International Immunology, Vol. 4, No. 4, pp. 509-517 © 1992 Oxford University Press

Intoxication of high affinity IL-2 receptorpositive thymocytes blocks early stages ofT cell maturation

W. Maslinski, J. R. Murphy1, and T. B. Strom

Department of Medicine, Beth Israel Hospital, 330 Brookline Avenue and Harvard Medical School, Boston,MA 02215, USA'The University Hospital, Boston University Medical School, Boston, MA 02118, USA

Key words: cytokines, diphtheria toxin fusion protein, interleukins, T cell development

Abstract

Early murine fetal thymocytes express functional, high affinity IL-2 receptors as determined by:(i) the presence of IL-2R 0 chain (p75) mRNA; (II) IL-2 (10 U/ml) induced cell proliferation/cellularmaturation in lobe submersion cultures (LSC). Under the Influence of IL-2, early thymocytesdifferentiate In vitro into more mature, early single positive CD4~CD8+ followed In vivo by doublepositive CD4+CD8+ and single positive CD4+CD8"T and CD4CD8 + thymocytes. SpecificIntoxication of high affinity IL-2R positive thymocytes by recombinant lnterleukin-2-diphtheriatoxin-related fusion protein (DAB4W-IL-2) results In transient, dose dependent blockade ofIn vivo and In vitro thymocyte maturation. DABug-IL-2 Induced effects upon In vivo maturationare reversible within 2 weeks after cessation of drug administration. Taken together, theseresults demonstrate the expression of functional, high affinity IL-2 receptors on earlythymocytes. Elimination of high affinity IL-2 receptor positive thymocytes with DAB466-IL-2results in transient blockade of T cell maturation. Since D A B ^ - I L ^ Is now In clinical trial, it Isreassuring to note that It does not permanently disrupt thymlc maturation.

Introduction

The thymus serves as a nursery for the generation ofimmunocompetent T lymphocytes (1). During fetal and neonataldevelopment lymphoid cells migrate into the thymus. In thismicroenvironment lymphoid cells proliferate (2), rearrange theirclone specific T cell antigen receptor genes (3,4) and differentiateinto mature T cells (5,6). While 50% of thymocytes in adult micebear neither the CD4 nor CD8 proteins, day 14-15 fetalCD4-CD8" thymocytes express the IL-2R p55 chain (9,12,15).CD4"CD8" thymocytes can differentiate into each of themature thymocyte subsets (16). While some workers believe thatthe maturation of early thymic T cells may result from stimulationof mterieukin-2 receptor (IL-2R) bearing immature thymic blastsby IL-2 (7 -14), the senstivity of the IL-2R expresed upon fetalCD4"CD8" thymocytes to IL-2 has not been unequivocallydemonstrated. Some report that CD4~CD8~ thymocytesrespond to IL-2 in vitro (9,10); others disagree (11,17). Thesediscrepancies may rise due to complexity regarding the structureand function of the multimeric IL-2R. Recent evidence hasdemonstrated that at least two distinct IL-2 binding polypeptidechains are present in the high affinity IL-2R complex: the well-

defined p55 protein (Tac, a chain) (18 - 23) and the more recenttydiscovered p75 chain (fi chain) (24-28). The p55 chain bindsIL-2 with low affinity (Kd = 10~8 M). The p75 chain binds IL-2with intermediate affinity (Kd •= 5 x 10"'° to 10~9 M) (25-27).The high affinity IL-2 R (Kd = 10"11 M) is formed by the non-covalent association of p55 and p75 chains (25-26). The p55chain has a rapid IL-2 association and dissociation rate while thep75 chain exhibits a slow association and dissociation rate (29).The high affinity binding of multimeric IL-2R to IL-2 occurs withrapid association (p55) and slow dissociation (p75). Binding ofIL-2 to high or intermediate affinity IL-2R sites results in inter-nalization of a ligand - receptor complex (30), and activationsignals for LAK, NK and T cells, and a prdiferative signal for LAKand T cells (31). In contrast, the low affinity site does not inter-nalize IL-2 or transduce IL-2 derived activation signals into theceil (28,29). A mAb that defines the binding domain of the murinep55 IL-2 binding protein blocks maturation of T cells in fetalthymus organ cultures (32). Moreover, treatment of pregnant miceon days 12 - 1 5 of gestation with this mAb blocks T cell matura-tion in feta) mice (33). However, recent studies showing 'normal'

