Immunology Letters 62 (1998) 165–170

Reconstitution of immune systems in RAG2−/− mice by transfer withinterleukin-12-induced splenic hematopoietic progenitor cells

Takashi Yahata a, Kiyoshi Ando b,g, Kazuhito Watanabe a, Takehiko Mori c, Akio Ohta a,Yasushi Ohmi a, Kenji Iwakabe a, Soichi Kuge d, Minoru Nakui d, Mamoru Ito e,

Sonoko Habu a, Shuhei Yasuda f, Takashi Nishimura a,g,*a Department of Immunology, Tokai Uni6ersity School of Medicine, Bohseidai, Isehara 259-11, Japan

b Department of Internal Medicine, Tokai Uni6ersity School of Medicine, Bohseidai, Isehara 259-11, Japanc Di6ision of Hematology, Department of Internal Medicine, Keio Uni6ersity School of Medicine, Shinjuku-ku, Tokyo 160, Japan

d Department of Surgery, Tokai Uni6ersity School of Medicine, Bohseidai, Isehara 259-11, Japane Laboratory of Immunology, Central Institute for Experimental Animals, Kawasaki, Kanagawa 216, Japan

f Department of Clinical Pharmacology, Yamanouchu Pharmaceutical Co. Ltd., Itabashi-ku, Tokyo 174, Japang Research Center for Genetic Engineering and Cell Transplantation, Bohseidai, Isehara 259-11, Japan

Received 12 February 1998; received in revised form 12 March 1998; accepted 16 March 1998

Abstract

The administration of a high dose of IL-12 into the mice resulted in the induction of splenomegaly. From the flow cytometryanalysis of cellularity in an enlarged spleen, it was demonstrated that Thy1.2−CD45RB−c-Kit+Sca-1+Lin− hematopoieticprogenitor cells markedly increased in IL-12-administered mouse spleen compared with untreated mouse spleen. The IL-12-in-duced hematopoietic progenitor cells showed a greatly enhanced colony-forming activity in CFU-granulocyte/macrophage(CFU-GM), blast-forming units-erythroid (BFU-E) and CFU-spleen (CFU-S) assay. Moreover, it was initially demonstrated thatthe transfer of IL-12-induced splenic hematopoietic progenitor cells into immunodeficient RAG2−/− mice caused a completereconstitution of their immune functions including T- and B-cell-mediated immunity. Thus, the evidence that IL-12 has acapability of inducing hematopoietic progenitor cells possessing stem cell-like activity in vivo, indicated another importantimmunomodulating activity of IL-12 in immunotherapy. © 1998 Elsevier Science B.V. All rights reserved.

Keywords: IL-12; Hematopoietic progenitor; RAG2−/− mice; Colony-forming activity

1. Introduction

The IL-12 consisting of p35 and p40 heterodimericsubunits has been demonstrated to show multiple im-munoregulatory functions both in vitro and in vivo[1,2]. Especially, it is now well accepted that IL-12reveals a potent therapeutic activity against tumor andinfectious diseases via the activation of Th1-dominantimmunity in various mouse models [3–7].

In addition, recently, several investigators have re-ported that IL-12 has an ability to stimulate hemato-poietic progenitor cells in vitro in combination withother cytokines such as stem cell factor (SCF) [8,9].Moreover, it was demonstrated that in vivo administra-tion of IL-12 caused the activation of mobilization ofhematopoietic progenitor cells into peripheral circula-tions [10,11]. However, it remains unclear whether IL-12-induced splenic hematopoietic progenitor cells candifferentiate into functional myeloid and lymphoid cellsin vivo. If the IL-12-induced progenitor cells have astem cell-like activity to differentiate into functional T-and B-cells in vivo, it is of great benefit for considering

* Corresponding author. Tel.: +81 463 931121; fax: +81 463965438.

0165-2478/98/$19.00 © 1998 Elsevier Science B.V. All rights reserved.PII S0165-2478(98)00044-3

T. Yahata et al. / Immunology Letters 62 (1998) 165–170166

the application of IL-12 to cancer therapy combinedwith bone marrow transplantation (BMT) or periph-eral blood stem cell transplantation (PBSCT) [12,13].

