Immunogenegcs 3~ 95-101, 1993 111111111110- geneties

© Springer-Verlag 1993

Original articles

Different rates of HLA class I molecule assembly which are determined by amino acid sequence in the a2 domain

Ann Hill l' 2, Masafumi Takiguchi 3, and Andrew Mc Michael 1

t Molecular Immunology Group and 2ICRF, Institute of Molecular Medicine, John Radcliffe Hospital, Oxford, UK 3 Department of Tumour Biology and Immunology, University of Tokyo, Japan

Received February 6, 1992; revised version received April 27, 1992

Abstract. Assembly of HLA class I molecules was studied using pulse-chase labeling of B-lymphoblastoid cell lines with 35S-methionine, immunoprecipitation with antibodies detecting free or ~2-microglobulin-associated heavy chain and isoelectric focusing. Marked differences between the products of different class I alleles were noted. HLA-B51 assembled very inefficiently, with con- siderable free heavy chain still detected in an unsialated form after a four hour chase. The closely related molecule HLA-B35 was in contrast rapidly assembled, all newly synthesized heavy chain being detected in a ~2m- associated, sialated form within 30 minutes. Analysis of naturally occurring variants related to HLA-B35 and HLA-B51 localized the region determining assembly ef- ficency to the c~2 domain, in which these molecules differ at eight amino acid residues. The effect was not due to a linked dominant gene, as both patterns of assembly were observed in a single cell line.

Introduction

Major histocompatibility complex (MHC) class I genes are among the most polymorphic known. By increasing the variety of peptides able to bind to MHC molecules, the polymorphism gives a population the best chance of mounting a cytotoxic T lymphocyte (CTL) response in the face of new intracellular pathogens (Doherty and Zinker- nagel 1975). Supporting this view of the significance MHC polymorphism, the crystallographic structure of HLA-A2 revealed that the most highly polymorphic

Correspondence to: A. Hill, Molecular Immunology Group, Institute of Molecular Medicine, John Radcliffe Hospital, Headington, Oxford OX3 9DU, UK.

amino acid residues are found around the peptide binding cleft (Bjorkman et al. 1987). Other residues are highly conserved, preserving the tertiary structure required for interaction with fl2-microglobulin (/32m) and the T-cell receptor (Tcr; Bjorkman et al. 1990). As had been ex- pected from the extent to which class I alleles determine epitopes recognized by CTLs (Doherty et al. 1978; McMichael et al. 1986; Pala et al. 1986), peptides with distinct profiles are eluted from different class I molecules (Falk et al. 1991).

Surprisingly, class I molecules have also been found to display considerable diversity in aspects of their biochemical behavior not obviously related to peptide binding. Allelic products have been noted to have dif- ferent half lives at the cell surface (Emerson et al. 1980), and to have different levels of steady state cell surface expression (Weis et al. 1985). Studying MHC class I molecules as a model for intracellular transport of glycoproteins, Williams and co-workers (1985) noted that all newly synthesized H-2K k molecules reached the cell surface within an hour, whereas H-2D k was transported slowly with a T 1/2 of 4-5 h. The rate-limiting step was in the pre-Golgi compartment.

The behavior of the mouse class I molecule L d is par- ticularly interesting. Transport of L d is very slow and hybrid molecules show that the rate of transport is depen- dent on the c~l and c~2 domains (Weis and Murre 1985; Beck et al. 1986). L d shares with D b the unusual property of being able to reach the cell surface without having associated with /32m (Allen et al. 1988; Hansen et al. 1988). Much L a at the cell surface is not found to be associated with/32m either because of direct transport of free heavy chains or because of dissociation at the cell surface (Myers et al. 1989). Incubation of cells with pep- tide specific for L d increases the normally low steady state L d expression to a level approximately equal to

96 A. Hill et al.: HLA class I assembly rate controlled by c~2 domain

o the r class I m o l e c u l e s (Lie et al. 1990). M u c h o f the

unusua l b e h a v i o r o f L a has b e e n a t t r ibu ted to a p r e s u m e d

low avidi ty f o r / 3 2 m (Beck et al. 1986; Lie et al. 1990).

