Proc. Natl. Acad. Sci. USAVol. 89, pp. 8337-8341, September 1992Immunology

Role for major histocompatibility complex class I in regulatingnatural killer cell-mediated killing of virus-infected cells

(histocompatibility antigens/herpes simplex virus/cell-mediated cytotoxicity/lymphocytes)

DAN S. KAUFMAN, RENEE A. SCHOON, AND PAUL J. LEIBSONDepartment of Immunology, Mayo Clinic and Foundation, Rochester, MN 55905

Communicated by Janet D. Rowley, June 4, 1992

ABSTRACT Target structures important for natural killer(NK) cell recognition of virally infected cells are not welldefined. Since major histocompatibility complex (MHC) classI molecules bind viral peptides during acute infection, weevaluated whether an interaction between MHC and virusmight influence the susceptibility of infected cells to NKcell-mediated lysis. To control for MHC class I expression ontarget cells, we used either HLA class I-deficient CiR cells orCiR sublines expressing transfectedHLA class I gene products.Human NK cells were unable to preferentially lyse classI-deficient COR cells after infection with herpes simplex virus(HSV). In contrast, HLA class I transfectants were significantlymore susceptible to NK cell-mediated cytotoxicity after HSVinfection. This occurred for HSV-infected CiR cells expressingany of the three HLA class I gene products tested (i.e.,HLA-B27, HLA-A3, or HLA-Aw68), indicating that NK cellrecognition in this system does not require "self' MHC and isnot unique for a single haplotype. Productive HSV infection isrequired for the increased killing, since inoculation with UV-inactivated virus did not lead to increased lysis. In addition,since HSV infection of the transfectants did not significantlyalter the level of class I expression, the change in susceptibilityappears to be due to qualitative changes in the target structureson HSV-infected, HLA class If targets. These results demon-strate a role for MHC class I in regulating NK cell-mediatedkilling of virus-infected cells.

Natural killer (NK) cells are a subpopulation of lymphocytesthat can preferentially lyse certain virus-infected cells, andthis antiviral activity appears to function as an early immunedefense against viruses in vivo (1-7). However, it remainsunclear as to what target structures on virally infected cellscan be recognized by NK cells and confer increased sensi-tivity to NK cell-mediated cytotoxicity. The paradigm forT-lymphocyte recognition of virus-infected cells does notdirectly relate to NK cell recognition-i.e., there is noevidence for clonotypic receptors on NK cells that arerestricted to recognizing viral peptides on "self' majorhistocompatibility complex (MHC) molecules. Yet, recentreports suggest that MHC molecule expression on tumortarget cells can potently influence their susceptibility to NKcell-mediated lysis (8-11). Specifically, tumor cells withreduced or absent MHC class I expression are preferentiallylysed by NK cells ("missing self' model) (9-23). In addition,exogenous loading of "foreign" peptides onto MHC classI-bearing targets can increase their sensitivity to killing (24,25). These observations suggest that antitumor NK cell-mediated cytotoxicity can be influenced by either quantita-tive changes in MHC class I expression on the targets or byaltering peptide-MHC ligands presented to potential NK celleffector lymphocytes.

The relevance of MHC expression on virus-infected cellsto their susceptibility to NK cell-mediated lysis remainsunclear. Although certain viruses have the potential to quan-titatively reduce MHC class I expression during viral infec-tion (26, 27), NK cells can preferentially lyse infected cellswith unaltered amounts of cell-surface MHC class I antigens(28). However, we hypothesized that MHC class I expressionmight still be a critical determinant for antiviral NK cellkilling. We reasoned that if exogenous peptide loading ofMHC could modulate antitumor cytotoxicity (24, 25), aphysiologic correlate might be association ofMHC moleculeswith endogenously synthesized viral peptides. Our hypoth-esis predicted that whereas MHC class I-deficient cellsinfected with virus might not be preferentially killed by NKcells, transfection of MHC class I genes would "reconsti-tute" the ability of virally infected cells to be selectivelylysed.We have evaluated, therefore, the influence ofMHC class

