0014-2980/99/0808-2579$17.50+.50/0© WILEY-VCH Verlag GmbH, D-69451 Weinheim, 1999

Selective amino acid substitutions of asubdominant Epstein-Barr virus LMP2-derivedepitope increase HLA/peptide complex stability andimmunogenicity: implications for immunotherapy ofEpstein-Barr virus-associated malignancies

Fabiola Micheletti1, Remo Guerrini2, Annarita Formentin1, Alessandro Canella1, MauroMarastoni2, Martina Bazzaro1, 2, Roberto Tomatis2, 3, Serena Traniello1 and RiccardoGavioli1

1 Department of Biochemistry and Molecular Biology, University of Ferrara, Ferrara, Italy2 Department of Pharmaceutical Sciences, University of Ferrara, Ferrara, Italy3 Biotechnology Center, University of Ferrara, Ferrara, Italy

The latent membrane protein 2 is an immunogenic antigen expressed in Epstein-Barr virus(EBV)-associated tumors and consequently it may represent a target for specific cytotoxic Tlymphocyte (CTL)-based immunotherapies. However, the efficacy of such a therapy is lim-ited by the poor immunogenicity of the protein that induces weak CTL responses directed tothe CLGGLLTMV (CLG) epitope only in the minority of EBV-seropositive donors. We havenow demonstrated that selective peptide stimulation of peripheral blood lymphocytesinduced CLG-specific CTL in all donors, suggesting that this epitope can be a suitable targetfor specific immunotherapies. We found that the CLG peptide has a low affinity for HLA-A*0201 and does not produce stable complexes, both factors that are likely to determine thestrength of CTL responses to this epitope. Therefore, we synthesized and tested CLG analo-gues carrying single or combined amino acid substitutions to increase HLA/peptide stability.Among the analogues tested we identified two peptides which, compared to the natural epi-tope, showed higher affinity for HLA-A*0201 molecules, and produced stable complexes.These peptides demonstrated a potent, specific stimulatory capacity and could be used forselective CTL-based therapies.

Key words: HLA class I / Epstein-Barr virus / Cytotoxic T lymphocyte / Epitope

Received 10/3/99Revised 19/5/99Accepted 26/5/99

[I 19451]

Abbreviations: BL: Burkitt’s lymphoma NPC: Nasopharyn-geal carcinoma HD: Hodgkin’s disease LCL: Lymphoblas-toid cell line LMP: Latent membrane protein

1 Introduction

EBV is a widespread lymphotropic herpes virus whichcauses infectious mononucleosis and is associated withan increasing number of human malignancies includingendemic Burkitt’s lymphoma (BL), undifferentiated naso-pharyngeal carcinoma (NPC), Hodgkin’s disease (HD)and immunoblastic B cell lymphomas of immunosup-pressed patients [1–3]. This broad disease association is

in sharp contrast to the spread of the virus in all humanpopulations and its capacity to persists as a life-longasymptomatic infection.

Primary EBV infection of immunocompetent hosts leadsto the establishment of a life-long carrier state wherebythe virus persists within the B cell compartment ofhealthy carriers. Infected B lymphocytes can proliferatein vitro, giving rise to lymphoblastoid cell lines (LCL)which express at least eight latency-associated viralantigens, the nuclear antigens EBNA1 to -6 and thelatent membrane proteins (LMP)1 and 2 [4].

EBV induces long-lasting CTL memory, which is believedto play an important role in controlling EBV-carrying cellsin the infected host. Thus, EBV-specific CTL precursorscan be reactivated in a relatively large number from theperipheral blood of EBV-seropositive individuals by in

Eur. J. Immunol. 1999. 29: 2579–2589 LMP2-derived improved epitopes 2579

vitro stimulation with autologous virus-infected B cells[5], or by selective stimulations [6–8]. Recent studieshave shown that, in each individual, such CTL responsesare a composite of several virus-induced reactivities[9–12]. Each reactivity is directed against one or other ofthe transformation-associated viral proteins presented inthe context of particular HLA class I restricting determi-nants. In particular, EBNA3, EBNA4 and EBNA6 repre-sent immunodominant targets of CTL responses over awide range of HLA backgrounds, while EBNA2, EBNA5,LMP1 and LMP2 represent subdominant targets and arepresented in the context of a limited number of HLAtypes [13]. CTL responses directed to EBNA1, whichcontains a glycine-alanine repeat sequence that blocksits processing and presentation [14], are not capable ofrecognizing naturally infected cells expressing EBNA1[15].

