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T, B, and Dendritic Cell MaturationResponses in Humanized Mice by Promoting GM-CSF and IL-4 Stimulate Antibody

and Jianzhu ChenQingfeng Chen, Fang He, Jimmy Kwang, Jerry K. Y. Chan

http://www.jimmunol.org/content/189/11/5223doi: 10.4049/jimmunol.1201789October 2012;

2012; 189:5223-5229; Prepublished online 22J Immunol 

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9.DC1http://www.jimmunol.org/content/suppl/2012/10/22/jimmunol.120178

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The Journal of Immunology

GM-CSF and IL-4 Stimulate Antibody Responses inHumanized Mice by Promoting T, B, and Dendritic CellMaturation

Qingfeng Chen,* Fang He,† Jimmy Kwang,†,‡ Jerry K. Y. Chan,x,{,‖ and Jianzhu Chen*,#,**

Engraftment of human hematopoietic stem cells into immunodeficient mice that lack T cells, B cells, and NK cells results in re-

constitution of human blood lineage cells, especially B cells, in the recipient mice. However, these humanized mice do not make any

significant level of IgG Ab in response to Ag stimulation. In this study, we show that in humanized mice, B cells are immature, and

there is a complete deficiency of CD209+ (DC-SIGN) human dendritic cells. These defects can be corrected by expression of human

GM-CSF and IL-4 in humanized mice. As a result, these cytokine-treated humanized mice produced significant levels of Ag-

specific IgG after immunization, including the production of neutralizing Abs specific for H5N1 avian influenza virus. A significant

level of Ag-specific CD4 T cell response was also induced. Thus, we have identified defects in humanized mice and devised

approaches to correct these defects such that the platform can be used for studying Ab responses and to generate novel human

Abs against virulent pathogens and other clinically relevant targets. The Journal of Immunology, 2012, 189: 5223–5229.

Adoptive transfer of human hematopoietic stem cells(HSCs) into immunodeficient mice that lack T cells,B cells, and NK cells can lead to stable engraftment of

human HSCs (1–5). Differentiation of the transferred HSCs givesrise to different lineages of human blood cells in the recipient mice,which are often referred to as humanized mice. Because human-ized mice enable in vivo study of human hematopoiesis, infectiousdiseases (especially those caused by pathogens that only infecthuman blood lineage cells), and human blood cell diseases (such asleukemia and lymphoma), the platform has significant applicationsin both basic and translational biomedical research (1, 2, 6).In humanized mice, human B cells and T cells are the most

abundantly reconstituted cell types among all blood lineage cells.Despite this, humanized mice do not make a significant Ab re-

sponse after immunization with different Ags in the presence ofvarious adjuvants and through various routes (1, 3, 7). For example,SCID mice that were transplanted with human thymus, bonemarrow, and skin failed to generate any Ag-specific IgG after im-munization with tetanus toxoid (TT) vaccine (7). Although addi-tional transplantation of human lymph nodes into the recipientmice improved Ab response, the level of TT-specific human IgGwas still very low (,0.2 mg/ml) (7). When BALB/c-Rag22/2

Il2rg2/2 mice were used as recipients of human blood cells, theresulting mice had around 37 mg/ml and 191 mg/ml of circulatinghuman IgM and IgG, respectively, which were increased to 173mg/ml and 459 mg/ml after TT immunization. However, the levelof Ag-specific human IgM was barely detectable, and no Ag-specific IgG was detected (8). When the same BALB/c-Rag22/2

Il2rg2/2 recipient mice were treated with recombinant IL-15/IL-15Ra agonist, the total human IgG level increased to ∼700 mg/ml.After TT immunization, TT-specific human IgG was significantlyelevated (0.5 IU/ml) compared with the untreated but immunizedhumanized mice (0.1 IU/ml) (5). Furthermore, when NOD-scid,Il2rg2/2 (NSG) mice were engrafted with human HSCs, no Ag-specific human IgG was elicited upon TT immunization, evenafter transplantation of human fetal liver and thymus (9). Simi-larly, humanized NOD-Rag12/2 Il2rg2/2 mice had only a lowlevel of human IgM (6 mg/ml) and failed to produce any Ag-specific IgG after TT immunization (10). The poor Ab responsein humanized mice has significantly limited the potential of theplatform for studying human B cell responses to pathogens andgeneration of Ag-specific human Abs.Induction of an IgG Ab response requires cognate interactions

between APCs and CD4 T cells and between CD4 T cells andB cells. Many factors are thought to contribute to the poor humanAb responses in humanized mice, including poor reconstitution ofmyeloid APCs, mismatch between human TCR and mouse MHCmolecules, deficiency in human cytokines, and block of T and Bcell maturation in humanized mice (1, 3, 4, 11, 12). For example,myeloid cells, including dendritic cells (DCs), are poorly recon-stituted because of a lack of appropriate human cytokines (11).As a result, CD209+ DCs, which are critical for Ag capture andpriming of T cell immune responses (13–15), are completely ab-

*Interdisciplinary Research Group in Infectious Diseases, Singapore–MIT Alliancefor Research and Technology, Singapore 138602; †Animal Health Biotechnology,Temasek Life Sciences Laboratory, National University of Singapore, Singapore117604; ‡Department of Microbiology, Faculty of Medicine, National Universityof Singapore, Singapore 117597; xExperimental Fetal Medicine Group, Depart-ment of Obstetrics and Gynecology, National University of Singapore, Singapore119228; {Department of Reproductive Medicine, Kandang Kerbau Women’s andChildren’s Hospital, Singapore 229899; ‖Cancer and Stem Cell Biology Program,Duke–National University of Singapore Graduate Medical School, Singapore169857; #Koch Institute for Integrative Cancer Research, Massachusetts Instituteof Technology, Cambridge, MA 02139; and **Department of Biology, Massachu-setts Institute of Technology, Cambridge, MA 02139

Received for publication June 27, 2012. Accepted for publication September 11,2012.

