Scand. J. Immunol. 25, 555-565, 1987

Inhibitory Effects of Anti-HLA-A, B, C HeavyChain and Anti-/^. Microglobulin MonoclonalAntibodies on Alloantigen and MicrobialAntigen-Induced Immune Responses in Vitro

G. C. S P A G N O L I . C. M. A U S I E L L O . A. C A S S O N E . C. U. C A S C I A N I ,0 . BELLONE & F. MALAVASiIstilutt) CNR 'Tipizzazionc Tissut;i!c c Problcmi dclhi Dialisi" L'Aquihi. Laboratorio cliBattcriolngia e Micologia Mcdica. Istiluto Superiorc di Sanitii. Catledra di Clinica Chirurgica. IIUniversita, Rome, and Laboraioriu di Biologia Cellularc. Dipartimento di Gcnetica. Biologia eChimica Medica. University of Turin, Italy

Spagnoli. G.C.. Ausiello. CM.. Cassone. A.. Caseiani. C.V.. Hellone. G & Malavasi. F.Inhibitory Effects of Anti-HLA-A. B. C Heavy Chain and Anti-//. Microglobulin MonoelonalAntibodies on Alloaniigcn and Microbial Aniigen-Induced Immune Responses in Vilro.Scand. J. Immunol. 25, S.S?-.%5. 19S7

The role of lU.A class I subunits in class Il-rcstrietcd immune responses was investigated bymeansof a panel of monoclonal antibodies (MoAb) recognizing MLA-A. lJ,Cheavy chain anddifferent /i. mitroglobulin (/i.m) epitopes. MoAh against either class I subunit stronglyinhibited mixcti lymphocyte cullures. generation of cyioto\ic T lymphocyte cultures, genera-tion of eytotoxie T lymphwytes or natural killer-like activity, and lymphoprolileration inresponse lo soluhle or piirtieulate microbial antigens derived from CimiJulu alhicom. Ingeneral. anli-//;ni MoAb were more efficient inhibitors than anti-HLA-A. B, C heavy chainMoAb. The inhibitory effects were specific, in ihat the parental myelt)ma ascitic lluid or a low-affinity MoAb auainst fi.m. or MoAh directed against non-HLA surface structures did notaffeci any t)f the immune responses studied. The MoAli-iniluced inhibition eould no! beattributed lo nonspecific loxic effects, since PHA-induced blasiogcnesis and lL-2-dcpendentproliferation of mixed lymphoeyli; euluire (MLC) blasts were not inhibited. Furthermore.exogenous IL-2 did not reverse the block of MLC and microbial antigcn-mduced proliferativeresponses by MoAb. lakcn together, these data suggest an Involvemeni ol both subunils ofclass 1 antigens in class Il-resiticted inmiune reponscs.

Giulio C. Spagnoii. .Ml). Laboriiiory of Biulerioln^y and Medical Mycology, tsdtutoSuperioredi Saniia. Viale Regina Elena. 299. 00161 Rome. Italy

Surface metnbrane molecules encoded by genesof the major histocumpalibiliiy complex (MliC)play a key role in the regulation of the imtnuneresponse. It is generally accepted that elass Ideterminants restrict cytolytic T lymphocyteresponses (2^1. whereas class II molecules are(he main stimulating dcterminanis in mi.>ted lym-phocyte cultures (MLC) [24| ami restrict interac-tions between antigen-presenting eells andT-heiper lymphocytes in antigen-induced lym-phoproliferatiuti [5. 7[.

Failure to express HLA elass I determinants

on cell surfaces in vivo have been observed in arare immune disorder, the so-called 'bare lym-phocyte" syiuirotne. which is characterized bysevere T-cell dysfunction and hypogamma-globulinaemia [25]. The fact that the expressionof class II molecules in this syndrome is oftennormal, supports a major rolcof cla.ss 1 determi-nants in the immune response, even though themechanisms by which these molecules influencelymphocyte functions are not fully tindcrstood.

i ILA class 1 molecules are heterodimers witha heavy ehain-bearing the allotypie determinants

555

556 G. C. Spagnttii et a!.

non-covalently associated withy : microglobulinifiim) [15]. Since surface//:m exceeds HLA-A.B.C molecules [8]. it is believed thatyV^m may beexpressed either in a free form or associated withadditional class 1 determinants, analogous to TLand Oa antigens in mice (12. 13. 27].

