Differential effects of US2, US6 and US11 human cytomegalovirus proteins on HLA class Ia and HLA-E...

Differential effects of US2, US6 and US11 humancytomegalovirus proteins on HLA class Ia andHLA-E expression: impact on target susceptibilityto NK cell subsets

Manuel Llano2, Monica Guma1, Mara Ortega2, Ana Angulo3 and Miguel Lopez-Botet1

1 Molecular Immunopathology Unit, DCEXS, Universitat Pompeu Fabra, Barcelona, Spain2 Servicio de Inmunologıa, Hospital Universitario de la Princesa, Madrid, Spain3 IDIBAPS, Barcelona, Spain

We compared in an inducible expression system the individual effect of US2, US6 and US11human cytomegalovirus (HCMV) proteins on HLA-E and HLA class Ia surface expression,assessing in parallel their influence on target susceptibility to NK cell clones. To this end, theRPMI 8866 B lymphoma cell line (HLA-A2, HLA-A3, HLA-B7, HLA-Cw7, HLA-ER, HLA-EG)was stably cotransfected with the ecdysone receptor, together with the US sequences underthe control of an ecdysone-inducible promoter. Biosynthesis of viral proteins was turned onby incubating transfectants with Ponasterone A. US6 down-regulated expression of allclass I molecules, hampering target resistance to NK cell clones controlled by the CD94/NKG2A, KIR2DL2 and/or CD85j (ILT2 or LIR-1) inhibitory receptors. By contrast, US11reduced the surface levels of class Ia molecules but preserved HLA-E; this rendered US11+

cells sensitive to NK clones under the control of KIR2DL2 and/or CD85j, while their resis-tance to CD94/NKG2A+KIR2DL2– effector cells was maintained. US2 preserved as well HLA-E expression but selectively targeted class Ia molecules; in fact, HLA-A and HLA-C allotypeswere down-modulated whereas HLA-B7 remained unaltered. US2+ targets became sensitiveto KIR2DL2+ cells but remained resistant to CD94/NKG2A+CD85j+ NK clones. The differentialeffects of US proteins on HLA class Ia and HLA-E likely reflect the evolutionary adaptation ofHCMV to counteract NK-mediated surveillance.

Key words: Cytomegalovirus / HLA / HLA-E / NK / Cytotoxicity

Received 16/5/03Revised 18/7/03Accepted 5/8/03

[DOI 10.1002/eji.200324182]

Abbreviations: HCMV: Human cytomegalovirus Pon A:Ponasterone A Luc: Luciferase TAP: Transporter associatedwith antigen processing ER: Endoplasmic reticulum

1 Introduction

Effective defense against cytomegalovirus (CMV) infec-tions involves both innate and adaptive immuneresponses [1, 2]. Reciprocally, these herpesviruses haveadopted along evolution a variety of strategies to evadeimmune surveillance [3, 4]. Several human CMV (HCMV)proteins down-modulate MHC class I expression,impairing antigen presentation to T lymphocytes. Amongthem, US6 binds to transporter associated with antigenprocessing (TAP) hampering peptide transport to theendoplasmic reticulum (ER) [5, 6]; US2 and US11 trans-locate class I heavy chains from the ER to the cytosol [7,8]; finally, US3 is an immediate-early protein that retains

class I molecules at the ER [9]. These viral proteins pre-dictably render infected cells vulnerable to NK cells,which are repressed by inhibitory receptors specific forMHC class I molecules [10]; hence, CMV presumablyuses other mechanisms to escape from NK cell control.

NK cells play an important role in the innate immuneresponse to CMV, discriminating infected cells by meansof activating and inhibitory receptors. NKG2D [11, 12]and the murine Ly49H [13, 14] lectin-like moleculesrespectively recognize stress-inducible molecules andthe m157 murine CMV (MCMV) glycoprotein. On theother hand, an NK cell response is promoted when inhib-itory receptors lose cognate interaction with MHC class Imolecules, down-regulated in CMV-infected cells [4].Members of the murine Ly49 and human KIR receptorfamilies, CD85j [Ig-like transcript (ILT)2 or leukocyte Ig-like receptor (LIR)-1], as well as the CD94/NKG2A inhibi-tory killer lectin-like receptor (KLR) fulfil this surveillancerole [10, 15–17].

2744 M. Llano et al. Eur. J. Immunol. 2003. 33: 2744–2754

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

Fig. 1. Pon A-dependent induction of US6 and US11expression in transfected RPMI 8866 cells. RPMI 8866 cellswere stably transfected to co-express the ecdysone recep-tor together with either the US6 or US11 HCMV proteins.Transfectants incubated with Pon A were harvested at differ-ent time points, lysed and analyzed by Western blotting withspecific antisera for the US proteins or an anti-tubulin anti-body as a loading control.

Two different strategies potentially employed by CMV tosubvert NK cell responses involve inhibitory receptors.First, viral MHC class I surrogates, such as the HCMVUL18 and MCMV m144 glycoproteins, constitute putativedecoy molecules that might limit the NK response againstinfected cells by engaging MHC class I-specific inhibitoryreceptors [18–20]; UL18 binds to CD85j [18] whereas aninhibitory receptor for m144 has not yet been identified.Another mechanism potentially employed by HCMV toevade NK cell responses targets the CD94/NKG2A inhibi-tory receptor, conserved in rodents and primates [10, 21].Human CD94/NKG2A interacts with HLA-E [22, 23],which presents in a TAP-dependent manner hydrophobicpeptides derived from the leader sequences of a subsetof class I molecules [24, 25].Thus, detection of HLA-E bythe CD94/NKG2A receptors represents a sensor mecha-nism whereby the status of the HLA class I biosynthesisis broadly probed. A peptide derived from the leadersequence of the UL40 HCMV protein was shown to bepresented by HLA-E, inhibiting cytotoxicity of a CD94/NKG2A+ cell line [26, 27]. Binding of HLA-E to the UL40-derived nonamer appeared to be TAP-independent andinsensitive to US6 HCMV protein, which converselyinhibited the constitutive expression of HLA-E [26]. On anindirect basis, UL40-dependent HLA-E expression wassuggested to be resistant to US2, US3, and US11.

We have used an inducible expression system to directlystudy the individual effect of US6, US2 and US11 viralproteins on endogenous HLA-E surface expression, ascompared to class Ia molecules. In addition, the impacton NK cell response against US-transfected target cellswas also assessed. Our results lend further support tothe hypothesis that the selective action of HCMV pro-teins on subsets of HLA class I molecules reflects theevolutionary pressure to escape from NK cell-mediatedsurveillance.

