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. 2005 Sep 27;55(2):178–184. doi: 10.1007/s00262-005-0063-0

Analysis of a rare melanoma patient with a spontaneous CTL response to a MAGE-A3 peptide presented by HLA-A1

Takeshi Hanagiri 1,2, Nicolas van Baren 1,3, Bart Neyns 4, Thierry Boon 1,3, Pierre G Coulie 1,✉
PMCID: PMC11030742  PMID: 16187089

Abstract

We describe an HLA-A1 melanoma patient who has mounted a spontaneous cytolytic T cell (CTL) response against an antigenic peptide encoded by gene MAGE-A3 and presented by HLA-A1. The frequency of anti-MAGE-3.A1 CTLp was 5×10−7 of the blood CD8 cells, with a dominant clonotype which was present in six out of seven independent anti-MAGE-3.A1 CTL clones. After vaccination with a recombinant poxvirus coding for the MAGE-3.A1 antigen, the blood frequency of anti-MAGE-3.A1 CTLp increased tenfold. Twenty-two independent CTL clones were derived. Surprisingly, only one of them corresponded to the dominant clonotype present before vaccination. Two new clonotypes were repeated 12 and 7 times, respectively. Our interpretation of these results is that the spontaneous anti-MAGE-3.A1 CTL response pre-existing to vaccination was polyclonal, and that the vaccine restimulated only some of these clones. To estimate the incidence of spontaneous anti-MAGE-3.A1 CTL responses in melanoma patients with a tumor expressing gene MAGE-A3, we measured the blood frequency of anti-MAGE-3.A1 T cells in 45 patients, and found only two clear responses.

Keywords: Melanoma, Vaccination, MAGE, Cytolytic T lymphocyte

Introduction

Among the tumor antigens that are presented to human cytolytic T lymphocytes (CTL) by HLA class I molecules, several constitute safe targets for immunotherapy because they are absent from normal tissues. An important category of such tumor-specific antigens are those encoded by the ‘cancer germline’ genes, such as the MAGE, BAGE, GAGE and LAGE/ESO-1 gene families [1–3, 5, 8]. These genes are expressed in several types of tumors but not in adult tissues with the exception of spermatogonia, which do not carry HLA molecules and therefore cannot present antigens to T cells.

Many antigenic peptides encoded by cancer-germline genes have been identified [15]. One of them is peptide MAGE-3168–176, which is encoded by gene MAGE-A3 and presented by HLA-A1 and HLA-B35 molecules [6, 12]. The MAGE-3.A1 peptide has been used for small-scale therapeutic vaccination trials of melanoma patients with detectable disease [10, 13, 14]. In one of these trials, 37 melanoma patients with advanced cancer were vaccinated with ALVAC mini-MAGE-1/3, a recombinant canarypox virus of the ALVAC type containing a minigene coding for peptides MAGE-3.A1 and MAGE-A1161–169, which is also presented by HLA-A1 [11, 14]. Vaccination included four injections of ALVAC mini-MAGE-1/3 followed by three booster injections of the two corresponding peptides. Among the 30 melanoma patients evaluable for tumor response, six showed some evidence of tumor regression [14].

To establish whether there was a correlation between these regressions and the anti-vaccine T cell responses, we set up a system to detect anti-MAGE-3.A1 CTL. Our approach is based on an in vitro stimulation of PBMC with the antigenic peptide over 2 weeks, followed by labeling with A1/MAGE-3 tetramers. To evaluate precursor frequencies, these mixed lymphocyte-peptide cultures (MLPC) are conducted under limiting dilution condition. Cells that are labeled with the tetramer are cloned and their diversity is analyzed by TCR sequencing [4]. Among four melanoma patients who showed tumor regression after ALVAC mini-MAGE-1/3 vaccination, three monoclonal anti-MAGE-3.A1 CTL responses were found, with post-vaccination CTLp frequencies of 3×10−7, 3×10−6, and 3×10−3 of the blood CD8 T cells, respectively [7]. No anti-MAGE-1.A1 CTL response was observed. Among 11 vaccinated patients who did not display tumor regression, only one mounted an anti-vaccine response [9], whereas two others had an anti-MAGE-3.A1 CTL response pre-existing to vaccination. We describe here the frequency and diversity of anti-MAGE-3.A1 CTL in one of these two patients, and provide an estimation of the incidence of such spontaneous responses in melanoma patients.