Conventional antisera or monoclonal anti-bodies (MoAb) specific for HLA determinantshave been used to dissect the functional roles ofdifferent molecules in immune responses.Contrasting data have been reported on theability of anti-class I reagents to inhibit MLC andantigeti-induced lymphoproliferation [11. 14.21. 23. 26]. The experiments reported in thispaper were performed to analyse the functionalroie of IILA class I determinants in antigen-induced lymphocyte stimulation. Since /:/;m isnot only associated with HLA-A. B. C heavychain but also with other determinantsexpressed on subsets of resting and activatedimmunocompetent cells [12.13. 17. 27]. we alsosought for possible differential effects of MoAbdirected against different epitopes of/?2ni andHLA-A. B. C heavv ehain.

MATERIALS AND METHODS

Production and characterization of MoAb. P4S,P.S6. RI.3t). AC4.25. ACl.(iK. and Ol.65 MoAb wereproduced in different fusions obtained after iminu-niziition with cither peripheral blood mononuclearcells (PBMC) from healthy donors or witli human celllines [17].

Screening of positive eulttires was earricd out usingPBMC". purifa'd '-''l-^^m. iind coniiniious cell lineswilh selected eharitcteristics [18]. infUiding Daudicells, which urcfi^m negative and consequently unableto express surface HLA elass I molecules,

The selected pritiKiry cultures were eloncd three-times by limiting diUiiion imd subsequently grown astumour ;iscites in pristane-prinied mice. The aseitictluids thus obtained were used throughout the presenistudy. Mean protein content wiis approxim;itely Img/inl tor all the MoAh under investigation and cytt)-toxie titres. as measured hy stiindard NIII technique.ranged lunween l:ll)()Oand l;2(l,tHK).

The seleetcd MoAb were used for immuiio-prccipiiation experiments with '-"M-labclled/)';m andlectin-purified glycoproteins. Experimental pro-cedures have been dcscriheti in detail elsewhere jlfi].

Epiiope competition cxperitiients were performedby using '-'M-labelled MoAb anil eolil (unlabelled)MoAb as inhibitors [14). The Clit)4 and P5l conirolMoAb were obtained and selected after immunize I ionof mice with PMBC. while A HI was selected in a fusionwhere the splenocytes had been immunized withDaudi eells. All MoAb containing ascttic tluids were

extensively dialysed againsl RP.MI. filtered through().22-//m membranes (Millipore. Molsheitn. France)and added to ihc cultures at the final dilution (i.e. 10,//g/ml). on the indicated days, unless otherwisespecified.

Cell M'paraiion. Blood samples were oblained fromadult healthy volunteers on preservalive-free heparin(Eparina Vitrum. Como. Italy). 20 IU/ml. PBMCwere isolated by centrilugation on density gradient(Lymphoprep. Nyegard. Oslo. Norway) according toBoyum [6]. and washed twice in RPMI metlitim(Gibeo, Grand Island. New York. USA). Cells werethen resuspended in RPMI medium, and supple-mented with [U'i pooled inaetivated AB serum and.mtibiolics (penicillin lt)(J IU/ml. streptomycin 0.1mg/ml. Cjiheo). hereafter referred lo as completemedium.

Proliferation studies. One-way mixed lymphocytecullures (MLC) were set up by coculiuring 2xUPPBMC frotn one donor with the same number ofallogencie irradiated (42O() nid) PBMC m 0.2 ml ofeomplete medium, in flat-bottomed y6-microwelltrays (Nune. Roskilde. Denmark) [I]. In some experi-ments irradiated Daudi lymphoma cells were used as astimulator. All tests were performed in triplicate. Thetrays were incubated in a 5% CO. incubator at 37"Cand cultures were harvested on day d. Eighteen hoursbefore harv'csting, radiolabellcd ihymidine [meihyl-['H]thymidine. 2^ Ci/mmol. 1 mCi/mi. Amersham.Bucks, UK), was added to a final eoneentration of 0.5uCi per well. Cultures were harvested by Titertek cellharvester (Skatrtin, C!)slo, Norway) and |'H]thymidineincorporation was measured by Ihe two-channel ratio,using Ihe external standard method, with a Beckman(Irvine. Calif., USA) l.S SlOO liquid seintillalionsystem.

The microbial antigen-induced lymphoproliterationwas stimulated by materials prepared from Candidaalbican.s yeast, using either glutaralilehyde fixed C.alhicans whole eells (CA) or a soluble phosphorylatedgluco-nuinnan-piotein eomplex (CiMP) purified fromthe yeast cell wall [. . 4. 4].

2x 10' PBMC in eomplete medium were stimulatedwith CA or GMP fraction at the opitimal concentra-tion, as indieatod in single experiments and['M]thymi(Jine inct>rporatioii was measured on day 6.

IL-2-dependent proliferation was measured asdescribed by Moretta ei al. [20]. BrieRy, 2x UV lym-phoblasts from 7-day MLC were eultured in 0,2 ml ofcomplete medium in flat-bottomed W-well trays(Nune) in the presence of W^r (v/v). human delccti-nated IL-2 preparation (Cellular Products, Buffalo.NY. USA). ['Hjthymidine incorporation wasmeasured after 4S h of euliure.

