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Discordance between transferrin receptor

expression and susceptibility to lysis by natural

killer cells.

K R Bridges, B R Smith

J Clin Invest.

1985;76(3):913-918. https://doi.org/10.1172/JCI112089.

Expression of the transferrin receptor on target cell lines has recently been implicated as a

determinant of susceptibility to cytolysis by natural killer (NK) lymphocytes. We have

examined this proposed relationship in several ways. First, K562 (a cell line highly

vulnerable to NK lysis) cells were grown for 24 h in the iron chelator desferrioxamine. Under

these conditions, the cells doubled their surface transferrin receptor expression as

determined both by radioligand binding and surface binding of the OK-T9 monoclonal

anti-transferrin receptor antibody. In contrast, cells grown for the same period of time in hemin

halved their receptor expression. This fourfold change in transferrin receptor expression

between the desferrioxamine-treated and hemin-treated cells produced no change in

susceptibility to NK cytolysis. Second, HeLa (a cell line which in its native state is very

resistant to NK cytolysis) cells were compared with K562 cells with respect to surface

transferrin receptor expression. The difference in NK susceptibility of the two cell lines was

not reflected in differences in transferrin receptor expression: the K562 cells expressed

approximately 1.5 X 10(5) receptors per cell while HeLa cells expressed 2.0 X 10(5)

receptors/cell. Third, infection of HeLa cells by measles virus greatly increased their

susceptibility to NK lysis but produced no change in surface transferrin receptor expression.

Furthermore, when measles-infected HeLa cells were grown for 6 d […]

Research Article

Find the latest version:

(2)

Discordance between Transferrin

Receptor Expression

and

Susceptibility

to

Lysis

by

Natural Killer Cells

Kenneth R. BridgesandBrian R.Smith

LaboratoryofHoward Hughes Medical Institute,DivisionofHematology, Brigham andWomen'sHospital;DivisionofPediatric

Oncology, DanaFarberCancerInstitute;andDepartmentsofMedicineandPediatrics,Harvard MedicalSchool,Boston,Massachusetts 02115

Abstract

Introduction

Expression of the transferrin receptoron target cell lines has recently been implicated as a determinant of

susceptibility

to

cytolysis bynaturalkiller(NK)lymphocytes.We have examined thisproposed relationship in several ways.First, K562(acell linehighly vulnerabletoNKlysis)cellsweregrownfor24 h in theiron chelatordesferrioxamine. Under these

conditions,

the cells doubled their surface transferrin receptor

expression

as

determined both byradioligand bindingand surfacebindingof the OK-T9 monoclonal anti-transferrin receptor

antibody.

In contrast, cells grown for thesameperiodoftime in hemin halved

their receptor expression. This fourfold

change

in transferrin receptor expression between the

desferrioxamine-treated

and

hemin-treated cells producednochange in

susceptibility

toNK

cytolysis.

Second,

HeLa

(a

cellline whichin itsnativestateis veryresistantto NKcytolysis)cellswerecompared withK562

cellswith respecttosurface transferrinreceptorexpression.The

differencein NKsusceptibility ofthetwocelllineswasnot

re-flected in differences in transferrin receptor

expression:

the K562 cellsexpressed -1.5X 105 receptors per cell while HeLa cells expressed 2.0 X 105

receptors/cell.

Third, infection ofHeLa cells by measles virusgreatly increased their susceptibility to

NKlysisbut producednochangeinsurface transferrinreceptor

expression.

Furthermore,

whenmeasles-infectedHeLacellswere

grownfor6dinmedium supplemented with iron-saturated human transferrin theyunderwenta50% reductionin receptor

expres-sionbutnochangeinNKsusceptibility. Finally, possible alter-ations in thesurface expression ofNK targetantigensonmodified

cellswerefurther assayed by their abilitytoserveascold-target inhibitorsof cytolysis of NK-sensitivetarget cells.Weexamined

two groupsofcells inwhich transferrinreceptorexpression was reduced. These were the transferrin-treated, measles-infected HeLa cells with the50% receptor reduction, and K562 cells grown in mediumcontaining heminand iron salts where the re-ductionwasfive- tosixfold relativetocontrol. Inneithercase was there a change in the apparent expression of NK target

antigen(s).

We conclude that there is a discordance between transferrin receptor expression and susceptibility to NK cytolysis in the model systems examined. Therefore, it is unlikely that the

trans-ferrinreceptor perseisthe target

recognition

structurefor human

NKcells, althougharoleinconcert with other, as yet undefined molecules, cannot be excluded.

Receivedforpublication20September1984 and inrevisedform15 March 1985.

Natural killer(NK)' cellsare apopulation of lymphocytes de-finedbytheirabilitytokill certain virus-infected cells and tumor cells withoutprior antigenexposure (1, 2). Althoughthe exact function of NK cells isnot certain, speculation centers on a

possiblerolein immune surveillance of transformedortumor cells (3, 4). The target antigen(s) for the NK cell is unknown. Several studies, however, suggest that the transferrin receptor is therecognitionsite for NK cells(5, 6, 7, 8).This receptorserves

asthe physiological mode of cellular iron uptake by binding

iron-laden transferrin (9). Iron isanessential element in

metab-olism,as acomponent of heme in enzymes such asperoxidases

and cytochromes, and alsoas acomponent of nonheme, iron-containing proteins. Proliferating cells have increased numbers oftransferrin receptors when compared with cells in restingphase (10, 11), which presumably reflects the increased iron

require-ment of growing cells. That the transferrin receptor might be the target in proliferating tumor cells recognized by NK cells is then, atleast, plausible. This interaction would allow the NK cellstodestroytumorcellsat anearly stage in their growth and thereby retard the development of malignancy.