2 Results

2.1 Effects of US11 and US6 on HLAclass I expression and susceptibility toNK-mediated cytotoxicity

To evaluate the effect of US6 and US11 on endogenousHLA class I expression, we stably expressed the viralproteins in the RPMI 8866 B cell line under the transcrip-tional control of an ecdysone-inducible promoter; a lucif-erase (Luc)-expressing transfectant was generated as acontrol. The RPMI 8866 cell line was selected as a targetbased on preliminary experiments, because it constitu-tively expressed surface HLA class Ia (HLA-A2, HLA-A3,HLA-B7, HLA-Cw7) as well as HLA-E molecules at levelsthat conferred partial resistance against NK cell clones

bearing inhibitory receptors for HLA-E (CD94/NKG2A),HLA-C (KIR2DL2) or class I molecules (CD85j) (notshown). The expression of US6 and US11 was detectedby Western blotting with specific antisera in cellularlysates of the corresponding transfectants treated withPonasterone A (Pon A), but not under basal conditions(Fig. 1). Pon A-mediated induction of Luc was analyzedwith a luminometer (data not shown).

US11 mediates export of heavy chain class I moleculesfrom the ER to the cytoplasm of CMV-infected cells,whereas US6 interferes with TAP-dependent transport ofantigenic peptides to the ER [4]. At different time pointsbetween 24 and 72 h after induction with Pon A, US+ andLuc+ transfectants were analyzed by flow cytometry forsurface HLA class I expression. Indirect immunofluores-cence staining was carried out with the HP-1F7 mAbreactive with all class I molecules, and with antibodiesspecific for HLA-A, HLA-B, HLA-C and HLA-E; CD19expression was analyzed as a control. Pon A treatmentdid not modify in Luc+ cells the levels of either CD19 orHLA class I (not shown). By contrast, induction of US11+

and US6+ cells selectively reduced class I expression,without altering CD19 (Fig. 2). In both cases, down-regulation of HLA class I was observed by staining withthe HP-1F7 mAb, which reacts with all class I molecules,as well as with antibodies specific for HLA-A, HLA-B orHLA-C molecules. The effects were perceived at 24 h(Fig. 2), and progressively increased at 48 (not shown)and 72 h (Fig. 2). In contrast to US6, which down-modulated all class I molecules, US11 did not reduceHLA-E expression even at 72 h post-induction.

Considering the different actions of US6 and US11 CMVproteins on HLA class I, we analyzed their impact

Eur. J. Immunol. 2003. 33: 2744–2754 HCMV and NK recognition of class I molecules 2745


Fig. 2. Expression of HLA class Ia and HLA-E in US6+ andUS11+ RPMI 8866 cells. US6+ (A) and US11+ (B) RPMI 8866transfectants were cultured in the absence (solid line) orpresence of Pon A for 24 h (dotted line) or 72 h (dashed line).Subsequently, cells were stained by indirect immunofluores-cence and analyzed by flow cytometry with antibodies spe-cific for HLA-A, HLA-B, HLA-C, HLA-E, HLA class I andCD19. Background staining is included for comparison(black histogram). Variations in the expression of the differ-ent HLA molecules are shown to illustrate the impact of theUS proteins along time.

Fig. 3. Susceptibility of US6+ and US11+ cells to KIR2DL2+ orCD94/NKG2A+ NK clones. Luc+, US6+ and US11+

RPMI 8866 transfectants were incubated with Pon A for72 h and used as targets in a 51Cr-release assay. NK cloneswere selected according to their surface phenotype (CD94/NKG2A+KIR2DL2– or KIR2DL2+CD94/NKG2A–) and low abil-ity to spontaneously kill RPMI 8866 cells. In every case, theeffects of anti-CD94/NKG2A, anti-KIR2DL2 or anti-HLAclass I mAb were compared to an anti-CD56 mAb as a con-trol. Data are representative of clones that displayed compa-rable response patterns.

on target susceptibility to NK lysis. To this end, weselected a group of CD94/NKG2A+KIR2DL2–CD85J– andKIR2DL2+CD94/NKG2A–CD85J– NK clones that dis-played low levels of spontaneous cytotoxicity againstRPMI 8866 cells. Both types of effector cells were testedin 51Cr-release assays against the US11+ and US6+

RPMI 8866 transfectants, cultured in the absence orpresence of Pon A for 72 h; Pon A-treated Luc+ cellswere included as a control. To estimate the level of pro-tection conferred by the interaction of inhibitory recep-tors and class I molecules, the antagonistic effects ofanti-HLA class I (HP-1F7), CD94/NKG2A (Z199), orKIR2DL2 (GL183) mAb were tested in parallel and com-pared to a control mAb (CD56).

Induction of US6+ cells with Pon A rendered them sensi-tive to lysis by both CD94/NKG2A+KIR2DL– andKIR2DL2+CD94/NKG2A– NK clones (Fig. 3), consistentwith the observed down-regulation of all HLA class Imolecules; cytotoxicity levels were comparable to thoseattained in the presence of mAb specific for either HLA

class I molecules or the corresponding inhibitory recep-tors. By contrast, US11+ cells became sensitive to lysisby KIR2DL2+ clones but maintained their resistance toCD94/NKG2A+ cells, which was reversed by anti-HLAclass I or CD94/NKG2A mAb (Fig. 3). In time courseexperiments, the increased susceptibility to lysis wasalready detected at 24 h post-induction (data notshown). These results supported that persistent HLA-Eexpression was sufficient to confer protection againstCD94/NKG2A+ NK cell subsets.

As NK cells may display variable combinations of inhibi-tory receptors, we analyzed the susceptibility of US6+

and US11+ cells to NK clones that co-expressed CD94/NKG2A together with KIR2DL2. Despite that they dis-played CD94/NKG2A, these effectors efficiently killedboth US6+ and US11+ targets after treatment with Pon A(Fig. 4). Moreover, only anti-KIR2DL2 but not anti-CD94/NKG2A mAb induced lysis of transfectants under basalconditions, further supporting that KIR2DL2 acted as thedominant inhibitory receptor in these NK clones. In the



Fig. 4. NK clones co-expressing KIR2DL2 and CD94/NKG2A inhibitory receptors can efficiently lyse US6+ and US11+ cells. US6+

and US11+ RPMI 8866 transfectants were incubated with Pon A for 72 h and tested in a 51Cr-release assay as targets for NKclones that displayed both CD94/NKG2A and KIR2DL2 inhibitory receptors. The effects of mAb specific for CD94/NKG2A and/orKIR2DL2 were compared to an anti-HLA class I mAb; anti-CD56 mAb was included as a negative control.

