lnternatimal lmmunology, W. 9, No. 3, pp. Wffi

Stimulation of human cytotoxic T cells withHIV-1 -derived peptides presented byrecombinant HLA-A2 peptide complexes

Jrirgen B. Watter, Christian Branderl, Mathai Mammen2, David N. Garboczi3,Spy-ros A. Kalamsl, George M. Whitesides2, Bruce D. Walkerl andHerman N. Eisen

Center for Cancer Research and Department of Biology, Massachusetts lnstrtute of Technology,Cambridge, MA 02139, USAltnfect ious Disease Unit , Massachusetts Generai Hosprtal and Harvard Medical School , Boston, MA0 2 1 1 4 , U S AzDepartment of Chemistry and Chemrcal Brology, Harvard Universi ty, Cambridge, MA02138, USA3Department of Moleculai and Cel lu lar Bioiogy, Harvard Universrty, Cambrrdge, MA 02138, USA

Keywords: cytotoxic T cells, HlV, HL-A-A2

Abstract

HLA-A2 heavy chain and fo-microglobulin were expressed In Escherichla coll, and refolded In the

presence of peptldes derived from HIV-1 RT and gag proteins. When recomblnant HLA-A2

molecules were at tached to cel ls lacking Hl A-A2, the cel ls became suscept ib le to lysis by HLA-A2

restr icted cytotoxic T lymphocyte (CTL) c lones speci f lc for pept ldes der ived from RT and gag

proteins. L imit ing di lut lon analyses of per ipheral b lood mononuclear cel ls f rom HIV-1- lnfected

lndiv iduals showed that the recombinant HLA-A2 pept lde complexes covalent ly immobi l ized on

microspheres stimulated the development of HLA-A2 peptide-specific CTL. Preformed HtA-peptide

complexes may provide an al ternat ive to immunizat ion procedures that depend upon intracel lu larprocessing of ant igen to el ic i t T cel l responses-

lntroduct ion

MHC-encoded class I molecules serve as pept lde binding,transport and display proterns on the cer l sunace, and can

evoke T cet l responses when recogntzeo bv ant lgen-soecrf lcreceptors on cytotoxrc T lymphocytes (CTL). l '"4HC class Imolecules are glycoproteins constst tng oi f 'vo non-covalent lyassoclated polypeptrde chains, a larger or heavy chatn and

a smal le r o r l igh t cha in , te rmed p2-mrcroQlobu l rn (1 ) . Human

class I molecules have been refolded f rom suounrts proouced

in Escherrchra col i in associatron wrth syntnet lc pepttdes

(2). The three-dimenstOnal StrUctures of severat recombtnant

complexes, determined by X-ray crystal lography (3), are

vrr tual ly ident ical to the Structure of the c laSS I molecule

isolated from human cel ls (4). However the MHC protelns

produced in E. col ido not contatn the ccmprex carbonyoratethat is normal ly at tached to asoaragrne 86. The aim of thtsr to rk i s to de termtne l f an MHC c lass I o ro ie rn , speCt f i ca l l yHLA-A2 (A2), produced in non-glycosylateo form rn E. colr

and assembled w i th svn the t ic nonapeot loes can ( i ) e l i c t t

@ 1996 Oxfotd Uniwrsitv Press

cytolyttc responses by CDB* rT cell clones arising tn thecourse of natural infect ion wrth HIV-1 and ( i i ) st imulate lheprolr feratron of pept ide-specrf ic CDB* T cel ls in perrpheral

blooo lymphocytes of HIV-1 seropostt tve donors.The HLA-A2 (HLA-A'0201) heavy chain and p2-mrcro-

globul in l ight chain v{ere assembled in the presence of fwoanttgenrc pept ides ber ived l rom HIV- ' l : a pQtide from reversetranscr lptase (RT-1V9, amrno actds 4THB4,ILKEPVHGV) (5)and a peptrde from the gag protein (Gag-SL9, amrno acros7745, SLYNTVATL) (6) Both peptrdes represent diseaseresponse targets in HLA-A2* HIV-1- infected pat ients (7),

conform to the H[-A-A2 consensus motif (8), and have theoptrmal sequence recognized by RT-|V9- 'and Gag-SL9-specrf ic CTL clones obtained from asymptomdtic HIV-1-sero-posrtrve pat ients (9,10). Both pept ides are recogntzed bycytotoxic T cel l c lones at ptcomolar concentrat ions, wi th SD.6values (r .e. peptrde concentrat ion givrng hal f -maximal specrf rclysis) of -3 pM for Gag-SL9 and -0 1 pM for Ff f - lV9 (5,9)

Corresponclence to: J. B. Walter

Transmrfttne edtor. D. Y. Loh Recetved 19 Seotember 1996, accepted 5 December 1996