Recently developed techniques allow a modulation of the expression of transferrin receptors in cells in tissue culture. HeLa cells grown in iron-supplemented medium or in medium

con-taining human transferrin undergo a reduction in transferrin receptor expression, as do K562 cells grown in hemin (12, 13). Incontrast, K562 cells grown in the presence of the iron chelator desferrioxamine show a marked increase in transferrin receptor expression (14, 15). This ability to modulate transferrin receptor expression in a single cell type provided an opportunity to test foracorrelation between receptor expression and susceptibility

tolysisby NKcells,particularly since theK562 linehas been widely used as a target for NK studies (1). Inaddition, compar-isons could be made of the transferrin receptor expression in NK-sensitive K562 cells, in NK-resistant HeLa cells, and in HeLa cells modified by measles virus infection, which are thereafter rendered NK-sensitive(2).

Methods

Cells. K562 cellswere grown at370Cina5%CO2 atmosphere in RPMI 1640medium (M.A. Bioproducts, Walkersville,MD)supplementedwith

L-glutamineand10%ofetalbovineserum(FBS;Gibco Laboratories, Grand Island, NY). The cellsweremaintained ina log-phase growth at a

con-centration of 5X 105 cells/mlbydaily dilutionswithan equal volume

of medium.HeLacellsweremaintained inthe samemediumand were

keptat subconfluent levels. MonolayerHeLa cells were convertedto

suspensions by removing the growth mediumandexposingthe cellsfor

15 minat roomtemperature to 10 mM EDTAin phosphate-buffered

saline(PBS).Afterthe HeLa cells were loosened from the plates,they

l-Abbreviationsusedinthispaper: NK, natural killer. J. Clin. Invest.

©TheAmericanSociety for Clinical Investigation,Inc.

0021-9738/85/09/0913/06 $1.00

(3)

were returned to the RPMI/FBS mediumfor assay of surface transferrin receptors orsusceptibilityto NKcelllysis(see below).

Transferrin saturation and radiolabeling. Apotransferrin (Calbi-ochem-Behring Corp.,LaJolla, CA)wassaturated with iron as previously

described using nitrilotracetateand ferricchloride (SigmaChemicalCo.,

St. Louis,MO)(16). The diferric transferrin was separated from unin-corporatediron on a PD-10 column (Pharmacia Fine Chemicals, Pis-cataway, NJ). A ratio of A 465 nm/A 280 nm of 0.045 indicated full

saturation,and preparations with a ratio of <0.040 were discarded. Di-ferric transferrin was labeled with125Iusing lodogen beads (Pierce Chem-ical Co., Rockford, IL) aspreviouslydescribed (14). The specific activities rangedfrom 5,000 to 15,000cpm/pmolof protein.

Transferrin binding studies. Cell surfacetransferrin binding studies

were performed as previously described(17).Inbrief, cells suspended at aconcentration of107/mlin RMPI 1640 medium containing 1% FBS were chilled to40Cat which time '251-diferrictransferrin was added to afinal concentration of 100nM.After a20-minincubation, the cells were pelleted through an oil cushion of dibutylphthalate/mineraloil (9: 1ratio) and the tube tips containing the cell pellets and the cell surface-bound'25I-transferrinwere severed and countedusing a gamma counter (8000; Beckman Instruments, Inc., Fullerton, CA). Nonspecific binding was determined with a parallel set of tubes towhich unlabeled diferric

transferrinwas added to aconcentration of 10 ,M 15minbefore adding

'251-diferrictransferrin. The upper aqueous phase containing free125[. diferrictransferrin was also counted in some cases.

QK-T9

antibody surface binding

studies. Anti-transferrin receptor

OK-T9 monoclonal antibody (Ortho, Raritan, NJ) was resuspended in 1 ml of physiological saline. -2 X 106 cells werepelleted at 200 g in 12

X75 mm polystyrene tubes in an RC-2Brefrigeratedcentrifuge (Beckman Instruments, Inc.) and were placed on ice after the removal of the

su-pernatant.TheOK-T9antibodywasdiluted1:5 and 50

Al

ofcold antibody solution was added to the cells with vortexing and a 30-min incubation onice. The cells were washed three times, each with 1 ml of ice-cold PBS. Thesolutionwas decanted after the last wash and 100Mlof

fluo-rescein-isothiocyanate-conjugated goat anti-mouse antibody

(Becton-Dickinson& Co., Oxnard, CA) was added withvortexing and a second 30-min incubation onice.Thecells were washed twice with cold PBS, thenfixedbyadding0.5 mlofPBS and 0.5 ml of 2% paraformaldehyde. The cells were vortexed and stored at4°Cuntil the fluorescence pattern couldbeanalyzed withthe flowcytometer (FACS Analyzer,

Becton-Dickinson& Co.). The amountsofOK-T9 antibody and fluorescent goatanti-mouse antibodyused weretitered andfoundto besaturating fortheirrespectiveantigens.

AssaysforsusceptibilitytoNKcytolysis. Human blood cellswere

collected inpreservative-free, heparinized tubes from normal donors.

