MHC Class II Presentation of gp100 Epitopes in Melanoma Cells Requires the Function of Conventional 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; ;

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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). 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 (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 , 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, -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 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 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 300 ␮g/ml Zeocin (Invitrogen). HLA-DRB1*0401 ␣-chain and cessed to a 34- to 38-kDa molecule (30), and forms the fibrillar ␤-chain expression constructs, a gift from J. Gorski (Medical College of scaffolding characteristic of stage II melanosomes and on which Wisconsin, Milwaukee, WI) were used for transient transfection of the melanin is deposited (29). In contrast, gp100 accumulates in con- HLA-DR-negative melanomas 1011mel and MNT-1. Expression of gp100 was measured by intracellular staining after permeabilization with BD ventional late endosomes and lysosomes when expressed in non- Perm/Wash (BD Pharmingen) using the gp100-specific Abs HMB45 or melanocytic cells (24). This targeting is mediated by sequences HMB50 (Lab Vision) and PE-conjugated goat anti-mouse IgG (Jackson encoded in the gp100 lumenal domain, the removal of which re- ImmunoResearch Laboratories). Expression of HLA-DRB1*0401 was sults in predominant retention of the protein in early endosomes measured by surface staining using the mAb L243 (45) or the HLA-DR4- Downloaded from (31, 32). Two alternative mechanisms for gp100 intracellular sort- specific Ab NFLD.D10 (46). Data were acquired on a flow cytometer and analyzed using CellQuest software (BD Pharmingen). ing have been proposed: a mechanism directly from the trans- ϩ Golgi network, and the other indirectly via endocytosis from the HLA-DR*0401 gp10044–59-specific CD4 T cell lines plasma membrane (33–35). Thus, the of this system ϩ Human CD4 T cell lines specific for HLA-DR*0401 gp10044–59 (6) were offers a unique opportunity to investigate the compartments with stimulated with a combination of irradiated (10,000 rad) HLA-DR*0401ϩ http://www.jimmunol.org/ class II processing capabilities in melanomas and the impact of BLCL, gp10044–59-expressing tumor cells (1102mel), and nonmatched subcellular trafficking of an intact membrane protein on its pro- PBMC in T cell culture medium. Cells were fed after 5 days using fresh cessing for Ag presentation. medium supplemented with 20 U/ml IL-2 and used in Ag presentation assays after another 2–3 days. Two distinct Ag processing pathways have been defined that ϩ Murine CD4 T cells specific for HLA-DR*0401 human gp10044–59 enable presentation of a broad range of peptides derived from ex- were generated in DR4-IE transgenic mice as previously described (6). For ogenous proteins entering endocytic compartments. In the classical Ag presentation assays, T cells were used 6 or 7 days after restimulation. pathway, epitopes require the proteolytic processing capacity of the highly acidic late endocytic compartments, where nascent class Confocal microscopy II molecules exchange CLIP for antigenic peptide (36). The pre- Cells were grown on coverslips, washed with PBS, fixed for 20 min at by guest on October 1, 2021 sentation of such epitopes is sensitive to depletion of newly syn- room temperature in 4% paraformaldehyde in PBS, and permeabilized with ␮ thesized MHC-II molecules through the use of protein synthesis 200 l BD Perm/Wash (BD Pharmingen) on ice. Cells were incubated for 1 h with Abs specific for gp100 (HMB45 or HMB50), HLA-DR (L243) or inhibitors and ablation of Ii chain and HLA-DM functions (37, 38). lysosome-associated membrane protein 1 (LAMP-1) (BD Pharmingen). In the alternative pathway, epitopes are generated within mildly These primary Abs were detected with Alexa Fluor 488 anti-mouse IgG1 proteolytic conditions of early endosomes and are loaded on ma- and Alexa Fluor 594 anti-mouse IgG2 conjugates (Molecular Probes). In ture MHC-II molecules recycling through this compartment, inde- some experiments, late endosomes or lysosomes were stained by incubat- ing cells for 60 min at 37°C with 60 ␮g/ml Lysotracker-Alexa Fluor 594 pendently of Ii chain and HLA-DM (37, 39–42). Which of these (Molecular Probes) resuspended in phenol red-free HBSS. Early endocytic pathways is involved in processing of endogenously synthesized compartments were labeled by incubation with 40 ␮g/ml transferrin-Alexa gp100 protein leading to MHC-II presentation in melanoma cells Fluor 594 (Molecular Probes) for 10 min at 37°C. Cells were washed with has not been fully investigated. PBS containing 5% serum, and fixed, permeabilized, and stained with In the current work we investigated the mechanisms for endog- gp100-specific Abs as described. To examine internalization of gp100, cells were incubated with HMB50 enous presentation of the HLA-DR*0401- restricted gp10044–59 Ab for 30 min at 4°C, and then shifted to 37°C for 90 min in the presence epitope. We used melanoma cells that express MHC-II molecules of Alexa Fluor 594-conjugated Lysotracker. After fixation and permeabi- constitutively and gp100 mutants targeted to early or late endo- lization, cells were stained with Alexa Fluor 488 donkey anti-mouse sec- somes to identify the endosomal compartments with roles in gen- ondary Ab. To investigate the intracellular accumulation of newly formed peptide-MHC-II complexes, DM331 cells transfected with mock or AP-2- erating this epitope and the processing requirements for its pre- specific small interfering RNA (siRNA) 3 days earlier were exposed to sentation. By expressing MHC-II molecules in pigmented brefeldin A at a final concentration of 3 ␮g/ml in complete medium for melanoma cells, we also investigated the processing capabilities of 12 h. Cells were washed three times in PBS and chased for 0 or 4.5 h at melanosomes. Finally, we established that pigmented melanoma 37°C before fixation in 4% paraformaldehyde. Mature MHC-II molecules cells displayed lower levels of gp100 epitope compared with their were detected with L243 mAb. Samples were mounted on glass slides with Vectashield (Vector Labo- de-differentiated counterparts that do not contain melanosomes. ratories), visualized using an Olympus confocal microscope, and processed Our results emphasize that the presentation of gp100 epitopes by with Adobe Photoshop 7.0. MHC-II molecules is influenced by its unique cell biology, and that the alteration of this biology during malignant transformation RNA-mediated interference modulates its recognition by the immune system. Expression of the ␮2-subunit of the AP-2 adaptor protein was blocked by transfecting cells twice at 4- or 72-h intervals with the siRNA duplexes Materials and Methods (Qiagen) to the sequence GUGGAUGCCUUUCGGGUCA using Oligo- Melanoma cell lines and transfectants fectamine (Invitrogen) at 20 nM siRNA/transfection. An irrelevant siRNA duplex was used as control. Cells were analyzed for depletion of AP-2 at The nonpigmented melanoma cell lines DM331 (gp100-negative) (43) and 48–72 h after the second round of transfection using flow cytometry or 1102mel (gp100-positive), a gift from S. L. Topalian (National Cancer immunofluorescence microscopy. The Journal of Immunology 7845

