proliferation and the cells that actually proliferate. Animals. Rats (LEW and DA strains and LEWxDA F, animals) were obtained from the Institut

Vol.30, No. 3 INFECTION AND IMMUNrry, Dec.1980,p.741-752

0019-9567/80/12-0741/12$02.00/0

In

Vitro

Proliferation of

T

Lymphocytes

from

Listeria-Infected

Rodents: Assay Conditions for Rat Peritoneal

Exudate

Cells and Characterization

of an

Inhibitor

THOMAS W. JUNGI

Swiss Research Institute, Medical Department, CH-7270 Davos, Switzerland Conditions favorableto[3H]thymidine incorporation into antigen-stimulatedT

lymphocytes from Listeria-infected rats have been established. In cultures of

peritoneal exudate (T) lymphocytes purified twice with nylon-wool vigorous

antigen-specificproliferationwasobservedwithin 2 days.Culturesoflymphocytes

from nodes drainingasubcutaneousListeria-infectionsite differed in that

back-ground proliferation was higher than for peritoneal exudate lymphocytes, and

[3H]thymidine incorporationwasmaximalatday3. Acritical factor for therate

of proliferation was the lymphocyte-to-macrophage ratio; optimal cultures of peritoneal exudate lymphocytes contained 2 to5% macrophages. Macrophages exceedingaproportionof 10% strongly, ifnotcompletely, inhibited[3H]thymidine incorporation into antigen-stimulated lymphocytes. Inhibition was associated

withmononuclearcells, adherenttoplastic ornylon-wool, of the stimulated or

unstimulated peritoneal cavity. It was neither attributable to release of cold thymidine from macrophagesnortorapid degradationof particulate antigen by

macrophages.Thedegreeof inhibitionreflected the metabolicactivity of

macro-phages;onacell-for-cellbasis,heat-killed andglutaraldehyde-fixedmacrophages

were less inhibitory, and stimulated macrophages were more inhibitory than macrophages from the unstimulatedperitoneal cavity.

The experimental infection of rodents with the facultative intracellular parasite Listeria

monocytogeneshasachieved thestatusofa clas-sical model forTcell-mediated defense(22).The mannerwhereby restimulatedTcells and

effec-tormacrophages collaborateappearstobe

rele-vantforawidespectrum of infections (32) and

for certain types of antitumoral immunity as well. These mechanismshavebeenanalyzedin vivo by Mackaness (17), McGregor et al. (18,

19), and North (22), who measured acquired

resistance in an adoptivetransfersystem.

How-ever, for dealing with avariety ofissues, it is

desirable to evaluate resistance in vitro inas-muchas asmaller number of cells suffices fora

largenumber ofparalleldeterminations. More-over, appropriate in vitro correlates offer the promise ofanalyzingindividual definedstepsin the chain ofeventsthatultimatelyleadto bac-terialelimination.

Thisis theinitialreport inaseriesand ana-lyzesthymidineincorporation by

antigen-stim-ulatedListeria-sensitivelymphocytes, anin

vi-troparameter representing T-celltriggering. It establishes the experimentalconditions forthe measurementofproliferation byrat peritoneal

exudateandlymphnode cells.Subsequent stud-ies will address the tissue disposition and

sub-populationrelationshipsof thecellsthat induce

proliferation and the cells thatactually prolif-erate.

MATERIALS AND METHODS

Animals. Rats (LEW and DA strains and LEWxDA F, animals)wereobtained fromthe Institut

fur Biologisch-Medizinische Forschung, Fiillinsdorf,

Switzerland and were used either directly or after

breeding foronemoregeneration.

Microorganisms. Listeria monocytogenes were

grownandquantifiedasreported previously(11, 18).

Particulate antigen (LMA) wasprepared by ethanol treatmentof fullygrowncultures and lyophilizationof

thewashed dead bacteria (12).Antigen was suspended

in tissueculture medium and brieflysonicated before use.

Francisella tularensis, usedasspecificity control,

was prepared and quantified as reported elsewhere

(15). A Francisella antigen (FTA) was made in a

mannersimilartothatforLMA.

Rats were immunizedbychallengewith 5x106live

bacteriainmultiple hindquartersites.

Cell collection. Peritoneal exudateswereinduced byintraperitonealinjection of50jugofLMA (orFTA,

respectively) in rats that had been infectedwiththe

homologous parasite6days previously.Exudates were collected in ice-cold Hanksbalancedsaltsolution

con-taining2U ofheparinper ml 24 hafter intraperitoneal stimulation. Thecellswerewashed once in medium

199supplemented with 1%newborncalfserumbefore column fractionation.

