Directed evolution of a soluble human DR3 receptor for the inhibition of TL1A induced cytokine secretion.

Itay Levin,Amir Aharoni,Marianna Zaretsky,Jagadeesh Bayry

doi:10.1371/journal.pone.0173460

Abstract

TNF-like 1A (TL1A) is a cytokine belonging to the TNF superfamily that promotes inflammation in autoimmune diseases. Inhibiting the interaction of TL1A with the endogenous death-domain receptor 3 (DR3) offers a therapeutic approach for treating TL1A-induced autoimmune diseases. Here, we generated improved DR3 variants showing increased TL1A binding affinity and stability using a directed evolution approach. Given the high cysteine content and post-translational modification of DR3, we employed yeast surface display and expression in mammalian cell lines for screening, expression and characterization of improved DR3 variants. A cell-based assay performed with the human TF-1 cell line and CD4+ T cells showed that two improved DR3 mutants efficiently inhibited TL1A-induced cell death and secretion of IFN-γ, respectively. These DR3 mutants can be used as drug candidates for the treatment of inflammatory bowel diseases and for other autoimmune diseases, including rheumatic arthritis and asthma.

Highlights

The mammalian immune system is a complex network of cells regulated by signals transmitted by many secreted and receptor proteins
We found that the addition of increasing concentrations of soluble domain receptor 3 (DR3) to PBL or CD4+ T cells together with tumor necrosis factor (TNF)-like 1A (TL1A)/IL-12/IL-18 led to a gradual decrease in TL1A-induced IFN-γ secretion (Fig 3A)
To obtain insight into the possible location of the mutations in DR3 structure, we generated a structural model of the extracellular domain (ECD) using the I-TASSER server [32]

Summary

Introduction

The mammalian immune system is a complex network of cells regulated by signals transmitted by many secreted and receptor proteins. These include the human tumor necrosis factor (TNF) superfamily that comprises at least 19 members and represents a major class of stimulatory proteins. TL1A is currently the only known ligand for death-domain receptor 3 (DR3), which is predominantly expressed by activated T cells and endothelial cells [4,5]. Binding of TL1A to DR3 triggers proliferative signals, most likely via the activation of NF-κB-associated pathways [4]. Subsequent studies have shown that TL1A is PLOS ONE | DOI:10.1371/journal.pone.0173460 March 9, 2017

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Results

Conclusion