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Major Histocompatibility Complex Class I Restricted Cytotoxic T Cells Specific for Natural Melanoma Peptides Recognize Unidentified Shared Melanoma Antigen(s)¹

Laboratory of Tumor Immunology [M. A. I., S. M., G. C., M. B., C. R., M. P. P.], Gene Therapy Program [V. R., C. T.], Universita Vita-Salute [C. R.], and Cancer Immunotherapy and Gene Therapy Program [M. A. I., S. M., V. R., G. C., M. B., C. R., C. T., M. P. P.], Scientific Institute H. San Raffaele, 20132 Milan, Italy

	ABSTRACT

Top ABSTRACT Introduction Materials and Methods Results and Discussion REFERENCES

 
CTLs were generated in vitro from two healthy donors and one melanoma patient by stimulation of CD8⁺ T cells with autologous dendritic cells pulsed with natural melanoma peptides (NMPs), obtained by acid treatment of HLA-matched melanoma cells. CTLs showed MHC class I-restricted melanoma-specific cytolytic activity. Importantly, CTLs from the patient, induced with NMPs obtained from an allogeneic HLA-A-matched melanoma, killed the autologous tumor. COS-7 cells cotransfected with the cDNA of 13 melanoma antigens and the HLA-A1-restricting allele did not induce cytokines release from NMP-specific CTLs, suggesting that they recognize unidentified shared melanoma antigens and that they may be valuable for identification of new tumor antigens. These results strongly support the use of autologous and/or allogeneic NMP-pulsed dendritic cells as cancer vaccines in patients whose neoplasms do not express or have lost expression of known tumor antigens.

	Introduction

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Several TAAs³ and related epitopes, recognized by autologous CTLs, have been identified (reviewed in Refs. 1 and 2 ). On the basis of their pattern of expression, TAAs are classified as: (a) tumor-specific antigens shared by tumors of different histotypes; (b) tissue-specific antigens shared among melanomas and melanocytes; or (c) unique tumor antigens, resulting from point mutations (1 , 2) . The existence of a fourth class of TAAs, shared among melanomas but not expressed in melanocytes or tumors of different histotypes has been suggested (3) , and a melanoma antigen belonging to this class has been cloned recently (4) .

The identification of TAAs has opened new perspectives in the treatment of cancer patients and clinical trials based on the use of synthetic peptides corresponding to sequence segments of known TAAs are ongoing. However, several requirements/concerns may limit their use as cancer vaccines.

(a) The expression of TAAs by the patients’ tumors and their role/immunodominance in the induction of tumor immunity must be demonstrated (5) . Indeed, a study (3) in which the antigen specificity of a panel of CTL clones from melanoma patients was evaluated showed that most of them were not directed against tissue or tumor-specific antigens. This observation suggests that still unidentified antigens could be effective in eliciting tumor immunity in those patients and that the repertoire of melanoma antigens may be larger than that which is currently known.

(b) Antigen loss tumor variants during progression of the disease and/or vaccination (6 , 7) may occur, making multiple determinations of antigen expression over time necessary.

(c) The TAAs are applicable to a restricted number of patients expressing HLA alleles for which synthetic sequences are available. This limitation may be overcome in the future because an increasing number of epitopes and their HLA-restricting alleles are being identified (reviewed in Ref. 8 ).

An alternative source of tumor antigens is the mixture of NMPs obtained by acid treatment of tumor cells (9) . Preclinical studies, both in vitro in humans and in vivo in animals, have documented the efficacy of natural tumor peptides in inducing antitumor responses (reviewed in Ref. 9 ). Recently, melanoma patients were treated with autologous DCs pulsed with tumor lysate, obtained from the supernatants of autologous melanoma cells by freeze/thaw cycles (10) . Although the number of patients was very small, this is the first report of treatment of cancer patients with unfractioned tumor antigens.

Here, autologous DCs pulsed with NMPs extracted from allogeneic HLA-matched melanoma cells were used in vitro to stimulate CD8⁺ T cells from two healthy donors and one melanoma patient. The induced CTLs were characterized for killing and tumor antigen specificity. The results demonstrate that NMPs obtained from an allogeneic melanoma are able to activate an antitumor response against the autologous tumor in the melanoma patient. Moreover, the study of the tumor antigen specificity shows that NMPs contain still unidentified melanoma peptides and supports their use as cancer vaccines and for identification of new TAAs.

