Cell culture and transfection

All cell lines were obtained from the American Type Culture Collection (Manassas, VA) and have been independently validated using STR DNA fingerprinting at MD Anderson, and tests for mycoplasma contamination were negative. These cells were grown in DMEM/F12 or RPMI 1640 medium supplemented with 10% fetal bovine serum. PD-L1-stable transfectants in MDA-MB-231, MDA-MB-468, BT549 and HEK 293T cells were selected using puromycin (InvivoGen, San Diego, CA, USA). For transient transfection, cells were transiently transfected with DNA using SN liposomes36 and lipofectamine 2000 (Life Technologies, Carlsbad, CA, USA).

Animal treatment protocol

All BALB/C mouse (6–8-week-old female; Jackson Laboratories) procedures were conducted under the guidelines approved by the Institutional Animal Care and Use Committee at MD Anderson Cancer Center. Mice were divided according to the mean value of tumour volume in each group. 4T1-Luc cells (5 × 104 cells in 50 μl medium mixed with 50 μl Matrixgel Basement Membrane Matrix (BD Biosciences, San Jose, CA, USA) were injected into the mammary fat fad. For treatment with antibody, 100 μg anti-PD-1 antibody (RMP1-14, Bio X Cell, West Lebanon, NH, USA) or rat IgG (Bio X Cell) as control was injected intraperitoneally on days 3, 6, 9 and 12 after 4T1 cell inoculation. For drug treatment, mice were treated with daily oral doses of 10 mg kg−1 gefitinib for 2 weeks (5 days per week). Tumour was measured weekly with a caliper, and tumour volume was calculated using the formula: π/6 × length × width2.

Tumour infiltration lymphocyte profile analysis

Mice receiving a 5 × 104 4T1-Luc cell challenge were treated with anti-PD-1 and/or gefitinib as discussed in the previous section. Excised tumours were digested in collagenase/hyalurinidase (Stemcell Technologies, Vancouver, BC, Canada) and DNase (Sigma), and lymphocytes were enriched on a Ficoll gradient (Sigma) and then T cells were isolated using Dynabeads untouched mouse T-cell kit (Invitrogen). T cells were stained using CD3-PerCP (BioLegend, San Diego, CA, USA), CD4-FITC (eBioscience, San Diego, CA, USA), CD8-APC/Cy7 (BioLegend), CD45.1-PE (BioLegend) and IFNγ-Pacific Blue antibodies. Stained samples were analysed using BD FACSCanto II (BD Biosciences) cytometer.

Generation of stable cells using lentiviral infection

The lentiviral-based shRNA (pGIPZ plasmids) used to knock down expression of human or mouse PD-L1 (ref. 37) was purchased from the shRNA/ORF Core Facility (UT MD Anderson Cancer Center). On the basis of knockdown efficiency of PD-L1 protein expression in MDA-MB-231 or A431 cells, we selected two shPD-L1 clones for this study. The mature antisense sequences are as follows: 5′-TCAATTGTCATATTGCTAC-3′ (shPD-L1 #1), 5′-TTGACTCCATCTTTCTTCA-3′ (shPD-L1 #5). Using a pGIPZ-shPD-L1/Flag-PD-L1 dual expression construct to knock down endogenous PD-L1 and reconstitute Flag-PD-L1 simultaneously, we established endogenous PD-L1 knockdown and Flag-PD-L1 WT, 3SA, or 4NQ mutants expressing cell lines. To generate lentivirus-expressing shRNA for PD-L1 and Flag-PD-L1, we transfected HEK 293T cells with pGIPZ-non-silence (for vector control virus), pGIPZ-shPD-L1 or pGIPZ-shPD-L1/PD-L1 WT, pGIPZ-shPD-L1/PD-L1 4NQ mutant or pGIPZ-shPD-L1/PD-L1 3SA mutant with FuGENE 6 transfection reagent. Twenty-four hours after transfection, the medium was changed, and then it was collected at 24-h intervals. The collected medium containing lentivirus was centrifuged to eliminate cell debris, and was filtered through 0.45-μm filters. Cells were seeded at 50% confluence 12 h before infection, and the media were replaced with a medium containing lentivirus. After infection for 24 h, the medium was replaced with fresh medium and the infected cells were selected with 1 μg ml−1 puromycin (InvivoGen).

