Chimeric antigen receptor (CAR) T cell therapies reprogram a patient’s immune cells to recognize and attack the patient’s cancer. They are a major advance in treating blood cancers such as leukemia and lymphoma. However, clinical outcomes remain variable, making it critical for researchers to understand how manufacturing influences CAR T cell composition and function. To better understand when CAR T cells acquire peak functionality, researchers at the University of Southern California (USC) Keck School of Medicine developed a spectral flow cytometry system to profile CAR T cells across the manufacturing timeline. When they used the flow cytometer to analyze CAR T cells, the researchers made an important discovery — CAR T cells are better able to fight cancer after a five-day expansion process than at the 10-day mark. “My lab has been on a mission to understand how to improve CAR T cell performance in cancer patients,” professor Mohamed Abou-el-Enein, MD, said. “We now have a much clearer picture of when these cells are at their strongest — and a tool to help us act on that information.” Designed as an integrated, single assay, the spectral flow cytometry system simultaneously captures high-dimensional immunophenotyping and in vitro cytotoxicity, generating a detailed fingerprint of the CAR T cell product. “Just as every person has a fingerprint that identifies them, T cells also have fingerprints,” Abou-el-Enein said. “By measuring the expression of markers on a cell’s surface, we can learn more about what distinguishes one CAR T cell therapy from another.” The spectral flow cytometry system analyzes the physical and chemical properties of individual cells. The researchers tag the cells with fluorescent markers that bind to the molecules comprising the cell’s fingerprint. As the cells pass through the cytometer’s laser beam, the beam excites their fluorescent tags, which emit light that is detected and measured by the cytometer. The way in which the tags emit light indicates whether a specific molecule is present and how strongly it is expressed. Unlike standard tools, which can only measure about 10 markers at a time, the high-dimensional spectral flow cytometer simultaneously captures data on 36 characteristics from each cell, providing a comprehensive, holistic view of the cell’s behavior. The 36 markers capture a range of T cell characteristics, including activation, metabolism, memory, and cytotoxicity, that relate to the T cell’s ability to identify and kill cancer. The researchers paired each marker with a fluorescent tag and used mathematical modeling to ensure that each tag was detected separately during analysis. In experiments, the team observed key biological transitions in CAR T cells that could guide manufacturing optimization and product design. The researchers collected data on CAR T cells at day five and day 10 in the manufacturing process. They found that day-five cells more closely resembled stem-like cells and had higher metabolic activity than those tested on day 10. While both products retained potent killing capacity against the model, the day-five cells were more consistent with features linked to sustained in vivo efficacy. By characterizing the impact of manufacturing variables on CAR T cells, spectral flow cytometry could help researchers align product features with specific therapeutic goals. “This work fills a critical gap in our understanding of how manufacturing conditions shape the therapeutic potential of CAR T cells,” Abou-el-Enein said. “By pinpointing when CAR T cells acquire — or lose — functional fitness, we can now tailor the timing of cell manufacturing, which could have an immediate impact on clinical decision-making and patient outcomes.” The spectral flow cytometry platform has the potential to help optimize the manufacturing process in other ways — for example, it could be used to compare standard viral vector technology with alternate ways to engineer CAR T cells. In the future, the spectral flow cytometry platform could be used in clinical settings to identify predictive biomarkers that link cell characteristics to patient outcomes, guiding the next generation of cell therapy design. Clinical trial centers could use the system to track the evolution of CAR T cells during and after treatment to gain insight into the factors that contribute to long-term success. Researchers could also use the spectral flow cytometry system to study the behavior of other cell types and compare different gene editing technologies and production platforms. “This is just the beginning,” Abou-el-Enein said. “Our platform is not only designed for discovery — it’s built for scalability, collaboration, and clinical translation. We’re excited to open new avenues for partnership with academic and industry partners who are committed to advancing next-generation immunotherapies.” The research was published in Molecular Therapy (www.doi.org/10.1016/j.ymthe.2025.04.006).
