ARTHRITIS & RHEUMATISMVol. 56, No. 7, July 2007, pp 2232–2243DOI 10.1002/art.22725© 2007, American College of Rheumatology

Two HLA–B27 Alleles Differently Associated WithSpondylarthritis, B*2709 and B*2705, Display Similar

Intracellular Trafficking and Oligomer Formation

Benoit Giquel,1 Sophie Carmouse,1 Celine Denais,1 Aıcha Cherfa,1 Maria-Sole Chimenti,2

Ingrid Fert,1 Cecile Hacquard-Bouder,2 Maxime Breban,2 and Claudine Andre1

Objective. To examine whether and to what extentthe intracellular trafficking features of HLA–B*2705,which is associated with the development of spondylar-thritis (SpA), differ from those of HLA–B*2709 andHLA–B*0702, which are not associated with SpA.

Methods. HeLa cells were transfected with com-plementary DNA encoding for HLA–B proteins fused toRenilla luciferase or yellow fluorescent protein. Thesubcellular distribution of properly folded andunfolded/misfolded HLA–B proteins was examined byflow cytometry and confocal microscopy of cells labeledwith ME1 and HC-10 antibodies, respectively. HLA–B/HLA–B interactions were monitored in endoplasmicreticulum (ER)– and plasma membrane–enriched sub-cellular fractions, by bioluminescence resonance energytransfer (BRET).

Results. All 3 HLA–B alleles displayed a similardistribution pattern (properly folded heavy chain at thecell surface, unfolded/misfolded proteins only in thecytoplasm). By means of BRET, we provided evidencethat both HLA–B*2705 and HLA–B*2709 formed moreoligomers in the ER and the plasma membrane than didHLA–B*0702. The propensity of HLA–B*2705 to form

oligomers in the ER was partly attributable to residueCys67 of the molecule. For all 3 alleles, increasedexpression of HLA–B proteins was associated withintracytoplasmic accumulation of unfolded/misfoldedproteins and intracellular vesicles, probably corre-sponding to expanded ER–Golgi intermediate compart-ments, in which these proteins accumulated togetherwith the stress sensor BiP.

Conclusion. Our results suggest that the differ-ence in disease susceptibility conferred by HLA–B*2705and HLA–B*2709 cannot be explained by their differentpropensity to form dimers or misfolded proteins, thuspresumably implicating other, still unknown factors.

The class I major histocompatibility complex(MHC) allele HLA–B27 is strongly associated with thedevelopment of a variety of chronic inflammatory rheu-matic disorders known as spondylarthritis (SpA) (1–4).A direct role of HLA–B27 in the disease process wasfurther demonstrated by the observation that in trans-genic rats and mice that overexpress HLA–B27, diseaseresembling SpA develops (5). Although it has beenshown that dendritic cells from rats overexpressingHLA–B27 display a diminished capacity to form conju-gates with and stimulate T cells (6), the exact role ofHLA–B27 in such dysfunction remains unknown.

In the last few years, attention has focused on thepeculiar immunobiologic behavior of the HLA–B27molecule (7,8). Hence, it has been shown that folding ofthe HLA–B27 heavy chain is remarkably slow, probablyleaving unpaired residue Cys67 of the molecule exposedand favoring the formation of disulfide-bonded HLA–B27 heavy chain dimers in the endoplasmic reticulum(ER) (9,10). At least part of these molecular species arethought to be misfolded �2-microglobulin (�2m)–freedimers that cannot even get through the ER–Golgibarrier. Furthermore, recent findings showed that such

Supported by a grant from the “Societe Francaise de Rhu-matologie.” Dr. Giquel’s work was supported by a fellowship from theFrench Ministry of Research and Technology.

1Benoit Giquel, PhD, Sophie Carmouse, BS, Celine Denais,MS, Aıcha Cherfa, MS, Ingrid Fert, BS, Claudine Andre, PhD: InstitutCochin, Universite Paris Descartes, CNRS (UMR 8104), Paris,France. Inserm, U567, Paris, France; 2Maria-Sole Chimenti, MD,Cecile Hacquard-Bouder, MD, PhD, Maxime Breban, MD, PhD:Institut Cochin, Universite Paris Descartes, CNRS (UMR 8104), Paris,France. Inserm, U567, Paris, France. AP-HP, Service de Rhumatolo-gie, Hopital Ambroise Pare, UVSQ, Boulogne-Billancourt, France.

Address correspondence and reprint requests to ClaudineAndre, PhD, Institut Cochin, Departement d’Immunologie, 27 Rue duFaubourg Saint Jacques, Pavillon Hardy A, 75014 Cedex 14 Paris,France. E-mail: claudine.andre@cochin.inserm.fr.

Submitted for publication August 3, 2006; accepted in revisedform March 29, 2007.

2232

misfolded HLA–B27 dimers bind to the protein BiP(GRP78), supporting the idea that these dimers couldbe prone to an unfolded protein response (UPR) thatcould interfere with cell function in the context ofinflammation (10–13). Moreover, misfolded �2m-freedimers, which are thought to be formed as a conse-quence of endocytic recycling of HLA–B27/�2m het-erodimeric complexes, were identified at the cellsurface (14). The production of HLA–B27 dimers wasobserved to increase as a function of HLA–B27 over-expression (11).

Several findings suggest that the pathogenicity ofHLA–B27 could be facilitated by HLA–B27 overexpres-sion. Indeed, only transgenic rats that carry a highHLA–B*2705 gene copy number develop a variety ofpathologic effects closely resembling those associatedwith SpA (15). Moreover, the expression of HLA–B*2705 was shown to be increased on peripheral bloodmononuclear cells from patients with SpA, as comparedwith healthy controls (16). Last, recent findings showedthat the expression levels of �2m might play an impor-tant role in the pathogenic effects of HLA–B27 (17).

Currently, 27 HLA–B27 subtypes have beenidentified. The majority of the most frequent subtypes,including HLA–B*2705, have been shown to be associ-ated with SpA (18). One of the few HLA–B27 subtypesnot associated with SpA is HLA–B*2709 (19), theencoding sequence of which differs from that of HLA–B*2705 by a single substitution at amino acid residue116. The difference in disease susceptibility borne by the2 HLA–B27 subtypes remains unexplained today, and itis also not known whether and to what extent theirimmunobiologic characteristics may differ.

Resonance energy transfer (RET)–based tech-niques allow the analysis of in situ protein–proteininteractions. By means of fluorescence RET (FRET),which is based on nonradioactive energy transfer be-tween a fluorescent energy donor and a fluorescentenergy acceptor, it has been demonstrated that severalclass I MHC molecules can form oligomers/clusters insitu (20–23). These can be detected in the ER at steadystate (20) and, when formed in the plasma membrane,may be essential to the activation of T cells (21). Untilnow, HLA–B27 dimers and multimers have been mainlyassessed in vitro using metabolic labeling, immunopre-cipitation, and Western blot analyses, which allow dem-onstration of only strongly associated protein complexes.However, in situ analysis of HLA–B27 oligomer forma-tion has not yet been achieved.

Here, we used the bioluminescence RET(BRET) technique (24,25) to monitor HLA–B/HLA–B

interactions in the ER and the plasma membrane of cellsoverexpressing HLA–B*2705, HLA–B*2709, and HLA–B*0702. According to this technique, a luminescentenergy donor (Renilla luciferase [Rluc]) oxidizes itssubstrate, coelenterazine, releasing photons. The fluo-rescent energy acceptor (yellow fluorescent protein[YFP]), when in close enough physical proximity to thedonor (�100 Å), can thus be excited to a higherenergetic state, emitting photons with longer wave-lengths. The key advantage of BRET over FRET is thevery high signal-to-noise ratio gained from luminescencedetection, which is unaffected by photobleaching orother optical effects and allows protein interactions tobe detected even at physiologic expression levels (26).

