Stable isotope labelling of amino acids in culture (SILAC) SPPLAT methodology is schematically outlined in Fig. S1.

Human breast adenocarcinoma SK-BR-3 cells (ATCC) were grown in McCoy's medium or RPMI-1640 containing 2 mM L-glutamine and supplemented with 10% foetal bovine serum (Invitrogen; Thermo Fisher Scientific, Inc.). The medium was changed every 2-3 days. For SILAC experiments, the RPMI-1640 heavy medium contained ¹³C₆ labelled L-lysine and L-arginine (K₆R₆), while the light medium was RPMI-1640 containing non-labelled L-lysine and L-arginine (K₀R₀), both of which were supplemented with 10% dialyzed foetal bovine serum (all from Dundee Cell Products), 2 mM L-glutamine, 100 U/ml penicillin and 100 µg/ml streptomycin (all from Invitrogen; Thermo Fisher Scientific, Inc.).

The cells were washed with PBS and were detached from tissue culture flasks by accutase (Gibco; Thermo Fisher Scientific, Inc.). Cells were incubated with LIVE/DEAD^® fixable violet dead cell stain (20 min, 4°C, Thermo Fisher Scientific, Inc.), washed with flow cytometry buffer (eBiosciences), and stained with AF647 (Thermo Fisher Scientific, Inc.)-conjugated anti-B7-H4 (MedImmune) or isotype control (clone R347, MedImmune) antibodies for 30 min at 4°C. Cells were washed with flow cytometry buffer and fixed with 1% paraformaldehyde for 20 min at 4°C before analysis. Compensation was performed using antibody-stained AbC beads (Invitrogen; Thermo Fisher Scientific, Inc.) and LIVE/DEAD^® violet-stained ArC beads (Invitrogen; Thermo Fisher Scientific, Inc.). Flow cytometry was performed on an FACS Canto II with 405 and 633 nm lasers (BD Biosciences), with data analysis in FlowJo v10.0.6 software (Tree Star, Inc.).

SK-BR-3 cells were spun onto coverslips pre-coated with 0.05% poly L-lysine, fixed with 4% parafor-maldehyde at room temperature for 10 min, washed in PBS and incubated with anti-B7-H4-488 (MedImmune) for 1 h at room temperature. To determine all locations of B7-H4, the cells were permeabilised with 1% saponin for 30 min prior to antibody incubation and imaged at ×60 magnification using an Olympus Fluoview IX81 laser scanning confocal microscope (Olympus Corporation).

The biotin tyramide reagent was prepared as previously described (21). A total of 1 mg each of Human IgG1 anti-B7-H4 monoclonal antibody (in-house; MedImmune) and anti-B7-H1 (PD-L1) monoclonal antibody (in-house; MedImmune) were conjugated with HRP using the EZ-link activated peroxidase kit at pH 9.4 (cat. no. 31497; Thermo Fisher Scientific, Inc.) and purified by gel filtration. This resulted in ~1:1 stoichiometry. Exponentially growing SK-BR-3 cells were pelleted and washed in PBS at room temperature. Approximately 1×10⁶ live cells were incubated with end-over-end rotation for 2 h with either 20 µg/ml human anti-B7-H4-HRP, 20 µg/ml human anti-B7-H4 with 200 nM free HRP (BioRad Laboratories, Inc.), or 20 µg/ml human anti-B7-H1-HRP in 20 ml PBS with 10% BSA (Sigma-Aldrich; Merck KGaA) at 4°C. For SILAC SPPLAT ~5×10⁸ heavy (K₆R₆) cells were incubated with end-over-end rotation for 2 h with 20 µg/ml HRP-conjugated human anti-B7-H4 and 5×10⁸ light (K₀R₀) with 20 µg/ml human anti-B7-H4 and 200 nM free HRP.

Cells were pelleted and re-suspended in 10 ml tyramide-labelling buffer (50 mM Tris HCl pH7.4, fresh 0.03% H₂O₂ and 80 µg/µl tyramide biotin label (Thermo Fisher Scientific, Inc.) and incubated with end-over-end rotation at room temperature for 5 min. After incubation, 100 U/ml cata-lase (Sigma-Aldrich; Merck KGaA) was added and incubated with the samples for a further 5 min to quench H₂O₂. Cells were washed gently with 45 ml antibody strip buffer [50 mM glycine (pH 3.0), 150 mM NaCl, 0.9 mM CaCl₂, 0.5 mM MgCl₂] and left on ice for 5 min. The extent of biotinylation was assessed by immunofluorescence using non-permeabilised cells stained with Streptavidin-488 (at 1:100) for 1 h at room temperature.