To resolve these issues, it was investigated whetherIL-12-induced peripheral hematopoietic progenitorcells could reconstitute immune function of im-munodeficient RAG2−/− mice by their cell transfer.The present paper clearly demonstrated that IL-12has a capability of inducing hematopoietic progenitorcells possessing stem cell-like activity in vivo.

2. Materials and methods

2.1. Treatment of mice with IL-12

The C57BL/6 mice were purchased from CharlesRiver Japan (Yokohama 222, Japan). All mice werefemale and used at 5–8 weeks of age. Generally,C57BL/6 mice were subcutaneously (S.C.) injectedwith 30 mg/kg recombinant mouse IL-12 (kindly do-nated by Genetics Institute, MA) three times a weekat intervals of 2 days. At 8–10 days after IL-12 treat-ment, the mice were sacrificed and their immunologi-cal parameters were examined as described in thefollowing.

2.2. Phenotypic analysis of lymphocytes by flowcytometry

The analysis of cell surface markers was carried outby FACScan using the Consort 30 program. ThemAbs used in this experiment (PE-conjugated antiCD8 mAb, PE-conjugated anti CD45RB mAb, PE-conjugated anti CD4 mAb, PE-conjugated antiCD45B220 mAb, FITC-conjugated anti CD4 mAb,FITC-conjugated anti Thy 1.2 mAb, FITC-conjugatedanti Sca-1 mAb, FITC-conjugated anti CD11b mAb,FITC-conjugated anti CD45B220 mAb, FITC-conju-gated TER-119 mAb, biotinylated anti c-kit mAb,purified anti CD3 mAb) were purchased fromPharMingen (San Diego, CA). The detailed procedurefor staining was described in a previous paper [14].Fluorescence data were collected with logarithmic am-plification. For each sample, data from 10000 vol-ume-gated viable cells were collected. TheLin−c-Kit+Sca-1+ hematopoietic progenitor cellswere isolated from nylon wool column-passed spleencells by sorting with FACStar. The FITC-conjugatedmAbs against CD4, CD8, CD11b, CD45B220 andTER119 were used for staining Lin+ cells. The purityof Lin−c-Kit+Sca-1+ cells were over 98%.

2.3. Colony-forming cell assay

The CFU-granulocyte/macrophage (CFU-GM) and

blast-forming units (BFU-E) assay of hematopoieticcells was determined by plating spleen cells (105 cells)in methylcellulose using Methocult M3430 Kit (StemCell Technologies, Vancouver, B.C.). Briefly, 1 mlculture medium contained spleen cells, 0.9% methyl-cellulose, 1% bovine serum albumin, 30% fetal bovineserum, 0.1 mM 2-mercaptoethanol, 2 mM L-glu-tamine, 3 units of erythropoietin and 2% pokeweedmitogen-stimulated spleen cell condition medium. Theculture was prepared in 35 mm petri dish (Becton-Dickinson, Lincoln Park, NJ) and incubated at 37°Cin 5% CO2 humidified atmosphere. The number ofBFU-E and CFU-GM were enumerated under themicroscope. To determine the value of CFU-S in sa-line- or IL-12-treated mouse spleen cells, spleen cells(104 cells) were i.v. transferred into lethally irradiatedC57BL/6 mice (8 Gy) a day before the experiment.At day 14 after the cell transfer, the mice were sac-rificed and the surface colonies of their spleen werecounted to determine the total number of CFU-spleen(CFU-S) per donor spleen cells. To rule out the pos-sibility of autologous colony formation after irradia-tion, the control mice were equally irradiated andalso received the same volume of PBS.

2.4. Repopulation assay

The immunodeficient C57BL/6 background RAG2knock out mice used in this experiment wereoriginally donated by Dr Yoichi Shinkai [15] andmaintained in Central Institute for ExperimentalAnimals (Kawasaki, Japan). The recipient RAG2−/−

mice were exposed to 3 Gy from 60Co source a daybefore the experiment. The irradiated mice weretransferred i.v. with 8×104 c-Kit+Sca-1+Lin−

hematopoietic progenitor cells, which were isolatedfrom IL-12-treated C57BL/6 mouse spleen cells bysorting with FACStar. As a control, unfractionatedspleen cells obtained from saline-treated mice wereused. At 2 months after the cell transfer, the micewere sacrificed and the proportion of myeloid andlymphoid cells was determined by flow cytometry.The T- cell or B-cell function in the reconstitutedcells was also measured by culturing spleen cells(5×105 cells) with anti-CD3 mAb (10 mg/ml) or LPS(20 mg/nl) for 36 h, respectively. The cell proliferativeactivity was determined by pulsing of stimulatedspleen cells with [3H]TdR (3.7 MBq) for 4 h beforethe cell harvest. The detection of RAG2 gene byPCR was carried out to confirm whether thereconstituted cells in RAG2−/− mice were derivedfrom C57BL/6 mouse donor spleen cells expressingRAG2 gene.

