation of Fascin Inhibition in the BM-DC Treated with

An tisense Oligonucleo tides .......................................................... 81

Discussion .................................................................................................. 84

4.1 Fascin Expression in Human DC ...................................................... 85 1.2 Fascin Expression in Mouse DC ....................................................... 86

.............. 4.3 Fascin Espression in In vitro Generated Mouse BM-DC 88

4.4 Correlation between the De~elopmenr of Fascin and

hff-fC class I I Expression on BM-DC during Maturation .............. 76

............................. 4.5 H7-2 Espression on BM-DC during Maturation 1W 4.6 Fascin and bII-IC class II Expression on BM-DC in thc Presencc

O f TGF-P and T N F-a ........................................................................ 1.7 The Effcct of Fascin Level on BM-DC Allo-stimulatory hctivity .

4.8 Isolation of Fascin as a Mediator OF DC rUlo-stimulaton- Activity

................................................................. 3.5 Surnrnary m d Conclusions

LIST OF FIGURES

Page Number

Fi y re 1 .

Figure 2 .

Figure 3 .

Figure 4 .

Figurc 5 .

Figure 6 .

Flow diagnm of bone marrow DC-precursor cell isolation from mouse bone rnarrow ..............................................

T ce11 purity after enrichment through nyion wool CO 1 umn .....................................................................

Immunoperosidase esamination of human s h n for fascin

Fascin espression in mouse lymph nodes ..............................

Expression of DEC-205 in mouse lvmph nodes .......................

Expression of fascin and hEIC class II in mouse epidermal skui sheet ................................................................. 41

Figurc 7 . Dm-elopment of BM-DC from bone marrow DC-precursors in the presence of GM-CSF ............................................. 45

Figurc 9 . Fascin expression in dav 9 BM-DC ..................................... 49

I;igurc 10 . Esamination of dar 9 BM-DC for IDC ............................... 50

I*ïCgurc 1 1 . MHC class 11 expression on dav 9 BM-DC .......................... 51

Figure 1 2 Fascin expression in BM-DC during maturation .................... 54

. .............. Fikwrc 13 kfHC class II espression on DM-DC during maturation 57

Figurc 14 . Fascin expression in KEI-II cells ....................................... 62

Figure 15 . Examination of WEHI ceils for fascin expression using flow cytometry ................................................................

Figurc IG . Dar 9 BM-DC double stained for fascin and MHC class II .........

Figure 17 . Correlation between hscin and L W C class II expression ............

Figure 18 . B7 expression on dav 9 BM-DC .........................................

Figure 19 . B7-2 espression on BM-DC during maturation .......................

Fi y r c 20 . Influence of different growth factor combinations on Fascin expression dunng BM-DC maturation ................................

Figure 21 . Influence of different growth factor combinations on h M C class II ................................ csprcssion during BM-DC maturation 74

Figure 22 . Comparison of the ailostirnulatory acti\-itv between day 6 and da- 9 BM-DC .................................................................. 76

Figure 23 . Alostimulatorv actix-itv of da y 9 BM-DC culnired under differcnt ................................................................ conditions 78

I'igurc 24 . Al10 stimulaton- activitv of BM-DC after fascin inhibition ........... 80

FiLprc 25 . Fascin espression in BM-DC cultured in the presence of GM-CSF +control oligonucleotides or GM-CSF +antisense oligonucleotides ......................................................... 83

ABSTRACT

Dendritic ceil (DC) maturation is a criucal process diat results in their devclopmcnt into potent mtigen-presenting c d s (.-WC). In mice, however, the study of rliis proccss has been limited by the lack of a single ce11 marker that pemits differentiation of mature from immature DC. Current methods to assess the degree of DC mahirity are based cidier on thc level of expression of a panel o f markers indudiig .\MC dass II. costirnulaton- ruid adhesion molecules or on their do-stimulaton- activio- in a functiond assa.. ï h e currcnt studv evaluates a single cell protein, fascin, that k esprmsed only in rnature DC. l l ie hinCU0na.l role of fascïn in facilitaring DC-T ce11 interactions k also csamiricd.

~ l o u s c ll-mph node-sections, epidermal skin sheets, and bone marrow-derived L X (B.\[-DC) were esarnined for fascin expression by immunoq-tochcmistry. M'e found strong fascin expression in interdigitating DC (IDC) of the 1 pli node (mature), veq- fc\v Fascin-positive Langerilans cells in the epidermai skin shect (immature), and iiicrmzcd fascin cspression in BU-DC upon maturation. Hom- ntomemc analysis of BAI-DC rcvealcd an up-regdation of fascui expression dong witli .\MC dass 11 and B7- 2 on DC during m a ~ c i t i o n in the presence of GAI-CSF. Fascin and .\MC class II csprcssion werc decreased when DC maturation was suppressed by the addition of TGF-p to the culture and were increased wlien DC maturation \vas enhaiiccd by tlie addition TSF-a. In a mlred lymphoqte reacaon F E R ) assa?. increased allo- stimulaton activin. was found to correlate with increased numbers of fascin-positive DC. .-Il togetiicr, phenonpical and hnctional csnminîtions demons trated a strong corrclation benvîen fascin espression and DC maturih.

The diffcrcntial espression of fascïn in mature IDC (T ccil arnsj of the 1:mph riode suggcsted a role for fascin in antigen presentation. whicli is tlie hdlmark of maturc DC. \Y-c csamincd thc do-stimulatory activitv and morpholog)- of Bll-DC following fascin inhibition uoing antisense oligonudeotides. In an . \KR asra-, anasense oligonuclcotidc treated-DC were less potent in stimulating T ce11 proliferntion tlian the non-trmted- or control oligonucleotide treated-DC. Immunoq-toclicmical csamliation rcvcded ïnliibition of the level of fascin expression in the aritiscnec oligonucleotide trcatcd-DC. but no difference in numbers of fascin expressing cells. This stud!. c o r i h s tliat bscin cspresrion in DC is directlr involved in r c g u l a ~ g their do-stimulatory am\-ih-.

Tlii-: study links fascin expression with DC maturation and demonstratcc a direct iiirolvcmcnt of this protein in thc hnction of mature DC in antigen prcscntation. :\ bcttcr understanding of DC maturation and the mechanisms that regdate tliis process mat- lmd to tlierapeutic advances in infcctious diseases and cancer therapv.

ABBREVIATIONS

BM-DC

CCR

FCS

ht igen presenting ceil

Bone marrow-derived dendritic celi

Bovine serum albumin

CC chemobe receptor

Clus ter of di fferentiation

C!totosic T lymphocyte

Dendritic ce11

Deionized, purîfied uTater

Fetal calf serum

Fluorescein isothiocvmate

Granulocvte/macroph~e colonv stimulating factor

In tercellular adhesion molccule

Interdigitating dendritic ceIl

Immunoglobulïn

Interleukin

Interleuhiin receptor

Langerhans ce11

Leukonte hnctional antigen

h~lonocyte chernotac tic protein

S

hWIC

AlII'

MLR

mIw A

PBS

PEI

SCF

SDC

'1'D C

'I'G F

T h

'f N F

hlajor histocompatibility comples antigen

hhcrophage in flamrnatory pro tein

blised lvmphocvte reaction

hkssenger RNA

Phosphate buffered saline

Ph ycoerythrin

Stem cc11 factor

S tromd cell-derived factor

'hvmic dendritic ce11

Tram forming growth factor

T helper ceIl

Tumor necrosis factor

ACKNOWLEDGMENTS

1 would like to express my sincere thanks to mv supervisor Dr. Tirnothy

Lxc, my CO-supen-isor Dr. Kenneth \Test, as wen as mv cornmittee members Dr.

Gcoffrcy Rowden and Dr. David H o s h for their patience, guidance, support

and uschi discussion through this degrec.

Specid thanlis to: Dr. Geoffrey Rowden and Dr. Kenneth U e s t for their

assistmcc, endless humor and escellent ideas throughout this degree, and hls

I'atricia Colp for her technical assistance.

T h m k vou to al1 mv colleagues Li the Department of Microbiologu m d

Immunology parricularly those who work in the Transplant Lab for thcir

assistmcc and support

To mv mothcr and mv f a d y , thank !ou for the support that o u

pro\-idcd in imrncasurable wavs.

Finallv, 1 wodd like to thank the Saudi government, rcpresented bu thc

Saudi [irabian Cultural Mission in Ottawa, for thcir continuous efforts to

imprm-c and support education.

sii

I N T R O D U C T I O N

1 . Dendritic cells:

Dendritic cells (DC) are a O E bone marrow derived leukocb-tes

characrerized bu a highly irregdar shape with numerous cell membrane processes

(1). 'lhcr wi-ere first descrîbed, by Steinman and Cohn in 1972, from murine

splccn and called DC because of their chanctenstic morpholog- (2). I t took

immunologists a long tirne before the. appreciatcd the significant role that thcse

cclls plav in the immune system. Subsequent studies have shown chat DC are

u-idclr. distributed in the bodv esccpt in the brain, comea of the cyc and testes

(3,4). [ [ o n ~ w r , they are relatively rare cells accounting for less thm 1" O of the

total lcukocvte population in most tissues studied (5). The paucity of DC, dong

u-ith thc difficult) in the isolation of thesc cells, has hampered extensive studv of

thcir rolc 1n immune modulation.

it is likcly that thc role of DC in the immunc system has been ~ignificantl~

undcrcstimatcd. Thc isolation of thcsc cclls has bccn difficult due to thcir lou-

numbcrs and the absence of DC spccific markers. Furthcr, man. immunologists

h a w rcprded thc macrophage as thc major professional antigcn prescnting cclls

(AIX). i\lso, it has becn difficult to understand how thcsc rare cells, even though

csccllcnt ,WC, codd be involvcd in the induction of T-cell-mcdiirtcd immunin-.

1

2 This decade has witnessed tremendous progress in the study of DC and

their role in disease- This progress has come as a result of the success achie\-ed in

chc field of DC isolation and enrichment in v i ~ o and in vitro. Studies havc

shown that tissue DC are heterogeneous with regard to their orïgin, maturation

state and ratc of turnover (6). This heterogeneiw has been reflected in the

molccules they espress and the nature of h c t i o n s the- perform. Not

u-ithstanding their heterogcnein-, there is a generd agreement that DC are

deri\-cd from bone marrow. Howcver, their lineage derivation remains

controvcrsid. There is growing evidence that DC c m be subdk-idcd h t o

in\-cloid-derk-ed and lymphoid-derired populations. Thc bulk of the evidcnce for

the mycloid origins of DC cornes from in vitro studics in which DC ha\-c bccn

grown from bone marrow, cord blood progenitors, and pcripherd blood using

granuloc~-tc/macrophage colon- stimulating Factor (Ghl-CSF; 7,8,9). Thc

conccpt that DC are of lymphoid origin comes from studics on the th\-mus

u-here thci- have bcen found to esprcss markers norrnally associatcd u-ith

Ii-mphoid cclls, including CD8, CD2, BPI, and CD25 (10,ll). Furthemiore,

l\.mphoid-derived DC lack a rcquircment for GhI-CSF, an csscntial cvtohne

rcquired for the developmcnt of myeloid DC, to stimulate thcir proliferation and

diffcrcntiation (12,13).

3 DC differ not only in their limage deriration but also in the nature of

function that they perform. FoUicular dendritic cells (FDC), for esample, are DC

that are present in the germinal center of lymph nodes. The origin of thesc cells

is still controversial but thev are believed to be derived from a different lineage

than the T cell associated DC. Seved studies suggested that FDC are of non-

hcmatopoictic origin and are derk-ed from mesenchvmal reticular fibroblasts

( 1 , 1 5 6 ) . Hoarever, other snidies have suggested that FDC are derivcd from

hcmatopoictic progenitors (17). FDC play an esscntial rolc in maintaining the

- of the gemllnal center. Thev have been shown to directl\- contributc to

B cc11 prolifcration and to maturation into memon- cells through CD40 ligauon

(18,19,20).

Thvmic denditic cells CïDC) are mother DC tvpe that arc bclievcd to be

Jcrivcd from lvmphoid precursors. This conclusion \vas based on thc absence of

thvmic DC in Ikaros mutant micc, u-hich lack d l lrmphoid cclls (21). Thc

hnction of thesc cells is not to initiatc a T ce11 rcsponsc, likc intcrdigitating

Jcndritic cclls (IDC), or a B ce11 responsc, like FDC, but rather to inducc thc cc11

Jcath of anv of the developirig T cells n-hich arc potentiallv reactkc with self-

antigcns (2571). Thcr mm ovcr rapidly, as Fast as the developing T cc11

population, and this is more cornpatiblc \\-ith thcir h c t i o n in negatil-c selcction

4 Langerhans cells (LC) are the best known member of the DC familv. Thev

wçre first described bv Paul Langerhans in 1868, as cells of neuronal origin. In

1968, Breathnach rt Ir( d e d out the neuronal origin of LC (25). The

dcmonstntion of Birbeck granules in LC confirmed a migration of LC from s k

to affcrent lymphatics (26). Es vivo migration from the epiderrnis of skin

through the dcrmïs and into culture medium has also been demonstnted (27.

Migration of LC into the T ce11 areas of lvmph nodes, wherc they acquire the

characteristic appearance of 1 DC, has becn dcmonstratcd using radiolabeled LC

(28). Cpon in vitro culture, they mature into IDC-like cells (29). LC xrc ofttri

rcfcrred to as the "classical" immature DC. Their differcntiation has bccn

rcportcd to bc largel~r dependent ori the presence o f tram forming growth factor-

bcta CrGF-p). TGF-p-/- mice have lymphoid DC, but Iack LC (30).

Myeloid denved IDC:

Throughout this thesis, \i-c \ d l Cocus on studying thc mveloid-dcrivcd

IDC. l'hcsc cells arc prescnt in thc T cc11 arcas of lymph nodc u-here thei-

* . . scnsitizc and iniuate prima? imrnunc responscs. IDC arc no\\- rccognized as the

most potcnt profcssional APC involvcd in engagement of T hclpcr (.Th) ccUs

(-31). A numbcr of studics havc confirmed that mature IDC are the most active,

if not the only W C , that arc ablc to intcract with and sensitize naiw Ih cclls

(32,33). Although B cells and macrophages present antigen effectively to

scnsitized T cells in a seconda^ response, responses appear to require

thc presence of DC. Recent studies have shown that DC, in the second-

lymphoid organs, espress high level O f the C-C chemokine, DC-CKI , which has

bccn shou-n to preferentiallv attract naïve (CD45RA3 T cells (34). Furthermore,

csprcssion of MHC class II molecules is 10-100 timcs higher on DC than on

othcr :WC (35). Mature DC hm-e also been found to resist the supprcssiw

c ffccts of IL- 10 and sccrete high lei-els of IL-12, a potent cytokine that s h v s thc

immunc rcsponsc toward type 1 immunitv (36,37). Mature DC have also been

shown to espress high levels of the costirnulato~ moleculcs B7 and CD40 as

\\-cil as thc intercellular adhcsion molecule (ICrLbf)-1, ICAhI-3 and the leukocytc

Functional antigen &Fr\)-3. ru1 of these are required for efficient T-cc11

a&\-ation (38,39). Taken togcther, these propertïes suggest that DC might act as

a potcn tiai therapeutic \-cctors that have the capability to inducc strong immunc

rcsponscs against cancer o r infectious discases. Indeed, mm\- studics harc

dcmonstrated that DC pulsed in vitro with antigenic protein (40,41), peptidcs

(42,33), or nimor ce11 fragments (44,15) have the abdity to stimulate active cc11

mcdiatc immunin- (ChlI) and engender mti-tumor immunity. Furthermore,

microscopie infdtration of DC into Nmors has been shown to be associated with

rcduccd metastatic disease and prolonged sun-ival in humans (46,47,48,49).