Using HLA–B*0702Y67C and HLA–B*2705C67S mutants, we further examined, by means ofBRET, the importance of Cys67 in oligomer formation.Finally, we made use of the HLA–B-YFP fusion proteins(HLA–BYFP) to study the distribution of misfolded/unfolded and properly folded HLA–B proteins and theirinteraction with the BiP protein in intact cells.

MATERIALS AND METHODS

Antibodies. HC-10 is an IgG2a mouse monoclonalantibody (mAb) recognizing HLA–B free heavy chains andhomodimers (27). ME1 is an IgG1 mouse mAb recognizingproperly folded HLA–B27 and HLA–B7 proteins (28). Anti-BiP (GRP78) and anticalnexin antibodies were purchasedfrom Stressgen (San Diego, CA), anti–human CD55 (anti-hCD55) antibodies were from R&D Systems (Abingdon, UK),anticalreticulin antibodies were from Affinity BioReagents(Golden, CO), anti–ER–Golgi intermediate compartment–53(anti–ERGIC-53) mAb were from Alexis Biochemicals(Lausen, Switzerland), anti-Rab6 antibodies were from SantaCruz Biotechnology (Santa Cruz, CA), anti-CD71 antibodieswere from Zymed (Burlingame, CA), fluorescein isothiocya-nate (FITC)–conjugated anti–HLA–B27 mAb (clone FD707-9E1E10) were from One Lambda (Canoga Park, CA), anti–green fluorescent protein (anti-GFP) antibodies were fromInvitrogen (Cergy-Pontoise, France), and Cy5-labeled anti-mouse IgG antibodies were from KPL (Gaithersburg, MD).

Plasmid constructions. HLA–B*2705 and HLA–B*0702 coding sequences were amplified by reversetranscription–polymerase chain reaction (RT-PCR) of HLA–B*2705– and HLA–B*0702–transgenic rat spleen RNA (L33.3and 120-4L, respectively) (15), using sense and antisenseprimers harboring Bam H1 sites and overlapping the start andstop codons, respectively. The amplified fragments were theninserted into the Bam H1 cloning site of the pcDNA3 expres-sion vector, in frame with the coding sequence of Rluc orYFP, to give the pcDNA3–HLA–B*2705Rluc, pcDNA3–HLA–B*2705YFP, pcDNA3–HLA–B*0702Rluc, and pcDNA3–HLA–B*0702YFP constructs. The stop codons were deleted by site-directed mutagenesis using a QuickChange Site-DirectedMutagenesis kit (Stratagene, La Jolla, CA). The HLA–

INTRACELLULAR TRAFFICKING OF HLA–B*2705 AND HLA–B*2709 2233

B*2705C67S, HLA–B*2705D116H (HLA–B*2709), andHLA–B*0702Y67C mutants were obtained by site-directedmutagenesis. To prepare the pcDNA3 construct encoding theFas/APO-1 (CD95) protein fused to Rluc (PcDNA3-FasRluc),the Fas coding sequences were amplified by RT-PCR of totalRNA from C57Bl/6 mouse liver and inserted into thepcDNA3–Rluc cloning site as described above. The pcDNA3–Rluc and pcDNA3–YFP constructs were generously providedby Dr. R. Jockers (Department of Cellular Biology, CochinInstitute, Paris).

Cell cultures and transfections. HeLa cells were grownin Dulbecco’s modified Eagle’s medium supplemented with5% (volume/volume) fetal bovine serum, 100 units/ml penicil-lin, and 0.1 mg/ml streptomycin. For transient transfections,2 � 106 cells were seeded in 100-mm–diameter dishes, and thetransfections were performed 24 hours later with 5 �g of eachplasmid/dish using the classic calcium phosphate transfectionprotocol (29). The cells were harvested 24 hours after trans-fection by means of trypsin–EDTA and reseeded (100,000cells/cm2) in Lab-Tek Permanox chamber slides (Nunc, Naper-ville, IL) for confocal microscopy, or harvested 48 hours aftertransfection and immediately used for flow cytometry orsubcellular fractionation.

Subcellular fractionation. Approximately 3 � 107 cellswere washed 3 times with phosphate buffered saline (PBS) andfinally resuspended in 1 ml of ice-cold hypotonic buffer (20mM HEPES, pH 7.4, 2 mM EDTA) containing proteaseinhibitors (Complete; Roche, Basel, Switzerland). The celllysate was homogenized with 50 strokes at 300 revolutions perminute in a mechanical homogenizer. Cellular debris andnuclei were removed by centrifuging at 1,000g for 5 minutes.Sucrose was added to the supernatant to obtain a finalconcentration of 0.2M sucrose. The supernatant was thenplaced on top of a discontinuous sucrose step gradient com-posed of the following final molar concentrations: 2M (1 ml),1.75M, 1.5M, 1.35M, 1.2M, and 0.9M (all at 1.8 ml), and finally0.5M (1 ml). All sucrose solutions were prepared in thehypotonic buffer. The samples were centrifuged for 16 hours at28,000 rpm in a Beckman SW41 rotor (Beckman Coulter,Villepinte, France), and 24 fractions (500 �l each) werecollected starting from the bottom of the tube. All manipula-tions were performed at 4°C.

Flow cytometric analysis. For assessment of �2m-bound or �2m-free HLA–B fusion proteins at the cell surface,cells were incubated with ME1 or HC-10 antibodies, respec-tively, for 20 minutes in PBS. The cells were then washed twicewith PBS, incubated for 20 minutes at 4°C with Cy5-labeledanti-mouse IgG antibodies, and again washed twice with PBS.To assess intracellular expression of HLA–B fusion proteins,transfected cells were first fixed with 4% paraformaldehyde(10 minutes at room temperature), washed twice with PBS, andpermeabilized with PBS containing 0.5% saponin (PBS-S) (10minutes at room temperature). The permeabilized cells werethen labeled with HC-10 or ME1 antibodies as indicated fornonpermeabilized cells, with incubations and washings per-formed in PBS-S. All labeling procedures were performed at4°C. The antibodies were used at a final concentration of 10�g/ml. The analyses were performed using a Cytomics FC 500flow cytometer (Beckman Coulter) and WinMDI software(online at: http://facs.scripps.edu/software.html).

Confocal microscopy. Cells grown in Lab-Tek chamberslides were washed twice in PBS, fixed with 4% paraformalde-hyde, and then permeabilized or not permeabilized withPBS-S, as described above for the visualization of intracellularand/or membranous proteins. Incubations with HC-10, ME1,FITC-conjugated anti–HLA–B27 mAb, anticalreticulin, anti-BiP, anti-Rab6, anti-CD71, and anti–ERGIC-53 antibodiesand with Cy5-labeled secondary antibodies were performed asdescribed above. Mounting was performed in Vectashieldmedium (Vector, Burlingame, CA) containing 4�,6-diamidino-2-phenylindole. Observations were performed with a confocallaser-scanning microscope (TCS SP2; Leica, Cambridge, UK),using a 63� oil-immersion objective. Quantification histo-grams were obtained with ImageJ software (online at: http://rsb.info.nih.gov/ij/).

