Selective Thymus Settling Regulated by Cytokine and Receptors Benjamin A. Schwarz, Arivazhagan Sambandam, Ivan Maillard, Benjamin C. Harman, Paul E. Love and Avinash This information is current as Bhandoola of September 27, 2021. J Immunol 2007; 178:2008-2017; ; doi: 10.4049/jimmunol.178.4.2008 http://www.jimmunol.org/content/178/4/2008 Downloaded from

References This article cites 65 articles, 27 of which you can access for free at: http://www.jimmunol.org/content/178/4/2008.full#ref-list-1 http://www.jimmunol.org/ Why The JI? Submit online.

• Rapid Reviews! 30 days* from submission to initial decision

• No Triage! Every submission reviewed by practicing scientists

• Fast Publication! 4 weeks from acceptance to publication

by guest on September 27, 2021 *average

Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts

The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2007 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Selective Thymus Settling Regulated by Cytokine and Chemokine 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 stem cells were excluded from the thymus, whereas downstream multipotent progenitors (MPP) and common lymphoid progenitors rapidly generated T cells following i.v. transfer. This cellular specificity correlated with the expression of the CCR9 by a subset of MPP and common lymphoid progenitors but not hemopoietic stem cells. Furthermore, CCR9 expression was required for efficient thymus settling. Finally, we demonstrate that a prethymic signal through the fms-like tyrosine kinase receptor-3 was required for the generation of CCR9-expressing early lymphoid progenitors, which were the most Downloaded from efficient progenitors of T cells within the MPP population. We conclude that fms-like tyrosine kinase receptor-3 signaling is required for the generation of T lineage-competent progenitors, which selectively express molecules, including CCR9, that allow them to settle within the thymus. The Journal of Immunology, 2007, 178: 2008–2017.

cells develop in the thymus (1). However, the thymus (CLP), which were originally identified as lymphoid committed

