Keratinocyte growth factor (KGF) enhances postnatal T-cell development via enhancements in proliferation and function of thymic epithelial cells

doi:10.1182/blood-2006-10-049767Prepublished online January 9, 2007;2007 109: 3803-3811   

 Andrei V. Gudkov, Yousuke Takahama, Werner Krenger, Bruce R. Blazar and Georg A. HolländerSimona W. Rossi, Lukas T. Jeker, Tomoo Ueno, Sachiyo Kuse, Marcel P. Keller, Saulius Zuklys, epithelial cellsdevelopment via enhancements in proliferation and function of thymic Keratinocyte growth factor (KGF) enhances postnatal T-cell

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IMMUNOBIOLOGY

Keratinocyte growth factor (KGF) enhances postnatal T-cell development viaenhancements in proliferation and function of thymic epithelial cellsSimona W. Rossi,1 Lukas T. Jeker,1 Tomoo Ueno,2 Sachiyo Kuse,2 Marcel P. Keller,1 Saulius Zuklys,1 Andrei V. Gudkov,3

Yousuke Takahama,2 Werner Krenger,1 Bruce R. Blazar,4 and Georg A. Hollander1

1Laboratory of Pediatric Immunology, Center for Biomedicine, Department of Clinical-Biological Sciences, University of Basel and The University Children’sHospital (UKBB), Basel, Switzerland; 2Division of Experimental Immunology, Institute for Genome Research, University of Tokushima, Tokushima, Japan;3Department of Molecular Genetics, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, OH; 4University of Minnesota Cancer Center andDepartment of Pediatrics, Division of Bone Marrow Transplantation, Minneapolis, MN

The systemic administration of keratino-cyte growth factor (KGF) enhances T-celllymphopoiesis in normal mice and micethat received a bone marrow transplant.KGF exerts protection to thymic stromalcells from cytoablative conditioning andgraft-versus-host disease–induced in-jury. However, little is known regardingKGF’s molecular and cellular mecha-nisms of action on thymic stromal cells.Here, we report that KGF induces in vivo atransient expansion of both mature and

immature thymic epithelial cells (TECs)and promotes the differentiation of thelatter type of cells. The increased TECnumbers return within 2 weeks to normalvalues and the microenvironment dis-plays a normal architectural organization.Stromal changes initiate an expansion ofimmature thymocytes and permit regularT-cell development at an increased rateand for an extended period of time. KGFsignaling in TECs activates both the p53and NF-�B pathways and results in the

transcription of several target genes nec-essary for TEC function and T-cell devel-opment, including bone morphogeneticprotein 2 (BMP2), BMP4, Wnt5b, andWnt10b. Signaling via the canonical BMPpathway is critical for the KGF effects.Taken together, these data provide newinsights into the mechanism(s) of actionof exogenous KGF on TEC function andthymopoiesis. (Blood. 2007;109:3803-3811)

© 2007 by The American Society of Hematology

Introduction

Decreased T-cell cellularity and a skewed TCR repertoire arehallmarks of an immune deficiency commonly observed in old age,as a consequence of general infectious diseases and aggressivelymphocyte-depleting therapies for diverse malignancies.1-4 Theregeneration of a phenotypically and functionally normal T-cellcompartment is curtailed for an extended period of time in patientsreceiving a hematopoietic stem cell transplant (HSCT).5-7 This lackin T-cell reconstitution is associated with opportunistic infections,the reactivation of latent viral and parasitic infections, chronicinflammation, and autoimmunity.3,4

Following cytoablative therapy, the recovery of the T-cellcompartment relies on 2 independent pathways, that is, theexpansion of peripheral T cells and, alternatively, the de novoproduction of T cells in the thymus.1,2,7-10 The latter assures thegeneration of a population of naive T cells expressing a diverserepertoire of TCR specificities.5,7,8,10,11 The extent of thymus-dependent T-cell reconstitution correlates directly with thymic sizefollowing immune ablation and hematopoietic stem cell (HSC)–derived reconstitution7,12 but is inversely related to age andtransplant-related toxicities such as graft-versus-host disease(GVHD).10,13-17 The generation of new T cells of donor origindepends on the migration of hematopoietic precursors to thethymus. Normal thymic T-cell development is in turn contingent onthe regular maintenance of the stromal microenvironment. How-ever, age-related thymic involution18 and injury from radiation,19

GVHD,20 chemotherapy,12,21 or infection3,4,12,18-23 preclude normal

thymopoiesis to occur as they directly affect thymic epithelialcells (TECs). There has been considerable interest in identifyingstrategies to prevent TEC injury. Recently, robust T-cell lympho-poiesis has been maintained in myeloablated HSCT recipientsby pretransplantation administration of different factors such asIL-7,24,25 androgen antagonists,26 and fibroblast growth factor 7(Fgf7; aka, keratinocyte growth factor [KGF]).20,27-29 KGFbelongs to the family of the structurally related Fgfs and is apotent epithelial cell mitogen.27,30 KGF is expressed underphysiological conditions within the thymus both by mesenchy-mal cells and by T cells at specific developmental stages. Toexert its biologic activity, KGF activates the IIIb variant of theFgfR2 receptor (FgfR2IIIb), which is expressed within thethymus exclusively on TECs.31 Experiments using mice defi-cient for FgfR2IIIb or the removal of mesenchyme from normalembryos revealed the importance of Fgf signaling during earlythymus organogenesis.32 The postnatal thymic epithelial compart-ment may continue to require growth-regulating signals includ-ing possibly endogenous KGF, whose thymic expression issustained throughout life.28

Although of considerable therapeutic potential, little is knownregarding KGF’s mode of action on adult thymopoiesis and thethymic microenvironment. Here, we report on the cellular andmolecular response of adult TECs to a systemic treatment withrecombinant human KGF and how the ensuing changes enhancethymopoiesis.

