αvβ3-dependent cross-presentation of matrix metalloproteinase–2 by melanoma cells gives rise to a new tumor antigen

A large array of antigens that are recognized by tumor-specific T cells has been identified and shown to be generated through various processes. We describe a new mechanism underlying T cell recognition of melanoma cells, which involves the generation of a major histocompatibility complex class I–restricted epitope after tumor-mediated uptake and processing of an extracellular protein—a process referred to as cross-presentation—which is believed to be restricted to immune cells. We show that melanoma cells cross-present, in an αvβ3-dependent manner, an antigen derived from secreted matrix metalloproteinase–2 (MMP-2) to human leukocyte antigen A*0201-restricted T cells. Because MMP-2 activity is critical for melanoma progression, the MMP-2 peptide should be cross-presented by most progressing melanomas and represents a unique antigen for vaccine therapy of these tumors

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

Detection of putative cryptic promoter activity in intercistronic region (IR) and upstream region (UR) of <i>meloe</i>.

<p>The SV40 promoter region of PGL4-13 vector was replaced by fragments of interest, i.e intercistronic region (IR_666–1490) between MELOE-1 and MELOE-2, MELOE-2 Upstream Region (UR_1–545), or the 600 bp melanA promoter as positive control. PGL4-13 without the SV40 promoter was used as negative control. A Renilla luciferase expression plasmid was co-transfected as a positive control of transfection. Renilla luciferase (black bars) and Firefly luciferase (white bars) activities were measured after 48 h and expressed as arbitrary units. Data are expressed as mean +/− SEM from four distinct experiments.</p

Expression of <i>meloe</i> mRNA and MELOE antigens in SW480 line transfected with full-length <i>meloe</i> cDNA.

<p>A. <i>Meloe</i> relative expression measured by RT-qPCR using 3′DR primers on SW480 cells wild type (wt-SW480) or transfected (T-SW480) with full-length <i>meloe</i> cDNA. M113 cells were used as positive control. B. RT-PCR products amplified from RNA of SW480 cells transfected or not with full-length <i>meloe</i> cDNA. Reverse transcriptions were performed on total RNA samples followed by PCR using 5′end and 3′end primers described on <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075233#pone-0075233-g001" target="_blank">Figure 1C</a>. C. TNF secretion by MELOE-1 (M170.48) and MELOE-2 (M170.51) T cell clones in response to SW480 cell line. Co-cultures were performed at 1∶2 clone:SW480 cells ratio and responses were assessed by TNF-α intracellular staining.</p

Analysis of IRES activity in upstream region and within the intercistronic region of <i>meloe</i> mRNA.

<p>M113 melanoma cells were transfected with the pRF bicistronic vector either empty (control vector) or containing the viral EMCV IRES (positive control) or various fragments of <i>meloe</i> mRNA: UR_1–545, UR_262–545 IR_666–1215, IR_1268–1490, IR_1391–1490. Renilla luciferase (black bars) and Firefly luciferase (white bars) activities were determined 48 h after transfection and expressed as arbitrary units. Data are expressed as mean +/− SEM from four distinct experiments. ***p<0.001, *p<0,05, ns (non significant) by ANOVA and Dunnett post-test.</p

<i>Meloe</i> silencing in melanoma cells with a short interfering RNA (siRNA) localized upstream of MELOE-2 ORF.

<p>A. M88 melanoma cells were transfected with 50(control si) or siRNA hybridizing 55 bp upstream of MELOE-2 ORF (<i>meloe</i> si) along with a GFP reporter plasmid. Relative expression levels of <i>meloe</i> mRNA were evaluated on GFP+ sorted cells by qPCR with MELOE-1 primers. B. TNF secretion by MELOE-1 (M170.48) and MELOE-2 (M170.51) T cell clones in response to GFP+ sorted M88 cells transfected with the different siRNAs. Co-cultures with specific clones were performed at 1∶2 clone to melanoma cell ratio and response of each clone was assessed by TNF-α intracellular staining.</p