Developmental Biology & Stem CellsDNA Methylation and Hydroxymethylation

Epigenetics Matter: Ensuring Functional Stability of Regulatory T Cell Therapy Products

shutterstock_183635540 (1)Many autoimmune diseases are characterized by an imbalance between the effector and regulatory arms of the immune system: auto-reactive effector T cells are hyper-activated, while their regulatory counterparts are impaired, or at least insufficient to keep harmful effector responses in check. Regulatory T cell (Treg) cell therapy is a promising therapeutic approach aiming at re-establishing the balance in favor of the regulatory arm. To this aim, Treg cells can be either purified from peripheral blood and expanded in vitro, or induced from circulating naive T cells upon in vitro culture with appropriate differentiation factors. However, it is still unclear which protocol will yield Treg cells that will remain functionally stable upon reinfusion into the patient. In their recent paper, Rossetti et al. looked at methylaytion status of the FOXP3 gene – a gene involved in immune system responses – to compare ex vivo expanded Treg (eTreg) cells and induced Treg (iTreg) cells to select the most suitable cell type for clinical implementation.

Three readouts are commonly used to define bone fide Treg cells: (1) expression of the hallmark transcription factor FOXP3, (2) in vitro suppressive function and (3) demethylation of the TSDR, a regulatory region of the FOXP3 gene. Although iTreg cells expressed FOXP3 and displayed suppressive function after one round of differentiation, they lost these regulatory features upon withdrawal of differentiation factors. In contrast, eTreg cells that were expanded in the presence of rapamycin maintained their regulatory properties upon re-stimulation. The level of methylation at the TSDR predicted the loss of regulatory properties: indeed, iTreg cells were as hypermethylated as naïve T cells, while eTreg cells retained the TSDR demethylation status that is typical of freshly isolated Treg cells.

Importantly, functionally stable eTreg cells could be obtained from patients affected by an autoimmune disease, namely rheumatoid arthritis (RA), demonstrating the feasibility of this approach in a pathological context. In addition, expansion of RA Treg cells with rapamycin endowed them with the ability to suppress hyper-activated effector T cells coming from the inflamed site – an ability that freshly isolated RA Treg cells lack.

These data not only support the clinical application of eTreg cells as therapy for rheumatological diseases, but also demonstrate that assessing TSDR demethylation is a necessary quality control step before infusion to ensure their functional stability.

Original Article:

Rossetti, M., Spreafico, R., Saidin, S., Chua, C., Moshref, M., Leong, J., Tan, Y., Thumboo, J., van Loosdregt, J., & Albani, S. (2014). Ex Vivo-Expanded but Not In Vitro-Induced Human Regulatory T Cells Are Candidates for Cell Therapy in Autoimmune Diseases Thanks to Stable Demethylation of the FOXP3 Regulatory T Cell-Specific Demethylated Region The Journal of Immunology, 194 (1), 113-124 DOI: 10.4049/jimmunol.1401145

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Maura Rossetti

Maura Rossetti