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.
Major barriers in using classical FOXP3+ regulatory T cells (Tregs) in clinical practice are their low numbers in the circulation, the lack of specific cell surface markers for efficient purification and the loss of expression of Treg signature molecules and suppressive function after in vitro expansion or in a pro-inflammatory microenviroment. A surface molecule with potent immunosuppressive function is the human leukocyte antigen-G (HLA-G), which is normally expressed in placenta protecting the "semi-allogeneic" fetus from maternal immune attack. Because HLA-G expression is strongly regulated by methylation, we asked whether hypomethylating agents (HA) may be used in vitro to induce HLA-G expression on conventional T cells and convert them to Tregs.



Human peripheral blood T cells were exposed to azacytidine/decitabine and analyzed for HLA-G expression and their in vitro suppressor properties.



HA treatment induces de novo expression of HLA-G on T cells through hypomethylation of the HLA-G proximal promoter. The HA-induced CD4+HLA-GposT cells are FOXP3 negative and have potent in vitro suppression function, which is dependent to a large extent, but not exclusively, on the HLA-G molecule. Converted HLA-Gpossuppressors retain their suppressor function in the presence of tumor necrosis factor (TNF) and preserve hypomethylated the HLA-G promoter for at least 2 days after azacytidine exposure. Decitabine-treated T cells suppressed ex vivo the proliferation of T cells isolated from patients suffering from graft-versus-host disease (GVHD).



We propose, in vitro 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 that can be easily purified for adoptive immunotherapy.
Bone Marrow Transplantation Unit, Medical School, University of Patras, Patras, Greece.
Panagiota
P
Stamou
Bone Marrow Transplantation Unit, Medical School, University of Patras, Patras, Greece.
Dimitra
D
Marioli
Department of Physiology, Medical School, University of Patras, Patras, Greece.
Alexandra L
AL
Patmanidi
Biology Lab, School of Science and Technology, Hellenic Open University, Patras, Greece.
Argyro
A
Sgourou
Bone Marrow Transplantation Unit, Medical School, University of Patras, Patras, Greece.
Angeliki
A
Vittoraki
Bone Marrow Transplantation Unit, Medical School, University of Patras, Patras, Greece.
Efthymia
E
Theofani
Molecular Haematology and Immunogenetics Center, Karaiskakio Foundation, Nicosia, Cyprus.
Chryso
C
Pierides
Department of Physiology, Medical School, University of Patras, Patras, Greece.
Stavros
S
Taraviras
Molecular Haematology and Immunogenetics Center, Karaiskakio Foundation, Nicosia, Cyprus.
Paul A
PA
Costeas
Bone Marrow Transplantation Unit, Medical School, University of Patras, Patras, Greece. Electronic address: spyridonidis@upatras.gr.
Alexandros
A
Spyridonidis
D001374:Azacitidine / Q000031:analogs & derivatives / Q000494:pharmacology; D018929:Cell Culture Techniques; D060846:Cell Engineering / Q000379:methods*; D002478:Cells, Cultured; D019175:DNA Methylation / Q000187:drug effects; D000077209:Decitabine; D005786:Gene Expression Regulation / Q000187:drug effects; D006086:Graft vs Host Disease / Q000276:immunology / Q000628:therapy*; D059951:HLA-G Antigens / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D016219:Immunotherapy, Adoptive / Q000379:methods*; D050378:T-Lymphocytes, Regulatory / Q000166:cytology / Q000276:immunology / Q000378:metabolism* / Q000637:transplantation*
D059951:HLA-G Antigens; D000077209:Decitabine; D001374:Azacitidine
adoptive cell therapy; graft-versus-host disease; human leukocyte antigen-G; hypomethylation; regulatory T cells