Fig. 5. Susceptibility of US6+ and US11+ cells to NK cells co-expressing the CD94/NKG2A and CD85j inhibitory receptors. US6+

and US11+ RPMI 8866 transfectants were incubated with Pon A for 72 h and tested in a 51Cr-release assay as targets for differentNK cells that displayed both CD94/NKG2A and CD85j inhibitory receptors. The antagonistic effects of mAb specific for CD94/NKG2A and/or CD85j were compared to those mediated by an anti-HLA class I mAb; a CD56-specific mAb was included as acontrol. Results are representative of the different patterns of response observed.

same line, we also studied cells co-expressing CD94/NKG2A and the CD85j inhibitory receptor, which broadlyinteracts with all class I molecules. The inhibitory effectof CD85j prevailed over CD94/NKG2A in the NKL cell line(Fig. 5) whereas CD94/NKG2A dominated in otherclones (Fig. 5, clone MLV56). These data indicated thatthe persistence of HLA-E expression did not confer pro-tection against every CD94/NKG2A+ NK cell. The molec-ular basis underlying the dominant role for a given inhibi-tory receptor at the clonal level is uncertain, and did notappear related to different surface expression levels.Attempting to solve this intriguing issue would require anextensive biochemical and functional analysis of NKclones.

2.2 US2 preserves HLA-E and HLA-B7expression maintaining target resistance toCD94/NKG2A+CD85j+ NK cells

Similarly to US11, US2 translocates class I moleculesfrom the ER to the cytoplasm; yet both proteins utilizedifferent targeting mechanisms for class I degradation[28]. RPMI 8866 cells were transfected with US2 asdescribed above; HLA expression was tested afterinduction of US2 expression with Pon A. As shown inFig. 6, US2+ cells displayed a decrease of HLA-A andHLA-C levels, whereas neither HLA-E nor HLA-B7 werealtered even at 72 h post-induction. Persistence of HLA-B explained the modest impact of US2 on total class I



Fig. 6. Expression of HLA class Ia and HLA-E in US2+

RPMI 8866 cells. US2+ RPMI 8866 transfectants were cul-tured in the absence (solid line) or presence of Pon A for48 h (dotted line) or 72 h (dashed line). Subsequently, cellswere stained by indirect immunofluorescence and analyzedby flow cytometry with antibodies specific for HLA-A, HLA-B, HLA-C, HLA-E, HLA class I and CD19. Background stain-ing is included for comparison (black histogram).

Fig. 7. US2 confers target susceptibility to KIR2DL2+ NKcells. US2+ RPMI 8866 cells were incubated with Pon A for72 h and tested in a 51Cr-release assay as targets for NKclones that displayed the KIR2DL2+CD94/NKG2A– pheno-type. The effects of an mAb specific for KIR2DL2 were com-pared to anti-HLA class I and CD56 mAb (negative control).Data corresponding to representative NK clones are dis-played.

Fig. 8. Engagement of CD94/NKG2A and CD85j inhibitory receptors contributes to maintain resistance of US2+ targets againstNK cells. US2-transfected RPMI 8866 cells were incubated with Pon A for 72 h and tested in a 51Cr-release assay as targets fordifferent NK cells that displayed both CD94/NKG2A and CD85j inhibitory receptors. The effects of mAb specific for CD94/NKG2A and/or CD85j were compared to anti-HLA class I and CD56 mAb (negative control). Results are representative of the pat-terns of response observed in different clones.

expression, as detected with the HP-1F7 mAb. Theseresults revealed important differences between theaction of US2 and US11 on the expression of class Iamolecules.

As shown in Fig. 7, Pon A-treated US2+ cells becamesensitive to KIR2DL2+CD94/NKG2A–CD85j– NK clones,consistent with down-regulation of HLA-C. By contrast,the US2+ transfectant remained partially resistant toCD94/NKG2A+CD85j+ NK cells (Fig. 8). As mentionedabove, CD85j broadly interacts with all class I moleculesand is the only defined inhibitory receptor that directly

recognizes Bw6+ HLA-B allotypes (i.e. HLA-B7). In theseexperiments, cytotoxicity was induced by antibodiesagainst HLA class molecules (HP-1F7) or by a combina-tion of anti-CD94/NKG2A and anti-CD85j mAb, thussupporting that both ligands (i.e. HLA-E and HLA-B7)contributed to target protection. As noticed above,CD85j appeared to be dominant in some effectors (i.e.NKL cells; Fig. 8), whereas the inhibitory action of CD94/NKG2A prevailed in other clones (i.e. MLV24; Fig. 8).



3 Discussion

In the present study we provide data supporting that thedifferential actions of US HCMV proteins on HLA class Iaand HLA-E molecules determine a variable degree of tar-get susceptibility to NK cell subsets bearing distinctinhibitory receptors. The possibility that HCMV mightpreserve HLA-E to evade NK cell responses was basedon observations pointing out that a nonamer derivedfrom the leader sequence of the UL40 HCMV proteincould replace the HLA class I-derived endogenous pep-tides that normally stabilize surface HLA-E [26, 27].Thus, synthesis of UL40 during viral infection could indi-rectly prevent activation of CD94/NKG2A+ effector cells,provided that HLA-E molecules were resistant to theeffect of the HCMV proteins which down-regulate HLAclass I molecules. Although HLA-E expression was origi-nally reported to be TAP- and tapasin-dependent [24,25], Tomasec et al. [26] observed that HLA-E presenta-tion of the UL40-derived nonamer was TAP-independentand refractory to the US6 HCMV protein. Moreover, asexpression of HLA-E was detected in HCMV-infectedcells, it was indirectly assumed that the class Ib mole-cule should be also resistant to US3, US11 and US2 mol-ecules.