	Materials and Methods

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Subjects and Cells.
PBMCs were obtained from two healthy donors and one melanoma patient (subject 1, subject 2, and patient 1). The melanoma cell lines SK-Mel-24, HT144, SK-Mel-28, and SK-Mel-5 were from the American Type Culture Collection (Manassas, VA). The melanoma cell lines SK23-Mel, LB33-Mel, and MZ2-Mel.2.2ET.1 (hereafter referred to as ET1) were kindly provided by Prof. T. Boon (Ludwig Institute for Cancer Research, Brussels, Belgium). The OI-TC and VC-TC cell lines were established in our laboratory from melanoma cells isolated from cutaneous metastases. ME-180, a cervix epidermoid carcinoma cell line, was a generous gift from Interlab Cell Line Collection (CBA, Genoa, Italy). VR-LCL and FR-LCL (from patient 1) are EBV-transformed LCLs. All cell lines were cultured in RPMI 1640 (Life Technologies, Inc., Grand Island, NY), containing 2 mM L-glutamine, 100 units/ml penicillin, 50 mg/ml streptomycin (BioWhittaker, Walkersville, MD), and 10% FCS (Life Technologies, Inc., Paisley, Scotland). The HLA types of the donors and the cells used in this study, identified by standard serologic or molecular typing, are reported in Table 1 .

Preparation of Melanoma Peptides and Generation of DCs.
NMPs were obtained by acid treatment of SK-Mel-24 cells, followed by gel filtration on a Sephadex G-25 column, as described previously (Ref. 11 ; 28–30 mg from 5 x 10⁹ cells). DCs were generated as described previously (12 , 13) . Briefly, PBMCs (obtained from 50–100 ml of blood) were resuspended in RPMI 1640 supplemented with 10% heat-inactivated pooled HS, 2 mM L-glutamine, 100 units/ml penicillin, and 50 mg/ml streptomycin (TCM; Technogenetics, Milan, Italy) and plated at the concentration of 5 x 10⁶ cells/ml in six-well plates for 2 h at 37°C. Nonadherent cells were then removed, and the adherent cells were cultured for 7 days with TCM (1% HS), in the presence of granulocyte macrophage colony-stimulating factor (800 units/ml; Mielogen, Schering Plough, Milan, Italy) and IL-4 (500 units/ml, kindly provided by Dr. Latini, Schering Plough). Half of the supernatant was replenished with fresh TCM and cytokines every other day. At day 7, DCs were retrieved on a Percoll (Pharmacia, Milan, Italy) gradient (1–5 x 10⁶ cells), as described previously (14) , and used for phenotypic analysis and for pulsing with melanoma peptides. DCs were incubated with NMPs (75–125 µg/ml) for 3–4 h at 37°C before they were admixed with CD8⁺ T lymphocytes.

CTL Induction.
CD8⁺ T cells were purified from the nonadherent fraction of PBMCs, using microbeads coated with anti-CD8 mAb and a miniMACS magnetic sorter (Miltenyi Biotec GmBH, Bergisch Glsadback, Germany), mixed with peptide-pulsed DCs at a 10:1 ratio, and cultured in TCM for 7 days. At day 7, activated cells were isolated on a Percoll gradient, as described previously (14) , and expanded in the presence of TCGF (Lymphocult-T; Biotest Diagnostic, Inc., Dreieich, Germany) for a further 5–7 days. At day 14 and then at weekly intervals, CD8⁺ cells were restimulated with irradiated (4000 rad) SK-Mel-24/B7-2 cells (i.e., SK-Mel-24 cells transfected with the B7-2 molecule, described in Ref. 15 ). The next day, effector cells were cleaned on a Ficoll gradient and re-expanded in TCGF. CD8⁺ T-cell clones were obtained by limiting dilution as described previously (16) . Clones were restimulated every other week with irradiated (4000 rad) allogeneic PBMCs and LCLs.

