an improved assembly assay for peptide binding to hla-b*2705 and h-2kk class i mhc molecules

Ž .Journal of Immunological Methods 209 1997 25–36

An improved assembly assay for peptide binding to HLA-B) 2705and H-2K k class I MHC molecules

Linda Tan a,1, Mads Hald Andersen b,1, Tim Elliott a, John S. Haurum b,)

a The Nuffield Department of Clinical Medicine, John Radcliffe Hospital, Oxford OX3 9DU, UKb Institute of Cancer Biology, Danish Cancer Society, StrandbouleÕarden 49, 2100 Copenhagen OE, Denmark

Received 4 February 1997; revised 7 August 1997; accepted 19 August 1997

Abstract

Ž .The assembly assay for peptide binding to class I major histocompatibility complex MHC is based on the ability tostabilise MHC class I molecules from mutant cell lines by the addition of suitable peptides. Such cell lines lack a functional

Ž .transporter associated with antigen presentation TAP and as a result accumulate empty, unstable class I molecules in theER. These dissociate rapidly in cell lysates unless they are stabilised by the addition of an appropriate binding peptide duringlysis. The extent of stabilisation of class I molecules is directly related to the binding affinity of the added peptide. However,some MHC class I molecules, including HLA-B)2705 and H-2K k are unusually stable in their peptide-receptive statemaking them inappropriate for analysis using this assay or assays which depend on the ability of peptides to stabilise MHCclass I molecules at the cell surface. Here we present an improved method that permits reliable measurements of peptidebinding to such class I MHC molecules that are unusually stable in the absence of peptide. Cells are lysed in the presence ofpeptide and incubated at 48C. After 2 h, during which peptide binding to empty MHC molecules occurs, the lysate is heatedto a temperature which preferentially destabilises those MHC molecules that remain empty. We have used this technique toassay peptide binding to HLA-B)2705, as well as to the murine allele H-2K k which also displays a stable phenotype whentransfected into TAP-deficient T2 cells and show that this method represents a marked improvement over previous methodsin terms of lower background signal and higher recovery of peptide bound molecules. q 1997 Elsevier Science B.V.

Keywords: MHC I; Peptide binding; HLA-B) 2705; H-2K k

1. Introduction

Peptide binding to class I major histocompatibilityŽ .complex MHC molecules is an important event in

Abbreviations: FCS, Fetal calf serum; HPLC, High perfor-mance liquid chromatography; HIV, Human immunodeficiencyvirus; IEF, Isoelectric focusing; e.o.e., End over end rotation

) Corresponding author. Tel.: q45-3525-7378; fax.: 45-3525-7721; e-mail: [email protected]

1 L.T. and M.H.A. contributed equally to the study.

the selection of peptide epitopes for MHC classŽI-restricted antigen presentation Elliott et al., 1993;

.Heemels and Ploegh, 1995 .Studies on peptides eluted from class I MHC

molecules have advanced our understanding of thenature of peptides presented by class I MHC in vivoŽRotzschke et al., 1990; van Bleek and Nathenson,

.1990; Falk et al., 1991; Jardetzky et al., 1991 . Thesestudies established that peptides presented by a givenMHC class I allele share a sequence motif corre-sponding to two or more essential amino acid side

0022-1759r97r$17.00 q 1997 Elsevier Science B.V. All rights reserved.Ž .PII S0022-1759 97 00142-7

( )L. Tan et al.rJournal of Immunological Methods 209 1997 25–3626

Ž .chains the so-called peptide anchor residues in theŽcontext of an 8–10 amino acid long peptide re-

viewed in Rammensee et al., 1995; Davenport and. )Hill, 1996 . Peptides which bind to HLA-B 2705

tend to be 9 amino acid long with Arg at P2 andpositively charged or hydrophobic residues at P9Ž . kJardetzky et al., 1991 , whereas H-2K preferen-tially binds 9-mers with Glu or Asp at P2 and

Žhydrophobic residues at P9 Norda et al., 1993;.Brown et al., 1994 . The solution of X-ray crystallo-

graphic structures of MHC class I-peptide complexesprovided a structural basis for understanding howthese anchor residues make specific contacts with the

ŽMHC molecule Bjorkman et al., 1987; Saper et al.,.1991; Madden, 1995 . It was revealed that depres-

sions or pockets in the MHC peptide binding grooveŽ Ž ..designated A to F, Saper et al., 1991 preferen-tially accommodate particular amino acids corre-sponding to the anchor residues of the peptide lig-and.

There is a long-recognised correlation betweencertain HLA-B27 subtypes and susceptibility to

Žspondyloarthropathies Brewerton et al., 1973;.Schlosstein et al., 1973 . Transgenic rats expressing

HLA-B) 2705 spontaneously develop spondy-Žloarthritic inflammatory disease Hammer et al.,

.1990 and HLA-B27-restricted cytotoxic T-lympho-Ž .cytes CTL have been isolated from the synovial

Ž .fluid of arthritic patients Hermann et al., 1993 .These findings have led to the hypothesis that HLA-B27 binds and presents certain microbial peptideswhich induce activation of autoreactive CTL withcross-reactivity towards homologous self-peptides,

Žthe so-called arthritogenic peptide hypothesis Be-.njamin and Parham, 1990 .

Given the potential link between peptide presenta-tion by HLA-B27 and its role in disease, there hasbeen considerable interest in identifying epitopesrecognised by autoreactive CTL and also in attempt-ing to find microbial peptide antigens carrying theB27 binding motif which may be responsible forinitiating the autoreactive CTL response.