Monocyte-depleted mononuclearcellswereprepared byincubationof

thewholebloodwith carbonyliron (Lymphocyte Separator Reagent;

Technicon InstrumentsCorp., Tarrytown,NY)at37°Cfor 1 hfollowed

by

layering

on a

Ficoll-Hypaque

cushion,centrifugationfor 40min at

1,200rpm, and removalofthemononuclear cellsattheinterface removed aspreviously described(18).Thesecellswereusedaseffector

lymphocytes.

Target cells(HeLa, measles-infectedHeLa,and

K562)

werechromated for 1 h at370C with

5"Cr

(New

England Nuclear, Boston, MA)

ata

concentration of 0.1 mCi/106cellsandwashedfourtimesbeforeuse.

Targetcells were plated in round-bottom microwell tissue culture plates (FlowLaboratories, McLean,VA)at aconcentration of 2.5X I04

cells/well.Effectorlymphocyteswereaddedat

effector-to-target

ratios of 1:1, 2.5:1, 5:1, 10:1, and20:1 intriplicatewells for each cellline;the volume wasadjusted to200 ulby

adding supplemented

RPMI 1640

medium;and theplateswereincubated for 6or24 h at37°C.Background

and 100%chromiumreleaseweredetermined

by respective

incubation oftarget cells either with medium aloneorwith 1% TritonX-100

(IG-EPALCA-630; GAFCorp, New York,NY) detergent.

Supernatants

wereharvestedusingasupernatantcollection system (Titer-tek;Flow

Laboratories, McLean,VA)andchromiumreleasewascounted witha

spectrometer(Biogamma II; BeckmanInstruments,Inc.).Chromate

re-lease into the supernatant was a measure of target cell

lysis.

Percent

cytolysiswascalculated

according

tothe formula:

(cpm

-

background

Figure 1. Cell surface transfer-°03_rinreceptor expressionin

con-"Z9

trol,

desferrioxamine-treated,

--I

rand

hemin-treated K562 cells.

Desferrioxamineandhemin

2-f wereaddedtogroups ofcells,

intriplicate,tofinal

concentra-tions of60 and20gM, respec-I tively. Athird group of cells

E

| servedascontrols. The cells

wereincubated for 24 hat

- 370C.

They

werethen

pelleted

<c-_S< a -- at

200

g,thesupernatantwas

removed,and the cellswere

re-suspendedin ice-cold RPMI 1640 mediumcontaining1% FBS. The

bindingof'251-diferrictransferrintosurfacereceptorswasperformed asoutlined in thetext.Theplottedvaluesarethemean±SD.

cpm)/(detergentcpm-background cpm)X 100.Spontaneouslysisin

allexperimentswas<5%.

Results

K562

cell

studies. K562 cells were grown for24 h in

control

medium orinmedium

supplemented

either with 60 uM des-ferrioxamine orwith 20 uMhemin. Thesetwoagents had no

effectontherateof cell

growth during

this

time,

but

markedly

altered transferrinsurface

binding capacity (Fig.

1).

Therewas afourfold differenceintransferrin receptor

expression

between the

desferrioxamine

and thehemin-treated cells. Therewere no

morphological

alterations inthecells.

The

transferrin

surface

binding capacity

isafunctional mea-sureofreceptor number. Itwas

possible, however,

thatNKcells

might

recognize functionally

inactive transferrin receptors on

thecell surface. The OK-T9 monoclonal

antibody

bindstothe

transferrin receptoratasite differentthan the

transferrin-binding

domain

(19).

This

antibody

wasusedtodetermine whetherthe hemin-inducedanddesferrioxamine-induced

changes

in

func-tionalreceptor

expression

reflected

changes

in

immunoreactive

receptoronthe

cell

surface.

Cellsgrown for24 h incontrol medium and in

desferriox-amine-supplemented

or

hemin-supplemented

medium were

chilled, washed,

and labeled witha

saturating

concentrationof

OK-T9

antibody,

followed

by

fluorescein-labeled

goat

anti-mouse

antibody.

Fig.

2shows the resultsof suchan

analysis

of

desferrioxamine-treated

and hemin-treatedcells. Mean

log

flu-orescence

intensity

inthehemin-treated cellswas90

(arbitrary)

Uvs. 133 in the desferrioxamine-treated cells. The

logarithmic

Figure2. Cellsurfacetransferrinreceptor

an-H D

tigen

in desferrioxamine-treated and

hemin-treatedK562 cells. Cellswereincubatedwith

(4)

amplifierinthisexperimentwas setsuchthat achangeof 20 U

in themeanfluorescence correspondedto atwofold changein theabsolutelevel offluorescence. The 43-U differencebetween the hemin-treated anddesferrioxamine-treated cells thus

indi-catedafourfold changeinthemeanlevelofantigenicreceptor

expression. This correlatedwell with thechangesseen in

func-tionalreceptorexpression. Thus,thechangeintransferrin surface

binding capacityseen inthe treated cells reflected achangein the amount of receptorproteinonthe cell rather thanan

acti-vationorinactivation ofreceptors.

