regulation of perforin-independent nk cell-mediated cytotoxicity

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© 2003 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Regulation of perforin-independent NK cell-mediated cytotoxicity Robert P. A. Wallin 1,2 , Valentina Screpanti 2 , Jakob Micha ¨ elsson 1 , Alf Grandien 2 and Hans-Gustaf Ljunggren 2 1 Microbiology and Tumor Biology Center, Karolinska Institutet, Stockholm, Sweden 2 Center for Infectious Medicine, Department of Medicine, Karolinska Institutet, Huddinge University Hospital, Stockholm, Sweden Natural killer (NK) cells have been thought to depend largely on perforin-mediated mecha- nisms for the induction of cell death in targets. However, this view has more recently been challenged. It is now clear that NK cells are capable of using death ligands like Fas ligand (FasL) or tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) to induce cytotox- icity. Still, relatively little is known about the control of these “perforin-independent” cell death eliciting reactions, for example, the regulation of FasL expression on NK cells. In the present study, we confirm the ability of NK cells to mediate target cytotoxicity in the absence of perforin, in vivo and in vitro. We show that the induction of perforin-independent NK cell- mediated cell death is prevented by inhibiting signals mediated by MHC class I recognition. Furthermore, we demonstrate in vitro that cross-linking of the activation receptor NK1.1 on NK cells leads to the up-regulation of FasL on the cell surface. However, simultaneous engagement of an MHC class I binding inhibitory receptor prevents the externalization of FasL. These results provide a mechanistic explanation for the MHC class I-dependent regu- lation of perforin-independent cytotoxicity. Key words: NK cells / Cytotoxicity / MHC class I / FasL / Apoptosis Received 3/4/03 Revised 2/7/03 Accepted 5/8/03 [DOI 10.1002/eji.200324070] Abbreviations: B6: C57BL/6 TAP: Transporter associated with antigen processing 2 m: 2 -microglobulin MMP: Ma- trix metalloproteinase 1 Introduction Natural killer (NK) cells are innate immune cells with capacity to kill cells infected with certain viruses and intracellular bacteria as well as some tumor cells [1]. NK cells also reject MHC class I-mismatched bone marrow grafts [2]. Additionally, NK cells are potent producers of inflammatory cytokines, including tumor necrosis factor (TNF)- and interferon (IFN)- , which are important dur- ing both the early and late phases of an immune response [3, 4]. The activation of NK cells occurs through the engage- ment of activation receptors on the cell surface, the nature and specificity of which have recently been eluci- dated [5]. The binding of ligands to their cognate activa- tion receptors on the NK cells leads to the initiation of effector functions, including cytotoxicity and secretion of cytokines. As some cells in normal tissue also express ligands for activation receptors, additional signals are needed to regulate the effector functions of NK cells. This is mediated by inhibitory receptors recognizing self MHC class I molecules, as originally predicted by the “missing-self” hypothesis [6, 7]. Several MHC class I- binding inhibiting receptors have been cloned and char- acterized in detail [8]. When these receptors bind to their ligands, they recruit and activate intracellular phospha- tases (e.g. SHP-1), leading to dephosphorylation of mol- ecules that signal activation [9, 10]. The inhibition of NK cell-mediated cytotoxicity by MHC class I does not seem, however, to interfere with early signals in the tar- get recognition and adhesion event [11]. The by far most investigated effector mechanism of cytotoxicity by NK cells is the perforin-mediated granule exocytosis pathway. When activated by a target cell, NK cells deliver lytic granules in a highly directed manner towards the intracellular junction formed between the two cells [12]. The released perforin is thought to enable uptake and activity of several cytotoxic proteases, e.g. granzymes, into the target cell, leading to the induction of apoptosis [13]. The importance of perforin-mediated cytotoxicity in NK cell killing has been studied in several model systems including perforin-deficient mice [14–16]. This has led to the conclusion that perforin-mediated Eur. J. Immunol. 2003. 33: 2727–2735 Perforin-independent NK cell cytotoxicity 2727 © 2003 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

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Page 1: Regulation of perforin-independent NK cell-mediated cytotoxicity

© 2003 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Regulation of perforin-independent NKcell-mediated cytotoxicity