contains no long-term self-renewing progenitors. Instead, (14), and cells downstream of the CLP such as the CLP-2 (15, 16). http://www.jimmunol.org/ T lymphopoiesis throughout adult life is maintained by Of these progenitors, HSC and MPP are also known to circulate in T 4 the periodic importation of bone marrow (BM) hemopoietic pro- the blood of adult mice (17, 18). However, it is unclear whether all genitors that reach the thymus via the bloodstream (2–6). The of these cells can physiologically settle within the thymus from the number of progenitors that enter the thymus each day is estimated bloodstream or if thymus settling is selective. to be exceedingly small (6–9), precluding their direct identifica- The molecular basis for progenitor entry into the thymus is tion within the thymus. Therefore, which cells physiologically mi- poorly understood. This process is likely to be analogous to the grate from the BM to the thymus to generate T cells in adult mice homing of mature leukocytes, which involves -mediated is unknown. weak adhesion to vasculature endothelium, followed by chemo- by guest on September 27, 2021 Multiple progenitors within the BM have T lineage potential kine signaling, strong adhesion through integrins, and transmigra- (10) demonstrated experimentally by the ability to generate T cells tion (19–21). For thymus settling, both CD44 (22, 23) and P- following intrathymic injection (2). These progenitors include he- selectin (24) have been shown to be important. Recent work mopoietic stem cells (HSC), which can produce all blood lineages suggests that CCR9 may also play a role in progenitor migration to and have the ability to self-renew (11), multipotent progenitors the thymus. CCR9 is the receptor for CCL25 (25), which is highly (MPP), which can generate all hemopoietic lineages but have lost expressed by thymic stroma (26). Although CCR9Ϫ/Ϫ mice have self-renewal capacity (12, 13), common lymphoid progenitors no obvious defect in T cell development or thymic cellularity (27, 28), an early defect in T cell development has been revealed in *Department of Pathology and Laboratory Medicine, University of Pennsylvania competitive mixed BM chimeras (29, 30). Migration of CLP-2 School of Medicine, Philadelphia, PA 19104; †Division of Hematology-Oncology, University of Pennsylvania School of Medicine, Philadelphia, PA 19104; and ‡Lab- cells into the thymus has been found to be CCR9 dependent (31). oratory of Mammalian and Development, National Institute of Child Health Furthermore, blocking CCL25 results in reduced migration of pro- and Human Development, National Institutes of Health, Bethesda, MD 20892 genitors into fetal thymic lobes in vitro (32). However, this same Received for publication October 26, 2006. Accepted for publication December group reported that whereas there is a role for CCR9 in the mi- 1, 2006. gration of progenitors into the fetal thymic anlage, there was no The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance role for CCR9 in adult thymic settling (33). Therefore a require- with 18 U.S.C. Section 1734 solely to indicate this fact. ment for CCR9 in the homing of T lineage progenitor to the adult 1 This work was supported by the National Institutes of Health Grants AI059621 (to thymus remains controversial. A.B.) and T32-AI-055428 (to B.A.S.) and Damon Runyon Cancer Research Foun- Recently, an analysis of Ccr9-GFP reporter mice revealed that dation Grant DRG-102-05 (to I.M.). A.B. is the recipient of a Career Development Award from the Leukemia and Lymphoma Society. the earliest identified progenitors in the thymus express high levels 2 B.A.S. and A.S. contributed equally to this work. of GFP (34), which is consistent with the idea that progenitors that ϩ 3 Address correspondence and reprint requests to Dr. Avinash Bhandoola, Department enter the thymus are CCR9 . A similar population was identified of Pathology and Laboratory Medicine, 264/266 John Morgan Building, 37th and in the thymus using the cytokine receptor fms-like tyrosine kinase Hamilton Walk, University of Pennsylvania School of Medicine, Philadelphia, receptor-3 (Flt3) (35, 36), suggesting that thymus-settling progen- PA 19104. E-mail address: [email protected] itors may also be Flt3ϩ (37). Flt3 is expressed by multiple BM 4 Abbreviations used in this paper: BM, bone marrow; CLP, common lymphoid pro- genitor; DN, CD4ϪCD8Ϫ double negative; DP, CD4ϩCD8ϩ double positive; ELP, progenitors with T lineage potential, including MPP, CLP, and early lymphoid progenitor; ETP, early T lineage progenitor; Flt3, fms-like tyrosine CLP-2-like cells, whereas HSC are Flt3Ϫ (12, 13, 16, 38). Fur- kinase receptor-3; Flt3L, Flt3 ligand; HSC, hemopoietic stem cell; Lin, lineage thermore, Flt3 is required for efficient generation of CLP and B Ag; MPP, multipotent progenitor; Sca-1, stem cell Ag-1; WT, wild type; LSK, Ϫ Ϫ LinϪSca-1highc-Kithigh. cells (38, 39). For T cell development, Flt3 / mice have only a www.jimmunol.org The Journal of Immunology 2009 modest reduction in thymic cellularity, but a requirement for Flt3 of gamma irradiation, recipient mice were given 500 rad of irradiation 4–6 Ϫ Ϫ and Flt3 ligand (Flt3L) was revealed in competitive-mixed BM h before i.v. injection. To prevent rejection, in transfers of Ccr9 / BM i.v. chimeras (35, 39). The importance of Flt3 signaling was most ev- 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- ident at the early stages of T lymphopoiesis (35). These results after. Effective depletion of CD4 splenocytes was confirmed at the time of suggest that Flt3 and Flt3L may be important for the generation of analysis. Equivalent chimerism between Ccr9Ϫ/Ϫ and wild-type (WT) cells thymus-settling cells. in the BM of recipient mice further confirmed that rejection had not oc- In this study, we inquired whether thymus settling is selective. curred. Control recipients were either administered anti-CD4 or PBS alone, with no difference in thymic or BM engraftment. Intrathymic injections We found that HSC injected into the blood of unirradiated mice were done as described previously (2). Unfractionated BM (1 ϫ 106 cells) were unable to settle within the thymus, whereas downstream MPP or freshly sorted progenitors from B6 BM (2000 cells) were injected in- and CLP both rapidly generated T lineage cells following i.v. trathymically into unirradiated anesthetized B6.Ly5SJL recipients. The ϩ ϩ transfer. This selectivity correlated with the expression of CCR9 number of donor-derived CD4 CD8 double-positive (DP) , by a subset of MPP and CLP but not HSC. Furthermore, CCR9 following i.v. or intrathymic transfers, was calculated by multiplying the total number of thymocytes with the frequency of donor DP thymocytes expression was important for thymus settling. We next found that ϩ determined by flow cytometry. The number of donor-derived early T lin- Flt3 signaling was required for the generation of CCR9 progen- eage progenitors (ETP) or CD4ϪCD8Ϫ double-negative (DN)3 thymocytes itors, including early lymphoid progenitors (ELP) (40). In the ab- were determined by multiplying (total thymic cellularity) ϫ (frequency of Ϫ Ϫ ϩ ϩ sence of prethymic Flt3 signaling, thymus settling by i.v. injected CD4 CD8 cells) ϫ (frequency of ETP or DN3 within the CD4 CD8 - depleted fraction of thymocytes). To generate mixed BM chimeras, recip- progenitors was impaired. We conclude that thymus settling is ient mice were irradiated with 900 rad and then injected i.v. with a mixture selective and is regulated by Flt3 signaling and consequent gen- of 1–2 ϫ 105 Ly5B6 BM cells from WT or knockout mice and 2 ϫ 105 eration of CCR9-expressing ELP. B6.Ly5SJL BM cells. Downloaded from Materials and Methods Real-time RT-PCR Mice RNA was isolated from sorted cells using the RNEasy (Qiagen), and cDNA was prepared with the Superscript II kit (Invitrogen Life Technol- C57BL/6 (B6) and B6.Ly5.2 (B6.Ly5SJL) mice were purchased from the ogies). Real-time RT-PCR was performed with TaqMan Universal PCR Ϫ Ϫ National Cancer Institute animal facility or Taconic Laboratories. Flt3l / Master Mix (Applied Biosystems) and analyzed on an ABI Prism 7900 Ϫ/Ϫ mice were purchased from Taconic Laboratories, and Il7ra mice were (Applied Biosystems). Primer and probe combinations were purchased http://www.jimmunol.org/ Ϫ Ϫ purchased from The Jackson Laboratory. Flt3 / mice were obtained from from Applied Biosystems to assess Ccr9 (Mm02528165_s1), Rag2 I. Lemischka (Princeton University, Princeton, NJ). NG-BAC mice (41) (Mm00501300_m1), and Tdt (Mm00493500_m1) expression. The primers were obtained from M. Nussenzweig (Rockefeller University, New York, for Hprt were 5Ј-CTCCTCAGACCGCTTTTTGC-3Ј and 5Ј-TAACCTG Ϫ Ϫ Ϫ Ϫ NY) and crossed with Flt3l / mice. We previously generated Ccr9 / GTTCATCATCGCTAATC-3Ј, and the probe sequence was VIC-CCGT mice (29). All mice were backcrossed at least four generations onto the B6 CATGCCGACCCGCAG-TAMRA. Relative expression levels were nor- background. Mice used as donors or for analysis were females of 4–9 wk malized with Hprt and calculated using the 2Ϫ⌬⌬CT method. of age. Recipient mice were all 4.5-wk-old females. All live animal ex- periments were performed according to protocols approved by the Office of Results Regulatory Affairs of the University of Pennsylvania in accordance with Selectivity of thymus settling guidelines set forth by the National Institutes of Health. The ability of BM hemopoietic progenitors to settle within the by guest on September 27, 2021 Cell preparations, flow cytometry, and cell sorting thymus has been evaluated previously using mainly irradiated re- BM isolated from both femurs and tibias was treated with ACK lysis buffer cipient mice (4, 7, 8, 42). The advantage of irradiation is that it (Cambrex) to remove RBC. Thymocytes were prepared as a single-cell depletes most host-type hemopoietic cells, vacating hemopoietic suspension. To enrich for early progenitors, CD4- and CD8-expressing thymocytes were depleted using subsaturating concentrations of anti-CD4 niches in the BM and thymus, and allows for efficient generation (GK1.5) and anti-CD8␣ (53.6-7), followed by removal of Ab-coated cells of donor-derived cells. However, hemopoiesis following irradia- with magnetic beads conjugated to goat anti-rat IgG (Polysciences). tion is not physiological. Irradiation causes extramedullary hemo- Cell preparations were stained with optimal dilutions of Ab. Abs in the poiesis in the spleen and other sites (43, 44), increased levels of lineage mixture included anti-B220 (RA3-6B2), anti-CD19 (1D3), anti- cytokines in the thymus and circulation (45), vascular damage, and 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- the loss of normal competitor cells. Therefore, we attempted to 5.8), anti-TCR␤ (H57), and anti-TCR␥ (GL-3). Additional Abs used in- evaluate thymus settling in unirradiated, unmanipulated WT adult cluded anti-Ly5B6 (104), anti-Ly5SJL (A20), anti-stem cell Ag-1 (Sca-1) recipient mice. For these experiments, we used 4.5-wk-old recip- ␣ (E13-161.7), anti-c-Kit (2B8), anti-Flt3 (A2F10.1), anti-IL-7R (A7R34), ients because this is an age at which the thymus has been reported anti-CD4 (RM4-5), anti-CD25 (PC61), and anti-CCR9 (242503). All Abs were directly conjugated to FITC, PE, PE-Cy5.5, PE-Cy7, allophycocya- to be most receptive to settling by progenitors from blood (5, 46). nin, allophycocyanin-Cy7, or biotin and purchased from BD Pharmingen We found that donor progeny could be detected in the thymus after or eBioscience, with the exceptions of anti-Sca-1 PE-Cy5.5 (Caltag Lab- injecting 5 ϫ 107 BM cells into the bloodstream. Donor-derived oratories) and anti-CCR9 (R&D Systems). Biotinylated Abs were revealed DP thymocytes were first seen at 2 wk following i.v. transfer, with streptavidin PE-Texas Red (Caltag Laboratories) or streptavidin pa- whereas large numbers of DP thymocytes were first generated at cific blue (Molecular Probes). For progenitor sorts, BM from 10 mice (6 ϫ 108 cells) was used to 3 wk (Fig. 1A). collect 5 ϫ 104 HSC, 105 MPP, and 105 CLP. Sort gates are indicated in We next fractionated BM into progenitor subsets to identify Fig. 1B. For experiments studying MPP subsets, Flt3low, Fltmed and Flt3high which BM progenitors, when placed in the blood of unirradiated MPP were sorted as indicated in Fig. 7A, top panel. Cells were sorted recipient mice, were competent to generate DP thymocytes at 3 wk on the FACSAria (BD Biosciences) or analyzed on the LSR-II (BD Biosciences). Cell suspensions were pretreated with 4Ј,6Ј-diamidino-2- (Fig. 1B). Candidates included HSC, MPP, and CLP, each of phenylindole for dead cell exclusion. Doublets were excluded using which has T lineage potential in irradiated recipient mice (11–14, forward side scatter-height vs forward side scatter-width and side scat- 47). HSC can be identified in the BM by their lack of mature ter-height vs side scatter-width parameters. Data were analyzed using lineage Ag (Lin) markers, high-surface expression of Sca-1, and FlowJo (Tree Star). the cytokine receptor c-Kit (LinϪSca-1highc-Kithigh; LSK), but Intravenous and intrathymic transfers lack of surface Flt3 expression (12, 13, 48–50); MPP are pheno- ϩ Ϫ ␣ϩ ϩ ϫ 7 typically LSKFlt3 (12, 13); and CLP are Lin IL-7R Flt3 c- Unfractionated BM (5 10 cells) or freshly sorted progenitors from B6 low low BM (5 ϫ 104 cells) were injected i.v., by the retro-orbital route, into un- Kit Sca-1 (14, 38). HSC, MPP, or CLP, as well as the “other” Ϫ manipulated B6.Ly5SJL recipients. For experiments examining the effects remaining Lin c-Kitneg/low cells, were sorted from the BM of 10 2010 SELECTIVE THYMUS SETTLING BY LYMPHOID PROGENITORS

though MPP were 50-fold more efficient than CLP at this time point. 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 the number of cells injected (data not shown). Apart from MPP and CLP, no other BM populations gave rise to T lineage progeny in this assay, including the remaining LinϪc-Kitneg/low fraction of BM or the LinϪc-Kitneg/lowB220ϩ population, which contains CLP-2 progenitors (15, 16) (Fig. 1D). This suggests that, although CLP-2 cells can efficiently settle within the thymus (31), they are inefficient progenitors of T cells (15, 16). Alternatively, these neg- ative results may be due to the low frequency of T competent progenitors within the donor population. Despite the potent T lin- eage potential of HSC, this population also failed to produce any DP thymocytes. These data indicate that the early 3-wk wave of T cell production, 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 irradiated

recipient mice. Three weeks after i.v. transfers into irradiated re- Downloaded from cipients, both HSC and MPP generated donor-derived DP thymo- cytes, as expected from past work (data not shown; Refs. 12 and 47). These results indicate that although HSC have T lineage po- tential, as revealed using irradiated recipients, HSC do not rapidly generate T cells in unirradiated recipient mice.