Correspondence to: W. Maslinski

Transmitting editor: C. Terhorst Received 26 August 1991, accepted 31 December 1991

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510 High affinity IL-2 receptors in T cell maturation

maturation of thymocytes in IL-2 deficient mice generated bytargeted homologous recombination have questioned the centralrole of IL-2 in T cell maturation (50). The role of cytokines in Tcell development has been reviewed (35).

In the present study we have analyzed the expression and therole of high affinity IL-2R bearing thymocytes in the process ofT cell differentiation. Using polymerase chain reaction (PCR)assisted reverse transcription we have defined the kinetics of p75chain of IL-2R expression in thymocytes in lobe submersioncultures (LSC). The functional role of the ligand stimulated IL-2Rwas tested by means thymocyte growth and IL-2 stimulatedexpression of surface antigens. Moreover, the importance of thehigh affinity IL-2R bearing cells in maturation/differentiation ofthymocytes was studied. For this purpose, an interleukin-2diphtheria toxin-related fusion protein (DAB4B6 —IL-2) whichselectively eliminates cells expressing high, but not low or'intermediate, affinity IL-2Rs was used as a probe (36-38).

Methods

Animals

Six to eight week old C57BL/6J (males) and BALB/c (females)mice were purchased from Charles River Laboratories (MA).Embryos were obtained from timed matings. The day of detectionof a vaginal plug was designated as gestation day 0. Pregnantfemale mice were killed by decapitation and thymuses dissectedfrom the embryos.

Protocol of in vivo treatment

Six-week old, male BALB/c mice were injected subcutaneouslywith medium, 5 ^g or 15 /*g of D A B ^ - IL-2 daily for a periodof 1 week. Thymocytes isolated 1 or 8 days after cessation ofDAB488-IL-2 administration were stained for flow cytometricanalysis of surface antigens as described below.

Cultivation and elimination of cells expressing high affinity IL-2receptors in LSC

Individual fetal thymus lobes were washed twice in RPMI-1640medium, placed in 180 /J of culture medium [RPMI-1640, 10%FCS, 1 mM sodium pyruvate, non-essential amino acids, 40 nMmercaptoethanol-2, and penicillin (0.5 U/ml)/streptomycin(0.5 /ig/ml)]. Twenty microliters of either culture medium orDAB488-IL-2 fusion protein (final concentration 1-10nM)diluted in culture medium was added, and the plates wereincubated for 6 h at 37°C in 5% CO2. The medium wassubsequently aspirated, and the lobes were washed once withculture medium, followed by addition of 200 y\ of culture medium+ / - human recombinant IL-2 (10U/ml). The medium waschanged every 3 days. Under these conditions lobe submersioncultures can be maintained for longer than 1 month. The cellsprepared for FACS analysis were harvested after 7 days inculture.

Flow cytometric analysis

This was performed using a FACS 440 flow cytometer coupledto a Consort 40 computer (Becton Dickinson; Mountain View,CA). The following mAbs were used: anti-CD4 (L3T4), biotinconjugated anti-CD8 (Lyt-2), and biotin conjugated Thy-1,2,

Fluorescein conjugated rabbit anti-rat Ab and phycoerythrin -streptavidin were used as developing reagents (all reagents werepurchased from Becton-Dickinson, La Jolla, CA). H597, antimurine cdfl TcR was a gift from Dr Ralph Kubo (National JewishHospital, Denver, CO). Anti-IL-2R (PC61) was isolated from PC61cells (American Type Culture Collection, ATCC) by protein Acolumn affinity purification. Immunofluorescent staining of cellswas carried out according to protocols recommended by Becton-Dickinson.