T. Yahata et al. / Immunology Letters 62 (1998) 165–170 167

Fig. 1. Induction of splenomegaly by IL-12 administration. The effectof various doses of IL-12-administration on spleen was determined bymeasuring the tissue weight and the cell number of the spleen. Thedata was represented by stimulation index (SI). The spleen weight SI() and cell number SI ( ) was calculated by the following formula:spleen weight SI=mean tissue weight of spleens obtained fromIL-12-treated mice/mean tissue weight of spleens obtained from sa-line-treated mice; cell number SI=mean cell number of spleens fromIL-12-treated mice/mean cell number of spleens obtained from saline-treated mice. The data represents mean of spleens derived from fivemice. The same results were obtained in three different experiments.

in Fig. 1, a marked splenomegaly was observed inIL-12-treated mice in a dose-dependent manner. Theweight and the number of cells in the spleen showed about5–6-fold increase in IL-12-treated mice compared withthose in saline-treated mice. Here, the effect of IL-12against spleen in normal mice was shown. However, thesame enlargement was also demonstrated in lymph nodesbut not the thymus by IL-12 injection (data not shown).In a previous paper [16], it was also demonstrated thatsuch significant enlargement of spleen and lymph nodeswas detected in IL-12-treated tumor-bearing mice as wellas the case in normal mice.

3.2. Induction of hematopoietic progenitor cells inspleen by IL-12 administration

The phenotypic analysis of spleen cells obtained fromsaline- or IL-12-treated mice, was carried out by flowcytometry. As clearly demonstrated in Fig. 2, the percent-age of both CD8+ T-cells and CD4+ T-cells in total cellsmarkedly decreased in spleen by IL-12 administrationthough the absolute number of T-cells in the spleen wasnot changed so much (Fig. 2B). Instead, abnormal cellpopulations increased in the spleen by IL-12-administra-tion (Fig. 2D). Namely, the cells expressing Thy1.2−

CD45RB− antigens, which could be detected in bonemarrow but were rarely demonstrated in normal mousespleen, were enriched in the spleen of IL-12-treated mice(Fig. 2C–F). Further flow cytometric analysis demon-strated that spleen cells obtained from IL-12 administered

3. Results

3.1. Induction of splenomegaly by administration of IL-12

The C57BL/6 mice were treated with i.p. injection ofIL-12 (30 mg/kg) three times a week at intervals of 2 days.The mice were sacrificed to examine the effect of IL-12on the spleen, 8 days after IL-12 administration. As shown

Fig. 2. Disappearance of mature CD4+ T-cells and CD8+ T-cells and accumulation of immature hematopoietic progenitor cells in spleen ofIL-12-treated mice. The spleen cells were prepared from saline-treated mice (A) or IL-12-treated mice (B). The cells were stained withPE-conjugated anti-CD8 mAb plus FITC-conjugated anti-CD4 mAb to determine the effect of IL-12 on CD4+ T-cells and CD8+ T-cells. Forthe analysis of hematopoietic progenitor cells, spleen cells (C, D, G, H) and bone marrow cells (E, F, I, J) were obtained from saline-treated mice(C, E, G, I) or IL-12-treated mice (D, F, H, J). Unfractionated spleen cells (C, D) or bone marrow cells (E, F) were stained with PE-conjugatedanti-CD45RB mAb plus FITC-conjugated anti-Thy1.2 mAb. The existence of c-Kit+Sca-1+cells in Lin− spleen cell populations (G, H) or bonemarrow (I, J) were analyzed after staining of the cells with FITC-conjugated mAbs against lineage markers (MAC-1, Gr-1, B220, TER119 andCD3), PE-conjugated anti-Sca-1 mAb and biotinylated anti-c-Kit mAb plus streptavidin-RED670. After the Lin− cells were gated, the expressionprofile of c-Kit and Sca-1 was determined by FACScan.

