A long peptide from MELOE-1 contains multiple HLA class II T cell epitopes in addition to the HLA-A*0201 epitope: an attractive candidate for melanoma vaccination

International audienceCD4(+) T cells contribute importantly to the antitumor T cell response, and thus, long peptides comprising CD4 and CD8 epitopes may be efficient cancer vaccines. We have previously identified an overexpressed antigen in melanoma, MELOE-1, presenting a CD8(+) T cell epitope, MELOE-1(36-44), in the HLA-A*0201 context. A T cell repertoire against this epitope is present in HLA-A*0201+ healthy subjects and melanoma patients and the adjuvant injection of TIL containing MELOE-1 specific CD8(+) T cells to melanoma patients was shown to be beneficial. In this study, we looked for CD4(+) T cell epitopes in the vicinity of the HLA-A*0201 epitope. Stimulation of PBMC from healthy subjects with MELOE-1(26-46) revealed CD4 responses in multiple HLA contexts and by cloning responsive CD4(+) T cells, we identified one HLA-DRβ1*1101-restricted and one HLA-DQβ1*0603-restricted epitope. We showed that the two epitopes could be efficiently presented to CD4(+) T cells by MELOE-1-loaded dendritic cells but not by MELOE-1+ melanoma cell-lines. Finally, we showed that the long peptide MELOE-1(22-46), containing the two optimal class II epitopes and the HLA-A*0201 epitope, was efficiently processed by DC to stimulate CD4(+) and CD8(+) T cell responses in vitro, making it a potential candidate for melanoma vaccination

Overexpression of meloe gene in melanomas is controlled both by specific transcription factors and hypomethylation.

The melanoma antigens MELOE-1 and MELOE-2 are encoded by a messenger, called meloe, overexpressed in melanomas compared with other tumour cell types and healthy tissues. They are both able to elicit melanoma-specific T cell responses in melanoma patients, and MELOE-1-specific CD8 T cells have been involved in melanoma immunosurveillance. With the aim to develop immunotherapies targeting this antigen, we investigated the transcriptional mechanisms leading to the preferential expression of meloe messenger in the melanocytic lineage. We defined the minimal promoter region of meloe gene and identified binding motifs for a set of transcription factors. Using mutagenesis, co-transfection experiments and chromatin immunoprecipitation, we showed that transcription factors involved in meloe promoter activity in melanomas were the melanocytic specific SOX9 and SOX10 proteins together with the activated P-CREB protein. Furthermore, we showed that meloe promoter was hypomethylated in melanomas and melanocytes, and hypermethylated in colon cancer cell lines and mesotheliomas, thus explaining the absence of P-CREB binding in these cell lines. This was a second key to explain the overerexpression of meloe messenger in the melanocytic lineage. To our knowledge, such a dual transcriptional control conferring tissue-specificity has never been described for the expression of tumour antigens

Overexpression of meloe cDNA in the melanocytic lineage.

<p>Eight melanocyte samples, twelve melanoma, three colon cancer (CC), four mesothelioma, two lung cancer (LC), one renal carcinoma (RC), one breast cancer (BC) and one neuroblastoma (NB) cell lines were tested by qPCR for the expression of <i>meloe</i>. RPLPO and cyclophilin-A gene expression were used as internal controls. The relative expression of <i>meloe</i> was calculated after normalization on the efficiency of PCR reaction and the mean expression of these two housekeeping genes, reported to its normalized expression in a mixture of eight distinct melanocyte samples. Results are from three independent experiments.</p

Validation of SOX9, SOX10 and CREB binding in vivo by ChIP.

<p>(A) <i>meloe</i> promoter region amplified on immunoprecipitated DNA. Forward and reverse primers used for PCR amplification are indicated in italics and underlined, and CREB and SOX binding sites are framed. (B) Chromatin Immunoprecipitation assay was performed on three cell lines: M113, Meso163 and SW480. Immunoprecipitations were performed with anti-P-CREB, anti-SOX9 and anti-SOX10 antibodies or with anti-GFP antibody as negative control. PCR amplification was performed on the immunoprecipitated DNA or on the input sample (positive control of non precipitated DNA) using primers spanning from −137 to +89.</p

Minimal promoter definition and TF binding sites.