In the present study we directly confirmed this latterhypothesis for US2 and US11, by assessing the individ-ual effect of HCMV proteins on endogenous HLA class Iexpression of US-transfected RPMI 8866 cells and onthe NK-mediated response against these targets. Induc-tion of US2 and US11 preserved surface HLA-E expres-sion while reducing HLA-C. Thus, under the influence ofUS11, RPMI 8866 cells became sensitive to KIR2DL2+

NK clones but remained resistant to CD94/NKG2A+KIR2DL2– clones. Remarkably, preservation ofHLA-E expression was not protective against someCD94/NKG2A+KIR2DL2+ clones, in which the action ofthe receptor specific for HLA-Cw7 appeared dominant. Itis of note that HLA-E levels under the threshold requiredto effectively engage CD94/NKG2A may be relevant forTCR-mediated recognition by CD8+ T cells [29, 30].

US11 down-modulated HLA-A, HLA-B and HLA-C,whereas US2 selectively targeted class Ia moleculespreserving HLA-B7 expression. Our observations are inline with other studies showing that the US2 proteinbound to HLA-A but to neither HLA-E, HLA-B7 nor HLA-B27 molecules [31]; according to this work, US2 did noteither interact with HLA-Cw4. In our hands, endogenousHLA-Cw7 expression was found to be down-regulated inUS2+ cells by two different criteria: direct immunofluo-rescence staining and loss of resistance to KIR2DL2+

clones. Thus, the possibility that US2 may also selec-tively target some HLA-C allotypes should be envisaged.

In the same line, a variable susceptibility of H2 class Iamolecules to US2 has been reported [7].

In agreement with our data, Huard and Früh [32]described that US11+ targets became sensitive toKIR2DL+ NK cells, indirectly supporting that HLA-Cexpression was reduced; by contrast, HLA-C appearedresistant to US2 and US11 in a trophoblast cell line [33].The basis for this discrepancy is uncertain, though pre-sumably related to the distinct experimental systemsemployed. Differences between the action of US2 andUS11 have been described at the molecular level [34],and further studies are required to determine whichstructural features render HLA-E refractory to the viralproteins. US2 and US11 alter HLA class Ia expressionacting at a post-translational level; thus these viral pro-teins are not expected to influence the input of class Ialeader sequence-derived nonamers that, together withvirus-derived peptides (i.e. UL40), may contribute to sta-bilize surface HLA-E expression in HCMV-infected cells.

According to our results, US6 down-modulated theexpression of all class I molecules, and hampered targetresistance to NK cells under the control of CD94/NKG2Aor KIR2DL2. In agreement with Tomasec et al. [26], weobserved that US6 inhibited endogenous HLA-E, consis-tent with TAP-dependent expression of the class Ib mol-ecule. These authors also reported that HLA-E presenta-tion of an UL40-derived peptide was TAP-independentand refractory to the US6 protein; this fact, together withthe observed resistance of HLA-E to US11 and US2,may contribute to maintain cell surface expression of theclass Ib molecule during HCMV infection [26]. It is ofnote that HLA-EG has been shown by Ulbrecht et al. tobe insensitive to US6 in transfected K562 cells [35], andother differences between the HLA-E alleles have beenrecently reported by Strong et al. [36]. As the RPMI 8866cell line is heterozygous (i.e. HLA-EG/HLA-ER), our exper-imental system did not enable to discriminate whetherUS6 differentially affected both alleles. In case constitu-tive expression of HLA-EG is confirmed to be resistant toUS6, surface levels of the class Ib molecule should bemaintained in HCMV-infected cells from HLA-EG homo-zygous individuals and thus the HLA-E haplotype mightinfluence the NK response to HCMV.

An interesting feature of our approach to dissect the indi-vidual role played by HCMV proteins is that it enables tofollow up their effects on endogenous class I moleculesand target sensitivity to NK-mediated lysis in the samecells. Certainly, the system does not provide informationon how these and other concomitant events are dynami-cally integrated along the course of viral infection. In thisregard, a recent report [37] analyzed the NK responseagainst fibroblasts infected with HCMV mutants lacking



the UL40 gene and/or US2-US11 region; the impact ofthese gene deletions on HLA class I expression and theresponse of two tumor NK cell lines was studied. In con-trast to the work of Tomasec et al. [26], the authors con-cluded that UL40 was insufficient to preserve HLA-Eexpression and resistance against the CD94/NKG2A+

NKL cell line. Moreover, they proposed that the US2-US11 region entirely governed the loss of HLA class Iaand HLA-E molecules, conferring susceptibility to bothCD94/NKG2A+ and KIR2DL2+ NK cell lines.

Though this study apparently questions that surfaceHLA-E may be effectively maintained along HCMV infec-tion to evade the NK response, several caveats shouldbe underlined. First, the US2-US11 deletion mutantlacked the whole set of genes that alter class I expres-sion. Therefore, these experiments did not discriminateto what extent individual US molecules may differentiallyinfluence HLA-E expression and target susceptibility toCD94/NKG2A+ cells along the viral replication cycle. Theanalysis of HLA-E was only shown after 72 h of infectionand thus does not rule out that HLA-E may be main-tained at earlier stages. Furthermore, the NKL cell lineemployed for functional assays displays, together withCD94/NKG2A, the CD85j inhibitory receptor, whichbroadly interacts with class I molecules, thus complicat-ing the interpretation of experiments in which infectedfibroblasts became sensitive to the NKL cell line. Finally,NK-mediated response against infected fibroblasts is acomplex process that does not simply depend on theirclass I levels [38, 39]. Additional variables such asexpression of ligands for activating receptors [12] maycondition as well the susceptibility to lysis during viralinfection, altering the balance of inhibitory and stimula-tory signals.

While the work by Falk et al. [37] does not formally ruleout a dissociated action of HCMV on HLA class Ia andHLA-E expression, their results point out that this puta-tive evasion mechanism can be overcome, as infectedcells were efficiently killed by some NK cells subsets.Our observations on the reactivity of KIR2DL2+ clonesagainst US+ cells also supported this concept. Alto-gether, these studies are consistent with the conven-tional idea that a heterogeneous distribution of inhibitoryreceptors on NK cell clones enables the immune systemto efficiently discriminate between variable alterations ofMHC class I expression [15].

Cohen et al. [40] reported that HIV down-modulates HLAclass I but preserves HLA-C and HLA-E, thus maintain-ing resistance of infected cells to both CD94/NKG2A+

and KIR2DL+ NK cells. Though HCMV partially counter-acts the control by CD94/NKG2A and CD85j inhibitoryreceptors, it is uncertain whether it may also evade KIR-

mediated surveillance. Our observations on KIR2DL2+

clones and the results of Falk et al. [37] would argueagainst this possibility, whereas the reported insensitivityof HLA-C to US6 and US2 in trophoblast cells [33] indi-rectly supported that this might occur in some cell types.