We next analyzed the kinetics with which HSC, MPP, and CLP http://www.jimmunol.org/ generate early thymic progenitors during the first 4 wk following i.v. transfer (Fig. 2, A and C). Within the thymus, ETP are phenotypically Linneg/lowc-KithighCD25Ϫ (47). This is followed by the Linneg/low c-KithighCD25ϩ DN2 stage and then the LinϪc-Kitneg/lowCD25ϩ DN3 stage (51, 52). Progenitors next down-regulate CD25 and up-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 were first detected 8 days after transfer, all of which were c-Kithigh by guest on September 27, 2021 CD25Ϫ ETP. By day 15, most progenitors had up-regulated CD25 expression and differentiated into DN3 cells. Large numbers of DP thymocytes were first seen at day 22, which is consistent with the kinetics of unfractionated BM (Fig. 1A), which is dominated by this MPP subset. Similar kinetics were observed following direct intrathymic transfer of 2000 MPP (Fig. 2, B and D). FIGURE 1. Differential ability of BM progenitors to generate DP thy- CLP injected i.v. had accelerated T lineage kinetics but gener- mocytes following i.v. transfer. A, Kinetics of intrathymic T lineage dif- ated fewer peak progeny for a shorter period of time than MPP ferentiation following i.v. transfer of 5 ϫ 107 whole BM cells into unir- (Fig. 2, A and C), as expected from past work with irradiated radiated mice. Shown are the means of three to five mice at each time recipient mice (14, 40, 47, 54). Interestingly, although CLP are point Ϯ SEM. B, BM cells were stained to identify HSC, MPP, and CLP. phenotypically c-Kitlow, they up-regulated c-Kit to generate Ϫ neg/low The remaining Lin c-Kit population was designated “other.” C, c-Kithigh ETP and DN2 thymocytes by day 8 after transfer. Sig- Ability of BM progenitor populations to generate DP thymocytes at 3 wk nificant numbers of DN3 cells were also present at this time. By following i.v. transfer into unirradiated recipients. Shown are representa- high tive FACS plots of thymi from recipient mice receiving the indicated donor day 15, all c-Kit cells had disappeared, and DN3 and DP thy- population. D, Number of donor-derived DP thymocytes at 3 wk following mocyte cellularities reached their peak. The peak number of DP i.v. injection of sorted HSC, MPP, CLP, “other” LinϪc-Kitneg/low cells, or thymocytes derived from CLP was ϳ10-fold lower than that of neg/low B220ϩLinϪc-Kitneg/low cells into unirradiated recipient mice. Anti-B220 MPP 1 wk later. By day 22, no Lin progeny from CLP was excluded only from the Lin mixture used to identify the B220ϩ remained, and the number of DP thymocytes was declining. Sim- subset containing CLP-2. Shown are the means of three to five mice per ilar kinetics were observed following direct intrathymic transfer of group Ϯ SEM. CLP (Fig. 2, B and D). These results indicate that both MPP and CLP, injected into unirradiated mice, can generate T cells through the conventional pathway, but with different kinetics and efficiencies. B6 donors and 5 ϫ 104 cells from each population were injected For both MPP and CLP, intrathymic transfers were always more i.v. into one unirradiated B6.Ly5SJL-congenic recipient. From each efficient than i.v. transfers (Fig. 2). One likely reason is that only sort, sufficient numbers of progenitors were obtained to inject one a small fraction of i.v. injected progenitors circulates through the recipient with HSC, two recipients with MPP, and two recipients thymus and settles within it. Intravenously injected progenitors with CLP. Three weeks after i.v. injection of purified progenitors circulate for a very short period of time, and the thymus is a small into mice, recipient thymi were analyzed for donor-derived DP organ estimated to receive only 1/400 of the cardiac output (17, thymocytes (Fig. 1, C and D). In all experiments, both MPP- and 18). Therefore, i.v. injected progenitors may lodge in other sites, CLP generated donor thymocytes, most of which were DP, al- including the BM, instead of the thymus. HSC, MPP, and CLP The Journal of Immunology 2011 Downloaded from http://www.jimmunol.org/

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 mice were injected either i.v. (5 ϫ 104 cells) or intrathymically (2000 cells) into unirradiated B6.Ly5SJL recipients. FACS plots are gated on donor-derived Linneg/low thymocytes at the indicated time after i.v. transfer (A) or intrathymic transfer (B). The absolute number of donor-derived ETP, DN3, or DP thymocytes is plotted as a function of time following i.v. (C) or intrathymic transfer (D) of the indicated progenitor populations. Numbers at each time point for each progenitor population are the mean of two to seven independent experiments Ϯ SEM. by guest on September 27, 2021 populations each gave rise to donor-derived cells in the BM at each thymus. Indeed, donor-derived DP thymocytes were detected 8 wk time point analyzed (data not shown). An additional possibility is after i.v. transfer of HSC (data not shown). These results indicate that the MPP and CLP populations may be heterogeneous with that HSC generate T cells with a significant delay relative to MPP only a small fraction of cells competent to settle within the thymus. and CLP, consistent with indirect thymic colonization from HSC In contrast to MPP and CLP, HSC injected i.v. failed to give rise via downstream progenitors. to any Linneg/low thymocytes during the first 4 wk after transfer Taken together, these experiments demonstrate that thymus set- (Fig. 2, A and C). This indicates that the inability of HSC to gen- tling is selective. HSC cannot directly settle within the adult thy- erate DP thymocytes in unirradiated recipients by 3 wk (Fig. 1, C mus, whereas downstream progenitors can. Therefore, the ability and D) is not due to a delay in intrathymic differentiation. Instead, of progenitors to settle within the thymus must be acquired in the HSC placed in blood do not establish in the thymus of unirradiated BM by progenitors downstream of HSC, at the MPP or CLP stage. recipients during this time period. This suggests that HSC either cannot directly settle within the thymus or alternatively that HSC CCR9 expression by BM progenitors that settle cannot compete with endogenous cells in a nonirradiated We next investigated the molecular basis for selective thymus set- thymus. To differentiate between these possibilities, we analyzed the tling. One molecule proposed to play a role in thymus settling is ability of HSC to generate T lineage progeny following intrathymic the chemokine receptor CCR9 (29–33). Furthermore, a recent transfer into unirradiated recipient mice (Fig. 2, B and D). Intrathymic analysis of Ccr9 reporter mice demonstrated that a subset of BM transfers differ from i.v. transfers in that they bypass a requirement for LSK progenitors and CLP express CCR9 (34). Therefore, we thymic entry. HSC injected intrathymically generated large numbers asked whether CCR9 is selectively expressed by MPP and CLP, of T lineage progeny, reaching a similar peak number of donor-de- which can rapidly generate T lineage cells following i.v. transfer, rived DP thymocytes as MPP at 4 wk after transfer. These results but not HSC, which cannot settle within the thymus. We found that show that HSC, when placed within the thymus, can successfully HSC lacked surface expression of CCR9, whereas a subset of both compete with endogenous progenitors to generate T cells. Therefore, MPP and CLP expressed surface CCR9 (Fig. 3A). Ccr9 mRNA the absence of any T lineage progeny from HSC within the thymus was also significantly increased in sorted MPP and CLP compared during the first 4 wk following i.v. transfer demonstrates that HSC with HSC (Fig. 3B). Altogether, the expression pattern of CCR9 is cannot settle within the thymus. consistent with the cellular specificity of thymus settling. The i.v. injection of HSC into unirradiated recipients always resulted in HSC engraftment of the BM (data not shown). There- Role of CCR9 in thymus settling fore, we expected that HSC injected i.v. would indirectly give rise We next evaluated the role of CCR9 in early T cell development to T lineage progeny in the thymus at later time points by first by generating competitive BM chimeras using a 1:1 mixture of producing progenitors in the BM capable of directly colonizing the either CCR9Ϫ/Ϫ or control B6 BM (Ly5B6) and host-type BM 2012 SELECTIVE THYMUS SETTLING BY LYMPHOID PROGENITORS

FIGURE 3. CCR9 expression by BM progenitors. A, BM was stained to identify CCR9 expression on gated HSC, MPP, and CLP. B, Ccr9 and Hprt1 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 Ϯ SEM; p values were determined by Downloaded from Student’s t test.

