A lineage-specific methylation pattern controls the transcription of the polycistronic mRNA coding MELOE melanoma antigens

International audienceWe recently characterized two melanoma antigens MELOE-1 and MELOE-2 derived from a polycistronic RNA overexpressed in the melanocytic lineage. This transcription profile was because of hypomethylation of the meloe proximal promoter in melanomas and melanocytes. Here, we investigated whether this demethylation was restricted to the meloe promoter or was linked to a general lack of methylation at the meloe locus in the melanocytic lineage. We established the methylation pattern of the locus spanning more than 40 kbp, focusing on CpG islands, using DNA bisulfite conversion and pyrosequencing. The study was carried out on cultured cell lines (melanoma, melanocyte, colon cancer, and mesothelioma cell lines), healthy tissues (skin and colon), and melanoma tumors. Demethylation, specifically observed in the melanocytic lineage, involves a large promoter area and not the entire meloe locus. This enables updating a tight regulation of meloe transcription in this lineage, suggesting tissue-specific epigenetic mechanisms. Associated with the previously described translational mechanisms, leading to the specific expression of MELOE-1 and MELOE-2 in melanomas, this makes MELOE-derived antigens a relevant candidate for immunotherapy of melanoma. Melanoma Res 00:000–00

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

PD-1 expression conditions T cell avidity within an antigen-specific repertoire

International audienceDespite its negative regulatory role on tumor specific T cells, PD-1 is also a marker ofactivated tumor-infiltrating T cells. In cancer, PD-1 blockade partially reverses T celldysfunction allowing the amplification of tumor reactive T cells. Here we investigated therole of PD-1 signaling on effector/memory human T cells specific for shared melanomaantigens, derived from blood. We documented for the first time the existence of melanomaspecific T cell clones unable to express PD-1. This stable feature was due to the persistentmethylation of the PDCD1 promoter. PD-1pos counterparts, suggesting that high affinity specific T cell clones unable to express PD-1 are not or rarely present in peripheral blood, as they are probably eliminated by negativeselection, due to their high reactivity. We also documented the existence of such PD-1neg Tcell clones in melanoma TIL, which also exhibited a lower functional avidity than PD-1pos TILclones. This clearly shows that PD-1 expression identifies antigen-specific T cell clonotypes ofhigh functional avidity. Finally, we demonstrated that PD-1 blockade during the in vitroselection process of Melan-A specific T cells favored the amplification of higher avidity T cellclonotypes. This preferential amplification of high avidity memory T cells upon PD-1blockade resonates with the expansion of reactive T cells, including neo-antigen-specific Tcells observed in anti-PD-1 treated patients. This feature should also be a useful biomarkerof clinical efficiency, while providing new insights for adoptive transfer treatments

Soluble HLA-I/Peptide Monomers Mediate Antigen-Specific CD8 T Cell Activation through Passive Peptide Exchange with Cell-Bound HLA-I Molecules

International audienceAccumulating evidence that serum levels of soluble class I HLA molecules (sHLA-I) can, under various pathological conditions, correlate with disease stage and/or patient survival, has stimulated interest in defining whether sHLA-I can exert immunological functions. However, despite a mounting number of publications suggesting the ability of sHLA-I to affect immune effectors in vitro, the precise underlying mechanism still remains controversial. In this article, we address potential functions of both classical and nonclassical sHLA-I, using soluble recombinant HLA-I/peptide monomers, and clearly demonstrate their ability to trigger Ag-specific activation of CD8 T cells in vitro. Furthermore, we provide strong evidence that this behavior results from the passive transfer of peptides from monomers to T cell-bound HLA-I molecules, allowing for fratricide representation and activation. Hence, we proposed a unifying model of T cell activation by HLA-I/peptide monomers, reappraising the potential involvement of sHLA-I molecules in the immune response

IRES-dependent translation of the long non coding RNA meloe in melanoma cells produces the most immunogenic MELOE antigens

International audienceMELOE-1 and MELOE-2, two highly specific melanoma antigens involved in T cell immunosurveillance are produced by IRES-dependent translation of the long « non coding » and polycistronic RNA, meloe. In the present study, we document the expression of an additional ORF, MELOE-3, located in the 5′ region of meloe. Data from in vitro translation experiments and transfection of melanoma cells with bicistronic vectors documented that MELOE-3 is exclusively translated by the classical cap-dependent pathway. Using a sensitive tandem mass spectrometry technique, we detected the presence of MELOE-3 in total lysates of both melanoma cells and normal melanocytes. This contrasts with our previous observation of the melanoma-restricted expression of MELOE-1 and MELOE-2. Furthermore, in vitro stimulation of PBMC from 6 healthy donors with overlapping peptides from MELOE-1 or MELOE-3 revealed a very scarce MELOE-3 specific T cell repertoire as compared to the abundant repertoire observed against MELOE-1. The poor immunogenicity of MELOE-3 and its expression in melanocytes is consistent with an immune tolerance towards a physiologically expressed protein. In contrast, melanoma-restricted expression of IRES-dependent MELOE-1 may explain its high immunogenicity. In conclusion, within the MELOE family, IRES-dependent antigens represent the best T cell targets for immunotherapy of melanoma

The melanoma antigens MELOE-1 and MELOE-2 are translated from a bona fide polycistronic mRNA containing functional IRES sequences.

Our previous studies on melanoma antigens identified two new polypeptides, named MELOE-1 and MELOE-2, that are involved in immunosurveillance. Intriguingly, these antigens are coded by distinct open reading frames (ORF) of the meloe mRNA which is significantly expressed only in the melanocytic lineage. In addition, MELOE-1 and -2 specific T cell clones recognized melanoma cells but very poorly normal melanocytes suggesting differential translation of meloe in normal vs tumor cells. This prompted us to elucidate the mechanisms of translation of these antigens in melanoma cells. We first demonstrated that no splicing event or cryptic promoter could generate shorter meloe transcripts containing only one of the two ORFs. Triggering meloe RNA degradation with a siRNA close to the ORF coding for MELOE-2 abrogated expression of both MELOE-1 and MELOE-2, thus confirming that the two ORFs are always associated. Next we showed, in a bicistronic reporter system, that IRES activities could be detected upstream of MELOE-1 and MELOE-2 and finally confirmed their translation from full length meloe cDNA in melanoma cells with eGFP constructs. In conclusion, meloe is a polycistronic mRNA that generates both MELOE-1 and MELOE-2 antigens through IRES-dependent translation in melanoma cells and that may explain their tumor specificity

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

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