Results

Detection of an anti-MAGE-3.A1 CTL response pre-existent to vaccination

Melanoma patient VUB39 received four injections of the ALVAC mini-MAGE-1/3 vaccine. To estimate the frequencies of anti-vaccine CTLp, i.e. CTLp recognizing either the MAGE-1.A1 or the MAGE-3.A1 peptide, PBMC collected before and after vaccination were restimulated in vitro with the two antigenic peptides, in a medium containing IL-2, IL-4 and IL-7. These MLPC were conducted in limiting dilution condition, with 2×105 or 2.5×105 PBMC/microwell. For the experiments shown in Fig. 1, 132 and 178 microcultures were set up with pre- and post-vaccination PBMC, respectively. On day 7 the cells were restimulated by the addition of peptide MAGE-3.A1. One day later all cultures were split and peptide MAGE-1.A1 was added. All microcultures were split again on day 12. On day 14 the cells of one set were labeled with an anti-CD8 antibody coupled to FITC, an A1/MAGE-1 tetramer coupled to allophycocyanin, and an A1/MAGE-3 tetramer coupled to phycoerythrin. The cells were analyzed for the presence of anti-MAGE-3.A1 CD8 T cells using flow cytometry. To increase sensitivity, analysis of the data included a procedure to gate out autofluorescent cells, as detailed previously [7].

Fig. 1.

Fig. 1

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Procedure to estimate the frequency of anti-MAGE-3.A1 CTLp frequencies in MLPC set up with PBMC collected from patient VUB39 before and after vaccination. Lymphocytes were restimulated on days 0 and 7–8 with peptides and cytokines, and labeled on day 14 with anti-CD8 antibodies and HLA-A1 tetramers containing either the MAGE-A1161–169 or the MAGE-A3168–176 peptide. None of the microcultures contained cells that were specifically stained by the A1/MAGE-1 tetramer. Those that contained CD8+ cells stained by the A1/MAGE-3 tetramer are indicated by grey disks. Representative plots show only CD8+ lymphocytes. The proportions of CD8+ lymphocytes that are stained by the A1/MAGE-3 tetramer but not the A1/MAGE-1 tetramer are indicated

Typical results are shown in Fig. 1. Five out of the 132 microcultures set up with pre-vaccination PBMC contained CD8 T cells labeled with the A1/MAGE-3 tetramer. In a second experiment, 3/92 microcultures were positive (data not shown). Considering that 32% of the PBMC were CD8 T cells, 8/224 positive microcultures leads to a frequency estimate for precursors of anti-MAGE-3.A1 T cells of 5.6×10−7 of the CD8 cells. This frequency is similar to that found in individuals without cancer, i.e. about 4×10−7 of the CD8 cells [9], suggesting that patient VUB39 had not responded to the MAGE-3.A1 antigen before vaccination.

We then examined the TCR diversity of these anti-MAGE-3.A1 T lymphocytes by producing T cell clones and obtaining their TCR sequences. Tetramer-positive cells from seven microcultures were sorted at one cell/well and restimulated weekly with HLA-A1+ EBV-transformed B cells that were incubated with peptide MAGE-3.A1. Stable anti-MAGE-3.A1 CTL clones were derived, that lysed the allogenic HLA-A1+ MAGE-A3+ melanoma cells EB81-MEL but did not lyse HLA-A1+ EBV-transformed B cells, unless these targets were pulsed with the antigenic peptide (Fig. 2). These results indicated that the tetramer-positive cells detected in the MLPC were CTL capable of recognizing tumor cells that naturally expressed the MAGE-3.A1 antigen. The TCRα and β sequences of the seven CTL clones were obtained. Six clones expressed the same receptor (Fig. 3 and Table 1), and the frequency of this clonotype can therefore be estimated to be about 5×10−7 of the blood CD8 cells.

Fig. 2.

Fig. 2

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Lytic activity of anti-MAGE-3.A1 CTL clones from patient VUB39. 51Cr-labeled targets included the allogenic HLA-A1+ melanoma cells EB81-MEL, which express gene MAGE-A3, and the allogenic HLA-A1+ EBV-transformed B cells CP64-EBV-B, incubated or not for 30 min with peptide MAGE-3.A1 at 10 μM before addition of the CTL at the indicated E/T ratios. Chromium release was measured after 4 h

Fig. 3.

