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A case of human B cell leukemia that implicates

an autocrine mechanism in the abnormal

growth of Leu 1 B cells.

N Kawamura, … , H Kikutani, T Kishimoto

J Clin Invest.

1986;

78(5)

:1331-1338.

https://doi.org/10.1172/JCI112719

.

Production of B cell growth factor (BCGF) from B-chronic lymphocytic leukemia (B-CLL)

cells was demonstrated. Freshly isolated monoclonal B-CLL cells expressed surface mu,

delta, B1, and Leu 1, but not Ba (an antigen expressed only on activated B cells). Upon

stimulation with anti-IgM, they secreted BCGF, which could act on anti-IgM-stimulated

autologous leukemic cells as well as anti-IgM-stimulated normal B cells. Cell lines

established from these leukemic cells also constitutively secreted BCGF. The BCGF from

B-CLL cells or established cell lines induced neither proliferation nor enhanced HLA-DR

expression in resting B cells. These results show the presence of B cell-derived BCGF,

which is distinct from BSF-1 and effective only on activated B cells. They also suggest that

an autocrine mechanism may operate in the growth of B-CLL cells.

Research Article

Find the latest version:

http://jci.me/112719/pdf

(2)

A

Case of Human

B

Cell

Leukemia That

Implicates

an

Autocrine

Mechanism in the Abnormal

Growth of Leu

1

B

Cells

NobuakiKawamura, Atsushi Muraguchi, AkiraHod,Yasuhiro Horii, Seiji Mutsuura, Richard R. Hardy, Hitoshi Kikutani, and Tadamitsu Kishimoto

Institutefor Molecular and Cellular Biology, Osaka University, 1-3, Yamada-Oka,Suita City, Osaka 565, Japan

Abstract

Production ofBcellgrowth factor

(BCGF)

from B-chronic

lym-phocytic

leukemia (B-CLL)

cells was demonstrated. Freshly isolated monoclonal

B-CLL

cellsexpressed surface A,

6,

Bi,

and Leu 1, but not Ba (an antigen expressed

only

on activated B

cells). Upon

stimulation with

anti-IgM, they

secreted BCGF, which could act on

anti-IgM-stimulated

autologous leukemic cells as wellas

anti-IgM-stimulated

normalB cells.

Cell

lines established from these leukemic cells also

constitutively

secreted BCGF. The BCGF from B-CLL cellsorestablished cell lines induced neither

proliferation

norenhanced HLA-DR

expression

in resting B cells. These results show the presence ofB cell-derived

BCGF,

which

is

distinct from BSF-1 and

effective only

on

activated

Bcells.

They

also suggest thatan

autocrine

mech-anism may operate in the growth of B-CLL cells.

Introduction

Recent

studies

have demonstrated the

involvement

of several soluble factors with different functions in the

growth

and

dif-ferentiation of

antigen-stimulated

Bcells

(

1-3).

Several ofthese

factors have been well

characterized

andthe gene

encoding

B

cell

stimulatory factor-I (BSF- 1),' which

was

originally

desig-nated Bcell growth

factor-I (BCGF-I)

has been

cloned

(4, 5).

BSF-l

shows synergy with

anti-IgM

in the

proliferation of resting

Bcells. BSF- 1

is

not

strictly

agrowth

factor;

however,

it

is

re-sponsible for

the

activation of resting

Bcells

(6-8). Therefore,

agrowth

factor for

Bcells comparable to

interleukin

2 (IL-2) for T cells

has

yettobe

identified.

Our

previous

report(9) as well as several

other

studies

(10-14) showed that

activated

B

cells

or

transformed

Bcell

lines

secreted B cell growth

factor(s)

that couldinduce the

proliferation of activated,

but not

resting

Dr.Mutsuura's address is Nakamura Municipal Hospital, 1-1-27,

Hi-gashimachi, Nakamura-City,Kohchi, 787, Japan.

Receivedfor publication 19May 1986.

1.Abbreviationsused in this paper: anti-s,anti-IgM;BCGF, B cell growth

factor; BCDF, Bcelldifferentiation factor; BSF-1, B cell stimulatory

factor-i;

CLL, chronic lymphocytic leukemia; EBV, EpsteinBarrvirus; EBNA,Epstein Barr nuclear antigen; FACS, fluorescence activatedcell sorter;FCS, fetal calfserum;FITC,fluoroisothiocyanate; y-IFN,gamma

interferon;HTLV,human T cell leukemia virus;IL-I,2, interleukin 1

or2;ISaIg, insoluble anti-immunoglobulin; MNC, mononuclearcells;

PBL, peripheralbloodlymphocytes; PHA,phytohemagglutinin; SAC,

StaphylococcusaureusCowanI;6MPDR, 6-methylpurine deoxyriboside; TdR,[3H]thymidine;TR, Texas Red.

B

cells, suggesting

an

autocrine

mechanism inBcell

proliferation

asin thecasefor T cell

growth.

In this

study,

wereportthe likelihood thata B

cell-derived

B cell growth factor (B-BCGF)maybe involvednotonlyin the

regulation

of normalBcells growth but also in the

proliferation

of

neoplastic

B cells. The results show that leukemic B cells

freshly

isolated froma

patient

with B-chronic

lymphocytic

leu-kemia (B-CLL) secrete B-BCGFupon stimulation with

anti-IgM

and thesameleukemic B cellsareresponsivetotheirown

BCGF

only following

anti-IgM stimulation. Although the

study

wasconductedonleukemic cells only fromasingle CLL

patient,

such results wouldaccountfor the

relatively benign

nature of B-CLL, since only in vivo antigen stimulation could induce ab-normal proliferation of those B cells. In this regard, the

likely

autospecificity

ofLeu I B cells and B-CLL is discussed.