T cell assays CD4 T cells (5 ϫ 104) were incubated with 5 ϫ 104 melanoma cells in U-bottom 96-well plates (Costar) for 16 h. Culture supernatants were as- sayed using ELISA kits for murine IFN-␥ or GM-CSF (eBioscience) or human IFN-␥ (Endogen). The data presented are average values of dupli- cate wells, with error bars indicating SDs. To assess the ability of inhibitors to block endogenous Ag presentation, cell surface HLA-DR molecules were denatured by incubating melanoma cells in mild acid buffer (47) at room temperature for 3 min. Acid-stripped cells were incubated with drugs (100 ␮M chloroquine, 0.5 mM leupeptin, 100 ␮M primaquine) for 16 h, fixed for 10 min on ice with 1% paraformaldehyde, and then incubated with CD4 T cells for 16 h before analysis of cell supernatants by ELISA. Doses of inhibitors were chosen because they showed maximal inhibition of pre- sentation without affecting cell viability.

Western blot analysis Cell pellets were solubilized in 10 mM Tris-HCl (pH 7.5), 0.5% deoxy- cholate, 1% Igepal, 5 mM EDTA, 4 mM PMSF, 10 ␮g/ml aprotinin, 10 ␮M pepstatin A, 10 ␮g/ml leupeptin, and 100 ␮M iodoacetamide. Where indicated, melanoma cells were lysed using premixed M-PER (Pierce), Downloaded from which allows recovery of gp100 from stage II melanosomes. After cen- trifugation at 21,000 ϫ g for 30 min, supernatants were separated by SDS- PAGE on 8–16% Tris-glycine gels (Invitrogen) and transferred to Immo- bilon-P membranes (Millipore). Blots were blocked in 10% (w/v) nonfat dry milk in PBS with 0.05% Tween 20 and incubated with first Abs diluted in the same buffer plus 2% nonfat dry milk for2hatroom temperature (␣Pep13h) or overnight at 4°C (HMB45). Blots were incubated with HRP- linked anti-rabbit (␣Pep13h) or anti-mouse (HMB45) whole Abs (The http://www.jimmunol.org/ Jackson Laboratory) and detected by ECL (Amersham Biosciences). FIGURE 1. The gp100 protein localizes in melanosomes of pigmented melanoma, and in conventional endosomes of nonpigmented melanoma. Pigmented 1011mel-DR4 and nonpigmented 1102mel and DM331-GP Results melanoma cells were fixed, permeabilized, and stained for LAMP-1 and gp100 (HMB50) (left column) or HLA-DR (L243) and gp100 (HMB45) gp100 accumulates in melanosomes of pigmented melanoma (right column), and analyzed by laser scanning confocal microscopy. Mag- and in conventional endosomes of melanoma that do not nification ϫ3(inset) of the same field. express other MDPs

