and Tissues of Transgenic Mice Dendritic Cell Subsets in Peripheral ...

The Journal of Immunology

Conditional Expression of Murine Flt3 Ligand Leads to Expansion of Multiple Dendritic Cell Subsets in Peripheral Blood and Tissues of Transgenic Mice1 Denise J. Manfra, Shu-Cheng Chen, Kristian K. Jensen, Jay S. Fine, Maria T. Wiekowski, and Sergio A. Lira2 The analysis of the development and function of distinct subsets of murine dendritic cells (DC) has been hampered by the limited number of these cells in vivo. To circumvent this limitation we have developed a conditional transgenic mouse model for producing large numbers of DC. We used the tetracycline-inducible system to conditionally express murine Flt3 ligand (FL), a potent hemopoietic growth factor that promotes the differentiation and mobilization of DC. Acute treatment (96 h) of the transgenic animals with the tetracycline analog doxycycline (DOX) promoted an ⬃200-fold increase in serum levels of FL without affecting the number of circulating DC. However, within 1 wk of DOX treatment, the relative number of DC in peripheral blood increased from ⬃8 to ⬃40%. Interestingly, both the levels of FL and the number of DC remained elevated for at least 9 mo with continual DOX treatment. Chronic treatment of the mice with DOX led to dramatic increases in the number of DC in multiple tissues without any apparent pathological consequences. Most DC populations were expanded, including immature and mature DC, myeloid (CD11cⴙCD11bⴙCD8aⴚ), lymphoid (CD11cⴙCD11bⴚCD8aⴙ), and the recently defined plasmacytoid (pDC) subsets. Finally, transplantation of BM from green fluorescent protein-expressing mice into lethally irradiated transgenic mice followed by subsequent DOX treatment led to expansion of green fluorescent protein-labeled DC. The transgenic mice described here should thus provide a readily available source of multiple DC subsets and should facilitate the analysis of their role in homeostasis and disease. The Journal of Immunology, 2003, 170: 2843–2852.

D

endritic cells (DC)3 are critical mediators of the immune response (1). They capture and present Ag to naive T cells and influence both the adaptive and the innate immune response. This critical role is mediated by multiple DC subsets that phagocytose and present Ag, secrete specific molecules, and migrate to specific tissues. Although significant progress has been made recently in identifying these DC subsets, there is still much to be learned with respect to their migratory properties, the molecules they express in vivo, and the mechanism(s) by which they differentially affect the immune response. A major obstacle in these analyses has been the low number of DC in the circulation and in tissues. Most of the studies to date have relied on DC expanded in vitro by treating BM-derived cells ex vivo with various growth factors and cytokines, such as GMCSF, TNF-␣, or IL-4 (2–5). However, the number of DC subsets expanded under these conditions is restricted to one or a few subsets. A second approach for expansion of DC involves the use of human FL (hFL). FL is a hemopoietic growth factor that promotes

Department of Immunology, Schering-Plough Research Institute, Kenilworth, NJ 07033 Received for publication February 25, 2002. Accepted for publication January 6, 2003. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 S.A.L. is an Irene Diamond Associate Professor of Immunology, and this work was supported in part by the Irene Diamond Fund. 2

Address correspondence and reprint requests to Dr. Sergio A. Lira, Mount Sinai School of Medicine, Immunobiology Center, 1425 Madison Avenue, Box 1630, New York, NY 10029-6574. E-mail: [email protected] 3 Abbreviations used in this paper: DC, dendritic cells; BM, bone marrow; DOX, doxycycline; FL, Flt3 ligand; ␤-gal, ␤-galactosidase; GFP, green fluorescent protein; h, human; m, murine; PB, peripheral blood; Tg, transgenic; pDC, plasmacytoid DC.

Copyright © 2003 by The American Association of Immunologists, Inc.

the differentiation and mobilization of hemopoietic progenitors and stem cells in vitro (6, 7) and in vivo (8, 9). Treatment of mice with hFL has been shown to lead to the expansion of DC subsets in multiple tissues and peripheral blood (PB) (5, 10 –12). However, this method involves the daily administration of a foreign protein and manipulation of mice. To overcome these limitations and to provide a model for studying DC biology in vivo, we developed a noninvasive method for expansion of DC subsets. This method consists of conditional expression of the physiological ligand, murine FL (mFL), in multiple tissues of transgenic (Tg) mice.

Materials and Methods Transgenes To generate a bidirectional responder transgene, the cDNA encoding mFL (13) was cloned into pBI-G (Clontech, Palo Alto, CA). The mFL cDNA used in this experiment encodes a functionally active type I transmembrane protein that can be processed to a soluble ligand (14 –16). To generate the activator transgene, cDNA for rtTA was subcloned from pTet-On plasmid (Clontech) into the EcoRI sites of an expression vector containing the hCMV enhancer/chicken ␤-actin promoter and the rabbit ␤-globin polyadenylation signal (17). Separation of the transgene from vector sequence was accomplished by zonal sucrose gradient centrifugation as previously described (18). Fractions containing the transgene were pooled, microcentrifuged through Microcon-100 filters (Amicon, Beverly, MA), and washed five times with microinjection buffer (5 mM Tris-HCl (pH 7.4), 5 mM NaCl, and 0.1 mM EDTA).

Mice Ggeneration of mice that carry the activator transgene (19) or the responder transgene was performed using standard techniques as previously described (20). Identification of Tg founders was conducted by PCR analysis using tail DNA, as previously described (21). The activator and responder mice were crossed to generate mice carrying both transgenes. Identification of double-Tg mice was accomplished by amplification of a segment of the rtTA transgene using the following primers: (5⬘-CGGGTCTACCATC 0022-1767/03/$02.00

2844 GAGGGCCTGCT-3⬘) and (5⬘-CCCGGGGAATCCCCGTCCCCCAAC3⬘) and amplification of a segment of the ␤-galactosidase (␤-gal) gene (5⬘-ACCAGCGAATACCTGTTCCGTCATAGC-3⬘ and (5⬘-AGTAAGG CGGTCGGGATAGTTTTCTTGC-3⬘). Primers for the endogenous ZP3 gene (5⬘-CAGCTCTACATCACCTGCCA-3⬘ and 5⬘-CACTGGGAAGA GACACTCAG-3⬘) were used as an internal control for the amplification reaction. These primers amplify a 242-bp segment of the rtTA transgene, a 791-bp segment of the ␤-gal gene, and a 511-bp segment of the ZP3 gene. PCR conditions were 95°C for 30 s, 60°C for 30 s, and 72°C for 60 s for 30 cycles. The resulting Tg mice were kept under specific pathogen-free conditions. All experiments with mice followed institutional guidelines.