Robert P. A. Wallin1,2, Valentina Screpanti2, Jakob Michaelsson1, Alf Grandien2 andHans-Gustaf Ljunggren2

1 Microbiology and Tumor Biology Center, Karolinska Institutet, Stockholm, Sweden2 Center for Infectious Medicine, Department of Medicine, Karolinska Institutet, Huddinge

University Hospital, Stockholm, Sweden

Natural killer (NK) cells have been thought to depend largely on perforin-mediated mecha-nisms for the induction of cell death in targets. However, this view has more recently beenchallenged. It is now clear that NK cells are capable of using death ligands like Fas ligand(FasL) or tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) to induce cytotox-icity. Still, relatively little is known about the control of these “perforin-independent” celldeath eliciting reactions, for example, the regulation of FasL expression on NK cells. In thepresent study, we confirm the ability of NK cells to mediate target cytotoxicity in the absenceof perforin, in vivo and in vitro. We show that the induction of perforin-independent NK cell-mediated cell death is prevented by inhibiting signals mediated by MHC class I recognition.Furthermore, we demonstrate in vitro that cross-linking of the activation receptor NK1.1 onNK cells leads to the up-regulation of FasL on the cell surface. However, simultaneousengagement of an MHC class I binding inhibitory receptor prevents the externalization ofFasL. These results provide a mechanistic explanation for the MHC class I-dependent regu-lation of perforin-independent cytotoxicity.

Key words: NK cells / Cytotoxicity / MHC class I / FasL / Apoptosis

Received 3/4/03Revised 2/7/03Accepted 5/8/03

[DOI 10.1002/eji.200324070]

Abbreviations: B6: C57BL/6 TAP: Transporter associatedwith antigen processing I 2m: g 2-microglobulin MMP: Ma-trix metalloproteinase

1 Introduction

Natural killer (NK) cells are innate immune cells withcapacity to kill cells infected with certain viruses andintracellular bacteria as well as some tumor cells [1]. NKcells also reject MHC class I-mismatched bone marrowgrafts [2]. Additionally, NK cells are potent producers ofinflammatory cytokines, including tumor necrosis factor(TNF)- § and interferon (IFN)- + , which are important dur-ing both the early and late phases of an immuneresponse [3, 4].

The activation of NK cells occurs through the engage-ment of activation receptors on the cell surface, thenature and specificity of which have recently been eluci-dated [5]. The binding of ligands to their cognate activa-tion receptors on the NK cells leads to the initiation ofeffector functions, including cytotoxicity and secretion ofcytokines. As some cells in normal tissue also express

ligands for activation receptors, additional signals areneeded to regulate the effector functions of NK cells.This is mediated by inhibitory receptors recognizing selfMHC class I molecules, as originally predicted by the“missing-self” hypothesis [6, 7]. Several MHC class I-binding inhibiting receptors have been cloned and char-acterized in detail [8]. When these receptors bind to theirligands, they recruit and activate intracellular phospha-tases (e.g. SHP-1), leading to dephosphorylation of mol-ecules that signal activation [9, 10]. The inhibition of NKcell-mediated cytotoxicity by MHC class I does notseem, however, to interfere with early signals in the tar-get recognition and adhesion event [11].

The by far most investigated effector mechanism ofcytotoxicity by NK cells is the perforin-mediated granuleexocytosis pathway. When activated by a target cell, NKcells deliver lytic granules in a highly directed mannertowards the intracellular junction formed between thetwo cells [12]. The released perforin is thought to enableuptake and activity of several cytotoxic proteases, e.g.granzymes, into the target cell, leading to the inductionof apoptosis [13]. The importance of perforin-mediatedcytotoxicity in NK cell killing has been studied in severalmodel systems including perforin-deficient mice [14–16].This has led to the conclusion that perforin-mediated

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Fig. 1. Perforin-deficient mice reject MHC class I-deficientbut not MHC class I-expressing tumors. Perforin-deficientmice were injected s.c. with 1×102 RMA or RMA-S tumorcells, and tumor growth was monitored. Percent of mice withdetectable tumors of total number of mice injected is shown.p X 0.0001 when the two groups were compared.

cytotoxicity is essential for the clearance of target cells,including tumor cells, although it has been known forsome time that NK cells also express and use deathligands, such as FasL and TRAIL [17–19]. Moreover, werecently reported that death receptor-mediated apopto-sis plays a more prominent role in the clearance of NK-sensitive tumors than previously suggested [20].

