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Cited by 5,294T-cell and B-cell ImmunologyCancer Immunotherapy and BiomarkersImmunotherapy and Immune Responses

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Engagement of the Pd-1 Immunoinhibitory Receptor by a Novel B7 Family Member Leads to Negative Regulation of Lymphocyte Activation

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Freeman, Gordon J., Long, Andrew J., Iwai, Yoshiko, Bourque, Karen, Chernova, Tatyana, Nishimura, Hiroyuki, Fitz, Lori J., Malenkovich, Nelly, Okazaki, Taku, Byrne, Michael C., Horton, Heidi F., Fouser, Lynette, Carter, Laura, Ling, Vincent, Bowman, Michael R., Carreno, Beatriz M., Collins, Mary, Wood, Clive R., Honjo, Tasuku·2000·The Journal of Experimental Medicine
DOI 10.1084/jem.192.7.1027PMC PMC2193311
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Abstract

PD-1 is an immunoinhibitory receptor expressed by activated T cells, B cells, and myeloid cells. Mice deficient in PD-1 exhibit a breakdown of peripheral tolerance and demonstrate multiple autoimmune features. We report here that the ligand of PD-1 (PD-L1) is a member of the B7 gene family. Engagement of PD-1 by PD-L1 leads to the inhibition of T cell receptor-mediated lymphocyte proliferation and cytokine secretion. In addition, PD-1 signaling can inhibit at least suboptimal levels of CD28-mediated costimulation. PD-L1 is expressed by antigen-presenting cells, including human peripheral blood monocytes stimulated with interferon gamma, and activated human and murine dendritic cells. In addition, PD-L1 is expressed in nonlymphoid tissues such as heart and lung. The relative levels of inhibitory PD-L1 and costimulatory B7-1/B7-2 signals on antigen-presenting cells may determine the extent of T cell activation and consequently the threshold between tolerance and autoimmunity. PD-L1 expression on nonlymphoid tissues and its potential interaction with PD-1 may subsequently determine the extent of immune responses at sites of inflammation.

