A HLA-Cw*0701 restricted Melan-A/MART1 epitope presented by melanoma tumor cells to CD8+ tumor infiltrating lymphocytes

Abstract

Melan-A/MART1 is a melanocytic differentiation antigen recognized on melanoma tumor cells by CD8+ and CD4+ T cells. In this study, we describe a new epitope of this protein recognized in the context of HLA-Cw*0701 molecules by a CD8+ tumor infiltrating lymphocyte (TIL) clone. This CD8+ TIL clone specifically recognized and killed a fraction of melanoma cells lines expressing Melan-A/MART1 and HLA-Cw*0701. We further show that the Melan-A/MART1(51-61) peptide is the optimal peptide recognized by this clone. Together, these data significantly enlarge the fraction of melanoma patients susceptible to benefit from a Melan-A/MART1 vaccine approach.

Fig. 1

Enrichment of CD8+ T cells specific for Melan-A/MART1_51–63 and Melan-A/MART1_51–73 peptide from a polyclonal TIL population. a T cells were obtained from TIL of a melanoma patient. T cells were cultured with or without Melan-A/MART1_51–63 or Melan-A/MART1_51–73 peptide in presence of Brefeldin A at 37°C. After 6 h, cells were stained for surface CD8, fixed, and then stained for intracytoplasmic IFN-γ. Fluorescence was analyzed by flow cytometry with a gate set on CD8+ T cells. b CD8+ T cells specific for Melan-A/MART1_51–63 peptide were enriched by IFN-γ magnetic beads cell sorting and were expanded during 2 weeks. The sorted TIL were then cultured with or without Melan-A/MART1_51–63 and Melan-A/MART1_51–73 peptide in presence of Brefeldin A at 37°C during 6 h. Cells were stained for surface CD8, fixed, and then stained for intracytoplasmic IFN-γ. Fluorescence was analyzed by flow cytometry with a gate set on CD8 + T cells

Fig. 2

The Melan-A/MART1_51–63 specific CD8+ T cell clone, M74.19, is restricted by the HLA-Cw*0701 molecules. a M74.19 CD8+ T cell clone was cultured alone or with 10 μM of Melan-A/MART1_51–63 peptide in presence of Brefeldin A at 37°C. Inhibitory mAb against HLA class I, HLA-B/C or HLA-A2 molecules or isotype control mAb were added to cultures at different concentrations. After 6 h, cells were fixed, permeabilized and stained for intracytoplasmic IFN-γ. Fluorescence was analyzed by flow cytometry. b M74.19 T cell clone was cultured alone or with 10 μM Melan-A/MART1_51–63 peptide pulsed HLA-Cw*0701+ or HLA-Cw*0701− B-LCL. After 6 h, TNF was measured in co-culture supernatants. c COS-7 cells were co-transfected with plasmid encoding the HLA-B8, HLA-B*1401, HLA-Cw*0701, HLA-Cw*0702 or HLA-Cw*0802 and with or without plasmid encoding Melan-A/MART1. 24 h after transfection, COS-7 cells were either unpulsed or pulsed with 10 μM of Melan-A/MART1_51–63 peptide and washed. Then, they were co-cultured with M74.19 CD8+ T cell clone. After 6 h, TNF was measured in co-culture supernatants

Fig. 3

Melan-A/MART1_51–61 peptide is the optimal epitope recognized by M74.19 CD8+ T cell clone. M74.19 CD8+ T cell clone was cultured with different concentrations of a panel of truncating peptides located in Melan-A/MART1_51–73 region. After 6 h, TNF was measured in co-culture supernatants

Fig. 4

The Melan-A/MART1 epitope recognized by M74.19 CD8+ T cell clone is presented by Melan-A/MART1+ HLA-Cw*0701+ melanoma tumor cells. a Untreated, or IFN-γ treated melanoma tumor cell line M74 was stained with unlabeled primary mAb produced in mouse, specific for HLA class I or HLA B/C molecule or Melan-A/MART1 protein. Then melanoma tumor cell lines were stained with PE conjugated goat anti-mouse mAb. Fluorescence was analyzed by flow cytometry, b M74.19 CD8+ T cell clone was cultured with untreated, or IFN-γ treated melanoma tumor cell lines pulsed or not with Melan-A/MART1_51–63 peptide. After 6h, TNF was measured in co-culture supernatants. c Cytolytic activity of M74.19 CD8+ T cell clone against untreated or IFN-γ treated melanoma tumor cell lines unpulsed or pulsed with Melan-A_51–61 peptide, was assessed by 4 h ⁵¹Cr release assay. ND not done