Enhance Immunology Research With Real-Time Metabolic Insights

Live Cell Metabolic Analysis for Immunology Research Agilent Seahorse XF technology Immune cells are able to sense a variety of stimulus and signals from their environment and integrate those signals to tune intracellular processes and functions to deliver an integrated immune response. Analytical approaches such as immune cell marker expression assays, cytokine release assays, and pathway profiling provide a conventional categorization of immune cells, especially after the cell is committed to a particular lineage or activation state. However, pathway intervention and modulation to control immune cell function requires a more comprehensive view of immune cell fate and function, including an understanding of the upstream drivers of immune cell processes. Metabolic programs are critical determinants of immune cell outcomes. Agilent Seahorse XF technology provides kinetic, functional metabolic measurements for a more direct measure of immune cell processes as they are happening, in real time. Metabolic programs are upstream determinants of immune cell outcomes. Immune cell processes—including activation, proliferation, and memory development—are all driven by metabolic reprogramming, which can be modulated to enhance performance. Metabolic analysis not only delivers real-time kinetics but provides a rich set of pathway targets to modulate immune cell biology. Direct the Immune System by Modulating the Metabolic Pathways That Regulate Cell Fate and Function Figure 1. Measurement of immune cell metabolic phenotypes, illustrating a relationship between Glycolysis/OXPHOS Ratio and cell fate, fitness, and function. T cell fate Naïve Effector Glycolysis Glycolysis/OXPHOS ratio OXPHOS Time Exhaustion Intervention T cell fitness T cell fate T cell activation Memory Chronic 3 Agilent Seahorse XF technology provides real-time measurement of cellular bioenergetics. Bioenergetic parameters such as mitochondrial activity, glycolysis, and ATP production rates deliver information about the immediate-to-early functions of immune cells for a more complete understanding of immune cell function that can be modulated to enhance or predict performance. Agilent Seahorse XF Cell Analysis Solutions for Immunology Research Generate real-time functional data from live cells Immunology researchers are using Agilent Seahorse XF cell analysis technology to investigate: – Immune cell life cycle, activation, and lineage commitment – Memory phenotyping, fitness, exhaustion, and ability to sustain function – Metabolic fuels requirements – Metabolic signaling, pathways, checkpoints, and blockades – Host-pathogen metabolism in infectious disease State-of-the-art data analytics tools Real-time calculation of results Tested kits, media, and reagents Label-free pH and O2 sensor cartridge More relevant injection ports for real-time modulation Live-cell analysis with 2D and 3D plate options Find out more about the Agilent Seahorse XF Pro, click here. Minutes Minutes CD28, TCRζ 4-1BB, TCRζ Minutes Day 0 Day 7 Day 21 Oligomycin Oligomycin Oligomycin FCCP FCCP FCCP Rotenone Rotenone Rotenone + Antimycin A + Antimycin A + Antimycin A OCR (pmol/min) OCR (pmol/min) OCR (pmol/min) 2500 2500 2500 2000 2000 2000 1500 1500 1500 1000 1000 1000 500 500 500 0 0 0 0 50 100 150 0 50 100 150 0 50 100 150 4 Discover strategies to perturbate pathways and control immune cell response to advance cell therapy developments The goal of immune cell-based therapies is to enhance the performance of native immune cells by expanding or modifying immune cells to alter relevant signaling pathways that results in change in the cellular function. XF analysis provides critical measurements of live cells in real time, revealing the functional outcome of modulation strategies. Discover how modulation of immune cell responses via signaling, checkpoint blockade, or pathway perturbation is "functionalized" through changes in metabolic programming. Spare respiratory capacity (SRC) is a reliable measure of mitochondrial function representing the ability of the cells to increase mitochondrial energy production if required. For expanded CAR T cells, an increase in SRC correlates strongly with T cell memory or T stem cell memory phenotypes. Kawalekar et al. used the Seahorse XF assays to show that the choice of CAR signaling domain determines the bioenergetic phenotype of CD8+ CAR T cells postantigen stimulation. Over a 21-day period, CAR T cells containing the 4-1BB signaling costimulatory domain progressed towards exhibiting