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Figures

Figure 1 Amino acid sequence alignment of murine and human B7 gene family members. Identical amino acids are boxed in black and conservative substitutions in gray. Predicted signal and transmembrane domains of PD-L1 are underlined. Amino acids comprising the binding site of human B7-1 are indicated with a dot (reference 26). The sequences of the human (reference 11) and murine (references 17 and 18) ligands of ICOS (ICOS-L) have been reported previously. The full-length cDNA sequences of the human and murine PD-L1 cDNAs have been deposited with EMBL/GenBank/DDBS under accession nos. AF233516 and AF233517, respectively.
Figure 1 Amino acid sequence alignment of murine and human B7 gene family members. Identical amino acids are boxed in black and conservative substitutions in gray. Predicted signal and transmembrane domains of PD-L1 are underlined. Amino acids comprising the binding site of human B7-1 are indicated with a dot (reference 26). The sequences of the human (reference 11) and murine (references 17 and 18) ligands of ICOS (ICOS-L) have been reported previously. The full-length cDNA sequences of the human and murine PD-L1 cDNAs have been deposited with EMBL/GenBank/DDBS under accession nos. AF233516 and AF233517, respectively.CC BY-NC-SA · PubMed Central
Figure 2 The binding of PD-1 to PD-L1. (A) CHO cells stably transfected with human or murine PD-L1 or vector alone were stained with hPD-1.Ig(γ2a), mPD-1.Ig(γ1), hCTLA-4.Ig(γ2a), hCD28.Ig(γ1), or hICOS.Ig(γ2a) (species matched), and developed with goat anti–murine IgG2a-PE or anti–human IgG-FITC antisera. (B) PD-L1.Ig was tested for binding to immobilized mPD-1.Ig(γ1) (gray bars), hPD-1.Ig(γ1) (black bars), and hCTLA-4.Ig(γ1) (white bars) using surface plasmon resonance on a BIAcore 2000 instrument. Receptor-Fc fusion proteins were immobilized on a CM5 dextran chip by amine coupling with normal human serum/N-ethyl-N′-(dimethylamino)propyl I carbodiimide hydrochloride (EDC) in 10 mM sodium acetate, pH 4.0, as described (reference 27). The amounts of protein immobilized were 5,383 response units (RU) for mPD-1.Ig(γ1), 5,416 RU for hPD-1.Ig(γ1), and 11,493 RU for hCTLA-4.Ig(γ1). Concentrated COS-conditioned medium from hPD-L1.Ig(γ2a)–transfected cells was analyzed with (+) or without (−) coinjection of 100 μg/ml of soluble mPD-1.Ig, hPD-1.Ig, or hCTLA-4.Ig for competition. Binding was quantified as an increase in RU at 60 s after the end of injection compared with a baseline established 20 s before injection.
Figure 2 The binding of PD-1 to PD-L1. (A) CHO cells stably transfected with human or murine PD-L1 or vector alone were stained with hPD-1.Ig(γ2a), mPD-1.Ig(γ1), hCTLA-4.Ig(γ2a), hCD28.Ig(γ1), or hICOS.Ig(γ2a) (species matched), and developed with goat anti–murine IgG2a-PE or anti–human IgG-FITC antisera. (B) PD-L1.Ig was tested for binding to immobilized mPD-1.Ig(γ1) (gray bars), hPD-1.Ig(γ1) (black bars), and hCTLA-4.Ig(γ1) (white bars) using surface plasmon resonance on a BIAcore 2000 instrument. Receptor-Fc fusion proteins were immobilized on a CM5 dextran chip by amine coupling with normal human serum/N-ethyl-N′-(dimethylamino)propyl I carbodiimide hydrochloride (EDC) in 10 mM sodium acetate, pH 4.0, as described (reference 27). The amounts of protein immobilized were 5,383 response units (RU) for mPD-1.Ig(γ1), 5,416 RU for hPD-1.Ig(γ1), and 11,493 RU for hCTLA-4.Ig(γ1). Concentrated COS-conditioned medium from hPD-L1.Ig(γ2a)–transfected cells was analyzed with (+) or without (−) coinjection of 100 μg/ml of soluble mPD-1.Ig, hPD-1.Ig, or hCTLA-4.Ig for competition. Binding was quantified as an increase in RU at 60 s after the end of injection compared with a baseline established 20 s before injection.CC BY-NC-SA · PubMed Central
Figure 3 Expression of PD-L1 in antigen-presenting cells and murine tissues. (A) Northern blot analysis of total RNA of human peripheral blood monocytes stimulated with 500 U/ml human IFN-γ, 100 U/ml human TNF-α, or media alone, and anti-Ig–activated human B cells, with human PD-L1, B7-1, B7-2, and β-actin cDNA probes. Cells were prepared and stimulated as described (reference 28). Act.B, actinomycin B. (B) Isolated human peripheral blood dendritic cells (DC) were cultured in either human GM-CSF alone (gray bars) or in GM-CSF, LPS, and IFN-γ (black bars) for 4 or 20 h, after which RNA was isolated for quantitative (real time) PCR analysis. Fluorescence is plotted as a ratio of PD-L1, B7-1, or B7-2 signal to the GAPDH signal. (C) Northern blot analysis of human keratinocyte total RNA with a human PD-L1 cDNA probe. Keratinocytes were isolated and activated with PMA and IFN-γ, as described previously (reference 10). (D) Northern blot analysis of murine tissue polyA+ RNAs (Ambion) with a murine PD-L1 cDNA probe. (E) The human PD-L1 gene was amplified by PCR from monochromosomal somatic cell hybrid genomic DNAs containing the indicated human chromosome as well as hamster, murine, and human genomic DNAs.
