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:
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
IntroductionExpression of the transferrin receptoron target cell lines has recently been implicated as a determinant of
susceptibility
tocytolysis 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 receptorexpression
asdetermined both byradioligand bindingand surfacebindingof the OK-T9 monoclonal anti-transferrin receptor
antibody.
In contrast, cells grown for thesameperiodoftime in hemin halvedtheir receptor expression. This fourfold
change
in transferrin receptor expression between thedesferrioxamine-treated
andhemin-treated cells producednochange in
susceptibility
toNKcytolysis.
Second,
HeLa(a
cellline whichin itsnativestateis veryresistantto NKcytolysis)cellswerecompared withK562cellswith 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 105receptors/cell.
Third, infection ofHeLa cells by measles virusgreatly increased their susceptibility toNKlysisbut producednochangeinsurface transferrinreceptor
expression.
Furthermore,
whenmeasles-infectedHeLacellsweregrownfor6dinmedium 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 humanNKcells, 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
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 receptorOK-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 100Mloffluo-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 aFicoll-Hypaque
cushion,centrifugationfor 40min at1,200rpm, and removalofthemononuclear cellsattheinterface removed aspreviously described(18).Thesecellswereusedaseffector
lymphocytes.
Target cells(HeLa, measles-infectedHeLa,andK562)
werechromated for 1 h at370C with5"Cr
(NewEngland Nuclear, Boston, MA)
ataconcentration 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 ulbyadding supplemented
RPMI 1640medium;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.
Percentcytolysiswascalculated
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 cellswereincubated for 24 hat
- 370C.
They
werethenpelleted
<c-_S< a -- at200
g,thesupernatantwasremoved,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 incontrol
medium orinmedium
supplemented
either with 60 uM des-ferrioxamine orwith 20 uMhemin. Thesetwoagents had noeffectontherateof cell
growth during
thistime,
butmarkedly
altered transferrinsurface
binding capacity (Fig.
1).
Therewas afourfold differenceintransferrin receptorexpression
between thedesferrioxamine
and thehemin-treated cells. Therewere nomorphological
alterations inthecells.The
transferrin
surfacebinding capacity
isafunctional mea-sureofreceptor number. Itwaspossible, however,
thatNKcellsmight
recognize functionally
inactive transferrin receptors onthecell surface. The OK-T9 monoclonal
antibody
bindstothetransferrin receptoratasite differentthan the
transferrin-binding
domain
(19).
Thisantibody
wasusedtodetermine whetherthe hemin-inducedanddesferrioxamine-inducedchanges
infunc-tionalreceptor
expression
reflectedchanges
inimmunoreactive
receptoronthe
cell
surface.Cellsgrown for24 h incontrol medium and in
desferriox-amine-supplemented
orhemin-supplemented
medium werechilled, washed,
and labeled withasaturating
concentrationofOK-T9
antibody,
followedby
fluorescein-labeledgoat
anti-mouse
antibody.
Fig.
2shows the resultsof suchananalysis
ofdesferrioxamine-treated
and hemin-treatedcells. Meanlog
flu-orescence
intensity
inthehemin-treated cellswas90(arbitrary)
Uvs. 133 in the desferrioxamine-treated cells. Thelogarithmic
Figure2. Cellsurfacetransferrinreceptor
an-H D
tigen
in desferrioxamine-treated andhemin-treatedK562 cells. Cellswereincubatedwith
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 containingNKcells. In three separateexperiments,thesampleswereassayed forchromium release as a measure of target celllysisafter 6 and 24 h.Fig. 3 isa
representative
6-hkilling
assayshowing
thatthe K562 cellswerehighly susceptibletolysis under theseconditions. Anincrease intransferrin
receptornumberby desferrioxamineor 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 cellsarerelatively
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
differenceex-isted inreceptor
affinity
for transferrin. Attempts to increase transferrinreceptorexpression
inHeLacells by desferrioxaminetreatment 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 amountoftransferrin-receptor
antigenonthesurfaces ofthesethree groups of cells was also measured by OK-T9 antibody
binding
andFACSanalysis.
