Administration of recombinant IL-12 to normal mice enhances cytolytic lymphocyte activity and...

International Immunology, Vol. 6, No. 1, pp. 157- 167 © 1994 Oxford University Press

Administration of recombinant IL-12 tonormal mice enhances cytolytic lymphocyteactivity and induces production of IFN-7in vivoMaurice K. Gately, Rajeev R. Warrier, Sharmlla Honasoge, Daisy M. Carvajal,Denise A. Faherty, Suzanne E. Connaughton, Timothy D. Anderson1,Ulla Sarmiento1, Brian R. Hubbard2, and Molly Murphy2

Departments of Inflammation/Autoimmune Diseases and 11nvestigative Toxicology, Hoffmann-La RocheInc., Nutley, NJ 07110, USA2Genetics Institute Inc., Cambridge, MA 02140, USA

Key words: cytolytic T lymphocytes, immunotherapy, interferon, mterleukin, natural killer cells

Abstract

IL-12 Is a heterodlmerlc cytoklne that has been shown to enhance natural killer (NK) andcytotoxic T lymphocyte (CTL) responses, and to Induce IFN-7 production In vitro. In this study,we have examined the effects In vivo of administering purified murine rlL-12 to normal mice.Dally Injections of riL-12 I.p. (1 ng to 10 /ig/day) caused dose-dependent enhancement of NK celllytic activity In the spleens and livers of treated mice. Histologlc examination of the livers ofIL-12-treated mice revealed focal mononuclear cell infiltrates, and flow cytometry studiesIndicated that the livers of IL-12-treated mice contained Increased numbers of NK cells, CD8+ Tcells, and monocytes. Liver and splenic lymphold cells from IL-12-treated mice, unlike liver andsplenic lymphoid cells from control mice, spontaneously secreted IFN-7 In vitro, suggesting thatthey had been Induced by IL-12 to produce IFN-7 In vivo. Consistent with this, IFN-7 could bedetected In the serum of IL-12-treated mice. In mice which had been immunized by footpadInjection of allogenelc splenocytes, dally administration of rlL-12 I.p. was shown to enhance thespecific CTL response In the draining lymph nodes. Thus, these studies demonstrate that IL-12can enhance NK and CTL activity and Induce IFN-7 production In vivo, as well as In vitro, andsuggest possible mechanisms by which IL-12 may exert therapeutic effects in the treatment ofsome tu more and Infectious diseases^

Introduction

IL-12 [originally called natural killer (NK) cell stimulatory factor(1) or cytotoxic lymphocyte maturation factor (2)] is a hetero-dimeric cytokine which can affect both the growth and functionof T and NK cells Human IL-12 is composed of two disulfide-bonded subunits having molecular masses of 40 and 35 kDa(1 - 4). These two subunits are unrelated to each other (3,4) andare encoded by genes located on two separate chromosomesin man (5). The p40 subunit bears sequence homology to theIL-6 receptor (6) and the ciliary neurotrophic factor receptor (7),whereas the p35 subunit is distantly related to IL-6 and granulo-cyte colony stimulating factor (8). In vitro studies on human IL-12have shown that it can enhance the lytic activity of NK/Iympho-kme-activated killer (LAK) cells (1,9-11), facilitate specific

cytotoxic T lymphocyte (CTL) responses (11), act as a growthfactor for activated T and NK cells (9,12,13), induce the secretionof IFN-7 from resting and activated T and NK cells (1,14), promotethe development of Th1 cells (15,16), and inhibit IL-4-inducedIgE synthesis by both IFN-7-dependent and IFN-y-independentmechanisms (17). These activities suggest that human rlL-12could have utility in the treatment of some cancers, infectiousdiseases, and IgE-mediated allergic disorders.

In order to test these possibilities in rodent models it wasnecessary to clone and express murine IL-12 since human IL-12was not biologically active on murine lymphocytes (7). Compar-ison of the amino acid sequences of the murine and human IL-12subunits revealed a high degree of conservation. The amino acid

Correspondence to: M. K. Gately

Transmitting editor. E. R. Unanue Received 20 August 1993, accepted 7 October 1993

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158 Effects of IL-12 in vivo

sequences of the human and murine p40 and p35 subunits were70 and 60% identical respectively (7). Furthermore, murine IL-12was found to be active in vitro in causing proliferation of activatedmouse T cells, enhancement of mouse NK activity, and IFN-7secretion by murine splenocytes (7). We have now preparedhighly purified mouse rlL-12 to permit evaluation of its activitiesin vivo. We herein report that in vivo, as in vitro, IL-12 is capableof enhancing NK cell lytic activity, promoting allogeneic CTLresponses, and inducing the secretion of IFN-7.

Methods

Mice

C57BL/6 and BALB/c mice were obtained from the JacksonLaboratory (Bar Harbor, ME), and were between 8 and 12 weeksof age when used in these studies.