Bone marrow (BM) transfers BM from green fluorescent protein (GFP) Tg mice (22) was harvested, and RBC were lysed using RBC lysing reagent (Sigma-Aldrich, St. Louis, MO). Ten million GFP⫹ BM cells were injected i.v. into lethally irradiated (1100 rad) mice.

Flow cytometry and cell sorting Single-cell suspensions were prepared from individual tissues by passage through a 100-␮m pore size, nylon cell strainer (Falcon, BD Biosciences, Mountain View, CA) in RPMI medium containing 10% FCS. Tissue cell suspensions and PBMC cells were analyzed after lysing the RBC. Cells (105–106) were incubated with 5 ␮g/ml Fc block (BD PharMingen, San Diego, CA) and 300 ␮g/ml mouse IgG (Pierce, Rockford, IL). Cells were stained with Abs in 1⫻ PBS, 1% BSA, and 0.1% sodium azide for 20 min at 4°C in the dark. mAbs to the following mouse surface markers were used: CD8␣ (53-6.7), pan NK (DX5), CD45R/B220 (RA3-6B2), CD11c (HL3), CD11b (M1/70), Ly6G/C (Gr-1, RB6-8C525-9-17), MHC II (IAb), CD3 (145-2C11), CD8b.2 (53-5.8), CD19 (1D3), CD86 (GL1), Ter119 (TER-119), CD4 (L3T4), CD90.2 (Thy1.2, 30-H12), and CD25 (IL-2Ra, PC61; all were purchased from BD PharMingen). F4/80 was purchased from Accurate Chemical and Scientific (Westbury, NY). To determine viability, samples were subsequently stained with 20 ␮l of 5 ␮g/ml propidium iodide (Calbiochem, La Jolla, CA). Events were acquired on a FACSCalibur (BD Biosciences) and analyzed using CellQuest software (BD Biosciences). For the identification and isolation of plasmacytoid cells, cells were stained with Abs against Ly6G/C, CD11c, and the lineage markers CD3, CD8b.2, CD11b, Ter119, and CD19. Cells were electronically gated on lineage-negative cells and screened for expression of CD11c and Ly6G/C. For isolation of plasmacytoid cells, CD11clowLy6G/Clow cells were sorted after electronic depletion of lineage-positive cells using a FACSVantage (BD Biosciences).

Mixed lymphocyte reaction Allogeneic T and DC were isolated for an MLR. For the isolation of DC from non-Tg B6D2 mice, spleens were harvested, single-cell suspensions were prepared as described above, and cells were stained with Abs against CD11c and the lineage markers CD3, CD4, Thy1.2, IL-2R ␣, B220, Gr-1, CD11b, F4/80, and Ter119. Lin⫺ CD11c⫹ cells were sorted after electronic depletion of lineage-positive cells using a FACSVantage (BD Biosciences). For the isolation of DC from Tg mice (H2b/d), splenocytes were stained with Abs against CD11b, CD11c, and the lineage markers NK1.1, Thy1.2, Ter119, and B220. Cells were electronically gated on lineagenegative cells and screened for the expression of CD11c and CD11b. Lin⫺ CD11chigh CD11blow/⫺ and Lin⫺ CD11chigh CD11b⫹ cells were sorted after electronic depletion of lineage-positive cells using a FACSVantage (BD Biosciences). Allogeneic CD4⫹ T cells were isolated from B10RIII (H2r) using Miltenyi MACS columns (Miltenyi Biotec, Bergisch Gladbach, Germany). Peripheral (axial, brachial, and inguinal) lymph nodes were harvested, and single-cell suspensions were prepared as described above. The cells were stained with biotinylated Abs against CD11b, CD8␣, B220, and IAd (MHC II), followed by incubation with streptavidin microbeads. CD4⫹ T cells were isolated to 95% purity by depleting the Abcoated cells using a magnetic column according to the manufacturer’s recommendations (Miltenyi Biotec). The MLR reaction was performed in a 96-well, round-bottom tissue culture plate. Allogeneic CD4⫹ T cells (1⫻ 105) were incubated with varying amounts of Tg DC (Lin⫺ CD11chigh CD11blow/⫺ and Lin⫺ CD11chigh CD11b⫹) or control DC (CD11c) in 0.2 ml of DMEM containing penicillin/streptomycin, L-glutamine, 10% FCS, and 10⫺2 M 2-ME in humidified 10% CO2 in air for 5 days. The cultures were pulsed with 1 ␮Ci of [3H]thymidine for 16 h, and the cells were harvested onto glass-fiber sheets for counting in a liquid scintillation counter (205 Betaplate; Wallac, Turku, Finland). Counts for the T cells, non-Tg DC, and Tg DC were between 200 and 300 cpm.

CONDITIONAL EXPRESSION OF FL IN Tg MICE Cytospins Spleens were harvested from control and Tg mice, single-cell suspensions were prepared, and Tg (CD11chigh CD11blow/⫺ and Lin⫺ CD11chigh CD11b⫹) and control (CD11c⫹) DC were sorted from total splenocytes as previously described. Cytospins were prepared by cytospinning 5 ⫻ 104 cells onto slides for 10 min at 500 rpm.

Viral stimulation of plasmacytoid cells Sorted plasmacytoid cells were plated at 105 cells/well in 60 ␮l of RPMI medium containing penicillin/streptomycin, L-glutamine, and 10% FCS in a 96-well, round-bottom tissue culture plate. To stimulate IFN-␣ production, 3 ⫻ 106 PFU of UV-inactivated HSV-1 virus was added to the plasmacytoid cells, and the incubation was continued for 24 h at 37°C in humidified 10% CO2 in air for 24 h. The culture supernatants were harvested, and the amount of IFN-␣ was determined by ELISA as described below.

