T lymphocyte cloning from rejected human
kidney allografts. Growth frequency and
functional/phenotypic analysis.
J F Moreau, … , C Desgranges, J P Soulillou
J Clin Invest.
1986;
78(4)
:874-879.
https://doi.org/10.1172/JCI112674
.
Mechanically harvested lymphocytes invading an irreversibly rejected human kidney
allograft were seeded at limiting dilution to calculate the frequency of growing precursors.
Optimal growth frequency (1/13) was obtained when Epstein-Barr virus (EBV)-transformed
donor B lymphocytes were used as stimulators (D-BLCL) in the presence of interleukin 2
(IL-2). The 55 clones analyzed were all T11+ and T3+, and all expressed DR antigens (45%
were T8+ and 55% T4+). Only one clone had a double-labeled (T4+ T8+) surface. All cells
proliferated significantly against D-BLCL, although T4+ clones had a significantly shorter
average doubling time than T8+ clones. Nearly all T8+ clones were specifically cytotoxic for
D-BLCL, while both T4 and T8 did not react against K562, autologous EBV-BLCL, and
third-party EBV-BLCL. Detectable IL-2 was found in the culture supernatants of only a
minority of clones (all T4+).
Research Article
Find the latest version:
T Lymphocyte Cloning
from Rejected
Human Kidney
Allografts
Growth Frequency and
Functional/Phenotypic AnalysisJ. F.Moreau, M. Bonneville, M. A. Peyrat, A.Godard,Y. Jacques, C. Desgranges,* and J. P.Soulillout InstitutNational de laSanteetde la Recherche Medicale, UniteU211,Facultede Medecine,Nantes 44035 Cedex; *Centred'Epidemiologieetd'Immunovirologie, Facultede Medecine Alexis Carrel,Lyon 69008Cedex;
andtCentre Hospitalier Regional de Nantes, Nantes, 44035 Cedex, France
Abstract
Mechanically harvested lymphocytes invading an irreversibly rejectedhuman kidney allograft were seeded at limiting dilution tocalculate the
frequency
ofgrowing precursors.Optimal
growthfrequency
(1/13) was obtained when Epstein-Barr virus(EBV)-transformed
donor Blymphocytes
were used as stimulators(D-BLCL)
in the presence ofinterleukin
2 (IL-2). The 55 clonesanalyzed
were all T1 andT3+,
and all expressed DR antigens (45% wereT8+
and 55% T4+). Only one clone had a double-labeled (T4+T8+)
surface. All cells proliferated significantlyagainst
D-BLCL,
although T4+ clones had a significantly shorter averagedoubling
time than T8 clones. Nearly all T8 clones werespecifically cytotoxic for D-BLCL, while both T4 and T8did
not reactagainst K562,
autologous EBV-BLCL, and third-partyEBV-BLCL.
Detectable IL-2 wasfound
in the culture su-pernatantsof
only aminority of
clones(all
T4+).
Introduction
The
histological hallmark of
organrejection is lymphocytic
in-filtration.
Thesedirectly
committed
andrecruited
lymphocytes (1,2)
have beenstudied for
years at afunctional
andphenotypic
levelin
tissue
orcoated
spongematrix
allografts (3, 4).
Inanimals,
transfer
experiments have shown activated
Tlymphocytes
tobedirectly involved
inrejection,
andpurified lymphocyte
subsetscarrying the helper phenotype
areapparently
able to mediategraft destruction
inimmunosuppressed recipients
(5).However,
nodefinitive conclusions
canbe drawn
inthe
absenceof modelsusing totally unresponsive
hosts(see
review[6]).
Another
direct approach
to therejection mechanism
is tolook
at thephenotypic
andfunctional
characteristics of cellswithin the rejected graft.
The useof
monoclonal antibodies(MAb)'
specifically directed against surface
membrane antigens hasallowedprecise enumeration of these
lymphocytes (7, 8);however,
allfunctional
studies
todate
have beenperformed
oncrude
lymphocyte
suspensions
extracted from
rejected
organsReceivedfor publication21January 1986 andinrevised form30May
1986.
