Molecular Immunology 66 (2015) 418–427

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A conserved WW domain-like motif regulates invariant chain-dependent cell-surface transport of the NKG2D ligand ULBP2 Franziska Uhlenbrock ∗ , Esther van Andel, Lars Andresen, Søren Skov ∗ Laboratory of Immunology, Section for Experimental Animal Models, Faculty of Health and Medical Sciences, University of Copenhagen, Stigbøjlen 7, DK-1870 Frederiksberg, Denmark

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Article history: Received 24 September 2014 Received in revised form 16 April 2015 Accepted 22 April 2015 Available online 18 May 2015 Keywords: NKG2D ligands ULBP2 Invariant chain WW domain Cell-surface transport

a b s t r a c t Malignant cells expressing NKG2D ligands on their cell surface can be directly sensed and killed by NKG2D-bearing lymphocytes. To ensure this immune recognition, accumulating evidence suggests that NKG2D ligands are trafficed via alternative pathways to the cell surface. We have previously shown that the NKG2D ligand ULBP2 traffics over an invariant chain (Ii)-dependent pathway to the cell surface. This study set out to elucidate how Ii regulates ULBP2 cell-surface transport: We discovered conserved tryptophan (Trp) residues in the primary protein sequence of ULBP1-6 but not in the related MICA/B. Substitution of Trp to alanine resulted in cell-surface inhibition of ULBP2 in different cancer cell lines. Moreover, the mutated ULBP2 constructs were retained and not degraded inside the cell, indicating a crucial role of this conserved Trp-motif in trafficking. Finally, overexpression of Ii increased surface expression of wt ULBP2 while Trp-mutants could not be expressed, proposing that this Trp-motif is required for an Ii-dependent cell-surface transport of ULBP2. Aberrant soluble ULBP2 is immunosuppressive. Thus, targeting a distinct protein module on the ULBP2 sequence could counteract this abnormal expression of ULBP2. © 2015 Elsevier Ltd. All rights reserved.

1. Introduction NKG2D ligands are cell-surface molecules, which in humans belong to either the MIC (MICA and MICB) or the UL16-binding protein (ULBP1-6) family (Champsaur and Lanier, 2010). The upregulation of NKG2D ligands signals cellular distress and evokes immune responses mediated by several immune cells expressing the activating receptor NKG2D. The recognition of eight different ligands by one single receptor is unusual and the functional as well as evolutionary diversity of these ligands has been studied intensively. An imbalanced cell-surface expression of NKG2D ligands is associated with virus infection, tumor development and certain autoimmune diseases (Groh et al., 2001, 2002, 2003; Raffaghello et al., 2004; Routes et al., 2005). Indeed, it has been shown that different members of the NKG2D ligand families hallmark these diseases (Li et al., 2009; Nuckel et al., 2010; Paschen et al., 2009). Hence, it is of particular interest to understand and characterize the regulatory mechanisms, which control the cell-surface expression of each NKG2D ligand. We have previously described that the NKG2D ligand ULBP2, in contrast to the ligands MICA/B, traffics over

∗ Corresponding authors. Tel.: +45 353 33126. E-mail addresses: [email protected] (F. Uhlenbrock), [email protected] (S. Skov). http://dx.doi.org/10.1016/j.molimm.2015.04.022 0161-5890/© 2015 Elsevier Ltd. All rights reserved.

an endosomal pathway to the cell surface. Moreover, ULBP2 cellsurface transport is dependent on invariant chain (Ii) expression (Uhlenbrock et al., 2014). One of the roles of Ii is to function as an ER chaperone: Ii assembles MHC class II (MHC II) ␣ and ␤ subunit molecules into heterodimers by binding to their peptide binding grooves (Amigorena et al., 1995; Anderson and Miller, 1992). Interaction with Ii also changes the conformation of MHC II molecules, which has been suggested to promote intracellular transport and MHC II receptor assembly (Amigorena et al., 1995; Verreck et al., 2001). In addition, Ii possesses an endosomal targeting motif that guides MHC II molecules to endocytic compartments containing internalized antigens. The release of Ii in these compartments leads to binding of antigen peptides to the class II binding groove (Bakke and Dobberstein, 1990; Bremnes et al., 1994; Roche and Cresswell, 2011). Notably, Ii is not completely digested. Instead, an Ii fragment, referred to as class II associated invariant chain peptide (CLIP), remains associated to the MHC II binding groove, which has been suggested to regulate assembly and intracellular transport of MHC II heterodimers (Ghosh et al., 1995; Romagnoli and Germain, 1994). Non-CLIP sites are also critical for the interaction between Ii and MHC II molecules. In this respect, Stumptner and Benaroch (1997) showed that a proline-rich sequence of Ii stabilizes its binding to MHC II molecules. How this proline-rich sequence interacts with MHC II molecules remained unclear until Neumann and Koch

