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IL-7EnhancesThymicHumanTCellDevelopmentin"HumanImmuneSystem"Rag2(-/-)IL-2Rgamma(-/-)(c)MicewithoutAffectingPeripheralTCellHomeostasis

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ImpactFactor:4.92·DOI:10.4049/jimmunol.0902019·Source:PubMed

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IL-7 Enhances Thymic Human T Cell Development in“Human Immune System” Rag2!/!IL-2R"c

!/! Mice withoutAffecting Peripheral T Cell Homeostasis1

Anja U. van Lent,* Wendy Dontje,2* Maho Nagasawa,* Rachida Siamari,* Arjen Q. Bakker,†

Stephan M. Pouw,* Kelly A. Maijoor,* Kees Weijer,* Jan J. Cornelissen,‡ Bianca Blom,*James P. Di Santo,§ Hergen Spits,*† and Nicolas Legrand3*

IL-7 is a central cytokine in the development of hematopoietic cells, although interspecies discrepancies have been reported.By coculturing human postnatal thymus hematopoietic progenitors and OP9-huDL1 stromal cells, we found that murine IL-7is !100-fold less potent than human IL-7 for supporting human T cell development in vitro. We investigated the role ofhuman IL-7 in newborn BALB/c Rag2!/!"c

!/! mice transplanted with human hematopoietic stem cells (HSC) as an in vivomodel of human hematopoiesis using three approaches to improve IL-7 signaling: administration of human IL-7, ectopicexpression of human IL-7 by the transplanted human HSC, or enforced expression of a murine/human chimeric IL-7receptor binding murine IL-7. We show that premature IL-7 signaling at the HSC stage, before entrance in the thymus,impeded T cell development, whereas increased intrathymic IL-7 signaling significantly enhanced the maintenance of im-mature thymocytes. Increased thymopoiesis was also observed when we transplanted BCL-2- or BCL-xL-transduced humanHSC. Homeostasis of peripheral mature T cells in this humanized mouse model was not improved by any of these strategies.Overall, our results provide evidence for an important role of IL-7 in human T cell development in vivo and highlight thenotion that IL-7 availability is but one of many signals that condition peripheral T cell homeostasis. The Journal ofImmunology, 2009, 183: 7645–7655.

I nterleukin 7 is a major cytokine for the control of lymphocytedifferentiation, the maintenance of cell viability, and the reg-ulation of lymphocyte homeostasis (1, 2). The receptor for

IL-7 comprises two chains, the IL-7R !-chain (IL-7R!/CD127)and the common "-chain ("c

4/CD132) that is shared by the recep-tors for IL-2, IL-4, IL-9, IL-15, and IL-21. IL-7 is produced in aconstitutive manner by stromal cells located in a variety of non-lymphoid and lymphoid tissues, including thymus, spleen, bonemarrow, lymph nodes, skin, and intestine, but is not expressed byhematopoietic cells (3). Lymphopenia in mice, humans, and non-

human primates is associated with elevated levels of IL-7 in theblood and in tissues (4–7). In contrast, the expression of CD127 istightly regulated during development and maturation of hemato-poietic cells, leading to the concept that availability of IL-7 iscritically conditioned by its utilization rather than regulation of itsproduction (8).

The expression of CD127 is mostly restricted to hematopoi-etic cell lineages, e.g., lymphoid progenitors, developing T andB cells, mature T cells, and bone marrow-derived macrophages(3, 8 –10). The expression of CD127 is dynamically regulatedduring hematopoiesis. Constitutive expression of CD127 im-pacts negatively on thymopoiesis, probably due to binding ofIL-7 by double-positive (DP) thymocytes leading to starvationof their double-negative (DN) progenitors (11). Mice transgenicfor IL-7 exhibit enhanced numbers of T and B lymphocytes inperipheral lymphoid organs (12, 13) and can eventually developB cell lymphomas (14). Consequently, IL-7 has been implicatedin the induction of cell proliferation, control of cell metabolism,maintenance of cell survival, and Ag receptor V(D)J rearrange-ments in developing T and B cells (3, 8, 15). As far as lym-phocyte survival is concerned, IL-7 enhances B cell lymphoma(BCL)-2 expression in freshly isolated mouse T cells (16), andmouse T cell cultures are protected from dexamethasone-in-duced apoptosis in the presence of IL-7, correlating with en-hanced expression of BCL-2 and BCL-xL antiapoptotic factors(17). Overall, mouse experiments indicate that the homeostaticmaintenance of naive T cells is strongly dependent on IL-7levels, especially naive CD8" T cells, but other signals are alsoproven important, e.g., TCR-MHC interactions (1, 18, 19).

Genetic ablation of IL-7 or CD127 expression in mice leads tosevere reduction in the number of thymocytes, B cell progenitorsin the bone marrow, and consequently peripheral T and B cells

*Department of Cell Biology and Histology, Center for Immunology of Amsterdam,Academic Medical Center of the University of Amsterdam, The Netherlands; †AIMMTherapeutics, Amsterdam, The Netherlands; ‡Department of Hematology, ErasmusMedical Center, Rotterdam, The Netherlands; and §Cytokines and Lymphoid Devel-opment Unit, INSERM Unite 668, Department of Immunology, Institut Pasteur, Paris,France

Received for publication June 24, 2009. Accepted for publication October 6, 2009.

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 grants from The “Grand Challenges in Global Health”Program (GC4—“Human Vaccine Consortium”), the Wijnand M. Pon Foundation,and the Landsteiner Foundation for Blood Research (Grant 2003-1365).2 Current address: Division of Clinical Immunology and Rheumatology, AcademicMedical Center of the University of Amsterdam, Amsterdam, The Netherlands.3 Address correspondence and reprint requests to Dr. Nicolas Legrand; Department ofCell Biology and Histology, Academic Medical Center, Meibergdreef 15, 1105 AZAmsterdam, The Netherlands. E-mail address: n.legrand@amc.uva.nl4 Abbreviations used in this paper: "c, common "-chain; HIS, human immune system;HSC, hematopoietic stem cell; IRES, internal ribosomal entry site; hu, human; mu,murine; PNT, postnatal thymus; FL, fetal liver; pDC, plasmacytoid dendritic cell;TSP, thymic seeding progenitor; DP, double positive; DN, double negative; SP, singlepositive.