NK inhibitory receptor gene families include memberswith activating function whose physiological role stillremains unclear [15]. It has been hypothesized thatKIR2DS/3DS and CD94/NKG2C receptors might triggercytotoxicity and cytokine production, provided that con-trol by inhibitory receptors would fall beneath a criticalthreshold. Since the affinity of stimulatory NKR for class Imolecules appears lower than that of their inhibitorycounterparts, only NK clones expressing pairs of recep-tors with non-overlapping class I specificities would beexpected to operate in that way. Moreover, either prefer-ential down-modulation of the inhibitory ligand and/oran increase of the activating NKR avidity should berequired. CD94/NKG2C+ cells, included amongKIR2DL2+CD94/NKG2A– NK clones, were directly identi-fied with the anti-NKG2A/C p25 mAb (data not shown).The possibility that the activating KLR might participatein the lysis of US11+ cells that maintained HLA-E expres-sion was considered. However, such experiments wereinconclusive as it became hard to discern to what extentCD94/NKG2C contributed to the enhanced lysis takingplace upon concomitant down-regulation of HLA-Cexpression and loss of KIR2DL2 engagement. Withregard to the second possibility (i.e. increased avidity oftriggering NKR), there is as yet no evidence for the exis-tence of class I-peptide complexes recognized withhigher affinity by the CD94/NKG2C activating receptoras compared to the inhibitory NKR.

Recently, an HSP60-derived peptide has been shown topotentially compete with endogenous class I-derivednonamers for binding to HLA-E in transfectants [41].Such MHC/peptide complex was not recognized byCD94/NKG2A thus rendering stressed cells vulnerable tothe NK-mediated attack; yet, no information on the puta-tive recognition by CD94/NKG2C was provided. Furtherstudies are required to determine whether that mecha-nism operates in a general way on other normal andpathological cell types upon stress-induced synthesis ofHSP60.

The existence of different HCMV molecules which redun-dantly down-regulate MHC class I expression underlinestheir importance for the virus, and suggests that theyhave sequentially evolved to counteract the pressure ofhost defense mechanisms. Even the roles of the homolo-gous US2 and US11 molecules are only partially overlap-ping, as both potentially contribute to maintain the resis-tance of infected cells to CD94/NKG2A+ NK cell subsets



but differently interfere with class Ia expression. On theother hand, the diversity of CMV strategies to subvert theimmune response goes beyond the control of HLAclass I expression. Considering the crucial role of trigger-ing receptors in the response to CMV, attention shouldbe paid to qualitative/quantitative variations of theirligands in cells infected by different HCMV strains [39].

4 Materials and methods

4.1 Cells and antibodies

RPMI 8866 cells were grown in RPMI 1640 supplementedwith 10% (v/v) heat-inactivated fetal calf serum. HLA class Ialleles expressed in the RPMI 8866 cell line were determinedby standard typing procedures using serologic and/ormolecular probes. NK clones were prepared by limiting dilu-tion from freshly isolated NK cells as previously described[42]; all microcultures were systematically phenotyped byflow cytometry analysis with a panel of receptor-specificmAb [43]. Functional analysis of inhibitory receptors wastested in redirected lysis assays against the P815 mastocy-toma [42]. The NKL cell line, kindly provided by Dr. M.Robertson (Dana-Farber, Boston, MA), was maintained inRPMI 1640 supplemented with 10% (v/v) heat-inactivatedhuman AB serum and 100 U/ml IL-2 (EuroCetus).

HP-3B1 anti-CD94, HP-1F7 anti-HLA class I, HP-3E4 anti-KIR2DL1/S1/S3 and HP-F1 anti-CD85j were generated inour laboratory and have been described elsewhere [44, 45].Z199 anti-CD94/NKG2A, C218 anti-CD56 and GL183 anti-KIR2DL2/S2 mAb were generously provided by Dr. A.Moretta (University of Genoa, Italy). DX9 and 5.133 anti-KIR3D mAb were, respectively, provided by Drs. L. Lanier(UCSF, San Francisco, CA) and M. Colonna (University of St.Louis, MO).

The HLA-E-specific 3D12 mAb [22] was kindly provided byDr. D. E. Geraghty (Fred Hutchinson Cancer Center, Seattle,WA). The anti-HLA-C human antiserum was a gift from Dr. AAlonso (Hospital Carlos Haya, Malaga, Spain); for the pur-pose of our study the specificity of this antiserum waschecked by flow cytometry analysis with a panel of HLAclass I transfectants in the 721.221 LCL including: HLA-A*0301, HLA-B*0702 and HLA-Cw*0702; these cell lineswere provided by Dr. R. Biassoni (Centro BiotecnologieAvanzate, Genoa, Italy), Dr. P Parham (Standford University)and Dr. D. Schendel (University of Munich), respectively.ME1 anti-HLA-B mAb [46] was provided by Dr. J. A. Lopezde Castro (Centro de Biologıa Molecular Severo Ochoa,Madrid, Spain); 1082C5 anti-HLA-A mAb [47] was providedby Dr. G. Ercilla (Hospital Clinic, Barcelona). Rabbit antiseraspecific to HCMV proteins (US6 and US11) were generouslyprovided by Dr. K. Früh (R. W. Johnson PharmaceuticalResearch Institute, La Jolla, CA). Anti-human § -tubulin mAbwas purchased from Sigma (St. Louis, MO). Anti-CD19 mAb

BU-12 was provided by Dr. M. Johnson (University of Bir-mingham, GB).

4.2 Plasmids

Plasmids pVgRXR and pInd were purchased from Invitrogen(Carlsbad, CA). pVgRXR constitutively expresses an ecdy-sone chimeric receptor and contains a Zeocin resistant genefor metabolic selection. pInd has an inducible expressioncassette formed by an ecdysone-inducible promoter fol-lowed by a multicloning site and a polyadenylation signal,and also contains a G418 resistant gene for metabolic selec-tion. The ecdysone-inducible promoter is composed of aminimal promoter associated to five copies of a hybridsequence formed by ecdysone and glucocorticoid responseelements. These sequences enable transcriptional regula-tion of the minimal promoter by the chimeric ecdysonereceptor coded in pVgRXR. The ecdysone receptorbecomes transcriptionally active following dimerization inthe presence of ecdysone or its analogues. The cDNA forthe viral proteins US2, US6 and US11 were cloned HindIII/BamHI into pInd. The cDNA for Luc was obtained from Dr.M. Rincon (University of Vermont, VT) and cloned HindIII/HpaI into pInd as a control.