The a s s e m b l y o f h u m a n class I mo lecu le s has b e e n less

wel l s tudied. H o w e v e r , d i f f e rences in the ra te o f a s s e m b l y

and t r anspo r t o f H L A class I mo lecu l e s w e r e o b s e r v e d

by Neef jes and c o - w o r k e r s (1988) ; H L A - A 2 , H L A - A l l ,

and H L A - B 2 7 w e r e a s s e m b l e d wi th i n t e rmed ia t e ef f ic ien-

cy, H L A - B 3 5 was a s s e m b l e d ve ry rap id ly . H L A - C w 4

was i ncomple t e ly a s s e m b l e d d u r i n g the p e r i o d o f the six

h o u r pu l se chase , thus s how i ng some s imi lar i ty to the

m o u s e L d molecu le . H o w e v e r , the low sur face expres -

s ion (Snary et al. 1977), less ex tens ive p o l y m o r p h i s m , and

dif f icul ty in iden t i fy ing H L A - C molecu l e s as r e s t r i c t ion

e l emen t s has led some au thors to sugges t tha t they m a y

b e re la t ive ly n o n f u n c t i o n a l ( G u s s o w et al. 1987; L a w l o r

et al. 1990).

In s tudy ing a s sembly and t r a n s p o r t o f H L A class I

molecu les , u s ing an i soe lec t r ic focus ing ( IEF) t echn ique ,

we w e r e a lso s t ruck by the ex ten t o f al lel ic d ivers i ty . W e

desc r ibe h e r e c o m m o n H L A - A and H L A - B molecu l e s

w h i c h a re ve ry s lowly a s sembled , and ut i l ize na tu ra l ly oc-

cu r r ing va r i an t s to loca l ize the r eg ion o f the mo lecu l e con-

t ro l l ing a s sembly ef f ic iency.

Materials and methods

Cell lines. The Epstein-Barr Virus (EBV)-transformed human B-lym- phoblastoid cell lines (BCL) used in these studies and their sources are listed in Table 1.

Pulse chase labeling. 108 cells in log phase of growth were centrifuged and resuspended in 2 ml methionine-free RPMI with 5 % fetal calf serum (FCS) and incubated at 37 °C in a small flask for 30-40 min. 0.5 mCi 35S-methionine (Flow Translabel, ICN Flow, High Wycombe, UK) was then added. After 15 min (30 rain for Figures 1 and 3), labeling was stopped by the addition of 300 ~tl aliquots to either 20 ml ice cold phosphate buffered saline (PBS; 0 time point sample) or to 5 ml RPMI with 10% FCS and 2 mM cold methionine at 37 °C (other time points).

Table 1. BCLs used in this study.

Cell line HLA class I typing Source

Hom-2 A3, B27 BASILIO A2, B51 KOSE A2, B35 KA263 A2, A23, Bw53, B58

AKIBA A24, B52 Khaled BCL A30, A36, B7, B78

(B'SNA') DukVM A2, A30, B52, Bw53,

Cw4 EVA1224 A1, A2, B37, B51

S. Marsh S. Marsh S. Marsh [derived from peripheral blood lymphocytes (PBL) from a Gambian individual; a gift from A. Hill] S. Marsh M. Andrien

F. Ward

W. Bodmer

These were removed from the incubator at the intervals shown and im- mediately added to 20 ml ice cold PBS. The cells in PBS were centrifuged at 4 °C, and the pellet lysed in 1 ml lysis buffer [0.5% NP40, 20 mM Tris pH 8, 10 mM ethylenediaminetetraacetate (EDTA) pH 8, 0.1 M NaC1, 1 mM phenylmethylsulfonyl fluoride (PMSF)]. After 30 rain on ice, the nuclei were sedimented at 14000 rpm for 5 rain at 4 °C. The lysates were stored on ice until all samples were ready for preelearing (maximum time 4 h). For the experiments in Figures 1 and 3 lysates were frozen at - 7 0 °C overnight for use the next day. Lysates were precleared with 150 ~tl 10% Staph-A cells (Pansorbin) for 1 h, centrifug- ed, and the supernatant divided into two equal aliquots for im- munoprecipitation. Monoclonal antibodies (mAb) were used as ascites (3-5 ~tl) or purified antibody to a final concentration of 10-15 ~tg/ml. Rabbit anti-heavy chain serum was used at 3 ~tl per 500 ~tl lysate. Lysate was incubated with antibody for 1 h at4 °C, then 100 ~tl 10% protein-A sepharose (Sigma Chemicals, London, UK), or 50 ~tl 10% Pansorbin, in lysis buffer was added and samples mixed end over end for 30 min at 4 °C. Protein A Sepharose beads were then washed at 4 °C twice with 20 mM Tris pH 8, 10 mM EDTA pH 8, 0.1 M NaC1, 0.5% Nonidet 40 (NP40), 0.1% sodium dodecyl sulfate (SDS), 1% bovine serum albumin (BSA), once with 10% lysis buffer in 0.5 M NaC1, and once in lysis buffer. For the experiment in Figure 6 the samples were in- cubated with 20 ~tl neuraminidase (10 units/ml in 0.05 M EDTA pH 6.8) at 37 °C for 3 h, and then washed with lysis buffer. They were resuspended in 60 ~tl sample buffer (9.5 M urea, 2% NP40, 2% am- pholines 3.5-10, 5 % 2 ME) and incubated at room temperature for 30 min. The beads were sedimented by spinning briefly in a microcen- trifuge, and 40 ~tl of the supernatant (20 Ixl for Pansorbin-precipitated samples) was removed for analysis by IEF.