Peptide binding studies have been widely used toinvestigate the binding affinity of putative peptideepitopes. Some peptide binding assays have been

based on the binding of iodinated peptide to purifiedHLA-B27 molecules or B27 molecules expressed on

Ž .the surface of cells Benjamin et al., 1991 . How-ever, the use of 125I-labelled peptides is a majorlimitation when it comes to screening large numbersof peptides for binding.

An alternative peptide binding assay developed byTownsend and colleagues is based on the ability tostabilise MHC class I molecules isolated from mu-

Žtant cell lines by adding suitable peptides Townsend.et al., 1990; Elvin et al., 1993 . Such cell lines lack a

functional transporter associated with antigen pre-Ž .sentation TAP and as a result accumulate empty,

unstable class I molecules in the ER. These dissoci-ate rapidly in cell lysates unless they are stabilisedby the addition of an appropriate binding peptide

Žduring lysis Townsend et al., 1990; Elliott et al.,.1991 . The extent of stabilisation of class I molecules

is directly related to the binding affinity of the addedŽ .peptide Elvin et al., 1993 .

However, some MHC class I molecules, includingHLA-B) 2705 and H-2K k are unusually stable in

Žtheir peptide-receptive state Benjamin et al., 1991;.Colbert et al., 1994 making them inappropriate for

analysis using this assay. Attempts have been madeŽto circumvent this problem Bowness et al., 1994;

.Colbert et al., 1994 by introducing an additionalstep of heating the cell lysate in order to effect thecomplete dissociation of HLA-B) 2705 molecules,prior to the addition of peptide. The unfoldedmolecules were then allowed to reassemble de novowith test peptides as had previously been described

b Ž .for H-2D Elliott et al., 1991 . However, this methodled to very poor recovery of folded HLA-B) 2705posing difficulties in quantification.

Here we present an improved method that permitsreliable measurements of peptide binding to suchMHC class I molecules, which are unusually stablein the absence of peptide. The modified assemblyassay introduces a heating step in order to preferen-tially destabilise those MHC molecules which re-main empty. We have used this technique to examinepeptide binding to HLA-B) 2705 and to H-2K k bothof which displays a stable phenotype when trans-fected into TAP-deficient T2 cells.

( )L. Tan et al.rJournal of Immunological Methods 209 1997 25–36 27

2. Materials and methods

2.1. Cell lines

The mutant human cell line T2 is derived from ahybrid of the B-LCL .174 and the T-LCL CEMŽ .Salter and Cresswell, 1986 . It synthesises HLA-A) 0201 and HLA-B)5101 encoded by the parentB-LCL .174, but fails to express normal amounts ofclass I MHC at the cell surface due the lack of afunctional TAP heterodimer. T2 cells transfected

) Ž . k Ž k .with HLA-B 2705 T2-B27 or H-2K T2-Kwere a kind gift from P. Cresswell and were main-

Ž .tained in RPMI 1640 Gibco supplemented with10% FCS, glutamine and antibiotics.

2.2. Synthetic peptides

The peptides used in this study are listed in Table1. The HLA-B) 2705-restricted peptide epitopes, Flu

Ž .NP SRYWAIRTR and HIV-1 gag383 – 391 263 – 272Ž . kKRWIIMGLNK , as well as the H-2K -restricted

Ž .epitope FLU NP SDYGERLI were used as50 – 58

positive control peptides. The unknown peptidebinders tested in this study are normally phosphory-

Žlated in the source protein residue in italics see.Table 1 . The non-phosphorylated form of the pep-

tides P23, P24, P25, OP36, OP39 and OP51 wereincluded in the study as potential novel B) 2705binding peptide candidates which carry both a natu-ral phosphorylation site and a peptide binding motif

Table 1

Peptides used in this study

Peptide name Peptide sequence Source protein Binding assay Ref.

) Ž .Flu NP SRYWAIRTR influenza ArPRr8r34 virus HLA-B 2705 Huet et al. 1990383 – 391

nucleoprotein residues 383–391) Ž .HIV gag KRWIIMGLNK HIV-1 gag residues 263–272 HLA-B 2705 Phillips et al. 1991 ;

Ž .Nixon et al 1988k Ž .Flu NP SDYGERLI influenza ArPRr8r34 virus H-2K Gould et al. 199150 – 58

nucleoprotein residues 50–58

) ) Ž .P23 RRPTS PVSR adenovirus 5 E1A protein HLA-B 2705 Dumont et al. 1993

residues 215–223) Ž .P24 KRPSPKPER adenovirus 2 and 5 DNA HLA-B 2705 Cleghon et al. 1993

binding protein residues 89–97) Ž .P25 SRPPRGLQER HIV-2 nef protein HLA-B 2705 Bandres et al. 1994

residues 10–19) Ž .OP36 SRHKKLMFKT p53 residues 378–387 HLA-B 2705 Takenaka et al. 1995) Ž .OP39 SRHKKLMFK p53 residues 378–386 HLA-B 2705 Takenaka et al. 1995) Ž .OP51 LKLA S PELER c-Jun residues 69–78 HLA-B 2705 Smeal et al. 1992

k Ž .P9 SDEKAA SPI influenza ArPRr8r34 virus H-2K Kistner et al. 1989

nucleoprotein residues 467–475k Ž .P10 IDEKL SEIL human respiratory syncytial H-2K Villanueva et al. 1994

virus RNA polymerase

residues 138–146k Ž .P11 EEY MPMEDL murine polyoma virus middle H-2K Srinivas et al. 1994

T antigen residues 313–321k Ž .P49 DDSHFVS I influenza ArUdornr72 virus H-2K Holsinger et al. 1995

matrix protein M2

residues 87–94

) Residues in italics are normally phosphorylated in the source protein.