Methods

Patient. Apatient (K.H.) with B-CLL wasstudied. Accordingtothe

stagingclassification of Rai et al.(15),thepatientwas instage0. At the

timeofthestudy, this patient didnotreceive any treatment. A profile of the blood picture extending from 22 March 1985to23July 1985 is showninTable I.

Reagents. StaphylococcusaureusCowan I(SAC)wasobtained from Hoechst Japan, Ltd., (Tokyo). Phytohemagglutinin-P (PHA)was

pur-chased fromDifco Laboratories (Detroit, MI). F(ab')2 fragment of goat anti-human IgM (anti-a) was purchased from Cappel Laboratories

(Cochranville, PA). Recombinant IL-I(rIL- 1)was agift of Otsuka

Phar-maceutical Co., Ltd. (Tokushima, Japan). Recombinant IL-2 (rIL-2)

wasprovided from Ajinomoto Co., Ltd., Tokyo. 1 U ofIL-I orIL-2was

definedastheactivity that gave 50% of the maximum proliferative re-sponse.Bcell differentiation factor(BSF-2)waspurifiedin this laboratory

from a human T cellleukemia virus (HTLV)-transformed T cellclone,

Na-I,aspreviously described (16). 100 pM of purified BCDFgave

max-imum response inaBCDF assay, in which Ig secretion from EBV-trans-formedBcelllinesorSAC-activated B cellswasmeasured (16). B-BCGF

waspreparedfromaBcellline,RPMI1788,asdescribed inaprevious

report(9).PHA-conditioned medium from T cells (PHA-T supernatant)

waspreparedbyamethoddescribedpreviously (17). Biotinated anti-HLA-DR andanti-Leu 1 monoclonal antibodieswereobtained from BectonDickinson Monoclonal Center, Inc. (Mountain View, CA).

Pu-rified anti-Tac monoclonalantibody(18)waskindly provided byDr.T. Uchiyama (Kyoto University). Insoluble anti-Ig (ISaIg)wasprepared by coupling 1mgpurified anti-IgM antibodytoI ml ofcyanogenbromide

(CNBr)-activated Sepharose 4B beads (Pharmacia Fine Chemicals, Uppsala, Sweden).

Animalsand cell lines. Female Balb/c mice were purchased from Shizuoka Laboratory Animal Center(Shizuoka,Japan)and bredinour owncolony. AnIL,2-dependent murine cytotoxic T cellline,MTH41.16,

was akind gift of Dr. Hamuro (Ajinomoto Co. Ltd., Tokyo).The EBV-transformed B cell line, RPMI 1788,wasoriginallyobtainedfrom Dr.

P. Ralph(Cetus Co.,PaloAlto,CA).Three celllines (KHB1, KHB2,

andKHB3)wereestablishedfromperipheralblood lymphocytes (PBL) ofapatient (K.H.)asdescribed in thetext,maintained in RPMI 1640 medium (FlowLaboratories,Irvine, CA) supplementedwith 10% fetal calfserum(FCS)(645, Gibco,GrandIsland,NY), 100U/mlpenicillin,

J.Clin. Invest.

©TheAmericanSocietyfor Clinical Investigation,Inc.

0021-9738/86/11/1331/08 $1.00

(3)

Table

I.

Analysis

ofPBL ofthe B-CLL Patient (KH.)

Date 3/22/85 5/1/85 7/23/85

RBC(X10-4) 548 525 518

Platelet

(X104)

12.8 14.9 14.5

WBC 33,600 46,700 41,000

Lymph (%) 79 90 77

Neu(%) 19 9 22

Surfacephenotypesof leukocytes (3/22/85)*

T3 8.0% Ia 91.1%

T4 6.2%

is

29.2%

T8 8.0% B. 91.5%

T1 16.7%

* PBL were analyzed by FACS 440 for expression of cell surface anti-gens.

100

jg/ml

streptomycin,2 mML-glutamine, and 5 X 10-5 M

2-mer-captoethanol.Themediumwasreplacedevery 3-4 d. These cell lines

werefound to befreefrom mycoplasma when screened by mycoplasma detection kit(Gen-Probe,SanDiego, CA)andthemycoplasma-mediated 6-methylpurine deoxyriboside (6MPDR) toxicitytestdesciibedby Proust

etal.(19).

Cell preparation. Peripheral blood mononuclear cells(MNC) from

the B-CLLpatient (K.H.)ortonsillar MNC obtained frompatientswith

chronictonsilitiswereseparated bystandardHypaque-Ficoll gradient centrifugation.Bcell-enrichedpopulations (E- cells)and Tcell-enriched

populations (E+ cell)were separated byrosettingMNCtwicewith

2-amino-ethylisothiouroniumbromide

(AET)-treated

sheepred bloodcells. E-cellswerefurthertreated withanti-monocyte(M206)antibody(20) andnewborn rabbitcomplement.Cells recovered after suchtreatment

usuallycontained >90% sIg cells,<0.1% E+cellsand monocytes, and were used aspurifiedB cells. In someexperiments, freshlyprepared tonsillar B cells or in vitro activated B cells that had beenincubatedwith either SACoranti-uantibodyfor 3 dwereseparatedintohigh density

cells (small Bcells)and lowdensityBcells(Bcellblasts) by usinga

discontinuous Percollgradient centrifugation. About 2X I07B cells werelayeredonthe topofthegradientsand tubeswerecentrifugedat

1,280gfor 15 minat4°C.Human Bcellswith low andhigh density

wererecovered infractionsbetween40/50%and60/70%,respectively. Cell staining andFACS

analysis.