Chromium-Release Assay.
Effector CD8⁺ cells were tested for lytic activity in standard 4-h ⁵¹Cr release assays as described previously (15) . In inhibition experiments, different concentrations (final dilutions, chosen on the basis of surface staining, were 1:100 and 1:1000) of the mouse mAbs specific for the MHC class I molecules (W6/32) and the MHC-class II molecules (anti-DR; Becton-Dickinson) were added to the wells. The percentages of specific lysis and inhibition were calculated as described previously (15) .

RT-PCR Analysis.
Total RNA was extracted by the use of RNAzolTMB (Biotech, Houston, TX), according to manufacturer’s instructions. Single-stranded cDNA was synthesized from 2 mg of total RNA, by Moloney murine leukemia virus-derived reverse transcriptase (Life Technologies, Inc.), in the presence of 20 units of RNasin (Promega, Madison, WI). After 1 h of incubation at 42°C, reverse transcription products were stored at -80°C. cDNAs coding for tumor antigens were detected by PCR amplification. Reaction mixture contained 5 ml of cDNA, corresponding to 100 ng of total RNA, 4 ml of a 10 mM dNTPs mixture (containing each dNTP at 2.5 nM), 5 ml of 10x DNA polymerase buffer (Finnzymes Oy, Espoo, Finland), 2 units of DNAZyme DNA polymerase (Finnzymes Oy), and sterile water up to a 50-ml total reaction volume. For oligonucleotide primer sequences and PCR amplification programs, see Dalerba et al. (Ref. 17 ; MAGE-1, MAGE-3, BAGE, GAGE, tyrosinase, and Melan-A/MART-1) and Lupetti et al. (Ref. 5 ; TRP-2, TRP-2-INT2, and gp100). Samples were scored as positive when a band of the appropriate size was visible on a agarose gel in the presence of ethidium bromide.

Transfection of COS-7 Cells.
Transfections were performed by the DEAE-dextran-chloroquine method (18) . In brief, 1.2 x 10⁴ COS-7 cells were cotransfected with 100 ng of plasmid pcD-SRa containing the HLA-A1 gene (19) and 100 ng of pcDNAI/Amp (Invitrogen, San Diego, CA) or pcD-SRa plasmids containing the cDNA of one of the following melanoma antigens: Melan-A/MART-1; tyrosinase; gp100; MAGE-1, -2, -3, -4, -6, or -12; BAGE-1; GAGE-1 (kindly provided by Dr. P. van der Bruggen, Ludwig Institute for Cancer Research, Brussels, Belgium); TRP-1 (kindly provided by Dr. R. S. Wang, National Cancer Institute, NIH, Bethesda, MD); and TRP-2 (cloned as described in Ref. 5 ).

CTL Stimulation Assay.
Transfectants or melanoma cell lines were tested for their ability to induce the production of TNF- or IFN- by NMP-specific CTLs. Two thousand and 6000 CTLs for TNF- and IFN- release assays, respectively, were added in wells containing target cells in 100 µl of Iscove’s modified Dulbecco’s medium (BioWhittaker) supplemented with 10% HS and 30 units/ml IL-2 (EuroCetus, Amsterdam, the Netherlands). TNF- and IFN- release were measured after 20 h of incubation, using the TNF-sensitive murine fibrosarcoma line WEHI-164 clone, as described previously (20) , and a standard ELISA (BMB, Mannheim, Germany), respectively.

	Results and Discussion

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NMPs were obtained from SK-Mel-24 cells that share the HLA-A1 allele with subject 1, the HLA-A1 and -B14 alleles with subject 2, and the HLA-A1 and -A2 alleles with patient 1 (see Table 1 ). DCs were generated by culture of adherent cells in the presence of granulocyte macrophage colony-stimulating factor and IL-4. Their surface phenotypic analysis showed high levels of CD33, MHC class I and class II, CD80, CD86, and CD40 molecules; variable expression of the CD1a molecule; low-level expression of the CD83 molecule; and no expression of the CD14 molecule (data not shown). CD8⁺ T cells were mixed with NMP-pulsed DCs and cultured for 1 week; activated cells were then expanded in the presence of TCGF for further 5–6 days. CD8⁺ T cells were propagated in culture for several months by weekly restimulations. The percentage of CD8⁺ T cells in the CTL lines, tested after the first stimulation and periodically thereafter during propagation, was always >90%. CD8^+dim NK cells were almost undetectable in CTL lines obtained from the healthy donors and were found in frequencies of 13–15% in CTL lines from the patient (data not shown).