Statistical analysis All experiments were performed in duplicate or triplicate, with three to five mice per group. The statistical difference between the mean values of groups was evaluated with Student’s t test for comparisons between paired samples. Multiplicity was taken into account using the Bonferroni method. Results are presented as the mean ⫾ SD. Data were analyzed using PRISM 3.0 (GraphPad, San Diego, CA).

Histology Fresh mouse tissues were fixed immediately after necropsy in 10% buffered formalin and then processed for paraffin sections. Paraffin sections (5 ␮m) were prepared routinely and stained with H&E. For immunohistochemical staining, fresh-frozen sections were prepared and fixed with ice-cold acetone. Slides were then incubated with purified anti-CD11c (Chemicon International, Temecula, CA) for 1 h at room temperature, followed by incubation with biotinylated goat anti-hamster Ab (Vector Laboratories, Burlingame, CA) for 30 min. After incubation with an avidin-biotin-HRP complex (Vectastain Elite ABC kit, Vector Laboratories) for 30 min, the tissue sections were stained with NovaRed (Vector Laboratories) and counterstained with hematoxylin. For ␤-gal histochemistry, fresh-frozen sections were fixed with 2% paraformaldehyde in PBS and then stained following a procedure previously described (23). Briefly, the slides were incubated overnight in the presence of 5-bromo-4-chloro-3-indolyl-␤-Dgalactopyranoside (X-gal; Roche, Indianapolis, IN) and counterstained with neutral red.

ELISA PB was collected, and the serum was separated using Multivette 600 LHGel (Sarstedt, Nu¨ mbrecht, Germany). The serum was diluted 1/25, and the mFL concentration was determined using an ELISA kit specific for mFL (R&D Systems, Minneapolis, MN). The level of sensitivity of this assay is 5 pg/ml. Medium from the cultured cells was harvested 24 h after coculture with HSV. The IFN-␣ concentration was determined using an ELISA kit specific for mIFN-␣ (R&D Systems).

Doxycycline (DOX) preparation For oral treatment, a 0.02 mg/ml solution of DOX (Sigma-Aldrich) was prepared by dissolving DOX in water containing 0.5% sucrose. The solution was placed in dark drinking water bottles and was changed every 4 days. For acute treatment, DOX (500 ␮g) was diluted in 0.2 ml of PBS and injected i.p.

Results Generation of Tg mice To generate Tg mice that conditionally express mFL, we used the tetracycline-dependent gene expression system originally described by Gossen and Bujard (24). In this bigenic system the tet-activator protein (rtTA) is expressed constitutively from the activator transgene (Fig. 1). In the presence of tetracycline (or one of its analogs, such as DOX), the rtTA protein binds to a tetracycline-responsive promoter element present on a responder transgene and induces transgene expression. The generation of mice carrying and expressing the activator transgene in multiple tissues was previously described (19). Mice carrying the responder transgene were generated by microinjection of mouse eggs with the


2845 in many reports in the literature. It is possible that the lower levels of expression of mFL in two of the four lines may be caused by integration of the transgene into different chromosomal regions (positional effects). Tg mice from line 1372 (referred to hereafter as Tg mice), which showed the highest induction of mFL, were selected for expansion and further studies. Uninduced mice did not show any gross abnormalities and had a normal life span. DOX treatment induces transgene expression in multiple tissues

FIGURE 1. Schematic representation of the activator and responder transgenes. In the activator transgene the hCMV enhancer (hCMVe) was juxtaposed to the chicken ␤-actin promoter (c␤Actpr) to control the expression of the transcription factor rtTA. The responder transgene encodes two genes (␤-gal and mFL) in opposite orientation. Because this transgene lacks a strong promoter of its own, its expression is often null or negligible. However, transcription of the ␤-gal and mFL genes can be strongly induced if DOX and rtTA are present. TRE, Tetracycline-responsive element; CMV, minimal CMV promoter; r␤glob, rabbit ␤-globin.

bidirectional transgene encoding ␤-gal and mFL as described in Fig. 1. Eleven founders carrying the responder transgene were crossed with Tg mice expressing the activator transgene in multiple tissues (CMTA line 72) (19). The offspring from these matings were screened for the presence of both transgenes by PCR. Eight of the 11 founders transmitted the responder transgene to their offspring, and animals carrying both activator and responder transgenes were selected for further analysis. To select a Tg line for further studies we determined mFL serum levels in wild-type and four Tg lines before and after DOX treatment (500 ␮g i.p.). Serum levels of mFL in controls and Tg mice before DOX treatment averaged 0.5 ng/ml. Twenty-four hours after DOX injection the serum levels of mFL were substantially elevated (⬎50 ng/ml) in Tg animals from two of the four lines tested. FL levels in the serum of control mice treated with DOX did not differ from those in untreated control mice. The levels of mFL in the circulation were only marginally elevated (⬍2-fold) in the serum of the two remaining lines (Fig. 2). Significant differences in the expression levels among Tg lines is a common finding

FIGURE 2. Levels of mFL in the serum of control and Tg mice. Levels of FL in control mice (C) did not differ from levels in Tg mice prior to (P) DOX treatment. Twenty-four hours after DOX injection (500 ␮g i.p.) the levels of mFL were dramatically elevated in the serum of animals in two of the four lines tested. Bars represent the mean ⫾ SD of three animals per group. The pre-DOX values were derived from animals in line 13.