1.Abbreviations used in this paper: A-BLCL, autologousBLCL; BRMP,
Biological Response Modifiers Program;BLCL,B
lymphoblastoid
cell line; CFC, colony-forming cells; CM, culturemedium;D-BLCL,
donor BLCL; EBV,Epstein-Barr virus;E/T,effector/target ratio; GIC, graft-invadingcells;IL-I andIL-2, interleukin 1 and2;LU 30,lyticunits,MAb, monoclonalantibody;PHA,phytohemaglutinin; rIL-2,pure re-combinantIL-2.
and have thus reflected many cell interactions. Nevertheless, there seems to be a preponderance of alloimmune cytotoxic or helper lymphocytes
in
human as well as in experimental animalgrafts
(3, 4, 6, 9, 10).The
limiting
dilution technique (1 1) can be used to clone large numbersof graft-invading
cells(GIG)
that can then befunctionally and phenotypically characterized.
Moreover, thespecific frequencies of each functional
or phenotypic kind ofinvading lymphocyte
canbeestimated.
This method was previously used by us (12, 13) to provide
direct evidence
for thespecific involvement of
several functional subsets ingraft rejection.
However, in these preliminary studies, theestimated
lymphocyte cloning frequency
was very lowcom-pared with
thatachieved
routinely with similarly processed
pe-ripheral
bloodlymphocytes (PBL), a result that cast doubt on therelevance of the
approach.This
paperdescribes
thechar-acteristics of clones
that werederived from
arejected
humankidney.
It waspossible
tomarkedly improve
growthfrequency
by
using
kidney
donor Blymphocytes immortilized by
Epstein-Barrvirus
(EBV)infection
as a(stimulatory) feeder
layerin
cul-ture.Thus,
cytotoxicity,
antigen-driven proliferation, cell-surface
phenotype, andinterleukin
2(IL-2) production
could bedeter-mined.
Methods
(a) Clinicalstatusofrecipient.A46-yr-oldwoman(HLAtyping HLA-A2, 3; B5, 7;DR2, W6) with end-stage renal failure caused byachronic interstitial nephropathyreceived acadavericgraft (HLA-A2, 1 1;B18,
22; DR5,8)onDecember28, 1983. She had been pregnant fivetimes and had received 21 blood transfusions. Before
grafting,
herserum con-tained antibodiesagainst40 and80%, respectively,ofTandBlympho-cytes ofaselected 20-memberpanel
(specificity:
anti-HLAAlO,
B12,B15, andDR7). She hadnoautoantibodies. Cross-matchwas
negative
with donorTlymphocytes andpositive(+++) with donorB
lymphocytes
asdeterminedusingcurrently
availablesera.Shewastreated withcy-closporineas
previously
described(14).
Herurine output remained below 1liter/24hand sherequiredhemodialysis
aftertransplantation.
On March 3,1984,cyclosporinewaswithdrawn withoutsubsequenteffectonurine outputorrenal function. Thekidney
wasremovedonMarch 27tofind symptoms ofacuterejectionsuperimposed
upontheprevious
clinical status.(b)
EBVtransformation ofB lymphocytes. Lymphocytes
wereisolated fromkidneydonorspleenorrecipientperipheral
bloodbyFicoll-Hypaque
densitygradient centrifugation. Cellswerewashedtwice, pelleted,
andresuspendedat
I07
cells/mlin supernatant culture medium(CM)
of
the B95.8 cell line(15)
that had been filteredthrough
a0.45-jum
filter. The cellswereincubatedat37°C
andkept
insuspension
by
regular agitation.
After2h,thecellswereagain pelleted, washed,and
resuspended
at 106cells/mlin fresh culture medium
(RPMI
1640plus
10% heat-inactivated fetal calfserum)supplemented
with0.1,Ig/ml
ofcyclosporine (Sandoz,
Basel,Switzerland).Thecultureswerethen incubatedat
37°C
ina hu-midifiedatmospherecontaining5%CO2andfedweekly
by replacement
of halfthesupernatant.
874 Moreau,Bonneville, Peyrat, Godard, Jacques, Desgranges, andSoulillou J.Clin.Invest.
©TheAmericanSocietyfor ClinicalInvestigation,Inc. 002
1-9738/86/10/0874/06
$1.00(c) Cell harvesting, limitingdilutionassays, and donalexpansion.
