TITLE: Ex vivo generation of innovative cellular immunotherapies by pharmacological epigenetic modulation

The present project aims to follow up the published work in Cytotherapy (2017;19:521–530) on the “Simple in vitro generation of human leukocyte antigen-G–expressing T regulatory cells through pharmacological hypomethylation for adoptive cellular immunotherapy against graft-versus-host disease”. Briefly, in this study we endeavored the development of an effective immunotherapy against Graft-versus-Host Disease (GvHD), a frequent and life-threatening complication of allogeneic hematopoietic stem cell transplantation (allo-HSCT) and for which current treatment approaches are far from satisfactory.

Our approach aspired to mimic the mechanism of the successful physiological immunotolerance during pregnancy where the HLA-G molecule expressed in placenta, a well-known immunoregulatory molecule, protects the “semi-allogeneic” fetus from maternal immune attack. Since the HLA-G gene is epigenetically repressed after prenatal life and the methylation status of HLA-G promoter regulates its transcriptional activity, we showed in small scale in vitro experiments that exposure of human peripheral T-cells to hypomethylating agents (HA)  induces a de novo and stable expression of HLA-G and converts them to regulatory cells (Treg) with in vitro immunosuppresive functions. Though the suppressor activity of HA-treated Tregs is exclusively within the HLA-G^pos compartment, their suppressor function is dependent to a large extent, but not exclusively, on the HLA-G molecule and probably additional immunsuppresive molecules are upregulated through HAs and contribute to the regulatory function of the HLA-G^pos Tregs. For the molecular and cellular characteriszation of the HLA-G^pos compertment we will be employing cutting-edge throughput technologies such as RNA-sequencing, scRNA-sequencing and deep immunophenotyping (full spectrum flow cytometry/mass cytometry) to further elucidating the identity of this population. HLA-G is not expressed in mice and therefore studies with the use of humanized mouse models are needed to demonstrate the in vivo suppressive abilities of these human HLA-G^pos Tregs. We propose the ex vivo generation of HLA-G-expressing T-cells through pharmacological hypomethylation as a simple, Good Manufacturing Practice (GMP)-compatible and efficient strategy to produce a stable Treg subset of a defined phenotype (HLA-G^pos ), which can be easily purified for adoptive immunotherapy (iG-Tregs).