The demonstration that the immunoblastic lymphomasoccurring in immunosuppressed individuals representthe in vivo outgrowth of EBV-positive LCL-like cells [16]emphasizes the role of immune surveillance in control-ling this potentially lymphomagenic virus. Indeed, EBVhas been used as target of specific CTL-based treat-ments that have been successfully applied in variouscases of immunoblastic B cell lymphomas [17–21], insevere chronic active EBV infection [22], and morerecently in HD [23]. The results of these studies suggestthat an antigen-specific CTL-based therapy may be ofbenefit in treating EBV-associated tumors such as NPCand HD that, among the immunogenic EBV antigens,express exclusively LMP1 and LMP2 [24–26]. NPC andHD are not associated with significant immunosuppres-sion, implying that escape from EBV-specific immunitymay play an important role in the development of thesetumors. In the case of HD, it has been demonstrated thatHodgkin’s cells secrete IL-10, a cytokine inhibitory to theinduction of CTL responses [27, 28]; this effect can beparticularly determinant in immune escape of HodgkinReed Sternberg cells, considering the low immunogenic-ity of LMP-derived epitopes.

In the present investigation we focused our analysis onLMP2, which represents the target of HLA-A*0201, HLA-A*0206 and HLA-A*0207-restricted EBV-specific CTLresponses. The peptide CLGGLLTMV (CLG) has beenmapped and one of the epitopes derived from LMP2[29]. Futhermore, it has been shown that only about 50 %of healthy donors respond to this epitope after in vitrostimulation of PBL with the autologous LCL, suggestingthat CLG represents a subdominant target of EBV-specific CTL responses [29].

We have now demonstrated that all HLA-A*0201-positive donors are capable of mounting significant CTL

respones to the CLG peptide after stimulation with APCpulsed with the synthetic peptide corresponding to thenatural epitope. However, we found that the CLG peptidedoes not produce stable complexes with HLA-A*0201and this may account for its low immunogenicity. Wetherefore synthesized and tested peptide analogues car-rying amino acid substitutions at non-anchor positions toincrease HLA/peptide stability. Peptides with increasedaffinity may be able to enhance stimulation of naturalepitope-specific CTL responses, and could prove usefulfor CTL-based immunotherapies.

2 Results

2.1 Detection of CLG-specific memoryresponses in HLA-A*0201 individuals

We investigated the presence of CLG-specific memoryCTL responses in a panel of HLA-A*0201 healthy EBV-seropositive individuals to identify specific responses inthose donors that did not respond to stimulation with theautologous LCL. For this purpose, isolated PBL werestimulated in parallel using the autologous B95.8 LCL orT2 cells pulsed with the CLG peptide, a stimulating pro-tocol that efficiently induces subdominant specificities[6]. All cultures were tested for specificity after the thirdstimulation. Out of 16 different cultures, 13 were MHCclass I restricted and EBV specific in that they lysed theautologous LCL and allogeneic LCL matched throughHLA class I alleles but did not lyse allogeneic HLA classI-mismatched LCL or untreated autologous PHA blasts(not shown). The presence of CLG-specific precursorswas evaluated by testing the EBV-specific culturesagainst HLA-A*0201-positive PHA blasts, either un-treated or pulsed with the CLG peptide (Table 1). None ofthe cultures killed untreated PHA blasts. Three CTL cul-tures out of those obtained by stimulation with the autol-ogous LCL contained a CLG-specific response. In con-trast, all cultures obtained by stimulation with CLG-pulsed T2 cells developed a significant CLG-specificresponse (Table 1). CTL obtained by peptide stimulationwere able to recognize the CLG epitope endogenouslyexpressed in HLA-A*0201-positive LCL, with killingwhich varied from 20 to 50 % specific lysis depending oneither the CTL culture and/or the target cells used in theassays. A representative result obtained with the EBV-specific CTL culture from donor RG is shown in Fig. 1.