Microplate BRET assays. Subcellular fractions (100�l) were distributed in a 96-well microplate, and coelentera-zine (h form; Interchim, Montlucon, France) was rapidlyadded to each well at a final concentration of 5 �M. Readingswere immediately performed at 25°C with a Fusion lumino/fluorometer (Perkin-Elmer, Waltham, MA), which allowedsequential integration of luminescence signals detected with 2filter settings (for the Rluc filter, mean � SEM 485 � 10 nm;for the YFP filter, 530 � 13 nm). Results are represented asthe BRET ratio, which is defined as follows: [(emission at510–590 nm) � (emission at 440–500 nm) � cf]/(emission at440–500 nm), where cf corresponds to emission at 510–590nm/emission at 440–500 nm for the Rluc construct expressedalone.

Immunoprecipitation assays. The subcellular fractionswere lysed for 30 minutes at 4°C in 20 mM Tris (pH 7.4), 150mM NaCl, 2 mM CaCl2, 0.5% Nonidet P40 buffer containingprotease inhibitors (Complete; Roche). Cell debris was re-moved by centrifugation at 17,500g for 20 minutes at 4°C. Thelysates were then incubated for 2 hours at 4°C with anti-GFPantibodies (5 �g/ml of lysate) and tumbled with proteinG–Sepharose for 2 hours at 4°C. The beads were then washed5 times with lysis buffer and finally suspended in 100 �l of PBS.For quantification of the immunoprecipitated Rluc-labeledproteins, samples of the bead suspension (50 �l) and the lysate(30 �l) were treated, as described above, with coelenterazine,and emission at 440–500 nm (Rluc filter) was immediatelymeasured in the Fusion lumino/fluorometer (Packard).

Western blot analysis. Samples (7.5 �l) of each frac-tion were subjected to sodium dodecyl sulfate–polyacrylamidegel electrophoresis and electrotransferred onto polyvinylidenedifluoride membranes. Blocking was for 1 hour at roomtemperature with PBS–Tween 20 (PBST) buffer (1� PBS,0.01% Tween 20) containing 5% dry milk, followed by incu-bation in PBST containing 2.5% dry milk for 16 hours at 4°Cwith anti-hCD55 antibodies (plasma membrane marker) orwith anticalnexin antibodies (ER marker). After being washedwith PBST, the blot was incubated for 1 hour at roomtemperature with a 1:10,000 dilution of anti-mouse or anti-rabbit IgG horseradish peroxidase–conjugated antibodies(Amersham, Buckingham, UK) in PBST containing 2.5% drymilk. Band detection was performed by enhanced chemilumi-nescence (Amersham).

Statistical analysis. To assess statistically significantdifferences between data sets, we used ordinary unpairedone-way analysis of variance with Bonferroni adjustment for

2234 GIQUEL ET AL

multiple comparisons. P values less than 0.05 were consideredsignificant.

RESULTS

Subcellular distribution of the HLA–B*2705,HLA–B*0702, and HLA–B*2709 fusion proteins. All ofthe studies described here were performed on HeLacells that were transfected with plasmids encodingHLA–B heavy chains fused at their C-terminal ending toRluc or YFP (Figure 1A). A first series of experimentswas conducted to verify whether fusion of the HLA–Bproteins to Rluc (HLA–BRluc) or YFP (HLA–BYFP)could alter their normal folding and exportation to theplasma membrane. HC-10 and ME1 antibodies wereused to discriminate between �2m-free and �2m-boundHLA–B proteins, respectively.

The HLA–B*2705YFP, HLA–B*0702YFP, andHLA–B*2709YFP fusion proteins displayed similar sub-cellular distribution patterns. The distribution of theHLA–B*2705YFP fusion protein is shown in Figures1Ba–c. As for the wild-type HLA–B proteins, the fusionproteins were expressed at the cell surface and wereabundant in the ER.

The folding state of the HLA–B proteins ex-pressed at the surface of HLA–B*2705YFP–, HLA–B*0702YFP–, and HLA–B*2709YFP–transfected cells wasexamined by flow cytometric analysis of nonpermeabi-lized cells that were labeled with either ME1 or HC-10antibodies (Figures 2b–d and e–g, respectively). Thesubcellular distribution of the ME1- and HC-10–reactiveHLA–B proteins was further visualized by confocalmicroscopy (for HLA–B*2705, Figures 1Bd–f and g–i,respectively; for HLA–B*2709 and HLA–B*0702, re-sults not shown). The results of the microscopic and flowcytometric analyses showed that for all 3 HLA–B sub-species, properly folded proteins were mainly expressedat the plasma membrane (Figures 1Bd–f and 2b–d), withmuch less expression in the ER (Figures 1Bd–f). Incontrast, �2m-free unfolded or misfolded proteins wereabundant in the ER (Figures 1Bg–i) but were undetect-able at the plasma membrane level (Figures 2e–g).

In HLA–B*2705Rluc–transfected cells, fusionproteins were abundant in the ER in a �2m-free form(Figures 1Ca–c) and were exported to the cell surface(Figures 1Ca and c), where they were expressed in aproperly folded form (Figure 1Dc). Moreover, the abun-dance of properly folded HLA–B proteins at the cellsurface appeared to be similar for HLA–B*2705YFP–and HLA–B*2705Rluc–transfected cells (Figures 1Daand 1Dc, respectively). Misfolded HLA–B*2705Rluc pro-

Figure 1. A, Schematic representation of the HLA–B constructs. EC �extracellular; TM � transmembrane; IC � intracellular; HC � heavychain. Ba–c, HLA–B–yellow fluorescent protein (YFP) fusion proteinsbehave as their wild-type counterparts. Fixed and permeabilized HLA–B*2705YFP–transfected cells were labeled with anticalreticulin (Clrt) andCy5-labeled secondary antibodies for visualization of the endoplasmicreticulum (ER). Confocal microscopy of HLA–B*2705YFP (a) and Clrt(b) labeling showed that the fusion proteins are abundant in the ER,and that some of them are exported to the cell surface. Bd–i,Subcellular distribution of correctly folded and unfolded/misfoldedHLA–B*2705YFP proteins as revealed by confocal microscopy. Thephotomicrographs show fixed permeabilized HLA–B*2705YFP–transfected cells that were labeled with ME1 (Bd–f) or HC-10 (Bg–i)monoclonal antibodies (mAb) and Cy5-labeled secondary antibodies.C and D, HLA–B*2705–Renilla luciferase (Rluc) behave as HLA–B*2705YFP. HLA–B*2705Rluc–transfected cells were labeled with flu-orescein isothiocyanate (FITC)–conjugated anti–HLA–B27 mAb tovisualize HLA–B*2705Rluc proteins at the cell surface (Ca), then werefixed, permeabilized, and labeled with HC-10 mAb, as described abovefor revelation of �2-microglobulin–free unfolded/misfolded HLA–B*2705Rluc, by means of confocal microscopy (Cb). Exposure ofproperly folded HLA–B*2705Rluc proteins at the cell surface (D) wasdemonstrated by flow cytometric analysis of nonpermeabilized HLA–B*2705Rluc–transfected cells that were labeled with ME1 (Dc) or withmouse IgG1 (isotypic control; Db) and Cy5-labeled secondary anti-bodies. ME1-labeled HLA–B*2705YFP–transfected cells (Da) wereanalyzed for comparison. Results in B–D are representative of at least3 individual series of experiments, each of which was performed for adifferent transfection assay. Arrows indicate HLA–*2705 proteinsexposed at the cell surface. Bars in B and C � 10 �m.

INTRACELLULAR TRAFFICKING OF HLA–B*2705 AND HLA–B*2709 2235

teins were not detected at the cell surface (results notshown).

In conclusion, the Rluc and YFP moieties of thefusion proteins did not hamper interaction of theHLA–B heavy chains with �2m nor their exportation tothe plasma membrane.