Murine rlL-12

The cDNAs for murine IL-12 p35 and p40 (7) were clonedseparately into the pED expression vector (18). The purifiedplasmids were mixed, transfected into Chinese hamster ovary(CHO) DUKX cells, and amplified to high copy number byselection in methotrexate (19). Murine rlL-12 was produced inserum-free medium by the transfected CHO cells and the cellswere removed by filtration. The culture supernatant was diluted3-fold with 20 mM Tris, pH 8.0, and applied to a Q SepharoseFast Row (Pharmacia, Piscataway, NJ) column equilibrated inthe same buffer. The column was washed with 10 columnvolumes of 20 mM Tris, pH 8.0, and then isocratjcally eluted with0.5 M NaCI in 20 mM Tris, pH 8.0. The protein peak was diluted10-fold with 20 mM sodium phosphate, pH 7.2, and applied toa Cellufine Surfate (Amicon, Lexington, MA) column equilibratedin the same buffer. The column was then washed with sevencolumn volumes of the equilibration buffer containing 0.1 M NaCI.IL-12 was eluted from the Cellufine Sulfate column with a lineargradient to 1.0 M NaCI in 20 mM sodium phosphate, pH 7.2,and the IL-12-containing fractions were pooled. This pool wasthen adjusted to 1.7 M ammonium surfate, 20 mM Tris, pH 7.6,and applied to a POROS 1 20 PE (PerSeptive Biosystems,Cambridge, MA) column equilibrated in 20 mM Tris, pH 7.6,containing 1.7 M ammonium sulfate. The POROS 1 20 PE columnwas washed with five column volumes of the equilibration bufferand then eluted with a linear gradient to 50% ethylene glycolin 20 mM Tris, pH 7.6. The fractions containing heterodimericIL-12 were pooled and dialyzed into Dulbecco's-PBS (D-PBS)Caz+/Mg2+-free, pH 7.2 (Hazleton Biologies, Lenexa, KS).

SDS-PAGE analysis of the purified IL-12 indicated it to be&95% pure with a small amount of contaminating IL-12 p40monomer. [Monomeric IL-12 p40 does not bind to the IL-12receptor (20) and lacks biologic activity (3,4).] Contamination byendotoxin, as assessed by the Limulus amebocyte assay, was<5 e.u./mg IL-12. The specific activity of purified murine IL-12,as determined by its ability to cause proliferation of human phyto-hemagglutinin-activated blasts (21), was - 7 x 106 units/mg andhence similar to that of human IL-12.

Treatment of mice with rlL-12

For administration to mice, murine rlL-12 was diluted in D-PBS(Gibco, Grand Island, NY) containing 1 % syngeneic mouse

serum. IL-12 was administered i.p., once daily, in a volume of0.2 ml, and animals were sacrificed - 2 4 h after the final dosewas given.

Cell lines

YAC-1 lymphoma cells, EL4 lymphoma cells, and P815 masto-cytoma cells were maintained in culture as previously described(22).

Culture media

The culture medium used for the cytolytic assays was RPMI1640supplemented with 100 U/ml penicillin, 100/ig/ml streptomycin,2 mM L-glutamine (all from Gibco) and 5% heat-inactivated fetalbovine serum (FBS) (Hyclone, Logan, UT). For in vitro productionof IFN-7, liver lymphoid cells were cultured in a 1:1 mixture ofRPMI 1640 and Dulbecco's modified Eagle's medium, supple-mented with 0.1 mM non-essential amino acids, 60 /ig/mlarginine-HCI, 10 mM HEPES buffer, 2 mM L-glutamine,100 units/ml penicillin, 100 /xg/ml streptomycin (all from Gibco),5 x 10~5 M 2-mercaptoethanol (Rsher Scientific, Fair Lawn, NJ),1 mg/ml dextrose (Fisher), and 10% heat-inactivated FBS.

Preparation of lymphoid cells

Spleens from vehicle- or rlL-12-treated mice were minced andthe resulting cell suspensions were washed three times withserum-containing medium before assay. Lung and liver lymphoidcells were harvested and prepared as described by Talmadgeetal. (23). Peritoneal cells were harvested in heparin-containingHank's buffered salt solution and washed once in culture mediumprior to assay.

Cytolytic assays

Cytotytic assays using 51Cr-labeled YAC-1, P815, and EL4target cells were performed as previously described (22). Thepercent specific 51Cr release was calculated as [(e - c)/(100 -c)] x 100, where e is the percentage of s1Cr released fromtarget cells incubated with lymphocytes and c is the percentageof 51Cr released spontaneously from target cells incubatedalone. Lymphocyte populations were assayed in triplicate (CTLassays) or quadruplicate (NK assays). Spontaneous 51Cr releasefor YAC-1, P815, and EL4 varied from 11 to 33, 17 to 23, and18 to 3 1 % respectively. Lytic units, based on the number ofeffectors required to cause 30% specific 51Cr release, werecalculated as described (24).

Production of IFN^y in vitro and assay for IFN-y

Liver lymphoid cells or splenocytes, 5x 106 cells/1 ml culture,were incubated in Costar 3524 tissue culture plates for 48 h at37°C. At the end of the culture period, the supernatant fluid washarvested from each culture by centrifugation and stored at 4°Cuntil assayed for IFN-7 by means of an ELISA.

