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. 2002 Apr;9(2):133-45.
doi: 10.1038/sj/mn/7800123.

Expression and regulation of the PD-L1 immunoinhibitory molecule on microvascular endothelial cells

Michael J Eppihimer  1 , Jason Gunn, Gordon J Freeman, Edward A Greenfield, Tetyana Chernova, Jamie Erickson, John P Leonard
Affiliations

Expression and regulation of the PD-L1 immunoinhibitory molecule on microvascular endothelial cells

Michael J Eppihimer et al. Microcirculation. 2002 Apr.

Abstract

Objective: To evaluate the expression and regulation of a novel B7-like protein, PD-L1, the ligand for the immunoinhibitory receptor PD-1 expressed on activated T-cells, on microvascular endothelial cells (ECs)

Methods: PD-L1 expression on ECs in vitro and in vivo was quantified by using a dual radiolabeled antibody technique after treatment with interferons (IFN) and IL-12, respectively. Changes in the level of PD-L1 mRNA were determined by using RT-PCR.

Results: PD-L1 was observed to be present on ECs under basal conditions. Treatment of ECs with IFN-alpha, -beta and -gamma, but not LPS, was observed to induce elevations in the mRNA and surface expression of PD-L1 on ECs. By using a dual radiolabeled monoclonal antibody (mAb) technique, PD-L1 expression in various tissues of control and IL-12 challenged wild-type and IFN-gamma-deficient mice was measured. A significant increase in PD-L1 expression was observed in tissues at 24 hours after IL-12-challenge, with peak levels of PD-L1 occurring 72 hours after IL-12 challenge. IL-12 was not effective at inducing PD-L1 expression in tissues of IFN-gamma-deficient mice.

Conclusions: These data show the expression of a novel B7-like molecule on murine ECs that is mediated by IFN-alpha, -beta, and -gamma, and suggest a potential pathway by which ECs may modulate T-cell function.

