Application Note


Agilent Seahorse XF Technology sheds light on immune cell kinetics and activation requirements


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ecent advances in the field of immunology identify cellular metabolism as a primary driver and regulator of immune cell function and differ- entiation. Many immune cell types show increased glycolysis upon activation, which is required for differentiation and for instructing immune cell fate and function. This Application Note demonstrates how Agilent Seahorse XF Technology can be used in a live-cell, real-time activation assay to detect and monitor immune cell activa- tion, illustrated by immune cell models including T cells and M1 macrophages. In this study, XF technology was used to simul- taneously measure the two major metabolic pathways that regulate immune cell func- tion – glycolysis and respiration. Notably, results herein report on glycolytic flux mea- surements accurately quantified by real-time proton efflux rates (PER). This assay can be adapted for routine studies on activation of T cells and macrophages and for the other immune cell types to investigate activation requirements and cell signalling.


Signalling requirements in glycolytic activation of T cells T cell activation is driven by a rapid switch towards glycolysis for cellular energy pro- duction1. The XF T cell activation assay can be adapted to understand cellular and sig- nalling requirements that establish activa- tion of an effector response. In this study, naïve CD8+ T cells activated with anti- CD3/CD28 bead injection showed an imme- diate glycolytic response, while naïve T cells receiving vehicle injection show no activa- tion-associated glycolytic response. The acti- vation-associated glycolytic response was blocked by PI3K inhibitor (LY294002) and Akt inhibitor (Akti-1/2), but not by mTOR inhibitor (rapamycin), indicating that T cell activation-associated glycolytic response requires PI3k-Akt signalling (Figure 1).


Drug Discovery World Winter 2017/18


Bi-phasic M1 macrophage activation revealed by long-term kinetic analysis


Naive CD8 T cells 120


40 80


0 0 40 80 Time (min) 120


Activation Rapamycin


LY294002 Akti-1/2


No Activation


Real-time kinetic detection of the early and continued glycolytic response can be used as a rapid and reliable method for immune cell activation detection and to monitor subsequent activity. Murine RAW264.7 macrophages co-stimulated by lipopolysac-


Figure 1: T cell activation requires PI3K-Akt signalling. Naïve T cells were activated by injection of anti-CD3/CD28 beads with or without pretreatment with inhibitors


A


200 400 600


0 B


1400W Control


LPS+IFNγ LPS+IFNγ+1400W


charide (LPS) and interferon gamma (IFNγ) show bi-phasic glycolytic responses, with an immediate glycolytic rate increase within 1hr, and a secondary increase caused by suppression of mitochondrial respiration after 4hrs (Figure 2). The mitochondrial down-regulation was preceded by inducible nitric oxide synthase (iNOS) elevation (data not shown) and blocked by iNOS inhibitor (1,400W). However, the immediate early glycolytic response was not affected by the inhibitor at all, demonstrating iNOS-medi- ated sequential regulation of macrophage metabolism and function.


Summary


0 120 240 360 480 600 Time (min)


100 150 200


50 0


1400W Control


LPS+IFNγ LPS+IFNγ+1400W


0 120 240 360 480 600 Time (min)


Figure 2: Bi-phasic changes in glycolysis (A) and OXPHOS (B) upon the activation of


macrophages by LPS/IFNɤ (A). RAW264.7 macrophages were activated by LPS-IFNγ co-injection (black arrow) with or without 1400W, iNOS inhibitor injection (gray arrow)


Reference 1 Gubser, PM et al. Rapid effector function of memory CD8 T cells requires an immediate-early glycolytic switch. Nat Immunol. 2013. 14: 1064-72.


The XF real-time activation assay pro- vides time-resolved information enabling a better understanding of the dynamic activation mechanism and continued response. As demonstrated in the T cell activation experiment, XF Technology provides a routine analysis for measuring cell activation requirements with early detection, within minutes instead of instead of hours/days through typical markers (eg CD-69, IL-2). For the macrophage activation assay, the capabil- ity to detect the magnitude and duration of the activation-associated bi-phasic response elucidates critical cell processes that endpoint assays would have missed. In this study, XF kinetic measurements informed the potential for iNOS activity, highlighting the added value of continu- ous kinetic measurements post-activation.


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PER (pmol/min/200k cells) OCR (pmol/min) PER (pmol/min)


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