Analysis of HLA–B protein–protein interactionsin the ER and plasma membrane by means of the BRETtechnique. The excessive formation of heavy chaindimers/multimers in the ER is generally considered to bea hallmark of the HLA–B*2705 subtype that could leadto dysfunction of antigen-presenting cells that overex-press HLA–B*2705 (7,9,10,30). However, it is not wellknown whether and to what extent the HLA–B*2709

subtype, which is not associated with SpA, forms heavychain oligomers. Therefore, we monitored the homolo-gous HLA–B heavy chain interactions of both HLA–B27subtypes, using HLA–B*0702 as control, at the level ofthe ER and the plasma membrane by means of theBRET technique (Figures 3A–C). Because the BRETtechnique is not well adapted to perform such studies inintact cells by microscopy, we performed BRET mea-surements on ER and plasma membrane fractions ob-tained by subcellular fractionation, using discontinuoussucrose gradients as described previously (31,32).

For these experiments, we used cells cotrans-fected with HLA–B*0702Rluc and HLA–B*0702YFP

(HLA–B*0702Rluc/YFP), HLA–B*2705Rluc and HLA–B*2705YFP (HLA–B*2705Rluc/YFP), or HLA–B*2709Rluc

and HLA–B*2709YFP (HLA–B*2709Rluc/YFP). Cells co-transfected with HLA–B*2705YFP and the apoptosis-inducing receptor FasRluc, which is expressed at theplasma membrane, were used as controls for plasmamembrane labeling and BRET signaling specificity. Cal-nexin and CD55 were used as markers for the ER(fractions 5–11) and the plasma membrane (fractions13–20), respectively (Figure 3A).

Measurement of Rluc activity in each fractionshowed that the HLA–B*0702Rluc, HLA–B*2705Rluc,and HLA–B*2709Rluc proteins were abundant in boththe ER- and the plasma membrane–enriched fractions(Figure 3B). As expected, FasRluc was much more abun-dant in the plasma membrane–enriched fractions than inthe ER-enriched fractions.

BRET measurements were performed on eachfraction, to examine interactions between the fusionproteins (Figure 3C). Significant BRET signals wereraised by all 3 HLA–B subtypes, and, moreover, thesesignals were higher in the ER- than in the plasmamembrane–enriched fractions. In contrast, BRET sig-nals measured in fractions of FasRluc/HLA–B*2705YFP–transfected cells were low (for fractions 6–16, P � 0.001versus all 3 HLA–B subtypes), demonstrating thatBRET signaling was specific for HLA–B/HLA–B inter-actions and was not attributable merely to overexpres-sion of fusion proteins.

Interestingly, BRET signals emitted by the sub-cellular fractions of HLA–B*2705Rluc/YFP–transfectedcells were considerably higher than those emitted bysubcellular fractions of HLA–B*0702Rluc/YFP–transfected cells, in both the ER and plasma membranefractions (for fractions 5–19, P � 0.01 versus HLA–B*0702) (Figure 3C). The BRET signal pattern ofHLA–B*2709 also significantly differed from that ofHLA–B*0702 (for fractions 5–19, P � 0.01) but largely

Figure 2. Characterization of HLA–B*0702, HLA–B*2705, andHLA–B*2709 protein expression at the cell surface. Fixed nonperme-abilized HLA–B*0702YFP–, HLA–B*2705YFP–, and HLA–B*2709YFP–transfected cells were labeled with ME1 or HC-10 mAb for revelationof �2-microglobulin (B2m)–bound correctly folded (b–d) or �2m-freeunfolded/misfolded (e–g) HLA–B proteins. Analysis of HLA–BYFP

expression at the cell surface was performed by flow cytometric gatingon HLA–BYFP–expressing cells, as illustrated for HLA–B*2705 (a).Cells were labeled with nonspecific mouse IgG1 or IgG2a and Cy5-labeled secondary antibodies as isotypic controls for ME1 and HC-10,respectively (b–g; grey peaks). Results shown are representative of atleast 3 individual series of experiments, each of which was performedfor a different transfection assay. See Figure 1 for other definitions.

2236 GIQUEL ET AL

superposed onto that obtained for HLA–B*2705 (forfractions 5–20, P � 0.05).

We next examined whether the difference be-tween BRET signals might reflect a difference in oli-gomerization. We prepared lysates from fractions 11(ER-enriched, high BRET signals) and 18 (plasmamembrane–enriched, low BRET signals) of HLA–B*0702Rluc/YFP–, HLA–B*2705Rluc/YFP–, and HLA–B*2709Rluc/YFP–transfected cells, then immunoprecipi-tated the HLA–BYFP proteins from these lysates bymeans of anti-GFP antibodies, and finally quantifiedHLA–BYFP/HLA–BRluc complexes in the immunopre-cipitates by measuring the luminescence emitted by Rluc(Figure 3D). The results provided evidence that HLA–Bcomplexes were more abundant in the ER than in theplasma membrane, and, moreover, that HLA–B*2705and HLA–B*2709 were significantly more abundantthan HLA–B*0702 in both subcellular fractions.

Having shown that use of the BRET techniqueallows the demonstration of oligomer formation in sub-cellular fractions, we used this technique to examinewhether and to what extent substitution of Cys67 forSer67 in the HLA–B*2705 fusion proteins (HLA–B*2705C67S) or substitution of Tyr67 for Cys67 in theHLA–B*0702 fusion proteins (HLA–B*0702Y67C) af-fects oligomer formation (Figure 3C).

The distribution pattern of these mutants amongthe fractions very closely resembled the patterns of thenonmutated HLA–B proteins, i.e., considerable expres-sion in both the ER- and the plasma membrane–enriched fractions (data not shown). However, bothtypes of mutation resulted in changes in BRET signaling(Figure 3C). Indeed, BRET values were significantlylowered by the C67S mutation in HLA–B*2705 (forfractions 5–10, P � 0.001; for fractions 11–17, P � 0.01)while BRET values appeared to be increased by theY67C mutation in HLA–B*0702 (for fractions 5–8 and13–16, P � 0.05). Nevertheless, BRET signaling ob-served with HLA–B*0702Y67C remained lower thanthat observed with HLA–B*2705 (for fractions 5–10,P � 0.01; for fractions 15–17, P � 0.05). The meanBRET values obtained for HLA–B*2705C67S were sig-nificantly higher than those obtained for HLA–B*0702only at the level of fractions 14–19 (P � 0.05).

