0014-2980/99/0202-537$17.50+.50/0© WILEY-VCH Verlag GmbH, D-69451 Weinheim, 1999

Cell surface expression of HLA-E: interaction withhuman I 2-microglobulin and allelic differences

Matthias Ulbrecht1, Andrea Couturier1, Silvia Martinozzi1, Marika Pla2, RakeshSrivastava3, Per A. Peterson3 and Elisabeth H. Weiss1

1 Institut für Anthropologie und Humangenetik, Ludwig-Maximilians-Universität München,München, Germany

2 Mouse Immunogenetics, INSERM U462, Institute of Hematology, Saint-Louis Hospital, Paris,France

3 Department of Immunology, IMM 8, The Scripps Research Institute, La Jolla, USA

The formation of a trimeric complex composed of MHC class I heavy chain, g 2-microglobulin ( g 2m) and peptide ligand is a prerequisite for its efficient transport to the cellsurface. We have previously demonstrated impaired intracellular transport of the humanclass Ib molecule HLA-E in mouse myeloma X63 cells cotransfected with the genes for HLA-E and human g 2m (h g 2m), which is most likely attributable to inefficient intracellular peptideloading of the HLA-E molecule. Here we demonstrate that cell surface expression of HLA-Ein mouse cells strictly depends on the coexpression of h g 2m and that soluble empty com-plexes of HLA-E and h g 2m display a low degree of thermostability. Both observations implythat low affinity interaction of HLA-E with g 2m accounts to a considerable extent for theobserved low degree of peptide uptake in the endoplasmic reticulum. Moreover, we showthat the only allelic variation present in the caucasoid population located at amino acid posi-tion 107 (Gly or Arg) greatly affects intracellular transport and cell surface expression upontransfection of the respective alleles into mouse cells. No obvious difference was found withregard to the sequence of the peptide ligand.

Key words: HLA-E / Allele / Antigen presentation / g 2-microglobulin / Peptide

Received 19/2/98Revised 30/10/98Accepted 3/11/98

[I 18065]

Abbreviations: I 2m: g 2-microglobulin h I 2m: human g 2-microglublin B-LCL: B lymphoblastoid cell line ER: Endo-plasmic reticulum HLA-EG: HLA-E polypeptide containingGly at amino acid position 107 HLA-ER: HLA-E polypeptidecontaining Arg at amino acid position 107

1 Introduction

MHC class Ia molecules with specific bound peptidesare the ligands for antigen-specific CTL [1] which allowthe recognition of virally infected or transformed cells aswell as the selection of the T cell repertoire [2]. Stable cellsurface expression requires the assembly of a trimolecu-lar complex composed of MHC class I heavy chain, g 2-microglobulin ( g 2m) and peptide within the endoplasmicreticulum (ER) [3]. The peptide component, 8–10 aminoacids in length, is usually generated in the cytosol by theaction of proteasomes and transported into the ER by aheterodimeric transporter [4–6].

A second group of class I molecules (Ib) can be definedwhich is characterized by limited polymorphism in theligand binding pocket and generally low and restrictedexpression. The function of these class Ib moleculesremains largely unknown, although a role for these mole-cules in antigen presentation is most likely (for reviewsee [7]). In the mouse the majority of these molecules areencoded in the Q, T and M subregions of the MHC [8, 9].They comprise the Qa-2 molecules which bind nona-meric peptides [10], the Qa-1 antigen, capable of inter-acting with mycobacterial heat shock protein 65 [11] andpresenting MHC class I leader sequence-derived pep-tides to alloreactive CTL [12], as well as the H-2M3 anti-gen which selectively binds bacterial peptides withunique chemical properties [13, 14]. In man, class Ib mol-ecules comprise the low polymorphic HLA-E, -F, -G, andCD1 antigens, in addition to the highly polymorphic MIC-A and -B molecules [15]. Of these, HLA-G was shown tobind a wide range of nonameric peptides [16] and toinhibit NK cells [17], whereas CD1b has been implicatedin the recognition of mycobacterial lipid and lipoglycancompounds by CD4–8– § g + T cells [18]. HLA-E is the onlyclass Ib gene which is transcribed in all human tissues

Eur. J. Immunol. 1999. 29: 537–547 HLA-E: cell surface expression and alleles 537

and cell lines [19, 20]. In the caucasoid population onlytwo alleles have been described which differ only inamino acid position 107 (Arg or Gly) [21]. We have previ-ously reported that the HLA-E antigen is weaklyexpressed at the cell surface upon cotransfection withthe gene for human g 2-microglobulin (h g 2m) into themouse myeloma cell line P3X63Ag8.653 (X63) [20]. Inthese transfectants the intracellular transport of the HLA-E molecule was impaired most likely due to an inefficientpeptide loading within the endoplasmic reticulum (ER)[22]. Recent in vitro studies have shown that ligands forHLA-E are contained within the leader peptides of cer-tain MHC class I molecules as well as within viral pro-teins [23, 24]. From these studies it appeared unlikelythat the cell surface expression of HLA-E is solely con-trolled by a restricted ligand repertoire. We thereforeinvestigated which additional factors might limit theintracellular transport of HLA-E. Since the two peptidebinding studies were performed on HLA-E alleles differ-ing at amino acid position 107 we also addressed theeffect of this position on intracellular transport and cellsurface expression.