IEF. One dimensional IEF was performed according to the method of Neefjes and co-workers (1986). The composition of the gel was 4.5% polyacrylamide gel (30:1.6 monoacrylamide:bisacrylamide; BioRad, Richmond, CA), 9 M urea, 2% NP40, 4% ampholyte 5-7 (LKB In- struments, Bromma, Sweden), and 1% ampholyte 3.5-10 (LKB). To polymerize 30 ml gel solution, 60 txl freshly prepared 10% w/vol am- monium persulphate and 30 Ixl TEMED were used. 16 mm gel plates were run on a Protean II aparatus (BioRad). The upper buffer was 50 mM NaOH, and the lower buffer 20 mM H3PO 4. A constant voltage of 880 V was set, with the current limited to 12 mA per gel and power to 8 W, allowing maximum voltage to be acheived over several h. The run was continued for 13-16 h, the gels fixed in 10% acetic acid for 30 min, then exposed to Amplify for 30 min, dried and exposed to Kodak XAR film, usually overnight.

Antibodies. The antibodies used were HC10, recognizing free B and C locus heavy chain (Stam et al. 1986, 1990; a gift from H. Ploegh), W6/32, recognizing/32m-associated class I molecules (Barustable et al. 1978; a gift from J. Bodmer), and a polyclonal rabbit anti-human heavy chain serum (Stam 1990; a gift from H. Ploegh).

Results

Pulse chase studies reveal wide variation in assembly kinetics between different HLA class I allelic products. B C L s w e r e s tudied by pu lse chase l abe l ing and ana lyzed

on 1D IEF gels as d e s c r i b e d in Ma te r i a l s and me thods .

M a r k e d d i f f e rences in the k ine t ics o f a s s e m b l y w e r e no ted

b e t w e e n d i f fe ren t HLA class I al leles. T h r e e r ep re sen -

ta t ive gels a re s h o w n in F i g u r e 1. F i g u r e l a shows a pulse

chase o f H o m - 2 , a cel l l ine exp re s s ing HLA-A3 and

B2705. B2705-free h e a v y cha in is still de tec tab le at 30 m i n o f chase bu t no t the rea f te r . /32m-associa ted ( W 6 / 3 2

A. Hill et ah: HLA class I assembly rate controlled by c~2 domain 97

Fig. 1. a Pulse chase labeling of three BCLs, analysed by IEF. After 15 min labeling with 3SS methionine, cells were lysed at the time points shown. For each time point samples were immunoprecipitated with polyclonal rabbit anti-heavy chain serum ( = H in a) or HC10, recogniz- ing free B and C locus heavy chain ( = H in b and c), and W6/32, recognizing/32m-associated class I (=W). The unsialated, ER form of each molecule is marked 0. The acquisition of sialic acid with complex glycosylation in the Golgi causes band shift towards the anode; the 1 and 2 sialic acid forms are marked 1 and 2 respectively. The lines studied are: a Horn-2 (HLA-A3, HLA-B27); b BASILIO (HLA-A2, HLA- B51). Only the HLA-B51 portion of the gel is shown c KOSE (HLA-A2, HLA-B35). Only the HLA-B35 portion of the gel is shown. The bands just below the HLA-B51 0 sialic acid band in b and just above the 2 sialic acid form of HLA-B35 in c are presumed to be untyped C locus products.