Origin and maturation of DC:

The past few vears have witnessed an increase in the number of studies

that focus on characterizing DC-committed precursors and the growth factors

that are required for their maturation. These studies lead to the conclusion that

DC arc a ven. heterogeneous population with regard to their differentiation (see

above). tIowe\-er, a bone marrow origin has been proven for a varien of DC

including 1 DC (2,5O,5 1,52,53).

Among the different cplÿncs studied, GM-CSF has been shown to be

cssentid not onlv for DC maturation but dso to maintain their \-iabili~ in vitro

(54). Thcrcfore, dl subsequent studies were focused on esmnining thc effects of

othcr cytohes, when added to GM-CSF, on DC maturation. Ghl-CSF plus

tumor nccrosis factor alpha FNFa) is a particularly important combination in

driving immature bone marrow progenitors to acquire DC charmenstics

(8,3,56). Addition of stem ce11 hctor (SCF) to CM-CSF plus TNFa

supplemcnted cultures hrthcr increases DC yicld from bone marrou-

progcnitors (57,58). Addition of IL-4 to G M-CSF plus T N F a supplementcd

culturcs cnhances DC maturation while it rcduccs thc perccntagc of

macrophages (53,60). Crosslinking of CD40 on DC br antibodies or CDW

lipnd has Lem shown to sclecti\-elv inducc DC maturation from bone marrow

prccursors (61). FLT? ligand is a reccntly discovercd cvtokine that has bcen

7 shown to increase the number of DC both in vivo (in lvmphoid tissues) and in

vitro (G2,63). Other cytokines have also been shown to enhance DC maturation

from the bone marrow progenitors. These include IL-13, which has been shown

to subs t i~re for IL-4 (64) as well as IL4 (65), IL-3 (12) and IL-6 (66).

TGFP has been shown to be important for the development of some

DC (-50). The effect of TGF-P on progenitor cells \vas found to be largely

dcpcndcnt on their statr of differentiation and the prescnce or absence of other

growh factors. I t has bcen shown that this cvtoknc has stimulaton cffects on

the prolifcration and differentiation of the more mature, lincage-committed

progcnitor cells (67). On immature progenitor cells, howcver, TGF-P has been

shou-n to csert mainlv inhibitcn- effects. Indecd, this cvtokine has been rcported

to supprcss DC maturation from progenitor cells (GS). I t has been clearly shou-n

that thc prescnce of diffcrent growth factor combinations in thc

microcn\-ironment during DC dcvclopmcnt is important in detcrrnining thcir

stage of maturi@ and the immune responsc tlicv triggcrcd. For csamplc, thc

prcscncc of IL-IO during DC maturation has been reported to skcw subsequent

immune rcsponses toward n-pe 2 irnmuniw bv . suppressing DC IL-12 production

during antigcn prcsentation (69,7(!,7 1).

?Lhcn DC-precursors leaw the bonc marrow, thcy circulate in the blood

and csist in pcnpheral tissues in an immature fom. Thesc immature DC are

8 characterizcd bv a low level of REEC, adhesion, and costirnulaton- molecule

cspression. But, these cells have a r e q - active endocytic and processing capacitv

(3 1,12,73). Esposure o f these immature DC to aritigen and cvtoliines results in

their maturation and migration to draining Iymph nodes (71,75,76). DC

maturation is associated with a reduced antigen processing abilin- and enhanced

csprcssion of h,EIC, adhesion, and the costirnulatory molecules (31). Thus, DC

maturation results in the conversion O f cells carrying out antigen sun-eillance into

a highly efficient r\PC with the abilitv to activate resting T ceus. In this view, the

distribution of immature DC in the sites O € high antigen esposure such as the

&in, the gastrointestinal tract, the ainïays and the interstitial spaces of mosc

orLpns cspccidly suited their hnction to capture antigens. On the othcr hand,

thc localization of mature DC in the T ce11 area of the lvmphoid organs is

compatible with thcir function in antigen presentation and T activation.

Chemokine and chemokine receptors on DC:

Onc of the hallmarks of DC is their capacîty to rnigratc from the sitc of

inflammation, N-hcrc thev pick up the antigens, into the lymphoid organs, whcrc

thcv prcscnt antigen and activate T cells. DC migration is closclr- associatcd with

thcir maturation (77). In thc pcriphcn-, DC arc present in an immature fom with

thc nbilitv to capturc and proccss antîgcn. Inflammatoq- signals such as TNF-a

9 and IL-1, or bacterial products such as LPS, induce DC maturation and

migration to the seconda- lymphoid o r p s , where thev acquire the capacity to

stimulate naive T cells. There is accumulating evidence that chemoliines plav an

import'mt role in trafficliing of DC (78,79). Chemolcines mediate their effect

through their binding to shared, or unique, cell surface receptors which belong to

chc se\-en transmembrane, G protein-coupled Rhodopsin superfadv (80)). Mmy

bclicvc that the changes in migratory behavior of DC are associated with a

sm-itch in the expression of the chemokùie receptors, which alleu- thcm to

rcspond to different chemohes . To this end, several espctiments a-cre

conductcd where they esamined the type of c h e m o b e receptors DC espress at

diffcrent stage of maturation, which allow the t raff ichg of immature DC into

the inflammation sitcs and mature DC into the lymph node. Immature DC, for

csampic, have bccn rcported to express hi& levels of the chemokuic rcceptors

(:CR1 and CCR5, which dnmatically dccrease upon maturation (81). Thc

csprcssion of CCRl and CCR5 make them rcsponsivc to SC\-cd chcmokincs

such as monocvte chernotactic protcin ( C ) - 3 , NCP-4, macrophage

in flmmatoq- protcin (hW)- 1 a, LWP-1 p, KANTES, stromal cell-dcri\-cd factor

(';III?- 1 and thymus-espressed chemokine @cl+ Thcse chemohes have becn

shoan to bc prcscnt in high le\-els at sites of inflammation, which mar csplain

the prcscnce of their ligancis on immature DC. I-Io\vevcr, thev have becn

10 rcported to esert chemotactic activity on other leukocvtes that dso express their

corresponding ligands and thus thev are not solelv chemotactic for DC.

Recendy, t\vo chemohne receptors, CCRG and CCR7, have been

dcscribcd to be present only on DC (82,83). These chemokine receptors are the

o n l ~ ho\i;n receptors for blIIP-3~t and hLIP-3P, respectkely. Their espression

has been found to be regulated during DC maturation (84,85). Immature DC

csprcss high Ic\-els of CCRG mRNA and migrate in rcsponse to hW-3a. Cpon

maturation, the! sharply down-regulate the csprcssion of CCRG W i \ and up-

rcgulate the cspression of CCR7. This is linhd to the loss of responsc to h W -

3a and the acquisition of a sustained responsiveness to ~ I I P - ~ B . DC not only

rcspond to diffcrent c h e m o ~ e signals, but they also sccrete various chcmohnes

such as hIIP-la, i \ , l ~ ~ - l P , IL-TES, MCP-1 and IL-8 (86). This suggcsts that

chemoliincs rcgulatc the trafficliing of DC, \\-hich in mm secrctc diffcrcnt

chcmoLncs to facilitate thc migration of 0 t h 1cukoc)-tes. In surnman-,

maturation influcnccs the expression of diffcrent c h e m o h c receptors on DC

and this in mm plays a critical rolc in regulating thcir trafficking from the site of

inflammation into the secondan- . Ivmphoid . organs, whcre they stimulatc T cc11

prolifcration.

Cytokines and cytokine receptors on DC:

DC, lke manu leukoqtes, produce a wide range o f cytokines and espress

different cytokine receptors. Given the effects of GhCCSF on DC growth, it is

not surprishg that they have been found to espress high ievels of GM-CSFR

(around 3,c)OO sites/cells) (87). However, thev produce onlv very small amounts

of GbI-CSF, suggesting that cher relr on other cells as sources of this cytokine.

The Leratinocytes in sL.n are obvious esample in this respect. Similarly, DC

csprcss high levels of IL-IR, but thev do not produce IL-1, an important

cvtohne for their maturation (88).

Depending on the stimuli, DC have been reported to secrctc variable

amounts of TNF-a (86). This cytokine has been shown to enhance DC

maturation, up-rcgulatc their do-stimulatow responses and provide a stimulus

for LC migration (87). Furthemore, DC have been reported to express TNF-

al<. DC also producc IL-6, an important cvtohnc in allogcneic cvtotosic T-

l!-mphocvtc rcsponses wherc responses ma! bc blocked by anti-IL4 antibody

(70). :\ctivated DC havc been shown to sccrete IL-?, which is important for ?'

cc11 stimulation in do-MLR assays (71). Compared to othcr M C , upon

maturation DC produce large amounts of IL-12. In fact, i t has bcen suggcstcd

that gcneration of this cvtoliine, which skews the immune rcsponse into type 1

12 rcsponses, is what makes DC such powerW agents in the immunocherap)- of

cancer and viral infections (92,93).

In s u m n . , DC respond to different cytokines and produce a number of

cytokines that regulate their maturation (GhI-CSF, TNF-a), migration FNF-a)

and allo-stimulation (IL-12, IL6 and IL-7). The production of these mtokines

bas bccn reportcd to bc dependent on thc parricular stimulus and the stage of

maturation.

Markers of DC:

I t h a become obvious that the distinction between mature and immaturc

DC is vcq- important as the? have becn shown to perform distinct specialized

functions at diffcrent stages of differentiation. hIany reports harc illustrated the

hcterogcncit) of DC population, which has becn reflectcd in the protein profdc

thcr csprcssed and the h c t i o n s they perform. In thc splccn, for csample, nvo

L X populations with distinct phcnon-pes and localizatïon hm-c bccn dcsctibed

(sec ho\-c; 10). Furthemore, thrcc populations of DC have becn identificd in

thc lymph nodes based on their phcnotr-pe (34,75). DC have becn shown to

csprcss a range of molecules common to othcr leukocvtcs such as CD45

(icukoci-te common mtigcn), LMC, adhcsion and costirnulaton molecules.

1 lou-cwr, DC express 10-100 Urnes higher level of MHC class I I thm B cells or

13 macrophages (35). The expression of these molecules on DC can reach >106

molccules per ce11 (96). Furthermore, they retain iLMC class II antigenic peptide

complcscs for prolonged penods (1-2 davs) in culture, whereas macrophages

have a turnover measured in hours (97,98).

Immature DC espress very low levels of the costimulatory molecules, BI-

1 (CDgi.)), B7-2 (CD86) and CD40 Khen maturc, thcl- up-regulate thc

csprcssion o f these costimulatory molecules, which provide an essential signal

for 1' cc11 stimulation (99,100). Furthermore, crosslinking of C D 4 has becn

rcpoacd to rescue DC from apoptosis, br up-replating Bcl-2 (100,101), and

pro\-idc important signals for IL-12 production (36,37). DC have also bccn

rcportcd to csprcss a variety of adhesion molecules including ICAhI-1, ICAM-3

and 1 Chhl-3 adhcsion molecules, the ligands for LFA-1. Furthermore, they

express L.Fr\-3 achesion molecules which bind to the CD2 molecules on T cells.

'L'hcsc adhcsion molecules arc vcry important for DC-T cc11 adhcsion.

:\ntilodics to LFh-1 and LFA-3 have becn reporteci to inhibit DC-stimulatcd

diogcncic T' cc11 @ER) responscs (1 02).

hfousc DC espress high l c d s of CD 1 1 c (PZ integrin) which is rccognizcd

bi- thc hamstcr N418 monoclonal antibodv (103). DC in T cell areas have becn

shou-n to csprcss DEC-205, which is rccognized br the rat NLDC-145

monoclonal mtibody (104). DEC-205 is an integral mcmbrane protcin

14

homologous to the macrophage mannose receptor and relatcd receptors which

arc able to bind carbohvdrates and mediate endocytosis. Despite their high

espression on DC, DEC-205 and C D l l c have been shown to be espressed to

somc estent on other cells such as macrophages. Regardless of this lack of

absolutc specificity, somc of these markers are currentlv used to assess the

dcgcc of DC maturity. For esample, TvLHC class II, costirnulatory and adhesion

molcculcs have becn shown to be up-regulated on DC during maturation.

I [ou-evcr, thcse markers are also up-regulatcd to a lesser degree on the other

profcssiond APC upon activation (105). Furthcrmore, DC maturih has becn

asscssed bu means of MLR An increase in DC maturation has becn reported to

bc dirccdv linlied with incrcased T ce11 stimulation. As thcsc methods providc

onli- limitcd information regarding DC maturin-, manv studics have focuscd on

clucidating easicr ways to distinguish between mature and immaturc DC. Such

studics have achicvcd a major step fonvard in human material, where a specific

rnarkcr that is prcscnt onlv on DC at a certain stage of maturation has becn

idcntificd. CD83 moleculcs arc only prcsent on mature DC (Wb), u-hcreas

LIirbcck panulcs are present onlr in immature DC such as LC (1(!/,108). Thesc

obscn-ations facilitate the studv o f DC maturation and diffcrcntiation in man.

In micc, which arc thc esperimental mode1 used to studr DC maturation

and diffcrcntiation, markers that can differcntiate maturc from immaturc DC arc

15 not vct a ~ d a b l e . The identification of such a marker would greatl- facilitate the

study of DC differentiation and maturation. This in mm rnay improve the

mcthods of identification, isolation and use of DC in the irnrnunotherapy of

cancer and infectious diseases.

1.2 Fascin as a potential marker of DC matunty:

Fascin is a 55-kD;i, actin-bundling protein, that regulatcs the

rcarrmgcment of the cytoskcletal elements and the interaction bcm-cen the

cytoskclcton and the ce11 membrane in response to extra- or intracellular signals

(103). I t is an intracellular protcin and \vas first isolated from cytoplasmic

cstncts of sea urchïn eggs (110). A few vears later, fascin homologous proteins

u-crc idcntified and characterïzed in Drosophila (1 1 l), Xenopus (1 l?), rodents

(1 l3), m d humans (1 14,115). Fascin cDNA w x cloned from mouse tissues in

1335 (1 13). I t mas found to share a high degree of conservation with other

specics (1 16).

I:sprcssion of fascin has bcen reportcd in various normal and

trnnsformcd cells. Fascin is abundantlv csprcsscd in tissues such as brain and

splccn md, at the cellular level, in specific tvpcs of cclls such as neuronal and

glial cclls, microcapillary endothelid cells, and DC (1 11,115,117). 1-iigh le\-els of

fascin csprcssion arc also obsen-cd in manr transforrned cclls, including virus-

16

trans formed fibroblasts, HeLa cells, and Epstein-Barr virus-infected B

1:-mphocytes (1 1 8,115). A morphological characteris tic common to the normal

cclls that express high lerels of fascin is the derelopment of many membrane

protrusions. Fascin espression, as assessed by immunocpchernistry and

immunofluorescence staining, is found to be localized in the membrane niftles,

microspikes, and stress fibers (1 17)).

This protein has been reported to be involred in a varie- of cellular

pmccsscs including sel1 locomotion, cytoplasmic stre:mulg and transport,

sccrc tion, phagocytosis and cytokinesis (1 20). Morc ;riportantly, it appears to

ha\-c a rolc in dendrite formation. Edwards et al have reported the disappcarance

of filopodia in cultured rat neurons when fascin espression \vas inhibited (1 16).

Fascin expression in hurnan DC:

In humans, fascin espression has only been dctccted in DC, and not in

an!- othcr Icukocrtcs esarnincd, including monocrtes, B cells and T cells.