2 Results

2.1 Interaction of HLA-E and h I 2m

In mouse myeloma X63 cells cotransfected with h g 2mand cosmid cd3.14 encoding HLA-EG with Gly in aminoacid position 107 (X63MEG), the majority of synthesizedHLA-E heavy chains are retained within the ER and mole-cules reaching the cell surface are rapidly degraded [22].From the stabilizing effect of exogenously added pep-tides a limited availability of endogenous ligands forHLA-E appeared to restrict the intracellular transport ofHLA-E. The different HLA-E ligands identified so far dis-play neither extraordinary chemical properties nor ahighly stringent motif [23, 24]. Also, due to its associationwith TAP, HLA-E should receive appropriate peptides.We therefore sought for additional restrictions in the for-mation of a cell surface expressed HLA-E complex. Inparticular, we addressed the possibility that the associa-tion of HLA-E with g 2m may further limit its cell surfaceexpression since the exogenous addition of h g 2m stabi-lized HLA-E molecules at the cell surface of X63MEG

cells. We therefore generated X63 transfectants express-ing HLA-E in the presence or absence of endogenoush g 2m. All transfectants were screened for the expressionof HLA-E and h g 2m RNA by Northern blot analysis. Onlytransfectants containing comparable amounts of HLAclass I and h g 2m mRNA levels were further analyzed.Only double-positive X63MEG cells expressed detect-able amounts of HLA-E molecules at the cell surfacewhich could be further increased by the addition of

exogenous h g 2m, detectable with the conformation-dependent mAb B9.12.1, specific for HLA class I/ g 2mdimers and the conformation-independent mAb A1.4,specific for HLA class I heavy chains (Fig. 1). In singlyHLA-E expressing X63EG cells no HLA-E antigen couldbe detected on the cell surface even upon adding exoge-nous h g 2m. This result indicates that no HLA-E mole-cules detectable with HLA class I-specific mAb aretransported to the cell surface in the absence of endoge-nous h g 2m. In contrast, HLA-B27 could be detected onX63 transfectants in the presence or absence of coex-pressed h g 2m. Furthermore, the HLA-B27 cell surfaceexpression on either X63B or X63BM cells could not beenhanced by the addition of exogenous h g 2m, althoughbinding of h g 2m to these cells could be demonstrated bythe increase of staining with BBM.1.

To address the functional significance of this strictdependence of HLA-E cell surface expression on thepresence of h g 2m in mouse cells for the assembly ofHLA-E molecules, we investigated the thermostability ofsoluble, empty HLA-E/h g 2m complexes secreted bytransfected D SC2 Drosophila melanogaster cells. Asshown in Fig. 2a these complexes are largely unstable at37 °C and almost entirely dissociate at 30 °C already inthe presence of 1% NP40. Dissociation of HLA-E/h g 2mcomplexes is reduced in the presence of additionalh g 2m. In contrast, soluble empty HLA-A2/h g 2m com-plexes display a much higher degree of thermostabilityas demonstrated by only partial dissociation after expo-sure to 42 °C even in the presence of detergent (Fig. 2b).These results demonstrate a weak interaction of HLA-Eheavy chains with h g 2m.

2.2 HLA-E alleles and cell surface expression

The allelic variation of the HLA-E polypeptide describedin the caucasoid population affects amino acid position107 which is either occupied by G (HLA-EG) or R (HLA-ER). Since single amino acid substitutions even locatedoutside the peptide binding groove may influence pep-tide binding and intracellular transport [25] we investi-gated the effect of the allelic substitutions of HLA-E atamino acid position 107 with regard to cell surfaceexpression and intracellular transport. We thus gener-ated X63 transfectants which in addition to theexpressed h g 2m transgene either contained HLA-EG

(X63MEG) or HLA-ER (X63MER). These transfectants wereagain matched for expression levels of h g 2m (Fig. 3a)and HLA-E mRNA (Fig. 4). The clones X63MEG

1 andX63MER

1 were chosen for further analysis and are calledfurther on X63MEG and X63MER. Whereas the cell sur-face of X63MEG cells stained positive for HLA-E mole-cules no expression of HLA-E was detectable on