Copyright © 2009 by The American Association of Immunologists, Inc. 0022-1767/09/$2.00

The Journal of Immunology

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(20, 21). The peripheral B and T cell lymphopenia observed inCD127#/# mice can be at least partially recovered by a Bcl-2transgene (22, 23). In contrast, human patients with deficienciesfor the expression of CD127 or the downstream JAK3 completelylack T cells, but exhibit normal or increased numbers of NK cellsand circulating B cells (24–28). Altogether, these discrepanciesbetween mouse and human studies highlight the existence of majorinterspecies differences for the role of IL-7 in lymphocyte devel-opment and homeostasis. To further clarify the role for IL-7 inhuman lymphopoiesis, in vitro experimental models such as fetalthymic organ cultures (29) or Notch ligand-expressing stromalcells (OP9-hDL1) (30) have been used. Alternatively, the role ofIL-7 can be addressed in vivo using mouse models of human he-matopoiesis and immunity (31–33). We and others have con-structed such “human immune system” (HIS) mice by transplant-ing human hematopoietic stem cells (HSC) into conditionednewborn BALB/c Rag2#/#"c

#/# recipient mice and the resultingHIS (BALB-Rag/") mice exhibit multilineage reconstitution byhuman lymphoid and myeloid cells, although in a suboptimal fash-ion (34, 35). Limited de novo human thymopoiesis and peripheralT cell accumulation is a common feature of several mouse modelshumanized for the immune system. Peripheral human T cell num-bers represent !1% of what is found in Rag2#/#"c

#/# mice re-constituted with murine fetal liver (FL) stem cells (36). To someextent, the suboptimal amount of human T lymphocytes in HIS(BALB-Rag/") mice resembles what is reported in mice with de-ficient IL-7 signaling (20, 21).

In this study, we examined the in vivo role of IL-7 on humanhematopoiesis, and particularly T cell development and peripheralmaintenance, in HIS (BALB-Rag/") mice. We asked whether poorcross-reactivity of murine (mu) IL-7 in vivo could explain at leastpartially the suboptimal levels of human reconstitution in mousexenograft recipients. We therefore used several approaches to en-hance IL-7 signaling in the human cells generated in the HIS(BALB-Rag/") mice, and we analyzed their respective impact ongeneration and maintenance of human immune cells.

Materials and MethodsConstructs and production of viral supernatants

The cDNA sequence encoding human CD127 and a chimeric murine-hu-man CD127 were inserted into the multiple cloning site of the LZRS vectorupstream of an internal ribosomal entry site (IRES) and enhanced GFP oryellow fluorescent protein, respectively (37). Control vectors were emptyLZRS IRES-GFP and LZRS IRES-yellow fluorescent protein. Retroviralsupernatants were produced as previously described (38) using the 293T-based Phoenix packaging cell line (39). Similarly, the cDNA sequenceencoding human BCL-2 or BCL-xL was inserted into the LZRS vector. ThecDNA sequence encoding human (hu) IL-7 was inserted into the multiplecloning site of a modified pCDH1 self-inactivated lentiviral vector (SystemBiosciences), downstream of the EF1! promoter and upstream of IRES andGFP sequences. Lentiviral supernatants were produced on 293T cells aspreviously reported (40, 41). Lentivirus stocks were concentrated with ul-tra-concentrator columns (Amicon), aliquoted. and stored at #80°C.

Isolation of human hematopoietic progenitors

Human thymic progenitors were isolated from postnatal thymus (PNT)obtained from children ($3 years of age) undergoing open heart surgery.Human hematopoietic progenitors were isolated from FL tissue samplesobtained from elective abortions, with gestational age of 14–18 wk. Theuse of these human tissues was approved by the Medical Ethical Commit-tee of the Academic Medical Center of the University of Amsterdam andwas contingent on informed consent.

Single-cell suspensions were prepared from PNT and FL tissues, andprogenitor cell populations were isolated as previously described (42, 43).In brief, the CD34" cells were enriched by immunomagnetic cell sortingusing either the direct (PNT) or the indirect (FL) CD34 human progenitorcell isolation kit (Miltenyi Biotec). The CD34"CD1a#CD56#BDCA2#

(further referred to as CD34"CD1a#) PNT cells and the CD34"CD38# FL

cells were further sorted using a FACSAria (BD Biosciences), to purityalways #99%.

Transduction of human hematopoietic progenitors

Human thymic or hematopoietic progenitors were transduced with retro-viral or lentiviral vectors before OP9 cocultures or inoculation into recip-ient animals. The CD34"CD1a# PNT progenitors were cultured overnightin IMDM (Invitrogen) supplemented with Yssel’s medium (44), 5% nor-mal human serum, 20 ng/ml human stem cell factor (PeproTech), and 20ng/ml huIL-7 (Cytheris). The following day, cells were incubated for 6–8h with virus supernatant in fibronectin-coated plates (30 $g/ml; TakaraBiomedicals). An identical procedure was used with FL CD34"CD38#

cells, except that the medium was supplemented with 20 ng/ml humanthrombopoietin (PeproTech).

Cocultures of human progenitor and OP9 cells

Murine bone marrow stromal OP9 control and OP9-huDL1 cell lines (45)were maintained in MEM! (Invitrogen) supplemented with 20% FCS (Hy-Clone). Development of human T cells was assessed by coculturing 5 %104 PNT CD34"CD1a# progenitor cells with 5 % 104 OP9-huDL1 cells inMEM! (Invitrogen) with 20% FCS (HyClone), 5 ng/ml human Flt-3 ligand(PeproTech) and variable amounts of huIL-7 (Cytheris) or muIL-7 (Pep-roTech). Cocultures were supplemented every 2–3 days with fresh mediumand progenitor cells were transferred to fresh stromal cells every 4–5 daysof culture (45).

Generation of HIS (BALB-Rag/") mice and huIL-7 inoculations

BALB/c H-2d Rag2#/#"c#/# mice (46) were bred and maintained in self-

ventilated isocages and fed autoclaved food and water. Mice with a HIS(BALB-Rag/") were generated with minor modifications as compared withpreviously described (34, 35, 42). Newborn ($1 wk old) Rag2#/#"c

#/#

mice received sublethal (3.5 Gy) total-body irradiation with a 137Cs sourceand were injected intrahepatically with 5–10 % 104 sorted CD34"CD38#

human FL cells. When FL cells were transduced with retroviral or len-tiviral vectors, the bulk cell culture was injected intrahepatically to thenewborn recipients immediately after transduction. All manipulationsof HIS (BALB-Rag/") mice were performed under laminar flow.