In some experiments. IL-2 preparation was addedfrom the beginning to MLC or nticrohial antigen-.stimulated cullures. in the presence of the appropriateMoAb.

Cyloto.xiclty snidlcs. CyU)toxic V lymphocytes(Cn . ) were generated during MLC tests in flat-bottomed 24-well trays (Nunc) in the presence ofappropriate dilutions of MoAb. After fi days theallosensitized cells were extensively washed and resus-pended in RPMI medium supplemented with 10%

HLA Class I Subunits in Immune Re.spon.\e 557

letal ealf scrum (FCS) (Gibco) (FCS medium).Specific cytouixic activity was assessed hy ;i 'Crrelease assay againsl PHA-stimulated PHMC of thespecific MLC stimulator, while natural killer-like(NK-like) activity was measured against K.'iCC targetcells.

Tho cytotnxic assay wa^ perlortiied as previouslydeseribed \2\ I arget cells were labelled wiih 15ll,i(Ciof sodium "ehromate INEN. Dreieieli. H<ti) tnO.2mIof FCS medium for I h al 37 C, and subsequentlywashed twice m cold medium Ihc cytotoxicity testswere cirriedout m triplicate in t' I ml of FCS mediumin round-hoiiomed W-niicrowell trays.

5 X l(t' target cells were added lo var> ing numhcrs ofeffector cells. Each sample wns tested at threedifferent effector lo target (IH'!) ratios. After briefccntrifugvition tlie irays were eultured for 4 h at .17't ina humidified meiilialor. then ihe su[iernatants wereharvesied with a Iileriek sujiernatant collection sys-tem tSkatrtin. Oslo. Norwayl anil "'( r release wasmeasured using .in LKH (Wallac. FinLmd) gaimma-countcr. Tlic pereentage of killing was calculated as

^ killingsTest rclcasc-spmitancous release , . j . ,

maximum rclease-sporitancous release

Ri-:sin.is

MoAb ilutracterization and spccificitv

Ihc MoAh selected for this study.IMN(|gCi2;t). P56(lgG2a). Rl ..•^OdgGIb).AC4.25(|gM). ACI.6S(IgCi2b). iiml 01.65

(IgM) reacted positively in itidircct inimutii)-fluoresccncc with y(l-l(>0';? of PBMC of alldotiors lested as well as with the iiKijiirity ofn.pstcin-I3arr virus (EBV)-tr;itislortiicct cells.whereas they were not reactive wiih Daudi lym-photna cells. Moreover. P4K. P5h. R1.30.AC4.25. and AC1.A.S were able to react withsoluble ''•'I-labflled /^-ni pttrificil from urine(Fig. 1; the purified/i;ni was a kind gift from DrM. D. PouUk. see also Ref. 10). These resultscould he taken as a rough indication of theaffinity of the individual Mo.Ah in the system,wlierc the highest score was atirihtiicd to MoAbKl..'*t)atui AC.4.25 MoAh ()I,(O failed to reactwith the purified/y:m. whereas it was ahle toprecipitate a dimer of 4f;.t)()(t-12.0()(l from '-M-lahelled membranes (Fig. 2). thus indicating thatthis MoAb does indeed react with I ILA-.A. B. Cheavy chain.

Immunoprecipitation with '-'I-labclled sur-face molecules of EBV-iransfortncil lytnpho-blastoid cell lines expressing both class I andclass II pnidticts invariably yielded 4.'>.1KHI12.(K)U dime IS tor MoAb Ol .fi5 as well as for theanti/y.ni MoAb. The fact thai OI.65 reagentdoes not react with purified//ni is an indicationthat the target epittipe is located on the 45.fHKlheavy chain. On the other hand. MoAbAA3.K4. under the same experimental condi-

P56

RI.30

FIG. I. Immunoprecipitation of '-'•l-/i.m hy the MoAb tested. AA.VS4 (anti-HLA DR) was included asnegative control. SO.tKHlcpm '-'I-^- were mtubated wiih a fised amount of MoAb (the concentrationof ihe individual reagents wascquali/ed to 1 nig;ml) anil the mimune complexes preeipitated with anitntnunoadsorbcnt (rabbit anti-mouse Ig plus Siaphyloi:oiru.s mai'us Cowan I strain). Tot furtherdetails see Materials and Methods,

558 G. C. Spagnoli ei <il.

0165 P56 R130 P48 AA384KD

04

68

43

30

21

Fid. 2. Immunoprecipitaiion of '-'I-^^Tn by anii-HLAclass 1 MoAb Mo.^b A>\^.M (anii-IILA-DR) wasused ;is negative comrol ltic molecule (45.(HHt-n.lMKlJ recognized hy MoAb ()l,(i5 was ineluded as jmigration murker. Tor further details sec Materialsand Methods.

tions as above, yielded a .35.nO(V-3O,n()ll dimersluivving the same etectrnphdrelic mobility ashuiiuin l:i (for lull details, see Rcl. 17 and IIWorkshop on HLA class II antigens. Edin-burgh. UK. September 1983: Disease Markers.vol . : . 19S4).