L'~irthcrrnorc, fascin cspression aa s found to be rcstricted to IDC which are

prcscnt in the T cc11 areas of the lvmph nodes. Fascin-positive DC wcrc not

found in thc B ce11 areas of human lvmph nodes (1 19). Taken together with data

abour its rolc in dendtitc formation, this suggcsts involvement of fascui

csprcssion in the process of DC maturation and T ccll activation.

17

Objective of the study

The objectives of this study were to assess the correlation benvecn fascin

espression and DC maturation and the potential role of fascin in facilitating DC-

-1' cc11 interactions.

This involved esamination of fascin distribution on mouse: i ) Ivmph

nodes, where mature DC are present and iii epidemial shn sheets, where

immature DC are present (LC). Fascin espression \vas also esamuied in DC

gncratcd in vitro where it can be correlated with other markers of DC rnaturit-y

as wcll as stimulaton. activity. For this purpose, DC were gcnerated from bonc

marrou DC-precursors (BM-DC) in short term culture supplcmented with GM-

CSF. In BM-DC, the development of fascïn was esarnined either donc or dong

with hWC class II and B7-2 espression, which are h o w n to be up-regulated on

DC upon maturation. Furthemore, the level of fascin and h.EK class I I was

csamincd in DC generated from bone marrow DC-precursors in the presence of

TG F-P and IN F-a which suppress and enhance DC maturation, respcctively.

Thcse growth factor combinations arere also employed to esamine the cffcct of

fascin lcwls on BM-DC do-stimulaton. a c t i v i ~ in m MLR assav.

The precisc role that fascin rnight play in facilitating DC-T ce11

in tcrac tions was dso inres tigated. This \vas achieved using an tiscnsc

olipnuclcotidcs to inhibit fascin expression in BhI-DC during maturation. Thc

18 cfTcct of this on fascin espression and the allo-stimulaton- activity of these cells

\vas compared with BM-DC treated with control oligonucleotides.

The results of

of mature DC. I t \id

DC hinction as rWC.

this study provide an important tool for the identification

also advance Our understanding of the role of dendrites in

M A T E R I A L S A N D M E T H O D S

2.1 Animals:

ilduit BhLB/c and C57BL/G rnice were purchased from Charles River

Cmada and housed in the Carleton Animal Care Facility (Sir Charles Tupper

hlcdical Building, Dalhousie Cniversity, Halifax).

rUl animals were housed in cornpliance with the guidelines establishcd bv

thc Cmadian Council on Animal Care and werc gi\-en st,mdard rodent chow and

N- a t c r ud J b i / ~ z

2.2 Media:

RPhLI-164~) liquid medium (Sigma-Aldrich Canada Ltd.) \vas

supplcmcntcd with 5"41 heat-inactivatecl (30 min, 6 5 ' ~ ) fetal calf scrum (FCS;

Gibco URL Inc.), 1 ~)(!C/ml pcnicillin, 100ug/ml strcptomycin, 5mM of 2-

mcrcaptocthanol (BD H Inc) under aseptic conditions. This cornpicte RPhII

164) u-as store at 4%. Purificd and dcionized \i7atcr (df1,O; M i l l i - ~ ~ ~ \V-atcr

Systcm, Corning) \vas stcrilized by autoclaring in glass botdes. Phosphate

buffcrcd saline (PBS; pH 7.4) \vas prepared as a 25s stock containing 3 . lM

NaCl (BD H Inc), 1.1 h l K,I-IPO, (Sigma-Aldrich Canada Ltd.) and U9mhl

NatI,I>O,.I-I,O (Fishcr Scientific) in dH20. Thc 25X stock \vas storcd at room

19

20 temperature- Before use, the 25X stock \vas diluted 25 tirnes in stede dH,O and

the 1 X PBS stored at room temperature.

2.3 Reagents:

Solutions of 1°'o (w/v) pparaformaldehvde (J.T.Baker) in PBS, 1% (u-Il-)

csscntially IgG Free bovine serum albumin (BSA; Sigma-Aldrich Canada Ltd.) in

PBS, i i . lO/b saponin (Sigrmi-Aldrich Canada Ltd.) in PBS were fdtered with

\Y hatrnan #1 fdter papers (Fisher Scientific) and stored at 4OC. The 1% BSA

solution was made fresh on the day of use, whercas the 1°.,(o paraformddehydc

and the 0. l 0 , o saponin were discarded after approsimately tsro wcelis. A solution

of TBS-Twcen 70 (pH 7.1) was prepared br adding 20mhI Tris-base (Boehringer

hhnnheim Corporation), 137miM NaCl (BDH Inc), 27mii1 KCl (BDH Inc) and

5x1 ( " J O Xi-cen 20 (BIO-RAD) and stored at 4% l(1C)miikI acetatc buffer

solution (pH5.2) was prepared by mking liiOrnii1 CH3.COONa3H,0 (Sigmi-

:\Idrich Canada Ltd.) and I O ~ h k I CH,.COOH (Fisher Scientific) and stored at

4°C. lhc I O O m h l acctate buffer \vas prepared fresh cven week Glycine-Lysine

buffcr \vas prepared bv mising IOmiiI of L-Lj-sine (Sigma-Aldrich Canada Ltd.)

and 1 i i( )mhI of Glycinc (Gibco BRL) in 1 liter of dtI,O. Destran-Tris buffer pl-I

7.1 avas prcpared bv dissolving 130pilI of destran (PharMacia Finc Chcmical) in

1 litcr of 5 ( h i i I Tris-base Poehringer hhnnheim Corporation). The Glr-cine-

21 Ixsine buffer and the Destran-Tris buffer were kept nt 4OC and discarded after

approsimately a month. 1O0.'o acetate buffered formalin \vas prepared br mising

XK!rnl of 370to formaldehvde and 350mi'i'I CH,.COONn3HZ0 in 2 liters of

d1-120.

2.2 Antibodies:

A panel of mouse, rat, or hamster antibodics \vas used in thc

immunocvtochcrnist~ and flow cytometrv studies. Al antibodies arcre titcred

and thc optimal dilution \vas uscd. rlnti-hscin monoclonal antibodr (mousc

I$X) \vas a generous gifi bv Dr. Erik Langhoff, Massachusetts G e n c d

I Iospital, Boston, CS A. rhti-DEC-205 (NLDC 115; rat IgC2a), anti-CD 1 1 c

(N318; hamster IgG), and mti-Ia (mousc IgGl), anti-CD1 l b (Mac- 1; rat IgC2b),

anti-Fcy RII/III (CD32/CDlG; mouse IgGZa) and anti-Thyl.2; (CD90; mousc

I@b) monoclonal antibodies werc purchascd €rom Cedarlanc Canada LtJ.

Puri ficd mousc IgG 1, Phvcocn-th& (PE) -conjugatcd rat IgG-2a and fluorcsccin

isothiocyanatc (F1TC)-labclcd strcptavidin, FITC-labeled rabbit anti-rat werc also

purchascd from Cedarlanc Canada Ltd. PE-conjugatc anti-1-Ad/ 1 -Ed

monoclonal antibodv (rat I & 2 ) was purchased from Pharhfingcn, San Diego,

California. Thc hvbridoma, GLI, producing anti-B7-2 (nt IgG2a) monoclonal

antibod\. was purchased from the Amcrican Typc Culture Collcction (ATCC3

22 1-IU-253). The Cn^r\-41g hision protein (human IgGl ) \vas a generous gift from

Bristol-hlvers Squibb PharMaceutical Research Institute, P ~ c e t o n , New Jersey

(1 2 1). The FITC-labeled goat anti-hurnan Ig antibodv was purchased from T a p

Immunological Inc. For negative controls, either isotvpe-specific IgG or PBS

supplementcd with 1 ° h BSA were substituted for the priman- antibodv.

2.2 Growth factors:

The recombinant murine granulocyte macrophage colon!-stimulating

factor (mGhL-CSQ was purchased from Cedarlane Canada Ltd. and uscd at

3 )C/ml. n i c recombinant murine m o r necrosis factor-a (rmTN F-a) \vas

purchascd from Gibco BRL Inc and used at 2OOC/ml. The recombinant human

tram forming growth factor-pl (rhTGB-p 1) was purchased from CaiBiochcm-

No\-aBiochem Corporation and uscd at OSng/ml. LUI growth factors were uscd

at thc optimal concentration (7,8,68).

2.2 Epidermal skin sheet preparation:

Epidcrmal skin sheets wcrc prepared essentidlr as prm-iouslr descnbcd bv

Baker r / . d (122). Briefl!., a mouse car \17as dissected and rubbed with the back of

a pair of forceps to separatc the hvo halres. The sepantcd shn w-as thcn cut into

srnall squares (3-4 mm3 and incubated in 3.8% NFISCN (Sigma-Adrich Canada

23 Ltd.) for 30 min at 37 '~ . The s h was then washed in PBS and the epidermis

scparatcd from the derrnis under a dissecting microscope. The epidemiis was

t h m f~xed in cold (-20'~) acetone @DM Inc) for 5 min and stored in PBS at

4 " ~ until stained Ail staining of the epidermal s h sheet \i7as perfomed in 1 . h l

micro fùgc vials (O. H. Johns Company Ltd-).

2.7 IgG coated-petri dish prepuation:

A to ta1 of 50ml of Fdtered-sterilized 0.02 acetate buffer containing hurnan

IL.(; (Sigma-Adrich Canada Ltd.), diluted at lug/ml, \vas uscd to coat a 150~15

mm pctri dish (Fisher Scientific Co.). Antibodies were allowed to bind br-

incubating the dish ovemight at 4'C. Before adding cells, the dish was washcd

nvicc u-ith TI3S-Tween buffer. This petrî dish \vas used to deplete FcR-positi\-c

cclls, n-hich arc mostlv monocvtes and macrophages. Bonc marrow cclls wcrc

incubatcd at 3 7 ' ) ~ for 1 h after which the non-adhercnt cells werc recovcrcd.

2.8 Preparation of DC from bone marrow:

DC \\-crc prcpared from BALB/c bonc marrow esscntially as prm-iousl!-

dcscribcd (7; sec fig. 1). Brieflr, mice were sacrificed by cervical dislocation and

clippcd in 70°k cthanol. Afier removing al1 musclcs from the femurs m d tibias,

thc bones uere dippcd in 70% ethmol for 1 min. The bones werc thcn washed

24 u-ith PBS and transferred hto a fresh dish with RPMI 1641. Both ends of the

bones wcre cut and the marrow flushed out usïng RPbiI. The cells werc then

suspcnded and passed through nvlon mesh to remove s m d pieces of bonc

marron. and debris. The cells were washed and red blood cells Ivsed with G d of

1)-sing buffer, contahing 1 55mi. i N H,CI in 1 O l M Tris-CICL buffer, (Sigma-

rildrich Canada Ltd.) for 2 min at room temperamrc. FcR-positive cells wcrc

dcplcted br. incubation (at 3 7 ' ~ for lh) on a petri dish coated with IgG. hftcr

n-ashing, lvmphocytes and Ia-positil-e ceils wcrc rcmol-ed bv incubation with a

cocktail of monoclonal antibodies for 60 min at 4OC followed by Low-Tos rabbit

complemcnt (Cedarlane) for 60 min at 37OC. The monoclonal antibodies werc

GKl.5 anti-CD4, M02.2 mtiCD8, B21-2 anti-Ia, and RA3-3AllG.l anti-

1322O/CD45R CrIB 207, 150, 229, and 146 rcspecti~elr; ATCC). The cclls

(5s106) wcrc cultured in 5Cknl flasks (Nunc International) in 5mI medium

supplcmcntcd with either 50C/mi rmGM-CSF + /-2OOC/rnl mTNF-a , or

aspirating 85O.0 of the medium and adding back fresh medium with growth

factors.

BALB/c mice Cut both ends of Flush cells out with media femur and tibia

Deplete T and B cells by antibody and rabbit

complement

Precursor cells

Pan macrophages and monocytes on IgG coated plate

Seed cells with growth factors

Fig.1 Hom diagram of bone marrow DC-precursor ceIl isolation from mouse borie marrow.

2.9 Immunostaining:

Immunostaining was perfomed on hurnan s l i i n sections, mouse Iymph

node sections, mouse epidermal skin sheets, or BkI-DC c-tospins. DC were

han-cs tcd on di fferen t days and cosp in s made. For cytospin preparation, 7s 1 O'

cells \i-ere cvtocentrihged for 7 min at 91.45sg on polv-L-lysine (Sigma Canada

1.td.) -coatcd slides. CeUs were then fked in cold (-20'~) acetone for 2 min and

storcd at - 2 0 ' ~ until use.

For fascin staining, where mouse anti-fascin monoclonal antibody werc

uscd, slidcs wcrc L ~ e d in 10"'o acetate buffcred fornidin and incubated in a

rcagent (signet Kit, IDlabs, ON) designed to block nonspecific binding of mousc

antibod!- to mouse tissue. For all other staining, slides were f~xed in cold ( -20 '~ )

acetonc (1 i) min), 9 0 paraformaldehvde (2 min), Destran-Tris buffer (1 5 min)

and Glycinc-Li-sinc buffer (1 5 min). These slides wcrc thcn incubated (3 min) in

-3" l l 1-I,O, - - to block the endogenous pcrosidase followed br 1 h in horse serurn to

block the nonspccific binding. Titcred plima. antibodv a-as then added to thc

sldcs for incubation over night-at room temperature foliowed by the appropriatc

hotin!-latcd sccondary antibody for 1 h at room temperature. The antibody \vas

locdized using strcpta\-idin-horscradish pcrosidase (signet Kit, IDlabs, ON) for

1 h at room temperature and 3-rù-nino-9-Ethvl-Carbozolc (AEC; Sigma Canada

Ltd.) as a chromogen.

2.10 Flow Cytomemc analysis:

Cultured DC were han-ested on different davs and suspended in 1 0 0 ~ 1 of

PBS su~plemented with 1°'o BSA. The ceIl suspension was then incubated with

the appropriate prima? antibody. Al1 antibodies were incubated for 30 min at

4 C . The FITC- and PE-labeled antiibodies were incubated in the dark

For singlc staining, cclls were directlv sciincd \rith mti-B7-3 and anti-

B220 priman- monoclonal antibodies followed bv FITC-labeled rabbit anti-rat

sccondary mtibody and CTLA-4Ig fusion protein-followed b y FITC-labelcd goat

anti-human Ig mtibody. The cells were also stallicd ~71th FITC-conjugatcd Mac-

1 (an ti-CD 1 1 b).

For doublc staining of fascin and bEIC class II, permeabilization was

rcquircd prior to the addition of the anti-fascin monoclonal antibody to stain for

fascin, u-hich is a a-toplasmic protein. For permeabilization, ceUs were incubatcd

m-ith 100" O methanol (BDI-I Inc) for 30 min at room tcmperaturc. Aftcr

pctmcabilization, cclls wcre stained with anti-fascin monoclond antibodv

follou-cd bv bio tinylated-anti-mouse secondan antibodr and s treptavidin FITC-

labclcd or FITC-conjugatcd goat anti-mousc. PE-conjugatcd anti-hDfC class II

monoclonal antibody \vas then addcd as a second mtibodv.

Cclls ucrc \rashcd nvicc with I0'oBSA-PBS aftcr each step m d fiscd with

1" O paraformddhydc-PBS. Fluorescence and mean channel numbcr were

28 analrzed on a total of 10,000 cells per sample using a flow cytometer (Becton

Dickinson FrKScan). \\/EH1 164 cells (ATCC; CRL-1751) were maintauied in

the lab and used as a positive control for hscin (123).

