A Link between PDL1 and T Regulatory Cells in Fetomaternal Tolerance 1 Antje Habicht,* Shirine Dada,* Mollie Jurewicz,* Brian T. Fife, † Hideo Yagita, ‡ Miyuki Azuma, § Mohamed H. Sayegh,* and Indira Guleria 2 * Acceptance of the fetus expressing allogeneic paternal Ags by the mother is a physiologic model of transplantation tolerance. Various mechanisms contribute to fetal evasion from immune attack by maternal leukocytes. We have recently demonstrated that the inhibitory costimulatory molecule PDL1 plays a critical role in fetomaternal tolerance in that PDL1 blockade or deficiency resulted in decreased allogeneic fetal survival rates. CD4  CD25  T regulatory cells (Tregs) have also been demonstrated to play an important role in fetomaternal tolerance. Since PDL1 is expressed on Tregs, we explored the interactions between PDL1 and Tregs in vivo in a mouse model of fetomaternal tolerance. Depletion of CD25  T cells abrogated the effect of anti-PDL1 Ab indicating that the effect of PDL1 is possibly mediated by CD25  Tregs. Adoptive transfer of Tregs from wild-type but not PDL1-deficient mice into PDL1-deficient recipients significantly improved fetal survival. The frequency, phenotype and placental trafficking of Tregs from PDL1-deficient mice were similar to those of wild-type controls, but were defective in inhibiting allo- reactive Th1 cells in vitro. This is the first report providing evidence for a link between PDL1 and T regulatory cells in mediating fetomaternal tolerance. The Journal of Immunology, 2007, 179: 5211–5219. T he development of fetus in the mother can be considered as an in vivo model of allograft tolerance. Study of tol- erance mechanisms enabling a state of pregnancy could therefore provide additional insight to our understanding of im- munologic tolerance as a whole. Many factors promoting immune evasion have been explored in recent years (1). We have recently proposed and tested the hypothesis that cells present at the utero- placental interface expressing PDL1, a negative costimulatory molecule, protect murine allogeneic concepti from maternal T cell mediated immunity. We observed that pregnant CBA mice treated with a blocking anti-PDL1 mAb lost allogeneic but not syngeneic concepti. Similarly, a deficiency of PDL1 resulted in substantial increase in the rate of spontaneous fetal resorption and decrease in fetal survival (2). This increase in the rate of resorption was cou- pled with increase in T cell infiltration in the placenta, thereby suggesting that PDL1-expressing decidual cells inhibit PD-1-ex- pressing T cells that infiltrate the placenta in response to paternal Ags. Recent studies have highlighted the significance of regulatory T cells during immune interactions (3–5). The CD4 + CD25 + regu- latory T cell (Treg) 3 subset has been found to suppress autoim- mune responses and to play a role in tolerating allogeneic organ grafts (3– 6). The acceptance of paternally derived tumor cells dur- ing pregnancy (7) suggests the involvement of systemic regulatory processes in pregnancy. Aluvihare et al. (8) have recently shown that Tregs expand in murine pregnancies and are essential for a successful allogeneic pregnancy. In another study involving CBAxDBA abortion prone model, accumulation of paternal al- loantigen specific Th1 cells is suggested to be due to insufficient generation of pregnancy induced Tregs (9). The data in mouse are consistent with a similar function for Tregs in human preg- nancy. It has been reported that decidual and/or peripheral blood CD4 + CD25 +high T cells increased during early preg- nancy (10, 11) and returned to lower levels postpartum in the subjects studied (12). Naturally occurring Tregs are characterized by the surface ex- pression of CD4 and CD25 (13). In addition to sustained high surface expression of CD25, CTLA4, and glucocorticoid-induced TNFR-related protein (GITR) expression are features of suppres- sive Tregs (14, 15). Expression of the transcription factor Foxp3 has been shown to be confined to Tregs in mice and is critical to their development and regulatory function (15). Unlike other Treg cell markers, Foxp3 expression has been reported as not being up-regulated in conventional mouse T cells upon their activation (16, 17). Recently mice that express normal Foxp3 linked to a fluorescent reporter (18, 19) have been generated. These mice would be a very useful in tracking and studying Tregs, since these cells can be isolated live by flow sorting and then used in tracking or functional studies. The PD-1 receptor and its ligands, PDL1 and PDL2, define a novel regulatory pathway with potential inhibitory effects on T, B, and monocyte responses (20 –23). In addition to the markers for Tregs discussed above, PD-1 mRNA is highly expressed in *Transplantation Research Center, Brigham and Women’s Hospital and Children’s Hospital Boston, Harvard Medical School, Boston, MA 02115; † University of Cali- fornia San Francisco Diabetes Center, University of California, San Francisco, San Francisco, CA 94143-0540; ‡ Department of Immunology, Juntendo University School of Medicine, Bunkyo-ku, Tokyo, Japan; and § Department of Molecular Immunology, Graduate School, Tokyo Medical and Dental University, Bunkyo-ku, Tokyo, Japan Received for publication April 3, 2007. Accepted for publication August 20, 2007. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported by National Institutes of Health Grant R01 AI051559 and P01 AI056299 (to M.H.S.), American Society of Transplantation Basic Scientist Faculty Grant (to I.G.), and National Kidney Foundation Research Fellowship Grant (to A.H.). 2 Address correspondence and reprint requests to Dr. Indira Guleria, Transplantation Research Center, 300 Longwood Avenue, Boston, MA 02115. E-mail address: indira.guleria@tch.harvard.edu 3 Abbreviations used in this paper: Treg, regulatory T cell; GITR, glucocorticoid- induced TNFR-related protein; dpc, days post coitum; WT, wild type. Copyright © 2007 by The American Association of Immunologists, Inc. 0022-1767/07/$2.00 The Journal of Immunology www.jimmunol.org