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Prepublished online as a Blood First Edition Paper on September 19, 2002; DOI 10.1182/blood-2002-03-0933.
IMMUNOBIOLOGY
From the Laboratory of Tumor Immunology, Cancer
Immunotherapy and Gene Therapy Program, Functional Proteomics Centre
HRS-IFOM, Università Vita-Salute San Raffaele, and MOLMED, SpA,
DIBIT, Scientific Institute H. San Raffaele, Milan, Italy;
Department of Genomics and Information Sciences, Hoffmann-La Roche Inc,
Nutley, NJ; and Department of Medicine, Division of Hematology,
Oncology, and Transplantation, University of Minnesota, Minneapolis,
MN.
The molecular characterization of the
CD4+ T-cell epitope repertoire on human tumor antigens
would contribute to both clinical investigation and cancer
immunotherapy. In particular, the identification of promiscuous
epitopes would be beneficial to a large number of patients with
neoplastic diseases regardless of their HLA-DR type. MAGE-3 is a
tumor-specific antigen widely expressed in solid and hematologic
malignancies; therefore, is an excellent candidate antigen. We used a
major histocompatability complex (MHC) class II epitope prediction
algorithm, the TEPITOPE software, to predict 11 sequence segments of
MAGE-3 that could form promiscuous CD4+ T-cell
epitopes. In binding assays, the synthetic peptides
corresponding to the 11 predicted sequences bound at least 3 different
HLA-DR alleles. Nine of the 11 peptides induced proliferation of
CD4+ T cells from both healthy subjects and melanoma
patients. Four immunodominant regions (residues 111-125, 146-160, 191-205, and 281-295), containing naturally processed epitopes, were
recognized by most of the donors, in association with 3 to 4 different
HLA-DR alleles, thus covering up to 94% of the alleles expressed in
whites. On the contrary, the other promiscuous regions
(residues 161-175 and 171-185) contained epitopes not
naturally processed in vitro. The immunodominant epitopes identified
will be useful in the design of peptide-based cancer vaccines and in
the study of the functional state of tumor-specific CD4+ T
cells in patients bearing tumors expressing MAGE-3.
(Blood. 2003;101:1038-1044) Evidence for a critical role of CD4+ T cells in
orchestrating the antitumor response is increasing (for reviews, see
Pardoll and Topalian1 and Toes et al2).
CD4+ T cells provide help for induction and maintenance of
antitumor CD8+ T cells and may exert effector functions,
both indirectly via macrophage and eosinophil activation, and directly
when inflammatory responses produce cytokines such as interferon MAGE-3 is a tumor-specific antigen widely expressed in solid tumors
such as melanoma, lung carcinoma, head and neck carcinoma, and
hematologic malignancies, including T-cell leukemias and myeloma but
not in normal tissues (with the exception of testis).7 The
MAGE gene family comprises several related genes divided into 3 clusters, named MAGE-A, -B, and -C.7 The gene previously known as MAGE-3 belongs to the MAGE-A cluster and it has received the
official name MAGE-A3. We will refer to it from now on as MAGE-3, in
accordance with our previous report.8 Clinical trials in
patients with metastatic neoplastic disease using the
HLA-A1-restricted MAGE-3 epitope yielded some clinical
results.9-13 However, the immune responses were too weak
and transient to eradicate tumor cells in the majority of immunized
patients. The additional use of CD4 epitopes might help the induction
of a memory antitumor response and improve the clinical efficacy of vaccines.
Several allele-specific CD8 and CD4 epitopes of MAGE-3 have been
identified.14 Promiscuous epitopes, that is, those able to
bind to different HLA-DR alleles, are crucial to increase the number of
patients eligible for peptide-based cancer vaccination. Up to now, only
MAGE-3146-160 has been described by us8 and others15 as a promiscuous epitope presented in association
with HLA-DR11, HLA-DR4, and HLA-DR7.
In this paper, we report the results of the analysis of the MAGE-3
tumor antigen for promiscuous CD4 epitopes by a combined approach of
computational identification of candidate T-cell epitopes, followed by
in vitro biologic validation analysis of the predicted sequences as
forming natural epitopes recognized by CD4+ T cells.
We identified 4 immunodominant regions (residues 111-125, 146-160, 191-205, and 281-295), containing naturally processed epitopes, that
were recognized by most donors in association with 3 to 4 different
HLA-DR alleles. These epitopes, potentially recognized by more than
90% of whites, will be useful for both clinical investigation and
cancer immunotherapy.