Submitted October 2, 2006; accepted December 22, 2006. Prepublishedonline as Blood First Edition Paper, January 9, 2007; DOI 10.1182/blood-2006-10-049767.

An Inside Blood analysis of this article appears at the front of this issue.

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Materials and methods

Animals

Female C57BL/6 and B6.SJL-PtprcaPep3b/BoyJ (B6.CD45.1; CD45.1�)mice were purchased from Charles River (Lyon, France) and the JacksonLaboratories (Bar Harbor, ME), respectively. Mice were 6 weeks of age at thetime of KGF administration. Animals were kept under specific pathogen-freeconditions and in accordance with federal regulations. [Smad4lox/lox: Foxn1-cre]F2 mice were generated by crossing B6.129Smad4lox/lox mice (a gift from C.Deng, Bethesda, MD) to B6;D2-Tg(Foxn1-cre)8Ghr transgenic mice that expressthe Cre-recombinase in TECs (L.T.J. and G.A.H., manuscript in preparation).

In vivo and in vitro KGF treatment

Mice were injected intraperitoneally for 3 days (days 0, 1, and 2) withHanks balanced salt solution (HBSS) or recombinant human KGF (palifermin,generously provided by Amgen, Thousand Oaks, CA) solubilized in HBSS at adose of 5 mg/kg per day. For in vitro studies, thymic stromal cell preparationstaken from E15.5 fetal thymic lobes were cultured for the indicated times inmedia supplemented with KGF (100 ng/mL) or HBSS (vol/vol).

Flow cytometry

For flow cytometric analyses and cell purifications, fluorochrome-conjugated or unconjugated moAbs against TCR� (clone H57-592), CD8(53-6.7), CD4 (RM4-5), CD3 (145-2C11), CD44 (IM7), CD25 (PC61),CD45 (30-F11), CD45.1 (A20), CD45.2 (104), I-Ab (AF6-120.1), CD117(2B8), and CD127 (A7R34) were used (BD Biosciences, San Diego, CA;eBioscience, San Diego, CA; Caltag Laboratories, Burlingame, CA). Toreveal biotinylated moAbs, streptavidin-conjugated Cy5, PerCP, Cy-Chrome, phycoerythrin, and APC (Zymed Laboratories, San Francisco,CA; and Caltag Laboratories) were used. Three-color analyses wereperformed using a FACSCalibur (Becton Dickinson, Mountain View, CA).The different medullary (m) and cortical (c) TEC subpopulations wereidentified in stromal cell preparations by flow cytometry by use of the lectinUEA-1 and antibodies specific for the surface molecule MTS24 (a TECprecursor marker33,34) and the cytokeratin 5 (K5) and K18.20,35-37 To thisend, thymic stromal cells were stained with a moAb to K18 (Ks18.04;Progene, Heidelberg, Germany), polyclonal antibody to K5 (Covance,Princeton, NJ), biotinylated UEA1 lectin (Vector Laboratories, Burlingame,CA), and the MTS24 moAb (a gift from R. Boyd, Melbourne, Australia).Large granular cells (FSChighSSChigh) present in the freshly isolated thymicstromal cell preparations were gated for analysis. Early thymic progenitors(ETPs) were identified and purified (� 98%) by cell sorting as Lin� (that is,TCR��, CD3�, CD4�, CD8�) CD44�CD25�CD117high lymphoid cells.

Thymic stromal cell preparations and culture

To generate thymic stromal cell preparations, thymic cell suspensions from adultC57BL/6 mice (6 weeks old) were digested with collagenase D and DNAse I for3 cycles, as described.33,38 For quantification, CD45�I-Ab int�high stromalcells present in each cell fraction were counted by flow cytometry.CD45

�I-Ab int�high stromal cells were sorted to purity (� 99%) using a

FACSVantage cell sorter (Becton Dickinson). Thymic stromal cells fromfetal (E14.5 or E15.5) C57BL/6 mice were isolated and depleted fromlymphoid cells with 2-deoxyguanosine, as described.39 Purified adult TECsor fetal thymic stromal cells were starved for 24 hours in IMDM/1% FCS,and subsequently stimulated with KGF. Stromal cell proliferation wasassessed by 3H-thymidine incorporation. E15.5 fetal thymic stromal cellswere cultured in the presence or absence of the selective I�B kinaseinhibitor PS1145 (10 mM; a gift from Millennium, Cambridge, MA), thefarnesyltransferase inhibitor L-778123 (1 mM; Merck Research Laborato-ries, Rahway, NJ), and the p53 inhibitor PFT102 (10 mM; provided by A. V.Gudkov, Cleveland, OH).

Assessment of TEC proliferation in vivo

Adult C57BL/6 mice (6 weeks old) were injected intraperitoneally with5�-bromo-2�-deoxyuridine (BrdU; 1 mg in 0.2 mL PBS; Sigma, Buchs,

Switzerland) 48 and 24 hours before being killed and analyzed for BrdUincorporation, as described.40 DNA-synthesizing cells were represented asfrequencies of BrdU� cells (in %) among total FSChighSSChighCD45�I-Ab�

cells present in the thymic stromal cell preparations.

Detection of recent thymic emigrants

Anesthetized adult mice treated with either KGF or HBSS were injected inone thymic lobe with 10 �L FITC (125 �g/mL diluted in PBS; Sigma).Splenic FITC�CD4� and CD8� lymphocytes were detected by flowcytometry 16 hours after intrathymic injection.

Transfer of ETPs

ETPs were purified from adult B6.CD45.1 mice on day 6 after initiation ofKGF or HBSS treatment and transferred intrathymically (1000 ETPs/lobe)into C57BL/6 (CD45.2�) recipients that had received either HBSS or KGF15 days earlier.

