Chimeric antigen receptor (CAR)-T cell immunotherapy has considerably changed the landscape of treatment options for blood cancers. CAR-T therapy aims to redirect a patient’s T cells to recognize and destroy cancer cells. T cells are genetically altered to express CARs that combine an antigen recognition domain with intracellular T cell signaling components to deliver T cell specificity and functionality. By using CAR T cells, researchers can specifically target antigen-expressing cells and then initiate signaling pathways that trigger effector T cell function. In theory this seems great, right? But T cells come in different flavors such as its differentiation stage which affects its proliferative and survival abilities which are essential for their antitumor activity.
“Chimeric antigen receptor (CAR)-T cell therapy is one such therapy in which a patient’s own immune cells are collected, genetically modified, expanded in culture, and then transferred back into patients,” explained Dr. Sam Fiorenza ( a physician-scientist formerly at Fred Hutch), one of the co-first authors of a study recently published in Nature Communications. “It’s known that the differentiation status of the circulating immune cells contributes to CAR-T cell efficacy: starting products composed of less differentiated naïve and central memory (CM) T cells tend to give better therapeutic outcomes than those from more differentiated effector memory (EM) cells,” Fiorenza added.
To explore the underlying differences between these T cell subsets, the team performed RNA sequencing, which measures the RNA molecules in the cells as an approximate of gene expression activities, and CUT&RUN to look at two specific histone marks, H3K27me3 and H3K4me2, representing polycomb repressed regions and enhancer/promoter regions, respectively. They found that histone mark analysis was better at detecting differences between these subsets compared to RNA-seq alone, especially between naïve and EM cells. They also examined the association between histone marks and transcript abundance. Many genes followed expected patterns, with the most active genes being associated with H3K4me2, while inactive genes were marked by H3K27me3. However, they also discovered several genes that did not follow the common pattern, revealing a more complex relationship between gene activity and histone modifications. “Our study demonstrates that histone marks provide valuable insights into T cell differentiation and CAR-T functionality beyond what is possible with transcriptomics alone,” noted Dr. Ye Zheng, the second co-first author of this article. “Single H3K4me2 and H3K27me3 marks and changes in patterns of combined H3Kme2 and H3K27me3 marks readily distinguish human naïve, central memory, and effector memory CD8 + T cells and CAR-T manufactured using similar, yet distinct, starting CD8 + T cell populations.”
By further analyzing genes known to change in expression and those that do not, they discovered that many genes displayed variations in histone mark patterns when comparing naïve T cells to CM and EM T cells. Interestingly, there were more genes showing these histone changes that did not have altered expression levels, especially when comparing CM and EM T cell subsets. These findings suggest that examining histone mark patterns can offer valuable insights into the differences between T cell subsets, even when RNA-seq readouts remain invariant.
When manufacturing CAR-T cells, researchers typically rely on RNA-seq to approximate and quantify gene activity and T cell differentiation status. Analysis of histone marks revealed key differences in CAR-T cells made from less mature cells (which tend to perform better in patients) versus more mature, potentially exhausted cells. By focusing on histone marks, the team could pinpoint genes related to how well these CAR-T cells might fight cancer, including genes involved in memory, exhaustion, and survival. Their approach highlighted important differences when comparing CAR-T cells made from patients with large B-cell lymphoma (LBCL) and healthy donors, showing that cells from LBCL patients had more signs of exhaustion. These findings highlight the importance of using histone mark analysis to improve CAR-T therapy and potentially enhance treatment outcomes for patients.
Notably, the team identified a transcription factor, KLF7, that correlated with improved CAR-T therapy outcomes in patients with LBCL. KLF7 is known to promote T cell proliferation. When analyzing samples from LBCL patients treated with CAR-T therapy, they found that the CAR-T cells with higher levels of KLF7 were associated with stronger cell expansion and accumulation in the body, which is vital for the therapy's success. When KLF7 was present, the CAR-T cells produced more IL-2, a protein secreted by activated T cells that boosts the immune response. Importantly, KLF7+ cells also had a greater ability to expand without being overactivated, maintaining a memory-like state that enhances long-term immunity. “By examining CAR-T products from a clinical trial in lymphoma, we find a novel association between the activity of the transcription factor KLF7 with in vivo CAR-T accumulation in patients and demonstrate that over-expression of KLF7 increases in vitro CAR-T proliferation and IL-2 production,” Fiorenza said. “Epigenomic profiling of the CAR-T cells offers a new pathway to improve CAR-T cell manufacturing and therapy,” Fiorenza added.
“Transduction of KLF7 during manufacturing to generate less-differentiated CAR-T cells with enhanced proliferative potential is an attractive strategy that could be investigated to improve CAR-T efficacy,” Fiorenza commented. However, many questions remain: “what would the mechanisms by which KLF7 enhances CAR-T proliferation and IL-2 production be? Can KLF7 overexpression be safely integrated into clinical CAR-T production?” Zheng also emphasized the need for further studies to determine whether KLF7 can improve CAR-T proliferation and durable survival without compromising function. Zheng is particularly interested in using other epigenomic profiling tools, like CUT&Tag, to investigate mechanisms of exhaustion in CAR-T cells. “Analyzing histone marks in T cells and CAR-T cells will uncover new genes for further investigation and modification to improve CAR-T immunotherapy outcomes. Can we profile the spatial epigenomic signatures of CAR-T cells to study their interactions with the tumor microenvironment? Can we leverage single-cell technology to map the trajectory changes as CAR-T cells respond to tumor antigens?” Zheng concluded.
The spotlighted worked was funded by the National Institute of Health, the Haematology Society of Australia and New Zealand Clinical Fellowship, Research Training Programme Stipend from the Australian Government Department of Education, the Fred Hutch Immunotherapy Integrated Research Center, the CLEARBridge Foundation and Juno Therapeutics.
Fred Hutch/University of Washington/Seattle Children's Cancer Consortium members Drs. Jay Sarthy, Jordan Gauthier, Alexandre Hirayama, Stan Riddell, Qian Wu, David Maloney and Steve Henikoff contributed to this work.
Fiorenza S, Zheng Y, Purushe J, Bock TJ, Sarthy J, Janssens DH, Sheih AS, Kimble EL, Kirchmeier D, Phi TD, Gauthier J, Hirayama AV, Riddell SR, Wu Q, Gottardo R, Maloney DG, Yang JYH, Henikoff S, Turtle CJ. Histone marks identify novel transcription factors that parse CAR-T subset-of-origin, clinical potential and expansion. Nat Commun. 2024 Sep 27;15(1):8309. doi: 10.1038/s41467-024-52503-2.
Joss Landazuri is a PhD candidate at the University of Washington in the Microbiology program working at the intersection of biomedical science, public policy, and science diplomacy. As a Latina scientist, communicator, and policy advocate, she is passionate about leveraging her academic training, personal background, and cultural heritage to engage underserved communities in both science and the policymaking process.
For the Media