Theprocedurehas beenpreviouslydescribedindetail(1 1-13). Briefly, after mechanical disruption ofthe rejectedkidney,thegraft-infiltrating
cellsobtainedwereplatedatdensitiesrangingfrom 5to 160cells/well
inthelimiting dilutionassayinthepresenceofI03 irradiated(10,000
rad)stimulator cells.PurerecombinantIL-2(rIL-2),kindlyprovided
Biogen(Geneva, Switzerland),wasaddedtoeach cultureata concen-tration of 150 BiologicalResponse ModifiersProgram (BRMP) U/ml
(seesectiong).Theemptywellswerecountedafter10to 15dofculture,
andthegrowthfiequenciescalculated(11). Onlymonoclonalcellcultures (probability>97%)werefurtherexpandedinIL-2-supplemented
(150 BRMPU/ml)withoccasionaladditionof irradiatedstimulator
(every5-7d).
(d) Markeranalysisofcolonycells.Cellswerefirstincubatedfor45
min at4VCwith thevariousmurineMAb(OKT8-OKT4-OKT3-OKT1I
and OKIa)dilutedto1:100.Afterthreewasheswithmedium
(phosphate-buffered saline [PBS]-I0% humanserum-0.I% sodiumazide),thecells
wereincubatedfor30 min at4VCwithfluoresceinisothiocyanate-labeled
goatanti-mouse Ig (NordicLaboratory, Tilburg, Netherlands) to1:40. Theywerethenwashed andresuspendedin
fixative (PBS-0.37%formaldehyde). Thepercentageof stained
thendeterminedunderan ultravioletmicroscope.
(e) Cell-mediatedcytotoxicityassays. A4-h"Cr-releaseassay(13)
wasusedtomeasurethecytotoxiccapacityof clones.Ineachexperiment, atleast foureffector/target ratios (E/T) were tested in triplicate.
targets usedwere donor, autologous, and one third-party B
blastoidcellline(BLCL)(HLA-A2;B12, 17;DR4,7),andtheK562
line. Resultsareexpressedas percentagesof specificchromium
Thespontaneousreleaseforanytargetusedneverexceeded
imum release. Individualwellswerescoredpositivewhentheir weregreaterthanthemean spontaneous"Cr-release value
plus threestandarddeviations.
(f) Proliferationassayand IL-2productionofclones.4or7dafter
the last addition of the irradiated donor B lymphoblastoid
(D-BLCL),clonedcellswerewashedthreetimesinCMand
aFicoll-Hypaque cushion toremovedead cells. Aftertwo washes,respondingcellswerecountedandviablecells(>98%) toadensity of 5X 105cells/mi.Irradiated(10,000rad)BLCLstimulators
(donorand autologous)werealsoadjustedto 5X 105 cells/ml,
ml ofresponding cellswasaddedintriplicatein round-bottom
either0.1 ml CMalone orto stimulator cells.24 hlater, ml supernatantwasusedtomeasure lymphokineproduction.The
0.05-mlcellsuspensionwasculturedforanother24h.Proliferation
assessedbytritiatedthymidineincorporation(0.05mi ofa
oftritiated thymidinestocksolution(TRK 61; 20-30
Amersham, Amersham,UnitedKingdom). Aftera4-hpulse,
harvestedontofiberglassdisks(Skatron,Oslo,Norway),
scintillationfluid(OCS, Amersham)andcountedinabeta
tillationcounter.
(g) IL-2 assay. 5,000 CTLL-2 weregrown in thepresence
dilutionsofputativeIL-2-containingmediuminflat-bottomed
plates(Falcon,Grenoble,France)withatotalvolume mi
well. 36h later, 0.5
gCi
of[3H]TdR(TRK61; 20-30 Ci/mMAmersham)wasaddedto eachwell.4h later, thecells were
ontofiberglassdisks,putin2mlof scintillationfluid(OCS,
and counted inaliquidscintillationcounter.IL-2units
aftera previouslydescribed method(16) using a standard
150 U/mi of IL-2, as defined by the BRMP ofthe National
Institute (BiologicalResourcesBranch, FrederickCancer
cility, Frederick,MD).
Results
(a) Frequencyofcolony-forming cells(CFC) among
graft-infil-trating cells. Our preliminary experiments showing
quencies obtained with a poolof 10 normal individuals'
diatedPBLas afeederlayerhavebeenpreviouslyreported(13).