538 M. Ulbrecht et al. Eur. J. Immunol. 1999. 29: 537–547

Figure 1. Dependence of HLA-EG cell surface expression on coexpression of h g 2m. X63 cells expressing HLA-EG (X63EG), h g 2m(X63M), HLA-B27 (X63B), HLA-EG and h g m2 (X63MEG) or HLA-B27 and h g 2m (X63MB) were stained with the mAb A1.4, specificfor HLA class heavy chains regardless of their conformation, B9.12.1, specific for HLA class I g 2m complexes, or BBM.1, specificfor h g 2m. Each transfectant was stained untreated or following an incubation with exogenous h g 2m at 1 ? M for 6 h at 37 °C. Ineach diagram the staining profiles obtained with (stippled lines) or without (solid lines) the addition of h g 2m are compared to thatof the isotype control (dotted lines).

X63MER cells (Fig. 3). Neither adding exogenous h g 2m(Fig. 3) nor reduction of the culture temperature (data notshown) led to a detectable cell surface expression of HLA-ER molecules. HLA-E cell surface expression on X63MEG

cells could also be enhanced by adding various knownpeptide ligands for 6 h at 37 °C: HLA-A23–11, HLA-B83–11,HLA-Cw33–11, and BZLF-139–47 (Fig. 3). All peptidesenhanced the cell surface expression of HLA-EG to thesame extent except HLA-A23–11 which effect was approxi-mately twice as strong. In contrast, incubation of X63MER

cells with the same peptides resulted in no detectableexpression of HLA-ER by flow cytometry analysis.

2.3 Peptide binding to HLA-E alleles in lysates

To rule out the possibility that the lack of HLA-ER cell sur-face expression on X63MER cells is caused by a loweraffinity of this allele for peptides compared to HLA-EG weanalyzed the ability of HLA-A23–11 and BZLF-139–47 to sta-bilize HLA-E molecules in cell lysates of the respectiveX63 transfectants. As shown in Fig. 5, HLA-A23–11 pre-vented the heat-induced dissociation of B9.12.1 precipi-table HLA-E/ g 2m complexes in lysates of both transfec-tants at a concentration as low as 3 ? M. In contrast, theBZLF-139–47 peptide was unable to stabilize HLA-E mole-

Eur. J. Immunol. 1999. 29: 537–547 HLA-E: cell surface expression and alleles 539

Figure 2. Different thermostability of soluble empty HLA-E/h g 2m and HLA-A2/h g 2m dimers. The supernatants of D. melanogas-ter SC2 transfectants expressing soluble empty HLA-EG/h g 2m (a) or HLA-A2/h g 2m (b) dimers were diluted 1:20 in PBS and sup-plemented with 1 % (w/v) NP40 and/or h g 2m at 2 ? M where indicated. Aliquots of 1 ml of these dilutions were heated to the indi-cated temperatures for 1 h. After subsequent immunoprecipitation of HLA class I/h g 2m dimers with mAb W6/32, immunoprecipi-tates were separated by SDS-PAGE on a 10% polyacrylamide gel and blotted onto nitrocellulose membranes. HLA class I §chains were visualized using mAb A1.4 and the Western-LightTM chemiluminescence detection system (Tropix). In a controlexperiment the temperature shift was omitted and the reaction was kept at 4 °C for the same time period. The positions of HLA-EG and HLA-A2 § chains, respectively, as well as of W6/32-light chains, which appear as doublets, are indicated.

cules under these experimental conditions even at 100-fold higher concentrations. The stronger signal for HLA-A23–11 induced HLA-ER complexes can be explained by ahigher labeling efficiency of this polypeptide comparedto HLA-EG as demonstrated by the amount of HLA-Eheavy chains precipitated by A1.4 (Fig. 5, lane 2).Thus,both the allelic HLA-E heavy chains do not differ signifi-cantly in peptide specificity nor in the affinity for pep-tides. In addition, both heavy chains are able to formcomplexes with g 2m in the presence of the leadersequence peptide.

2.4 HLA-E alleles and intracellular transport

To correlate cell surface expression of the two investi-gated HLA-E alleles with their biosynthesis, intracellulartransport and stability, we performed pulse chase exper-iments on X63MEG and X63MER cells using X63MB cellsas a positive control for regular transport of HLA class Imolecules. At the onset of the chase experiment at leastsimilar amounts of HLA-E heavy chains were precipitableby mAb A1.4 from lysates of X63MER and X63MEG cells(Fig. 6a and b). Although the majority of polypeptides ofboth HLA-E alleles did not egress the ER (Fig. 6a and b),at least some HLA-EG molecules acquired a mature(Endo H resistant) glycosylation after a 1-h chase(Fig. 6a, black arrowhead), whereas no processed, EndoH-resistant HLA-ER polypeptides could be precipitatedeven after 2-h chase, a period sufficient for HLA-B27 incontrol transfectants to be entirely processed (Fig. 6c).