PCR assisted detection of IL-2R0 (p75) mRNATotal cytoplasmic RNA was extracted from thymocytes bystandard procedures (39). Briefly, thymocytes (10®) werewashed twice with PBS, resuspended in 200 yi of IsoHi buffer(140 mM NaCI, 1.5 mM MgCI2, 10 mM TRIS-CI, pH 8.6,0.5% NP40, vanadyl ribonuclease complex 20 mM (Gibco-BRL,Gaithersberg, MD) and RNA extracted on ice for 5 min, followedby rapid centrifugation (5 min, 10 000 g). The supernatant fluidwas treated with SDS (2%), EDTA (25 mM), NaCI (300 mM),Protemase K (200 mg/ml) (Gibco-BRL) and digested for 30 minat 37°C followed by phenol/chloroform/isoamyl alcohol (50:50.1)extraction. RNA was precipitated from the agarose phase,washed with 99% ethanol and air dried. Total RNA (300 ng) wasused for cDNA preparation using digo(dT) primers and 200 UM-MLV reverse transcriptase (BRL). A oligonucleotide coding foran intracytoplasmic domain of p75 IL-2R protein was amplifiedby the use of 21 mer ('sense') oligonucleotide (codons 311-317)and 18mer ('antisense', codons 246-251) synthesized on anApplied Biosystems 381A DNA synthesizer. Actin-relatedoligonucleotides were also synthesized that enable amplificationof a 349 bp fragment of the actin transcript. A fourth of the entirevolume of the amplified cDNA (5 jd) was then combined in a 50 /Jfinal reaction volume with 0.25 /xM of each oligonucleotide primerand 1.5 U of Taq polymerase (Perkin-Elmer Cetus, Norwalk, CT).Each PCR cycle consisted of a 60 s period for denaturation at94°C, 45 s for annealing at 55°C and 30 s for enzymatic primerextension at 72°C. The specificity of the amplified IL-2R p75 andactin oligonucleotides were verified by Southern blot analysisutilizing ^P-labeled 24mer ('antisense1 codons 294-301) and300mer 0 actin probes respectively.

Results

IL-2 stimulates fetal thymocytes growth/maturation in LSC

Intact murine thymic lobes taken from day 13-15 embryos wereplaced in LSC. Intact thymic lobes harvested at day 14 ofgestation were individually placed in 96-well flat-bottomed platesin 200 fd of culture media supplemented with 1-10 U/ml humanrecombinant IL-2 (10-100 pM). In cultures lacking IL-2, very fewcells, primarily fibroblasts, grow outside the lobe (Fig. 1A). Asthymocyte proliferation ensued, the cells propagate as outgrowthfrom the lobes and form a monolayer culture covering the surfaceof the well (Fig. 1b). If the IL-2 laden medium is changed very3 - 4 days, the LSC were maintained for longer than 1 month.Thymocyte proliferation was far more vigorous in 96-well thanin 24-well plates. These results suggest that cultured thymusesrelease cytokines whose concentration is too low to supportvigorous cell growth in the larger, 24-well plates. Cellular

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High affinity IL-2 receDtors in T cell maturation 511

Fig. 1. IL-2 supports mouse thymocyte growth in the LSC system. Intact, gestaton day 14, mouse thymuses were cultured in lobe submersionculture in the absence (A) or presence (B) of IL-2 (10 U/ml) for 7 days. Magnification x28.

Table 1.