When indicated, 2- and 10-wk-old HIS (BALB-Rag/") mice received200–250 $g huIL-7/kg (Cytheris) i.p. every other day (three injections perweek), i.e., 1–5 $g of huIL-7 per injection depending on the age. In someexperiments, 10-wk-old HIS (BALB-Rag/") mice received a 5-fold higherdose (&1 mg huIL-7/kg; 25 $g per injection). The huIL-7 was diluted inPBS and control animals were injected with the same volume of PBS(100–200 $l depending on the size of the animals).

Flow cytometry analysis for cell surface markers

Cell suspensions were labeled with FITC, PE, PerCP-Cy5.5, PE-Cy7,allophycocyanin, Alexa Fluor 647, allophycocyanin-Cy7, or Alexa Fluor700 coupled anti-human mAb targeting the following cell surface markers:CD3 (SK7), CD4 (SK3), CD8 (SK1), CD11c (B-Ly6), CD19 (HIB19),CD34 (8G12), CD38 (HB7), CD45 (2D1), CD45RA (HI100), CD123/IL-3R! (9F5) (BD Biosciences), BDCA2 (AC144) (Miltenyi Biotec), CD127/IL-7R! (eBioRDR5; eBioscience), and CD1a (T6-RD1; Beckman Coulter).Dead cells were excluded based on 4',6-diamidino-2-phenylindole incorpora-tion. All washings and reagent dilutions were done with PBS containing 2%FCS and 0.02% NaN3. All acquisitions were performed with an LSR-II cy-tometer interfaced to a FACSDiva software system (BD Biosciences).

Statistical analyses

Data were subjected to two-tail unpaired Student’s t test analysis whereindicated in the figure legends. The obtained p values were consideredsignificant when p $ 0.05.

ResultshuIL-7 is 100-fold more potent than mouse IL-7 in promotinghuman hematopoietic progenitor cell proliferation anddifferentiation in vitro

muIL-7 binds to the huIL-7 receptor (32), thereby allowing somedegree of functional redundancy in chimeric experimental systemssuch as human/mouse fetal thymic organ culture (29). We ana-lyzed to which extent muIL-7 was able to sustain the developmentof human lymphoid precursors in the presence of mouse bone mar-row stromal OP9 cells expressing the human NOTCH ligand

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Delta-like 1 (Fig. 1A). Human CD34!CD1a" PNT lymphoid pro-genitors cultured in these conditions expand extensively andprogress toward the T cell lineage (43, 45).

OP9-huDL1/PNT cocultures were supplemented either withmuIL-7 or with huIL-7 at concentrations ranging from 0.5 to 50ng/ml. In contrast to huIL-7, muIL-7 induced limited expansion ofthe human PNT progenitors in the coculture assay over a 3-wkperiod (Fig. 1B). We used the expression of CD1a and CD3 toevaluate the degree of T cell commitment induced by muIL-7 andhuIL-7 in the coculture assay. Acquisition of CD1a expressionis associated with T cell commitment and human pro-T cells(CD1a!CD3") further differentiate into immature thymocytes(CD1a!CD3!) and mature thymocytes (CD1a"CD3!) (10). Asalready described (47), the frequency of human progenitors com-mitted into the T cell lineage (expressing CD1a and/or CD3) andthe number of CD3!CD1a" mature T cells were increased byhuIL-7 in a concentration-dependent manner (Fig. 1C). In contrast,muIL-7 enhanced T cell commitment only at high doses (50 ng/ml), leading to a modest accumulation of mature T cells (Fig. 1C).The extent of cell expansion and T cell differentiation was similarwhen 50 ng/ml muIL-7 and 0.5 ng/ml huIL-7 were used, suggest-ing that huIL-7 is #100-fold more potent than muIL-7 to promotehuman PNT proliferation and differentiation.

Heterogeneous expression of huIL-7 receptor by human cells inHIS (BALB-Rag/!) mice

To study the role of IL-7 in human lymphoid development andmaintenance in vivo, we transplanted human HSC into sublethallyirradiated immunocompromised newborn BALB/c Rag2"/"!c

"/"

mice to generate HIS (BALB-Rag/!) mice (Fig. 2A) that are mul-tilineage repopulated by human immune cells in primary and sec-ondary lymphoid organs, but in a suboptimal fashion. We askedwhether poor cross-reactivity of muIL-7 in vivo could explain atleast partially the suboptimal levels of human reconstitution inhuman HSC-transplanted mouse recipients.

We analyzed CD127 expression on human cells found in HIS(BALB-Rag/!) mice 2, 6, and 10 wk after human HSC transplan-tation. At 2 wk after graft, human cells are mostly immature pro-genitors located in the liver and the bone marrow, with limitedcolonization of the mouse thymic rudiments. At 6 wk of age, HIS(BALB-Rag/!) mice exhibit robust human B cell development inthe bone marrow and human T cell differentiation in the thymus.Circulating mature human T and B lymphocytes were observed inthe blood and secondary lymphoid organs starting 6–8 wk afterHSC transplantation (34, 35). CD127 was expressed on a limitedproportion (1.7 $ 0.8% at week 2; 5.5 $ 0.5% at week 6; 4.3 $ 0.9%

FIGURE 1. In vitro developmentof human T cells in the presence ofmuIL-7 or huIL-7. A, Human CD34!

CD1a" lymphoid progenitors werepurified from postnatal thymocytesand cocultured with stromal OP9 cellsexpressing the huDL1 ligand ofNOTCH. The cocultures were supple-mented with huFlt-3L and variableamounts of either huIL-7 or muIL-7.B, The graphs show the global humancell expansion in the cocultures overtime, relative to the original CD34!

CD1a" PNT progenitor input (50 %103 cells, rate & 1). Typical cell ex-pansions obtained with no IL-7 (E),0.5 ng/ml (‚), 5 ng/ml (!), and 50ng/ml (") of either huIL-7 (left) ormuIL-7 (right) are shown. C, Thefraction of human cells committed tothe T cell lineage (expressing CD1aand/or CD3) was followed over timeand is shown in the left graphs. Theaccumulation of mature T cells (CD3!