''^Mabelled cell surfiicc molecules fromDaudi cells did not provide class I products, butthey gave a 35.0(X)-3(l.tHK) dimer which immunopreeipitated with MoAb AA3,84 (see Ref. 19).

Ihe molecules characterized by theseimmunoprecipitation techniques were alsoanalysed by t\v(»-dimensioniil gel electro-phoresis; they showed the characteristics of dis-tinct Hl-A products. Finally, ihe specificity ofthe MoAb used throughout was checkedthrough binding-inhibition experiments byselected polyclonal and monoclonal antibodies,as fully described m Rcfs 17 and IS. Thespecificity of the MoAb used as controls hasbeen described in detail elsewhere: CB04 recog-nized the complement receptor 1 (CRl) {l^\.Aid recognized a molecule similar to Till [17|.whereas V5\ reacted with a moiumuirphic cpi-tope delcclable on the surface of all the humancells .so far tested (F. Malavasi. unpublishedobser\aiions).

Inhihition of proliferative response to allogeneiccelh

The effects of anti-HLA MoAb underinvestigation were assayed on one-way MLC.MoAb were added to the microwells at Ihebeginning tif the cultures at final dilutions oftheasciticlluidof 1:111(1 to I: !O.(KH)(averagestarlingpmtein concentration 1 mg/ml). Table I showsdata from three experiments invo|\ ing differentpaired combinations of responding/stimulatingcells, while Fig. ? collecIivcK d<Kuments thecomparative inhibitory effect of ihe anti-HLAclass 1 MoAb studied in all the MLC performed.

Anti/J^m reagents (R 1.30. AC 4.25. P48. P56). independently of their Ip class, strongly

I. Eftecis ol anli-HLA elass I MoAb on mixed lymphocyte culture

MoAb added*

NoneRI.30At4.25P4&P56ACI.6801.65CB.(MA inP5 l

Expt 1

98.4±I3.2(—)$25.5±4.7(25.9)21.8±4.U(22.1)

N.D.N.D.

1H(J.5± 14.7 (102.!)29.912.7 (30.3)

106.715.7(108.4)110.1+13.2(111.8)

N.D.

|'H|thvmidine incorporationt(tlpmxIO 'ISO)

Expt 2

166.214.8 (—)IO.2i5.9(6.1)5.3±O.fi(3.2)

20.5±A.3(12.3)3.210.4 (l.y)

N.D.N.D.

189.613.6(114.1)156.2±7.3 (93.9)172.4±K.9 (103.7)

Expt 3

173.8121.3 (—).14.1+2.(1(19.6)2O.7±2.4(1I.9)

N.D,N.D.

l72.t±lO.lM'>9.0)77.6114,1 (44.6)

130.7113.7(75.2)176.9+7.7(101.7)163.7±17.7(<W.2)

• MoAb were iidded at ihe beginning of ihe cultures.t ('HJlhymidinc incurpor.itmn wab measured on day 6.t Percentage of tontrul value.N.D.=not done. For further details, sec Maicriab and Methods,

HLA Class I Subunits in Immune Respon.w 559

120-

100-

8a

f 60

4b

20

RIM ac4,25 P ie P56 0l,6S Ac16B

Ki(.. .V I-ITccis of anti-HLA elaw 1 MoAh on MLC.2x|0*' PBMC were euUurcd with 2x10' irradiated;i!l<))iL-nn.' I'BMC, MoAh were addi-d to llic mierowelhal ilie hegmning of the ctiliures. Proliferation wasassev-icd by [ 'I I |lhymidine incorpoialion on d;iy A afterun tS-h isotope pulse. Data, expressed :is a pereeniageof L'oniFol viilues. refer u> all MI.(" experiment-, per-formed lluoughout ihis study.

inhibited iilloyenic praliferalivc responses, thedegree of inhihition riitiginii from 55 to 95'> ofa)ntii>l \;ilufs. Tlie ami III.A-A. B. C heavychain MoAb (01.65) also exerted an inhibitorycflect on MLC. but the degree ot inhibitionvaried from 20 to 75* ; (Table 1 and Fig. ^). Thelow-aftinity anti*/^:!!! MoAb (AC1.6S) had no

effect on allogenic response and«as regarded asa control reagent (Table I and Fig. .1), Similarly,negative results were obtained with the controlascitic fluid oblained with the parental myelomaXft.^.AgS(data not shown), iis well ;ts the MoAbspecil'ic for nun-I II.A surfjiee striK'turcs (C'R()4,AH). P5I).