We investigated the intracellular localization of gp100 and Access of gp100 to the endosomal pathway is required for by guest on October 1, 2021 MHC-II molecules in melanoma cells displaying different pigmen- human gp100 epitope production and MHC-II presentation tation phenotypes. The nonpigmented melanoma cell line 1102mel 44–59 expresses gp100 but not Tyrp1, and tyrosinase in these cells is MHC-II-restricted epitopes from different endogenous proteins retained in the due to a genetic polymor- have been shown to arise from proteolytic processing either by the phism (V. Robila and V. H. Engelhard, unpublished data). The in the or by proteases in endocytic com- nonpigmented DM331 fails to express tyrosinase, gp100, Tyrp1, or partments (49–51). To test which of these mechanisms was in- volved in processing of the HLA-DR*0401-restricted gp100 MART-1 (43). Both of these cell lines constitutively express HLA- epitope gp100 , surface MHC molecules of nonpigmented DRB1*0401. The pigmented melanoma 1011mel expresses all of 44–59 1102mel and DM331-GP cells were first denatured by mild acid these MDPs but not MHC-II molecules. To analyze the relation- buffer treatment. Then new MHC-peptide complexes were allowed ship between MHC-II molecules, gp100, and melanosomes, to form for6hinthepresence of lactacystin, a specific proteasome 1011mel was transfected to express HLA-DR*0401 (1011mel- inhibitor, or chloroquine, which neutralizes endosomal pH and in- DR4), whereas DM331 was stably transfected with a plasmid en- hibits resident cathepsins. Lactacystin did not inhibit gp100 coding wild-type gp100 (DM331-GP). Using laser scanning con- 44–59 presentation to gp10044–59-specific CD4 T cells (Fig. 2, A and B), focal microscopy and HMB50, an Ab that detects gp100 in both although it stabilized two gp100 degradation intermediates in the stage I and stage II melanosomes, we found a marked segregation (data not shown) and, as expected, abrogated presentation between gp100 and LAMP-1, a marker of endosomes and lyso- of Tyr369, a proteasome-dependent (52, 53) HLA-A*0201-re- somes in 1011mel-DR4 cells (Fig. 1). This is consistent with pre- stricted epitope derived from tyrosinase (Fig. 2C). In contrast, vious work (16, 48) that has demonstrated in pigmented melano- whereas presentation of Tyr369 by HLA-A*0201 was not affected mas, gp100 is principally localized to stage I and stage II by chloroquine, this inhibitor blocked gp10044–59 epitope presen- melanosomes and excluded from conventional endosomes. Using tation, indicating that its formation required the functions of acid- HMB45, an Ab that recognizes a proteolyzed fragment of gp100 ified endosomes. characteristic of stage II melanosomes (30), we observed a similar To test whether gp100 localization to endosomal compartments segregation between gp100-positive and MHC-IIϩ vesicles. In was essential for MHC-II presentation of gp10044–59, we used a contrast, there was significant colocalization of gp100-positive gp100 mutant, DEL, in which both the transmembrane and cyto- with both LAMP-1 and MHC-II in intracellular vesicles in non- plasmic domains were deleted. DEL expressed in DM331 cells pigmented DM331 and 1102mel melanomas (Fig. 1). This indi- showed no vesicular distribution and failed to localize to cates that, in these de-differentiated cells, gp100 molecules are LAMP-1ϩ compartments (Fig. 3A). The range of expression of routed to conventional late endosomal compartments, including DEL was very broad in these transient transfectants, and the mean those where MHC-II molecules also reside. intensity fluorescence was approximately two-thirds that of cells 7846 MHC-II EPITOPE PROCESSING IN MELANOMA CELLS

FIGURE 2. The gp10044–59 epitope processing requires endosomal pro- teolysis. 1102mel (A) DM331-GP (B), and DM331-tyrosinase (C) cell lines were acid-stripped to remove existing MHC-peptide complexes and then incubated in presence or absence of lactacystin or chloroquine for 6 h. Downloaded from Following fixation with paraformaldehyde, cells were incubated with gp10044–59-specific murine CD4 T cells and epitope presentation evaluated based on GM-CSF secretion (A and B), or Tyr369-specific CD8 T cells and epitope presentation evaluated based on IFN-␥ secretion (C). Data repre- sent average value of duplicate wells, with error bar indicating SD. expressing wild-type gp100 (Fig. 3B). However, these cells failed http://www.jimmunol.org/ to present the gp10044–59 epitope to any significant extent (Fig. 3C). Thus, the presentation of gp10044–59 strongly correlates with the presence of full-length gp100 protein in late endosomes and lysosomes.

The gp10044–59 epitope can be produced in both early and late endosomal compartments Distinct MHC-II-restricted epitopes are produced in either early by guest on October 1, 2021 or late endosomes based on differences in protease content,

FIGURE 4. The gp10044–59 epitope can be produced both in early and late endosomes of DM331 melanoma. A, DM331 cells were transfected with wild-type gp100 (GP), ⌬PKD or ⌬RPT. Five days later, cells were incubated in medium containing transferrin-Alexa Fluor 594 for 10 min or Lysotracker-Alexa Fluor 594. Cells were fixed, permeabilized, stained with gp100 Abs HMB50 (GP, ⌬RPT) or HMB45 (⌬PKD), and analyzed by laser scanning confocal microscopy. B, DM331 melanoma cells were in- cubated with leupeptin or chloroquine to inhibit endosomal proteases, and transfected with wild-type (GP) or ⌬PKD gp100. Incubation in the pres- ence of inhibitors was continued for 16 h, after which cells were fixed with

paraformaldehyde and evaluated for epitope presentation to gp10044–59- specific murine CD4 T cells. C, Cell surface expression of MHC-II in DM331-GP, DM331-PKD, or DM331-RPT after inhibitor treatment was analyzed by staining with L243 Ab and flow cytometry (open histogram). Gray-filled histogram represents staining of untreated cells.

