CD73 contributes to anti-inflammatory properties of afferent lymphatic endothelial cells in humans and mice.

Dominik Eichin,Akira Takeda,Joni Laakkonen,Patrizia Stoitzner,Lydia Bellmann,Jing Tang,Sirpa Jalkanen,Matti Kankainen,Marko Salmi,Alberto Pessia,Beat A Imhof

doi:10.1002/eji.201948432

Abstract

CD73 is an important ectoenzyme responsible for the production of extracellular adenosine. It is involved in regulating inflammatory responses and cell migration and is overexpressed in various cancers. The functions of CD73 in blood endothelial cells are understood in detail, but its role on afferent lymphatics remains unknown. Moreover, anti‐CD73 antibodies are now used in multiple clinical cancer trials, but their effects on different endothelial cell types have not been studied. This study reveals that a previously unknown role of CD73 on afferent lymphatics is to dampen immune responses. Knocking it out or suppressing it by siRNA leads to the upregulation of inflammation‐associated genes on lymphatic endothelial cells and a more pro‐inflammatory phenotype of interacting dendritic cells in vitro and in vivo. In striking contrast, anti‐CD73 antibodies had only negligible effects on the gene expression of lymphatic‐ and blood‐endothelial cells. Our data thus reveal new functions of lymphatic CD73 and indicate a low likelihood of endothelial cell–related adverse effects by CD73 targeting therapeutic antibodies.

Highlights

CD73, a GPI-anchored ectoenzyme, dephosphorylates AMP into adenosine
To determine the role of CD73 in afferent lymphatics, we silenced the expression of CD73 in primary human lymphatic endothelial cells (LECs) with a pool of four siRNA constructs, CRISPR/Cas9, or a single siRNA
Three days after silencing with the siRNA pool, the gene expression of CD73 was on average reduced by more than 96% (76% after CRISPR/Cas9; 98% after single siRNA), while the protein expression went down by 88% (95% after CRISPR/Cas9; 96% after single siRNA; Fig. 1A and Supporting Information Fig. S1A and B; gating is shown in Supporting Information Fig. S2)

Summary

Introduction

CD73 (ecto-5 -nucleotidase), a GPI-anchored ectoenzyme, dephosphorylates AMP into adenosine. This generates an antiinflammatory and pro-angiogenic halo that can reduce immune activation and promote cancer progression [1, 2]. Due to its abundant expression on many types of tissues and cells throughout the body, CD73 is one of the most important producers of extracellular adenosine. Adenosine acts by engaging a variety of different adenosine receptors (A1, A2A, A2B, and A3) on the cell surface. The exact outcome of this interaction depends on the engaged receptor, and on the cellular partner [3,4,5]. As some cells express CD73 as well as adenosine receptors concomitantly, even self-regulating feedback loops are possible [6, 7]

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