TITLE: Ex vivo generation of innovative cellular immunotherapies by pharmacological epigenetic modulation
Αλέξανδρος Σπυριδωνίδης, Καθηγητής Αιματολογίας, Πανεπιστήμιο Πατρών, Διευθυντής Μονάδας Μεταμόσχευσης Μυελού των Οστών, Πανεπιστημιακό Γενικό Νοσοκομείο Πατρών
Κέντρο Γονιδιακής και Κυτταρικής θεραπείας, Therapy Center, Dpt. of Hematology, Hematopoietic Cell Transplantation Unit, George Papanicolaou Hospital, Thessaloniki, Greece (Δρ. Γιαννάκη) VUmc Cancer Center Amsterdam, Dpt. of Hematology, Amsterdam, Netherlands (Dr. Themeli)
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-Gpos 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-Gpos Tregs. For the molecular and cellular characteriszation of the HLA-Gpos 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-Gpos 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-Gpos ), which can be easily purified for adoptive immunotherapy (iG-Tregs).