I million cellswereincubated with

0.1 to I

jtg

of biotinated anti-HLA monoclonal

antibody

for 20mm,

washedthree times withstainingbuffer(RPMI 1640deficientofbiotin, riboflavin,andphenol red),incubated with Texas Red

(TR)-conjugated

avidinfor 20 min, and washed three times with

staining

buffer. Two-colorfluorescence-activatedcellsorter(FACS) analysiswascarriedout

bystaining cellswith

fluoroisothiocyanate

(FITC)-conjugated antibody

andbiotinated-antibodyfollowedbyTR-avidin. Stained cellswere

an-alyzedon aFACS440(BectonDickinsonCo.)

equipped

with bothan

argonionlaser

operating

at448nm

generating

forward scatter, fluores-cein,propidium iodide (PI),andlarge

angle

scatterandasecond argon ion laserpumpinganorganicdye lasercirculatingrhodamine 6G tuned

toemit 595nmlighttoexcite TR. List mode datawascollectedon 20-50XI03cellsfor eachsampleandanalyzedonaVAX 11/730computer

usingprograms

originally

developedatStanford

University (21).

Southern

blotting analysis. High

molecular

weight

DNAwas isolated

fromPBLof the B-CLLpatient (K.H.)orestablished cell lines and

di-gestedwith theindicated restriction enzymes.

Digested

DNA

fragments

weresize-fractionatedbyelectrophoresisin 0.5% agarose and transferred

tonitrocellulosefiltersbythe Southern

blotting

technique (22).

Filter-bound DNAfragments were thenhybridized to nick-translated

(32P)

probeandvisualizedonautoradiograms (23).The human Ig geneprobe

used inthisstudywastheIgheavychain

joining region

JHprobe (3.5

kbembryonicEcoRI-HindIIIJH-containing

fragment)

provided byT.

Honjo (KyotoUniversity) (24).

In vitroculture.To assayproliferation,cells (usually 105/well if not

specified)wereput in 100

id

of serum-free HBI01 medium (HanaMedia,

Inc., Berkeley, CA) inflat-bottomed microtiterplates(2596, Costar Data

Packaging, Cambridge, MA) and 100y1 of variousconcentrations of

IL-1, IL-2, SAC,anti-s,BCGF, BCDF,or testsamples were added. All cultureswereincubated for 60-70 hasindicated in thelegends.To assay

IL-I, thymocytesfrom 6-wk-old Balb/c mice were culturedfor3 dat a

celldensityof 2X 106/wellwith I

$&g/ml

PHAin the presence oftest

samples. To assay IL-2, an IL-2-dependent murine T cell line, MTH41.16,wasculturedat acelldensityof2X103/wellwithtestsamples for 24 h. All cultureswerepulsedwith I

AtCi

of

[3H]thymidine

(TdR) (15.1Ci/mmol:NewEngland Nuclear,Boston,MA)atvariousperiods

asindicatedinfigure legends,followedbyharvestingonglass filterpaper

byanautomatedcellharvester(Labo MashScience Co.,Tokyo)and

incorporationofradioactivitywasmeasuredbyaBeckmanscintillation

counter(Beckman Instruments, Inc., Fullerton, CA). All assayswere

performedintriplicate.

Preparation

of

culturesupernatant. Culture supernatants from PBL of the B-CLL

patient

(K.H.)wereobtained afterculturingthecells(106/ ml)for 48to72 hinmediumalone,withinsoluble anti-Ig(ISa Ig: anti-Ig coupledonbeads),orwith control beads(CB)in I mlofserumfree HBIO1 mediumina15.5-mmdiamdish(3047,FalconLabware).Culture supernatants from established cell lines(KHB1,KHB2,andKHB3)were

obtained by

culturing

the cells

(106/ml)

for 48 h in HBI01medium.The collected supernatantswerepassed

through

a

0.45-jsm

filter,followedby

dialysis against

PBS.Sampleswerefrozenat-20'C beforeuse.

Assays

for

'y-interferon

(IFN).

'y-IFN

activity wasassayed by the methodof

Yip

etal.(25)withsomemodifications,inwhichinhibition

of the

cytopathic

effect of Sindbis virus in human FL5. 1 cellswas

mea-sured.

Detection

of

EBNA.EpsteinBarrvirus nuclearantigen (EBNA)was

detected

using

astandardanti-EBNAsera

(26).

Freshly separatedPBL from K.H.wasfoundtobe

negative

withtheanti-EBNAsera,whileall threeestablishedcelllinesfromPBLofthe

patient

werefoundtobe

positive

with theanti-EBNAsera.

Results

Analysis ofsurface phenotypes

and Ig gene configuration ofPBL

from

the B-CLL patient

(K.H.).

The

surface

phenotype of the

PBL

from

K.H.

is summarized in Table I.