Time-lapse visualization of T-cell precursor attraction

Thymocyte-depleted fetal thymic stromal cell preparations (E14.5) that hadbeen exposed for 24 hours to either KGF (100 ng/mL) or HBSS werecocultured with T-cell precursors. Cell culture, visualization, reaggregatepreparations, and data acquisition were carried out as published.41

Immunohistofluorescence confocal microscopy

For analysis of mTECs and cTECs by immunohistology, frozen thymicsections (6 �m) were fixed with 4% paraformaldehyde/PBS and stainedwith a panel of reagents that was also used for flow cytometry (above; Gillet al33; and Bennett et al34) and the cytokeratin 5 (K5) and K18.20,35-37 Inaddition, moAbs against MTS10 (BD Biosciences) and polyclonal anti-FgfR2IIIb Ab (R&D Systems, Minneapolis, MN) were used. Images werecaptured on a Zeiss LSM 510 confocal microscope with a 20�/0.5 NAobjective (Carl-Zeiss AG, Feldbach, Switzerland). Overlays of blue (Cy5),red (Alexa555), and green (Alexa488) stainings were colored by computer-assisted management of data using the Zeiss LSM 510 software version 3.2.

Quantitative PCR

cDNA was generated from total RNA and amplified using primers designedfor real-time polymerase chain reaction (PCR). Primer sequences for IL-7,GAPDH, Wnt5b, Wnt10b, BMP2, BMP4, CCL21, and CCL25 are avail-able from the authors. RNA transcription levels in TECs from KGF-treatedmice or stromal culture levels were normalized to GAPDH and comparedwith their expression in HBSS-treated control mice or thymic stromalcontrol cultures.

Statistical analysis

Groups were compared by one-way ANOVA and Bonferroni/Dunn posthoctest. The overall statistical significance level was set to 5%. For 2-groupcomparisons, the Mann-Whitney test was applied. StatView from SASInstitute (Cary, NC) was used for statistical analysis.

Results

KGF increases thymocyte numbers by differentially stimulatingcell proliferation

We first determined in adult C57BL/6 mice the consequences ofKGF signaling for thymopoiesis. Following treatment for 3 consecu-tive days (days 0, 1, and 2) with KGF, the total thymic cellularitywas transiently diminished (days 4-8) but recovered to higher thannormal values on and beyond day 11 (Figure 1A). Supranormalnumbers of thymocytes were sustained for no fewer than 2 monthsthereafter (data not shown). The increase in total thymocytecellularity noted beyond day 11 was initially caused by an

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expansion due to cell proliferation of the most immature, CD3/CD4/CD8 triple-negative (TN), thymocytes (Figure 1B-J). These cellssequentially gave rise to cells with a more mature phenotype. Onday 4, the absolute number of all TN thymocytes was comparablefor both treatment groups, although TN1 and TN2 cells werealready increased due to a higher proliferation rate. Over the nextfew days, TN3 and TN4 followed suit. Thus, KGF affected themost immature thymocytes, resulting in a developmental wave ofnew T cells that followed the correct sequence of TN maturationalstages. KGF treatment also resulted in a transient decrease ofmature CD4�CD8� and CD4�CD8� thymocytes (Figure 1L-M).Absolute numbers of mature thymocytes stably rose above levelsfound in HBSS-treated mice, however, once a higher number ofimmature CD4�CD8� thymocytes had been achieved (ie, at day 11and thereafter).

An increased thymic size in KGF-treated mice correlateswith enhanced thymic T-cell export

Next, we quantified thymic export in response to KGF treatment bydetection of intrathymically FITC-labeled T cells that have recentlyemigrated into the spleen. In agreement with the reduction ofmature CD4�CD8� thymocytes at 8 days after initiation of KGFtreatment (Figure 1L), we found in KGF-treated mice a significantdecrease in the export of CD4� cells to the periphery (Figure 2A).Thymic export of CD4�CD8� cells was similarly affected. Sixweeks later (day 45), thymic export of CD4� and CD8� cells to thespleen was significantly increased in the KGF-treated mice (Figure2B), corresponding to the overall increase in mature thymocytes.These results demonstrated during steady-state conditions ofthymopoiesis a direct correlation between thymic cellularity, theabsolute increase in mature thymocytes, and the number of recentthymic emigrants.

KGF affects numbers and thymic entry of earlyT-cell precursors

As the absolute number of TN1 cells had increased by 4 days afterKGF treatment (Figure 1C), we investigated whether this effectwas due to expansion of the most immature intrathymic cellprecursors (early thymic progenitors [ETPs]). These cells residewithin the population of TN1 cells and display aLin�CD44�CD25�CD117� phenotype.42,43 Although the fre-quency of ETPs was expanded to supranormal values on day 30,their number was decreased early (ie, 4 to 11 days) after initiationof KGF treatment (Figure 3A). To investigate the cause for thistransient reduction, we sought to quantify the attraction andsubsequent entry of hematopoietic precursors to the thymus. Thiswas most informatively achieved by time-lapse video microscopyof fetal thymic lobes as previously demonstrated by us.41 Following

Figure 1. KGF increases thymocyte numbers via induction of TN cell division. Adult naive C57BL/6 mice were treated with KGF (5 mg/kg, u) or HBSS (f) on 3consecutive days (days 0, 1, and 2), and thymi were analyzed by flow cytometry at the indicated days. (A) Total thymocyte numbers (cells � 10�6). (B-F) TN cell numbers perthymus (� 10�6). TN indicates CD3�4�8�; TN1, CD44�CD25�; TN2, CD44�CD25�; TN3, CD44�CD25�; and TN4, CD44�CD25� cells. (G-J) BrdU incorporation into TN cellsas a measure of cell division. The data represent the fraction (in %) of cells among total thymocytes that have incorporated BrdU. (K-M) Total numbers per thymus ofCD4�CD8�, CD4�CD8�, and CD4�CD8� cells. Three independent experiments were performed, and one representative experiment is shown. Mean SD; *P .05 versusHBSS controls, with 5 mice per group and time point.