Themeanvaluesof logfluorescence
intensity
forthecontrol andmeasles-infected HeLacells were 127 and 123 U, respectively. The human transferrin-treatedHeLacells 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. Cellsweregrown 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 untreatedmeasles-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 theexpressionof 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
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 betweentransferrin
receptorexpression
andNKsusceptibility
previously noted (5), indicated that this interdependency held only to acertain level
of
receptorexpression.
A strongpositive correlation existed for cells withfewer
receptors than K562, but beyondthis NK susceptibility reached aplateau. Any testof this
hy-pothesis
wouldthereforerequire experiments
inwhich changes inNKsusceptibilityweresought inresponse todecreases inthelevel of transferrin receptor expression to below that seen in K562 cells.
Aseries of
experiments
wasperformed
wherein cells whosesurface
receptorshad beendown-regulated
wereused ascom-petitive
"cold-target inhibitors". Here the ratio ofNKeffector cells to labeled target cells is held constant whileincreasing
numbersofagroupof unlabeled secondtargetcellswereadded. The
ability
ofthe unlabeled cellstopreventthelysis
ofthe labeledtargetcells is
thought
tobe afunction
of thequantity
of thespecific
NK targetantigen(s)
ontheunlabeled cells.Therefore,
thismaybeamore
sensitive
way toexamine
NK targetantigen
expression,
since itseparates targetrecognition
from celllysis.
Asshown
earlier,
HeLa,measles-infected
HeLa, and K562 cells haveroughly
the same number of transferrin receptors.Measles-infected
HeLa cells in which transferrin receptorexpressionwas 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 arelationshipapparent, a seriesof
experiments
wasperformed in whichre-ceptor-modulated and unmodifiedK562 cells were used as cold-targetinhibitors.Toattainagreaterdegreeofreduction of
trans-ferrinreceptorexpression, cellswere grownfor5din medium
supplemented
with 10tM
hemin and 5,g/ml
ferric ammoniumcitrate.
Thecombined
useof hemin and the iron saltproduced a reduction in receptorexpression from
1.72 X 10 to 0.33X
10
percell inthe first series of experiments (fivefold), andfrom 1.63 X 105to0.27X
105
percell(sixfold) inthe second. Asshownin Table II, for each experimentuntreated K562 cells were used as "hot" targetsinoneinhibition series,whilehemin/
iron treated cellswereusedas"hot"targets toanother. Inhibition
of
lysis of
untreated K562targetcellsby treated and untreated coldtargets wascomparabledown to acold/hottargetratio of4:1 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, thecold-target inhibition
by the treated andun-treatedcellswasidentical.
Discussion
TheNKcell isa memberofthe immune system's
armamen-tarium;
itsexactrole isuncertain. One hypothesis is that NKcells
provide
animmune surveillance mechanism thatcandes-troy tumorcellsat anearlystageofdevelopment(3, 4). Eradi-cation oftumorsismoredifficult with
large
cellburdens,
sothat TableI. "Cold-target Inhibition"of
NK-mediatedK562Cytolysis by
UnlabeledMeasles-infected
HeLa CellsE/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).
Table II. NK-mediated Cytolysis ofNative K562TargetCells
("Untreated') Vs.K562 Target Cells
Modified
byTreatment withHemin 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 byinterferon,
which is produced by virally infected cells(23).
Recent datasuggestthatNKcellsmay, in addition, playarole inregulating
hematopoiesis. Studies showinganin vitro inhibition of granulopoiesisandsuppression oferythroid stem cellproliferation
by NK cells supportthis concept (24, 25, 26).Despite extensive
investigations,
however, the structure ofthe NKtarget cells thatmarkthem for
cytolysis
hasyet to bedefinitively
identified. Somerecent studies have suggested that thetransferrin
receptor maybethetargetantigen
forNKcellcytolysis(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 fromhighly
sensitive to insensitive. Inaddition,
apurified trypsin-cleavage fragment
ofthetransferrinreceptor could
partially
block thecytolytic
activity
ofthe NKcells.