More than 90% of PBL

(26,500/mm3)

were Bcells as

judged by

BI

expression

and

the

percentage

of

T

cells

was

less than 10%

as

judged

by T3

expression. Two-color

FACS

analysis,

shown

in

Fig.

1,

A-D,

demonstrated that >90% of PBL expressed the

B1

antigen, but

didnotexpress the Ba

antigen (27).

Very few cells expressed the

Tac

antigen

orthe Leu 4

antigen.

Of

note

is

that

the

majority

of the cells

strongly expressed

both

IgM

and

IgD

on

their surface,

which is unusual

in the case

of B-CLL.

All

surface

IgM'

cells

expressed

Leu

1,

which isacommon

feature of B-GLL (28).

In

order

to

study

the

clonality

of the

B

cells, the

rearrange-ment

of

Ig

geneswas

examined.

The

DNA

from

patient

PBLor

from human

placenta

was

digested

with

Hind

IIIor

Xba

I

and

the

fragments

were

hybridized

with

the

3.5-kb

JH

Hind

III/Eco

RI

probe

(Fig. 2,

lane 1and

2).

The

Hind III restriction

fragments

of

patient

PBL gave a 6.0-kb

(kilobase)

band

(lane

2),

while

those

of

placenta

showed

an

expected

10.9-kb band

(lane 1).

The

Xba I

fragments

of

patient

PBL gavetwobands

of 6.6

and

6.0-kb,

respectively,

(lane 2),

whereas

thoseof

placenta

showed the

expected

6.6-kb germ line band

(lane 1).

Theresults show the monoclonal nature of the B cells in

this

B-GLL

patient (K.H.).

In vitro

proliferative

response

of

the

B

cells

from

the B-CLL

patient

(K.H.)

tovarious

stimuli. The

Bcells

from

the

patient

(4)

B

c1

e

I,lie lies

.1' 'l -I1 1,0

Leu4

.~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~

LB~ ~~Lu

F

Bi

IgM

Figure 1. Two-color FACS analysis of PBL of the B-CLL patient (K.H.). PBL separated by Ficoll-Hypaque gradient

centrifugationfrom the blood ofaB-CLL patientwere

stained with biotinated anti-Ba andFITC-anti-Bl.(A) Biotinated anti-Tac andFITC-anti-Leu4; (B)biotinated

anti-IgMand

FITC-anti-IgD,

(C) biotinated anti-IgM and FITC-anti-Leu 1 (D) and followed by TR-coupled avidin.

Anestablished B cell line (KHB1) was also stained with biotinatedanti-Leu 1 andFITC-anti-IgM(E); biotinated anti-Ba andFITC-anti-BI

(F),

followed by TR-coupled avidin.Analysis wasperformedon a FACS 440 witha

dual laser systemasdescribed in Methods. Contour plots represent the correlated expression of two-surface antigens byshowingpeak linesenclosingequal percentages of cells within the two-parameter distribution.

with anti-it, SAC, rIL-l, rIL-2,orBcell-derivedBCGF (9),and

theproliferativeresponsesweremeasured. As listed in TableII,

rIL-1, rIL-2,rIL- I plus rIL-2, SAC, and BCGF didnotinduce

anysignificant proliferationofthose B cells. Someproliferative

response wasobservedby anti-Igstimulation and the addition

of B-BCGF with anti-M synergistically augmentedthis

prolifer-ation.It isnoteworthythat Bcellsfreshlyisolated from thepatient

didnotexpressBa(Fig. 1A),which isexpressedonactivated B

cellsatGI phase,butnotonrestingBcells(27). These results

indicate thatfreshlyisolated leukemic B cellsareattheresting

stage andsodo notrespondtogrowth factors.However,

anti-Ig stimulationisabletoactivatetheseleukemic B cellstoastage

responsivetoBCGF.

Growth

factor production by

leukemicBcells

from

the

B-CLL

patient (K.H.).

Anti-M stimulation of the leukemicBcells

inthe absence of BCGF inducedasignificantproliferation (Table II), suggestingthepossibilitythat these cells couldsecreteBCGF

uponstimulation with

anti-is.

Tostudythispossibility, highly

purifiedBcellswereculturedinserum-free medium with

anti-Ig-coupled Sepharose beads (ISaIg) and culturesupernatantwas

examined for thepresenceof thegrowthfactoractivityon

au-tologousleukemic Bcells. Supernatant from such

ISaIg-stim-ulatedleukemic Bcells inducedproliferation of

anti-A-stimu-latedautologousBcells,whereassupernatantfromleukemic B cells incubated with control beads or medium alone did not

induceanyproliferation of anti-M-stimulatedleukemic B cells.

Representative data ofthree differentexperimentswith similar resultsareshown inFig.3.Itshould bepointedoutthat

super-natantfrom ISaIg-stimulatedleukemic B cells didnotinduce

anyproliferationofnonstimulatedleukemic B cells. The results

indicate that anti-M stimulation of leukemic B cells induces BCGFproductionaswellastheexpressionofBCGFreceptors,

thusleadingtoproliferationinanautocrinefashion.

Inordertoexamine whether the supernatant from

anti-Ig-stimulated leukemicBcells inducesproliferationof normal B

orTcells, purifiedBorTcells fromtonsilswereculturedwith

BCell Leukemia and B Cell Growth Factor 1333

1.1 i t2 t toe

Bl liOe

E

C

E

A

J)

I

(5)

T---A B

1 2 3 4 5

6.1

6-0-4 a s fit. 6.