T-cell epitope prediction and peptide synthesis
Peptide-binding assays
Subjects and cells Peripheral blood mononuclear cells (PBMCs) were obtained from 4 healthy subjects (donors 1, 2, 3, and 4) and 2 patients with melanoma (donors 5 and 6). Melanoma cell lines MD TC, OI TC, and GF TC were established from 2 cutaneous metastases and one lymph node metastasis, respectively, from 3 patients with melanoma, and line SK-Mel-24 was purchased from the American Type Culture Collection (Rockville, MD). MAGE-3 expression by patients' biopsies and melanoma cell lines was verified by reverse transcriptase-polymerase chain reaction (RT-PCR) and in melanoma cells also by intracytoplasmic staining (data not shown). The HLA-DR types of donors and melanoma cell lines were identified by molecular or serologic typing and are reported in the figures. Homozygous LCLs used were Bor (DR11) and Mun (DR3), established in our laboratory; Leis (DR4), kindly provided by F. Marincola (National Institutes of Health, Bethesda, MD); BM21 (DR11) purchased from ATCC; and Pitout (DR7), purchased from the European Collection of Cell Culture (ECCC; Salisbury, United Kingdom).Propagation of CD4+ T cells Synthetic peptides corresponding to pool I and pool II (Table 1) were used to stimulate the PBMCs from the different donors. Briefly, 20 × 106 PBMCs were cultured for 7 days in RPMI 1640 (Gibco), supplemented with heat-inactivated human serum (10%), L-glutamine (2 mM), penicillin (100 U/mL), and streptomycin (50 µg/mL; Biowhittaker, Walkersville, MD), containing pool I or pool II (1 µg/mL of each peptide). The reactive blasts were isolated on a Percoll gradient,22 expanded in interleukin 2 (IL-2)-containing medium (10 U/mL; Lymphocult, Biotest Diagnostic, Dreieich, Germany), and restimulated at weekly intervals with the same amount of peptides plus irradiated (4000 rad) autologous PBMCs as antigen-presenting cells (APCs).
Flow cytometry Cytofluorometric analyses were performed on a FACStarPlus (Becton Dickinson, Sunnyvale, CA). We used the following monoclonal antibodies (mAbs): anti-CD4-phycoerythrin (PE) and anti-CD8-fluorescein isothiocyanate (FITC; Becton Dickinson); L243 (anti-MHC class II; ATCC); 57B (originally described as an anti-MAGE-3 mAb,23 and later identified as an anti-MAGE-A4 mAb that cross-reacts with several MAGE-A proteins24) kindly provided by G. Spagnoli (Basel, Switzerland); and 20.4 (mAb that recognizes the truncated form of the human low-affinity nerve growth factor receptor, NGFr; ATCC). FITC-rabbit antimouse immunoglobulin antibody (Dako, Glostrup, Denmark) was used as second-step reagent in indirect immunofluorescence stainings. For intracytoplasmic staining, melanoma cells were fixed in
3.7% paraformaldehyde, permeabilized in 0.05% NP40, and then stained
as described.25
RT-PCR analysis Total RNA was extracted by the use of RNAzolTMB (Biotech, Houston, TX), according to the manufacturer's instructions. Single-stranded cDNA was synthesized from 2 µg total RNA, by Moloney murine leukemia virus-derived reverse transcriptase (Life Technologies, Milan, Italy), in the presence of 20 U RNasin (Promega, Madison, WI). cDNA coding for MAGE-3 was detected by PCR amplification as described.26 Samples scored positive when a band of the appropriate size was visible on an agarose gel in the presence of ethidium bromide.Proliferation assay CD4+ T cells and autologous irradiated PBMCs or HLA-DR-matched homozygous LCLs as APCs were diluted at a 1:10 or 1:5 ratio, respectively, and used as we described previously.8 Stimulants are indicated in the figure legends, where appropriate. In inhibition experiments, mAb L243 or an isotype-matched irrelevant mAb was added at the concentrations indicated in the figure legends. After 48 hours, the cultures were pulsed for 16 hours with [3H]-thymidine (TdR; 1 µCi [37 Bq], well, Amersham, Milan, Italy). The cells were collected with a Skatron Titertek multiple harvester (Skatron, Sterling, VA) and the thymidine incorporated was measured in a liquid scintillation counter.Selection of peptide-specific CD4+ T cells by magnetic separation To select MAGE-3161-175-specific CD4+ T cells from donor 5 from the polyclonal cell line, we first incubated the cells with the peptide in the presence of homozygous DR4-PBMCs and then we used the magnetic activating cell sorting (MACS) cytokine secretion assay-cell enrichment and detection kit (Miltenyi Biotec, Bergisch Gladback, Germany), following the manufacturer's instructions. MAGE-3161-175-specific CD4+ T cells were then restimulated weekly in the presence of the peptide (1-5 µg/mL) and DR4-PBMCs as APCs.Recombinant viruses and infection of LCLs The cDNA encoding Ii.MAGE-3,27 a fusion protein between the human invariant chain (Ii) and MAGE-3, was kindly provided by P. van der Bruggen (Brussels, Belgium). The retroviral vectors M3-CSM and IiM3-CSM, which encode theLNGFr, and either the
