Flow Cytometry Case Study

Revealing the Immune Response to CAR-T Cell Therapy

T cells genetically engineered to express a lab-designed chimeric-antigen receptor, or CAR, offer many patients with blood cancer a new treatment option. But while these T cells may have lifesaving anti-cancer effects, many patients experience a toxic side effect called cytokine release syndrome, or CRS, a body-wide inflammatory response triggered by the engineered T cells. Dr. Sylvain Simon, a postdoctoral fellow in the Hutch’s Riddell Lab, aims to develop safer and more efficient next-generation CAR-T cell therapies that are less likely to cause CRS.

First, he must understand the cellular and molecular changes that underlie CRS. To do so, Simon is studying the blood of CAR-treated patients who experience CRS. One of his main goals is to define the changes in immune cell activity and gene expression associated with CRS development and severity.
 

The Challenge

In this study, blood samples are taken longitudinally from cancer patients undergoing CAR-T cell therapy. Because patients who receive CAR-T cell therapy also undergo a lymphodepleting chemotherapeutic regimen prior to receiving their engineered T cells, their blood samples often contain few immune cells. To maximize the information obtained from each incredibly valuable sample, he needed an extensive flow cytometry panel that could delineate as many immune-cell subsets as possible. He also needed to create a rigorous protocol that would enable him to generate consistent, comparable results from samples taken over an extended period. 
 

The Approach

Staff members in the Flow Cytometry core helped Simon identify the best markers and antibodies to include in his panel and create a strategy to assign the right fluorochrome to each marker. Because of their deep experience with the flow cytometer, the shared resource team members were also able to help Simon create a standardized assay. When he saw that certain reagents resulted in poor resolution, the Flow Cytometry team helped him troubleshoot and optimize his panel.

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Development of a flow panel to describe immune cell perturbations associated with CRS declaration and severity in CAR treated patients. Image by Flow Cytometry shared resource
Defining the populations of immune cells in CAR-treated patients through the use of flow cytometry.
Defining the populations of immune cells in CAR-treated patients through the use of flow cytometry.

The Outcome

Aided by the Flow core, Simon developed a 27-color flow cytometry panel to quantify and phenotype a large array of immune cells from the blood of CAR-treated patients who developed CRS. He is now applying this method to whole blood samples collected longitudinally using a standardized procedure. With these robust and consistent data, Simon is now building a dataset that will help him better understand how variations in immune cells’ frequencies associate with development and severity of CRS.

Discussions with the core’s staff also stimulated new ideas. To complement the flow cytometry panel, they helped Simon develop a broad, 48-marker CyTOF panel to further characterize immune cells from cryopreserved blood samples. With assistance from the Flow Cytometry core, Simon is also designing an even larger flow cytometry panel for the next generation of spectral flow machines, which will be used to study the immune responses of patients in additional immunotherapy trials. 
 

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Learn more about the Riddell Lab's research.

“The expertise from people in the Flow Cytometry core was absolutely key to succeed in designing the optimal methodology to address our scientific questions in the best way. They were very keen in sharing their experience and knowledge, which ends up being the best training someone can get.”

– Dr. Sylvain Simon, postdoctoral fellow, Riddell Lab, Fred Hutch