In the present study, we set out to study non-perforin-dependent (perforin-independent) cytotoxicity by NKcells, with a particular emphasis on its regulation by acti-vating and inhibitory ligands on target cells.

2 Results

2.1 MHC class I-deficient tumors are rejected byNK cells in perforin-deficient mice

To test if the rejection of tumor cells in the absence ofperforin is controlled by recognition of MHC class I mole-cules on target cells, perforin-deficient mice were graftedwith MHC class I-deficient and corresponding MHCclass I-expressing tumors (Fig. 1). While only 14% of themice inoculated with MHC class I-deficient RMA-S cellsdeveloped tumors, 100% of the mice inoculated withMHC class I-expressing RMA cells developed tumors(p X 0.0001). The rejection of RMA-S tumor cells was NKcell-dependent, since RMA-S cells readily grew inperforin-deficient mice depleted of NK cells (data notshown). These results demonstrate that MHC class Imolecules protect tumor targets from NK cell-mediatedperforin-independent cytotoxicity.

2.2 MHC class I-deficient tumor cells arerapidly eliminated by NK cells in the lungsof perforin-deficient mice

One mechanism for the protection against circulatingtumor cells is rapid elimination by NK cells [21]. Perforin-expressing NK cells in the capillary beds of the lung havea high cytotoxic activity and are able to rapidly clear i.v.injected MHC class I-deficient tumor cells. To testwhether perforin-deficient NK cells are also capable ofrapidly eliminating MHC class I-deficient tumor cells,and to test whether they can distinguish between MHCclass I-deficient and MHC class I-expressing tumorcells, we injected such tumor cells i.v. into perforin-deficient and C57BL/6 (B6) control mice. MHC class I-deficient RMA-S and C4.4.25– tumor cells were rapidlyeliminated in lungs of perforin-deficient mice, whereasthe corresponding MHC class I-expressing RMA and EL-4 parental tumor cell lines were not cleared (Fig. 2A, B).Elimination of MHC class I-deficient tumor cells was NKcell-dependent, as these tumor cells were not eliminatedin lungs of perforin-deficient mice depleted of NK cells(Fig. 2C). These observations suggest that perforin-deficient NK cells are capable of efficiently eliminatingMHC class I-deficient tumor cells in the lung, and thatthey can discriminate between MHC class I-deficientand -expressing tumor cells.

2.3 MHC class I-deficient bone marrow graftsare rejected by NK cells in perforin-deficientmice

NK cells can reject MHC class I-mismatched and MHCclass I-deficient bone marrow grafts [2, 22]. To investi-gate if rejection of bone marrow cells also occurs in theabsence of perforin, we injected MHC class I-deficient or-expressing bone marrow into lethally irradiated perforin-deficient mice. MHC class I-deficient bone marrow cells(TAP-1/ g 2m-deficient), but not corresponding MHCclass I-expressing bone marrow cells, were rejected inperforin-deficient mice (Fig. 3). This rejection was NKcell-dependent since TAP-1/ g 2m-deficient bone marrowwas accepted by NK cell-depleted, perforin-deficientmice (Fig. 3). These results support and extend therecent observation by Taylor et al. [23], demonstratingthat perforin-deficient NK cells are capable of rejectingMHC class I-mismatched bone marrow grafts.

2.4 MHC class I-deficient tumor cells are killedby perforin-deficient NK cells in vitro

Perforin-deficient NK cells have been claimed to belargely deficient in cytotoxic potential in vitro [14]. How-ever, while perforin-deficient NK cells do not kill RMA-S

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Fig. 2. NK cells in perforin-deficient mice rapidly eliminateMHC class I-deficient but not MHC class I-expressing tumorcells in the lungs. B6 or perforin-deficient mice were injectedi.v. with radiolabeled MHC-expressing or -deficient tumorcells. The injected mice were sacrificed after 12 h, lungswere removed, and remaining radioactivity was analyzed. (A)Elimination of RMA-S and RMA tumor cells in B6 andperforin-deficient mice. (B) Elimination of C4.4.25– and EL-4cells in B6 and perforin-deficient mice. (C) Elimination ofC4.4.25– cells in untreated and NK-depleted perforin-deficient mice. Experiments were performed with at leastfour mice in each group and repeated at least three timeswith similar results. One representative experiment is shown.Error bars indicate standard deviations.