Hind III

1 2 3 4 5

Xba

C Human Placenta

H X B H B X

I

I

I

1Kb J H probe

Figure 2. Immunoglobulin heavy-chain gene analysis of the

B-CLL-PBLand established cell lines. High molecular weight DNA isolated from patient PBL or established cell lines was digested with Hind III (A) or Xba I (B). Digested DNA was size-fractionated and hybridized with a 32P-labeled 3.5 kb Eco RI-Hind III JH probe. Lane 1, human placenta; lane 2, B-CLL-PBL; lane 3, cell line KHB1; lane 4, cell line KHB2; lane 5, cell line KHB3. Numbers indicate fragment sizes in kilobases (kb). (C) Restriction map of human placental JH region. The restriction endonuclease cleavage sites are shown as H (Hind III), X

(Xba I),and B(Bgl II).

the

supernatant in the presence or absenceofsuboptimal

con-centrations of mitogens.

The supernatant did not induce any

proliferation of nonstimulated

or PHA-stimulated T cells

(data

not

shown).

In contrast,

it induced

a

significant proliferation of

anti-is-stimulated

B cells in a dose-dependent fashion, but no response

with nonstimulated

B

cells (Fig.

4). The control su-pernatant

obtained

from leukemic

B cellsthat had been

incu-bated with control beads

didnot induce any

proliferation

of

anti-A-stimulated

Bcells. These results

again

demonstratethat

activated leukemic

B

cells

secrete

BCGF,

whichcanact

only

on

activated

B

cells.

It

has been

reported

that

IL-i, IL-2,

or

y-IFN

have significant

effect

onthe

proliferation

ofB cells(29-3 1).In order to determine

whether the supernatant from

anti-Ig-stimulated

leukemic B

cells

contained

any IL-IorIL-2

activity,

various concentrations

of the supernatant

from the

ISaIg-stimulated

leukemic B cells

Table II.

Effect of

Mitogens and

Lymphokines

on

the

Proliferation of

Leukemic

B

Cells

Proliferation

Stimulant* (StimulationIndex)t

Nil 1.0

IL-l

1.6

IL-2 2.6

IL-1+

IL-2

1.6

anti-p

4.6

BCGF 1.6

anti-p

+BCGF 24.0

SAC 1.6

* 1X

l05

cellswerestimulated withrecombinantIL-I(IU/ml),

re-combinant

IL-2

(1 U/ml),

anti-p

antibody (10

pg/ml),

BCGF(20%

vol/vol),

orSAC

(0.01% vol/vol).

i Cellswerepulsedwith 1

pCi/well

of

[3H]TdR

overthe last 16 h ofa

60-hculture.Backgroundcountsper minute obtainedbycells cul-tured inmediumalonewas1,230±56.

30

E

0

x

= 20_

0

4

_

0 0

00

10~~~~~~~~~~~

I-I

Figure 3. Effectofthe supernatant from ISaIg-stimulated leukemic B cellsontheproliferationof autologous leukemic B cells. Supernatant

wasobtained by incubatingB-CLLcells with ISaIg or control beads

(CB).The supernatant was addedtothe culture offreshlyseparated autologous leukemicBcells ataconcentration of 30% vol/vol in the presence or absence of

anti-p

antibody (10

pg/ml).

Cultures were

pulsedwith I

pCi [3H]TdR

over16 h ofa68-h incubation and

[3H]TdR

uptakewasdetermined. All data are shown as means±SEM

ofthetriplicatecultures.

E

a

I?

0

x

-c

0

0

0 0 c

I-I cr

10~

5

f~~~

I--

-~~~--E

I

I---zzL-o 10 20 30 40

% Supernatant

Figure4.Effect of the supernatant from ISaIg-stimulated leukemicB

cellsontheproliferationof normal B cells.Highly purified

tonsillar

B cells

(105/well)

werecultured with(a)variousconcentrations of the

su-pernatant from insolubleanti-Ig-stimulatedleukemicBcells in the presence(-

*)

orabsence(---A) of 10

pg/ml

anti-;

antibody;

(b)thesupernatant obtained from controlbeadsstimulatedleukemic Bcells in the presence (o o)orabsence(A- -

A)

ofI0O g/ml

anti-p-antibody.

Cultureswerepulsedwith 1

pCi [3H]TdR

over16h

ofa60-hincubationand

[3H]TdR

uptakewasmeasured. Resultsare

(6)

were

added

to

PHA-stimulated

murine

thymocytes

or to an

IL-2-dependent

T

cell

line, MTH 41.16 cells, and

proliferative

re-sponses

were

measured

(Fig.

5). Virtually

no

growth

activity

on

thymocytes

or a T

cell line

was detected in the supernatant

ob-tained from

the

ISaIg-stimulated leukemic

Bcells. The super-natant did not

contain

any

detectable -y-interferon

(data not

shown).

In

addition,

no inhibitory activity for IL-1, IL-2, or

y-IFN was

detected

in the

supernatant,

since the

addition

ofthe

supernatant did

not

diminish

those

activity

(data not

shown).

Establishment of

cell lines

from

the

patient's

PBL.

