Reagents
The antibodies and reagents were purchased as follows: anti-IL-2 (JES6-1A12, e-Bioscience, 14-7022-85, RRID:AB_468406), biotin-labeled anti-IL-2 (JES6-5H4, e-Bioscience, 13-7021-85, RRID:AB_466899), PE–anti-I-A/I-E (M5/114.15.2, BioLegend, 107608, RRID:AB_313323), PE–anti-mPD-1 (29F.1A12, BioLegend, 135206, RRID:AB_1877231), anti-mPD-1 (29F.1A12, BioLegend, 135248, RRID:AB_2783091), anti-hPD-L1 (29E.2A3, BioLegend, 329746, RRID:AB_2783199), and anti-hPD-L2 (24F.10C12, BioLegend, 329624, RRID:AB_2819957), PE–anti-hPD-1 (MIH4, BD bioscience, 557946, RRID:AB_647199), PE–anti-hPD-L1 (MIH1, BD bioscience, 557924, RRID:AB_647198), PE–anti-hPD-L2 (MIH18, BD bioscience, 558066, RRID: AB_647197), Alexa Fluor 647–labeled anti-pCD3z (K25-407.69, BD bioscience, 558489, RRID: AB_647152), and Alexa Fluor 647–labeled anti-pSLP-76 (J141-668.36.58, BD bioscience, 558438, RRID:AB_647159), rabbit polyclonal anti-SHP1 (C-19, Santa Cruz Biotechnology Inc., sc-287, RRID:AB_2173829) and mouse anti-SHP2 (B-1, Santa Cruz Biotechnology Inc., sc-7384, RRID:AB_ 628252), anti-Erk (Cell Signaling Technology, 4695S, RRID: AB_390779), anti-pErk (Cell Signaling Technology, 4370S, RRID: AB_2315112), anti-PLCg (Cell Signaling Technology, 5690S, RRID: AB_10691383), anti-pPLCg (Cell Signaling Technology, 8713S, RRID: AB_10890863), anti-Akt (Cell Signaling Technology, 4691S, RRID: AB_915783), anti-pAkt (Cell Signaling Technology, 4060S, RRID: AB_2315049), and HRP-anti-rabbit IgG polyclonal Abs (Cell Signaling Technology, 7074S, RRID: AB_2099233), HRP-anti-mouse IgG polyclonal Abs (Cappel, 55550), pembrolizumab (MCE, HY-P9902), nivolumab (MCE, HY-P9903), durvalumab (MCE, HY-P9919), and atezolizumab (MCE, HY-P9904), APC-anti-human IgG (H+L) (Jackson Immuno Research, 705-136-147, RRID: AB_2340407), DyLight 650 and 549 labeling kits (Thermo Fisher Scientific, 84535 and 53044), HaloTag (HT) STELLA Fluor 650 and TMR ligands (Promega, GCKA308-01 and G8252), SNAP-Cell 647-Sir (New England BioLabs, S9102S), MCC88-103 (ANERADLIAYLKQATK, GenScript), and OVA257-264 (SIINFEKL, GenScript) peptides. A B cell hybridoma producing anti-CD28 (PV-1) was provided by R. Abe (Tokyo University of Science, Noda, Japan); anti-CD3z (145-2c11) by J. Bluestone (University of California, San Francisco, USA); anti-TCRb (H57-597) by R. T. Kubo (Cytel Corp., CA, USA); and anti-I-Ek (14-4-4) and anti-ICAM-1 (YN1/1.7.4) by M. L. Dustin (University of Oxford, UK); anti-hPD-L1 (MIH1)24 and anti-hPD-L2 (MIH18)24, 25 by M. Azuma (Tokyo Medical and Dental University, Tokyo, Japan).
Mice and cells
AND TCR-Tg mice were provided by Dr. S. M. Hedrick (University of California San Diego, San Diego, CA); Rag2-/- mice by Dr. F. Alt (Boston Children’s Hospital, Boston, MA); OT-I TCR-Tg Rag2-/- mice by Dr. W. Health (University of Melbourne, Melbourne, Australia); and Pdcd1-/- mice from RIKEN BRC. Mice were maintained in specific pathogen-free conditions at Tokyo Medical University. All experiments were performed in accordance with a protocol approved by the Animal Care and Use Committee of Tokyo Medical University (H30-0044, H31-0065, R2-0001). The DC-1 fibroblast cell expressing I-Ek and ICAM-1 was provided by J. Kaye (Cedars-Sinai Medical Center, Los Angeles, CA). PLAT-E, the retrovirus packaging cell line, was provided by G. Nolan (Stanford University, Stanford, CA). BHK, EL-4, and E. G7-OVA Cell Line were purchased from ATCC (ATCC, ACC-61, RRID:CVCL_1915; ATCC, TIB-39, RRID:CVCL_0255; ATCC, CRL-2113, RRID:CVCL_3505). The T cell hybridoma expressing the AND-TCR (AND-TCR T cell hybridoma, 2D12) was established by cell fusion of activated AND TCR-Tg CD4+ T cells with lymphoma cell line, BW514726. We completely deleted mPD-1 on EL-4, E.G7, and 2D12 cells by CRISPR-Cas9 system (PX458, addgene, 48138).