Fig. 3. NK cells in perforin-deficient mice reject MHC class I-deficient but not MHC class I-expressing bone marrowgrafts. Lethally irradiated mice were injected i.v. with1×106 bone marrow cells from B6 or TAP-1/ g 2m-deficient (T/g –/–) mice and engraftment was measured by assessing pro-

liferation of grafted cells in the spleen on day 5 after bonemarrow transfer. NK cell-dependent rejection of bone mar-row grafts was studied by comparing bone marrow rejectionin untreated mice and mice depleted of NK cells. One repre-sentative experiment of three is shown. Error bars indicatestandard deviations.

cells in a regular 5-h cytotoxicity assay, they readily killRMA-S cells in a 18-h cytotoxicity assay (Fig. 4 and [20]).The killing of RMA-S cells by perforin-deficient NK cellsin the 18-h cytotoxicity assay was FasL-dependent sinceaddition of blocking antibodies against FasL abrogatedkilling of the target cells (Fig. 4 and [20]). Blocking FasLon B6 NK cells did not have any effect on the killing ofRMA-S. As illustrated in Fig. 5, we extended theseobservations by showing that perforin-deficient NK cellsdistinguished MHC class I-deficient from MHC class I-expressing tumor cells in a 18-h 51Cr-release assay invitro. While MHC class I-deficient C4.4.25– or RMA-S

cells were readily killed by perforin-deficient NK cells, thecorresponding MHC class I-expressing EL-4 or RMAcells were not (Fig. 5).

2.5 FasL is stored intracellularly in IL-2-activatedNK cells and is up-regulated upon cross-linking of the activation receptor NK1.1

The foregoing results suggest that NK cell-mediatedcytotoxicity is regulated by MHC class I recognition,even in the absence of perforin. Several death ligandshave recently been demonstrated to be expressed by NKcells. Therefore, we hypothesized that activating signalsmight regulate the expression of such ligands on NKcells. We chose, as a model death receptor ligand, tostudy the regulation of FasL. Only low levels of FasLwere detectable on the cell surface of IL-2-activated NKcells, even in the presence of inhibitors of MMP (Fig. 6A).However, permeabilization of the cells revealed intracel-lular storage of FasL (Fig. 6B), in agreement with recentpublications [24, 25]. Since degranulation triggered byionomycin has been shown to elicit translocation of FasLto the cell surface [24, 25], we hypothesized that recep-tors known to trigger NK cell effector function might alsocause such translocation. To test this possibility, we acti-vated NK cells in vitro by cross-linking the activationreceptor NK1.1 (CD161, NKR-P1C). Upon activation ofNK cells by NK1.1 cross-linking, FasL was up-regulatedin a dose-dependent fashion (Fig. 7A-C). Up-regulation

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Fig. 4. Perforin-deficient NK cells kill MHC class I-deficienttumor cells in vitro. Cytotoxicity of purified, IL-2-activatedNK cells from (A, B) B6 mice and (C, D) perforin deficientmice. 51Cr-release was measured after (A, C) 5 h and (B, D)18 h against RMA-S target cells. In (B, D) anti-Fas ligandantibodies were used to asses the involvement of FasL inthe in vitro cytotoxicity.

Fig. 5. Perforin-deficient NK cells kill MHC class I-deficientbut not MHC class I-expressing tumor cells in vitro. Cytotox-icity of purified, IL-2-activated NK cells against (A) RMA andRMA-S or (B) EL-4 and C4.4.25– cells, was measured in an18-h 51Cr-release assay.

was rapid (Fig. 8A) and reached a maximal level afterapproximately 90 min (Fig. 8B). It should be noted thatwhile the experiments shown were performed with B6NK cells, control experiments with perforin-deficient NKcells displayed similar up-regulation of FasL (data notshown).