Mouse sera and culture supernatant fluids were assayed forIFN-7 protein by means of a sandwich ELISA. Rat anti-mouseIFN-7 mAbs XMG-1.2 and AN-18 were supplied by Dr GianniGarotta (F. Hoffmann-La Roche, Basel, Switzerland). ELISA plates(Nunc MaxiSorp, Thousand Oaks, CA) were coated with purifiedAN-18 (5 /ig/ml; 100 /il/well) at room temperature for 16 h andblocked with 1 % BSA in PBS, pH 7.4, for 1 h at 37°C. Serialdilutions of purified murine rlFN-7 (Genzyme, Cambridge, MA)and of the unknown samples diluted in D-PBS with 5% FBS were

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Effects of IL-12 in vivo 159

added to the plates and incubated for 3 h at room temperature.The plates were then washed with distilled water, and biotinylatedXMG-1.2, 400 ng/ml, was added to each well and incubated for1 h at 37°C. Following another wash, peroxidase-conjugatedstreptavidin (Sigma, St Louis, MO), 1 /ig/ml, was added andincubated for 30 min at 37°C. After washing, the plates weredeveloped with ABTS (Sigma), 1 mM; and the absorbance at405 nm was determined with a V^ Kinetic Microplate Reader(Molecular Devices, Palo Alto, CA). The sensitivity of the assaywas -500 pg/ml.

Cell surface marker analysis

FITC-conjugated mAb to mouse CD3, CD4, and CD8, phyco-erythrin (PE)-conjugated mAb to mouse B220, and biotm-conjugated mAb to mouse NK1.1 were all from Pharmingen (SanDiego, CA). FITC- or PE-conjugated mAb to murine Mac-1 wasfrom Boehringer Mannheim (Indianapolis, IN) and FITC-conjugated F4/80 mAb was from Harlan Bioproducts (Indian-apolis, IN). Control FITC-, PE-, or biotin-conjugated rat IgG wasfrom Pharmingen. Aliquots of 100 /d of lymphoid cells (107

cells/ml staining buffer consisting of D-PBS with 2% heat-inactivated FBS and 0.1% sodium azide) were mixed with 25 jdof rat IgG (Sigma), 1 mg/ml in staining buffer, and incubated for15 min on ice. Aliquots of 25 ^l of the various mAbs at optimalconcentrations (determined in preliminary experiments) were thenadded and the mixtures were incubated for an additional 30 minon ice. The cells were then washed, those cells which had beenincubated with biotinylated mAb were stained by furtherincubation with PE-conjugated streptavidin (Fisher Biotech,Pittsburgh, PA) followed by washing, and all samples wereanalyzed by flow cytometry using a FACScan (Becton-Dickinson,Mountain View, CA).

Histologic studies

Livers, lungs, and spleens were fixed in 10% neutral bufferedformalin, embedded in paraffin, sectioned at 6 nm, and stainedwith hematoxylm and eosin.

In vivo generation of CTL

CTL responses were elicited by footpad injection of 107

allogeneic splenocytes, and CTL activity in the draining popliteallymph nodes was measured as previously described (25). Lymphnode cell populations were assayed for lytic activity at 10 serial2-fold dilutions, beginning at an effectortarget ratio of 80:1.Lymph nodes were harvested on the indicated number of days(usually 4 or 5) after immunization.

Treatment of cells with anti-CD8 plus complement (C)

Treatment of C57BU6 liver lymphoid cells or lymph node cellswith anti-CD8 (anti-Lyt-2.2 from Accurate Chemical & Scientific,Westbury, NY; clone AD4 used at a dilution of 1 /100) plus C wasperformed as previously described (22).

Results

Enhancement of NK cell activity in vivo by IL-12

Because IL-12 is known to enhance NK cell activity in vitro, weevaluated whether IL-12 could also augment NK activity in vivo.In initial experiments, such as the one shown in Fig. 1 and

100

EffectonTarget Ratio

FJg. 1. Effect of IL-12 on NK activity in vivo. C57BL/6 mice received1 jig IL-12 (solid symbols) or a comparable amount of vehicle (opensymbols) i.p. daily for 4 days. Lymphoid cells were harvested from thespleen (squares), peritoneal cavity (diamonds), lungs (circles), and liver(triangles) on day 5, and assayed for their ability to lyse YAC-1 targets.There were six mice per treatment group and lymphoid cells from a givenanatomical site were pooled for all six mice. Values shown are the meansof quadruplicate samples, and all standard errors were s 3 % specificrelease. The spontaneous 51Cr release was 27%.

Table 1 . In vivo effects of IL-12 on NK activity of mouse splenic,peritoneal, liver, and lung lymphoid cells*

Cellsource

SpleenSpleen

PeritoneumPeritoneum

LiverLiver

LungLung

IL-12

i +

+

+

+

Cells permouse (x iO" 6 )

79175

41

0.21.4

0 140 28

NK activity

LU/106 cells

0.80.9

0.76 7

1.48.8

09.1

LU/mouse

63163

2.86.7

0312.3

02.5

aC57BL/6 mice received 1 M9 IL-12 or a comparable amount ofvehicle i.p. daily for 4 days. Cells were harvested on day 5. Lytic activitywas measured using YAC-1 targets.