102 lnduction of CTL responses by recombinant HU-42 peptide complexes

V V V

(v

Log I r l uo rescence In tens i t y

Flg. 2. Adsorptron of purrf ied soluble H[-A-AZ peptide complexes tothe surface of Ht-A-A2-negaltve cells monrtored by FACS anatysrs.MHC class I expressron on unmoorf ied HLA-A2-negative C1R cetts(HLA-8,C) was determtned wirh mAb 887.7-FITC conjugate (doneoline, 887.7 antr.HLA-A,B,C). To adsorb the Ht-A+€ptide comptexeswe covalently anached mAb BBMl (antr-pTmicroglobul in) and mAbMA2.1 (antr-HLA-A2,B 1 7) to the cel l sur{ace using a heterobifunctronalcross-lrnker (SPDP). Sudace bound mAb-SPDP conjugates weredetected using FITC-goat anti-mouse F(ab)r fragments (dasheC l ine)Adsorption of HLA-A2 peptrde complexes to mAb-coated cetts wasshown by an increase in MHC class I expression using mAb 887.7-FITC conlugate (sol id l ine).

mAb W6/32 (11) ( lgG2a specrf ic for HI-A-A,B,C) and PA2.1(12) ( lgG1 specr i ic for HLA-M ano A2B), both reactrve wrrhnatrve Ht-A-A2 complexes, wts detected with goat antr-mouselg conjugated to horseradish peroxrdase (1 :5000: Amersham)After washing the ni t rocei lu lose was incubated for 1 mrn wrthreagents for ECL immunodetectron (1:1; Amersham) andexposeo to X-ray fi lm. HLA-A2 peptrde concentrations weremeasureo by mrcro-BCA (bic inchonrntc acid) assay (Pierce,Rockford, lL) , uslng BSA as a stanoard:

Covalent attachment of mAb to the cell sur't'ace

mAb BBMl (13) (an t r - p2-mrcrog iobu i ln ) and mAb MA2.1 (14)(antr-HLA-A2, 817) were covalent ly at tached to cel ls usrng aheterobi funct lonal cross- l inker, SPDP IMsuqcinimidyl 3-(2-pyr idyldi th io)propionate, Piercel , as descr ibcd (15). SPDP(50 pl, 20 mM in ethanol) was added to mAb in PBS, pti 7 2,(1 mg/ml) for 30 min at room temoerature and then dialyzedagainst PBS Cel ls (106) were reduceo with 50 pM di th iothrertolfor 30 mrn, washed twrce with P8S and incubated wrth 200pl of mAb-SPDP at 0 5 mg/ml (1 h at room temperature),wasned and sublected to FACS analvsis.

Adsorption of soluble A2-pepttde complexes to mAb pre-coated cells

Cel ls (106) wi th covalent ly at tacheo mAb (using SPDP anddi th iothrei to l , see above) were incubated with 10 pg solubleHLA-A2 peptide complexes in 500 pl PBS for 30 min at 4'C,washed in 10 ml of cold RPMI, counted and adjusted to theappropnate cell number.

o!

oo

L 9

oc r . i

r \

cot^l

l 5 0

m A b W 6 / 3 :

m A b P A l . I

Flg. 1. Gel lr itratron HPLC profi les of HI-A-A2 peptrde complexes.HPLC prolries of Ht-A-M reconstttutton lrom recombrnant healy charnand p2-mrcroglobuiln in associatron wrth (a) HIV-1 Gag-SL9 peptrde77-€5, SLYNTVATL and (b) HIV-'l RT-lvg peptrde 47G-484,ILKEPVHGV Specrfic brnding of mAb W6/32 and mAb PA2.1 (bothspecrlically reactrve with nattve HLA-A2 complexes)to HPLC fractionswas used to monitor refolded A2-peptrde complexes.

Methods

CTL clones and cell lines

CTL clones from asymptomatic HIV-1 seroposrt ive patrentsand the corresponding (autologous) Epstein-Barr vrrus-trans-formed B lymphoblastoid cel l l ines (B-LCL) were establ ishedand marntained as descnbed prevrously (6,7). Subiects '115i ,

15760 and 16' lJ were previously shown to have srgnrf icantgagl .specrfrc CTL actrv i fy (6 and unpubl ished). HlA-typrngwas performed by the Massachusetts General Hospi ta lTlssueTyping Laboratory usrng standard serologrcal technrques(sub jec t 161J : A2, 43 ; 87 , 860; C3; DR2, DR4: sub jec t 010-1 1 5 i : A 2 , A 2 B ; B 1 a , 8 5 2 ; C w B ; D R 1 , 2 ; D O 1 a n d s u b y e c t15760: A2,844,851; C3; DR11) RPMI 16a0 (S igma, S t Lou is ,MO) contarning 20 % (vlv) heat- inact ivated FCS (Sigma) wassupplemented with u-glutamine (2 mM), penic i l i in (50 U/ml) ,streptomycrn (50 Fg/ml) and used for al l cel l l ines.

