Advances in Immunometabolism: From Bulk Samples to Single Cells

Since the 1960s, it has been observed that white blood cells enlarge dramatically before cell division, suggesting major shifts in metabolism. “Immune cells, especially T cells, undergo striking changes in their metabolic phenotype during activation and differentiation,” explains Alice Gao, Ph.D., a Product Manager in the Cell Analysis Division of Agilent Technologies. “This impacts how they fight pathogens and cancer cells.”

The field of immunometabolism, which has been accelerating rapidly over the past few years, involves the integration of metabolism with immunity. But what is the best way to understand the metabolic states of different immune cells―such as T cells, B cells, dendritic cells, and macrophages?

Over the past few decades, various techniques have been pioneered for the study of immunometabolism, including flow cytometry, extracellular flux analysis, mass spectrometry, single-cell RNA sequencing, and mass cytometry. However, these techniques―applied to analyze everything from proteins to metabolites to lipids to RNA―vary significantly in terms of cost and feasibility. Biocompare recently interviewed several companies to understand what state-of-the-art technologies they are developing to advance this emerging new field.

Investigating metabolism in live cells

“Over the past ten years, we have seen tremendous progress within the immunometabolism field,” Dr. Gao notes. “The Seahorse XF Analyzer has contributed significantly toward building a lot of this foundational knowledge.”

The Seahorse, which is an extracellular flux analyzer, measures the two major energy pathways of the cell―mitochondrial respiration and glycolysis. This is achieved with fluorophores that simultaneously analyze oxygen consumption rates and proton release in the medium immediately surrounding the cell. Ultimately, these methods enable researchers to understand the bioenergetic status of living cells without having to add labels.

Seahorse technology allows for the easy and reasonably affordable profiling of cells in 96-well plates. It can also elucidate the immediate effects of metabolic modulations using built-in injection ports. In addition, Gao emphasizes, “The Seahorse Analyzer provides richer functional data that many other technologies cannot match.”

She also highlights that, compared with mass spectrometry and other proteomic approaches, Seahorse XF assays are easy to run and provide results quickly―often within a day or two. Finally, when combined with the Agilent XF T Cell Metabolic Profiling Kit, the Seahorse provides multiparametric outputs that completely characterize the T cell metabolic profile. “This information can be incorporated as a routine assay to optimize the design and manufacture of T cell‑derived therapeutics,” says Gao.

Mass spectrometry for proteins, metabolites, and lipids

The advantages of mass spectrometry for proteomics studies include its specificity and sensitivity down to the level of single molecules. High-resolution mass spectrometry, in particular, can now detect the mass-to-charge ratio down to four decimals. Over the past couple of years, high-resolution mass spectrometry studies of the whole proteome have yielded insight into how T cells shift between glucose and fatty acid metabolism over the course of an immune response.

Thomas Moehring is the Senior Director of Omics Applications at Thermo Fisher Scientific. He explains that discovery-based quantitative proteomics focuses on identifying proteins and measuring changes in relative abundance across multiple sample sets. Moehring points out that several discovery-based techniques are being employed in immunometabolism studies, including stable isotope labeling by amino acids in cell culture (SILAC), isobaric labeling with tandem mass tags (TMT), and label-free quantitation (LFQ). The advantage of metabolic labeling is that it can determine protein changes with a precision as high as a few percent. Furthermore, TMT methods allow researchers to multiplex up to 18 samples in a single run.

Finally, Moehring notes that mass spectrometry is well-suited for the high-throughput identification and quantification of metabolites and lipids. “High-resolution and accurate instruments like Orbitrap-based mass spectrometers are ideal here,” he explains. “When coupled with a separation method such as liquid chromatography or gas chromatography, they can provide a sensitive and robust set of data from which metabolites and lipids can be identified and quantified in a reproducible way.”

Profiling individual cells

While many key immunometabolism studies have involved analysis of bulk samples, researchers are also interested in how individual cells behave within a larger population. This is where single-cell applications come into play. According to Marla Lubinsky, Director of Proteomics Product Marketing at Standard BioTools™, “Single-cell analysis provides a greater understanding of what is occurring within specific cell types when they are introduced to a disease or treatment.”

One well-established single-cell approach involves single-cell RNA sequencing (scRNA-seq), which can now detect up to 5,000 genes in each cell. However, the method can be very expensive, and transcriptional data are arguably a less direct proxy for metabolism.

The high-throughput analysis of proteins within single cells is still in its infancy. However, mass cytometry—which has been used in over 200 clinical trials―has led to exciting advances in recent years.

Mass cytometry is a type of flow cytometry, in which labeled antibodies are employed to characterize the presence of specific antigens in large populations of cells. However, mass cytometers use time-of-flight (TOF) mass analyzers and detectors. And while flow cytometry relies upon fluorescent labels, mass cytometry involves the measurement of rare earth metal isotope tags. “Given that mass cytometry is not impacted by autofluorescence or spectral overlap like fluorescence flow cytometry, it can capture more reliable insights for high-parameter immune profiling,” explains Lubinsky.

Finally, Lubinsky notes that researchers can easily add immune profiling to their investigation with the Maxpar^® Direct™ Immune Profiling Assay™, a validated 30-marker immune profiling assay with a 5-minute analysis time. “This comprehensive and reliable approach to immune profiling reduces time to results and lowers the cost for large studies.”

Determining the best method

Ultimately, Gao stresses the importance of corroborating findings using different techniques. For instance, recent studies on T cells have demonstrated consistency between metabolic changes measured with the Seahorse Analyzer and changes observed on the protein level with mass cytometry. “These different technologies offer different insights, so they complement each other and provide a more complete story. So, they are all important approaches,” concludes Gao.