Cells were re-suspended in 15 ml cell lysis buffer [20 mM Tris-HCl (pH 7.5), 5 mM EDTA, 1X protease inhibitor cocktail (Roche Diagnostics), 150 mM NaCl, 1% v/v Triton X-100, 0.1 M sodium thiocyanate (Sigma-Aldrich; Merck KGaA)] and incubated for 30 min on ice. Insoluble material was removed by centrifugation at 10,000 × g for 10 min at 4°C, and the protein-containing soluble fraction was recovered for streptavidin-bead capture.

For the initial SPPLAT, 1 ml lysate from 2×10⁶ SPPLATed cells was added to 100 µl slurry of high-capacity Neutravidin resin (Thermo Fisher Scientific, Inc.), incubated with end-over-end rotation for 1 h at 4°C, washed three times with lysis buffer containing sodium thiocyanate to reduce non-specific interactions, then biotinylated proteins were eluted with lysis buffer containing 10 mM biotin. Eluted proteins were separated by SDS-PAGE (10%) and stained with Simply Blue™ SafeStain Coomassie (Invitrogen; Thermo Fisher Scientific, Inc.) for 1 h at room temperature prior to dividing and excising equally into 16 bands. Gel slices were de-stained in ddH₂O and 20 mM NH₄HCO₃, reduced with 2 mM DTT and alkylated with 10 mM iodoacetamide prior to overnight digestion with 2 µg sequencing grade trypsin (Promega Corporation). Peptides were extracted with acetonitrile and 1% formic acid and re-suspended in water with 1% formic acid after vacuum-drying.

For SILAC SPPLAT, 5 ml cell lysates were quantified and equal amounts (1 mg) of protein from both heavy and light cell lysates were added to 0.5 ml slurry of high-capacity Neutravidin resin and incubated as mentioned above. Reciprocal labelling (specific-light, control-heavy) and affinity purification was also performed. Two rounds of 200 µl eluted proteins were combined and precipitated with 80% ice cold acetone, re-suspended in 1X SDS sample buffer and separated by SDS-PAGE (10%) as before, but were separated for only 1 cm; 4 bands per lane were excised for mass spectrometry (MS) analysis and prepared as before.

All liquid chromatography (LC)-MS/MS SPPLAT experiments were performed using a NanoAcquity UPLC system (Waters Corp.) and an LTQ Orbitrap Velos hybrid ion trap mass spectrometer (Thermo Fisher Scientific, Inc.). Separation of peptides was performed by reverse-phase chromatography using a Waters reverse-phase nano column (BEH C18, 75 µm i.d. x250 mm, 1.7 µm particle size) at a flow rate of 300 nl/min. Peptides were initially loaded onto a pre-column (Waters UPLC Trap Symmetry C18, 180 µm i.d. x20 mm, 5 µm particle size) from the NanoAcquity sample manager with 0.1% formic acid for 3 min at a flow rate of 10 µl/min. After this period, the column valve was switched to allow the elution of peptides from the pre-column onto the analytical column, where a linear gradient of increasing acetonitrile (5-35%) over 60 min was employed. The LC eluant was sprayed into the mass spectrometer by means of a nanospray source. All m/z values of eluting ions were measured in the Orbitrap Lumos Velos mass analyser, set at a resolution of 30,000. Data-dependent scans (top 10) were employed to automatically isolate and generate fragment ions by collision-induced dissociation in the linear ion trap, resulting in the generation of MS/MS spectra. Ions with charge states of ≥2+ were selected for fragmentation. The raw MS data files were converted to mgf files and searched against the Swissprot Human database (accessed in May 2017; 71,567 entries) using the Mascot search algorithm (version 2.3.02, Matrix Science) with methionine oxidation (M) as a variable modification and cysteine carbamidomethylation (C) as a fixed modification, allowing 2 missed cleavages, a peptide mass tolerance of ±1 Da and a fragment mass tolerance of 0.8 Da. Single-peptide hits were removed from the lists.

Quantitation was performed using MaxQuant (version 1.6.0.1; https://www.maxquant.org/). Raw data were searched using Andromeda (http://www.coxdocs.org/doku.php?id=maxquant:andromeda), with Arg-6 and Lys-6 set as heavy labels, methionine oxidation and N-acetylation as variable modifications, and cysteine carbamidomethylation as a fixed modification. The proteins were identified if there was at least one unique peptide and quantified if there were at least two unique peptides. Only unique peptides were used for quantitation.

SK-BR-3 cells (1×10⁵) were grown in either McCoy's medium or RPMI-1640 and lysed, and the soluble fraction was separated by 10% SDS-PAGE, Coomassie-stained as described above, and the entire lane was excised to allow the analysis of all proteins by MS. The stained bands were reduced and alkylated, and MS was performed as described above.