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(2006) identified a conserved WW-like motif within MHC II ␤ chains that associates with Ii. WW domains are one of the smallest known protein domains and named after the presence of two strictly conserved tryptophan (Trp, W) residues that are separated from each other by 20–22 amino acids. Their three-dimensional (3D) structure is characterized by a three-stranded, antiparallel ␤-sheet with two ligand binding grooves (Bork and Sudol, 1994; Macias et al., 1996; Sudol, 1996; Sudol et al., 1995a, 1995b). Functionally, a WW domain binds to proline-rich ligands and thereby establishes protein-protein interactions (Sudol, 1996). Interestingly, WW domains are present in multiple structural and signaling proteins that are important for the regulation of cellular processes ranging from RNA transcription and protein trafficking to receptor signaling (Salah et al., 2012; Sudol et al., 2005). In this study we searched for possible Ii-binding structures on the ULBP2 protein. Strikingly, we discovered a WW domain-like structure containing conserved Trp residues within the protein sequence of ULBP1-6. With regard to NKG2D ligand ULBP2, we found that substitution of these conserved Trp residues inhibited cell-surface expression of ULBP2. In addition, the interaction of these Trp residues and Ii was examined.

2. Materials and methods 2.1. Cell lines The Jurkat Tag-9 (acute human T cell leukemia) cell line was kindly provided by Dr. Carsten Geisler (Department of International Health, Immunology and Microbiology, University of Copenhagen, Denmark). Jurkat Tag-9 cells are stably transfected with the large T antigen from SV40. The HEK-293T (human embryonic kidney) cell line was kindly provided by Novo Nordisk, Denmark. The FM-86 (human melanoma) and the HT-29 (human colon adenocarcinoma) cell lines were kindly provided by Dr. Per thor Straten (CCIT, Herlev University Hospital, Denmark). The MDAMB-231 (human breast cancer) cell line was kindly provided by Dr. José Moreira (Department for Veterinary Disease, University of Copenhagen, Denmark). With the exception of HEK-293T and MDA-MB-231 cells, all cell lines were grown in 10%FBS/RPMI 1640 (Sigma, Brøndby, Denmark) supplemented with 2 mM glutamine, 2 mM penicillin/streptomycin. HEK-293T and MDA-MB-231 cells were grown in 10%FBS/DMEM +GlutaMAX with 2 mM penicillin and streptomycin.

2.2. Sequence alignment and crystal structure The primary protein sequence alignment was visualized with the CLC Sequence Viewer free software version 6.7.1 (EMEA, Aarhus, Denmark). The ULBP3 structure was based on the PDB ID 1KCG: (http://www.rcsb.org/pdb/explore.do?structureId=1KCG). Visualization and annotation was done using Discovery Studio 4.0 from Accelrys and Adobe Illustrator CS6.

