Generation of allo-antigen-specific induced Treg stabilized by vitamin C treatment and its application for prevention of acute graft versus host disease model

International Immunology, Vol. 29, No. 10, pp. 457–469 doi:10.1093/intimm/dxx060 Advance Access publication 4 November 2017 © The Japanese Society for Immunology. 2017. All rights reserved. For permissions, please e-mail: Generation of allo-antigen-specific induced Treg stabilized by vitamin C treatment and its application for prevention of acute graft versus host disease model 2 Correspondence to: A. Yoshimura; E-mail: Received 21 August 2017, editorial decision 26 October 2017; accepted 28 October 2017 Abstract Antigen-specific regulatory T cells (Tregs) possess the potential to reduce excess immune responses in autoimmune diseases, allergy, rejection after organ transplantation and graft-versushost disease (GVHD) following hematopoietic stem cell transplantation. Although in vitro-expanded antigen-specific induced Tregs (iTregs) have been considered to be a promising therapeutic agent against such excessive immune reactions, the instability of iTregs after transfer is a fundamental problem in their clinical application. In this study, we searched for the optimal way to generate stable iTregs for the prevention of the murine GVHD model, in which conventional iTregs are reported to be inefficient. Allo-antigen-specific iTregs were generated by co-culturing naive T cells with allogenic dendritic cells in the presence of TGF-β and retinoic acid. By examining various agents and genes, we found that vitamin C stabilized Foxp3 expression most effectively in adoptively transferred iTregs under a GVHD environment. Vitamin C treatment caused active DNA demethylation specifically on the conserved non-coding sequence 2 (CNS2) enhancer of the Foxp3 gene locus in allo-antigenspecific iTregs and reduced iTreg conversion into pathogenic exFoxp3 cells. Vitamin C-treated iTregs suppressed GVHD symptoms more efficiently than untreated iTregs. Vitamin C also facilitated induction of a FOXP3high iTreg population from human naive T cells, which was very stable even in the presence of IL-6 in vitro. The treatment of vitamin C for iTreg promises innovative clinical application for adoptive Treg immunotherapy. Keywords: DNA methylation, Foxp3, GVHD, immune tolerance, regulatory T cell Introduction Graft-versus-host disease (GVHD) remains a significant cause of morbidity and mortality following allogenic hematopoietic stem cell transplantation (HSCT), despite decades of research and trials in post-transplant immunosuppressive therapies (1). GVHD is initiated by the presentation of alloantigens by host antigen-presenting cells to donor CD4+ and CD8+ T cells; thereafter donor T cells expand and become alloreactive effector T cells (2). The alloreactive T cells then induce tissue injury and dysfunction through the production of inflammatory cytokines and cytotoxicity (3, 4). Regulatory T cells (Tregs) play essential roles in immunological tolerance and suppress excess immune responses. Tregs are specified by the expression of Foxp3, which is essential for the differentiation, maintenance and function of Tregs. Tregs include thymus-derived Tregs (tTregs) and induced Tregs (iTregs), which are generated from naive CD4+ T cells in vitro by the TCR stimulation in the presence of TGF-β (5). Induction of tolerance by antigen-specific Tregs has long been considered a promising therapeutic strategy to prevent GVHD following HSCT (6). Umbilical cord blood tTregs FEATURED ARTICLE Department of Microbiology and Immunology, Division of Hematology, Department of Medicine and 3 Department of Pathology, Keio University School of Medicine, Shinjuku-ku, Tokyo 160-8582, Japan 4 Department of Immune Regulation, Research Institute, National Center for Global Health and Medicine, Ichikawa, Chiba 2728516, Japan 5 Department of Pathology, Saitama Medical University, Iruma, Saitama 350-0495, Japan 1 Hidenori Kasahara1,2, Taisuke Kondo1, Hiroko Nakatsukasa1, Shunsuke Chikuma1, Minako Ito1, Makoto Ando1, Yutaka Kurebayashi3, Takashi Sekiya4, Taketo Yamada5, Shinichiro Okamoto2 and Akihiko Yoshimura1 458 Prevention of GVHD by stabilized iTregs Methods Mice We purchased 6- to 8-week-old male BALB/c AJc1 mice from Tokyo Laboratory Animals Science Co. Ltd (Tokyo, Japan). Foxp3hCD2-hCD52 (C57BL/6 genetic background) mice were kindly provided from the laboratory of Dr. S. Hori (The University of Tokyo, Tokyo, Japan). Mice were kept in conventional conditions in Keio University (Tokyo, Japan). All experiments using these mice were approved by the Institutional Animal Care and Use Committee (IACUC) (approved number 08004) of Keio University and performed according to the guidelines of IACUC. All experiments using these mice were also approved by and performed according to the guidelines of the Animal Ethics Committee of Keio University. Antibodies and reagents For the flow cytometry analysis, FITC, PE, peridinin chlorophyll protein-cyanine 5.5 (PerCP-Cy5.5), allophycocyanin (APC), PE-Cy7 and APC-Cy7-conjugated antibodies were purchased from BioLegend (San Diego, CA, USA) or eBioscience (San Diego, CA, USA). The following clones were used: anti-mouse CD4 (RM4-5), anti-mouse CD45.1 (A20), antimouse H-2Kb (AF6-88.5), anti-mouse TCRβ (H57-597), antimouse CD103 (2E7), anti-mouse CD25 (PC61.5), anti-mouse Thy1.1 (HIS51), anti-mouse Foxp3 (FJK16s), anti-mouse CTLA-4 (UC10-4F10-11), anti-mouse IFN-γ, (XMG1.2), antimouse IL-21 (mhalx21), anti-human CD2 (RPA2.10), antihuman CD4 (RPA-T4), anti-human CD25 (BC96), anti-human CD45RA (HI100), anti-human CD127 (A019D5), anti-human CCR7 (G043H7) and anti-human FOXP3 (236A/E7). Fixable Viability Dye eFluor 780 (FVD780) was used to remove dead cells. Murine IL-2, human IL-2, human IL-4, human IL-6 and human GM-CSF were purchased from Peprotech (Rocky Hill, NJ, USA); murine GM-CSF was purchased from Wako Pure Chemical Industries (Osaka, Japan); and human TGF-β1, anti-human IFN-γ antibody (B27) and anti-human IL-4 antibody (8D4-8), HumanTrustain TCX™ were purchased from BioLegend. ATRA, 5-aza-2′-deoxytidine (5-aza-dC) and l-ascorbic acid 2-phosphate sesquimagnesium salt hydrate (vitamin C) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Belinostat was purchased from Chemscene LLC (Monmouth Junction, NJ, USA). Normal human male serum was purchased from Innovative Research Inc (Novi, MI, USA). GVHD induction and prevention BALB/c host male mice (H-2Kd CD45.2+) were conditioned by myeloablative total body irradiation using a Faxitron X-ray system (Faxitron, Tucson, AZ, USA) at a dose of 8.0 Gy, split into two doses separated by at least 3 h. Within 4 h after irradiation, sex- and age-matched 5 × 106 B6.Ly5.1 (H-2Kb CD45.1+) bone marrow (BM) cells with 1 × 106 CD25+ cell-depleted conventional T cells isolated using magneticactivated cell sorting (MACS) from the spleen were injected via the tail vein in an SFO3 medium (Iwai Chemical, Tokyo, Japan). Usually, iTregs were transferred intravenously together with conventional T cells (1 × 106 cells) and BM cells (5 × 106 cells) int (...truncated)