I expression on NK cell-mediated killing of herpes simplexvirus (HSV)-infected cells. Our model focuses on HSVbecause of its well-characterized role in enhancing suscep-tibility to human NK cell-mediated cytotoxicity (6, 29-33).As targets, we have used the HLA class I-deficient B-lym-phoblastoid cell line C1R and stableHLA class I transfectantsof C1R expressing HLA-B27 (C1R-B27 cells), HLA-A3(C1R-A3 cells), or HLA-Aw68 (ClR-Aw68 cells) (15, 17, 19,20). Using this model, we demonstrate that human NK cells(isolated either from peripheral blood or as cloned homoge-neous populations of CD16+/CD3- cells) are unable topreferentially kill HSV-infected, class I-deficient CiR cells.In contrast, HSV infection of MHC class I-transfectantsincreases their sensitivity to NK cell-mediated cytotoxicity.The virus-induced modulation ofNK cell sensitivity requireslive virus replication and is not mediated by down-regulationofMHC class I expression. These results suggest that inter-actions between MHC and virus can potently influencesusceptibility to NK cell-mediated lysis.

MATERIALS AND METHODSEffector Cells. Human peripheral blood lymphocytes

(PBLs) were isolated from defibrinated blood of healthydonors or from Mayo Clinic Blood Bank buffy coats byFicoll/Hypaque density gradient centrifugation. For NKcell-depleted populations, PBLs were first incubated for 30min at 40C with the anti-CD16 monoclonal antibody (mAb)3G8 (34). The cells (10 x 106 per ml) were washed and thenincubated for 30 min at 40C with magnetic beads that had beenprecoated with goat anti-mouse IgG (50 1Ld of beads per 106

Abbreviations: HSV, herpes simplex virus; HSV-1 gC and gD, HSVtype 1 glycoproteins C and D; mAb, monoclonal antibody; MHC,major histocompatibility complex; NK cells, natural killer cells;PBL, peripheral blood lymphocytes; C1R-B27, C1R-Aw68, andC1R-A3, HLA class I-transfected C1R cells bearing the HLA-B27,HLA-Aw68, and HLA-A3 specificity, respectively.

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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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cells; Advanced Magnetics, Cambridge, MA). The beadswere then magnetically removed. Fluorescence-activatedcell sorter (FACS) analysis after depletion showed that <1%of CD16+ cells remained. Clonal human NK cell lines wereisolated and passaged as described (35). All NK cell linesexpressed surface markers consistent with the phenotype ofactivated human NK cells: CD16+, CD3, CD56+, CD11a+,CDllb+, CD2+, CD4-, and HLA-DR+.

Target Cells. The HLA class I-deficient CiR cell lineprovided by Peter Cresswell (Yale University School ofMedicine, New Haven, CT) was derived from the humanplasma cell leukemia line LICR-LON-HMy2 (36) after -t-ir-radiation and selection with anti-class I mAb and complement(15). C1R expresses no HLA-A molecules on its surface, lowlevels ofHLA-B35, and normal levels ofHLA-Cw4 (37). Theisolation and characterization of the HLA class I-transfectedsublines (C1R-B27, C1R-A3, and ClR-Aw68) have beendescribed (15, 17, 19, 20). All cell lines were free of myco-plasma (mycoplasma T.C. detection kit; Gen-Probe, SanDiego).

Virus. HSV type 1, KOS strain, was provided by A. R.Hayward (University of Colorado Health Sciences Center,Denver) and was passaged in rabbit skin cells (RSC; Amer-ican Type Culture Collection). For HSV infection, cells (3 X106 per ml) were inoculated at a multiplicity ofinfection of5: 1in RPMI 1640 medium (GIBCO). After 2 hr, the cells werediluted to 6 x 105 cells per ml in RPMI 1640 supplementedwith 10% (vol/vol) fetal calf serum (HyClone) and incubatedat 370C for an additional 16 hr.