CD1a") is detailed in the rightgraphs. The results show one repre-sentative experiment of three.

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at week 10) of CD34"CD38# bone marrow cells, which are enrichedfor HSC (Fig. 2B). A larger fraction of CD34"CD38" multilineage-committed precursor cells expressed CD127 at their surface (17.5 &8.7% at week 2; 32 & 3.7% at week 6; 29 & 12.4% at week 10l datanot shown). Approximately 20% of the CD19" B cell-committed cellpopulation in the bone marrow expressed CD127 on the cell surfaceat all analyzed time points (Fig. 2C). At week 2, the bone marrow ofHIS (BALB-Rag/") mice also contained a CD34"CD7"CD45RA"

cell population (4.5 & 2.1% of human cells), of which the majority(66 & 16%) was CD127 negative (data not shown). This CD34"

CD7"CD45RA"CD127# cell population might correspond to hu-

man thymic seeding progenitors (TSP) (10). In the thymus, a largefraction (50–75%, depending on the age) of the human thymocytesexpressed CD127 (Fig. 2D). Surface CD127 expression wasobserved on various thymocyte subsets such as immature CD3#

CD1a"CD4#CD8#, early thymic precursors, immature (CD3#

CD1a") single-positive (SP) CD4" cells, CD4"CD8" DPthymocytes, and SP (CD4"CD8# or CD4#CD8") mature CD3"

CD1a# thymocytes (data not shown). In peripheral lymphoidorgans 10 wk after graft, CD127 was expressed on the surface ofT cells, and not on other lineages such as B cells or plasmacytoiddendritic cells (pDCs) (Fig. 2, E and F).

FIGURE 2. Production and charac-terization of HIS (BALB-Rag/") mice.A, Schematic representation of stepsperformed to produce mice with a HISusing newborn BALB/c Rag2#/#"c

#/#

mice as hosts for human FL CD34"

CD38# HSC xenografts. B, The flowcytometry dot plots show one represen-tative example of staining for CD34and CD38 in the bone marrow of a6-wk-old HIS (BALB-Rag/") mouse(left) and CD127 (IL-7R! chain) ex-pression on the surface of CD34"

CD38# cells (right). Values in the dotplots are for the frequency of the cor-responding populations. The bottomgraphs give the frequency of CD34"

CD38# cells among human (hCD45-gated) bone marrow cells (left) and thefrequency of CD127 on their surface(right) at weeks 2, 6, and 10 after trans-plantation. Similar results were ob-tained with the CD34"CD38# cellsfrom the liver (data not shown). C, Thesame analysis was performed for B lin-eage-committed cells (CD19") in thebone marrow and D, total human cellsin the thymus. E, Typical CD127 stain-ings on the surface of spleen T cells, Bcells, and pDCs at week 10 after trans-plantation are shown in the flow cytom-etry dot plots and F, pooled results areshown in the graph. On all graphs, onedot represents one individual HIS(BALB-Rag/") mouse. Unpaired Stu-dent’s t test analysis: !, p $ 0.05; !!,p $ 0.01; and !!!, p $ 0.001.

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huIL-7 administered to HIS (BALB-Rag/") mice generates atransient increase in human thymocytes numbers

To investigate the role of IL-7 in early human thymopoiesis, wetreated 2-wk-old HIS (BALB-Rag/") mice with huIL-7 over 4 wk(Fig. 3A). Thymus cellularity was increased !3-fold after 2 wk ofhuIL-7 treatment, but was similar to PBS-injected animals atweeks 1, 3, and 4 of treatment (Fig. 3B). A more detailed anal-ysis of thymic subsets revealed that the absolute number ofCD4#CD8#CD3# triple-negative thymocytes was increased3-fold after 1 wk of huIL-7 treatment (Fig. 3C). The increase ofthymus size at 2 wk was due to the accumulation (3-fold in-crease) of CD4"CD8" DP thymocytes (Fig. 3C). After 4 wk ofhuIL-7 treatment, that is, in 6-wk-old HIS (BALB-Rag/") mice,we finally observed the accumulation of CD3"CD1a# maturethymocytes (Fig. 3C), which did not result in increased numbersof peripheral mature T cells (data not shown). In the bone mar-row, we did not observe any significant impact of repeatedhuIL-7 injection on B cell ontogeny (Fig. 3D), which is con-

sistent with the fact that B cell numbers are normal in CD127-deficient patients (24 –28). In contrast, we observed a transientaccumulation of human pDCs in the liver of huIL-7-treated HIS(BALB-Rag/") mice, which peaked at !2–3 wk after the onsetof treatment (Fig. 3E).

We asked whether an increased availability of huIL-7 wouldlead to accumulation of human cells in adult (#10 wk afterxenograft transplantation) HIS (BALB-Rag/") mice, which al-ready contain significant amounts of human cells in all periph-eral lymphoid organs. We injected adult HIS (BALB-Rag/")mice for 2 wk with high doses of huIL-7 (up to 25 $g/mouse,three injections per week) and analyzed the effect on human celldevelopment and peripheral homeostasis. The number of humancells did not increase in huIL-7-treated animals in the thymus(Fig. 4A), spleen (Fig. 4B), liver, bone marrow, or blood (datanot shown). No significant effect was observed on thymopoiesis(Fig. 4A) or the frequency of T cells (Fig. 4B), B cells, pDCs,NK cells, or myeloid cells (data not shown) in the periphery.

FIGURE 3. Injection of huIL-7 toyoung HIS (BALB-Rag/") mice. A,Schematic representation of the ex-perimental set up. Two weeks afterhuman HSC transplantation, the ani-mals received every other day 200–250 $g of huIL-7/kg i.p. over a 4-wkperiod (from 1 $g per injection dur-ing the first week to 4 $g during thefourth week). The control animals re-ceived the same volume of PBS.Some animals were sacrificed and an-alyzed every week after the onset ofthe treatment. B, The number of hu-man thymocytes was compared be-tween PBS (E) and huIL-7-treated(f) HIS (BALB-Rag/") mice. C, Adetailed analysis of the human thy-mopoiesis was performed for the im-mature triple-negative (TN; CD3#

CD4#CD8#), immature DP, andmature (CD3"CD1a#) SP subpopula-tions. D, The B cell development andE, the accumulation of human pDCswere analyzed in the bone marrowand liver, respectively. Three inde-pendent experiments were per-formed and the results were pooledto perform the statistical analysis.We used a total of 5–7 PBS-treatedHIS (BALB-Rag/") mice and 6 –10huIL-7-treated HIS (BALB-Rag/")mice per time point. Unpaired Stu-dent’s t test analysis: !, p $ 0.05and !!, p $ 0.01.

