Melanosomal Targeting Sequences from gp100 Are Essential for MHC Class II–Restricted Endogenous Epitope Presentation and Mobilization to Endosomal Compartments

Abstract

CD4+ T lymphocytes play an important role in CD8 + T cellmediated responses against tumors. Considering that f20% of melanomas express MHC class II, it is plausible that concomitant presentation by MHC class I and class II shapes positive (helper T cells) or negative (regulatory T cells) antitumor responses. Interestingly, gp100, a melanoma antigen, can be presented by both MHC class I and class II when expressed endogenously, suggesting that it can reach endosomal/MHC class II compartments (MIIC). Here, we showed that gp100 putative NH 2 -terminal signal sequence and the last 70 residues in COOH terminus are essential for MIIC localization and MHC class II presentation. Confocal microscopy analyses confirmed that gp100 was localized in LAMP-1 + / HLA-DR + endosomal/MIIC. Gp100 targeting sequences were characterized by deleting different sections in the COOH terminus (last 70 residues). Transfection in 293T cells, expressing MHC class I and class II molecules, revealed that specific deletions in COOH terminus resulted in decreased MHC class II presentation, without effects on class I presentation, suggesting a role in MIIC trafficking for these deleted sections. Then, we used these gp100 targeting sequences to mobilize green fluorescent protein to endosomal compartments and to allow MHC class II and class I presentation of minimal endogenous epitopes. We conclude that these specific sequences are MIIC-targeting motifs, which could be included in expression cassettes for endogenously expressed tumor or viral antigens for MHC class II and class I presentation and optimize in vivo T-cell responses or as an in vitro tool for characterization of new MHC class II epitopes.

Multidrug Resistance Decreases with Mutations of Melanosomal Regulatory Genes

“…The melanoma MelFB lost gp100/Pmel17 expression through immunoselection. The congenic cell line MelFB + gp100/Pmel17 was derived from MelFB by the transduction of gp100/Pmel17 into MelFB; another congenic cell line MelFB À gp100/Pmel17 was generated by transducing MelFB with a control vector encoding the green fluorescent protein (23). Figure 2 shows that absence of gp100/Pmel17 results in an increase in cDDP sensitivity compared with in the presence of gp100/Pmel17, providing the first direct evidence that disruption of the process of normal melanosome biogenesis increases sensitivity to cDDP.…”

Heat Shock Protein 90-Mediated Peptide-Selective Presentation of Cytosolic Tumor Antigen for Direct Recognition of Tumors by CD4+ T Cells

“…Nevertheless, direct recognition of MHC-II-expressing tumors by tumor Ag-specific CD4 + T cells has been reported, although the mechanism for the direct presentation of physiologically expressed tumor Ag on MHC-II remains largely unclear except for a melanosomal Ag, gp100, which is efficiently presented to gp100-specific CD4 + T cells. The presentation of gp100 to CD4 + T cells was completely abolished by removing a melanosomal targeting sequence in gp100, indicating the critical role of melanosomal localization for the MHC-II presentation (30). Because melanosomes are closely related to lysosomes and gp100 is localized to late endosomes in melanosome-negative tumors, it is reasonable that melanosomal Ags are naturally processed via an MHC-II processing pathway.…”

Proteasome-Independent Major Histocompatibility Complex Class I Cross-Presentation Mediated by Papaya Mosaic Virus-Like Particles Leads to Expansion of Specific Human T Cells

“…CD40-B were expanded and cultured from peripheral blood mononuclear cells (PBMCs) (13,18) by the addition of 500 ng/ml of soluble trimeric CD40L (Immunex Corporation, Seattle, WA) and 500 U/ml recombinant human interleukin 4 (IL-4) (Peprotech, Rocky Hill, NJ).…”

“…gp100-or influenza virus M1-specific T cells were added at 2 ϫ 10 4 to 10 ϫ 10 4 cells/well in complete media for 20 h. Culture supernatants were harvested, and IFN-␥ was evaluated by ELISA (18). In some experiments, APCs were pretreated for 1 h with 50 to 70 M chloroquine (Sigma), 20 to 25 g/ml lactacystin, or 1.3 to 3.3 M MG-132 (the last two from Calbiochem, San Diego, CA).…”