Keratinocyte Growth Factor Induces Expansion of Murine Peripheral CD4  Foxp3  Regulatory T Cells and Increases Their Thymic Output 1 Marieke Bruinsma,* Peter L. van Soest,* Pieter J. M. Leenen, † Bart N. Lambrecht, ‡ Tom Cupedo,* Bob Lo ¨wenberg,* Jan J. Cornelissen,* and Eric Braakman 2 * Keratinocyte growth factor (KGF) has been shown to reduce the incidence and severity of graft-versus-host disease by prevention of epithelial damage and by modulating alloreactivity. Since regulatory T cells (Treg) play a crucial role in immune modulation, we evaluated the effects of exogenous KGF on peripheral CD4  Foxp3  Treg and the generation of Treg in the thymus of normal mice. A 3-day course of KGF induced a rapid selective increase in the number of highly suppressive CD4  Foxp3  Treg. Blood Treg numbers remained elevated for >2 mo, but the frequency normalized after 2 wk due to a concomitant increase in CD4  Foxp3  T cells. Analysis of single joint TCR excision circles frequency and Ki-67 expression in peripheral blood Treg showed that the early selective increase of Treg was predominantly accounted for by peripheral expansion. Thymectomy before KGF administration did not affect the early selective increase of Treg but abrogated the late increase in CD4  T cell numbers, thereby showing its dependence on thymic output. Collectively, these results show that KGF induces an increase in blood CD4  Foxp3  Treg numbers via two independent mechanisms. First by selective peripheral expansion of Treg and thereafter by enhanced thymic output of newly developed Treg. The Journal of Immunology, 2007, 179: 7424 –7430. K eratinocyte growth factor (KGF), 3 also known as FGF-7, is a 28-kDa member of the fibroblast growth factor fam- ily (1). KGF is produced by cells of mesenchymal origin and binds to FGFR2IIIb that is expressed primarily by epithelial cells. Exogenous KGF has profound effects on the thymus of nor- mal mice and on the recovery of the thymus in bone marrow trans- plantation (BMT) recipients. It enhances thymopoiesis in normal mice, reverses age-related involution of the thymus, and protects KGF receptor-positive thymic epithelial cells (TEC) from radia- tion- and cytotoxic therapy-induced damage and from damage caused by graft-versus-host disease (GVHD) (2–5). Administra- tion of KGF before allogeneic bone marrow transplantation (allo- BMT) enhances thymic regeneration and peripheral T cell recon- stitution by these protective effects (4–6). Several studies in murine GVHD models have shown that KGF administration be- fore and shortly after BMT reduces GVHD severity (4, 7–9) and may improve survival (10). The beneficial effects of KGF are mainly attributed to its protective effects against conditioning- induced epithelial cell injury. However, GVHD studies in uncon- ditioned mice have shown that KGF reduces GVHD also by im- munological effects (8). KGF administration reduces the in vivo alloresponse and alters cytokine expression in acute GVHD, fa- voring the development of a mixed TH1/TH2 pattern in which TH2 cytokines, such as IL-4 and IL-13, predominate. In addition, lymphocytes isolated from KGF-pretreated alloimmunized mice were shown to exhibit reduced responsiveness in a secondary in vitro MLR. To date, it is unknown whether the immunomodulatory effects of KGF also involve regulatory T cells (Treg). Accumulat- ing data demonstrate that CD4 + CD25 + Foxp3 + Treg play an im- portant role in the prevention of GVHD (11–15). Adoptive transfer of large numbers of freshly isolated or in vitro-expanded donor- or host-type CD4 + CD25 + Treg at the time of allo-BMT were shown to reduce the mortality associated with GVHD in experimental models (11–14). Conversely, depletion of CD25 + cells from a do- nor T cell infusion increases GVHD lethality (11, 12). In addition, in vivo depletion of CD25 + T cells of the recipient before exper- imental BMT results in increased GVHD (11, 13). These results have underscored the pivotal role of Treg in the suppression of alloreactivity. Given the observation that KGF may modulate alloreactivity also by affecting immune responsiveness apart from epithelial protection, we set out to study the effects of KGF on Treg in normal mice. It is shown that KGF strongly affects the peripheral expansion of peripheral blood Treg as well as thy- mic output of Treg. Materials and Methods Mice C57BL/6-CD45.2 mice were purchased from Charles River Laboratories and housed under specific pathogen-free conditions in the Erasmus Med- ical Center Animal Center. Mice were used at 8 –12 wk of age. Animal experiments were performed in accordance with Dutch legal regulations, which include approval by the animal ethical committee. KGF treatment Palifermin, N23-KGF (KGF), was provided by Amgen. Mice were in- jected s.c. with 5 mg/kg per day KGF once daily for 3 consecutive days. *Department of Hematology, † Department of Immunology, and ‡ Department of Pul- monary Medicine, Erasmus University Medical Center, Rotterdam, The Netherlands Received for publication April 24, 2007. Accepted for publication September 12, 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 in part supported by the Landsteiner Foundation for Blood Trans- fusion Research (Grant 0221). 2 Address correspondence and reprint requests to Dr. Eric Braakman, Department of Hematology, Erasmus University Medical Center, P.O. Box 2040, 3000 CA Rotter- dam, The Netherlands. E-mail address: e.braakman@erasmusmc.nl 3 Abbreviations used in this paper: KGF, keratinocyte growth factor; BMT, bone marrow transplantation; GVHD, graft-versus-host disease; RTE, recent thymic emigrant; TEC, thymic epithelial cell; sjTREC, single-joint T cell receptor excision circle; Treg, regula- tory T cell; TSLP, thymic stromal lymphopoietin; DC, dendritic cell; SP, single positive. Copyright © 2007 by The American Association of Immunologists, Inc. 0022-1767/07/$2.00 The Journal of Immunology www.jimmunol.org