full-length MAGE-3 protein or the fusion protein Ii.MAGE-3,
respectively, were produced as reported previously.28 LCLs
were transduced by coculture with irradiated packaging cells producing
the M3-CSM or the IiM3-CSM vectors, produced by Phoenix cells (GP
Nolan, Stanford, CA). The percentage of infected cells was evaluated 48 hours after transduction by flow cytometry for LNGFr
expression with the mAb 20.4.28 The
LNGFr+ cells were purified by magnetic cell
sorting, using rat antimouse IgG1-coated beads (Dynabeads M-450, Dynal,
Oslo, Norway).
Cytotoxicity assay CD4+ T cells were tested for specific lytic activity in a standard 4-hour 51Cr release assay as described previously.29 The following targets were used: melanoma cells MD TC, OI TC, GF TC, and SK-Mel-24, LCL and LCL engineered to express MAGE-3 (LCL-M3). In cold-target competition assays, unlabeled target cells (cold targets) were added to CD4+ T cells (effectors) and 51Cr-labeled target cells (hot targets) at serial ratios of cold-to-hot target cells. The percentage of inhibition was calculated as follows: [(% specific lysis without cold target %
specific lysis with cold target)/(% specific lysis without cold
target)]× 100.
CD4+ T-cell stimulation assay LCL-M3, LCL-IiM3 (LCLs expressing Ii.MAGE-3), or melanoma cells were tested for their ability to induce the production of IFN- by
peptide-specific CD4+ T cells, after 24 to 48 hours of
incubation, using a standard ELISA (Biosource Europe, Nivelles,
Belgium), following the manufacturer's instructions.
We selected 11 MAGE-3 sequences, based on prediction by TEPITOPE, and used synthetic peptides corresponding to the selected sequences to confirm their binding capability in competition assays, using 7 common HLA-DRB1 alleles (Table 1). We grouped the 11 peptides into pool I and pool II, based on the results of the binding assays. Peptides forming pool I had the highest promiscuity, and peptides forming pool II bound well to at least 3 different HLA-DR alleles. We used pool I and pool II to propagate polyclonal T-cell lines from 6 donors. Total PBMCs were stimulated with pool I or pool II or both in independent cultures for 7 days, activated cells were enriched by a density gradient, expanded in the presence of IL-2, and restimulated weekly with irradiated peptide-pulsed autologous PBMCs. For 5 donors, after 2 cycles of stimulation, we obtained lines that comprised only CD4+ T cells. In the case of donor 1, a CD8+ T-cell depletion step was necessary to obtain more than 90% CD4+ T cells after 4 cycles of stimulation (data not shown). CD4+ T-cell lines from healthy donors 1, 2, and 4 and from donor/patient 5 could be propagated for several months. The lines from healthy subject 3 and donor/patient 6 could be propagated only for a few weeks and did not expand well in culture. Repertoire of HLA-DR-restricted epitopes recognized by polyclonal CD4+ T cells propagated with pool I and pool II We determined the epitope repertoire of the CD4+ lines from all donors by testing, after a few cycles of stimulation, their proliferative response to each single peptide forming the pools. Figure 1 summarizes the results obtained. Nine of 11 peptides tested elicited proliferation of CD4+ T cells in at least 2 donors. MAGE-3111-125, MAGE-3141-155, and the overlapping MAGE-3146-160, MAGE- 3191-205, MAGE-3281-295, and the overlapping MAGE-3286-300, were strongly recognized by most of the donors, consistent with a high degree of promiscuity. MAGE-3156-170 was weakly but significantly recognized by donor 1 and donor/patient 5, and the overlapping peptide MAGE-3161-175 was significantly and strongly recognized by donor 4 and donor/patient 5. MAGE-3171-185 was strongly recognized by donor 1 and donor/patient 5. Recognition of the MAGE-3 sequences was HLA-DR restricted as demonstrated in vitro by inhibition of the proliferation of CD4+ T cells to the peptides in the presence of an anti-DR antibody in the cultures (data not shown). MAGE-321-35 and MAGE-3251-265 were never recognized.