Gel filtration HPLC of HLA-A2 peptide complexes

Peptides were synthesized at the MIT Bropolymers laboratory.A2 refolding and complex format ion was in i t iated by di lut ionof the fwo oenatured subunrts in the presence of peptrdes asprevrously oescrtbed (2).

mAb bindtng to HPLC fracilons

mAb binorng to HPLC fractrons was used to monrtor refoldedA2-peptrde complexes. Samples of HPLC peak fract ions(20 trl) were pipetted onto a nitrocellulose fi l ter (Amersham,Santa Clearbrook, lL) and allowed to adsorb for 2 h at roomtemperature. The membrane was then incubated with 3%BSA in PBS followed by the respective mAb in 50 mM Tris-HCl, pH 8, 100 mM NaCl, 0 05% Tween. Specific binding of

(a) anti Gag/SLg clone D23

hjuction of CTLresponses by recombinant HLA-A| peptide complexes 103

(b) anti RT/[V9 clone 68A62

4

t(,

20

catn

3o'6q.)

v1

N

mCIR-mAtrA2-SL9

CIRA2+SL9 60

CIR-mAbAz-lvg

ClM2+lV9 {

P8l5-mAbA2-SL9

- O r : : '

#

- - c l - -

*

- - o - -

4-

- - - -E l - - " P8 l tmA-bA2- lV9

- - z - - P 8 l 5

(c ) anti Gag/SLg clone D23E :T

FACS staining

MHC c lass I express ion on unmodr f ieo C1R, K562 and PB15

cel ls was determined with mAb 887 7-FITC coniugate IB87'7( 1 6), ant i -HLA-A,B,Cl. Surface bound mAb-.SFDP coniugateswere detected using FITC-goat antr-mouse F(ab)z fragments(Jackson lmmunoresearch, Avondale, PA). Adsorptron of HLA-

A2 peptrde complexes to mAb-coated cel ls was shown by

an increase in MHC I expressron usrng the mAb 837.7-

FITC conjugate. lmmunof luorescent StalnrnQ was performed

according to standard protocols, usrng a FACScan f low

cytometer (Becton Dickinson, Mountarn Vierv ' CA).

Cytotoxicity assays

The slCr- labeled target cei ls (107 cel ls) lvere washed twrcewith cold PBS, incubated with 50 pM drthiotnrei to l for 30 min,

again washed with cold PBS ano resuspenoed in 200 pl of

SPDP-+nAb at 0.5 mg/ml. After 30 min, the cells were washed

twice with cold PBS, resuspenoed wrth 10 pg soluble HLA-

A2 peptide in 500 pl PBS at 4"C, and after 30 min cells were

E:T

*- C1R-MAFA2-SL9

-# I$62'mAbA2'SI-9

4- PsltmAbA2'SLg

- - o - - C I R ' E A b

- -.- - K562-EAb

- - X - - P 8 l 5 ' m A b

washed with cold RPMI and adiusted to 2X10s cet ls/mlCytotoxicity assays were performed in duplicate wtth CTLclones added at ef fector- to- target ratros (E:T) of 10, 5, 2 5and 1 25:1 to mAb-A2 pept ide-coated slCr- labeled cei lsand to pept ide-pulsed cel ls (prerncubated with a peptrde

concentration of 1 pg/ml for 30 mrn and *Ettiu-O) After 4 hat 37"C, cet l supernatants were assayed for 51Cr release.Total s lCr release was determtned by detergent lysis andpercent speci f ic lys is was calculated as [ (stCr ' samolerelease - Spontaneous release) i ( total re leaSe - spontaneous

release)]x100. Spontaneous release was always <209' . ofmaxrmal release.

Preparation of silica microspheres (beads) covalently mooiiedwith HlA-peptide complexes

We prepared si l ica beads (microspheres, 5 pm dtameter)whose surfaces are covaiently modified with the HLA-A2peptide complexes using either the RT ot gag nonapeptrdes.

Beginning with commercrally available (Bangs, LocatronU,

0 6 - n-

a a

v,

3(J

= {(J

ct

v,

S r (

O 6 - h

t r : r

Flg. 3. susceptibilaty ol target cells coareo wln oufified A2-peptide complexes lo lysis by RT- and Ga,q-specilic 9-T! clones. cytotoxrcrtva"i.vi n"i" ciri,ei out t",in the Gag-Sl9-specrtrc CTL clone D 23 (a_and c; ano ihe Rl'lvg-specriic ClL-rlone 18030 (b) on CIR cerls.precoated wrth mAb and loaded w(fi sotubte A2.oeptrde complexes (c1R-mAd-A2-peptide). as well as.c1R-A2-transfectants pulsed wrth

b"pr,o"lCrn-ez + peptroe. I [g/ml). CTL crones were arso tested agarnsr P815 cells and Pgl5-mAb-A2-peptrde as well as K562 and K562'mAO-n2-peptrde. nLe-A2 pepirO! complexes were bound to slCr-labeted larget cetls atter covalent anachmenl oi antl-HLA-A2-specrfic mAbMA2.l (a ano 3) or antr-F2-mrcrogtobutrn-soecrirc mAb BBMl to the cell sudace (c). Background lysrs for both T cell clones was determrneoby testr;g them agaanst u;ransle;ied clF and C1R cetls pulsed wrth the resoectve peptlde (data not shown)'

104 lnduction of CTLresponses by recombinant-r^-Ot peptide complexes

(a )

p sr.txtrfI lpirroIpcd

nqtap.pld.

o subLnl9lfrnorrrpo6rd

( c )

A2fc{tldo comfler

lN) aminopropyt s i l ica beads (4 NH2 groups/100 A2), we f i rst

prepareo an acuvated surface usrng the bis N-hydroxysuccini-

mide der ivatrve of a diacid of o l igoethyleneglycol (Fig. 4a).