Table 1, i.p. administration of 1 ̂ g IL-12 daily to C57BL/6 micefor 4 days was shown to enhance significantly NK cell activityin the lungs, liver, peritoneal cavity, and, to a lesser extent, spleen.Splenic NK activity was not significantly increased on a per cellbasis, but because IL-12 induced splenomegaly, the NK activityper spleen was increased in proportion to the increased numberof splenocytes. In the peritoneal cavity, the NK activity wassubstantially increased on a per cell basis in IL*12-treated mice,but the number of cells recovered per mouse was reproduciblydecreased. In contrast, in the lungs and liver IL-12 caused bothan increase in the NK lytic activity per 108 cells and an increasein the number of lymphoid cells recovered from these organs.As the number of lymphoid cells recovered from the lungs of

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Rg. 2. Kinetics of enhancement of splenic and liver NK activity by IL-12. C57BL/6 mice received 1 ng IL-12 or a comparable amount of vehicle(D-PBS plus 1 % syngeneic mouse serum) i.p. daily for 1 - 6 days as indicated. Splenic and liver lymphoid cells were harvested 24 h after the finalinjection and assayed for lytic activity on YAC-1 targets as described in Methods. The days on which mice began receiving IL-12 were staggeredso that all lymphoid populations were harvested and assayed on the same day. There were four mice per treatment group and splenic or liver lymphoidcells from all four mice in each group were pooled prior to assay. Solid bars indicate NK lytic activity and hatched bars indicate the average numberof lymphoid cells recovered per mouse. Similar results were obtained in two other experiments.

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100

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YAC-1Target*P816Targets

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160

100

60

Liver

YAC-1TargetsP816Targets

10"* 10"* 10-* 10"' 1

IL-1 2 Oosa (ug)10-" io-* 10-" io"

IL-1 2 Dose (ug)

Rg. 3. Dose - response for enhancement of splenic and liver NK activity by IL-12. C57BL/6 mice received 0.2 ml vehicle containing the indicatedamount of IL-12 i.p. daily for 2 days. Splenic and liver lymphoid cells were harvested 24 h after the second injection and assayed for lytic activityon YAC-1 cells (solid bars) or P815 cells (hatched bars). Splenic or fiver lymphoid cells from all mice in each treatment group (four per group) werepooled prior to assay.

IL-12-treated mice was low and variable, further experimentsfocused on the effects of IL-12 in the liver and spleen.

Kinetic studies revealed that in both the spleen and liver of micereceiving 1 /ig IL-12 daily, NK activity peaked after two injectionsand declined thereafter (Fig. 2). In both the spleen and liver, thenumber of lymphoid cells/organ increased between days 2 and6 of treatment, but the total lytic activity/organ decreased. Afterthe sixth injection, the NK activity per spleen in IL-12-treated micewas similar to, or in some experiments slightly less than, the NKactivity per spleen in vehicle-treated controls, whereas total liverNK activity following six daily injections of IL-12 remained at least5- to 10-fo)d greater than that observed in vehicle-treated controls.At 24 h after a single injection of 1 nQ IL-12, NK activity in boththe spleens and livers of IL-12-treated mice was significantlyincreased as compared with vehicle-treated controls. Themechanism causing the decrease in NK cell activity as a resultof continued daily administration of IL-12 is unknown. However,

a similar phenomenon has been previously observed in micereceiving recombinant IFN-a or IFN-7 (23,26).

Dose - response studies were performed in which NK activitywas measured following two daily injections of IL-12, the timeof the peak response. Virtually identical results were seen in twoseparate experiments, one of which is illustrated in Fig. 3. Dose-dependent enhancement of both splenic and liver NK activitywas observed over the dose range of 1 ng to 1 /ig IL-12 perinjection. Administration of 0.1 ng IL-12 did not significantly altereither the NK activity (Fig. 3) or the number of lymphoid cellsrecovered from the spleen or liver (data not shown). IL-12-activated splenic and liver lymphoid cells caused enhanced lysisof both NK-sensitive YAC-1 target cells and NK-resistant P815(Rg. 3). In three additional experiments in which NK activity wascompared in mice that were given either 1 or 10 HQ IL-12 dailyfor 2 days, it was found that the 10 ug dose did not result in asignificant enhancement ( s 1.5-fold) in liver or splenic NK activity

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b.0 10"* 10"* 10"' 1 10

IL-12 Administered In Vivo (ug)

Fig. 4. Production of IFN-7 in vitro by liver lymphoid cells andsptenocytes from IL-12-treated mice C57BL/6 mice received 0.2 mlvehicle containing the indicated amount of IL-12 i.p. daily for 2 days, andliver lymphoid cells and splenocytes were harvested 24 h after the finalinjection. Liver lymphoid cells (solid bars) or splenocytes (hatched bars)were cultured in medium without any added stimulants for 2 days; andthe amounts of IFN-7 secreted into the culture supernatant fluids weredetermined by an ELISA as described in Methods. There were six miceper treatment group and splenic or liver lymphoid cells from all mice ineach group were pooled Each culture contained 5 x 106 cells upon in-itiation.

70

60

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50

40

30

20

10

00 24 48 72 96 120

Hours after First Injection

Fig. 5. Detection of IFN-y in the serum of IL-12-treated mtce. C57BU6mice received 1 pg IL-12 (• ) or a comparable amount of vehicle (O)i.p. dairy for up to 5 days. Three mice were bled out at the indicatednumber of hours after the initial injection and serum IFN-7 levels weremeasured by an ELISA as described above. Each point represents themean ± SEM of values obtained for three separate sera. For pointslacking error bars, the bars fell within the point.

as compared with that observed in mice receiving 1(data not shown).

