Generation of a Jurkat-based fluorescent reporter cell line to evaluate lipid antigen interaction with the human iNKT cell receptor

Piotr Humeniuk,Sabine Geiselhart,Karin Hoffmann-Sommergruber,Wolfgang Paster,Tonya Webb,Peter Steinberger,Claire Battin

doi:10.1038/s41598-019-43529-4

Piotr Humeniuk, Sabine Geiselhart + Show 5 more

Open Access

https://doi.org/10.1038/s41598-019-43529-4

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Abstract

Invariant natural killer T (iNKT) cells are a specialized subset of T cells contributing to both, the innate and adaptive immune responses. In contrast to conventional T lymphocytes they recognize lipid antigens. The aim of the project is to establish a novel model system, to study iNKT-TCR – ligand interaction. An iNKT reporter cell line (JE6-1REP-iNKT) was engineered by introducing the human iNKT-TCR into a human leukemic T cell line carrying an NF-κB-driven fluorescent transcriptional reporter construct. Antigen presenting BWSTIM cells expressing human CD1d and CD80 were generated. Reporter induction in JE6-1REP-iNKT cells was assessed by flow cytometry. CRISPR/Cas9 was used for β2M knock out in JE6-1REP-iNKT cells to abrogate CD1d expression and thus excluding antigen self-presentation. Reporter cells were shown to specifically react with iNKT antigens presented via CD1d. Their sensitivity towards α-GalCer was comparable to a murine iNKT hybridoma cell line. In conclusion, we created a novel iNKT reporter platform which, compared to traditional iNKT cell assays, is characterized by a shorter turnaround time and lower costs. It thus facilitates the identification of antigenic structures that drive the activation of iNKT cells in health and disease.

Highlights

Invariant natural killer T cells are a specialized subset of T cells contributing to both, the innate and adaptive immune responses
Surface expression of the Invariant natural killer T (iNKT)-TCR in the JE6-1REP-iNKT reporter cell line was verified by positive staining with APClabelled α-GalCer loaded CD1d tetramers (Fig. 1B, upper panel)
The well-established murine hybridoma cell line DN32.D337 stained positive for both, the iNKT-TCR and CD28 (Fig. 1B – right panel), in contrast to the control cell line N37-1A1238, which stained positive only for CD28

Summary

Introduction

Invariant natural killer T (iNKT) cells are a specialized subset of T cells contributing to both, the innate and adaptive immune responses. Reporter cells were shown to react with iNKT antigens presented via CD1d. Their sensitivity towards α-GalCer was comparable to a murine iNKT hybridoma cell line. We created a novel iNKT reporter platform which, compared to traditional iNKT cell assays, is characterized by a shorter turnaround time and lower costs It facilitates the identification of antigenic structures that drive the activation of iNKT cells in health and disease. Recent findings indicate the participation of iNKT cells in the pathomechanisms of allergy and asthma[20,21] Whether they generally play a protective or pathological role is still under debate[13]

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