Cytotoxicity Assay. The 51Cr-release assay was performedas described (33, 35). Briefly, HSV-infected or uninfectedtarget cells were incubated for 60 min at 37°C with sodium[51Cr]chromate (50 p&Ci/106 cells; 1 ,uCi = 37 kBq) (Amer-sham). Labeled cells were washed, and 100-,ul aliquots (104cells per well) were pipetted into 96-well round-bottomplates. Non-MHC-matched effector cells (100 p1 per well)were added at the indicated effector: target ratios in triplicatewells. After 4 hr at 37°C, the plates were centrifuged, and the51Cr released into the supernatants was measured by Ycounting. Results are given as % specific cytotoxicity or aslytic units per 106 cells as described (35).Immunofluorecence. To quantitate cell-surface expression

ofHSV-1 glycoprotein C (HSV-1 gC), HSV-1 glycoprotein D(HSV-1 gD), or HLA class I antigen, aliquots of HSV-infected or uninfected cells were incubated with saturatingamounts (2 jig per 106 cells) of 19-S mAb (38) (provided byS. D. Showalter, Bio-Molecular Technology, Frederick,MD), 4-S mAb (38), or W6/32 mAb (39), respectively.Isotype-matched negative controls were included in all anal-yses. The cells were washed and then incubated for 30 min at4°C with fluorescein-conjugated goat anti-mouse IgG (BectonDickinson Monoclonal Center). The cells were then washedand paraformaldehyde-fixed, and 10,000 cells from eachsample were analyzed by flow cytometry.Conjugate Analysis. Formation of effector-target conju-

gates was quantified by flow cytometry as described (35).Briefly, NK effectors were labeled intracellularly for 1 hr at37°C with 100 ,uM sulfofluorescein diacetate (MolecularProbes), and the target cells were labeled intracellularly for1 hr at 37°C with 40 ug of hydroethidine (Polysciences) perml. The cells were then washed and resuspended at 5 x 106cells per ml. The effectors and targets (25 p1 of each) weremixed, pelleted, and then incubated at 37°C for 20 min. Thepellet was resuspended and transferred into 1 ml of ice-coldmedium. Conjugate formation (simultaneous green and redfluorescence) was quantitated by flow cytometry. Resultswere expressed as the percentage of total NK cells thatformed conjugates.

RESULTS

Susceptibility of Class I-Deficient C1R Cells and HLA ClassI-Transfected CIR Sublies to NK C-Mediated AntiviralJInnunity. To evaluate whether an interaction between MHCand virus might critically influence the susceptibility ofinfected cells to NK cell-mediated lysis, we infected classI-deficient CiR cells and HLA class I-transfected CiR sub-lines with HSV-1 for 18 hr and then tested these targets in astandard 5lCr-release cytotoxicity assay. Using freshly iso-lated PBLs as effectors, we found that HSV-infected CiRcells were no more sensitive to lysis than were uninfectedCiR cells (Fig. 1A). In contrast, HSV infection ofHLA classI-transfectants significantly increased their susceptibility tokilling (Fig. 1 B and C). Of note, the degree to which theseHSV-infected, HLA class I-transfectants were killed wassimilar to that of uninfected, class I-deficient CiR cells.These results suggest that not only does MHC class Iinfluence susceptibility to antiviral immunity, but virus-infected, MHC class I-bearing cells can have similar sensi-tivity to lysis as uninfected, class I-deficient targets.To determine whether the killing measured in these exper-

iments was due to NK cells, PBLs were selectively immu-nodepleted and then used as effectors in the 51Cr-releaseassay. Depletion of CD16+-bearing cells completely elimi-nated killing ofall four target types (Fig. 2). In contrast, mockdepletion with an irrelevant monoclonal antibody (Fig. 2) orT-cell depletion with anti-CD3 (data not shown) did notreduce the killing of these targets. These experiments dem-onstrate that CD16+ CD3 NK cells are the active effectorsin these PBL populations.

Influence of MHC on Antiviral Cytotoit Meed byCloned NK Cell Lines. Although the preceding experimentsindicated that CD16+ NK cells were required for the ob-

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FIG. 1. PBL lysis of HSV-infected or uninfected target cells.PBLs were incubated for 4 hr with either HSV-infected (closedsymbols) or uninfected (open symbols) 51Cr-labeled target cells. (A)HLA class I-deficient COR cells. (B) C1R-B27 cells (bearing HLA-B27). (C) C1R-Aw68 cells (bearing HLA-Aw68). (D) C1R-B27 cellsinoculated with live HSV (U), UV-inactivated HSV (A), or medium(o) for 16 hr prior to testing in the 51Cr-release assay. Each percent-age is the mean of triplicate wells. All SD were <10%. These resultsare representative of eight experiments done with 10 different PBLdonors.