IL-12

Induction of IFN-y secretion by IL-12

In addition to their enhanced NK rytic activity, liver lymphoid cellsand, to a lesser extent, splenocytes from IL-12-treated mice were

found capable of spontaneously secreting IFN-7 in culture. Whenliver lymphoid cells from mice given 2 daily injections of IL-12(^0.1 /xg/day) were incubated in vitro in the absence of anyadded stimulant, they were found to produce substantial amountsof IFN-7; whereas liver lymphoid cells from control, vehicle-treatedmice secreted none (Fig. 4). Splenocytes from mice given^ 1 /ig/day IL-12 also secreted small amounts of IFN-7 in vitroin the absence of any added stimulant (Fig. 4). The observationthat liver lymphoid cells and splenocytes from IL-12-treated micespontaneously secrete IFN-7 in vitro was reproducible in nineseparate experiments and suggests that they may also secreteIFN-7 in vivo. To further examine this possibility, sera fromIL-12-treated mice were tested for the presence of IFN-7. In eachof three experiments, such as the one shown in Fig. 5, IFN-7could be detected in the serum of mice given 1 /ig/day IFN-yand was first detected on day 2 after initiation of treatment.

Histologic and phenotypic changes induced by IL-12

Histologic examination of livers from IL-12-treated mice revealedsinusoidal and randomly distributed, multifocal parenchymalinfiltrates composed of cells of lymphocytic and/or monocyticlineages (Fig. 6). Occasional foci of extramedullary hematopoiesiswere also observed (not shown). Analysis by flow cytometry ofliver lymphoid cell subsets in mice that had received four dailyinjections of IL-12 revealed a substantial increase in the numbersof CD8+ T cells and NK1.1+ NK cells (Fig. 7). On the otherhand, the numbers of CD4+ T cells or B220+ B cells were notsignificantly altered. A significant increase in Mac-1 + cells wasalso observed in IL-12-treated mice. Although Mac-1 is a markerfound on monocytes and myeloid cells, it can also be expressedby NK cells (27). Dual-staining experiments indicated that theNK1 1 + cells from IL-12-treated mice were also Mac-1+ (datanot shown). Hence a substantial portion of the increase inMac-1 + cells may reflect the increase in NK cells. In twoadditional experiments, liver lymphoid cells from mice given 1 ugIL-12 or vehicle daily for 4 days were stained with mAb F4/80,which is more specific for macrophages (28). In these experi-ments, (2 7 ±0.2) x 106 (mean ± SD) F4/80+ cells/liver wereobserved in IL-12-treated mice, whereas livers from vehicle-treated mice contained an average of (1.3 ±0.2) x 106 F4/80+

cells. Thus, IL-12 treatment also appeared to increase the numberof F4/80+ macrophages recoverable from the liver. Phenotypicanalysis of liver lymphoid cells from mice treated with two dailyinjections of IL-12 showed increases in NK1.1+ cells andMac-1+ cell similar to that seen after four injections; however,there was little increase in the CD8+ cells after two injections(Fig. 7). Hence, in mice given IL-12, NK cells appear to infiltrateinto the liver early, followed later by CD8+ T cells. Phenotypicanalysis of liver lymphoid cells from mice treated with eight dailyinjections of IL-12 yielded results similar to those obtained afterfour daily injections (data not shown).

As noted above, normal mice given daily injections of IL-12developed marked splenomegaly. Histologic examination of thespleens of IL-12-treated mice revealed that the splenomegaly waslargely a result of increased extramedullary hematopoiesis of theerythroid, myeloid, and megakaryocytic lineages (data notshown). Phenotypic analysis of splenocytes from mice which hadreceived four daily injections of 1 fig of IL-12 revealed only minorchanges in the distribution of lymphoid cell subsets consisting

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Fig. 6. Microphotographs of livers from vehicle- and IL-12-treated mice. C57BL/6 mice received 1 ng IL-12 or a comparable amount of vehiclei.p. daily for 4 days, and livers were harvested 24 h later. (A) Normal liver from a vehicle-treated mouse. 95 x . (B) Liver from a mouse treated withIL-12. Note a focal cluster of mononuclear cells (arrow) in the hepatic parenchyma. 95 x . (C) Close-up of liver from (A) showing normal, vacuolated(glycogen-filled) hepatocytes. 285 x . (D) Close-up of liver from (B). At this site, mononuclear cell infiltration was associated with hepatocellular necrosis;however, hepatocellular necrosis was not observed in the majority of IL-12-induced mononuclear cell foci.

primarily of an increase in NK cells (Fig. 8). In four experiments,the percentage of NK-1.1+ cells increased from 1 ±0.3 invehicle-treated mice to 6±0.9 in IL-12-treated mice. In theseexperiments, the number of splenocytes recovered from IL-12-treated, as compared with vehicle-treated, mice was increasedby 1.6 ± 0.2-fold, and the percentage of splenocytes which werephenotypic T, B, or NK cells or macrophages was reduced from99 ± 1 % in vehicle-treated mice to 82 ± 1 % in IL-12-treated mice.This is compatible with the histologic observation thatsplenomegaly in IL-12-treated mice was due to increasednumbers of hematopoietic progenitor cells in the spleen.