T. Yahata et al. / Immunology Letters 62 (1998) 165–170168

Fig. 3. The IL-12-induced splenic hematopoietic progenitor cellsshowed enhanced colony-forming activity. The CFU-GM, BFU-E orCFU-S colony-forming activity in the spleen obtained from saline-treated mice () or IL-12-treated mice ( ) was determined by thestandard methods described in Section 2. The data represent mean9SE of three different experiments.

background RAG2-/− mice, which were pretreated witha low dose of irradiation (3 Gy) a day before theexperiment. As a negative control, RAG2−/− mice weretransferred with unfractionated spleen cells obtained fromsaline-treated mice, which contained few numbers ofLin−c-kit+Sca-1+populations (Fig. 2G). The mice weresacrificed 2 months later and the existence of CD4+andCD8+ T cells, B cells and MF in spleen and thymus wasexamined by flow cytometry (Fig. 4A). Although theRAG2−/− mice contained no significant numbers of Tcells and B cells in both thymus and spleen, the transferof progenitor cells prepared from IL-12-treated micecaused a complete reconstitution of thymic T cells, splenicT cells and B cells. The fact that the reconstituted cellswere demonstrated to express RAG2 gene by PCRindicated the cells were derived from C57BL/6 donormouse but not from the recipient RAG2−/− mouse (datanot shown). In contrast, mice treated with the transferof spleen cells obtained from normal mice containedneither CD4+CD8+thymocytes or B cells though slightnumbers of T cells, which might be derived from maturesplenic T cells but not from splenic stem-like cells, existedin both thymus and spleen. The absolute numbers of Tcells in mice transferred with untreated mice were less than1/10 compared with mice transferred with IL-12-inducedstem-like cells (data not shown). Moreover, it was clearlydemonstrated that the reconstituted spleen cells inRAG2−/− mice by cell transfer of IL-12-induced splenicprogenitor cells showed the same levels of T cell prolif-erative response to anti-CD3 mAb and B cell responseto LPS as well as spleen cells obtained from normaleuthymic C57BL/6 mice did (Fig. 4B). In contrast, spleencells obtained from RAG2−/− mice transferred with noneor normal mouse spleen cells showed neither T cellproliferative response or B cell proliferative response.These data initially demonstrated that peripherally in-duced Lin-c-kit+Sca-1+ progenitor cells by IL-12 havemultipotent progenitor-like activity which could com-pletely reconstitute immune system of immunodeficientRAG2−/− mice.

4. Discussion

An increasing body of recently published data suggeststhat IL-12 is a useful cytokine for the therapy of immunediseases including tumor, infectious diseases, allergy andautoimmune diseases [3–7,17]. This multifunction ofIL-12 is mediated by the activation of Th1-dominantimmunity which is essential for the induction of cell-me-diated immunity. The present paper further extends apotent biological activity of IL-12 concerning the induc-tion of peripheral hematopoietic progenitor cells.

Recently, IL-12 has been shown to induce the prolif-eration of hematopoietic progenitor cells in the presenceof other cytokines such as stem cell factor (SCF)

mice contained increased proportion of Lin−c-Kit+Sca-1+hematopoietic progenitor cells as well asbone marrow cells (Fig. 2G–J).

3.3. Enhanced colony-forming acti6ity of IL-12-inducedsplenic hematopoietic progenitor cells

To evaluate hematopoietic progenitor activity of thesecells, the quantitation of CFU-GM and BFU-E wascarried out by in vitro methylcellulose culture. The CFU-Sassay was also investigated by intravenous (i.v.) transferof 104 cells into irradiated C57BL/6 mice. As shown inFig. 3, spleen cells obtained from IL-12-administered micerevealed a greatly enhanced number of CFU-GM, BFU-Eand CFU-S compared with saline-treated mouse spleencells.

