Proc. Natl. Acad. Sci. USAVol. 91, pp. 12659-12663, December 1994Immunology

Interleukin 12 induces the differentiation of majorhistocompatibility complex class I-primed cytotoxicT-lymphocyte precursors into allospecific cytotoxic effectors

(cytokines/aflogeneic response)

SALEM CHOUAIB*, JIHED CHEHIMIt, LYNDA BANI*, NOELLE GENETET*, THOMAS TURSZ§, FRANCOISE GAY*,GIoRGio TRINCHIERI¶, AND FATHIA MAMI-CHOUAIB II*CJF 94-11 Institut National de la Sante et de la Recherche Medicale "Cytokines et Immunite Antitumorale," Institut Gustave Roussy 94805 Villejuif Cedex,France; tChildren's Hospital, Philadelphia, PA 19104; *Groupe Universitaire de Recherche en Immunologie, Universite de Rennes I, 35016 Rennes Cedex,France; §Unite de Recherche Associee 1156, Institut Gustave Roussy, 94805 Villejuif Cedex, France; lWistar Institute, Philadelphia, PA 19104; andI'Unitd 333 Institut National de la Sante et de la Recherche Medicale, Institut Gustave Roussy, 94805 Villejuif Cedex, France

Communicated by Jean Dausset, September 22, 1994

ABSTRACT The production of interleukin 12 (IL-12) fol-lowing allogeneic stimulation and its involvement in the differ-entiation of allospecific cytotoxic T lymphocytes (CTLs) havebeen investigated. Supernatants of mixed lymphocyte cultureshad detectable levels of IL-12 p40 which were completelyabrogated after depletion of responder cells from monocytes.While addition to the culture of anti-IL-12 neutralizing anti-bodies partially inhibited the allogeneic proliferative responseand the subsequent CTL activity, addition of IL-12 stimulatedboth responses, suggesting that endogenously produced IL-12plays a role in the development of alloreactivity. Furthermore,using primary mixed cultures of lymphocytes from majorhistocompatiblity complex-recombinant siblings identical forclass II antigens and displaying class I disparity, we demon-strated that addition of recombinant IL-12 at the sensitizingphase of the primary mixed lymphocyte culture induced CTLactivity. Under these stimulation conditions, addition of re-combinant IL-12 also triggered cell proliferation, indicatingthat IL-12 provides both growth and differentiation signals.The mechanism underlying this process does not appear torequire IL-2, since IL-12-mediated CTL generation was notabrogated by anti-IL-2 a-chain antibodies. IL-12 increasedgranzyme B and perforin mRNA accumulation in major his-tocompatibility complex class I-primed lymphocytes, suggest-ing that this cytokine activates these two genes in CTL pre-cursors. We conclude that IL-12 can stimulate the generationof alloreactive CTLs. We suggest that IL-12 may play a role inhelper cell-independent CTL generation.

After exposure to foreign histocompatibility determinants, Tlymphocytes acquire cytolytic capacity toward cells bearingthe specific sensitizing alloantigen. These cytotoxic T lym-phocytes (CTLs) play a major role in the protection andrecovery from certain viral, bacterial, and parasitic infectionsand mediate allograft rejection. The differentiation of CD8+CTLs is a complex process that may require or may beregulated by a heterogenous combination of signals. Cyto-kines play an important functional role in the development ofcell-mediated cytotoxicity. Indeed, CTL generation is de-pendent on the activation of their precursors by majorhistocompatibility complex (MHC) class I disparity to a stateof responsiveness to soluble mediators and subsequent trig-gering by these factors. While MHC class I-restricted CTLsrequire an exogenous source of cytokines in addition to theantigenic stimulus, MHC class II-restricted CTLs do not (1).Previous studies have clearly indicated that interleukin 2

(IL-2) promotes the proliferation ofCTL precursors and havesuggested the involvement of various CTL differentiationfactors (2, 3).

IL-12 is a 75-kDa heterodimeric cytokine composed oftwocovalently linked glycosylated chains, a heavy chain of 40kDa (p40) and a light chain of 35 kDa (p35), encoded by twoseparate genes (4-7). Originally identified as a product ofEpstein-Barr virus-transformed B-cell lines, IL-12 is pro-duced by phagocytic cells and possibly other accessory cells(4, 5, 8). IL-12 acts on T and natural killer (NK) cells byinducing proliferation and production of cytokines (9-14) andby enhancing the generation as well as the activity of cyto-toxic lymphocytes (15-20). This cytokine appears to be animportant inducer ofTh, helper T-cell responses produced byaccessory cells during early antigenic stimulation (21, 22),and it has been suggested that a defect in its production mightbe a factor contributing to immune depression (23, 24).

