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Cytokine and Chemokine ReceptorsSelective Thymus Settling Regulated by

BhandoolaMaillard, Benjamin C. Harman, Paul E. Love and Avinash Benjamin A. Schwarz, Arivazhagan Sambandam, Ivan

http://www.jimmunol.org/content/178/4/2008doi: 10.4049/jimmunol.178.4.2008

2007; 178:2008-2017; ;J Immunol 

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Selective Thymus Settling Regulated by Cytokine andChemokine Receptors1

Benjamin A. Schwarz,2* Arivazhagan Sambandam,2* Ivan Maillard,† Benjamin C. Harman,*Paul E. Love,‡ and Avinash Bhandoola3*

To generate T cells throughout adult life, the thymus must import hemopoietic progenitors from the bone marrow via the blood.In this study, we establish that thymus settling is selective. Using nonirradiated recipient mice, we found that hemopoietic stemcells were excluded from the thymus, whereas downstream multipotent progenitors (MPP) and common lymphoid progenitorsrapidly generated T cells following i.v. transfer. This cellular specificity correlated with the expression of the chemokine receptorCCR9 by a subset of MPP and common lymphoid progenitors but not hemopoietic stem cells. Furthermore, CCR9 expression wasrequired for efficient thymus settling. Finally, we demonstrate that a prethymic signal through the cytokine receptor fms-liketyrosine kinase receptor-3 was required for the generation of CCR9-expressing early lymphoid progenitors, which were the mostefficient progenitors of T cells within the MPP population. We conclude that fms-like tyrosine kinase receptor-3 signaling isrequired for the generation of T lineage-competent progenitors, which selectively express molecules, including CCR9, that allowthem to settle within the thymus. The Journal of Immunology, 2007, 178: 2008–2017.

T cells develop in the thymus (1). However, the thymuscontains no long-term self-renewing progenitors. Instead,T lymphopoiesis throughout adult life is maintained by

the periodic importation of bone marrow (BM)4 hemopoietic pro-genitors that reach the thymus via the bloodstream (2–6). Thenumber of progenitors that enter the thymus each day is estimatedto be exceedingly small (6–9), precluding their direct identifica-tion within the thymus. Therefore, which cells physiologically mi-grate from the BM to the thymus to generate T cells in adult miceis unknown.

Multiple progenitors within the BM have T lineage potential(10) demonstrated experimentally by the ability to generate T cellsfollowing intrathymic injection (2). These progenitors include he-mopoietic stem cells (HSC), which can produce all blood lineagesand have the ability to self-renew (11), multipotent progenitors(MPP), which can generate all hemopoietic lineages but have lostself-renewal capacity (12, 13), common lymphoid progenitors

(CLP), which were originally identified as lymphoid committed(14), and cells downstream of the CLP such as the CLP-2 (15, 16).Of these progenitors, HSC and MPP are also known to circulate inthe blood of adult mice (17, 18). However, it is unclear whether allof these cells can physiologically settle within the thymus from thebloodstream or if thymus settling is selective.

The molecular basis for progenitor entry into the thymus ispoorly understood. This process is likely to be analogous to thehoming of mature leukocytes, which involves selectin-mediatedweak adhesion to vasculature endothelium, followed by chemo-kine signaling, strong adhesion through integrins, and transmigra-tion (19–21). For thymus settling, both CD44 (22, 23) and P-selectin (24) have been shown to be important. Recent worksuggests that CCR9 may also play a role in progenitor migration tothe thymus. CCR9 is the receptor for CCL25 (25), which is highlyexpressed by thymic stroma (26). Although CCR9�/� mice haveno obvious defect in T cell development or thymic cellularity (27,28), an early defect in T cell development has been revealed incompetitive mixed BM chimeras (29, 30). Migration of CLP-2cells into the thymus has been found to be CCR9 dependent (31).Furthermore, blocking CCL25 results in reduced migration of pro-genitors into fetal thymic lobes in vitro (32). However, this samegroup reported that whereas there is a role for CCR9 in the mi-gration of progenitors into the fetal thymic anlage, there was norole for CCR9 in adult thymic settling (33). Therefore a require-ment for CCR9 in the homing of T lineage progenitor to the adultthymus remains controversial.

Recently, an analysis of Ccr9-GFP reporter mice revealed thatthe earliest identified progenitors in the thymus express high levelsof GFP (34), which is consistent with the idea that progenitors thatenter the thymus are CCR9�. A similar population was identifiedin the thymus using the cytokine receptor fms-like tyrosine kinasereceptor-3 (Flt3) (35, 36), suggesting that thymus-settling progen-itors may also be Flt3� (37). Flt3 is expressed by multiple BMprogenitors with T lineage potential, including MPP, CLP, andCLP-2-like cells, whereas HSC are Flt3� (12, 13, 16, 38). Fur-thermore, Flt3 is required for efficient generation of CLP and Bcells (38, 39). For T cell development, Flt3�/� mice have only a

*Department of Pathology and Laboratory Medicine, University of PennsylvaniaSchool of Medicine, Philadelphia, PA 19104; †Division of Hematology-Oncology,University of Pennsylvania School of Medicine, Philadelphia, PA 19104; and ‡Lab-oratory of Mammalian Genes and Development, National Institute of Child Healthand Human Development, National Institutes of Health, Bethesda, MD 20892

Received for publication October 26, 2006. Accepted for publication December1, 2006.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.1 This work was supported by the National Institutes of Health Grants AI059621 (toA.B.) and T32-AI-055428 (to B.A.S.) and Damon Runyon Cancer Research Foun-dation Grant DRG-102-05 (to I.M.). A.B. is the recipient of a Career DevelopmentAward from the Leukemia and Lymphoma Society.2 B.A.S. and A.S. contributed equally to this work.3 Address correspondence and reprint requests to Dr. Avinash Bhandoola, Departmentof Pathology and Laboratory Medicine, 264/266 John Morgan Building, 37th andHamilton Walk, University of Pennsylvania School of Medicine, Philadelphia,PA 19104. E-mail address: bhandooa@mail.med.upenn.edu4 Abbreviations used in this paper: BM, bone marrow; CLP, common lymphoid pro-genitor; DN, CD4�CD8� double negative; DP, CD4�CD8� double positive; ELP,early lymphoid progenitor; ETP, early T lineage progenitor; Flt3, fms-like tyrosinekinase receptor-3; Flt3L, Flt3 ligand; HSC, hemopoietic stem cell; Lin, lineageAg; MPP, multipotent progenitor; Sca-1, stem cell Ag-1; WT, wild type; LSK,Lin�Sca-1highc-Kithigh.