3.4. Reconstitution of immune system of RAG2−/− miceby IL-12-induced splenic hematopoietic progenitor cells

In a preliminary experiment, it was demonstrated thatunfractionated spleen cells obtained from IL-12-injectedmice but not saline-treated mice could reconstitute thethymus if they (107 cells) were transferred into RAG2−/−

mice. Therefore, finally, it was investigated whetherIL-12-induced Lin−c-Kit+Sca-1+stem-like cells couldsystemically reconstitute T- and B-cell-mediated immunefunctions in immunodeficient RAG2−/− mice. Thespleen cells (8×104 cells) obtained from IL-12-treatedmice were i.v. transferred into H-2 compatible C57BL/6

T. Yahata et al. / Immunology Letters 62 (1998) 165–170 169

Fig. 4. The IL-12-induced splenic hematopoietic progenitor cells completely reconstituted immune system of immunodeficient RAG2−/− mice.(A), C57BL/6 background RAG2−/− mice, which were exposed to 3 Gy irradiation, were transferred with none (a, d, g), unfractionated spleencells obtained from saline-treated mice (b, e, h) or Lin−c-Kit+Sca-1+cells separated by FACStar from IL-12-treated mice (c, f, i). The existenceof CD4+ T-cells and CD8+ T-cells in the thymus (a–c) and in the spleen (d–f) was determined 2 months after cell transfer, by analyzingCD4/CD8 expression pattern. The proportion of B cells and macrophages in spleen (g–i) was also determined by analyzing CD45B220/MAC-1expression profile. (B), Spleen cells derived from RAG2−/− mice, which were treated with cell transfer with none (a), saline-treated mouse spleencells ( ) or IL-12-induced c-Kit+Sca-1+spleen cells () were stimulated with either none, anti-CD3 mAb (2C11) or LPS for 36 h and theirproliferative responses were determined by pulsing with [3H]TdR for 4 h. As a control, untreated C57BL/6 mouse spleen cells ( ) were used. Thesame results were obtained in two separate experiments.

and IL-3 [8,9,18]. Moreover, it has been shown thatIL-12-administration stimulates the mobilization ofbone marrow hematopoietic progenitor cells into spleenand peripheral blood [11]. Here, it is further demon-strated that IL-12-induced splenic hematopoietic pro-genitor cells have a capability of reconstituting both T-and B-cell-mediated immunity in RAG2−/− mice.

The major mechanisms of IL-12 in immunomodula-tion in vivo generally mediated by IFN-g production ofimmunoregulatory cells including Th1 cells [19,20].However, in the case of hematopoietic stem cell activa-tion, IL-12-induced IFN-g appears not to be involvedbecause anti-IFN-g mAb administration with IL-12-treatment causes no significant inhibition ofsplenomegaly and the induction of colony-forming cellsthough CTL activation is blocked (data not shown).Judging from the basic experiments concerning IL-12-induced stem cell proliferation in vitro [8,9,17,21], it ispossible to speculate that some other cytokines inducedby IL-12 in vivo may be also involved in the accumula-tion of hematopoietic progenitor cells in the spleen.Therefore, it still remains unclear how IL-12 induceshematopoietic progenitor cells in vivo.

It has been documented that the absence of graft-ver-sus-host response, which occurs due to syngeneic or Tcell-depleted BMT, in leukemia patients increases therisk of a relapse because of a weak induction of graft-versus-leukemia (GVL) effect [22,23]. Therefore, the

fact that IL-12 can activate both the induction ofantitumor effector cells and the mobilization of hema-topoietic progenitor cells may provide a new strategyfor the therapy of leukemia coupled with hematopoieticstem cell transplantation [24]. Moreover, the evidencethat IL-12 induces the mobilization of hematopoieticstem cells into spleen strongly suggests that it may beuseful for the PBSCT [25]. Thus, the present data thatIL-12-induced spleen cells contain stem-cell like activityin vivo strongly suggests its usefulness for the applica-tion to a new therapy of malignant diseases and im-mune diseases.

Acknowledgements

The authors would like to thank Drs MichikoKobayashi and Steven H. Herrmann for their kind giftof IL-12. This work was supported in part by a Grant-in-Aid for Scientific Research on Priority Areas fromthe Ministry of Education Science and Culture, Japan,a Grant-in-Aid for Cancer Research from the Ministryof Health and Welfare, CREST (Core Research forEnvironmental Science and Technology) of JST (JapanScience and Technology Corporation) and a Grant-in-Aid for the IL-12 project of Tokai University School ofMedicine.

T. Yahata et al. / Immunology Letters 62 (1998) 165–170170

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