Popliteal and axillary lymph nodeswereremoved aseptically and teased withforceps in Hanks balanced salt solution plus heparin, and the dissociated cells werefiltered throughadouble layer of sterile gauze and washed several times beforeuse.

Theperitonealcavity of normalrats(irritated either with casein or untreated controls) was ravaged to

obtain cell populations rich in macrophages. They werewashed three times beforeuseinculture.

Cell separation. Adherent cells (macrophages, neutrophils, and B lymphocytes) were removed by filtrationthroughnylonwool(10).Nylon-wool swabs (0.6 g, dry weight) (Fenwal, Illinois, code 4C2906), boiled indistilledwaterfor1hbefore use,wereplaced intosyringes, rinsed thoroughly with saline and sepa-rationmedium, and then dropwiseoverlayered with2

ml ofcellsuspension and1 ml of medium. Columns weresealed and incubated for1hat370C,after which nonadherentcellswerewashedoutbydropwise addi-tion ofseparation medium(medium199supplemented with 0.01 M N-2-hydroxyethylpiperazine-N'-2-ethane-sulfonic acid [HEPES], 50U ofpenicillin per ml, 50

pg

ofstreptomycin per ml, and 10% serum). Fetal calf

serum wasfirstused, butwaslater replaced by

new-borncalfserum.Unless otherwiseindicated,50x 10' to 70x 106peritoneal exudate cells (PEC) (roughly

oneexudate equivalent)wasapplied percolumn for initial purification. For subsequent filtration, the cell number percolumn varied dependingonthenumber of animals per group. These conditions consistently provided maximal [3H]thymidine uptake by nonad-herent PEC enriched in Tlymphocytes (PETL).

Unpurifiedexudatesgenerallyconsisted of approx-imately 16%lymphocytes, 18%granulocytes,and 66% macrophages. Eachpurification reduced the number of phagocytes (neutrophils and macrophages) by a

factor offrom4 to 6. Immunoglobulin-bearing cells

werereducedto<5% in PEC purified once with nylon wool andto<3% inPECpurified twice in nylon-wool

(ofteneven to<1%).

Immunofluorescence. Insomecases, the propor-tionof B cells(seeabove)wasdeterminedby indirect immunofluorescence. 3 x 10' to 5 x 106 PEC were

treated with theFab fraction ofsheepanti-rat

immu-noglobulinG antibodies that cross-reacted with other

rat immunoglobulin classes (Nordic, Biogenzia Le-mania, Lausanne/Switzerland; code ShARa/Fab).

This was followed by treatment with fluorescence-conjugated rabbitanti-sheepantibodies(Nordic,code RASh/FITC). Cellswereexamined under a Wild M20

microscopeequipped withaZeissIV FLreflectedlight condenser,KP 490andKP 500exciterfilters,anFT

510reflector, and an LP520barrierfilter. Controls includedwerenormalsplenocytes; thoracic duct lym-phocytes, which wereunpurified and didnot adhere

tonylon-wool; thymocytes; anda setoftestcellsthat weretreated withonly the secondantibody.

Tissue culture. Cellswereculturedin a volume of 0.2ml in96-well, flat-bottomed,microtiterplates (Cos-tar, Cambridge, Mass., catalogno.3596). Theywere

stimulated either with2jigofLMAor 6,g ofFTA. In

all experiments, four (or more) cell concentrations were tested intriplicate orquadruplicate, thus per-mitting the plotting of a curve for theproliferative capacity.

In mostexperiments, culture medium consisted of RPMI1640supplemented with2ml of essential amino acids(Flowlab, catalogno. 16-011-49),2ml of nones-sential amino acids(Flowlab, catalog no. 16-810-49),1

mlof glutamine (200mM; GIBCOLaboratories, Grand Island, N.Y.), 1 mlofpenicillin-streptomycin (50U/

ml and 50ug/ml, respectively; Novo Industri A/S., Copenhagen,Denmark),1mlofHEPES (1 M),0.5ml of2-mercaptoethanol (0.01 M), and homologous serum (5% serum was used in early experiments, but was later reduced to 1%).

Cells were cultured for 1, 2, or 3 days at 37°C in sealed boxes gassed with a mixture of 7% 02, 10%CO2,

and 83%N2.

Determination ofthymidine incorporation. For the terminal4hofculture, each wellwaspulsed with

0.5 uCi of [3H]thymidine (CEA, Gif-sur Yvette, France); (specific activity,5Ci/mmol). Cells were then harvested withamultiple cell harvester (Skatron Ti-tertek cellharvester) onglass fiber disks which were counted with Econofluor (New England Nuclear Corp., Boston, Mass.) ina Packard Prias PL liquid scintillation spectrophotometer. Counts per minute werecorrected for quenching (cpm). In most assays, standard deviation was less than 10% of the mean countsper minute.

