March 15, 2017 (Vol. 37, No. 6)

Rapid, Real-Time Detection and Monitoring of T Cell Activation for Immunotherapy

Recent clinical successes in targeting the immune system instead of cancer cells have generated a resurgence of effort to harness the immune system more routinely for therapeutic intervention. A key success factor in these efforts will be the ability to control the qualitative and quantitative aspects of immune-cell activation. However, current approaches for detecting and monitoring T cell activation are almost exclusively single time-point measurements, performed hours to days after the initial stimulation.

This time lag prevents the opportunity to measure and monitor the earliest phases of activation, which have profound effects on the type of response, effectiveness, and durability. For example, immediately upon activation immune cells undergo dynamic changes in the metabolic pathways engaged, their kinetics, and magnitude. The interplay and coordination of these initiating factors significantly influence the resulting response and outcome. Having a better understanding and ability to manipulate these early events will play a significant role in the development of a diverse and effective array of immunotherapeutic modalities.

This tutorial describes a novel assay that measures immune-cell activation within minutes of activation, throughout the fully developed activated phenotype, in both a qualitative and quantitative manner. This assay provides a sensitive window of opportunity to identify and characterize attributes that can drive immune responses to the desired outcome. The tremendous early clinical successes of immunotherapy approaches, especially in cancer, have created vast opportunities and need for tools to determine optimal immune-cell activation, leading to desired cell fates and clinical responses in this rapidly developing field.

Energy Metabolism

Energy metabolism reacts instantaneously to T cell activation in both a qualitative (metabolic pathways engaged) and quantitative (level of energetic activity) manner. Equally relevant is the metabolic “wiring” before activation.1,2 This metabolic wiring of relevant cell lineages profoundly influences, if not dictates, the type of response that follows. The ability to measure these energetic changes in real time would enable scientists to follow immune responses from initiation, and help immunotherapy developers tailor immune responses to desired outcomes.

The first demonstration of this principle was published by Gubser et al. in Nature Immunology in August 2013.1 An “in-Seahorse activation system” was used to investigate energy metabolism in quiescent naïve and effector memory CD8+ killer T cells, after stimulation via the T cell receptor and its co-receptor CD28. In the assay, they injected into the T cell culture monoclonal antibody (mAb) to CD3 (T cell receptor) and mAb to CD28 (co-receptor), while monitoring the glycolytic rate in real time. They were able to demonstrate how both the wiring and early response kinetics between the two functional subsets differed, and more importantly how this influenced the outcome of the immune response. This research was the first formal demonstration of the requirement for an immediate-early glycolytic energy metabolic switch to achieve rapid killing function of memory vs. naïve CD8+ killer T cells.

The Gubser et al. paper provided the starting point for the design and development of an application that enables the immediate and kinetic measurement of T cell activation, within a few minutes of stimulation. Scientists can now reveal in a single, rapid assay both the qualitative (pathway contributions of mitochondrial respiration and glycolytic flux) and quantitative (rate of energy metabolism) aspects of T cell activation. A powerful tool for optimizing immune-cell activation, this will enable a better fundamental understanding of immune-cell function, as well as improved design, development, and potentially the improved production of immune therapies.

Figure 1. XFp-based detection of CD4+ T cell activation. (A) XFp T cell Activation Assay strategy. (B) Kinetic trace of ECAR vs. time, demonstrating increases in ECAR upon injection (arrow) of anti-CD3/CD28 beads onto human T cells; naïve T cells activated with anti-CD3/CD28 bead injection (•, 4:1 bead-to-cell ratio); naïve T cells receiving vehicle injection show no activation (•).