Plasmids

The human PD-L1 clone was obtained from the shRNA/ORF Core Facility (UT MD Anderson Cancer Center, Houston, TX, USA) and cloned into pCDH lentiviral expression vectors to establish PD-L1-Flag or PD-L1-Myc expression cell lines. In addition, it also cloned into pEGFP-N1 and pCMV-HA mammalian cell expression vectors for transient transfection. Using the pCDH/PD-L1-Flag expression vector as a template, we made PD-L1-Flag NQ mutants (N35Q, N192Q, N200Q, N219Q), and 4NQ (N35Q/N192Q/N200Q/N219Q) were developed by performing a site-directed mutagenesis (Supplementary Table 1). To create a pGIPZ-shPD-L1/Flag-PD-L1 dual expression construct to knock down endogenous PD-L1 and reconstitute Flag-PD-L1 simultaneously, first, we selected a shPD-L1 construct (shPD-L1 #5) that targets the 3′-untranslated repeat region of PD-L1 mRNA. And then Flag-PD-L1 WT or 4NQ mutant were cloned into pGIPZ-shPD-L1 (Thermo Scientific, Pittsburgh, PA, USA), expressing shRNA for endogenous PD-L1. To create a pGIPZ-shmPD-L1/Flag-mPD-L1 (mouse PD-L1) or a pGIPZ-shGSK3β/Flag-GSK3β dual expression construct, we used the same method as the pGIPZ-shPD-L1/Flag-PD-L1 dual expression construct. All constructs were confirmed using enzyme digestion and DNA sequencing. Detailed information is available upon request.

qRT–PCR assays

Quantitative reverse transcriptase PCR (qRT–PCR) assays were performed to measure the expression of mRNA37,38. Cells were washed twice with PBS and immediately lysed in QIAzol. The lysed sample was subjected to total RNA extraction using the RNeasy Mini Kit (Qiagen, Hilden, Germany). To measure the expression of mRNA, cDNA was synthesized from 1 μg purified total RNA with the SuperScript III First-Strand cDNA synthesis system using random hexamers (Life Technologies) according to the manufacturer’s instructions. qPCR was performed using a real-time PCR machine (iQ5, Bio-Rad, Hercules, CA, USA) with the following primers: 5′-TCACTTGGTAATTCTGGGAGC-3′ (PD-L1 forward), 5′-CTTTGAGTTTGTATCTTGGATGCC-3′ (PD-L1 reverse), 5′-GCAAAGACCTGTACGCCAACA-3′ (β-actin reverse) and 5′-TGCATCCTGTCGGCAATG-3′ (β-actin reverse). All the data analyses were performed using the comparative Ct method. Results were first normalized to internal control β-actin mRNA.

Antibodies and chemicals

The following antibodies were used: Flag (F3165; Sigma-Aldrich, St Louis, MO, USA; 1:5,000), Myc (11667203001; Roche Diagnostics, Indianapolis, IN, USA; 1:5,000), HA (11666606001; Roche Diagnostics; 1:5,000), PD-L1 (13684; Cell Signaling Technology, Danvers, MA, USA; 1:1,000), PD-L1 (329702; BioLegend; 1:1,000), PD-L1 (GTX117446; GeneTex, Irvine, CA, USA; 1:1,000), PD-L1 (AF156; R&D Systems, Minneapolis, MN, USA; 1:1,000), PD-1 (ab52587; Abcam, Cambridge, MA, USA; 1:2,000), Granzyme B (ab4059; Abcam; 1:1,000), EGFR (4267; Cell Signaling Technology; 1:1,000), GSK3β (9315; Cell Signaling Technology; 1:1,000), phospho-GSK3β Ser 9 (9336; Cell Signaling Technology; 1:1,000), β-TrCP (4394; Cell Signaling Technology; 1:1,000), α-Tubulin (B-5-1-2; Sigma-Aldrich; 1:5,000), β-Actin (A2228; Sigma-Aldrich; 1:10,000). EGF, cycloheximide and TM were purchased from Sigma-Aldrich. Gefitinib, erlotinib, lapatinib, cetuximab and AG1478 were obtained from Calbiochem Corp (Billerica, MA, USA).