3 Discussion

We have previously reported the impaired intracellulartransport of HLA-E in mouse X63 cells cotransfectedwith the genes for HLA-E and h g 2m [22]. We hereaddressed the question which factors might reduce therate of intracellular processing of HLA-E. One explana-tion for this feature is the low affinity for the HLA-E heavychain for g 2m. We find that expression of HLA-E inmouse cells strictly depends on the coexpression ofh g 2m and that soluble empty complexes of HLA-E andh g 2m are less stable than comparable HLA-A2/h g 2mdimers. Whereas HLA class Ia cell surface expression isalso reduced when complexed with mouse g 2m [26], theinability of the HLA-E polypeptide to interact with mouseg 2m in a way that would allow cell surface transport is

unusual and probably reflects on an overall weak associ-ation with the light chain. The strict dependence of HLA-E cell surface expression on the presence of h g 2m is notunique to X63 transfectants but can also be observed inspleen cells and PBL of HLA-E transgenic mice (tgm),when detected with HLA class I-specific mAb. In HLA-E-tgm splenocytes cell surface expression of HLA-E canbe recovered on hybridoma cells after fusion with X63M,and double HLA-E/h g 2m-positive tgm express HLA-Emolecules on the cell surface of lymphocytes (data notshown). The inability of HLA-E to interact with mouseg 2m is confirmed by previous results that exogenous

mouse g 2m in contrast to human and bovine g 2m is notcapable of stabilizing HLA-E at the cell surface ofX63MEG cells [22]. An alignment of the amino acidsequences for human, bovine and mouse g 2m and com-paring the residues with contact to the HLA heavy chain

540 M. Ulbrecht et al. Eur. J. Immunol. 1999. 29: 537–547

Figure 3. Different cell surface expression of HLA-EG andHLA-ER on X63 transfectants. X63 cells cotransfected eitherwith HLA-EG and h g 2m or HLA-ER and h g 2m were stainedwith the mAb A1.4, specific for human HLA class heavychains regardless of their conformation (shaded histogramand solid lines in a–d) and BBM. 1, specific for h g 2m(dashed lines in a). Cells were either stained untreated(shaded histograms in a–d, BBM.1 staining in a) or followinga 6-h incubation with either h g 2m at 1 ? M [thin solid lines ina–d or peptides at 150 ? M (thick solid lines: (a) HLA-A23–11,(b) HLA-B83–11, (c) HLA-Cw33–11, and (d) BZLF-139–47)]. Ineach histogram the staining of the isotype control is given asa dotted line.

Figure 4. Comparison of HLA-E mRNA expression inX63MEG and X63MER transfectants. X63M cells expressingh g 2m were further transfected with HLA-EG or HLA-ER. Ofthe resultant clones 10 ? g of total RNA per lane were sepa-rated on a formaldehyde agarose gel and hybridized with adigoxigenin-labeled probe generated from exon 3 of HLA-Eby PCR (a). Hybridization was detected by chemilumines-cence. The parental X63M cells (lane 1) served as negativecontrol. The amount of RNA loaded is visualized by the ethi-dium bromide staining of the 28 S rRNA (b). The clonesX63MEG

1, and X63MER1 having similar level of HLA-E mRNA

expression were chosen for further analyses and designatedX63MEG and X63MER.

according to Saper et al. [27] reveals three residuesunique to mouse g 2m (K3, Q6 and M55) that might beresponsible for the impaired interaction. On the otherhand, the known contact sites of the HLA class I heavychain with h g 2m are conserved in the HLA-E polypep-tide. As based on its three-dimensional structure HLA-Ebinds g 2m like HLA-A2, it is not possible to explain theselectivity of HLA-E for human g 2m on the basis of thecrystal structures [28]. In terms of biological significancea weak association of HLA-E with h g 2m as reflected bythe low thermostability of the empty complex, may pro-vide a means to regulate the cell surface expression ofHLA-E in that only high affinity peptides may compen-sate for the small contribution of h g 2m to the assemblyof a processable trimeric complex. Moreover, it is plausi-ble that HLA-E has to compete for h g 2m with other HLAclass I polypeptides as has been proposed for H-2 class

I molecules [29]. If such competition would be operativein the ER the cell surface expression of HLA-E shouldinversely correlate with the expression of other HLAclass I molecules. The observation that coexpression ofHLA-class I molecules in the cell line 721.221 onlyslightly increases cell surface expression of HLA-E givessupport to this conclusion [30–32]. However, theseresults can also be explained either by a lower amount ofHLA-E compared to HLA class Ia molecules, or by thefact that the supply of peptides is limiting; indeed it is notknown how MHC leader sequence peptides are pro-cessed and whether this processing is efficient. Analysisof human cells with the antibody 3D12 recognizing theHLA-E molecule, indicated that HLA-E is expressed onvirtually all peripheral blood mononuclear cells [33]. Thelevels varied over an order of magnitude with B lympho-cytes and monocytes showing the highest expression.