2.11 T ce11 isolation:

T cells were ennched from the spleen bv filtration through nvlon wool as

prcl-iously dcscribed (124). Briefly in a laminar flow hood, Ig of sterilized nylon

\i-ool (Ch-cns-Fiberglass Corp.) was packed to the 9-ml mark of a plugged IOml

plastic syringe (Becton Dickinson 81 companv). Afier the addition of 7ml of

RPhII, the column was kept at 37OC for 1 h. During the incubation, 3-4

C57BL/6 rnice \vere sacrificed as above. In a larninar flow hood, spleens wcre

rcmol-cd and homogenized with the stede back end of the srringc plunger. Cells

\i-erc \\-ashcd with RPhlI and red blood celis lrsed as above. After n-ashing twicc

\\.;th RPhlI, thc ce& \r-ere suspended in a total volume of 4ml RPiLII. Before

loading cclls into thc column, the column \vas flushed with lOm1 of w a m RPhU.

Cclls \vcrc thcn loadcd into the colurnn and 4ml of fluid allowed to pass through.

.in cstra 2ml of RPMI \ras then addcd and 2ml of fluid allo\red to pass through.

*l'hc v d ï c \vas thcn closcd and lm1 of RPhII gently added to the top of the

column. Thc column \vas then wrapped in sterilc tinfoil and incubatcd at W°C

for 1 h. Cells wcre collccted at 1 drop per 2 scc whde adding warrn 10% FCS

29 media to the top of the column. The first 13rnl were coilected and pelleted at

U)(!sg for 10 min at 4 ' ~ . This process removed approsimately 7O0<0 of the

slpenic B cells. The remauiuig B cells were depleted by complement mediated

I\-sis (as above) using anti-B220 monoclonal antibody. Viable cells were counted

and the percentqe of T cells \ras assessed bv Thy1.2 stainïng using flow

crtometry. T ce11 pu r i - \vas routinelv benveen 80°'o-8G0-'o (Fig.2).

2.12 Mixed lymphocyte reaction (MLR):

BM-DC were han-ested on day 9 and treated with 25pg/ml mitomvcin C

(Sigma-Aldrich Canada Ltd.) for 30 min at 37OC. Treated DC were then applied

in gradcd doses to 2x10' nylon wool-enriched allogeneic T cells for 4 days.

Culturcs were maintained, in C-shape 96-well plate (Nunc), in 200rnl RPhII 1640

supplcmented with 1 0°,0 fetd cdf serum, 5OuhI/ml 3-mercaptoethanol,

1 ( )( )C /ml penicillin, and lC)Opg/ml streptomvcin. The cells merc pulsed with

l u C i / d of ['[-TI thvmidinc (ICN PharMaccutical, Inc.) in thc last 18 h of

incubation. T ce11 proliferation \vas assessed bv han-esting the cells on fdtcrrnats

~ising a ce11 han-ester (Skatron Instruments Inc.) and measuring the thvmidine

uptake in a liquid scintillation counter (Bechan).

Fig.2 T ceil puris- after e ~ c h r n e n t through nylon wool colurnn. At the end of the enrichment, T ceiis were coilected and stained with the ami-Thyl.2 monoclonal antibody (red line) or no prirnary antibody (blue line) followed by a secondary FITC-labeled goat anti-mouse antibody. Fluorescence of 10" ceUs was then analyzed by flow cytometry.

3 1 2.U Cultivation of bone marrow-precursors with antisense

oligonucleotides:

Fascin antisense oligonucleotides and matchcd control oligonucleo tides of

thc samc length (17 base pairs) were purchased from Cniversity Core DNA

Sen-ices, Cniversih of Calgary. Three base pairs at the 3' and the 5' end of the

antiscnsc and the control oligonucleo tides were phosphorothioatcd. The

scqucncc of the control oligonucleotides was: CCGGCACCATGACCGCC and

thc antiscnsc oligonucleotides was: GGCGGTCATGGTGCCGG. Bone

marrou--prccursors \t7erc culti\-atcd in cRPhfI supplcmented G h [-CS F donc,

oligmucicotidcs (2phI)- At dav 3 and 6, the ceils were fcd with ncw mcdia

supplcmented with GM-CSF +/-oligonucleotidcs. On da\- 9, DC wcre

han-csted, c~rosplns made for rnorphological esamination, and an Ml.R assav

u-as carricd o u t to investigate thc impact of inhibiting filscli csprcssion on DC

allos tirnulaton- a&-in..

RESULTS

3.1 Fascin expression in human DC:

As dcscnbed abovey fascin is in\-ol\-ed in dendrite formation and thus we

hypothesized that there wodd be an up-regulation of fascin expression during

DC maturation and achievement of DC hnctional capacitv as iWC. Sincc fiiscin

h;is alreadv bcen characterized in mature h m a n lymph node-DC (1 13), as a first

stcp to address this hvpothesis ure csamined fascin expression in human LC in

s k n scctions. In the epidermis, LC are b o w n as the classical immature DC. \ K é

found no fascln espression at this site (Fig. 3). In contnst, LC in the demis arc

considcrcd to be undergoing maturation as they migratc Çrom the epidcrmis.

chis site u-c found considerablv more &scin positirc LC.

In the humans, the presence of fascin in lymph nodcs is rcstncted to IDC,

u-hich arc mature DC. Fascui esprcssion is not seen in the epidcrmal LC, which

arc classical immature DC. These Fuidings suggcsted that fascin esprcssion is

linkcd to maturation. Furthemore, the restncted expression of this protcin to

DC in the T ce11 zone OF the lymph node (125) suggested that it ma? have a rolc

in thc arca of T cc11 regdation.

33 Bascd on these preliminary results, we asked the folloming questions: 1) 1s

thcre a correlation between fascin expression md DC maturation? and 2) what is

thc significance of fascin espression on DC function as rZPC?.

As a first step to address these questions, we developed an animal mode1

that dlowed us to in\-estigate fascin expression at different stages of maturation

as u-el1 as to studr its functional significance. \Ve chose a mouse svstem since

mouse DC are the most estensi\-elv studied and mm!- reagents are availablc for

this s\-stcm.

Fig.3 Imrnunoperoxidase examkation of human skin for fascin expression. Formaiin-hed skin section was reacted with anti-fascin antibody foilowed by a secondvy biotinyiated goat anti-mouse antibody and detected by the horseradish peroxidase system.

3.2 Characterization of fascin in mouse DC in vivo:

(a) Lyrnph node:

hIouse anti-human fascin (monoclonal) antibody \vas used to detcct

human fascin. The reactiviv of this rnouse antibody with mouse fascin has not

bccn assessed. To determine whether this mtibodv urould cross-rcact \\-ith

mousc fascin, WC esarnined fascin cspression on histologicd sections of

Fortnalin-€ixed lymph nodcs from BhLB/c mouse. Fascin binding \iras detected

br immunohistochemist~ using biotinylated anti-mouse as secondan- antibody

and strcptal-idk-horseradish perosidase for visualization. The rcsults (Fig. 4)

rcvcaled strong fascin staining of DC in the T cell-depcndent areas of the Ivmph

no& (IDC). tlowever, in the germinal ccntcr (B ceIl) areas of thc lymph nodc,

thc I'DC wcrc essentiallv fascin ncgativc. h similar distribution of fascïn

csprcssio n has been rcported prc\-iously in human Ivmph nodcs (1 25).

1.0 confimi that the fascin-positive cells were indecd IDC, we staincd

histoiogïcal scctions of mousc lymph nodc with another DC markcr cdlcd

N1.DC-115. This monoclonal antibody is hou -n to stain only IDC and is

Jcscribcd as "anti-interdigitating D C antibody" (104). Thc rcsults (Fig. 5)

shou-cd that thc distribution of cclls staincd n-ith NLDC-135 and the mti-fascïn

36 monoclonal antibody was identical, confirming that the fascin-positive cells were

IDC.

These results confirrn that anti-human fascin monoclonal mtibodv cross-

reacts with the mouse fascin. Furthemore, they showed that the espression of

this protcin in the mouse Iymph node is s i d a r to that in humans in that it is

rcstrïctcd to IDC. This supports a role for this protein in T' ce11 engagement in

both spccics.

Fig.4 ~asc in expression in mouse lymph nodes. FormalLi-fked lpmph node section was reacted with anti-fascin antibody followed by a secondary bio~ylated goat anti-mouse antibody and detected by the ho rseradish peroxidase system.

Fig.5 Expression of DEC-205 in mouse lymph nodes. Fmzm lpmph node section was reacted with the NLDC-145 antibody foiiowed by a secondaq bio tinylated nbbit anti-nt antibody and detected by die horsardish peroxidase system.

(b) Epidemal skin sheet:

In human si&, LC arc h o w n as immature DC. Following interaction

u-ith antigen, LC undergo maturation, characterked by up-regdation of hMC

class I I , costimulaton~ and adhesion molecules, and espression of long dendites

(58,126,102). To confirm the relevance of the mouse model, nre esamined fascin

csprcssion on the epidcrmd LC of mice. Compared to the human, mouse s h is

vcn. thin and onlv yen few LC can be \-isualized in histological sections. To sec

large nnmbers of LC we esamined epidcrmd s h sheets.

\Vhen the epiderrnal skin sheets were stained with the anti-fascin

monoclonal antibodv, onlr x-ery few fascin-positive LC \vere dctected and they

appcarcd adcndritic in nature (Fig. Ga). Howe\-er, this might bc due to thc

prescnce of few LC in the epiderrnd s h shcet we preparcd. To confirm the

prcscncc of fascin-negatix-e LC, w-e stained thc sheet \r.ith an anti-hIHC class II

monoclonal antibodv n-hich is known, from carlier u-ork, to stain LC stronglv

(1 27,128). The obscn-arion of many LC staining with this monoclonal antibody

(1.-igbb) confimicd the prescncc of sigrtificanr numbcrs of fascin-ncgatiw LC.

Th& is consistent with our earlier rcsults on human skin.

Altogcthcr, the in x-ivo esaminations demonstratcd strong fascin

csprcssion by IDC of the mouse 1 mph node (mature) but onlv \-en- few fascin-

positi\-e LC in the mousc epidermd skin sheet (immature). Thesc observations

40

are consistent with the human results and suggest that the expression of fascin in

DC mar be regulated during maturation.

Fig.6 Expression of fascin and hMC class II in mouse epidermal s h n sheet. An cpidermal skin sheet was stained with the anti-fascin (A) or anti-hIHC class II (B) antibodr follomed by a seconda. biotinylated goat anti-mousc antibody and detected bv the honeradish perosidase sustem. The staining \vas pcrformcd in microfuge tube and the sheet was then mounted on slide for microscopie esamination.

3.3 Characterization of fascin in mouse DC in vitro:

(a) Generation of DC from mouse bone mvrow DC-precursors:

The ultimate goals were to investigate the correlation betsveen fascin

csprcssion and DC maturation and the fùnctional significance of fascin

esprcssion in antigen presentation. For this purposc, the use of in vitro-

gcncratcd DC is essential since it ad1 d o m us: 1) to compare fascin lercls dong

u-ith other markers that are up-regulated during DC maturation; 2) to examine

hou- fiscin espression is influenced bv different gromth factor combinations

kno\r-n to influence DC maturation and 3) to esarnine if fascin le\-cls effect thc

ah-stimulatory a c t i v i ~ of DC.

To this end, we generated bone marrow deri\-ed-DC (BM-DC) (127).

Uonc marrow DC-precursors were isolated from mouse femur and tibia and

n-cre gron-n, in vitro, for 9 davs in cRPMI supplcrncnted n-ith Ghl-CSF. This

rcgimc pushcs DC precursors to maturc into DC (65,130,7). During the 9 davs,

cclls \vcrc monitored in culture bv light microscopy. Aftcr 3 days, cclls a-crc

obscn-ed to Fom clustcrs (Fig-Ta). By day 6, cells u-ith typicd morpholog- of DC

\\-crc rclcased from the clusters (Fig. B). O n dav 9, celis were han-estcd and thcic

ultra-stmcturc esmincd by electron rnicroscopy. Cnder thc clectron

microscope, the day 9 DC displayed typical features of DC with irregular nuclci

U

and many thil cytoplasmic processes (Fig. 8). Light and electron microscope

csarninations demonstrated cells ~ 7 1 t h typicd morphologv of DC ac this Ume.

Fig.7 Dcvclopment of BM-DC €rom bone marron- DC-precursors in the prescnce of GM-CSF. A) proliferating cellular aggregates at da^ 3 o f culture and B) cclls with typical morpholog o f DC at dar 6 of culture.

Fig.8 Ultrastructure of day 9 BM-DC. Ele-n microscopie micsograph of BM-DC at day 9 shows rnany cytophsmic veils and irreguiar nuclei.

48

(b) Immunocytochernical examination of fascin expression in BM-DC:

\Xe nest esamined whether these in vitro-generated DC would also

cspress fascin. Bone marrow DC-precursors were grown as above for 9 davs and

then han-ested for cytocentrihgation. \Then cytospins were stained for fascïn

and esarnined br immunoc)~ochemistn-, we obsen-ed that a significant nurnber

of cells stained positive for hscin (Fig. 9). The staining \vas el-enlr distributcd

throughout the cvtoplasm. Cells that stained positively for fascin aTere large and

displayxi long dendrites, features normallr seen in mature DC. Cvtospins were

also staincd \rith othcr DC markers such as NLDC-145 or anti-kEEC class I I

monoclonal antibodies and esamined by immunocytochemistry. Fig. 1 0

dcmonstrates cells stîining positive aith NLDC- L 45 monoclonal antibody,

rcflccting the prcsence of IDC-like DC in the Bhl-DC population. Furthetmore,

cclls n-erc obsen-ed to st'wi stronglv positive for hLHC class I I reflecting their

dcgcc of maturîty (Fig. 11). This data demonstrated that mature BhI-DC csprcss

Ç'scin, hlI-IC class I I and DEC-205.

Fig.9 Fascin expression in day 9 BM-DC. Bone m w DC-precursors were cultured in the presence of GM-CSF. On &y 9, cells were harvested for c y t o c e n ~ t i o n . The cytospin was stained with the anti-fiscin antibody foiiowed by a secondary b i o ~ y h t e d goat anti-mouse antibody and detected by the horseradish peroxdase system.

Fig.10 Examination of day 9 BM-DC for IDC. M3 wue generated fiom bone mvrow DC-precursors culnved in the presence of GM-CSF. On day 9, cells weze harvested for cytocenmfug?tion. The cytospin was then stained with the NLDC-145 antibody followed by a secondary biotinylated rabbit anti-rat antibody and detected by the horseradish peroxidase systan.

Fig.11 MHC class II expression on &y 9 BM-DC. Cytospin was made on DC generated frorn bone rmnow DC-precursors that were dtured in the pressence of GM-CSF for 9 days. The cytospin was then stained with the ana-LWC dass II antibody followed by a seconday biotinyhted p t mti- mouse antibody and detected by the horseradish peroidase system

52

(c ) Examination of fascin and MHC class II expression on BM-DC

duhg maturation:

To confirm the correlation benveen fascin espression and DC maturation,

\vc esamincd fascin espression throughout the maturation process of BhI-DC.

As a control \ire esarnined bDIC class 11 expression, a molecule known to be up-

rcçulated during DC maturation (131). To this end, bone rnarrow DC-precursors

\i-crc grown as above and cells were han-ested afier 3, G and 9 days for

cvtoccntrifug;ition. Cvtospins from day 3, 6 and 7 werc then stained for fascïn

and csamined by immunocytochemistn.

At da\- 3, when DC are still immature, onlv \-en- few fascin-positk-e DC

\i-crc dctected (Fig. 12a). The number of fascin-positive DC \vas markcdly

increascd on dav 6 and 9 as more DC reached maturin- (Fig. 12b&c).

I~urthcrrnorc, fascin-positive DC on da- 6 and 9 \r-erc obsen-ed to be largc a-ith

cstcndcd dcndritcs, typical of thc morpholog of mature DC (Fig. 124.

