Coat color dilution in mice because of inactivation of the melanoma antigen MART-1.

Melanoma antigen recognized by T cells 1 (MART-1) is a melanoma-specific antigen, which has been thoroughly studied in the context of immunotherapy against malignant melanoma and which is found only in the pigment cell lineage. However, its exact function and involvement in pigmentation is not clearly understood. Melanoma antigen recognized by T cells 1 has been shown to interact with the melanosomal proteins Pmel17 and OA1. To understand the function of MART-1 in pigmentation, we developed a new knockout mouse model. Mice deficient in MART-1 are viable, but loss of MART-1 leads to a coat color phenotype, with a reduction in total melanin content of the skin and hair. Lack of MART-1 did not affect localization of melanocyte-specific proteins nor maturation of Pmel17. Melanosomes of hair follicle melanocytes in MART-1 knockout mice displayed morphological abnormalities, which were exclusive to stage III and IV melanosomes. In conclusion, our results suggest that MART-1 is a pigmentation gene that is required for melanosome biogenesis and/or maintenance.

Melan-A/MART-1-specific CD4 T cells in melanoma patients: identification of new epitopes and ex vivo visualization of specific T cells by MHC class II tetramers.

Over the past decade, many efforts have been made to identify MHC class II-restricted epitopes from different tumor-associated Ags. Melan-A/MART-1(26-35) parental or Melan-A/MART-1(26-35(A27L)) analog epitopes have been widely used in melanoma immunotherapy to induce and boost CTL responses, but only one Th epitope is currently known (Melan-A51-73, DRB1*0401 restricted). In this study, we describe two novel Melan-A/MART-1-derived sequences recognized by CD4 T cells from melanoma patients. These epitopes can be mimicked by peptides Melan-A27-40 presented by HLA-DRB1*0101 and HLA-DRB1*0102 and Melan-A25-36 presented by HLA-DQB1*0602 and HLA-DRB1*0301. CD4 T cell clones specific for these epitopes recognize Melan-A/MART-1+ tumor cells and Melan-A/MART-1-transduced EBV-B cells and recognition is reduced by inhibitors of the MHC class II presentation pathway. This suggests that the epitopes are naturally processed and presented by EBV-B cells and melanoma cells. Moreover, Melan-A-specific Abs could be detected in the serum of patients with measurable CD4 T cell responses specific for Melan-A/MART-1. Interestingly, even the short Melan-A/MART-1(26-35(A27L)) peptide was recognized by CD4 T cells from HLA-DQ6+ and HLA-DR3+ melanoma patients. Using Melan-A/MART-1(25-36)/DQ6 tetramers, we could detect Ag-specific CD4 T cells directly ex vivo in circulating lymphocytes of a melanoma patient. Together, these results provide the basis for monitoring of naturally occurring and vaccine-induced Melan-A/MART-1-specific CD4 T cell responses, allowing precise and ex vivo characterization of responding T cells.