Differences were also observed in the potencyof inhibition hy MLC anli-class I MoAb. asdocumented in Fig. 4, A 10 'fimil dilution (i.e. 1Hg ml) of all //:ni-•specific reagents still deter-mined relevant inhibition of MI.( \ while at thesame dilution the ami HLA-A. B. C Ol.ft5MoAh was completely ineffective. To investig-ate whether the anti-HLA class I MoAh exertedtheir ellects on MLC responder or on stimulatorcells, the two eell populations were separatelytreated with the different MoAb. When the res*ponder eells were precultured wlih anti-/?:m(RI.30. P4S)or;mti-HLA-A. B.C heavy chain(C)Lh5) MoAh for 2 h. then washed and incu-hated with untreated stimulators, strtmg inhihi-tion was still ohserved. On the oihcr hand, whenthe stimulators were pretreated with MoAb. noor minimal inhibition was observeil (Table II).These data suggest that the anti-MLA class IMoAb act primarily on responder eells. which isalso indicated hy MLC experiments performedwith class l-defieient Daudi cells as stimulator.In the latter ease, a marked proliferative

ISO

? aoco

" 60

ICl" 10'

Fic>. 4. Dosc-resp<tnse inhibition of MLC hy anii-I ILAelass I MoAh. 2xlO' PBMC were awuiturcd with2x u r irradiated allogenie PBM( MoAh were addedto Ihe mierowells at the beginning ol ihe cultures.Proliferation was assessed hy [iljlhymiJinc inctir-poration on day ft after an tH-h isorope pulse .MLCcontrol value was 45.1 ±5.4x10 * dpm Symbols: • .R1..M); • . AC4.25; A . 01.65: A. P4K. For furtherdetails see Materials and Methods,

Culture eonditions"

DI+D2X

TAHI.II II. Anti-HLA class I MoAh and mixed lympho-cyte culture: effects on responder or stinuilal<ir eel!populations

I'Hjthyniidinciiieurpoiiitiont

(dpmxlU '±SD)

91.3+1. .5 (^ iT33.2±2.4 (36.3)35.4+6.4(38.7)78.7111.5(86.7)17.713.4(19.3)29.6 + 7.4 (32.4)

|y.litl.7 (2().y)2ft.Illl.m2Hp5)76.3113.4(83.5)

(DI+P4«|-KD2x + P(DH-P4K) + D2xDU(D2x4P4H)

• MoAh (l:l()(l final dilution) were added lo Ihccells from responder (Dl) or siimulaior (I)2x) or loamixture o( rcs|>iinder-siimu!;iior eells. Two-hour prc-cultures were performed at 37''C and the tells werewashed Ihree times hefore MLC leM.

^ ['l(|thymidine ineorporalion was measured onday ft.

X Percentage of control value. For further details,sec Materials and Methods.

560 G. C. Spagnoli et al.

sol-

o 30

5 20

10

(bl

10:1 40; I 10; 1 20M 40; I

Fici. 5. lnhibilii>nofi!t:iiL'r;iliiiiii»(CTL(a)and NK-likL-acUviiicMn MLClh) hy ;inii l l t-A C1;ISN IMuAh. Onc-viiiy hulk MLC' were sccdfd ;n ix|()",'rnt rcsptinilcr cell ci>m.cnit;ilitin in ll;tl-hdtuimed 2-l-\vcll pliitcs. at ;i 1:1 rcsptindcr losliniul;ilor railo M<iAb were added to ihc wells althe K'ginning u( ihc ciillurcs al a llnal dilution of hHXI. Aflcr 6 days cells were washed andassayed in a 4-h -'(.'r release icsl agamsl labelled >pei.iric iilloyenic target cells (CTl.) or K5(>2cellline (NK-like). Data are expressed as a percentage of the kiUtng activity. Symbols: _ .control: D.P 56: A . P4K: • . RI.30: A: 01.65. For further details see Materials and Melhods.

response was observed, which was blocked byMoAb lo iin extent simihir to the MLC per-formed with allogenic ^tiniulator (data notshown).

Inhibition of generation of cytotoxic effector cells

MLC induce the generation of cylotoxic effec-tor cells active against specific ailogenic targetsor nonspecific NK-scnsitive targets. As shown in

Fig. 5. anti-HLA class I MoAh inhibited thegeneration of both CTL (panel A) iind NK-like(panel B) effector cells \shen used at the dilu-tions itihihitini; lymphoproliferation In MLC. Atthe highest F. T ratio, inhibition ranged fmm 40to 'X l ' i . depending on the MoAb used. No sub-stantial difterences between effects on thegeneration tif specific and nonspecific effectorswere recorded, and neithei was any differenceobstMveit between anti-//.ni and anti-A. B, Clicavy chain MoAb in their inhibitory effects.

