1. Volume 20, Issue 5, Pages 1017-1028 (August 2017)
Blockage of Core Fucosylation Reduces Cell-Surface Expression of PD-1 and Promotes Anti-tumor Immune Responses of T Cells 
Masahiro Okada, Shunsuke Chikuma, Taisuke Kondo, Sana Hibino, Hiroaki Machiyama, Tadashi Yokosuka, Miyako Nakano, Akihiko Yoshimura 
Cell Reports 
Volume 20, Issue 5, Pages (August 2017)
DOI: /j.celrep
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2. Cell Reports 2017 20, 1017-1028DOI: (10.1016/j.celrep.2017.07.027)
Copyright © 2017 The Authors Terms and Conditions

3. Figure 1
Overview of Screening for Genes that Positively Regulate PD-1 Expression
(A) Schematic representation of the loss-of-function programmed cell death 1 (PD-1) expression screen using the mouse genome-wide knockout library T cell lines stably expressing Cas9-P2A-Puro (68-41-Cas9) were transduced with a lentiviral gRNA library (∼87,898 gRNAs/∼19,150 genes). PD-1low fractions were sorted and expanded four times. The bulk gRNA sequences extracted from the genome DNA of the PD-1low cells were read by high-throughput sequencer.
(B) Representative flow cytometry analysis showing gating strategy of first sort and purification of post-fourth sort.
(C) Representative sequence analysis of the number of read counts and gRNA sequence rank score.
(D) Average of PD-1 mean fluorescence intensity (MFI) in each gRNA-positive cell Cas9 was individually transduced with gRNA against candidate gene (nos. 1–3). gRNA-targeted genes were listed in order of false discovery rate (FDR) compared with cells transduced with control gRNA (Luciferase and EGFP). The dashed red line indicates the border of FDR < The genes involved in the core fucosylation pathway are emphasized in red. PD-1 and control gRNA are emphasized in orange and blue, respectively. Data are shown as mean ± SD of gRNA nos. 1–3.
(E) Pathway of core fucosylation and GDP-L-fucose biosynthesis. Gmds, Tsta3, Slc35c1, and Fut8 encode GMDS, FX, FUCT1, and FUT8, respectively.
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4. Figure 2
Validation of the Selected Genes Involved in the Core Fucosylation Process
(A and B) Left: representative flow cytometry analysis of Cas9 cells individually transduced with gRNA targeting the Luciferase or Fut8 genes showed expression of core fucose (Lens culinaris agglutinin [LCA]) binding and PD-1 (A) or PD-L1 Ig binding (B). Right: PD-1 (A) or PD-L1 Ig (B) MFI in cells gated on LCA(+) or LCA(−) were plotted. Note that Tsta3-targeted gRNA no. 3 yielded few LCA(−) cells, which could not be analyzed. Data are pooled from 2 independent experiments and shown as mean ± SD.
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5. Figure 3
N-Linked Glycosylation Analysis Determines Core Fucosylation Sites of PD-1 and Its Biological Function
(A) Average MS spectrum of desialo-glycopeptides containing N74-glycans. Base peak chromatogram (BPC) used for obtaining the average MS spectrum was shown in Figure S3A. MS/MS spectrum of monofucosylated glycans was shown in Figure S3B to show presence of core fucose. Average MS spectrum of glycopeptides containing N49 glycans and N116 glycans and summary of relative glycan structures were shown in Figure S3C.
(B) Schematic diagram of PD-1-GFP and construction of its glycosylation site mutants. EC, TM, and IC refer to the extracellular, transmembrane, and intracellular domain, respectively.
(C) Lectin and western blot analysis of immunoprecipitated PD-1-GFP and its glycosylation site mutants. Densitometry analysis of Aspergillus oryzae L-fucose-specific lectin (AOL)/GFP is plotted. Data are shown as mean ± SD; n = 3.
(D) Expression of rescued PD-1 wild-type (WT) or its glycosylation mutants in PD-1 knockout or PD-1 and Fut8 double-knockout Cas9 cells were analyzed by flow cytometry. Relative PD-1 MFI was plotted. NS means not statistically significant. Data are pooled from 3 independent experiments and shown as mean ± SD.
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6. Figure 4
Blocking Core Fucosylation Downregulates PD-1 Expression and Enhances T Cell Activation
(A–C) MFI of PD-1 (A) or percentages of interferon-gamma (IFN-γ)(+), interleukin-2 (IL-2)(+) (B), and memory fractions (C) were plotted. Data are representative of 2 independent experiments and shown as mean ± SD.
(D and E) Flow cytometry analysis of T helper cell 1 (Th1)-skewed CD4 cells treated with DMSO or 100 μM 2F-Fuc shows LCA and PD-1 (D) or indicates cell-surface markers (E). The numbers on the indicated panel show MFI.
(F) Flow cytometry analysis as in (E) shows surface and intracellular expression of PD-1. The numbers on the indicated panel show MFI.
(G) MFI of PD-1 and its relative percentage of initial MFI after brefeldin A chase experiments in Th1-skewed CD4 cells treated with DMSO or 100 μM 2F-Fuc. Data are pooled from 5 independent experiments and shown as mean ± SD.
(H) MFI of PD-L1 Ig was plotted. Data are pooled from 5 independent experiments and shown as mean ± SD.
(I) Flow cytometry analysis of carboxyfluorescein diacetate succinimidyl diester (CFSE)-labeled CD4 T cells activated by allogeneic dendritic cells (DCs) under Th1-skewing condition shows CFSE dilution and IL-2 and IFN-γ production.
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7. Figure 5 Blocking Core Fucosylation Strengthens Anti-tumor Immunity
(A) Flow cytometry analysis of tumor-infiltrating lymphocytes (TILs) in B16 melanoma model 2 weeks after transplantation shows LCA binding gated on indicated exhaustion markers. LCA MFI was plotted. Data are shown as mean ± SD; n = 6.
(B) Experimental scheme of B16 melanoma mice model. Eleven days after tumor inoculation, activated CD8 OT-I cells treated with DMSO or 100 μM 2F-Fuc were intravenously transferred into B16-ovalbumin (OVA)-transplanted mice, and the mice were immunized with OVA emulsified with incomplete Freud’s adjuvant (IFA/OVA).
(C) Tumor volume was measured every 2 days after transfer of the OT-I T cells. Data are pooled from 4 independent experiments and shown as mean ± SEM.
(D) Transferred OT-I T cells in TIL and lymph nodes were analyzed 6 days after transfer. Upper: flow cytometry analysis shows granzyme B, tumor necrosis factor-α (TNF-α), and IFN-γ. Lower: total cell numbers or percentages of indicated cell fractions were plotted. Data are pooled from 2 independent experiments and shown as mean ± SEM; n = 9.
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