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modest reduction in thymic cellularity, but a requirement for Flt3and Flt3 ligand (Flt3L) was revealed in competitive-mixed BMchimeras (35, 39). The importance of Flt3 signaling was most ev-ident at the early stages of T lymphopoiesis (35). These resultssuggest that Flt3 and Flt3L may be important for the generation ofthymus-settling cells.

In this study, we inquired whether thymus settling is selective.We found that HSC injected into the blood of unirradiated micewere unable to settle within the thymus, whereas downstream MPPand CLP both rapidly generated T lineage cells following i.v.transfer. This selectivity correlated with the expression of CCR9by a subset of MPP and CLP but not HSC. Furthermore, CCR9expression was important for thymus settling. We next found thatFlt3 signaling was required for the generation of CCR9� progen-itors, including early lymphoid progenitors (ELP) (40). In the ab-sence of prethymic Flt3 signaling, thymus settling by i.v. injectedprogenitors was impaired. We conclude that thymus settling isselective and is regulated by Flt3 signaling and consequent gen-eration of CCR9-expressing ELP.

Materials and MethodsMice

C57BL/6 (B6) and B6.Ly5.2 (B6.Ly5SJL) mice were purchased from theNational Cancer Institute animal facility or Taconic Laboratories. Flt3l�/�

mice were purchased from Taconic Laboratories, and Il7ra�/� mice werepurchased from The Jackson Laboratory. Flt3�/� mice were obtained fromI. Lemischka (Princeton University, Princeton, NJ). NG-BAC mice (41)were obtained from M. Nussenzweig (Rockefeller University, New York,NY) and crossed with Flt3l�/� mice. We previously generated Ccr9�/�

mice (29). All mice were backcrossed at least four generations onto the B6background. Mice used as donors or for analysis were females of 4–9 wkof age. Recipient mice were all 4.5-wk-old females. All live animal ex-periments were performed according to protocols approved by the Office ofRegulatory Affairs of the University of Pennsylvania in accordance withguidelines set forth by the National Institutes of Health.

Cell preparations, flow cytometry, and cell sorting

BM isolated from both femurs and tibias was treated with ACK lysis buffer(Cambrex) to remove RBC. Thymocytes were prepared as a single-cellsuspension. To enrich for early progenitors, CD4- and CD8-expressingthymocytes were depleted using subsaturating concentrations of anti-CD4(GK1.5) and anti-CD8� (53.6-7), followed by removal of Ab-coated cellswith magnetic beads conjugated to goat anti-rat IgG (Polysciences).

Cell preparations were stained with optimal dilutions of Ab. Abs in thelineage mixture included anti-B220 (RA3-6B2), anti-CD19 (1D3), anti-CD11b (M1/70), anti-Gr-1 (8C5), anti-CD11c (HL3), anti-NK1.1 (PK136),anti-Ter119 and anti-CD3 (2C11), anti-CD8� (53-6.7), anti-CD8� (53-5.8), anti-TCR� (H57), and anti-TCR� (GL-3). Additional Abs used in-cluded anti-Ly5B6 (104), anti-Ly5SJL (A20), anti-stem cell Ag-1 (Sca-1)(E13-161.7), anti-c-Kit (2B8), anti-Flt3 (A2F10.1), anti-IL-7R� (A7R34),anti-CD4 (RM4-5), anti-CD25 (PC61), and anti-CCR9 (242503). All Abswere directly conjugated to FITC, PE, PE-Cy5.5, PE-Cy7, allophycocya-nin, allophycocyanin-Cy7, or biotin and purchased from BD Pharmingenor eBioscience, with the exceptions of anti-Sca-1 PE-Cy5.5 (Caltag Lab-oratories) and anti-CCR9 (R&D Systems). Biotinylated Abs were revealedwith streptavidin PE-Texas Red (Caltag Laboratories) or streptavidin pa-cific blue (Molecular Probes).

For progenitor sorts, BM from 10 mice (6 � 108 cells) was used tocollect 5 � 104 HSC, 105 MPP, and 105 CLP. Sort gates are indicated inFig. 1B. For experiments studying MPP subsets, Flt3low, Fltmed and Flt3high

MPP were sorted as indicated in Fig. 7A, top panel. Cells were sortedon the FACSAria (BD Biosciences) or analyzed on the LSR-II (BDBiosciences). Cell suspensions were pretreated with 4�,6�-diamidino-2-phenylindole for dead cell exclusion. Doublets were excluded usingforward side scatter-height vs forward side scatter-width and side scat-ter-height vs side scatter-width parameters. Data were analyzed usingFlowJo (Tree Star).

Intravenous and intrathymic transfers

Unfractionated BM (5 � 107 cells) or freshly sorted progenitors from B6BM (5 � 104 cells) were injected i.v., by the retro-orbital route, into un-manipulated B6.Ly5SJL recipients. For experiments examining the effects

of gamma irradiation, recipient mice were given 500 rad of irradiation 4–6h before i.v. injection. To prevent rejection, in transfers of Ccr9�/� BM i.v.into unirradiated recipients, some recipients were treated with anti-CD4(GK1.5), 0.5 mg i.v. at the day before BM transfer and once a week there-after. Effective depletion of CD4 splenocytes was confirmed at the time ofanalysis. Equivalent chimerism between Ccr9�/� and wild-type (WT) cellsin the BM of recipient mice further confirmed that rejection had not oc-curred. Control recipients were either administered anti-CD4 or PBS alone,with no difference in thymic or BM engraftment. Intrathymic injectionswere done as described previously (2). Unfractionated BM (1 � 106 cells)or freshly sorted progenitors from B6 BM (2000 cells) were injected in-trathymically into unirradiated anesthetized B6.Ly5SJL recipients. Thenumber of donor-derived CD4�CD8� double-positive (DP) thymocytes,following i.v. or intrathymic transfers, was calculated by multiplying thetotal number of thymocytes with the frequency of donor DP thymocytesdetermined by flow cytometry. The number of donor-derived early T lin-eage progenitors (ETP) or CD4�CD8� double-negative (DN)3 thymocyteswere determined by multiplying (total thymic cellularity) � (frequency ofCD4�CD8� cells) � (frequency of ETP or DN3 within the CD4�CD8�-depleted fraction of thymocytes). To generate mixed BM chimeras, recip-ient mice were irradiated with 900 rad and then injected i.v. with a mixtureof 1–2 � 105 Ly5B6 BM cells from WT or knockout mice and 2 � 105

B6.Ly5SJL BM cells.