Little is known about the effect of IL-12 in alloreactivityand in the development of antigen-specific T-cell-mediatedcytotoxicity. We have therefore investigated the involvementof IL-12 in the induction of the human allogeneic response.To accurately delineate the functional relevance of IL-12 inthe development of CTL activity, we took advantage of anexperimental system in which the endogenous secretion ofcytokines is undetectable (25). By using MHC-recombinantsibling cells identical for MHC class II but displaying MHCclass I disparity in the primary mixed lymphocyte culture(MLC), we demonstrated that IL-12 by itselfwas sufficient toinduce the differentiation ofCTL precursors into fully matureCTLs. This further confirms that CTLs can be induced in theabsence of helper mechanisms and emphasizes the role ofIL-12 as a CTL differentiation factor involved in the gener-ation of MHC class I-restricted CTLs.

MATERIALS AND METHODSCell Isolation. Peripheral blood mononuclear cells (PBMCs)

obtained from batch leukopheresis of normal adult volunteers(Banque du sang, H6pital Saint Louis, Paris, and Centre deTransfusion Sanguine, Creteil, France) were isolated byFicoll/Hypaque density gradient centrifugation. The cellswere washed twice with RPMI 1640 medium and were thensuspended in medium supplemented with penicillin (100 units/ml), streptomycin sulfate (100 ,ug/ml), and L-glutamine (2mM). Adherent cells were removed by two rounds of adher-ence to plastic followed by immunodepletion with magnetic

Abbreviations: IL, interleukin; CTL, cytotoxic T lymphocyte; MLC,mixed lymphocyte culture; MHC, major histocompatibility complex;mAb, monoclonal antibody; NK, natural killer; PBMC, peripheralblood mononuclear cell; PHA, phytohemagglutinin.

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The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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beads (Dynal, Oslo) using MY4 (anti-CD14) monoclonal an-tibody (mAb). The cells were typed for the expression ofMHCclass I and class II antigens by the standard National Institutesof Health two-stage complement-dependent microcytotoxic-ity test with a battery ofwell-characterized anti-HLA antisera.PAT and PHI are two siblings who are genotypically identicalfor HLA class II but differ for the HLA-A of one haplotype,as a result ofa recombimation between the MHC class II locusand the MHC class I HLA-B locus (Fig. 1). MHC class IIalleles were assigned by genomic DNA typing using reversedot blot hybridization (Innolipa kit) after specific PCR ampli-fication of HLA-DRB, -DQB, and -DPB polymorphic secondexons (26).MLC. For MLC, 105 PBMCs were incubated with 105

irradiated (2500 rad; 1 rad = 0.01 Gy) stimulating PBMCs. Allcultures were in triplicate in round-bottomed microwellplates (Nunc) in a final volume of 200 ,ul of RPMI 1640(GIBCO) supplemented with L-glutamine (1 mM), penicillin(100 units/ml), streptomycin sulfate (100 ,ug/ml), and 15%heat-inactivated pooled human serum. After 6 days of incu-bation at 37°C in 5% C02, the cultures were incubated with2 ,Ci of [methyl-3H]thymidine (57 mCi/mmol; Amersham; 1,uCi = 37 kBq) for 6 hr at 37°C and harvested onto fiter paperwith a Skatron harvesting apparatus (Skatron, Lier, Nor-way). Thymidine incorporation was measured in a 3 scintil-lation counter (LKB) and the results from triplicate wellswere expressed as mean cpm. Cell-free supernatants wereharvested at various time intervals and were stored at -80°Cuntil the assay for IL-12.

Cytokines and mAbs. Highly purified recombinant humanIL-2 (>95% pure; specific activity, 23.3 x 106 units/mg ofprotein) used in this study was kindly provided by Sanofi(Bio-Recherches, Labege, France). Recombinant humanIL-12 (5 x 106 units/mg) was kindly provided by S. Wolf(Genetics Institute, Boston). Neutralizing anti-IL-12 mAbsC8.6 and C11.79 were used as described (8). Anti-IL-2receptor a-chain (anti-Tac, anti-CD25) mAb was provided byT. Waldmann (National Institutes of Health, Bethesda).