2.6 MHC class I-mediated inhibiting signalsabrogate up-regulation of FasL surfaceexpression on activated NK cells

Given the previous results, we subsequently investigatedwhether the ligation of an inhibitory MHC class I bindingreceptor would prevent the up-regulation of FasL elicitedby NK1.1 cross-linking. We, thus, activated NK cells bycross-linking the activation receptor NK1.1 and simulta-neously engaged inhibiting receptors with recombinantH-2Db. This resulted in the abrogation of FasL cell sur-face up-regulation in a subpopulation of NK cells (Fig. 9).In parallel, control experiments revealed that inhibitoryreceptor engagement by H-2Dd reduced NK1.1 inducedproduction of IFN- + (Fig. 9D), as previously observed by

Kambayashi et al. [26]. The fact that FasL expressionwas inhibited on only in a subpopulation of the NK cellswas entirely as expected, since only a subpopulation ofall NK cells expresses inhibitory receptors recognizingH-2Db [27]. Taken together, this observation provides apossible mechanism for the regulation of perforin-independent NK cell-mediated cytotoxicity.

3 Discussion

In the present study, we first confirm the induction of NKcell-mediated target cell death in the absence of perforinin several in vivo and in vitro assays of NK cell activity.We show that inhibiting signals mediated by MHC class Irecognition prevent perforin-independent NK cell-mediated cell death induction. Furthermore, we demon-strate that cross-linking of the activation receptor NK1.1on NK cells leads to the up-regulation of FasL on the cellsurface, while simultaneous engagement of an MHCclass I-binding inhibitory receptor prevents expression ofFasL.

Fas-FasL interactions regulate tissue homeostasis inmany physiological systems and also play importantroles in immune regulation and immune effector func-

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Fig. 6. Expression of FasL in IL-2-activated NK cells. Theexpression of FasL was studied using flow cytometry. (A)Cell surface expression. (B) Intracellular expression. All mAbincubations and washes were done in the presence of MMPinhibitors to prevent processing of FasL to its soluble form.

Fig. 7. Up-regulation of FasL on IL-2-activated NK cellsupon cross-linking of NK1.1. IL-2-activated NK cells wereincubated for 1 h in tissue culture plates coated with mAbNK1.1. (A) FasL expression on untreated NK cells (thin line);NK cells activated with 0.125 ? g/well NK1.1 (dashed line),and NK cells activated with 1 ? g/well of NK1.1 (thick line). (B)Percentage of FasL high NK cells after cross-linking withindicated amounts of plate-bound NK1.1. The FasL highgate was set as in Fig. 9A–C. (C) The same NK1.1 titrationwas used as in (B), but FasL expression is indicated by thegeometric mean of fluorescence intensity.

Fig. 8. Kinetics of FasL up-regulation on IL-2-activated NKcells. IL-2-activated NK cells were incubated in tissue cul-ture plates coated with mAb NK1.1. (A) FasL expression onNK cells incubated for indicated periods of time in NK1.1-coated wells (thick lines) compared with untreated NK cells(thin lines). (B) Percentage of NK cells with high FasL stain-ing after incubation in wells coated with 0.5 ? g of NK1.1(filled symbols) or incubated in untreated wells (open sym-bols) for indicated periods of time. The FasL high gate wasset as in Fig. 9A–C. Data from three independent experi-ments are plotted.

tions. The death receptor Fas has widespread, some-times constitutive, expression in many tissues, anddeath signal induction is likely to be governed by the reg-ulation of FasL expression. Non-triggered effector cellsgenerally express low steady-state cell surface levels of

FasL. Swift up-regulation of FasL on NK cells should bea prerequisite to rapidly eliminate target cells via thispathway. The present data demonstrate rapid up-regulation of FasL upon triggering of NK1.1 on NK cells.In line with our results, Bossi et al. as well as Kojima et al.[24, 25] found that T and NK cells store FasL in intracellu-lar granules, which can be exocytosed upon nonspecific(PMA/ionomycin) triggering of the cells. Activation of NKcells with cytokines and/or triggering of activation recep-tors have previously also been shown to increase thesynthesis of FasL mRNA [28, 29]. It is likely that the rapidexternalization of FasL described herein results fromexocytosis of preformed FasL, whereas the long-termup-regulation upon continuous stimulation with NK1.1might also involve de novo synthesis of FasL. It has been