Susceptibility to lysis byNKcells wasdetermined for the

variousK562 cell groupsby achromium releaseassay. As

de-scribed inMethods,target cells labeled with

5"Cr

wereincubated with enriched populations ofhuman lymphocytes containing

NKcells. In three separateexperiments,thesampleswereassayed forchromium release as a measure of target celllysisafter 6 and 24 h.Fig. 3 isa

representative

6-h

killing

assay

showing

thatthe K562 cellswerehighly susceptibletolysis under theseconditions. Anincrease in

transferrin

receptornumberby desferrioxamine

or adecreaseby heminproducednochange inNKcell

suscep-tibility.Allthreeexperiments produced identical results. Given the fourfold differenceinthe level of receptorexpressionbetween

the desferrioxamine-treated and hemin-treated cells, the data

suggest that this receptor is not the sole determinant of NK cell

susceptibility.

HeLa

cell studies.

HeLa cellsare

relatively

resistanttolysis byNKcellsexceptwhen alteredby measles virus infection (2).

When soinfected,a two- tofourfold increased susceptibilityto

lysis byNKcellsis observed. The levelsof transferrin surface

receptorexpressionweretherefore compared inaHeLacontrol celllineand ameasles-infected HeLa cell line

(Fig.

4). The bind-ingcurvesperformed withincreasing concentrations of

'251-di-ferric transferrin showed that bothgroupshad -2X 105 recep-tors per cell. The fact that the curves plateaued at the same

transferrin concentrations indicatedno

significant

difference

ex-isted inreceptor

affinity

for transferrin. Attempts to increase transferrinreceptor

expression

inHeLacells by desferrioxamine

treatment wereunsuccessfulasthe cellsfailedtosurviveexposure tothechelator.Measles-infectedHeLacellsgrowninthe presence

of humantransferrin showedasignificant reduction in transferrin

receptorexpressionto -1 XI05receptors percell (Fig. 4), similar toresults

previously

notedfor noninfected HeLa cells(12).The amountof

transferrin-receptor

antigenonthesurfaces ofthese

three groups of cells was also measured by OK-T9 antibody

binding

andFACS

analysis.

Themeanvaluesof log

fluorescence

intensity

forthecontrol andmeasles-infected HeLacells were 127 and 123 U, respectively. The human transferrin-treated

HeLacells had a mean valueof

101

U,indicatinga 50%

reduc-tionintransferrinreceptorexpression.Aswiththe K562cells,

Figure 3.NKcelllysisof

con-trol(o),

desferrioxamine-treated(-), andhemin-treated

(A)

K562 cells. Cellswere

grown for 24 h incontrol,

des-ferrioxamine-treated,or hemin-treatedmediumasinFig.1.At that timetheywereassayed for

susceptibilitytolysisbyNK

cellsatvaryingNKeffector to K562 target cell ratios as de-scribed in thetext.

^ 25

20

15 -.A

~10

57.

10 20 40 60 80 100 120

-'5I-TRANSFERRINCONCENTRAT/OW(nM)

Figure 4. Transferrinsurfacebindingprofiles ofHeLacells,

measles-infected HeLa cells, and humantransferrin-treated, measles-infected

HeLacells. Humandiferric transferrinwasadded tosubconfluent

cul-turesofmeasles-infectedHeLacellsto afinal concentration of 7.5

MM.

The cellswereincubatedat37°C for 6d,alongwith untreated

measles-infectedHeLa cells anduninfectedHeLacells. The culture

media werechangedevery2 d. After 6d, all the cellswerewashed

with RPMI 1640medium with 10% FBS andwereallowedto

incu-bate foranadditional 4 h. Themediumwasthen removed and the

cells weredetachedfromtheplate withanEDTA/PBSsolution. The

cellswerethenresuspendedat aconcentration of107cells/mlin

RPMI 1640/1% FBS. 1/2-mlaliquots ofthe cellswereincubated inice

withincreasing concentrations of'25-differictransferrin,andsurface bindingwasdeterminedasdescribedinthetext.(o), uninfected HeLa

cells;(.),measles-infected HeLa cells; (A), humantransferrin-treated,

measles-infected HeLa cells.

thechangesinthe level oftransferrin-receptor antigencorrelated wellwith thechangesintransferrinbinding capacity.

HeLacellinfection by the virus drastically increasesNKcell

susceptibility as shown in Fig. 5 A. And yet, the number of

transferrinreceptorsexpressedinHeLa cells andmeasles-infected HeLacellswasidentical

(Fig.

4). Viral infectionproduces sub-stantial changes in the cell membrane,

particularly

with the

expressionof new surface glycoproteins. Therewas, however,

nochange in theexpression of the transferrin receptor, either

(1)

-.1

Q(.2

K

(Z5

100

80_

60_

40_

20_

2.5:1 5:1 10:1 20:1

EFFECTOR/TARGET CELL RATIO

Figure 5.NKcelllysisofgroupsof HeLa cells.(A) Lysisofuninfected

HeLa cells(o),measles-infected HeLa cells(.),and K562 cells(A)at

varyingNKeffector cell-to-target cell ratios.Theassays aredescribed

inthetext.(B)NKcelllysisof measles-infected HeLa cells(-)and of measles-infected HeLa cellsexposedtohuman transferrin(A).