TABLF i n . Effects of anii-MLA class I MoAh on microhial antigen-indua-d tymphnprolircriition*

MoAb addcd+

NoneRL30AC4.25P48P56O1.65CB.(MA 10P5l

I'nstimulatedcultures

1

.6±0.1,5 ±0.5'.9 ±0.1.7±(3".3.3±a.4.3 ±0.2

».9±0.5.9±0.3.5±(t.2

I'M]thyiniUine incurporatton^(dpmxlO'+SD)

39.7±4.7(—)§9.5±3.2(24.I)7.4±2.4(1S,I)3.3 ±0.3(8.3)3.4±(l.y (8.(1)

20.6±3.(i(5I.S)39.1 ±4.2 (W 4)4O.2±5.4 (101.2135.ft±9.3 (89.6)

GMP

25.7±1.2(—)§0.2±tI.0t (0.7)0.5±t).()6(l.y)5.8±().3 (22.5)0.310.07(1.1}3.9±0.5(I5.2)

N.D.N.D.N.D.

10' PBMC were vultiired in ihe presenceof t>ptimal doses of antigenie preparation (CA: 5//g'ml: GMP: 50in tlat-Koiiomed 'Ki-wcll niicioplates.

+ MoAb were ackletl at iKc beginning nf the cuUuiest |'H]thymidine incorporalion was ttieasured on d;iy d.§ Percentage of contnil vakic.N.D.. not done. For fiirlhcr details, see Materials and Methods.

HLA Class I Subunits in Immune Response 561

Effects of MoAb on lymphoproliferation inducedby microbial antigens or PHA

The effects of anti-HLA class I MoAb otiblastogcnic response to soluble and paniculatemicrobial antigens were also studied. PBMCwere stimuhited with opiinial doses of Candidaalhicans preparations, capable of inducingstrong proliferative responses in ali normalindividuals (3. 4|.

Table III shows data from one representativeexperiment out of the five carried out. Bothanti-//:m and anti-HLA-A,B.C heavy chainMoAb strongly inhibited and in some cases vir-tually abolished the proliferative responses toboth whole cells of C. albicans (CA) and a solu-ble microbi:il antigen of C. alhicans cell wall(GMP). Control reagents, such as MoAb anti-non-HLA membrane antigens, did not show sig-nificant inhibition in CA-indiieed lympho-proliferation.

On the other hand. PHA-induccd blasto-genesis was not substantially affected by theaddition of anti-HLA class 1 MoAb. In fact,using tbe same MoAb dilutions that almost com-pletely blocked CA-induced lymphoprolifera-tion, the inhibition, of PHA-stimulated culturesnever exceeded W/t of control values (data notshown),

Kinetics of MoAb-induced inhibition of MLCand microbial antigen-inducedlymphoproliferation

To further analyse the inhibition of immuneresponses by MoAb, anti-HLA class I reagentswere added to MLC or to CA-stimulated cul-tures at different times (Fig. 6). Maximumdegrees of inhibition for both allogenic andmicrobial antigen-induced lympboproliferationwere observed when MoAb were added withinthe first 2 days of culture. Similar effects wereobtained with dnU-fi^m or anti-HLA-A, B, Cheavy chain (O1.65) reagents.

Effects of exogenous IL-2 on inhihiiion of MLCand microbial antigen-induced lymphoprolifera-tion by anti-HLA class I MoAb

In further experiments we sought for a possi-ble reversion of anti-class I MoAb-indueedinhibition of MLC and of microbial aiitlgen-stimulated lympboproliferation by delectinatedIL-2.

The addition of exogenous IL-2 to MLC wasnot able to revert the inhibition by anti-class Ireagents (Table IV). vSimilar results wereobtained in cultures stimulated wtih GMP whenthe same MoAb were added. These experiments

100

eci

60

40

20

(ol

Ooy

Fui. f>. Effefisol' the addiiiuii uf anii-ULA ckiss I MoAb on different days in MLC (panel ;i) orCA stimulaicd cullures (panel b). 3x 111'- PBMC were cultured in nat-boitomcd microwells inihc presence of either 2x l(l'irr;idialcdalk)ucnicPBMCor.'i//g.'mlt)f CA. MdAb were added tothe cultures on the days indicated at a final dilution of I: KHI. C"ell proliferation was measured bypHJlhymidine incorporation on day () after an IS-h piilse (orboih tests. Duia arc expressed as apercentage of control values. Control values; MLC. 94.8+8.5x 10 ' dpra; CA, 66.y-l-C).4x 10"dpm. Symbols: • , R1.3(); • . AC4.25: A. OL65.