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 receptorplayed
arole in the affector-targetinteraction,though,interestingly,
nontrans-formed L-cells alsoproducedsignificantinhibition. Incontrast
tothe datainhuman systems, therewasnocorrelation between mousetransferrinreceptor
expression
and NKrecognition
inamurine system.
Sincethe transferrin receptor is the
physiologic
mode of ironacquisitionby cells, theimplications of these studies are
far-reaching. The receptor isexpressedon
proliferating
cells such as tumor cells inculture,
culturedfibroblasts,
anderythroid
pre-cursors, as well as nonproliferating cells such asreticulocytes
andhepatocytes
(10,
11,
27-30).
If the transferrinreceptorwasthetarget structureforNK
cells,
these youngreticulocytes
wouldbein gravedangerof destruction.
Here,the
relationship
between transferrin receptorexpression
andNK
cytolysis
wasexamined in several ways. TheNK-sen-sitive
K562 cell has beenwidely
usedas astandard targetin NKstudies. The transferrin receptor
expression
on the surface ofthesecells was increasedbytreatmentwith desferrioxamine or
decreased
by
treatment with hemin. A fourfold difference inboth functionaland
antigenic
receptorexpression
wasachievedbetween the desferrioxamineand the hemin-treated cells.
None-theless, nodifference intheextentof
susceptibility
toNKcy-tolysiswasobserved.A recent
study
inwhichtransferrin
receptor expressionwasreduced in K562 cellsby
severalmanipulations
alsofailedtoshow a
corresponding
reduction in NKsuscepti-bility(3 1).
The degree of differencein
transferrin
receptorexpression
in the desferrioxamine and hemin-treated cells wassimilarto
that between the cell lines with different NK
susceptibility
re-ported
previously (5).
Thesedata,
then,
suggestthat thetrans-ferrin receptor isnotthetarget
antigen
for NK cells. Thepos-sibility
remained, however,
that thedegree
of modulation of receptorexpression
was insufficient to cause achange
in NKsusceptibility
in theK562 cells.The HeLacell
line,
whichwasresistanttoNKcytolysis,
wasexamined 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).
Thisincreasedsensitivity
toNKcytolysis,
however,wasnotreflective ofanincrease in transferrinreceptor
expression.
Control and measles-infected HeLa cells have thesamenumberof functional and
antigenic
transferrin
receptors. Since it waspreviously reported
that a strong correlationbetweenthe leveloftransferrinreceptor
expression
andNKsus-ceptibility existed only with lines
having
fewerreceptors than K562(5), itwasimportanttodeterminewhethershifts ofreceptorexpression in thisrange
produced
acovariance in NKsuscep-tibility.
Thecold-target inhibitionmethodwasusedtoexamineNKtargetantigenexpressionon the cells.
Measles-infected HeLa cells grown for6 d in
medium-sup-plementedhumantransferrinshoweda50%reductionin
trans-ferrin receptorexpression, both functionally and
antigenically,
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 anduntreated K562 cells to serve as
cold-target
inhibitors. Theexpression ofNK target
antigen
on thesetwogroups of cells, then, appearedtobe equal.NKcelltargeting is, doubtless, acomplexaffair andmany parameters maybeinvolved. The
factors controlling
thetargeting
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
forananchorage-dependent
cellline but not for K562 cells(32). It islikely
that this subset of NKcells
recognizes
a targetantigen
other than thetransferrinreceptorsince theK562 cells express this receptor in abundance. In ag-gregate, these data show
minimal,
if any, correlation betweentransferrin receptor
expression
andsusceptibility
to NK lysisover awiderange of receptor
expression.
Thisstrongly
implies that thetransferrin receptoris notthe sole targetstructurefor NKcellrecognition.
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