MELOE-1 is a new antigen overexpressed in melanomas and involved in adoptive T cell transfer efficiency - PubMed (original) (raw)

Comparative Study

. 2008 Oct 27;205(11):2673-82.

doi: 10.1084/jem.20081356. Epub 2008 Oct 20.

Affiliations

Comparative Study

MELOE-1 is a new antigen overexpressed in melanomas and involved in adoptive T cell transfer efficiency

Yann Godet et al. J Exp Med. 2008.

Abstract

A cytotoxic T lymphocyte (CTL) clone was derived from a tumor-infiltrating lymphocyte (TIL) population infused to a melanoma patient who remained relapse free for 10 yr after this adoptive transfer. This clone recognized all melanoma cell lines tested and, to a lower extent, melanocytes, in the context of human histocompatibility leukocyte antigen A2 (HLA-A2), but it did not recognize other tumor cell types. The gene coding for the antigen recognized by this clone was identified by the screening of a melanoma complementary DNA expression library. This antigen is overexpressed in melanomas, compared with other cancer cell lines and healthy tissues, and was thus called melanoma-overexpressed antigen (meloe). Remarkably, the structure of meloe was unusual, with multiple short open reading frames (ORFs). The peptide recognized by the CTL clone was encoded by one of these ORFs, called MELOE-1. Using a specific HLA-A2/peptide tetramer, we showed a correlation between the infusion of TILs containing MELOE-1-specific T cells and relapse prevention in HLA-A2 patients. Indeed, 5 out of 9 patients who did not relapse were infused with TILs that contained MELOE-1-specific T cells, whereas 0 out of the 21 patients who relapsed was infused with such TIL-containing lymphocytes. Overall, our results suggest that this new antigen is involved in immunosurveillance and, thus, represents an attractive target for immunotherapy protocols of melanoma.

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Figures

Figure 1.

Figure 1.

T cell clone selection and characterization. (A) Percentage of TNF-producing T cells and of HLA-A2/Melan-AA27L tetramer–positive T cells in the M170 TIL population in response to the autologous melanoma cell line. 105 TILs and 2 × 105 melanoma cells were incubated for 5 h in the presence of Brefeldin A, stained with HLA-A2/Melan-AA27L tetramer, fixed, and stained with anti-TNF antibody in a permeabilization buffer. 104 T cells were then analyzed by flow cytometry. (B) TNF secretion by the M170.48 T cell clone in response to the autologous melanoma cell line. 104 CTLs were added to 3 × 104 M170 melanoma cells in the presence of blocking antibodies directed against class I, A2, and B/C HLA, diluted to 1:50 (shaded bars), 1:500 (hatched bars), and 1:5,000 (open bars). CTL clone reactivity was assessed by a TNF release assay. (C) TNF response of the M170.48 CTL clone to HLA-A*0201 tumor cell lines. The M6 cell line (HLA-A2 negative) was used as a negative control. (D) IFN-γ response of the M170.48 CTL clone (shaded bars) and of a Melan-A/A2–specific CTL clone (hatched bars) to HLA-A*0201 melanocytes. The M170 cell line was added as a positive control.

Figure 2.

Figure 2.

Characterization of the cDNA coding for the recognized antigen. (A) M170.48 TNF responses to COS-7 cells (E/T ratio = 1:3) transfected with the indicated plasmids. The T cell clone was added 2 d after the transfection, and the CTL clone reactivity was assessed by a TNF release assay. (B) Comparison of the nucleotide sequences of meloe and BC008026 cDNAs and the localization of this sequence on the HDAC-4 gene. The indicated nucleotides correspond to SNPs between the meloe sequence isolated from M134 and A498 tumor cell lines and the meloe sequence isolated from the M117 and SW480 cell lines, and the BC008026 cDNA sequence.

Figure 3.

Figure 3.

Characterization of _meloe_-derived peptide recognized by the M170.48 T cell clone. (A) Structure of meloe cDNA. Boxes illustrate ORFs >120 bp present along the meloe sequence, and black boxes correspond to ORFs tested for recognition by the CTL clone. (B) M170.48 TNF responses to COS-7 cells (E/T ratio = 1:3) transfected with the indicated plasmids. The T cell clone was added 2 d after the transfection, and the CTL clone reactivity was assessed by a TNF release assay. (C) Nucleotide and amino acid sequences of the ORF 1,230–1,370 of meloe isolated from the M134 cDNA library. The two candidate peptides are bolded. (D) Cytotoxicity of the M170.48 CTL clone against peptide-pulsed T2 cells. Target cells were 51Cr-labeled for 60 min and incubated for 30 min with a range of the indicated peptides. The M170.48 T cell clone was added (E/T ratio = 10:1), and chromium release was then measured after a 4-h incubation period.

Figure 4.

Figure 4.

Preferential expression of meloe cDNA in melanoma cell lines measured by qPCR, and impact of meloe expression on specific CTL clone activation. (A) Four melanoma, one breast cancer, two renal carcinoma, and one lung cancer cell lines were tested by qPCR for the expression of meloe. RPLPO and β2-microglobulin gene expression were used as internal controls. The relative expression of meloe was calculated after normalization on the efficiency of the PCR reaction and the mean expression of these two housekeeping genes, reported to its normalized expression in melanocytes. (B) TNF secretion by the M170.48 CTL clone in response to HLA-A2 tumor cell lines nontransfected (open bars) or transfected with meloe (hatched bars) or _meloe_-1 (shaded bars) expression plasmids. Tumor cells were transiently transfected with 100 ng of each plasmid with a lipofectamine reagent kit. 104 CTLs were added to 3 × 104 target cells, and the CTL clone reactivity was assessed by a TNF release assay. (C) meloe relative expression measured by qPCR in 16 human healthy tissues.

Figure 5.

Figure 5.

Detection of MELOE-1/A2–specific CTLs in TILs infused to relapse-free melanoma patients and analysis of their repertoire diversity. (A) HLA-A2 TIL populations labeled with the A2/MELOE-136-44 tetramer. (top) TILs infused to relapse-free patients. (bottom) TILs infused to patients who relapsed. TILs were coincubated with MELOE-1 tetramer and anti-CD8 mAb. Values indicate the percentage of tetramer-positive cells among CD8+ TILs. (B) Repertoire diversity of multimer-sorted populations was evaluated by labeling with 25 anti-Vβ mAbs. Insets illustrate the purity of each sorted TIL population, assessed by MELOE-1–specific tetramer labeling.

Figure 6.

Figure 6.

Reactivity of MELOE-1/A2–specific TILs against HLA-A2 tumor cell lines. (A) Lysis of the M170 melanoma cell line (closed circles) and of the 1355 lung carcinoma cell line (open circles) by the M170.48 CTL clone and MELOE-1–specific TIL populations. 51Cr-labeled tumor cells were co-cultured with T cells at various E/T ratios. Chromium release in the supernatants was measured after a 4-h incubation period. (B) Cytokine production by the M170.48 CTL clone and MELOE-1–specific TIL populations in response to M170 melanoma cells. Effector and target cells were incubated at a 1:2 ratio in the presence of Brefeldin A and stained with anti-TNF antibody (open bars), anti– IFN-γ antibody (hatched bars), or anti–IL-2 antibody (closed bars), and 104 T cells were analyzed by flow cytometry.

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