Thistechnique,limitedbytheshortageof available donorcells,
allowed
the
growth
of outof139 cellsand outof767 cellswith and without phytohemaglutinin
(PHA)in theCM.ThelowCFC
frequency
aswellasthesubsequent slowgrowthratemayhave been related eithertothefeedercellswhich, for example,
may not
have
born sufficient donor antigen, or tothe usesemi-purified
IL-2 obtainedfromPHA-stimulatedPBL (17).an
effort
tosolve
thisproblem,anEBV-positive D-BLCLderivedfrom the
kidney
donor's
splenocyteswasused,afterirradiation,in place of the
PBL pool.Autologous Bcellsof recipient originwere
also
transformedinthisway.Fig.
indicates
thatCFCfrequencyfromGIC couldbedra-matically increased
byusingD-BLCLas irradiated feederintheabsence
of
anyadded
PHA.Undertheseconditionstheestimatedfrequency
was 1:13; whereasonly 1:2,190 cellsgrewwhenau-tologous
BLCL
(A-BLCL)were usedas feeder layer. Althoughrecombinant (Escherichia
coli),pureIL-2wasusedintheseandall subsequent
experiments, the considerable improvement inCFC
frequency
wasnot duetothe growth factorsource (asdicated by
controlculturewithrIL-,2andsemipurifiedIL-2,datanot
shown)
buttoD-BLCLfeedercellswhich,aswillbe indicatedlater,
provide increased
alloantigen expression and may haveproduced
IL-I
or other nonspecific growth activation factors(18).
Moreover, the subsequent long-term growth rate ofex-panded clones wasalso dramatically increased, with doubling
times of '24
h comparedwith 54 h when pooled PBLfeederwasused. In severalinstances, abillioncellscouldbegrown
twoweeks.
(b)
Colony phenotypes.
Inbulk GIC fromfourrejected kid-neysstudiedsofar,an average of 71.5% cells havebeen TI 1,61%
T8+,
and 14.5% T4+ (TableI).OnlyexperimentsdonewithGIC
from
kidney No. 4 (TableI) are reported in this paper,since
both donorandrecipientBLCLwereonlyavailableinthiscase. The colonies obtained from this graft were grown with
irradiated
D-BLCL in the presence of 150 BRMP U ofrIL-2per milliliterwith no lectin added. Colonies with >97%
prob-ability
of being
monoclonal were studied and phenotyped attimes ranging
from40dto14 ofculture.TableII summarizesthe results: 33/55 (60.0%) ofthe colonies were T4+, whereas
22/55(40.0%)wereT8+;these Ti andT3+.
100
Q)
0
2
0 0
50-
37-10.
10 2040
1C0
8C Cells cultured well320
Figure1.Limitingdilutionanalysisofgraft-infiltratingcells.Each
pointrepresentsthepercentofwells inwhichgrowthwasnot
forthemeannumberofcellsper wellindicatedontheabscissa. wereculturedwith(C) 1,000irradiated(10,000rad)A-BLCL
with(o) 1,000 pooled PBLirradiated(3,000rad)perwell, (A&)
1,000 irradiated(10,000rad) D-BLCLperwell.
f=1/2490
f=1/282
/
_.A
fa
H*1A13
k
TableI.
Surface
MarkerPatternofGraft-infiltrating CellsPercentage of stainedcells amongGIC
MAbtested No. I No. 2 No. 3 No. 4
OKTI1 66 74 57 89
OKT3 88
OKT8 63 60 51 71
OKT4 18 11 13 16
Cellsinfiltrating four different
kidneys
wereextracted and labeledwithmouseMAb
OKTl
1,OKT3,OKT8,and OKT4.colonies
weretyped severaltimes
and,except for one(1Fl
1),
showed
exclusive expression of
T4 or T8 antigens. Althoughdouble-labeling experiments
were notperformed
onlFl
1, 95%of the cells
wereT8V
and40%
T4+;therefore,
at least 35% wereboth
T4'
and
T8'.
This clone
wasincluded in
theT8'
subgroup asmostof
the cells expressed T8. Class II antigenexpression,
astested
using
the OKIAI MAb,
varied in
all clonesfrom
100
to30%
of
positive
cells
depending
onthe
time of testing.
(c) Cytotoxicity. All long-term cultured clones
(55 clones todate)
weretested
against donor, autologous,
and third-partyBLCL
(all these
targetssharing
only the HLA-A2 antigen) as well asagainst
theK562
cell
line. No cloneswere
cytotoxic forK562.
Furthermore, these
clonesdid
notkill
autologous or third-party5"Cr-labeled
BLCL,
which emphasized
that cells obtainedwith this
system were notcommitted
tonon-donor-specifican-tigens such
asEBV
antigens.