(Ly5SJL) to reconstitute irradiated mice. At 10 wk after transfer, the BM and thymi of these mice were analyzed for donor-derived Ϫ/Ϫ cells (Fig. 4A). CCR9 progenitors efficiently engrafted in the http://www.jimmunol.org/ BM and generated HSC and MPP. However, the chimerism of all thymic subsets derived from CCR9Ϫ/Ϫ BM, beginning at the ETP stage, was significantly less than the chimerism in the BM HSC compartment. These data extend previous work (29, 30), indicating an early requirement for CCR9 in mixed BM chimeras, and indi- cate that CCR9 confers a competitive advantage at or before the ETP stage of T cell development. To determine whether CCR9 is important for efficient thymus settling or for the intrathymic development of ETP, we injected by guest on September 27, 2021 BM from either WT or CCR9Ϫ/Ϫ mice i.v. or intrathymically into Ϫ Ϫ unirradiated recipients (Fig. 4, B and C). We found that CCR9 / FIGURE 4. Requirement for CCR9 in thymus settling. A, Mixed BM BM injected i.v. was significantly less efficient at generating ETP chimeras were generated by combining BM from either CCR9Ϫ/Ϫ or B6 than control BM (Fig. 4C, left panel). This difference was evident mice with BM from B6.Ly5SJL mice. At 10 wk after transfer, BM and by day 8 after transfer (Fig. 4B, left panels), a time at which both populations were analyzed for Ly5B6 chimerism. The mean chi- Ϫ/Ϫ Ϯ CLP and MPP contribute to the generation of ETP (Fig. 2). merism from 13 CCR9 or 10 WT chimeras SEM is shown. Asterisks Ͻ CCR9Ϫ/Ϫ BM continued to produce less ETP chimerism than WT indicate 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 or BM at day 22 (Fig. 4B, right panels), when the generation of ETP Ϫ Ϫ CCR9 / BM into unirradiated B6.Ly5SJL-recipient mice. Plots are gated is driven by MPP (Fig. 2). This defect was specific to the thymus. on LinϪ thymocytes. C, Number of donor-derived ETP 3 wk following the Ϫ/Ϫ LSK progenitors from CCR9 BM had no defect in engrafting transfer of either WT or CCR9Ϫ/Ϫ BM i.v. (left panel) or intrathymically the BM (Fig. 4C, middle panel). The reduction in donor-derived (right panel) into unirradiated recipient mice. The percent donor chimerism ETP was not due to an intrathymic defect because there was no within the BM LSK population, following i.v. transfer, was also deter- Ϫ Ϫ significant difference in the ability of WT or CCR9 / BM in- mined (middle panel). Shown is the mean of three to five separate exper- jected intrathymically to generate ETP (Fig. 4C, right panel). iments Ϯ SEM; p values were determined by Student’s t test. D, Analysis Ϫ Ϫ These results indicate that CCR9 is important for efficient thymus of CCR9 / thymi. The numbers of ETP, DN2, and DN3 thymocytes per Ϫ/Ϫ settling. However, CCR9 expression was not absolutely required, thymus from WT or CCR9 thymi are shown. Results are the mean of Ϯ as some T lineage progeny were detected in the thymi of mice three mice per group SEM; p values were determined by Student’s t test. receiving CCR9Ϫ/Ϫ BM, although considerably reduced in num- ber relative to WT controls. If CCR9 is important for thymus settling, why do the thymi of P-selectin (24), the requirement for CCR9 in thymus settling was CCR9Ϫ/Ϫ mice have essentially normal cellularity? To gain in- most clearly revealed in competitive assays (Fig. 4, A–C). Taken sight into this issue, we analyzed the earliest progenitor subsets in together, our results demonstrate a role of CCR9 in efficient thy- CCR9Ϫ/Ϫ thymi (Fig. 4D). We found a reduced number of ETP mus settling. Ϫ/Ϫ and DN2 cells in CCR9 thymi compared with aged-match WT ϩ controls, whereas the number of DN3 thymocytes was normal. Flt3 signaling requirement for generation of CCR9 ELP This is similar to the phenotype of P-selectin glycoprotein ligand- We next investigated the cytokine requirements for the generation 1Ϫ/Ϫ mice, which are also thought to have a thymus settling defect of CCR9-expressing progenitors in early hemopoiesis. For lym- (24). These data suggest that under noncompetitive situations, the phocyte development, the cytokine receptors c-Kit, Flt3, and thymus may be able to compensate for a reduction in thymus set- IL-7R are each known to be important at stages before lineage tling by the DN3 stage. Similar to what was seen in the case of commitment (55). Of these, only Flt3 is expressed by MPP and The Journal of Immunology 2013

T lineage potential is the ELP (40). ELP are the earliest progenitors in hemopoiesis to be lymphoid specified—expressing lymphocyte- specific genes, including Rag1, Rag2, and Tdt (40). Therefore, we asked whether CCR9 expression by MPP correlates with lymphoid specification. We found that CCR9 expression within the MPP population was restricted to a subset of ELP, identified as GFPϩ MPP in the BM of NG-BAC reporter mice, which express GFP under the control of Rag2 cis-elements (Fig. 5B). This suggests a common program for lymphoid specification and CCR9 expres- sion within the BM MPP population. This differentiation program precedes lineage commitment, as both ELP and CCR9ϩ LSK cells have been shown to be multipotent (34, 40). Additionally, both ELP and CCR9ϩ LSK cells are known to circulate in blood and thus have physiological access to the thymus (18, 34). Since CCR9 expression within the BM LSK population was restricted to the lymphoid specified ELP subset, we next asked whether Flt3 signaling was important for the generation of ELP in addition to CCR9 expression. We found that Flt3LϪ/Ϫ NG-BAC ϩ Rag2-GFP reporter mice lacked the GFP ELP subset of MPP Downloaded from (Fig. 5C). Furthermore, the reduced MPP frequency in Flt3LϪ/Ϫ mice could be accounted for by the lack of ELP. These data dem- onstrate that the defect in lymphocyte development, in the absence of Flt3 signaling (35, 38, 39), is evident at an earlier stage of lymphopoiesis than previously appreciated. Consistent with these