FIGURE 3. The gp10044–59 epitope presentation correlates with gp100 protein localization in the endosomal pathway. A, 1102mel and DM331 loading on mature or newly synthesized or mature MHC-II mol- cells with wild-type (GP) or mutant (DEL) gp100 5 days posttransfection ecules or the presence or absence of HLA-DM (37, 41, 54, 55). were fixed, permeabilized, stained for LAMP-1 and gp100 (HMB50), and To investigate whether gp100 epitope could be produced analyzed by laser scanning confocal microscopy. B, Expression of gp100 in 44–59 in early endosomes, we used another gp100 mutant, ⌬PKD, DM331-GP (solid line histogram) and DM331-DEL (dotted line histo- gram) was analyzed by intracellular staining using HMB50 Ab and flow which lacks aa 243–293 in the lumenal domain and accumulates cytometry. Gray-filled histogram represents isotype control staining. C, in early endosomes (31). In DM331 transfectants, wild-type gp100 colocalized with Lysotracker, a marker of late endo- The gp10044–59-specific CD4 T cells were incubated with 1102mel, DM331-GP, and DM331-DEL. Epitope presentation was evaluated based somes, but not with transferrin internalized for 10 min, an early on IFN-␥ secretion by the T cells and measured by ELISA. endosomal marker (Fig. 4A). Conversely, the ⌬PKD mutant The Journal of Immunology 7847 colocalized partially with internalized transferrin, and was largely ex- cluded from late endosomes. Nonetheless, DM331 cells expressing ⌬ PKD were efficiently recognized by gp10044–59-specific T cells (Fig. 4B). To investigate whether localization of gp100 in early or late endosomes correlated with epitope production in that compart- ment, we treated cells with leupeptin, which inhibits late endoso- mal serine and cysteine proteases, or chloroquine, to deacidify these compartments (51, 56). Treatment with these agents did not affect the expression of MHC-II or transfected gp100 molecules (Fig. 4C). DM331 also does not express the invariant chain (see Fig. 7A), and thus, leupeptin cannot alter epitope expression by blocking invariant chain degradation. As with wild-type gp100, ⌬ chloroquine inhibited gp10044–59 production from the PKD mu- tant, indicating that it also required intracellular processing in acid- ified endosomes (Fig. 4B). However, gp10044–59 presentation from wild-type gp100 was inhibited by leupeptin, whereas presentation of this epitope from the ⌬PKD mutant was not inhibited. This difference might have been due to the large deletion in ⌬PKD, Downloaded from which could render gp100 more susceptible to proteolytic degra- dation and thus less sensitive to inhibition. To further investigate FIGURE 5. The gp10044–59 epitope can be produced from wild-type this possibility, we analyzed epitope presentation from a second gp100 in early as well as late endosomes of DM331 melanoma. DM331 gp100 mutant, ⌬RPT, which has a deletion encompassing aa 314– cells expressing ⌬PKD (top left), GP (top right), or tyrosinase (bottom left) 424, but displays the same intracellular localization as wild-type were acid-stripped to remove existing peptide-MHC complexes and incu- gp100 (31). When expressed in DM331, ⌬RPT colocalized with bated for 16 h with the indicated inhibitors. DM331 cells were incubated http://www.jimmunol.org/ Lysotracker but not with internalized transferrin, indicating its dis- in the presence or absence of inhibitors together with 100 ␮g/ml gp10044–59 synthetic peptide (bottom right). Following fixation with para- tribution in late but not early endosomes (Fig. 4A). As with GP, 369 ⌬ formaldehyde, cells were incubated with Tyr -specific murine CD8 T presentation of gp10044–59 from RPT was inhibited by both chlo- cells (bottom left) or gp10044–59-specific murine CD4 T cells (bottom right roquine and leupeptin (Fig. 4B). These results indicate that and top panels) and epitope presentation was evaluated based on IFN-␥ gp10044–59 can be generated in either early or late endosomes secretion measured by ELISA. Data represent average value of duplicate depending on the primary intracellular localization of the source wells, with error bar indicating SD. One experiment representative of four protein. is shown. by guest on October 1, 2021