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Figures

Figure 1
Figure 1
CHO and EL4 PD-L1 transfectants, CHO-mPD-L1 and EL4-mPD-L1, respectively, were made by coelectroporation of linearized mPD-L1 cDNA in the pAXEF mammalian expression vector and a plasmid containing the puromycin resistance gene. Puromycin-resistant cells were stained with PD-1-Ig, sorted twice, and cloned. (A) CHO cells (B) mock-transfected EL4 cells, (C) CHO-mPD-L1 cells, and (D) EL4-mPD-L1 cells were stained with 10 μg/mL 10F.9G2 (solid line) or rat IgG2b isotype control (dash line) and goat anti-rat IgG-FITC or goat anti-rat IgG-PE and analyzed by flow cytometry. 10F.9G2 was observed to bind to CHO-mPD-L1 and EL4-mPD-L1 cells but not the mock-transfected cells, indicating the specificity of 10F.9G2 for mPD-L1.
Figure 2
Figure 2
Dose-response effect of IFN-γ-induced PD-L1 expression on microvascular endothelial cells. FACs analysis of PD-L1 expression on MS1 ECs demonstrates an appreciable level of PD-L1 present on resting ECs. At 24 hours after activation, IFN-γ was observed to elevate the expression of PD-L1 in a dose-dependent manner.
Figure 3
Figure 3
PD-L1 expression on microvascular endothelial cells after activation with LPS and IFN-γ. (A) MS1 ECs were grown to confluency in 12-well tissue culture plates and treated with IFN-γ (100 ng/mL) and/or LPS (1 μg/ mL) for 24 hours, and the expression of PD-L1 was measured by using FACs analysis. (B) Relative levels of PD-L1 mRNA in MS1 ECs after 2 and 6 hours of activation with IFN-γ (100 ng/mL) and/or LPS (1 μg/mL). LPS had no effect on PD-L1 mRNA levels.
Figure 4
Figure 4
Kinetics of (A) PD-L1 surface expression and (B) PD-L1 mRNA in MS1 ECs after activation with IFN-α, -β, and -γ. Confluent MS1 ECs in 48-well tissue culture plates were treated with IFN-α (1000 U/mL), IFN-β (1000 U/mL), and IFN-γ (100 ng/mL), and PD-L1 mRNA and PD-L1 surface expression was measured by using RT-PCR and radiolabeled mAbs, respectively, as described in Materials and Methods. Values are means ± SEM and represent n = 4. *Significantly different from constitutive levels (t = 0), p < 0.05.
Figure 5
Figure 5
Kinetics of PD-L1 expression in the lung and heart of IL-12-challenged wild-type mice was determined after the administration of radiolabeled PD-L1 mAb. Within 24 hours after IL-12 challenge, a significant accumulation of PD-L1 mAb was measured in the heart and lung (p < 0.05). Peak expression of PD-L1 was observed between 48 and 72 hours after IL-12 challenged. By 120 hours, PD-L1 expression was observed to decline but remained significantly above baseline values (p < 0.05). Values shown are means ± SEM.
Figure 6
Figure 6
Immunohistochemical analysis of PD-L1 expression in unstimulated and IL12-challenged murine brains. (A) Frozen tissues sections of unstimulated brains from mice were stained with rat IgG2b and rabbit Ig. (B) Unstimulated and Il-12-challenged brains were stained for the presence of PD-L1 (green) and vWF (red). Colocalization of PD-L1 and vWF indicate that PD-L1 is expressed solely on ECs in these tissues, as indicated by white arrows. (C) A more intense fluorescent intensity of PD-L1 (green) was observed in vessels from IL-12-challenged mice.
Figure 7
Figure 7
Ratio of PD-L1 to PECAM-1 expression in various tissues under control conditions and after IL-12-challenge. Under control conditions, the organs of the gastrointestinal tract were observed to three- to sixfold greater expression of PD-L1 than the lung and heart, as evidence of a greater PD-L1 to PECAM-1 ratio. After IL-12-challenge (72 hours), an invariance in PD-L1 expression was observed between tissues, suggesting that the peak expression of PD-L1 was not different among tissues. Values shown are means ± 95% confidence intervals.
Figure 8
Figure 8
Constitutive and IL-12-induced expression of PD-L1 in wild-type and IFN-γ deficient mice after the administration of a radiolabeled PD-L1 mAb. In comparison, the constitutive expression of PD-L1 was not significantly different between wild-type and IFN-γ-deficient mice. However, administration of IL-12 induced a significant accumulation of PD-L1 mAb in wild-type mice. In contrast, IL-12 administration had no effect on PD-L1 expression in IFN-γ-deficient mice, suggesting that IFN-γ was responsible for mediating the elevation in PD-L1 expression in wild-type mice. Values shown are means ± SEM.
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References

    1. Arbiser JL, Moses MA, Fernandez CA, Ghiso N, Cao Y, Klauber N, et al. Oncogenic H-ras stimulates tumor angiogenesis by two distinct pathways. Proc Natl Acad Sci USA. 1997;94:861–866. - PMC - PubMed
    1. Billiau A, Heremans H, Vandekerckhove F, Dijkmans R, Sobis H, Meulepas E, Carton H. Enhancement of experimental allergic encephalomyelitis in mice by antibodies against IFN-gamma. J Immunol. 1988;140:1506–1510. - PubMed
    1. Briscoe DM, Henault LE, Geehan C, Alexander S, Lichtman AH. Human endothelial cell costimulation of T cell IFN-gamma production. J Immunol. 1997;159:3247–3256. - PubMed
    1. Dalton DK, Pitts-Meek S, Keshav S, Figari IS, Bradley A, Stewart TA. Multiple defects of immune cell function in mice with disrupted interferon-g genes. Science. 1993;259:1739–1742. - PubMed
    1. Duong TT, St Louis J, Gilbert JJ, Finkelman FD, Strejan GH. Effect of anti-interferon-gamma and anti-interleukin-2 monoclonal antibody treatment on the development of actively and passively induced experimental allergic encephalomyelitis in the SJL/J mouse. J Neuroimmunol. 1992;36:105–115. - PubMed

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