Enhancement of specific CTL responses in vivo by IL-12

To examine whether administration of IL-12 could enhanceallogeneic CTL responses in vivo, mice were immunized withallogeneic splenocytes injected into the footpads and then givendaily i.p. injections of IL-12, beginning on the day of immunization.Administration of >1 /ig IL-12 daily for a total of 4 - 5 daysresulted in a marked enhancement in the cytolytic activity oflymphocytes harvested from the draining lymph nodes (Fig. 9).In 12 experiments, administration of IL-12 enhanced the specificcytolytic lymphocyte response to the allogeneic lymphocytes by

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H l l Vehicle

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CD3 +

CD4 +

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NK1.1 +

B220 +

Mac-1+

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50


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Vehicle

IL-12

10 20 30 40

Cells/Liver x 10"

Fig. 7. Effects of IL-12 on liver tymphotd cell subset distribution. C57BL/6 mice received 1 jig IL-12 (solid bars) or an equivalent volume of vehicle(hatched bars) i.p. dairy for (A) 2 or (B) 4 days. Liver lymphoid cells were harvested 24 h later and analyzed by ftow cytometry as described inMethods. The values shown represent means ± SEM of three separate experiments.

an average of 14-fold (mean ± SEM: 14.4±4.2). The cytolyticlymphocytes in the draining lymph nodes were specific in theirlytic activity since lymph node cells from H-2b mice immunizedwith H-2d splenocytes ± IL-12 lysed H-2d P815 target cells butnot syngeneic EL4 cells whereas lymph node cells from H-26

mice immunized with H-2b splenocytes ± IL-12 lysed H-2b EL4target cells but not H -^ P815 cells (Table 2). Lymph node cellsfrom mice which were given IL-12 but no allogeneic splenocyteslysed neither P815 nor EL4 cells (Table 2). The specific cytolyticactivity of lymph node cells from IL-12-treated mice was mediatedby CD8+ T cells since it could be completely abolished bytreatment of the lymph node cells with anti-CD8 plus C, whereassimilar treatment had no effect on the NK activity of liver iymphoidcells from IL-12-treated mice (data not shown). IL-12 did not alterthe kinetics of the cytdytic lymphocyte response to the allogeneicsplenocytes. No enhancement of the cytolytic lymphocyteresponse by IL-12 was observed until the fourth day afterimmunization and the response peaked on day 5, the same dayon which the CTL response was maximum in vehicle-treated mice(data not shown). Dose - response studies indicated that little orno enhancement of the specific CTL response was seen whenmice were given IL-12 for 5 days at doses s 10 ng/day (Fig. 9).Hence the amount of IL-12 required to enhance a specific CTLresponse in vivo was greater than the amount required toenhance the nonspecific lytic activity of NK cells (see above,Fig. 3).

Discussion

In studies performed in vitro, IL-12 was previously shown toenhance both specific and non-specific cytolytic lymphocyteresponses (1 -3 ,7 ,9-11) and to induce the secretion of IFN-7

by T and NK cells (1,3,7,14). The studies reported in thismanuscript represent an initial evaluation of the biologic activitiesof IL-12 in vivo in normal mice and demonstrate that in vivo, asin vitro, IL-12 is a very potent immunomodulatory agent

Like IFN-a, IFN^, and IL-2 (22,23,26,29,30), IL-12 can enhanceNK lytic activity at several different anatomical sites in vivowhen administered to normal mice. Enhancement of NK cell

Vehicle

IL-12

0 10 20 30 40 50 60 70 80 90

Cells/SDleen x 10 " '

Fig. 8. Effects of IL-12 on splenocyte subset distribution. C57BU6 micereceived 1 HQ IL-12 (solid bars) or an equivalent volume of vehicle(hatched bars) i.p. dairy for 4 days. Splenocytes were harvested 24 hlater, and analyzed by flow cytometry as described above. The datashown represent means ± SEM of four separate experiments.

activity was dose-dependent over a range of 1 ng to 10 HQIL-12 per dose. In mice receiving 1 ^g IL-12 daily, NK activityin both the spleen and liver was maximum following the secondinjection and declined thereafter. A similar decline in NK cellactivity following prolonged daily administration was previouslyreported for IFN-a and IFN-7 (23,26). The reason for this declineis unclear. It is unlikely that neutralizing antibodies were formedso early after beginning treatment with IL-12 and, indeed, anti-IL-12 antibodies have not been detected in mice receiving murineIL-12 for as long as 3 weeks (M. Brunda et a/., unpublishedresults). Moreover, IL-12-induced increases in liver and spleniclymphoid cell numbers, IFN-7 levels in serum, and CTL tyticactivity are seen beyond day 3. Redistribution of lymphoid cellsalso seems to be an unlikely explanation for the observeddecrease in liver and splenic NK activity between days 2 and6 of IL-12 treatment since the number of NK1.1 + cells in theseorgans appeared to increase, rather than decrease, over this time

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IL-12 (ugh G

Ly«i« olP81S

E--12 (ugh 0 10"* 10"' 1

Lymph Nod*C«ll»/Mou««

Lysi* ofEL4

Lymph NodeCells/Mouse

Fig. 9. Enhancement of allogeneic CTL responses by IL-12. C57BL/6 mice were immunized by footpad injection of BALB/c splenocytes (b antkJ)or BALB/c mice were immunized by footpad injection of C57BL/6 splenocytes (d anti-b). Mice received 0.2 ml vehicle containing the indicated amountof IL-12 i.p. daily for 4 days beginning on the day of immunization. Lymph node cells from draining popliteal lymph nodes were harvested 24 hafter the final injection and tested for their ability to lyse P815 targets (b anti-d effectors) or EL4 targets (d anti-b effectors). Lymph node cells fromfour mice per treatment group were assayed separately and the results shown are average values for the four mice in each group. Solid bars indicatehylic activity, and hatched bars indicate the average number of lymph node cells recovered per mouse.