Formation of misfolded HLA–B*2705, HLA–B*0702, and HLA–B*2709 proteins as a consequence ofoverexpression. By gating on cell populations that dis-played high or low expression levels of HLA–BYFP

proteins (Figure 4Aa), we examined HC-10 labeling inpermeabilized HLA–B*2705YFP–, HLA–B*0702YFP–,and HLA–B*2709YFP–transfected cells, by means offlow cytometry (Figures 4Ab–d). The results provided

Figure 3. Monitoring of HLA–B oligomers in subcellular fractions bymeans of the bioluminescence resonance energy transfer (BRET)technique. Approximately 3 � 107 HLA–B*2705Rluc/YFP–, HLA–B*2709Rluc/YFP–, HLA–B*0702Rluc/YFP–, or FasRluc/HLA–B*2705YFP–cotransfected cells were homogenized and resolved on a discontinuoussucrose gradient to give 24 fractions (500 �l each). A, Identification ofER- and plasma membrane (PM)–enriched fractions. Immunoblotanalyses were performed on 7.5-�l aliquots of each fraction usinganti-CD55 or anticalnexin (Clnx) antibodies for revelation of ER- andPM-enriched fractions, respectively. Results are representative of 15series of analyses, each of which was performed on a different set offractions. B, Distribution of the HLA–BRluc proteins among ER- andPM-enriched fractions. Rluc activity was measured in 30-�l aliquots ofeach fraction, as described in Materials and Methods, for determina-tion of the HLA–BRluc protein distribution profiles. Results arerepresentative of at least 4 sets of triplicate measurements for eachHLA–B subspecies, with each set being performed on a differentfractionation. C, BRET signaling in the subcellular fractions of HLA–B*2705Rluc/YFP–, HLA–B*2709Rluc/YFP–, HLA–B*0702Rluc/YFP–,HLA–B*2705C67SRluc/YFP–, HLA–B*0702Y67CRluc/YFP–, andFasRluc/HLA–B*2705YFP–cotransfected cells. BRET measurementswere performed on 50–100-�l aliquots of each fraction, as described inMaterials and Methods. Results are presented as mBRET units(mBU), where 1 mBU corresponds to the BRET ratio value � 1,000.D, Analysis of HLA–B*0702, HLA–B*2705, and HLA–B*2709 oli-gomers in ER- and PM-enriched fractions. Immunoprecipitation ex-periments were performed on fractions 11 and 18 from HLA–B*0702Rluc/YFP–, HLA–B*2705Rluc/YFP–, and HLA–B*2709Rluc/YFP–cotransfected cells, using FasRluc/HLA–B*2705YFP–cotransfected cellsas a control. Anti–green fluorescent protein antibodies were used toprecipitate HLA–BYFP–bound protein complexes. The measurementof Rluc activity in the immunoprecipitates allowed quantification ofHLA–BYFP/HLA–BRluc complexes. Values in C and D are the mean �SEM of at least 4 independent experiments. � � P � 0.05; �� � P �0.01, versus HLA–B*0702. See Figure 1 for other definitions.

INTRACELLULAR TRAFFICKING OF HLA–B*2705 AND HLA–B*2709 2237

evidence that for each HLA–B subspecies, HC-10 label-ing was much higher in the population that expressedhigh levels of HLA–BYFP than in the population thatexpressed lower levels.

HLA–B overexpression leading to accumulationof misfolded proteins in intracellular vesicles. Examina-tion of HLA–B*2705YFP–, HLA–B*2709YFP–, or HLA–B*0702YFP–transfected cells by microscopy revealed

that strong overexpression of these proteins was accom-panied by the appearance of large vesicles heavily la-beled with YFP (Figure 4B). We further examined theextent to which �2m-free unfolded/misfolded or properlyfolded HLA–B proteins could be present in these vesi-cles. The cells were therefore permeabilized and labeledwith either HC-10 (Figures 4Ba–i) or ME1 (Figures4Bj–r) and examined by confocal microscopy. For all 3HLA–B species (HLA–B*2705, HLA–B*2709, andHLA–B*0702), HC-10–labeled proteins accumulated tovery high densities in the vesicles (Figures 4Ba–c, d–f,and g–i, respectively). Compared with the HC-10 label-ing, the vesicles were labeled only faintly by ME1(Figures 4Bj–l, m–o, and p–r, respectively).

Association of accumulation of unfolded HLA–Bproteins in the intracellular vesicles with increased BiPexpression. It has been reported that in transgenic ratsoverexpressing HLA–B*2705, the expression of BiP isincreased (12,13). Consequently, we labeled permeabil-ized cells expressing HLA–B*2705YFP by means of anti-BiP antibodies and monitored, in cells displaying strongor weak HLA–B*2705YFP expression levels (Figures5Cb1 and Cb2, respectively) and in untransfected cells(Figure 5Cb3), the intensity of BiP labeling (Figures5D). The antibodies faintly labeled untransfected cells(Figures 5Cb3 and D3) and cells with low HLA–B*2705YFP expression levels (Figures 5Cb2 and D2) butmore intensely labeled cells with high HLA–B*2705YFP

expression levels (Figures 5Cb1 and D1). Most interest-ingly, BiP labeling was particularly intense in the intra-cellular vesicles (Figures 5Ca–c).

In an attempt to identify the intracellular vesicles,we labeled permeabilized HLA–B*2705YFP–expressingcells with anti-Rab6 (Figures 6a–c), anti-CD71 (Figures6d–f), anticalreticulin (Figures 6g–i), and anti–ERGIC-53 (Figures 6j–l) antibodies, which are Golgi-,early endosome–, ER- and ERGIC-specific markers,respectively. The vesicles were revealed to be labeledwith the anticalreticulin and the anti–ERGIC-53 anti-bodies only (Figures 6g–i and j–l, respectively).

Similar characteristics were observed for HLA–B*0702– and HLA–B*2709–containing intracellular ves-icles; these vesicles were intensely labeled by anti-BiPantibodies (Figures 5A and B, respectively) and wererecognized by anti–ERGIC-53 antibodies (Figures 6p–rand 6m–o, respectively).

DISCUSSION

Several studies have shown that the slow foldingof HLA–B*2705 is probably the origin of its propensity

Figure 4. Overexpression of HLA–B heavy chains induces formationof unfolded/misfolded HC-10–reactive HLA–B proteins. A, Fixed andpermeabilized cells expressing HLA–B*2705YFP (a and b), HLA–B*0702YFP (c), and HLA–B*2709YFP (d) were labeled with HC-10 forrevelation of unfolded/misfolded HLA–B proteins (solid lines) ormouse IgG2a antibodies (isotypic control; broken lines). Cells wereanalyzed by flow cytometry for HC-10 labeling, gating on low or highHLA–BYFP expression levels, as shown in a. Results are representativeof 3 individual experiments. B, Overexpression of HLA–B heavy chainsinduces formation of intracellular vesicles in which mainly misfoldedHLA–B accumulate. Fixed and permeabilized cells expressing highlevels of HLA–B*2705YFP (a–c and j–l), HLA–B*2709YFP (d–f andm–o), or HLA–B*0702YFP (g–i and p–r) and displaying stronglyYFP-labeled vesicles were labeled with HC-10 and ME1, as shown inFigure 1, for revelation of unfolded/misfolded (a–i) and properlyfolded (j–r) HLA–B proteins. Results are representative of at least 3individual series of experiments, each of which was performed on cellsissuing from a different transfection assay. Arrowheads indicate intra-cellular vesicles. Bars in Ba–c and j–r � 10 �m; bars in Bd–i � 15 �m.See Figure 1 for definitions.

2238 GIQUEL ET AL

to form misfolded proteins and disulfide-linked heavychain dimers in the ER (10,13,33). Although it is thoughtthat these particularities might alter the function of cells,it has not clearly been established to what extent thisbehavior differs from that of HLA–B alleles that are notassociated with SpA, such as the HLA–B*2709 subtypeor the HLA–B*0702 allele, which are structurally veryclose to HLA–B*2705. Here, we have explored theintracellular trafficking of these 3 HLA–B alleles usingexperimental cell models consisting of HeLa cells thatexpress functional HLA–B*2705, HLA–B*2709, or

HLA–B*0702 proteins fused at their C-terminal to Rlucor YFP.

