Pegah Rahmati (Hannover/ DE), Agnes Bonifacius (Hannover/ DE), Dr. Anna Christina Dragon (Hannover/ DE), Chiara Malinconico (Hannover/ DE), Prof. Dr. Rainer Blasczyk (Hannover/ DE), Prof. Dr. Michael Hudecek (Würzburg/ DE), Stefan Floess (Braunschweig/ DE), Jochen Huehn (Braunschweig/ DE), Prof. Dr. Britta Eiz-Vesper (Hannover/ DE)
Background
Despite the promising results of CAR-T-cell therapy, poor in vivo persistence and low potency in the treatment of solid tumors are challenges that remain to be addressed. It has been shown that the efficacy of adoptively transferred and genetically modified T cells can be modulated by ex vivo culture conditions. Vitamin C (vitC) is a micronutrient that influences the immune system by mechanisms such as the regulation of epigenetic processes and reactive oxygen species-induced oxidative stress.
Methods
In this study, we investigated the impact of vitC pre-conditioning on the phenotype and functionality of CD19-targeting CAR-T cells (vitC-CAR19-Ts vs. CAR19-Ts) in co-culture with CD19+ Nalm-6 cells. We also generated CD19 knockout (CD19-) Nalm-6 cells using CRISPR/Cas9 as a negative control. Using multicolor flow cytometry, bulk-RNA transcriptomics, metabolic and multiplex assays, we analysed the activation and regulatory status as well as phenotypic characteristics and cytotoxic capacity of (vitC‑)CAR19-Ts during generation and upon target cell encounter.
Results
Strikingly, vitC-CAR19-Ts had a prominent effector memory phenotype and displayed increased long-term mitochondrial fitness when compared to CAR19-Ts. Moreover, vitC-CAR19-Ts showed significantly enhanced cytotoxicity towards CD19+ but not CD19- Nalm-6 cells. In line, their granulysin release was significantly increased, which was confirmed to originate from both CD4+ and CD8+ T cells by intracellular staining. RNA sequencing of (vitC-)CAR19-Ts after target cell encounter revealed upregulation of GNLY (granulysin) as well as genes involved in T-cell activation, metabolism and epigenetic reprograming. Furthermore, vitC-CAR19-Ts showed increased cytotoxicity upon repetitive target cell encounter when compared to CAR19-Ts.
Conclusion
In conclusion, we could show in this proof-of-principle study that vitC pre-conditioning leads to CAR19-T-cell products with significantly enhanced cytotoxicity and fitness, which is potentially epigenetically imprinted. Therefore, vitC pre-conditioning is a promising strategy to generate improved T-cell products with superior long-term cytotoxic and persistence capacity.
Offenlegung Interessenkonflikt:
The authors declare no conflict of interest.
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