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Fig. 9. Inhibition of FasL up-regulation on IL-2-activated NKcells by ligation of inhibitory receptors with H-2Db. IL-2-activated NK cells were incubated for 2 h in tissue cultureplates that were: (A) uncoated, (B) coated with mAb NK1.1(0.5 ? g/ well) and control protein and (C) coated with mAbNK1.1 (0.5 ? g/ well) and soluble H-2Db. Histograms fromduplicate cultures are shown. (D) ELISA of IFN- + produced insupernatants from the NK cells used in the FasL up-regulation experiment shown in A-C. (E) Collected data fromeight experiments are expressed as % NK cells in the FasLhigh marker gate shown in A-C. Control protein (ovalbuminor bovine serum albumin) is denoted as c.p.

observed that NK cells must “rearm” after killing a targetcell [30]. It is still unclear whether FasL is translocateddirectly to the cell surface, under conditions of continu-ous stimulation, or if it is transported to intracellular cyto-toxic granules, which are later exocytosed. In the presentstudy, we demonstrated that MHC class I-recognitioninhibited the externalization of FasL. This outcome pro-vides a novel mechanism for NK cell-mediated cytotox-icity of MHC class I-deficient (or -mismatched) targetcells.

After externalization of FasL, its half life on the cell sur-face is tightly controlled in part through the rapid cleav-age by MMP that release soluble FasL, which has lowerapoptosis-inducing capacity than the membrane-boundform [31]. NK cells express several MMP and also regu-lators of MMP activity [32–34]. This could explain the factthat, when the activity of MMP is not inhibited, little FasLcan be observed on the cell surface, even after activa-tion. Regulation of MMP activity during contact with the

effector cell might also play a role in controlling deathligand-mediated NK cell cytotoxicity.

Perforin-mediated cytotoxicity has been described asthe major mechanism for rejection of NK-sensitivetumors in vivo. Furthermore, earlier studies indicated thatperforin-deficient NK cells are impaired in cytotoxicfunction and, therefore, not capable of rejecting largedoses of injected tumor cells, preventing metastases orkilling susceptible targets in vitro [15, 16, 35]. Our datasuggest that NK cells lacking perforin are, indeed, capa-ble of rejecting tumor cells. Our data also show thatperforin-deficient NK cells display cytotoxic activitytowards sensitive target cells in the circulation. Oneexplanation for the fact that perforin-independent cyto-toxicity has been underestimated or in part beenneglected in cytotoxic assays performed in vitro, may bethe fact that this killing operates under different kineticsthan perforin-mediated killing. Indeed, most in vitroassays are optimized to measure the rapid perforin-dependent cytotoxicity, usually operating over 4 h.Finally, in light of the present results and data publishedpreviously (claiming co-localization of perforin and FasLin cytotoxic granules), care has to be taken in interpret-ing results obtained when using inhibitors of granuleexocytosis, e.g. EGTA. The possibility exists that suchinhibitors might also inhibit FasL up-regulation, andmight, thus, inhibit Fas-mediated cytotoxicity.

Our present data suggest that the previously reported“deficiency” of NK cell-mediated cytotoxic activity inperforin-deficient mice is relative rather than absolute.The present data implicate that cytotoxic effector mech-anisms mediated by NK cells, irrespective of beingperforin-dependent or not, have in common that they areregulated by ligands binding to activating and inhibitoryreceptors.

4 Materials and methods

4.1 Reagents and antibodies

The metalloproteinase inhibitor KB8301, the intracellularstaining kit as well as mAb against FasL (MFL3-PE), NK1.1(PK136-FITC), and CD3 (145-2C11-FITC) were purchasedfrom BD PharMingen (San Diego, CA). The matrix metallo-proteinase (MMP) inhibitors, MMP inhibitor II, MMP inhibi-tor III and MMP-MMP-9/MMP-13 inhibitor I, were pur-chased from Calbiochem (San Diego, CA). Ovalbumin waspurchased from Sigma (St. Louis, MO) and bovine albuminfraction V solution (7.5%) was purchased from GibcoBRL(Life Technologies, Inc. Fredrick, MD). Na51CrO4 and5’-[125I]iodo-2’-dUrd were purchased from Amersham Bio-sciences (Uppsala, Sweden). 5’-fluoro-2’-dUrd was pur-chased from Calbiochem.