Wild-type gp100 is processed both in early and late endosomal compartments The major source protein for gp10044–59 presented by Despite prevalent localization of wild-type gp100 to late endo- HLA-DR*0401 is internalized from the plasma membrane by AP-2 ϳ somes, gp10044–59 presentation was only 55–60% reduced after The protein gp100 might reach endosomal compartments for pro- treatment with leupeptin. This result, together with the demonstra- cessing either directly from the Golgi or by transiting to the cell ⌬ tion that gp10044–59 is produced in early endosomes from PKD surface followed by internalization (16, 34, 57). The clathrin coat gp100, led us to hypothesize that wild-type gp100 is also processed component AP-2 has been implicated in endosomal targeting of in this compartment. Primaquine prevents re-expression of inter- newly synthesized proteins by endocytosis from the plasma mem- nalized recycling MHC-II molecules and inhibits presentation of brane (58–61), and previous work was consistent with the possi- epitopes produced in early endosomes (42). Treatment of ⌬PKD- bility that gp100 was an AP-2 cargo protein (33). To test this idea expressing cells with primaquine inhibited gp10044–59 presenta- directly, we ablated AP-2 expression in DM331-GP using siRNA tion to CD4 T cells to a similar extent as chloroquine (Fig. 5, top oligonucleotides directed to the ␮2 subunit of the complex (59). In left), whereas leupeptin had no effect either alone or in combina- keeping with earlier work (58, 60), AP-2 depletion led to a 5-fold tion with primaquine. Primaquine did not reduce surface MHC-II increase in surface expression of LAMP-1, whereas the expression expression (data not shown), HLA-A*0201 presentation of the of mature MHC-II molecules was unchanged (Fig. 6A). AP-2 de- Tyr369 epitope (Fig. 5, bottom left), or presentation of exogenous pletion also led to a significant increase in cell surface gp100 while gp10044–59 peptide pulsed on DM331 (Fig. 5, bottom right). In having no effect on total cellular gp100 content (Fig. 6A). By pre- keeping with the localization of the ⌬PKD mutant protein and its labeling cell surface gp100 molecules with specific Ab, we also insensitivity to leupeptin, this result suggests that the gp10044–59 found that their subsequent internalization into endosomal com- derived from this molecule is presented by MHC-II molecules re- partments was substantially inhibited in AP-2-depleted cells (Fig. cycling through early endosomes. In four independent experi- 6B). In AP-2-depleted cells, gp10044–59 presentation was substan- ments, primaquine treatment also reduced gp10044–59 presentation tially inhibited (Fig. 6C), suggesting that gp100 internalized from by DM331 cells expressing wild-type gp100 by 30–50% relative the plasma membrane is a major source of protein for epitope to chloroquine (Fig. 5, top right and data not shown). The effects production. of leupeptin and primaquine on presentation by these cells were Although AP-2 deficiency did not alter the overall level of additive, and equivalent to treatment with chloroquine. These re- MHC-II expression (Fig. 6A), it has been shown to direct inter- sults suggest that gp10044–59 is also produced from wild-type nalization of newly synthesized MHC-II molecules to endosomes gp100 in both late and early endosomes. by interaction with the Ii chain (58). Thus, impaired gp10044–59 7848 MHC-II EPITOPE PROCESSING IN MELANOMA CELLS Downloaded from http://www.jimmunol.org/

FIGURE 7. AP-2 depletion does not impair intracellular accumulation FIGURE 6. The gp100 protein internalized from the plasma membrane of new MHC-II-peptide complexes in DM331 melanoma. A, Expression of Ii chain by DM331 (left), 1011mel cells (middle), and BLCL (right) was by AP-2 is the major source protein for gp10044–59 processing. DM331-GP by guest on October 1, 2021 cells were transfected with control or AP-2-specific siRNAs, and cultured evaluated by intracellular staining with LN2 (specific for the lumenal side for 72 h (A–C)or48h(D). A, Cells were stained for cell surface expression of the Ii chain) Ab (open histogram). Gray-filled histogram represents iso- of LAMP-1 (LAMP-1 Ab), mature MHC-II (L243 Ab), and gp100 type control. B, DM331-GP cells transfected with control or AP-2-specific (HMB50) and of total gp100 (HMB50) after permeabilization. Solid line siRNA were incubated with 3 ␮g/ml brefeldin A for 12 h, washed, and histogram represents cells transfected with control siRNA. Dotted line his- chased for 0 or 4.5 h at 37°C. Cells were then fixed, permeabilized, stained togram represents cells transfected with AP-2 siRNA. Gray-filled histo- for mature MHC-II molecules with L243 Ab, and analyzed by confocal gram shows isotype control. B, Cells were allowed to bind HMB50 for 30 microscopy. min at 4°C, washed, and then incubated for 90 min at 37°C in the presence of Lysotracker-Alexa Fluor 594 (red). After washing and fixation, HMB50 Ab was detected with Alexa Fluor 488-conjugated second Ab (green). C, and AP-2-depleted cells (Fig. 7B, right). Thus, AP-2 is not essen- tial for endosomal trafficking of newly synthesized HLA-DR*0401 The indicated number of cells was incubated with gp10044–59-specific mu- rine CD4 T cells and epitope presentation evaluated based on IFN-␥ se- molecules and formation of new peptide-MHC-II complexes in cretion measured by ELISA. Data represent average value of duplicate DM331 melanoma, presumably because of the absence of Ii chain. wells, with error bar indicating SD. D, Cells were stripped, washed, and We conclude that the reduction of gp10044–59 epitope presentation incubated in the presence of the indicated inhibitors for 16 h. Fixed cells after AP-2 knockdown is a consequence of preventing access of were incubated with gp10044–59-specific murine CD4 T cells, and epitope cell surface gp100 to MHC-II processing compartments. presentation evaluated based on IFN-␥ secretion measured by ELISA.