Two in vitro studies performed with different HLA-Ealleles report on the binding of either viral peptides toHLA-EG [24] or of MHC class I leader sequence-derivedpeptides to HLA-ER [23]. We analyzed peptide bindingand intracellular processing of both alleles in transfectedX63 cells coexpressing h g 2m. The peptides present in

Eur. J. Immunol. 1999. 29: 537–547 HLA-E: cell surface expression and alleles 541

Figure 5. Peptide-induced stability of the two HLA-E allelesin cell lysates. X63 transfectant clones expressing eitherh g 2m and HLA-EG (a, X63MEG) or h g 2m and HLA-ER (b,X63MER) were metabolically labeled for 60 min. Equal ali-quots of cells were lysed in plain lysis buffer (lanes 1 and 2)or lysis buffer supplemented with 10 ? g of mAb B9.12.1(lane 3) or with the peptides HLA-A23–11 (lanes 4 and 5) andBZLF-139–47 (lanes 6–8) at the concentrations indicated. Thelysates were heated at 45 °C for 2 min (lanes 2–8) or kept at4 °C (lane 1) until standard immunoprecipitation was per-formed using either mAb A1.4 specific for HLA class I heavychains (lane 1) or mAb B9.12.1 specific for HLA class I com-plexes (lanes 2–8).

Figure 6. Impaired processing of the HLA-EG and HLA-ER

class I heavy chains. X63 transfectant clones expressingeither h g 2m and HLA-EG (a, X63MEG), h g 2m and HLA-ER (b,X63MER) or h g 2m and HLA-B37 (c, X63MB) were metaboli-cally labeled for 15 min and chased for the times indicated at37 °C. HLA class I molecules were immunoprecipitated withmAb A1.4 specific for HLA class I heavy chains. Equal ali-quots were either mock (–) or Endo H (+) digested and sepa-rated by SDS-PAGE using 10 % gels. The autoradiograms ofthe X63MEG and the X63MER precipitates were equallyexposed for 25 days whereas the exposure of the autoradio-gram of the X63MB precipitates was 5 days. The positionsof the mature (arrowheads) HLA-E § chains are indicated.

HLA-class I signal sequences very efficiently stabilizedthe HLA-EG molecule on the cell surface of the transfec-tants. The increase in cell surface staining in the pres-ence of HLA-A23–11 was at least threefold higher com-pared to the stabilizing effect of the viral peptide BZLF-139 47 indicating a higher affinity of this ligand for HLA-EG.As both antibodies A1.4 (see Fig. 3) and B9.12.1 (datanot shown) gave the same staining profiles we concludethat the HLA-E antigen stabilized at the cell surface in thepresence of the peptide ligands is complexed withh g 2m.

To our surprise the HLA-ER antigen is not detectable atthe cell surface of the transfectant cells compared to adistinct expression of HLA-EG on these cells. Thesequence of the HLA-E mRNA expressed in the HLA-ER

transfectant cells was determined to exclude any muta-tion in the phygER expression vector. Direct sequencingof the amplified HLA-E cDNA confirmed the correctsequence encoding R in position 107. An increase inHLA-ER cell surface expression in the presence of itspeptide ligands was not observed or is only marginal(HLA-A23–11). Due to the low expression level of HLA-ER

on X63MER cells binding of BZLF-139–47 cannot be dem-onstrated by this experimental approach. In vitro assem-

bly assays performed with lysates of X63MEG andX63MER cells revealed no difference of the two alleles inpeptide affinity or specificity. In addition, these experi-ments demonstrate that both alleles associate equallywell with g 2m in the presence of the leader sequencepeptide ligand. The results also show that the BZLF-1peptide cannot stabilize an HLA-E trimolecular complexunder the experimental conditions and confirm that thisviral peptide does not bind HLA-E as well as most HLA-class I leader sequence peptides. It has recently beenshown with HLA-E-RMA-S transfectant cells that HLA-Ecomplexes with the BZLF-1 peptide are considerablyless stable and that the CD94/NKG2A receptor cannotinteract with HLA-E complexed to the BZLF-1 peptide[34]. The HLA-E sequence is conserved in primates [35].Interestingly, all known primate MHC-E alleles encodeglycine in amino acid position 107, thus the substitutionby arginine has occurred late in evolution and is specifi-cally found only in the human HLA-E allele. Based on thefrequencies of the two HLA-E alleles it has been pro-posed that this polymorphism has been maintained indiverse populations by stabilizing selection [36].