<p>(A) Sequence of the 5′ flanking region of <i>meloe</i>. Consensus sequences for TF are framed and flags illustrate positions of truncated promoters. The transcriptional start site and the putative TATAbox are underlined. (B) 5′ truncated or (C) mutated promoters were transfected into M113 melanoma cell line together with the <i>Renilla</i> luciferase pRL-CMV. The pGL4.10 empty vector was used as negative control and a Melan-A promoter as a positive one (black bar). Results, presented as <i>Firefly/Renilla</i> luciferase ratios (FF/R), are mean with SD from three independent experiments, performed in triplicate. Statistical analysis was performed using one-way ANOVA followed by a Dunnett's multiple comparison test, with P-1565 as reference for truncated promoters and P-644 for mutated ones (** p<0.01, ***p<0.001).</p

Methylation status of meloe region [−270–+544] in melanocytes, melanoma, mesothelioma and colon carcinoma cell lines.

<p>Each of the 26 CpG motifs included in the explored region was illustrated by empty circles (upper panel). The CpG island included in the CREB binding site is indicated by an arrow. DNA treated by bi-sulfite conversion was amplified, cloned and sequenced. Methylation status of this region in each cell line was represented by a grid where each line corresponds to one allele and each column to one CpG motif, methylated (black) or unmethylated (white).</p

Class II epitopes are naturally processed from MELOE-1 whole antigen.

<p>Autologous DC, were loaded (before or after fixation) with MELOE-1_2–46 (1 µM) or, as a negative control, with Melan-A_16–40L peptide (1 µM), and matured. T cell clones were then stimulated with DC at a ratio 1/1, during 5 h in presence of BFA. Reactivity was assessed by double staining TNF-α/CD3, and analyzed by flow cytometry. Histograms illustrated the percentage of TNF-α producing cells among CD3 positive T cells.</p

MELOE-1 specific responses in healthy donors.

<p>(A) Dot plot illustrating peptide-specific TNF responses, in MELOE-1 stimulated microcultures from HD9 and HD28. Fourteen days after PBMC stimulation with MELOE-1 whole polypeptide (2–46), microcultures were re-stimulated or not with each indicated peptide during 5 h. Reactivity was then assessed by double staining TNFα/CD4. Percentages indicate the fraction of TNF producing T cells, among CD4 T cells. (B) Frequency of microcultures containing CD4 T cells specific for MELOE-1 peptides. A microculture was considered as positive when the fraction of TNF producing T cells was 2-fold higher upon peptide stimulation than in unstimulated cultures. (C) Percentages of TNF-α producing CD4 T cells upon peptide-stimulation, in positive microcultures from all donors.</p

Assessment of MELOE-1 CD4 T cell responses in PBMC from healthy donors.

<p>PBMC from healthy donors were stimulated with 10 µM of MELOE-1. After 14 days, the presence of CD4 T cells specific for the different regions of MELOE-1 was assessed by re-stimulating cells with MELOE-1_2–21, MELOE-1_11–30, MELOE-1_18–37 and MELOE-1_26–46 peptides, followed by CD4/TNF-α double staining and flow cytometry analysis. Between brackets is indicated the mean % of TNF-α producing CD4 T cells, in positive microcultures. NA: not available.</p

Assessment of MELOE-1 CD4 T cell responses in PBMC from melanoma patients.

<p>PBMC from melanoma patients were stimulated with 10 µM of MELOE-1. After 14 days, the presence of CD4 T cells specific for the different regions of MELOE-1 was assessed by re-stimulating cells with MELOE-1_2–21, MELOE-1_11–30 and MELOE-1_22–46 peptides, followed by CD4/IFN-γ double staining for the detection of Th1 responses, and by CD4/IL4 double staining for Th2 responses. Between brackets is indicated the mean % of cytokine producing CD4 T cells, in positive microcultures.</p