Table 2. Specificity of cytolytjc lymphocyte response in mice immunized with allogeneic lymphocytes and treated with IL-12 orvehicle*

Experiment Strainimmunized

Imrnunogen IL-12 LU/mouse assayed onb

EL4 (H-2")

<4C

<4

NDNDNDND

88±13471 ±28

<4<417±4

343±40

PBIStH-^

17±5296±60

<4<4107±20

1104±120

<4<4

NDNDNDND

C57BL/6C57BL/6

C57BL/6C57BL/6C57BU6C57BL/6

BALB/cBALB/c

BALB/cBALB/cBALB/cBALB/c

BALB/cBALB/c

NoneNoneBALB/cBALB/c

C57BL/6C57BL/6

BALB/cBALB/cC57BL/6C57BL/6

aC57BL/6 (H-26) or BALB/c ( H ^ mice were immunized by footpad injection of 107 allogeneic or syngeneic splenocytes, and draining popliteallymph nodes were harvested on day 4 (experiments 1 and 4) or day 5 (experiments 2 and 3) after immunization. The lymph node cells were testedfor their ability to lyse EL4 and/or P815 target cells as described in Methods. Mice received either 1 /ig IL-12 or an equivalent volume of vehiclei.p. daily beginning on the day of immunization.

''Lymph node cells from three or four individual mice or three or four pools of mice were assayed separately. Values in the table representmeans ± SEM.

than 10% specific 51Cr release was observed at the highest effectortarget ratio tested (80:1).

span. A more likely possibility is that the decline in NK activityin mice receiving more than two daily injections of IL-12 was dueto the suppressive effects of other cells and/or mediators. Forexample, activated macrophages, including hepatic Kupffer cells,have been shown to be capable of suppressing NK/LAK activityvia the production of prostaglandins (31,32). The IFN-y producedin response to IL-12 may result in macrophage activation, releaseof prostaglandins and/or other suppressive mediators, andconsequent down-regulation of NK activity. It is of interest to note

that IFN-y first becomes detectable in the serum of IL-12-treatedmice on day 2 following the initiation of treatment and NK lyticactivity decreases as serum IFN-y levels increase over the next4 days. Whether IFN-7 produced in response to IL-12 plays anyrole in the initial enhancement of NK lytic activity by IL-12 isunclear. However, neutralizing antibodies to IFN-7 had little orno effect on the activation of NK/LAK cells by IL-12 in vitro(1,10,11).

In comparison to levels of NK activity previously observed by

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ourselves and others in IL-2-treated mice (22,29,30), the levelsof NK activity elicited by IL-12 are rather low. This is consistentwith the in vitro observations that optimal concentrations of IL-12elicited only 20 - 50% of the maximum NK/LAK activity that couldbe induced by IL-2 (10,11) and that IL-12, unlike IL-2, induceslittle or no proliferation of resting NK cells (9,10). In addition, atleast in our experience (22), daily administration of IL-2 toC57BU6 mice for as long as 7 days did not result in a declinein NK activity such as that seen after day 2 in IL-12-treated mice.

Recent studies on cytokine production by lymph node cellsfrom mice infected with Leishmania major showed that cells frominfected mice receiving IL-12-treatment produced more IFN-7 inresponse to L. major antigen in vitro than did lymph node cellsfrom infected mice that had not received IL-12 (33,34). Thepresent studies extend those results and prior in vitro observa-tions (1,14,35,36) by showing that administration of IL-12 tonormal mice resulted in IFN-7 production in vivo in the absenceof an infectious agent as costimulus. Although there wassubstantial individual-to-individual variation in the serum IFN-ylevels observed in IL-12-treated mice, the biphasic time courseshown in Fig. 5 was observed reproducibly in each of threeseparate experiments. Both NK and T cells have been shownto produce IFN-y in response to IL-12 in vitro (14). One possibleexplanation for the biphasic time course of IL-12-induced IFN-7production in vivo is that it reflects differences in the kinetics ofinduction of IFN-7 production by NK and T cells. This possibilityis consistent with the observations that resting NK cells (37) butnot resting T cells (38) display detectable IL-12 receptors andthat NK and T cells differ in the kinetics with which they infiltratethe liver in IL-12-treated mice. Studies are in progress tocharacterize further the pharmacodynamics and cellular source(s)of IFN-7 production in IL-12-treated mice

In the present studies, IL-12 induced focal mononuclear cellinfiltrates in the hepatic parenchyma. Similar mononuclear cellinfiltrates have been described in the livers of mice treated withIL-2 (22,29,30). IL-2 also was found to induce substantialperivascular mononuclear cell infiltrates in the lungs (22,29); incontrast, IL-12 induced only minimal mononuclear cell infiltratesin the lungs, even when given at lethal doses (T. Anderson andU. Sarmiento, unpublished results). Flow cytometry analysis ofliver rymphoid cells from IL-12-treated and control mice suggestedthat the mononuclear cells infiltrating the livers of IL-12-treatedmice were primarily NK cells, CD8+ T cells, and macrophages.No increase in CD4+ T cells was observed in the livers ofIL-12-treated mice. Similarly, it was previously reported that themononuclear cell infiltrates in the livers and lungs of IL-2-treatedmice were composed largely of NK cells and CD8+ T cells(22,30). IL-12, like IL-2, is capable of causing the proliferationof both CD4+ and CD8+ T cells in vitro (12). It is unclear whyboth IL-2 and IL-12 in vivo elicit lymphoid cell infiltrates whichare largely lacking in CD4+ T cells. In IL-2-treated mice whichhad been depleted of NK cells by administration of anti-asialoGM,, pulmonary and hepatic infiltrates containing both CD4+

and CD8+ T cells but few or no NK cells were observed (22).Hence cytokine-activated NK cells may in some way prevent theinfiltration of CD4+ T cells into the tissues of IL-2- orIL-12-treated mice.