positive) unsialated B2 705 is detectable for 60 min chase. Fully glycosylated, t32m-associated B2705 is first detected at 15 min chase and increases thereafter. The assembly kinetics of B2 705 were typical of many alleles. One extreme pattern was shown by HLA-A3 (Fig. la). HLA-A3 free heavy chain and/32m-associated molecules were still detected at the end of the 3 h pulse period, although even from the beginning of the pulse chase most unsialated HLA-A3 was detected in a /32m-associated form. A slightly different pattern was seen with HLA-B51 (Fig. lb). A significant proportion of newly synthesized HLA-B51 had also failed to become sialated during the pulse chase. However, in contrast to HLA-A3, most un- sialated HLA-B51 was detected as free heavy chain rather than as/32m-associated. HLA-B51 showed a similar pat- tern of assembly in a further three lines studied (data not shown). The other extreme of assembly was noted with

HLA-B35 (Fig. lc). Most newly synthesized HLA-B35 was already/32m-associated at the end of the 15 min pulse period, and some sialated form was already detected at this time. After a further 15 min of chase, most of the newly synthesized heavy chain was already fully assembl- ed and sialated.

To exclude the possibility that the inefficient assembly of HLA-B51 was in some way a consequence of EBV transformation, a polyclonal alloreactive T-cell line, maintained in interleukin 2 (IL-2), was studied (Fig. 2). HLA-B51 showed a similar pattern of inefficient assembly in this line.

HLA-B35/HLA-B51 cross-reactive epitope group (CREG) variants localize the region controlling assembly efficien- cy to the c~2 domain. HLA-B35 and HLA-B51 are closely related HLA molecules, differing only in the c~1 and c~2 domains. The existence of several variants, listed in Table 1, within this CREG, which are in effect naturally occur- ring exon shuffles, has allowed us to map more finely the region controlling assembly efficiency. Autoradiographs of IEF gels from pulse chase studies were scanned on an Image analyzer (Bio Image, Ann Arbor, MI), allowing assessment of band intensity by optical density (OD). The OD of free heavy chain, unsialated (where detectable), and fully sialated /32m-associated forms were plotted against time. Studies of five members of the HLA- B35/HLA-B51 CREG are shown in Figure 3. HLA-B35 and HLA-B53, which differ in the o~1 domain but are iden- tical in the o~2 domain (Ooba et al. 1989; Hayashi et al. 1990; Allsopp et al. 1991) both assemble rapidly and com- pletely. HLA-B51, HLA-B52, and HLA-B78 (B'SNA'), which are identical in the o~2 domain (Hayashi et al. 1989; Sekimata et al. 1990), all fail to assemble completely new-

Fig. 2. Pulse chase labeling of a polyclonal alloreactive T-cell line ex- pressing HLA-A2, -B7, and -B51. The conditions of the pulse chase are as for Figure "1; immunoprecipitation was with HC10 (H), and W6/32 (w).

98 A. Hill et al.: HLA class I assembly rate controlled by c~2 domain

( a ) 600.

500 •

400 '

5 300"

200 '

1 O0 '

(c ) 400 '

300

6 200

100

k_ _

100 200 300 minutes of chase

B 5 1

, i • i , i

1 O0 200 300

minutes of chase

( b ) 120.

1 O0 -

80-

6 60-

40"

20-

lOO 200 300

minutes of chase

( d ) 3 0 0 = B 5 2

0 100 200 300 minutes of chase

(e) 200 "

~ HClO

B78

1 O0 - / ~ 4 W6/32-2 siaHc acid

~ II W6/32-unsialated

0 1 O0 200 300

minutes of chase

Fig. 3a-e. Pulse chase labeling studies of five BCLs expressing members of the HLA-B5/HLA-B35 CREG. The cells were pulsed with 35S methionine for 15 min, chased with 2 mM cold methionine and lysed at the time points shown. HLA class I molecules were im- munoprecipitated with He10 (free heavy chain) and W6/32 (/32m associated class I) for each time point shown. The autoradiographs were scanned on an image analyser (Bio Image), and integrated optical density (IOD) of the relevant band is plotted against the vertical axis. The cell lines are a KOSE (HLA-B35), b KA263 (HLA-B53), e BASILIO (HLA- B51), d AKIBA (HLA-B52), and e Khaled [HLA-B78 (B'SNA')]. For HLA-B51 and HLA-B52 the 0 sialic acid W6/32 positive bands were too weak to allow accurate measurement; for these lines only the 2 sialic acid band is shown.

