Induction of Regulatory T Cells and Its Regulation with Insulin-like Growth Factor/Insulin-like Growth Factor Binding Protein-4 by Human Mesenchymal Stem This information is current as Cells of September 27, 2021. Ippei Miyagawa, Shingo Nakayamada, Kazuhisa Nakano, Kaoru Yamagata, Kei Sakata, Kunihiro Yamaoka and Yoshiya Tanaka J Immunol published online 19 July 2017 Downloaded from http://www.jimmunol.org/content/early/2017/07/19/jimmun ol.1600230 http://www.jimmunol.org/ Supplementary http://www.jimmunol.org/content/suppl/2017/07/19/jimmunol.160023 Material 0.DCSupplemental Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision by guest on September 27, 2021 • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2017 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published July 19, 2017, doi:10.4049/jimmunol.1600230 The Journal of Immunology Induction of Regulatory T Cells and Its Regulation with Insulin-like Growth Factor/Insulin-like Growth Factor Binding Protein-4 by Human Mesenchymal Stem Cells Ippei Miyagawa,* Shingo Nakayamada,* Kazuhisa Nakano,* Kaoru Yamagata,* Kei Sakata,*,† Kunihiro Yamaoka,‡ and Yoshiya Tanaka* Human mesenchymal stem cells (MSCs) are multipotent and exert anti-inflammatory effects, but the underlying mechanism re- mains to be elucidated. In the current study, we investigated the regulatory mechanism of regulatory T cell (Treg) induction through the growth factors released by human MSCs. Human naive CD4+ T cells were stimulated with anti-CD3/28 Abs and cocultured with human MSC culture supernatant for 48 h. The proliferation and cytokine production of CD4+ T cells and surface molecule + + expression on CD4 T cells were evaluated. The proliferation of anti-CD3/28 Abs–stimulated CD4 T cells was suppressed by the Downloaded from addition of human MSC culture supernatant; in addition, the production of IL-10 and IL-4 increased. The human MSC culture supernatant induced CD4+FOXP3+ Tregs that expressed CD25, CTLA-4, glucocorticoid-induced TNFR-related protein, insulin- like growth factor (IGF)-1R, and IGF-2R, showing antiproliferative activity against CD4+ T cells. In addition, the induction of Tregs by human MSC culture supernatant was enhanced by the addition of IGF and suppressed by the inhibition of IGF-1R. In contrast, a significant amount of IGF binding protein (IGFBP)-4, an inhibitor of IGF action, was detected in the human MSC culture supernatant. After neutralization of IGFBP-4 in the human MSC culture supernatant by anti–IGFBP-4 Ab, Treg numbers http://www.jimmunol.org/ increased significantly. Thus, our results raise the possibility that human MSC actions also involve a negative-regulatory mech- anism that suppresses Treg proliferation by releasing IGFBP-4. The results of this study suggest that regulation of IGF may be important for treatments using human MSCs. The Journal of Immunology, 2017, 199: 000–000. heumatoid arthritis (RA) is a systemic autoimmune disease chondrocytes, and adipocytes (4, 5). Therefore, they are regarded that is characterized by synovitis and subsequent joint de- as potential therapeutic tools for promoting tissue repair at various R struction (1). Recently developed biological agents, such as sites, including joints. MSCs also have the capacity to modulate TNF-a inhibitors and IL-6R Abs, and JAK inhibitors have proven to immune responses. Studies have shown MSCs to be effective in sup- be more effective than conventional treatments (2, 3); however, some pressing disease progression in animal autoimmune disease models, by guest on September 27, 2021 patients fail to respond to these newer treatments. In addition, no ef- as well as in graft-versus-host disease (GVHD) patients receiving fective treatment for repairing damaged joints has been established. bone marrow transplantation (6–9). However, the mechanism of im- Mesenchymal stem cells (MSCs) are multipotent and exert munosuppression by human MSCs (hMSCs) has not been adequately anti-inflammatory effects. MSCs can differentiate into osteoblasts, explored. We reported previously that hMSCs inhibited osteoclast differ- *First Department of Internal Medicine, University of Occupational and Environ- entiation by producing osteoprotegerin and were able to differentiate mental Health, Kitakyushu 807-8555, Japan; †Mitsubishi Tanabe Pharma, Yokohama, into osteoblasts and chondrocytes under the influence of IL-1 and Kanagawa 227-0033, Japan; and ‡Division of