Administration of IL-2 to normal mice was reported to causemassive proliferation of lymphoid cells and in particular NK cells(29). It is not clear whether IL-12 causes similar lymphoprolifera-

tion in vivo. The splenomegaly induced by IL-12 was due largelyto extramedullary hematopoiesis rather than to the proliferationof mature lymphoid cells, and the lymphoid infiltrates in the liversof IL-12-treated mice may simply reflect redistribution of lymphoidcells from other anatomical compartments. Consistent with thispossibility, mice given 1 /ig/day IL-12 were found to developsignificant lymphopenia and neutropenia within 2 days after theinitiation of IL-12 treatment (M. Gately and T. Anderson,unpublished results). In wiro, human IL-12 caused only minimalproliferation of resting human PBMC (1,12).

Like IL-2 (39), IL-12 can enhance specific allogeneic CTLresponses in vivo. The dose - response and kinetics of IL-12-mediated enhancement of CTL responses differed significantlyfrom the dose - response and kinetics of IL-12-induced enhance-ment of NK activity. Whereas the IL-12-induced enhancementof NK activity was dose-dependent over the dose range 1 ng/dayto 10 ̂ g/day, little or no IL-12-mediated enhancement of specificCTL responses was observed at doses < 100 ng/day. Likewise,IL-12-mediated enhancement of NK activity was significant aftera single injection and maximum after two injections whereasIL-12-mediated enhancement of CTL responses was notobserved until day 4 after initiation of IL-12 treatment and wasmaximum on day 5. Interestingly, the recovery of cells from thedraining popliteal lymph nodes of IL-12-treated, immunized micewas reproducibly decreased by 25 -50% as compared to thecell recovery from the nodes of vehicle-treated, immunized mice(Fig. 9) despite the fact that the total CTL activity per mouse wasincreased by an average of 14-fold in the IL-12-treated animals.One possible explanation for this decrease in lymph node cellsis an IL-12-induced alteration in the distribution of rymphoid cellsbetween various anatomical compartments. Alternatively, the highlevels of IFN-7 produced in response to IL-12 might, either directlyor indirectly (40,41), cause a partial suppression of alloantigen-induced lymph node cell proliferation. In unimmunized mice,administration of IL-12 resulted in lymph node enlargement withincreased cell recovery (D. Faherty and S. Connaughton,unpublished observations).

The majority of studies in this manuscript were performed withmice that had received IL-12 at a dose of 1 /ig/day. This doseis sufficient to cause an optimum therapeutic effect in micefnfected with L. major (33,34) and in murine tumor models (42).Mice treated for 6 - 8 days with this dose displayed some furruffling and mild lethargy. No IL-12-associated mortalities wereobserved in these studies. However, in other studies it has beenshown that treating mice with 10 fig IL-12 daily is lethal whencontinued for longer than 1 week (T. Anderson, unpublishedresults). The primary toxicities of IL-12 in mice are hepatotox'icityand anemia (T. Anderson and J. Lipman, unpublished results).The hepatotoxicity, which is characterized by focal hepatocytenecrosis (e.g. see Fig. 6) and elevation of hepatic transaminases,is similar to that previously seen in IL-2-treated mice (22), but theanemia in IL-12-treated mice is more severe than that previouslyobserved with IL-2 (22). Unlike IL-2-treated mice (22), IL-12-treated mice did not develop pulmonary edema when given lethaldoses of cytokine, correlating with the lack of mononuclear cellinfiltrates in the lungs (T. Anderson, unpublished results). WhereasIL-12-induced enhancement of NK and CTL activity plateauedat a dose of 1 /ig/day, the amount of IFN-7 produced by liverand splenic lymphocytes was substantially greater in mice thathad received 10 /igVday IL-12 as compared with 1 ^g/day (Fig. 4).

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This observation suggests that the toxicities observed in micereceiving 10 ̂ g/day IL-12 may result, either directly or indirectly,from the high levels of IFN-7, and probably other cytokines, whichare produced.

Recently, IL-12 has been shown to have strong antitumor andantimetastatic effects in several murine tumor models (42), to cureL major infections in BALB/c mice (33,34), and to prolong survivalof scid mice infected with T. gondii (36). The results reported inthis manuscript suggest possible mechanisms by which IL-12may exert therapeutic effects against some tumors and infectiousagents.

Abbreviations

CCHOCTLD-PBSFBSLAKNKPBMCPE

References

complementChinese hamster ovarycytolytic T lymphocyteDulbecco's phosphate-buffered salinefetal bovine serumlymphokine-activated killernatural killerperipheral blood mononuctear cellphycoerythrin

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