) Ž .for HLA-B 2705 Haurum, 1996 . The non-phos-phorylated form of the peptides P9, P10, P11, P49were included in the study as potential novel H-2K k-binding peptide candidates which carry both a

natural phosphorylation site and a peptide bindingmotif for H-2K k.

The positive control peptides and P23, P24 andP25 were synthesised on an automatic peptide syn-


Ž .thesiser Zinsser Analytical, Maidenhead, UK usingconventional Fmoc chemistry. The peptides OP36,OP39 and OP51 were purchased from Research Ge-netics, Huntsville, USA. All peptides were more than95% pure by reverse phase HPLC, dissolved in PBS,sterile filtered and stored at y208C. Peptide concen-trations were determined by measuring absorbanceof tyrosine or tryptophan residues in the peptide

Ž .dissolved 1r50 in 6 M guanidine hydrochlorideŽ . Ž .Pierce according to Edelhoch 1967 and using the

Ž .equation OD s N = ´ q N = ´ = l = cTrp Trp Tyr Tyr

where c is the peptide concentration, N and NTrp Tyr

is the number of Trp and Tyr residues per peptideand l is the length of the light path. Extinctioncoefficients used at 280 nm were ´s1280 My1 fortyrosine and ´s5690 My1 for tryptophan. Concen-trations of peptides that did not contain either tyro-sine or tryptophan residues were estimated using

Ž .HPLC ls220 nm by comparison of peak areawith known peptide standards.

2.3. Antibodies

The HLA class I specific conformation dependentŽmonoclonal antibody W6r32 Barnstable et al.,

.1978 was used for immunoprecipitation of correctlyfolded, peptide-associated MHC molecules in theHLA-B) 2705 assembly assay. H100-5R, specific for

k Ž . kH-2K Lemke et al., 1979 was used for H-2Kassays. Both antibodies were purified from hy-

Ž .bridoma supernatants Ey et al., 1978 .

2.4. Assembly assay for peptide binding to MHC

The assembly assay protocol was adapted fromŽ .Elvin et al. 1993 . The TAP deficient cell line T2

) Ž . ktransfected with HLA-B 2705 T2-B27 or H-2KŽ k . w35 xT2-K were metabolically labelled with S -

Ž .methionine Amersham : Briefly, cells growing inŽ 6 .the log-phase 5=10 cells per sample were washedŽ . Ž .once in PBS 378C and starved 45 min, 378C in

Ž .methionine free RPMI Gibco with 10 mM Hepes,Ž10% FCS previously dialysed against PBS to re-

. 7move free methionine and 2 mM glutamine at 10Žcells per ml. The cells were harvested 1200 rpm, 5

.min, 378C throughout and resuspended in freshŽ 8 .methionine free medium 10 cells per ml together

35 Ž 7 .with S-methionine 100 mCi per 10 cells before aŽ .final incubation 30 min, 378C . The labelling was

stopped by addition of ice-cold PBS and all subse-quent steps were carried out at 48C. Aliquots contain-ing 5=106 cells were lysed on ice in 0.5 ml lysis

Žbuffer 150 mM NaCl, 50 mM Tris–HCl, 0.5%Ž .NP-40 Fluka, Buchs, Switzerland , 5 mM EDTA,. Ž .pH 7.5 with 0.5% Mega-9 Sigma, St. Louis, USA

Žin the presence of protease inhibitors 2 mM PMSF,5 mM iodoacetamide, 2 mgrml pepstatin, 2 mgrml

.leupeptin with or without synthetic peptide. CellŽ .nuclei were pelleted after 20 min 5 min, 10,000 g .

In the case of samples subjected to a heating step,the lysate was incubated for 2 h at 48C before being

Ž )heated 5 min at 50, 55, 60 or 658C for T2-B 2705k .and 2 min at 45, 50, 55 or 608C for T2-K . The

heating step was carried out in a pre-equilibratedwater bath using a floating rack for Eppendorf tubes,ensuring complete submersion of the sample volume.Upon completion of the heating step, the samplevials were transferred directly onto ice and all sam-

Žples were precleared overnight at 48C by addition ofŽ .50 ml 10% vrv freshly washed Staphylococcus

Žaureus organisms Pansorbin, Calbiochem, Notting-.. Ž .ham, UK with end over end rotation e.o.e. . Pan-

sorbin was removed the next day by centrifugationŽ .10 min, 15,500 g and the appropriate monoclonalantibody added at 10 mg per ml for 90 min, followed

Žby addition of protein A-Sepharose beads 75 ml,

Fig. 1. Optimisation of conditions for HLA-B) 2705 peptide binding assay. Lysates of metabolically labelled T2-B27 cells were incubated at) Ž .48C for 2 h in the presence of 0, 20, 2, 0.2 or 0.02 mM HLA-B 2705 binding HIV-1 gag peptide KRWIIMGLNK as indicated on263 – 272

Ž . Ž . Žthe x-axis. One set of samples were left at 48C panel a , whereas the remaining samples were heated 5 min at 508C panel b , 558C panel. Ž . Ž . Ž .c , 608C panel d or 658C panel e . Finally, one set of samples were heated for 20 min at 608C prior to the addition of peptide panel f .