This work was supported by the National Research Foundation Singapore through theSingapore–MIT Alliance for Research and Technology’s Interdisciplinary ResearchGroup in Infectious Disease research program. J.K.Y.C. received salary support fromthe National Medical Research Council, Singapore (NMRC/CSA/012/2009).

Address correspondence and reprint requests to Prof. Jianzhu Chen, MassachusettsInstitute of Technology, 77 Massachusetts Avenue, 76-261, Cambridge, MA 02139.E-mail address: jchen@mit.edu

The online version of this article contains supplemental material.

Abbreviations used in this article: Anhui, A/Anhui/1/05 H5N1 virus isolate; DC,dendritic cell; HK, A/Hong Kong/213/03 H5N1 virus isolate; HSC, hematopoieticstem cell; Idn, A/Indonesia/CDC669/06 H5N1 virus isolate; MDCK, Madin–Darbycanine kidney; NP-KLH, 4-hydroxy-3-nitrophenyl acetyl–keyhole limpet hemocya-nin; TCID50, virus median tissue culture infectious dose; TT, tetanus toxoid.

Copyright� 2012 by The American Association of Immunologists, Inc. 0022-1767/12/$16.00

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sent in humanized mice. Similarly, development and maturation ofhuman B cells is blocked in humanized mice, and T cell functionis impaired (12). Transgenic expression of human MHC class Ior II molecules in the recipient mice has been shown to enhanceHLA-restricted cytotoxic T cell responses (6). However, Ab re-sponses in these mice either remain very poor (9, 10) or have notbeen reported.We have previously developed a simple and efficient method to

enhance human immune cell reconstitution in humanized mice byexpressing appropriate human cytokines via hydrodynamic in-jection of cytokine-encoding plasmids (11). In particular, we foundthat expression of human GM-CSF and IL-4 significantly in-creases reconstitution of human myeloid cells, including DCs.In the current study, we show that GM-CSF and IL-4 treatmentpromotes development of human CD209+ DCs and maturation ofhuman B cells and T cells. Upon immunization, Ag-specific Abresponses are greatly improved in the cytokine-treated humanizedmice, including induction of human neutralizing Abs againstH5N1 influenza viruses. These findings show that the poor Abresponses in humanized mice are partly due to a lack of humanCD209+ DCs and a block in B and T cell maturation attributed tolack of appropriate human cytokines. Provision of these humancytokines helps overcome these defects and improves Ag-specificAb responses in humanized mice.

Materials and MethodsHSC isolation, construction of humanized mice, andhydrodynamic gene delivery

Human fetal livers (n . 8) were obtained from aborted fetuses at 15–23 wkof gestation, in accordance with the institutional ethical guidelines of theNational University Hospital of Singapore. All women gave written in-formed consent for the donation of their fetal tissue for research. Fetuseswere collected within 2 h of the termination of pregnancy. Fetal liver tissuewas initially cut into small pieces and digested with 2 mg/ml collagenase VI(prepared in DMEM) for 15 min at 37˚C with periodic mixing. A single cellsuspension was prepared by passing the digested tissue through a 100-mmcell strainer (BD Biosciences). Umbilical cord blood was obtained from theNational Disease Research Interchange or the Singapore Cord Blood Bank.CD34+ cells from both fetal liver and cord blood were purified with the useof a CD34+ selection kit (Stem Cell Technologies, Vancouver, BC, Canada);the purity of CD34+ cells was 90–99%. Viable cells were counted by trypanblue exclusion of dead cells. All cells were isolated under sterile conditions.To construct humanized mice, 13 105 to 23 105 CD34+ fetal liver or cordblood cells were injected into pups within 48 h of birth intracardially. Ten totwelve weeks after engraftment, mice were analyzed for reconstitution byflow cytometry analysis of peripheral blood. Mice that had 40% or morehuman leukocyte reconstitution were used in subsequent experiments.Hydrodynamic injection of plasmid DNA was exactly as described (11).Briefly, human GM-CSF and IL-4 genes were cloned separately intopcDNA3.1(+) vector (Invitrogen). Plasmid DNAwas purified by Maxi-prepKit (Qiagen). For hydrodynamic gene delivery, 10- to 12-wk-old humanizedmice were injected with 50 mg of each plasmid in a total of 1.8 ml salinewithin 7 s using a 27-gauge needle. All research with human samples andmice was performed in accordance with institutional guidelines.