Purified

B

cells from

the

patient's

PBL were seeded at a cell density of

106/ml

in 10% FCS-containing

RPMI

1640 medium in culture

flasks

and

maintained

over

several months. Although

no

increase

in cell number

was

observed

in

cultured

cells over the first 3

wk,

a

population of cultured

cells started to grow with

doubling

time of 7-10 d hereafter. The growth

rate

gradually accelerated

and the doubling time reached

to48-72 h after 3 mo.

Three

different cell lines, referred

to as

KHB1,

KHB2, and

KHB3,

were

examined for

Ig gene

configuration

by Southern

blot

anal-ysis.

The rearrangement patterns of the Ig

gene in

all

three

cell

lines

were

exactly identical

to

that of the original leukemic

B

cells

(Fig.

2, lanes 3-5). FACS

analysis

revealed that all three

cell lines expressed Bl

as

well

as Leu 1

antigens

as

the original

leukemic

B

cells did, although the

expression

of

IgM

became

dull

(Fig.

1, E

and F). Of

note

is the fact

that

established

Bcell

lines expressed the

Ba

antigen, although

the

original leukemic

B

cells did

not. From these data,

it could

be

concluded

that these

cell lines

were

derived from

the

original

leukemic B cells, but atan

activated stage.

Production of

BCGF

by

the

established

cell lines. Cell-free

supernatants from cultures of the established

cell

lines (KHB1,

KHB2, and KHB3)

were

collected and examined for the presence

of BCGF activity.

As

shown in

Fig. 6,

the

supernatants

from

the

cell lines induced the

proliferation

of anti-j-stimulated normal

tonsillar

B

cells in

a

dose-dependent fashion.

It

is noteworthy

to

point

out

that these culture supernatants did

not

induce

sig-A

I05

!v v

I I I I25I 25 2 ~ co

B

2

Dilutions of Supernat

nificant

proliferation

of

B

cells in

the absence

of

anti-M

stimu-lation. Furthermore,

no

IL-l

or IL-2

(Fig.

4)

y-IFN

or BCDF

(BSF-2) activity

(data not

shown)

was

detected.

These data dem-onstrate

that these

established

B

cell lines

constitutively

produce

Bcell

growth factor,

which acts on

activated

Bcells.

Stimulation

of

the

established

B

cell

lines

with

ISaIg or

SAC did

notaugment the

production of BCGF (data

not

shown).

To

delineate

the stage at

which

KHB

1-derived

BCGF acts

during the cell cycle of normal

B

cells, small resting

B

cells

or

activated

B

blast cells

were

cultured with

KHB1

supernatant

and the

proliferative

response was

measured (Fig. 7).

KHB1

supernatant induced the

proliferation

of

B

blast

cells, which had

been

obtained by

anti-iu

or SAC

stimulation, but

not

resting

B

cells, suggesting

that BCGF in

KHB1 supernatant served

as a

progression signal in

the B

cell cycle.

Recent

studies

have

shown

that murine BSF-1 induces enhanced

Ia

expression

on

resting

murine

Bcells (6). In order to study whether

BCGF in

KHB1

supernatant could

act on

resting

B

cells, changes

in HLA-DR

expression

were

investigated.

Purified

B

cells

were

incubated

with

KHB1

supernatant

or

control supernatants and

HLA-DR

expression

was

measured by FACS

analysis (Fig.

8).

Therewas

no

increase

in the

expression of

HLA-DR in B

cells cultured

with

KHB1

supernatant,

whereas

there

was a

significant

en-hancement of

the HLA-DR

expression

on

resting

B

cells cultured

with

anti-iu

or PHA-T

supernatant

as

previously reported (9).

These data

indicate

that BCGF in

KHB1

supernatant delivers

a

progression

signal for

proliferation,

but notacompetence

signal

responsible for

the

activation of resting

B

cells.

Inorder to

study

whether the

established

cell

lines

are

able

to

respond

to

autologous growth factor,

KHB1

cells

were

cultured

with various concentrations of

KHB1

supernatant

and the

pro-liferative responses

were

measured (Fig. 9).

There was no

aug-mentation of

DNA

synthesis

in

KHB1

cells

by

the

addition of

autologous culture supernatant,

whereas

the

same

supernatant

could induce

or

augment the

proliferation

of

anti-,i-stimulated

B

blast cells

or

that of

a B

lymphoblastoid

cell

line,

RPMI

1788.

Figure 5.

IL-I

and IL-2activitiesin the supernatant from

ISaIg-stimu-lated leukemicBcellsorKHBI

cells.(A) Balb/c thymocytes (2

X

106/well)

werecultured with 0.02% PHA-Pplus

various

concen-trationsof the supernatantfrom ISaIg-stimulated patientBcells

(A A)or

KHB1

cells(a

*-).

MediumcontainingrIL- 1(30 U/ml)

wasusedasacontrol (o o). Cultureswerepulsedwith 1,uCi

[3H]TdR

overthe last 16 hofa64-h

incubationand

[3H]TdR

was deter-mined. (B) 5X 103ofan

I-2-de-pendentTcell

line,

MTH41.16,

O,

were cultured with various concen-trations ofthesupernatant from ISaIg-stimulated leukemicBcells (A A)or KHBIcells

(u

- *). Medium containing rIL-2 (2U/ml)

<^

wasusedas acontrol (o o). t-t 4.4*Cultureswerepulsedwith 1

MACi

[3H]TdR

over last 4 h of a24-h in-23 25 27

Sfl~,,e

cubation anduptakeof

[3

H]dR

23 25 27 c

wasdetermined. All dataare repre-sentedas

means±SEM

of

triplicate

a n t cultures. E

0. 0

0

co

0

0._

0 0 C

cc

(7)

&-Ea 0.

