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.

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.

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.

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.

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.