RESULTS

Antigen-stimulated proliferation in vitro by Listeria-induced exudate cells. In rats infected withL. monocytogenes, largenumbers

of specifically sensitized lymphocytes migrate intoantigen-induced exudates (14).Assuch cells confer antimicrobial resistance tonaive

recipi-ents, it islogicaltopostulate thatthey have a

functioninthe antimicrobial defenseoncethey have reached the focus of the bacterial chal-lenge. Accordingly, the capacity to proliferate

upon LMA-stimulation in vitro was tested for PEC induced with LMA in L.

monocytogenes-immune rats.The relatively low proportion of

lymphocytesinsuch exudateswasincreased by the removal of adherent cells on nylon-wool

columns.

Figure 1 showsthat PECenriched in nonad-herentPETL responded by vigorous prolifera-tion when stimulated with LMA, reaching a

peakatday2 of culture. In contrast,little thy-midinewasincorporatedin the absence ofadded

LMA,althoughthe sameantigenhad been used for exudate induction. This was surprising in

view of earlier reports that lymphoblasts are

privileged in their localization in

antigen-in-duced exudates (14). Detailed comparisons

re-vealed that theproliferationrateof PETL was

indistinguishable in LMA-supplemented cul-turesand cultures without added LMA atday0 ofculturing(4,000to6,000 cpm/50x104PETL).

Whereas theproliferationratedroppedto

back-ground levels (ca. 300cpm) atday1incultures freeofLMA,it rosesharplyinLMA-containing

LISTERLA-SENSITIZED RAT T-CELL PROLIFERATION 743

wells within the first 24 h (>20,000 cpm/50 x

104 PETL). Nonpurified PEC failedtorespond

invitro. LMA-induced proliferation ofratPETL

depended upon preinfection of the cell donors

with L.monocytogenes(Table1). Thus,whereas PETLfromratsthat had beeninfected 1, 2, or 4 weeksbefore cell collection showed vigorous

[3H]thymidine

incorporation after2days of

cul-ture with LMA, similar cells from uninfected

rats or ratsinfected with an unrelated parasite, F. tularensis, displayed only modest

prolifera-tion. Likewise, peritoneal resident lymphocytes

25

0

50

25

a..

C.)

C)_0~

0

75

50

25

0

from preinfected rats showed significantly

higher[3H]thymidine incorporation than similar cells from normal donors. Results similar to those withPETL were obtained withunpurified

lymphnode cells draining the site of L. mono-cytogenesinfection,exceptthatbackground

pro-liferation (in the absence ofadded LMA) was higher than for PEC, andmaximal

[3H]thymi-dineuptakewasnotedonday3.

Assessment of media additives in PETL

proliferation test. Subsequent experiments

wereaimedatoptimizingtheconditions for the

50

25

12.5

6.25

3.13

10

4

PEC

FIG. 1. Antigen-induced[3H~thymidineincorporationintoPECfromListeria-immune rats. PEC collected from24-h exudates induced with LMA in L. monocytogenes-immune rats were tested either unpurifiedor

afterone ortwonylon-woolpurificationstoremoveadherent cells.Cellswereculturedatvarious concentra-tions either in thepresence(closedsymbols)orabsence(opensymbols)of2jigofLMA per well.Exp.1,cells derivedfrom DArats; exp2, cellsfrom bothDA and LEW rats weretested. d, Days.

INFECT. IMMUN.

proliferation assay with L.

monocytogenes-im-munePETL.Serumsources andconcentrations

werefirstexploredtominimizebackground

pro-liferationand toincrease antigen-induced

stim-ulation.

Figure 2 shows that serum wasrequired,

al-though relatively low concentrations (i.e., 1%) weresuperior to larger amounts.Fetalcalfserum gave superior stimulation but induced

signifi-cantlyhigher background proliferationthan did

homologousserum and wastherefore excluded insubsequentexperiments.

Thecapabilityof2-mercaptoethanoltoassist proliferationwasdetermined next(Fig. 3).

Evi-dently, 2-mercaptoethanolwasrequired,the op-timal dosebeing5 x

10'

M.