results, the mRNA expression of Ccr9, as well as the lymphoid- http://www.jimmunol.org/ specific genes Rag2 and Tdt, were significantly reduced within LSK cells from Flt3LϪ/Ϫ 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 that expresses CCR9. FIGURE 5. Flt3 requirement for the generation of CCR9-expressing Flt3 requirement for thymus settling progenitors. A, The expression of Flt3 and CCR9 is shown on LSK-gated progenitors from the BM of WT, Flt3LϪ/Ϫ, or IL-7R␣Ϫ/Ϫ mice. B, The Based on the lack of ELP, CLP (38), and CCR9 expression in the expression of Flt3 and CCR9 is shown on the GFPϪ subset and GFPϩ absence of Flt3 signaling, we predicted that Flt3 signaling would by guest on September 27, 2021 (ELP) subset of MPP from BM of NG-BAC reporter mice. C, The expres- be required for the generation of efficient thymus settling progen- sion of Flt3 and GFP is shown on LSK-gated progenitors from the BM of itors. To test this hypothesis, we first analyzed at what stages of T Ϫ Ϫ NG-BAC or NG-BAC Flt3L / mice. D, Mean Ccr9, Rag2, Tdt, and lineage development Flt3 is important by generating mixed BM Hprt1 gene expression determined by real-time RT-PCR, using RNA de- chimeras using BM from either Flt3Ϫ/Ϫ or control Flt3ϩ/Ϫ mice Ϫ/Ϫ Ϯ rived from sorted LSK progenitors from either WT or Flt3L BM (Ly5B6) mixed with WT BM (Ly5SJL) and injected into irradiated SEM; p values determined by Student’s t test. Ly5SJL hosts (Fig. 6A). At 10 wk after transfer, the BM and thymi of these mice were analyzed for donor (Ly5B6)-derived cells. Whereas Flt3Ϫ/Ϫ progenitors efficiently engrafted in the BM and CLP but not HSC (12, 13, 38), corresponding with the expression generated LSK cells, Flt3Ϫ/Ϫ progenitors had a competitive dis- pattern of CCR9. Flt3 is first expressed at the MPP stage of he- advantage in the thymus, which was evident at the earliest ETP mopoiesis and is important for the generation of CLP from MPP stage. There was no further requirement for Flt3 downstream of (38). Furthermore, roles for Flt3 in early B lineage differentiation ETP. The chimerism beyond this stage remained stable, which is and thymocyte production have been revealed using competitive- consistent with the lack of Flt3 expression by thymocytes down- mixed BM chimeras (39). Therefore, we asked whether Flt3 sig- stream of ETP (35). This early requirement for Flt3 in T cell de- naling was required for the generation of CCR9-expressing BM velopment was most clearly revealed under competitive condi- progenitors. Within the BM LSK compartment of WT mice, CCR9 tions. These results demonstrate a requirement for Flt3 signaling at was selectively expressed by a subset of Flt3ϩ MPP. However, in or before the generation of ETP. Flt3LϪ/Ϫ mice, BM LSK cells lacked CCR9 surface expression Flt3 signaling may be important either prethymically and/or in- (Fig. 5A). The same result was found in Flt3Ϫ/Ϫ mice (data not trathymically. To determine whether Flt3 signaling was required in shown). This result was specific to Flt3 signaling because mice the BM for generation of efficient thymus-settling progenitors, we lacking the ␣-chain of the IL-7 cytokine receptor had no defect in assayed the ability of progenitors from Flt3L-deficient BM, which CCR9 expression (Fig. 5A). Additionally, although Notch signal- have developed in the absence of Flt3 signaling, to settle within the ing is critical for regulating T cell development (36, 56, 57), block- thymus (Fig. 6B). Such Flt3LϪ/Ϫ progenitors are competent to ing Notch signaling in BM progenitors with dominant-negative receive Flt3 signals when adoptively transferred into Flt3L-suffi- mastermind like-1 (35) had no effect on CCR9 expression (data not cient mice. We injected whole BM from either Flt3LϪ/Ϫ or WT B6 shown). mice i.v. into unirradiated WT B6.Ly5SJL recipients. Three weeks Flt3LϪ/Ϫ mice also had less MPP, evident by the reduced fre- later, the recipient thymi were analyzed for donor-derived ETP quency of LSK cells that expressed Flt3 (Fig. 5A). This suggests (Fig. 6B, left). BM from Flt3LϪ/Ϫ mice gave rise to significantly that Flt3LϪ/Ϫ mice might be missing a subset of MPP that ex- fewer ETP compared with WT BM. All downstream progenitors presses CCR9. One subset of MPP that is known to possess potent within the thymus were similarly reduced (data not shown). This 2014 SELECTIVE THYMUS SETTLING BY LYMPHOID PROGENITORS

FIGURE 6. Flt3 signaling requirement before thymus settling. A, Mixed BM chimeras were generated by combining BM from either Flt3Ϫ/Ϫ or ϩ/Ϫ SJL Flt3 mice with B6.Ly5 BM. At 10 wk after transfer, BM and thy- Downloaded from mocyte populations were analyzed for Ly5B6 chimerism. The mean chi- merism from four Flt3Lϩ/Ϫ or four Flt3Ϫ/Ϫ chimeras Ϯ SEM is shown. Asterisks indicate p Ͻ 0.05 (Student’s paired t test) relative to HSC chi- merism. B, Number of donor-derived ETP 3 wk following the transfer of either WT or Flt3LϪ/Ϫ BM i.v. (left panel) or intrathymically (right panel) into unirradiated recipient mice. The percent donor chimerism within the BM LSK population, following i.v. transfer, was also determined (middle http://www.jimmunol.org/ panel). Shown is the mean of three to five separate experiments Ϯ SEM, and p values were determined by Student’s t test. defect was specific to the thymus. LSK progenitors from Flt3LϪ/Ϫ FIGURE 7. Flt3high ELP are competent T lineage progenitors. A, GFP BM had no defect in engrafting in the BM (Fig. 6B, middle). Fur- expression on Flt3low, Flt3med, and Flt3high MPP from NG-BAC BM is thermore, the reduction in donor-derived ETP was not due to an shown. B, Number of donor-derived DP thymocytes at 3 wk following i.v. ϫ 4 low med high intrathymic defect because there was no significant difference in transfer of 5 10 Flt3 , Flt3 , and Flt3 MPP into unirradiated Ϫ Ϫ Ϯ recipient mice. Shown is the mean of three mice per group SEM. The by guest on September 27, 2021 the ability of WT or Flt3L / BM injected intrathymically to gen- number of DP thymocytes derived from Flt3high MPP was significantly erate ETP in a Flt3L-sufficient environment (Fig. 6B, right). In the different (p Ͻ 0.05, Student’s t test) from the number of DP thymocytes same experiment, generation of downstream DN2, DN3, and DP derived from Flt3low or Flt3med MPP. C, GFP expression on ETP from Ϫ/Ϫ thymocytes was also comparable between WT and Flt3L BM NG-BAC thymi (filled histogram) or B6 control thymi (dashed histogram). injected intrathymically (data not shown). These results demon- D, Ccr9/Hprt1 gene expression was determined by real-time RT-PCR from strate that Flt3 signaling is required in the BM for the generation RNA derived from sorted Flt3ϩ ETP, Flt3low ETP, and DN2 thymocytes. of efficient thymus-settling cells. Shown is the mean relative expression ratio Ϯ SEM.

T lineage competence of MPP subsets ϩ Our results suggest that the most efficient thymus-settling progen- markers. Indeed, we found that ETP are entirely GFP in NG- itors within the MPP population are ELP, as these progenitors are BAC Rag2 reporter mice (Fig. 7C). This differs from a previous absent in the BM of Flt3LϪ/Ϫ-deficient mice (Fig. 5C). Therefore, result (54) using a different reporter mouse that had GFP knocked we directly compared the ability of ELP and the remaining MPP into the Rag1 , indicating that these two reporter mouse subsets to generate T cells 3 wk after i.v. transfers into unirradiated strains are not identical. Recently, a subset of ETP has been shown recipients. For these experiments, we were unable to use NG-BAC to express high levels of GFP in a Ccr9-GFP reporter mouse (34) ϩ donors, as GFP can cause rejection in immunocompetent recipient and a parallel study identified the Flt3 subset of ETP (35). Both mice (58). Instead, we used Flt3 expression to identify ELP. The studies concluded that their populations contained the earliest pro- top 20% of Flt3-expressing LSK progenitors (Flt3high MPP), also genitors identified in the thymus (34–37). However, the overlap referred to as lymphoid-primed MPP (59, 60), were enriched for between these two populations has not been examined previously. ϩ ELP, whereas the Flt3low and Flt3med fractions of MPP contained Therefore, we analyzed Ccr9 gene expression of sorted ETP Flt3 fewer ELP (Fig. 7A). Whereas the Flt3med fraction did contain cells compared with ETP Flt3low cells and downstream DN2 thy- some GFPϩ cells, these appeared dull for GFP expression com- mocytes (Fig. 7D). We found that Ccr9 was highly expressed by ϩ pared with the Flt3high cells. When purified Flt3high, Flt3med, and ETP Flt3 compared with ETP Flt3low and DN2 thymocytes. This ϩ Flt3low MPP were injected i.v. into unirradiated recipients, Flt3high demonstrates that Flt3 ETP and Ccr9-GFPhigh ETP are overlapping MPP gave rise to ϳ10-fold more DP thymocytes at 3 wk than populations, which is concordant with the idea that progenitors enter either of the other subsets (Fig. 7B). We conclude that the early the thymus expressing Flt3 and CCR9 in addition to Rag2. 3-wk wave of T cell development, following i.v. transfer into un- irradiated recipients, is driven by the ELP subset of MPP. Discussion Our findings indicate that physiological thymus-settling progen- In this study, we investigated whether thymus settling is selective. itors express Rag2, Flt3, and CCR9. Therefore, we expected that We found that, whereas HSC have potent T lineage potential, re- the earliest progenitors in the thymus would also express these vealed by direct intrathymic injection, they fail to settle within the The Journal of Immunology 2015 thymus when placed in the blood of unirradiated recipient mice. delay in their ability to generate thymus-settling progenitors in Downstream MPP and CLP both rapidly generate T cells follow- unirradiated recipient mice. Importantly, our findings reveal that ing i.v. transfer. This selectivity corresponded with the expression the study of physiological thymus settling requires the use of un- of the chemokine receptor CCR9 by a subset of MPP and CLP but irradiated recipient mice. not HSC. We demonstrated that CCR9 is important for efficient A variety of BM progenitors downstream of HSC may physio- thymus settling, indicating that CCR9 is one of the molecules that logically settle the thymus ranging from MPP subsets to CLP and regulate the cellular specificity of thymus settling. Additionally, CLP-2 cells (10). The differential ability of these progenitors to we found that CCR9 was selectively expressed by ELP but not contribute to T lymphopoiesis will depend on their ability to mi- other MPP subsets. Furthermore, Flt3 expression and signaling grate from the BM to the bloodstream (18), their capacity to settle was required for the generation of ELP. Consistent with the role of within the thymus from the bloodstream, and their efficiency in Flt3 in the generation of thymus-setting cells, progenitors from generating T cells once within the thymus. MPP and CLP each Flt3LϪ/Ϫ BM lacked the ability to efficiently settle within the thy- rapidly generated T lineage progeny in the thymus following i.v. mus, whereas sorted Flt3high MPP, enriched for ELP, were efficient transfer, whereas the remaining lineage-negative BM progenitors progenitors of T cells following i.v. transfer into unirradiated re- did not. MPP were also the most efficient progenitor for reconsti- cipients. We conclude that thymus settling is selective. We further tuting T lymphopoiesis in both irradiated (44) and unirradiated conclude that progenitors downstream of HSC acquire competence mice (the present study). MPP are present in the bloodstream and to settle within the thymus through expression of homing mole- thus have physiological access to the thymus (18). Furthermore, cules that include CCR9. the kinetics of T lineage differentiation from MPP suggests that