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2.4. Mutagenesis Site-directed mutagenesis was performed using the QuickChange Lightning Multi Site-Directed Mutagenesis kit (Agilent technologies, Glostrup, Denmark) following the manufacturer’s instructions for templates larger than 5 kb. The ULBP2-GFP-myc construct was used as a template for the generation of the ULBP2 mutations, in which single tryptophan amino acids (position: 139, 152 or 172) or the combination of all three were substituted for an alanine, respectively. Positions were chosen with respect to the first methionine. Primer design was performed according to the instructions in the mutagenesis protocol using the free web-based QuickChange Primer Design program (Agilent technologies, Glostrup, Denmark). The following primers were purchased from Eurofins MWG Operon, Germany: W139A: 5 -GAAGGACACAGCAGTGGATCTGCGCAGTTCAGTTTCG-3 ; W158A: 5 -GACTCAGAGAAGAGAATGGCGACAACGGTTCATCCTGG-3 ; W172A:5 -CCAGAAAGATGAAAGAAAAGGCGGAGAATGACAAGGTTGTGG-3 . Selection was performed on Luria Broth (LB) agar plates with 100 ␮g/mL ampicillin (Bistrol-Myers Squibb, Lyngby, Denmark). Single colonies were picked for growth. Plasmid DNA was purified using the NucleoSpin Plasmid QuikPure kit (Macherey-Nagel, Düren, Germany) and subsequently sequenced at Eurofins MWG Operon, Germany using the following sequencing primer: SP6: 5 -CA TTT AGG TGA CAC TAT AG-3 . Sequences were analyzed by using the free software CLC Sequence Viewer version 6.7.1 (EMEA, Aarhus, Denmark). Plasmids containing the correct mutations were amplified through growth of bacteria in selective medium and purified using the NucleoBond Xtra Midi EF kit (Macherey-Nagel, Düren, Germany). 2.5. Transient transfection Transient transfections of Jurkat Tag-9, HT-29, FM-86, MDAMB-231 and HEK-293T cell lines were performed using the Amaxa Nucleofector Device (Lonza, Copenhagen, Denmark) according to the manufacturer’s protocol. In short, for each cell line: Jurkat Tag-9; 2 × 106 cells were resuspended in 100 ␮L of Ingenio Electroporation Solution (Mirus Bio LLC, Madson, US-Wi), mixed with 1 ␮g of plasmid DNA/1 × 106 cells, and pulsed using the Nucleofector program G-010. HT-29; 1.5 × 106 cells were resuspended in 100 ␮L Cell Line Nucleofector Solution R (Lonza, Copenhagen, Denmark), mixed with 2 ␮g of plasmid DNA/1.5 × 106 cells, and pulsed using the Nucleofector program Q-009. FM-86; 2.5 × 106 cells were resuspended in 100 ␮L of Nucleofector solution T (Lonza, Copenhagen, Denmark), mixed with 1 ␮g of plasmid DNA/1 × 106 cells, and pulsed using the Nucleofector program U-020. MDA-MB-231; 2.5 × 106 cells were resuspended in 100 ␮L Cell Line Nucleofector Solution V (Lonza, Copenhagen, Denmark), mixed with 1 ␮g of plasmid DNA/1 × 106 cells, and pulsed using the Nucleofector program X-013. HEK-293T; 1.5 × 106 cells were resuspended in 100 ␮L Cell Line Nucleofector Solution T (Lonza, Copenhagen, Denmark), mixed with 1 ␮g plasmid DNA/1.5 × 106 cells and pulsed using Nucleofector program Q-001. Cells were resuspended in fresh medium and plasmid expression was analyzed 22–24 h post transfection. 2.6. Flow cytometry

2.3. Plasmids The generation of the ULBP2-GFP-myc construct was previously described (Uhlenbrock et al., 2014). The construct contains the full-length ULBP2 gene downstream a generic leader, a GFP cassette and a myc tag. The invariant chain construct (Ii31(33)) was kindly provided by Dr. Norbert Koch (University of Bonn, Germany) (Neumann and Koch, 2006).

Antibody cell-surface staining and intracellular staining experiments were done as previously described (Andresen et al., 2007; Jensen et al., 2013). Cell-surface expression of the wild type (wt) and mutated ULBP2-GFP-myc fusion proteins were detected by using an anti-myc tag antibody (clone 4A6, #05-724, Millipore, Billerica, USA-MA). Binding of recombinant hNKG2D-Fc (#1299-NK, R&D Systems, Abingdon, UK) to the different ULBP2-GFP-myc fusion

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proteins was detected by using an APC-conjugated mouse antihNKG2D antibody. Overexpression of intracellular Ii was detected by a mouse anti-hCD74 (PIN.1, # sc-47742, Santa Cruz, Heidelberg, Germany) antibody. APC-conjugated goat anti-mouse IgG (#405308, Biolegend, London, UK) was used as secondary antibody and isotype control. Flow cytometry data acquisition was performed with a BD Accuri C6 Flow Cytometer and CFlow software, while analysis of the collected data was done in FCSexpress 3.0 (De Novo, Glendale, USA-CA). Gating on transfected cells was carried out on viable cells (Gate 1) according to a GFP-negative control plasmid (pUC18). Transfection efficiency was determined by GFP intensity and surface expression of the different ULBP2-GFPmyc fusion proteins was investigated by myc-tag surface staining of GFP positive cells.

2.9. Statistics Data in Figs. 2B and 4B were analyzed by one-way analysis of variances with Tukey’s multiple comparisons test to determine significant differences among groups. The level of ULBP2 construct cell-surface expression in Jurkat Tag-9 cells was analyzed by the same statistical method (Fig. 2C left). Unpaired, two-sided t-tests were applied to investigate cell-surface expression of wt ULBP2 vs. 3xW ULBP2 in HT-29, FM-86 and MDA-MB-231 cells (Fig. 2C, graphs 2–4), as well as the difference in MFI of myc-positive cells transfected with or without the li-construct for the different ULBP2 fusion proteins (Fig. 5B). Data are presented as mean ± s.d. and the level of statistical significance was set at p < 0.05 for all experiments. The statistical analysis and graph preparation was performed using the software package Prism, version 6 (GraphPad Software Inc.; La Jolla (Ca), USA).