Real-time RT-PCR

RNA was isolated from sorted cells using the RNEasy kit (Qiagen), andcDNA was prepared with the Superscript II kit (Invitrogen Life Technol-ogies). Real-time RT-PCR was performed with TaqMan Universal PCRMaster Mix (Applied Biosystems) and analyzed on an ABI Prism 7900(Applied Biosystems). Primer and probe combinations were purchasedfrom Applied Biosystems to assess Ccr9 (Mm02528165_s1), Rag2(Mm00501300_m1), and Tdt (Mm00493500_m1) expression. The primersfor Hprt were 5�-CTCCTCAGACCGCTTTTTGC-3� and 5�-TAACCTGGTTCATCATCGCTAATC-3�, and the probe sequence was VIC-CCGTCATGCCGACCCGCAG-TAMRA. Relative expression levels were nor-malized with Hprt and calculated using the 2���CT method.

ResultsSelectivity of thymus settling

The ability of BM hemopoietic progenitors to settle within thethymus has been evaluated previously using mainly irradiated re-cipient mice (4, 7, 8, 42). The advantage of irradiation is that itdepletes most host-type hemopoietic cells, vacating hemopoieticniches in the BM and thymus, and allows for efficient generationof donor-derived cells. However, hemopoiesis following irradia-tion is not physiological. Irradiation causes extramedullary hemo-poiesis in the spleen and other sites (43, 44), increased levels ofcytokines in the thymus and circulation (45), vascular damage, andthe loss of normal competitor cells. Therefore, we attempted toevaluate thymus settling in unirradiated, unmanipulated WT adultrecipient mice. For these experiments, we used 4.5-wk-old recip-ients because this is an age at which the thymus has been reportedto be most receptive to settling by progenitors from blood (5, 46).We found that donor progeny could be detected in the thymus afterinjecting 5 � 107 BM cells into the bloodstream. Donor-derivedDP thymocytes were first seen at 2 wk following i.v. transfer,whereas large numbers of DP thymocytes were first generated at3 wk (Fig. 1A).

We next fractionated BM into progenitor subsets to identifywhich BM progenitors, when placed in the blood of unirradiatedrecipient mice, were competent to generate DP thymocytes at 3 wk(Fig. 1B). Candidates included HSC, MPP, and CLP, each ofwhich has T lineage potential in irradiated recipient mice (11–14,47). HSC can be identified in the BM by their lack of maturelineage Ag (Lin) markers, high-surface expression of Sca-1, andthe cytokine receptor c-Kit (Lin�Sca-1highc-Kithigh; LSK), butlack of surface Flt3 expression (12, 13, 48–50); MPP are pheno-typically LSKFlt3� (12, 13); and CLP are Lin�IL-7R��Flt3�c-KitlowSca-1low (14, 38). HSC, MPP, or CLP, as well as the “other”remaining Lin�c-Kitneg/low cells, were sorted from the BM of 10

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B6 donors and 5 � 104 cells from each population were injectedi.v. into one unirradiated B6.Ly5SJL-congenic recipient. From eachsort, sufficient numbers of progenitors were obtained to inject onerecipient with HSC, two recipients with MPP, and two recipientswith CLP. Three weeks after i.v. injection of purified progenitorsinto mice, recipient thymi were analyzed for donor-derived DPthymocytes (Fig. 1, C and D). In all experiments, both MPP- andCLP generated donor thymocytes, most of which were DP, al-

though MPP were 50-fold more efficient than CLP at this timepoint. Indeed, DP progeny were easily detected from 104 MPP(data not shown). For i.v. delivered MPP ranging from 104 to 105

cells, we found the T lineage chimerism was proportional to thenumber of cells injected (data not shown). Apart from MPP andCLP, no other BM populations gave rise to T lineage progeny inthis assay, including the remaining Lin�c-Kitneg/low fraction ofBM or the Lin�c-Kitneg/lowB220� population, which containsCLP-2 progenitors (15, 16) (Fig. 1D). This suggests that, althoughCLP-2 cells can efficiently settle within the thymus (31), they areinefficient progenitors of T cells (15, 16). Alternatively, these neg-ative results may be due to the low frequency of T competentprogenitors within the donor population. Despite the potent T lin-eage potential of HSC, this population also failed to produce anyDP thymocytes. These data indicate that the early 3-wk wave of T cellproduction, following the injection of unfractionated BM i.v. into un-irradiated recipients, is predominantly driven by the MPP subset.

These results differ from a similar experiment using irradiatedrecipient mice. Three weeks after i.v. transfers into irradiated re-cipients, both HSC and MPP generated donor-derived DP thymo-cytes, as expected from past work (data not shown; Refs. 12 and47). These results indicate that although HSC have T lineage po-tential, as revealed using irradiated recipients, HSC do not rapidlygenerate T cells in unirradiated recipient mice.

We next analyzed the kinetics with which HSC, MPP, and CLPgenerate early thymic progenitors during the first 4 wk following i.v.transfer (Fig. 2, A and C). Within the thymus, ETP are phenotypicallyLinneg/lowc-KithighCD25� (47). This is followed by the Linneg/low

c-KithighCD25� DN2 stage and then the Lin�c-Kitneg/lowCD25�

DN3 stage (51, 52). Progenitors next down-regulate CD25 andup-regulate CD4 and CD8 to generate DP thymocytes (10, 53).MPP injected i.v. progressed through this conventional pathway(Fig. 2, A and C). Small numbers of Linneg/low donor progeny werefirst detected 8 days after transfer, all of which were c-Kithigh

CD25� ETP. By day 15, most progenitors had up-regulated CD25expression and differentiated into DN3 cells. Large numbers of DPthymocytes were first seen at day 22, which is consistent with thekinetics of unfractionated BM (Fig. 1A), which is dominated bythis MPP subset. Similar kinetics were observed following directintrathymic transfer of 2000 MPP (Fig. 2, B and D).