Amajority
were cytotoxic forD-BLCL (Table
II).
Inthese experiments, four
E/T
were done(0.5:1, 1:1, 5:1,
and
10:1),
allowing the
calculation
of lytic units
(LU 30).
However, someclones
did
not reachthe
30%specific
targetlysis,
although they repeatedly lysed the donor
target
sig-nificantly (i.e., above the
threshold
ascalculated in
Methods,section e)
atthe
highest ratios. Therefore,
in
Fig.
2, the 1:1 ratio
wasselected
becauseit
gavethe best
discrimination of
cytotoxicactivity
between
clones and the
sameranking
asLU30. Some
clones
werehighly
efficient killers,
with >50% of specific
5'Cr-release
at a1:1 E/T
(Fig.
2). Almost all
T8+
clones (except
1E6)
were
cytotoxic against
D-BLCL, with
14(out
of
22) exhibiting
>20%
specific
'1Cr-release.
However,
only
afew T4+ cells (2
outof 33) evidenced
prominent
(>20%
specific 5'Cr-release
at a 1: 1E/T)
cytolytic activity,
eventhough 14/33 clones
exhibited
modest
cytotoxic activity
(i.e.,
above the threshold
asdefined
in
Methods).
Allclones
sequentially
assayed have remained
simi-larly cytotoxic for D-BLCL
tothe
presenttime (>
1 yrin
somecases).
Table II.Phenotypic and Functional Characterization
of
SS TCell ClonesPhenotypesof clones Cytotoxicpotential
against D-BLCL T4+ T8+
No.of positive clones 17/38 21/38
(38/55)
(44.74%)
(55.26%)
No. of negative clones 16/17 1/17
(17/55) (94.12%) (5.88%)
(d) Proliferation and IL-2 production. All clonesweretested for their
proliferative responses against A-BLCL, D-BLCL, and
third-party BLCL. Proliferative
responses againstthird-party
BLCL
werenegative (data
notshown).
Severaldifferences
of behaviorbetween
T4+ and T8+ clones were notedand
are il-lustratedin
Fig.3. "Spontaneous"
proliferation,
astested inme-dium
alone,
wasmuch
higher
with
T4+ thanwith T8+
clones when thesecultures
were tested 6dafter alloactivation
(4 d, plus 2dof
test),whereas
9
dafter stimulation
(7 d,plus 2 dof
test) both types ofclones proliferated poorly (comparison between
Fig. 3,
Aand B, in
medium).
Secondly, proliferation in
the pres-enceof
D-BLCL
wasalso
morepronounced with
T4+ clones
atday 6;
however, stimulation indices
werehigher for T8+ clones
due
tolower
"spontaneous"
proliferation. Thirdly, when
A-BLCL
wereadded,
the resulting
proliferations
weresignificantly
higher
than spontaneousproliferations, indicating either
that somedeterminants
wererecognized with
aweak
affinity
or more probably thatthey
reflected
anonspecific feeder effect from
BLCL, in
this particular
caseA-BLCL.
Fourthly, themagnitude
ofproliferation varied with the responder/stimulator ratio. Fig.
4illustrates this feature in
arepresentative
experiment usingclone
1E7. The lower the
ratio, the better the
response,until
aplateau
is reached
at aratio of 1:6. All the above observations
were made atratios
of
1:1.Supernatants
of
thesecultures
werescreened
for
IL-2bioactivity using
the CTLL-2
assay.Only
five
(all T4+) of 55
cloneshad
released detectable IL-2
uponantigenic
stimulation (Table III). Clones
grownin
media
alone,
orwith
A-BLCL,
neverelaborated IL-2.
Inaddition,
nocorrelation
wasfound between
IL-2production
and
proliferative
response.Discussion
This
paperdescribes
amethod
for efficient
cloning of
T lym-phocyteswhich invade
arejected
kidney allograft and
somecharacteristics of the clones obtained. This method
canof
coursebe
applied
toother tissue
allografts
ortumors.Our data indicate
that
it
is nowpossible
toobtain
cytotoxic
orhelper
clones in
large numbers and alsoprovide further information
on phe-notypes,functions,
and
phenotype/function
relationships
of GIC
in
adirect approach
tothe
immunological
eventsleading
toallograft
rejection.
As
compared with
previousattempts toclone GIC
using
apool of normal
PBLasfeeder
layer
(13),
useof EBV-transformed
BLCL
derived from the
graft
donor
dramatically
improved
the
frequency
of IL-2-dependent CFC
(i.e.
from 1:767
to1:13).