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Altogether, these results show that supplementation of youngHIS (BALB-Rag/") mice with huIL-7 enhanced human T cell andpDC development in a transient manner, whereas huIL-7 treatmenthad no effect in adult animals.

Enforced expression of huIL-7 increases human thymopoiesis,but does not affect peripheral human cell homeostasis

Considering that exogenous delivery of huIL-7 to HIS (BALB-Rag/") mice might not result in optimal IL-7 bioavailability, weassessed whether constitutive expression of huIL-7 by the hu-man cells would be more effective. We therefore generated HIS(BALB-Rag/") mice using HSC transduced with a huIL-7-encod-ing lentiviral vector (Fig. 5A). An aliquot of transduced HSC wasmaintained in culture to measure the expression of the reporterGFP (transduction efficiency & 20% for both control and huIL-7vectors). When comparing the transduction efficiency with the fre-quency of GFP" cells in HIS (BALB-Rag/") mice 8–10 wk aftertransplantation, we observed a competitive advantage of huIL-7-expressing thymocytes (supplemental Fig. 1A).5 The tendency to-

ward increased thymus size in huIL-7-expressing animals corre-lated with higher numbers of CD1a"CD3# T cell-committedprogenitors and CD4"CD8" thymocytes (Fig. 5B). In contrast, thefrequencies of peripheral human CD45" cells and T cells wereunaltered in adult huIL-7-expressing HIS (BALB-Rag/") mice(Fig. 5C), as well as the CD4:CD8 T cell ratio (data not shown).Overall, we conclude that cell-autonomous huIL-7 expression re-sults in a selective advantage for the transduced cells, with specificbenefits for subsets of immature thymocytes.

Transplantation of HSC overexpressing a chimericmu/huCD127 results in enhanced recovery of human cells,except T cells

We next assessed whether muIL-7, which is abundantly availablein recipient BALB/c Rag2#/#IL-2R"#/# mice due to the absenceof mature murine lymphocytes and their precursors (6, 8), canpromote human lymphopoiesis in HIS (BALB-Rag/") mice. Toallow optimal interaction of muIL-7 with human cells, we trans-duced the human progenitors with a retroviral vector encoding achimeric mu/huCD127 composed of mouse CD127 extracellulardomain and huCD127 transmembrane-cytoplasmic domain. Wetransduced human PNT progenitors with the mu/huCD127-encod-ing vector and cultured them on OP9-huDL1 cells (Fig. 1A). Theresponsiveness of human cells to muIL-7 was markedly enhanced,with a 15-fold increase in cell expansion and mature T cell gen-eration compared with control-transduced PNT (supplemental Fig.2). These results indicate that the chimeric mu/huCD127 sustainedproper binding of muIL-7 and enhanced signal transduction withinhuman cells.

We next investigated whether endogenous muIL-7 could en-hance the human hematopoiesis in HIS (BALB-Rag/") mice byinjecting human HSC transduced with the chimeric mu/huCD127-expressing vector into newborn BALB/c Rag2#/#"c

#/# mice. Weproduced control groups either with an empty vector or with avector expressing huCD127. The resulting HIS (BALB-Rag/")mice were analyzed !10 wk after xenograft transplantation, whenhuman cell repopulation in peripheral lymphoid organs was opti-mal. Human CD45" leukocyte reconstitution was equivalent in thethymus of all groups (Fig. 6A), whereas it was significantly im-proved in the spleen (Fig. 6B), the bone marrow, liver, and pe-ripheral blood (data not shown) of HIS (BALB-Rag/") mice in-jected with mu/huCD127-transduced HSC. Improved humanreconstitution in these organs correlated with selective advantagefor the GFP" mu/huCD127-expressing human cells, whereas noeffect was noted in the thymus (supplemental Fig. 1B). Engraft-ment of mu/huCD127-transduced HSC resulted in increased num-bers of human CD11c#BDCA2" pDCs, CD11c"BDCA2# cells,which contain conventional dendritic cells and monocytes, andCD19" B cells in the bone marrow, spleen, liver, and blood ofthese animals (Fig. 6, C and D, and data not shown). The numbersof peripheral T cells in HIS (BALB-Rag/") mice injected withHSC transduced either with the chimeric mu/huCD127, thehuCD127, or the control GFP- expressing vectors were similar(Fig. 6E). Consistently, thymopoiesis was not enhanced in mu/huCD127-expressing HIS (BALB-Rag/") mice at 4 wk after HSCtransplantation, whereas a significant increase in peripheral (non-Tcell) reconstitution was already apparent at this early time point(supplemental Fig. 3A). The enforced expression of the chimericmu/huCD127 chain resulted in a reduction of the frequency ofCD3#CD1a" thymocytes (12% in GFP# vs 5% in GFP" on av-erage, p $ 0.05), suggesting that commitment to the T cell lineagewas impaired (supplemental Fig. 3B).

Overall, increasing the responsiveness of human cells to theenvironmental IL-7 (muIL-7) globally enhanced the levels of5 The online version of this article contains supplemental material.

FIGURE 4. Injection of huIL-7 to adult HIS (BALB-Rag/") mice. Ten-week-old HIS (BALB-Rag/") mice were treated for 2 wk with PBS or 5–25$g of huIL-7 every other day. A, The number of human thymocytes wascompared between PBS (f) and huIL-7-treated (Œ) HIS (BALB-Rag/")mice at the end of the treatment time frame (top). The relative frequenciesof thymic cellular subsets, based on CD4/CD8 (middle) or CD3/CD1astainings (bottom), are shown (DN, DP, and SP). B, The number of humansplenocytes is shown (top) as well as the frequency of mature T cells inspleen (bottom). No difference was observed between animals receivingeither 5 or 25 $g of huIL-7 and these animals are pooled together. Twoindependent experiments were performed and the results were pooled toperform the statistical analysis.

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human hematopoietic reconstitution in HIS (BALB-Rag/") mice,with the exception of thymocytes and peripheral T cells.