CLP injected i.v. had accelerated T lineage kinetics but gener-ated fewer peak progeny for a shorter period of time than MPP(Fig. 2, A and C), as expected from past work with irradiatedrecipient mice (14, 40, 47, 54). Interestingly, although CLP arephenotypically c-Kitlow, they up-regulated c-Kit to generatec-Kithigh ETP and DN2 thymocytes by day 8 after transfer. Sig-nificant numbers of DN3 cells were also present at this time. Byday 15, all c-Kithigh cells had disappeared, and DN3 and DP thy-mocyte cellularities reached their peak. The peak number of DPthymocytes derived from CLP was �10-fold lower than that ofMPP 1 wk later. By day 22, no Linneg/low progeny from CLPremained, and the number of DP thymocytes was declining. Sim-ilar kinetics were observed following direct intrathymic transfer ofCLP (Fig. 2, B and D). These results indicate that both MPP andCLP, injected into unirradiated mice, can generate T cells through theconventional pathway, but with different kinetics and efficiencies.

For both MPP and CLP, intrathymic transfers were always moreefficient than i.v. transfers (Fig. 2). One likely reason is that onlya small fraction of i.v. injected progenitors circulates through thethymus and settles within it. Intravenously injected progenitorscirculate for a very short period of time, and the thymus is a smallorgan estimated to receive only 1/400 of the cardiac output (17,18). Therefore, i.v. injected progenitors may lodge in other sites,including the BM, instead of the thymus. HSC, MPP, and CLP

FIGURE 1. Differential ability of BM progenitors to generate DP thy-mocytes following i.v. transfer. A, Kinetics of intrathymic T lineage dif-ferentiation following i.v. transfer of 5 � 107 whole BM cells into unir-radiated mice. Shown are the means of three to five mice at each timepoint � SEM. B, BM cells were stained to identify HSC, MPP, and CLP.The remaining Lin�c-Kitneg/low population was designated “other.” C,Ability of BM progenitor populations to generate DP thymocytes at 3 wkfollowing i.v. transfer into unirradiated recipients. Shown are representa-tive FACS plots of thymi from recipient mice receiving the indicated donorpopulation. D, Number of donor-derived DP thymocytes at 3 wk followingi.v. injection of sorted HSC, MPP, CLP, “other” Lin�c-Kitneg/low cells, orB220�Lin�c-Kitneg/low cells into unirradiated recipient mice. Anti-B220was excluded only from the Lin mixture used to identify the B220�

subset containing CLP-2. Shown are the means of three to five mice pergroup � SEM.

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populations each gave rise to donor-derived cells in the BM at eachtime point analyzed (data not shown). An additional possibility isthat the MPP and CLP populations may be heterogeneous withonly a small fraction of cells competent to settle within the thymus.

In contrast to MPP and CLP, HSC injected i.v. failed to give riseto any Linneg/low thymocytes during the first 4 wk after transfer(Fig. 2, A and C). This indicates that the inability of HSC to gen-erate DP thymocytes in unirradiated recipients by 3 wk (Fig. 1, Cand D) is not due to a delay in intrathymic differentiation. Instead,HSC placed in blood do not establish in the thymus of unirradiatedrecipients during this time period. This suggests that HSC eithercannot directly settle within the thymus or alternatively that HSCthat settle cannot compete with endogenous cells in a nonirradiatedthymus. To differentiate between these possibilities, we analyzed theability of HSC to generate T lineage progeny following intrathymictransfer into unirradiated recipient mice (Fig. 2, B and D). Intrathymictransfers differ from i.v. transfers in that they bypass a requirement forthymic entry. HSC injected intrathymically generated large numbersof T lineage progeny, reaching a similar peak number of donor-de-rived DP thymocytes as MPP at 4 wk after transfer. These resultsshow that HSC, when placed within the thymus, can successfullycompete with endogenous progenitors to generate T cells. Therefore,the absence of any T lineage progeny from HSC within the thymusduring the first 4 wk following i.v. transfer demonstrates that HSCcannot settle within the thymus.

The i.v. injection of HSC into unirradiated recipients alwaysresulted in HSC engraftment of the BM (data not shown). There-fore, we expected that HSC injected i.v. would indirectly give riseto T lineage progeny in the thymus at later time points by firstproducing progenitors in the BM capable of directly colonizing the

thymus. Indeed, donor-derived DP thymocytes were detected 8 wkafter i.v. transfer of HSC (data not shown). These results indicatethat HSC generate T cells with a significant delay relative to MPPand CLP, consistent with indirect thymic colonization from HSCvia downstream progenitors.

Taken together, these experiments demonstrate that thymus set-tling is selective. HSC cannot directly settle within the adult thy-mus, whereas downstream progenitors can. Therefore, the abilityof progenitors to settle within the thymus must be acquired in theBM by progenitors downstream of HSC, at the MPP or CLP stage.

CCR9 expression by BM progenitors

We next investigated the molecular basis for selective thymus set-tling. One molecule proposed to play a role in thymus settling isthe chemokine receptor CCR9 (29–33). Furthermore, a recentanalysis of Ccr9 reporter mice demonstrated that a subset of BMLSK progenitors and CLP express CCR9 (34). Therefore, weasked whether CCR9 is selectively expressed by MPP and CLP,which can rapidly generate T lineage cells following i.v. transfer,but not HSC, which cannot settle within the thymus. We found thatHSC lacked surface expression of CCR9, whereas a subset of bothMPP and CLP expressed surface CCR9 (Fig. 3A). Ccr9 mRNAwas also significantly increased in sorted MPP and CLP comparedwith HSC (Fig. 3B). Altogether, the expression pattern of CCR9 isconsistent with the cellular specificity of thymus settling.

Role of CCR9 in thymus settling

We next evaluated the role of CCR9 in early T cell developmentby generating competitive BM chimeras using a 1:1 mixture ofeither CCR9�/� or control B6 BM (Ly5B6) and host-type BM

FIGURE 2. Kinetics of T lineage development following i.v. or intrathymic transfer of HSC, MPP, and CLP. HSC, MPP, and CLP from B6 donor micewere injected either i.v. (5 � 104 cells) or intrathymically (2000 cells) into unirradiated B6.Ly5SJL recipients. FACS plots are gated on donor-derivedLinneg/low thymocytes at the indicated time after i.v. transfer (A) or intrathymic transfer (B). The absolute number of donor-derived ETP, DN3, or DPthymocytes is plotted as a function of time following i.v. (C) or intrathymic transfer (D) of the indicated progenitor populations. Numbers at each time pointfor each progenitor population are the mean of two to seven independent experiments � SEM.