As indicated in Fig. 1, the responder transgene encodes both mFL and ␤-gal. To test whether DOX treatment induced ␤-gal expression we treated Tg mice with DOX and examined the expression of ␤-gal in several tissues, including spleen, muscle, thymus, pancreas, lymph nodes, brain, lung, heart, liver, kidney, intestine, trachea, and tongue. No ␤-gal expression was detected before DOX treatment (Fig. 3, A, C, and E), but it could easily be detected in multiple tissues after DOX treatment. The most intense ␤-gal staining was detected in the exocrine pancreas (Fig. 3F) and skeletal muscle (Fig. 3B). ␤-Gal staining was also detected in kidney (Fig. 3D) and liver (not shown), but not in hemopoietic organs, such as BM, spleen, and thymus (not shown). The expression of ␤-gal in the kidney was detected in the cortex. The likely source of transgene expression in this organ is the podocyte, since a recent study by Imai and coworkers (25) using the same promoter as that employed here demonstrated expression of GFP in this cell type. This expression pattern of ␤-gal described here is similar to that observed previously using a different responder transgene, but the same activator line (19). Thus, the expression of the responder transgene in multiple tissues after DOX treatment is likely to account for the high levels of mFL in serum. Conditional expression of FL leads to expansion of CD11c⫹ cells in blood It is well established that exogenous treatment of mice with hFL induces the expansion of DC in blood and several tissues (5, 10, 11). Therefore, having detected expression of the responder transgene in multiple tissues and high circulating levels of mFL, we examined the DC numbers in Tg mice. We first determined the number of DC in PB of animals treated with DOX. Acute treatment (96 h) induced dramatic changes in the circulating levels of mFL, which were, on the average, 200-fold higher than the values found in DOX-treated controls or nontreated Tg mice (Fig. 4A), but did not affect the number of CD11c⫹ cells in the circulation (Fig. 4B). However, chronic treatment of the Tg mice with DOX was associated with significant changes in the number of circulating DC. By 1 wk of DOX treatment, DC represented ⬃40% of the cells in the PB. Interestingly, the high levels of mFL and the elevated number of DC in circulation persisted in treated animals throughout the course of the experiment (27 wk). There were no changes in the number of DC in the blood of nontreated Tg mice at any of the time points examined (data not shown). These results show that DOX treatment of Tg mice induces a dramatic elevation in the level of mFL and the number of DC in the circulation. Next we determined whether the significantly increased number of DC would revert to baseline levels after removal of DOX. After 2 wk of DOX treatment, DC represented ⬃40% of the PBL. DOX was then withdrawn, which led to a decrease in the level of mFL (data not shown) and in the relative number of DC in the circulation (Fig. 5). Within 5 wk of DOX removal, the number of DC had approached baseline levels. Interestingly, at this point, readministration of DOX to the Tg mice led to an increase in serum mFL levels and a blunted, but still significant, DC expansion. These results showed that it is possible to modulate the number of DC in

2846

CONDITIONAL EXPRESSION OF FL IN Tg MICE

FIGURE 3. DOX-dependent expression of ␤-gal in tissues from Tg mice. The ␤-gal staining of fresh frozen tissues prepared from untreated Tg mice (⫺DOX) or from Tg mice treated with DOX (0.02 mg/ml orally) for 2 wk (⫹DOX) is indicated. The expression of ␤-gal is detected in the presence, but not the absence, of DOX. A and B, Skeletal muscle; C and D, kidney; E and F, pancreas.

the circulation by altering the concentration of DOX in the drinking water.

immune diseases; blood cell counts and blood chemistry parameters of hepatic, pancreatic, and renal function were normal.

DOX treatment leads to expansion of cells expressing CD11c in multiple tissues of Tg mice

Expansion of the myeloid and lymphoid DC subsets

Next we examined whether mFL expression would also lead to the expansion of DC in tissues. We examined tissue sections from untreated Tg mice and from Tg mice treated with DOX (0.02 mg/ml orally) for 6 wk. Analysis of H&E-stained paraffin sections showed a significant mononuclear cellular infiltrate in multiple tissues from Tg mice after DOX treatment (data not shown). These infiltrates, which were most abundant in lymphoid organs such as the spleen, lymph nodes, and thymus, were not associated with overt inflammation or necrosis. Phenotypic analysis of these infiltrates using immunohistochemistry indicated that the infiltrates consisted primarily of CD11c⫹ cells. In the thymus, CD11c⫹ cells could be found in both cortex and medulla (Fig. 6B). CD11c⫹ cells were also found in the portal region and the parenchyma of the liver (Fig. 6D), in the epithelium of the small and large intestines (Fig. 6F), in the pancreas (Fig. 6H), and in tongue striated muscle (Fig. 6J). With the exception of the thymus, where they accumulated in the medulla, CD11c⫹ cells were only rarely detected in similar tissues obtained from untreated Tg mice (Fig. 6, A, C, E, G, and I). Despite the elevated levels of mFL and the increased numbers of DC, there was no evidence of any abnormalities or auto-

DC are frequently classified as lymphoid or myeloid based on their expression of the markers CD8␣ and CD11b, respectively. These DC populations apparently differ in their functions: lymphoid DC are less phagocytic, generally secrete IL-12, and initiate a Th1 response, whereas myeloid DC generally are more phagocytic and initiate a Th2 response (1). DC can also be classified on the basis of MHC class II expression, which distinguishes mature and immature DC. To further characterize the DC subsets present in lymphoid and hemopoietic tissues, we used flow cytometry. We observed dramatic changes in the number of DC in these tissues. In the spleen, for instance, there was a significant expansion of both immature (CD11c⫹IAb⫺/low) and mature (CD11c⫹IAb⫹) DC, and of myeloid or CD11c⫹CD8␣⫺CD11b⫹ and lymphoid or CD11c⫹CD8␣⫹CD11b⫺ DC subsets (Fig. 7A). The CD11c⫹ IAb⫹ DC also expressed CD86 and varying levels of CD11b, but they did not express CD3, Gr-1, or Ter119 and were essentially negative for B220 (Fig. 7B). There was also a dramatic expansion in the relative (Table I) and absolute (Table II) numbers of various DC subsets in the thymus, mesenteric lymph node, BM, and PB. Most DC subsets were expanded in most tissues except the thymus, where primarily lymphoid (CD11c⫹CD8␣⫹CD11b⫺) DC


2847 an MLR assay. The stimulatory capacity of DC from Tg mice were compared with those of control (non-Tg) Lin⫺ CD11c⫹ DC. DC from the Tg mice were as efficient as control DC in their ability to stimulate allogeneic T cells (Fig. 8). Expansion of plasmacytoid DC In humans, plasmacytoid DC are the major producers of IFN-␣ in response to viruses or CpG stimulation (26, 27). Recently, the murine counterpart to the hIFN-␣-producing plasmacytoid DC has been identified (28, 29). Plasmacytoid cells are CD11c⫹, B220⫹, and Ly6G/C⫹, are negative for CD19 and CD11b, and express low levels of MHC class II. Asselin-Paturel and coworkers (28) showed that these cells are lineage negative for Ter119, CD3, and CD8b.2. Therefore, to determine whether plasmacytoid cells were expanded in Tg mice after DOX treatment, we analyzed Ter119, CD8b.2, CD3, CD19, and CD11b lineage-negative PBL for the expression of Ly6G/C, CD11c, and B220. This DC subset constituted 5% of the lineage-negative cells before DOX treatment. After 7 wk of DOX treatment, the relative and absolute numbers of this cell population were increased in blood (Fig. 9A), BM, mesenteric

