New understanding of cancer treatment could lead to more personalized treatment
Researchers in the lab of Assoc. Prof. Jun Huang at the UChicago Pritzker School of Molecular Engineering have found that CAR T-cells multiply in two waves, which could lead to more personalized cancer treatment (Photo by Jason Smith)
CAR T-cell therapy, which engineers a patient’s own white blood cells to fight their cancer, has emerged in the last decade as a potent treatment option.
The therapy involves extracting T cells from a patient and genetically engineering them to express a receptor—chimeric antigen receptor (CAR)—that allows them to find and kill cancer. The cells are infused back into the patient, where they multiply and destroy cancer cells.
Many scientists assumed that when these CAR T-cells multiply, they do so in one big wave.
But using advanced analytic techniques, University of Chicago Pritzker School of Molecular Engineering (UChicago PME) scientists and engineers in the lab of Assoc. Prof. Jun Huang have found that this expansion happens in two waves.
The first expansion of CAR T-cells in the body are the activated killers, while a second wave—which happens a week or two after the first—are the persistent inspectors. These cells aren’t actively killing cancer, but have the ability to, should the cancer recur.
The results, published in Nature Communications, offers a new view into the biology of the therapy that could have implications for more personalized treatments.
“We have always known that the more CAR T-cells expand and the longer they stay, the better clinical outcomes the patient will have,” Huang said. “But now we know that we have two groups of cells that are different biologically. This could allow us to better engineer CAR T-cells in the future to get better responses from patients.”
“This could allow us to better engineer CAR T-cells in the future to get better responses from patients.”
To study the biology of CAR T-cells, Huang’s team examined blood samples from patients with diffuse large B-cell lymphoma, the most common non-Hodgkin’s lymphoma in the United States.
This blood cancer involves malignant B cells that proliferate and is often cured with chemotherapy. But patients who do not respond to chemotherapy or relapse have limited treatment options.
Since 2017, lymphoma patients have had access to CAR T-cell therapy, which has eliminated cancer for more than 40 percent of patients with this type of lymphoma. Still, the majority of patients don’t respond to the treatment, and those who do face toxic side effects, like inflammatory responses or neurotoxicity.
“Many scientists have tried to figure out why it fails for certain patients,” said Yifei Hu, an MD/PhD student who conducted the research. “Is it because the cells can’t kill properly? Is it because they can’t persist? But before we can figure that out, we needed to analyze the cells in the blood at different points in time.”
The team examined blood samples from lymphoma patients who underwent CAR T-cell therapy between 2019 and 2021. These samples were taken between 8 and 14 days after infusion, then again on day 21 and on day 28.
The team performed single-cell sequencing for each CAR T-cell, which revealed that the genes are being expressed at the RNA level. They also examined the cells’ T-cell receptor (TCRs), an important step toward understanding CAR T-cell differentiation. When CAR T-cells expand, they do so by dividing into “daughter” cells. These daughter cells retain the same TCR sequencing as their parent cell. That allowed the team to track the clonal dynamics of the expansion.
“Other studies of CAR T-cells haven’t tracked these clonal dynamics,” Hu said. “But it’s like an ID for every CAR. It tells you where they came from.”
When they integrated that data with RNA sequencing, they found that what they thought was one big expansion and contraction was actually two separate waves. Not only that, those two waves were distinct CAR T-cells with different jobs.
“That was a key discovery,” said Tony Pan, a graduate student who conducted the research. “The first group expands, actively kills the tumor, and then contracts, then the second group expands and persists for longer. They have the ability to kill, but are not actively killing.”
That second wave exists at least until day 28. It’s possible that they persist much longer. Other studies have found that CAR T-cells persist in patients even 10 years after infusion.
“There is a strong possibility that people who don’t do well with the therapy have a failure of wave one or wave two.”
Huang’s team is now studying to see if this phenomenon occurs with different types of CAR T-cell therapies and in other cancers, like multiple myeloma. Early results indicate that it does.
The results could ultimately affect how patients are treated in the future. “There is a strong possibility that people who don’t do well with the therapy have a failure of wave one or wave two,” Hu said.
It could also potentially help with potential toxic side effects, like cytokine release syndrome, which triggers an inflammatory response. Perhaps those with side effects have too many of the second cohort CAR T-cells that persist for too long.
The team is currently developing tests to predict how many of each cohort are within each engineered T-cell sample. They’ve also identified protein markers for each, which will allow other labs to study this response in other cancers. These markers could also be the basis of future clinical blood to detect the amount of each cohort in a patient’s body after infusion.
“If we can predict how many of each group is in a patient from the very beginning, then continue to test that throughout treatment, then we potentially have a solution for non-responders,” Hu said.
Other authors on the paper include Guoshuai Cao, Erting Tang, Nicholas Asby, Thomas Althaus, Jun Wan, Peter A. Riedell, Michael R. Bishop, and Justin P. Kline.
DOI: Cao, G., Hu, Y., Pan, T. et al. “Two-stage CD8+ CAR T-cell differentiation in patients with large B-cell lymphoma.” Nature Communications April 16, 2025 DOI: https://doi.org/10.1038/s41467-025-59298-w
FUNDING: National Institutes of Health, American Cancer Society, Phi Beta Psi, the Ullman Fund in Cancer Immunology, the Hoogland Lymphoma Research Pilot Projects, Chicago Immunoengineering Innovation Center