After an overnight incubation at 48C, HLA-B) 2705 heavy chains were immunoprecipitated and subjected to IEF electrophoresis andŽ . Ž . Ž .autoradiography boxed inserts or quantification on a phosphorimager histograms . Panel g illustrates the improvement in signal-to noise

Ž .ratio by centrifuging the samples at 15,500 g for 10 min lanes 3 and 4, without and with peptide HIV-1 gag compared to 9,000 g263 – 272Ž .for 5 min lanes 1 and 2, without and with peptide . The markings 0, 1 and 2 refers to the number of sialic acid residues attached to the

carbohydrate side-chain of metabolically labelled HLA-B) 2705 heavy chains.


. Ž .10% vrv and incubation e.o.e. for 1 h 48C . Afterimmunoprecipitation, the beads were washed 4 times

Žwith lysis buffer without Mega-9 or protease in-.hibitors and stored at y208C until analysis by

electrophoresis.Assembly assays were also carried out according

to the previously published protocol by Colbert et al.Ž .1994 . Cells were metabolically labelled and lysedas above but in the absence of peptide. After addition

Ž .of Pansorbin 50 ml, 10% vrv the lysates wereheated to 608C for 20 min. The heat treated celllysates were then added to 50 ml volumes of peptideto give the appropriate final peptide concentration

Ž .and incubated overnight at 48C e.o.e . The assayproceeded the following day as described above.

2.5. Electrophoresis

In the case of HLA-B) 2705 assembly assays,Žsamples were eluted in reducing buffer 9.5 M urea,

2% NP-40, 5% 2-mercaptoethanol, 2% ampholines,ŽpH range 3.5–9.5 Pharmacia Biotech, Uppsala,

..Sweden and focused for 13–16 h at 880 V on 5.5%Ž .polyacrylamide isoelectric focusing IEF gels as

Ž . kdescribed Neefjes et al., 1986 . Samples from H-2K

Ž .assembly assays were eluted by boiling 5 min inŽSDS reducing buffer 50 mM Tris–HCl, pH 6.8, 2%

SDS, 5% 2-mercaptoethanol, 10% glycerol, 2.5%.bromophenol blue and run on 12% SDS-PAGE gels

Ž .around 1 h at 200 V . All gels were fixed in 10%acetic acid with 5% methanol and dried down on

Ž .3MM paper Whatman and exposed to Phosphorim-ager screens. MHC heavy chain bands were quanti-fied using the Imagequant Phosphorimager programŽ .Molecular Dynamics . The intensity of the band isdirectly related to the amount of peptide-bound classI MHC complex recovered during the assay.

3. Results

3.1. Conditions for peptide binding to B)2705

The B) 2705-restricted epitope KRWIIMGLNKwas used as a positive control when establishingoptimal heat treatment protocols for the T2-B27assembly assay. Peptide binding was assessed at a

Žrange of temperatures 4, 50, 55, 60 and 658C, each.for 5 min after incubation with 0, 0.02, 0.2, 2 or 20

mM peptide for 2 h. In addition, some cell lysates

) ) ŽFig. 2. Identification of HLA-B 2705 binding peptides. Using the two known HLA-B 2705 binding peptides Flu NP SRYWAIRTR,383 – 391. Ž .Flu NP from Influenza ArPRr8r34 virus nucleoprotein and HIV-1 gag KRWIIMGLNK, HIV Gag as positive controls, the263 – 272

binding of six peptides containing the HLA-B) 2705 peptide binding motif were tested in the modified assembly assay using the conditionsŽ . Ž . Ž .shown in Fig. 1d heating at 608C for 5 min . The peptides P23 RRPTSPVSR adenovirus 5 E1A ; P24 KRPSPKPER adenovirus 2215 – 223

Ž . Ž . Ž .and 5 DNA binding protein ; P25 SRPPRGLQER HIV-2 nef ; OP36 SRHKKLMFKT p53 ; OP39 SRHKKLMFK89 – 97 10 – 19 378 – 387Ž .p53 and OP51 LKLA SPELER c-Jun were tested at 20, 2, 0.2 and 0.02 mM in their non-phosphorylated form.378 – 386 69 – 78


were heated at 608C for 20 min before addition ofpeptide.

Ž . )As illustrated in Fig. 1 inserts , the B 2705heavy chain from T2 cell lysates migrates as a tripletin IEF electrophoresis. Multiple bands correspondingto the same MHC class I heavy chain are routinelyobserved after IEF electrophoresis for a number ofMHC alleles. Often, this is simply due to glycosyla-tion heterogeneity and in particular due to differingnumbers of sialic acid residues in the N-linked gly-

Ž .can Neefjes et al., 1986 .When the assay was performed at 48C, the major-

ity of HLA-B) 2705 could be recovered as assem-

bled, W6r32-reactive complexes even in the ab-Ž .sence of any added peptide Fig. 1a . This increased

background effectively reduces the sensitivity of theassembly assay for HLA-B) 2705 and, while it wouldnot prohibit the identification of high affinity bindingpeptides, weaker binders may easily go undetected.Heating of the cell lysate for 20 min before additionof peptide resulted in thermal denaturation of themajority of class I molecules and a very low recov-

Ž .ery of peptide-bound complexes Fig. 1f .By taking advantage of the differential suscepti-

bility of peptide-bound and ‘empty’ class I moleculesŽ .to thermal denaturation Fahnestock et al., 1992 , we

