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By Melanosomes Conventional Endosomes and Is Influenced In MHC Class II Presentation of gp100 Epitopes in Melanoma Cells Requires the Function of Conventional Endosomes and Is Influenced by Melanosomes This information is current as of October 1, 2021. Valentina Robila, Marina Ostankovitch, Michelle L. Altrich-VanLith, Alexander C. Theos, Sheila Drover, Michael S. Marks, Nicholas Restifo and Victor H. Engelhard J Immunol 2008; 181:7843-7852; ; doi: 10.4049/jimmunol.181.11.7843 Downloaded from http://www.jimmunol.org/content/181/11/7843 References This article cites 72 articles, 38 of which you can access for free at: http://www.jimmunol.org/content/181/11/7843.full#ref-list-1 http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists by guest on October 1, 2021 • Fast Publication! 4 weeks from acceptance to publication *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2008 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology MHC Class II Presentation of gp100 Epitopes in Melanoma Cells Requires the Function of Conventional Endosomes and Is Influenced by Melanosomes1 Valentina Robila,* Marina Ostankovitch,* Michelle L. Altrich-VanLith,2* Alexander C. Theos,3‡ Sheila Drover,† Michael S. Marks,‡ Nicholas Restifo,§ and Victor H. Engelhard4* 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- Downloaded from -1high/MHC-II؉ late endosomes. Using mutant gp100 molecules with altered intracellular trafficking, we demonstrate that endo somal 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 http://www.jimmunol.org/ 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 Journal of Immunology, 2008, 181: 7843–7852. elanoma immunotherapy strategies developed over sponse to IFN-␥ induction (11–13), the extent to which they the last several years have been focused on cyto- present MHC-II-associated epitopes derived from endog- toxic CD8 T cells that recognize defined peptide enously synthesized MDPs and the pathways leading to this M by guest on October 1, 2021 Ags presented by MHC class I molecules on tumor cells and presentation are not well understood. IFN-␥ treatment of mel- widely recognized by T cells from patients (1, 2). Melanocyte anoma cells results in down-regulation of MDP expression (14 differentiation proteins (MDPs)5 represent a majority of these and J. Fortini and M. S. Marks, unpublished data) and there shared melanoma Ags. Several groups have also identified appears to be an inverse correlation between expression of MHC class II (MHC-II)-associated peptide epitopes derived MHC-II molecules and MDPs (15). It remains unclear whether from MDPs (3–9), and some of these have more recently been this correlation also prevents presentation of MDP-derived included in vaccination regimens (1, 10). Although many mel- epitopes in MHC-IIϩ melanoma cells. Alternatively, if presen- anoma cells express MHC-II molecules constitutively or in re- tation occurs, the common intracellular compartment where MHC-II molecules and melanosomal proteins meet for Ag pro- cessing remains to be identified. *Department of Microbiology and Carter Immunology Center, University of Virginia School of Medicine, Charlottesville, VA 22908; †Division of Biomedical Sciences, Pigmented melanoma cells contain conventional endosomes as Memorial University of Newfoundland, St. John’s, Newfoundland, Canada; ‡Depart- well as the melanosomes that synthesize and store the pigment ment of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia, PA 19104; and §National Cancer Institute, National Institutes melanin (16). In contrast to melanosomes, which are distinct or- of Health, Bethesda, MD 20892 ganelles, the compartments in which MHC-II processing occurs Received for publication May 5, 2008. Accepted for publication September 22, 2008. correspond to conventional endosomes and lysosomes, modified The costs of publication of this article were defrayed in part by the payment of page by expression of MHC-II, invariant (Ii) chain, and HLA-DM (17). charges. This article must therefore be hereby marked advertisement in accordance Despite the fact that melanosomes share a number of characteris- with 18 U.S.C. Section 1734 solely to indicate this fact. tics with conventional endosomes (18), their MHC-II processing 1 This work was supported by Grants AI20963 and AI33134 from the U.S. Public abilities have not been investigated. In addition, many melanomas 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.). lose their ability to synthesize pigment and no longer contain iden- 2 Current address: IBT Laboratories, 11274 Renner Boulevard, Lenexa, KS 66219. tifiable melanosomes (19, 20). These changes are partially due to 3 Current address: Department of Human Science, School of Nursing and Health down-regulation of expression of MDPs and other components of Studies, Georgetown University, Washington, DC 20057. the intracellular machinery involved in maintaining the identity of 4 Address correspondence and reprint requests to Dr. Victor H. Engelhard, Carter melanosomes within the endocytic pathway (21, 22). Given that Immunology Center, University of Virginia School of Medicine, Box 801386, Char- MDPs expressed in nonmelanocytic cells localize to conventional lottesville, VA 22908. E-mail address: [email protected] late endosomes (23–25), it is conceivable that their intracellular 5 Abbreviations used in this paper: MDP, melanocyte differentiation protein; MHC-II, MHC class II; siRNA, small interfering RNA; LAMP, lysosome-associated mem- localization will also be perturbed in melanoma cells that display brane protein; Ii, invariant chain. this de-differentiated phenotype. www.jimmunol.org 7844 MHC-II EPITOPE PROCESSING IN MELANOMA CELLS Protein gp100 (also called Pmel17 or Silver) is an MDP that Institute, Bethesda, MD), both express HLA-DR*0401 constitutively. The plays a critical role in melanosome formation (26), and is also a pigmented melanoma cell lines 1011mel and MNT-1 (16) expresses all tumor Ag expressed by more than 75% of human melanomas (27). MDPs but not MHC-II molecules. DM331, 1011, and MNT-1 all fail to express significant amounts of the invariant chain. DM331 cells transfected The gp100 protein expressed endogenously in both melanoma and to express tyrosinase (DM331-tyrosinase) have been previously described nonmelanoma cells is processed for presentation of multiple (44). Melanoma cells were grown in RPMI 1640 (Invitrogen) supple- epitopes by MHC-II molecules (7, 28). This activity suggests that mented with 5% heat-inactivated FBS (Valley Biologicals) and 2 mM gp100 is targeted to MHC-II processing compartments via an in- glutamine. The wild-type gp100 gene and the DEL, ⌬PKD, and ⌬RPT gp100 mu- tracellular pathway. In melanocytes and pigmented melanoma, tants (31) were subcloned in pcDNA3.0 (Invitrogen) and transfected in gp100 is localized to the most immature (stage I and stage II) DM331 melanoma using Fugene6 (Roche Diagnostics) or a Nucleofection melanosomes and poorly represented in late endosomes and lyso- system (Amaxa Biosystems). The high efficiency of the latter system fa- somes (16). During transit through stage I melanosomes, the lu- cilitated experiments with long timeframes or requiring cotransfection of menal domain of gp100 is cleaved by a proprotein convertase into multiple gene constructs. A bulk stable line expressing wild-type gp100 (DM331-GP) or short-term transfected lines were obtained by culturing 26- and 70-kDa fragments (24, 29). The latter can be further pro- with
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