HLA-DR restriction molecules for the immunodominant sequences To identify the HLA-DR-restricting allele for the immunodominant sequences, CD4+ T cells from donors 1 and 2 and donor/patient 5 were challenged in microproliferation assays with APCs, homozygous for each of the 2 HLA-DRB1 alleles expressed by the donor, pulsed with individual peptides (Figure 2).
We did not perform HLA-DR restriction on donor 4 because donor 4 is HLA-DR11 homozygous, nor on donor 3 and donor/patient 6, because CD4+ T cells did not expand in culture enough to allow further characterization. MAGE-3111-125 was recognized in association with HLA-DR1, HLA-DR4, and HLA-DR11 (Figure 2A). Moreover, MAGE-3114-127, which largely overlaps MAGE-3111-125, was shown by van der Bruggen and colleagues27 to be recognized in association with HLA-DR13. MAGE-3146-160 was recognized in association with HLA-DR1, HLA-DR4, HLA-DR11, and HLA-DR7 (Figure 2B). MAGE-3191-205 was recognized in association with HLA-DR1, HLA-DR4, and HLA-DR11 alleles (Figure 2C). MAGE-3281-295 was recognized in association with HLA-DR1, HLA-DR4, and HLA-DR11 (Figure 2D). The same immunodominant sequence could be recognized in different donors with either one or both of the HLA-DRB1 alleles expressed. Moreover, different donors sharing the same alleles might preferentially recognize the same epitope with different alleles, probably due to a difference in precursors' frequency. Immunodominant sequences contain naturally processed epitopes MAGE-3114-127, largely overlapping MAGE-3111-125, MAGE- 3146-160, and MAGE-3281-295 were already described to contain naturally processed epitopes recognized in association with HLA-DR13,27 HLA-DR4 and HLA-DR7,15 and HLA-DR11,8 respectively. MAGE-3114-127 contains a natural epitope that is formed after processing through the exogenous pathway28; we could not test the natural processing pathways for MAGE-3111-125, which most likely contain the same natural epitope, because we failed to obtain CD4+ T cells recognizing exclusively that sequence. MAGE-3146-160 was formed after both endogenous and exogenous pathways.15 In the present work, we found that MAGE-3146-160 is recognized by CD4+ cytotoxic T cells (CTLs) from donor 2 (HLA-DR3, HLA-DR7) when expressed on HLA-DR7-matched melanoma cells (GF TC) and DR7-LCL engineered to express MAGE-3 (data not shown). MAGE-3281-295 was shown by us8 to contain an epitope formed after both endogenous and exogenous pathways and recognized by CD4+ CTLs.To verify whether MAGE-3191-205 contains a natural
epitope, we used polyclonal CD4+ T cells from donor 4 after
9 weeks of propagation with pool II, when the cells strongly and
exclusively recognized MAGE-3191-205 (Figure
3A lower panel). Recognition was HLA-DR
restricted as shown in Figure 3B. DR11-LCLs were engineered to express
MAGE-3 (DR11-LCL-M3) and used in cytotoxicity assays. As shown in
Figure 3C, MAGE-3191-205-specific CD4+ T cells
killed DR11-LCL-M3, but not wild-type DR11-LCLs. Moreover, CD4+ CTLs killed the MD TC and OI TC melanoma cells, which
express MAGE-3 and HLA-DR11, but not SK-Mel 24, expressing unrelated
HLA-DR (Figure 3D). In addition, we performed cold-target inhibition experiments, in which cold targets (DR11-LCLs) preincubated with peptide MAGE-3191-205 were added to the hot tumor targets.
