MHC Class II Presentation of gp100 Epitopes in Melanoma Cells Requires the Function of Conventional Endosomes and Is Influenced by Melanosomes

Abstract

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.

Disulfide Reduction in the Endocytic Pathway: Immunological Functions of Gamma-Interferon-Inducible Lysosomal Thiol Reductase

“…As class II-expressing melanoma cells can present epitopes from melanoma-associated antigens (51,52,65,68), they can potentially stimulate anti-melanoma CD4 þ T cell responses. However, class II expression in melanoma correlates with advanced disease and poor survival (54,71).…”

Gamma-interferon-inducible lysosomal thiol reductase is upregulated in human melanoma

“…Melanoma cells, APCs and keratinocytes may contribute to MHC class II-restricted presentation in melanoma tumors. Melanoma cells may express MHC class II and are capable of presenting endogenous membrane bound and cytoplasmic antigens on MHC class II [14, 15]. Melanoma tumor cells directly presenting antigen are capable of activating naïve T cells [12].…”

N-terminal Domains Elicit Formation of Functional Pmel17 Amyloid Fibrils

“…Pmel17 is synthesized as a type 1 integral membrane glycoprotein featuring a single transmembrane domain and a large lumenal domain exposed within the lumen of the endoplasmic reticulum (12). After terminal glycosylation in the Golgi apparatus, Pmel17 is eventually delivered to early endosomes (13), most likely indirectly after delivery to the plasma membrane and subsequent internalization; internalization is facilitated by interaction of a dileucine-based signal in the Pmel17 cytoplasmic domain with the clathrin adaptor AP-2 (14,15). Within endosomes, Pmel17 partitions to membrane microdomains that invaginate to form intralumenal vesicles (ILVs) 3 (1,13).…”