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(Ly5SJL) to reconstitute irradiated mice. At 10 wk after transfer,the BM and thymi of these mice were analyzed for donor-derivedcells (Fig. 4A). CCR9�/� progenitors efficiently engrafted in theBM and generated HSC and MPP. However, the chimerism of allthymic subsets derived from CCR9�/� BM, beginning at the ETPstage, was significantly less than the chimerism in the BM HSCcompartment. These data extend previous work (29, 30), indicatingan early requirement for CCR9 in mixed BM chimeras, and indi-cate that CCR9 confers a competitive advantage at or before theETP stage of T cell development.

To determine whether CCR9 is important for efficient thymussettling or for the intrathymic development of ETP, we injectedBM from either WT or CCR9�/� mice i.v. or intrathymically intounirradiated recipients (Fig. 4, B and C). We found that CCR9�/�

BM injected i.v. was significantly less efficient at generating ETPthan control BM (Fig. 4C, left panel). This difference was evidentby day 8 after transfer (Fig. 4B, left panels), a time at which bothCLP and MPP contribute to the generation of ETP (Fig. 2).CCR9�/� BM continued to produce less ETP chimerism than WTBM at day 22 (Fig. 4B, right panels), when the generation of ETPis driven by MPP (Fig. 2). This defect was specific to the thymus.LSK progenitors from CCR9�/� BM had no defect in engraftingthe BM (Fig. 4C, middle panel). The reduction in donor-derivedETP was not due to an intrathymic defect because there was nosignificant difference in the ability of WT or CCR9�/� BM in-jected intrathymically to generate ETP (Fig. 4C, right panel).These results indicate that CCR9 is important for efficient thymussettling. However, CCR9 expression was not absolutely required,as some T lineage progeny were detected in the thymi of micereceiving CCR9�/� BM, although considerably reduced in num-ber relative to WT controls.

If CCR9 is important for thymus settling, why do the thymi ofCCR9�/� mice have essentially normal cellularity? To gain in-sight into this issue, we analyzed the earliest progenitor subsets inCCR9�/� thymi (Fig. 4D). We found a reduced number of ETPand DN2 cells in CCR9�/� thymi compared with aged-match WTcontrols, whereas the number of DN3 thymocytes was normal.This is similar to the phenotype of P-selectin glycoprotein ligand-1�/� mice, which are also thought to have a thymus settling defect(24). These data suggest that under noncompetitive situations, thethymus may be able to compensate for a reduction in thymus set-tling by the DN3 stage. Similar to what was seen in the case of

P-selectin (24), the requirement for CCR9 in thymus settling wasmost clearly revealed in competitive assays (Fig. 4, A–C). Takentogether, our results demonstrate a role of CCR9 in efficient thy-mus settling.

Flt3 signaling requirement for generation of CCR9� ELP

We next investigated the cytokine requirements for the generationof CCR9-expressing progenitors in early hemopoiesis. For lym-phocyte development, the cytokine receptors c-Kit, Flt3, andIL-7R are each known to be important at stages before lineagecommitment (55). Of these, only Flt3 is expressed by MPP and

FIGURE 3. CCR9 expression by BM progenitors. A, BM was stained toidentify CCR9 expression on gated HSC, MPP, and CLP. B, Ccr9 andHprt1 gene expression determined by real-time RT-PCR, using RNA de-rived from sorted HSC, MPP, or CLP. Shown is the mean relative Ccr9/Hprt1 ratio from three separate sorts � SEM; p values were determined byStudent’s t test.

FIGURE 4. Requirement for CCR9 in thymus settling. A, Mixed BMchimeras were generated by combining BM from either CCR9�/� or B6mice with BM from B6.Ly5SJL mice. At 10 wk after transfer, BM andthymocyte populations were analyzed for Ly5B6 chimerism. The mean chi-merism from 13 CCR9�/� or 10 WT chimeras � SEM is shown. Asterisksindicate p � 0.05 (Student’s paired t test) relative to HSC chimerism. B,FACS analysis of thymi at the indicated time after i.v. transfers of WT orCCR9�/� BM into unirradiated B6.Ly5SJL-recipient mice. Plots are gatedon Lin� thymocytes. C, Number of donor-derived ETP 3 wk following thetransfer of either WT or CCR9�/� BM i.v. (left panel) or intrathymically(right panel) into unirradiated recipient mice. The percent donor chimerismwithin the BM LSK population, following i.v. transfer, was also deter-mined (middle panel). Shown is the mean of three to five separate exper-iments � SEM; p values were determined by Student’s t test. D, Analysisof CCR9�/� thymi. The numbers of ETP, DN2, and DN3 thymocytes perthymus from WT or CCR9�/� thymi are shown. Results are the mean ofthree mice per group � SEM; p values were determined by Student’s t test.

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CLP but not HSC (12, 13, 38), corresponding with the expressionpattern of CCR9. Flt3 is first expressed at the MPP stage of he-mopoiesis and is important for the generation of CLP from MPP(38). Furthermore, roles for Flt3 in early B lineage differentiationand thymocyte production have been revealed using competitive-mixed BM chimeras (39). Therefore, we asked whether Flt3 sig-naling was required for the generation of CCR9-expressing BMprogenitors. Within the BM LSK compartment of WT mice, CCR9was selectively expressed by a subset of Flt3� MPP. However, inFlt3L�/� mice, BM LSK cells lacked CCR9 surface expression(Fig. 5A). The same result was found in Flt3�/� mice (data notshown). This result was specific to Flt3 signaling because micelacking the �-chain of the IL-7 cytokine receptor had no defect inCCR9 expression (Fig. 5A). Additionally, although Notch signal-ing is critical for regulating T cell development (36, 56, 57), block-ing Notch signaling in BM progenitors with dominant-negativemastermind like-1 (35) had no effect on CCR9 expression (data notshown).