Othermedium components tested were mix-tures of essential aminoacids, astheyare used

forMishel-Dutton typemedia (21), and nones-sential amino acids(datanotshown).The

pres-ence of either essential or nonessential amino acids or bothdid notimprove lymphocyte pro-liferation in thepresence of 5%serum; but,the

addition of essential and nonessential amino

TABLE 1. Effects of L. monocytogenes (LM) and F. tularensis (FT) preinfection on LMA- and FTA-induced proliferation of nylon-wool-purified nonadherent PETL and resident peritoneallymphocytesa

[3H]thyiidine

incorporationbylymphocytesat 2days after cell collection

LM or FTpreinfec- LMA orFTAinduc- (cpm)

tionof cell donors tion of exudate (days Cultures with LMA Cultures withoutantigen (days before cell col- beforecell

collec-lection) tion) 50x104cellsper 25x104cellsper 50x104cells 25x104cells

well well perwell perwell

LM (27) LMA(1) 26,372b 12,648 553 1,056 LM (13) LMA(1) 36,878 21,661 139 330 LM (6) LMA(1) 31,840 25,933 918 319 LMA(1) 4,309 3,091 NDc ND FT (6) FTA (1) 3,660 1,881 ND ND LM (27) 157,352 173,628 5,848 12,083 LM (13) 92,819 146,265 3,944 2,679 LM (6) 138,339 42,132 679 422 5,241 4,790 ND ND

a

Peritoneal

cellswerecollectedonday0.

bMean cpm of

triplicate

wells.

'ND, Not done. 00 U FC NBCS NO SERUM 50/0. 10/00 " 20%o. "A i

*/.a

-

m

"\

50/e-____m

I 50 25 12.5 6.25 50 * ", RAT SERUM 10/0 * .-60/0 *-l. "% N._ 201.*N * ' 25 12.5 6.25

104 PWc

FIG. 2. EvaluationofvariousserumconcentrationsforLMA-induced [3H~thymidineincorporation into L.

monocytogenes-immune PETL. Closedsymbols, wellscontaining2pgof LMA;opensymbols,noexternal

LMA added. 75 50 C-) (7) ° 25 0 I I

LISTERIA-SENSITIZED RAT T-CELL PROLIFERATION 745

acids to mediumcontaining1%serumdid signif-icantly enhance LMA-induced proliferation,

particularlyforsmall cell inocula.

This evaluation of additives resulted in the formulation of the medium for ratlymphocyte proliferationassaysasgiven above.

Antigenspecificity of lymphocyte prolif-eration in vitro. In view of the report that

Listeria-derived antigens are polyclonal B-cell activators(3), antigen specificityof the lympho-proliferative response had to be secured by

30 20 C-) °10 0o 50 25

meansof anothernon-cross-reactingparasite, F.

tularensis(Fig. 4).

Whereas L. monocytogenes-immune PETL displayed vigorous [3H]thymidine uptakeover a

broad range of LMA concentrations (6 to0.06

jigperwell), they showed littleproliferation in

thepresenceofsimilaramountsof FTA.

Accord-ingly, F. tularensis-immune PETL responded welltoFTA (20to2jigperwell), but

incorpo-rated little [3H]thymidine in the presence of

LMA.Antigen specificitywas confirmed inthe

12.5 6.25

104

TWICE PURIFIED PEC

FIG. 3. Requirement of 2-mercaptoethanol (2-ME) for optimal[3Hjthymidine incorporation into LMA-stimulated(2pgperwell)PETLfromL.monocytogenes-immunerats.

40 30 20 101 0 20 6.32 2 0.63 0.2 G06

ANTIGEN

(jpg/well)

FIG. 4. Antigen specificity of[3Hjthymidine incorporation into L. monocytogenes (LM)-immune or F.

tularensis(FT)-immunePETL. Cells stimulated with thehomologousantigen(closed symbols)respondmuch

betterinthymidine uptakethan those stimulated withanunrelatedantigen (open symbols).

*o 5x10-5M 104 M A 2.5x 1 M\ A

K

-NO

2-ME _ A__Z_ mum=m==_ _O _

.=

0-C-) CV) 0D .0 LM-immune *: FT-immune

' -

0---

-*

/

**

A.0

0o/ 0 _ IMA * * FTA 0 °: =d 9~ I I 9 VOL. 30,1980

experiment shown in Fig. 5, in which optimal

amounts of antigenswereculturedwithvarious amounts of eitherimmunePETLorunpurified

axillary andpopliteallymph node cellsdraining

the site ofL. monocytogenes infection. It was

not determined whether moderate stimulation inwellscontainingthe heterologous antigenwas

duetocross-reactivityor to amitogenic effectof thebacterialantigens (3). Inanyevent, intense

proliferation ensued only when sensitized lym-phocyteswere exposedtohomologous antigen,

afinding thatis notsurprisinginlight of thelow

degree ofcontaminationofPETLpreparations

withimmunoglobulin-bearingcells.

Inhibitionoflymphocyteproliferation by

peritonealwashout cells. It was nextsought

toadjust theassayforacceptable

reproducibil-ity.Inparticular,itwasappreciatedthat factors

unrelatedtonumbersoractivity of sensitizedT

cells could influence the level ofthymidine

in-corporation.Accessorycellsmayinfluence

anti-genpresentationandpromotionandinhibition of T-cell proliferation and differentiation.