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* ClR0 C1R +HSVo B27

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FIG. 2. CD16-depleted PBLs are unable to kill HSV-infected or

uninfected targets. Unfractionated PBL, CD16-depleted PBL (3G8mAb + goat anti-mouse IgG-coated magnetic beads), or mock-depleted PBL (irrelevant murine IgG myeloma protein + goatanti-mouse IgG-coated magnetic beads) were incubated for 4 hr withthe indicated 51Cr-labeled targets. Cytotoxicity is expressed as lyticunits per 106 cells. B27, C1R-B27 cells.

served killing, they did not address whether NK cells alonecould mediate the preferential killing of the HSV-infected,class I+ targets. Previous reports indicated that, dependingon the cell type infected by virus, NK cells have twoalternative mechanisms of antiviral cytotoxicity (33, 40-42).One is dependent on interferon production by accessory

cells, whereas the other does not require any accessory cellsor factors. Using homogeneous cloned populations ofCD16+/CD3- NK cells, we found the same pattern of killing(Fig. 3) as was seen for PBL effectors (Fig. 1)-i.e., whereasNK cells were unable to preferentially lyse HSV-infectedclass I-deficient C1R cells, HSV infection of class I-trans-fected sublines increased their sensitivity to NK cell-mediated killing. These results suggest that accessory cellsare not required for the antiviral killing measured in thisexperimental system and that the differential effects ofMHCon this process directly occur at the level of the NKcell-target cell interaction.Use of NK clones also permitted us to evaluate whether

CD8 expression on NK cells is required for the MHC-dependent killing of HSV-infected cells. Since 30-50%o ofCD16+ NK cells coexpress CD8 (43) and since CD8 can bindto nonpolymorphic portions ofMHC class I molecules (44),CD8 could potentially influence the interaction of NK cellswithMHC class I-bearing targets. However, CD8+ and CD8-NK cell lines were both able to preferentially lyse HSV-infected, class I-bearing targets (Table 1). In addition, CD8+NK cells preferentially lysed C1R-Aw68 cells, which bearHLA-Aw68 (Fig. 3C), in spite of the inability of CD8 to bindHLA-Aw68 (44). Thus, the NK cell recognition and lysis ofvirus-infected class I-bearing C1R sublines were independentof CD8 expression on NK cells.

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FIG. 3. Lysis of HSV-infected or uninfected targets by clonedNK cells. Cloned CD16+/CD8+/CD3- NK cells (CJ7) were incu-bated for 4 hr with either HSV-infected (closed symbols) or unin-fected (open symbols) target cells. (A) Class I-deficient CiR cells. (B)C1R-B27 cells. (C) C1R-Aw68 cells. (D) C1R-A3 cells. Each per-centage is the mean of triplicate wells. All SD were <10o. Theseresults are representative of eight experiments with eight differentNK cell lines.

Increased Susceptibility ofHSV-Infected, Class I+ Targets toNK Cell Lysis Is Not Due to Differences in Infection Rate or Dueto Enhanced Effector-Target Conjugate Formation. AlthoughMHC molecules can affect the binding of certain viruses(e.g., cytomegalovirus) to uninfected cells, similar processeshave not been described for HSV. To be sure that comparablenumbers of class I-deficient C1R cells vs. C1R-B27 cells wereinfected with HSV, immunofluorescent analysis ofHSV-1 gCand HSV-1 gD expression was performed. After inoculationwith HSV, similar proportions of C1R and C1R-B27 becameinfected (Fig. 4 Top and Middle). Furthermore, these targetsexpressed comparable levels of HSV-1 gC (Fig. 4 Top andMiddle) and HSV-1 gD (data not shown). We then separatelyanalyzed whether enhanced susceptibility to killing was dueto increased effector-target conjugate formation. While therewere clonal differences in conjugate formation (ranging from3.4% to 21.9o), in no case did NK cells form conjugates withHSV-infected cells more efficiently than with uninfectedtargets (Table 2). This analysis supports the notion thatalthough conjugate formation is required for NK cell-

Table 1. Preferential lysis of HSV-infected C1R-B27 targets is independent of CD8 expression onNK cells