IL-12 Assay. The production of IL-12 p40 (either alone orassociated with p35) in cell-free culture supernatants wasmeasured by specific radioimmunoassay (RIA) (8) using themAb pair C11.79/C8.6. In brief, C11.79 antibodies wereadsorbed [5 mg/ml, in 0.1 ml of 0.1 M sodium carbonatebuffer (pH 9.5) per well] for 24 hr at 4°C to 96-well plates(Costar). The plates were washed three times with phos-phate-buffered saline plus 0.05% Tween 20, and standardantigen and culture supernatants were added to the plates (0.1ml per well) for 24 hr at 4°C. The plates were washed and C8.6antibodies (0.1 ,g/ml in 0.1 ml per well) labeled with 1251(chloramine-T method) were added to the plates. After 18 hrof incubation, plates were washed, and radioactivity wasquantitated in a 'y counter (Packard).

Cytotoxicity Assay. Cells were harvested from MLC andsuspended in medium. Serial dilutions of effector cells weredistributed in duplicates (0.1 ml per well) into round-bottomed microwell plates in RPMI 1640 medium supple-mented with 10% fetal bovine serum. Target cells [2 x 106

a A29.812.DR7. D02(DP1101)b A2. B12. DR1301. DQ1 (DP0201)

phytohemagglutinin (PHA)-stimulated (3 days) blasts] in 0.2ml of medium were labeled with 200 ,uCi of Na251CrO4 (5mCi/ml; Amersham) for 1 hr at 37°C and then washed threetimes. Aliquots (0.1 ml) oftarget cells (5000 cells per well) andeffector cells (at the indicated ratio) were distributed inround-bottom microwells. After 4 hr at 37°C, the plates werecentrifuged at 2000 x g for 2 min. The supematants wereharvested with a Skatron device (Skatron, Lier, Norway) and51Cr release was measured with a Kontron counter. Sponta-neous release was determined by incubating target cells inmedium alone. Maximum release was determined by adding0.1 ml of 1 M HCI to 0.1 ml of the target cell suspension.Percent specific lysis was calculated as [experimental 51Crrelease - spontaneous 51Cr release)/(maximum 51Cr release- spontaneous 51Cr release)] x 100.Northern Blot Analysis. Total cellular RNA was extracted

from control and stimulated cells by an RNAzol (Bioprobesystem, Montreuil Sous Bois, France) procedure based onthe method of Chomczynski and Sacchi (27). RNA (10 ,g perlane) was size-fractionated in 1.5% agarose/formaldehydegels, transferred by capillarity to nylon membranes (Amer-sham), and hybridized with cDNA probe corresponding togranzyme B or perforin (kindly provided by E. R. Podack,University of Miami). A cDNA probe specific for T-cellreceptor /-chain mRNA was used as a control to quantitatethe RNA level. The integrity and amount ofRNA loaded perlane were visualized by ethidium bromide staining. cDNAprobes were labeled with [a-32P]dCTP (DuPont/NEN) byrandom hexamer priming. Hybridizations were performed asdescribed (28).

RESULTSIL-12 Production in Allogeneic MLC. Initial experiments

were performed to examine whether IL-12 secretion occursfollowing alloantigen stimulation. The time course of IL-12p40 induction in primary MLCs was assessed. PBMCs fromdonor A were stimulated with irradiated PBMCs from anunrelated donor, B. The supematants were collected andassayed for IL-12 p40 by RIA. IL-12 p40 was detected within1 day of allogeneic stimulation (400 pg/ml) and reached apeak (700 pg/ml) after 6 days of incubation (Fig. 2). Depletionof monocytes from responder cells with anti-CD14 mAbeliminated IL-12 p40 production, suggesting that monocytesmight be the major source of IL-12 during the allogeneicresponse.