4.3 Ecdysone-inducible cell lines

RPMI 8866 cells were transfected using a Bio-Rad ElectroCell manipulator BTX 600 (Bio-Rad, Hercules, CA). Briefly,107 cells were electroporated (250 V and 1,300 ? F) in 0.4-cmgap cuvettes (Bio-Rad) in 400 ? l PBS containing 10 ? g ofplasmid DNA, and 24 h later plated in selection culturemedium.

We first generated a cell line expressing the RXR and VgEcRsubunits of the ecdysone receptor by transfectingRPMI 8866 cells with the plasmid pVgRXR. Cells wereselected in culture medium supplemented with 60 ? g/mlZeocin (Invitrogen). Zeocin-resistant clones were evaluatedfor the presence of a functional ecdysone receptor by tran-sient transfection with the reporter plasmid pInd/Luc. Fol-lowing electroporation with the reporter plasmid the cellswere split and treated or not with 10 ? M of an ecdysone ana-log, Pon A (Invitrogen), for 24 h. Luc activity was evaluatedwith a Luc assay system (Promega, Madison, WI) in celllysates using a luminometer Lumat LB9501 (Berthold, Ger-many). A subclone, RPMI 8866/6, expressing high induciblelevels of Luc activity, was selected for subsequent transfec-tions with plasmids expressing the viral proteins or the fireflyLuc control protein under the control of the ecdysone induc-ible promoter (pInd constructs).

RPMI 8866/6 cells were electroporated as above with differ-ent pInd constructs and selected in the presence of 0.5 mg/ml G418 (Life Technologies, Eggenstein, Germany) and60 ? g/ml Zeocin. G418-resistant clones were studied for the



ecdysone-regulated viral proteins or Luc expression. AsUS2, US6 and US11 CMV proteins have been described todown-regulate HLA class I expression, HLA class I levelswere evaluated by flow cytometry in G418-resistant clonesthat were treated or not with 10 ? M Pon A for 48 h. Clonesshowing Pon A-induced HLA class I down-regulation werelater screened for the presence of US6 or US11 by Westernblotting with specific rabbit antisera, kindly provided by Dr.Klaus Früh (R. W. Johnson Pharmaceutical Research Insti-tute). Induction of US2 expression was checked by reversetranscription PCR with specific primers. Luc expression wasevaluated in G418-resistant clones treated or not with Pon Ausing the Luc assay system described above.

4.4 Flow cytometry

Staining by indirect immunofluorescence was carried out asdescribed [44]. Briefly, cells were pretreated with saturatingconcentrations of human aggregated Ig to block FcR andsubsequently incubated with the different mAb, followed bywashing and labeling with FITC-tagged F(ab’)2 goat anti-mouse Ig antibody (Dakopatts, Glostrup, Denmark). For indi-rect immunofluorescence staining with the human anti-HLA-C antiserum, the procedure was carried out employing arabbit serum preparation to block FcR and an FITC-taggedrabbit anti-human Ig (PharMingen, San Diego, CA). Sampleswere analyzed on a FACScan flow cytometer (Becton Dick-inson, Mountain View, CA).

4.5 Cytotoxicity assays

Effector cells were tested in a 2-h 51Cr-release assay [44]against transfectants of the RPMI 8866 cell line treated ornot with Pon A (20 ? M); the E/T ratios employed ranged from10:1 to 2:1. The role of inhibitory receptors specific for HLAclass I was evaluated by pre-incubating for 10 min the effec-tor cells with anti-receptor mAb or the target cells with ananti-HLA mAb; the mAb were maintained along the assay.Specific lysis was calculated as previously described [44].

4.6 Western blotting

RPMI 8866 transfectants treated or not with Pon A (10 ? M)were lysed at different time points in TBS containing 1% Tri-ton X-100 and protease inhibitors. After SDS-PAGE, proteinswere transferred to nitrocellulose using a Bio-Rad Transblot.Membranes were blocked with TBS-10% nonfat dried milk,pH 7.2, and incubated with rabbit antisera specific for US6or US11 diluted 1:1,000 in TBS-5% nonfat dried milk or ascontrol with anti-human § -tubulin mAb (1 ? g/ml). Boundantibodies were detected with horseradish peroxidase-conjugated anti-rabbit Ig antisera (PharMingen) followed bydevelopment with ECL reagents (Pierce, Rockford, IL).

Acknowledgements: We are grateful to Dr. Klaus Früh (R.W. Johnson Pharmaceutical Research Institute, La Jolla, CA)for providing the US plasmids and specific antibodies. Wethank as well Dr. Jose Luis Vicario (Centro de Transfusiones,CAM, Madrid) for HLA typing of RPMI 8866 cells, Dr. F.Navarro (Hospital de la Princesa, Madrid) for providing NKclones, Dr. Carlos Vilches (Clınica Puerta de Hierro, Madrid)for helpful advice, and all colleagues who provided us withreagents. This work was supported by Fundacio la Maratode TV3 (00510), Fondo de Investigaciones Sanitarias (FIS00/0181) and European Commission (QLRT-2001–01112).M.G. is a recipient of a fellowship from Fondo de Investigaci-ones Sanitarias.

References

1 Biron, C. A., Nguyen, K. B., Pien, G. C., Cousens, L. P. andSalazar-Mather, T. P., Natural killer cells in antiviral defense:function and regulation by innate cytokines. Annu. Rev. Immunol.1999. 17: 189–220.

2 Welsh, R. M. and Selin, L. K., No one is naive: the significance ofheterologous T cell immunity. Nat. Rev. Immunol. 2002. 2:417–426.

3 Alcami, A. and Koszinowski, U. H., Viral mechanisms ofimmune evasion. Immunol. Today 2000. 21: 447–455.

4 Tortorella, D., Gewurz, B. E., Furman, M. H., Schust, D. J. andPloegh, H. L., Viral subversion of the immune system. Annu. Rev.Immunol. 2000. 18: 861–926.

5 Lehner, P. J., Karttunen, J. T., Wilkinson, G. W. and Cresswell,P., The human cytomegalovirus US6 glycoprotein inhibits trans-porter associated with antigen processing-dependent peptidetranslocation. Proc. Natl. Acad. Sci. USA 1997. 94: 6904–6909.