Immunization and ELISA

Ten to twelve weeks after engraftment, mice were immunized with TTvaccine (Tetavax; Sanofi Pasteur). Specifically, 10 ml of the vaccine, rep-resenting 1/50 of the recommended vaccination dose for an adult human,was mixed with 90 ml aluminum hydroxide gel (Sigma) and then injectedi.p. Mice were boosted twice with the same dose 3 wk apart. After im-munization, sera were analyzed by human TT-specific IgG ELISA (MPBiomedicals Germany GmbH). Total human IgM and IgG levels wereanalyzed with human IgM and IgG kit (Bethyl Laboratories). When4-hydroxy-3-nitrophenyl acetyl–keyhole limpet hemocyanin (NP-KLH)(Bioresearch Technologies, Novato, CA) was used as Ag, 100 ml of1 mg/ml NP-KLH was mixed with 100 ml aluminum hydroxide gel and usedfor immunization as above. Blank ELISA plates (Bethyl Laboratories)were coated with NP-KLH, and sera from immunized mice were added.HRP-conjugated anti-human IgG Ab (Bethyl Laboratories) was used as

secondary Ab. To induce Ab response to H5N1 avian influenza viruses,humanized mice were immunized twice 2 wk apart by i.p. injection. Eachimmunization comprises 100 ml of inactivated H5N1 influenza virus (HAtiter 28) with 100 ml of adjuvant (Seppic). Sera were collected 14 d afterthe second immunization by facial vein bleeding. H5N1 influenza virusesused for the immunization were inactivated A/Anhui/1/05, A/Indonesia/CDC669/06, and A/Hong Kong/213/03.

Single-cell preparation, Abs, and flow cytometry

Preparation of mononuclear cells from liver, lung, spleen, and bone marrowof humanized mice was described previously (11). The following Abs wereused: CD20 (2H7), CD10 (HI10a), CD5 (UCHT2), CD27 (M-T271), IgM(G20-127), IgD (IA6-2), CD3 (HIT3a), CD4 (RPA-T4), CD40L (TRAP1),CD25 (2A3), CD19 (HIB19), HLA-DR (L243), CD209 (DCN46), CD45(2D1), CD69 (L78), CD33(WM53) from BD Biosciences; and CD40(5C3), CD80 (2D10), CD86 (IT2.2), mouse CD45.1 (A20) from Bio-Legend. Cells were stained with appropriate Abs in 100 ml PBS containing0.2% BSA and 0.05% sodium azide for 30 min on ice. Flow cytometry wasperformed on an LSR II flow cytometer using the FACSDiva software (BD,Franklin Lakes, NJ). Ten thousand to 1 million events were collected persample and analyzed using the FlowJo software.

ELISPOT assay

After the third immunization, single-cell suspensions were prepared fromspleens for ELISPOT assay (R&D Systems, Minneapolis, MN). Splenocytescontaining 1 3 105 CD3+ cells were plated per well in medium alone or inthe presence of either 10 ng/ml of PMA or 0.5 mg/ml of a tetanus toxinpeptide (Genscript). Spots were counted using an ImmunoSpot S5 VersaAnalyzer (Cellular Technology) and analyzed with ImmunoCapture soft-ware (Analysis Software).

Immunofluorescence assay

Madin–Darby canine kidney (MDCK) cells cultured in 96-well plates wereinfected with H5N1 subtype viruses. The cells were rinsed with PBS andfixed with 4% paraformaldehyde 24 h postinfection. Fixed cells were in-cubated with sera from humanized mice (1:50 in PBS) or H5 mAb 2D9 at37˚C for 1 h, rinsed with PBS, and then incubated with a 1:500 dilution ofFITC-conjugated rabbit anti-human or anti-mouse IgG (Dako). Ab bindingwas evaluated by wide-field epifluorescence microscopy (Olympus IX71).

Microneutralization assay

mAb (2D9) and polyclonal sera from humanized mice were serially diluted2-fold and incubated with 100 virus median tissue culture infectious dose(TCID50) of H5N1 virus isolates for 1 h at room temperature and plated induplicate onto MDCK cells grown in a 96-well plate. The TCID50 of eachH5 strain in MDCK cell culture was determined by the Reed and Muenchmethod. The neutralizing titer was assessed as the highest Ab dilution inwhich no cytopathic effect was observed by light microscopy.

Statistical analysis

Data are presented as mean and SEM. Differences between groups wereanalyzed via Student t test. A p value of ,0.05 was considered statisticallysignificant. All calculations were performed using the Origin 8.0 softwarepackage.

ResultsGM-CSF and IL-4 stimulate production of human CD209+

DCs and maturation of human B and T cells in mice

Humanized mice were constructed by adoptive transfer of CD34+

HSCs isolated from human cord blood or fetal liver into suble-thally irradiated NSG newborn pups. Twelve weeks after HSCtransfer, PBMCs of recipient mice were stained for human andmouse CD45 to determine the reconstitution level of human leu-kocytes [% human CD45+ cells/(% human CD45+ cells + %mouse CD45+ cells)]. Mice with 40% or more human leukocytereconstitution were used in this study. To stimulate human DCreconstitution, humanized mice between 12 and 16 wk of age werehydrodynamically injected with vector plasmid (control) or plas-mids encoding human GM-CSF and/or IL-4. Seven days afterinjection, splenocytes were stained for Abs specific for humanCD45, CD11c, and CD209 and analyzed by flow cytometry. The

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frequency of CD11c+ cells within the human CD45+ leukocytepopulation was increased significantly in mice treated with GM-CSF (∼17%) or GM-CSF plus IL-4 (∼20%) compared with con-trol mice (∼6%) or IL-4–treated mice (∼7%) (Fig. 1A, 1B). Aprominent population of human CD209+ DCs was detected onlyin GM-CSF plus IL-4–treated mice, reaching ∼30% of CD45+

CD11c+ human cells. To investigate whether the cytokine-inducedCD209+ DCs are functional, 7 d after injection of GM-CSF andIL-4 plasmids, humanized mice were challenged with LPS.Twenty-four hours later, splenocytes were stained for humanCD45, CD209 plus CD40, CD80, CD86, or HLA-DR. AlthoughCD40 expression did not change significantly after LPS stimula-tion, expression of CD80, CD86, and HLA-DR on CD209+ DCswas significantly upregulated (Fig. 1C). When human CD209+

