Single and combined effect of retinoic acid and rapamycin modulate the generation, activity and homing potential of induced human regulatory T cells

RESEARCH ARTICLE Single and combined effect of retinoic acid and rapamycin modulate the generation, activity and homing potential of induced human regulatory T cells Enzo Candia1, Paz Reyes2, Camila Covian3, Francisco Rodriguez3, Nicolas Wainstein3, Jorge Morales1, Claudio Mosso1, Mario Rosemblatt2,3,4, Juan Alberto Fierro1* a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 1 Centro de Trasplantes, Clinica Las Condes, Santiago, Chile, 2 Fundación Ciencia & Vida, Santiago, Chile, 3 Facultad de Ciencias Biologicas, Universidad Andres Bello, Santiago, Chile, 4 Departamento de Biologı́a, Facultad de Ciencias, Universidad de Chile, Santiago, Chile * Abstract OPEN ACCESS Citation: Candia E, Reyes P, Covian C, Rodriguez F, Wainstein N, Morales J, et al. (2017) Single and combined effect of retinoic acid and rapamycin modulate the generation, activity and homing potential of induced human regulatory T cells. PLoS ONE 12(7): e0182009. https://doi.org/ 10.1371/journal.pone.0182009 Editor: Vassiliki A. Boussiotis, Beth Israel Deaconess Medical Center, UNITED STATES Received: July 1, 2016 Accepted: July 11, 2017 Published: July 26, 2017 Copyright: © 2017 Candia et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Adoptive transfer of CD4+CD25+FOXP3+ regulatory T cells (Treg cells) has been successfully utilized to treat graft versus host disease and represents a promising strategy for the treatment of autoimmune diseases and transplant rejection. The aim of this study was to evaluate the effects of all-trans retinoic acid (atRA) and rapamycin (RAPA) on the number, phenotype, homing markers expression, DNA methylation, and function of induced human Treg cells in short-term cultures. Naive T cells were polyclonally stimulated and cultured for five days in the presence of different combinations of IL-2, TGF-β1, atRA and RAPA. The resulting cells were characterized by the expression of FOXP3, activation, surface and homing markers. Methylation of the Conserved Non-coding Sequence 2 was also evaluated. Functional comparison of the different culture conditions was performed by suppression assays in vitro. Culturing naive human T cells with IL-2/TGFβ1 resulted in the generation of 54.2% of Treg cells (CD4+CD25+FOXP3+) whereas the addition of 100 nM atRA increased the yield of Treg cells to 66% (p = 0.0088). The addition of RAPA did not increase the number of Treg cells in any of these settings. Treg cells generated in the presence of atRA had an increased expression of the β7 integrin to nearly 100% of the generated Treg cells, while RAPA treated cells showed enhanced expression of CXCR4. The differential expression of homing molecules highlights the possibility of inducing Treg cells with differential organ-specific homing properties. Neither atRA nor RAPA had an effect on the highly methylated CNS2 sites, supporting reports that their contribution to the lineage stability of Treg cells is not mediated by methylation changes in this locus. Treg cells generated in the presence of RAPA show the most potent suppression effect on the proliferation of effector cells. Funding: This work was supported by Fondecyt 1120731, Conicyt Proyecto basal PFB-16, Direccion Academica Clinica Las Condes 2011, Julio Krauss Rotter. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. PLOS ONE | https://doi.org/10.1371/journal.pone.0182009 July 26, 2017 1 / 32 Human regulatory T cells induced ex vivo Competing interests: The authors have declared that no competing interests exist. Introduction The discovery, isolation, and generation of CD4+CD25+FOXP3+ regulatory T cells (Treg cells) represent a remarkable breakthrough in modern immunology[1,2]. Regulatory T cells (Treg cells) are crucial players for the maintenance of peripheral tolerance, controlling activation and expansion of autoreactive cells, therefore performing a vital contribution to the control of autoimmunity [3]. Since it is possible to expand Treg cells ex vivo, an increasing number of preclinical and clinical trials have evaluated the administration of Treg cells in autoimmune diseases such as type 1 diabetes [4], systemic lupus erythematosus [5], rheumatoid arthritis [3] and Crohn’s disease [6,7]. Furthermore, in the context of organ and bone marrow transplantation, Treg cells contribute critically to the acceptance of allogeneic implants [8]. In this regard, the adoptive transfer of Treg cells in animal models of transplantation has shown to improve graft survival [9,10]. In humans, the administration of Treg cells in the setting of hematopoietic cell transplantation resulted in a reduced incidence and severity of graftversus-host disease [11–13]. Thus, the manufacture and use of Treg cells for the advance of solid organ transplantation is being evaluated in a large cooperative project, the “ONE Study” [14,15]. Several limitations must be overcome to convert human Treg cell therapy into an efficient and safe procedure [16]. These include; the production of a high, clinically useful number of Treg cells while maintaining a stable lineage with very low, ideally absent, level of contaminating cells, the suitability of providing Treg cells with specific homing receptors depending on the clinical context, and providing Treg cells with antigen specificity. Additionally, the production of Treg cells should proceed in adherence to “good manufacturing practices,” which also relates to regulatory issues. Currently, two main strategies to harvest Treg cells ex vivo appear suitable for clinical applications: the expansion of differentiated Treg cells, and the induction of Treg cells starting from naïve T cells (Tn cells). To date, it is not clear which would be the most effective and safe strategy, since both paths have potential benefits and shortcomings. Because Treg cells are scarce in peripheral blood, the generation of Treg cells from Tn cells offers the advantage of initiating the expansion from a larger cell number. Additionally, the differentiation process of Tn cells may facilitate the manipulation of cells to pursue the expression of defined homing receptors depending on the desired clinical application. Moreover, it has been reported that induced Treg cells, under certain conditions, may offer greater stability and functionality in comparison with natural Treg cells, [17]. Our study represents an effort to define the optimal conditions for the generation and expansion of Treg cells from Tn cells. TGF-β1 and IL-2 have been shown to act as key cytokines on the differentiation of naïve T lymphocytes into Treg cells [18]. However, Treg cells generated ex vivo w (...truncated)