0

I0

x

C 0

0

0

0.

0 0 a

20

10

Vl

:-1

Ant*- + - + + - + +

12 12 25 25 12 12 25 25 12 12 25 25

Sup(%.) I

KHB-1 I L KHB-2 _J

L.

KHB-3

-Discussion

could

in(

leukemi(

This study

demonstrates

that

monoclonal

leukemic

B

cells de-

These re

rived

from

a

B-CLL

patient

secrete a

BCGF

upon

stimulation

could in(

with

anti-u.

Moreover, the BCGF

derived from leukemic

B

cells

without,,

Exp Cells Sup [3H]TdR Incorporation (cpmx103)

5 10

A RestingB

+

B Activated B

+

Figure7.Effect ofKHBl-supernatantontheproliferationofrestingB cellsvs.activatedBcells. SmallrestingBcellsseparated from tonsillar

Bcellfraction(ExpA),activated blast cellswhichhad been prepared byincubatingsmallrestingBcellswith

anti-n

antibody(Exp B),were

culturedwith orwithout 50%vol/volof KHB

l-supernatant

at acell densityof

105/well.

Cultures werepulsed with

[3H]TdR

(I

gCi/well)

overlast 16 hofa64-hincubation and

[3H]TdR

uptakewas

mea-sured. Resultsareshownasmeans±SEM oftriplicatecultures.

Figure 6. Effect of the supernatantsfromKHB1,

KHB2, and KHB3 cells on the proliferation of normal Bcells.Purifiedtonsillar B cells(105/well)were cul-tured with 12or25%of the supernatantsfrom KHB1,

KHB2,orKHB3 cells in the absence or presence of

anti-u

antibody(10 ug/ml). Cultures were pulsed with

['H]TdR

(1

gCi/well)

overthe last 16 hof a 60-h

in-cubationand

['H]TdR

uptakewasmeasured. Results

areshownasmeans±SEMoftriplicatecultures.

duce the

proliferation

ofanti-u-stimulated

autologous

c B cells aswell as

anti-ug-stimulated

normal

B

cells.

sults may

explain

the reason

why

anti-u stimulation

duce

a

significant proliferation

of the leukemic B

cells

addition of

growth factors;

anti-uinduced BCGF

pro-in the leukemic

Bcells and also

activated the cells

to a

ponsive

to

BCGF.

The

freshly

isolated leukemic cells

respond

toany

growth

factors in the absence

of

anti-u

ion,

indicating that the cells

were at

the

Go

resting

stage. Face

analysis with FACS

also showed that Ba

molecules,

re

expressed only

onactivated B cellsatG1

phase (27),

tdetectable onthese cells. This

might explain

the

rela-nign

nature of

B-CLL;

it is

likely

that stimulation of

B-Is

with the

appropriate antigen might

induce proliferation

cells

in

vivo. The

present

study

was

conducted

on

leu-ells from a

single

patient. Therefore,

while the results

rmative for

the

autocrine mechanism

in the

growth of

ells,

wehavetoadmit that it isnotclear how

applicable

Its are to other

CLLs.

B-CLL

cells in this

study coexpressed

B1 and Leu 1

(Fig. 1).

The latter

is

a

65-69-kD

glycoprotein

that was

oly

thought

tobe

expressed only

oncells of the T

lineage

iwever, the presence of Leu 1

positive

Bcells in normal tals

(33)

andthe

high

frequency

of

Leu

1-positive

Bcells in

autoimmune

patients (34)

has been demonstrated. CLL cells seem to

belong

to the category of Leu 1 B

). Lyl

Bcells in the mouse, which may be the counterpart

(8)

A B C

Fluorescence Intensity

Figure 8. Effect ofthe KHBl-supernatantonthe expression of

HLA-DRantigen. Highly purified tonsillarBcellswerecultured for48 h

with mediumalone ( ); 10

Ag/ml

of

anti-A

(-- -)(A); 25% vol/vol

ofPHA-stimulated T-supernatant(- -) (B),or25% vol/volof

KHBl-supernatant(-- -) (C).Culturedcellswerestained with

biotin-atedanti-HLA-DRmonoclonalantibody, followingby

TR-conju-gated avidine. Stainedcellswereanalyzed by FACS 440. Histograms representtherelativenumberof cellsasafunctionoffluorescence

in-tensity withinaparticulargated population.