Effect of Exogenous IL-12 and Anti-IL-12 mAbs on theAllogeneic Proliferation and CTL Activity in the PrimaryMLC. To test whether exogenous IL-12 plays a role in thedevelopment and regulation of allogeneic response, exoge-nous IL-12 was added at the initiation of MLC. Addition ofIL-12 (0.5 U/ml) at the time of stimulation of the culturesresulted in a significant enhancement of cell proliferation asmeasured by [3H]thymidine incorporation (Fig. 3). The lyticactivity of the specific CTLs against PHA-stimulated blasttargets derived from the sensitizing cells (donor B) was alsoincreased. When nonrelated target cells were used in the

c A3. B7. DR4. D03d A2. B21. DR7. DQ2 (DPO301)

b A2. 812. DR1301. DOBI 0603. DPO201

d A2. B21. DR7. DOBi 0201. DPO301

FIG. 1. Pedigree of family Mes. F, father; M, mother. PAT and PHI are two siblings used for this study.

alb A29. 812. DR1301. DOBI 0603. DP0201

d A2. 821. DR7. DOBi 0201. DP0301

12660 Immunology: Chouaib et al.

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E

a

° 400aC\

II

1 2 3 4 5 6Time, days

FIG. 2. Kinetics of IL-12 production in the primary MLC. PBMC(106) or peripheral blood lymphocytes (monocyte-depleted PBMCs)(106) from donor A were cocultured with irradiated (2000 rad)stimulator cells from donor B. On days 1-6 of MLC, IL-12 p40 wasquantitated in culture supernatants by RIA. IL-12 p40 was notdetected in cultures containing only responder or stimulator cells.Bars show mean and SE of five experiments with triplicate deter-minations.

cytotoxic assay, no killing was observed (data not shown)further stressing that IL-12 induced the differentiation ofspecific cytotoxic effectors. Following monocyte depletionfrom responder cells, the effect of IL-12 on specific lysis andproliferation was more pronounced (Fig. 3B). Addition of aneutralizing anti-IL-12 mAb (C8.6) inhibited significantlyboth the proliferative and specific cytotoxic responses (Fig.3A).

IL-12 Induces the Differentiation ofCTL Precursors Primedby MHC Class I Disparity. Two siblings (PAT and PHI)genotypically identical for HLA class II but different forHLA-A of one haplotype were studied. Stimulation of PATlymphocytes by PHI cells did not result in IL-12 production(data not shown). To study the effect of added IL-12 on CTLtriggering, cells from PAT were stimulated with irradiatedcells from PHI in the absence or presence of IL-12. After 6days of culture, effector cells were assessed for their lyticactivity against PHA-blast targets derived from the sensitiz-ing PHI cells. In the absence of exogenous cytokine, theprimed responder cells did not exhibit specific CTL activityagainst PHI cell targets (Fig. 4). When exogenous IL-12 (0.5unit/ml) was added to the culture at the sensitizing phase, alytic activity of PAT effector cells against PHI cell targetswas induced. When unrelated target cells were used in thecytotoxic assay, no killing was observed (<5% at a 25:1effector/target ratio), further stressing that IL-12 induced thedifferentiation of specific cytotoxic effectors. The generatedCTLs could be distinguished from nonspecific lymphokine-activated killer cells on the basis of their specificity becausethey do not kill Daudi target cells. When IL-12 was added tothe MHC class I-primed lymphocytes, an increased prolifer-ative response was observed as compared with control cul-ture (14,680 cpm vs. 1200 cpm), suggesting that IL-12 acts asa growth factor for allospecific CTLs.The Effect of IL-12 Is Independent from the IL-2 Pathway.

To determine whether the observed effect ofIL-12 was director not, we performed neutralization studies using anti-IL-2receptor a-chain (anti-Tac, anti-CD25) mAb. Addition of thismAb did not alter IL-12-induced CTL generation upon MHCclass I allosensitization (Fig. 5). Moreover, the failure of

O

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(cpm)Medium 41980 + 5140AIL-12 63100 + 5020+anti-IL-12 Ab 39800 ± 3160+Isotype IgG 43020 ± 4630