Figure 2. Thymocyte numbers correlate with T-cell exit into the periphery. AdultC57BL/6 mice were first treated either with KGF (u) or HBSS (f) as in Figure 1 andthen intrathymically injected with FITC (10 �L) 7 or 44 days later. After 24 hours of invivo labeling, the export of thymus-derived cells into the periphery was assessed byenumeration of FITC-positive cells (� 10�6) on day 8 (A) and day 45 (B). Mean SD;*P .05 versus HBSS controls, with 6 mice per group and time point.

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the exposure of alymphoid E14.5 thymic lobes to KGF, both thenumber of precursors attracted to and the number of precursorsthen entering into the lobes were significantly reduced (Figure 3B).Several chemokines, including CCL21 and CCL25, have beenimplicated for the colonization of hematopoietic precursor cells asthey are expressed both in the combined fetal thymus/parathyroidanlage and in the postnatal thymus.41,44-46 The exposure of fetalthymic lobes to KGF caused an up-regulation of CCL21 but areduction in CCL25 transcripts (Figure 3C). Thus, the decrease inCCL25 correlated with the decreased seeding of T-lymphoidprecursors to the thymus.

The effect of KGF on early T-cell precursors isnot T-cell autonomous

The effect of KGF on early thymocyte proliferation may have beendriven by the acquisition of a cell-autonomous capacity of TN cellsto proliferate better or, alternatively, by an improved thymicstromal microenvironment via the creation of additional develop-mental niches. We therefore measured the extent of donor-derivedthymopoiesis in recipients of intrathymically injected, congenicETPs. We purified ETPs from B6.CD45.1 donors and intrathymi-cally transferred them to KGF-treated C57BL/6 mice. In a secondgroup, C57BL/6 recipients received ETPs isolated from KGF-treated B6.CD45.1 mice. One week after injection, these mice werecompared with a control group where neither donors nor hosts hadbeen treated with KGF. The thymic cellularity was enhanced only

under conditions where the C57BL/6 recipient thymic stromalenvironment had been exposed to KGF (Figure 4A dashed bar).Here, the absolute numbers of intrathymic CD45.1� cells (ie, ETPprogeny) were found to be the highest among the 3 transplantationgroups (Figure 4B). In contrast, ETPs isolated from mice treatedwith KGF did not exhibit an increased growth rate upon intrathy-mic transfer into HBSS-injected recipients (gray bars). Thus, thetreatment with KGF resulted in an improved stromal microenviron-ment able to increase thymopoiesis, but the stroma did not imparton ETPs a cell-autonomous capacity for enhanced reconstitution.

Exogenous KGF is mitogenic for all TEC subpopulationsin adult mice

Our data indicated that KGF triggered a temporary deficiency toattract hematopoietic precursor cells but eventually may havecreated new developmental niches for T lymphopoiesis or, alter-natively, enhanced the ability of TECs to support thymopoiesis.To examine whether KGF differentially acted on the known TECsubpopulations, we first determined in adult C57BL/6 mice theFgfR2IIIb expression among major cortical (K18�K5�

MTS 10�UEA1�), minor cortical (K18�K5�), major medullary(K5�MTS10�), and minor medullary (K18�UEA1�) TECs as wellas MTS24� TEC precursors. Using immunohistofluorescenceconfocal microscopy, we found that a minority of the K18� cTECsand K5� mTECs expressed FgfR2IIIb (Figure 5A-B), whereasalmost all UEA1� and the large majority of MTS24� epithelialcells were positive for FgfR2IIIb (Figure 5C-D). Thymocytes didnot express FgfR2IIIb (data not shown, and Rossi et al20). To testthe in vivo responses of the diverse TEC subpopulations to KGF,adult mice were treated for 3 consecutive days (days 0, 1, and 2)with KGF or HBSS. On day 3, thymic tissue was collected andstromal cell preparations were subjected to flow cytometric analy-sis. Following treatment with KGF, the relative frequencies of thedifferent TEC subsets increased each by at least 2-fold whencompared with HBSS-treated mice (Figure 5E-H). However, thearchitectural organization of all tested TEC populations appearednormal on day 15 by immunohistofluorescence analysis (Figure

Figure 4. Non–cell-autonomous effect of KGF on early thymic progenitors.ETPs (Lin�CD44�CD25�CD117hi cells) were purified on day 6 after initiation oftreatment of B6.CD45.1 donors (CD45.1�) that had received KGF (5 mg/kg per day,u) or HBSS (f, o) on days 0, 1, and 2. Afterward, ETPs (1 � 103) from either groupwere transferred separately to a single thymic lobe of adult C57BL/6 recipients(CD45.2�) that had received 15 days earlier either HBSS (f, u) or KGF (5 mg/kg perday, o) on 3 consecutive days. The panels depict recipient thymic total cellularity (A)and CD45.1� congenic ETP progeny (B) that were determined 7 days afterintrathymic (i.t.) injection. Mean SD; *P .001 versus HBSS controls (black bars),with 5 mice per group and time point.