562 Ci. C. Spagnoli et al.

TABU IV. Effects of exogenous IL-2 on inhibition of MLC and (IMP induced lymphoproliferiition dclcrmlncd byanti-HLA class I MoAb

MoAb added*

NoneRI.30P56P48OLft5

-IL-223.0±.V4 ( - )55.410.7 (2.V5)3.2+1.ft (L1.9)

11.6 + 2.0(50.7)11.3 + 0.2(49.1)

l'H]ihymidinc incorptiraiiont (dpmxio ' + SD)

MLC

+IL-2$

4I.O+5.I (—I5.9±O.6(I4.4)4.7 + 1.0(11.5)

21.1+3.7(51.4)4.810.6(11.7)

GMP-rL-2

2O.Si2.7(—)1.7 + 0.3 (K.O)0.5+0.1 (2.4)

N.D.1,'»±|.2(').I)

+IL-2I50.3 + 5.5 (—)0.410.1 (0.7)1.310.9(2.5)

N.D.12.3 + 3.5(24.4)

• MoAb were added lo ihe microwells ;n the beginninp of ihc ciillures :ii J final dilution of 1:100.t [iljlhyinidine incor|xir;tli»)n was measured on d.iy fii;. IX-left united IL-2 w;is iidded to the microwelis at ihe bejiinniiiji of the cultures ;it a fin;il dilution of 1:10.S IVreentaiie of control. For further iletails. see Miiterials and Mflhods.N.n . not done.

TABLI. V. Effects ot anti-HLA class I MoAb on lL-2-dcpcndent proliferation ofMLC blasts*

MoAbaddedt

Exogenous ['H)thymidine incorporation^IdpmxlO '±SD)

NoneNoneR1..WAC.425O1.65ACL68

55.711.1 (—)

61.615.8(110)72.2±5.K(I2V)

• 2xHH 7-day MLC blasts per wvll were cultured in nal-txittomed 9f>-wcHmicroplates.

^ MoAb were added at the beginning of the cultures at a nnnl dilution of 1:1(X).t Delectinated !L-2wasadded to the microwclls at the beginning of the cultures at

a final dilution oi 1:10.^ ['Hl'fiyni't^iii' ineorporaiion was measured on day 3 of culture alter an IS h

pulse.1 Percentage of control. Kor further details, sec Materials and Methods

were repeated three limes with different donorsand in all cases comp«r;iblc results wereobtained. The IL-2 preparation used throtighoutthis study was repeatedly checked for its ahilityto enhance the prolitei;iiioii nl either MLCblasts or CrMP-indiieeii lynipliDhlasts. con-stantly giving positive results (see also Table V).It was also demonstrated that this IL-2 prepara-tioti was able to revert the inhibition of MLC andCiMP-induced proliferalinn caused by anii-HLAelass l[ MoAb (data not shown).

Tlie effects of the MoAb under investigationwere also studied on IL-2-dependent prolifera-tion of MLC lymphoblastN. It was observed thatno reagent among those used was able to affect

exogenous IL-2-indueed proliferation of theseblasts, as shown in Table V.

DISCUSSION

We have reported here that MLA classIl-restricted imtnune responses may be inhibitedby MoAb specific for class 1 determinants. Bothanti-/>:m and anti-HLA-A. B. C" heavy chainreagenis inhibited MLt" in a dose-responsefashion. Although direct evidence for the site ofaction ot MoAb is still lacking, some data seemto support the idea tluit the jiiliihitory eftccl isdue to the aetioti of MoAb on responder cells. In

HLA Class I Subunits in Immune Response 563

tact we i>bserved ;t significant inhibition whenthe responder cells were precultured with MoAband incubaicd with untreated stiniuialor forMl.( \ whereas the inhibitory effect was minimalwhen MoAb were prereacted wilh stimuUiti'rs.

Moreover, all MoAb still sironyly inhibitedthe proliferative response when the allogenicstimulation was provided by an HLA elass[•deficieni cell line (Daudi).

Allospccific and nonspecific cylotoxieactivities generared in MLC were also markedlyinhibited by anti-class I MoAb. No dissociationbetween the effects on tbe proliferaiion andgciier;itiiin ot cytotoxicity was observed at ail.suggesting that inhibition of the eytotoxieactivity could be a consequence of (be block oflymphoproliferiition and not ot a masking ofclass I target molecules [2^).