This study is the first in which BRET technologywas used to monitor HLA–B/HLA–B–protein interac-tions in ER- and plasma membrane–enriched subcellu-lar fractions of HLA–BRluc/YFP–transfected cells. For all3 HLA–B subtypes, BRET signals measured in the ERwere much higher than those measured in the plasmamembrane. Furthermore, BRET signals were signifi-

Figure 5. A–C, Strong overexpression of HLA–B*0702, HLA–B*2709, and HLA–B*2705 induces BiP expression and BiP accumu-lation in intracellular vesicles. Fixed and permeabilized HLA–B*0702YFP–, HLA–B*2709YFP–, and HLA–B*2705YFP–transfectedcells displaying large intracellular vesicles were labeled with anti-BiPand Cy5-labeled secondary antibodies and examined by confocalmicroscopy. D, Intensity of BiP labeling (shown in Cb) of cellsdisplaying strong or weak expression of HLA–B*2705YFP (Cb1 andCb2, respectively) and an untransfected cell (Cb3) was calculated pixelper pixel using ImageJ software, to produce the graphs in D1, D2, andD3, respectively. Results are representative of at least 3 individualseries of experiments, each of which was performed on cells issuingfrom a different transfection assay. Arrowheads show intracellularvesicles. Bars in A–C � 10 �m. Fm � fluorescence mean (see Figure1 for other definitions).

Figure 6. a–i, HLA–B heavy chain overexpression–induced vesiclesbelong to the ER network. Fixed and permeabilized HLA–B*2705YFP–transfected cells were labeled with anti-Rab6, anti-CD71,anticalreticulin, or Cy5-labeled secondary antibodies, for revelation ofGolgi apparatus (a–c), early endosomes (d–f), and ER (g–i), byconfocal microscopy. Vesicles are labeled with only anticalreticulinantibodies. j–r, For revelation of ER–Golgi intermediate compart-ments (ERGICs), fixed permeabilized HLA–B*2705–, HLA–B*2709YFP–, or HLA–B*0702–transfected cells were labeled withanti–ERGIC-53 antibodies and Cy5-labeled secondary antibodies (j–l,m–o, and p–r, respectively). Results are representative of at least 3individual series of experiments, each of which was performed on cellsissuing from a different transfection assay. Arrowheads indicate intra-cellular vesicles. Bars � 10 �m. See Figure 1 for other definitions.

INTRACELLULAR TRAFFICKING OF HLA–B*2705 AND HLA–B*2709 2239

cantly higher for both HLA–B27 subtypes than forHLA–B*0702, with no remarkable difference betweenHLA–B*2705 and HLA–B*2709. We used the fusionproteins to directly demonstrate and quantify HLA–BRluc/HLA–BYFP oligomers in immunoprecipitation as-says. From these experiments, it can be concluded thatthe differences in BRET signals reflect, at least in part,differences in the abundance of HLA–B oligomers.Taken together, these observations indicate that notonly HLA–B*2705 but also HLA–B*2709 and evenHLA–B*0702 form oligomers mainly in the ER and to alesser extent at the cell surface. The finding that oli-gomer formation is not a property of the HLA–B27alleles is consistent with earlier studies, which haveshown that other HLA alleles (including HLA–B*0702)form homodimers despite the absence of an unpairedcysteine residue (10,34). Furthermore, by means of theFRET technique, it has been shown that HLA moleculescan form oligomers in both the ER and at the plasmamembrane (20–23).

We observed that HLA–B*2705 and HLA–B*2709 form more oligomers than are formed by HLA–B*0702, not only in the ER but also in the plasmamembrane. Because it has been suggested that forma-tion of HLA oligomers in the plasma membrane mightplay a role in the activation of T cells, our observationsraise the question of whether the propensity of HLA–B27 to oligomerize might be implicated in the alterationof the functionality of antigen-presenting cells thatoverexpress HLA–B27. BRET and immunoprecipitationexperiments showed similar levels of oligomerization forHLA–B*2709 and for HLA–B*2705, raising questionsabout the relevance of HLA–B27 oligomers in diseasesusceptibility. However, it cannot be excluded that dif-ferences between both HLA–B27 subtypes have to besought at the level of the conformation and/or the nature(multimer/dimer) of the oligomers.

If, as shown here, the BRET technique proves tobe a very sensitive tool for demonstrating HLA–Boligomer formation in subcellular fractions, it still doesnot allow discrimination between properly folded andmisfolded HLA–B oligomers. Flow cytometric and con-focal visualization studies were therefore undertaken tofurther examine the production of ME1-reactive prop-erly folded and HC-10–reactive �2m-free unfolded/misfolded HLA–B proteins in HLA–B*2705YFP–, HLA–B*2709YFP–, and HLA–B*0702YFP–transfected cells.

All 3 alleles were shown to be expressed asME1-reactive proteins at the cell surface and mainly asHC-10–reactive proteins in the cytoplasm. Furthermore,we demonstrated that overexpression induced the pro-duction of unfolded/misfolded heavy chains for all 3

HLA–B subspecies. Interestingly, however, the in-creased production was not greater for HLA–B*2705than for HLA–B*2709 or HLA–B*0702, and in neithercase did the overexpression lead to expression of mis-folded proteins at the plasma membrane. This findingcontrasts with those of earlier studies showing expres-sion of not only properly folded but also unfolded/misfolded HLA–B27 proteins at the surface of humanC1R, tapasin-deficient 721.221, or transporter associ-ated with antigen processing–lacking T2 lymphoblastoidcells (35,36). However, HLA–B*2705 and HLA–B*2709displayed similar behavior in these cells.

Considering the results of the BRET experi-ments, our observations further suggest that the partic-ularity of the HLA–B*2705– and the HLA–B*2709–overexpressing cells versus the HLA–B*0702–overexpressing cells is not to produce higher amounts ofmisfolded/unfolded proteins but rather to display higherproportions of oligomers in the ER and the plasmamembrane. Moreover, the labeling patterns observedwith the ME1 and HC-10 antibodies suggest that theoligomers in the ER-enriched fractions should be mainlyunfolded/misfolded proteins, while those from theplasma membrane–enriched fractions should ratherconsist of properly folded HLA–B molecules. This ideais supported by the observation that also in lymphoblas-toid cells, part of the HLA–B27 dimers have been shownto be properly folded and to be exported from the ER(10). In contrast to what was observed in our cellsystems, the appearance of misfolded dimers was in-duced by HLA–B27 overexpression at the plasma mem-brane of dendritic cells (37). Taken together, these datalead us to conclude that the extent of production ofmisfolded/unfolded HLA–B proteins and the export ofthese proteins to the cell surface not only can bemodulated by the folding kinetics of these proteins butmost probably implicate intervention of other intracell-ular components, which may vary from one cell type tothe other. Among putative candidates are the differentintracellular proteins that participate in the correctfolding and exportation of HLA–B proteins.

Numerous class I MHC proteins possess un-paired cysteines. Only a few of them, such as H-2Ld (38),HLA–G (39), and HLA–B*2705 have been shown toform disulfide bond dimers. The HLA–B*2705 heavychain dimerization is by far the most studied. It isthought that slow folding of the HLA–B27 heavy chainfavors the exposure of its unpaired Cys67 in the �1-helical region of the peptide binding groove and therebyleads to disulfide-bonded heavy chain dimer formation(9–11,13,14). In our cell systems, BRET signals mea-sured in the ER considerably decreased when amino

2240 GIQUEL ET AL

acid Cys67 of HLA–B*2705 was replaced by a serine,while inversely, BRET signals increased when aminoacid Tyr67 of HLA–B*0702 was replaced by a cysteine.Nonetheless, BRET signaling observed for this lastmutant remained significantly lower than that observedfor HLA–B*2705, supporting the idea that not onlyCys67 but probably also other factors (such as otheramino acids, peptide loading, interaction with chaper-ones) promote oligomer formation. Recent studies sug-gested that Cys164 could also be implicated in theformation of disulfide-bonded HLA–B27 homodimers,and that perhaps Cys67-flanking amino acids such asLys70 could be implicated in the modulation of ho-modimer formation (11).