Whether HSC directly settle the adult thymus was previously some of these cells directly enter the thymus (46) (the present Downloaded from unresolved. HSC have potent T lineage potential and are present in study). There is also evidence for a T cell developmental pathway blood, thus having access to the thymus (17, 18). However, self- independent of CLP intermediates, again supporting direct thymus renewal is the hallmark of stem cell activity, and no self-renewing settling by MPP (10, 47). Additionally, multipotent cells have been progenitors have been identified in the thymus (3, 4). Furthermore, identified in the thymus, at the single-cell level, and this population when BM was injected i.v. into irradiated mice, donor-derived has been proposed to contain thymus-settling cells or the direct

cells in the thymus lacked the ability to self-renew, indicating that progeny of thymus-settling cells (34, 37). Therefore, it is probable http://www.jimmunol.org/ HSC are either excluded from the thymus or that the thymic en- that a subset of MPP can physiologically settle within the thymus. vironment induces the rapid loss of self-renewal from these pro- The ability of CLP to contribute to T lymphopoiesis through the genitors (42). In this study, we found that HSC injected i.v. into conventional pathway has been questioned recently (63). We unirradiated mice failed to generate any donor progeny within the found that c-Kitlow CLP, injected either i.v. or intrathymically, thymus for the first 4 wk after transfer, whereas the same cells rapidly up-regulate c-Kit surface expression to generate c-Kithigh injected into the thymus generated large numbers of DP thymo- ETP by day 8 after transfer. This was likely due to Notch signaling cytes within this timeframe. MPP were similarly efficient to HSC within the thymus (56) because Notch signals have been shown to upon direct intrathymic injection, but unlike HSC, MPP also rap- up-regulate c-Kit expression on progenitors in vitro (63–65). Our idly and efficiently generated T cells following i.v. transfer. Even results indicate that CLP can transit through the conventional path- by guest on September 27, 2021 CLP, which were inferior to MPP and HSC upon intrathymic way of T lineage differentiation. We conclude that CLP placed in transfer, could be clearly shown to generate donor-derived thymo- blood can settle within the thymus. However, can CLP physiolog- cytes following i.v. transfer. The i.v. injection of HSC did lead to ically enter the bloodstream? We were unable to find CLP in the BM engraftment and subsequent T cell development by 8 wk after bloodstream of adult mice (18). However, a new study suggests transfer. These experiments establish that HSC cannot physiolog- that low numbers of CLP-like cells may circulate (54). Therefore, ically settle within the thymus of adult mice. One advantage of this CLP may directly contribute to T cell development, although they may be that the development of specific T competent progenitors are much less efficient T lineage progenitors than MPP. downstream of HSC in the BM allows for the regulation of thymus Since thymus settling is selective and HSC cannot physiologi- settling independently of development of other lineages. This may cally enter the thymus, it follows that progenitors in the BM must be important in situations such as crisis hemopoiesis or in aging in acquire the ability to settle within the thymus while still retaining which T lymphopoiesis is decreased relative to myeloid and ery- T lineage potential. We refer to this process as the acquisition of throid development. We conclude that thymus settling is selective T lineage competence. One molecule that has been implicated in and prethymic differentiation steps are necessary to generate phys- the acquisition of T lineage competence is the chemokine receptor iological thymus-settling progenitors. CCR9. We found that HSC lack CCR9 expression, whereas down- The inability of HSC to settle within the thymus was only ev- stream progenitors, including a subset of MPP and CLP, express ident using unirradiated recipient mice. In irradiated recipients, surface CCR9. Therefore, CCR9 expression correlates with the HSC injected i.v. rapidly and efficiently generated donor-derived ability of progenitors to rapidly reconstitute the thymus. However, DP thymocytes by 3 wk after transfer. It is unclear why irradiation a functional role for CCR9 in thymus settling has been controver- has this effect. One possibility is that irradiation makes the thymus sial. Recently, two studies used short-term thymus settling assays receptive to HSC settling, perhaps through vascular damage or the to determine whether CCR9 is important in adult thymus settling, induction of in the thymus that allow for HSC entry with conflicting results (31, 33). Furthermore, neither of these (45). A second explanation is that irradiation can lead to alternative studies could determine whether the cells entering the thymus were pathways of T cell differentiation. Recent evidence indicates that relevant T lineage progenitors. We instead investigated the devel- following irradiation the spleen and lymph nodes become permis- opment of T lineage progeny from CCR9-deficient progenitors. sive for the development of pre-T cells, which could then settle For these studies, we used unfractionated BM to avoid presump- within the thymus (44, 61). A third possibility is that T cell dif- tions about the identity of thymus-settling progenitors. CCR9Ϫ/Ϫ ferentiation occurs normally following irradiation but is acceler- progenitors had comparable BM engraftment to WT control BM. ated. HSC are normally quiescent, but following irradiation, these Additionally, the development of MPP and CLP was normal from cells cycle more rapidly to reconstitute the ablated hemopoietic CCR9Ϫ/Ϫ progenitors, and MPP continued to circulate at normal system (62). This quiescence of HSC may account for the large levels in the blood of CCR9Ϫ/Ϫ mice (data not shown). Instead, 2016 SELECTIVE THYMUS SETTLING BY LYMPHOID PROGENITORS