Figure 3. KGF inhibits the capacity of the thymic microenvironment to attractT-cell precursors. (A) Adult C57BL/6 mice were treated either with KGF (u) or HBSS(f) as in Figure 1, and the numbers of ETPs (Lin�CD44�CD25�CD117hi) weredetermined at the indicated days. (B) Time-lapse video microscopy recorded E14.5fetal thymocytes that migrated toward (open bars) and then entered (filled bars) analymphoid E14.5 fetal thymic lobe that had been pre-exposed for 24 hours to KGF(100 ng/mL, u) or HBSS-supplemented medium (f) before the start of the migrationexperiments. The graph depicts the numbers of cells that migrated in a directedfashion and that reached the thymic lobe. Statistical significance was evaluatedindividually for open bars (�) and filled bars. (C) Changes in chemokine expressionfollowing exposure to exogenous KGF. Chemokine mRNA levels were determined byquantitative reverse-transcription PCR qRT. The y-axis shows the relative transcriptlevels (the group with maximal chemokine expression was set as 100%, which isdisplayed as value of 1.0). Mean SD; *P .05 versus HBSS controls in panel A,with 5 mice per group and time point. Mean SEM; *P .01 versus HBSS controlsin panel B where data are representative of 10 independent in vitro experiments.

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5I-N). To determine TEC proliferation in vivo, adult mice weretreated with KGF and then injected with BrdU. The frequency ofTECs (CD45�I-Ab�) incorporating BrdU was increased by morethan 10-fold in mice injected 3 days earlier with KGF whencompared with animals treated with HBSS (Figure 5O). Thisproliferative response was transient as the frequency of BrdU�

TECs on day 6 was enhanced by only 3-fold in response to KGF.The increase in BrdU incorporation, as observed 3 days afterinitiation of KGF treatment, translated at day 6 into enhanced TECnumbers. While HBSS-treated 6-week-old normal C57BL/6 miceheld 3.6 0.7 � 105 CD45�I-Ab� TECs per thymus, the cellular-ity for both immature (MTS24�) and mature (MTS24�) TECs roseto 5.4 0.7 � 105 cells upon KGF administration (Figure 5P). Thenumber of TECs returned to normal values by day 15 afterinitiation of KGF treatment in parallel with a shift in the ratio ofMTS24� to MTS24� TECs (Figure 5P). Taken together, these datademonstrated a transitory effect of pharmacological doses of KGFon TEC proliferation in vivo leading to an early expansion of bothmature and immature TECs, and on the transition of epithelialprecursors to more mature TECs.

Bone morphogenetic proteins play a role in adult TECsin their response to KGF

Early TEC development as well as the survival, proliferation,differentiation, and migration of thymocytes are under the

precise control of different molecules produced by TECs,including Wnt glycoproteins and the bone morphogeneticproteins BMP2 and BMP4.47-51 We therefore tested whetherKGF affected the expression of these factors in TECs from adultmice. CD45�I-Ab� TECs were isolated on day 7 from adultC57BL/6 mice treated with either KGF or HBSS. As shown inFigure 6A, transcripts for Wnt5b, Wnt10b, BMP2, and BMP4were up-regulated in vivo by 2- to 5-fold above normal inresponse to KGF. To determine a definitive role for BMPmedi-ated signaling in the TEC response to KGF, we next investigatedmice that lacked Smad4 specifically in TECs. CytoplasmicSmad4 acts as an indispensable partner in the canonical BMPsignaling pathway leading to target gene transcription.52 Whencompared with B6.129Smad4lox/lox controls, [Smad4lox/lox: Foxn1-cre]F2 mice displayed a smaller thymus whose cellular composi-tion and architectural organization were proportionally normal(L.T.J. and G.A.H., manuscript in preparation). However, thelatter failed to increase their thymic cellularity in response toKGF treatment (Figure 6B). This deficit was independent of theage of the mouse (data not shown). Hence, the KGF-mediatedincrease in thymic cellularity was dependent on signals in TECsthat engaged Smad4, thus indicating a definitive role of BMPs inthe response of thymic epithelium to KGF.

As freshly isolated TECs are not suited to manipulations in vitrodue to their rapid loss of TEC-specific features, we used E15.5 fetal

Figure 5. KGF enhances TEC numbers via induction of cell division in adult mice. (A-D) Adult TECs express the receptor for KGF. Thymic sections from 6-week-old,naive C57BL/6 mice were analyzed by confocal immunohistofluorescence for the expression of FgfR2IIIb on K18� epithelial cells (A), K5� cells (B), MTS24� cells (C), and onTECs binding UEA1 (D). The colocalization denotes distinct TEC subpopulations expressing the KGF receptor (arrows). A total of 3 experiments were performed, providingcomparable results. (E-H) TECs expand in response to KGF in vivo. Adult C57BL/6 mice were treated with KGF (5 mg/kg, u) or HBSS (f) on 3 consecutive days (days 0, 1, and2). Frequencies of the 4 TEC subpopulations were assessed on day 3 by flow cytometry. Relative frequencies are given (x-fold changes KGF vs HBSS treatment, wherebyfrequencies [in %] for HBSS-treated mice were set as 1.0 and shown as dashed lines). Mean SD. *P .01 versus HBSS. (I-N) KGF administration to adult mice does notalter the normal architecture and epithelial composition of the thymus. Confocal microscopy analysis was performed 15 days after treatment of adult C57BL/6 mice with HBSS(I-K) or KGF (L-N) for 4 TEC subpopulations, as indicated. mTEC indicates medullary TEC; cTEC, cortical TEC. (O) Adult C57BL/6 mice were treated as described in panelsE-H and then injected with BrdU 48 and 24 hours before killing. BrdU� cells among FSChighSSChighCD45�I-Ab� cells present in freshly isolated thymic stromal cells (days 3 and6) were analyzed (x-fold change KGF vs HBSS, whereby frequencies [in %] in HBSS-treated mice were set as 1.0 [dashed lines]). Mean SD; *P .05 versus HBSS controls,with 6 mice per group and time point. A total of 3 experiments were performed, providing comparable results. (P) Absolute TEC numbers on days 6 and 15 following treatment ofmice with either KGF (u) or HBSS (f). Total CD45�I-Ab int�high stromal cells were counted by flow cytometry. Relative frequencies of total TECs are given, whereby the valuesfor HBSS-treated mice were set as 1.0. The fractions of MTS24� and MTS24� cells (separated by horizontal lines) among total TECs and their absolute cell numbers are alsoindicated. Mean SD; *P .01, total TEC numbers and #P .02, total MTS24� cells, respectively, in KGF-treated mice versus those in HBSS controls. A total of 12 individualmice were analyzed.