DCs were purified from peripheral blood and spleen by cellsorting and stimulated with LPS in vitro, a significant level ofhuman IL-712p70 was detected in the culture supernatant (Fig.1D). These results show that expression of GM-CSF and IL-4 inhumanized mice stimulates reconstitution of functional humanCD209+ DCs.Expression of human GM-CSF and IL-4 in humanized mice also

has a significant effect on the maturation status of human T andB cells. Seven days after injection of vector plasmid or GM-CSFand IL-4 plasmids, splenocytes from humanized mice were stainedfor human CD45, CD19 plus CD5, CD40, CD20, HLA-DR, CD10,IgM, CD80, or CD86. After IL-4 or GM-CSF plus IL-4 treatment,the levels of CD40, IgM, and CD86 on CD45+CD19+ B cells weresignificantly elevated, whereas the levels of CD5 and CD20 werereduced (Fig. 1E, Supplemental Fig. 1A, 1B). The expression ofHLA-DR, CD10, and CD80 did not change. The frequency ofCD19+CD27+ memory B cells was low (∼1%) in humanized micewithout cytokine treatment (Supplemental Fig. 1A). After IL-4 orIL-4 plus GM-CSF treatment, the frequency was increased ∼10-fold. Similarly, the proportion of human CD45+CD3+ T cells thatexpressed CD25 and the proportion of CD45RA2CD3+ T cellswere significantly increased after IL-4 or GM-CSF plus IL-4treatment (Fig. 1F, Supplemental Fig. 1C, 1D). Notably, mostCD3+ T cells that expressed CD25 were CD4+ T cells but notCD8+ T cells (Fig. 1G, 1H), suggesting the development of CD4+

CD25+ regulatory T cells in humanized mice. Thus, expression ofIl-4 alone or GM-CSF plus IL-4 in humanized mice also stim-ulates T and B cell maturation.

GM-CSF and IL-4 treatment significantly enhances Ag-specificIgG responses in humanized mice

To determine the effect of GM-CSF and IL-4 treatment on Ag-specific Ab responses, we immunized mice 7 d after injection ofvector plasmid or GM-CSF and/or IL-4 plasmids with TT vaccinei.p. three times every 3 wk (Fig. 2A). Two weeks after the thirdimmunization, mice were bled for measuring total human IgM andIgG and TT-specific human IgG in the sera by ELISA. Beforeimmunization, the levels of human IgM were similar (∼120 to∼170 mg/ml) in humanized mice injected with vector controlplasmid and cytokine plasmids (Fig. 2B, left panel). Althoughafter injection of IL-4 or GM-CSF plasmid the levels of human

FIGURE 1. GM-CSF and IL-4 stimulate production of human CD209+

DCs and maturation of human B and T cells in mice. (A and B) Induction

of human DCs by cytokine treatment. Humanized mice were hydrody-

namically injected with empty pcDNA plasmid (Control) or pcDNA vec-

tors expressing human GM-CSF and/or IL-4. Seven days later, splenocytes

were stained for human CD45, CD11c, and CD209. (A) Representative

CD11c versus CD209 plots gating on human CD45+ cells from one of

three mice per group. (B) Comparison of frequencies of CD11c+ and

CD11c+CD209+ cells within the human CD45+ cell population between

nontreated and cytokine-treated mice. The values represent mean 6 SEM

of three mice per group. *p , 0.05. (C) Activation of human CD209+ DCs

by LPS. Seven days after injection of GM-CSF and IL-4 plasmids, hu-

manized mice were injected with 10 mg LPS. Twenty-four hours later,

splenocytes were stained for human CD45, CD209 plus CD40, CD80,

CD86, or HLA-DR. Histograms show CD40, CD80, CD86, or HLA-DR

expression by human CD45+CD209+ DCs without LPS (thin line) and with

LPS treatment (thick line). Representative data from one of the three mice

per group are shown. (D) Induction of IL-12 expression by human CD209+

DCs by LPS in vitro. Seven days after injection of GM-CSF and IL-4

plasmids, CD45+CD33+CD209+ DCs were purified from PBMCs and

splenocytes by cell sorting. Purified DCs (2 3 105) were cultured alone

(Control) or in the presence of LPS (1 mg/ml). Supernatants were analyzed

24 h later for human IL-12p70 by ELISA. (E) Maturation of human B cells

after GM-CSF and IL-4 treatment in humanized mice. Seven days after

injection of empty plasmid or GM-CSF and IL-4 plasmids, splenocytes

were stained for human CD45, CD19, plus the indicated markers. Histo-

grams compare CD40, IgM, CD86, CD5, CD20, HLA-DR, CD10, and

CD80 expression by human CD45+CD19+ B cells in mice treated with

empty plasmid (thin line) or GM-CSF and IL-4 plasmids (thick line).

Representative data from one of the seven mice per group are shown. (F)

Effect of GM-CSF and IL-4 treatment on T cells in humanized mice. Seven

days after injection of empty plasmid or GM-CSF and IL-4 plasmids,

splenocytes were stained for human CD45, CD3 plus CD25, C45RA,

CD40L, or HLA-DR. Histograms compare CD40L, CD25, CD45RA, or

HLA-DR expression by human CD45+CD3+ T cells in mice treated with

empty plasmid (thin line) or GM-CSF and IL-4 plasmids (thick line). Rep-

resentative data from one of the seven mice per group are shown. (G and H)

Staining profiles of CD4 and CD8 versus CD25, CD40L, and HLA-DR

gating on CD4+ (G) and CD8+ (H) human T cells, respectively. Representative

data from one of seven mice per group are shown. The numbers indicate

percentages of cells in the gated quadrants. Ctrl, Vector plasmid-treated mice.