To confirm the co-localisation of B7-H4 with integrins, HLA-E or plexin, SK-BR-3 cells (~1×10⁴) were spun onto 13 mm coverslips, fixed, but not permeabilised, washed, blocked and incubated with goat anti-B7-H4 polyclonal IgG Ab (1:250; cat. no. PA5-47261; Thermo Fisher Scientific, Inc.), and then either integrin α1 (mouse anti-human IgG₁ at 2 µg/ml; cat. no. MAB1973; EMD Millipore), HLA-E (mouse anti-human IgG₁ at 1:50; cat. no. sc-71262; Santa Cruz Biotechnology, Inc.) or plexin B2 (mouse anti-human IgM at 1:50; cat. no. sc-373969; Santa Cruz Biotechnology, Inc.) for 1 h at room temperature. Integrin α1 incubations were also performed at 4 and 37°C. Incubations with anti-goat IgG-488 (1:1,000; cat. no. A-11078; Thermo Fisher Scientific, Inc.) and anti-mouse IgG-647 (1:1,000; cat. no. A-21239; Thermo Fisher Scientific, Inc.) for 1 h at room temperature were performed to visualise the cell surface proteins using confocal microscopy, as mentioned before.

SK-BR-3 cells (~1×10⁴) were spun onto coverslips pre-coated with 0.05% poly L-lysine. Live cells were washed and blocked in 10% BSA in PBS and incubated with goat anti-B7-H4 (2.5 µg/10⁶ cells) in 10% BSA for 30 min at room temperature to induce clustering, then fixed with 4% paraformaldehyde at room temperature for 10 min and washed in PBS. For controls, cells were first incubated in antibodies against integrin α1 (2 µg/ml; cat. no. MAB1973; EMD Millipore), HLA-E (1:50; cat. no. sc-373969; Santa Cruz Biotechnology, Inc.) or plexin B2 (1:50; cat. no. sc-373969; Santa Cruz Biotechnology, Inc.) for 1 h at room temperature. Controls included fixing cells prior to B7-H4 antibody incubation and omission of the primary antibodies. Sigma PLA probes (goat plus and mouse minus; Sigma Aldrich; Merck KGaA) were used and the ligation and amplification (far red) was performed according to the manufacturer's instructions. Cells on coverslips were mounted onto slides and imaged using an Olympus Fluoview IX81 laser scanning confocal microscope (Olympus Corporation) at x60 magnification.

SK-BR-3 cells were seeded at 1×10⁴ cells/well in an E-plate (Roche Applied Science) pre-coated with either 10 µg/ml fibronectin or 10 µg/ml collagen peptides or 5% albumin. The wells were incubated with 20 µg/ml human anti-B7-H4 (MedImmune), or non-specific human IgG (1:100 dilution of ascitic fluid; in-house; MedImmune). To quantitate cell-line binding to collagen peptides, the impedance-based xCELLigence system (Acea), which allows label-free, dynamic monitoring of cell adhesion in real time, was used (23). The assay system expresses the adhesion-dependent rise in well impedance in units of cell index (CI), defined as (R_n-R_b)/15, where R_n is the electrical impedance of each cell-containing well and R_b is the background impedance of the well with medium alone. CI was measured for up to 2 h. All experiments were performed in triplicate in the presence of either 5 mM Mg²⁺ or 5 mM EDTA. Data are presented as the mean ± standard deviation and were analysed for statistical significance using one-way ANOVA followed by Tukey's post hoc test using the GraphPad Prism software package (version 7.0d, GraphPad Software, Inc.).

Total RNA was extracted from ~5×10⁶ SK-BR-3 cells using an RNA extraction kit (Qiagen GmbH). RT-PCR was carried out with 1 mg of RNA, using the One-Step RT-PCR kit (Qiagen GmbH), following the manufacturer's instructions. The amplification primers used were as follows: Human integrin α1, forward 5′-GGT GAA TCA TTA CCT TGC GT-3′ and reverse 5′-AGC ACA TCT CCA GAA GAA GC-3′; human integrin β1, forward 5′-AGG AAC AGC AGA GAA GCT CA-3′ and reverse 5′-CAT TTT CTT CAA TTT TCC CC-3′.