Plasmid construction
EGFP-tagged hPD-1, mSHP1, and mSHP2 were generated by polymerase chain reaction (PCR) and subcloned into retroviral vector, pMXs and/or pMCs (kindly provided by Dr. T. Kitamura, University of Tokyo, Japan)27. SNAP-tag (New England BioLabs, N9183) or Renilla luciferase (RLuc) 8 and/or HaloTag (Promega, G9651)-tagged hPD-1, hPD-L1, or hPD-L2 were generated by PCR and subcloned into the pMXs retroviral vector. pMXs-RLuc8 was constructed by PCR using Yellow Nano-lanterns (kindly provided by Dr. Y. Okada, Riken, Japan)28 as a template.
Primary cell culture and transduction
Retroviral vectors were transiently transduced into a packaging cell, PLAT-E (provided by G. Nolan, Stanford University, Stanford, CA) using Lipofectamine 2000 (Invitrogen, 11668019). The supernatants were concentrated 40-fold by centrifugation at 8,000 g for 12 h. AND TCR-Tg CD4+ T cells were purified from AND TCR-Tg Pdcd1-/- Rag2-/- mice and stimulated with 5 μM MCC88-103 and irradiated spleen cells from B10.BR mice. OT-I TCR-Tg CD8+ T cells were purified from OT-I TCR-Tg Pdcd1-/- Rag2-/- mice and stimulated with 100 nM OVA257-264, 200 U/mL recombinant mouse IL-2 (Peprotech, 212-12), and irradiated spleen cells from B6 mice. One day after stimulation, the cells were suspended in retroviral supernatant with 10 μg/mL polybrene (Sigma-Aldrich, TR-1003) and 200 U/mL mouse IL-2 and centrifuged at 2,600 rpm for 90 min at 37°C. On day 2 or later, the cells were sorted to obtain the populations with homogeneous fluorescence intensity and were then maintained in RPMI1640 medium (Sigma-Aldrich, R6504-10L) containing 10% FCS (Thermo Fisher Scientific, 10270106) and mouse IL-2.
Microscopy
The cells expressing the proteins tagged with GFP and/or HaloTag stained by fluorescent-labeled H57 Fab and/or TMR-(Promega, G8252) or Stella650-labeled HaloTag ligand (Promega, GCKA308-01) were allowed to settle onto an SLB and visualized by confocal microscopy. The image acquisition and the image processing were described previously6.
Glass-supported lipid planer bilayers
The purification and fluorescent labeling of GPI-anchored proteins have been established according to the protocols7. BHK cells (ATCC, ACC-61, RRID:CVCL_1915) highly expressing hPD-L1–GPI or hPD-L2–GPI were established. hPD-L1–GPI and hPD-L2–GPI were purified from the lysates by affinity column with 29E.2A3 (BioLegend, 329746, RRID:AB_2783199, anti-hPD-L1) and MIH18 (provided by M. Azuma, anti-hPD-L2)24, 25, respectively. The expression level of each GPI-anchored protein on the planar bilayer was quantified using silica beads with a diameter of 5 μm (Bangs Laboratories, SS05N)11. The densities were calculated based on the standard beads, Quantum FITC-5 MESF (Bangs Laboratories, 555p), and adjusted to the approximate concentration by comparison with natural APCs: I-Ek, 200 molecules/μm2; mICAM-1, 150/μm2; hPD-L1, 17.25–600/μm2, and hPD-L2 200/μm2. The planar bilayers were loaded with 1 μM MCC88-103 (GenScript) in citrate buffer, pH 4.5, for 24 h at 37°C, blocked with 5% nonfat dried milk (Cell Signaling Technology, 9999S) in PBS for 1 h at 37°C, and left to stand in the assay medium (Hepes-buffered saline, Sigma-Aldrich, H3375-250G) containing 1% FCS (Thermo Fisher Scientific, 10270106), 2 mM MgCl2, and 1 mM CaCl2.
T cell–APC conjugation assay
DC-1 or DC-1 cells expressing hPD-L1-HaloTag or SNAP-tag and/or hPD-L2-HaloTag were prepulsed with 1 μM MCC88-103 overnight at 37°C and prestained with SNAP-Cell 647-Sir (New England BioLabs, S9102S, red) and/or HaloTag ligand-TMR (cyan). mPD-1-deleted 2D12 cells, the T cell hybridoma expressing the AND-TCR, expressing hPD-1-EGFP were cultured with DC-1 cells with or without 0.1 or 10 mg/mL anti-hPD-1 and/or anti-hPD-L1 and/or anti-hPD-L2 antibody. mPD-1-deleted 2D12 cells expressing hPD-1-HaloTag and EGFP-mSHP1 or -mSHP2 were cultured with DC-1 cells. The conjugates were visualized by confocal microscopy.