Fig. 3

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Frequencies and repertoire of anti-MAGE-3.A1 CTLp in patient VUB39. Frequencies measured by MLPC-tetramer in blood collected before and after the 4 vaccinations with ALVAC mini-MAGE-1/3 are indicated in the top panel. The bottom panel represents anti-MAGE-3.A1 CTL clones, with different numbers for each TCR sequence

Table 1.

Repertoire of anti-MAGE-3.A1 CTLp in patient VUB39

Clones Vαa CDR3 Jα Vβ CDR3 Jβ
1 5 CAE SPRAGTASKL TFG 44 20-1 CSA ASGPYNEQ FFG 2-1
2 12-1 CVV SSNTGKL IFG 37 10-3 CAI SEPGQGQPQ HFG 1-5
3 12-1 CVV NKRNDYKL SFG 20 29 CSV NNNEQ FFG 2-1
4 17 CAT DDPL VFG 57 15 CAT RWDRGYEQ YFG 2-7
5 8-6 CAV SDSSYKL IFG 12 12-3 CAS STRGRSNEKL FFG 1-4
6 14 CAM REVLYNFNK FYG 21 3 CAS SPGAGVATDTQ YFG 2-3
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a V and J designation according to the nomenclature available at http://imgt.cines.fr

The presence of this repeated clonotype contrasts with what is found in the anti-MAGE-3.A1 naïve TCR repertoire [9]. In a non-cancerous individual with a frequency of anti-MAGE-3.A1 CTLp of 6×10−7 of the blood CD8 T cells, the diversity of the anti-MAGE-3.A1 TCR repertoire was analyzed by examining the TCR sequences of 23 independent anti-MAGE-3.A1 CTL clones. All but two clones had different TCR sequences, and a similar diversity was observed in melanoma patients also. The presence of a single repeat in a series of 23 clones makes it possible to estimate that the diversity of the naïve anti-MAGE-3.A1 TCR repertoire is most likely to be around 250. Assuming a diversity of only 50, there is a <5% probability of finding the same clonotype twice in a series of not >8 clones, and the probability of finding the same clone six times among seven clones is 3×10−9. We conclude that the repeated clonotype results from an in vivo amplification. Therefore, even though the pre-vaccination frequency of anti-MAGE-3.A1 CTL in the blood of patient VUB39 is not higher than that found in non-cancerous individuals, the analysis of the diversity of these CTL indicates that the patient produced a spontaneous anti-MAGE-3.A1 T cell response prior to vaccination.

Anti-MAGE-3.A1 CTL in patient VUB39 after vaccination with ALVAC mini-MAGE-1/3

In MLPC set up with post-vaccination lymphocytes, 62/178 microcultures contained cells labeled with the A1/MAGE-3 tetramer (Fig. 1), leading to an anti-MAGE-3.A1 CTLp frequency of 6.1×10−6 of the blood CD8 cells. This represents a tenfold increase compared to the pre-vaccination level, suggesting that the patient may have responded to the vaccine.

The CTL clones were derived from 22 positive microcultures. They showed specificity for the MAGE-3.A1 antigen (Fig. 2). The TCR analysis indicated that only one of these CTL clones expressed TCR 1, the clonotype that was repeated among the pre-vaccination clones (Fig. 3). Instead, two new clonotypes were repeated among the post-vaccination CTL clones.

Frequency of melanoma patients with a spontaneous anti-MAGE-3.A1 CTL response

Frequencies of anti-MAGE-3.A1 T cells were estimated by the MLPC/tetramer approach in blood collected before vaccination from 45 HLA-A1+ patients (Fig. 4). Six patients had no sign of tumor after resection of a primary melanoma, while the others had metastatic disease. Two patients were found to have anti-MAGE-3.A1 T cells at frequencies of 3 and 7×10−5 of the CD8 cells, i.e. 75 and 175 times above the naïve level [9], indicating that they had mounted a spontaneous response. Another patient had a frequency of 2.6×10−6, i.e. six times above the naïve level, suggesting but not proving that he had a response. All the other patients had frequencies similar to those found in individuals without cancer (Fig. 4). These results indicate that a spontaneous and detectable anti-MAGE-3.A1 CTL response is a rare event in melanoma patients, occurring in about 5% of cases. However, some of the patients may have mounted a response without a detectable increase of frequency, as did patient VUB39.