80

h 60

40 20 28

B TRANSFERRINTREATED

2.5:1 5.1 10.1 20:1

122 51 5:1 10:1 20:1

,-FF,-croR/TAR6,-rCELRArlo

(5)

functionally orimmunologically. This isstrong evidence that

thetransferrin receptor aloneisnot the targetforthe NK cell. As shown inFig. 4,growth of measles-infected HeLa cells in

medium containinghumantransferrin for6 dreduced by half

the leveloftransferrinreceptorexpression. This change,however, produced no alterationinthe levelofNK cellsusceptibility, as

shown inFig.5 B.Theassayfortargetcelllysiswas conducted

after 6 and 24 h of incubation in medium containingno human

transferrin. Under these conditions there could have been re-covery

of

transferrin receptor expression in the transferrin-treated, measles-infectedHeLa cells. These cells were,therefore, assayedfor transferrin surface binding capacity6 and 24 hafter the removal of themedium containingthe humantransferrin.

Atthe6-hassaypointthecellshad 40%ofthe control level of

transferrinreceptors,whileat24hthe value wasstillonly 72%

of control.

Cold-target

inhibition studies. The correlation between

transferrin

receptor

expression

andNK

susceptibility

previously noted (5), indicated that this interdependency held only to a

certain level

of

receptor

expression.

A strongpositive correlation existed for cells with

fewer

receptors than K562, but beyond

this NK susceptibility reached aplateau. Any testof this

hy-pothesis

wouldtherefore

require experiments

inwhich changes inNKsusceptibilityweresought inresponse todecreases inthe

level of transferrin receptor expression to below that seen in K562 cells.

Aseries of

experiments

was

performed

wherein cells whose

surface

receptorshad been

down-regulated

wereused as

com-petitive

"cold-target inhibitors". Here the ratio ofNKeffector cells to labeled target cells is held constant while

increasing

numbersofagroupof unlabeled secondtargetcellswereadded. The

ability

ofthe unlabeled cellstopreventthe

lysis

ofthe labeled

targetcells is

thought

tobe a

function

of the

quantity

of the

specific

NK target

antigen(s)

ontheunlabeled cells.

Therefore,

thismaybeamore

sensitive

way to

examine

NK target

antigen

expression,

since itseparates target

recognition

from cell

lysis.

Asshown

earlier,

HeLa,

measles-infected

HeLa, and K562 cells have

roughly

the same number of transferrin receptors.

Measles-infected

HeLa cells in which transferrin receptor

expressionwas halved by priorgrowth withsaturated human

transferrinwereusedin cold-target inhibition studiesalongwith unmodified, measles-infected cells.As showninTableI,the two groupsof cellswere equallyeffective as cold-target inhibitors.

Thereduction inreceptorexpressionto below theK562 "stan-dard" in the transferrin-modified, measles-infectedHeLa cells wouldhave placed them inarange where covariance of

trans-ferrin receptor expression and NK susceptibility should have

occurred.Thiswas not seen.

Since

itwaspossible that the level ofreceptorreduction in thisexperimentwasstill insufficienttomake such arelationship

apparent, a seriesof

experiments

wasperformed in which

re-ceptor-modulated and unmodifiedK562 cells were used as cold-targetinhibitors.Toattainagreaterdegreeofreduction of

trans-ferrinreceptorexpression, cellswere grownfor5din medium

supplemented

with 10

tM

hemin and 5

,g/ml

ferric ammonium

citrate.

The

combined

useof hemin and the iron saltproduced a reduction in receptor

expression from

1.72 X 10 to 0.33

X

10

percell inthe first series of experiments (fivefold), and

from 1.63 X 105to0.27X

105

percell(sixfold) inthe second. Asshownin Table II, for each experimentuntreated K562 cells were used as "hot" targetsinoneinhibition series,while

hemin/

iron treated cellswereusedas"hot"targets toanother. Inhibition

of

lysis of

untreated K562targetcellsby treated and untreated coldtargets wascomparabledown to acold/hottargetratio of

4:1 in bothexperiments. The treatedcells were less potent in-hibitorsat an8:1 ratio.Atwo-tailedchi-squareanalysis of the inhibition series showed that therewas nosignificant difference betweenthe treated and untreated cellsascold-target inhibitors (P<0.05).

Similarly,

when the treatedK562 cellswereusedas

"hot"

targets, the

cold-target inhibition

by the treated and

un-treatedcellswasidentical.

Discussion

TheNKcell isa memberofthe immune system's

armamen-tarium;

itsexactrole isuncertain. One hypothesis is that NK

cells

provide

animmune surveillance mechanism thatcan

des-troy tumorcellsat anearlystageofdevelopment(3, 4). Eradi-cation oftumorsismoredifficult with

large

cell

burdens,

sothat TableI. "Cold-target Inhibition"

of

NK-mediatedK562

Cytolysis by

Unlabeled

Measles-infected

HeLa Cells

E/T=10:1 E/T=5:1

Ratio of unlabeled

"cold"targetcells "Cold"target "Cold"target tolabeled K562 "Cold"target =transferrin-treated "Cold"target =transfemin-treated targetcells = measles-infectedHeLacells measles-infectedHeLacells =measles-infected HeLacells meades-infectedHeLacells

6-hassay

0.5:1 46.5±5.9 45.2±2.8 24.5±3.4 25.1±3.1

1:1 36.8±4.0 36.4±3.8 18.1±1.8 20.7±1.8

2:1 31.8±4.2 34.4±4.3 19.6±3.1 22.0±2.1

4:1 24.9±3.4 25.1±3.2 14.4±2.2 14.2±2.4

8:1 21.2±2.3 23.0±2.6 10.2±2.2 12.5±1.7

24-hassay

0.5:1 55.1±3.7 54.8±5.8 35.6±3.5 29.4±7.9

1:1 60.9±7.9 52.8±5.6 27.9±2.3 31.1±2.7

2:1 45.0±5.2 45.2±5.0 25.0±2.9 25.7±2.5

4:1 35.3±3.1 36.8±4.5 17.5±2.7 18.9±2.2

8:1 30.2±4.0 29.1±3.3 19.1±2.7 21.0±2.1

Asdescribed in the text,increasingnumbersof "cold" measles-infected HeLa cells(eitherunmodulatedorcultured intransferrin)wereadded to 6-or24-hcytolysisassaysmeasuringtheprogressiveinhibition of thelysisof labeled K562 cells(targets) byhuman blood NK cells(effectors).