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IgG were elevated 4- to 6-fold compared with mice injected withvector plasmid, the absolute levels of IgG remained low (1–13 mg/ml, Fig. 2B, right panel). After immunization, the levels of the totalIgM did not change much in mice injected with vector or GM-CSFor IL-4 plasmid alone but were increased ∼2.5-fold in miceinjected with GM-CSF and IL-4 plasmids (Fig. 2C, left panel).Similarly, the levels of total IgG did not change significantly inmice injected with vector or GM-CSF plasmid. However, the levelsof IgG were increased ∼28-fold in IL-4–treated mice, reaching anaverage of ∼120 mg/ml (Fig. 2C, middle panel). In GM-CSF andIL-4–treated mice, the levels of IgG were increased 55-fold,reaching ∼700 mg/ml. Furthermore, TT-specific IgG (∼0.14 IU/ml)was readily detected in GM-CSF and IL-4 plasmid injected miceafter immunization but not in other groups of mice (Fig. 2C, rightpanel). Among the serum IgG isotypes, the predominant isotypewas IgG1, followed by IgG2 and IgG4 (Supplemental Fig. 2A).These results show that GM-CSF and IL-4 treatment greatlystimulates Ag-specific IgG responses in humanized mice.

GM-CSF and IL-4 treatment promotes B and T cell responsesafter immunization

To investigate the cellular mechanisms of the enhanced Ab re-sponses in GM-CSF and IL-4–treated mice, we analyzed T andB cell responses in the spleen 2 wk after the third immunization.An immediate difference that was noticed was the enlargedspleens from immunized mice that have been injected with GM-CSF and IL-4 plasmids compared with mice injected with vector,GM-CSF, or IL-4 plasmids (Fig. 3A). Flow cytometry analysisrevealed that nearly 90% of cells in the spleen of GM-CSF and IL-4–treated mice were human CD45+ (Supplemental Fig. 2B). The

total numbers of human leukocytes (CD45+) in the spleen of GM-CSF and IL-4–treated mice were 52.7 6 12.5 3 106, representing∼10-fold increase over the numbers in the spleen of mice injectedwith vector plasmid (4.1 6 0.4 3 106), IL-4 plasmid (5.2 6 2.2 3106), or GM-CSF plasmid (7 6 3.1 3 106) (Fig. 3B), correlatingwith the sizes of the spleens. Within the human CD45+ cells,∼30% was CD19+ B cells and ∼60% was CD3+ T cells in GM-CSF and IL-4–treated mice (Supplemental Fig. 2C, 2D). Incomparison, ∼65% were CD19+ B cells and ∼25% CD3+ T cellsin vector plasmid-treated (control) mice. As a result, the numberof B cells was increased ∼5 times in the spleen of mice injectedwith GM-CSF and IL-4 plasmids compared with mice injectedwith vector plasmid, IL-4 plasmid, or GM-CSF plasmid (Fig. 3B).Even more dramatically, the number of CD4+ T cells in the spleenwas increased .12 times in mice injected with GM-CSF and IL-4plasmids compared with mice injected with vector plasmid, IL-4plasmid, or GM-CSF plasmid. Although the number of CD8+

T cells was also increased in the spleen of mice injected with GM-CSF and IL-4 plasmids, most splenic T cells were CD4+ T cells(Supplemental Fig. 2D).We further analyzed B cells in immunized mice by staining

splenocytes for human CD45, CD19 plus CD40, IgM, CD86, CD5,CD20, HLA-DR, CD10, or CD80. In mice injected with vectorplasmid, the phenotype of CD45+CD19+ human B cells did notchange significantly after immunization: 80% of CD19+ humanB cells expressed CD5 in humanized mice (Fig. 1E) and vectorplasmid-injected humanized mice (Fig. 3C). After immunization,80% of CD19+ human B cells were CD52 in GM-CSF and IL-4–treated mice (Fig. 3C), indicating an induction of conventional(CD52) B cells. Furthermore, most B cells in the spleen of GM-CSF and IL-4–treated mice exhibited an elevated expression ofCD40, IgM, HLA-DR, CD80, and CD86, but reduced expressionof CD19, CD20, and CD10 (Fig. 3C, Supplemental Fig. 2B),suggesting maturation of human B cells. Thus, the enhanced Ag-specific IgG response in GM-CSF and IL-4–treated mice is as-sociated with induction of mature conventional B cells.We also analyzed T cells in immunized mice by staining

splenocytes for human CD45, CD3 plus CD25, CD45RA, CD40L,or HLA-DR. Compared to the vector plasmid-injected humanizedmice, CD3 T cells upregulated CD40L and HLA-DR, and a smallfraction of the T cells was also positive for CD25 in humanizedmice injected with GM-CSF and IL-4 plasmids (Fig. 3D, Sup-plemental Fig. 2D), suggesting that T cells were further activatedafter immunization. The proportion of CD45RA2 T cells alsoincreased significantly in cytokine-treated mice. HLA-DR wasupregulated in both CD4+ and CD8+ T cells (Fig. 3E, 3F). Whensplenocytes were stimulated with a dominant CD4 peptide(QYIKANSKFIGITE) from tetanus toxin, no IFN-g or IL-4 se-cretion was detected by ELISPOT assay in vector plasmid-injected(control) mice. In contrast, TT-specific IFN-g– and IL-4–secretingT cells were readily detected in the splenocytes of GM-CSF andIL-4–treated mice, reaching 1.0 6 0.2% and 1.8 6 0.5%, re-spectively (Fig. 3G). As controls, no IFN-g or IL-4 secretion wasdetected without TT peptide stimulation and much higher fre-quencies of IFN-g– or IL-4–secreting T cells were detected afterPAM stimulation. These results show that GM-CSF and IL-4stimulate T cell maturation and Ag-specific responses in human-ized mice.To distinguish the effect of cytokines versus immunization on