a greater SRC, which culminated in enhanced in vitro persistence and increased central memory differentiation relative to CAR T cells containing the CD28-signaling costimulatory domain. Metabolic Analysis Reveals a Rich Set of Pathway Targets to Advance New Therapeutic Opportunities in Immuno-Oncology Figure 2. T cell engineering and bioenergetics affects "T cell fate" favoring toward effector or memory population. Figure 3. Adapted from Kawalekar, O. et al. Distinct Signaling of Coreceptors Regulates Specific Metabolism Pathways and Impacts Memory Development in CAR T Cells. Immunity 2016, 44(2), 380–90. Metabolic analysis advances adoptive immunotherapy development by revealing a critical metabolic link between CAR T coreceptors and effector versus memory cell response Cell Fate Tuning CAR Engineering CAR Engineering CD28, TCRζ 4-1BB TCRζ Immune Memory Persistence Central Memory Oxidative Phosphorylation Tumor Killing Effector memory Aerobic glycolysis T cell Cell Fate Tuning 5 Seahorse XF assays were used in studies by Zandberg, D. et al. to show that oxidative metabolism is upregulated as tumors become resistant to anti-PD-1 blockade. They demonstrated that the metabolic status of the tumor microenvironment can be predictive of tumor response to anti-PD-1 therapy. Notarangelo et al used Seahorse XF technology to show that tumor derived d-2-hydroxyglutarate (D-2HG) leads to altered metabolic poise in CD8+ T cells with increased reliance on oxidative phosphorylation for ATP generation, which results in impaired proliferation, cytokine production, and cytotoxicity. Figure 4. Adapted from Zandberg, D. et al. Tumor hypoxia is associated with resistance to PD-1 blockade in squamous cell carcinoma of the head and neck. J ImmunoTher Cancer 2021, 9(5), e002088. Figure 5. Adapted from Notarangelo, G. et al. Oncometabolite d-2HG alters T cell metabolism to impair CD8+ T cell function. Science 2022, 377(6614), 1519–1529. Figure 6. Adapted from Li, L. et al. Loss of metabolic fitness drives tumor resistance after CAR-NK cell therapy and can be overcome by cytokine engineering. Science Advances 2023, 9, eadd6997 Monitor metabolic changes in tumor cells to better characterize the tumor microenvironment and exploit checkpoint therapies Assess T cell bioenergetic poise for unique insights into cellular phenotype and function Evaluate the impact of metabolic fitness on immune cell function Li et al. used Seahorse XF technology to show that engineering CAR19 NK cells to express interleukin 15 (IL-15) results in enhanced metabolic fitness with improved glycolytic activity compared to controls. Their study showed that the antitumor effects of CAR NK cells can be improved by increasing their metabolic fitness. 6 Functional Seahorse XF measurements provide insights into the critical factors affecting immune cell response, fate, and function Metabolism as an indicator of phenotype and inflammatory function in macrophages Metabolic measurements provide a sensitive indication of how immune cells respond under various conditions to meet the integrated and multifaceted demands associated with immune responses. Researchers are applying Seahorse XF technology to investigate questions relating to inflammation, autoimmunity, and immunosuppression, along with substrate dependencies and immunosuppressive effect of the tumor microenvironment. Both pro‐inflammatory (M1) and anti‐inflammatory (M2) polarization of macrophages causes an increase in the ATP production rate, but energy metabolism responds in dramatically different ways. The anti‐inflammatory M2 phenotype is strongly characterized by oxidative phosphorylation, whereas macrophages of the pro‐inflammatory M1 phenotype increase glycolysis and redirect their aerobic metabolism towards the generation of reactive oxygen species and pro‐inflammatory metabolites such as succinate and itaconate. Real-Time, Quantitative Measurements Provide a Comprehensive Picture of Immune Cell Function Figure 7. The Agilent Seahorse XF Real-Time ATP rate assay reveals that macrophage polarization is driven by two distinct cellular metabolic programs. From Divakaruni et al. (2018) Cell Metabolism and Desousa et al. (2023) EMBO Reports. 