% positive

Thymocyte surface

Thy 1,290-98

antigens expressed

IL-2R (p55)15-25

after one week of

CD4"CD8+

20-30

culture in the

CD4

1

presence

+CD8"

ot IL-2 in LSC

CD4+CD8+

2

TCR/330-40

outgrowths from lobes cultured in the presence of IL-2 formmulticellular clumps after 3 days in culture and continue to growin this manner for at least 1 week (Fig. 1B, black dots). FACSanalysis of IL-2 supplemented day 7 cultures reveals the

phenotype of the cultured cells: Thy-1 + (98%), IL-2R p55 +

(15%), CD8+ (40%), TCRa/3+ (40%). Very few cells (<2%)express both CD8+ and CD4+ and < 1 % of cells areCD4+CD8" (Table 1). Since exogenous IL-2 promotes

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512 High affinity IL-2 receptors in T cell maturation

differentiation, at least some thymocytes bear functional IL-2receptors. Because monomeric IL-2R p55 binding site does nottransduce IL-2 signals into mature T cells (40,41), we examined

Day of gestation

IL-2(10U/ml)

p75

B-actin

Fig. 2. IL-2R p75 chain transcription in fetal mouse thymocytes cultivatedin LSC. cDNA was prepared from intact, gestation day 14-16, thymusescultured for 7 days in LSC in the presence or absence of exogenousIL-2 PCR amplification of the intracytoplasmic domain of IL-2R p-75 wascarried out as described in Methods.

14 14 15 15 16 16

early thymocytes for expression of IL-2R p75 mRNA and theirsensitivity to DAB486-IL-2 which selectively intoxicates cellsexpressing high affinity IL-2R (38).

Expression of IL-2 p75 mRNA on thymocytes from LSC

In order to determine whether fetal thymocytes express IL-2Rp75 mRNA, we applied PCR methodology (see Methods) to RNAextracted from thymocytes cultured in LSC with/withoutexogenous IL-2 for a period of 7 days. IL-2R/3 encoding mRNAis first detected in thymocytes at the 15th/16th day of gestation(Fig. 2). Cultivation of thymic lobes in the presence of IL-2(10 U/ml) induces low levels of p75 RNA in thymocytes from the14th day of gestation and amplifies p75 mRNA expression inthymocytes from days 15th and 16th of gestation.

Intoxication of high affinity IL-2 receptor positive thymocytesblocks the early stages of T cell maturation in vitro

In order to probe the role of high affinity IL-2R bearing thymocytesin the process of in vitro maturation, we assessed the impact ofDAB486-IL-2 in the LSC system. Thymic lobes were incubatedfor 10 h in the presence of this fusion toxin. At low molar

B

Fig. 3.(B, 5 x

~ IL-2 fusion protein disturbs maturation of mouse thymocytes in vitro. Thymuses were incubated without (A) or with DAB,^ - IL-2; C, 10~8 M; D, 5 x 10" 8 M) for 6 h followed by washing, IL-2 addtion and culture for 7 days in LSC. Magnification x 104.

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High affinity IL-2 receptors in T cell maturation 513

wCL

WOz111oin ' aUJtrO C3

QO

1,

1,

1.

1.

1

-

1

1 '

s,

d lu m :

. . : : : : . . 4

1 ' 1 ' 1 ' 1 ' 130 ea a

D

-

-

1 1 ' 1

i

L'2 .-I

4

1 • 1 ' 1 ' 1

CD4 F L U O R E S C E N C E ( F I T C )

D A B 4 « « - ? « 1 0 M

^ rca

Fig. 4. DAB4B6-IL-2 blocks expansion of CD4~CD8+ cells in LSC. Expression of CD4/CD8 antigens on mouse thymocytes cultured in the absence(A) or presence (B-E) of IL-2 in LSC for 7 days. D A B ^ - I L ^ was present in c (5 x 10"9M), d (1CT8M) and e (5 x 10"8M)

2 0 0

THY 1.2 F L U 0 R E 8 C E N C E ( F I T C )

Fig. 5. DAB4a8- |L-2 blocks Thy-1 expression. Ceils were prepared as in Fig. 4. and stained with anti-Thy 1,2 antibodies.