Flt3L�/� mice also had less MPP, evident by the reduced fre-quency of LSK cells that expressed Flt3 (Fig. 5A). This suggeststhat Flt3L�/� mice might be missing a subset of MPP that ex-presses CCR9. One subset of MPP that is known to possess potent

T lineage potential is the ELP (40). ELP are the earliest progenitorsin hemopoiesis to be lymphoid specified—expressing lymphocyte-specific genes, including Rag1, Rag2, and Tdt (40). Therefore, weasked whether CCR9 expression by MPP correlates with lymphoidspecification. We found that CCR9 expression within the MPPpopulation was restricted to a subset of ELP, identified as GFP�

MPP in the BM of NG-BAC reporter mice, which express GFPunder the control of Rag2 cis-elements (Fig. 5B). This suggests acommon program for lymphoid specification and CCR9 expres-sion within the BM MPP population. This differentiation programprecedes lineage commitment, as both ELP and CCR9� LSK cellshave been shown to be multipotent (34, 40). Additionally, bothELP and CCR9� LSK cells are known to circulate in blood andthus have physiological access to the thymus (18, 34).

Since CCR9 expression within the BM LSK population wasrestricted to the lymphoid specified ELP subset, we next askedwhether Flt3 signaling was important for the generation of ELP inaddition to CCR9 expression. We found that Flt3L�/� NG-BACRag2-GFP reporter mice lacked the GFP� ELP subset of MPP(Fig. 5C). Furthermore, the reduced MPP frequency in Flt3L�/�

mice could be accounted for by the lack of ELP. These data dem-onstrate that the defect in lymphocyte development, in the absenceof Flt3 signaling (35, 38, 39), is evident at an earlier stage oflymphopoiesis than previously appreciated. Consistent with theseresults, the mRNA expression of Ccr9, as well as the lymphoid-specific genes Rag2 and Tdt, were significantly reduced withinLSK cells from Flt3L�/� BM compared with WT LSK cells (Fig.5D). Therefore, Flt3 signaling was required for lymphoid specifi-cation and generation of ELP, which is the only subset of MPP thatexpresses CCR9.

Flt3 requirement for thymus settling

Based on the lack of ELP, CLP (38), and CCR9 expression in theabsence of Flt3 signaling, we predicted that Flt3 signaling wouldbe required for the generation of efficient thymus settling progen-itors. To test this hypothesis, we first analyzed at what stages of Tlineage development Flt3 is important by generating mixed BMchimeras using BM from either Flt3�/� or control Flt3�/� mice(Ly5B6) mixed with WT BM (Ly5SJL) and injected into irradiatedLy5SJL hosts (Fig. 6A). At 10 wk after transfer, the BM and thymiof these mice were analyzed for donor (Ly5B6)-derived cells.Whereas Flt3�/� progenitors efficiently engrafted in the BM andgenerated LSK cells, Flt3�/� progenitors had a competitive dis-advantage in the thymus, which was evident at the earliest ETPstage. There was no further requirement for Flt3 downstream ofETP. The chimerism beyond this stage remained stable, which isconsistent with the lack of Flt3 expression by thymocytes down-stream of ETP (35). This early requirement for Flt3 in T cell de-velopment was most clearly revealed under competitive condi-tions. These results demonstrate a requirement for Flt3 signaling ator before the generation of ETP.

Flt3 signaling may be important either prethymically and/or in-trathymically. To determine whether Flt3 signaling was required inthe BM for generation of efficient thymus-settling progenitors, weassayed the ability of progenitors from Flt3L-deficient BM, whichhave developed in the absence of Flt3 signaling, to settle within thethymus (Fig. 6B). Such Flt3L�/� progenitors are competent toreceive Flt3 signals when adoptively transferred into Flt3L-suffi-cient mice. We injected whole BM from either Flt3L�/� or WT B6mice i.v. into unirradiated WT B6.Ly5SJL recipients. Three weekslater, the recipient thymi were analyzed for donor-derived ETP(Fig. 6B, left). BM from Flt3L�/� mice gave rise to significantlyfewer ETP compared with WT BM. All downstream progenitorswithin the thymus were similarly reduced (data not shown). This

FIGURE 5. Flt3 requirement for the generation of CCR9-expressingprogenitors. A, The expression of Flt3 and CCR9 is shown on LSK-gatedprogenitors from the BM of WT, Flt3L�/�, or IL-7R��/� mice. B, Theexpression of Flt3 and CCR9 is shown on the GFP� subset and GFP�

(ELP) subset of MPP from BM of NG-BAC reporter mice. C, The expres-sion of Flt3 and GFP is shown on LSK-gated progenitors from the BM ofNG-BAC or NG-BAC Flt3L�/� mice. D, Mean Ccr9, Rag2, Tdt, andHprt1 gene expression determined by real-time RT-PCR, using RNA de-rived from sorted LSK progenitors from either WT or Flt3L�/� BM �SEM; p values determined by Student’s t test.

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defect was specific to the thymus. LSK progenitors from Flt3L�/�

BM had no defect in engrafting in the BM (Fig. 6B, middle). Fur-thermore, the reduction in donor-derived ETP was not due to anintrathymic defect because there was no significant difference inthe ability of WT or Flt3L�/� BM injected intrathymically to gen-erate ETP in a Flt3L-sufficient environment (Fig. 6B, right). In thesame experiment, generation of downstream DN2, DN3, and DPthymocytes was also comparable between WT and Flt3L�/� BMinjected intrathymically (data not shown). These results demon-strate that Flt3 signaling is required in the BM for the generationof efficient thymus-settling cells.

T lineage competence of MPP subsets

Our results suggest that the most efficient thymus-settling progen-itors within the MPP population are ELP, as these progenitors areabsent in the BM of Flt3L�/�-deficient mice (Fig. 5C). Therefore,we directly compared the ability of ELP and the remaining MPPsubsets to generate T cells 3 wk after i.v. transfers into unirradiatedrecipients. For these experiments, we were unable to use NG-BACdonors, as GFP can cause rejection in immunocompetent recipientmice (58). Instead, we used Flt3 expression to identify ELP. Thetop 20% of Flt3-expressing LSK progenitors (Flt3high MPP), alsoreferred to as lymphoid-primed MPP (59, 60), were enriched forELP, whereas the Flt3low and Flt3med fractions of MPP containedfewer ELP (Fig. 7A). Whereas the Flt3med fraction did containsome GFP� cells, these appeared dull for GFP expression com-pared with the Flt3high cells. When purified Flt3high, Flt3med, andFlt3low MPP were injected i.v. into unirradiated recipients, Flt3high

MPP gave rise to �10-fold more DP thymocytes at 3 wk thaneither of the other subsets (Fig. 7B). We conclude that the early3-wk wave of T cell development, following i.v. transfer into un-irradiated recipients, is driven by the ELP subset of MPP.