Fig. 4.

Fig. 4

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Frequencies of anti-MAGE-3.A1 CTLp. Frequencies of precursors of anti-MAGE-3.A1 CD8 T cells were measured by the MLPC-tetramer method in blood collected from 45 melanoma patients and 5 individuals without cancer. Closed circles indicate frequencies established on the basis of the presence of at least one positive microculture in the experiment, whereas triangles indicate that no positive microculture was found, with a resulting frequency that is lower than the indicated value. The open circle represents the frequency observed in patient VUB39

Discussion

Our results indicate that melanoma patient VUB39 produced a spontaneous anti-MAGE-3.A1 T cell response, with a low frequency of 6×10−7 of the blood CD8 cells, and one dominant clonotype. Following vaccination with ALVAC mini-MAGE-1/3, the blood frequency of anti-MAGE-3.A1 T cells increased about tenfold, with two new dominant clonotypes. These results were surprising in several respects.

Patient VUB39 was the first in whom we found a spontaneous anti-MAGE-3.A1 CTL response, pre-existing to vaccination. The frequency of the main clonotype of this response was about 5×10−7 of the blood CD8 cells. This is clearly higher than the frequency of cells of a naïve anti-MAGE-3.A1 clonotype, which we estimated to be lower than 4×10−9 of the blood CD8 cells [9]. A frequency of 5×10−7 of the CD8 cells is clearly low, but with an estimated total number of 4×1010 CD8 lymphocytes in the human body, it corresponds to an appreciable amount of 20,000 cells, or 14 rounds of division following the activation of a single CTL precursor.

A spontaneous anti-MAGE-3.A1 CTL response is not a frequent event, being found in less than 5% of HLA-A1 patients with a melanoma expressing gene MAGE-A3. It is noteworthy that when patient VUB39 was diagnosed with melanoma she presented with a lymph node metastasis and no concomitant or previous detection of a primary tumor. The patient reported the spontaneous regression of a pigmented lesion in the region drained by the metastatic node several years before diagnosis. It is therefore possible that a spontaneous tumor-specific CTL response including anti-MAGE-3.A1 effectors occurred during progression of the primary tumor and participated in its rejection. We made the similar observation of an anti-MAGE-3.A1 CTL response associated with a spontaneous tumor regression in another melanoma patient, who was subsequently vaccinated with dendritic cells pulsed with the MAGE-3.A1 peptide (unpublished observation).

Another unexpected observation in the monitoring of patient VUB39 is that the anti-MAGE-3.A1 CTL clonotype 1, which was amplified prior to vaccination, did not appear to be restimulated by the ALVAC mini-MAGE-1/3 vaccines. These CTL may have become quiescent in vivo, as a result of their staying within tumors. It is noteworthy that in the other patient of this ALVAC mini-MAGE-1/3 vaccination trial who had a spontaneous anti-MAGE-3.A1 CTL response prior to vaccination, patient NAP37, the anti-MAGE-3A.1 CTLp frequency was 3×10−5 of the blood CD8 T cells before and 2×10−5 after vaccination [9]. In this patient also, the pre-vaccination CTL appeared resistant to restimulation by the vaccine.

The last unexpected result was the detection of two amplifed CTL clones after vaccination, which is at odds with what we observed in the three patients who produced an anti-MAGE-3.A1 CTL response after ALVAC mini-MAGE-1/3 vaccination [7]. In each of these cases the CTL response was monoclonal. A monoclonal response was also found in a few patients vaccinated with peptide MAGE-3.A1 administered without adjuvant [4, 9]. Our interpretation of these monoclonal responses is that the poor immunogenicity of the vaccines led at best to a single hit. It is possible that the anti-vaccine response of patient VUB39 was a small variation of this phenomenon, with two instead of one CTL precursor activated by the vaccine. However, it is tempting to link the polyclonality of the anti-vaccine response with the observation of a spontaneous CTL response pre-existing to vaccination. This spontaneous response was probably polyclonal, including clonotypes 1–4. Clone 1, but not the other clones, was amplified sufficiently to be detected among the 14×106 pre-vaccination CD8 cells that were used for the MLPC. The anti-vaccine response would then correspond to a secondary amplification of previously primed CTL clones 3 and 4, and not a primary stimulation of naïve precursors.