Antibody generation

The mouse anti-phospho-PD-L1 T180 antibody was raised against a phosphorylated synthetic peptide (C-QVLSGKTT(p)TTNSKREE-NH2), and the mouse anti-phospho-PD-L1 S184 antibody was generated from a phosphorylated synthetic peptide (C-GKTTTTNS(p)KREEKLF-NH2). Antibodies were generated as previously described39. Briefly, antibodies against T180 and S184 phosphorylation sites of PD-L1 were generated in LifeTein (Somerset, NJ, USA). Synthetic phosphorylated peptides representing portions of PD-L1 around either T180 or S184 were used as antigens for producing antibodies. The antibodies were then purified using a phosphopeptide column.

Western blot analysis and immunoprecipitation

Western blot analysis was performed as described previously39,40. Image acquisition and quantitation of band intensity were performed using Odyssey infrared imaging system (LI-COR Biosciences, Lincoln, NE, USA). For immunoprecipitation, the cells were lysed in buffer (50 mM Tris·HCl, pH 8.0, 150 mM NaCl, 5 mM EDTA and 0.5% Nonidet P-40) and centrifuged at 16,000g for 30 min to remove debris. Cleared lysates were subjected to immunoprecipitation with antibodies. To measure 2-DG incorporation on PD-L1 protein, cells were incubated with IRDye 800CW 2-DG Optical probe (LI-COR Biosciences) for overnight, and then we performed immunoprecipitation. Uncropped scans of the most important western blots are shown in Supplementary Fig. 10.

Immunocytochemistry

For immunocytochemistry, cells were fixed in 4% paraformaldehyde at room temperature for 15 min, permeabilized in 5% Triton X-100 for 5 min and then stained using primary antibodies. The secondary antibodies used were anti-mouse Alexa Fluor 488 or 594 dye conjugate and/or anti-rabbit Alexa Fluor 488 or 594 dye conjugate (Life Technologies). Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI; blue; Life Technologies). After mounting, the cells were visualized using a multiphoton confocal laser-scanning microscope (Carl Zeiss, Thornwood, NY, USA).

PD-L1 and PD-1 interaction assay

To measure PD-1 and PD-L1 protein interaction, cells were fixed in 4% paraformaldehyde at room temperature for 15 min and then incubated with recombinant human PD-1 Fc protein (R&D Systems) for 1 h. The secondary antibodies used were anti-human Alexa Fluor 488 dye conjugate (Life Technologies). Nuclei were stained with DAPI (blue; Life Technologies). And then we measured the fluorescence intensity of Alexa Fluor 488 dye using a microplate reader Synergy Neo (BioTeK, Winooski, VT, USA) and normalized to the intensity by total protein quantity. To take an image, after mounting, the cells were visualized using a confocal laser-scanning microscope (Carl Zeiss). To monitor a dynamic PD-1 protein binding on live cell surface, PD-L1 WT or 3SA-expressing BT549 cells were incubated with Alexa Fluor 488 dye conjugate PD-1 Fc protein and taken a time-lapse image at every hour using the IncuCyte Zoom microscope (Essen Bioscience).

T-cell-mediated tumour cell-killing assay

T-cell-mediated tumour-cell-killing assay was performed according to the manufacturer’s protocol (Essen Bioscience). To analyse the killing of tumour cells by T-cell inactivation, nuclear-restricted red fluorescent protein (RFP)-expressing tumour cells were co-cultured with activated primary human T cells (Stemcell Technologies) in the presence of caspase 3/7 substrate (Essen Bioscience). T cells were activated by incubation with anti-CD3 antibody (100 ng ml−1) and IL-2 (10 ng ml−1). After 96 h, RFP and green fluorescent (NucView 488 Caspase 3/7 substrate) signals were measured. Green-fluorescent cells were counted as dead cells.

Co-culture experiments and IL-2 expression measurement

Co-culture of Jurkat T cells and tumour cells and IL-2 expression measurement was performed as described previously41. To analyse the effect of tumour cells on T-cell inactivation, tumour cells were co-cultured with activated Jurkat T cells expressing human PD-1, which were activated with Dynabeads Human T-Activator CD3/CD28 (Life Technologies). Co-cultures at 5:1 (Jurkat: tumour cell) ratio were incubated for 12 or 24 h. Secreted IL-2 level in the medium was measured as described by the manufacturer (Human IL-2 ELISA Kits, Thermo Scientific).