T and B cell numbers, we analyzed humanized mice without im-munization 9 wk after injection with control plasmid, GM-CSF,and/or IL-4 plasmids. The numbers of human CD45+ leukocytesin the spleens were increased slightly in IL-4–treated (5.76 0.73106) and GM-CSF plus IL-4–treated (6.5 6 0.8 3 106) mice

FIGURE 2. GM-CSF and IL-4 treatment dramatically stimulates Ag-

specific IgG responses in humanized mice. (A) Experimental scheme. Ten-

to twelve-week-old humanized mice were hydrodynamically injected (h.i.)

with the vector plasmid or GM-CSF and/or IL-4 plasmids (50 mg each) on

day 0. Seven days later, mice were immunized with TT vaccine and then

boosted twice 3 wk apart. Two weeks after the third immunization, mice

were sacrificed and analyzed. (B and C) Total human IgG and IgM and TT-

specific human IgG were analyzed in sera from mice that were injected

with the vector plasmid (Ctrl), IL-4 plasmid, GM-CSF plasmid, or GM-

CSF and IL-4 plasmids before (B) (n = 6) and after (C) (n = 12) immu-

nization. Each symbol represents one mouse. The numbers indicate the

average level of IgM or IgG within the group. *p , 0.05.

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compared with control mice (4.5 6 0.43 106) (Supplemental Fig.3). The numbers of CD19+ B cells in the spleens did not changesignificantly after cytokine treatment. In contrast, the number ofCD3+ T cells was increased ∼2-fold after IL-4 or GM-CSF plusIL-4 treatment. Most of the increase in total T cell numbers wasdue to increase in CD4+ T cells (∼2.5 times). Because 9 wk afterimmunization the numbers of CD4+ T cells increased 28-fold inGM-CSF plus IL-4–treated mice compared with control mice(Fig. 3B), the observed effects are primarily due to the combi-nation of cytokine treatment and immunization.

GM-CSF and IL-4–treated mice are capable of generatingH5N1 virus-neutralizing Abs

To test whether GM-CSF and IL-4–treated mice produce humanAb responses to other Ags, we used NP-KLH, a commonly usedmodel Ag, and inactivated avian influenza virus H5N1. Cytokine-treated humanized mice were immunized three times at 3-wkintervals with NP-KLH, and sera were measured for total and NP-specific human IgM and/or IgG. Immunization of GM-CSF andIL-4–treated humanized mice with NP-KLH also led to elevatedlevels of total human IgM (∼445 mg/ml), IgG (∼770 mg/ml), andNP-specific IgG (∼1350 ng/ml) (Supplemental Fig. 4). Similarly,cytokine-treated humanized mice were immunized twice at 2-wkinterval with one of the three inactivated H5N1 virus isolates:

A/Anhui/1/05 (Anhui), A/Indonesia/CDC669/06 (Idn), and A/HongKong/213/03 (HK). Seven days after the second immunization,sera were collected and assayed for the presence of anti-viral Absusing two methods. In the immunofluorescent assay, MDCK cellswere infected with Anhui, Idn, or HK viruses, fixed, incubatedwith sera from the respective virus-immunized mice, and thendeveloped with labeled Abs specific to human IgG or mouse IgG.As shown in Fig. 4A, no positive staining was detected when serafrom nonimmunized GM-CSF and IL-4–treated (Ctrl) mice wereused. As a positive control, when a murine anti-H5N1 mAb 2D9(16) was used, positive signal was detected only with labeledsecondary Ab specific for the mouse, but not human, IgG. Incontrast, sera from all six H5N1-immunized GM-CSF and IL-4–treated mice (two for each viral isolate) reacted with infectedMDCK cells when labeled secondary Ab was specific to human,but not mouse, IgG. In the neutralization assay, 100 TCID50 ofvirus was incubated with different dilutions of sera or 2D9 mAband then analyzed for cytopathic effect on MDCK cells. Whereascontrol serum did not show any neutralizing activity, 2D9 neu-tralized viruses at a maximum dilution factor of 128 (starting with100 mg/ml of Ab) (Fig. 4B). Sera from GM-CSF and IL-4–treatedand immunized mice also exhibited significant neutralizingactivity against respective strains of H5N1 virus isolates withmaximum dilution factors of 18 for the Anhui isolate, 14 for the

FIGURE 3. Enhanced B and T cell responses in GM-CSF and IL-4–treated mice after immunization. (A) Comparison of the sizes of spleens of hu-

manized mice that were injected with vector (Ctrl), IL-4, GM-CSF, or GM-CSF plus IL-4 plasmids 2 wk after the third immunization. (B) Comparison of

the total human CD45+ leukocytes, CD19+ B cells, CD4+ T cells, and CD8+ T cells in the spleens of immunized humanized mice that were injected with

vector (Ctrl), IL-4, GM-CSF, or GM-CSF plus IL-4 plasmids. Single-cell suspensions of spleens were prepared, counted, and analyzed by flow cytometry

for human CD45, CD19, CD3, CD4, and CD8. The numbers of human cells were calculated by multiplying the total cell numbers with the frequency of the

specific cell type. Shown are mean6 SEM (n = 3 to 5). (C) Flow cytometry analysis of human B cells. Splenocytes were analyzed for human CD45, CD19,

plus the indicated markers. Histograms compare CD40, IgM, CD86, CD5, CD20, HLA-DR, CD10, CD80, and CD19 expression gating on CD45+CD19+

cells between mice injected with vector plasmid (thin line) and GM-CSF and IL-4 plasmids (thick line). (D) Flow cytometry analysis of human T cells.