2.7. Western blot All cell lines were lysed in RIPA buffer (1% NP40, 20 mmol/L Tris–HCl (pH 8.0), 140 mmol/L NaCl, 10% glycerol, 1 mmol/L PMSF, 1 mmol/L Na3 VO4 , 10 mmol/L NaF, 1 mmol/L IAA, 5 mmol/L EDTA, and 7.5 Ag/mL aprotinin) for 30 min at 4 ◦ C. Cell lysates were clarified by centrifugation at 16 000 × g for 15 min at 4 ◦ C. Samples were mixed with 1×NuPAGE LDS sample buffer (Invitrogen Life Technologies, Naerum, Denmark) and incubated at 70 ◦ C for 10 min. Proteins were separated in NuPAGE Novex 4–12% Bis–Tris Midi gels (Invitrogen Life Technologies, Naerum, Denmark) with 1×NuPAGE MOPS SDS running buffer (Invitrogen Life Technologies, Naerum, Denmark) and afterwards transferred to nitrocellulose using an iBlotTM Device (Invitrogen Life Technologies, Naerum, Denmark), according to the manufacturer’s protocol The following antibodies were used in this study: ant-GFP rabbit polyclonal antibody (#3999-100, Biovision, Milpitas, USA-CA), anti-GFP rat monoclonal antibody (#3H9, Chromotek, München, Germany), anti-CD74 antibody (#NBP1-33109, Novus Biologicals, Cambridge, UK), antiERK1 rabbit polyclonal antibody clone C-16 (#Sc-93, Santa Cruz, Heidelberg, Germany), peroxidase-conjugated swine anti-rabbit immunoglobulins (#P0399, Dako, Copenhagen, Denmark), IRDye 800CW Goat anti-Rat IgG Secondary antibody (#32219, LI-COR Biosciences, Cambridge, UK) and IRDye 680 LT Donkey anti-Rabbit IgG Secondary antibody (#68023, LI-COR Biosciences, Cambridge, UK). Protein visualization was performed by either ECL detection or using an Odyssey Fc Imager (LI-COR Biosciences, Cambridge, UK).

2.8. Microscopy Jurkat Tag-9 cells were transiently transfected with either the ULBP2-GFP-myc wt or the W139A mutant construct as described above. Post transfection, cells were pelleted for 10 min at 90 × g and washed with PBS. Cells were fixed in 2% PFA for 15 min. After fixation, cells were three times washed with PBS followed by blocking with 4% BSA for 45 min. To stain for construct cell-surface expression, cells were incubated with an anti-myc-tag (clone 4A6, #05-724, Millipore, Billerica, USA-MA) antibody at 4 ◦ C overnight. The next day, cells were again blocked with 4% BSA for 30 min followed by cell incubation with an Alexa-Fluor 565-conjugated goat anti-mouse secondary antibody (Invitrogen Life Technologies, Naerum, Denmark). Cells were stained for 45 min at RT. Cell nuclei were stained with Hoechst dye (Sigma, Brøndby, Denmark). Fixed and stained cells were mounted using ProLong® Gold reagents (Invitrogen Life Technologies, Naerum, Denmark) and sealed. Confocal microscopy was performed with an LSM 780 on an inverted Zeiss AxioObserver Z1 (Carl Zeiss Microscopy GmbH, Germany) with a 40×/1.3 objective.

3. Results 3.1. NKG2D ligands ULBP1-6 display tryptophan residues in their protein sequence in contrast to NKG2D ligands MICA/B Searching for putative binding domains for Ii, we identified a conserved WW-like structure in the primary sequence of the human NKG2D ligands ULBP1-6. This motif was also present in the murine homologs RAE-1 beta and RAE-1 gamma (Fig. 1). Each sequence contained Trp residues at position 139, 158 and 172 with respect to the first methionine of ULBP2. Trp139 and Trp158 are separated by 20 amino acids, whereas Trp158 and Trp172 are only separated by 14 amino acids (Fig. 1). The primary sequence of the NKG2D ligands MICA/B contained no conserved Trp residues (not shown). Next, we used the crystal structure of ULBP3 (55% homologous to ULBP2 Cosman et al., 2001) to visualize the different Trp residues (highlighted in yellow). As seen in Fig. 1B (left) Trp139 and Trp158 are located within the ␤ sheet, whereas Trp172 is part of the ␣ sheet of the protein. Notably, the Trp residues are exposed and located outside the NKG2D binding area (Fig. 1B right). 3.2. Substitution of conserved tryptophan residues inhibits surface transport of ULBP2 in several cancer cell lines Next, we investigated whether the three conserved Trp residues are important for the cell-surface transport of ULBP2. To this end, Trp residues at position 139, 158 and 172 were substituted for alanine (Ala) residues, respectively by site directed mutagenesis in a plasmid encoding wild type (wt) ULBP2 fusion protein. Ala residues were chosen because Ala is known as a neutral, nonreactive amino acid (Matthews et al., 1987). The new constructs, ULBP2 W139A, ULBP2 W158A and ULBP2 W172A together with the original plasmid (ULBP2 wt), were transiently expressed in Jurkat Tag-9 cells. The empty plasmid pUC18 was used as transfection control. The effect of the mutations on cell-surface expression of the ULBP2 GFP-myc fusion protein was analyzed by flow cytometry (Fig. 2A). Transfection efficiency was determined as GFP intensity and surface expression of the different constructs was investigated by myc-tag surface staining of GFP positive cells. As shown in Fig. 2A, substituting the conserved Trp residues either at position W139, W158 or W172 resulted in a robust abrogation of cellsurface expressed ULBP2. This suggests that all three Trp residues are important for ULBP2 cell-surface transport. As expected, no expression of GFP and myc-tag was measured in pUC18-transfected control cells. In addition, no variation in intracellular GFP/myc-tag could be detected (data not shown). To exclude the possibility that inhibition was due to a cell line-specific regulation, we performed a similar experiment in HT-29 colon cancer, FM-86 melanoma