Patient VUB39 had no detectable tumor regression following vaccination, even though she was primed to the MAGE-3.A1 antigen already before the vaccinations, and very probably had vaccine-induced stimulation of anti-MAGE-3.A1 CTL. It is possible that the spontaneous anti-MAGE-3.A1 response contributed to select tumor cells that did not express the antigen. A tumor sample resected 3 weeks after the first vaccination was found to express gene MAGE-A3, but no analysis of the expression of the HLA-A1 gene by the tumor cells could be performed on this material.

Materials and methods

Patient

Melanoma patient VUB39, a 73-year-old female, had axillary lymph node metastases, as well as mediastinal and lung metastases, when she received the ALVAC/MAGE vaccines. The primary tumor had not been detected. During vaccination, the size of all metastases increased slowly and the patient died from tumor progression 11 months after the onset of vaccination.

MLPC-tetramer analysis

Mixed lymphocyte-peptide cultures (MLPC) were performed as described previously [7]. Briefly, PBMC were thawed and resuspended at 107 cells/ml in Iscove’s medium supplemented with 1% human serum (HS) and divided into two fractions. One was incubated for 60 min at room temperature with peptide MAGE-1.A1 (20 μM), and the other with peptide MAGE-3.A1. These peptide-pulsed PBMC were washed, pooled, and distributed at 2.5×105 cells/0.2 ml in round-bottom microwells, in Iscove’s medium with 10% HS, IL-2 (20 U/ml), IL-4 (10 ng/ml), and IL-7 (10 ng/ml). On day 7, half of the medium was replaced by fresh medium containing IL-2, IL-4, IL-7, and peptide MAGE-3.A1 (20 μM). Peptide MAGE-1.A1 was added to all cultures one day later. During the second week of stimulation, the cultures were split according to proliferation, in medium containing IL-2 alone. Tetramer labeling was performed on day 14 on aliquots of the microcultures. Cells were washed, resuspended in PBS with 1% HS, and incubated for 30 min at 37°C with A1/MAGE-1 and A1/MAGE-3 tetramers (20 nM each), coupled to phycoerythrin and allophycocyanin, respectively. Anti-CD8 antibodies coupled to FITC (SK1; BD Biosciences, Mountain View, CA, USA) were then added. After a further incubation for 30 min at 37°C, the cells were washed, fixed with 0.5% formaldehyde, and analyzed on a FACS Calibur flow cytometer (BD Biosciences) using the CellQuest software (BD Biosciences).

T cell clones

To derive stable CTL clones from the populations of tetramer-positive cells, cells stained by tetramer were seeded at one cell per well in round-bottom microplates using a FACS Vantage cell sorter (BD Biosciences). Sorted cells were stimulated by the addition of irradiated (100 Gy) allogenic PBMC (8×104/well) as feeder cells and irradiated allogenic HLA-A1 EBV-B cells (2×104/well) incubated with the MAGE-3.A1 peptide (20 μM), and washed in culture medium with IL-2 (100 U/ml), IL-4 (10 ng/ml), and IL-7 (10 ng/ml). The CTL clones were restimulated weekly by the addition of feeder cells and peptide-pulsed EBV-B cells in medium with growth factors. After about 3 weeks, they were transferred into 2-ml wells and maintained by weekly restimulations with allogenic LG2-EBV-B cells and peptide-pulsed HLA-A1 tumor cells. For TCR analysis, total RNA from CTL clones was extracted with the RNeasy Mini Kit (Qiagen, Crawley, UK) and converted to cDNA as described [7]. This cDNA served as a template for a PCR amplification using panels of Vα- or Vβ-specific forward primers and a reverse Cα or Cβ primer. The PCR products were purified and sequenced to obtain a complete identification of the CDR3 region.

Acknowledgements

This work was supported by the Belgian Programme on Interuniversity Poles of Attraction initiated by the Belgian State, Prime Minister’s Office, Science Policy Programming, and by grants from the Fonds J. Maisin (Belgium), the Fédération Belge contre le Cancer (Belgium), the Fondation Salus Sanguinis (Belgium), and the Fonds National de la Recherche Scientifique (Belgium).

Abbreviations

CTL

Cytolytic T lymphocytes

MLPC

Mixed lymphocyte peptide culture

TCR

T cell receptor

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