Differentiation of induced pluripotent stem cells (iPSCs) into specific T cell subtypes using epigenetic editing
Immuno-suppressive CD4+ regulatory T cells (Tregs) are the main natural preventer of auto-immune diseases and chronic inflammation and are known to support tissue regeneration after injury and immuno-pathology. Therefore, Tregs are currently being extensively studied as "living drugs" in adoptive cellular therapy against pathogenic inflammation and to foster tissue regeneration.
Classical approaches of adoptive Treg therapy, which are based on the extensive in vitro expansion of pre-existing Treg populations, are currently being hampered by several obstacles:
1) the acquisition of proliferation-induced cellular senescence during in vitro expansion resulting in limited survival;
2) a functional instability of Tregs during inflammatory conditions;
3) the absence of pre-existing functional Treg populations in certain auto-immune disease patients and
4) a lack of pre-existing Treg populations displaying the required antigen-specificity or migration capacity, as the antigen-specific T cells in auto-immune patients have acquired a (pathogenic) pro-inflammatory phenotype.
Our group is trying to address these limitations from a new molecular angle: from epigenetics. We have identified several critical epigenetic control elements ('Epi-stabilizers') in T cells which are involved in maintaining a given T cell phenotype (Durek et al, Immunity 2016). The best characterized one is the so-called 'Treg-specific demethylated region – TSDR' in the FOXP3 gene, which is determining Treg function and cellular identity (Huehn et al, Nat Rev Immunol., 2009). To date, the demethylated state of the TSDR is the most reliable biomarker for human Tregs.
Previous work in the group established a CRISPR/Cas9 based system of 'epigenetic editing' allowing the targeted switching of methylation states at Epi-stabilizer elements. With this, Epi-stabilizers can be switched on (=demethylation) and off (=methylation) at will. While this system was successful to switch DNA methylation states and with that, regulate expression of the associated gene, the functional phenotype of the T cells was not completely switched probably due to the remaining pre-imprinted T cell epigenetic landscape.
This is why I now suggest to take a small detour in the functional re-programming of T cell phenotypes and include a re-programming step towards induced pluripotent stem cells (iPSCs). During this, the original T cells can be rejuvenated (addresses obstacle 1 of current Treg therapy, above), while the original TCR can be maintained (obstacle 4). This approach would allow Treg therapy even in Treg-deficient patients (obstacle 3). The resulting iPSCs can be grown in virtually unlimited numbers without senescence acquisition (obstacle 1), are easy to manipulate by epi-/genetic editing and can be stored for repetitive transfusions if needed (obstacle 1). In addition, iPSCs can also be generated from other cellular sources but still be re-differentiated into T cells. The epigenetic editing will be introduced during the T cell differentiation step from iPSC-derived hematopoietic precursor cells, which mimics the normal T cell development in the thymus (7) and thus, is a promising approach to induce stable functional T cell subsets (obstacle 2).
With this project, we expect to establish a system, with which large numbers of storable, fully functional T cell populations can be generated displaying defined advantageous characteristics (e.g. functional stabilization, re-juvenation, selected TCR-specificity). This system can be extended to induce features like a defined migration behavior (obstacle 4) or cytokine expression profile, since Epi-stabilizers in homing receptor (Szilagyi et al., Mucosal Immunol., 2017; Pink et al., J Immunol., 2016) and cytokine genes (Lee et al., Immunity, 2006) have also been identified. With this, we assume to break down several road blocks on the way towards a successful clinical application of adoptive T cell therapies.
1. P. Durek et al., Epigenomic Profiling of Human CD4(+) T Cells Supports a Linear Differentiation Model and Highlights Molecular Regulators of Memory Development. Immunity 45, 1148-1161 (2016). [PubMed]
2. J. Huehn, J. K. Polansky, A. Hamann, Epigenetic control of FOXP3 expression: the key to a stable regulatory T-cell lineage? Nat Rev Immunol 9, 83-89 (2009). [PubMed]
3. B. A. Szilagyi et al., Gut memories do not fade: epigenetic regulation of lasting gut homing receptor expression in CD4(+) memory T cells. Mucosal Immunol 10.1038/mi.2017.7 (2017). [PubMed]
4. M. Pink et al., Imprinting of Skin/Inflammation Homing in CD4+ T Cells Is Controlled by DNA Methylation within the Fucosyltransferase 7 Gene. J Immunol 10.4049/jimmunol.1502434 (2016). [PubMed]
5. G. R. Lee, S. T. Kim, C. G. Spilianakis, P. E. Fields, R. A. Flavell, T helper cell differentiation: regulation by cis elements and epigenetics. Immunity 24, 369-379 (2006). [PubMed]