The
improved
cloning efficiency
maybe due
to one or morenon-specific
factors
produced by
EBV-transformed
BLCL,
such
asIL- I
(18-20).
Although
suchnonspecific factors
mayhave
contributedtothe high CFC
frequency
observed, they
appear tobe effective
only
inconjunction
with
antigen,
since
autologous
feeder
yielded
only
a1:2,190
colony
frequency. Furthermore,
theavailability
of D-BLCL allows indefinite
useof
specific
donor
antigens
in-stead of
third-party
cellsbearing
certain shared
HLAantigens.
This
antigen
presentation
wasof
major
importance
in
ourstudy,
since A-BLCL
yielded
avery lowfrequency
of
CFC(1:2,190),
indicating that
nearly
all testedcolonieswerealloresponsive
and notresponsive
against
EBV-encodedsurface
determinants
pre-sentedby
self-major
histocompatibility
complex
molecules.Apart
from their
efficiency
in
thelimiting
dilution
phase
of
cloning, immortilized
D-BLCL were alsorequired
in
large
T4
% specific 51Cr release
.I
10 20 30
I I I 2B4 2F 7
2E5
1D6 16B 1G2
IClO 2C10 1F5
1B3
165
1E8
2E6 Cicl
IE6 2E4
1B4 2D4
2B3 1B4
1C3
*1Fl1
.
T8
% specific 51Cr release
20 40 60 80
-I
I I IEs
El
E.
El~~-M~
a
El
El
Figure 2.Cytotoxic potential of 33T4+
clones and of 22 T8+ clones. Each bar indi-catesthepercentageof specific
5"Cr-release
observedontheD-BLCLastarget atan E/T of 1:1 (see Results, section c). Clone IFI1 (*)was amixture ofT4+,T8+, and
T4+ T8+ cells (see Methods).Thevertical linerepresentsthe cutoff for positives and negativesasdefinedin Methods(section e).
quantitiestoobtainsimilarlylarge numbers (greaterthan one billionalloreactiveclonal cells within 2 wkof cultureinseveral instances) of clonally derived cells needed for functionalor
mo-lecularcharacterization of the derived clones.
Theidentified phenotypes ofthe cloned cellsweresomewhat
curious,sinceT4+cloneswere more numerousthanT8+ clones, whereasourprevious studies(12, 13) and others (21, 22) have indicatedthat thereverseprofile is commonly observed within rejected kidneys assessed either intissue sectionsorfrom fine needleaspiration biopsies. Indeed, the starting GIC bulk material
used inthese experiments also containedamajority of T8+ cells
(71%). As T8+clones hada consistently lowerdoubling time
thanT4+in similar invitro culture conditions, itmaybe
sup-posed that the rejected kidney microenvironment providesone ormorefactors(lackinginthe culture vessel) that enhance T8+
cellgrowthlocallyortrapT8+ cellsingrafts. Thesefactorsmay
beproduced by regulatory cells such asT4+ lymphocytes and
would thusbe absent from the clonalculturesystemanddistinct
fromIL-2. Alternatively,ahigher proportion of T8+ GICthan
T4+cellsmaynotbespecifically committedtothedonorantigens
and hence failtogrowwhenchallenged with D-BLCL.
Exper-imentsusing T4+clone culture supernatants inT8+cultures(in
thepresenceofrIL-2)todetect eventualgrowth-enhancing
fac-tor(s) produced by T4+culturesareinprogress.Onlyafew clones
produced detectable amounts ofIL-2 when stimulated by
D-BLCL, although theywerestrongly inducedtoproliferateinthe absence ofexogenousrIL-2 bythe stimulator cells. The most likely explanation that would fitwith the accepted model of
antigen-driven lymphocyte growth involvingautocrine produc-tion/consumptionof IL-2(23)is that the IL-2produced bythe clone isimmediatelyboundtothe receptors thatappearat
ex-tremely high density. Only quantitative studyof related mes-sengerRNA(mRNA)-IL-2 production after D-BLCL challenge
will, however, provide definitive evidence forthishypothesis.