Fig. 3. Optimisation of conditions for H-2K k peptide binding assay. Lysates of metabolically labelled T2-K k cells were incubated at 48C fork Ž .2 h in the presence of 0, 20, 2, 0.2 or 0.02 mM H-2K binding peptide NP SDYGERLI from Influenza ArPRr8r34 virus50 – 58

Ž .nucleoprotein as indicated on the x-axis. One set of samples were left at 48C panel a , whereas the remaining samples were heated for 2Ž . Ž . Ž . Ž . kmin at 458C panel b , 508C panel 3c , 558C panel d , or 608C panel e . After an overnight incubation at 48C, H-2K heavy chains were

Ž . Ž .immunoprecipitated and subjected to SDS-PAGE and autoradiography boxed inserts or quantification on a phosphorimager histograms .


found that heating the cell lysates after incubationŽ .with peptide Fig. 1b–e reduced background values

while maintaining a high recovery of correctly foldedpeptide-stabilised MHC molecules. The optimal heat

Ž .treatment was judged to be 608C for 5 min Fig. 1d .This produced low background and a highly repro-ducible and titratable stabilisation of heavy chainfrom 20 mM to 20 nM.

We observed that introducing a heating step in theprotocol caused protein aggregation to occur whichappeared as novel, peptide independent bands in the

ŽIEF gels Fig. 1g, lanes 1 and 2, without and with.peptide, respectively . The majority of these aggre-

gates could be sedimented by centrifugation at15,500 g for 10 min after the overnight pre-clearing

Žstep Fig. 1g, lanes 3 and 4, without and with.peptide . This step was adopted in all subsequent

assays.

3.2. Identification of HLA-B)2705 binding peptides

One important application of this assay is theidentification of peptides which are able to bind classI MHC and thus have the potential to elicit specificCTL. This approach has led to the identification of

epitopes derived from P. falciparum, which areŽpotentially protective against severe malaria Hill et

.al., 1992 . Our own research interest is the identifica-tion of novel post-translationally modified peptideepitopes derived from normal, viral and oncogenic

Žproteins Haurum et al., 1994; Haurum et al., 1995;.Haurum, 1996 . We have therefore used the modi-

fied binding assay to screen candidate peptides fortheir ability to bind to HLA-B) 2705.

Based on the published HLA-B) 2705 peptidebinding motif which consists of an Arg at P2 and an

ŽArg or a Lys residue at the C-terminus Jardetzky et. )al., 1991 , potential B 2705 binding peptides were

Žselected from viral phosphoproteins P23, P24 and.P25 , as well as from oncogene phosphoproteins

Ž .OP36, OP39 and OP51 and their binding comparedwith that of natural HLA-B) 2705-restricted CTLepitopes from influenza virus nucleoprotein and HIVgag using the modified assembly assay describedabove.

The data presented in Fig. 2 shows that, as ex-pected, the two known B) 2705 restricted peptideepitopes from influenza virus nucleoprotein NP383 – 391Ž . ŽSRYWAIRTR and HIV-1 gag KRWI-263 – 272

. )IMGLNK bound with high affinity to B 2705. In

k k Ž .Fig. 4. Identification of H-2K binding peptides. Using the H-2K binding peptide NP SDYGERLI as a positive control, the binding50 – 58

of four peptides containing the H-2K k peptide binding motif were tested in the modified assembly assay using the conditions shown in Fig.Ž . Ž . Ž .3d heating at 558C for 2 min . The peptides P9 SDEKAA SPI influenza nucleoprotein NP ; P10 IDEKLSEIL human respiratory467 – 475

Ž . Ž .syncytial virus RNA polymerase ; P11 EEY MPMEDL murine polyoma virus middle T antigen and P49 DDSHFVSI138 – 146 313 – 321

influenza matrix protein M2 were tested at 20, 2, 0.2 and 0.02 mM in their non-phosphorylated form.87 – 94


addition, it is seen that the adenovirus 5 E1A 215 – 223Ž .derived peptide P23 RRPTSPVSR as well as the

Ž .p53 derived peptide OP39 SRHKKLMFK378 – 386

bound with a similar high affinity, while the HIV-2Ž .nef peptide P25 SRPPRGLQER bound only10 – 19

Ž .weakly and the peptides P24 KRPSPKPER , OP36Ž . Ž .SRHKKLMFKT , or OP51 LKLA SPELER didnot show any appreciable binding to B) 2705. Thisexperiment illustrates the usefulness of this assay inscreening large numbers of potential peptide epitopesfor their ability to bind to HLA-B) 2705 and that theassay is able to distinguish between strong and weakbinders.

3.3. Assay for peptide binding to H-2K k

When transfected into the TAP negative humancell line T2, the murine allele H-2K k displays asimilar stable phenotype in the absence of peptide. Inthis case, the stable phenotype of empty MHCmolecules may be due to the fact that murine heavy

Žchain has a high affinity for human b m Hochman2.et al., 1988; Baas et al., 1992 . We next sought to

establish whether the same method could be appliedto H-2K k. As seen in Fig. 3, without peptide, theH-2K k heavy chain remains associated with b m in2

the absence of heating. As for HLA-B) 2705, differ-ent heat treatments were carried out to optimise forthe preferential denaturation of peptide-unbound H-2K k, using the H-2K k-restricted epitope from in-

Ž .fluenza virus nucleoprotein NP SDYGERLI .50 – 58

Milder conditions were required to achieve the de-naturation of ‘empty’ H-2K k molecules, comparedto B) 2705. Fig. 3 shows that heating cell lysates at558C for 2 min resulted in a quantifiable titration ofpeptide-induced stabilisation and negligible back-ground class I stability in the absence of peptide.