Epitope gp10044–59 presentation is reduced in pigmented melanomas that contain melanosomes in addition to presentation in AP-2-deficient cells could be due to limited avail- conventional endosomes ability of newly synthesized HLA-DR*0401 molecules in endo- We next investigated whether the segregation of gp100 into mela- somes. However, DM331 melanoma fails to express significant nosomes in pigmented cells reduced presentation of gp10044–59 amounts of Ii chain (Fig. 7A). To directly test whether AP-2 relative to depigmented melanoma cells in which gp100 localizes knockdown decreased endosomal availability of newly synthe- to MHC-IIϩ endosomes. For this evaluation, we used 1011mel and sized HLA-DR*0401 molecules in DM331, we treated control and a second pigmented melanoma cell line, MNT-1. As with DM331, AP-2-deficient cells with brefeldin A for 12 h to trap newly syn- neither of these cells expresses Ii chain (Fig. 7A and data not thesized MHC-II complexes in the cis-Golgi or medial-Golgi and shown). Both were transfected to express HLA-DR*0401 and clear late endosomal compartments of mature MHC-II molecules sorted by flow cytometry to select cells expressing levels of sur- (detected with L243 mAb) (Fig. 7B, left). When brefeldin A was face MHC-II molecules similar to DM331-GP (Fig. 8A). The washed out and the cells incubated for 4.5 h, mature MHC-II mol- gp10044–59 epitope presentation was markedly reduced in both ecules were detected in endosomal compartments of both control 1011mel-DR4 and MNT-1-DR4, requiring approximately eight The Journal of Immunology 7849

FIGURE 9. In pigmented melanoma, gp10044–59 epitope is produced in early but not late endosomes. A, 1011mel pigmented melanoma were trans- fected DRB1*0401 ␣-chain and ␤-chain and control or AP-2-specific siRNA. After 72 h of transfection, the indicated number of cells was in-

cubated with gp10044–59-specific murine CD4 T cells and epitope presen- tation evaluated based on IFN-␥ secretion measured by ELISA. B, 1011mel pigmented melanoma cells were transfected with DRB1*0401 ␣-chain and ␤-chain. Twelve hours after transfection, cells were incubated in the pres- ence of the indicated inhibitor for 16 h. After fixation with paraformalde- Downloaded from

hyde, the indicated number of cells was incubated with gp10044–59-specific murine CD4 T cells and peptide presentation evaluated based on IFN-␥ secretion measured by ELISA. Data represent average value of duplicate wells, with error bar indicating SD. http://www.jimmunol.org/

full-length gp100 and a 26-kDa fragment that can be produced by proteolysis in either endosomes or early stage melanosomes (24, 29) (Fig. 8E). However, both MNT-1 and 1011mel have much higher levels of the mature 34- to 38-kDa gp100 fragment pro- duced only in melanosomes and detected by mAb HMB45 (30), as compared with DM331-GP (Fig. 8F and data not shown). Al-