Killing of MD TC and OI TC was inhibited by addition of peptide-pulsed DR11-LCLs (up to 75% and 98%, respectively), suggesting that this peptide contains an epitope that is presented by the melanoma cells
(Figure 3E-F). The percentage of inhibition of DR11-LCL (Figure 3E) or
DR11-LCLs pulsed with an irrelevant peptide (Figure 3F) was comparable
in the 2 cases, and up to 25% to 30%. The high cold/hot ratio (50%)
required to observe a specific inhibition of MD TC killing, compared to
OI TC, might be related to different binding affinity of the peptide
for the 2 subtypes of DR11-LCLs (DR*1104 and DR*1101) used. In the case
of MD TC, competition for MHC binding to the cold or hot targets of
free peptide released from the LCLs in culture might reduce the ability
of these LCLs to inhibit the killing.
MAGE-3161-175 and MAGE-3171-185 contain epitopes not naturally processed in vitro To verify whether MAGE-3161-175 contains a natural epitope, we used polyclonal CD4+ T cells from donor/patient 5, after selection of peptide MAGE-3161-175-specific CD4+ T cells by magnetic sorting (Figure 4B). CD4+ T cells did not secrete IFN- after challenge with
melanoma cells or DR4-LCLs engineered to express MAGE-3 (DR4-LCL-M3),
nor DR4-LCLs engineered to express Ii.MAGE-3 (DR4-LCL-IiM3), suggesting
that MAGE-3161-175 does not contain a natural epitope
produced after processing through both the endogenous and the exogenous
pathways (Figure 4C).
To characterize the recognition of peptide MAGE-3171-185,
we used polyclonal CD4+ T cells from donor/patient 5, after
12 weeks of propagation in culture, when they strongly and exclusively
recognized MAGE-3171-185 (Figure
5B). CD4+ T cells did not
recognize melanoma cells or DR4-LCL-M3, nor DR4-LCL-IiM3, suggesting
that this sequence does not contain a natural epitope as well (Figure
5C). To verify that DR4-LCL-M3 and DR4-LCL-IiM3 express a sufficient
amount of the MAGE-3 protein to be recognized by CD4+ T
cells, we tested its expression by flow cytometry. MAGE-3
intracytoplasmic staining in transduced DR4-LCLs and DR11-LCLs
recognized by CD4+ T cells specific for
MAGE-3191-205 (Figure 3D) showed that they contain a
comparable amount of MAGE-3 (data not shown).
In this study, we describe a successful strategy to identify promiscuous CD4 epitopes on candidate tumor antigens. Using a combined approach of computational prediction for promiscuous MHC class II epitopes and in vitro validation by biologic assays, we identified 4 immunodominant regions of MAGE-3, which contain naturally processed epitopes presented by several different HLA-DR alleles. For most of the predicted epitopes, we found an excellent correlation
between prediction and the ability of the peptides to bind
promiscuously to HLA-DR molecules and to stimulate CD4+ T
cells from different donors. This was especially striking for overlapping sequences MAGE-3141-155 and
MAGE-3146-160, and overlapping sequences
MAGE-3281-295 and MAGE-3286-300. On the
contrary, MAGE-3111-125 and MAGE-3191-205 that,
as predicted, bound HLA-DR*1101 but not HLA-DR*0101 and HLA-DR*0401,
could strongly stimulate proliferation of CD4+ T
cells in association with all the 3 HLA-DR alleles, pointing to the
importance of the in vitro studies for validation of the predicted
sequences. MAGE-3251-265, which was predicted to bind 48%
of the alleles, did not bind well to the HLA-DR molecules that we
tested (Table 1) and was never recognized by CD4+ T cells.
MAGE-3251-265 partially overlaps MAGE-3243-258,
which is recognized in association with HLA-DP4 but not with
HLA-DR.30 MAGE-321-35, which was predicted to
bind poorly, never elicited CD4+ T-cell responses. In
agreement, Kobayashi et al15 also reported that the
overlapping peptide sequence MAGE-322-36 could stimulate T
lymphocytes from different donors in association with HLA-DR4, but the
CD4+ T cells did not expand in culture.15 All
the other sequences activated CD4+ T cells from at least 2 donors and did so in association with 2 to 4 different alleles (Figure
1; Table 2), confirming the ability of
TEPITOPE to predict promiscuous epitopes.