B35, B51,B52

B53 B78 • 9 4 1 T

• 95 I W

~ 9 7 R T

~ 1 0 3 L V

• 114 D N

O 116 S Y

@ 1 5 2 V E

171 Y H

Fig. 4. Location in the antigen binding cleft of class I molecule of the eight residues in the c~2 domain which control assembly efficiency in the HLA-B35/HLA-B51 CREG.

markedly the rate of intracellular transport of a rat class I MHC molecule (Livingstone et al. 1989; Powis et al. 1991). To exclude the possibility that the efficiency of assembly was controlled by a gene in strong linkage dis- equilibrium with HLA-B, a cell line expressing both HLA-B52 (inefficient) and HLA-B53 (efficient) was studied. As shown in Figure 5, in this cellular environ- ment HLA-B52 still failed to assemble completely, whereas HLA-B53 was rapidly and completely assembled.

interferon (~-IFN) does not improve the efficiency of assembly of HLA-B51. Assembly deficient mutants in which assembly of class I heavy chain and/32m do not occur in the absence of ,y-IFN have been described, and imply the existence of a 3,-IFN-induced gene product in- volved in assembly (Klar et al. 1989). We cultured EVA1224 in the presence of 250 u/ml 3,-IFN for 48 h prior to a pulse chase. For this experiment the samples were treated with neuraminidase to remove sialic acid before running on the gel. Figure 6 shows that 3,-IFN did not alter the assembly kinetics of HLA-B51.

ly synthesized heavy chain during the course of the 4 h chase. Thus assembly efficiency in this group is governed by the c~2 domain, where there are differences in eight amino acid residues. The position of these residues inthe antigen binding cleft of the class I molecule is shown in Figure 4.

Both assembly patterns can occur in the same cell. A dominant MHC-linked gene has been shown to affect

Fig. 5. Pulse chase of DukVM (HLA-A2, -A30, -B52, -Bw53, -Cw4). Conditions are the same as for Figure 1. H=HC10 W=W6/32

A. Hill et al. : HLA class I assembly rate controlled by e~2 domain 99

Fig. 6a, b. Pttlse chase of EVA1224 (HLA-A1, -A2, -B37, -B51). a cultured in normal medium, b cultured for the previous 24 h in the presence of 250 u/ml 7-IFN. The cells were labeled for 30 rain, chased with cold methionine and immunoprecipitated with HC 10 (H) or W6/32 (W) at the time points shown. Samples were treated with neuraminidase to remove sialic acid.

Discussion

The marked difference in assembly efficiency caused by alteration in eight amino acids in the c~2 domain in the HLA-B35/HLA-B51 CREG may shed light on the pro- cesses of class I assembly./32m, heavy chain and peptide assemble in the endoplasmic reticulum (ER) and in most cases only fully assembled trimers leave the ER for com- pletion of N-linked glycosylation and cell surface expres- sion (Nuchtern et al. 1989; Townsend et al. 1989). Studies of assembly in cell lysates show that two routes of assembly are thermodynamically possible: free heavy chain may fold in the presence of high affinity peptide without/32m (Elliot et al. 1991), or peptide may stabilize preformed loosely assembled heavy chain/32m complexes (Townsend et al. 1990). Most protein oligomerization is thought to occur in vivo with the assistance of other molecules (Gething et al. 1992). Degen and Williams (1991) have identified an 88 kD protein which is induced to coprecipitate with heavy chain by chemical cross-link- ing. Ability to coprecipitate this protein disappears with acquisition of Endo-H resistance by class I molecules, suggesting that it plays a role in assisting correct assembly in the ER.

Several possible explanations for the difference in assembly efficiency of members of the HLA-B35/HLA- B51 CREG are apparent. The efficient assemblers could have a higher overall affinity for available peptides or for t32m; more peptides of appropriate motif may be available for these alleles; or they may utilize more effectively ap- propriate chaperones. The difference at amino-acid 171, tyrosine in the efficient assemblers and most class I molecules and histidine in the inefficient assemblers, seemed to provide a plausible explanation for the phenomenon. Position 171 is located in the A pocket, and