We first verified the induction of CTLs by testing, at day 4 or 5 after the first restimulation, their killing activity against autologous DCs, unpulsed or pulsed with the NMPs (Fig. 1) . The CD8⁺ T cells strongly recognized NMP-pulsed DCs, thus demonstrating that DCs, after incubation with the NMP, express MHC-NMP complexes that are able to activate a specific CTL response.

View larger version (14K): [in this window] [in a new window] [Download PPT slide]   Fig. 1. CD8+ cells from subject 1 stimulated with melanoma peptides extracted from SK-Mel-24 cells are NMP specific. CD8+ T cells were cultured for 7 days with NMP-pulsed DCs, expanded in TCGF, and restimulated as described in "Materials and Methods." The lytic activity was tested at day 4 after the first restimulation in a 51Cr release assay. Target cells were: NMP-pulsed DCs () and unpulsed DCs (). The data are representative of two experiments.

CTLs from all subjects recognized SK-Mel-24 from which the NMPs were extracted. Moreover, CTL-lysed melanoma cells sharing HLA alleles with donors’ lymphocytes (HT144 sharing the HLA-A1 allele with all donors, OI-TC sharing the HLA-A1 allele with subject 1 and the HLA-A1 and -B14 alleles with subject 2; (Fig. 2, A–C) . Importantly, CTLs from the patient recognized the autologous tumor (RF-TC; Fig. 2C ), demonstrating that NMPs contained peptides shared between the allogeneic HLA-matched melanoma and the autologous tumor. This is a relevant finding of this study; in fact, a limitation in the use of NMPs as cancer vaccines is the need for a large number of tumor cells from which to extract the peptides. The possibility to use allogenic NMPs instead or in combination with the autologous NMPs would make the use of natural peptides for vaccination protocols more widely applicable. Indeed, strong evidence is now available on CTL priming across allogeneic barriers by engaging DCs in situ (21 , 22) . Furthermore, the use of allogeneic NMP might favor the activation of precursor CTL specific for epitopes that are dominant in the allogeneic but subdominant or cryptic in the autologous tumor and therefore eventually more relevant in antitumor immunity.

View larger version (24K): [in this window] [in a new window] [Download PPT slide]   Fig. 2. NMP-specific CTLs recognize epitope(s) shared among melanoma cells and the autologous tumor. The data are representative of (n = x) experiments performed with multiple CTL lines. A, subject 1, n = 12; B, subject 2, n = 6; C, patient 1, n = 7. The lytic activity was tested in a 51Cr release assay against the following targets: HLA-A1-matched melanoma cells (SK-Mel-24, HT144, OI-TC, VC-TC, and RF-TC); HLA-A1+ cells of nonmelanoma origin (ME-180, VR-LCL, and RF-LCL); and non-HLA-related melanoma cells (SK-Mel-28, SK-Mel-5, and M14). Target cells symbols, along with their HLA type, are indicated at the bottom. HLA types of donors’ lymphocytes are reported in Table 1 . Right, percentage of inhibition of the lytic activity of CTLs from subjects 1 and 2 against SK-Mel-24 [D (n = 4) and E (n = 2)] and of CTLs from patient 1 against the autologous tumor [F (n = 3)] with W6/32 mAb. Final dilutions, chosen on the basis of surface staining, were 1:100 and 1:1000. The percentages of lysis against SK-Mel-24 (E:T = 10:1), in the absence of mAb, for CTLs of subjects 1 and 2 were 17 and 33%, respectively. The percentage of lysis against RF-TC (E:T = 10:1), in the absence of mAb, for CTLs of patient 1 was 26%.