It was previously reported that in tap1-deficientHLA–B27–positive animals, HLA–B27 proteins fail toassemble correctly, and that these misfolded proteinsaccumulate in the ERGIC (40). Here, visualization ofthe proteins fused to YFP by means of confocal micros-copy showed that strong overexpression of HLA–B*2705proteins is accompanied by formation of intracellularvesicles in which the misfolded proteins accumulatetogether with BiP. These vesicles were only faintlylabeled by ME1 antibodies and were not labeled byeither anti-Rab6 antibodies (Golgi) or by anti-CD71antibodies (early endosomes). However, they were la-beled by anti-calreticulin and anti–ERGIC-53 antibod-ies. These findings strongly suggest that the observedvesicles belong to the ER/ERGIC network and areprobably not ER-derived misfolded protein–containinginclusion bodies such as Russell bodies, which have beenshown to be devoid of BiP (41). Microscopically per-formed observations of our cell systems clearly showedthat an overall increase in BiP expression occurs only athigh HLA–B*2705 expression levels, when vesicle for-mation is also observed. However, for similar HLA–Bexpression levels, the non–disease-associated HLA–B*2709 and HLA–B*0702 alleles displayed the samebehavior as that displayed by HLA–B*2705.

In conclusion, we successfully used the BRETtechnique to study in situ HLA–B oligomer formation.Hence, we demonstrated that, despite displaying a verysimilar subcellular distribution pattern of misfolded/unfolded and properly folded proteins, both HLA–B*2709 and HLA–B*2705 form more dimers thanHLA–B*0702, in both the ER and the plasma mem-brane. In our cell systems, however, overexpression ofthe HLA–B27 proteins did not induce expression ofmisfolded/unfolded HC-10–reactive proteins at the cellsurface. Moreover, HLA–B*0702 as well as HLA–

B*2705 or HLA–B*2709 overexpression was accompa-nied by the formation of intracytoplasmic vesicles inwhich HC-10–reactive heavy chains and BiP accumulate.

Because increased BiP levels are characteristic ofUPRs (8), these findings raise doubts about the linkageof UPRs induced by the overexpression of HLA–B*2705proteins to the pathogenicity of HLA–B*2705. This issupported by the recent finding that in transgenic ratsthat have a low copy number of the HLA–B*2705transgene and normally do not display an SpA pheno-type, the overexpression of �2m triggered the appear-ance of arthritis and spondylitis but reduced the slowfolding of the HLA–B*2705 heavy chain as comparedwith transgenic rats that have a high copy number of theHLA–B*2705 transgene and do display the SpA pheno-type (17). Similarly, HLA–B*2709 was found to have thesame propensity to form dimers as HLA–B*2705, raisinga question about the extent to which HLA–B27 dimersmay play a role in the pathogenicity of HLA–B27. Itshould be kept in mind that the susceptibility HLA–B27alleles and the nonsusceptibility allele HLA–B*2709 arecarried by distinct haplotypes, leading to the hypothesisthat other genes within the HLA region besides HLA–B27 may play some role in conferring susceptibility toSpA (42).

ACKNOWLEDGMENT

We thank P. Bourdoncle (Cochin Institute confocalplatform, Paris, France) for teaching confocal microscopy.

AUTHOR CONTRIBUTIONS

Dr. Andre had full access to all of the data in the study andtakes responsibility for the integrity of the data and the accuracy of thedata analysis.Study design. Giquel, Breban, Andre.Acquisition of data. Giquel, Carmouse, Denais, Cherfa, Chimenti,Fert, Hacquard-Bouder, Andre.Analysis and interpretation of data. Giquel, Carmouse, Denais,Cherfa, Chimenti, Fert, Hacquard-Bouder, Breban, Andre.Manuscript preparation. Giquel, Breban, Andre.Statistical analysis. Andre.

REFERENCES

1. Brewerton DA, Caffrey M, Nicholls A, Walters D, James DC.Acute anterior uveitis and HL-A 27. Lancet 1973;2:994–6.

2. Schlosstein L, Terasaki PI, Bluestone R, Pearson CM. Highassociation of an HL-A antigen, W27, with ankylosing spondylitis.N Engl J Med 1973;288:704–6.

3. Amor B, Feldmann JL, Delbarre F, Hors J, Beaujan MM, DaussetJ. The HL-A W27 antigen: its incidence in ankylosing spondylar-thritis and the Fiessinger-Leroy-Reiter syndrome. Nouv PresseMed 1974;3:1373–4. In French.

INTRACELLULAR TRAFFICKING OF HLA–B*2705 AND HLA–B*2709 2241

4. Reveille JD. The genetics of ankylosing spondylitis. Bull RheumDis 2001;50:2–3.

5. Breban M, Hacquard-Bouder C, Falgarone G. Animal models ofHLA-B27-associated diseases. Curr Mol Med 2004;4:31–40.

6. Hacquard-Bouder C, Falgarone G, Bosquet A, Smaoui F,Monnet D, Ittah M, et al. Defective costimulatory function is astriking feature of antigen-presenting cells in anHLA–B27–transgenic rat model of spondylarthropathy. Arthri-tis Rheum 2004;50:1624–35.

7. Smith JA, Marker-Hermann E, Colbert RA. Pathogenesis ofankylosing spondylitis: current concepts [review]. Best Pract ResClin Rheumatol 2006;20:571–91.

8. Colbert RA. The immunobiology of HLA-B27: variations on atheme [review]. Curr Mol Med 2004;4:21–30.

9. Allen RL, O’Callaghan CA, McMichael AJ, Bowness P. Cuttingedge: HLA-B27 can form a novel �2-microglobulin-free heavychain homodimer structure. J Immunol 1999;162:5045–8.

10. Dangoria NS, DeLay ML, Kingsbury DJ, Mear JP, Uchanska-Ziegler B, Ziegler A, et al. HLA-B27 misfolding is associated withaberrant intermolecular disulfide bond formation (dimerization)in the endoplasmic reticulum. J Biol Chem 2002;277:23459–68.

11. Antoniou AN, Ford S, Taurog JD, Butcher GW, Powis SJ.Formation of HLA-B27 homodimers and their relationship toassembly kinetics. J Biol Chem 2004;279:8895–902.

12. Turner MJ, Sowders DP, DeLay ML, Mohapatra R, Bai S, SmithJA, et al. HLA-B27 misfolding in transgenic rats is associated withactivation of the unfolded protein response. J Immunol 2005;175:2438–48.

13. Tran TM, Satumtira N, Dorris ML, May E, Wang A, Furuta E, etal. HLA-B27 in transgenic rats forms disulfide-linked heavy chainoligomers and multimers that bind to the chaperone BiP. J Immu-nol 2004;172:5110–9.

14. Bird LA, Peh CA, Kollnberger S, Elliott T, McMichael AJ,Bowness P. Lymphoblastoid cells express HLA-B27 homodimersboth intracellularly and at the cell surface following endosomalrecycling. Eur J Immunol 2003;33:748–59.

15. Taurog JD, Maika SD, Satumtira N, Dorris ML, McLean IL,Yanagisawa H, et al. Inflammatory disease in HLA-B27 transgenicrats [review]. Immunol Rev 1999;169:209–23.

16. Cauli A, Dessole G, Fiorillo MT, Vacca A, Mameli A, Bitti P, etal. Increased level of HLA-B27 expression in ankylosing spondy-litis patients compared with healthy HLA-B27-positive subjects: apossible further susceptibility factor for the development of dis-ease. Rheumatology (Oxford) 2002;41:1375–9.