Rheumatology, Department of Internal IL-6 (10–12). We also demonstrated that implantation of hMSCs Medicine, Keio University School of Medicine, Tokyo 160-8582, Japan with a poly-lactic-co-glycolic acid scaffold significantly suppressed ORCID: 0000-0002-8125-8630 (K. Yamagata). arthritis and joint destruction in the collagen-induced arthritis rat Received for publication February 8, 2016. Accepted for publication June 24, 2017. model and reported that their immunomodulatory effect was medi- This work was supported in part by research on rare and intractable diseases and ated by suppression of lymph follicles, splenomegaly, and anti–type Research Grants-in-Aid for Scientific Research by the Ministry of Health, Labor and Welfare of Japan, the Ministry of Education, Culture, Sports, Science and Technol- II collagen Abs through TGF-b (13). Therefore, we hypothesized ogy of Japan, the Japan Agency for Medical Research and Development, the Uni- that hMSCs might have potential as novel therapeutic tools for tissue versity of Occupational and Environmental Health, Japan, and a University of repairandimmunosuppressioninRApatients. Occupational and Environmental Health Grant for Advanced Research. Regulatory T cells (Tregs) are important for immune tolerance (14– I.M. contributed to the study design, overall review, and writing of the manuscript; the other authors were involved in the performance of the study and review of the 16). TGF-b reportedly plays a role in the mechanism of Treg induc- manuscript; and Y.T. and S.N. participated in the study design and coordination. All tion; however, it is known that MSCs produce growth factors other authors read and approved the final manuscript. than TGF-b (17, 18). In the current study, we found that hMSCs in- Address correspondence and reprint requests to Prof. Yoshiya Tanaka, First Depart- duced Tregs by releasing insulin-like growth factors (IGFs). We also ment of Internal Medicine, School of Medicine, University of Occupational and Environmental Health, 1-1 Iseigaoka, Yahata-nishi, Kitakyushu 807-8555, Japan. found that this induction was negatively regulated, in part, by hMSCs E-mail address: [email protected] releasing IGF binding protein (IGFBP)-4, an inhibitor of IGF action. The online version of this article contains supplemental material. Abbreviations used in this article: GITR, glucocorticoid-induced TNFR-related pro- tein; GVHD, graft-versus-host disease; hMSC, human MSC; IGF, insulin-like growth Materials and Methods factor; IGFBP, IGF binding protein; IGFR, IGF receptor; MSC, mesenchymal stem Preparation of hMSCs cell; RA, rheumatoid arthritis; Treg, regulatory T cell. Bone marrow–derived hMSCs were cultured with MSCGM Mesenchymal Copyright Ó 2017 by The American Association of Immunologists, Inc. 0022-1767/17/$30.00 Stem Cell Growth Bulletkit Medium (both from Lonza, Walkersville, MD) www.jimmunol.org/cgi/doi/10.4049/jimmunol.1600230 2 INDUCTION OF Tregs WITH IGF/IGFBP-4 + 5 in culture flasks at 37˚C under 5% CO2 atmosphere. They were used in Ab-stimulated CFSE-labeled human CD4 T cells (2 3 10 cells) for 96 h experiments after 75–85% confluence after one or two passages expanding (1:10). The proliferation of CD4+ T cells was evaluated using a CFSE within 3–7 d. Ethylene diamine tetraacetic acid 0.01%/trypsin was used to dilution assay. Levels of IL-10 in the culture supernatant were determined release the cells from the culture flasks. hMSCs were seeded directly onto using a BD Cytometric Bead Array (CBA) Human Flex Set, according to 12-well plastic plates at a density of 4 3 104 cells per well and cultured in the manufacturer’s instructions (BD Pharmingen). In addition, the con- 1000 ml of RPMI 1640 (Wako Pure Clinical Industries, Osaka, Japan) centration of soluble CD25 was measured by ELISA as a surrogate marker supplemented with 10% FCS (Tissue Culture Biologicals, Tulare, CA), of T cell activation (IL-2Ra, soluble, human, ELISA Kit, Quantikine, 100 U/ml penicillin, and 100 U/ml streptomycin (Invitrogen, New York, RSD). In addition, CFSE-labeled CD4+CD25+ T cells (CD4+FOXP3+ NY). After 4 d of culture, hMSC supernatant was collected for later use. T cells) were cocultured with plate-bound anti-CD3 (2 mg/ml) and soluble CD28 (1 mg/ml) Ab-stimulated human CD4+ T cells (2 3 105 cells) for + CD4 T cell isolation and culture conditions 96 h (1:10) to evaluate the proliferation of Tregs during coculture. The proliferation of CD4+CD25+