Chimeric antigen receptor (CAR) T cells represent the next generation of therapeutic interventions and are a major advancement in personalized disease treatment. CAR T-cell therapies utilize a patient’s own T cells to recognize and destroy cancer cells or other disease-causing cells. Find out how the ZE5 Cell Analyzer has helped overcome some of the challenges associated with development of these powerful new therapies.

CAR T-cell therapies: How flow cytometry empowers breakthroughs.

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CAR T cells are typically generated by genetic modification of a patient’s own T cells to express a synthetic receptor called a CAR, which specifically recognizes proteins found on the surface of cells that need to be destroyed. The CAR T cells either directly kill these cells or orchestrate an immune response to target them to ensure their destruction. While CAR T-cell therapies have revolutionized cancer treatment, there are still significant challenges associated with CAR T-cell research and production, which cause barriers to effective CAR T-cell therapy. These challenges include validation of experimental models, proving potency and efficacy, assessing the quality of the therapeutic product, and monitoring potential off-target effects.

One technology that is helping to drive CAR T-cell therapy forward is flow cytometry. Flow cytometry can deliver exceptionally detailed and quantitative information in a wide variety of applications and is an integral part of cell-based therapies research.

Recent advances have allowed flow cytometry analysis to be performed at even greater speeds, which means even greater utility to CAR T-cell therapy development. In this article, we examine recent publications demonstrating how flow cytometry and the ZE5 Cell Analyzer can empower the development of CAR T-cell therapies.

Demonstrating Protein Expression

The verification that a protein is or is not expressed by a cell may be a critical step needed to validate a method and progress in CAR T-cell therapy design. One of the initial steps taken by Cooper et al. when designing a CAR T-cell strategy was to develop a method of producing T cells that do not express CD7 (Cooper et al. 2018). CD7 is a surface marker expressed by many T-cell malignancies, making it an attractive target for immunotherapy. The problem is that normal T cells, including those used to generate CAR T-cells, also express CD7. CRISPR-Cas9 technology can be used to disrupt expression of CD7 on CAR T-cells, ensuring that they are not self-reactive. Following gene editing, flow cytometry can be used to assess CD7 expression on T cells, as it provides direct evidence of cell surface protein expression.

Assessing Tumor Infiltration of CAR T Cells

Getting CAR T-cells into a solid tumor presents some challenges. Multiple factors such as the tumor stroma and the tumor microenvironment can make it difficult for T cells to penetrate or be retained.  Demonstrating effective tumor infiltration requires a way to quantitatively measure the number of T-cell in a tissue. Because flow cytometry is designed to identify specific cell types in a heterogenous mixture, it is well-suited to fill this role. This was demonstrated by Mishra et al. when they used flow cytometry to assess the infiltration of a CAR T cell therapy into solid tumors in a mouse model. By dissociating the cells and measuring the proportion of T cells — identified by the expression of CD3 — they were able to show that the CAR T cells that they generated were better at infiltrating tumors than normal T-cells were (Mishra et al. 2021).

Assessing the Ability of CAR-T Cells to Deplete a Target Population In Vivo

Eliminating cancer cells is often the ultimate goal of CAR T-cell therapies, but this technology has applications for diseases other than cancer, due to its ability to precisely target desired cell types. For example, in lupus, B cells are central players of disease as they produce autoantibodies that recognize and destroy the patient’s own cells, causing organ damage. CAR T-cell therapy can be used to reduce problematic B cells through a CAR engineered to recognize them. In a model of lupus, Kansal et al. demonstrated near total B-cell aplasia from CAR T-cell therapy, with this result demonstrated throughout the course of experiments — even more than one year post–administration of transduced T cells (Kansal et al. 2019). In this example, flow cytometry provided an excellent means of assessing the number and proportion of specific cell types in blood (such as B cells), making it an important technology for monitoring CAR T-cell therapy effectiveness.

Monitoring Composition and Potency

Being able to monitor the composition and potency of a therapeutic product is critical in gaining regulatory approval (U.S. Department of Health and Human Services 2011). O’Neal et al. used flow cytometry to measure the proportion of T-cell subsets following the development of a CAR T-cell therapy that specifically targets CD319 (O’Neal et al. 2022). They were concerned that because CD319 is expressed by some CD8+ T cells that they may induce a degree of self-killing, or fratricide, resulting in a less effective product. They did observe a drop in the proportion of CD8+ T cells in the CAR T cells generated, compared to their proportion in native T-cells, but by deleting the CD319 gene they were able to stabilize the proportion of CD8+ CAR T cells and this had no effect on overall efficacy.

Monitoring Off-Target Effects

Careful monitoring of drug side effects, or off-target effects, is one of the central tenants of drug development (Ahmad et al. 2003). Where therapies are intended to deplete a particular set or subset of cells, it is critical to ensure that other cell types are not inadvertently depleted at the same time. Mishra et al developed a CAR T cell that targeted CD126, a marker expressed by some solid tumors. To assess the likelihood of any unwanted cells being targeted by their approach, Mishra et al. used the ZE5 Cell Analyzer to study different immune cell subsets, including T cells, B cells monocytes, and natural killer cells (Mishra et al. 2021). They saw no depletion of total peripheral blood mononuclear cells or any subset, providing evidence that CD126-expressing cells can be safely targeted without killing immune cells and paving the way for in vivo studies.


CAR T-cell therapy holds a tremendous amount of promise as a new pillar in cancer treatment. But many challenges still remain, especially with regards to efficacy in treating solid tumors, toxicity, and the substantial cost associated with these treatments. Flow cytometry has been and will continue to be a key technology empowering researchers to develop new strategies and potential solutions in order to bring about safer and more effective future therapies.

See more publications featuring the ZE5 Cell Analyzer in CAR T-cell therapy development.


Ahmad SR (2003). Adverse drug event monitoring at the Food and Drug Administration. J Gen Intern Med 18, 57–60.

Cooper M et al. (2018). An “off-the-shelf” fratricide-resistant CAR-T for the treatment of T cell hematologic malignancies. Leukemia 32, 1,970–1,983.

Kansal R et al. (2019). Sustained B cell depletion by CD19-targeted CAR T cells is a highly effective treatment for murine lupus. Sci Transl Med 11, eaav1648.

Mishra AK et al. (2021). Preclinical development of CD126 CAR-T cells with broad antitumor activity. Blood Cancer J 11, 3.

O’Neal J et al. (2022). CS1 CAR-T targeting the distal domain of CS1 (SLAMF7) shows efficacy in the high tumor burden myeloma model despite fratricide of CD8+CS1 expressing CAR-T cells. Leukemia 36, 1,625–1,634.

U.S. Department of Health and Human Services, U.S. FDA. (2011). Final guidance for industry potency tests for cellular and gene therapy products., accessed January 6, 2023.

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