Overexpression of antiapoptotic genes in HSC does not improvetheir T cell differentiation capacity

We previously reported that human T cells in HIS (BALB-Rag/")mice exhibit a high turnover rate and susceptibility to apoptosis(48), suggesting that low T cell numbers may result at least partlyfrom limited survival capacity. We hypothesized that it might bethe consequence of an inadequate balance between proapoptoticstimuli and IL-7-induced antiapoptotic factors. To test this possi-bility, we investigated the impact of human BCL-2 or BCL-xL geneoverexpression on human hematopoiesis in the HIS (BALB-Rag/") mouse model.

Ectopic expression of BCL-2 in human HSC favored the sur-vival of human cells in the thymus, as illustrated by an increasedrecovery of GFP" cells (Fig. 7A). In particular, the maintenance ofCD3#CD1a" T-committed progenitors and more specifically im-mature CD4"CD8" DP thymocytes was favored by BCL-2, al-though the total number of human thymocytes was not enhanced(Fig. 7A). No effect of BCL-2 gene overexpression was observedon the numbers of total human leukocytes, T cells, or B cells in theperipheral lymphoid organs (Fig. 7B). In contrast, overexpressionof the BCL-xL gene resulted in a 2- to 3-fold increase of the thy-mus, which was almost fully repopulated by BCL-xL-transducedhuman cells ((90% GFP"), indicating a strong competitive ad-vantage of BCL-xL-transduced HSC (Fig. 7C). Consequently, pe-ripheral T cells were mostly GFP" BCL-xL-expressing cells andthe frequency of T cells among BCL-xL GFP" cells was signifi-cantly enhanced (Fig. 7D). However, there was no significant in-crease of the total number of human peripheral T cells since onlyrare T cells were observed in the GFP# cellular fraction (data notshown). We conclude that the high frequency of BCL-xL GFP" T

cells in the periphery is due to the overrepresentation of GFP"

cells in the thymus and not to a selective survival advantage ofBCL-xL GFP" cells in the periphery.

DiscussionIn this study, we explored the dependency of human hematopoiesis onIL-7 using an experimental system mimicking HIS development invivo. In adult HIS (BALB-Rag/") mice, the generation and mainte-nance of human lymphoid and myeloid cell lineages are suboptimal,in particular for human thymocytes and peripheral T cells. AlthoughmuIL-7 interacts with the huIL-7 receptor (32), it is !100-fold lesspotent than huIL-7 to promote human PNT progenitor proliferationand differentiation toward the T cell lineage in vitro. However, asshown here, this low efficacy cannot explain the suboptimal human Tcell development observed in vivo in HIS (BALB-Rag/") mice.

Using several approaches to improve IL-7 signaling in the de-veloping human cells, we made the following observations: 1) en-forced expression of the mu/huIL-7R! chain, which ensures con-stitutive binding of muIL-7 in an optimal manner, gave rise toenhanced production of B cells, pDCs, and myeloid cells, but notthymocytes or peripheral T lymphocytes, suggesting a role for IL-7in the generation of non-T cell-committed progenitor cells; 2) IL-7supplementation (either injected or overexpressed) and promotionof human cell survival (overexpression of BCL-2 or BCL-xL genes)consistently improved human thymopoiesis, in particular by en-hancing the maintenance of immature thymocyte subsets (CD1a"

CD3# T cell-committed progenitors; CD4"CD8" DP thymo-cytes), confirming a role for IL-7 in early intrathymic T celldevelopment; and 3) the number of human peripheral T cells wasnot increased by any of the approaches tested. This observationmight indicate that IL-7 alone is not sufficient to sustain the sur-vival of human peripheral T cells in HIS (BALB-Rag/") mice, forreasons that are potentially intrinsic to this model.

FIGURE 5. Enforced expressionof huIL-7 in HIS (BALB-Rag/")mice. A, Experimental scheme in-cluding HSC manipulation by lentivi-ral transduction before xenografttransplantation. B, The number of hu-man thymocytes was compared be-tween HIS (BALB-Rag/") mice gen-erated with control-transduced (f) orhuIL-7-transduced (Œ) HSC 8–10 wkafter transplantation (left). The rela-tive frequencies of thymic cellularsubsets are shown (middle and rightgraphs), as depicted in the legend ofFig. 4. C, The number of humansplenocytes is shown (left) as well asthe frequency of mature T cells inspleen (right). Two independent ex-periments were performed and the re-sults were pooled to perform the sta-tistical analysis. Unpaired Student’s ttest analysis: !, p $ 0.05; !!, p $0.01; and !!!, p $ 0.001. Ctrl,Control.

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Premature expression of the mu/huCD127 chain in HIS (BALB-Rag/") mice hinders the engraftment of the thymus by humancells, as indicated by the effect on early, committed T cell precur-sors (CD3#CD1a"), whereas non-T cell lineages benefit from in-creased access to muIL-7. This observation suggests that the main-tenance and expansion of an early hematopoietic progenitor cellpopulation is mediated by IL-7, and/or that premature expressionof the IL-7 receptor directs these progenitors away from the T celllineage. Alternatively, the reduced frequency of mu/huCD127" Tcell lineage-committed (CD1a"CD3#) thymocytes might reflect

an intrinsic defect of mu/huCD127" TSP to enter the thymus. Inhumans, little is known about the nature of the TSP, largely due toexperimental limitations. It was initially proposed that B, NK, andT lymphoid cells derive from a CD127" common lymphoid pro-genitor (49), but this concept is now challenged by the recent iden-tification of CD127# intrathymic T cell progenitors (50, 51).Haddad et al. (51) reported that the most immature (CD34high

CD1a#) human fetal thymocytes do not express CD127, like fetalbone marrow CD34highCD45RAhighCD7" hematopoietic progen-itor cells, suggesting that the most efficient T cell progenitor seed-ing the thymus is CD127# and is not a progeny of the commonlymphoid progenitor (52). As shown in mice, enforced expressionof CD127 from the immature DN thymocyte stage results in adecreased thymic cellularity (11), which is not corrected by Bcl-2-enforced expression (53). It was speculated that the constitutiveIL-7 consumption by CD127-transgenic CD4"CD8" thymocytesdeprives the early CD4#CD8# progenitor compartment from IL-7,therefore resulting in impaired thymopoiesis (11). In the context ofHIS (BALB-Rag/") mice, an alternative, nonexclusive explanationto reduced thymopoiesis might be that the premature, ubiquitousexpression of mu/huCD127 leads to hindered generation of TSP orto impaired TSP migration into the thymus. Overall, these obser-vations highlight the fact that a strict regulation of CD127 expres-sion is a key element for proper T cell development (54).