These findings demonstrate that the lack of CLG-specific responses in cultures obtained by stimulationwith the autologous LCL is not due to the absence ofCTL percursors, but that this specificity can be selec-tively reactivated in all individuals by specific peptidestimulation.

2580 F. Micheletti et al. Eur. J. Immunol. 1999. 29: 2579–2589

Figure 1. Cytotoxic activity of a CTL culture obtained bystimulation with CLG-pulsed T2 cells. Freshly isolated PBLfrom donor RG (A2, B8,44) were stimulated with CLG-pulsedT2 cells. Cytotoxic activity was assayed after three consecu-tive stimulations against the indicated target cells. Resultsare expressed as % specific lysis recorded at E/T ratios of20:1, 7:1 and 2:1. One representative experiment out of fiveis shown.

Table 1. CLG-specific CTL responses in different EBV-seropositive donors

Stimulation with:

Autologous LCL T2/CLG

Donora) % Target cell lysis % Target cell lysis

Unpulsed CLG-pulsed Unpulsed CLG-pulsed

ADP 0 0 ND ND

CB 0 0 1 24

CHM 0 1 0 18

FT 0 1 2 39

FW 0 0 0 34

LDS 1 2 ND ND

MON 0 2 1 38

MT 0 0 0 40

MR 6 34 3 83

RG 2 27 3 75

RON 0 30 2 80

SH 0 0 1 50

ST 0 0 ND ND

a) CTL cultures from different donors tested after the thirdstimulation at 10:1 E/T ratio against HLA-A*0201matched PHA blasts.

2.2 Affinity of the CLG epitope for HLA-A*0201

To explore the reasons for the low immunogenicity of theCLG epitope, we evaluated the ability of the CLG peptideto associate with HLA-A*0201 and the stability of thecorresponding complexes in intact cells.

The mutant T2 cells were treated at 26 °C for 18 h inserum-free medium to induce high levels of empty mole-cules at the cell surface, and then exposed to 10–4 MCLG peptide at 37 °C for 1 h. The cells were extensivelywashed to remove the unbound peptide, and the surfaceexpression of HLA class I complexes was evaluated inkinetic experiments by immunofluorescence using mAbW6-32, which recognizes HLA class I molecules inde-pendently of the associated peptides. Parallel experi-ments were performed with the synthetic GILGFVFTLpeptide, derived from the influenza virus matrix antigen,which was previously shown to have high affinity forHLA-A*0201 [30].

The results indicate that the HLA-A*0201/CLG com-plexes are not stable at the cell surface: in fact, after 4 ha net decrease in expression was detected, and virtuallyall complexes were disrupted after 24 h. In contrast, theGIL peptide induced a higher number of complexes thatwere stably expressed for up to 24 h, and were stilldetectable at 50 % after 48 h (Fig. 2). We next sensitizedPHA blasts with the synthetic CLG peptide across a 10–4

to 10–12 M range of concentrations and evaluated spe-cific killing by CLG-specific effectors (Fig. 3). Half-maximal lysis was obtained with 10–9 M peptide, a ratherhigh concentration as compared to that required by syn-thetic peptides corresponding to immunodominant epi-topes which are able to induce half-maximal lysis of tar-get cells at 10–12 to 10–14 M [11, 30].

2.3 Peptides derived from CLG by single aminoacid substitution

We sought to increase the affinity and immunogenicity ofthe CLG peptide by changing non-anchor amino acids inorder to obtain modified epitopes with the capacity toform stable complexes with HLA-A*0201, and to effi-ciently stimulate CLG-specific CTL precursors.

We synthesized a series of CLG analogues differing fromthe original sequence by single amino acid substitutionsin positions 1, 3, 4 and 5 (Table 2). The amino acids tyro-sine (Y) and serine (S) were selected since they havebeen demonstrated as preferred non-anchor residues atthese positions [31–33]. All peptides were tested, in par-allel experiments, for their ability to stably associate withHLA-A*0201 molecules expressed at the surface of T2cells. The 3Y and 4S peptides exhibited an increased

Eur. J. Immunol. 1999. 29: 2579–2589 LMP2-derived improved epitopes 2581

Figure 2. HLA-A*0201/peptide complex stability. T2 cellswere treated as described in Sect. 4.3. At the indicated timepoints the surface expression of HLA class I molecules wasdetected by indirect immunofluorescence using the W6-32mAb. Results are expressed as % increase of HLA class Iexpression calculated with respect to untreated T2 cells.The mean of four different experiments is shown.