in the crystallographic structure of HLA-B27 the tyrosine side chain was hydrogen bonded to the amino terminus of the bound peptide (Madden et al. 1991). The loss of this one hydrogen bond could lead to a significant decrease in affinity of peptide binding, resulting in slower overall assembly. However, HLA-Aw33, and HLA-B18, which also have H171, assemble quickly (data not shown), and a site-directed mutant of HLA-B35 with H 171 transfected in to C1R still assembled quickly (data not shown). The other polymorphic peptide-facing residues may therefore be important in determining the assembly rate, possibly selecting a rare sequence motif for the slow assemblers. /32m affinity could be involved, as in addition to interac- ting with the c~3 domain t32m also interacts with the under- side of the/3-pleated sheat formed by the c~l and o~2 do- mains. Residue 94 contacts /32m and could affect the affinity of binding, as could alteration of tertiary structure in this region. The possibility that interaction with a resi- dent ER protein may be responsible for our findings is raised by the finding that the c¢2 domain controls the abili- ty of murine class I molecules to be bound by the adenovirus E3/19K protein, which is responsible for retention of class I heavy chains in the ER. Alterations affecting E3/19K binding also altered assembly kinetics (Jeffries et al. 1990). Intriguingly, an E3/19K reactive mAb precipitates an 88 kD protein, which may be a cellular homologue or ancestor of the E3/19K protein (Cox et al. t991).

In this study, the slow assembly of HLA-B51 shows some similarity to the mouse L d molecule. However, we find no evidence of transport of HLA-B51 to the cell sur- face in the absence of ~2m. Small amounts of sialated free heavy chain can sometimes be detected. However, these bands are best seen late in the chase when the two sialic acid band, precipitable by the W6/32 mAb, may already be decreasing, suggesting that the sialated free heavy chain is a result of dissociation of less stable complexes at the cell surface or in the lysate.

The biological significance of the biochemical dif- ferences we have described is unknown. Despite its slow assembly, L d is the immunodominant restricting element in the CTL response to several viruses. Similarly, HLA- B51 was shown to be the immunodominant restricting ele- ment in the CTL response to EBV in one individual (Chen et al. 1989). HLA-B51 and HLA-B52 are common class I alleles; 16% of Caucasians and 29% of Orientals have HLA-B51 or HLA-B52. Similarly, HLA-A3, which also shows slow assembly, is present in 25 % of Caucasians (Scheisle 1987). Thus it is likely that these slow assemblers have conferred protective advantages at some point of human evolution. Whether slow assembly is an incidental by-product of changes whose selective advan- tage is the ability to bind certain peptides, or whether this pattern of assembly itself confers advantages in the presence of some parasites requires further investigation.

100 A. Hill et al.: HLA class I assembly rate controlled by a2 domain

Acknowledgments. A.H. is an Oxford Dominions Medical Fellow of the Nuffield Trust. We also thank the Imperial Cancer Research Fund and the Medical Research Council for support. We are grateful to Dr. H. Ploegh and Dr. L. Pazmany for helpful discussion and advice regar- ding the IEF technique, and to Dr. J. Bodmer, Dr. F. Ward, Dr. A. Hill, S. Marsh, Dr. A. Rickinson, and C. Allsopp for generous gifts of cell lines and tissue typing, and to Dr. W.F. Bodmer for support and advice. We are especially grateful to Dr. T. Elliot for many helpful discussions.

References

Allen, H., Fraser, J., Flyer, S., Calvin, S., and Flavell, R.: /32-microglobulin is not required for cell surface expression of the murine class I histocompatibility antigen H-2D b or of a truncated H-2D b. Proc Nad Acad Sci USA 79: 7447, 1988

Allsopp, C. E. M., Hill, A. V. S., Kwiatowski, D., Hughes, A., Bunce, M., Taylor, C. J., Pazmany, L., Brewster, D., McMichael, A. J., and Greenwood, B. M.: Sequence analysis of HLA-Bw53, a com- mon West African allele, suggests an origin by gene conversion of HLA-B35. Hum Immunol 30: 105-109, 1991

Barnstable, C. J., Bodmer, W., Brown, G. E. A., Galfre, G., Milstein, C., Williams, A.F . , and Ziegler, A.: Production of monoclonal antibodies to group A erythrocytes, HLA and other human cell sur- face antigens-new tools for genetic analysis. Cell 14: 9, 1978

Beck, J. C., Hansen, T. H., Cullen, S. E., and Lee, D. R.: Slower pro- cessing, weaker/32m association, and lower surface expression of H-2L d are influenced by its amino terminus. J Immunol 137: 916-923, 1986

Bjorkman, P.J. and Parham, P.: Structure, function, and diversity of class I major histocompatibility complex molecules. Annu Rev Biochem 59: 253-288, 1990