(6)

Table II. NK-mediated Cytolysis ofNative K562TargetCells

("Untreated') Vs.K562 Target Cells

Modified

byTreatment with

Hemin Plus Iron ("Treated'): "Cold-Target Inhibition"*"

Unlabeled("cold")

targetinhibitor "Cold:hot"

Killing of K562 target ratio Untreated Treated ("hot")targetcells (unlabeled/labeled) K562 K562

Experiment 1

Untreated 0:1 49.7±5.9

0.5:1 36.4±4.5 39.0±4.0

1:1 23.9±3.2 22.9±4.0

2:1 25.9±3.2 30.4±4.1

4:1 19.8±2.6 26.5±5.5

8:1 11.6±2.4 21.6±2.3

Hemintreated 0:1 39.8±2.0

0.5:1 35.2±5.0 32.0±4.4

1:1 18.0±5.2 23.8±3.0

2:1 30.0±3.4 22.7±1.9

4:1 22.9±5.2 15.7±2.7

8:1 18.0±3.7 15.0±2.6

Experiment 2

Untreated 0:1 36.8±6.7

0.5:1 29.8±4.4 36.4±0.5

1:1 24.5±2.5 22.4±4.0

2:1 19.8±4.7 21.8±4.9

4:1 10.5±2.5 18.1±4.2

8:1 7.0±1.4 16.3±2.7

Hemin treated 0:1 81.9±30.6

0.5:1 72.4±21.4 78.5±26.5

1:1 53.1±18.8 47.7±16.5

2:1 48.6±17.9 35.2±13.9

4:1 39.7±12.5 46.4±14.5

8:1 25.1±9.0 26.0±9.2

* Effector/target ratio,

10:

1.

amechanismthatdestroyedsuchcellsearly in their growth would providea valuabledefense against neoplasia (20).NK cells may

alsoprovideafirst-line host defense

against

viralinfections (21, 22). Thisconceptissupported bythefactthat NKcytotoxicity is augmented by

interferon,

which is produced by virally infected cells

(23).

Recent datasuggestthatNKcellsmay, in addition, playarole in

regulating

hematopoiesis. Studies showinganin vitro inhibition of granulopoiesisandsuppression oferythroid stem cell

proliferation

by NK cells supportthis concept (24, 25, 26).

Despite extensive

investigations,

however, the structure of

the NKtarget cells thatmarkthem for

cytolysis

hasyet to be

definitively

identified. Somerecent studies have suggested that the

transferrin

receptor maybethetarget

antigen

forNKcell

cytolysis(5,6,7, 8). Several celllineswereexamined to discern the number ofcells intheir populationsthat expressed the

trans-ferrinreceptor and thedegreeof NKsusceptibility oftheline.

Thosecelllines with few receptor-positivecells wereresistantto NKcytolysis (5).However, in cell linescontaining>50%

recep-tor-positivecells intheir populations,there was a rangeof

sus-ceptibility

to NKlysis from

highly

sensitive to insensitive. In

addition,

a

purified trypsin-cleavage fragment

ofthetransferrin

receptor could

partially

block the

cytolytic

activity

ofthe NK

cells.

Inanotherstudy,mouseL-cellsweretransfected with sheared humanDNA,and cellsthatsubsequently expressedthe human transferrin receptor on their surfacewereselected for and cloned (8). These transformed mouse L-cells could

partially

inhibit cytolysis of labeled K562 target cellsbyhumanperipheralblood lymphocytes when used inacold-targetinhibition assay. This suggested that the human transferrin receptor

played

arole in the affector-targetinteraction,though,

interestingly,

nontrans-formed L-cells alsoproducedsignificantinhibition. Incontrast

tothe datainhuman systems, therewasnocorrelation between mousetransferrinreceptor

expression

and NK

recognition

ina

murine system.

Sincethe transferrin receptor is the

physiologic

mode of iron

acquisitionby cells, theimplications of these studies are

far-reaching. The receptor isexpressedon

proliferating

cells such as tumor cells in

culture,

cultured

fibroblasts,

and

erythroid

pre-cursors, as well as nonproliferating cells such as

reticulocytes

andhepatocytes

(10,

1

1,

27-30).

If the transferrinreceptorwas

thetarget structureforNK

cells,

these young

reticulocytes

would

bein gravedangerof destruction.

Here,the

relationship

between transferrin receptor

expression

andNK

cytolysis

wasexamined in several ways. The

NK-sen-sitive

K562 cell has been

widely

usedas astandard targetin NK

studies. The transferrin receptor

expression

on the surface of

thesecells was increasedbytreatmentwith desferrioxamine or

decreased

by

treatment with hemin. A fourfold difference in

both functionaland

antigenic

receptor

expression

wasachieved

between the desferrioxamineand the hemin-treated cells.