542 M. Ulbrecht et al. Eur. J. Immunol. 1999. 29: 537–547

The HLA-E polypeptide shares several amino acid sub-stitutions with the mouse Qa-1 antigen in comparisonwith classical MHC class I [37]. A possible homologousfunction for these class Ib molecules is supported by thefinding that both bind signal sequence-derived peptides,in particular the Qdm epitope contained in the leader ofH-2Dd (see Fig. 3, and [23]). Evidence has been providedthat the known Qa-1 alleles present the identical pep-tides, and we show that both HLA-E variants bind thesame peptide [12].

As the X63 cells are derived from the BALB/c mousewhich is of the Qdm+ phenotype they should deliver theH-2Dd signal sequence-derived peptide and thus anHLA-E ligand into the ER. This peptide binds HLA-EG

expressed in RMA-S cells and the complex interactswith the CD94/NKG2A receptor [34]. Nevertheless, theavailability of this peptide apparently does not allow for astable cell surface expression of the HLA-ER antigendetectable by immunofluorescence staining. This sug-gests that the amount of the peptide is probably low orthat the ligand cannot interact efficiently with the assem-bling HLA-E heavy chain/h g 2m complex due to the pres-ence of the mouse homolog Qa-1b.

It has been proposed that MHC class I assembly is atwo-stage process: that an initial binding of suboptimalpeptides is followed by peptide optimization thatdepends on temporary ER retention [38]. The divergentbehavior of the HLA-E variants in trafficking to the cellsurface may be due to a difference in binding low-affinitypeptides that would only allow HLA-EG to be releasedinto the secretory pathway as peptide-receptive mole-cules as described for the mutant HLA-A*0201 moleculeT134K [38]. It is thus conceivable that indirect mecha-nisms account for the low cell surface expression of theHLA-ER molecule. A similar behavior of the two HLA-Ealleles with regard to cell surface expression has beenobserved by transfecting the genes into the human mye-loid cell line K562 (E. H. Weiss et al., manuscript in prep-aration). It is therefore unlikely that an inefficient interac-tion with mouse ER chaperones interferes with cell sur-face transport of HLA-ER. The observed dependence ofthe HLA-ER allele on human tapasin for loading of leadersequence-derived peptides might be more strict thanthat of HLA-EG [32]. For HLA-B alleles such a mechanismaccounts for marked differences in their dependence oncoexpression of human tapasin for high cell surfaceexpression in mouse L cells [39].

The Qa-1 molecule is associated with both § g and + ˇ Tcell recognition [40, 41]. Thus, it is conceivable that theHLA-E antigen controls similar responses in man. Thereported ability of HLA-E to bind viral peptides, althoughat a low affinity, suggests that it might on the one hand

restrict virus-specific CTL responses [24]. On the otherhand the non-polymorphic nature of the HLA-E antigenand its tight control of cell surface expression wouldfavor an exclusive function in recognition by NK cells.Several reports show that HLA-E complexed with HLAclass I signal sequence-derived peptides confers protec-tion to NK cell-mediated lysis via CD94/NKG2A recogni-tion [30, 33, 34, 42, 43]. As the CD94/NKG2A receptor isalso expressed on different subsets of T cells and hasbeen implicated in the modulation of their activity, HLA-Emight be involved in regulating additional effector func-tions of the immune system [44, 45].

In this context it might be of importance that a possiblecompetition of HLA-E with HLA class Ia molecules forh g 2m might regulate HLA-E’s cell surface expressionand thus immune recognition. Therefore, it is conceiv-able that under pathological conditions such as viralinfections or transformation where assembly of HLAclass Ia molecules is impaired, elimination of diseasedcells by NK cells may be inhibited by an increase of HLA-E cell surface expression. Finally, it should be of interestto investigate whether the observed difference in cellsurface expression of HLA-EG and HLA-ER may have bio-logical implications in shaping the NK repertoire.

4 Materials and methods

4.1 Antibodies

mAb used for immunofluorescence and immunoprecipita-tion were as described previously [22]: B9.12.1, W6/32, A1.4(United Biomedical Inc., Hauppauge, NY) and BBM.1. Assecondary reagents dichlorotriazinyl aminofluorescein(DTAF)-conjugated goat anti-mouse IgG F(ab')2 fragments(Dianova, Hamburg, Germany) were used for immunofluo-rescence and alkaline phosphatase (AP)-labeled goat anti-mouse Ig (Tropix, Weiterstadt, Germany) were applied forimmunodetection of Western blots.

4.2 Peptides

The peptides HLA-A23–11 (VMAPRTLVL), HLA-B83–11 (VMA-PRTVLL), HLA-Cw33–11 (VMAPRTLIL), and BZLF-139–47 (SQA-PLPCVL) were custom synthesized and RP-HPLC-purifiedby Dr. Georg Arnold (Genzentrum, Munich). The peptidespurity was G 95 % as assessed by analytical RP-HPLC andtheir identity was confirmed by mass spectrometry.