The beaOs were first washed twice wrth deiontzed water,

twice wi th ethanol , then resuspended in a smal l amount of

anhydrous ethanol and added to a tube containing a solut ion

of 10 mM bis(NHS)ol igoethyleneglycoldicarboxyl ic acid (a

mixture of d iesters contatning seven to 14 etnyleneglycol

uni ts) ano 10 mM tr iethyl amtne in anhydrous drmethyl for-

mamide. The react ion tube was inverted gent ly ( for mixrng)

for 24 h at room temperature. The beads were then wasneo

three trmes with deionized water and modrf ied in one of

two ways:( i )The act ivated beads were converted into non- interact tng

control beads by reacting them as above with 10 mM amtno-

ethanol, yielding a OH-terminated oligoethyleneglycol-derivat-ized surface. lt has been shown prevlously that surfaces

o'o

oN

( ) oo

oOJ

'o

(.)o

F G .

(.)r \

ol

.,o2I r tc

Log Fluorescence IntensitY

Fig.4. Schematic represenratton oi beads covalently modilied with HLA-A2 peptrde complexes and FACS analysls HLA-peplide complexes

,u"-r" io*rJnty corpred to 5lm srtica oeioidenvatized with actve (NHS) ester orouos at the temrnal posmdns ol oligoethylensgtycol {seven

to 14 EG orougs per chain). The drstnbutron ol potentrally reactlve e'amrno grouoiol.lysne residues oilhe HlA-A2__peptide complex is shown

on tt" ,lg;t. fh" bornons ol the tysrnes were rdentrlied usrng 15e structure ol lhe HLA-A2 peptide comptex deposrled in the ptotein data banK

iili "rii"L,v"t,i-rir;g

ouanta. HLA-A2 peptrde-coaled beads wefe prepared lor FAcs analvsrs exaclly as were the cells These beads

(5x105) were lncubalecl wrth a porycronat ,"bb't serum (11200) agalnst heavy chatn (b) and_1 ltg ot HLA-A2-soecrfiC mAb MA2 1 (c) ano

ii"in"o'r",rn irrc-go"t anrr+auoit Fiab)zlrag-"nt. 1"otid tin", b) aino goat anir-mouse F(ab)z kagments (solid.line. c) , Beads harborrng onlv

a OH-lermrnared olrgoetny€negtycor wlre useo as a negatNe control ano slarned as deacrrbed above (dashed line in b and c) The

lluorescence Inlensity oi c1R ce s wrth r;c;i-J rulic ir""ir "rpression

(HLA-B.C) starned with polyclonal rabbrt serum-('l-:2OO) against heavv

chain is srmrlar to (b) (data not shown)

r 0 4',03ro2r o lroot 0 4, 0 3' 'o t. ,00

modrfied covalently with short oligoethyleneglycols are oftenhighly resistant to protein adsorptron (17).

(i i) The activated beads were reacted with either the A2-

RT pept ide complex or the A2-gag pept ide complex usrng-50 pg of e i ther of the A2-pept ide complexes (1 nmol)

dissolved in PBS (1.5 mi) wi th ' l mM tr iethylamine. Approxr-

mately 3 mg of the actrvated beads (10 nmol) were useoper reactton mrxture. ASSumrng a maximum COVerage ol i

complex/3g nmz, at most 0.1 nmol of protein would form a

monolaver on the sudace of 3 mg beads; therefore, theprotetn was added at 1O-fold molar eXceSS over reactrvegroups in the reaction. The solution was allowed to tnvertgently for 6 h at room temperature and the beads were

washed as described above. They were finally Stored at 4"C

in PBS. 0.1"/o sodium azide and 1 1r9/mt of the appropflalepeptide. The residual concentration of soluble peptide aher

washing and resuspension of the beads was calculated to

(b )

A - l

l r/ rt 1

t l

,- t

tniuction of CTLresponses by recombinant HLA-A2 peptide canplexes

(a) HLA-A2 peptide coated Beads (b) peptide pulsed autologous PBMC

c e l l s / w e l l c e l l s / w e l l

t 0 0 0 0 t 5 0 0 0 o 5 0 0 0 t 0 0 0 0 t t c o c0 5 0 0 0

1 0 5

o'aa

oazxtl

aa

oa

x

( c )

pep t ide pu lscd au to logous PBI \ tCHLA-A2 pep t lde coa ted Beads

pep t ide pu lscd C IR-A2 ce l l sHLA-A2 pep t idc coe ted Beads

pcpt ide pulscd autotogous PBI\ ICHLA-A2 pcp t idc coa ted Beads

precursor CTL per 106 PRMC

I (N ) 19 (12 -31 ) 326 (24e428)| (o-8) 2 (0{) 1e8 ( ts} '2s6)