25 12.5 6Effector/target ratio

3

FIG. 3. Effect of exogenous IL-12 on allogeneic proliferation andCTL generation. PBMCs (106) (A) or monocyte-depleted cells (106)(B) from donor A were cocultured with irradiated allogeneic PBMCsfrom donor B (106) in the absence (i) or presence of IL-12 (0.5unit/ml) (o), anti-IL-12 antibody (Ab) (15 pg/ml) (v), or isotypecontrol IgG (o) for 6 days. After the 2-hr preincubation, the cells werewashed and cytotoxicity against the specific sensitizing target cells(PHA blasts) was tested in a 4-hr 51Cr release assay at the indicatedeffector/target ratio. Each data point is the average of duplicates.(When PHA blasts from an unrelated donor or Daudi cells were usedas control targets, the percent specific lysis at a 25:1 effector/targetratio was <5%.) Proliferation was assessed by determining [3H]thy-midine incorporation (cpm) after 5 days of culture. Data are ex-pressed as mean ± SD of triplicate samples. Similar results wereobtained in each of four experiments.

anti-Tac mAb to affect IL-12-induced proliferation suggeststhat endogenous IL-2 is not required in our system. Simul-taneous addition of IL-12 and IL-2 at the initiation of theMLC significantly enhanced specific cytotoxic activity ofMHC class I-primed lymphocytes. Addition of anti-Tac mAbabrogated the effect of IL-2 and IL-2/IL-12 combination onCTL differentiation and allogeneic proliferative response(data not shown).

Effect of IL-12 on Perforin and Granzyme B Gene Expres-sion. Northern blot analysis was performed with RNA ex-tracted from MHC class I-primed lymphocytes cultured for 6days in the absence or presence ofIL-12, IL-2, or both. In theabsence of exogenous cytokine, no granzyme B or perforinmRNA was observed (Fig. 6). Addition ofrecombinant IL-12or IL-2 induced accumulation of both granzyme B andperforin mRNA. When IL-12 and IL-2 were added simulta-neously, granzyme B (Fig. 6A) as well as perforin (Fig. 6B)mRNA were significantly increased. Hybridization of the

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FIG. 4. Effect of exogenous IL-12 on the geMHC class I-primed lymphocytes. PAT cells (Ifor 6 days with irradiated PHI stimulator cells (11or presence of IL-12 (0.5 unit/ml) (e) or IL-2 (0.preincubation, the cells were washed and cyt(specific sensitizing target cells (PHA blasts) wasrelease assay at the indicated effector/target celblasts from an unrelated donor or Daudi cells v

targets, the percent specific lysis at a 25:1 effec<5%.) [3H]Thymidine (3HTdR) incorporation w3.

blots with T-cell receptor (chain probe (Ithat similar amounts of RNA had been loz

DISCUSSION

The aim of this study was to further elucirequirement for the induction of humanactivity. It is known that soluble factoressential second signal in the developmen'

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FIG. 5. The effect of IL-12 is independent o:Conditions for culture and assay were identicalCytokines without or with anti-Tac (anti-CD2added at the initiation of the culture. Data aretative experiment of three.

A

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0.9 kb-o- ga:g

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C CM1-

CM +

-'- -4

I1 ..- .I cMX. *_@4 _2.9 kb '""~-# *4e w1 kb::::: I:

FIG. 6. Effect of IL-12 on granzyme B and perforin gene expres-sion in MHC class I-primed lymphocytes. Total RNA was extractedfrom 6-day MHC class I-primed lymphocytes stimulated or not byIL-12 (0.5 unit/ml), IL-2 (3 units/ml), or IL-12 and IL-2. RNA blotswere sequentially hybridized to cDNA probes specific for perforin(A), granzyme B (B), and T-cell receptor ,3 chain for normalization(C).

response. Understanding the cytokine requirement and cy-tokine repertoire is critical to the regulation of allogeneicproliferative response and subsequent cytolysis. The effect ofIL-12 on the development of the allogeneic response has so

I 1.5 far not been established. We therefore studied the involve-ment of IL-12 in the human alloreactive response, withspecial attention to allospecific CTL differentiation.

neration of CTLs by We first demonstrated that IL-12 was produced following106) were cocultured allogeneic stimulation (class I and class II disparity) and that06) in the absence (o) monocytes were the major source of IL-12 in the primary.5 unit/ml) (m). After MLC. Although produced at low levels, IL-12 appears to play)toxicity against the a role in the induction of the allogeneic proliferative responsetested in a 4-hr 5Cr and in the increase in cytotoxic effector function. The lowwere used as control amounts of IL-12 detected may interact with endogenously:tor/target ratio was produced IL-2 to elicit a synergistic effect. This wouldtas assayed as in Fig. explain why IL-2 neutralization by specific antibodies abro-

gates this synergistic effect and therefore possibly downreg-ulates the IL-2/IL-12 responsiveness. Because monocytes

Fig. 6C) indicated appear to be the major source of IL-12 produced during theaded in each lane. primary allogeneic response, our data are consistent with the

existence of cross talk between monocytes and T lympho-cytes during a specific alloreactive response in addition to theknown one during natural and adaptive immune responses

date the cytokine (29).alloreactive CTL Genetic studies have established that MHC class II incom-

rs function as an patibility is mainly implicated in the proliferative allogeneict of the allogeneic stimulation and lymphokine release in primary MLC (30).