though this gp100 fragment does not encompass the gp10044–59 peptide, its prevalence indicates that the relative paucity of by guest on October 1, 2021 gp100 presentation is also not due to a lack of source protein FIGURE 8. The gp10044–59 epitope presentation is dramatically re- 44–59 duced in pigmented melanoma cells. 1011mel and MNT-1 pigmented mel- expression. Instead, it suggests that this limitation is a consequence anoma cells were transfected with genes encoding DR*0401 ␣-chain and of source protein localization. ␤ -chain (A–C) or AAD (a chimeric molecules that has the HLA-A2 peptide The gp10044–59 epitope presentation is completely abolished in binding site) (B–D), and incubated for 48 h. A, DR*0401 transfectants were AP-2-depleted 1011mel-DR4 (Fig. 9A), demonstrating that gp100 stained using the HLA-DR4-specific mAb NFLD.D10, and electronically internalized from plasma membrane is the only source protein for sorted to select cells expressing the same level of HLA-DR4 as DM331-GP production of the gp10044–59 epitope in pigmented melanoma. To cells. Postsort mean intensity fluorescence (MFI) of each cell line. B, The test whether gp100 is produced in conventional early or late indicated number of sorted DR4*0401 transfectants was incubated with 44–59 endosomes, despite the under-representation of gp100 localization gp10044–59-specific murine CD4 T cells and epitope presentation measured high by IFN-␥ secretion. C, AAD transfectants were stained using the HLA- in LAMP-1 compartments, we analyzed the effect of prima- A2-specific mAb CR11–351. D, The indicated number of AAD transfec- quine or leupeptin treatment on epitope presentation. 1011mel tants was incubated with Tyr369-specific CD8 T cells, and epitope presen- were transfected with DRB1*0401 and expression allowed to oc- tation was measured by IFN-␥ secretion. E, The indicated melanoma cells cur in the presence of inhibitors, then fixed and incubated with ϫ 5 were solubilized and 5 10 cell equivalent of melanoma cell was sepa- murine CD4 T cells specific for gp10044–59/DRB1*0401. Leupep- rated by SDS-PAGE, followed by immunoblotting with Abs specific for tin had no effect on gp10044–59 presentation, whereas primaquine the C terminus of gp100 (␣Pep13h). F, Melanoma cells were lysed using treatment led to a substantial reduction (Fig. 9B). These results M-PER, which allows recovery of gp100 from stage II melanosomes. SDS- indicate that, in pigmented melanomas, gp10044–59 is generated PAGE and immunoblotting were performed using lumenal gp100-specific entirely within early endosomes or stage I melanosomes and not in mAb HMB45. late endosomal compartments. times as many cells as DM331-GP to achieve T cell comparable Discussion activation (Fig. 8B). Although both of these cells expressed lower In the current work, we investigated the mechanism of presentation levels of HLA-A2 than DM331-GP (Fig. 8C), 1011mel presented of an MHC-II-restricted epitope from gp100 (Pmel17), a cellular Tyr369 to specific CD8 T cells comparably to DM331-GP, whereas protein with an endosomal or melanosomal localization. Using MNT-1 was about half as effective (Fig. 8D). Thus, the substantial melanoma cells that have down-regulated components of the mela- differences in presentation of gp10044–59 were not a consequence nosomal pathway, but constitutively express HLA-DR*0401 mol- of general differences in the ability to stimulate T cells. ecules, we established that the majority of gp100 is sorted to Western blot analysis using the C-terminal specific Ab ␣Pep13h LAMP-1high/MHC-IIϩ conventional endosomes. In these cells, showed that all three melanoma lines express similar levels of presentation of the gp10044–59 epitope required localization of 7850 MHC-II EPITOPE PROCESSING IN MELANOMA CELLS gp100 source protein to endosomes and processing by acidic pro- epitopes generated in these two compartments are antigenically teases. We also found that similar antigenic epitopes were pro- indistinguishable, our data do not exclude the possibility that struc- duced in both early and late endosomes, despite differences in turally different peptides containing the epitope are generated with processing requirements. Epitope presentation depends in large greater or lesser efficiency in either compartment. part on gp100 trafficking to the plasma membrane and internaliza- The inhibition of epitope presentation by siRNA-mediated de- tion mediated by the AP-2 adaptor protein. We also established pletion of AP-2 suggests that the major forms of gp100 that serve that the presentation of this epitope was dramatically reduced in as substrates for gp10044–59 epitope presentation were internalized pigmented melanomas that contain melanosomes in addition to from the plasma membrane. This observation supports and extends conventional endosomes, consistent with the localization of gp100 previous work demonstrating a correlation between gp100 cell sur- to melanosomes and not to LAMP-1high MHC-IIϩ late endosomes. face expression and presentation of a DR*0701- restricted epitope, ϩ In these cells, gp10044–59 presentation still depends on protein which led to the suggestion that gp100 accessed MHC-II pro- internalization from the cell surface via AP-2, but processing oc- cessing compartments by endocytosis from the plasma membrane curs entirely in early endosomes or stage I melanosomes and not (66). AP-2 directs cargo internalization by recognition of tyrosine- late endosomes. Collectively, our results emphasize that: process- or di-leucine-based sorting signals located in cytoplasmic domains ing of gp100 epitopes is directly tied to intracellular targeting of (61, 67), and a di-leucine-based signal in the cytoplasmic domain the source protein; melanosomes are relatively poor compartments of gp100, which is deleted in the natural Silver mutation of mouse for MHC-II processing, despite their endosomal origin; and de- Pmel17, has been shown to be required for its efficient internal- differentiation of melanoma cells, resulting in loss of melano- ization and accumulation in melanosome precursors (34). These somes, augments presentation of epitopes from those MDP that data suggest that AP-2 facilitates internalization by interaction continue to be expressed due to alterations in their endosomal with the di-leucine-based motif. Interestingly, LePage and Downloaded from targeting. Lapointe (66) reported that deletion of the gp100 cytoplasmic do- Previous studies demonstrated that endogenous cytoplasmic main reduced presentation of the DRB1*0701-restricted