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514 High affinity IL-2 receptors in T cell maturation

concentrations, D A B ^ - I L ^ has been shown to bind to andbe internalized by high, but not low or intermediate, affinity IL-2R(38). Cells bearing high affinity IL-2R are intoxicated by the

catalytic core of diphtheria toxin (fragmented A) and result in ADP-ribosylation of EF-2 (36,37). Whole thymic lobes were incubatedfor DAB4S6-IL-2 for 10 h, followed by washing and subsequent

Table 2. The effect of DAB4a6-IL-2 fusion protein on number of thymocytes cultivated in the presence of IL-2 in LSC

No IL-2

1-5%

IL-2(10U/ml)

100%

DAB486-IL-2

5 x 10~9M

70-80%

10"8M

30-40%

5 x 10"8M

20-30%

100% = 250 000 cells/well.

Table 3. Thymus weight (mg) after DAB,^ - IL-2 in vivo administration

Days after last D A B ^ - IL-2administration

18

Dose ot DAB486

0

408 (±47)374 (±24)

- IL-2 (ug/day/mouse)

5

325 (±21)332 (±40)

15

376 (±10)358 (±10)

A «

oo3

u.O

ocUJmS

UJ>

. . . . . . i . , n TTT.• • • • I I I • I | I I I I |1QQ

O A B 4 * 6 - 5 u Q / d t y

2 0 0

THY 1,2 FLUORE8CENCE (FITC)

B

Ulo.

UluzUloCOU loco3

OSD

u

3-

am

•

•

' 1 '

t

• a I n *

f. . . .'.."i . b

| 1 1 ' I

a

' l ' l3B

•Ba •

CD4 FLUORE8CENCE (FITC)

Fig. 6. DAB486-IL-2 intoxicates mouse thymocytes in vivo. Expression of CD4/CD8 (A) and Thy-1 (B) antigens on thymocytes isolated fromDAB.486-IL-2 treated mice.

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High affinity IL-2 receptors in T cell maturation 515

incubation with IL-2 for 7 days. Incubation with DAB486-IL-2causes a dose dependent decrease of cell number in LSC (Table2, and Fig. 3) and perturbs the pattern of T cell maturation (Figs4 and 5). The proportion of cells bearing Thy-1 and CD8 proteinswere decreased by - 6 0 % and 80%, respectively, inDAB436- IL-2 treated cultures. The inhibitory effects of the toxinwere prevented by the addition of a molar excess of IL-2(5 x 10"8 M), thereby confirming specificity of DAB486-IL-2 forthe IL-2R.

Intoxication of high affinity IL-2 receptor positive thymocytestransiently blocks the early stages of T cell maturation in vivo

The importance of cells bearing high affinity IL-2 receptors in theprocess of T cell maturation in vivo was probed 1 week follow-ing cessation of DAB466-IL-2 treatment. Elimination of cellsbearing high affinity IL-2 receptors with DAB486-IL-2 results inthymus weight loss (Table 3) and a decreased number ofCD4+CD8+ double positive thymocytes (Fig. 6). The proportionof cells bearing more mature, single positive CD4+ or CD8+

proteins increased. These changes are transient; one week aftercessation of toxin administration weight and CD4/CD8 patternsof saline or toxin treated mice are similar (Tables 3 and 4).

Discussion

Other workers have demonstrated transient expression of theIL-2R p55 chain by murine fetal thymocytes on day 14-16 ofgestation (9,12,15). Since gestationaJ thymocytes also produceIL-2 (5,42,43), the IL-2/IL-2R autocrine pathway may participatein thymic maturation. This hypothesis has been studied in vitroand in vivo by several groups. Thymocytes isolated from newbornmice born to anti-IL-2R mAb-treated mothers do not containmature CD4+CD8~ or CD8+CD4" T cells (42). On the otherhand CD4+ and CD8+ thymocyte subsets do mature in IL-2deficient mice (50). Hence, factors other than IL-2 can drivethymic maturation. Exponential growth of early thymocytes(13- 16th day of gestation) discerned in the thymus of normalmice is driven by other than IL-2 growth factors such as IL-3,IL-4, or IL-7 are utilized.