NK % specific cytotoxicitycell line CD8 C1R C1R + HSV C1R-B27 C1R-B27 + HSV

CH9 + 38.5 (3.9) 38.3 (0.2) 21.6 (1.7) 32.3 (2.0)CJ6 + 40.5 (1.4) 48.7 (3.0) 26.4 (8.1) 43.9 (1.0)CL3 - 39.0 (5.5) 36.3 (2.4) 8.9 (1.9) 22.2 (5.4)CL9 - 32.7 (3.3) 42.4 (2.4) 12.9 (1.5) 41.0 (0.5)Clonal CD16+ CD3- NK cells were incubated for 4 hr at 370C with the indicated 51Cr-labeled target

cells. Percent specific cytotoxicity was measured at a 1: 1 effector: target ratio. Each percentage is themean of triplicate wells, with the SD given in parentheses. All four clones more effectively lysedHSV-infected C1R-B27 than uninfected C1R-B27 (P < 0.05).

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FIG. 4. Surface expression of HSV-specific proteins and HLAclass I antigens on target cells. Target cells were stained by indirectimmunofluorescence and analyzed by flow cytometry. (A) HSV-infected (solid line) or uninfected (dashed line) C1R cells werestained with anti-HSV-1 gC mAb. (B) HSV-infected (solid line) oruninfected (dashed line) C1R-B27 cells were stained with anti-HSV-1gC mAb. (C) HSV-infected C1R-B27 cells (dashed line), uninfectedC1R-B27 cells (solid line), or uninfected C1R cells (dotted line) werestained with anti-HLA class I mAb.

mediated killing of virus-infected cells, differences in degreedo not account for their preferential lysis.HSV Infection Increases Target Cell Susceptibility to NK

Cell-Mediated Lysis Without Down Modulation of Class I.Although inoculation of HLA class 1-transfected C1R cellswith live HSV increased their susceptibility to NK cell-mediated lysis, inoculation with UV-inactivated HSV had nosignificant effect (Fig. 1D). This suggested that viral replica-tion was required for the observed effects. Since the repli-cation of certain viruses (e.g., cytomegalovirus and adeno-virus) has been associated with down-modulation in class Iexpression (26, 27), we investigated whether similar eventsoccurred during HSV infection of C1R class I transfectants.

Table 2. Conjugate formation between cloned NK cells andeither uninfected or HSV-infected C1R-B27 target cells

% NK cells formingconjugates

NK C1R-B27cell line CD8 C1R-B27 + HSV

CG4 + 5.9 3.4CJ6 + 5.0 4.9CK1 - 5.1 2.3CL10 - 21.9 17.6

CD8+ orCD8- NK cell lines were incubated with either uninfectedor HSV-infected C1R-B27 target cells. Conjugate formation wasmeasured by two-color flow cytometry as described in text.

This was especially important since decreases in MHC classI expression might also account for their increased suscep-tibility to lysis. FACS analysis of MHC class I expressionwas performed 18 hr after HSV infection to coincide with thetime ofthe cytotoxicity assay. This analysis indicated that theHLA class I transfectants, such as C1R-B27, expressedapproximately equal levels ofclass I antigens before and afterHSV infection (Fig. 4 Bottom). Thus, although HSV infectionof C1R-B27 cells resulted in increased sensitivity to NKcell-mediated lysis, the level of MHC class I expression ontheir surface remained unchanged. These results suggest thatqualitative, rather than quantitative, changes in MHC ex-pression on HSV-infected C1R transfectants altered theirsusceptibility to NK cell-mediated lysis.

DISCUSSIONMajor advances in our understanding of how T lymphocytescontrol viral infections resulted from the identification oftarget structures on infected cells. Specifically, it becameclear that T-cell antigen receptors were restricted to recog-nizing viral peptides presented by cell surface MHC mole-cules. Although NK cells also participate in antiviral immu-nity, it has remained unclear as to what target structures onvirus-infected cells they recognize. NK cells can preferen-tially lyse virally infected cells from different individuals,indicating that this process is not restricted to target cellsbearing "self' MHC. This observation suggests a departurefrom the T-cell paradigm in which T-cell antigen receptorscan only recognize foreign peptides on "self' MHC. How-ever, the fact that NK cells can kill infected, allogeneictargets in no way excludes a role for MHC in the recognitionprocess. To address this question, one ideally would like touse an experimental system in which direct comparisons canbe made with targets differing only in MHC expression. Wereport here that human NK cells are unable to preferentiallykill HSV-infected C1R cells deficient in MHC class I expres-sion. In contrast, HSV infection ofC1R class I-transfectantsincreases their susceptibility to NK cell-mediated cytotox-icity. These results suggest a role for MHC class I inregulating NK cell-mediated killing of virus-infected cells.