FIGURE 4. Kinetics of mFL expression and DC accumulation in the blood of Tg mice after DOX treatment (0.02 mg/ml orally). A, mFL levels in controls (C) or in Tg mice before treatment (0). After 96 h of DOX treatment the circulating levels of mFL were ⬃200-fold higher than baseline levels. Continued exposure to DOX resulted in chronically elevated levels of mFL. B, Relative numbers of CD11c⫹ cells in the circulation as determined by flow cytometry. Similar relative numbers of DC were detected in controls, nontreated Tg, and Tg mice that had received DOX for 96 h. Significant differences in the proportions of these cells in the circulation were observed within 1 wk of treatment. Bars represent the mean ⫾ SD (n ⫽ 5).

were expanded. The apparent difference in cellularity of the thymus was not statistically significant between the controls and Tg mice treated with DOX. Expanded DC are functional To determine whether Tg DC were functional, we sorted Lin⫺ CD11chigh CD11blow/⫺ and Lin⫺ CD11chigh CD11b⫹ DC and tested them for the ability to stimulate allogeneic CD4⫹ T cells in

FIGURE 5. Modulation of the relative number of CD11c⫹ cells in the circulation as a function of DOX treatment. Mice were given 0.02 mg/ml DOX in the drinking water for 2 wk. At wk 2, DOX was eliminated from the drinking water (Stop). DOX was restarted at wk 9 (Start). Values represent the mean ⫾ SD of five or six mice per point. f, control; ⽧, Tg mice treated with DOX. ⴱ, p ⬍ 0.001 for DOX-treated Tg vs no treatment (0); ⴱⴱ, p ⬍ 0.01 for DOX-treated Tg at 14 and 16 wk vs Tg at 9 wk or no treatment.

FIGURE 6. DOX treatment induces the accumulation of DC in various tissues of Tg mice. Immunohistochemical staining for CD11c in freshfrozen tissues from untreated or DOX-treated Tg mice (0.02 mg/ml orally) for 6 wk. In DOX-treated Tg mice, CD11c⫹ accumulated in the medulla and cortex of the thymus (B), around the portal regions (arrow) and in the parenchyma of the liver (D), in the epithelium of the large intestine (F), around the ductal regions (arrow) and in the exocrine tissue of the pancreas (H), and in the muscle layers of the tongue (J). Sections shown in A, C, E, G, and I represent, respectively, thymus, liver, large intestine, pancreas, and tongue of nontreated Tg mice. I, islets of Langerhans; m, medulla; c, cortex; v, villi; sm, smooth muscle.

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FIGURE 7. Flow cytometric and morphological characterization of CD11c⫹ splenocytes from Tg mice before (⫺DOX) or after DOX treatment (⫹DOX). A, Cells were stained with Abs against CD11c, CD8␣, and CD11b. B, Profiling of the CD11chigh IAbhigh DC subpopulation. Cells were stained with Abs against CD11c, IAb, CD86, B220, Ter119, Ly6G/C, CD3, or CD11b. CD11chigh IAbhigh were screened for the expression of CD86, B220, Ter119, Ly6G/C, CD3, or CD11b. Thin line is control IgG; thick line is specific Ab. C, Cytospins were prepared by cytospinning 5 ⫻ 104 cells onto slides showing the morphological profile of the CD11chigh IAbhigh and CD11chigh IAb⫺ DC. Results are representative of three independent experiments (three to five animals per experiment).

lymph node, spleen, and thymus (Table III). A characteristic of both human (26) and murine (28, 29) plasmacytoid cells is that they secrete IFN-␣ upon viral stimulation. To confirm that the Lin⫺Ly6G/ClowCD11clow cells in Tg mice were similar to the previously described plasmacytoid cells, we cultured them with UVirradiated HSV for 24 h and measured IFN-␣ in the culture supernatants. No IFN-␣ could be detected in the supernatants of isolated cells in the absence of HSV. In contrast, IFN-␣ could be readily measured in the supernatants of Lin⫺Ly6G/ClowCD11clow cells exposed to HSV (Fig. 9C). Only minimal expression (⬍10 pg/ml) of IFN-␣ was detected in Ly6G/C⫺CD11c⫺ cells cultured in the absence or the presence of HSV. These results conclusively demonstrate that in vivo expression of mFL can induce the expansion of a DC subset showing the characteristics of plasmacytoid cells. Expansion of GFP-expressing DC To determine whether the Tg mFL could drive DC proliferation from transplanted BM, we developed BM chimeras. These chimeras were generated by grafting BM from Tg mice expressing GFP in hemopoietic cells (22) into lethally irradiated mFL Tg mice. One week after BM transplantation, ⬎95% of the PB cells of the host were GFP⫹. Treatment of these Tg mice with DOX caused a significant increase in the relative number of GFP⫹ DC in circulation. In nontreated mice GFP⫹ DC constituted 2% of all PBL, and in the treated mice they constituted 16% of the PBL (Fig. 10).

These results indicate that expression of mFL by the host induced the expansion of DC precursors present in the grafted BM.