Limited de novo human thymopoiesis is a common feature ofmany humanized mouse models (32, 33, 48). It is known that IL-7administration in conditioned recipients receiving a bone marrowtransplant improves T cell production (55, 56). Similarly, intrathy-mic T cell development was consistently enhanced in HIS (BALB-Rag/") mice with improved IL-7 signaling, in particular throughbetter maintenance of immature T cell subsets (CD4"CD8" andCD3#CD1a"). Enforced expression of genes that recapitulate theantiapoptotic effects of IL-7 also had a positive outcome on humanthymopoiesis, although BCL-2 and BCL-xL differentially impactedon this process. When a survival advantage was provided throughBCL-xL gene overexpression, which should decrease T cell sus-ceptibility to apoptosis (17), human thymocytes (this work) andNK cells (57) were selectively favored. BCL-2 increased the pro-portion of immature thymocytes, most obviously CD3#CD1a"

thymocytes, whereas BCL-xL enhanced all thymic subsets equally.Exogenous huIL-7, however, induced a transient wave of thy-mocyte generation in young HIS (BALB-Rag/") mice. Such aphenomenon was also observed in mice with nonoptimal thy-mopoiesis, e.g., in newborn animals or during recovery afterbone marrow transplantation. For instance, in 2-wk-old micereceiving muIL-7 daily during 3 wk, the number of thymocyteswas increased transiently 1 wk after onset of treatment (58).The transient effect observed in HIS (BALB-Rag/") mice maybe the consequence of proliferation and/or differentiation ofhuIL-7-responsive human thymic progenitors, which have be-come huIL-7 insensitive and/or are insufficiently replenishedwith TSP from the bone marrow.

Thymic subsets recovered from adult (8- to 10-wk-old) HIS(BALB-Rag/") mice contain relatively high numbers of mature Tcells, supporting the notion that the early thymic progenitor supplyis suboptimal in the HIS (BALB-Rag/") mice. In line with thisobservation, exogenous huIL-7 administration to adult HIS(BALB-Rag/") mice (1–25 $g every other day) was ineffective. Incontrast, in the presence of constitutive huIL-7 expression, we ob-served a long-term enhancement of human thymopoiesis. One ex-planation may be an insufficient bioavailability of exogenoushuIL-7 in the thymus, e.g., due to short half-life in vivo. It isreasonable to hypothesize that IL-7 bioavailability is higher inyoung animals after huIL-7 inoculation (as compared with adult

FIGURE 6. HIS (BALB-Rag/") mice with enforced expression of a chi-meric mu/huCD127. A, The graph shows the number of human thymocytesharvested from adult HIS (BALB-Rag/") mice (#10 wk after transplantation)produced either with control-transduced, huCD127-transduced, or chimericmu/huCD127-transduced human HSC. The frequency of huCD45" cells in thethymus was always #95%. B, The frequency (top) and total number (bottom)of human cells in the spleen are shown. The number of human cells generatedby the transduced progenitors (GFP"-gated) is given for the following humancell subsets: C, pDCs (top) and conventional dendritic cells/monocytes (bot-tom; data from the liver); D, B cells (data from the bone marrow); and E, Tcells (data from the spleen). To correct for variations in transduction efficien-cies between retroviral supernatants and experiments, the results are normal-ized for a fixed (30,000) amount of GFP" progenitors. Unpaired Student’s ttest analysis: !, p $ 0.05; !!, p $ 0.01; and !!!, p $ 0.001. Ctrl. Control.

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mice) or in the presence of a steady source of huIL-7 (enforcedhuIL-7 expression). Alternative strategies for IL-7 delivery in vivomake use of IL-7/anti-IL-7 mAb complexes or IL-7-Fc fusion pro-teins, which intrinsically possess improved biological activityand/or stability compared with IL-7. Two to three injections ofIL-7/anti-IL-7 mAb complexes (1.5 $g plus 15 $g every other dayi.p.) are sufficient to transiently enhance thymopoiesis in wild-typemice (15–20% increase in thymus size) or in IL-7#/# lymphopenicmice (50- to 100-fold increase in thymus size) (59). HumanizedNOD-scid IL-2R"null mice transplanted with human CD34" mo-bilized peripheral blood cells and immediately treated with IL-7-Fc fusion protein (20 $g weekly i.v.) also exhibited enhancedthymopoiesis, with marked maintenance of CD4"CD8" immaturethymocytes (60). In comparison to these quantities of inoculatedhuIL-7, it was reported that CD127#/# mice, which cannot con-sume IL-7, exhibit a muIL-7 concentration of 40 pg/ml in theirserum (6). Of note, we have observed in cocultures of humanCD3"CD1a" PNT and OP9-huDL1 cells that huIL-7 (5 ng/ml)induces the generation of mature T cells even in the presence ofhigh amounts of muIL-7 (50 ng/ml), indicating that muIL-7 isinefficient at blocking the binding of huIL-7 to its receptor (sup-plemental Fig. 4).

Despite numbers of studies linking peripheral IL-7 levels and Tcell survival (8, 19, 56, 61), we did not observe enhanced humanperipheral T cell accumulation in HIS (BALB-Rag/") mice by ge-netically providing huIL-7 or survival advantage, even when hu-man thymopoiesis was improved. It is likely that, once they have

left the thymus, mature human T cells lack factors required fortheir survival in the mouse peripheral lymphoid organs (48). Weshow that IL-7 alone is not enough to overcome this human T cellsurvival defect, which probably results from a combination ofmissing factors. It may be that molecules involved in cell migra-tion between lymphoid organs, cell adhesion, costimulation, orcross-talk with epithelial and stromal components function in asuboptimal fashion due to interspecies incompatibilities, therebynegatively impacting on the generation of lymphoid progenitorcells, T cell differentiation, and mature T cell survival. As shownhere for IL-7, cross-reactivity between mouse cytokines and thecorresponding human receptors is not optimal (32). Similarly, hu-man NK reconstitution in HIS (BALB-Rag/") mice is hindered bypoor reactivity of human cells to mouse IL-15, which can be over-come by IL-15/IL-15R! treatment (57). The construction of xe-nograft-permissive mouse recipients expressing human cytokinesinstead of the corresponding mouse molecules (gene swapping)will provide valuable insights on their functions, but our resultsalready indicate that the combination of cytokines will be neces-sary to obtain significant effects on the extent of human reconsti-tution. Considering that MHC-TCR interactions are required foroptimal survival of murine naive T cells (61), humanized mousemodels transgenic for human MHC molecules (e.g., HLA-A2,HLA-DR2) are good candidates to dissect the mechanisms drivingthe peripheral maintenance of human T cells. Similarly, improvinghuman dendritic cell accumulation in HIS (BALB-Rag/") miceshould be beneficial to human T cell homeostasis. Human T cells