4.3 Cosmids and generation of expression constructs

The cosmid cd3.14 which encodes the HLA-E*01033 allelewith Gly at amino acid position 107 has been described ear-lier [20]. A Hind III/Bgl II fragment thereof was cloned into a

Eur. J. Immunol. 1999. 29: 537–547 HLA-E: cell surface expression and alleles 543

derivative of the Cos203 vector [46], in which the EBVsequences and Cos site had been deleted by Eca digestiongenerating phygEG. Two strategies were used to generate anHLA-E allele encoding Arg at amino acid position 107. (1) InphygEG an Xmn I/Bgl II fragment spanning amino acid posi-tion 7 to 141 was replaced by the corresponding fragment ofthe cDNA clone LG2-C1 isolated from the B-LCL LG2 (whichis homozygous for HLA-E*01020 containing an Arg at aminoacid position 107 and AAA at codon 77) [47]. The resultingconstruct phygcER, contained no intron 2 and a silent substi-tution (from AAC to AAA) in codon 77. (2) The same Xmn I/Bgl II fragment of phygEG was replaced by the correspond-ing fragment obtained by restriction of an amplificat gen-erated with the primers E § (1)5’ (5’-GGCCTCTACCGGG-AGTAGAGA-3’, binding to intron 1) and E § (2)3’ (3’-GAGAGTAGCCCTGTGGACCCT-3’, binding to intron 3) fromthe genomic DNA of the B-LCL LG2. The resulting constructwas designated phygER. Both constructs phygcER andphygER encode HLA-E transcripts with Arg at amino acidposition 107. HLA-E alleles encoding R107 will be called fur-ther on HLA-ER and those with 107G HLA-EG.

4.4 Northern blot analyses

RNA from cell lines was prepared by acid phenol extraction[48]. A total of 10 ? g of total RNA was separated on formal-dehyde agarose gels and transferred to Hybond-N+ mem-branes (Amersham Buchler, Braunschweig, Germany). Adigoxigenin-labeled probe for exon 3 of HLA-E was gener-ated by PCR from cd3.14 as the template with the primersE § (2)5* (5’-CCAAAATGCCCACAGGGTGGT-3’) and E § (2)3’by using the PCR-Dig-labeling kit of Boehringer Mannheim(Mannheim, Germany). Hybridization was performed follow-ing the protocol of Engler-Blum et al. [49] and visualized bychemiluminescence with CSPD® (Tropix) as the substrate.

4.5 Cell culture and transfectants

The transfection of X63 cells with either h g 2m (X63M), h g 2mand the HLA-EG encoding cosmid cd3.14 (X63MEG), orh g 2m and HLA-B*2705 (X63MB27) has been describedelsewhere [20]. X63M cells were further transfected by stan-dard electroporation procedures (Gene Transfector 300; BTXInc., San Diego, CA) with 20 ? g of either phygEG, phygcER orphygER linearized with Sal I. Transfectant clones (X63MEG

and X63MER) were selected in medium containing hygromy-cin B. In addition the same protocol was used to introducethe Cla I-linearized cosmid cd3.14 into X63 cells by electro-poration and to isolate transfectant clones (X63EG) resistantto G418.

The expression of soluble HLA-E and HLA-A2 molecules inD. melanogaster cells has been described elsewhere [24,50].

4.6 Reverse transcription-PCR and sequencing

Reverse transcription was performed on 1 ? g of RNA iso-lated from X63MEG and X63MER transfectants usingExpandTM reverse transcriptase (Boehringer Mannheim) in atotal volume of 20 ? l according to the manufacturer’s recom-mendations. The primers E5’UT (5’-CCACCATGGTAGA-TGGAACCCTC-3’) and E 3’UT (5’-GCTTTACAAGCTGTG-AGACTC-3’) were used to amplify the entire coding regionof the HLA-E cDNA by standard PCR. PCR products werepurified by use of the High PureTM PCR Product Purifica-tion Kit (Boehringer Mannheim) and sequenced using theDyeDeoxy Terminator Cycle Sequencing Kit (Perkin Elmer)and the following primers: E 5’UT, E 3’UT, A47 (5’-GAGACACGGAGCCCAGGGAC-3’, aa 63–69) and A46 (5’-GCTCGGGTAGCCCCTCATGCT-3’, aa 262–269).

4.7 Thermal stability assay for soluble HLA class Imolecules

The themostability assay performed on supernatants oftransfected SC2 D. melanogaster cells which were inducedto express soluble dimers of either HLA-E and h g 2m dimersor HLA-A2 and h g 2m diluted 1:20 with PBS and either sup-plemented with NP40 to 1 % (w/v) or used directly has beendescribed elsewhere [24].