Bjorkman, P. J., Saper, M.A. , Samraoui, B., Bennett, W. S., Strum- inger, J. L., and Wiley, D. C.: The foreign antigen binding site and T cell recognition regions of class I histocompatibility antigens. Nature 329: 512-518, 1987

Chen, B.P. , Lain, V., Kraus, E.E. , DeMars, R., and Sondel, P.M.: Restriction of Epstein-Barr Virus-specific cytotoxic T cells by HLA-A, -B, and -C molecules. Hum Immuno126: 137-147, 1989

Cox, J. H., Bennlnk, J. R., and Yewdell, J. W.: Retention of Adenovirus El9 Glycoprotein in the endoplasmic reticulum is essential to its ability to block antigen presentation. J Exp Med 174: 1629-1637, 1991

Degen, E. and Williams, D. B.: Participation of a novel 88-kD protein in the biogenesis of murine class I histocompatibitity molecules. J Cell Biol 112: 1099-1115, 1991

Doherty, P. C. Biddison, W. E., Bennink, J. R., and Knowles, B.B.: Cytotoxic T cell responses in mice infected with influenza and vac- cinia viruses vary in magnitude with H-2 genotype. J exp Med 148: 534-543, 1978

Doherty, P. C. and Zinkernagel, R. M.: A biological role for the major histocompatibility antigens. Lancet i: 1406-1409, 1975

Elliot, T., Cerundolo, V., Elvin, J., and Townsend, A.: Peptide-induced conformational change of the class I heavy chain. Nature 351: 402-406, 1991

Emerson, S.G., Murphy, D.B., and Cone, R.E.: Selective turnover and shedding of H-2K and H-2D antigens is controlled by the major histocompatibility complex. J Exp Med 152: 783-795, 1980

Falk, K., Rotzschke, O., Stevanovic, S., Jung, G., and Rammensee, H.-G.: Allele specific motifs revealed by sequencing of self peptides eluted from MHC molecules. Nature 351: 290-296, 1991

Gething, M.J. and Sambrook, J.: Protein folding in the cell. Nature 355: 33-45, 1992

Gussow, D., Rein, R. S., Meijer, I., de Hoog, W., Seemann, G. H. A., Hochstenbach, F. M., and Ploegh, H. L.: Isolation, expression, and

the primary structure of HLA-Cwl and HLA-Cw2 genes: evolu- tionary aspects. Immunogenetics 25: 313-322, 1987

Hansen, T. H., Myers, N. B., and Lee, D. R.: Studies of two antigenic forms of L d with disparate/32-microglobnlin (/32m) associations suggest that t32m facilitates the folding of the cd and ~2 domains during de novo synthesis. J Immunol 140: 3522-3529, 1988

Hayashi, H., Ennls, P. D., Ariga, H., Salter, R. D., Parham, P., Kano, K., and Takiguchi, M.: HLA-B51 and HLA-Bw52 differ by only two amino acids which are in the helical region of the alpha 1 do- main. J Immunol 142: 306-311, 1989

Hayashi, H., Ooba, T., Nakayama, S., Sekimata, M., Kano, K., and Takiguchi, M.: Allospecificities between HLA-Bw53 and HLA-B35 are generated by substitution of the residues associated with HLA-Bw4/Bw6 public epitopes. Immunogenetics 32: 195-199, 1990

Jeffries, W.A. and Burgert, H.-G.: E3/19K from Adenovirus 2 is an immunosubsersive protein that binds to a structural motif regulating the intracellular transport of major histocompatibility complex class I proteins. J Exp Med 172: 1653-1664, 1990

Klar, D. and Hammerling, G. J.: Induction of assembly of MHC class I heavy chains with/32 microglobulin by interferon-'),. EMBO J 8: 475-481, 1989

Lawlor, D. A., Zemmour, J., Ennis, P. D., and Parham, P.: Evolution of Class-I MHC genes and proteins: from natural selection to thymic selection. Annu Rev Immunol 8: 23-63, 1990

Lie, W.-R., Myers, N. B., Gorka, J., Rubocki, R. J., Connolly, J. M., and Hansen, T. H.: Peptide ligand-induced conformation and sur- face expression of the L d class I MHC molecule. Nature 344: 439-441, 1990

Livingstone, A. M., Powis, S. J., Diamond, A. G., Butcher, G. W., and Howard, J.C.: A trans-actiug major histocompatibility complex- linked gene whose alleles determine gain and loss changes in the antigenic structure of a classical class I molecule. J Exp Med 170: 777-795, 1989