In an in vitro single cell suspension system, double negativeCD4-CD8" thymocytes fail to respond to IL-2 alone (9,11), butproliferate vigorously in IL-2-dependent fashion if also providedmitogenic lectins (9,10). In the thymus organ culture system,whole undisrupted lobes were placed on the surface of floatingsponges (Millipore Corp.) and submerged in culture medium.In this system, CD4~/CD8~ double negative cells differentiateinto CD4+ or CD8+ single positive as well as the doublenegative cells that are present in normal adult mouse thymus (44).Double positive cells were also detected (45). In this system anti-Table 4. DAB486 fusion protein induced intoxication ofCD4+CD8+ double positive cells is time and dose dependent(expressed as a percentage of maximal CD4+CD8+ doublepositive thymocytes decrease)

Days after last D A B ^ -admini strati on

18

IL-2 Dose of

0

00

DAB48S-IL-2

5

590

(pg/day/mouse)

15

10050

IL-2R mAb inhibits thymocyte growth (32); however, others failedto reproduce this finding (46). In addition, IL-2 stimulatesexpansion of cytotoxic LGL from thymus and causes a markeddecrease of other cell types (47,48).

Another in vitro system, LSC provides a different environment.The culture conditions dilute the endogenous thymic factors.Hence, the LSC system is more sensitive to exogenous factorsthan organ culture (OC). In this system, lower IL-2 concentra-tions (1 - 10 U) (see Figs 1,2) versus 500 U in organ culture (48)are sufficient to stimulate cell growth and a quantitatively differentpattern of thymocyte subpopulations is evident. As shown hereinand elsewhere (45,49) in the LSC system, IL-2 clearly promotesproliferation of thymocytes (Figs 1 and 2) and maturation ofCD8+/CD4- single positive Thy-1,2\ IL-2R + cells. Sincethymocyte growth occurs at IL-2 concentrations of 1 -10 U/ml(a concentration that stimulates cells with high, but not inter-mediate, affinity receptors) and since proliferation and matura-tion was inhibited by the intoxication of cells bearing high affinityIL-2Rs, we conclude that these early thymocytes bear functionalhigh affinity IL-2 receptors. The same conclusion was recentlyreported by others on the basis of proliferation data in LSC, aswell as by Scatchard analysis of [125l]IL-2 binding to thymocytesin LSC (43). We have confirmed and extended these observationsby Southern blot analysis showing the presence of IL-2R p75mRNA in thymocytes isolated from the 15th and 16th days ofgestation. The presence of IL-2 amplifies/induces IL-2R p75mRNA in thymocyte cultures harvested at day 14 of gestationand amplifies its level in thymocytes taken at day 15 and 16 ofgestation (Fig. 2).

In short, we conclude that fetal thymocytes (days 14-16 ofgestation) express functional, high affinity IL-2Rs. Activation ofthese receptors may be important for early stages of exponentialthymocyte growth resulting in generation of single positive(CD8+) cells. These single positive thymocytes further differen-tiate into CD4+/CD8+ double positive and CD4+ single positivethymocytes as has been shown following intrathymic injection(34). Intoxication of cells bearing high affinity IL-2 receptorsdisturbs thymocytes maturation resulting in decrease ofCD4"/CD8" double negative cells. Intoxication is reversible invitro. Since D A B ^ - I L ^ is now in clinical trial, it is reassuringto note that it does not permanently disrupt thymic maturation.

Acknowledgements

This work was supported in part by grant NIH UO1 CA48626. W.M. isa recipient of a Fogarty International Fellowship, Bethesda, MD, USA.

Abbreviations

DAB486-IL-2

IL-2IL-2RLSCTCR a,0

recombinant interteukin-2 diphtheria toxin-relatedfusion proteinlnterleukin-2Interteukin-2 receptorlobe submersion cultureT-ceH receptor a. 0, subunits

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