4.8 Pulse chase experiments andimmunoprecipitations

X63MEG, X63MER and X63MB transfectants were kept at2.5 × 107/ml in 4.5 ml methionine- and cysteine-free DMEMsupplemented with 2 mM L-glutamine, 10 % dialyzed FCS,1 mM sodium pyruvate, 50 U/ml penicillin, 50 ? g/ml strepto-mycin (all Bioproducts) and 2.5 × 10–5 M 2-ME for 1 h at37 °C and 5 % CO2. Thereafter, each reaction was supple-mented with 500 ? Ci of Trans35S-label (ICN, Eschwege, Ger-many). After 15 min of labeling the reactions were chased.Aliquots of 5.7 × 106 cells were removed after 0-, 1- and 2-hchase, washed twice with 7 ml PBS/5 % FCS and placed onice. The cells were lysed for 30 min at 4 °C in 600 ? l of TBS(10 mM Tris/HCl pH 7.5, 150 mM NaCl) supplemented with1 % NP40, 1 mM PMSF and 1 % (v/v) TrasylolTM. Afterremoval of debris by centrifugation 60 ? l of FCS were addedand lysates were precleared overnight at 4 °C by adding 4 ? lor normal rabbit serum and 20 ? l of protein A-Sepharose.After further preclearing at 4 °C for 1 h with 3 ? g of purifiedIgG2a mAb (ICN) and 20 ? l of protein A-Sepharose half ofthe lysates were immunoprecipitated by the addition of150 ? l of A1.4 followed by incubation for 40 min at 4 °C andthen the addition of 30 ? l of protein A-Sepharose followed byincubation for 20 min at 4 °C. Immunoprecipitates wererecovered by centrifugation and washed five times with 1 mlof 0.2 % NP40/0.025 % SDS/TBS. They were divided in twoand either mock or Endo Hf (2 kU per aliquot, New EnglandBiolabs, Schwabach, Germany) digested according to the

544 M. Ulbrecht et al. Eur. J. Immunol. 1999. 29: 537–547

manufacturer’s protocol. Aliquots equivalent to 2.3 × 106

cells were analyzed by SDS-PAGE using 10 % gels. Gelswere treated with AmplifyTM (Amersham Buchler), dried andfluorographed at –80 °C.

4.9 Assembly assays

A total of 6.5 × 107 X63MEG and X63MER transfectantsstarved for 1 h in methionine- and cysteine-free mediumwere labeled for 1 h with 1.2 mCi Trans35S-label (ICN) in atotal volume of 12 ml as described above. Cells were col-lected, washed twice with PBS/5 % FCS and divided intoaliquots of 6.5 × 106 cells. Each aliquot was lysed for 30 minat 4 °C in 300 ? l of lysis buffer (50 mM Tris/HCl pH 7.5,150 mM NaCl, 5 mM EDTA, 5 mM iodoacetamide and 2 mMPMSF) in the presence or absence of different concentra-tions of peptide or in the presence of mAb B9.12.1 (10 ? g).Following removal of debris by centrifugation supernatantswere heated for 2 min at 45 °C except for one unsupple-mented aliquot of each transfectant. Thereafter 15 ? l of pro-tein A-Sepharose were added to the supernatants exceptthose containing mAb B9.12.1 to allow preclearing overnightat 4 °C. The next day lysates were supplemented with BSAto a final concentration of 1 % (w/v) and standard immuno-precipitation was performed using 5 ? g of B9.12.1 and 15 ? lof protein A-Sepharose for each aliquot. The unsupple-mented, unheated aliquots were subjected to a secondround immunoprecipitation by mAb A1.4. Samples wereanalyzed by SDS-PAGE using 10 % gels. Gels were treatedwith AmplifyTM (Amersham Buchler), dried and fluoro-graphed at –80 °C.

4.10 Immunofluorescence staining and flow cytometryanalysis

X63 transfectants grown to subconfluency were incubatedat 1×105–2× 105 cells per 96-well microtiter plate well with150 ? M peptides or 1 ? M h g 2m for 6 h at 37 °C in completemedium prior to staining by indirect immunofluorescencewith mAb B9.12.1 and A1.4 and flow cytometry analysis asdescribed elsewhere [22].

Acknowledgment: This work was supported by theDeutsche Forschungsgemeinschaft (SFB 217).

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Correspondence: Elisabeth H. Weiss, Institut für Anthropo-logie und Humangenetik, Richard Wagner Str. 10/I, D-80333Munich, GermanyFax:+49-89-5203-266e-mail: e.h.weiss — lrz.uni-muenchen.de

Eur. J. Immunol. 1999. 29: 537–547 HLA-E: cell surface expression and alleles 547