Madden, D. R., Gorga, J. C., Strominger, J. L., and Wiley, D. C. : The structure of HLA-B27 reveals nonamer "self-peptides" bound in an extended conformation. Nature 353: 321-325, 1991

McMichael, A., Gotch, F., and Rothbard, J.: HLA-B37 determines an influenza A virus nucleoprotein epitope recognized by cytotoxic T lymphocytes. J Exp Med 164: 1397-1406, 1986

Myers, N. B., Lie, W.-R., Nett, M., Rubocki, R. J., and Hansen, T. H.: The conformation of L d induced by 132-microglobnlin is fixed dur- ing de novo synthesis and are reversible by exchange or dissocia- tion. J Immunol 142: 2751-2758, 1989

Neefjes, J. J., Breur-Vriesendorp, B. S., van-Seventer, G.A., Ivanyi, P., and Ploegh, H. L.: An improved biochemical method for the analysis of HLA-Class I antigens. Definition of new HLA-Class I subtypes. Hum Immunol 16: 169-181, 1986

Neefjes, J.J. and Ploegh, H.L.: Allele and locus-specific differences in cell surface expression and the association of HLA class I heavy chain with /32-microglobulin: differential effects of inhibition of glycosylation on class I subunit association. Ear J lmmunol 18: 801-810, 1988

Nuchtern, J .G. , Bonifacino, J .S. , Biddison, W.E. , and Klausner, R.D.: Brefeldin A implicates egress from the endoplasmic reticulum in class I-restricted antigen presentation. Nature 339: 223-226, 1989

Ooba, T., Hayashi, H., Karaki, S., Tanabe, M., Kano, K., and Takiguchi, M. : The structure of HLA-B35 suggests that it is derived from HLA-Bw58 by two genetic mechanisms. Immanogenetics 30: 76-80, 1989

Pala, P. and Askonas, B.A.: Low responder MHC alleles for Tc recognition of influenza nucleoprotein. Immunogenetics 23: 379-384, 1986

Powis, S. J., Howard, J. C., and Butcher, G.W.: The major histocom- patibility complex class II-linked cim locus controls the kinetics of

A. Hill et al. : HLA class I assembly rate controlled by c~2 domain 101

intracellular transport of a classical class I molecule. J Exp Med 173: 913-921, 1991

Sekimata, M., Hiraiwa, M., Andrien, M., Dupont, E., Karaki, S., Yamamoto, J., Kano, K., and Takiguchi, M.: Allodeterminants and evolution of a novel HLA-B5 CREG antigen, HLA-B SNA. J Im- munol 144: 3228-3233, 1990

Scheisl, B. : HLA-Typing: Problems and Solutions. Biotest Diagnostics (Biotest-Mitteilungen, Biotest, Dreieich, FRG), 1987

Snary, D., Barustable, C.J., Bodmer, W.F., and Crumpton, M.J.: Molecular structure of human histocompatibility antigens: the HLA-C series. Eur J Immunol 7." 580-585, 1977

Stam, N., Vroom, T., Peters, P., Pastoors, E., and Ploegh, H.: HLA-A and HLA-B specific monoclonal antibodies reactive with free heavy chains in Western blots, in formalin-fixed paraffin-embedded tissue sections and in cryo-immuno-electron microscopy. Int Immunol 2: 113-125, 1990

Stare, N. J., Spits, H., and Ploegh, H. L.: Monoclonal antibodies raised

against denatured HLA-B locus heavy chains permit biochemical characterization of certain HLA-C locus products. J lrnmunol 137: 2299, 1986

Townsend, A., Elliott, T., Cerundolo, V., Foster, L., Barber, B., and Tse, A.: Assembly of MHC class I molecules analyzed in vitro. Cell 62: 285-295, 1990

Townsend, A., Ohlen, C., Bastin, J., Ljunggren, H.G., Foster, L., and Karre, K.: Association of class I major histocompatibility heavy and light chains induced by viral peptides. Nature 340: 443-448, 1989

Weis, J.H. and Murre, C.: Differential Expression of H-2D d and H-2L a Histocompatibility Antigens. J Exp Med 161: 356-365, 1985

Williams, D.B., Swiedler, S.J., and Hart, G.W.: Intracellular transport of membrane glycoproteins: two closely related histocom- patibility antigens differ in their rates of transit to the cell surface. J Cell Biol 101: 725-734, 1985