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thymic epithelial cultures to investigate in more detail 2 importantissues related to our findings in adult mice: (1) the regulation in theexpression of growth and differentiation factors; and (2) the signaltransduction pathways in TECs activated by KGF. As demonstratedin Figure 7A, the in vitro exposure to KGF (100 ng/mL) robustlyincreased TEC proliferation. Similar to the results obtained withadult TECs, the exposure of fetal TECs to KGF resulted in anup-regulation of BMP2, BMP4, and Wnt10b (Figure 7B). Thesignaling pathways previously shown to mediate KGF activityhave included Ras/MAP kinases, phosphatidylinositol 3�kinase(PI3K), and p53.53-57 We therefore assessed the effect of KGF onTECs by independently blocking these different signaling path-ways (Figure 7C). As Wnt molecules provide signals critical fornormal TEC development,49 Wnt10b mRNA expression was usedas a quantifiable readout for our experiments. The blocking of p53with the inhibitor PFT102,58 and that of NF-�B (a downstreamtarget of PI3K) with the I�B kinase inhibitor PS1145,59 suppressedthe up-regulation of Wntl0b, suggesting that this effect was highly

dependent on signaling via both NF-�B and p53. In contrast, usingthe farnesyltransferase inhibitor L-778123,60 the activation of theRas pathway was not found to be obligatory to mediate theobserved KGF effect in fetal TECs.

Discussion

Administration of KGF has been among the therapeutic strategiesto promote thymic function.20,28 Here, we have detailed themolecular and cellular mechanisms by which KGF stimulatesthymic T lymphopoiesis in adult mice. We show that the KGF-specific receptor, FgfR2IIIb, is expressed by both mature corticaland medullary as well as immature TECs (Figure 5). Uponexposure to exogenous KGF, these stromal cells proliferate andexpress several growth and differentiation factors, including differ-ent members of the family of Wnt and BMP molecules (Figures5-6). In consequence, a robust and sustained increase in thymopoi-esis occurs, which is initiated as a single wave affecting first themost immature T-cell precursors (Figure 1). This effect of enhancedthymopoiesis is uniquely dependent on the exposure of the stromalmicroenvironment to KGF as the transfer of ETPs from KGF-treated donor mice to naive recipients fails to impart an enhance-ment in thymopoiesis (Figure 4). The increased thymic cellularityleads to an enhanced export of mature T cells to the periphery(Figure 2). Thus, exogenous KGF displays a strong pharmacologi-cal activity in the thymus of naive adult mice that assures avigorous and enduring increase in thymic T-cell development.

Here, we report the normal TEC cellularity of 6-week-oldC57BL/6 mice (3.6 � 105 cells; see also Gray et al61) cantransiently be expanded by exogenous KGF (Figure 5). In parallelwith a higher frequency of mature (that is, MTS24�) TECs, thetotal number of TECs returned at day 15 after initiation of KGFtreatment to normal values. These kinetics are consistent with thenoted physiological turnover of TECs in adult mice.61,62 Due to anincreased proliferation rate following KGF stimulation, the result-ant TEC expansion has likely an initial but nonetheless decisiveimpact on lymphoid cellularity after day 11. It is conceivable thatenhanced TEC numbers create a “larger” microenvironment able toaccommodate increased amounts of early-stage thymocytes. Due to

Figure 6. Wnts and BMPs are target genes of KGF. (A) Analysis of genetranscription by qRT-PCR in adult TECs. Six-week-old C57BL/6 mice were treatedwith KGF or HBSS as described in Figure 1 and sorted CD45�I-Ab� TECs wereanalyzed on day 7 for mRNA expression of Wnt5b, Wnt10b, BMP2, and BMP4.Expression levels in KGF-treated mice (u) were compared with those in control micetreated with HBSS (x-fold change KGF vs HBSS, whereby the expression levels inthe latter were set as 1.0; dotted line). (B) The increase in thymic cellularity inresponse to KGF depends on signals mediated by Smad4. Adult wild-typeB6.129Smad4lox/lox mice (� Cre) and [Smad4lox/lox: Foxn1-cre]F2 mice (� Cre) weretreated with KGF (u) or HBSS (f). Six and 15 weeks after the initiation of treatment,total thymocyte cellularity was determined. Absolute thymocyte numbers (� 10�6)are shown. Mean SD versus HBSS controls. For data shown in panel A, 3experiments were performed whereby material from 15 mice was pooled for eachexperiment.