4 (1 -11 ) 2 (0 -s ) l l 0 (84 '144)il (620) 6 (3-14) s2 (37-72)

r 9 (t t -31) t2 (&22) /t '19 (r!6.591)20 (1233) 6 (914) ZE (7enn

su bject

1 6 r J

1 15i

1 t/60

lv-9 sL.9

Flg. S- Ouantrtation ol Gag-SLg- and RT-lvg-specric CTL strmulated bymultivalenl recombinant HLA'A2 peptrde complexes' Limiting dilullon

"ni'r,, n lr p"Ao,."O oi pgpl6 lrom tl,iee ilv-l-rnfecled sublects. eAUC lZSO-l OOOO per well. 24 wells Aach) were stimulated rh vitto wth

HLA-A2 Deotrde complexes covalenlly anached to beads {15.OOO HLA-A2 SLg beads or HLA-A2 lvg betds) and alternatvely w$ peplroe-

pulsed {SLg or lvg) l-rr.adrated autologous PBMC of ceptrde-pulsed trrraoraled CIR'A2 translectanls (10 Fg/ml lor t h} lL'2 was added al

l i ig "i"

i,n"r concenrrarron of so U/;t The srrmurareo cTL were rested 12 days later against srcr-labeled auloiogous B-LCL alone ia) or

ir-riotogous a-rcr incubaled wft 1 pg/ml sLg (c) or lv9 (O) peptlde. Fleg.essron lrnes were calculated by the melhod ol maximum lrkelrhood

fr dl n'ou""t "orp"nson

ot limrring dlluro; aniryies oeaormeo w'th HLA:A2 geptrde-coated beaos (a) and pepttde-pulsed autologous PBMc

ioj' i" ano*n lor subjecl 15760. precu,st, irequenc,"s oi CTL delermrneo In tnree suoiecte are summanzed below Sanges are grven In

parenlhesrs.

be 1QQ-fold below the minimum concentratron reqUlre6 to

St imulate the corresponding T cel ls wnen aoded to these

cel ls in the presence of feeder cel ls.

FACS staining of A2'pepttde-coated beaos

A2-oeptrde-coated beaOs were prepareo for FACS analysis

exact ly as were cel ls. Beads were washec In cold RPMI and

a l iquots o f 5x10s beaOs were incubateo In a to ta l vo iume o f

100 p l fo r 30 min w i th a po lyc iona l raobr t serum (1 :200)

against HLA heavy charns or wrth A2-specr i ic mAb MA2' 1(1 pg), lvashed, and f inal ly starneo wrth FITC-goat ant i -

rabbi t F(ab)z f ragments or goat antr-mouse F(ab)z f ragments(Jackson) for 30 min at 4"C. Beacjs harborrnQ only the OH-

terminated oligoethyleneglycol chain were useO aS a negatlve

control and Stained as described above. FACS analysis was

performed as with cells.

Limiting ditution analysis of precursor CTL (O,Cfq silmulatton

by recombrnant A2-peptide complexes on {nQrospheres

Numbers of pCTL specific for peptides frcm the HIV-1 gag(sL9) and RT ( lvg) proteins were determined by l tmitrng

dr lut ion analysis (18). Per ipheral b lood mononuclear cel ls(PBMC) from HIV-1-posrt ive subjects were st imulated in vr t ro

usrng sr l ica beads to which A2-pept ide complexes were

covaleni ly at tached. PBMC vrere cul tured at 250 -16,000

cel ls/wel l tn 24 repl icate lvel ls in 96-wel l microtr ter p lates

(costar, cambridge, MA) wi th erther 15,000 beads/wel l coateowith A2-peptrde, ai ternat lvely the PBMC were St imulated wrth

25,000 peptrde-pulsed autologous ant igen-presentrng cel is

[y- i r radiated (3000 rad) PBMC pulsed with 10 [g/ml peptroe

for t h foilowed by removal of unbound peptidel To eacrr

well was added 2.5x 10a y-irradiated (3000 rad) PBMC 'f eeder

cells {rom an HIV-1-seronegative Hl-{-mismatched donor and

106 lnduction of CTL responses by recombinant HIA-42 peptide complexes

IV9/SL9 Beads+ fecder cclls

IV9 Beads+ feeder cells

SL9 Bcads+ feeder cells

IV9/SL9 Beads

soluble pcpt lde/SL9( 5 n g m l ' l ) + f e e d e r c e l l s

or ig inal Beads unmodi f icd+ feeder cel ls

Bcads modi f ied ( l inkcr)+ feeder ccl ls

7. specilic kllllng

Flg. 6. T cert stimutation by multivalent recombinant HLA-A2 peptide complexes is specilic. PBMC (20.000 'vell) from HlV.1-positive subjectt5760 were ptaced in culture along with the 5 F n srlrca beads (20,000/well) modilied covalenlly wath the HLA-A2 peptid€ complexes in thegresence or absence ol HLA-mismatched y-rradrated feeder cells (7x lor/well). lL-2 was added at day 4 at a linal concentration ol 50 U/mlAfter 12 davs culture. lyltc activtty was assayed agarnst 5lCr-labeled autologous 8-LCL alone (solrd bars) or autologous B-LCL incubateo wnh1 frg/mt lvg (open bars) or SLg (shaded bars) peptde. Data are expressed as a percenlage of specrlic srcr release and values.epresentthe average ol lour welis.