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Fig. 1. NKG2D ligands ULBP1-6 display tryptophan residues in their protein sequence in contrast to NKG2D ligands MICA/B. (A) A list of human NKG2D ligands ULBP 1-6 and murine RAE-1 beta and gamma is shown. Sequences were obtained from the UniProt or NCBI database. The positions of conserved Trp (W) residues are framed and labeled with black arrow heads. The shown positions are with respect to the immature ULBP2 protein (first Met). (B) The crystal structure of ULBP3 is shown to indicate the three different Trp residues: W139, W158 and W172 (highlighted in yellow) on the protein alone (left) or within the binding complex of ULBP3 and NKG2D (right). The crystal structure was modified with Swiss-Deep view3.7 and visualized with Viewer Lite5.0.

and MDA-MB-231 breast cancer cell lines (Fig. 2B). Again, the cell-surface expression of ULBP2 mutations was strongly inhibited compared to ULBP2 wt expression. In addition, we designed a threefold-mutated construct in which all three Trp residues were substituted with Ala (ULBP2 3xW). As seen in Fig. 2C, cell-surface expression of ULBP2 was more reduced compared to each of the single-mutated constructs and wt ULBP2. Twofold-mutated constructs (W139/W156; W139/W172 and W156/W172) inhibited ULBP2 cell-surface expression as expected (data not shown). These results further strengthen our observation that these Trp residues are important for ULBP2 cell-surface transport. 3.3. Inhibition of ULBP2 surface expression after tryptophan substitution in several cancer cell lines Transfection with all constructs resulted in a positive GFP signal measured by flow cytometry analysis. This suggested that the ULBP2 mutations are intracellular retained but not degraded during the timeframe of the assay. To test this, we performed Western blot experiments of whole lysates from all transfected cell lines, detecting the ULBP2 fusion proteins using a GFP-specific antibody (Fig. 3A). The expected molecular mass of the ULBP2 wt fusion protein is 57 kDa. As seen in Fig. 3A, the electrophoretic mobility of the W139A, W158A and W172A constructs were not altered in Jurkat Tag-9, HT-29 and FM-86 cells due to site directed mutagenesis. The expression of the W139A construct in MDA-MB-231 was

marginally decreased. Importantly, after Western blotting no free GFP (30 kDa) was detected in all tested cell lines (data not shown), indicating that the observed cell-surface inhibition was not caused by substantial degradation. Next, the most abrogated monosubstituted mutant (ULBP2 W139A) in Jurkat Tag-9 cells and ULBP2 wt were chosen to be further investigated by fluorescence microscopy. During the staining procedure, cells were fixed but not permeabilized. Hence, cells are only positive for myc in case the construct is expressed on the cell surface. As seen in Fig. 3B, the ULBP2 wt construct was expressed on the cell surface of Jurkat Tag-9 cells (top, myc channel) and the staining co-localized with GFP expression (top, merge channel). Jurkat Tag-9 cells transiently transfected with ULBP2 W139A were negative for myc staining (bottom, myc channel) and the GFP signal was intracellular located (bottom, GFP channel). These results further demonstrate that the mutated constructs are translated but intracellular retained. Taken together, these data demonstrate that substituting the conserved Trp residues affects cell-surface expression of ULBP2 but it does not involve protein degradation. 3.4. Tryptophan-substituted ULBP2 is functionally recognized by NKG2D The transient overexpression of the different ULBP2 constructs in HEK-293T cells led to an interesting observation: Like ULBP2