Figure 7. KGF signaling engages NF-�B and p53 in TECs. (A) Proliferation of cells present in thymocyte-depleted E15.5 fetal thymic stromal cell preparations was assessedin culture by 3H-thymidine incorporation (cpm). The x-axis indicates the time (in hours) after exposure to exogenous KGF (100 ng/mL, E) or HBSS (f); n � 6 experiments.Mean SD; *P .05. (B) Transcripts for Wnt5b, Wnt10b, BMP2, and BMP4 were quantified by qRT-PCR after 24 hours of culture. Expression levels in KGF-treated mice (u)were compared with those in control mice treated with HBSS (x-fold change KGF vs HBSS, whereby the expression levels in the latter were set as 1.0; dotted line). A total of 3experiments were performed, whereby material from 15 mice was pooled for each experiment. (C) Transcripts for Wnt10b were quantified by qRT-PCR in alymphoid E15.5 fetalthymic lobes exposed in culture for 24 hours to either KGF (u) or HBSS (f) in the presence or absence of the following inhibitors: the selective I�B kinase (IKK) inhibitorPS1145, the farnesyltransferase (FTase) inhibitor L-778123, which blocks the Ras pathway, or PFT102, a specific small-molecular inhibitor of p53. Transcription levels werecompared with control cultures exposed to HBSS but not supplemented with any of the inhibitors (dotted line � 1.0). Three independent experiments were performed; mean SD. *P .05 versus HBSS control, with 6 lobes examined per time point (A), 3 lobes per group and experiment (B, total of 3 experiments), and 4 experiments (C).

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the time required for the transition from TN1 to mature thymo-cytes,63-65 an appreciable increase in total thymocyte cellularitysecondary to quantitative TEC changes may be noted only aftersuch a maturational progression has occurred (that is, after about 13days). With respect to our results at and beyond day 15, we wouldargue for a qualitative rather than a quantitative change in theability of TECs to support thymopoiesis. Such an interpretation isin keeping with Wnt data shown in Figure 6A, the reported role ofWnts in thymopoiesis,66-68 and the changes in the ratio of MTS24�

to MTS24� TECs.The stromal response to KGF allowed for an early and

sequential increase in TN thymocytes (Figure 1), suggesting thatthe transient expansion of the thymic epithelial scaffold renderedthe microenvironment more proficient to support early thymopoi-esis. This interpretation is consistent with previous work demonstrat-ing that thymic size is limited by the availability of stromal nichesfor TN thymocytes.69 Cells at and beyond the CD4�CD8� stagewere, in contrast, decreased during this initial phase followingKGF treatment. This finding implied a transient but substantialchange in the potential of the microenvironment to sustain thesurvival and/or the differentiation of more mature thymocytes. Alikely explanation for this finding may be the observation thatproliferating TECs lose their capacity to support CD4�CD8�

thymocytes since this deficit correlated with TEC proliferation inresponse to KGF. Independent of the immediate changes inthymocyte cellularity following KGF treatment, a robust andlong-lasting increase in the cellularity of all thymocyte subpopula-tions was eventually achieved. The transient increase of CD4�CD8�

cells at day 6 was caused by immature single-positive thymocytes,while the delayed appearance of CD4�CD8� cells could besecondary to the decreased MHC class II expression of KGF-treated cortical TECs as previously reported.70 Our results are inagreement with a recent report28 and with the observations that thenumber of CD4�CD8� thymocytes is proportional to the numberof TN cells present.71 Thus, the increase in thymic size togetherwith a restoration of the different thymocyte subpopulations in aproportionally correct fashion reflect the establishment of a regularthymic homeostasis 4 weeks after treatment with KGF.

As the thymus does not contain hematopoietic stem cells withan unlimited self-renewal capacity, T-cell progenitors need to berecruited from the blood to maintain thymopoiesis. Several mecha-nisms may account for the decrease in ETPs following thetreatment with KGF, including the possibility that KGF affects thenumber of T-cell precursors homing to the thymus. The necessarysteps in the homing process are initiated in vessels at thecorticomedullary junction and involve not only the expression ofdifferent adhesion molecules on endothelial cells but also thepresence of different chemokines41,44,46,72 Recent data suggest thatCCL25 controls the seeding of hematopoietic precursors to bothfetal nonvascularized as well as adult thymic tissue.46,73 Theexposure of the thymic microenvironment to KGF not onlydecreased the attraction of T-cell precursors to the thymus but alsoreduced their entry. This fact was correlated with a decrease inCCL25 expression by the fetal thymic microenvironment exposedto KGF. A causal link between these 2 findings is possible becauseage-matched embryos deficient for CCL25 display a considerabledecrease in thymocyte numbers,44 and the use of neutralizingantibodies against CCL25 in adult mice decreases the thymicaccumulation of T-cell precursors.46 Thus, the down-regulationof CCL25 expression by TECs exposed to KGF may, at leastpartly, account for the observed paucity of ETPs present earlyafter treatment.

In addition to a phase during which the thymus may have beenpartially inaccessible for blood-borne T-cell precursors, changes inthe kinetics of early thymocyte development could further affectthe thymic entry of precursors and/or their developmental expan-sion in KGF-treated mice. It appears that thymic size is controlledby the number of TN thymocytes, which in turn depends on theavailability of developmental niches.69 In KGF-treated mice, TN1and TN2 cells were indeed significantly increased as early as day 4after the initiation of KGF treatment and displayed a notably higherproliferation rate when compared with the same cell populations incontrol mice. Yet, all TN subpopulations and their proliferativeresponses returned to normal values within 2 weeks after initiationof KGF treatment. At this point, the number of CD4�CD8�

thymocytes had increased above the values noted in controlanimals. However, the kinetics of the changes in the number of TNthymocytes in response to KGF (Figure 1) was in keeping with thetime reported that TN cells require to transit to CD4�CD8�

thymocytes,63-65 and hence excludes a decrease in the TN transitiontime as an explanation for the observed increase in cellularity. Ourfindings, however, suggest that the early and sequential increase inthe different TN subpopulations was caused by a single developmen-tal wave initiated by ETPs or their immediate progeny. Beyond day15 after KGF treatment, increased and proportionally normalthymopoiesis was present for an extended period of time.