Most importantly, the immunodominant epitopes identified here are recognized in association with HLA-DR1, HLA-DR4, HLA-DR11, and HLA-DR7 that, with the addition of HLA-DR13,27 cover up to 94% of the alleles expressed in whites (Table 2). We cannot exclude that HLA-DRB3 or HLA-DRB4 molecules in addition to HLA-DRB1 can also present the identified sequences. The immunodominant regions contain epitopes that are naturally processed from different sources of tumor antigen and by different APCs. In our present and previous studies,8 sequences MAGE-3141-160, MAGE-3191-205, and MAGE-3281-295 were recognized by cytolytic CD4+ T cells after endogenous processing of MAGE-3 by LCLs and melanoma cells. Moreover, sequence MAGE-3281-295 was recognized by CD4+ T cells after processing and cross-presentation of the recombinant protein by autologous APCs.8 MAGE-3141-160-reactive T-helper cells were also shown to recognize various forms of the MAGE-3 protein (tumor cell lysate, dead/apoptotic tumor cells, or recombinant protein).15 MAGE- 3111-125 largely overlaps MAGE-3114-127, which is produced after processing and presentation of recombinant MAGE-3 protein by autologous dendritic cells and by melanoma cells that express an invariant chain-MAGE-3 fusion protein in the endosomal/lysosmal compartment.27 On the contrary, MAGE-3161-175-specific and MAGE-3171-185-specific CD4+ T cells did not recognize in vitro the MAGE-3 protein after processing through either the endogenous or the exogenous pathways. A possible explanation for this finding is that we failed to detect recognition of the native epitope because peptides bind to HLA molecules in a different conformation when they are loaded exogenously or when they are generated endogenously, as shown by Unanue and colleagues.31,32 In contrast with "naturally processed" epitopes that derive from processing of the whole protein antigen, "cryptic" epitopes are presented after the exposure to the antigen in peptide form, but not after processing of the whole protein antigen.33 Protease activity as well as the nature of the APCs during the processing of the protein may have a major influence on the repertoire of naturally processed or cryptic epitopes displayed for T-cell recognition.33 It is interesting that, in addition to the immunodominant epitopes, these sequences were strongly and predominantly recognized after long-term culture by CD4+ T cells from donor/patient 5, who had metastatic melanoma. We cannot completely exclude that priming of CD4+ T cells recognizing these sequences occurred in vivo as a consequence of epitope spreading during inflammatory conditions and operated by APCs not tested in our in vitro studies, leading to the formation of "cryptic" epitopes. The use of pools of peptides for CD4+ T-cell stimulation has the advantage that competition among peptides with different binding affinity for the presenting alleles mimics in culture what occurs in vivo, where different processed peptides compete for MHC class II binding and T-cell-receptor recognition. Therefore, the finding that some peptides are predominant in our cultures may be related to their higher affinity for the MHC class II molecules, or to the presence of peptide-specific CD4+ T cells expressing T cell receptor with high affinity for the peptide/MHC class II complexes. The finding that CD4+ T cells specific for MAGE-3191-205 and MAGE-3281-295 proliferate at peptide concentrations as low as 1 to 10 ng/mL (data not shown) supports the latter possibility. The predominant recognition of different immunodominant regions in different donors may also be related to their precursor repertoires. Animal models have shown that tumor-specific CD4+ T cells, in addition to playing a role in the induction of the antitumor immune response, have direct effector functions.1-4 The results of the present and previous studies by us and others8,33-36 strongly support a potential role of human tumor-specific CD4+ T cells as effectors of the antitumor immune response. Although the significance of the CD4+ T cells compared with CD8+ T cells in vivo remains to be clarified, lytic function of CD4+ T cells may be relevant when neoplastic cells, constitutively or as a consequence of inflammatory milieu, express MHC class II molecules. It will be interesting to verify the expansion of CD4+ CTLs ex vivo from cancer patients, as it has been shown,37 where CD4+ perforin-positive T cells were expanded in donors with chronic viral infections. Clinical trials of vaccination with synthetic peptides corresponding to the HLA-A1-restricted MAGE-3 epitope recognized by CD8+ T cells have started in patients bearing MAGE-3+ tumors.9-13 The additional use of peptides corresponding to the immunodominant CD4 epitopes identified here would increase the magnitude and life span of the antitumor immune response in these patients. Moreover, the identification of these epitopes will help in the study of the number and function of MAGE-3-specific CD4+ T cells, and the reliable monitoring of the immune responses in cancer patients before and after vaccination.
We thank Cinzia Arcelloni for high-performance liquid chromatography analyses, and Paolo Dellabona, Matteo Bellone, and Angelo Manfredi for enjoyable discussions and critical reading of the manuscript.
Submitted March 26, 2002; accepted September 12, 2002.
Prepublished online as Blood First Edition Paper, September 19, 2002; DOI 10.1182/blood-2002-03-0933.