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Fig. 2. Substitution of conserved tryptophan residues inhibits surface transport of ULBP2 in several cancer cell lines. (A) Jurkat Tag-9 cells were transiently transfected with an empty control plasmid (pUC18), or plasmids encoding GFP-myc-tagged ULBP2 fusion proteins (ULBP2 wt, W139A, W158A and W172A). Cell-surface expression of the different fusion proteins was investigated by flow cytometry. Gating was carried out according to Section 2. GFP intensity is shown in the second row, whereas the third row shows myc surface staining of GFP-positive (Gate1 + 2) cells. Mean fluorescent intensity (MFI) and % positive cells are shown where appropriate. GFP-positive cells (Gate1 + 2) stained with an isotype control are illustrated in the last row. The dot plot data is a representative of at least four different experiments. (B) HT-29, FM-86 and MDA-MB-231 cells were transiently transfected with an empty control plasmid (pUC18), or plasmids encoding for GFP-myc-tagged ULBP2 fusion proteins (ULBP2 wt, W139A, W158A and W172A). Cell-surface expression of the different fusion proteins was investigated by flow cytometry and gating was performed as described under 2A. Data from four different experiments are shown as bar graphs (mean ± s.d.). (C) Jurkat Tag-9 cells, HT-29, FM-86 and MDA-MB-231 cells were transiently transfected with an empty control plasmid (pUC18), or plasmids encoding for GFP-myc-tagged ULBP2 fusion proteins (ULBP2 wt, W139A, W158A, W172A or 3xW). Cell-surface expression of the different fusion proteins was investigated by flow cytometry and gating was performed as described under 2A. Data from four different experiments are shown as bar graphs (mean ± s.d.). *** p < 0.0001, ** p < 0.01, * p < 0.05.

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Fig. 3. Inhibition of ULBP2 surface expression after tryptophan substitution in several cancer cell lines. (A) Jurkat Tag-9, HT-29, FM-86 and MDA-MB-231 cells were transiently transfected with an empty control plasmid (pUC18) or with plasmids encoding for GFP-myc-tagged ULBP2 fusion proteins (ULBP2 wt, W139A, W158A and W172A). Whole cell lysates were prepared and used for Western blotting. Construct translation was detected by an anti-GFP antibody (57 kDa). An anti ERK1 antibody was used as loading control (44 kDa). Western blots represent data from three different experiments. (B) Jurkat Tag-9 cells were transiently transfected with ULBP2 wt or ULBP2 W139A fusion proteins. Post transfection cells were fixed using PFA and construct cell-surface expression was investigated by anti-myc staining. Cell nuclei were visualized using Hoechst stain. Confocal microscopy was performed with an LSM 780 on an inverted Zeiss AxioObserver Z1 with a 40×/1.3 objective. Data represent two different experiments.

wt, all mono substituted mutations were highly expressed in HEK293T cells, but only transfection with ULBP2 3xW resulted in a reduced ULBP2 cell-surface expression (Fig. 4A). This proposes that HEK-293T cells regulate ULBP2 similarly compared to the other examined cell lines, but with less stringency. The HEK-293-T cell line is a good tool to investigate the functional interaction between the NKG2D receptor and the mono substituted mutations as they are expressed on the cell surface comparable to the wt construct. Therefore, transiently, with W139A, W158A, W172A and wt ULBP2, transfected HEK-293T cells

were cell-surface stained with NKG2D-Fc. As shown in Fig. 4B, NKG2D-Fc recognized all three mutants. This implies that the conserved Trp residues are crucial for ULBP2 sequestration but do not disturb recognition by NKG2D. In addition to NKG2D-Fc, an anti-ULBP2/5/6 antibody was also able to bind to all three ULBP2 mutants (data not shown). Moreover, we confirmed that the cellsurface expression of endogenous NKG2D ligands was not altered during the course of the experiment (data not shown). These results indicate that ULBP2 maintains its functional NKG2D ligand structure after mutating single Trp residues.

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Fig. 4. Tryptophan-substituted ULBP2 is functional-recognized by NKG2D. (A) HEK-293T cells were transiently transfected with an empty control plasmid (pUC18), or with plasmids encoding GFP-myc-tagged ULBP2 fusion proteins (ULBP2 wt, W139A, W158A, W172A and 3xW). Cell-surface expression of the different fusion proteins was investigated by flow cytometry. Gating was carried out according to Section 2. GFP intensity is shown in the second row, whereas the third row shows myc surface staining of GFP-positive (Gate1 + 2) cells. Mean fluorescent intensity (MFI) and % positive cells are shown where appropriate. GFP-positive cells (Gate1 + 2) stained with an isotype control are illustrated in the last row. Data is a representative of four different experiments. (B) HEK-293T cells were transiently transfected with an empty control plasmid (pUC18), or with plasmids encoding GFP-myc-tagged ULBP2 fusion proteins (ULBP2 wt, W139A, W158A and W172A). Post transfection, cells were stained with NKG2D-Fc and receptor binding was detected using an anti-NKG2D antibody. Gating was carried out on the isotype control. The pUC18-transfected cells are positive after staining with recombinant NKG2D-Fc due to their endogenous MICA/B and ULBP2 cell-surface expression. Data from four different experiments are shown as bar graphs (mean ± s.d.). *** p < 0.0001.

