No abstract

CD4+ regulatory T cells (Tregs) are key mediators of immunological tolerance and promising effector cells for immuno-suppressive adoptive cellular therapy to fight autoimmunity and chronic inflammation. Their functional stability is critical for their clinical utility and has been correlated to the demethylated state of the TSDR/CNS2 enhancer element in the Treg lineage transcription factor FOXP3. However, proof for a causal contribution of the TSDR de-methylation to FOXP3 stability and Treg induction is so far lacking. We here established a powerful transient-transfection CRISPR-Cas9-based epigenetic editing method for the selective de-methylation of the TSDR within the endogenous chromatin environment of a living cell. The induced de-methylated state was stable over weeks in clonal T cell proliferation cultures even after expression of the editing complex had ceased. Epigenetic editing of the TSDR resulted in FOXP3 expression, even in its physiological isoform distribution, proving a causal role for the de-methylated TSDR in FOXP3 regulation. However, successful FOXP3 induction was not associated with a switch towards a functional Treg phenotype, in contrast to what has been reported from FOXP3 overexpression approaches. Thus, TSDR de-methylation is required, but not sufficient for a stable Treg phenotype induction. Therefore, targeted demethylation of the TSDR may be a critical addition to published in vitro Treg induction protocols which so far lack FOXP3 stability.

The Rap G protein signal regulates Notch activation in early thymic progenitor cells, and deregulated Rap activation (Raphigh) results in the development of Notch-dependent T-cell acute lymphoblastic leukemia (T-ALL). We demonstrate that the Rap signal is required for the proliferation and leukemogenesis of established Notch-dependent T-ALL cell lines. Attenuation of the Rap signal by the expression of a dominant-negative Rap1A17 or Rap1GAP, Sipa1, in a T-ALL cell line resulted in the reduced Notch processing at site 2 due to impaired maturation of Adam10. Inhibition of the Rap1 prenylation with a geranylgeranyl transferase inhibitor abrogated its membrane-anchoring to Golgi-network and caused reduced proprotein convertase activity required for Adam10 maturation. Exogenous expression of a mature form of Adam10 overcame the Sipa1-induced inhibition of T-ALL cell proliferation. T-ALL cell lines expressed Notch ligands in a Notch-signal dependent manner, which contributed to the cell-autonomous Notch activation. Although the initial thymic blast cells barely expressed Notch ligands during the T-ALL development from Raphigh hematopoietic progenitors in vivo, the ligands were clearly expressed in the T-ALL cells invading extrathymic vital organs. These results reveal a crucial role of the Rap signal in the Notch-dependent T-ALL development and the progression.

The concept of a "topographical memory" in lymphocytes implies a stable expression of homing receptors mediating trafficking of lymphocytes back to the tissue of initial activation. However, a significant plasticity of the gut-homing receptor αβ was found in CD8 T cells, questioning the concept. We now demonstrate that αβ expression in murine CD4 memory T cells is, in contrast, imprinted and remains stable in the absence of the inducing factor retinoic acid (RA) or other stimuli from mucosal environments. Repetitive rounds of RA treatment enhanced the stability of de novo induced αβ. A novel enhancer element in the murine Itga4 locus was identified that showed, correlating to stability, selective DNA demethylation in mucosa-seeking memory cells and methylation-dependent transcriptional activity in a reporter gene assay. This implies that epigenetic mechanisms contribute to the stabilization of αβ expression. Analogous DNA methylation patterns could be observed in the human ITGA4 locus, suggesting that its epigenetic regulation is conserved between mice and men. These data prove that mucosa-specific homing mediated by αβ is imprinted in CD4 memory T cells, reinstating the validity of the concept of "topographical memory" for mucosal tissues, and imply a critical role of epigenetic mechanisms.