Similad!-, cytospins from da. 3, 6 and 9 BM-DC wcrc staincd for hIHC class I I

and csarnincd bv immunoc~ochemistn.. WC obsen-ed an increase in the numbcr

of RGIC class II-positivc cclls that correlated closelr uith thc increasc in DC

maturation (Fig 13 a-c).

Thcsc rcsults confirmed that there \vas a parallel incrcasc in hll-iC class I I

csprcssion and fascin cspression a-hich closcly correlated with DC maturation.

53 This is in line urith the in vivo results and hrther suggests an association hem-een

fascin espression and DC maturation.

Fig.l2(A-D) Fascin expression in BM-DC duhg maturation. Cpospins \i-erc made at dars 3, 6 and 7 on DC generated from bone marrow DC-prccursors cultured in presence of GM-CSF. The cvtospins uTere then stained with the anti-fascin antibodv followed bv a seconda? biotinylated goat anti-mouse antibody and detected by the horseradish perosidase sustem. A) day3 B) dayG C) dav9 and D) da 9 (higher magni fication).

Fig.l.3 (A-C) iLMC class II espression on BM-DC dunng maturation. Cytospins were made at days 3, 6 and 9 on DCs generated from bone marrow DC-prccursors cultured in presence of GM-CSF. Cytospins were stained with thc anti-MI-IC class II antibody followcd b y a secondas- biotinylated goat anti- mouse antibodv and detected b v the horseradish perosidase system. A) day3 B) dav6 and C) day9.

60

(d) Double color flow cytometric anaiysis of BM-DC for fascin and

MHC class II coexpression:

To confirm and estend our immunocvtochernical data, we employed flow

ci-tometric malysis of BM-DC. Flow cvtometm allowed us to double stain for

hscin and hLHC class II and thus both esamine their level on DC during

diffcrent stages of maturation and confimi that bEIC class II and fascin are

present on the same mature DC ce11 population.

1 t is important to mention that although fascin is a CL-toplasrnic protein,

pcrmeabilization \vas not required for staining on cytospins or sections. This is

duc co the damage that occurred to the ce11 membrane during ntocentrifigation

or scctioning. To confirm this, we made cvrospins from \\EH1164 cells which

are known to be fascin positive (123) and esarnined them bv

immunoc~-tochernistn-. In our hands, al1 WEHI-164 cells stained positive for

Fascin (Fig. 11). For flow c~romctric analvsis, live cells are used a-hcre the ce11

mcmbranc rcmains intact Thus, pemcabilization is nccessan- to permit thc anti-

fascin monoclonal antibodv to gain access to the cells. This was confirmed uskg

li\-c \\Ei-11-164 cclls uhich stained negative b y flow mtometric malysis (data no

ç hown) unless permeabilized (Fig. 1 5) .

\\;C applicd the samc technique to staïn BhI-DC for fascin. DC wcrc

gencrated from bone marrow DC-prxursors as abore. On day 9, cclls werc

61 harvestcd, permeabilized and double stained for fascin and hMC class II. As a

control, the priman- antibodies were substituted with non-specific isot-ype-

matched IgG. Fascin was detected by FITC-labeled antibody and the MHC class

I I monoclonal antibody \vas dire+- labeled with PE. By tlow q-tometric

analvsis, we noted that al1 fascin-positive cells are bfHC class I I positive (Fig. lu).

Furthemore, dl fâscin-positive DC were hfHC class I I bright, reflecting their

rnaturin.

Fig.14 Fascin expression in WEHI cells. Cytospin was prepared h m WEHI cells and s h e d with the anti-fascin antibody foiiowed by secondary biotinylated p a t anti-mouse antibody and detected by the horseradish peroxidase systea

Fig.15 Examination of WEHI164 cells for fascin expression using flow cytometry. 10' N'EH1164 ceiis were coiiected and permeabilized Mth 100°/o methanol. The cells were then stained with the anti-fascin monoclonal antibody (red iine) or control isotype (blue line) foiiowed by a secondary b io~y la ted goat mu-mouse antibody and FITC-labeled streptavidin. The fluorescence of 1O4WEHI cells was then analyzed by flow cytometry.

Fig.16 Day 9 BM-DC double stained for fascin and lWIC class II. DC were generated from bone marrow DC-progenitors cultured in presence of GN-CSF for 9 days. On day 9, ceiis were colîected and permeabilized with 100% methanol. The cells were then stained with the ami-fascin monoclonal antibody or isotypic control followed b y a secondary bioth~lated goat ami-mouse antibody and FITC-labeled streptavidin. Next, cells were incubated with a PE-conjugated ami-MHC class II or PE- conjugated isotypic control. For flow cytomemc analysis, IO4 celîs were collected. Dot plots represent the coexpression of fascin and L W C class II on the sarne BM-DC population.

65

(e) Investigation of the correlation between fascin and MHC class II

expression on BM-DC during maturation:

In the previous esperïment, we successfullv double stained mature BM-

DC for fascin and MHC class 11. To confirm that this linked espression

correlates with matun-, DC werc generated from bone marrow DC-precursors

culnircd in cRPhfl supplemented with GM-CSF. The cells u7ere han-ested on

days O (before the addition of GM-CSF), 3, 6 and 9, double stained for fascin

and LU-IC class II and esamined bu flow cvtometry. Thc results demonstrated

that al1 fascin-positive cells were MHC class II-bright at al1 tirne points tested.

This sugges ts that fascin-posi tive cells are mature DC.

Consis tent with Our earlier observation by immunocytochemsitry

csamination, verv few fascin- and bfHC class II-positk-e cells were obsen-ed on

da^ 0 and da? 3, when most DC wcre still immature bu morphologv (Fig. 17).

\'ihcrcas on da!- G and 9, are obsen-ed a dramatic increase in fascin- and a parallcl

incrcasc in MHC class II-positive ceils, when more DC reached maturiry. During

thc 9 da\- csamination, there \vas a direct correlation bmveen the level of fascin

cspression and hlHC class II expression with a correlation coefficient of 0.93 as

calculated from Fig. 17. This highly significant correlation strongly links fascin

cspression with DC maturation.

Fig.17 Correlation behveen fascin and bEIC class II csprcssion. DC wcrc çcneratcd from bone marrow DC-progenitors culturcd in presencc of GM- CSF. At days O, 3, 6 and 9 cclls were han-ested and permeabilized. The cells wcrc then double stained for fascin and MHC class II and analvsed b v llour cytomcry. This figure displavs the number of fascin-positi\-c and h E K class II-positiw cclls out of IO' collected. The correlation coefficient as calculated €rom this figure is 0.39.

67

(f) Examination of B7-2 expression on BM-DC dunng maturation:

In short terni culture, \vc demonstrated a strong correlation benveen

fascin and R.MC class II expression and suggested that this correlates with

maturation. To confirm this, we esamined a second marker of DC maturation,

B7-2. It is a costirnulaton. molecule known to be up-regulated during DC

maturation (99). \Té therefore esamined the l e~e l of B7-2 molecules on Bhf-DC

during maturation. As a control, we first used a CTLA-41g hsion protein that is

knon-n to bind both B7-1 and 87-3 molecdes with high avidity (132). \lé

gcncntcd DC from bone marrow DC-prccursors grown in clWhII

supplcmcntcd with Ghf-CSF. On dar 9, cells han-ested and stained with

rhc =LA-lIg h i o n protein followed bu a secondan FITC-labeled goat anti-

human antibodv. :\n.zlysis br flow cl-tometq- as illustrated in Fig 18 clcarly

demonstrates B7-positive staining on dav 9 BM-DC. This suggested that our

Bhf-DC csprcss B7 molccuies, but docs not diffcrcntiatc ben\-een B7-1 and B7-2

lcvcl.

In order to spccificallr determine 87-2 levels on BM-DC durinç diffcrcnt

s t q p o f maturation, we used an anti-B7-2 spccific monoclonal mtibody. As

bc fo rc, bonc marro\r7 precursors were cul turcd in cRP MI supplrmcn tcd wi th

C hl-CSF and DC wcrc hanested at days 0, 3, 6 and 9. Cclls wcrc staincd \\sith

anti-137-2 monoclonal antibodv followed by a seconda. FITC-labeled rabbit

68

mti-rat antibody and esamùied by flow cytometry. The results (Fig 1%-d)

confimi up-regdation of B7-2 on the BM-DC d u ~ g maturation.

Fig.18 B7 expression on day 9 BM-DC. Day 9 BM-DC were incubated with (red Line) o r without @lue h e ) the CTLA-41g fusion protein foliowed by a FITC-labeled goat snti-hurnan Ig antibody. The fluorescence of 10" cells was then analyzed by flow cytometry.

Fiuorescence intensity

Fig.19 B7-2 expression on BM-DC during maturation. DC were generated h m bone marrow DC-precursors culnired in the presence of GM-CSF. Ceiis were then harvested at days 0 , 3 , 6 and 9, and stained Mth the mu-B7-2 monoclonal antibody (red line) or no primary antibody (blue line) followed by a secondary FITC-labeled rabbit ami-rat anabody. Fluorescence on a total of 10" cells was then analyzed by flow cytometry.

71

(g) Effect of TGF-B and TNF-a on fascin and MHC class II expression

on BM-DC duMg maturation:

N é nest tested whether it \vas DM-DC maturation, or simplv time in

culture, that resulted in fascin espression. To accomplish this we suppressed, or

enhmccd, Bhl-DC maniration with additional gron& factors and csamined

thcm for fascin and hII-fC class II cspression.

The growth factor combinations wc used were: i ) GhI-CSF donc; ii) GA,[-

CSF~TGF-P which has been reported to suppress DC maturation from bonc

mnrrow-progcnitors (68); or üi) GM-CSF+TNF-a which has been shown to

cnhmcc DC maturation from bone marrow-progenitors (G8,8). To this end,

bonc marrow DC-przcursors were grown in cRPhn supplemented with the

diffcrcnt growth factor combinations and cells u-ere han-ested on days ( 1 @efore

the addition of growth factors), 3, O, and 9 from each group. Cells u-cre then

double staincd for fascin and &WC class II and analvzed by flow cvtomctn-.

In dl groups, thcre mas an increase in fascin espression over timc and al1

fascin-positive DC were obsen-cd to bc MI-IC class II-bright. Thc numbcr of

fascin-positil-c DC in thc group treatcd with GM-CSF+TGF-P how-CL-er \vas

markedlr rcduced compared to those treated with Ghf-CSF alone (Fig. 20). The

cclls in this group werc smdl and only few DC-like cells werc obscn-cd when

csamincd in culture by light microscopy. The numbcr of fascin-positiw DC was

72

higher in the group treated with GM-CSF+TNF-a when compared to those

treatcd with GhI-CSF alone (Fig. 20). Morphological esaminations, in culture bv

light microscopy, demonstrated more DC-like cells in this group. This figure

clearly demonstrated that enhancement of BiM-DC maturation with GM-

CSFtTNF-a treatment increased Çascin espression. In contnst, suppression of

B h 1-DC maturation with GM-CSF+TGF-p trcatmen t marked- reduced fascin

cspression.

Sirnilar effects on bMC class I I espression wcre obsen-cd on BhI-DC

uhcn trcated with thcse growth combinations (Fig. 21). These results proïide

strong cvidcnce of a 1ink benveen fascin expression and DC maturi&.

Fig.20 Influence of diffcrcnt grou-th factor combinations on fascin csprcssion during BAI-DC maturation. DC wcrc gencrated from bone marrow DC-prccursors culturcd in the prescncc of GM-CSF donc

(diamond), GM-CSF+TNF-CL (circle) or GM-CSF+TGF-P (trïanglc). Thc cclls uxxc han-ested on d a n 0 (without gro\rth factors), 3, G and 9, pcrmcabilized and double staincd for fascin and hhEIC class II. This figure \vas obtaincd from flow c~tometric analrsis and represents thc numbcr of hscin-positive cells out of 10' collectcd. The results are espressed as mcan

of fascin-positive cells I SE and are represcntative of 3 indcpendcnt cspcrimcnts.

Fig.21 Influence of different grou-th factor combinations on hDIC class I I csprcssion du&g BkI-DC maturation- DC were gencrated frorn bone marrow DC-prccursors culturcd in the presence o f GR1f-CSF donc (diamond), GhI- C S F+TN F-a (circle) or G M-CS F+TG F-p (triangle). Thc cclls \wre han-cstecl on da!-s 0 (\vithout growth hctors), 3, 6 and 9, pcmcabilized and double staincd for fascin and MFEC class 11. This figure was obtained from flow c!-tomctnc analysis and represents the number of hn-IC class 1 I -positive cclls out of IO" collected. Thc resuits are espressed as mean of MHC class I I - positive cclls + SE and are rcprcscntatire of 3 indepcndcnt csperïmcnts.

-- 13

3.3 Investigation of the effect of fascin level on BM-DC allostirnulatory

activity:

(a) Cornparison of the allo-stimulatory activity between day 6- and day

9-DC:

Compared to immature DC, mature DC have been demonstrated in

scvcrd studics to be superior in stimulating naire T ce11 proliferation in a mised

lvmphocrte reaction @KR) assay (77,31). Since we obsen-ed an increase in

fascin expression on DC durirtg maturation, we esamined whether this incrcase

n-ould bc associated with enhanced do - s timulato ry activitv.

To this end, bone marrow DC-precursors were grown in cRPkil

supplemcnted with GM-CSF. Day G- and da)- 9-DC were then added (as

stimulator cells) in graded doses to a f ~ x d number of naive allogeneic T cells (as

rcspondcr cclls) and thcir do-stimulatory activity assessed in 4 days LfLR ssay.

Rcg;irdless of the responder to stimulator ratio, the do-stimulaton activity of

dav 6-DC, which have fewer fascin-positk-e DC, \vas markedly lower than that of

da? 9-DC (Fig. 22). This suggcsted highcr T ce11 proliferation with incrcxed

numbcrs of fascin-positk-c DC. I t also functionally links fascin cspression n-ith

LX maturation.

Fig.22 Cornparison of the allostirnulaton- activin- betwecn da! 6 and dai- 9 Bhl- L X . DC werc generated from bone marrow DC-precursors cultureci in thc prcscnce of GM-CSF. BhI-DC werc collectcd at day 6 (open triangle) or dar 9 (closc triangle) and treated with rnitomycin C for 30 min at 37'C and addcd in gradcd doses to nai1-e allogcneic 2~10' T cells in a 96 wells plate. Thc cells wcrc incubatcd for 4 days and pdsed with ['FU thymidine in the last 18 h. T cc11 proliferation \vas assessed by measuring the ['Fu thymidine uptakc in a liquid scintillation counter. The results are espressed as mcan DPM + SE and arc rcprcscntative of three independent csperiments.

(b) The effect of fascin levels on BM-DC aiio-stimuhtory activiy

To confirm the link behveen fascin expression and DC do-stimulaton

actix-i~, we used different growth factor combinations that we have shown

carlier to affect fascin levels. To this end, bone tnarrow DC-precursors were

gron-n in cRPblI supplementec~ with GM-CSF alone, CSF+TNF-a, or GkI-

CSF+TGF-P for 9 days (as above). On day 9, DC werc han-ested fcom the

different groups and added in p d e d doses to a f~xed nurnber of naive allogcneic

-1' cells. T ceil proliferation \vas then assessed in a 4 dar MLR assay. In al1

groups, there \vas an increase in T ce11 proliferation when DC numbers increased

(Fig 23). 1-Iowever, the do-stimulatory actiritv of DC treated with GM-

CSF~TGF-P, which we ha\-e shown to posses lower fascin levels, \vas markedlv

lowcr t h m thosc treated with GM-CSF done. Furthermore, DC treated with

Ghl-CSPTNF-a , whïch we have shown to cnhance fascin levels, wcre bettcr

s tîmulators than those treated with G M-CS F alone.