7 Seahorse XF technology provides insights into essential pathways, signaling, and nutrient requirements for antitumor immune responses relevant to the tumor microenvironment Metabolic measurements provide a sensitive indication of how immune cells respond under various conditions to meet the integrated and multifaceted demands associated with immune responses. Researchers are applying Seahorse XF technology to investigate questions relating to inflammation, autoimmunity, and immunosuppression, along with substrate dependencies and immunosuppressive effect of the tumor microenvironment. Seahorse XF assays were used by Poznanski et al., to generate insights into the mechanisms of NK cell dysfunction in the tumor microenvironment (TME). The study showed that reprogramming NK cells to increase their metabolic substrate flexibility resulted in sustained metabolic fitness and increased antitumor activity in the hostile TME. Figure 8. Targeting oxidative stress restores natural killer cell metabolism and function in the immunosuppressive tumor microenvironment Adapted from Pozanski, S. et al. Metabolic flexibility determines human NK cell functional fate in the tumor microenvironment. Cell Metab 2021, 33, 1205–1220. 8 Real-time analysis of immune cell activation offers insights into metabolic requirements, signaling, pathways, and checkpoints Measure metabolism as an indicator of immune cell type, function, and lineage commitment Metabolic phenotyping provides insights into immune cell function Activation, proliferation, engagement of effector functions, and return to homeostasis are all crucial aspects of the immune cell life cycle—with lineage and immune cell identity constraining the ability of immune cells to function in these roles. Seahorse XF technology provides real-time functional measurements for a comprehensive picture of immune cell function and insights into pathways and targets to modulate immune cell responses. The XF T cell Metabolic Profiling kit reveals the complete bioenergetic profile, including glycolytic and mitochondrial bioenenergetic activity, metabolic poise as well as mitochondrial bioenergetic capacity. It provides a unique insight into cellular phenotype and function at different cellular differentiation states. Naïve T cells have a quiescent phenotype with a low reliance on glycolysis, upon activation by antigen encounter there is an increase in glycolytic activity. When the infection is resolved a small number of these cells generate a memory population and glycolytic activity decreases again. Any Change in Immune Cell Function Requires a Change in Bioenergetics Redirection of glucose metabolismDCA T cell receptor signaling LcK1 inhibitor Cell signaling pathway, GSK-3beta inhibitor Figure 9.The Agilent Seahorse XF Hu T Cell Activation Assay reveals metabolic and signaling requirements critical for T cell activation, allowing you to interrogate the effect of T cell modulators. From Kam et al. Determining the Metabolic Consequences of Pharmacological Modulation of T Cell Activation. Agilent Technologies Poster, ASGCT, 2020. Figure 10. T cells undergo major metabolic changes upon activation, switching to glycolysis to support effector functions and rapid cell growth. Human panT cells were activated with anti CD3/CD28 for 3 days and expanded after in cell culture media. The XF T Cell Metabolic Fitness Assay of naive T cells (before activation) and T cells activated and expanded for 6 days (Effector) and 15 days (Memory) was performed using the Agilent Seahorse XFPro Analyzer. Agilent Technologies unpublished data. 0 100 200 300 PER (%) Time (min) B 0 20 40 60 80 100 120 CD3/CD28 Activator 2-DG Control Vehicle 0 100 200 300 PER (%) Time (min) A 0 20 40 60 80 100 120 CD3/CD28 Activator 2-DG Control Vehicle 0 100 200 300 PER (%) Time (min) A 0 20 40 60 80 100 120 CD3/CD28 Activator 2-DG Control Vehicle 0 100 200 300 PER (%) Time (min) B 0 20 40 60 80 100 120 CD3/CD28 Activator 2-DG Control Vehicle 0 100 200 300 PER (%) Time (min) A 0 20 40 60 80 100 120 CD3/CD28 Activator 2-DG Control Vehicle 0 100 200 300 PER (%) Time (min) A 0 20 40 60 80 100 120 CD3/CD28 Activator 2-DG Control Vehicle 0 100 200 300 PER (%) Time (min) B 0 20 40 60 80 100 120 CD3/CD28 Activator 2-DG Control Vehicle 0 100 200 300 PER (%) Time (min) A 0 20 40 60 80 100 120 CD3/CD28 Activator 2-DG Control Vehicle 0 100 200 300 PER (%) Time (min) A 0 20 40 60 80 100 120 CD3/CD28 Activator 2-DG Control Vehicle Lck inhibitor TWS119 Naive Naive glycoATP production rate (pmol/min/1x105 cells) %ATP from glycolysis OCR (pmol/min/1x105 cells 1200 1000 800 600 100 80 60 40 20 0 80 60 40 20 0 400 300 200 100 0 0 20 40 60 Time (minutes) Naive Effector Memory 80 Effector Effector Memory Memory Oligomycin A Rotenone/ BAM15 Antimycin A 9 Generate Powerful Insights into Host-Pathogen Metabolic Response Figure 11. Norovirus infected murine macrophages increase their dependence on glycolysis for ATP