Glycosylation analysis of PD-L1

To confirm glycosylation of PD-L1 protein, we treated the cell lysates with PNGase F, Endo H and O-glycosidase (New England BioLabs, Ipswich, MA, USA) as described by the manufacturer. To stain glycosylated PD-L1 protein, we stained purified PD-L1 protein using the Glycoprotein Staining Kit (Pierce/Thermo Scientific) as described by the manufacturer.

IHC staining of human breast tumour tissue samples

Human breast tumour tissue specimens were obtained following the guidelines approved by the Institutional Review Board at MD Anderson Cancer Center, and written informed consent was obtained from patients in all cases at the time of enrolment. IHC staining was performed as described previously39,42. Briefly, tissue specimens were incubated with antibodies against PD-L1 (Cell Signaling Technology, #13684, 1:30 dilution), p-EGFR (Y1068; #3777, Cell Signaling Technology, 1:200 dilution), p-GSK3β (S9; #9323, Cell Signaling Technology, 1:200 dilution) or Granzyme B (ab4059, Abcam, 1:100 dilution) and a biotin-conjugated secondary antibody and then incubated with an avidin–biotin–peroxidase complex. Visualization was performed using amino-ethylcarbazole chromogen. For statistical analysis, Fisher’s exact test and Pearson χ2-test were used and a P value less than 0.05 is considered statistically significant. The intensity of staining was ranked into four groups: high (+++), medium (++), low (+) and negative (−) according to histological scores.

Identification of N-glycopeptide

Purified Flag-tagged PD-L1 protein was reduced by 10 mM dithiothreitol for 1 h at 37 °C, and then alkylated by 50 mM iodoacetamide for 1 h in the dark at room temperature, both in 25 mM ammonium bicarbonate buffer. Reduced and alkylated PD-L1 was then treated with sequencing grade trypsin (1:50, enzyme-to-substrate) at 37 °C overnight, after which the digested products were diluted with formic acid to a final concentration of 0.1% and desalted with ZipTip C18 (Millipore) before nano LC-MS/MS analysis on an Orbitrap Fusion Tribrid fitted with an UltiMate 3000 RSLCnano System (Thermo Scientific). Briefly, the peptide mixture was loaded on an Acclaim PepMap 100 (2 cm × 100 μm i.d; Dionex) trap column and then separated on an Acclaim PepMap RSLC 25 cm × 75 μm i.d. column (Dionex) by a gradient of 5–35% acetonitrile/0.1% formic acid in 60 min at a flow rate of 500 nl min−1. MS and MS/MS data were acquired under the higher-energy collisional induced dissociation (HCD) product ion trigger collision-induced dissociation (CID) mode, using a 3-s top-speed-mode cycle time. Collision energy used for HCD (fourier transform mass spectrometry (FTMSn)) was 28% stepped at 5% and the product ions m/z 204.0867 or 366.1396 were used to trigger CID (ion trap mass spectrometery (ITMSn)) at 30% collision energy. The raw data obtained were then converted to the Mascot generic format using Proteome Discoverer 1.4, and the resulting HCD MS2 data were searched for tentative glycopeptide matches using Byonic v. 2.2.9 (Protein Metrics) with the following parameters: peptide tolerance, 2 p.p.m.; fragment tolerance, 6 p.p.m.; missed cleavages, 1; modifications, carbamidomethyl cysteine (fixed), methionine oxidation (common2) and deamidation at N (rare 1). The returned positive glycopeptide hits were then further validated manually by considering both HCD and CID MS2 results.

Statistical analysis

Data in bar graphs represent the mean fold change relative to untreated or control groups with s.d. of three independent experiments. Statistical analyses were performed using SPSS (Ver. 20, SPSS, Chicago, IL). The correlation between protein expression was analysed using Pearson χ2-test and Mann–Whitney test. Student’s t-test was performed for experimental data. A P value<0.05 was considered statistically significant.

Data availability

All data supporting the findings of this study are available with the article, or from the corresponding author upon reasonable request.