. r o 807060504 03020' t0

recombrnant lL-2 (50 U/ml added on day 3) After 12 dayseach weil was split and assayed for cytotoxtcity on slcr-

labeled autologous B-LCL that were ei ther Incubated withsynthet ic SL9 or lV9 pept ide (1 pg/ml peptrde for t h andwashed) or B-LCL without added peptrde. The fractron ofnon-responding wel ls was the number of wet ls in which s lCr

release did not exceed the mean + 3 SD of the release Inthe 24 control wel ls (no pept ide) div ided by the total numberof assayed wel ls (19,20). pCTL freeuency was estrmated bythe maxrmum l ikel ihood method (18).

T cell stimulation by recombinant Hl-A-peptide complexes

PBMC (20,000/wel l ) f rom HIV-1-posi t ive subiect 15760 wereplaced in 96-wel l mtcrot i ter p lates along wrth the 5 pm st l icabeads (20,0004,,/ell) modified covalently wrth the A2-peptrdecomplexes tn the presence or absence of 1- i r radiated HLA-mismatched feeder cel ls (3000 rad, 7x t04/wel l ) . lL-2(Hoffman La Roche) was added at day 3 at a f inal concentra-tron of 50 U/ml. Af ter 12 days lyt ic actrvrry was assayed onpept ide-pulsed slcr- labeled target cel ls (autologous B-LCLpulsed wrin 1 Fg/ml pept ide as descrrbeo above).

Resul ts

Refolding of HIA-A2 in the presence of HIV-1-derived pepttdes

HLA-A2 complexes with RT-lvg (A2-lV9) and Gag-Slg (A2-

SL9) were formed by dilution of recombinant heavy chain(1 pM) and p2-microglobulin (2 pM) in the presence of each

peprrde (100 pM) (2). The complexes appeared as sharppeaks on HPLC gel f i l tration at €lutron times consistent wrththe mot wt of the ternary complex (Fig. 1). HPLC fractions wereplpened onto ni t rocel lu lose and stained with mAb specrfrcfor c iass I MHC proterns (MHC l) The f i rst peak fractron,corresponding to the fo lded complex, was recognized bymAb W6/32 (11), whrch recognrzes human MHC I moleculesin general , and by'mAb PA2.1 (12), whictr recognrzes thenatrve HLA-A2 molecule specifically Thdrecognition by mAbsensttrve to conformatron rmpl ies that the recombinant com-plexes contain native epitopes, consrstent with the presenceof correct ly fo lded molecular complexes.

Adsorptton of A2-peptide complexes to target cells

To determine if the A2-peptide complexes assembled in vrtrocan activate Az-restricted T cell clones, we coated a panelof A2-negatrve cel ls wi th the complexes and used them astargets for RT-|V9 and Gag-SL9 specrf ic T cel l c lones derrvedfrom HIV-1- infected indiv iduals (c lone 68462: antt-A2- lvg anoclone D23: ant i -A2-SLg) in cytotoxrc T cel l assays Themoorfiecj target cells were the Ht-A-A negative human B celll i ne C1R (21) , the MHC Inegat rve numan ce l l l i ne K562, anoP815, a mouse mastocytoma cel l l rne. To bind the A2-peptrdecomplexes on these cells, we first covalently attached theA2-soecrf ic mAb MA2.1 (14) to the surface of d i th iothrertol-t reated C1R and PB15 cel ls using the heterobi funct ional cross-linker SPDP. The antibody coated cells were subsequentlyincubated with a saturating concentration of soluble A2-

ndlrtion of crL

peptide comptexes. The binding of A2 to the cell surface was

demonstrated byflowcytometry, using mAb B87'7 (16)' which

reacts wrth a combinatorial determlnant of H|'-A-A,B,C heavy

chain ano p2-microglobulin (Fig. 2).

specific lysis of A2-peptide-coated target cells by cTL clones.

As shown in Fig. 3, the cTL ctones lysed c1R cells coated

with the relevant A2-peptide complexes. rndicatrng that the

Hlv-1 pepttdes were effectively presenteo and recogntzed by

T ceils bearing receptors of the approprrate specificiry. ClR

cells coated with the reconstituted A2'pepttde complexes

were lyseo essentially as rvell aS were C1R cells transfected

with A2 and pulsed with pept ides (1 pg/ml) ' Both T cel l c lones

also lysed the murine P815 cel ls to whrch the approprrate A2-

pept ide complex had been adsorbed. al though to a lesser

extent than human target cel ls modrf ieo wrth the same com-

plexes. As expected. the T cel ls far led to lyse unrrrodrf ied

P815 cer ls and c1R-A2 t rans fec tan ts pu lsed wt th an

irrelevant pePt ide.Recognrt ion of A2-pept ide complexes by TCR was evident ly

not imparred by the binding of these complexes to mAb

MAz.1, even though this ant ibody brnos to the polymorphtc

regron of HI-A-A2 close to the peptrde brnding groove (22)'

HLq-nz pept ide molecules held by mAb MA2.1 at the cel l

surface mrght not be accessrble for optrmal engagement wi th

the TCR. Accordingly mAb BBM1, an ant i -p2-microglobul in

ant ibody (13) , was cova len t ly l inked to C1R, K562 and P815

cei ls. and used to bind refolded complexes. A2-pept ide

complexes on each of these target cel ls were iust as suscept-

ib le to lysrs (Fig. 3c) as target cel ls that made use of the

MA2.1 mAb.