This data links the increase in fascin levels with strongcr naive allogeneic

-1' cc11 activation. It should be noted, however, that u-e can not attributc the

incrcasc in the allo-stimulaton- activity onlv to increased fascin level, sincc hII-IC

class I I Icl-els \i7erc also up-regulated on these ceus.

1 :a

DC:T cell

Fig.23 Ailostirnulatory activity of dav 9 Bhl-DC cultured under diffcrcnt conditions. DC a-erc generated from bone marrow DC-precursors culmred in thc prcscncc of GM-CSF donc (circle), GM-CSF+TNF-a (diamond) or G h l - CSF+TGF-P (triangle). BhI-DC were harvestcd at day 9 and trcatcd a-ith rnitomycin C for 30 min at 3 7 ' ~ and added in gradcd doscs to 2x10' naivc allogcncic 7 cclls in a 96 wells plate. Thc cells wcre pulscd with ['E-q thymidine in thc last 18 h of the 1 day incubation. T cc11 prolifération \vas asscsscd br measuring the ['i-rl thymidine uptakc in a liquid scintillation countcr. Thc results arc cspresscd as mcan D P M f. SE and are representatiw of thrcc indcpcndcn t espcriments.

3.4 Investigation of the d e of fascin in DC-T ceîî interactions

(a) Examination of the effect of fascin inhibition in mature BM-DC on

their aiio-stimulatory activity

To isolate the effects of fascin on BM-DC do-stimulaton- actiritv, we

used antiscnse oligonudeotides to inhibit fascin expression during maturation.

To this end, bone marrow precursors were seeded in cRPh411 supplementcd with

G hI-CSF done, GM-CS F+antisense oligonucleo tides (2p?vl) or G LI-

CSF+control oligonucleotides (2plLi). On day 9, DC were han-ested and added

in graded doses to a f~xed number of naire dillogeneic T cells for 1 days. T ce11

prolifcration was then assessed by measurïng thymidine-uptAe in the last

18h. In al1 groups, there was an increase in T ce11 proliferation with an increasc in

thc number of DC (Fig. 24). Fürthermore, we obscn-ed no significant difference

in the do-stimulaton. activin- between DC treated with GM-CSF alonc and

thosc treated with GM-CSF+control oligonuclcotidcs. However, thc allo-

stimulaton- a&-itv of DC treated with the GM-CSF+antiscnse oligonuclcotides

\ras marbdly suppressed compare to those treated with thc GM-CSFicontrol

oligonuclcotides or GM-CSF alonc. This result demonstratcd that inhibition of

fascin expression in mature DC dramaûcally reduces their do-stimulaton.

ac tivitn-.

Fig.24 Mo-stimulatom activie of BM-DC after fascin inhibition. Bonc marrow DC-prccursors u-crc treated with GM-CSF alone (circlc), Ghf-CSF+ control oligonuclcotidc (square) or antisensc oligonucleotide (diamond). O n day 9, cells uvcrc han-cstcd and treated wîth mitom-cin C and added in graded doses to

nail-c dlogcncic 2x10' T cells in a 96-wells plate. Thc cclls wcre thcn pulsed with 1'1 11 thymidine in the last 18 h of the 4 day incubation. T ce11 prolifcration was asscsscd b~ mcasurïng the [)fil thymidine in a liquid scintillation countcr. The rcsults arc csprcssed as mean DPM t SE and are rcprescntativc o f thrcc indcpcn Jcnt espcrimcnts.

81

(b) Confirmation of fascin inhibition in the BM-DC treated with

antisense oligonucleotides:

In the previous esperiment, we demonstrated inhibition of manire BM-

DC dlo-stimuiaton: activitc- that have been treûted with fascin aritisense

oliçonuclcotides. To confimi that this inhibition is indeed the result of reduced

fascin expression, we compared fiscin level of Bkl-DC treated uith antisense

oligonucleotides to those treated with control oligonucleotides. To this end,

bone marrow DC-precursors urere grown in cRPhfI supplemented with Gh.1-

CSF plus cither antiscnse oligonucleotides (2pM) or control oligonucleotides

(2ph4). On day 9, cells \vere han-ested from each group for cvtocentrihgation.

\\;hcn cosp ins \rere stained for fascin and cxamined by irnmunocytochemistry,

a e obsen-cd no sipiticance diffcrcncc in the numbcr of fascin-positk-c ceUs

bcnvccn the two groups. However, while the control cells espresscd high la-els

of fascin, cvenlv distnbuted throughout the cytoplasm of DC, the DC treatcd

u-ith mtisensc oligonucleotides n-erc obsen-ed to have lower fascin levels and

nrens where fascin cspression is almost absent (Fig-ZSaFrb). hloreover, the DC in

culturcs u-ith antiscnse oligonucleotides were obsen-cd to bc smaller comparcd

to thosc trcatcd with the control oligonucleotides.

h similar dccrease in the levcl of fiiscin esprcssion a7as obscn-cd bu

immuno fluoresccncc staining (data not sho\vn). Thc intensitt- of fascin staining

bv immunocvtochernis tn- and immun0 fluorescence esaminations demonstrated

an inhibition of fascin espression in mature Bhl-DC treated with antisense

oligonucleotides. The fact that al1 groups possess equal number of fascin-positi\-e

DC, confimis the presence of similar numbers of mature DC. Furtherrnore, are

obscn-cd high lerels of iLMC class II espression on the group treated \rith

antisense oligonucleotides, indicating that the antisense oligonucleotides

spccifically cffected fascin expression.

~Utogether, this data confirms that the inhibition of Bh1-DC d o -

stimulaton ac t i~ ih that we obsen-cd, was due to rcduced fascin expression.

Fig.25 Fascin expression in BM-DC dtured in the presence of (A) GM- CSF +control oligonucleotides and (B) GM-CSF +adsense ohgonucleotides. Bone niurow DC-precursors cultured in the prcsence of GM-CSF +control okgonudeotides or GM-CSF +antisense oligonucleotides. On day 9, cells wre hrvested for cytocentnfbgation. The cytospins were thm stained with the anti-hscin antiboày foliowed by a secondvg biotinylated goat ak-mouse antibody and detected by the horseradish peroxidase system.

DISCUSSION

DC are the most potent APC. Their ability to act as a potcnt APC is

dcpendent on their degree of maniri-. In mice, isolation and chancterization of

maturc DC has been hampered by the lack of specific markers of maturi..

l'hcrcfore, manv esperirnents have focused on defming a marker that would

distinguish mature from immature DC. However, such esperirnents ha\-e shown

littlc success. Defming a specific marker of DC maturi? has important

implications for the study of therapeutic applications of mature DC in ncoplasms

and infcctious diseases (1 33,134)-

Fascin is a cvtoplasmic protein that has been shou-n to bc involvcd in

dcndnte formation in other ce11 types. Recently, it has also bcen suggcsted to be

in\-ol\-cd in dcndtitc formation in mouse epidemid LC. Therc is a gcncrd

agrccmcnt that DC originate from bonc marrow precursors, u-herc ther are

smallcr and lack dendrites (31). Cpon maturation, they incrcase in sizc md

csprcss long dclicate dendrites (102). \lé hypothesized that fascin u-ould bc up-

rcgulatcd in DC during maturation correlating with dendrite cspression. \Cc dso

h\*pothcsizcd, that because dmdritcs increasc the surface area of DC, thus

hcilitating DC-T cell interactions, that this protcin might plar a rolc in T cc11

activation bv DC.

85 To test these hypotheses, we studied fascin expression in vi\-O and in vitro

undcr different growth conditions. Fascin expression stronglv correlates with DC

rnairih- . bv . phenotypicd and hct ional esaminations. Furthemore, fascin \vas

found to lx specificdl y involved in DC d o - s tirnulaton. actk-ih-.

4.1 Fascin expression in human DC:

Nhcn this study started, fascin had onlv been dcscnbed in human DC.

Fascin expression was demonstrated in pcripherd blood-DC and Iymph node-

IDC, u-hcre DC arc known to interact with T cells and initiate immune

rcsponses (1 19). The significance of fascin \vas first illustrated by Edwards (1 10 ,

who dcmonstrated the disappearance of fdopodia and well-spread growth cones

n-hcn fascin cspression \vas inhibited in neuron cultures using antiscnse

oligonuclcotides. Recently, more evidence of fascin involvcmcnt in dendntc

Fornation \vas dcmonstntcd in hvo studies perfomed on mouse cpidermai LC

and n pig cpithclial ceIl line (1 23,135).

1s.ascin in\-olvement in dmdrite formation suggests that fascin rnight bc

up-rc'platcd in DC during maturation. In humans, fascin cxprcssion has alrcadv

bccn dcmonstrated in mature IDC. \T'e used thïs kno\vledge to compare the

maturc IDC with immature DC for thcir le\-cl of fascin cspression. For fascin

csarnination in immature DC, we used human s h sections u-hich contain largc

86

nurnbcrs of LC, the classicd immature DC (27). \Te found no fascin expression

in thc cpidermis, where DC are still immature. tIowe\-cr, in the demis, which

contains DC that have migrated from the epidermis en route to the regional

Iymph nodc and are presumed to be more mature (136), fascui-positive DC were

o bscn-cd.

P . I hcsc data support Our hvpothesis and suggest a link benveen fascin

csprcssion and DC maturity. Furthemore, the? confimi the restricted

csprcssion of fascin br IDC, which stronglr suggests a potential role for fascin in

the arca of T ceIl regdation.

1.2 Fascin expression in mouse DC:

Our fiiding with human DC promptcd Fuaher esamination of a

corrclation bcnveen fascin espression and DC maturation. \ X e thus chose to

invcstiptc this protcin in mousc DC, where markers of DC rnaturity arc not yct

availablc and whcrc chancterization of fascin during DC maturation would

pro\-ide a modcl that would allow thc in~cstigation o f the rolc of fascin in DC

function as :WC.

'l'hc anti-hscin mtibodv used to dctect humm fascin \vas mousc anti-

human fascin monoclonal antibody. The reactivin. o f this mtibody with mousc

Çascin had not been assessed. Thcreforc, the first qucstion u-e askcd \vas u-hcthcr

87 this nntibodv would cross react with the mouse fascin. Fascin is evolutionaq-

highlv consen-ed in amino acid sequence among different species (35O.b identity;

137). This increased the chance that this antibody would cross react with the

mouse fascin. Since fzcin espression is high in humm lvmph node IDC (1 13), it

was Iogical to esamine its espression in mouse lvmph node cells. \ X e found

strong fascin expression in the T celi-dependent areas of the mouse lvmph node,

u-hcrc iDC are found, but DC in the germinal center (B cell) arcas wcrc

csscntiall~ Çascin negative. This resul t demonstrated that the anti-human hscin

antibody not only cross reacts with the mouse fascin, but it reacts with the same

DC population (IDC) as in humans.

\ré confirmcd that the fascin-positive DC in the lvmph node werc indeed

IDC usïng an NLDC-145 monoclonal antibodr, u-hich reacts \rith onlr IDC.

Thc distribution of the cells labeled with the NLDC-145 and anti-fhscin

antibodics wcrc identical. The restricted expression of hscin to mature DC

found in the T cc11 areas of both human and mousc stronglv supports a rolc for

this protcin in T ce11 regdation.

1'0 determine whethcr fascin espression corresponded with DC maturih-

in thc mouse, we esamineci hscin espression in immature DC reprcscntcd br the

1 .C of thc s k n (1 38). tlowe\-cr, mouse s k i n , udikc human's, is \-en- thin and thus

it is difficult to \-isualizc manv LC on s h section. This problcm was

88 circum\-ented bv using an epidermal skin sheet, where many LC codd be

visudized. \\;hm epidermal skin sheet \vas stained for fascin we obsen-ed very

fcw fascin positive cells indicating that LC lack fascin expression- hlthough this

olsen-ation was in line with Our hypothesis, WC considered the possibilitv that

this might result frorn the presence of low numbers of LC in the preparation. To

rulc out this possibility, we stained epidermal skin sheets with anti-hDIC class 11

antibodr which staïns LC ve-en. well (127,128). \lé found many LC in each si&

shcct confirming the presencc of man- fascin-neetil-c LC in our prcpantion.

In vix-O, WC clearly demonstrated fascin expression in mature lvmph node

L X . Furthemore, we showed that fascin is selcctivelv espressed in IDC of thc

1)-mph node in both specics. Taken together, these rcsults support a hypothesis

that fascin expression is increased during mouse DC maturation and that it mar

ha\-c a rolc in the arca of ?' cc11 replation.

4.3 Fascin expression in in vitro generated mouse BM-DC:

Although thc in vivo rcsults suggcsted a corrclation bctwccn fascin

csprcssion and DC maturitv, wc necdcd to confirm this correlation in

association with othcr fcaturcs of DC maturim. ' ïhc use of an in \-itro modcl has

SC\-cral advmtages, as it do\i7s the evduation of fascin expression in an

89

indi\-idual cell. I t also allows the investig@on of Fascin espression in conjunction

u-ith DC morpholog, phenon-pe and fünction.

Athough DC c m be generated from manv sources, gencration of DC

from bonc marrow is the standard method to studv DC maturation in vitro (7).

Bonc marrow is the source of DC precursors which give tise to mature DC (31).

Othcr sources of DC arc much lcss useM for thc smdy of maturation. The DC

population in thc splcen, for instance, is mostlr mature and ei-en the lcss mature

oncs reach maturih after ovemight culture, which is part of the isolation

proccdure (139)). This makes the spleen an unsuitable source. rUthough LC arc

~mmaturc DC, thcir isolation remains faidv difficult. hloreovcr, LC mature

quicklv (3 days) in vitro (140). Bone marrow DC-prccursors require 7-9 days to

manirc into DC (7). This dlows the esamination of &scin cspression at diffcrent

stages of DC maturation. Furthetmore, it allowed the investigation of whcthcr

fnscin is csprcsscd earlr in maturation, which mav facilitatc DC migration from

thc intcrstitia into the lrmphatic tissue, or is maintaincd through maturation. This

u-as bascd o n thc cl-idence of the role of dendtitcs in facilitating DC migration

and a rcccnt study that has reportcd incrcased motilin- of cpithclid cells whcn

tram fcctcd with fascin (1 35).

i*'or DC gencration from bone marrow, WC followed thc protocol

Jcscribcd b y Inaba (7). The FcR-bcaring cclls, mostly macrophages and

90

monocrtcs, werc depleted by incubation on petri dish coated with I g G , whereas

B, l', and hfMC class II-positive cells were depleted bv incubation with specific

monoclonal antibodies and low-Tos rabbit complement. Subsequentlv, these

cclls wcre referred to as day U DC which probablr represent stem cells. \Ye

asscssed the cfficiencv of the depletion by stuning them with anti-B220, mu-

Thv 1.2 and anti-MI-IC class II monoclonal mtiidies. Bv flo~- cvtometric

csarnination, dmost complete depletion of B and T cells wcre obscncd.

t lo\re\-cr, we obsen-ed some cells still espressing low le\-cl of hfI-IC class 11

molcculcs. Based on the observation that manv of the cells \vcrc stained dim-

positivc with the anti-CD11 b (Mac-1) md with the anti-FcyR (CDlG/CD33), we

bclicvcd that these cclls are macrophages or immature DC. Although Mac-1 is

considcrcd to bc macrophage-specific (as is CD 16/32; 141,1421, immature DC

havc bccn reported to espress low levels of both CD1 l b and CDl6/32 and their

csprcssion is down-rcgulated upon maturation (143,144). This demonstrates thc

hctcrogcncin- of the bone marrow prccursors that wcre isolated.