Figure 3. Target cell sensitization by peptides. HLA-A*0201-positive PHA blasts were treated with a 10–4 to 10–12 concen-tration range of the indicated peptides and used as target ofCLG-specific CTL cultures. Results are expressed as %specific lysis recorded at an E/T ratio of 20:1. The mean ofthree different experiments performed in parallel is shown.

affinity since they induced high levels of HLA-A*0201molecules, and 50 % of the corresponding complexeswere still detectable after 48 h. In contrast, the 1Y and 5Ypeptides associated with HLA-A*0201 molecules with anefficiency comparable to that obtained using the CLGepitope (Fig. 2). We next titrated the CLG analogues onPHA blasts across a 10–4 to 10–12 M range of concentra-tions to determine the level of recognition by CLG-specific CTL cultures (Fig. 3). The 1Y analogue sensitizedtarget cells more efficiently than the CLG peptide, in thathalf-maximal lysis was obtained with a concentration of5 × 10–11 M. The 3Y and 5Y peptides were recognizedless efficiently than the natural epitope, implying that

these amino acid substitutions involved a key residue forTCR contact or induced a change in peptide conforma-tion affecting TCR recognition. The 4S peptide was notrecognized by CLG-specific CTL, suggesting that thissubstitution, which strongly increases the complex sta-bility, may induce a conformational change of the pep-tide unfavorable for CTL-mediated lysis.

The analogues were then tested for their capacity tostimulate CTL responses directed against the wild-typeepitope. PBL from the EBV-seropositive donors MT, FRand RG were stimulated, in parallel experiments, with T2

2582 F. Micheletti et al. Eur. J. Immunol. 1999. 29: 2579–2589

Figure 4. CTL reactivation by CLG analogues carrying sin-gle amino acid substutions. Freshly isolated PBL derivedfrom the EBV-seropositive donor RG (HLA-A2, -B8,44) werestimulated with T2 cells pulsed with 10–4 M CLG, 1Y, 3Y, 5Y,or 4S peptides. CTL cultures, obtained after three consecu-tive stimulations, were tested in cytotoxicity assays againstHLA-A*0201 single-matched PHA blasts treated or not with10–7 M CLG peptide for 1 h before the assay. The percentspecific lysis recorded at two E/T ratios (20:1, 7:1) in onerepresentative experiment is shown.

Table 2. Peptide sequences

cells pulsed with the synthetic peptides, and CTL cul-tures were tested after three consecutive stimulationsagainst HLA-A*0201 single-matched PHA blasts treatedor not with 10–7 CLG peptide. Fig. 4 shows one represen-tative experiment performed with CTL cultures obtainedfrom donor RG. Peptides 1Y and 3Y were able to reac-tive CLG-specific responses with an efficiency compara-ble to the wild-type epitope. In contrast, 5Y and 4S pep-

tides failed to induce CLG-specific responses. Similarresults were observed with CTL cultures obtained fromMT and FR cultures (not shown).

It is noteworthy that CTL cultures from some donors (RGand MT) developed a CLG-specific response after a sin-gle stimulation with the 1Y peptide, in contrast to CTLcultures stimulated with the CLG or 3Y peptides, whichshowed CLG-specificity only after the second stimula-tion (not shown).

2.4 Peptides derived from CLG by combinedamino acid substitutions

Since the 1Y peptide showed a slightly higher immuno-genicity as compared to the wild-type epitope, wedesigned peptides carrying combined substitutions. Allanalogues presented a Y at position 1 in combinationwith those amino acids that were shown to increase HLAcomplex stability, such as Y at position 3 and/or S atposition 4 (Table 2). These peptides exhibited a highaffinity for HLA-A*0201 molecules, since they inducedhigh levels of HLA-A*0201 molecules at the cell surfaceof T2 cells, and the relative complexes were stable for upto 48 h (Fig. 2).