Covalent coupling of A2-peptide complexes to mrcrospheres.

A c r u c i a | q u e s t i o n i s w h e t h e r t h e r e c o m b | n a n t m o i e c u | e s c a nst imulate pCTL present in blood samples of HIV-1-seropostt tve

sublects, i .e. in natural ly prrmed indrvrouals. To examtne this

possrbi l i ty , we covalent ly coupled the A2-peptrde complexes

to s i l ica beads der ivat ized wrth act lve ester groups at the f ree

ends of o l rgoethyleneglycol charns tnat extenoed -3O-50 A

from the bead surface.Figure 4 shows the coupl ing proceoure ano the dlstr lbut ton

of potenttal ly react ive e-amrno groupS of lysrne resrdues of

the A2-peptrde complex. The partrcular amtno groups that

react with acttvated ester groups on tf ' le surface of the bead

determrnes the or ientat ton of the MHC complex. Posi t ive

staining rvr th 3 rabbi t serum against human heavy chain (Fig

4b) showed attachment of HLA complexes on the surface.

Stainrng wrth the ant i -Ht-A-A2 mAb MA2 1 (Fig 4c) indtcated

that a srgni f icant f ract ion of the HLA-peptrde complexes

matntaineo a nat ive conf igurat ion and were exposed to the

solvent, and are thus presumably accessrble to TCR on T cel ls.

Recombtnant HlA-pepttde complexes are rmmunogenic

To assess the immunogentc ef fectrveness of the A2-pepttde

complexes on beads, we used them to st tmulate PBMC of

th ree seroposr t rve sub lec ts (161J , 1151 and 15760) measur ing

the frequency of peptrde speci f ic pCTL af ter 12 days by

l imit ing or lut ion analyses (Fig. 5) . The resui ts show that the

1.ecorntinant A2-peptide complexes were effective and as

specific as pepttde-pulsed autologous antrgen-presentlng

responses by recombinant HU-A2 peptide complexes 107

cells in inducing functional responses that normally depend

upon ceil<ell interactions, i.e. they triggered antigen{epend-

ent responses of primed pCTL. A direct comparison of l imiting

dilution analyses performed with A2-peptide complexes dis-

played on beads (Fig. 5a) and autologous peptide-pulsed

PBMC (Fig. 5b) revealed that the responses were of compar-

able magnitude.

Expansion of pCTL is peptide specific

To rule out non-specific mitogenlc effects of the bead surface,

PBMC were stimulated with a mixture of A2-lV9- and M-sL9-

coated beads and tested for A2-lv9- and M-sL9-specific

responses (Fig. 6). Only SL9 specific responses were detect-

able. culture of the same PBMC with beads bearing only A2-

lV9 induced no response; only A2-sL9 beads stimulated a

specific SL9 response. The apparent absence of lV9-specificpcTL in the responder population was confirmed using the

same batch of beads as in the l imiting dilution analysis

(Fig. 5). The recovery of SL9-specific CTL suggests a true

expansron of peptrde-specrfic pcTL, confirming other data (6

and unpublished), that the dominant Gag-specific HLA-A2

restncted response is directed against the SL9 peptide in

these subjects.Titratron of peptide revealed that a peptide concentration

over 5 ng/ml SL9 was requrred to stimulate pcTL expansion

from PBMC in the presence of feeder cel ls (Fig. 6 and data

not shown). By contrast much less pept ide is needed when

it is presented by HI-A-A2 on the microspheres. Thus with

maximal packrng a 5 pm microsphere could accommodate

about 2x 106 HLA-A2 peptrde complexes in a monolayer. This

numoer corresponds to 0.35 ng/ml peptide if all the A2 on

the beads were loaded with the peptide originally and all the

peptrde adduct then dissociated (given 2X 101 beads, 0.2 ml l

wel l and a maxtmum of 2x103M-pept ide Complexes per

beao) By comparing the fluorescence intensity of HLA-A2

oeot ide-coated mtcrosoheres wi th C1R cel ls stained wrth

polyclonal antrbodres to MHc I heavy chain i t appears,

however, that the actual amount of surface-bound A2-pepttde

complexes on the pnrcrospheres is -50 t t rqes lower than the

calculated maxtmum. re. the f luorescence intensi ty ts stmt lar

to C1R ce ls v r i th -3x104 HLA 835 and Cw4 per ce l l (21)

Thus r t appears that the T cel l response was el ic i ted by far

less peptide associated wrth H[-A-A2 on the microspheres

than by pepttde addeo rn solut ion. Non-speci f ic mrtogenrc

effects of the beads modrfied wrth only OH-terminated oligo-

ethylenglycol groups were not observed (Fig ' 6) ', .