3.5. Overexpression of the invariant chain enhances ULBP2-wt but not tryptophan-substituted ULBP2 The results stated above clearly indicate that substitution of the conserved Trp residues in the ULBP2 sequence causes the cellsurface inhibition of ULBP2 fusion proteins. However, it is yet unclear whether this WW domain-like motif is important for Iidependent cell-surface transport of ULBP2. To further test this hypothesis, we confirmed by Western blotting that all used cell lines endogenously express Ii (Fig. 5A). Next, we transiently overexpressed ULBP2-wt, W139A, W158A or W172A alone as well as together with an Ii construct in Jurkat Tag-9 cells. We have previously shown that endogenous ULBP2, but not MICA/B, cell-surface expression is induced after Ii overexpression (Uhlenbrock et al.,

2014). As shown in Fig. 5B ULBP2 wt was significantly increased after Ii overexpression. However, cell-surface expression of the ULBP2 mutants was not increased after Ii overexpression. Overexpression of the Ii construct was verified by intracellular FACS staining (Fig. 5C). These data suggest that the WW domain-like motif on ULBP2 proteins is of functional importance for Ii-mediated cell-surface transport of ULBP2. 4. Discussion A tight regulation of NKG2D ligand surface expression is crucial as imbalance can lead to sustained virus infection, tumor development and autoimmunity (Groh et al., 2001, 2002, 2003; Raffaghello

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Fig. 5. Overexpression of the invariant chain enhances ULBP2-wt but not tryptophan-substituted ULBP2. (A) The expression of Ii (34 kDa) was verified by Western blotting of whole cell lysates from Jurkat Tag-9, HT-29, FM-86, MDA-MB-231 and HEK293T cells. ERK1 (44 kDa) was used as loading control. (B) Jurkat Tag-9 cells were transiently transfected with plasmids encoding for GFP-myc-tagged ULBP2 fusion proteins (ULBP2 wt, W139A, W158A and W172A) alone or in combination with an expression construct for Ii (differences in DNA concentration were adjusted by empty plasmid DNA). Cell-surface expression of the different fusion proteins was investigated by flow cytometry with one half of the transfected cells and gating was carried out as described under Section 2. Data from four different experiments are shown as bar graphs (mean ± s.d.). The dot plot data (MFI; Gate 1 + 2) is a representative of one of the four different experiments. (C) The second half of the transfected cells was fixed, permeabilized and endogenous expression and overexpression of Ii was visualized by intracellular staining using flow cytometry and an antibody against Ii. The histogram is a representative of four different experiments. * p < 0.05.

et al., 2004; Routes et al., 2005). Consequently, it is of profound interest to reveal mechanisms that regulate cell-surface expression of NKG2D ligands in order to better understand the different biological and biochemical features that modulate this diverse set of ligands. In previous work, we studied and compared the posttranslational cell-surface transport of the NKG2D ligands MICA/B and ULBP2 in various cancer cells. We discovered that the NKG2D ligand ULBP2, in contrast to MICA/B, traffics via an endosomal, Ii

dependent pathway to the cell-surface (Uhlenbrock et al., 2014). In this study sequence alignments revealed three, ULBP-family specific, conserved Trp residues, which are likely to interact with Ii. Upon mono-, two-, and threefold substitution of these Trp residues, ULBP2 cell-surface expression was robustly inhibited in different cancer cell lines. By using Western blot and confocal microscopy, we demonstrate that the Trp-mutated ULBP2 constructs were retained and not degraded inside the cell. In addition, we show that transfection with both ULBP2 wt and Ii caused increased