To confirm direct binding of α1 integrin and surface B7-H4, antibodies to α1 integrin (EMD Millipore) and B7-H4 (MedImmune) were coupled to separate preparations of Protein G beads (Thermo Fisher Scientific, Inc.) overnight at 4°C with gentle agitation. To assess only surface interactions of α1 integrin, IP was performed with live cells, as lysates would contain predominantly cytosolic B7-H4. Live cells (1×10⁶) were washed in PBS and allowed to bind antibody-bound Protein G beads. Control cells were first incubated in goat serum with a non-specific antibody, and then Protein G beads were added. Unbound beads were washed off and cells were then lysed in lysis buffer as before. Soluble lysates were separated by SDS-PAGE (10%), transferred to a nitrocellulose membrane and probed with either antibodies to α1 integrin (1:3,000; EMD Millipore) or B7-H4 (1:2,000; Thermo Fisher Scientific, Inc.) for the B7-H4 and α1 integrin co-IP, respectively.

A partial SK-BR-3 proteome was obtained, which enables estimation of the abundance of common proteins that may also reside in the footprint of the SPPLAT biotin label and thus specifically bind affinity resins. For a stringent list, single-peptide hit protein assignments were removed, enabling positive identification of 1,220 proteins that were ranked by empirical abundance using the protein EMPAI score (Table SI). These equated to ~0.77% coverage of the expected total human proteome (currently 159,552 protein entries) and were interpreted with caution if they appeared in SPPLAT downstream purifications. Gene Ontology (GO) annotation analysis identified no over- or under-representation of any 'cell components' compared with the human reference proteome, indicating successful solubilisation and extraction of all protein classes. These abundant proteins included keratins, histones, metabolic enzymes, structural and heat shock proteins. The target protein, B7-H4, was not identified as a high-abundance protein, as expected, thereby highlighting the fact that a method for specifically labelling these B7 cell surface membrane proteins is required to identify proximal proteins.

This analysis also compared SK-BR-3 cells grown in McCoy's medium and the RPMI-1640 medium required for SILAC labelling. There was a large (86%) overlap in identified peptides for the most abundant proteins from each condition, indicating that the culture of SK-BR-3 cells in RPMI-1640 medium for quantitative analysis would not result in a significant change in the proteome.

The localisation of B7-H4 in SK-BR-3 cells was first characterised and its weak plasma membrane expression compared with abundant intracellular expression was confirmed (Fig. 1A-C), suggesting its suitability for investigation via the SPPLAT method. Analysing B7-H4 3D structures using PDB structures identified B7-H4 only having two surface exposed tyrosine residues that are required for SPPLAT labelling. Confocal imaging and western blotting confirmed that B7-H4 and its neighbours were biotinylated (Fig. 1D and E). As a comparison, targeting B7-H1 resulted in biotinylation of a different, and smaller, set of proteins to B7-H4, as determined by western blotting (Fig. 1E). The negative control, using an HRP-tagged non-specific IgG, did not lead to biotinylation of any proteins.

This non-quantitative B7-H4 SPPLAT experiment followed by affinity purification (AP)-MS resulted in the identification of 2,004 biotinylated proteins (Table SII). The B7-H4 protein and its proximal proteins were enriched and identified, a number of which were not identified in the initial SK-BR-3 proteome screen. GO annotation revealed an enrichment of cell surface and peripheral membrane proteins compared with the control, as expected. The top 80 cell surface-associated proteins from the anti-B7-H4 SPPLAT experiment that include the target, VTCN1 (B7-H4), ICOS ligand (B7-H2), a number of integrins, plexins, cell adhesion molecules and proteins from the Ig superfamily are shown in Table I. A number of cytosolic proteins were also identified, suggesting that the biotin-tyramide can permeate the plasma membrane in the 5-min labelling time and/or that cytosolic proteins can be recruited to the plasma membrane upon B7-H4 activation by antibody. As a further comparison, B7-H1 was also labelled (Fig. 1E), and several immune signalling molecules were identified, a number of which were also associated with B7-H4 (Table SI), although the protein microenvironments differed. The negative control did not non-specifically label signalling molecules.