D iscuss ion

This is the first demonstratron of biological activity of E' co1-

derrved HLA-A2 peptroe complexes' In contrast to nattve

HLA-A2, the recombrnant protern used here ts not glycosylated

at Asn-86 and i ts p2-mrcroglobul in subuni t has an aodrt ional

N-termrnal methionrne resrdue not c leaved fol lOwing synthesrs

(3). These di f ferences have previously been shown not to

significantly affect the crystal structure of A2 in the form of

A2-pept ide complexes (3 '4) .We show here that these differences also do not prevent

recombinant A2-Oeptrde complexes from el ic i t ing spectf ic

cytolytic responses of CDB* CTL clones from HIV-1-infected

1OB tnduction of CTL responses by recombinant HtA-42 peptide complexes

individuals. Thus, the antigen-specrfic receptor (TCR)of these

clones can recognize recombinant E. colr-produced A2'proteins refolded in assoctation wrth approprlate peptides'

This study shows furthermore that mtcrospheres coated withrecombinant A2-peptide complexes can specifically stimulateT cel ls in PBMC from HIV-1- infected indivrduals. The st imulatedcells, presumably primed by natural infectron, develop into

an expanded population of CTL that specrfically responds tothe A2-peptide complex that trlggered this development.Since the Az-peptide-coated microspheres lack B7 and otherco-stimulatory molecules, their immunogentciN has to beconsidered in the l ight of the large body of evrdence that, in

addition to TCR ligation, the actrvation of quiescent T cellsdepends upon secondary signals that trrgger the expressronof lL-2 receptors and the production of lymphokrnes (35). In

the present system which makes use of PBMC from HIV-1-rnfected indiv iduals, i t is possrble that the responding T

cel ls already express lL-2 receptors and that the exogenousrecombrnant lL-2 substrtutes for endogenous production oflymphokines. In addi t ion, the HLA-mrsmatched feeder cel lsmight provide a co-strmulatory functron and, by elicit ing analloreactron, tnduce cells in the respondrng PBMC population

to produce endogenous lymphoktnes. In any event, wnatever

the detai led mechantsm may be, r t seems clear that the beadscoated covalently with A2-peptrde complexes are activeimmunogenical ly wi th PBMC of HIV-1- infected indiv iduals.

Several drfferent systems have been developed for stimulat-ing T cells wrth purif ied class I MHC molecules (24-26)

including al logeneic MHC proteins (27. 28) and var iousmethods have been used to assay T cell actlvation, e.g.release of BlT-esterase (29), production of lL-2 in T cellhybridomas and release of serotonrn in basophils transfectedwith a TCR-( fusion (30). The MHC-peptide complexes usedin these studies included purrfied native MHC I molecules,

cleaved from the cellular surface (28,29), and 'empty' MHC Imolecules produced in rnsect cel is and loaded with pept ides

of interest (31,32). Soluble H-2Ko-peptrde fusrons were alsoproduced in which heavy charn, p2-mrcroglobulrn and pept ide

have been fused Into a s ingle cnaln protetn and cel lsexpressing these constructs were shown to be highly immuno-genic (33). Genet ical ly engrneered soluble H-zOd moleculescoated on plast ic were also found to present HIV-1 envelopepept ide to an ant igen-specrf ic CTL clone, inducing i t toproduce IFN-y (3a).

Since the overexpression of the class I subunrts in the E. coli

system and the subsequent refolding provides an abundantsource of homogenous Ht-A-peptide complexes, the recom-

binant soluble complexes descrrbed here of fer new toolsfor explor ing drverse problems and tssues, such as s lgnal

t ransductron events el ic i ted by agonrst and antagonrst pept ide

l igands (36,37), the ident i f icatron ancj isolat ion of ant igen-speci f ic T lymphocytes ln complex cel l mixtures, and thedevelopment of vacctnes aimed at st tmulat tng part lcular antt-gen-speof ic T cel ls.

Acknowledgements

J. W. is a recrpient of a Mildred-Scheel fellowshrp from Germany andwas supported by grants lrom the NIH (Al 34247 and CA 60686).The efforts of C. 8. were supported by the Schwerzerische Stiftung

fOr medizinisch biologische Stipendien, M. M. was a Eli Lilly predoc-toral fellow and supported by an NIH grant (GM 30367). D. N. G.was supported by grant no. DAMD17-94'J'4060 from the Departmentof the Army and his eflorts were performed in the laboratory of D. C.Wiley who is a Howard Hughes Medrcal lnstitute Investigator.'Wethanx P. Cresswell lor provrdrng the C1R'A2 cells and A. Trocha iorexcerlent technical asststance.

Abbreviat ions

B.LCL

CTLPBMCSPDP

Epstein-8arr vrrus-transformed B lymphoblastoid celll inecytotoxic T lymPhocytepenpheral blood mononuclear cel lMsuccinimrdyl 3-(2-pyrrdyldithio)propionate

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