MHC class I molecules which are essential for target-cellrecognition by alloreactive and antigen-specific CTLs play a

Medium role in allogeneic activation (31-33). Furthermore, MHCIL-2 class I antigens do not represent a potential ligand forL-12 anti-MHC receptor specificity sufficient to trigger cytokineL12 + IL.2 release (1, 25). Although IL-12 production could not beL.12 + anti-CD25

triggered by MHC class I disparity, it seems likely that thisdisparity has the ability to drive the expression of functionalIL-12 receptor by CD8+ primed lymphocytes, since thesecells responded in a dose-dependent manner to recombinantIL-12. When W6/32, a mAb specifically directed against amonomorphic determinant on human class I HLA-A, -B, and-C, was added to the culture, the IL-12-induced enhancementof CTL precursor differentiation by MHC class I disparitywas abrogated (data not shown), further confirming previousstudies (1).The differentiation of CTLs is a complex process. Given

the variety of cytokines that can directly influence thedifferentiation of CTLs, it is often difficult to determinewhich factor is involved. Our experimental model, based on

.:5 . the use of lymphocytes from recombinant siblings, is poten-1.5 . tially useful for analyzing cytokine requirements in CTLdifferentiation which depends on the strength of the activa-

bfthe IL-2 pathway. tion signal delivered through the T-cell receptor. Previous[to those in Fig. 3. work has shown that IL-2 is the essential factor required for'5, 25 jig/ml) were the generation of CTL (1, 25); we now report that IL-12 byfrom one represen- itself also plays a role in the differentiation of specific CTLs

to alloantigen. This emphasizes the ability of human CD8+

ct( i

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precursors to undergo helper cell-independent CTL differ-entiation under some stimulation conditions. Gately et al. (19)have reported that IL-12 facilitates CTL response to alloge-neic melanoma cells. Recently, Bloom and Horvatah (34)have shown in a murine system that IL-12 acts on respondingT cells to augment antigen-specific CTL activity by CD8+ Tcells without inducing an increase in the proliferative re-sponse. The apparent discrepancy between our study andthat of Bloom and Horvatah could be explained by thedifferent stimulation conditions and a species difference.Brunda et al. (35) have reported that IL-12 has potent in vivoantitumor and antimetastatic effects against murine tumorsand have demonstrated the critical role ofCD8+ cells in theseprocesses.The effect of IL-12 on CTL generation is direct and does

not appear to require IL-2, suggesting the existence of anIL-2-independent pathway of alloreactive CTL differentia-tion. This is in concordance with the work of Mehrotra et al.(20) showing the effect of IL-12 on CTL generation followingactivation of CD8+ cells with immobilized anti-CD3 anti-body.Our data indicate that IL-12 induced the differentiation of

CTL precursors into lytically active CTL by a mechanisminvolving increased perforin and granzyme B gene expres-sion. Perforin and granzyme B mRNAs were not apparentlyinduced in CTL precursors following MHC class I recogni-tion on stimulator cells. It is possible that the expression ofboth genes requires in addition to the engagement of theT-cell receptor another stimulus provided by some cyto-kine(s). Salcedo et al. (36) have reported that IL-12 upregu-lates the expression of perforin and granzyme in NK cells.We suggest that the IL-12-dependent pathway of perforin andgranzyme B mRNA induction ofNK cells is also operative inantigen-specific CTLs.

We are grateful to the volunteers (donors, Banque du Sang,H6pital Saint Louis and Centre Transfusion Sanguine de Creteil)without whom this study would not have been possible. We thank C.Flament for technical assistance. This work was supported in part bygrants from the Institut National de la Sante et de la RechercheMedicale (CRE 920605), the Institut Gustave Roussy (CRC 93), andthe Association de la Recherche sur le Cancer.

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