Extracellular Flux Technology

The key to this application was the use of extracellular flux (XF) technology. XF technology follows the two major energy metabolism pathways, mitochondrial respiration and glycolysis, in real time. The rate of oxygen consumption (OCR) provides a measure of mitochondrial respiration, and the extracellular acidification rate (ECAR) is an indicator of glycolysis. As discussed above, and in additional publications, a correlation has been established between T cell activation and an acute increase in glycolysis as measured by ECAR.1–5

Proof of concept for in situ T cell activation by injection of immunogenic beads was illustrated through the design and use of several assays. Figure 1 shows a pairwise comparison between naïve CD4+ helper T cells and those that received an injection of anti-CD3/CD28 DynaBeads. Cells exposed to beads that activate the T cell receptor have an immediate and significant increase in ECAR, while ECAR in naïve cells remains at a basal level (Figure 1B).

Next, to ensure in situ-activated CD4+ helper and CD8+ killer T cells reached the same level of ECAR as T cells activated by a pretreatment of immunogenic beads, a pairwise comparison was made between these conditions (Figures 2A and 2E). Indeed, the ECAR values were of similar magnitude for both groups, as demonstrated in Figures 2B and 2F. To demonstrate that increases in ECAR upon T cell activation resulted in appropriate immune function, downstream production of both interleukin (IL-2) and interferon (INF-γ) was measured. The same cells that demonstrated ECAR increases after activation were cultured for an additional 48–72 hours to enable cytokine production. Figures 2C and 2G show similar levels of IL-2 and INF-γ expression, respectively, between in situ and pre-activated T cells 48 hours post-activation. Figures 2D and 2H further validate the assay by demonstrating a typical progression of morphological changes associated with T cell activation, including increases in cell diameter and the number of cells due to proliferation for in situ-activated T cells 72 hours post-activation.

In conclusion, the XF T cell activation assay allows for a real-time kinetic test of activation by monitoring ECAR, an indicator of glycolytic activity. The results of T cell activation may be observed within minutes, as compared to conventional detection methods, which can take hours or days and are typically “end point” in nature. This early, kinetic window significantly influences the effectiveness of the resulting immune response, affording those designing and developing immunotherapies a valuable tool to help determine how to tailor outcomes.

Figure 2. Stimulation of ECAR correlates with markers of T cell activation. (A) and (E) XFp T cell activation assay strategy. (B) and (F) kinetic traces of ECAR vs. time, showing that in situ-activation of naïve T cells by bead injection (•) reaches the same magnitude as T cells activated by conventional bead treatment (•, 1:1 bead-to-cell ratio, plated and incubated for 1 hour) prior to the XF assay. (C) and (G) ELISA assay showing comparative levels of IL-2 (ng/mL) and INF-? (ng/mL) production in the in situ-activated (•) and pre-activated (•) T cells. (D) and (H) images of T cells showing morphological changes on XFp plates days 0 and 3 post-in situ-activation by bead injection. Representative data for human CD4+ (upper panel) and CD8+ (lower panel) T cells.

David A. Ferrick ([email protected]) is senior director, cell analysis; Brian P. Dranka is manager of biology, cell analysis; Yoonseok Kam is an R&D scientist, cell analysis; Pamela M. Swain is an R&D Scientist, cell analysis; and Luke Dimasi is global product marketing manager, cell analysis, at Agilent Technologies.

1. Gubser, P. M.; et al. Rapid effector function of memory CD8+ T cells requires an immediate-early glycolytic switch. Nat. Immunol. 2013, 14, 1064 1072.
2. Sukumar M.; et al. Inhibiting glycolytic metabolism enhances CD8+ T cell memory and antitumor function. J Clin Invest. 2013 Oct;123(10):4479-88.
3. van der Windt, G. J.; Chang, C. H.; Pearce, E. L. Measuring Bioenergetics in T Cells Using a Seahorse Extracellular Flux Analyzer. Curr. Protoc. Immunol. 2016, 113, 3 16B 11-13, 16B 14.
4. Procaccini, C.; et al. The Proteomic Landscape of Human Ex Vivo Regulatory and Conventional T Cells Reveals Specific Metabolic Requirements. Immunity 2016, 44, 406-421.
5. Buck, M. D.; et al. Mitochondrial Dynamics Controls T Cell Fate through Metabolic Programming. Cell 2016, 166, 63-76.

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