Discussion Despite a large body of evidence supporting the existence of multiple DC subsets (for review, see Ref. 30), little is known about the differences in gene expression profiles, trafficking, and localization patterns of these subsets during immune responses and pathological conditions. Efforts in this direction have been hampered by the limited number of DC present in tissues and PB. In this manuscript we describe a novel Tg system for the conditional expansion of DC subsets in vivo. This system has the potential to greatly facilitate analysis of the biological role of the diverse DC subsets. The Tg mice described here conditionally express mFL in multiple tissues. These animals were generated by intercrossing lines of mice carrying a responder transgene (encoding both FL and ␤-gal) with a line of Tg mice expressing the trans-activator gene in a variety of organs (19). Animals carrying both transgenes were examined before and after DOX treatment, and a specific line (cross of activator line 72 with responder line 13) was chosen for the studies shown here. These animals were chosen because 1) we could not detect expression of mFL in serum or ␤-gal in tissues before DOX treatment (e.g., there was no “leakiness” in transcription of the target genes); and 2) they showed a dramatic response to DOX treatment; the levels of mFL in circulation were rapidly


2849

Table I. Relative number of DC subsets in tissues and peripheral blood from Tg mice after DOX treatmenta Thymus

CD11c CD11c⫹IAb⫺ CD11c⫹IAb⫹ CD11c⫹CD11b⫹ CD11c⫹CD8a⫹ Cells ⫻ 10⫺5

Spleen

mLN

Blood

C

Tg

C

Tg

C

Tg

1.4 ⫾ 1.1 0.6 ⫾ 0.5 0.8 ⫾ 0.6 1.3 ⫾ 0.5 1.7 ⫾ 0.8 994 ⫾ 230

5.5 ⫾ 3.8 3 ⫾ 0.5d 2.6 ⫾ 0.6 3.8 ⫾ 2.5d 8.5 ⫾ 3.7b 597 ⫾ 228

9.7 ⫾ 2 3.3 ⫾ 0.8 6.8 ⫾ 1.7 5.6 ⫾ 0.6 1.1 ⫾ 0.1 956 ⫾ 126

40 ⫾ 15b 15 ⫾ 7b 25 ⫾ 8b 19 ⫾ 7d 5.9 ⫾ 0.7c 1130 ⫾ 249

5.8 ⫾ 2 7.3 ⫾ 7 4.2 ⫾ 1.7 3.4 ⫾ 1.4 1.2 ⫾ 0.3 92 ⫾ 31

30 ⫾ 16b 15 ⫾ 7d 15 ⫾ 6b 14 ⫾ 7d 5.4 ⫾ 2.2b 116 ⫾ 58

C

BM Tg

7.9 ⫾ 2.9 38 ⫾ 19b 4.6 ⫾ 1.9 29 ⫾ 16d 3.8 ⫾ 1.9 9.1 ⫾ 4d 5.9 ⫾ 2.2 26 ⫾ 16b 0.2 ⫾ 0.1 1 ⫾ 0.3c 100 ⫾ 68 132 ⫾ 58

C

Tg

5.9 ⫾ 0.8 3.5 ⫾ 0.9 2.6 ⫾ 0.4 3.4 ⫾ 0.6 0.2 ⫾ 0.03 420 ⫾ 49

39 ⫾ 8.6c 29 ⫾ 9c 9.8 ⫾ 1.8c 15 ⫾ 5.8b 2.6 ⫾ 0.7c 450 ⫾ 121

a Blood, BM, thymus, mesenteric lymph nodes (mLN), and spleen were harvested from Tg mice 7 wk after DOX treatment. Single-cell suspensions were prepared by passage through 100-␮m pore size mesh, and the RBC were lysed. Cells were stained with Abs against CD11c, CD8␣, CD11b, and IAb (MHC II). Numbers represent the percentage of cells staining with the specific Abs based on total viable cells. The relative number of cells expressing CD11c in untreated Tg mice did not differ from that in controls. Data are the mean ⫾ SD of three Tg and five control mice and are representative of two independent experiments. Statistical analysis was performed using Student’s t test (two-tailed, paired). b p ⬍ 0.01. c p ⬍ 0.001. d p ⬍ 0.05.

and significantly elevated after a single DOX treatment. The elevated levels of mFL detected in the serum after DOX treatment were most likely caused by widespread expression of the transgene, as suggested by the pattern of ␤-gal expression. In addition to ␤-gal, the responder transgene used in our experiments encoded the natural transmembrane form of mFL, which can be processed to a soluble form (13). Because both the membrane-bound and soluble forms of mFL appear to be biologically active (14), either form may have contributed to the increased number of DC in multiple tissues. Our results confirm and extend several reports in the literature regarding the ability of mFL to induce DC expansion in vivo (for review, see Ref. 31). Treatment of mice with hFL leads to the expansion of DC subsets in blood, BM, lymphoid tissues, liver, and lung (10, 11). These effects are probably mediated by the interaction of hFL with its tyrosine kinase receptor (flt3R) expressed by hemopoietic progenitor cells (9). In agreement with these studies, we observed a time-dependent increase in the number of DC in blood and lymphoid organs as a function of mFL expression. The CD11c⫹ MHCII⫹ spleen-derived DC showed similar phenotypic characterization as those reported previously in mice administered exogenous hFL (10). However, despite the significant increase in the relative and absolute numbers of various DC subsets, tissue cellularity was only slightly affected in our study (Table I), in contrast to the previous reports (5, 10, 32). The effective amount of mFL in the circulation may be the reason for the discrepancy. In previous studies exogenous hFL was used at high concentrations (10 ␮g/day), which could have led to higher systemic levels of hFL

than those reported here. Unfortunately, a direct comparison between our studies is not possible because there is no information on the levels of circulating hFL or its half-life in the studies cited above. We should note that treatment of Tg mice with higher doses of DOX (2 mg/ml in food) leads to even higher levels of mFL and higher numbers of DC in PB and tissues (data not shown). Cessation of mFL induction led to normalization of DC numbers in blood. The number of DC gradually approached the baseline level after removal of DOX. Subsequent administration of DOX led to a second wave of DC expansion, indicating that the effect of mFL on DC expansion was reversible. In our study the number of DC in PB remained elevated for an extended period of time (⬎9 mo), a finding in contrast to that reported by Brasel et al. (9) and Shaw et al. (32). These authors have observed a decline in the number of DC in mice treated for ⬃3 wk with exogenous hFL and have suggested that this decline may have been caused by negative regulatory mechanisms or by a limited number of hemopoietic stem cells available for expansion (32). Our results do not support the concept that there is a limited number of hemopoietic stem cells available for expansion. Although the reasons for the discrepancy between our results and those described above remain unclear, we suggest that treatment of mice with hFL may have led to an immune response to this protein and subsequent inactivation of its biological activity. The functionality of the expanded DC was determined by performing an MLR assay. We have shown that two independent populations of DC sorted from Tg mice are capable of stimulating the proliferation of allogeneic T cells as efficiently as DC isolated