Figure 3 Expression of PD-L1 in antigen-presenting cells and murine tissues. (A) Northern blot analysis of total RNA of human peripheral blood monocytes stimulated with 500 U/ml human IFN-γ, 100 U/ml human TNF-α, or media alone, and anti-Ig–activated human B cells, with human PD-L1, B7-1, B7-2, and β-actin cDNA probes. Cells were prepared and stimulated as described (reference 28). Act.B, actinomycin B. (B) Isolated human peripheral blood dendritic cells (DC) were cultured in either human GM-CSF alone (gray bars) or in GM-CSF, LPS, and IFN-γ (black bars) for 4 or 20 h, after which RNA was isolated for quantitative (real time) PCR analysis. Fluorescence is plotted as a ratio of PD-L1, B7-1, or B7-2 signal to the GAPDH signal. (C) Northern blot analysis of human keratinocyte total RNA with a human PD-L1 cDNA probe. Keratinocytes were isolated and activated with PMA and IFN-γ, as described previously (reference 10). (D) Northern blot analysis of murine tissue polyA+ RNAs (Ambion) with a murine PD-L1 cDNA probe. (E) The human PD-L1 gene was amplified by PCR from monochromosomal somatic cell hybrid genomic DNAs containing the indicated human chromosome as well as hamster, murine, and human genomic DNAs.CC BY-NC-SA · PubMed Central
Figure 4 TCR activation of murine and human T cells in the presence of PD-L1 results in inhibition of T cell proliferation and cytokine production. (A) Splenic T cells from wild-type (open squares) and PD-1 −/− (filled circles) C57BL/6 mice were stimulated for 72 h with varying concentrations of precoated anti-CD3 Ab. (B) Splenic T cells from wild-type (squares) and PD-1 −/− (circles) C57BL/6 mice were stimulated for 72 h with 10 μg/ml of anti-CD3 mAb in combination with varying concentrations of hPD-L.Ig(γ2a) (filled symbols) or control mIgG2a (open symbols). [3H]Thymidine incorporation was measured in triplicate. These data are representative of four separate experiments. (C) Purified human CD4+ T cells were stimulated for 4 d with anti-CD3 mAb/control IgG–coated beads (stippled bars), anti-CD3 mAb/hPD-L1.Ig(γ2a)–coated beads (hatched bars), or medium alone (black bars). The bead/cell ratio was 1:1. Proliferation was determined by [3H]thymidine incorporation in triplicate wells. Supernatants were collected at 96 h after activation, and cytokine concentrations were measured using commercially available ELISA kits. The data are representative of two separate experiments.
Figure 4 TCR activation of murine and human T cells in the presence of PD-L1 results in inhibition of T cell proliferation and cytokine production. (A) Splenic T cells from wild-type (open squares) and PD-1 −/− (filled circles) C57BL/6 mice were stimulated for 72 h with varying concentrations of precoated anti-CD3 Ab. (B) Splenic T cells from wild-type (squares) and PD-1 −/− (circles) C57BL/6 mice were stimulated for 72 h with 10 μg/ml of anti-CD3 mAb in combination with varying concentrations of hPD-L.Ig(γ2a) (filled symbols) or control mIgG2a (open symbols). [3H]Thymidine incorporation was measured in triplicate. These data are representative of four separate experiments. (C) Purified human CD4+ T cells were stimulated for 4 d with anti-CD3 mAb/control IgG–coated beads (stippled bars), anti-CD3 mAb/hPD-L1.Ig(γ2a)–coated beads (hatched bars), or medium alone (black bars). The bead/cell ratio was 1:1. Proliferation was determined by [3H]thymidine incorporation in triplicate wells. Supernatants were collected at 96 h after activation, and cytokine concentrations were measured using commercially available ELISA kits. The data are representative of two separate experiments.CC BY-NC-SA · PubMed Central
Figure 5 TCR/PD-L1 activation in the presence of CD28 costimulation results in inhibition of T cell proliferation. Purified human CD4+ cells were stimulated for 4 d with anti-CD3 mAb/control mIgG–coated beads (gray bars), or anti-CD3 mAb/hPD-L1.Ig(γ2a)–coated beads (black bars) in the presence of various concentrations of soluble anti-CD28 mAb. Stimulations were performed at (A) suboptimal (1 μg/ml) and (B) optimal (2 μg/ml) concentrations of anti-CD3 Ab. The bead/cell ratio was 1:1. Proliferation was determined by [3H]thymidine incorporation in triplicate wells. The data are representative of two separate experiments.
Figure 5 TCR/PD-L1 activation in the presence of CD28 costimulation results in inhibition of T cell proliferation. Purified human CD4+ cells were stimulated for 4 d with anti-CD3 mAb/control mIgG–coated beads (gray bars), or anti-CD3 mAb/hPD-L1.Ig(γ2a)–coated beads (black bars) in the presence of various concentrations of soluble anti-CD28 mAb. Stimulations were performed at (A) suboptimal (1 μg/ml) and (B) optimal (2 μg/ml) concentrations of anti-CD3 Ab. The bead/cell ratio was 1:1. Proliferation was determined by [3H]thymidine incorporation in triplicate wells. The data are representative of two separate experiments.CC BY-NC-SA · PubMed Central

