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Culture expansion of CAR T cells evokes aberrant DNA methylation that is associated with adverse clinical outcome (Methylation)

ABSTRACT: Cellular immunotherapy using autologous, genetically modified T cells has delivered remarkable initial response rates in various hematological malignancies, including acute lymphoblastic leukemia (ALL) or lymphoma. Upon treatment with these chimeric antigen receptor (CAR) T cells, approximately 30–40% of patients show long-term remission. There is a growing perception that not only CAR constructs, but also in vitro manufacturing processes may determine therapeutic efficacy. However, little is known about how duration of culture expansion affects molecular and functional features of CAR T cells. A better understanding of molecular signatures, reflecting therapeutic activity of CAR T cells, may help to further optimize the manufacturing process. In this study, we therefore investigated DNAm changes during culture expansion of T cells and CAR T cells during a period of up to three weeks. The results of our current study indicate that culture expansion of CAR T cells leads to DNAm changes that are continuously acquired during culture expansion. Further analysis of culture-associated methylation profiles in CAR T cells from clinical trials support the notion that these chnages are disadvantageous with regards to therapeutic outcome. A shortened cultivation period to avoid dysfunctional methylation programs might be a promising manufacturing strategy to improve therapeutic efficacy of adoptive cell therapies. Our epigenetic signature for culture-associated DNAm may provide a biomarker for quality control of CAR T cell productions and to identify patients at risk for adverse events.

ORGANISM(S): Homo sapiens

PROVIDER: GSE220157 | GEO | 2023/07/20

REPOSITORIES: GEO

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Culture expansion of CAR T cells evokes aberrant DNA methylation that is associated with adverse clinical outcome (RNA-Seq)

Project description:Cellular immunotherapy using autologous, genetically modified T cells has delivered remarkable initial response rates in various hematological malignancies, including acute lymphoblastic leukemia (ALL) or lymphoma. Upon treatment with these chimeric antigen receptor (CAR) T cells, approximately 30–40% of patients show long-term remission. There is a growing perception that not only CAR constructs, but also in vitro manufacturing processes may determine therapeutic efficacy. However, little is known about how duration of culture expansion affects molecular and functional features of CAR T cells. A better understanding of molecular signatures, reflecting therapeutic activity of CAR T cells, may help to further optimize the manufacturing process. In this study, we therefore investigated DNAm changes during culture expansion of T cells and CAR T cells during a period of up to three weeks. The results of our current study indicate that culture expansion of CAR T cells leads to DNAm changes that are continuously acquired during culture expansion, which is also reflected on gene expression level. Further analysis of culture-associated methylation profiles in CAR T cells from clinical trials support the notion that these chnages are disadvantageous with regards to therapeutic outcome. A shortened cultivation period to avoid dysfunctional methylation programs might be a promising manufacturing strategy to improve therapeutic efficacy of adoptive cell therapies. Our epigenetic signature for culture-associated DNAm may provide a biomarker for quality control of CAR T cell productions and to identify patients at risk for adverse events.

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Project description:Culture expansion of primary cells evokes highly reproducible DNA methylation (DNAm) changes These changes might be directly regulated or caused by gradual deregulation of the epigenetic state, which is often referred to as “epigenetic drift” We have identified CG dinucleotides (CpGs) that become continuously hyper- or hypomethylated in the course of culture expansion of mesenchymal stem cells (MSCs) and other cell types During reprogramming into induced pluripotent stem cells (iPSCs) particularly the culture-associated hypomethylation is reversed simultaneously with age-associated and pluripotency-associated DNAm changes Bisulfite barcoded amplicon sequencing (BBA-seq) demonstrated that upon passaging the DNAm patterns of neighboring CpGs become more complex without evidence of continuous pattern development and without association to oligoclonal subpolulations of MSCs at later passages Circularized chromatin conformation capture (4C) revealed reproducible changes in nuclear organization between early and late passages, while there was no preferential interaction with other genomic regions that also harbor culture-associated DNAm changes Chromatin immunoprecipitation of CTCF did not show significant differences during long-term culture of MSCs, however culture-associated hypermethylation was enriched at CTCF binding sites and hypomethylated CpGs were devoid of CTCF Taken together, our results indicate that DNAm changes during culture-expansion resembles epigenetic drift

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