In a separate experiment, we found that overnightincubation of T2-K k cells at 208C in the presence ofpeptide dilutions were able to induce upregulation ofH-2K k expression, as detected by FACS-stainingusing the H100-5R antibody as primary antibody,with a half-maximal induction of H-2K k expression

Ž .at 10 mM peptide results not shown . Hence, usingT2-K k the FACS type peptide binding assay is 10–100 times less sensitive than the assembly assay. Asimilar comparison was not made for T2-B27, due toa lack of a HLA-B) 2705-specific antibody.

3.4. Identification of H-2K k binding peptides

Again we used the modified assembly assay totest the binding of candidate peptide epitopes derivedfrom viral phosphoproteins. Both P10 from humanrespiratory syncytial virus RNA polymerase138 – 146Ž .IDEKLSEIL and P49 from influenza matrix pro-

Ž .tein M2 DDSHFVSI were found to bind to87 – 94

H-2K k with high affinity, compared to the knownH-2K k-restricted influenza virus NP peptide50 – 58

Ž .epitope SDYEGRLI , whereas the influenza nucleo-Ž .protein NP peptide P9 SDEKAA SPI bound467 – 475

Ž .with intermediate affinity see Fig. 4 . No bindingwas observed for the murine polyoma virus middle T

Ž .antigen derived peptide P11 EEY MPMEDL ,313 – 321

despite its ‘strong’ H-2K k- specific binding motif.It is noteworthy that the binding of the immun-

odominant H-2K k-restricted influenza virus NP50 – 58Ž .peptide epitope SDYEGRLI seems somewhat

unimpressive when comparing with P10 and P49.However, this does not detract from its value asreference peptide.

4. Discussion

Peptide ligands play a central role in promotingthe intracellular assembly of MHC class I molecules.The binding of peptide by the class I MHC heavy

Žchain stabilises its interaction with b m Elliott et2

al., 1991; Townsend et al., 1990; Townsend et al.,.1989 . This provides the basic principle of the as-

sembly assay. In TAP deficient cell lines, class Imolecules lack an endogenous supply of peptideligands. As a consequence, they accumulate in theER as unstable, peptide receptive heavy chainrb m2

heterodimers. When released into detergent-contain-ing solution, these empty class I molecules will bindto peptides added to the cell lysates and becomestabilised. These can subsequently be detected byconformation dependent antibodies after sufficienttime has elapsed for the remaining empty MHCmolecules to dissociate.

Previously published data on the thermal stabilityof H-2Kd molecules showed that the addition ofpeptide to purified Kd molecules caused a large shiftin the transition temperature for heat induced denatu-

Ž .ration Fahnestock et al., 1992 . The unfolding tran-


sition temperature corresponding to simultaneousdissociation and unfolding of the Kd heavy chainwas found to be ;458C for empty Kdrb m het-2

erodimers, whereas peptide-filled heterodimers un-derwent thermal denaturation at ;588C. Thus, heattreatment of samples after the addition of peptidewould be expected to cause empty class I moleculesto dissociate whilst peptide-filled molecules remainresistant to denaturation.

Accordingly, in our modified protocol for peptidedependent assembly of class I MHC molecules, thecell lysates are first incubated in the presence ofpeptide for 2 h at 48C. This allows the binding ofpeptide to empty class I molecules. Following this,

Žthe cells lysates are heated briefly 608C for 5 min) k .for T2-B 2705 or 558C for 2 min for T2-K in

order to promote dissociation of empty MHC-b m2

heterodimers, the rationale being that heat treatmentmust be sufficiently mild so as not to also causethermal denaturation of peptide-filled molecules.Different class I alleles vary in their thermal stabil-ity, and thus a unique set of conditions will apply toeach allele.

Previously, the availability of a reliable assemblyassay has been limited to the study of those MHCalleles which are very unstable without bound pep-tide at 48C. The simple modification of the assaydescribed here provides a way of applying the sametechnique to other, more stable alleles. Two suchexamples are HLA-B) 2705 and H-2K k. In particu-lar, the stability of HLA-B) 2705 has hindered theidentification of candidate CTL epitopes which mayhave clinical relevance in a number of B27-associ-ated autoimmune diseases such as ankylosingspondylitis and reactive arthritis. We have shownthat this assay is well suited to the kind of peptide-screening procedure which is now an essential stepin identifying important CTL epitopes by ‘reverse

Ž .immunogenetics’ Hill et al., 1992 .

Acknowledgements

L.T. is the recipient of an Overseas ResearchStudentship award. M.H.A. is the recipient of aDanish Cancer Society scholarship. T.E. is a Well-come Senior Fellow in Basic Biomedical Science.This project was funded by the Alfred Benzon Foun-

dation, Fru Asid Thaysens Legat for Lægevidens-kabelig Grundforskning, Novo Nordisk Fonden, andthe Danish Medical Research Council.

References

Baas, E.J., van Santen, H.M., Kleijmeer, M.J., Geuze, H.J.,Peters, P.J., Ploegh, H.L., 1992. Peptide-induced stabilizationand intracellular localization of empty HLA class I complexes.J. Exp. Med. 176, 147.

Bandres, J.C., Luria, S., Ratner, L., 1994. Regulation of humanimmunodeficiency virus Nef protein by phosphorylation. Vi-rology 201, 157.

Barnstable, C.J., Bodmer, W.F., Brown, G., Galfre, G., Milstein,C., Williams, A.F., Ziegler, A., 1978. Production of mono-clonal antibodies to group A erythrocytes, HLA and otherhuman cell surface antigens: New tools for genetic analysis.Cell 14, 9.