The CLG analogues were then titrated on PHA blastsacross a 10–4 to 10–12 M range of concentrations to deter-mine the level of recognition by CLG-specific CTL cul-tures (Fig. 3). None of the peptides were recognized byCLG-specific CTL, suggesting that these substitutionsinduce a conformational change of the peptides andabolish CTL-mediated lysis.

The analogues were tested for their capacity to stimulateCTL responses directed against the wild-type CLG epi-tope. PBL from the EBV-seropositive donors MT, FR andRG were stimulated, in parallel experiments, withpeptide-pulsed T2 cells and then tested for CLG speci-ficity after three consecutive stimulations. None of thecultures developed a CLG-specific response (notshown).

2.5 Peptides derived from the CLG epitope bysingle or combined amino acid substitutionswith non-anchor residues at intermediateaffinity

To enhance HLA-A*0201/peptide complex stability andto maintain immunogenicity, we chose to synthesizeCLG analogues carrying amino acid substitutions at non-anchor positions with residues defined in “intermediateaffinity” molecules [31, 33]. We expected that these pep-

Eur. J. Immunol. 1999. 29: 2579–2589 LMP2-derived improved epitopes 2583

Figure 5. CTL reactivation by CLG peptide analogues.Freshly isolated PBL derived from the EBV-seropositivedonors MON (HLA-A2,24 -B44,51) and RG (HLA-A2, -B8,44)were stimulated with T2 cells pulsed with 10–4 M CLG, 3A,3L, 1Y-3A or 1Y-3L peptides. CTL cultures obtained afterthree consecutive stimulations were tested in cytotoxicityassays against HLA-A*0201 single-matched PHA blaststreated or not with 10–7 M CLG peptide for 1 h before theassay. The percent specific lysis recorded at two E/T ratios(20:1, 7:1) in one representative experiment is shown.

tides would form stable HLA-A*0201/peptide complexeswithout affecting the conformation of the antigenic com-plex, thus allowing recognition by CLG-specific T cells.

The amino acids alanine (A) and leucine (L) were placedat the third position of the peptide, alone or in combina-tion with a tyrosine residue at position 1 (Table 2). Allpeptides were tested for their capacity to produce stablecomplexes with HLA-A*0201 molecules. The 3A and 3Lpeptides produced complexes detectable for up to 48 hwhile the relative 1Y-3A and 1Y-3L analogues producedcomplexes stable for up to 24 h (Fig. 2). The four pep-tides were titrated on PHA blasts to determine the levelof recognition by CLG-specific CTL cultures. The 3Apeptide sensitized target cells as efficiently as the CLGpeptide, whereas the 3L, 1Y-3A and 1Y-3L peptides werenot recognized by CLG-specific CTL, confirming thatamino acid substitutions in the CLG antigenic sequenceoften induce conformational changes in the peptideaffecting target cell recognition (Fig. 3).

All analogues were then tested for their capacity to stim-ulate CLG-specific CTL responses. PBL from the EBV-seropositive donors MT, LP, MON and RG were stimu-lated with T2 cells pulsed with the relevant peptides.Fig. 5 shows representative experiments performed withCTL cultures obtained from two donors. 3A- and 1Y-3A-stimulated PBL showed significantly higher CLG-specific CTL reactivity as compared to CLG-stimulatedPBL. In contrast, the 1Y-3L peptide induced CLG-specific responses as did the wild-type epitope, whilethe 3L analogue stimulated very poor responses. Similarresults were consistently observed with CTL culturesobtained from MT and LP cultures (not shown).

2.6 Cytotoxic activity of CLG-specific CTLcultures toward EBV-infected LCL

We investigated whether CTL cultures obtained by stim-ulation with the CLG-derived 3A and 1Y-3A peptidescould also recognize the epitope naturally presented byHLA-A*0201-positive LCL. CTL cultures obtained by dif-ferent peptide stimulations were compared in cytotoxic-ity assays using as targets single HLA-A*0201-matchedLCL and mismatched LCL. The results, represented inFig. 6, demonstrate that all CLG-specific CTL culturesspecifically lysed HLA-A*0201-positive LCL and did notlyse mismatched LCL. Further confirming the efficiencyof CLG-specific CTL cultures obtained by stimulationwith 3A and 1Y-3A peptides, we observed an increase inspecific killing of HLA-A*0201-positive LCL.