Table II. Total number (⫻10⫺5) of DC subsets in tissues and peripheral blood from Tg mice after DOX treatmenta Thymus

CD11c CD11c⫹IAb⫺ CD11c⫹IAb⫹ CD11c⫹CD11b⫹ CD11c⫹CD8␣⫹ Cells ⫻ 10⫺5

Spleen

C

Tg

13 ⫾ 8 5.1 ⫾ 3.8 7.4 ⫾ 4.5 11 ⫾ 3.8 16 ⫾ 4.1 994 ⫾ 230

30 ⫾ 16 17 ⫾ 11 13 ⫾ 7 21 ⫾ 10 52 ⫾ 35d 597 ⫾ 228

C

MLN Tg

91 ⫾ 15 460 ⫾ 197 32 ⫾ 8 175 ⫾ 92d 64 ⫾ 12 283 ⫾ 105b 42 ⫾ 25 219 ⫾ 104d 10 ⫾ 1 68 ⫾ 19c 956 ⫾ 126 1130 ⫾ 249 b

Blood

C

Tg

5.4 ⫾ 3.3 7.6 ⫾ 8.9 4 ⫾ 2.6 3.2 ⫾ 2.2 1.1 ⫾ 0.4 92 ⫾ 31

40 ⫾ 36 21 ⫾ 22 19 ⫾ 14d 18 ⫾ 14d 7 ⫾ 5.6d 116 ⫾ 58

C

BM Tg

7.6 ⫾ 5.9 55 ⫾ 39 4.1 ⫾ 3.3 42 ⫾ 33 3.5 ⫾ 3 14 ⫾ 9 5.8 ⫾ 4.3 38 ⫾ 31 0.2 ⫾ 0.2 1.4 ⫾ 0.8d 100 ⫾ 68 132 ⫾ 58

C

Tg

25 ⫾ 4.5 15 ⫾ 4 11 ⫾ 2.3 14 ⫾ 3 0.8 ⫾ 0.1 420 ⫾ 49

172 ⫾ 44c 128 ⫾ 46c 43 ⫾ 10c 64 ⫾ 18c 12 ⫾ 5.3c 450 ⫾ 121

a Blood, BM, thymus, mesenteric lymph nodes (mLN), and spleen were harvested from Tg mice 7 wk after DOX treatment. Single-cell suspensions were prepared by passage through 100-␮m pore size mesh, and the RBC were lysed. Cells were stained with Abs against CD11c, CD8a, CD11b, and IAb (MHC II). Numbers represent the total number of cells staining with the specific Abs based on the total number of viable cells. The total number of cells expressing CD11c in untreated Tg mice did not differ from that in controls. Data are the mean ⫾ SD of three Tg and five control mice and are representative of two independent experiments. Statistical analysis was performed using Student’s t test (two-tailed, paired). b p ⬍ 0.01. c p ⬍ 0.001. d p ⬍ 0.05.

2850

FIGURE 8. DC from Tg mice are capable of stimulating allogeneic T cell proliferation. A, The distribution of CD11b and CD11c Ags on lineage (CD3, CD8b.2, CD11b, Ter119, CD19)-depleted splenocytes from Tg mice after 2 wk of DOX treatment (0.02 mg/ml orally). 1) Lin⫺ CD11chigh CD11blow/⫺ and 2) Lin⫺ CD11chigh CD11b⫹ DC were isolated. The stimulating capacity of 1) Lin⫺ CD11chigh CD11blow/⫺ and 2) Lin⫺ CD11chigh CD11b⫹ DC isolated from Tg was comparable to that of control DC isolated from non-Tg mice.

from control non-Tg mice. These data are consistent with those reported by Maraskovsky and coworkers (10), who found that similar populations of DC expressing high levels of CD11c and being CD11b⫹, CD11blow, or negative were capable of stimulating allogeneic or keyhole limpet hemocyanin-specific T cell proliferation. Despite the significantly elevated levels of mFL and the increased number of DC, the Tg mice described here have not shown any abnormal phenotype. Previously, Juan and coworkers (33) reported that mice grafted with BM transduced with a retrovirus encoding mFL developed severe anemia, abnormal cellular infiltrates, vascular occlusion of the spleen, and premature death (33). Most deaths were observed 10 –13 wk after transplantation. Our animals have now survived ⬎36 wk of oral DOX treatment, and we have not observed any of those changes. There are, however, significant differences in our approaches. Juan and coworkers (33) used transduced BM cells in a transplant setting. The Tg mice reported here express mFL in most tissues, but not BM cells or spleen. The expression of mFL in the context of hemopoietic tissues could potentially favor the development of a severe phenotype. Since the levels of mFL were not measured in that study, it remains possible that they were in vast excess over what was observed here. Thus, the conditional Tg system described here provides a method for continued expansion of DC for extended periods of time without deleterious effects on the health or survival of these mice. The system described here will be particularly useful for obtaining DC from PB without sacrificing the mice. One of the most important findings of our study was the demonstration that mFL expression can promote the expansion of plas-


FIGURE 9. Expansion of plasmacytoid cells in blood of Tg mice after DOX treatment. Mice were treated with 0.02 mg/ml of DOX in the drinking water for 6 wk. Cells were stained with Abs against CD11c and Ly6G/C and the lineage Ags CD3, CD8b.2, CD11b, DX5, and CD19. A, Distribution of Ly6G/C and CD11c on blood leukocytes after the electronic depletion of lineage-positive cells. Plasmacytoid cells expressing low levels of Ly6G/C are indicated in the boxed areas. B, Distribution of B220 on Lin⫺CD11clowLy6G/ Clow cells. C, IFN-␣ levels in supernatants from Lin⫺CD11clowLy6G/Clow cells cultured for 24 h in the presence or the absence of HSV-1. Values represent the mean ⫾ SD of three mice. These results are representative of three independent experiments.