17. Tran TM, Dorris ML, Satumtira N, Richardson JA, Hammer RE,Shang J, et al. Additional human �2-microglobulin curbsHLA–B27 misfolding and promotes arthritis and spondylitis with-out colitis in male HLA–B27–transgenic rats. Arthritis Rheum2006;54:1317–27.

18. Reveille JD. Major histocompatibility genes and ankylosing spon-dylitis. Best Pract Res Clin Rheumatol 2006;20:601–9.

19. Paladini F, Taccari E, Fiorillo MT, Cauli A, Passiu G, Mathieu A,et al. Distribution of HLA–B27 subtypes in Sardinia and conti-nental Italy and their association with spondylarthropathies. Ar-thritis Rheum 2005;52:3319–21.

20. Pentcheva T, Edidin M. Clustering of peptide-loaded MHCclass I molecules for endoplasmic reticulum export imaged byfluorescence resonance energy transfer. J Immunol 2001;166:6625–32.

21. Bodnar A, Bacso Z, Jenei A, Jovin TM, Edidin M, DamjanovichS, et al. Class I HLA oligomerization at the surface of B cellsis controlled by exogenous �2-microglobulin: implications inactivation of cytotoxic T lymphocytes. Int Immunol 2003;15:331–9.

22. Gaspar R Jr, Bagossi P, Bene L, Matko J, Szollosi J, Tozser J,et al. Clustering of class I HLA oligomers with CD8 and TCR:three-dimensional models based on fluorescence resonanceenergy transfer and crystallographic data. J Immunol 2001;166:5078–86.

23. Matko J, Bushkin Y, Wei T, Edidin M. Clustering of class I HLAmolecules on the surfaces of activated and transformed humancells. J Immunol 1994;152:3353–60.

24. Xu Y, Piston DW, Johnson CH. A bioluminescence resonanceenergy transfer (BRET) system: application to interacting circa-dian clock proteins. Proc Natl Acad Sci U S A 1999;96:151–6.

25. Milligan G, Bouvier M. Methods to monitor the quaternarystructure of G protein-coupled receptors [review]. FEBS J 2005;272:2914–25.

26. Boute N, Jockers R, Issad T. The use of resonance energy transferin high-throughput screening: BRET versus FRET. Trends Phar-macol Sci 2002;23:351–4.

27. Stam NJ, Spits H, Ploegh HL. Monoclonal antibodies raisedagainst denatured HLA-B locus heavy chains permit biochemicalcharacterization of certain HLA-C locus products. J Immunol1986;137:2299–306.

28. Ellis SA, Taylor C, McMichael A. Recognition of HLA-B27 andrelated antigen by a monoclonal antibody. Hum Immunol 1982;5:49–59.

29. Wigler M, Silverstein S, Lee LS, Pellicer A, Cheng Y, Axel R.Transfer of purified herpes virus thymidine kinase gene to culturedmouse cells. Cell 1977;11:223–32.

30. Lopez de Castro JA. HLA-B27 and the pathogenesis of spondy-loarthropathies. Immunol Lett 2007;108:27–33.

31. Issafras H, Angers S, Bulenger S, Blanpain C, Parmentier M,Labbe-Jullie C, et al. Constitutive agonist-independent CCR5oligomerization and antibody-mediated clustering occurring atphysiological levels of receptors. J Biol Chem 2002;277:34666–73.

32. Salahpour A, Angers S, Mercier JF, Lagace M, Marullo S, BouvierM. Homodimerization of the �2-adrenergic receptor as a prereq-uisite for cell surface targeting. J Biol Chem 2004;279:33390–7.

33. Mear JP, Schreiber KL, Munz C, Zhu X, Stevanovic S, Ram-mensee HG, et al. Misfolding of HLA-B27 as a result of its Bpocket suggests a novel mechanism for its role in susceptibility tospondyloarthropathies. J Immunol 1999;163:6665–70.

34. Nossner E, Parham P. Species-specific differences in chaperoneinteraction of human and mouse major histocompatibility complexclass I molecules. J Exp Med 1995;181:327–37.

35. Vazquez MN, Lopez de Castro JA. Similar cell surface expressionof �2-microglobulin–free heavy chains by HLA–B27 subtypesdifferentially associated with ankylosing spondylitis. ArthritisRheum 2005;52:3290–9.

36. Goodall JC, Ellis L, Hill Gaston JS. Spondylarthritis-associatedand non–spondylarthritis-associated B27 subtypes differ in theirdependence upon tapasin for surface expression and their incor-poration into the peptide loading complex. Arthritis Rheum2006;54:138–47.

37. Kollnberger S, Bird LA, Roddis M, Hacquard-Bouder C,Kubagawa H, Bodmer HC, et al. HLA-B27 heavy chain ho-modimers are expressed in HLA-B27 transgenic rodent models ofspondyloarthritis and are ligands for paired Ig-like receptors.J Immunol 2004;173:1699–710.

38. Capps GG, Robinson BE, Lewis KD, Zuniga MC. In vivo dimericassociation of class I MHC heavy chains: possible relationship toclass I MHC heavy chain-�2-microglobulin dissociation. J Immu-nol 1993;151:159–69.

39. Boyson JE, Erskine R, Whitman MC, Chiu M, Lau JM, KoopmanLA, et al. Disulfide bond-mediated dimerization of HLA-G on thecell surface. Proc Natl Acad Sci U S A 2002;99:16180–5.

40. Raposo G, van Santen HM, Leijendekker R, Geuze HJ, PloeghHL. Misfolded major histocompatibility complex class I moleculesaccumulate in an expanded ER-Golgi intermediate compartment.J Cell Biol 1995;131(6 Pt 1):1403–19.

41. Kopito RR, Sitia R. Aggresomes and Russell bodies: symptoms ofcellular indigestion? EMBO Rep 2000;1:225–31.

42. Fiorillo MT, Cauli A, Carcassi C, Bitti PP, Vacca A, Passiu G, et

2242 GIQUEL ET AL

al. Two distinctive HLA haplotypes harbor the B27 alleles nega-tively or positively associated with ankylosing spondylitis in Sardi-

nia: implications for disease pathogenesis. Arthritis Rheum2003;48:1385–9.

DOI 10.1002/art.22757

Clinical Images: Iloprost-induced vascular remodeling

The patient, a 29-year-old woman with a 3-year history of Raynaud’s phenomenon and positivity for antinuclear antibodies (titer1:2,500), presented to our clinic with tenderness, swelling, and cyanosis of the toes, which had developed in the previous 48 hoursafter exposure to low temperatures. Nailfold capillaroscopy performed at the time of presentation revealed capillary featurespathognomonic for active scleroderma, such as giant capillaries (A) and microhemorrhages, and occasional areas reminiscent of themore advanced late inactive pattern, with abnormal neoangiogenesis and decreased capillary density. Low-dose iloprost trometalol(a prostacyclin analog) was administered in a 24-hour/day continuous intravenous infusion (0.5 ng/kg/minute) for 10 consecutivedays, with excellent clinical response. Followup capillaroscopy performed 6 hours after initiation of the infusion (B), 3 days afterinitiation of the infusion (C), and after completion of the treatment (D), revealed an impressive increase in capillary density andstructural remodeling of the capillaries, as a direct consequence of the iloprost administration.

Athina Pyrpasopoulou, MD, PhDSpyros Aslanidis, MDHippokration General HospitalThessaloniki, Greece

INTRACELLULAR TRAFFICKING OF HLA–B*2705 AND HLA–B*2709 2243