None-theless, nodifference intheextentof

susceptibility

toNK

cy-tolysiswasobserved.A recent

study

inwhich

transferrin

receptor expressionwasreduced in K562 cells

by

several

manipulations

alsofailedtoshow a

corresponding

reduction in NK

suscepti-bility(3 1).

The degree of differencein

transferrin

receptor

expression

in the desferrioxamine and hemin-treated cells wassimilarto

that between the cell lines with different NK

susceptibility

re-ported

previously (5).

These

data,

then,

suggestthat the

trans-ferrin receptor isnotthetarget

antigen

for NK cells. The

pos-sibility

remained, however,

that the

degree

of modulation of receptor

expression

was insufficient to cause a

change

in NK

susceptibility

in theK562 cells.

The HeLacell

line,

whichwasresistanttoNK

cytolysis,

was

examined todetermine whetheran increased resistance

corre-latedwith thepresence of fewer transferrin receptorsthanwere

seen inK562 cells. TheHeLa cellswerefoundtohave

slightly

moretransferrinreceptorsthan theK562 cells. Measlesinfection ofHeLa cells caused this cell line to become sensitiveto

lysis

byhuman NKcells

(2).

Thisincreased

sensitivity

toNK

cytolysis,

however,wasnotreflective ofanincrease in transferrinreceptor

expression.

Control and measles-infected HeLa cells have the

samenumberof functional and

antigenic

transferrin

receptors. Since it was

previously reported

that a strong correlation

betweenthe leveloftransferrinreceptor

expression

andNK

sus-ceptibility existed only with lines

having

fewerreceptors than K562(5), itwasimportanttodeterminewhethershifts ofreceptor

expression in thisrange

produced

acovariance in NK

suscep-tibility.

Thecold-target inhibitionmethodwasusedtoexamine

NKtargetantigenexpressionon the cells.

Measles-infected HeLa cells grown for6 d in

medium-sup-plementedhumantransferrinshoweda50%reductionin

trans-ferrin receptorexpression, both functionally and

antigenically,

(7)

mea-sles-infected HeLa cells iscomparable, the transferrin-treated, measles-infected HeLacells wouldhavefallenin a range where apositive correlation betweenreceptorexpressionand NK

sus-ceptibilitywould have beenexpected.The K562cells,incubated for 5 d in medium supplemented with hemin and iron salts, underwent a five- to sixfold reduction in the level of transferrin

expression. Thesecellswouldhave fallen wellwithin therange where a linearcorrelation betweenreceptorexpressionand NK susceptibility was reported (5). Despite this marked reduction inreceptor expression, there was no evidence ofachange in levelofNK targetantigenasdetermined by cold-target inhibition (Table II). When untreated K562 cellswereusedastargets, the hemin/iron treated cellswerelesseffectiveascoldtarget

inhib-itors at the highest cold/hottargetratio. When the inhibition curves werestatistically analyzedas awhole, however, thetwo groups ofcellswere notdifferent. Inthe obverse experimental

series wherehemin/iron treated cells were used ashottargets,

there wasclearly no difference in the

capacity

of treated and

untreated K562 cells to serve as

cold-target

inhibitors. The

expression ofNK target

antigen

on thesetwogroups of cells, then, appearedtobe equal.

NKcelltargeting is, doubtless, acomplexaffair andmany parameters maybeinvolved. The

factors controlling

the

targeting

ofvirally infected cellsmay differ from those involved intumor

cellrecognition. Inaddition,NKcells may beaheterogeneous population grouped together

by

their functional definition.

Re-cently,a monoclonal antibodywasused toisolatea subset of NKcellsthatwas

cytotoxic

foran

anchorage-dependent

cellline but not for K562 cells(32). It is

likely

that this subset of NK

cells

recognizes

a target

antigen

other than thetransferrinreceptor

since theK562 cells express this receptor in abundance. In ag-gregate, these data show

minimal,

if any, correlation between

transferrin receptor

expression

and

susceptibility

to NK lysis

over awiderange of receptor

expression.

This

strongly

implies that thetransferrin receptoris notthe sole targetstructurefor NKcell

recognition.

References

1.Ortaldo,J.R., and R. B. Herberman. 1984. Heterogeneity ofnatural

killercells. Annu. Rev.Immunol.2:359-394.

2.Ault, K. A., and H. L. Weiner. 1979. Natural killing of

measles-infectedcellsby humanlymphocytes.J.Immunol. 122:2611-2616. 3.Herberman,R. B., editor. 1980. Natural CellMediated Immunity

Against Tumors. Academic Press, Inc., New York. 1321 pp.

4. Dennert, G. 1980. Cloned linesof natural killer cells. Nature

(Lond.).287:47-49.

5.Vodinelich,L., R.Sutherland,C.Schneider,R. Newman, and M. Greaves. 1983.Receptors fortransferrin may bea"target"structurefor

naturalkillercells. Proc.NatL. Acad.Sci. USA.80:835-839.

6.Baines,M.G.,F. L.Lafleur,andB. E.Holbein. 1983. Involvement oftransferrin receptors in human naturalkiller effectorntarget interactions.

Immunol. Lett. 7:51-55.