(35), suggestingthatthese cells include autoantigen-reactive B

cellclones. Ifthis should be thecasefor human Leu 1 B cells,

autoantigens might be responsible for the induction of

prolif-eration of B-CLLcellsinanautocrine fashion; the autoantigen

activates resting B-CLLcellstothe responsive stage toBCGF

andinduces the production of BCGF, although this is speculative

without anyevidence. McGrath and Weissman (36) proposed

asimilar possibilityin

leukemogenesis

with retrovirus; they

sug-gestedthat leukemic cells have antiviral specificity and the

en-A

E

CL

0

I

r,

B

10 20 40

C

30-20

10

10 20 40

% Supernatant

Figure9.Effect ofthe KHB1-supernatantonthe growth of normalB

blastcells,RPMI 1788 cellsand KHB1 cells. IO'ofnormal Bblast

cellsthat had beenincubated with

anti-A

for48 h (A); 103ofRPMI

1788cells,(B)or5 x 103 ofKHB1 cells, (C)werecultured with

me-dium alone(c);various concentrations ofthe KHB I-supernatant

(a .)orRPMI 1788-supernatant( AA).Cultureswere

pulsedwith[3H]TdR(1 MCi/well)overthe last 16 h ofa60-h incuba-tion and[3H]TdRuptakewasmeasured.Resultsareshownas

means±SEM oftriplicatecultures.

velope

of the

retrovirus stimulates those

lymphocytes

into

growth.

Incontrast tothe

freshly

prepared

leukemic B

cells,

the

es-tablished cell lines

expressed

Ba molecules and could

constitu-tively

secreteBCGF without any stimulation.

However,

BCGF didnot

augment

the

proliferation

of the established cell lines. The result doesnotprove but

strongly

suggests that the autocrine mechanism maynot

operate

any

longer

in the continuous

growth

of the established cell lines. Inan

IL-3-dependent myeloid

cell

line,

itwasshown that transformation with Abelson virus

ab-rogated

the factor

dependence

of those cells

(37).

In this

study,

leukemic B cells appeartobe transformed with

EBV,

since EBNA

wasdetected in these cell lines.

Therefore,

the situation may be thesame asthat observed with the

IL-3-dependent

cell lines. Growth factor

independence

andautonomous

growth

of

trans-formed cells

might

be dueto constitutive expression and acti-vation of the membrane receptor thatserves as atransducer of the extracellular

signal.

Alternatively,

it

might

be due to the continuous

expression

ofintracellular

signals

that

eventually

leadstocell division.

The BCGF derived from the leukemic B cells appearstobe different from IL- 1or

IL-2,

since therewas no

stimulatory

effect

onPHA-stimulated murine

thymocytes

or

IL-2-dependent

cell

line,

MTH 41.16 cells

(Fig. 5).

No

IL-l

orIL-2

inhibitor(s)

in the supernatant was

detected,

since the supernatant didnot in-hibit

IL-l-induced

growth

of

PHA-stimulated thymocytes

or

IL-2-dependent growth

of MTH41.16 cells

(data

not

shown).

Moreover,

recombinant IL- 1 orIL-2 didnotinduce any

prolif-eration of B-CLL cells in the absenceorpresence of anti-s

an-tibody (Table

I and

unpublished data).

A

previous study

(9)

demonstrated the secretion of BCGF from SAC-activated normal B cells orB

lymphoblastoid

cell lines. It showed that B cell-derived BCGF

(B-BCGF)

from those cells could induce

prolif-eration of SAC-or

anti-Ig-activated

B cells and sustain

growth

of activated B cells. B-BCGF from these cell lines could also

augment

the

growth

of

autologous cells;

itinduced

colony

for-mation of

autologous

cells in soft agar as well as

augmented

DNA

synthesis

of

autologous

cells. The presentaswellas

pre-vious results indicate that normal B

cells,

transformed B cell lines and leukemic B cells can secrete and respond to B cell

specific growth factor(s).

At

present,

it isnotclear whether the

same molecules are secreted from normal activated B

cells,

transformed B cell lines and the leukemic B cells.

BCGF(s)

seem

tobe very

heterogeneous

with regardtotheir molecular

weight

and isoelectric

point (9,

1

1).

It is thus unclear whether BCGF

activity

is associated witha

single

factoror

multiple

factors. This may be answered when the

gene(s)

for

BCGF(s)

is cloned. How-ever, the

immunological

functions of these factorsare

similar;

(a)

they

act

only

onactivated B cells and induce

proliferation

butnot

Ig

secretion, (b) they

donotinduce proliferation orIa

expression

of

resting

B

cells,

and

(c) they

donotshow any

activity

in

BCLI

assay.

The

present

as well as

previous

results

(9)

obtained with normal and leukemic B cells show that B cells can secrete their

own

growth

factors and

strongly

support the notion thatan

au-tocrine mechanism similarto the

growth

ofT cells might be

operating

in the growth of B cells.

Acknowledgments

We wish to thank Drs. K. Nakajimaand T. Hiranoof thislaboratory

for BCDF assay, Dr. Wada ofShionogiPharmaceutical Co., Osaka, for

,y-IFNassay, Dr. E. L. Barsumian forafruitful discussion and Ms.Kyoko

ID mE

._

z

0

0

(9)

Kubota, JunkoMori, and Masako Kawatafor their excellent editorial assistance.

This work was supportedby the grant from the Ministry of Education, Science, and Culture.

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Genetic factors that increase susceptibility to oxidative stress, endothelial dysfunction and, possibly, stroke include Angiotensin Converting Enzyme gene deletion

both KRAS -mut) cancer cell lines exposed to increasing concentrations of dabrafenib, trametinib, or their com- bination (at a fixed 1000:1 ratio) for 72 h, simultaneous inhibition

By combining in vitro analysis with the generation of conditional mouse models expressing the fusion kinase ITK-SYK in a cell-type specific manner, it is shown that ITK-SYK

As stated above we are interested, in this paper, on the propagation characteristics of the IA wave in a plasma composed of protons and electrons (of solar wind origin) and