3 Discussion

In the last few years there has been a great deal of inter-est in defining CTL-based therapies for the treatment ofhuman malignancies and many efforts have been madeto characterize tumor-associated epitopes. The LMP2-derived CLGGLLTMV (CLG) epitope may be regarded asa target of specific immunotherapies for the treatment ofEBV-associated tumors, since it (1) induces specific CTLresponses in EBV-seropositive donors [29], (2) is anti-genically conserved among EBV isolates derived fromdifferent populations, including viruses present in NPCand HD cell biopsies [12, 29], and (3) is presented at thecell surface even in tumor cells deficient for the trans-porter associated with antigen presentation (TAP) [34,35]. However, the feasibility of specific CTL therapy maybe limited by the poor immunogenicity of this antigen.

2584 F. Micheletti et al. Eur. J. Immunol. 1999. 29: 2579–2589

Figure 6. Cytotoxic activity of a CTL culture obtained by stimulation with peptide-pulsed T2 cells. Freshly isolated PBL fromdonors MON and RG were stimulated with T2 cells pulsed with CLG, 3A or 1Y-3A peptides. Cytotoxic activity of the cultures wasassayed after three consecutive stimulations against the HLA-A*0201 single-matched BN, MR and MT LCL and the HLA class I-mismatched BOL LCL. Results are expressed as % specific lysis recorded at E/T ratios 20:1 and 7:1. One representative experi-ment out of three is shown.

Indeed, CTL responses to LMP2 are weak in comparisonwith the immunodominant EBV-specific reactivities, andonly about 50 % of the donors mounted a CTL responsedirected to this epitope, following PBL stimulation withthe autologous LCL [9, 29].

In this investigation we first evaluated CLG-specificresponses in a group of HLA-A*0201-positive EBV-seropositive individuals. Our results demonstrate that alldonors have CLG-specific CTL precursors that can bereactivated by specific peptide stimulations, a findingwhich further favors the use of the CLG epitope as targetof EBV-specific immunotherapies and the employmentof specific stimulations to amplify a desired CTL popula-tion [6–8].

The affinity of a peptide for MHC class I molecules seemsto play an important role in determining CTL responsive-ness. In fact, it has been shown that the immunogenicityof peptide antigens depends on a low dissociation rate ofMHC/peptide complexes [36, 37], and that peptidesforming stable complexes represent immunodominanttargets of CTL responses [38, 39]. We have demonstratedhere that the CLG peptide has a low HLA-A*0201 bindingaffinity and the HLA-A*0201/CLG complexes are not sta-ble at the cell surface, factors which can strongly influ-ence CTL responses to a given epitope. Since it may bepossible to improve the immunogenicity of subdominantepitopes by replacing specific residues with amino acidsthat favor binding [40–43], we synthesized a panel ofCLG analogues carrying single or combined amino acidsubstitutions at putative non-anchor positions toincrease HLA/peptide stability.

The 1Y analogue was able to sensitize target cells moreefficiently than the CLG peptide, and induced a morerapid reactivation of CLG-specific precursors in somedonors. Since the presence of Y in position 1 does notaffect the peptide association with HLA-A*0201, the C toY substitution may improve the interaction with the TCRof CLG-specific CTL. In contrast, the substitution L to Yin position 5 is not suitable for the design of improvedCLG-derived epitopes since the L in 5 is necessary forTCR recognition.

Amino acid substution of glycines in positions 3 and 4produced CLG analogues carrying residues necessaryfor maintaining stable interactions with HLA-A*0201 mol-ecules (3Y and 4S peptides). It is noteworthy that thesenew interactions, in the case of 4S peptide, induce con-formational changes in peptide residues contacting theTCR of CLG-specific T cells, leading to loss of immuno-genicity. The 3Y peptide maintains a certain degree ofrecognition by CLG-specific effectors, but showed astimulatory capacity comparable to that of the naturalepitope. Combinations of amino acid substitutions thatfavor binding and TCR activation produced peptides thatstably bound to HLA-A*0201 molecules but with poorimmunogenicity.