Benjamin, R., Parham, P., 1990. Guilt by association: HLA-B27and ankylosing spondylitis. Immunol. Today 11, 137.

Benjamin, R.J., Madrigal, J.A., Parham, P., 1991. Peptide bindingto empty HLA-B27 molecules of viable human cells. Nature351, 74.

Bjorkman, P.J., Saper, M.A., Samraoui, B., Bennett, W.S., Stro-minger, J.L., Wiley, D.C., 1987. Structure of the human class Ihistocompatibility antigen, HLA-A2. Nature 329, 506.

Bowness, P., Allen, R.L., McMichael, A.J., 1994. Identification ofT cell receptor recognition residues for a viral peptide pre-sented by HLA B27. Eur. J. Immunol. 24, 2357.

Brewerton, D.A., Hart, F.D., Nicholls, A., Caffrey, M., James,D.C., Sturrock, R.D., 1973. Ankylosing spondylitis and HL-A27. Lancet 1, 904.

Brown, E.L., Wooters, J.L., Ferenz, C.R., O’Brien, C.M., Hewick,R.M., Herrmann, S.H., 1994. Characterization of peptide bind-ing to the murine MHC class I H-2Kk molecule. Sequencingof the bound peptides and direct binding of synthetic peptidesto isolated class I molecules. J. Immunol. 153, 3079.

Cleghon, V., Piderit, A., Brough, D.E., Klessig, D.F., 1993.Phosphorylation of the adenovirus DNA-binding protein andepitope mapping of monoclonal antibodies against it. Virology197, 564.

Colbert, R.A., Rowland Jones, S.L., McMichael, A.J., Frelinger,J.A., 1994. Differences in peptide presentation between B27subtypes: The importance of the P1 side chain in maintaininghigh affinity peptide binding to B) 2703. Immunity 1, 121.

Davenport, M.P., Hill, A.V., 1996. Peptides associated with MHCclass I and class II molecules. In: Browning, M., McMichael,

Ž .A. Eds. , HLA and MHC Genes, Molecules and Function.BIOS Scientific Publishers, Oxford, p. 277.

Dumont, D.J., Marcellus, R.C., Bayley, S.T., Branton, P.E., 1993.Role of phosphorylation near the amino terminus of aden-ovirus type 5 early region 1A proteins. J. Gen. Virol. 74, 583.

Edelhoch, H., 1967. Spectroscopic determination of tryptophanand tyrosine in proteins. Biochemistry 6, 1948.


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

Elliott, T., Smith, M., Driscoll, P., McMichael, A., 1993. Peptideselection by class I molecules of the major histocompatibilitycomplex. Curr. Biol. 3, 854.

Elvin, J., Potter, C., Elliott, T., Cerundolo, V., Townsend, A.,1993. A method to quantify binding of unlabeled peptides toclass I MHC molecules and detect their allele specificity. J.Immunol. Methods 158, 161.

Ey, P.L., Prowse, S.J., Jenkin, C.R., 1978. Isolation of pure IgG1,IgG2a and IgG2b immunoglobulins from mouse serum usingprotein A-sepharose. Immunochemistry 15, 429.

Fahnestock, M.L., Tamir, I., Narhi, L., Bjorkman, P.J., 1992.Thermal stability comparison of purified empty and peptide-filled forms of a class I MHC molecule. Science 258, 1658.

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

Gould, K.G., Scotney, H., Brownlee, G.G., 1991. Characterizationof two distinct major histocompatibility complex class I Kk-re-stricted T-cell epitopes within the influenza ArPRr8r34virus hemagglutinin. J. Virol. 65, 5401.

Hammer, R.E., Maika, S.D., Richardson, J.A., Tang, J.P., Taurog,J.D., 1990. Spontaneous inflammatory disease in transgenicrats expressing HLA-B27 and human beta 2m: An animalmodel of HLA-B27-associated human disorders. Cell 63, 1099.

Haurum, J.S., Arsequell, G., Lellouch, A.C., Wong, S.Y., Dwek,R.A., McMichael, A.J., Elliott, T., 1994. Recognition ofcarbohydrate by major histocompatibility complex class I-re-stricted, glycopeptide-specific cytotoxic T-lymphocytes. J.Exp. Med. 180, 739.

Haurum, J.S., Tan, L., Arsequell, G., Frodsham, P., Lellouch,A.C., Moss, P.A., Dwek, R.A., McMichael, A.J., Elliott, T.,1995. Peptide anchor residue glycosylation: Effect on class Imajor histocompatibility complex binding and cytotoxic Tlymphocyte recognition. Eur. J. Immunol. 25, 3270.

Haurum, J.S., 1996. Recognition of peptides carrying post-transla-tional modifications by class I MHC-restricted, cytotoxic T

Ž .lymphocytes CTL . Ph.D. Thesis. University of Oxford.Heemels, M.T., Ploegh, H., 1995. Generation, translocation and

presentation of MHC class I-restricted peptides. Annu. Rev.Biochem. 64, 463.

Hermann, E., Yu, D.T., Meyer zum Buschenfelde, K.H., Fleis-cher, B., 1993. HLA-B27-restricted CD8 T cells derived fromsynovial fluids of patients with reactive arthritis and ankylos-ing spondylitis. Lancet 342, 646.