FIGURE 7. Overexpression ofantiapoptotic BCL-2/BCL-xL genesin HIS (BALB-Rag/") mice. AdultHIS (BALB-Rag/") mice reconsti-tuted with BCL-2-transduced humanHSC were analyzed (#10 wk aftertransplantation). A, The two graphson the left show the number of humanthymocytes and the recovery ratio ofGFP" cells in the thymus comparedwith the original transduction effi-ciency (at 100%, the frequency ofGFP" cells in the animal is identicalto the GFP" frequency of originallytransplanted HSC). The two bargraphs on the right show the relativefrequencies of thymic cellular subsetsas depicted in the legend of Fig. 4. B,The two graphs on the top show thenumber of human splenocytes andthe recovery ratio of GFP" cells inthe spleen compared with the origi-nal transduction efficiency. The bargraph on the bottom shows the relativefrequencies of T cells (CD3"), B cells(CD19"), and other subsets in thespleen. C and D, The same analysis wasperformed with HIS (BALB-Rag/")mice containing BCL-xL overexpress-ing human cells. Three (BCL-2) to six(BCL-xL) independent experimentswere performed and the results werepooled to perform the statistical analy-sis. Unpaired Student’s t test analysis:!, p $ 0.05; !!, p $ 0.01; and !!!, p $0.001.

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in HIS (BALB-Rag/") mice could also be particularly sensitive toactive mechanisms of elimination by the remaining components ofthe murine immune system, e.g., macrophages or monocytes (48).Considering that defective interaction between human CD47 andmouse SIRP! expressed on phagocytes might be a major deter-minant of xenograft rejection (62), SIRP! humanization in mouserecipients should provide a more permissive background for hu-man HSC transplantation. These few examples illustrate that futuregenerations of HIS mice will have to combine the expression ofseveral human gene products, both soluble and on stromal cellularcomponents, to ensure improved levels of human cellular recon-stitution and proper function of human immune cells (63).

Overall, we conclude that IL-7 alone stimulates human T celldevelopment in HIS (BALB-Rag/") mice, but is by itself unable toadequately support peripheral T cell maintenance in such a xeno-geneic environment. Our results provide further evidence for animportant role of huIL-7 in human T cell development in vivo andhighlight the notion that IL-7 availability is but one of many sig-nals that condition peripheral T cell homeostasis.

AcknowledgmentsWe thank Mireille Centlivre for helpful comments on this manuscriptand Berend Hooibrink for cell sorting and maintenance of the AcademicMedical Center of the University of Amsterdam flow cytometry facility.We acknowledge the Bloemenhove Clinic (Heemstede, The Nether-lands) for providing fetal tissues and the staff of the Animal ResearchInstitute Amsterdam for animal care.

DisclosuresThe authors have no financial conflict of interest.

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Supplementary Figure 1. Recovery of GFP+ cells in HIS (BALB-Rag/!) mice

generated with huIL-7-transduced HSC or CD127-transduced HSC. Recovery of GFP+

cells (= % of GFP+ cells in harvested cells / % GFP+ cells transplanted into the mouse

recipients) in the thymus 8 to 10 week-old HIS (BALB-Rag/!) mice that received HSC

transduced either with (A) the huIL-7-GFP lentiviral vector, or (B) the mu/huCD127"GFP or

huCD127"GFP retroviral vectors. Control vectors only express GFP.

Supplementary Figure 2. In vitro development of human T cells with enforced

expression of a chimeric mu/huCD127. Control transduced or chimeric mu/huCD127-

transduced human CD34+CD1a- post-natal thymocytes (transduction efficiency was 17% and

10% respectively) were co-cultured with stromal OP9-DL1 cells. The co-cultures were

supplemented with huFlt-3L and variable amounts of either huIL-7 or muIL-7. (A) The graphs

show the total number of GFP+ cells developing in the co-cultures over time, starting from

10.000 GFP+ cells at day 0. Typical cell numbers obtained with 0.5ng/mL (closed diamonds)

huIL-7 and 5ng/mL (open triangles) or 50ng/mL (open squares) of muIL-7 are shown for

control transduced cells (left) and chimeric mu/huCD127 transduced cells (right). (B) The

accumulation of GFP+ mature T cells (CD3+CD1a-) over time. The results show one

representative experiment out of 3.

Supplementary Figure 3. Analysis of mu/huCD127-transduced HIS (BALB-Rag/!)

mice 4 weeks after HSC transplantation. (A) The left graph shows the number of human

thymocytes harvested from 4 week old HIS (BALB-Rag/!) mice produced either with control-

transduced or chimeric mu/huCD127-transduced human HSC. The graph on the right shows

the total number of human cells in the spleen. (B) This graph shows the proportion of thymic

early committed T cell precursors (CD3-CD1a+) in the populations with or without the

enforced mu/huCD127 expression in the HSC. Two independent experiments were

performed and the results were pooled to perform the statistical analysis. Student’s t-test

analysis: * p<0.05.

Supplementary Figure 4. In vitro development of human T cells in concomitant

presence of mouse and human IL-7. Human CD34+CD1a- lymphoid progenitors were

purified from post-natal thymocytes and co-cultured with stromal OP9-DL1 cells. The co-

cultures were supplemented with huFlt-3L and variable amounts of huIL-7 and muIL-7 (0,

0.5, 5 or 50ng/mL). The graph shows the amount of mature T cells (CD3+CD1a-) generated

after 3 weeks of co-culture in one representative experiment out of 2.