We conclude that the increase of peptide stability doesnot always allow the identification of “improved epi-topes”, since a high increase in affinity may induce con-formational changes affecting the immunogenicity ofpeptide antigens. To abolish the negative effect of HLA/peptide association on the peptide immunogenicity, wesynthesized CLG analogues carrying amino acid substi-

Eur. J. Immunol. 1999. 29: 2579–2589 LMP2-derived improved epitopes 2585

tutions at non-anchor positions with residues defined at“intermediate affinity” [31, 33]. Among the analoguestested we identified two peptides, 3A and 1Y-3A, whichshowed, as compared to the CLG peptide, higher affinityfor HLA-A*0201 molecules and produced more stablecomplexes. These peptides demonstrated a potent stim-ulatory capacity. Interestingly, the 1Y-3A peptide failed tosensitize PHA blasts to lysis by CLG-specific CTL, indi-cating that this analogue cannot sufficiently mimic theviral epitope on target cells to the point of inducing CLG-specific killing. We have previously shown that a singleconservative amino acid substitution is sufficient toabrogate the capacity of immunogenic epitopes to sen-sitize target cells to lysis by CTL specific for the wild-typeepitope but does not affect the ability to induce memoryCTL reactivation [44, 45].

In conclusion, the LMP2-derived CLG epitope may rep-resent a good candidate for use as a target of EBV-specific immunotherapies in the treatment of NPC andHD. As the poor immunogenicity of this epitope may bea limitation, we identified two peptides, 3A and 1Y-3A,which are “improved epitopes” able to produce HLAclass I complexes stably expressed at the cell surfaceof APC, and to induce strong CTL responses directedagainst the subdominant CLG natural epitope. Theseanalogues may be efficiently used for selective in vitroamplification of CLG-specific T cell precursors that canbe transferred to the patients, or else to pulse properAPC to boost in vivo CLG-specific CTL responses.

4 Materials and methods

4.1 Cell lines

The .174/T2 cell line (T2) was obtained by fusion of the pep-tide transporter mutant .174 LCL with the T cell line CEM.LCL were obtained by infection of lymphocytes from HLA-typed donors with culture supernatants of the virus producerB95-8 cell line, cultured in the presence of 0.1 ? g/ml of cyc-losporin A (Sandoz, Switzerland). Cell lines were maintainedin RPMI 1640 supplemented with 2 mM glutamine, 100 IU/mlpenicillin, 100 ? g/ml streptomycin, and 10 % heat-inactivated FCS (HyClone, UT). PHA-activated blasts wereobtained by stimulation of PBL with 1 ? g/ml purified PHA(Wellcome Diagnostics, Dartford, GB) for 3 days andexpanded in medium supplemented with IL-2 (Proleukin;Chiron, Milan, Italy) as described [11].

4.2 Peptide synthesis

The CLG peptide, corresponding to amino acids 426–434 ofLMP2, and the analogues were synthesized by a solid phasemethod using a continuous-flow instrument with on-line UVmonitoring.

The stepwise syntheses were carried out by Fmoc chemis-try. The Fmoc-MBHA resin (Novabiochem, Läufelfingen,Switzerland) was swelled in dimethylformamide (DMF) andpacked in the reaction column. Fmoc-amino acids werecoupled in a fourfold excess using diisopropylcarbodiimide(DIPCDI) in the presence of hydroxybenzotriazole (HOBt).The Fmoc group was cleaved with 20 % piperidine-DMFsolution. Protected peptides were cleaved from the resin bytreatment with reagent B (88 % trifluoracetic acid, 5 % H2O,7 % triethylsilane), and the resulting products were collectedby centrifugation. Crude deprotected peptides were purifiedby HPLC; purity was G 98 %. Structure verification wasachieved by elemental and amino acid analyses and MALDI-TOF mass spectrometry [46]. Peptide stocks were preparedin DMSO at a concentration of 10–2 M, kept at –20 °C, anddiluted in PBS before use.