Proliferative responses to both (/. albicanswhole cells and a soluble antigen extracted fromits cell wall were similarly inhibited.

ihe lack of effect on non-1 II.A-restrieted,PMA-iiuluccd blastogenesis sugjicsls ihai (i) theobserved inhibitory effects are probably no( dueto iispecific toxic mechanisms or mere technicalpitfalls, and (ii) that these events are specific, aspecificity which is also supported by ihe ineffec-tiveness of a low-affinity anti'/^.-m MoAb(ACl .68) or the irrelevant X(i3. AgS moniKlonaiprotein. To rule out a membrane effecC linked tothe binding to the cell surface structure ratherthan to an individual specificity, ihe ctmtrolreagents included MoAb rccogni/^iiig structuresnot coded for by genes ofthe HLA region. Noneof the reagents selected (anti-complementreceptor 1. anti-AUlITID] and anti-humanspecies) was able to mimic ihe ctfccts induced byanti-IILAclassI MoAb on tbe immune responsestudied. Furthermore, it is unlikely that sterichindrance plays any role in the inhibition of classll-restricled immune responses by anli-class IMoAb, since it is known thai binding til anii/y.mantibodies enhances the biinling of anti-class 11antibodies on cell surfaces [I()|.

Tlie inhibitory effects of anti /iitn reagentswere largel\ independent of their relativeaffinity or their Ig class. I-urihermtire. the factthat comparable inhibitory effects were deter-mined by MoAb directed againsl different epi-topes argues againsl the hypothesis of theexistence of a single functional epitope on the/^m molecule. Interestingly, a lower inhibiioryeffect was shown by O1.65 MoAb. although its

cytotoxic titre is higher tir ct>inparable to thoseofthe anti-light chain reagents.

Anti-HLA class I MoAb exerted Ihcir max-imum inhibitory effects by the first 2 days ofculture, as reported here, and ihey were alsoable Ui inhibit IL-2 production 111. 22|: for thesereasons we investigated whether the block inIL-2 production could account for our observa-tions. It was seen that addition of e.xogenousIL-2 to either MLC or microbial antigen-stimul-ated cultures never abrogated ibe inhibitoryeffects of any anti-class I MoAb. These findingsindicate tliai the effects of these rea^icnts cannotbe allributcd siilely to a decreused productinn oflL-2. suggesting a reduced expression of IL-2reeeptors on the surface i>f activated lympho-cytes or a defective receptor -iigaiui interaction.In this respect our results are consistent withthose reported by Sterkers ei al. |23| and Turcoei al. [2b]. but in contrast with those recentlyreported by Chouaibrr «/ | l 1|. who were able torestore MLC'-induced prolitcrati\e responsesblocked by anti-/^-ni Mo.'\t> by ibe addiliitn ofIL-2. These discrepancies, however, could be aresult of the rather different experimentalapproach to assess the effects of ;mli-/)-m MoAbon immune responses and possibly also of thedifferent nature of the MoAb used by theseauthors. It should be noted that proliferation oflL-2-dependent lymphoblasts induced byexogenous IL-2 was not affected by ihc additionof any anti-HLA class I MoAb. making itunlikely ihai these MoAb interfere wilh ihebinding of IL-2 to its receptor.

Taken together, our data suggest that MoAbdirected against both class I subunils may affectone or mi>re early steps of ;intigen-induced lym-phocyte activation, probably preceding theexpression of large amounts of IL-2 receptors|2fi| on responding cell surfaces. Even if thiseffect per se does not constitute any conclusiveevidence lor a direct involvement of I ILA class Ideterminants in the generation or regulation ofclass Il-rcstrictcd responses, our results suggestthat this may be a distinct possibility.

Future Investigations using a number ofMoAb against epitopes of HLA-A. B. C heavychains could assist in assessing the significance ofthe slight differenees noted in this study betweenHLA-A. B.("heavy chain-specific()l.(i5 MoAband anti-//7m reagents on atloantigcn or micro-bial antigen-induced lymphoproliferation. Suchdifferenees seem to indicate that^^m plays a role

564 O'. ('. Spagnoli et at.

in antigen-induced lymphoprotiferaiion. per-haps in association with HLA-A.B,C heavychains.

A C K N O W L E D G M E N T S

We arc grateful to Dr Annemarie Termijlelen(Immunohaematology Department. I'niversityof Leiden. The Netherlands) ft>r helpful sugges-tions. This work was supported in part by CNR(Ilaly) Special Project Oncologia' (asseg-nazione No. -M/«5()6S/SS Contralto No.85/02557/44) and grants to F.M, fromAssociazione Italiana Ricerca C"ancro (Italy).Thanks are also due to the 'Centro lransfu-sionale Ospedaliero'. ILS.L. RM3 lor supplyingblood samples from human volunteers.

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Received 2S April l^ShReceived in revised form 2 January 1987