I o push the bone marrow DC-prccursors to mature into DC, \vc ge\v

thcm in cIWhlI supplemcntcd with G hl-CSF. \\ hen esamincd in culture, many

cc11 clustcrs u-crc obscn-ed as early as dar 3. Othcr studies have demonstrated

that in vitro gcncntcd DC groar in tvpical clusters in response to GM-CSF

(1 3 ),65,7). Thc numbers of clustcr wcre decreascd and the numbcrs of cells with

91 the ~ p i c a l morphologv o f DC were increased bv dav 6. Consistent w-ith

pra-ious studics of in \-itro gcnerated DC, ive obsen-ed that day 9 cultures have

the largcst nurnber of DC-like cclls. Furthermore, we obsen-ed that the cells at

da!.s 6 and 9 were much larger compared to day 3 cultures.

Esamination of day 9 DC by electron rnicroscopy revedcd cells \rith

'picd delicnte dendrites and irregular nuclei, characteristic feanires of mature

DC.

Tb confirrn fascin espression in thesc mature Bhl-DC, cells u-ere stained

for fascin a-ith the antibodl- we previously used for immunohistochcrnistn-. \Y-c

found that man! of the cells in thc da) 9 culturcs staincd strongly positive for

fascin. hlorcovcr, thc fascin-positivc DC werc found to Le larger than the rcst of

thc cells. Fascin staining was locdized diffusely to thc crtoplasm and dong the

denciritic projections. LUI hscin-positive ceUs werc obsen-cd to displav . npicd .

morpholop of DC. \ F e also stained dav 9 Bhif-DC with an anti-MI-IC class I I

monoclonal antibodv and obscn-cd cclls staining strongly p o s i t i ~ for MI-IC class

I I . 'L'his suggcsts the presence of mature DC in out BiLI-DC.

Since WC obscn-cd that fascin is selcctiw-cly csprcsscd in 1 DC of the lvmph

nodc, u-c csamined whethcr our BM-DC contain IDC-likc cells. To this cnd, WC

staincd da? 9 BM-DC with the NLDC-145 monoclonal antibody that is spccific

to IDC and found manr cells staining positive for DEC-205. Takcn togcther,

92 thcsc data demonstrate the presence of fascin-positive, IDC-like, cclls in our

I) hl-DC. They also confirm the presence of mature DC in our BM-DC.

T o investigate a correlation between the obsen-cd fascin espression and

DC maturin-, we had to esamine its expression in BhI-DC during maturation.

Bonc marrow DC-precursors were grown as above, cells han-ested and

cvtoccntrifiged at dan 3, 6 and 9. The slides were then staincd for both fascin

and hlE-IC class II.

For ncgativc control, the primat). antibodr \vas substitutcd with PBS.

Although there s7as no staining on the negative control slides ofday 6 and 9, we

obscn-cd positive staîning on da. O and 3 of the negati\-e control slides. The

possibdih that this non-specific staining \vas caused by endogenous pcrosidasc

\\-as ruled out as no significant difference obsen-ed when alkdine phosphatasc

mcthods wcrc used. \V'e bcliel-e that this \vas caused bu the sccondan- mtl-

mousc antibodr that n-as uscd to detect fascin. In Our laboraton-, wc normall!.

use horse scmm to climinatc the non-specific staining. Han-e\-er, this cffort dicl

not rcducc the background staining in this inst'mcc. Funhcrrnorc, WC tricd goat

and mousc serum, but no significant diffcrence n7as obsen-cd. Intcrcstinglv, thc

nonspccific staining nppcared on the ce11 surfacc comparcd to fascin staining

\\-hich is crtoplasmic. Onc possibili is that the seconda. antibod

bound to IgG on B cclls. However, this is unlikelv since no BZO-postil-c cells

93 u-crc obsen-ed on the slides. Moreover, the conditioned medium used to grow

our BM-DC does not support B ce11 sun - id in vitro. The other possibili~ \vas

that the secondan- antibodv bound non-specificall to FcR on macrophages.

This is more lkelr, since GM-CSF is known to d n ~ e the precursor cells not onlr

ton-ard DC lineage but also toward macroph;ige and neutrophil lineages

(56,115,58)- \ T é finallv resolvcd this problem using a commercial kit designed to

block nonspecific binding of mouse antibodr to mouse tissue. \Ye believed chat

this kit has FcR blockers that eliminated the nonspecific binding of the anti-

fascin antibodv to the FcR. When this kit \vas used, we obscn-ed an increasc in

thc numbcr of fiscin-positive cells over the davs of esamination. To confirm

that the increase in hscin-positive cells \vas due increase in the number of

mamrc DC, thc slides were tested for hIHC class II csprcssion, which is kno\\m

to bc up-regulatcd on DC duting maturation (131). \Té Found an incrcasc in the

niimbcr of MI-IC class II-positive cells ovcr thc daïs of csarnination.

Collcctivelv, thcsc rcsults dcmonstnte an incrcase in fascin csprcssion and

a parailcl incrcasc in MHC class II espression during BAI-DC maturation. This is

in linc ni th thc in vivo results and Furthcr links Fascïn cspression with DC

maturih-.

To confirm and estend the imrnunocj-tochcmicd data, WC uscd flow

cvtomctric mdvsis of BM-DC. This allowed us to doublc stain for hscin and

94 MI-iC class 11 and thus esamine their coespression on DC d u ~ g different stages

of maturation. 1t also allowed us to confirm that MHC class II and fascin are

prcscnt on the sarne mature DC cell population.

Aithough fascin is a cvtoplasmic protein, its detection on a-tospins or

tissue sections did not require perrneabilization. This can simplv bc esplaincd bv

thc damage occurs to ceil membrane during sectioning or cvtocentrifiigation.

Iimhcrmore, al1 c~osp ins werc Fked in acetonc pnor to storage and acetonc has

bccn shown to have a weak perrneabilization ability (146). To investipte this, WC

madc cvtospins from REI-II cells which are h o w n to bc fascin positive (123)

and csamined them by irnrnunocytochemistn-. rUl \TEH1 cells were obsen-cd to

stain positk-c for fascin. This confirms that fasciri stauiing on cvtospins does not

rcquirc pcrrneabilization.

For flou- cvtometric anal+, lire cclls are used where the integriry of thc

cc11 membrane remains intact. Thus, WC prcsumed that permeabilization \vould

bc ncccssan- to allotv penetration of thc anti-fascïn antibodr into cclls. To

invcs tigatc this, we stained Live \T EH1 cells with and without pcrrneabilization

and csarnined thcm bv flou crtomctn-. Only uhcn the \Y-EH1 cells \r-ere

pcrrncabilizcd, fascin-positive staining \vas obscn-ed. This confirms the ncccssih-

o f pcrmcabilization to dctect fascin in livc ceUs.

93 For permeabilization, we tes ted differen t concentrations of saponin,

Tween-W and acetone, which are known to have a variable permeabilizing

abdih. \X ken these reagents were used, no fascin-positive staining \vas obsen-ed.

However, when the \\-EH1 cells were pemeabilized with lOW& methanol,

lascin-posi tive s taining was obsen-ed.

As a first step to understand the link between fiscin espression and DC

maturation, WC esmined fascin and bMC class II levels on the same Bhl-DC

population using the double staining technique. Fascin is an intraccllular marker

\\-hile hWC class II is a surface marker. In this casc, most double staining

tcchniqucs recornmend the staining of the surface marker first, pcmeabilization

and thcn staining of chc intracellular rnarker (147,148). \\;ben we used this

staining sequence, neither fascin nor kEIC class II \vas dctected. I t could bc that

the use of the permeabilization reagent following surface marker staining might

havc intcrfcrcd with the fluorescence of antibodies (149,150). Furthermorc, it

ma? bc that ïncubatïng thc ccUs at 4'C to stain thcrn for the surfxc marker first

rcndered thcm rcsistant to permeabilization, nhich is perforrned at room

tcmpcraturc. In our hmds, whcn the cells were permcabilized and stained for the

intraccllular m a r k first, a e obsen-ed positive st'aining for both marlicrs.

Consequcntly, bonc marrow DC-prccursors w r e grown as abow, han-csted on

dav 7 and double staincd for fascin and MHC class II. \\ken csmincd bv flan-

96 cïtomctry, we found that there are not hvo ce11 populations and that ail fascin-

positive DC are hMC class II-positi~e- More importantlv, we found a high l e d

of hIHC class II molecules on the fascin-positi\-e DC. This observation reflects

thc dcgree of matung of the fascin-positive DC and demonstntes a

cocspression of fascin and high levels of bCHC class II on the same mature Bhf-

DC population at dav 9.

4.4 Correlation between the development of fascin and MHC class II

expression on BM-DC dunng maturation:

Giwn that the le\-cls of W C class II molecules are up-regulated during

DC maturation (1 3 l), w e as ked how this correlates with the esprcssion of Fiscin

owr timc. By singlc color staining, n7e obscn-ed an increasc in hWC: class II and

a corrclatcd increasc in facin expression on BhI-DC during maturation. WC thcn

usecl doublc staining to in\-cstigate the corrclation bchvccn hscin and h I t K class

I I csprcssion on the samc BM-DC d u ~ g maturation.

Rcgardlcss of thc dav of esamination, al1 fascin-positk-c DC wcrc

obscn-cd to csprcss high Icvels of i\EK class II molccules, suggcsting that fascin

is cspresscd onlv in maturc DC. hIoreovcr, thcrc a-as a highlr- corrclatcd (0.99

corrclation cocffcicnt) incrcasc in hscin and MHC class II csprcssion on BhI-

DC during maturation. This strongly links fascin csprcssion with DC maturi-.

97 From the double staining esamuiations, ail fascin-positive cells were

dcmonstrated to be MHC class II-positive. However, there were manr hMC

class II-positive cells that were fascin-negative. Athough the identitv of these

cclls was not confirmed, four esplanations are feasible. Firstly, they might be DC

at intermediate stages of maturation that require the presence of another growth

fncror to inducc thcir fuial maturation. For esample, thc addition of TNF-a to

CM-CSF has been reported to enhance the maturation of DC-precursors into

DC (8). Furthcrrnorc, the addition of IL-4 to GM-CSF has becn reportcd to

inducc the differcntiation of monocvtcs into DC (134). Other growth factors

such as SCF and FLT3 ligand have been shown to enhance the response of DC-

prccursors to GM-CSF (58,G3). Secondly, it could be that part of this population

bclongs to a different DC lineage that u-as comrnitted to becomc fascin ncgatk-e.

I'otcntidly thcse DC may be FDC-likc cclls since FDC in the lvmph node of the

mousc and human arc fascin negativc (1 19). tIowe\-cr, thc support for this

possibili~. is a-cak sincc FDC have bccn rcportcd to differcntiate from non-bone

marron. prccursors, dthough this is still somcwhat contra\-crsiai (14)- Thirdv,

thc! could bc DC committed to home into particular organ as immaturc cclls

and in ordcr to maturc csposurc to particular c v t o h e s or pathogcns is rcquircd.

In s h n , LC diffcrcntiatc from bonc marrow precursors and upon csposurc to

proinflammaton- . cnoliincs . or pathogcns the. undergo maturation and miptc

98 into the regional lvmph node (151). We codd not confimi this due to the

uns\-ailabilin of a LC-specific marker in mousc. F o u d y , a part of this

population rnight be macrophages. Previous snidies demonstrated that GM-CSF

induce the differentiation o f bone marrow precursors not only to DC but also to

macrophages and neutrophils (56,145,152). 1 t is unlikelr that these cells wcre

neutrophds since there is no solid information showing MHC class II esprcssion

on thcsc cclls. These cells arc more likely to be macrophages that have been

induced to express hMC class II in the presence of Ghl-CSF.

DC and macrophages are believed to share transitional stages and the

c a p a c i ~ to derive DC from monoqtcs adds even more confusion as to how to

scparatc DC from monocytes (153). This close rclationship bem-een DC and

macrophages is reflected by a considerable phenohpicd o~crlap which m'akes

dis tinguishing between them more complicated (1 (14).

Athough several attempts werc camed out to identifi: thesc cclls, onlv

limitcd information was obtained. In one atccmpt, BM-DC wcrc stained u-ith a

numbcr of markers that are bclicred to be macrophage-spccific markers

including Mac-l (anti-CD1 1 b), CD32/CD16 (anti-FcyRII/II 1) and i\fOht.\-2

(anti-monoc~-tes/macrophages). Esamination br flow cvtometn. demonstrated

lou- (dim) cspression of thesc markcrs. It was difficult to interpret thcsc results,

99 sincc some studies have demonstrated weak expression of these markers on DC

hnother attempt involved the depletion of hIHC class II-cells that

dcvclop in response to GM-CSF; hoping that when the hIHC class II-negative

cells wcrc rc-cultured onlv DC-committed precursors would grow. In this

cspcrimcnt, Bhl-DC were han-ested on da- 3, h,U-iC class II-positive cells

dcplctcd, and thcn re-grown for 3 dars in thc presence of GM-CSF. Three days

latcr, cells wcre double stained for fascin and MHC class II and esamincd b y

flow cytomctq-. We found a slight decrease in the number of h,L-IC class II-

positk-c fascin-negatiw compared to thc non-depleted group. Ho~cver , thc

Jccrcase of hWIC class II-positive cells \vas accompanied by a decrease in the

numbcr of Çascin-positive DC. This suggests that the M-IC class II-positivc

fascin-ncgative cclls had gained the abilin- to grow W n .

FinaIl!-, u-c tricd to idcntifi thcsc cclls bv morphological csarnination. To

thk cnd, cells were han-csted on day 3, spun over 30' O-80° O pcrcoll and

colIcctcd from different lavers for cytoccntrihgation. Thc slidcs wcre thcn

staincd for Çascin and csamined by immunocvtochcrnistn-. Thc results

dcmonstratcs: i ) 1-c fca? cells passed through the 80'' 8 layer and the[- \rVerc smdl

and fascin ncgativc, similar to lymphocytes or ncutrophils in morphology; 2) few

cclls rcmaincd on the 30% laver and thcy werc large with fcw cspressing fascin

1 O0

but \vert obsen-ed to have vacuoles, suggesting that the- are dead DC; 3) most

cells were trapped in the 30°,'~-800/0 interface are of s d a r size but only feu7

express fascin. At present, non of the alternatives c m be escluded based on the

available data

4.5 B7-2 expression on BM-DC during maturation:

To confirm that the strong correlation benveen fascin and MHC class II

csprcssion \vas indeed correlated with DC maturation, w e esaMned a second

marker of DC maturit-y, BI-2, during Bhf-DC maturation. Bi-2 1s a co-

stimulatom molecule critical for T ce11 activation and is known to be up-regulated

during DC maturation (99)). To identifir the B7 family members, we used CTLA-

4Ig fusion protein which is known to bind both B7-1 and B7-2 molecules with

high al-idin (132). Dav 9 BM-DC were labelcd with the CTLh-41g and esamined

b!. flou- cvtomctq-.

\Yc found that dm- 9 BM-DC espress 87 molcculcs. However, this did

not diffcrcntiate between B7-1 and B7-2 expression. To determinc thc

csprcssion of 87-2 on BbI-DC during maturation, w e used an anti-B7-2

monoclond mtibody, and found an incrcase in B7-2 espression on BM-DC with

timc in culturc.

101 These data confirm that the increase ùi fascin espression, and the

corrclatcd increase in hfHC class II, was due to a n increase in the number of DC

that have rcached maturîty.