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4.2 Cell lines and mice

RMA and EL-4 cells originate from T cell lymphoma cells ofB6 mice origin. They are thought to be initially derived fromthe same cell line, but have probably been separated viagenetic drift for more than 30 years [36]. RMA-S is a TAP-2-deficient variant of RMA [6, 21, 37]. C4.4.25– is a g 2-microglobulin ( g 2m)-deficient variant of EL-4 [38]. Cell lineswere maintained in RPMI 1640 supplemented with 5% FCS,penicillin and L-glutamine (Life Technologies). B6, TAP-1/g 2m-deficient [39] and perforin-deficient [35] mice were bred

and maintained in the animal facility at MTC, KarolinskaInstitute and Stockholm University according to institutionalguidelines.

4.3 Subcutaneous tumor growth

Groups of five to eight mice were injected s.c. in the inter-scapular region with 1×102 RMA and RMA-S cells. In experi-ments performed with NK cell-depleted mice, animals wereinjected i.p. with 100 ? g anti-NK1.1 mAb 2 days prior totumor grafting, then 2, 7 and 12 days after tumor challenge.Tumors were monitored every second day by palpation, for8 weeks. Mice were killed with CO2 when tumors reached asize of approximately 1 cm3, as recommended by the Stock-holm Ethical Committee for Animal Experiments, or whenthe experiment was terminated. The p value (two-sided) wascalculated by use of a two-tailed student’s t-test.

4.4 Rapid tumor elimination assay

This assay was performed as described by Carlson et al. [40]with some modifications. Tumor cells were labeled with500 ? Ci Na51CrO4 in 500 ? l of LAK medium ( § MEM contain-ing penicillin, L-glutamine, 10 mM HEPES, 5×10-5 M 2-mercaptoethanol and 10% FCS, all reagents from Life Tech-nologies), for 1 h at 37°C. After washing, the cells wereresuspended in PBS at a concentration of 5×106 cells/ml.Cells (1×106) were then injected i.v. into B6 or perforin-deficient mice. The perforin-deficient mice were either leftuntreated or pretreated to deplete NK cells by injecting100 ? g and 50 ? g anti-NK1.1 mAb i.p. 2 days and 1 day,respectively, before grafting. At 12 h after injection of tumorcells, the mice were killed, and the lungs were removed. Theradioactivity in the lungs was measured in a gamma counterand the percent remaining radioactivity was calculated asfollows: [(CPM lung – CPM background)/(CPM of cellsinjected – CPM background)]×100.

4.5 Bone marrow graft assay

Bone marrow was harvested from B6 or TAP-1/ g 2m-deficient mice by flushing the femurs and tibia with PBS.Bone marrow cells (1×106) were injected i.v. into lethally irra-diated (900 rad) perforin-deficient mice either untreated or

pretreated with 100 ? g anti-NK1.1 mAb i.p. Two days afterbone marrow injection, the anti-NK1.1-treated groupreceived another dose of 50 ? g anti-NK1.1. After 5 days,0.5 ? Ci 5’-[125I]iodo-2’-dUrd was injected i.p. To inhibitendogenous thymidylate synthesis, 25 ? g 5’-fluoro-2’-dUrdin 200 ? l PBS was injected i.p. On day 6, mice were sacri-ficed, and incorporated radioactivity was measured in thespleens using a gamma counter (Wallac). The data areshown as the CPM of incorporated radioactivity.

4.6 Generation of effector cells

IL-2-activated NK cells were prepared according to the fol-lowing protocol: 25×106 erythrocyte-depleted splenocyteswere enriched for NK cells using an anti-DX5 mAb linked toMACS beads (Miltenyi Biotec, Bergisch Gladbach, Ger-many), according to the manufacturer’s recommendations.After MACS separation, the DX5-positive cells were resus-pended in LAK medium supplemented with 1,000 U/mlhuman rIL-2 (PeproTech inc., Rocky Hill, NJ), and cultured in25-cm2 tissue culture flasks in 10% CO2 at 37°C. After5 days, IL-2-activated NK cells were resuspended by pipet-ting and scraping and used for cytotoxicity assays and anal-ysis of FasL expression.