SK-Mel-24 expresses, albeit mainly at low levels, most of the known tumor- and tissue-specific tumor antigen genes, as detected by RT-PCR analysis (Table 2) . To identify the antigen(s) recognized by NMP-specific CTLs, we cotransfected COS-7 cells with the cDNAs encoding the HLA-A1 (i.e., a restriction allele for all CTLs tested, see Figs. 2 and 3 ) and the different tumor antigen genes expressed in SK-Mel-24 cells. Indeed, COS-7 cells after transfection with melanoma antigens and the HLA-A1 allele are able to synthesize, process, transport, and display melanoma peptides in association with the HLA-A1 allele, as demonstrated by their recognition by peptide-specific CTL clones (19 , 23) . CTLs from polyclonal lines were incubated with the transfected cells and assessed for IFN- release. The results obtained with CTL lines from the patient could not be evaluated because of the high background level of IFN- release, probably due to the presence of NK cells in the bulk populations. Therefore, CTLs from the patient were cloned by limiting dilution and CTL clones were screened for the absence of TNF- release in the presence of K562 cells (i.e, targets for NK cells). CTL clone E7, which recognizes SK-Mel-24 but not K562 cells (Fig. 3C) , was used to test the tumor antigen specificity. Fig. 3 shows the results of CTL stimulation assays with melanoma cell lines and transfectants for CTL polyclonal lines from subjects 1 and 2 (A and D and B and E, respectively) and CTL clone E7 from patient 1 (C and F). All CTLs strongly recognized SK-Mel-24; moreover, CTLs from subject 2 cross-reacted with the HLA-A1-matched SK23-Mel (Fig. 3B) , and CTL clone E7 cross-reacted with the HLA-A1-matched ET1 melanoma cells (Fig. 3C) . None of the TAAs tested were recognized by NMP-specific CTLs from all donors, indicating that they recognize still unidentified shared melanoma-specific antigen(s) (Fig. 3) .

View larger version (21K): [in this window] [in a new window] [Download PPT slide]   Fig. 3. NMP-specific CTLs do not recognize known melanoma antigens. Stimulation of CTL polyclonal lines from subjects 1 (A and D) and 2 (B and E) and of CTL clone E7 from patient 1 (C and F). The production of IFN- or TNF- by CTLs was measured after 20 h of coculture with stimulator cells (i.e., melanoma cell lines or COS-7 cells cotransfected with the cDNA of HLA-A1 and the indicated melanoma antigens), as described in "Materials and Methods." As controls, CTLs alone, CTLs in the presence of K562, COS-7 cells alone, and COS-7 cells transfected with the HLA-A1 cDNA were used. The data are representative of three experiments.

Recently, Reynolds et al. (24) reported, in melanoma patients vaccinated with a polyvalent vaccine containing multiple antigens, a HLA-independent heterogeneity of CD8⁺ T-cell responses. All responding patients reacted to different combination of peptide/antigens although they were all A*0201⁺ and the peptides were all A*0201⁺ restricted, suggesting "the need to construct vaccines of multiple peptides or antigens to maximize the proportion of responding patients." Toward this aim, the use of NMPs as cancer vaccines exactly fills this need. Indeed, the presence in the NMP mixture of a large number of peptides/antigens of different origins and binding affinities should increase the possibility of a broader recruitment of tumor-specific CTLs. Moreover, the use of multiple peptides of different tumor antigens origin might avoid the need to demonstrate the antigen immunodominance in the antitumor immune response.

Finally, in clinical trials using NMP-pulsed DCs, the risk of induction of immune response against normal tissues should be carefully monitored, although no evidence of autoimmunity has been reported in animals immunized with natural tumor peptides (9) and antinuclear and antithyroid antibodies were detected in melanoma patients after vaccination with synthetic peptide but not tumor lysate-pulsed DCs (10) .

	FOOTNOTES

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This work was supported by the Fondazione Centro San Raffaele del Monte Tabor, the Associazione Italiana Ricerca Cancro, the Comitato Nazionale Ricerche, and the Ministero dell’Università e della Ricerca Scientifica e Tecnologica. S. M. is a recipient of a fellowship from the Fondazione Italiana Ricerca Cancro.

² To whom requests for reprints should be addressed, at Laboratory of Tumor Immunology, Scientific Institute H. San Raffaele, Via Olgettina 60, 20132 Milan, Italy.

³ The abbreviations used are: TAA, tumor-associated antigen; NMP, natural melanoma peptide; DC, dendritic cell; PBMC, peripheral blood mononuclear cell; LCL, lymphoblastoid cell line; mAb, monoclonal antibody; HS, human serum; IL, interleukin; TCGF, T-cell growth factor; RT-PCR, reverse transcriptase-PCR; TNF, tumor necrosis factor; NK, natural killer.

Received 1/ 5/99. Accepted 3/31/99.

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