Our findings indicate that physiological thymus-settling progen-itors express Rag2, Flt3, and CCR9. Therefore, we expected thatthe earliest progenitors in the thymus would also express these

markers. Indeed, we found that ETP are entirely GFP� in NG-BAC Rag2 reporter mice (Fig. 7C). This differs from a previousresult (54) using a different reporter mouse that had GFP knockedinto the Rag1 locus, indicating that these two reporter mousestrains are not identical. Recently, a subset of ETP has been shownto express high levels of GFP in a Ccr9-GFP reporter mouse (34)and a parallel study identified the Flt3� subset of ETP (35). Bothstudies concluded that their populations contained the earliest pro-genitors identified in the thymus (34–37). However, the overlapbetween these two populations has not been examined previously.Therefore, we analyzed Ccr9 gene expression of sorted ETP Flt3�

cells compared with ETP Flt3low cells and downstream DN2 thy-mocytes (Fig. 7D). We found that Ccr9 was highly expressed byETP Flt3� compared with ETP Flt3low and DN2 thymocytes. Thisdemonstrates that Flt3� ETP and Ccr9-GFPhigh ETP are overlappingpopulations, which is concordant with the idea that progenitors enterthe thymus expressing Flt3 and CCR9 in addition to Rag2.

DiscussionIn this study, we investigated whether thymus settling is selective.We found that, whereas HSC have potent T lineage potential, re-vealed by direct intrathymic injection, they fail to settle within the

FIGURE 6. Flt3 signaling requirement before thymus settling. A, MixedBM chimeras were generated by combining BM from either Flt3�/� orFlt3�/� mice with B6.Ly5SJL BM. At 10 wk after transfer, BM and thy-mocyte populations were analyzed for Ly5B6 chimerism. The mean chi-merism from four Flt3L�/� or four Flt3�/� chimeras � SEM is shown.Asterisks indicate p � 0.05 (Student’s paired t test) relative to HSC chi-merism. B, Number of donor-derived ETP 3 wk following the transfer ofeither WT or Flt3L�/� BM i.v. (left panel) or intrathymically (right panel)into unirradiated recipient mice. The percent donor chimerism within theBM LSK population, following i.v. transfer, was also determined (middlepanel). Shown is the mean of three to five separate experiments � SEM,and p values were determined by Student’s t test.

FIGURE 7. Flt3high ELP are competent T lineage progenitors. A, GFPexpression on Flt3low, Flt3med, and Flt3high MPP from NG-BAC BM isshown. B, Number of donor-derived DP thymocytes at 3 wk following i.v.transfer of 5 � 104 Flt3low, Flt3med, and Flt3high MPP into unirradiatedrecipient mice. Shown is the mean of three mice per group � SEM. Thenumber of DP thymocytes derived from Flt3high MPP was significantlydifferent (p � 0.05, Student’s t test) from the number of DP thymocytesderived from Flt3low or Flt3med MPP. C, GFP expression on ETP fromNG-BAC thymi (filled histogram) or B6 control thymi (dashed histogram).D, Ccr9/Hprt1 gene expression was determined by real-time RT-PCR fromRNA derived from sorted Flt3� ETP, Flt3low ETP, and DN2 thymocytes.Shown is the mean relative expression ratio � SEM.

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thymus when placed in the blood of unirradiated recipient mice.Downstream MPP and CLP both rapidly generate T cells follow-ing i.v. transfer. This selectivity corresponded with the expressionof the chemokine receptor CCR9 by a subset of MPP and CLP butnot HSC. We demonstrated that CCR9 is important for efficientthymus settling, indicating that CCR9 is one of the molecules thatregulate the cellular specificity of thymus settling. Additionally,we found that CCR9 was selectively expressed by ELP but notother MPP subsets. Furthermore, Flt3 expression and signalingwas required for the generation of ELP. Consistent with the role ofFlt3 in the generation of thymus-setting cells, progenitors fromFlt3L�/� BM lacked the ability to efficiently settle within the thy-mus, whereas sorted Flt3high MPP, enriched for ELP, were efficientprogenitors of T cells following i.v. transfer into unirradiated re-cipients. We conclude that thymus settling is selective. We furtherconclude that progenitors downstream of HSC acquire competenceto settle within the thymus through expression of homing mole-cules that include CCR9.

Whether HSC directly settle the adult thymus was previouslyunresolved. HSC have potent T lineage potential and are present inblood, thus having access to the thymus (17, 18). However, self-renewal is the hallmark of stem cell activity, and no self-renewingprogenitors have been identified in the thymus (3, 4). Furthermore,when BM was injected i.v. into irradiated mice, donor-derivedcells in the thymus lacked the ability to self-renew, indicating thatHSC are either excluded from the thymus or that the thymic en-vironment induces the rapid loss of self-renewal from these pro-genitors (42). In this study, we found that HSC injected i.v. intounirradiated mice failed to generate any donor progeny within thethymus for the first 4 wk after transfer, whereas the same cellsinjected into the thymus generated large numbers of DP thymo-cytes within this timeframe. MPP were similarly efficient to HSCupon direct intrathymic injection, but unlike HSC, MPP also rap-idly and efficiently generated T cells following i.v. transfer. EvenCLP, which were inferior to MPP and HSC upon intrathymictransfer, could be clearly shown to generate donor-derived thymo-cytes following i.v. transfer. The i.v. injection of HSC did lead toBM engraftment and subsequent T cell development by 8 wk aftertransfer. These experiments establish that HSC cannot physiolog-ically settle within the thymus of adult mice. One advantage of thismay be that the development of specific T competent progenitorsdownstream of HSC in the BM allows for the regulation of thymussettling independently of development of other lineages. This maybe important in situations such as crisis hemopoiesis or in aging inwhich T lymphopoiesis is decreased relative to myeloid and ery-throid development. We conclude that thymus settling is selectiveand prethymic differentiation steps are necessary to generate phys-iological thymus-settling progenitors.