4.7 Cytotoxicity assay

Effector cells were washed once, resuspended and used aseffectors in a 51Cr-release assay. Briefly, target cells werelabeled with 150 ? Ci sodium [51Cr]chromate in 100 ? l for 1 hat 37°C and then washed. Cells (5×103) were incubated withtitrated numbers of effector cells in round-bottom 96-wellplates for 5 or 18 h at 37°C in 10% CO2. For blocking ofFasL, 10 ? g/ml of MFL3 antibody (BD PharMingen) wereadded to the assay media. After incubation, the superna-tants were harvested, and released radioactivity was mea-sured. Specific lysis was calculated according to the for-mula: % specific release = [(experimental release – sponta-neous release)/(maximum release – spontaneousrelease)]×100.

4.8 Treatment of IL-2-activated NK cells with anti-NK1.1 mAb and MHC class I molecules

This experimental setup was adapted from Kambayashi etal. [26]. The ligands for inhibiting NK cell receptors were gen-erated as follows: recombinant H-2Db was refolded with g 2mand the peptide KAVANFATC, by using the same protocol asdescribed for the generation of H-2Dd peptide complexes[41]. This protocol was also used to produce H-2Kb-SIINFEKL complexes. The refolded products were purifiedusing size exclusion chromatography. Anti-NK1.1 mAbalone or together with soluble MHC class I molecules orcontrol protein (OVA or BSA) was added to 24- or 12-welltissue culture plates at indicated concentrations and incu-bated overnight at 4°C. The wells were washed twice with

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PBS before adding the cells. IL-2-activated NK cells from B6mice were resuspended in LAK medium containing matrixmetalloproteinase inhibitor (KB8301, MMP inhibitor II or acombination of MMP inhibitor II, III and MMP9/13 inhibitor)at a concentration of 2×106 cells/ml and was added to eachpretreated well. NK cells incubated in untreated wells wereused as unstimulated controls. Cells were incubated for15 min to 3 h at 37°C, and in some experiments the cell-freesupernatants were harvested for ELISA. The IFN- + contentof the supernatants was determined using a commercialELISA kit (BD PharMingen).

4.9 Analysis of FasL expression by flow cytometry

NK cells cultured in treated wells were harvested, and Fcreceptors were blocked with supernatant from the HB197hybridoma (anti-CD16/32) containing also 1% normalmouse serum (DAKO) and 1% normal rat serum (DAKO).Cells were then stained with fluorochrome-conjugated anti-FasL, anti-CD3 and biotinylated Ly-49 mAb (BD PharMin-gen). The biotinylated antibodies were detected withstreptavidin-RED670 (Life Technologies, Rockville, MD). Allincubations were done on ice for 1 h in the presence of MMPinhibitors and sodium azide. Intracellular staining of FasLwas performed with unstimulated IL-2-activated NK cells.NK cells were incubated with anti-CD3 mAb (BD PharMin-gen) for 1 h, then permeabilized with “perm wash solution”(BD PharMingen) and incubated with anti-FasL mAb orisotype-control antibody for 1 h. The cells were washed andanalyzed on a FACScan (BD PharMingen) using CellQuestsoftware (BD PharMingen). Statistical analysis was per-formed using ANOVA.

Acknowledgements: We thank Drs. B. J. Chambers, K.Kärre, and T. Kambayashi as well as members of our respec-tive labs for fruitful discussions and methodological help.This work was supported by grants from the SwedishResearch Council, the Swedish Cancer Society, the SwedishFoundation for Strategic Research and the Karolinska Insti-tutet.

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Correspondence: Robert P. A. Wallin, Center for InfectiousMedicine, Department of Medicine, Karolinska Institutet,Huddinge University Hospital, 141 86 Stockholm, SwedenFax: +46-8-746-7637e-mail: robert.wallin — mtc.ki.se

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