In-deed, the results ofFig.1andotherexperiments

(notshown)suggestedthatpopulations enriched

in adherent cells,possibly in macrophages,

in-hibitLMA-induced proliferationof sensitizedT

cells. To explore this systemically, various

amounts of cells from macrophage-enriched populationswereaddedtopredetermined

num-bers ofsensitizedlymphocytes (Fig. 6).

Normal peritoneal washout cells (PWC)

75 -UNPURIFIED LNC 50 LI

a-L) 9-25 0

added to lymphocytes from nodes draining a

subcutaneous L. monocytogenes infection site inhibited not only antigen-induced thymidine

incorporationbysensitized lymphocytesbutalso their background proliferation, i.e., thymidine

incorporation in the absence ofexternal LMA (experiment 1). Inhibitionwassharplydose

de-pendent(experiment2).Whereashigh numbers ofPWCinhibitedantigen-induced proliferation

of immune PETL as compared with cultures without added PWC, low numbers enhanced this proliferation. Two lines of evidence argue against the proposition that

macrophage-de-rivedcoldthymidineacts as acompetitive

inhib-itoroftheradiolabeledisotope (experiment3).

First,even afivefoldincreaseofthepulsing dose didnotreduce thecomplete inhibition of

[3H]-thymidineincorporation.Second, removal of ex-tracellular thymidine by washing thecells and replacing the medium before pulsing did not

alter the degree of inhibition by PWC.

(Al-thoughunlikely,thepossibilitystill remains that

intracellularthymidine storesestablishedbefore washing and pulsing could later be used for incorporation in preference toextracellular la-beledmaterial.)

Furtherevidenceforactual inhibition of

pro-liferation bymacrophage-rich populationscame from determinationsofviable cells in cultures

supplemented by PWC (not shown). PETL to

whichrelativelyhigh numbers ofPWC had been addedcontained relativelyfewerviable

lympho-50

25

12.5

6.25

50

25

12.5

6.25

104

CELLS

FIG. 5. Antigen specificity of

[3HWthymidine

incorporation into L. monocytogenes (LM)-immune orF.

tularensis (FT)-immunePETLordraininglymph node cells (LNC).-,antigen-supplementedcultures; ---noantigen added.

LISTERIA-SENSITIZED RAT T-CELL PROLIFERATION 747 cytesafter2or 3days of culture than

unsupple-mentedcells.

Properties ofacellular inhibitor of lym-phocyte proliferation. Since the PWC popu-lation which proved to beinhibitorywas a mixed

cell population, the inhibitors were character-ized.PWCwerefractionatedonFicoll-Hypaque gradients intocellsof lower density (containing

macrophages andlymphocytes, <1%mastcells) andhigher density (containing 26%mastcells). Cells in various doseswereappliedtomicrotiter plates and incubated for 2 h. Thereafter, the

nonadherentcellsweretransferredto newwells, andanadditionalsetof wellscontainingthefull nominal cell numbers (adherentplus

nonadher-ent) was left untreated. Mediumwas removed

50 25j C-) o (" 150

C"

_olS

from all wells. Then, immune twice-purified

PEC andLMA wereadded.

Thevalues for thymidine incorporation after

2 days of culture are given in Table 2. It is obvious that those PWC adhering to plastic were moreinhibitory thanthose notadhering. More-over, removal of mast cells did not diminish inhibition. This pointed to inhibition by the macrophage, and evidence obtained inanother

experimentsupported this view. Here, PWC and casein-induced PECwerefractionatedonnylon wool columns and then added to cultures of immune PETL. Figure 7 shows that column fractionation removed the inhibitory cells in

both PWC and PEC populations. Moreover, it appeared that stimulated macrophages, on a

EXP

1

LNC: .+LMA o

w/o

LMA * *a LMA+5 xOPWC( 1

+LMA+

25xlOsPWU

0 50 25 125 625

1O4LNC

EXP

2

2xPURIFIED PEC(25

x1'4)

+PWC U

EXP 3

2xPURIFIE PEC (25x

104

) +Pwc 1001 504 0' iCi pulse 300 250 200 x

1

-) '150C CCi

pose

t100

40 2010 5 2.5 1.25 0 40 20 10 5 25 1.26

104

pWC

104

pWC

0 50

FIG. 6. InhibitionbyPWCof[3Hlthymidineincorporationinto L.monocytogenes-sensitizedcells.Exp.1: 2.5 to 5x105PWCcompletelyabolished[3Hlthymidineuptakeexertedby draining lymphnode cells(LNC) that wereeitherexternally stimulated with LMAor not.Exp. 2:Although higherdosesofPWC inhibited

[3HWthymidineuptake byimmuneperitonealexudatelymphocytes,lowercellnumbersenhancedit.Exp.3:

Inhibitionof[3Hjthymidineuptakeby highdosesofPWC(0-0) wasneitherabrogatedwhen thepulsing dosewasincreasedfivefold (W-U) norwhen the cells werewashedbefore pulsingto removeextracellular unlabeledthymidine(v----).[3H]ThymidineuptakedidnotoccurwhenLMAwasomitted, regardlessofthe numberof added PWC(O----0).