CCR9 was found to be important for T lymphopoiesis first at the within the bone marrow LinϪSca1ϩc-kitϩ stem cell compartment is accompanied by ETP stage. This was not due to an intrathymic requirement for loss of self-renewal capacity. Immunity 15: 659–669. 13. Christensen, J. L., and I. L. Weissman. 2001. Flk-2 is a marker in hematopoietic CCR9 because CCR9-deficient progenitors injected intrathymi- stem cell differentiation: a simple method to isolate long-term stem cells. Proc. cally generated normal numbers of ETP. Instead, our results are Natl. Acad. Sci. USA 98: 14541–14546. most compatible with a role for CCR9 in thymus settling. 14. Kondo, M., I. L. Weissman, and K. Akashi. 1997. Identification of clonogenic common lymphoid progenitors in mouse bone marrow. Cell 91: 661–672. Our work suggests that the acquisition of T lineage competence 15. Martin, C. H., I. Aifantis, M. L. Scimone, U. H. Von Andrian, B. Reizis, can occur through the expression of CCR9, as well as other mol- H. Von Boehmer, and F. Gounari. 2003. Efficient thymic immigration of B220ϩ lymphoid-restricted bone marrow cells with T precursor potential. Nat. Immunol. ecules important for migration to the thymus. Therefore we inves- 4: 866–873. tigated the generation of CCR9-expressing progenitors. We found 16. Balciunaite, G., R. Ceredig, S. Massa, and A. G. Rolink. 2005. A B220ϩ ϩ Ϫ that CCR9 expression was restricted to a fraction of Flt3high pro- CD117 CD19 hematopoietic progenitor with potent lymphoid and myeloid de- velopmental potential. Eur. J. Immunol. 35: 2019–2030. genitors. Furthermore, all progenitors able to rapidly reconstitute 17. Wright, D. E., A. J. Wagers, A. P. Gulati, F. L. Johnson, and I. L. Weissman. high high the thymus were Flt3 . Within the MPP population, only Flt3 2001. Physiological migration of hematopoietic stem and progenitor cells. Sci- ELP expressed CCR9 and Flt3 signaling was required for the gen- ence 294: 1933–1936. Ϫ/Ϫ 18. Schwarz, B. A., and A. Bhandoola. 2004. Circulating hematopoietic progenitors eration of ELP. Therefore, we predicted that Flt3L BM would with T lineage potential. Nat. Immunol. 5: 953–960. be deficient in T competent progenitors. Indeed, we found that Flt3 19. Cyster, J. G. 2005. Chemokines, sphingosine-1-phosphate, and cell migration in expression was required at or before the ETP stage of T cell de- secondary lymphoid organs. Annu. Rev. Immunol. 23: 127–159. Ϫ/Ϫ 20. Rosen, S. D. 2004. Ligands for L-selectin: homing, inflammation, and beyond. velopment, and Flt3L progenitors were defective in settling the Annu. Rev. Immunol. 22: 129–156. thymus. Furthermore, ELP were the most efficient progenitors of T 21. von Andrian, U. H., and T. R. Mempel. 2003. Homing and cellular traffic in cells within the MPP population. We conclude that Flt3 signaling lymph nodes. Nat. Rev. Immunol. 3: 867–878. Downloaded from 22. Lesley, J., R. Hyman, and R. Schulte. 1985. Evidence that the Pgp-1 glycoprotein is required for the generation of efficient thymus-settling progenitors. is expressed on thymus-homing progenitor cells of the thymus. Cell. Immunol. Consistent with this, we found that the earliest subset of ETP in the 91: 397–403. thymus expresses Flt3, Ccr9, and Rag2 (34, 35, 37). 23. Wu, L., P. W. Kincade, and K. Shortman. 1993. The CD44 expressed on the earliest intrathymic precursor population functions as a thymus homing molecule In summary, we established that thymus settling is selective and but does not bind to hyaluronate. Immunol. Lett. 38: 69–75. have uncovered two molecular determinants for this selectivity. 24. Rossi, F. M., S. Y. Corbel, J. S. Merzaban, D. A. Carlow, K. Gossens, J. Duenas, L. So, L. Yi, and H. J. Ziltener. 2005. Recruitment of adult thymic progenitors is The chemokine receptor CCR9 is important for the homing of http://www.jimmunol.org/ regulated by P-selectin and its ligand PSGL-1. Nat. Immunol. 6: 626–634. progenitors to the thymus. The cytokine receptor Flt3 regulates the 25. Zaballos, A., J. Gutierrez, R. Varona, C. Ardavin, and G. Marquez. 1999. Cutting generation of T competent progenitors, including CCR9-express- edge: identification of the orphan chemokine receptor GPR-9-6 as CCR9, the ing ELP. However, other molecules, which remain to be discov- receptor for the chemokine TECK. J. Immunol. 162: 5671–5675. 26. Vicari, A. P., D. J. Figueroa, J. A. Hedrick, J. S. Foster, K. P. Singh, S. Menon, ered, may also play a role in these processes. Further character- N. G. Copeland, D. J. Gilbert, N. A. Jenkins, K. B. Bacon, and A. Zlotnik. 1997. ization of these molecular determinants will help refine our TECK: a novel CC chemokine specifically expressed by thymic dendritic cells understanding of thymus-settling progenitors. and potentially involved in T cell development. Immunity 7: 291–301. 27. Wurbel, M. A., M. Malissen, D. Guy-Grand, E. Meffre, M. C. Nussenzweig, M. Richelme, A. Carrier, and B. Malissen. 2001. Mice lacking the CCR9 CC- Acknowledgments chemokine receptor show a mild impairment of early T and B cell development ␥␦ϩ We thank V. Zediak, M. Cancro, B. Kee, and D. Allman for critical comments; and a reduction in T cell receptor gut intraepithelial lymphocytes. Blood 98: by guest on September 27, 2021 2626–2632. M. Ve´lez for technical support; and R. Schretzenmair, R. Wychowanec, and H. 28. Benz, C., K. Heinzel, and C. C. Bleul. 2004. Homing of immature thymocytes to Pletcher of the Abramson Cancer Center Flow Cytometry and Cell Sorting the subcapsular microenvironment within the thymus is not an absolute require- Shared Resource for technical expertise. ment for T cell development. Eur. J. Immunol. 34: 3652–3663. 29. Uehara, S., A. Grinberg, J. M. Farber, and P. E. Love. 2002. A role for CCR9 in T lymphocyte development and migration. J. Immunol. 168: 2811–2819. Disclosures 30. Wurbel, M. A., B. Malissen, and J. J. Campbell. 2006. Complex regulation of The authors have no financial conflict of interest. CCR9 at multiple discrete stages of T cell development. Eur. J. Immunol. 36: 73–81. References 31. Scimone, M. L., I. Aifantis, I. Apostolou, H. von Boehmer, and U. H. von Andrian. 2006. A multistep adhesion cascade for lymphoid progenitor cell homing to the 1. Miller, J. F., and D. Osoba. 1967. Current concepts of the immunological func- thymus. Proc. Natl. Acad. Sci. USA 103: 7006–7011. tion of the thymus. Physiol. Rev. 47: 437–520. 32. Liu, C., T. Ueno, S. Kuse, F. Saito, T. Nitta, L. Piali, H. Nakano, T. Kakiuchi, 2. Goldschneider, I., K. L. Komschlies, and D. L. Greiner. 1986. Studies of thy- M. Lipp, G. A. Hollander, and Y. Takahama. 2005. The role of CCL21 in re- mocytopoiesis in rats and mice. I. Kinetics of appearance of thymocytes using a cruitment of T precursor cells to fetal thymi. Blood 105: 31–39. direct intrathymic adoptive transfer assay for thymocyte precursors. J. Exp. Med. 33. Liu, C., F. Saito, Z. Liu, Y. Lei, S. Uehara, P. Love, M. Lipp, S. Kondo, 163: 1–17. N. Manley, and Y. Takahama. 2006. Coordination between CCR7- and CCR9- 3. Scollay, R., J. Smith, and V. Stauffer. 1986. Dynamics of early T cells: prothy- mediated chemokine signals in prevascular fetal thymus colonization. Blood 108: mocyte migration and proliferation in the adult mouse thymus. Immunol. Rev. 91: 2531–2539. 129–157. 4. Shortman, K., and L. Wu. 1996. Early T lymphocyte progenitors. Annu. Rev. 34. Benz, C., and C. C. Bleul. 2005. A multipotent precursor in the thymus maps to J. Exp. Med. Immunol. 14: 29–47. the branching point of the T versus B lineage decision. 202: 21–31. 5. Foss, D. L., E. Donskoy, and I. Goldschneider. 2001. The importation of hema- 35. Sambandam, A., I. Maillard, V. P. Zediak, L. Xu, R. M. Gerstein, J. C. Aster, togenous precursors by the thymus is a gated phenomenon in normal adult mice. W. S. Pear, and A. Bhandoola. 2005. Notch signaling controls the generation and J. Exp. Med. 193: 365–374. differentiation of early T lineage progenitors. Nat. Immunol. 6: 663–670. 6. Donskoy, E., and I. Goldschneider. 1992. Thymocytopoiesis is maintained by 36. Tan, J. B., I. Visan, J. S. Yuan, and C. J. Guidos. 2005. Requirement for Notch1 blood-borne precursors throughout postnatal life: a study in parabiotic mice. signals at sequential early stages of intrathymic T cell development. Nat. Immu- J. Immunol. 148: 1604–1612. nol. 6: 671–679. 7. Wallis, V. J., E. Leuchars, S. Chwalinski, and A. J. Davies. 1975. On the sparse 37. Zediak, V. P., I. Maillard, and A. Bhandoola. 2005. Closer to the source: notch seeding of bone marrow and thymus in radiation chimaeras. Transplantation and the nature of thymus-settling cells. Immunity 23: 245–248. 19: 2–11. 38. Sitnicka, E., D. Bryder, K. Theilgaard-Monch, N. Buza-Vidas, J. Adolfsson, and 8. Kadish, J. L., and R. S. Basch. 1976. Hematopoietic thymocyte precursors. I. S. E. Jacobsen. 2002. Key role of flt3 ligand in regulation of the common lym- Assay and kinetics of the appearance of progeny. J. Exp. Med. 143: 1082–1099. phoid progenitor but not in maintenance of the hematopoietic stem cell pool. 9. Spangrude, G. J., and R. Scollay. 1990. Differentiation of hematopoietic stem Immunity 17: 463–472. cells in irradiated mouse thymic lobes: kinetics and phenotype of progeny. J. Im- 39. Mackarehtschian, K., J. D. Hardin, K. A. Moore, S. Boast, S. P. Goff, and munol. 145: 3661–3668. I. R. Lemischka. 1995. Targeted disruption of the flk2/flt3 gene leads to defi- 10. Bhandoola, A., and A. Sambandam. 2006. From stem cell to T cell: one route or ciencies in primitive hematopoietic progenitors. Immunity 3: 147–161. many? Nat. Rev. Immunol. 6: 117–126. 40. Igarashi, H., S. Gregory, T. Yokota, N. Sakaguchi, and P. Kincade. 2002. Tran- 11. Morrison, S. J., N. Uchida, and I. L. Weissman. 1995. The biology of hemato- scription from the RAG1 locus marks the earliest lymphocyte progenitors in bone poietic stem cells. Annu. Rev. Cell. Dev. Biol. 11: 35–71. marrow. Immunity 17: 117–130. 12. Adolfsson, J., O. J. Borge, D. Bryder, K. Theilgaard-Monch, I. Astrand-Grundstrom, 41. Yu, W., H. Nagaoka, M. Jankovic, Z. Misulovin, H. Suh, A. Rolink, F. Melchers, E. Sitnicka, Y. Sasaki, and S. E. Jacobsen. 2001. Upregulation of Flt3 expression E. Meffre, and M. C. Nussenzweig. 1999. Continued RAG expression in late The Journal of Immunology 2017