epitope, (49, 62) and transmembrane (50) proteins can be processed by the whereas deletion of the di-leucine motif did not, despite increased proteasome for presentation by MHC-II molecules. The resulting mutant protein localization to cell surface. However, that work peptides reach endosomal compartments through TAP (51) or used gp100 transfectants to assess this issue, whereas we have used LAMP-2 (63). However, high and sustained levels of cytosolic cell expressing the endogenous gp100 gene. Overexpression of http://www.jimmunol.org/ source protein may be required compared with class I-mediated rec- gp100 minimizes the influence of the endocytosis signal on effi- ognition (64, 65). In this study, we show that inhibition of protea- cient localization into endosomes (31, 34), perhaps because of AP- somes by lactacystin had no effect on presentation of gp10044–59, 2-independent internalization or direct transport from the trans- although it stabilized gp100 fragments in both membrane and cy- Golgi network, and may explain the apparent discrepancy of our tosol. This indicates that defective ribosomal products or poorly results and theirs. Our data do not exclude the possibility that a folded forms of the protein that were retro-translocated into the cohort of gp100 molecules traverses an alternative transport path- cytosol are inefficient substrates for MHC-II presentation, com- way from the trans-Golgi network directly to stage I melanosomes pared with full-length protein directly targeted to endosomes, even as has been proposed (33), but suggests that this is a minor path- by guest on October 1, 2021 though the latter are fully folded and thus presumably more resis- way at best. Thus, our results establish that an endosomal protein tant to proteolytic degradation. reaches MHC-II processing compartments by endocytosis mech- Using mutant forms of gp100 that localize to different compart- anisms resembling those responsible for internalization of exoge- ments, we established that antigenically indistinguishable epitopes nous proteins from the plasma membrane. are produced in both early and late endosomes. Thus, gp10044–59 Our results represent one of the first analyses of MHC-II pro- is among a limited number of epitopes for which presentation via cessing and presentation capabilities of melanosomes. Previous recycling MHC-II molecules in early endosomes has been dem- studies showed that melanosomes share a number of characteris- onstrated and the only one so far described to originate from an tics with conventional endosomes, including low luminal pH and endogenous protein with an endosomal or melanosomal localiza- the presence of lysosomal proteases and membrane proteins (18). tion; other endosomal recycling-dependent epitopes have been de- In contrast, we found that MHC-II molecules accumulated in con- rived from exogenous (37, 40–42) or cytosolic (51) proteins. ventional LAMP-1high late endosomes, and were largely excluded However, the involvement of AP-2-mediated internalization of from stage II melanosomes. In accordance with previous reports gp100 from the plasma membrane suggests that endocytosis is a (16), the majority of gp100 was localized in stage II melanosomes. common mechanism to deliver epitope source proteins, either en- In keeping with our results demonstrating that gp100 targeting to dogenous or exogenous, to early endosomes for presentation by endosomes was the primary determinant of the presentation of recycling MHC-II molecules. gp10044–59 by class II molecules, these cells presented this epitope Other studies have demonstrated that distinct MHC-II-restricted inefficiently, and the processing was confined to early endosomes. peptides are produced in early and late endosomal compartments. These data indicate that the MHC-II molecules and MDP intersect Epitopes produced in late endosomes require more substantial at the early or stage I melanosome, but are segregated source protein denaturation, whereas those produced in early en- thereafter, presumably via specific sorting signals. It will be inter- dosomes are produced by mild proteolysis, and may be destroyed esting to determine whether melanosomal targeting also dimin- in the harsher environment of late endosomes (37, 42). In this ishes the presentation of epitopes from other MDPs such as ty- study, we demonstrated that gp10044–59 is generated by leupeptin- rosinase or Tyrp1, particularly if these epitopes require the more sensitive proteases in late endosomes, and by leupeptin-insensitive robust processing environment of late endosomal compartments. proteases in early endosomes. This may be facilitated by the fact Conversely, it will also be interesting to determine whether muta- that the protein itself is a normal resident of endosomes, and thus tions that interfere with transit of these proteins to melanosomes able to resist denaturation. In contrast, the N-terminal region of (68–71) enhance presentation of epitopes from these proteins. gp100, which contains this epitope, seems to be dissociated or An inverse correlation between expression of MHC-II mole- degraded during the maturation of melanosomes (35, 57), suggest- cules and of MDPs has been previously reported (15). However, ing that it is accessible to endosomal proteases. Although the melanocytes and some melanomas express MHC-II after induction The Journal of Immunology 7851 with IFN-␥ (11–13). It remains to be determined whether IFN-␥ 16. Raposo, G., D. Tenza, D. M. Murphy, J. F. Berson, and M. S. Marks. 2001. stimulation also alters the localization of MDP from melanosomes Distinct protein sorting and localization to premelanosomes, melanosomes, and lysosomes in pigmented melanocytic cells. J. Cell Biol. 152: 809–824. to late endosomes. Conversely, the limited ability of pigmented 17. Pierre, P., L. K. Denzin, C. Hammond, J. R. Drake, S. Amigorena, P. Cresswell, melanoma cells to present MHC-II epitopes derived from MDPs is and I. Mellman. 1996. HLA-DM is localized to conventional and unconventional MHC class II-containing endocytic compartments. Immunity 4: 229–239. likely to make presentation of MDP-derived epitopes more reliant 18. Orlow, S. J. 1995. Melanosomes are specialized members of the lysosomal lin- on uptake by professional APC. Thus, the contribution of epitope eage of . J. Invest. Dermatol. 105: 3–7. presentation directly by tumor or mediated by professional APC 19. Hearing, V. J. 1999. Biochemical control of melanogenesis and melanosomal organization. J. Invest. 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