Studies on NK cell rejection of allogeneic bone marrowand lymphomas first raised the notion that MHC or MHC-linked gene products might influence the cytotoxic process(8-12, 45-47). These studies have been extended to suggestthat quantitative (8-23) or qualitative (19, 20, 24, 25) changesin MHC expression on tumor cells can alter their suscepti-bility to NK cell-mediated lysis. According to the "missingself' hypothesis, NK cells kill certain targets because theyfail to express adequate levels ofMHC class I gene products(8). Furthermore, even cells that express adequate levels ofcell surface MHC class I can become susceptible to lysis ifthey either bear "foreign" peptides in their antigen-bindingcleft (24, 25), are pulsed with "foreign" antigen (48), orexpress certain alloantigens (49, 50). However, it remainedunclear whether virus replication during acute infectionmight have similar MHC-dependent effects on the cytotoxicprocess. To address this question, we used targets differingin MHC class I expression. Using this model, we demon-strated that only the targets expressing MHC class I hadincreased susceptibility to killing after HSV infection. Ofnote, HSV-infected targets bearing different MHC class Imolecules (i.e., HLA-B27, HLA-Aw68, or HLA-A3) couldall be preferentially lysed by NK cells derived from a singledonor.These experimental results would be consistent with a

model in which the association between endogenously syn-thesized viral peptides and the MHC enhances susceptibilityto NK cell-mediated lysis. As extensions of previously de-scribed models (8), this could occur if viral peptide interac-

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tions with the MHC either unmasked additional target struc-tures or eliminated the negative regulatory influence of selfpeptide-MHC complexes. This model would provide anexplanation for two recent experimental observations: (i)expression of nonstructural, immediate-early genes is suffi-cient for induction ofNK cell-mediated lysis ofHSV-infectedfibroblasts (51); and (ii) preferential lysis of HSV-infectedcells can occur in the absence of altered levels of MHC classI expression (28). In each case, an interaction of a viralpeptide, whether structural or regulatory, with the antigen-binding groove of a class I molecule could directly modulatethe cytotoxic process.Although this report focuses on MHC-dependent changes

in susceptibility to NK cell-mediated killing of virus-infectedcells, we consider it unlikely that this is the sole mechanisminfluencing this process. Clearly, multiple factors influencethe control of virus infections by NK cells (5, 6); therefore,we support the "multiple choice" model (8, 10) in which,among the different regulatory influences, MHC is only one.This raises the possibility that in some systems MHC mayplay the predominant role, and in others additional factorswill critically influence the outcome. For example, the ex-perimental system here focuses on rapid interferon-independent mechanisms of target-cell lysis. Additional anal-yses will be required to determine its relevance to NKcell-mediated antiviral immunity that requires additional ac-cessory cells and cytokine release.Our results also focus attention on trying to identify NK

receptors that have the capacity to differentially bind either"self' peptide-MHC complexes or viral peptide-MHC com-plexes. Although CD8 can bind toMHC class I molecules, wedemonstrate here that both CD8+ and CD8- NK cells pref-erentially kill HSV-infected class I-bearing targets. In addi-tion, HLA class I transfection coding for HLA-Aw68 alone,which does not bind to CD8 (44), confers susceptibility toantiviral lysis. Therefore, different receptors with MHCbinding properties will need to be evaluated for their potentialinvolvement in NK cell-mediated antiviral immunity. Ourresults showing similar conjugate formation between NKcells and infected vs. noninfected targets also suggest that thecritical receptor-ligand interaction may involve signal-generating systems rather than purely adhesive interactions.Together, our results indicate a role for the MHC in regu-lating NK cell-mediated killing of virus-infected cells andprovide new directions for the evaluation of factors influenc-ing the control of virus infection.

We thank Peter Cresswell for generously providing the C1Rsublines, Carlos V. Paya and Larry R. Pease for their helpful critiqueof these studies, and Theresa Lee for her excellent assistance withthe preparation of this manuscript. This research was supported bythe Mayo Foundation and by National Institutes of Health GrantCA47752.

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