production. Reproduced under the creative commons license 4.0 from Passalacqua, K. et al. Glycolysis Is an Intrinsic Factor for Optimal Replication of a Norovirus. mBio 2019, 10(2), 02175–18. https://creativecommons.org/licenses/by/4.0/ Pathogens commonly target host cellular metabolic pathways to establish optimal conditions for infection. The host response may include large-scale changes in cellular metabolism, including hijacking central carbon metabolic pathways and changing mitochondrial function. A cascade of signaling networks and checkpoints also triggers the innate immune response for combating infection. Seahorse XF technology provides critical functional measurements for a comprehensive, systems-level view of the host response, providing insights into potential therapeutic targets. The Seahorse XF Real-Time ATP rate assay revealed that norovirus infection of murine macrophage cells results in an increase in overall ATP production rate, with increases in both glycolysis and OXPHOS but with an overall reprogramming to a more glycolytic phenotype, suggesting a critical role of host-glycolytic activity to sustain initial viral infection Find out more about how Agilent Seahorse XF can help you investigate immune metabolism at our dedicated webpage here. ATP production rate (pmol/min) Mitochondrial-ATP Production rate (pmol/min) Glycolysis-ATP production rate (pmol/min) mitoATP production rate/glycoATP (pmol/min) 1200 1000 800 600 400 200 0 800 600 400 200 mock 0 0 100 MNV +2DG MNV aerobic energetic quiescent glycolytic mock +2DG mock 200 300 400 mock 1 10 72% 28% 66% 34% 81% 81% 19% 19% RAW 264.7 ATP production RAW 264.7 energetic map XF ATP rate index RAW 264.7 Mitochondrial-ATP Glycolysis-ATP MNV MNV *** ns mock +2DG mock MNV +2DG +2DG MNV +2DG 10 XF T Cell Metabolic Profiling assays: Customized assays for cell therapeutics development With optimized reagents for different T cell and NK cell populations, these assays provide robust bioenergetic parameters linked to critical attributes for antitumor properties: cell persistence and metabolic fitness. A comprehensive view of cellular bioengergetics Optimized Assays for Measuring Immunometabolism Part number 103772-100 (XF Pro /XFe96) and 103771-100 (XF HS Mini and XFp) – Suitable for evaluation of construct design, engineering strategies, starting material selection, or metabolic conditioning during in vitro cell expansion – Applicable for use in assessing the capacity of T cells and NK cells to maintain metabolic fitness in tumor microenvironments – Includes BAM15 an improved uncoupler for more consistent and accurate measurements of T cell and NK cell mitochondrial function – Provides a comprehensive view of T cell and NK cell metabolism, including simultaneous quantification of both glycolytic and mitochondrial effects activity and bioenergetic capacity – Tested for both T cell and NK cell metabolic profiling 11 Agilent Seahorse XF real-time immune cell activation assays deliver a rapid and robust indicator of activation Immune cell activation is driven by a rapid switch towards growth-promoting pathways required for cellular proliferation. Agilent Seahorse XF real time immune cell activation assays enable the detection of activation responses in minutes upon stimulation, providing insights into immune cell activation, modulation and related metabolic reprogramming,. T Cell Activation Assay (Kit part number 103759-100 Macrophage Activation Assay LPS+IFNy Neutrophil Activation Assay Explore Agilent Seahorse XF assay kits here Immune cell specific: – Agilent Seahorse XF Hu T cell activation assay – Agilent Seahorse XF real-time macrophage activation assay – Agilent Seahorse XF real-time neutrophil activation assay 0 0 50 100 150 200 25 50 75 100 PER (pmol/min) Time (min) Activator or control 2-DG Baseline Activation Control Activator 0 50 100 150 200 250 0 100 200 300 400 Time (min) OCR (pmol/min) PMA Human neutrophils (4 x 104/well) Vehicle Rot/ AA PMA or Veh. Time (min) 0 30 60 90 120 150 LPS+IFNy Control PER (pmol/min) 0 100 200 300 Learn more: www.agilent.com/lifesciences/cellanalysis Buy online: www.agilent.com/lifesciences/store Find local Sales and Support in your country: www.agilent.com/lifesciences/contactus USA and Canada: 1-800-227-9770 agilentinquiries@agilent.com Europe info_agilent@agilent.com Asia Pacific inquiry_lsca@agilent.com For Research Use Only. Not for use in diagnostic procedures. RA45427.4037615741 This information is subject to change without notice. © Agilent Technologies, Inc. 2024 Published in the USA, October 2, 2024 5994-7553EN