Supported by the AIRC (Italian Association for Cancer Research), European Community (grant QLK2-CT2000-00470; M.P.P.), the MURST (Ministry of University and Scientific Research), the Ministry of Health, the National Heart, Lung, and Blood Institute (grant HL6192; B.M.C.-F.) and the National Institute of Neurological Disorders and Stroke (grant NS23919; B.M.C.-F.).
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Maria Pia Protti, Laboratory of Tumor Immunology, Cancer Immunotherapy and Gene Therapy Program, DIBIT, Scientific Institute H. San Raffaele, Via Olgettina 58, 20132 Milan, Italy; e-mail: m.protti{at}hsr.it.
1. Pardoll DM, Topalian SL. The role of CD4+ T cell responses in antitumor immunity. Curr Opin Immunol. 1998;10:588-594[CrossRef][Medline] [Order article via Infotrieve].
2.
Toes RE, Ossendorp F, Offringa R, Melief CJ.
CD4 T cells and their role in antitumor immune response.
J Exp Med.
1999;189:753-756
3.
Mumberg D, Monach PA, Wanderling S, et al.
CD4+ T cells eliminate MHC class II-negative cancer cells in vivo by indirect effects of IFN-
4.
Qin Z, Blankenstein T.
CD4+ T cell-mediated tumor rejection involves inhibition of angiogenesis that is dependent on IFN- 5. Pawelec G, Muller L, Wagner W. MHC class II-restricted tumor antigens and CD4+ T cells play a role in hematological malignancies as well as solid tumors. Trends Immunol. 2001;22:422-423[CrossRef][Medline] [Order article via Infotrieve]. 6. Wang R-F. The role of MHC class II-restricted tumor antigens and CD4+ T cells in antitumor immunity. Trends Immunol. 2001;22:269-276[CrossRef][Medline] [Order article via Infotrieve]. 7. Van der Eynde BJ, van der Bruggen P. T cell defined tumor antigens. Curr Opin Immunol. 1997;9:684-693[CrossRef][Medline] [Order article via Infotrieve]. 8. Manici S, Sturniolo T, Imro MA, et al. Melanoma cells present a MAGE-3 epitope to CD4+ cytotoxic T cells in association with histocompatibility leukocyte antigen DR11. J Exp Med. 1999;189:8871-876. 9. Marchand M, Weynants P, Rankin E, et al. Tumor regression responses in melanoma patients treated with a peptide encoded by gene MAGE-3. Int J Cancer. 1995;63:883-885[Medline] [Order article via Infotrieve]. 10. Marchand M, van Baren N, Weynants P, et al. Tumor regression observed in patients with metastatic melanoma treated with an antigenic peptide encoded by gene MAGE-3 and presented by HLA-A1. Int J Cancer. 1999;80:219-230[CrossRef][Medline] [Order article via Infotrieve]. 11. Nestle FO, Alijagic S, Gilliet M, et al. Vaccination of melanoma patients with peptide- or tumor lysate-pulsed dendritic cells. Nat Med. 1998;4:328-332[CrossRef][Medline] [Order article via Infotrieve].
12.
Thurner B, Haendle I, Roder C, et al.
Vaccination with MAGE-3A1 peptide-pulsed mature, monocyte-derived dendritic cells expands specific cytotoxic T cells and induces regression of some metastases in advanced stage IV melanoma.
J Exp Med.
1999;190:1669-1678
13.
Banchereau J, Palucka AK, Dhodapkar M, et al.
Immune and clinical responses in patients with metastatic melanoma to CD34(+) progenitor-derived dendritic cell vaccine.
Cancer Res.
2001;61:6451-6458 14. Cancer Immunity. Peptide database. Available at: http://www.cancerimmunity.org/peptidedatabase/tumorspecific.htm.
15.
Kobayashi H, Song Y, Hoon DSB, Appella E, Celis E.
Tumor-reactive T helper lymphocytes recognize a promiscuous MAGE-3 epitope presented by various major histocompatibility complex class II alleles.
Cancer Res.
2001;61:4773-4778 16. Sturniolo T, Bono E, Ding J, et al. Generation of tissue-specific and promiscuous HLA ligand database using DNA microarrays and virtual HLA class II matrices. Nat Biotechnol. 1999;17:555-561[CrossRef][Medline] [Order article via Infotrieve].
17.
Houghten RA.