-r

.- -. I I 7el . 7.4 .

cell-for-cell basis,were morestronglyinhibitory

thanunstimulatedperitonealcells.

Althoughinclusion of theinhibitor in the frac-tionadherenttonylon-wool would also applyto

Bcells, other evidencearguesagainstan

inhibi-toryeffect of B cells.Thus, inhibitorsarepresent

in high concentrations in peritoneal washouts andexudates, but theyseemtobeabsent from

lymphnodes and spleens. Addition of mitomycin C-treated lymph node cells does notalter the

proliferative rate of LMA-stimulated immune

PETL orlymph node cells. Inclusion of

mito-mycin C-treatedsplenocytes (upto25x 104per

well) even enhances thymidine uptake by L.

monocytogenes-immunelymphocytes (datanot

shown).

Attempts to elucidate themechanism of macrophage-mediated inhibition of T-cell

proliferation.Theabove dataqualify the

per-itoneal macrophageas astronginhibitorof

an-tigen-induced PETLtriggering, thus corrobor-ating thefindingsinothersystems(1, 2, 5, 9, 13, 20, 23-25, 27-30) but leaving the mechanism unexplained. One possibility is that the rela-tively high numbers of macrophages could

rap-idly degrade the antigen and render itnolonger

stimulatory for T cells. The hypothesis was

tested inmacrophage-containing cultures of 25

x104PETL where LMA had beenadministered

eitherattheonsetofcultureorinthreedosesat

times 0 and 18 and 30 h after administration. Thymidine uptake was measured between 44

and48hasusual.Evenrepeated administration

of LMA did notalleviate the strong inhibition

exerted by as few as 2.5 x 104 macrophages,

suggestingthatmacrophagespreventT-cell trig-gering byamechanismother thanrapidantigen

removal.

Whether macrophages need to be viable to inhibit LMA-induced lymphocyte proliferation

was next determined. Graded doses ofviable, heat-killed, or glutaraldehyde-fixed PWC were

incubatedwith 25x 104immune PECand LMA.

Untreated cells exerted strong inhibition,

whereas deadperitonealmacrophageswereless

inhibitory (Fig. 8). However,eventhe lattercells

inhibitedmorethanperitonealcells from which

adherentcellshad been removed(Fig.7). These experiments illustratethat the

macro-phage-to-lymphocyteratio isacriticalfactor in

the

[3H]thymidine

incorporationassaywith

lym-phocytesfromListeria-infectedrats.The inhib-itory effect is dissociated from rapid antigen

removal or cold-thymidine release by

macro-phages,and itsextentdependsonthe metabolic

activity of the macrophages, i.e., stimulated

macrophages inhibit more and dead

macro-phagesinhibit less than normalresting

macro-phages.

DISCUSSION

Determination of [3H]thymidine

incorpora-tion by antigen-stimulated T cells has been

adapted to the study of Listeria-infected rats with the intent to have available an in vitro

assayrepresenting T-cell triggering in listeriasis.

Several lines of evidence suggest that

[3H]thy-midine incorporation in lymphocyte cultures

TABLE 2. Characterizationofcells that inhibitthymidine incorporation byantigen-stimulated purified PECfrom immune rats

No.ofinhibitory cells added to wellscontaining all Cell fraction addedto 25 x10 Subfraction of cells added (adherentplusnonadherent)cells"

PETL 40x104 20 x104 ox104 5x104 PWCunfractionatedb Adherent' 250 476 332 5,643 Poorly adherent' 227 5,316 45,014 42,421 Both 159 245 157 1,227 PWCFicoll-Hypaque-purified

Top layer Adherent 180 446 417 994

Poorly adherent 328 19,449 57,097 51,198

Both 138 185 436 672

Bottomlayered Both 193 325 733 8,674

Nocells added 30,929 3,479

aDatarepresents counts perminuteperwell(meansoftriplicates).

bContained7% mastcells,17%lymphocytes,and 75%macrophages.