stages of B cell development and no apparent re-induction after immunization. 53. Petrie, H. 2003. Cell migration and the control of post-natal T cell lymphopoiesis Nature 400: 682–687. in the thymus. Nat. Rev. Immunol. 3: 859–866. 42. Mori, S., K. Shortman, and L. Wu. 2001. Characterization of thymus-seeding 54. Perry, S. S., R. S. Welner, T. Kouro, P. W. Kincade, and X. H. Sun. 2006. precursor cells from mouse bone marrow. Blood 98: 696–704. Primitive lymphoid progenitors in bone marrow with T lineage reconstituting 43. Till, J. E., and E. A. McCulloch. 1961. Direct measurement of radiation sensi- potential. J. Immunol. 177: 2880–2887. tivity of normal mouse bone marrow cells. Radiat. Res. 14: 213–222. 55. Singh, H., and J. M. Pongubala. 2006. Gene regulatory networks and the deter- 44. Maillard, I., B. A. Schwarz, A. Sambandam, T. Fang, O. Shestova, L. Xu, mination of lymphoid cell fates. Curr. Opin. Immunol. 18: 116–120. A. Bhandoola, and W. S. Pear. 2006. Notch-dependent T lineage commitment 56. Maillard, I., T. Fang, and W. S. Pear. 2005. Regulation of lymphoid development, occurs at extrathymic sites following bone marrow transplantation. Blood 107: differentiation, and function by the Notch pathway. Annu. Rev. Immunol. 23: 3511–3519. 945–974. 45. Zubkova, I., H. Mostowski, and M. Zaitseva. 2005. Up-regulation of IL-7, stro- 57. Rothenberg, E. V., and T. Taghon. 2005. Molecular genetics of T cell develop- mal-derived factor-1␣, thymus-expressed chemokine, and secondary lymphoid ment. Annu. Rev. Immunol. 23: 601–649. tissue chemokine gene expression in the stromal cells in response to thymocyte 58. Bubnic, S. J., A. Nagy, and A. Keating. 2005. Donor hematopoietic cells from depletion: implication for thymus reconstitution. J. Immunol. 175: 2321–2330. transgenic mice that express GFP are immunogenic in immunocompetent recip- 46. Porritt, H. E., K. Gordon, and H. T. Petrie. 2003. Kinetics of steady-state differ- ients. Hematology 10: 289–295. entiation and mapping of intrathymic-signaling environments by stem cell trans- 59. Adolfsson, J., R. Mansson, N. Buza-Vidas, A. Hultquist, K. Liuba, C. T. Jensen, plantation in nonirradiated mice. J. Exp. Med. 198: 957–962. D. Bryder, L. Yang, O. J. Borge, L. A. Thoren, et al. 2005. Identification of Flt3ϩ 47. Allman, D., A. Sambandam, S. Kim, J. P. Miller, A. Pagan, D. Well, A. Meraz, lympho-myeloid stem cells lacking erythro-megakaryocytic potential a revised and A. Bhandoola. 2003. Thymopoiesis independent of common lymphoid pro- road map for adult blood lineage commitment. Cell 121: 295–306. genitors. Nat. Immunol. 4: 168–174. 60. Lai, A. Y., and M. Kondo. 2006. Asymmetrical lymphoid and myeloid lineage 48. Okada, S., H. Nakauchi, K. Nagayoshi, S. Nishikawa, S. Nishikawa, Y. Miura, commitment in multipotent hematopoietic progenitors. J. Exp. Med. 203: and T. Suda. 1991. Enrichment and characterization of murine hematopoietic 1867–1873. stem cells that express c-kit molecule. Blood 78: 1706–1712. 61. Lancrin, C., E. Schneider, F. Lambolez, M. L. Arcangeli, C. Garcia-Cordier, 49. Spangrude, G. J., S. Heimfeld, and I. L. Weissman. 1988. Purification and char- B. Rocha, and S. Ezine. 2002. Major T cell progenitor activity in bone marrow- acterization of mouse hematopoietic stem cells. Science 241: 58–62. derived spleen colonies. J. Exp. Med. 195: 919–929. 50. Ikuta, K., and I. L. Weissman. 1992. Evidence that hematopoietic stem cells 62. Suda, T., F. Arai, and A. Hirao. 2005. Hematopoietic stem cells and their niche. Downloaded from express mouse c-kit but do not depend on steel factor for their generation. Proc. Trends Immunol. 26: 426–433. Natl. Acad. Sci. USA 89: 1502–1506. 63. Krueger, A., A. I. Garbe, and H. von Boehmer. 2006. Phenotypic plasticity of T 51. Godfrey, D. I., J. Kennedy, T. Suda, and A. Zlotnik. 1993. A developmental cell progenitors upon exposure to Notch ligands. J. Exp. Med. 203: 1977–1984. pathway involving four phenotypically and functionally distinct subsets of 64. Massa, S., G. Balciunaite, R. Ceredig, and A. G. Rolink. 2006. Critical role for CD3ϪCD4ϪCD8Ϫ triple-negative adult mouse thymocytes defined by CD44 and c-kit (CD117) in T cell lineage commitment and early thymocyte development in CD25 expression. J. Immunol. 150: 4244–4252. vitro. Eur. J. Immunol. 36: 526–532. 52. Pearse, M., L. Wu, M. Egerton, A. Wilson, K. Shortman, and R. Scollay. 1989. 65. Hoflinger, S., K. Kesavan, M. Fuxa, C. Hutter, B. Heavey, F. Radtke, and

A murine early thymocyte developmental sequence is marked by transient ex- M. Busslinger. 2004. Analysis of Notch1 function by in vitro T cell differentiation http://www.jimmunol.org/ pression of the interleukin 2 receptor. Proc. Natl. Acad. Sci. USA 86: 1614–1618. of Pax5 mutant lymphoid progenitors. J. Immunol. 173: 3935–3944. by guest on September 27, 2021