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Research works printed but not linked: 2

  1. Byrne, 2000, Preparation of mRNA for expression monitoring, 22
  2. Freeman, 1992, CTLA-4 and CD28 are coexpressed in most T-cells after activation, J. Immunol., 149, 3795, 10.4049/jimmunol.149.12.3795 · DOI

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Metadata

Authors
Freeman, G. J., Long, A. J., Iwai, Y., Bourque, K., Chernova, T., Nishimura, H., Fitz, L. J., Malenkovich, N., Okazaki, T., Byrne, M. C., Horton, H. F., Fouser, L., Carter, L., Ling, V., Bowman, M. R., Carreno, B. M., Collins, M., Wood, C. R., & Honjo, T.
Affiliations
  • Harvard University (US)
  • bGenetics Institute, Wyeth-Ayerst Research, Cambridge, Massachusetts 02140
  • Kyoto University (JP)
ORCID
Venue
The Journal of Experimental Medicine
Publisher
Rockefeller University Press
Published
2000-10-02
Volume
192
Issue
7
Pages
1027-1034
ISSN
  • 0022-1007 (ISSN-L)
  • 1540-9538
DOI
10.1084/jem.192.7.1027
PMC
PMC2193311
OpenAlex
W2118471749
Cited by
5,294
References
26
Citations by year
2026: 147 · 2025: 255 · 2024: 298 · 2023: 328 · 2022: 357 · 2021: 454 · 2020: 451 · 2019: 391 · 2018: 386 · 2017: 375 · 2016: 279 · 2015: 267 · 2014: 158 · 2013: 163 · 2012: 109
FWCI
18.27
Type
journal article
Language
en
Open access
Locations
  • The Journal of Experimental Medicine (publishedVersion)
  • PubMed Central (publishedVersion, cc-by-nc-sa)
  • PubMed (publishedVersion)
Funders
  • Association pour la Recherche sur le Cancer
  • Seerave Foundation
  • E-Rare
  • European Commission
  • Agence Nationale de la Recherche (ANR-16-RHUS-0008)
  • Fondation Leducq
  • Institut National de la Santé et de la Recherche Médicale
  • Fondation pour la Recherche Médicale
  • Ligue Contre le Cancer
  • Institut Universitaire de France
  • Institut National Du Cancer
  • National Institutes of Health (5p01ai041584-05)
  • Horizon 2020 Framework Programme (GDW20171100085, GDW20181100051)
  • High-end Foreign Experts Recruitment Plan of China (GDW20171100085, GDW20181100051)
  • Labex Immuno-Oncology
  • National Cancer Institute (CA 84500, R01CA084500)
  • National Institute of Allergy and Infectious Diseases (AI 39671, AI 41584, P01AI039671)
Topics
  • Life Sciences › Immunology and Microbiology › Immunology › T-cell and B-cell Immunology
  • Health Sciences › Medicine › Oncology › Cancer Immunotherapy and Biomarkers
  • Life Sciences › Immunology and Microbiology › Immunology › Immunotherapy and Immune Responses
Keywords
PD-1, PD-L1, B7 family, T cell activation, lymphocyte proliferation, cytokine production, CD28 costimulation, antigen-presenting cells, dendritic cells, monocytes, interferon-gamma, autoimmunity, peripheral tolerance
MeSH terms
Amino Acid Sequence, Animals, Antigen-Presenting Cells, Antigens, Surface, Base Sequence, Cell Division, Humans, Ligands, Membrane Glycoproteins, Molecular Sequence Data, T-Lymphocytes, Signal Transduction, Antigens, CD, Gene Expression, CD3 Complex, DNA, Complementary, CD28 Antigens, B7-1 Antigen, Apoptosis Regulatory Proteins, Mice, B7-2 Antigen, Programmed Cell Death 1 Receptor

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