Immunoprecipitation and Western blotting
DC-1 cells were prepulsed with 5 μM MCC88-103 overnight at 37°C and washed before the assay. 2 × 106 mPD-1-deleted 2D12 cells transduced with hPD-1 were stimulated with 2 × 106 DC-1 cells not transduced or transduced with hPD-L1. The cells were lysed with the lysis buffer (50 mM Tris-HCl, 50 mM NaCl, and 5 mM EDTA) containing 1% NP-40. Whole cell lysates (WCLs) or those immunoprecipitated by anti-GFP (MBL International, D153-11, RRID:AB_2893312) were blotted with anti-GFP (Miltenyi Biotec, 130-091-833, RRID:AB_247003), anti-mSHP1 (Santa Cruz Biotechnology, sc-287, RRID:AB_2173829), anti-mSHP2 (Santa Cruz Biotechnology Inc., sc-7384, RRID:AB_ 628252), anti-PLCg (Cell Signaling Technology, 5690, RRID:AB_10691383), anti-pPLCg (Cell Signaling Technology, 8713, RRID:AB_10890863), anti-Akt (Cell Signaling Technology, 4691, RRID:AB_915783), anti-pAkt (Cell Signaling Technology, 4060, RRID:AB_2315049), anti-Erk (Cell Signaling Technology, 4695, RRID:AB_390779), or anti-pErk (Cell Signaling Technology, 4370, RRID:AB_2315112) as a first antibody and HRP–anti-rabbit IgG polyclonal Abs (Cell Signaling Technology, 7074, RRID:AB_2099233) as a second one. Each intensity of band was calculated by ImageJ (RRID:SCR_003070).
Flow cytometry
Staining antibodies were used at a concentration of 0.01 to 100 μg/mL. A cell sorter, SH800S (Sony), was used for cell isolation and cell analyzers, FACS Canto II (BD, 07B1X00003000102) and Guava easyCyte (MERCK, 0500-5007JPK) were used for analysis. Data were depicted using FlowJo (RRID:SCR_008520).
T cell stimulation assay
2 × 104 mPD-1-deleted 2D12 cells or 1 × 105 Pdcd1-/- AND TCR-Tg CD4+ T cells were stimulated with 2 × 104 DC-1 cells expressing hPD-L1 and/or hPD-L2 with 1 μM MCC88-103 in the presence or absence of anti-hPD-1 and/or anti-hPD-L1 and/or anti-hPD-L2 antibody. The concentration of IL-2 was measured by ELISA 16 h after stimulation. All experiments were performed in triplicate.
CTL killing assay
RLuc8-introduced and mPD-1-deleted EL-4 cells (ATCC, TIB-39, RRID:CVCL_0255) were used as a target cell. At the indicated E/T ratios, hPD-1-transduced Pdcd1-/- OT-I TCR-Tg CD8+ T cells were cocultured with 1 nM OVA257-264 pulsed 1 × 105 EL-4 cells expressing hPD-L1 for 16 h in the presence or absence of anti-hPD-1 or anti-hPD-L1 antibodies. After treatment with coelenterazine, RLuc8 substrate (FUJIFILM Wako, 031-22993), the intensity of RLuc8 luminescence in live target cells was measured using a lumino image analyzer, ImageQuant LAS4000 mini (GE Healthcare). All experiments were performed in triplicate.
In vivo tumorigenicity assay
1 × 106 mPD-1-deleted E. G7-OVA Cell Line (ATCC, CRL-2113, RRID:CVCL_3505) reconstituted by hPD-L1 were subcutaneously inoculated in 100 μL PBS in the dorsal region of Rag2-/- mice. Tumors were allowed to grow for 8 to 10 days before treatments (tumor area between 90 and 400 mm2). Tumor size was blindly measured using calipers. Mice received 100 μL PBS containing 1.5 × 106 activated Pdcd1-/- OT-I TCR-Tg CD8+ T cells expressing hPD-1 by intravenous injection in the tail vein. Two days later, mice were injected intraperitoneally with nivolumab (MCE, HY-P9903, RRID:AB_2810223) or durvalumab (MCE, HY-P9919) at 0.001 mg/kg or 0.1 mg/kg four times every 2 to 3 days. Mice were sacrificed on day 18 to 20.
Statistics and reproducibility
Data were presented as the mean ± standard deviation (SD). Statistical analysis was performed by the Student’s t-test or one-way analysis of variance (ANOVA) using GraphPad Prism (RRID:SCR_002798) or KaleidaGraph (Synergy Software). p-values <0.05 were considered to be statistically significant. Reproducibility, including biological independent sample sizes and replicates, are stated in each figure legend.
Data availability
All data supporting the conclusions included in the manuscript are available within the paper and its supplementary information. Source data for figures and graphs in the main text can be found in Supplementary Data.