6 Hengel, H., Koopmann, J. O., Flohr, T., Muranyi, W., Goulmy,E., Hammerling, G. J., Koszinowski, U. H. and Momburg, F., Aviral ER-resident glycoprotein inactivates the MHC-encodedpeptide transporter. Immunity 1997. 6: 623–632.

7 Machold, R. P., Wiertz, E. J., Jones, T. R. and Ploegh, H. L.,The HCMV gene products US11 and US2 differ in their ability toattack allelic forms of murine major histocompatibility complex(MHC) class I heavy chains. J. Exp. Med. 1997. 185: 363–366.

8 Jones, T. R. and Sun, L., Human cytomegalovirus US2 destabi-lizes major histocompatibility complex class I heavy chains. J.Virol. 1997. 71: 2970–2979.

9 Ahn, K., Angulo, A., Ghazal, P., Peterson, P. A., Yang, Y. andFrüh, K., Human cytomegalovirus inhibits antigen presentationby a sequential multistep process. Proc. Natl. Acad. Sci. USA1996. 93: 10990–10995.

10 Lopez-Botet, M. and Bellon, T., Natural killer cell activation andinhibition by receptors for MHC class I. Curr. Opin. Immunol.1999. 11: 301–307.

11 Cerwenka, A. and Lanier, L. L., Ligands for natural killer cellreceptors: redundancy or specificity. Immunol. Rev. 2001. 181:158–169.

12 Groh, V., Rhinehart, R., Randolph-Habecker, J., Topp, M. S.,Riddell, S. R. and Spies, T., Costimulation of CD8alphabetaT cells by NKG2D via engagement by MIC induced on virus-infected cells. Nat. Immunol. 2001. 2: 255–260.

13 Arase, H., Mocarski, E. S., Campbell, A. E., Hill, A. B. andLanier, L. L., Direct recognition of cytomegalovirus by activating



and inhibitory NK cell receptors. Science 2002. 296:1323–1326.

14 Smith, H. R., Heusel, J. W., Mehta, I. K., Kim, S., Dorner, B. G.,Naidenko, O. V., Iizuka, K., Furukawa, H., Beckman, D. L., Pin-gel, J. T., Scalzo, A. A., Fremont, D. H. and Yokoyama, W. M.,Recognition of a virus-encoded ligand by a natural killer cell acti-vation receptor. Proc. Natl. Acad. Sci. USA 2002. 99: 8826–8831.

15 Vilches, C. and Parham, P., KIR: diverse, rapidly evolving recep-tors of innate and adaptive immunity. Annu. Rev. Immunol. 2002.20: 217–251.

16 Moretta, L., Biassoni, R., Bottino, C., Mingari, M. C. andMoretta, A., Human NK-cell receptors. Immunol. Today 2000. 21:420–422.

17 French, A. R. and Yokoyama, W. M., Natural killer cells and viralinfections. Curr. Opin. Immunol. 2003. 15: 45–51.

18 Cosman, D., Fanger, N. and Borges, L., Human cytomegalovi-rus, MHC class I and inhibitory signalling receptors: more ques-tions than answers. Immunol. Rev. 1999. 168: 177–185.

19 Lopez-Botet, M., Llano, M. and Ortega, M., Human cytomega-lovirus and natural killer-mediated surveillance of HLA class Iexpression: a paradigm of host-pathogen adaptation. Immunol.Rev. 2001. 181: 193–202.

20 Farrell, H. E., Vally, H., Lynch, D. M., Fleming, P., Shellam, G.R., Scalzo, A. A. and Davis-Poynter, N. J., Inhibition of naturalkiller cells by a cytomegalovirus MHC class I homologue in vivo.Nature 1997. 386: 510–514.

21 Vance, R. E., Kraft, J. R., Altman, J. D., Jensen, P. E. and Rau-let, D. H., Mouse CD94/NKG2A is a natural killer cell receptor forthe nonclassical major histocompatibility complex (MHC) class Imolecule Qa-1(b). J. Exp. Med. 1998. 188: 1841–1848.

22 Lee, N., Llano, M., Carretero, M., Ishitani, A., Navarro, F.,Lopez-Botet, M. and Geraghty, D., HLA-E is a major ligand forthe natural killer inhibitory receptor CD94/NKG2A. Proc. Natl.Acad. Sci. USA 1998. 95: 5199–5204.

23 Braud, V. M., Allan, D. S., O’Callaghan, C. A., Soderström, K.,D’Andrea, A., Ogg, G. S., Lazetic, S., Young, N. T., Bell, J. I.,Phillips, J. H., Lanier, L. L. and McMichael, A. J., HLA-E bindsto natural killer cell receptors CD94/NKG2A, B and C. Nature1998. 391: 795–799.

24 Lee, N., Goodlett, D. R., Ishitani, A., Marquardt, H. andGeraghty, D. E., HLA-E surface expression depends on bindingof TAP-dependent peptides derived from certain HLA class I sig-nal sequences. J. Immunol. 1998. 160: 4951–4960.

25 Braud, V. M., Allan, D. J., Wilson, D. and McMichael, A. J.,TAP- and tapasin-dependent HLA-E surface expression corre-lates with the binding of an MHC class I leader peptide. Curr.Biol. 1998. 8: 1–10.

26 Tomasec, P., Braud, V. M., Rickards, C., Powell, M. B.,McSharry, B. P., Gadola, S., Cerundolo, V., Borysiewicz, L. K.,McMichael, A. J. and Wilkinson, G. W., Surface expression ofHLA-E, an inhibitor of natural killer cells, enhanced by humancytomegalovirus gpUL40. Science 2000. 287: 1031.

27 Ulbrecht, M., Martinozzi, S., Grzeschik, M., Hengel, H.,Ellwart, J. W., Pla, M. and Weiss, E. H., Cutting edge: thehuman cytomegalovirus UL40 gene product contains a ligand forHLA-E and prevents NK cell-mediated lysis. J. Immunol. 2000.164: 5019–5022.

28 Rehm, A., Engelsberg, A., Tortorella, D., Korner, I. J.,Lehmann, I., Ploegh, H. L. and Hopken, U. E., Human cytomeg-alovirus gene products US2 and US11 differ in their ability toattack major histocompatibility class I heavy chains in dendriticcells. J. Virol. 2002. 76: 5043–5050.

29 Garcıa, P., Llano, M., de Heredia, A. B., Willberg, C. B., Capar-ros, E., Aparicio, P., Braud, V. M. and Lopez-Botet, M., HumanT cell receptor-mediated recognition of HLA-E. Eur. J. Immunol.2002. 32: 936–944.

