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* Department of Microbiology and Carter Immunology Center, University of Virginia School of Medicine, Charlottesville, VA 22908;
Division of Biomedical Sciences, Memorial University of Newfoundland, St. John’s, Newfoundland, Canada;
Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia, PA 19104; and
National Cancer Institute, National Institutes of Health, Bethesda, MD 20892
Many human solid tumors express MHC class II (MHC-II) molecules, and proteins normally localized to melanosomes give rise to MHC-II-restricted epitopes in melanoma. However, the pathways by which this response occurs have not been defined. We analyzed the processing of one such epitope, gp10044–59, derived from gp100/Pmel17. In melanomas that have down-regulated components of the melanosomal pathway, but constitutively express HLA-DR*0401, the majority of gp100 is sorted to LAMP-1high/MHC-II+ late endosomes. Using mutant gp100 molecules with altered intracellular trafficking, we demonstrate that endosomal localization is necessary for gp10044–59 presentation. By depletion of the AP-2 adaptor protein using small interfering RNA, we demonstrate that gp100 protein internalized from the plasma membrane to such endosomes is a major source for gp10044–59 epitope production. The gp100 trapped in early endosomes gives rise to epitopes that are indistinguishable from those produced in late endosomes but their production is less sensitive to inhibition of lysosomal proteases. In melanomas containing melanosomes, gp100 is underrepresented in late endosomes, and accumulates in stage II melanosomes devoid of MHC-II molecules. The gp10044–59 presentation is dramatically reduced, and processing occurs entirely in early endosomes or stage I melanosomes. This occurrence suggests that melanosomes are inefficient Ag-processing compartments. Thus, melanoma de-differentiation may be accompanied by increased presentation of MHC-II restricted epitopes from gp100 and other melanosome-localized proteins, leading to enhanced immune recognition.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 This work was supported by Grants AI20963 and AI33134 from the U.S. Public Health Service (to V.H.E.), Grants AR041855 and EY015625 (to M.S.M.), and Grant ROP-38369 from Canadian Institutes for Health Research (to S.D.).
2 Current address: IBT Laboratories, 11274 Renner Boulevard, Lenexa, KS 66219.
3 Current address: Department of Human Science, School of Nursing and Health Studies, Georgetown University, Washington, DC 20057.
4 Address correspondence and reprint requests to Dr. Victor H. Engelhard, Carter Immunology Center, University of Virginia School of Medicine, Box 801386, Charlottesville, VA 22908. E-mail address: vhe{at}virginia.edu
5 Abbreviations used in this paper: MDP, melanocyte differentiation protein; MHC-II, MHC class II; siRNA, small interfering RNA; LAMP, lysosome-associated membrane protein; Ii, invariant chain.
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