1.6 Fascin and MHC class II expression on BM-DC in the presence of

TGF-p and TNF-a:

One of the limitations of in vitro culture models of maturation is thc

conccrn that time in culture, rather thm maturation, could be rcsponsible for

obscn-cd cellular changes. To address this concem, we suppressed, o r enhanced,

B hl-DC maturation with additional growth factors. The prowth factor

combinations we used were: 1) GiLI-CSF alone, which pushes DC-precursors to

maturc into DC (65,130,1); 2) GhI-CSF +TGF-p, which has bcen shown to

supprcss DC maturation from bone marrow progenitors (68) and 3) Ghl-

CSF-TNF-ûr n-hich has b e m shon-n to enhance DC maturation from bonc

rnarrou. progenitors (68,8). For this purpose, bone marrow DC-precursors wcrc

Fou-n in thc prcsencc of the different growth factor combinations. Cells werc

thcn han-cstcd at days 0, 3, 6 and 9 and doublc stiuned for fascin and hIHC class

11. In al1 groups and at a n - tirnc o f csarnination, ail fascin-positi\-c DC werc

obscn-cd to cspress high lewA of MHC class II molccules, suggesting that thcse

102 fascin-positive cells are mature DC. Furthemore, fascin espression was

obsen-cd to increase on BM-DC over the time of esamination.

Similar effects on MHC class I I expression were obsen-ed on BM-DC

o-hen treated with these growth factor combinations. In line with their

irnmaturity, the group treated with GM-CSF+TGF-p vas obsen-ed to have a

mark reductïon in the nurnber of fascin- and PIMC class II-positk-e cells when

compared to those treated with GM-CSF alone. \Vhen esarnined in culture bv

light microscopy, the cells in this group were obsen-ed to be smaller and less

dcndritic than those treated with GM-CSF alone. The group treated with GM-

CSF+TNF-a, which enhances DC maturation, was obsen-ed to have more

fascin-positive DC when compared to the other nvo groups. Consistent with

their mahirïtr-, this group \vas obsen-ed to have more cells with typical DC

morpholog as assessed by light rnicroscopic esaminations in culture.

Although the number of MHC class II-positive cclls at days 3 and 6 \vas

more in thc group treated with GM-CSF+TNF-a, lesser numbers werc obsen-ed

on da! 9 comparcd to the group treated with GM-CSF done. Prcvious studics

havc shown that the addition of TNF-a to GM-CSF not only enhances DC

maturation from bone rnarrow prccursor cells but it suppresscs macrophage

diffcrentiation (1 54). In line with this, we obsen-ed that this group had more

fascin positil-e-cells and less hfiI-IC class II-positi\-c-cells on da? 9, suggcsting the

1 O3 prcscncc of more mature DC and a decreased nurnber of hDIC class II-positk-e

fascin-negative cells. Taken together, the results presented here provide strong

cvidcnces of a Iink between fascin espression and DC maturi. rather than

lcngth O f time in culture.

1.7 TheeffectoffascinlevelonBM-DCallo-stimulatoryactivity

From the above, strong evidence of a correlation benveen fascin

csprcssion and DC maturit-v ha\-e bcen provided. Furthemore, it has been

shonm in sel-cral pre\-ious studies that mature DC are superior in stKnulating T

c d prolifcration in hlLR assq-s (151). Given this, we asked whether chere is a

corrchtion bctween the nwnber of fascin-positive DC and the level of T ce11

stimulation. \Xc have already shown that da!- 9 cultures have more fascin-positive

DC than da- 6 cultures. As a first step to address this question, w e compared the

dlo-stimulaton- a & \ - i ~ of da! G and dar 3 DM-DC in an hLLR assa!. Prcvious

hUAR studics on mousc DC ha\-c measured naiw dogcncic T cc11 prolifcration

afrcr 1, 5 and 6 days of stimulation (63,155,156)- \'if pcrformcd a time course of

hU.R assars in Our laboratos and obsen-ed the highest I' ceIl proliferaaon aftcr

4 davs and thcrefore dl the MLR assays were measured on da!- 4. To this cnd,

bonc marrow DC-precursors were grown in the presence of GM-CSF and

han-cstcd on dars 6 and day 9 For hLLR assays. \Tc found the espccted increasc

1 O-l in ?' cc11 proliferatîon with increased stîmulator to responder ratios. Xe also

Found that the do-sbmulatow activitv of day 9-DC, which have more fascin-

positk-e cells, was markedly hîgher than that of da. 6-DC. This confimis the data

obtained From the prevîous expenments whîch link fascin espression with DC

manirih and suggests a potentid rok for this protein in the h c t i o n of DC as

--II) C.

\Yc hypothesized that increased numbers of Çiscîn-positive mature DC

a-ould correlate with stronger do-stimulatory activitv. To thîs end, Bhl-DC \i-ere

gcncrated in the presence of the different growth factor combinations studicd

aboi-e. TGF-p dl suppress fascin expression and DC maturation and TNF-a

u-il1 cnhance t h e m Dav 9 BM-DC, whîch showed the most sîgnificant difference

in the nurnber of hscin-positive DC after treatment with the additional

CI - tohcs , wcre added in graded doses to a f~xed number of naive ailogencic T

cclls. Regardess OF the treatment group, there was an increase in T cc11

prolifcration m-ith an increase în the number of fascin-positil-e DC. DC trcated

u-ith G hl-CSF+TN F-a were sho~vn to have stronger do-stimulatory a c t k - i ~

cornparcd to thosc trcated \ v î t . GM-CSF alone. In contrast, T ce11 prolifcntion

was markedly reduced in the group treated with GM-CSF+TGF-P, which WC

had shown to posses fcwer fascin-positive DC.

103 These data are consistent with a correlation bebveen fascin expression and

DC do-stimulation. It should not be forgotten that fascin up-regdation was

accompanied bu an increase in hLHC class II and B7-2 expression, the major

triggcrs for the initiation of MLR (1 57,158,l Sg,l6O,l6 1). Howcver, \ire might

nr'guc that it is fascin rather than hIHC class I I that correlates with the enhanced

DC do-stimulaton activity of the group treated with Ghf-CSF+TNF-a. This

group has more fascin-positiw and less hMC class II-positive cells than group

rrcatcd u-ith Ghl-CSF donc and -et wcre more potent do-stimulators.

[-Io\\-cver, one might dso argue that some of thc hItIC class II-positi\-e cells in

thc CM-CSF treated group might be macrophages which are not as potent [WC

as DC. Therefore, this fmding while demonstrating higher le\-els OF T cc11

prolifcration with an increasc in thc numbers of fascin-positive cell, docs not

cscludc thc contribution of MHC class II and B7-2 molcculcs in the initiation of

h LT,K

1.8 Isolation of fascin as a mediator of DC allo-stimulatory activity:

Thc data abore dcmonstrate a dircct corrclation bcnvccn fiscin

csprcssion and DC do-stimulation. Howcrcr, ther do not nilc out the

poss~bility that MtIC class II and B7-2 molecules might contrïbuted to initiatc T

cc11 prolifcration. To dcterminc the prccise rolc that fascin plars in DC dlo-

1 O6 stimulatom act iv i~ , we esamined the effect of a l t e ~ g its espression on DC

abdie to stimulate T proliferation in a . MLR assw. Fascin is involved in

dendrite development and the espression of long delicate dendrites is one of the

distinguishing features of DC. However, little yet is known about the rolc of

thcsc dendrites in DC-T ce11 interactions (102,123). K e hvpothesized that br

forming dendrites, DC may increase their surface a r a allowing morc T cclls to

bc cncountered. A single DC cm esprcss > 106 hMC class II molecules and

only intcracts with a maximum of 200-300 T cells. I t has also becn reported that

-1' ccils can be activated by a veq- small number of cornpleses of peptide with

MI-IC molecules displaycd on the surface of M C (162). Moreorcr, it sho\ved

that crosslinking of only \-en few T ce11 rcceptors is enough for T ce11 activation.

This suggcsts that there is morc than h[HC class II cspression involvcd in T

stimulation br DC. The best h o w n esample is LC which have been reported to

bc poor stimulators of T ce11 proliferation in scwral previous reports (163).

:\lthough these fascin-negative cclls espress MHC class II, the? do not possess

Jcndrites and, as such, they interact with fewer T cclls and harc reduced allo-

s tirnulatory activity.

To this end, BM-DC u-ere gencrated in thc presence of Ghl-

CS l'+antiscnsc oligonuclcotidcs, G hI-CS Ftcontrol oligonucleotides, or G hl-

(:SIc donc. The do-stimulatom activitv of day 9 BhI-DC from each group \vas

1 O 7 thcn compared in an hlLR assay. In al1 groups, we observed an increase in T ce11

proliferation with increasing DC numbers. Ho\vever, the do-stimulaton- actilitv

of the group treated with the GM-CSF-f-antisense oligonucleotides was markedlv

rcduccd compared to the other nvo groups. On the other hand, no significant

differcnce in the do-stimulation behveen DC treated with Ghf-CSF alone and

those trcated with GM-CSF+control oligonucleotides. This result demonstrates

that the inhibition of fascin expression in mature DC marliedly reduces their

allo-stimulaton- a&\- i . This confirms that fascïn plavs a significant role in

mcdiating the function of DC as M C .

f Ial-ing showed rcduced do-stimulatory activirv of the B hi-DC treated

with antiscnse oligonucleotides, we focused on confimiing the effect of the

antiscnsc oligonucleotides on façcin esprcssion. The usc of antiscnsc

oligonuclcotides has incrcased tremendously in the past Fe\\- )cars and has

bccomc onc of the top 10 emerging research areas (164,165). Thc exact

mcchanism of how antisense oligonuclcotides work is not Mly understood. Thc

nntiscnsc oligonuclcotides sclectivelv hybcidizes to its cognatc RNA and

intcrfcrcs with thc expression of the encodcd protein bu scverd difkrcnt

mcchmisms includuig blocking RN A transport, splicing, and translation (1 66). 1 t

is vcn. complicatcd process to design spccific antiscnse oligonucIcotides. Thc

scqucncc of the fascin antiscnsc oligonuclcotidcs WC used \vas obtained through

1 O8 a personal contact with Dr. Joseph Bn-an. Dr. Bn-an and his coworkes inhibited

fascin espression in rat neurons using 17 base pair antisense oligonucleotides

complementaw to the mouse fascin mRNA (116). \Xe needed, however to

confirm a decrease in fascin expression, using these antisense oligonucleotides, in

our B h 1-DC. To stabilize the antisense oligonucleotides, three base pairs at the 3'

and 5' cnd were phosphorothioated (167). As a control, w e used a reverse

scqucncc of the antisense oligonucleo tides.

To this end, BM-DC were generated in the presence GhI-CSF +control

oliçonuclcotides o r GbI-CSF tantisense oligonucleotidcs. Khen esamincd in

culture, WC obsen-ed less dendrites in the group treated with the antisense

oligonuclcotides. O n day 9, \ire compared the level o f fascin espression between

the ni-O groups using immunocvtochernistn Initidly, there was no significant

ciiffcrcncc in the number of fascin-positive cells ben\-een the antiscnse

oligc~onuclcotidcs-treated and the control oligonuclcotides-treated group.

Hou-cvcr, closer csarnination showed that DC treated u-ith mtiscnse

oligonucleotidcs cspress less fascin and are smaller than thosc treatcd with rhc

control oligonucleotides. This \vas reflectcd by generally lightcr fascin staining

and prcscnce of areas in the cvtoplasm devoid of fascin expression. Fascin \vas

also obscn-cd to bc clustcrcd into small circles within thc cytoplasm, suggesting

chat thc antiscnsc oligonucleotides ma!. alter the polymerization of thc actin

1 O9 filaments. In contrast, fascin \vas strongly espressed and erenlv distributed in the

cvtoplasm of the control oligonucleo tide-treated DC. This implies that the

antiscnse oligonucleotides have a partial nther than a complete effect on fascïn

espression.

Although higher concentrations of the antisense and the control

olipnuclcotides were tried, no differcnces were obsen-cd. This suggests that the

efÇcct of the antisense oligonucleotides may not be to inhibit fascin expression

but rather to interfere with the actin polvmerïzation, and thus change the

function of fascin. Furthemore, w-e esamined fascin espression bv

immuno fluorescence s taining, where FITC-labeled s trep tavidin \vas used instead

o Ç the streptaridin horseradish perosidase. The rcsults confirmed what has been

obscn-ed bv immunoc~tochemsitry esaminations.

\\'c nest esamined w-hether the antisense oligonucleotides affect hDIC

class I I csprcssion on the fascin-positive DC. The rcsults demonstrated no

sig-mificant diffcrences in bEIC class I I csprcssion among the three groups (data

not shown). Taken together, these rcsults demonstrated that the antisensc

oligonucicotidcs inhibit fascin expression but ha= no cffect on hIFIC class I I

csprcssion. This observation con fimis that the inhibition of the do-stimulaton.

actil-io- of thc antiscnsc oligonuclcotidcs treatcd Bhll-DC \vas indecd duc to

rcduccd fascin levels.

110 The results presented hereïn provide evidence that directly links fascùi

cspression in DC with stronger do-stimulaton- activitv. Athough the antisense

oliçonucleotides treated DC were observed to still espress MHC class II, thev

were less potent in the initiation of hlLR. This implies that although hMC class

I I is important for the initiation of MLR, the fornation of dendrites by DC is

esscntial to facilitate their interaction with T cells. Support for this is the

observation of a poor do-stimulatom actil-in. of immature DC, such as LC,

n-hich express MFIC class 11 but lack fascin (127,144). Thus it might be that

fascin cspccssion (dendrite formation) increases thc DC surface arca and

promotes thcir interaction with morc T cells. Moreover, it might bc that fascin

pro\-ides mechanical force through dlowing coordinated extension O f dendrites

to optirnixe DC interaction with T ceIl (103). In our laboraton-, u7e obscn-cd an

up-rcgdation of fascin expression in the DC that have intcracted with

parafomddhydc L ~ e d T cells, suggesting a cross talk behvcen DC and T cells.

Thc dendrite cstcnsion ma? play a rolc in stabilizing the interaction benvecn DC

and 1' cc11 that providc a sustaincd TCR signaling, an important stcp in T ce11

activation (168). It has been shown that T ce11 actin CI-toskclcton is a major

motor for sustaining signal transduction and driving TCR cross-linking (1 69).

Compatible with thcir fwiction as sentincl cclls, DC ma. esprcss dendrites to

coordinnte and direct their interaction with T cells (170).

111

FinalIr, the rearrangement of the actin cytoskeleton may be involved in

thc esprcssion of adhesion molecules which are important to ensure an intimate

-1' ccll contact or to facilitate DC migration (13). This possibility c m not be

cscluded as adhesion molecule espression \vas not investigated in this study. \lé

concluded from this esperiment that manipulation of fascin expression in DC

plays critical role in their h c t i o n as LWC.

4.9 Summary and conclusion:

DC, the most potent APC, have a unique ability to initiate a prirnaq-

immune response against tumors and infectious diseases. The ability of these

cclls to act as potent APC is criticaily dependent on their degree of maturity. In

this thesis, u-e ha\-e presented data shows that fascin is espressed in DC upon

maturation. \lé also providcd clidence that this protein plavs a significant role in

thc DC dlo-s tirnulatory actkin-.

In vivo, w-c demonstrated fascin espression in mature IDC of the lymph

nodc. Ho\i.e\-cr, fascin was essentially ne,gati\-e in immature DC (LC). In vitro,

n-c dcmonstrated an increase in fascin expression and a parallel increase of hMC

class I I and I37-2 expression on BM-DC dunng maturation. In an MLR

hnctional assar, \i-c showd stronger do-stimulaton- activitv with incrcase in thc

numbcr of fascin-positive-BM-DC. Morc specitically, we showed a mark

112

rcduction in the do-stimulatory activitv of BM-DC when fascin espression was

inhibited.

In conclusion, fascin espression is strongly linlied with DC maturation.

Inhibition of fascin expression in BM-DC dramaticailv reduces their d o -

stimulatory acti\-ity, reflecting the role of this protein in DC hnction as APC.

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