4.3 Detection of HLA-A*0201/peptide complex stability

Aliquots of 5 × 106 T2 cells were cultured overnight in 2 mlserum-free AIM-V medium containing 10–4 M of the indicatedpeptides. Cells were than extensively washed, treated withmitomycin C (Sigma-Aldrich, Milan, Italy) to avoid cell prolif-eration, divided in aliquots in serum-free AIM-V medium (LifeTechnologies, Milan, Italy) and maintained at 37 °C forkinetic experiments in AIM-V medium containing 1 ? g/mlbrefeldin A (Sigma-Aldrich) to block the egress of new MHCclass I molecules. Surface expression of HLA class I mole-cules was detected by indirect immunofluorescence usingthe mouse mAb W6-32, which recognizes HLA-A, -B, -Cmolecules irrespectively of the associated peptide [47].Mean logarithm fluorescence intensity was measured with aFACS analyzer (Becton Dickinson). Data are expressed as %increase HLA class I expression calculated with respect tountreated T2 cells.

4.4 Preparation of peptide-pulsed cells

Aliquots of 3 × 106 T2 cells were cultured overnight at 26 °Cin 1 ml serum-free AIM-V medium. Cells were then washed,treated with mitomycin C to avoid cell proliferation, andpulsed with the indicated concentrations of peptides for 3 hat 37 °C in AIM-V medium. After extensive washing the cellswere used as stimulators for the reactivation of memory CTLresponses [6].

4.5 Generation of CTL cultures

Monocyte-depleted PBL from the EBV-seropositive donorsADP (HLA-A2,11 -B35,44), BN (HLA-A2,30 -B7), CB (HLA-A2,32 -B18,51), CG (HLA-A2,3 -B51), CHM (HLA-A2,24-B44,51), FT (HLA-A2,24 -B35,73), FR (HLA-A2 -B18), FW(HLA-A2,3 -B51), LDS (HLA-A2,3 -B21,35), LP (HLA-A1,2-B7,18), MON (HLA-A2,24 -B44,51), MR (HLA-A2,3 -B7,18),MT (HLA-A2, -B18), RG (HLA-A2, -B8,44), RON (HLA-A2,24

2586 F. Micheletti et al. Eur. J. Immunol. 1999. 29: 2579–2589

-B18,51), SH (HLA-A2,3 -B18,35), ST (HLA-A2 -B18,21) andVA (HLA-A2,33 -B14,18) were stimulated with mitomycin-treated autologous LCL or T2 cells incubated overnight at26 °C and pulsed with 10–4 M synthetic peptides for 3 h at37 °C, at stimulator responder ratio of 1:20. The first stimula-tion was performed in RPMI 1640 containing 10 % FCS. Thesecond and third stimulations were performed under thesame conditions on day 7 and day 14. The medium wassupplemented from day 8 with 10 U/ml recombinant IL-2 [6].

4.6 Cytotoxicity tests

Cytotoxic activity was assayed in standard 5-h 51Cr-releaseassays [48]. LCL and PHA blasts were labeled with 0.1 ? Ci/106 cells of Na2

51CrO4 (NEN, Brussels, Belgium) for 90 min at37 °C. For the peptide sensitization assays, 4 × 103 PHAblasts were placed in triplicate wells of 96-well V-shapedwell plates. Peptides were added to each well, and theplates were incubated for 1 h at 37 °C before addition of theeffectors [11]. Peptide toxicities were checked in eachassay, and were always p 3 %. Percent specific lysis wascalculated as 100 × (cpm sample – cpm medium)/(cpm Tri-ton X-100 – cpm medium).

Acknowledgments: We are very grateful to the donorswhose CTL are described in this paper, and to Banca delSangue of Ferrara for supplying fresh blood. We thank Ter-esa Grappa for HLA typing, and Eva Reali for critical readingand comments; we also thank Mrs. Linda Bruce for Englishrevision of the manuscript. This investigation was supportedby grants awarded by the Ministero Dell’Universita e dellaRicerca Scientifica e Tecnologica (MURST), AssociazioneItaliana per la Ricerca sul Cancro (AIRC) and Istituto Super-iore di Sanita (progetto AIDS).

5 References

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Correspondence: Riccardo Gavioli, Dipartimento di Biochi-mica e Biologia Molecolare, Via Borsari 46, I-44100 Ferrara,ItalyFax: +39-0532202723e-mail: r.gavioli — unife.it

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