'After 1h ofincubationofindicatedcell numbers perwell, cells that did not adhere were gentlyaspirated

andtransferredtonewwells.Allwellswerewashedwith fresh medium,25 x 104PETL and LMA were added toeachwell.

dContained 1%mastcells.

LISTERMA-SENSITIZED RAT T-CELL PROLIFERATION 749 50 25 0

$250

25 0 40 20 10 5 2.5 1.25

104

PECCASEIN

FIG. 7. Mediation by nylon-wool-adherent cellsofPEC- orPWC-mediated inhibition of[3H~thymidine incorporation by antigen-stimulated lymphocytes.PWCorPEC(collected3days aftercasein-induction) from

normal animalswereadded invariousnumbers eitherbeforeorafterone ortwonylon-woolpurificationsto triplicatewells. Inaddition,eachwellreceived 25x104PETLfromL.monocytogenes-immuneratsand 2pg

of LMA.

stimulated with LMA depends upon specific

stimulation of sensitized T cells. Inacomparable

murine system, PEC adherent to nylon-wool from athymic mice do notrespond in vitro to

LMA, and PETL fail to proliferate in LMA-containing cultures aftertreatmentwith

mono-clonal anti-Thy 1 antibodies and complement

(Jungi,unpublisheddata). Duetolack of inbred athymic ratsandunavailabilityofhigh-density surface markersonratTcells, such studies have

not yetbeen confirmed in thepresent rat system. However,thedocumentation ofantigen specific-ity (Fig. 4 and 5) and the observed low-grade contamination with immunoglobulin-bearing

cells (<3%) of nylon-wool-purified lymph node cells and PEC wouldsuggestthatLMA-induced proliferation ofratlymphocytes purified by

ny-lon-wool represents triggering of sensitized T

cells.

In thepresent study, appropriate conditions forthestudy of peritoneal exudatelymphocytes

notadheringto nylon-wool withrespect to

in-cubationtime, culture medium (serum source,

concentrationof2-mercaptoethanoland

require-ment for amino acid additives), antigen dose, and cell purification procedure have been

de-vised. Purificationovernylonwoolwasessential

since it removed a cellular inhibitor of

LMA-induced proliferation (Fig. 7). This inhibitorwas

found inthe unstimulated peritoneal cavityof

normalrats.Itwaspresent withincreased

activ-ity, on a cell-for-cell basis, in casein- or

LMA-stimulatedperitonealexudates,butwas absent or present in much lower concentrations in

lymph nodes and spleensof normalorL.

mon-ocytogenes-infectedrats.Its adherence toplastic andnylon-wool,presencein theFicoll-Hypaque gradientlow-densityfraction (mononuclearcell fraction), and its tissue distribution

character-ized itas amacrophage.Other celltypessuchas

mastcellsorlymphocytesdid not interfere.

De-creaseofthymidine uptakewasassociatedwith

lx purif. 2x purif. unpurd 40 20 10 5 2.5 1.25 0

104 Pwc

2x

purif>,,,-

Ah

m*-,lx

purif.

A .

ulpu~re ~~

'7I 0 VOL. 30,1980

30~~~~~~

20 0 & 30' 560 a.~~~~~~~-t c

10

t

,GLu-al.

o /Untreated 10~~~~~~ 40 20 10 5 2.5 1.25 0.63

104

PWC

FIG. 8. Inhibition of

[3Hlthymidine

incorporation into LMA-stimulated PETL by viable,heat-killed, or glutaraldehyde-fixed unstimulated peritoneal cells. PWC from normal donorswereeither treatedfor30min

at560C, treatedfor30min at370C with 0.25glutaraldehyde (Glu-al) orleft untreated. From eachpool,

washed cells were added in varying amounts to triplicate wells containing 25 x 104 PETL from L. monocytogenes-infected donors and2pgofLMA. Inclusion of appropriate controls (notshown)indicated that neither viablenordead PWCincorporated[3Hjthymidineperse orstimulatedincorporationby PETL in the absenceof LMA.

alower number of viable cells in 2- and 3-day cultures of lymphocytes and macrophages when compared with cultures ofpurified lymphocytes alone. It seemedtobeunrelatedtorapid removal of theantigenicstimulus byexcessmacrophages

asrepeated administration of LMA didnot over-comeinhibition.