The KGF-mediated stimulation of fetal and adult TECs en-hanced BMP2 and BMP4 transcripts. Induction of BMP4 expres-sion is consistent with comparable findings in tracheal epitheliumcultures exposed to either KGF or Fgf10, which binds also toFgfR2IIIb. It is likely but not formally demonstrated by ourexperiments that enhancement of BMP2/4 mediated the increase inthymic cellularity as Smad4�/� mice failed to expand theirthymocyte numbers in response to KGF. Normal thymic cellularityis indeed dependent on the correct BMP dose, as mice eithertransgenic for the BMP antagonist, noggin, or deficient for theBMP4 inhibitor, twisted gastrulation, display a reduced thymic sizesecondary to microenvironmental changes.50,74 Even though ourresults demonstrated an increase in BMP2/BMP4 transcripts as aconsequence of KGF signaling, a previous report had placed theeffect of BMP4 on TECs upstream of KGF/Fgf10.51 This discrep-ancy is likely explained by differences in the experimental design,as the latter study investigated the effects of BMP4 on thymocytedevelopment, while our analysis examined the immediate transcrip-tional changes in TECs upon KGF signaling. However, it is alsoconceivable that KGF/Fgf10 are part of a positive feedback loopregulating BMP4 signaling in TECs.

KGF treatment of TECs also enhanced Wnt expression, albeit atdifferent magnitudes when comparing fetal and adult cells (Figures6-7). This divergence most likely reflects differences in maturationand thus function. Wnt molecules exert a profound effect on TECs(S.Z., M.P.K., and G.A.H., manuscript in preparation; and Balciun-aite et al49). We used Wnt10b as a readout in primary TECs andhave established a link between KGF signaling and the activationof the PI3K/Akt/NF-�B and the p53 pathways. Stimulation of thePI3K/Akt/NF-�B pathway constitutes a typical feature of KGFsignaling in some epithelial cells, but the activation of the p53pathway has not yet been linked to KGF signaling.56,57 A functionallink between the PI3K/Akt/NF-�B and the p53 pathways mayoccur indirectly since Akt can modulate p53 activity via a positivefeedback loop, thus coordinating p53 activity with other signalingevents including the Wnt pathway.75,76 Activation of the Ras/MAP

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kinase pathway was not required for the KGF-mediated up-regulation of Wnt10b. This finding was unexpected as Fgf signal-ing typically leads to the phosphorylation of the docking proteinFRS2� and the recruitment of multiple Grb2/Sos complexes,which eventually results in the activation of the Ras/MAP kinasepathway.53,54 KGF may elicit differential effects in TECs,whereby only some necessitate the activation of the Ras/MAPkinase pathway.

Taken together, the exposure of TECs to KGF has revealed 3specific aspects. First, KGF enhances in fetal and adult TECs theexpression of growth and differentiation factors previously demon-strated to affect TEC biology. In consequence, a transientlyenlarged but regularly structured thymic microenvironment isgenerated able to impart enhanced T-cell production. Second, theexposure of the thymic microenvironment to KGF decreased theexpression of the chemokine CCL25 in TECs and thus may haveaffected the homing of T-cell precursors. This effect was, however,temporary and a proportionally normal representation of thedifferent thymocyte subpopulations was re-established within 4weeks after the start of treatment. Third, KGF caused changes inthe stroma’s ability to support separate phases in thymocytematuration. While these changes allowed for an early and sequen-tially correct expansion of the TN thymocytes, the immediateconsequences of the KGF treatment precluded in parallel asimultaneous increase in CD4�CD8� and mature single-positivethymocytes. Transient alterations in the number, function, and/orcomposition of niches specific for the development and selection ofmore mature thymocytes could account for this finding. Thus, thetreatment of naive mice with KGF may serve as an informativemodel to assess the molecular regulation of distinct microenviron-mental niches necessary for mouse T-cell development.

KGF-induced thymopoiesis resulted in enhanced export ofmature thymic T cells to the periphery. As there is no evidence thatthe thymus senses peripheral lymphopenia and gauges its T-cellexport accordingly, and as changes in thymic output will contribute

to the replenishment of the peripheral T-cell compartment, anyincrease in thymopoiesis may therefore be beneficial for individu-als with lymphopenia. The enhanced thymic export of naive T cellswill also secure a diverse T-cell repertoire, as it will concurrentlyoffset the homeostatic expansion of a limited number of mature Tcells. Hence, the use of KGF in clinical medicine may produce anefficient restoration of the T-cell compartment and thus thecompetence for an effective adaptive immune response.

Acknowledgments

This work was supported by the Swiss National Science Founda-tion (grants 3100-68310.02 to G.A.H., and NFP46 to G.A.H. andW.K.), the European Community 6th Framework Program Euro-Thymaide Integrated Project (G.A.H.), the Basel Cancer League(W.K., M.P.K.), NIH grants R01-A1057477 (G.A.H) and R01-HL073794 (B.R.B), and the Dana Foundation.

Authorship

Contribution: S.W.R., W.K., and G.A.H. designed and performedwork, analyzed the data, and wrote the paper; T.U., S.K., Y.T.,L.T.J., M.P.K., and S.Z. designed and performed work; B.R.B.designed work and wrote the paper; A.V.G. provided materials.W.K. and G.A.H. share senior authorship.

Conflict-of-interest disclosure: The authors declare no compet-ing financial interests. Human recombinant KGF (palifermin) was agenerous gift of Amgen Inc (Thousand Oaks, CA). A.V.G. providedthe p53 inhibitor.

Correspondence: Georg A. Hollander or Werner Krenger,Laboratory of Pediatric Immunology, Center for Biomedi-cine, University of Basel, Mattenstrasse 28, 4058 Basel,Switzerland; e-mail: [email protected] or [email protected].

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EFFECT OF KGF ON ADULT THYMIC EPITHELIAL CELLS 3811BLOOD, 1 MAY 2007 � VOLUME 109, NUMBER 9

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