macytoid cells. Plasmacytoid cells or DC precursors are the primary cells producing IFN-␣ in the blood (26) and have a major impact on both innate and adaptive immune responses. The production of IFN-␣ by plasmacytoid cells leads to the mobilization and activation of macrophages and NK cells during the acute innate response (1, 26, 34, 35). Subsequently, these plasmacytoid cells differentiate and mature into DC, which stimulate T cells to differentiate into Th1 or Th2 subsets or into T cells that produce both IFN-␣ and IL-10 (for review, see Refs. 1 and 30). Recently, the murine counterpart of human plasmacytoid cells was identified (28, 29). In contrast to human plasmacytoid cells, which lack expression of CD11c (26), murine plasmacytoid cells

Table III. Relative and absolute number of plasmacytoid cells in tissues and peripheral blood from Tg mice after DOX treatmenta Relative No. (%)

Blood BM mLN Spleen Thymus

Absolute No. (⫻10⫺3)

C

Tg

C

Tg

5.1 ⫾ 1.9 3.4 ⫾ 0.8 2.7 ⫾ 0.7 5.9 ⫾ 2.1 0.7 ⫾ 0.8

31.3 ⫾ 16.7b 13 ⫾ 7.2b 19 ⫾ 13.7b 16.5 ⫾ 6.6b 10.5 ⫾ 7.4b

7.1 ⫾ 2.6 69 ⫾ 20 4.6 ⫾ 3.3 146 ⫾ 57 26.3 ⫾ 35.1

374 ⫾ 381 1225 ⫾ 422b 77 ⫾ 50 1927 ⫾ 1069b 185 ⫾ 150

a Blood, BM, thymus, mesenteric lymph nodes (mLN), and spleen were harvested from Tg mice 7 wk after DOX treatment. Single-cell suspensions were prepared by passage through a 100-␮m pore size mesh, and the RBC were lysed. Cells were stained with Abs against CD11c and Ly6G/C and the lineage Ags CD3, DX5, CD8b.2, CD11b, and CD19. The expression of Ly6G/C and CD11c was determined on lineage-negative cells by electronic depletion of lineage-positive cells. The relative and absolute numbers of lineage-negative cells expressing Ly6G/C and CD11c in untreated Tg mice did not differ from those in controls. Numbers represent the percentage or absolute number of cells staining with the specific Abs based on the total number of viable cells. Data are the mean ⫾ SD of three Tg mice and five control mice and are representative of two independent experiments. Statistical analysis was performed using Student’s t test (two-tailed, paired). b p ⬍ 0.05.


FIGURE 10. Generation of GFP-labeled DC. Lethally irradiated Tg mice were reconstituted with BM from GFP-expressing mice. The relative numbers of DC expressing GFP and CD11c in the blood were assessed 2 wk after DOX treatment. Results are representative of two independent experiments using three to five mice in each experiment.

express CD11c. These murine cells also express B220 and Ly6G/C and minimal levels of MHC class II, CD19, and CD11b, but are lineage negative for Ter119, CD3, and CD8b.2 (28). Here we show a dramatic expansion in PB and tissues of DOX-treated mice of a population of cells that is lineage negative (Ter119, CD8b.2, CD3, CD19, and CD11b), but expresses Ly6G/C, CD11c, and B220. Consistent with these cells being plasmacytoid cells, we detected a significant increase in IFN-␣ production upon exposure of these isolated cells to UV-inactivated HSV. IL-12 (p40) was also measured in the supernatants of plasmacytoid DC (pDC) cultures (data not shown). Similar to what was found by Gilliet and coworkers (36), we could not detect IL-12 in the absence of virus. However, we could not document the modest up-regulation of IL-12 by pDCs after viral stimulation as reported by those authors. The reasons for this discrepancy are not clear, but may stem from the source of the DC. We obtained our pDC from the spleens of mice chronically exposed in vivo to mFL, whereas Gilliet et al. (36) obtained pDC from mBM by stimulation in vitro with hFL. In both our report and that by Gilliet, however, there was irrefutable production of IFN-␣ by pDCs upon HSV-1 incubation. These results indicate that mFL expression in vivo can lead to the expansion of a DC subset with the characteristics of murine plasmacytoid cells in blood and multiple tissues. Thus, the Tg mice described here should aid in the characterization of this newly defined murine DC subtype.

2851 Finally, the Tg mice described here should prove very useful for the analysis of trafficking and localization patterns of DC. First by providing the source of a large number of DC of a given subtype that can be isolated and subsequently labeled and used in adoptive transfer studies, and second, by serving as a host for the expansion of genetically modified DC. We have shown here that grafting the FL-expressing mice with BM from GFP-expressing mice can induce the expansion of grafted DC. We anticipate that this approach will be particularly useful in defining the roles of specific molecules, such as chemokines, in DC trafficking. Chemokines induce the chemotaxis of DC in vitro, and chemokine receptors are differentially expressed by DC subsets (reviewed in Refs. 1 and 37– 39). Targeted deletion of the chemokine receptor CCR6 abolishes the ability of a specific subset of DC (CD11b⫹CD11c⫹) to localize within the subepithelial dome of Peyer’s patches (40), and inactivation of CCR7 reduces the migration of mature DC from the skin to the draining lymph nodes (41). These results strongly suggest that chemokines are important regulators of DC trafficking in vivo and that the migration of specific subsets may be controlled by specific chemokines. Experiments involving the transfer of GFP⫹ BM cells from mice lacking a particular chemokine receptor into lethally irradiated Tg mice should help elucidate the role of this chemokine receptor in DC trafficking and localization. In summary, we describe a novel method to generate a large number of mDC in vivo. The method described here is based on the induction of Tg mFL in multiple tissues by DOX. The method is noninvasive and reversible, does not require frequent manipulation of the mice, and is not associated with overt pathology, even after prolonged treatment with DOX. DOX treatment of Tg mice allows for the expansion of all DC subsets analyzed and for obtaining large numbers of DC from blood and tissues. This method should thus greatly facilitate gene expression profiling and trafficking studies of DC subsets and contribute to a better understanding of the role of DC in health and disease.

Acknowledgments We thank G. Zurawski for the mFL cDNA; Robert Chase for the HSV-1 stocks; and David Kinsley, P. Zalamea, B. Wilburn, W. Sharif, and Andrew Morschauser for excellent technical work. We thank Dr. Jun-ichi Miyazaki for the CMV enhancer/chicken ␤-actin promoter cassette.

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