Assessment of costimulation and coinhibition in a triple parameter T cell reporter line: Simultaneous measurement of NF-κB, NFAT and AP-1

Abstract

Engagement of the T cell receptor complex reprograms T cells for proliferation, cytokine production and differentiation towards effector cells. This process depends on activating costimulatory signals and is counteracted by coinhibitory molecules. Three transcription factors, namely NF-κB, NFAT and AP-1, have a major role in inducing the transcriptional program that is required for T cell activation and differentiation. Here we describe the generation of a triple parameter reporter based on the human Jurkat T cell line, where response elements for NF-κB, NFAT and AP-1 drive the expression of the fluorescent proteins CFP, eGFP and mCherry, respectively. The emission spectra of these proteins allow simultaneous assessment of NF-κB, NFAT and AP-1 activity in response to stimulation. Ligation of the TCR complex induced moderate reporter activity, which was strongly enhanced upon coengagement of the costimulatory receptors CD2 or CD28. Moreover, we have generated and tested triple parameter reporter cells that harbor costimulatory and inhibitory receptors not endogenously expressed in the Jurkat cells. In these experiments we could show that engagement of the costimulatory molecule 4-1BB enhances NF-κB and AP-1 activity, whereas coinhibition via PD-1 or BTLA strongly reduced the activation of NF-κB and NFAT. Engagement of BTLA significantly inhibited AP-1, whereas PD-1 had little effect on the activation of this transcription factor. Our triple parameter reporter T cell line is an excellent tool to assess the effect of costimulatory and coinhibitory receptors on NF-κB, NFAT and AP-1 activity and has a wide range of applications beyond the evaluation of costimulatory pathways.

A human monocytic NF-κB fluorescent reporter cell line for detection of microbial contaminants in biological samples

“…We have previously described the generation of Jurkat reporter cell lines based on a set of highly sensitive and selective fluorescent transcriptional reporter constructs for the activity of the transcription factors AP-1, NFAT and NF-κB [26, 31]. For the current study we aimed to employ our reporter technology for the development of a test system for ligands to toll-like receptors.…”

“…For the current study, we aimed to exploit the exquisite sensitivity and specificity of the TLR signalling machinery for the development of a cell-based, fluorescent reporter system. THP-1, a human monocytic cell line expressing a wide range of TLRs [33], was equipped with an NF-κB-driven reporter construct we have described recently [26, 31, 49]. Our reporter cells are amenable to high-throughput analysis of NF-κB-activity by flow cytometry.…”

“…Overall, by harnessing the evolutionary-conserved sensitivity of the TLR system and combining it with our recently described fluorescent reporter technology [26], we have generated a highly sensitive and versatile cellular test platform for microbial contaminants. We have successfully applied our THP-1 NF-κB-eGFP reporter cells to the detection of mycoplasma and endotoxin in biological samples.…”

“…The NF-κB-eGFP reporter construct was previously described [26]. The THP-1 cells were retrovirally transduced with the NF-κB-eGFP construct and resting cells were sorted to eGFP-low expression.…”

T Cells of Infants Are Mature, but Hyporeactive Due to Limited Ca2+ Influx

“…For T cell activation, three main signal transduction pathways are initiated by TCR/CD28 signaling which leads to expression and translocation of the transcription factors NF-κB (nuclear factor kappa-light-chain enhancer of activated B cells), AP-1 (activator protein 1), and NFATc (nuclear factor of activated T cells cytoplasmic) into the nucleus (see below) [16]. Their binding to the IL-2 promoter is an obligatory prerequisite for IL-2 transcription.…”