7.Alarcon, B., and M. Fresno. 1985.Specificeffectofanti-transfernn

antibodiesonnaturalkillercellsdirectedagainsttumorcells:evidence for the transferrin receptorbeingoneof the targetstructuresrecognized

byNKcells. J.Immunol. 134:1286-1291.

8.Newman, R.A., J. F.Warner, and G. Dennert. 1984.NK

rec-ognition of targetstructures: is the transferrin receptor the NK target

structure? J.Immunol. 133:1841-1845.

9.Aisen,P., andI.Listowsky. 1980. Iron transport and storage pro-teins.Annu. Rev.Biochem. 49:357-393.

10.

Larrick,

J.

W.,

and P.Cresswell. 1979. Modulation ofcell surface irontransferrin receptors

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density

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Supramol.

Struct. 11:579-586.

11.Frazier,J.C.,J.H.Caskey,M. Yoffee, and P. A. Seligman. 1982. Studies of thetransferrin receptoronbothhuman reticulocytes and

nu-cleated human cells in culture.J. Clin. Invest.69:853-865.

12.Ward,J.H.,J.P. Kushner, and J. Kaplan. 1982. Regulation of Hela Cell Transferrin Receptors. J. Biol. Chem. 257:10317-10323.

13. Pelicci, P. G., A. Tabilio, P. Thomopoulos, M. Titeux, W.

Vain-chenker,H. Ronchant, and U. Testa. 1982. Hemin regulates the expres-sion oftransferrin receptors in human hematopoietic cell lines. FEBS (Fed. Eur. Biochem. Soc.). Lett. 145:350-354.

14.Bridges, K. R., andA.Cudkowicz. 1984. Effect of ironchelators

onthe transferrin receptor in K562 cells. J. Biol. Chem. 259:12970-12977.

15. Mattia, E., R. Krishnamurty, D. S. Shapiro, H. H. Sussman, and R.D.Klausner. 1984. Biosynthetic regulation of the humantransferrin receptorby desferrioxamine in K562 cells. J. Biol. Chem. 259:2689-2692.

16. Zaman, Z., M.-J. Heynen,and R. L.Verwilghen. 1980.Studies

onthe mechanism of transferrin iron uptake byratreticulocytes.Biochim. Biophys. Acta. 632:553-561.

17. Klausner, R. D., J. Van Renswonde, G. Ashwell, C. Kempf, A. N. Schechter, A. Dean, and K. R. Bridges. 1983. Receptor-mediated endocytosis of transferrin in K562 cells. J.Biol. Chem. 258:4715-4724. 18. Timonen, T., C. W. Reynolds, J. R. Ortaldo, and R. B.

Herber-man. 1982. Isolation of human andratnatural killer cells. J. Immunol. Methods. 51:269-274.

19.Sutherland, R., D. Delia, C.Schneider,R. Newman, J. Kemshead, andM. Greaves. 1981. Ubiquitous cell-surface glycoproteinon tumor

cellsis proliferation-associated receptor for transferrin. Proc.Natd.Acad. Sci. USA. 78:4515-4519.

20. Durie, B. G., S. E. Salmon, and T. E. Moon. 1980. Pretreatment tumor mass,cellkinetics, and prognosis in multiple myeloma. Blood. 55:364-372.

21. Herberman, R. B., and J. R. Ortaldo. 1981. Natural killer cells: their role in defenses against disease. Science (Wash. DC). 214:24-30.

22.Bancroft,G. J., G. R.Shellam,and J. E.Chalmer. 1981.Genetic influenceson the augmentation of natural killer cells during murine cytomegalovirus infection: correlation with patterns of resistance. J. Im-munol. 126:988-994.

23.Einhorn, S., H. Blomgren, and B. Strander. 1978. Interferon and spontaneouscytotoxicityinman. 1.Enhancement of the spontaneous cytotoxicity of peripheral lymphocytes by humanleucocyteinterferon. Int. J. Cancer. 22:405-410.

24.Hansson, M., M. Beran, B. Andersson, and R.Kiessling. 1982. Inhibition of in vitro granulopoiesis by autologous allogeneic human natural killer cells. J. Immunol. 129:126-132.

25.Mangon, K. F., M. E. Hartnett, S.A.Matis, A.Winkelstein,and T. Abo. 1984. Natural killer cells suppress human erythroidstemcell proliferation in vitro. Blood. 63:260-269.

26.Holmberg,L.A.,B. A.Miller,and K. A.Ault. 1984. The effect of natural killer cellsonthedevelopmentofsyngeneichematopoietic

progenitors.J.Immunol. 133:2933-2939.

27.lacopetta,B.J., E. G.Morgan,and G. C. T. Yeoh. 1982. Trans-ferrin receptors and iron uptake during erythroid cell development.

Biochim. Biophys.Acta. 687:204-210.

28. Young, S. P.,and P.Aisen.1981. Transferrin receptors and the uptake and release of ironbyisolatedhepatocytes. Hepatology

(Balti-more). 1:114-119.

29.Nunez,M.-T.,and J. Glass. 1983. The transferrincycleandiron uptakeinrabbitreticulocytes.J.Biol. Chem. 258:9676-9680.

30.lacopetta,B. J., and E. H.Morgan. 1983. The kinetics of

trans-ferrinendocytosisandironuptakefrom transferrin in rabbitreticulocytes.

J.Biol. Chem. 258:9108-9115.

31.Dokhelar,M.-C.,D.Garson,U.Testa,and T. Tursz. 1984.Target

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References

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