The observedeffects of macrophages is remi-niscent ofmacrophage-mediated inhibition de-scribed in anumber ofsystems. These include thymidine uptake by nonadherent lymphoid cells(28) andlymphoblastoid and leukemic cell lines (13, 24),thymidine incorporation (23) and generation ofcytolytic effector cells (23, 29) in

primary mixed lymphocyte cultures, mitogen-andantigen-stimulation ofTcells (1, 2, 13, 27,

30), polyclonal B cell activation (1, 13), and

generationofantibody-forming cellsinprimary lymphocyte cultures (25). The difference be-tween the reported systems and the present assay lies in the degree ofsensitivity in T cell

inhibitionasreflectedinmorethan 90%

inhibi-tion ofproliferation byasfewas 2x 104 stimu-latedPEC (ca.8%macrophages added to a cul-ture containing optimal lymphocyte-to-macro-phage ratios). Thisexquisite sensitivity may be due to effects of theparticularbacterialantigens used, as they might stimulate macrophages thereby rendering them moreinhibitory for

T-cell proliferation. It is inkeeping with this

hy-pothesisthatstimulatedmacrophages,on a cell-for-cellbasis,are morestronglyinhibitory than residentperitoneal macrophages and that opti-malmacrophage-to-lymphocyte ratios for T-cell proliferation arehigher for cells collected from

the unstimulated peritoneal cavity when com-pared with antigen-elicited peritoneal cells

(Jungi,unpublished data).

The exactmechanism of T-cell inhibitionby macrophageshas not beendetermined,but it is

toberememberedthat the describedinhibitory

effects ofmacrophages fall broadly into three

categories: (i) adsorption to or degradation by

macrophages of T-cell-derived factors

promot-ing T-cell proliferation and differentiation (2,

25), (ii) release ofcytostatic factors of low and

high molecular weights (5, 13, 20,27), and (iii)

liberation by macrophages of thymidine

com-petingwith the radiolabeledproductfor incor-poration (23, 24).Althoughthe latterpossibility was ruled out, the results are compatible with the notion that macrophages actively release factorsinterferingwithlymphocyteproliferation

anddifferentiation as far as the extent of inhi-bition was related to the metabolic activity of themacrophages.However, the firstpossibility

cannot beexcluded,since heat-killed and glutar-aldehyde-fixed cells were still more inhibitory

thansimilar numbers of nonadherent cells. Besides the strong inhibition observed for

LISTERIA-SENSITIZED RAT T-CELL PROLIFERATION 751 highproportions of macrophages, it wasfound

thatverylowproportions of macrophages also did not allow optimal stimulation (data not

shown). This could be explained with the re-quirement by T cells of growth-promoting or

-stimulating factors from macrophages (4, 6).

Alternatively, itmaybe dueto alack of antigen-presentingcells, for ithas beenshown forsoluble antigens thatmacrophages areessentialfor

T-celltriggering in the presentation of antigen (26). Since thetwice-purified PEC still contained low numbers(2 to5%) of macrophages,anobligatory role ofmacrophages was not formally demon-strated in this system. However, others have found thatL.monocytogenes-immuneratTcells containing the W 3/25 surface marker require the presence ofIa-positive cells torespond by proliferation tosoluble L.

monocytogenes-anti-gen and interaction of T cells with antigen-pulsed spleen cells was subject to allogeneic

restriction (31).Inthemouse,I-A-region

homol-ogy isrequired for the generation ofmitogenic

protein in cultures containing immune Tcells, peritoneal macrophages, and heat-killed Liste-ria(7).ThesameH-2restrictionappliesfor the generation of tumoricidalmacrophages insuch

cultures, whereby restriction operates at the

levelofT-cellinteraction with antigen-present-ing cellsand notduringinteraction of lympho-kines with effectormacrophages (8).

Observing optimal T cell-to-macrophage ra-tios allows the determinationin relative terms

of the number of sensitized Tcellsresponding

to aparticulate bacterial antigen. Theassay is adaptableto a mousesystem(Jungi,manuscript

inpreparation). The sizeable difference between stimulated cultures and background prolifera-tion (30,000to100,000 cpmfor5 X 105cellsper

well) should favorapplicationoflimitingdilution conditions (16), thereby allowingthe quantita-tion ofresponding clonesinabsoluteterms.

Despite itsadvantage foreasyquantitation of sensitizedTcellsinagiven cellpopulation,the relation between thymidine incorporation and

functions manifested in antimicrobial defense

are notclear. Studiesonsubpopulation relation-ships and tissue distribution ofTcellsconferring

protection and T-cellproliferationin vitro are in progress. However, other lymphoid cell func-tionsexpressed invitro,such ascytolyticactivity

and quantitative assessment of the release of

lymphokines,should beincludedin theanalysis

ofListeria-induced T-cellresponses mediating antimicrobialresistance.

ACKNOWLEDGMENTS

Theexpert technical assistance of RuthJungiisgratefully acknowledged.I thank B. Rahn of theDepartmentof Exper-imentalSurgeryof this institute forlettingme usehis fluores-cencemicroscope.

This work was supported by grant 3.213.77 of the Swiss NationalScience Foundation.

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