Splenocytes were analyzed for human CD45, CD3, plus the indicated markers. Histograms compare CD25, CD45RA, CD40L, and HLA-DR expression

gating on CD45+CD3+ cells between mice injected with vector plasmid (thin line) and GM-CSF and IL-4 plasmids (thick line). (E and F) Splenocytes were

analyzed for human CD45, CD4, or CD8 plus the indicated markers. Shown are plots of CD4 (E) and CD8 (F) versus CD25, CD40L, and HLA-DR gating

on CD4+ and CD8+ human T cells, respectively. The numbers indicate percentages of cells in the gated quadrants. (G) ELISPOT analysis of TT-specific

human T cell responses. Total splenocytes (containing 1 3 105 CD3+ T cells/well) were incubated with medium, PMA, or TT peptide for 24 h and then

analyzed by ELISPOT assay. The numbers indicate frequencies of T cells that secreted human IFN-g or IL-4. Shown are mean 6 SEM (n = 4).

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Idn isolate, and 22 for the HK isolate. These results show thatGM-CSF and IL-4 treatment stimulates human Ab responses toa diverse set of Ags, including avian H5N1 influenza virus.

DiscussionAfter engraftment of human HSCs into immunodeficient recipientmice, B cells are the most abundantly reconstituted human bloodcell type. However, they do not make any significant IgG Ab re-sponse after immunization with specific Ag. Studies have sug-gested multiple defects contribute to the poor human Ab responses,including poor reconstitution of myeloid APCs, mismatch betweenhuman TCR and mouse MHC molecules, block of T and B cellmaturation, and deficiency in human cytokines in humanizedmice (1, 3, 4, 11, 12). In this study, we show that in humanizedmice, a majority of B cells express CD5 and are blocked at thetransition stage, and there is a profound deficiency of humanCD209+ DCs. More importantly, we show that these defects canbe partially corrected by expression of human GM-CSF and IL-4in mice. As a result, the cytokine-treated humanized mice makesignificant levels of Ag-specific IgG after immunization, includingneutralizing Abs specific for H5N1 avian influenza viruses.The vast majority of human B cells in humanized mice are IgM+

(8). Consistently, we found that .85% of human CD19+ B cellsare IgM+ in the spleens of normal humanized mice (Fig. 1E). Wenoticed that most of these B cells also express CD5, consistentwith a previous report (17), whereas CD5+IgM+ B cells are onlya minor fraction of the B cell population in human (18, 19). On thebasis of CD5 expression, human B cells can be divided into CD5+

B1 cells and CD52 conventional B2 cells. Serum IgM is thoughtto be secreted primarily by CD5+ B1 cells, whereas IgG Abresponses are mediated by CD52 conventional B2 cells. Therelatively high levels of serum IgM but little IgG in humanizedmice is consistent with this notion and raises the possibility thatthe observed defect in IgG Ab response is due to the compositionof B cells in humanized mice.CD5 is also expressed on transitional human pre-naive B cells,

which also express CD10 and a low level of CD40 (20). We foundthat the majority of human B cells (.50%) in humanized miceexhibited a phenotype of CD19+CD10+CD40lowIgM+IgD+ (Fig.1E), indicating that the development of human B cells in hu-manized mice is blocked at the transitional stage. Our findingis consistent with a previous study showing accumulation ofCD24int/hiCD38hi immature B cells, which composed 60% of thesplenic CD19+ cells in humanized mice using the NOG mice asrecipients (12). Our study extends the previous study by definingthe stage at which B cell development is blocked. Furthermore, weshow that maturation of B cells can be stimulated by the expres-sion of GM-CSF and IL-4 in humanized mice, as indicated by the

increased expression of IgM, CD40, CD86, and decreased ex-pression of CD20 (Supplemental Fig. 1). Notably, cytokinetreatment alone also results in a significant increase in the pro-portion of CD52 conventional B cells. The maturation of humanB cells and development of CD52 B cells are further stimulatedby immunization of cytokine-treated humanized mice. Besidesfurther upregulation of CD40 and downregulation of CD20, HLA-DR and CD80 are upregulated and CD10 and CD19 are down-regulated on human B cells in immunized mice (Fig. 3C), con-sistent with observation that expression of CD19 and CD20decreases during B cell Ab responses in human (21, 22). Mostprofoundly, the majority of B cells become CD52 after immu-nization. This is even more remarkable considering that totalnumbers of B cells increased around 6-fold in immunized GM-CSF and IL-4–treated mice compared with mice injected withcontrol vector. As CD10 disappears upon B cell maturation (21,23), these results suggest that B cell development in humanizedmice is likely blocked at the pre-naive B cell stage. Thus, thedeficiency in B cell maturation as well as preferential developmentof CD5+ B1 cells are likely contributing factors to the poor IgGAb response in humanized mice.Correlating with the maturation of B cells, GM-CSF and IL-4