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cell-surface expression of ULBP2. In contrast, overexpression of ULBP2 mutants together with Ii had no effect on ULBP2 cell-surface expression. It has been shown that Ii also facilitates cell-surface expression of MHC I molecules (Huang et al., 2008; Sille et al., 2011; Sloma et al., 2008). However, it has not been investigated, how these MHC I molecules interact with Ii. Due to the lack of CLIP, interaction of Ii and MHC I molecules, in this case ULBP2, possibly occurs at alternative sites. Interestingly, Neumann and Koch (2006) identified a conserved WW domain-like structure (named WWCII) on MHC II molecules that interacts with a proline-rich structure on the sequence of Ii. Based on this data and our current findings, we hypothesized that the conserved Trp residues found in ULPBs could be the basis for the formation of such a WW-domain like structure. Like WWCII domains, the here uncovered Trp-motif also has a different architecture compared to classical WW domains present in proteins such as dystrophin (Bork and Sudol, 1994): Trp residues are separated from each other by 14 and 20 residues and not 20–22 residues as proposed by Sudol et al. (Sudol, 1996). Thus, it seems likely that the proline-rich sequence of Ii is tolerant in its interactions with potential binding partners because it is also capable of associating with WW domain-like structures, which is supported by our own study. Our present experiments do not show a physical interaction of ULBP2 and Ii via these conserved Trp residues; however, the presented data strongly suggest that the Trp residues are important for a biological-relevant association of ULBP2 and Ii. So far, we did not succeed in studying the interaction of Ii and ULBP2 in co-immunoprecipitation experiments. Nonetheless, we think that additional biochemical and structural studies of the defined Trpmotif in the sequence of ULBP2 could help to further reveal the direct interaction of Ii and ULBP2. In all tested cancer cell lines, cell-surface expression of 3xW ULBP2 was nearly completely abrogated, whereas a low cell surface expression of mono substituted W139A-, W158A- or W172A ULBP2 could be detected in HT-29, FM-86 and MDA-MB-231 cells. This residual ULBP2 expression could be explained by the fact that a single-mutated Trp on ULBP2 protein is not enough to prevent the interaction of Ii and ULBP2. Moreover, there are numerous evidences that cancers develop different strategies to alter their cell-surface expression of NKG2D ligands and that NKG2D ligands traffic via an alternative pathway to the cell surface once their conventional pathway is blocked (Uhlenbrock et al., 2014; Fernandez-Messina et al., 2010, 2011, 2012). The interesting observation that only the 3xW ULBP2 construct caused an inhibited ULBP2 cell-surface expression in HEK-293T cells proposes that these cells regulate ULBP2 in a similar manner but with less stringency. Moreover, these experiments showed that the recognition by NKG2D is not disturbed upon mono substitution, which indicates that the Trp-motif is specifically important for ULBP2 cell-surface transport. Interestingly, the WW domain-polyproline interaction has been linked to several genetic disorders such as Liddle’s syndrome, Alzheimer’s disease, retroviral budding and oncogenic transformation (nicely reviewed in Sudol, 1996) and it has been suggested to therapeutically target the WW domain alone or together with its ligand by low molecular weight compounds (Sudol, 1996). Certain cancers produce aberrant soluble ULBP2, which is associated with immune inhibition and a poor prognosis (Li et al., 2009; Paschen et al., 2009; Wu et al., 2004). Inhibition of soluble ULBP2 secretion would be a tempting therapeutic approach against these tumors. We have previously shown that ULBP2 is transported to the surface by a specific endosomal and Ii-dependent pathway, and that this pathway can be targeted without affecting surface expression of the homologues MICA and MICB molecules as they are transported via a more conventional pathway to the cell surface (Uhlenbrock

et al., 2014). Though, inhibition of endosomal transport as such might be too substantial because it will likely also blunt cytokine secretion and degranulation of NK and CD8T cells. Our current findings suggest that it is possible to target ULBP2 more specifically e.g. by using peptides engineered to bind to the WW domain-like motif on ULBP2 protein. Depending on the peptide chemistry one could either specifically target ULBP2 or all ULBPs. In any case, ablation of ULBP2 might possess therapeutic potential towards tumors that survive because of massive production of soluble, immunosuppressive ULBP2.

Acknowledgements We thank Dr. C. Geisler (Department of International Health, Immunology and Microbiology, University of Copenhagen, Denmark) for providing the Jurkat Tag-9 cell line, Dr. P. thor Straten (CCIT, Herlev University Hospital, Denmark) for providing the FM-86 and HT-29 cell lines and Dr. J. Moreira (Department for Veterinary Disease, University of Copenhagen, Denmark) for providing the MDA-MB-231 cell line. We thank Dr. N. Koch (University of Bonn, Germany) for the invariant chain construct and the Core Facility for Integrated Microscopy (Faculty of Health and Medical Sciences, University of Copenhagen). This project is a part of the EU Marie Curie Initial Training Networks Biomedical engineering for cancer and brain disease diagnosis and therapy development: EngCaBra. Project no. PITN-GA-2010-264417 and the Danish Council for Independent Research/Medical Sciences; project number DFF1331-00169.

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