30 Romagnani, C., Pietra, G., Falco, M., Millo, E., Mazzarino, P.,Biassoni, R., Moretta, A., Moretta, L. and Mingari, M. C., Iden-tification of HLA-E-specific alloreactive T lymphocytes: a cellsubset that undergoes preferential expansion in mixed lympho-cyte culture and displays a broad cytolytic activity against alloge-neic cells. Proc. Natl. Acad. Sci. USA 2002. 99: 11328–11333.

31 Gewurz, B. E., Wang, E. W., Tortorella, D., Schust, D. J. andPloegh, H. L., Human cytomegalovirus US2 endoplasmicreticulum-lumenal domain dictates association with major histo-compatibility complex class I in a locus-specific manner. J. Virol.2001. 75: 5197–5204.

32 Huard, B. and Früh, K., A role for MHC class I down-regulationin NK cell lysis of herpes virus-infected cells. Eur. J. Immunol.2000. 30: 509–515.

33 Schust, D. J., Tortorella, D., Seebach, J., Phan, C. and Ploegh,H. L., Trophoblast class I major histocompatibility complex(MHC) products are resistant to rapid degradation imposed bythe human cytomegalovirus (HCMV) gene products US2 andUS11. J. Exp. Med. 1998. 188: 497–503.

34 Story, C. M., Furman, M. H. and Ploegh, H. L., The cytosolic tailof class I MHC heavy chain is required for its dislocation by thehuman cytomegalovirus US2 and US11 gene products. Proc.Natl. Acad. Sci. USA 1999. 96: 8516–8521.

35 Ulbrecht, M., Hofmeister, V., Yuksekdag, G., Ellwart, J. W.,Hengel, H., Momburg, F., Martinozzi, S., Reboul, M., Pla, M.and Weiss, E. H., HCMV glycoprotein US6 mediated inhibition ofTAP does not affect HLA-E dependent protection of K-562 cellsfrom NK cell lysis. Hum. Immunol. 2003. 64: 231–237.

36 Strong, R. K., Holmes, M. A., Li, P., Braun, L., Lee, N. andGeraghty, D. E., HLA-E allelic variants. Correlating differentialexpression, peptide affinities, crystal structures, and thermal sta-bilities. J. Biol. Chem. 2003. 278: 5082–5090.

37 Falk, C. S., Mach, M., Schendel, D. J., Weiss, E. H., Hilgert, I.and Hahn, G., NK cell activity during human cytomegalovirusinfection is dominated by US2–11-mediated HLA class I down-regulation. J. Immunol. 2002. 169: 3257–3266.

38 Carr, W. H., Little, A. M., Mocarski, E. and Parham, P., NK cell-mediated lysis of autologous HCMV-infected skin fibroblasts ishighly variable among NK cell clones and polyclonal NK cell lines.Clin. Immunol. 2002. 105: 126–140.

39 Cerboni, C., Mousavi-Jazi, M., Linde, A., Soderström, K.,Brytting, M., Wahren, B., Kärre, K. and Carbone, E., Humancytomegalovirus strain-dependent changes in NK cell recogni-tion of infected fibroblasts. J. Immunol. 2000. 164: 4775–4782.

40 Cohen, G. B., Gandhi, R. T., Davis, D. M., Mandelboim, O.,Chen, B. K., Strominger, J. L. and Baltimore, D., The selectivedownregulation of class I major histocompatibility complex pro-teins by HIV-1 protects HIV-infected cells from NK cells. Immunity1999. 10: 661–671.

41 Michaelsson, J., Teixeira de Matos, C., Achour, A., Lanier, L.L., Kärre, K. and Soderström, K., A signal peptide derived fromHsp60 binds HLA-E and interferes with CD94/NKG2A recogni-tion. J. Exp. Med. 2002. 196: 1403–1414.

42 Perez-Villar, J. J., Melero, I., Rodrıguez, A., Carretero, M.,Aramburu, J., Sivori, S., Orengo, A. M., Moretta, A. andLopez-Botet, M., Functional ambivalence of the Kp43 (CD94)NK cell-associated surface antigen. J. Immunol. 1995. 154:5779–5788.



43 Perez-Villar, J. J., Melero, I., Navarro, F., Carretero, M., Bellon,T., Llano, M., Colonna, M., Geraghty, D. E. and Lopez-Botet,M., The CD94/NKG2-A inhibitory receptor complex is involved innatural killer cell-mediated recognition of cells expressing HLA-G1. J. Immunol. 1997. 158: 5736–5743.

44 Navarro, F., Llano, M., Bellon, T., Colonna, M., Geraghty, D. E.and Lopez-Botet, M., The ILT2(LIR1) and CD94/NKG2A NK cellreceptors respectively recognize HLA-G1 and HLA-E moleculesco-expressed on target cells. Eur. J. Immunol. 1999. 29:277–283.

45 Colonna, M., Navarro, F., Bellon, T., Llano, M., Garcıa, P.,Samaridis, J., Angman, L., Cella, M. and Lopez-Botet, M., Acommon inhibitory receptor for major histocompatibility complexclass I molecules on human lymphoid and myelomonocytic cells.J. Exp. Med. 1997. 186: 1809–1818.

46 Ellis, S. A., Taylor, C. and McMichael, A., Recognition of HLA-B27 and related antigen by a monoclonal antibody. Hum. Immu-nol. 1982. 5: 49–59.

47 Lozano, F., Santos-Aguado, J., Borche, L., Places, L., Dome-nech, N., Gaya, A., Vilella, R. and Vives, J., Identification of theamino acid residues defining an intralocus determinant in thealpha 1 domain of HLA-A molecules. Immunogenetics 1989. 30:50–53.

Correspondence: Miguel Lopez-Botet, Molecular Immuno-pathology Unit, DCEXS, Universitat Pompeu Fabra, Dr.Aiguader 80, 08003 Barcelona, SpainFax: +34-93-5422802e-mail: miguel.lopez-botet — upf.edu

Manuel Llano’s present address: Molecular Medicine Pro-gram, Mayo Clinic, Rochester, Minnesota, USA



Differential effects of US2, US6 and US11 human cytomegalovirus proteins on HLA class Ia and HLA-E...

Documents

Transcript of Differential effects of US2, US6 and US11 human cytomegalovirus proteins on HLA class Ia and HLA-E...