General method for the rapid solid phase synthesis of large numbers of peptides: specificity of antigen-antibody interaction at the level of individual amino acids.
Proc Natl Acad Sci U S A.
1985;82:5131-5135 18. Raddrizzani L, Sturniolo T, Guenot J, et al. Different modes of peptide interaction enable HLA-DQ and HLA-DR molecules to bind diverse peptide repertoires. J Immunol. 1997;159:703-711[Abstract].
19.
Cammarota G, Scheirle A, Takas B, et al.
Identification of a CD4 binding site on the 20. Sinigaglia F, Romagnoli P, Guttinger M, Takas B, Pink JRL. Selection of T-cell epitopes and vaccine engineering. Methods Enzymol. 1992;203:370-386. 21. Roche PA, Cresswell P. High-affinity binding of an influenza hemagglutinin-derived peptide to purified HLA-DR. J Immunol. 1990;144:1849-1856[Abstract]. 22. Protti MP, Manfredi AA, Straub C, Wu X, Howard JF Jr, Conti-Tronconi BM. Use of synthetic peptides to establish anti-human acetylcholine receptor CD4+ cell lines from myasthenia gravis patients. J Immunol. 1990;144:1711-1720[Abstract].
23.
Kocher T, Schultz-Tjater E, Gudat F, Schaefer C, Heberer M, Spagnoli G.
Identification and intracellular location of MAGE-3 gene product.
Cancer Res.
1995;55:2236-2239 24. Landry C, Brasseur F, Soagnoli GC, et al. Monoclonal antibody 57B stains tumor tissues that express gene MAGE-A4. Int J Cancer. 2000;86:835-841[CrossRef][Medline] [Order article via Infotrieve]. 25. Imro MA, Dellabona P, Manici S, et al. Human melanoma cells transfected with the B7-2 costimulatory molecule induce tumor specific CD8+ cytotoxic T lymphocytes in vitro. Hum Gene Ther. 1998;9:1335-1344[Medline] [Order article via Infotrieve]. 26. Dalerba P, Ricci A, Russo V, et al. High homogeneity of Mage, Bage, Gage, Tyrosinase, and Melan-A/MART-1 gene expression in clusters of multiple simultaneous metastases of human melanoma: implication for protocol design of therapeutic antigen-specific vaccination strategies. Int J Cancer. 1998;77:200-204[CrossRef][Medline] [Order article via Infotrieve].
27.
Chaux P, Vantomme V, Stroobant V, et al.
Identification of MAGE-3 epitopes presented by HLA-DR molecules to CD4 (+) T lymphocytes.
J Exp Med.
1999;189:767-778
28.
Fleischhauer K, Tanzarella S, Russo V, et al.
Functional heterogeneity of HLA-A*02 subtypes revealed by presentation of a MAGE-3-encoded peptide to cytotoxic T cell clones.
J Immunol.
1997;159:2513-2521
29.
Protti MP, Imro MA, Manfredi AA, et al.
Particulate naturally processed peptides prime a cytotoxic response against human melanoma in vitro.
Cancer Res.
1996;56:1210-1213
30.
Schultz ES, Lethé B, Cambiaso CL, et al.
A MAGE-3 peptide presented by HLA-DP4 is recognized on tumor cells by CD4+ cytolytic T lymphocytes.
Cancer Res.
2000;60:6272-6275 31. Viner NJ, Nelson CA, Deck B, Unanue ER. Complexes generated by the binding of free peptides to class II MHC molecules are antigenically diverse compared with those generated by intracellular processing. J Immunol. 1996;156:2365-2386[Abstract].
32.
Peterson DA, Di Paolo RJ, Kanagawa O, Unanue ER.
Cutting edge: a single MHC anchor residue alters the conformation of a peptide-MHC complex inducing T cells that survive negative selection.
J Immunol.
2001;166:5874-5877 33. Sercarz EE, Lehmann PV, Amentani A, Benichou G, Miller A, Moudgil K. Dominance and crypticity of T cell antigenic determinants. Annu Rev Immunol. 1993;11:729-766[CrossRef][Medline] [Order article via Infotrieve]. 34. Takahashi T, Chapman PB, Yang SY, Hara I, Vijayasaradhi S, Houghton AN. Reactivity of autologous CD4+ T lymphocytes against human melanoma. J Immunol. 1995;154:772-779[Abstract].
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Abstract
Introduction
2 domain of HLA-DR molecules.
Nature.
1992;356:799-801