Hill, A.V., Elvin, J., Willis, A.C., Aidoo, M., Allsopp, C.E.,Gotch, F.M., Gao, X.M., Takiguchi, M., Greenwood, B.M.,Townsend, A.R., McMichael, A.J., Whittle, H.C., 1992.Molecular analysis of the association of HLA-B53 and resis-tance to severe malaria. Nature 360, 434.

Hochman, J.H., Shimizu, Y., DeMars, R., Edidin, M., 1988.Specific associations of fluorescent beta-2-microglobulin withcell surfaces. The affinity of different H-2 and HLA antigensfor beta-2-microglobulin. J. Immunol. 140, 2322.

Holsinger, L.J., Shaughnessy, M.A., Micko, A., Pinto, L.H.,Lamb, R.A., 1995. Analysis of the posttranslational modifica-

tions of the influenza virus M2 protein. J. Virol. 69, 1219.Huet, S., Nixon, D.F., Rothbard, J.B., Townsend, A., Ellis, S.A.,

McMichael, A.J., 1990. Structural homologies between twoHLA B27-restricted peptides suggest residues important forinteraction with HLA B27. Int. Immunol. 2, 311.

Jardetzky, T.S., Lane, W.S., Robinson, R.A., Madden, D.R.,Wiley, D.C., 1991. Identification of self peptides bound topurified HLA-B27. Nature 353, 326.

Kistner, O., Muller, K., Scholtissek, C., 1989. Differential phos-phorylation of the nucleoprotein of influenza A viruses. J.Gen. Virol. 70, 2421.

Lemke, H., Hammerling, G.J., Hammerling, U., 1979. Fine speci-ficity analysis with monoclonal antibodies of antigens con-trolled by the major histocompatibility complex and by theQarTL region in mice. Immunol. Rev. 47, 175.

Madden, D.R., 1995. The three-dimensional structure of peptide-MHC complexes. Annu. Rev. Immunol. 13, 587.

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

Nixon, D.F., Townsend, A.R., Elvin, J.G., Rizza, C.R., Gallwey,J., McMichael, A.J., 1988. HIV-1 gag-specific cytotoxic Tlymphocytes defined with recombinant vaccinia virus andsynthetic peptides. Nature 336, 484.

Norda, M., Falk, K., Rotzschke, O., Stevanovic, S., Jung, G.,Rammensee, H.G., 1993. Comparison of the H-2Kk- andH-2Kkm1-restricted peptide motifs. J. Immunother. 14, 144.

Phillips, R.E., Rowland-Jones, S., Nixon, D.F., Gotch, F.M.,Edwards, J.P., Ogunlesi, A.O., Elvin, J.G., Rothbard, J.A.,Bangham, C.R., Rizza, C.R., McMichael, A.J., 1991. Humanimmunodeficiency virus genetic variation that can escape cyto-toxic T cell recognition. Nature 354, 453.

Rammensee, H.G., Friede, T., Stevanovic, S., 1995. MHC ligandsand peptide motifs: First listing. Immunogenetics 41, 178.

Rotzschke, O., Falk, K., Deres, K., Schild, H., Norda, M., Met-zger, J., Jung, G., Rammensee, H.G., 1990. Isolation andanalysis of naturally processed viral peptides as recognized bycytotoxic T cells. Nature 348, 252.

Salter, R.D., Cresswell, P., 1986. Impaired assembly and transportof HLA-A and -B antigens in a mutant TxB cell hybrid.EMBO J. 5, 943.

Saper, M.A., Bjorkman, P.J., Wiley, D.C., 1991. Refined structureof the human histocompatibility antigen HLA-A2 at 2.6 Aresolution. J. Mol. Biol. 219, 277.

Schlosstein, L., Terasaki, P.I., Bluestone, R., Pearson, C.M., 1973.High association of an HL-A antigen, W27, with ankylosingspondylitis. N. Engl. J. Med. 288, 704.

Smeal, T., Binetruy, B., Mercola, D., Grover Bardwick, A.,Heidecker, G., Rapp, U.R., Karin, M., 1992. Oncoprotein-mediated signalling cascade stimulates c-Jun activity by phos-phorylation of serines 63 and 73. Mol. Cell. Biol. 12, 3507.

Srinivas, S., Schonthal, A., Eckhart, W., 1994. Polyomavirusmiddle-sized tumor antigen modulates c-Jun phosphorylationand transcriptional activity. Proc. Natl. Acad. Sci. USA 91,10064.

Takenaka, I., Morin, F., Seizinger, B.R., Kley, N., 1995. Regula-tion of the sequence-specific DNA binding function of p53 by


protein kinase C and protein phosphatases. J. Biol. Chem. 270,5405.

Townsend, A., Ohlen, C., Bastin, J., Ljunggren, H.G., Foster, L.,Karre, K., 1989. Association of class I major histocompatibil-ity heavy and light chains induced by viral peptides. Nature340, 443.

Townsend, A., Elliott, T., Cerundolo, V., Foster, L., Barber, B.,Tse, A., 1990. Assembly of MHC class I molecules analyzedin vitro. Cell 62, 285.

van Bleek, G.M., Nathenson, S.G., 1990. Isolation of an endoge-nously processed immunodominant viral peptide from theclass I H-2Kb molecule. Nature 348, 213.

Villanueva, N., Navarro, J., Mendez, E., Garcia Albert, I., 1994.Identification of a protein kinase involved in the phosphoryla-tion of the C-terminal region of human respiratory syncytialvirus P protein. J. Gen. Virol. 75, 555.

an improved assembly assay for peptide binding to hla-b*2705 and h-2kk class i mhc molecules

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