In order to quantify the enrichment of proteins in an anti-B7-H4-SPPLAT experiment compared with a negative control IgG, a SILAC SPPLAT analysis was performed, where cells cultured in isotope-labelled media were labelled via SPPLAT for 2 min to minimise membrane diffusion and cytosolic labelling. In performing two biological replicates and two reciprocal labellings, 938 proteins were identified, of which 452 were present in all four datasets (Table SIII). By performing control experiments with a non-specific IgG, a significant proportion (~90%) of proteins that demonstrated minimal expression changes between the anti-B7-H4 and non-specific IgG control were eliminated. Subsequently, the 13 proteins that demonstrated a log2 fold-change of >2 and significance of P>0.01 (log10 adjusted P-value of 2) between the test and control experiments were analysed; in addition to plasma membrane-localised proteins, there were some cytoplasmic and nuclear proteins, despite a short labelling time (Fig. 2). One identified plasma membrane protein, Enox1, an Ecto-NOX disulphidethiol exchanger, is involved in electron transport to the cell surface and has not previously been reported to be associated with B7-H family members. A total of 13 predicted binding partners of Enox1 were not found in our list, notably Protein GREB1, which is considered to play a role in oestrogen-stimulated cell proliferation by acting as a regulator of hormone-dependent cancer growth in breast and prostate cancers. A cytosolic candidate, interleukin enhancer-binding factor 3, interacts with and inhibits viral mRNAs (24); therefore, it was not further investigated. The remainder of increased fold changed proteins were metabolic or nucleus-localised and were also not further investigated. As B7-H4 was exclusively SPPLATed and not detected in the SILAC SPPLAT, the present study focused on the proteins that were present only in the B7-H4 labellings and absent in controls with relevant GO annotation, good peptide coverage and abundance, as indicated by EmPAI scoring or peak intensity from MaxQuant analysis (listed in Table I). Of particular interest were several integrins, HLA-E and plexins. These were further investigated using PLA, CoIP and binding studies.

The SPPLAT method can biotinylate proteins within a 100-nm footprint of the target HRP-coupled antibody-ligand interaction. PLA can validate our identified proteins and further positions within 40 nm. PLA was performed on integrin α1, HLA-E and plexin B2. As shown in Fig. 3, when SK-BR-3 cells are pre-incubated with B7-H4 antibody, this induces B7-H4 clustering at the cell surface, and both integrin α1 and HLA-E co-localise with B7-H4 forming distinct clusters compared to when no B7-H4 antibody incubation occurs (Fig. 3A-E). Plexin B2 was not found to co-localise with B7-H4 at the cell surface (Fig. 3F and G); however, standard immunofluores-cence demonstrated co-localisation just beneath the plasma membrane (Fig. 3H).

Several integrin subunits (α1, α2, α3, αV, β1 and β5) were identified in the B7-H4 SPPLAT AP-MS screen. Integrins function as αβ heterodimers in various combinations, and several, such as αV and β5, have been implicated in tumour growth, so were used as positive controls in validation experiments. Integrin α1 operates in partnership with β1 and β5. The PLA experiment demonstrated that integrin α1 was indeed within 40 nm of B7-H4 on the plasma membrane (Fig. 3A) and, in parallel, it was confirmed by RT-PCR that α1 and β1 are expressed in SK-BR-3 cells (Fig. 4A). To assess whether α1 and β1 interact with B7-H4 on the cell surface, integrin real-time binding assays were performed with known substrates of integrin heterodimers α1β1 (collagen receptor) and α5β1 (fibronectin receptor) in the presence and absence of anti-B7-H4 (blocking) antibody. The presence of B7-H4 in close proximity to integrins α1 and β1 could modulate integrin-substrate binding and, furthermore, the presence of a blocking antibody for B7-H4 may alter binding. Indeed, the presence of anti-B7-H4 reduced binding of SK-BR-3 cells, via α1, to collagen substrates at 0.5 h (Fig. 4B), whereas the cell binding response to fibronectin was significantly higher (P<0.0001) in the presence of an anti-B7-H4.

To ensure that integrins were not being non-specifically activated due to the PLA being performed at 37°C, antibody incubation at 4°C was also performed prior to fixation; no differences in the expression or internalisation were observed (Fig. S2A-D). To assess whether integrin α1 is a direct binding partner of B7-H4, co-IP was performed in SK-BR-3 cells using an antibody to B7-H4 to pull down B7-H4-bound proteins, and subsequent detection of integrin α1 by western blotting (Fig. S2E). The anti-B7-H4 pulled down integrins, whilst a control anti-B7-H4 Fab alone did not pull down B7-H4 with integrins α1 and β1, thus confirming that integrin α1 is in a complex with B7-H4 (Fig. S2E). Thus, multiple lines of evidence show that integrin α1 is found in a complex with B7-H4 on the cell surface.

HLA-E was another interesting find, as its role is typically in negative regulation of the immune system. Its close proximity to B7-H4 confirmed by SPPLAT and PLA (Fig. 3D and E) was further validated by co-IP and found to be in complex with B7-H4 at the cell surface (Fig. S2F).

Only a limited number of SPPLAT hits that had both high and low scoring EMPAI values were investigated. There were also several Ig superfamily members and antigens, such as CD58/LFA-3, ALCAM and L1CAM, EGFR and ST14 in the B7-H4 microenvironment that may play a role that would warrant further validation.