treatment stimulates a significant increase in serum IgG com-pared with nontreated humanized mice (Fig. 2B). After immuni-zation, the levels of total IgM and especially total IgG are furtherincreased, reaching a much significant level of 400–700 mg/ml,respectively. Importantly, a significant level of Ag-specific IgG isalso induced to immunizing Ags TT and NP-KLH (Fig. 2C, andSupplemental Fig. 4). The level of TT-specific human IgG is at theminimum protective level in humans. As in humans, the mostabundant Ig isotypes are IgG1, followed by IgG2, and then IgMand IgG4. In contrast to humanized mice that were not treatedwith GM-CSF and IL-4, the predominant Ig isotype is IgM. Inaddition, GM-CSF and IL-4–treated humanized mice were able toproduce neutralizing Abs against three different isolates of H5N1avian influenza virus. IL-4 is known to stimulate B cell Ab re-sponses and Ig class switching. It is notable that expression ofIL-4 alone is not sufficient to elevate serum IgG levels and Ag-specific IgG response, suggesting that combined GM-CSF andIL-4 treatment contributes to the improved Ab responses throughadditional mechanisms beyond stimulating B cell maturation.Another major contributing factor for the improved Ab responses

in GM-CSF and IL-4–treated humanized mice is the induction ofCD209+ myeloid DCs and the enhanced priming of CD4 T cells.The functions of human T cells, particularly CD4+ T cells, areknown to be impaired in humanized mice as they only developedvery weak responses to stimulations and immunizations (1, 3, 5,12). Specifically, it has been shown that human T cells in hu-

FIGURE 4. Induction of neutralizing Abs against H5N1 in GM-CSF and IL-4–treated mice. (A) MDCK cells were infected with H5N1 isolates Anhui,

Idn, or HK, then fixed, incubated with sera from the respective virus isolate-immunized mice, or control (Ctrl) sera from nonimmunized GM-CSF and IL-4–

treated humanized mice, or 2D9, a mouse mAb specific for H5N1. Binding of primary Ab was detected using FITC-conjugated anti-human or anti-mouse

secondary Abs. Two humanized mice were immunized with each H5N1 virus isolate, and representative data from one mouse are shown. Original

magnification 3100. (B) H5N1 virus isolates (100 TCID50) were incubated with different dilution of sera from corresponding virus isolate-immunized

mice, nonimmunized mice, or 2D9 mAb. The mixture was then added to MDCK cells, and cytopathic effect was observed by light microscopy. Shown are

the neutralizing titers at the highest serum or Ab dilution at which no cytopathic effect was observed. *p , 0.05.

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manized mice are unable to produce a detectable amount of IFN-gand IL-4 upon Ag restimulation (12). We have shown previouslythat GM-CSF and IL-4 treatment induces the generation of CD11c+

CD209+ cells in humanized mice (11). In this study, we furtherdemonstrated that these CD209+ DCs are functional as they up-regulated expression of costimulatory molecules CD80, CD86, andHLA-DR as well as secretion of IL-12 upon stimulation with LPS.Previous reports have suggested that CD209+ DCs are critical forthe initiation of T cell immune responses (15, 24), and their in-duction in cytokine-treated mice likely contributes to the elevatedAb responses in humanized mice through induction of CD4 T cellresponses. Expression of GM-CSF and IL-4 also increased theproportion of human T cells that were CD45RA2 (SupplementalFig. 1C, 1D). As human CD45RA2 T cells preferentially undergohomeostatic expansion and acquisition of memory-like phenotypein mice (25), the result suggests that expression of GM-CSF andIL-4 stimulates T cell maturation in humanized mice. Upon im-munization, T cells further upregulated CD40L and HLA-DR, witha small fraction of T cells still expressing the activation markerCD25 (Fig. 3D). Furthermore, a significant fraction of human CD4T cells responded to restimulation with TT peptide by secretingIFN-g and IL-4 as shown by ELISPOT assay (Fig. 3E). It is worthto note that in our ELISPOT assay, total splenocytes were stimu-lated with TT peptide QYIKANSKFIGITE. This peptide is knownto be a dominant CD4 epitope in human (26) but also can stimulateCD4+ T cell response in mice (27). Although it is not knownwhether the peptide was presented by human and/or mouse MHCin the ELISPOT assay, our findings clearly show that GM-CSF andIL-4 treatment stimulates induction of CD209+ DCs and effectiveCD4 T cell priming in humanized mice.In summary, the poor Ag-specific IgG Ab response in humanized

mice is correlated with a block of B cell development at the pre-naive B cell stage, preferential development of CD5+ B cells,and a lack of CD209+ DCs. The expression of GM-CSF and IL-4 inhumanized mice can correct these defects by stimulating B cellmaturation, generation of CD52 conventional B cells, and induc-tion of CD209+ DCs. As a result, these cytokine-treated mice canmake a significant Ag-specific IgG Ab response after immuniza-tion. We have now identified the specific defects in humanizedmice with regard to the generation of a physiological Ab responseand defined a novel approach to correct them such that this plat-form can be used to study Ab responses and generate novel humanAbs against virulent pathogens and other clinically relevant targets.

AcknowledgmentsWe thank Dr. William Hwang of the Singapore Cord Blood Bank for pro-

viding cord blood, Lan Hiong Wong and Hooi Linn Loo for technical as-

sistance, Drs. Maroun Khoury, Adam Drake, and Bettina Iliopoulou for

discussion and critical review of the manuscript, and Dr. Farzad Olfat

for general support.

DisclosuresThe authors have no financial conflicts of interest.

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