AsalloreactiveT8+andT4+ cellsarelikely effectorsinvolved
in class I and class II recognition, respectively (24), the high cytotoxicity score ofT8+clones suggestsa majorrole for this
lymphocytesubset in therejectionprocess;however, their role maybedependentonthehelpereffect of theneighboring T4+. No clonesdisplaying cytotoxicity to K562 were found. That
K562, inaddition tobeinga natural killer-sensitive target, is
lysed by lymphokine-activatedkillers reinforces thefindingthat 2B11
2C7
lE7 2D11
iF10
1C7 2F2 2E7 2B9 2D8 2F4
1F3
2D8 1C2 2F6 2E 10
2C2 IF5
103 2F5 3C8
IF4 1C4 1D7 2C5
2B5
2C3
1E3 1D2
1E2 1B2
1D9 2C6
R I I I I
El I
Pi
100-A
50
0
T-x
E
0)
0
-j
10-
5-
1-0.5.
I
I I I
MEDIUM D-SLCL A-BLCL
OKT4
I I I
MEDIUM D-SLCL A-BLCL
OKT8
0.1-B
I I I
MEDIUM D-BLCL A-BLCL
OKT4
1 I I
MEDIUM D-SLCL A-BLCL
OKT8
Figure 3. Proliferationresponseof clones 6d (A) and 9 d (B) after the last stimulationby the D-BLCL.
[3H]TdR
incorporation ofT4 andT8' cloneswasassayedinmedium alone,inpresenceof irradiated (10,000rad) D-BLCL, andinpresenceof irradiated(10,000 rad) A-BLCL,ataresponder/stimulator ratio of1:1.
-1-o
0.5-x
E
0.1-0 ,
0.05-E
0
0
-i 0.01.
1 i2 6 7
days after stimulation
Figure4.Effect of theresponder/stimulatorratioonthegrowthofone
clone(IE7).Identical numbers of 1E7 cells(5X10)wereseeded
without (1:0)orwithincreasing numbersofirradiated (10,000 rad)
D-BLCL(1:1.25, 1:2.5, 1:5,and1:10)attheinitiation of the cultures
(day zero).Cellcounts,expressedontheordinate,werefollowedfrom day3today7.
the clones
obtained
wereall
alloreactive.
Nevertheless,
the
fol-lowing
question
arises: what is the
natureofthe
remaining
lym-phocytes
(90%) that do
notgrow in this
system?
Apossible
ex-planation would
bethat
the
experimental
system is still unable
TableIII.IL-2 Titers
of
theFiveSecreting ClonesCulture conditions
Clones Medium D-BLCL A-BLCL
2C6 0 50 0
2B9 0 16.5 0
2D8 0 5.25 0
1C4 0 2.21 0
2F4 0 3.90 0
11-2activities in supernatants from clones either nonstimulated (me-dium)orstimulatedwith D-BLCL and with A-BLCLwereassayedon CTLL-2 andexpressedinBRMPunits per milliliter in the Methods section.
Bonneville, Peyrat, Godard,Jacques, Desgranges,andSoulillou
50-
10-
5- 1-0
x
E
0)cm
0 -j
0.5
0.1
100-to recruit
satisfactorily
all the committed cellsagainst
donorantigens.
However, with the same culture system (1 1),two-thirdsof the PBLfrom normal individualsin thepresenceof PHAand
with
autologouscells
asfeeder
cellsareabletogrow,whichsug-geststhat the culture conditionsare not
limiting.
It is alsopossible that the nongrowing cells may be committed against antigens absent from the surface of D-BLCL. Suchantigens
presentonkidney cells, whether restricted by major
histocompatibility
complex antigens ornot,have been described astargetsinal-lorejections (25-27). Conversely,
theseunresponsive
lympho-cytes arenot,perhaps, actually
committedagainst graft antigens,
but are recruitedby
other committed cells thatproduce
che-motactic factors
within therejected graft. Finally,
it mustbeemphasized that our data appliedto a case of definitive graft
rejection in
arecipient who
was withoutanimmunosuppressive
regimen
for weeks. TheGIC
frequency
and the functional pat-terns obtained may be somewhat different inothertypesofre-jection.
Nevertheless,
cellular infiltrates collectedby
the meanof thefine needle
aspiration biopsy
may be usedby
themethodwe
herein
described for the
analysis of rejection. Different
types ofrejection
and their evolution could thus be studied.Acknowledgments
We thank Biogen (Geneva, Switzerland) for supplying recombinant IL-2, Mrs. A. Bertho for her technical assistance, and Dr. J. Grayfor
reviewing the manuscript.
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