The inability of HSC to settle within the thymus was only ev-ident using unirradiated recipient mice. In irradiated recipients,HSC injected i.v. rapidly and efficiently generated donor-derivedDP thymocytes by 3 wk after transfer. It is unclear why irradiationhas this effect. One possibility is that irradiation makes the thymusreceptive to HSC settling, perhaps through vascular damage or theinduction of chemokines in the thymus that allow for HSC entry(45). A second explanation is that irradiation can lead to alternativepathways of T cell differentiation. Recent evidence indicates thatfollowing irradiation the spleen and lymph nodes become permis-sive for the development of pre-T cells, which could then settlewithin the thymus (44, 61). A third possibility is that T cell dif-ferentiation occurs normally following irradiation but is acceler-ated. HSC are normally quiescent, but following irradiation, thesecells cycle more rapidly to reconstitute the ablated hemopoieticsystem (62). This quiescence of HSC may account for the large

delay in their ability to generate thymus-settling progenitors inunirradiated recipient mice. Importantly, our findings reveal thatthe study of physiological thymus settling requires the use of un-irradiated recipient mice.

A variety of BM progenitors downstream of HSC may physio-logically settle the thymus ranging from MPP subsets to CLP andCLP-2 cells (10). The differential ability of these progenitors tocontribute to T lymphopoiesis will depend on their ability to mi-grate from the BM to the bloodstream (18), their capacity to settlewithin the thymus from the bloodstream, and their efficiency ingenerating T cells once within the thymus. MPP and CLP eachrapidly generated T lineage progeny in the thymus following i.v.transfer, whereas the remaining lineage-negative BM progenitorsdid not. MPP were also the most efficient progenitor for reconsti-tuting T lymphopoiesis in both irradiated (44) and unirradiatedmice (the present study). MPP are present in the bloodstream andthus have physiological access to the thymus (18). Furthermore,the kinetics of T lineage differentiation from MPP suggests thatsome of these cells directly enter the thymus (46) (the presentstudy). There is also evidence for a T cell developmental pathwayindependent of CLP intermediates, again supporting direct thymussettling by MPP (10, 47). Additionally, multipotent cells have beenidentified in the thymus, at the single-cell level, and this populationhas been proposed to contain thymus-settling cells or the directprogeny of thymus-settling cells (34, 37). Therefore, it is probablethat a subset of MPP can physiologically settle within the thymus.

The ability of CLP to contribute to T lymphopoiesis through theconventional pathway has been questioned recently (63). Wefound that c-Kitlow CLP, injected either i.v. or intrathymically,rapidly up-regulate c-Kit surface expression to generate c-Kithigh

ETP by day 8 after transfer. This was likely due to Notch signalingwithin the thymus (56) because Notch signals have been shown toup-regulate c-Kit expression on progenitors in vitro (63–65). Ourresults indicate that CLP can transit through the conventional path-way of T lineage differentiation. We conclude that CLP placed inblood can settle within the thymus. However, can CLP physiolog-ically enter the bloodstream? We were unable to find CLP in thebloodstream of adult mice (18). However, a new study suggeststhat low numbers of CLP-like cells may circulate (54). Therefore,CLP may directly contribute to T cell development, although theyare much less efficient T lineage progenitors than MPP.

Since thymus settling is selective and HSC cannot physiologi-cally enter the thymus, it follows that progenitors in the BM mustacquire the ability to settle within the thymus while still retainingT lineage potential. We refer to this process as the acquisition ofT lineage competence. One molecule that has been implicated inthe acquisition of T lineage competence is the chemokine receptorCCR9. We found that HSC lack CCR9 expression, whereas down-stream progenitors, including a subset of MPP and CLP, expresssurface CCR9. Therefore, CCR9 expression correlates with theability of progenitors to rapidly reconstitute the thymus. However,a functional role for CCR9 in thymus settling has been controver-sial. Recently, two studies used short-term thymus settling assaysto determine whether CCR9 is important in adult thymus settling,with conflicting results (31, 33). Furthermore, neither of thesestudies could determine whether the cells entering the thymus wererelevant T lineage progenitors. We instead investigated the devel-opment of T lineage progeny from CCR9-deficient progenitors.For these studies, we used unfractionated BM to avoid presump-tions about the identity of thymus-settling progenitors. CCR9�/�

progenitors had comparable BM engraftment to WT control BM.Additionally, the development of MPP and CLP was normal fromCCR9�/� progenitors, and MPP continued to circulate at normallevels in the blood of CCR9�/� mice (data not shown). Instead,

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CCR9 was found to be important for T lymphopoiesis first at theETP stage. This was not due to an intrathymic requirement forCCR9 because CCR9-deficient progenitors injected intrathymi-cally generated normal numbers of ETP. Instead, our results aremost compatible with a role for CCR9 in thymus settling.

Our work suggests that the acquisition of T lineage competencecan occur through the expression of CCR9, as well as other mol-ecules important for migration to the thymus. Therefore we inves-tigated the generation of CCR9-expressing progenitors. We foundthat CCR9 expression was restricted to a fraction of Flt3high pro-genitors. Furthermore, all progenitors able to rapidly reconstitutethe thymus were Flt3high. Within the MPP population, only Flt3high

ELP expressed CCR9 and Flt3 signaling was required for the gen-eration of ELP. Therefore, we predicted that Flt3L�/� BM wouldbe deficient in T competent progenitors. Indeed, we found that Flt3expression was required at or before the ETP stage of T cell de-velopment, and Flt3L�/� progenitors were defective in settling thethymus. Furthermore, ELP were the most efficient progenitors of Tcells within the MPP population. We conclude that Flt3 signalingis required for the generation of efficient thymus-settling progenitors.Consistent with this, we found that the earliest subset of ETP in thethymus expresses Flt3, Ccr9, and Rag2 (34, 35, 37).

In summary, we established that thymus settling is selective andhave uncovered two molecular determinants for this selectivity.The chemokine receptor CCR9 is important for the homing ofprogenitors to the thymus. The cytokine receptor Flt3 regulates thegeneration of T competent progenitors, including CCR9-express-ing ELP. However, other molecules, which remain to be discov-ered, may also play a role in these processes. Further character-ization of these molecular determinants will help refine ourunderstanding of thymus-settling progenitors.

AcknowledgmentsWe thank V. Zediak, M. Cancro, B. Kee, and D. Allman for critical comments;M. Velez for technical support; and R. Schretzenmair, R. Wychowanec, and H.Pletcher of the Abramson Cancer Center Flow Cytometry and Cell SortingShared Resource for technical expertise.

DisclosuresThe authors have no financial conflict of interest.

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