Flt-3 Ligand

Flt-3 Ligand Bali Pulendran* and Stephanie Dillon Baylor Institute for Immunology Research, 3434 Live Oak, Dallas, TX 75204, USA * corresponding author tel: (908) 541-5146, fax: (908) 231-4988, e-mail: [email protected] DOI: 10.1006/rwcy.2001.0909. Chapter posted 5 November 2001

SUMMARY

BACKGROUND

Flt-3 ligand (FL) is a member of a family of cytokines that stimulate the proliferation of hematopoietic cells. Other cytokines in this family include stem cell factor (SCF, also known as c-kit ligand, steel factor or mast cell growth factor) and colonystimulating factor 1 (CSF-1, also known as macrophage colony-stimulating factor (M-CSF)). All three of these cytokines belong to the `helical cytokine' family, whose members have -helical bundles in their structure. FL, SCF, and CSF-1 bind to type III tyrosine kinase receptors (FL with Flt-3 receptor; SCF with c-kit; and CSF-1 with c-fms), from the platelet-derived growth factor receptor (PDGFR) subfamily of tyrosine kinase receptors (Lyman, 1998; Lyman and Jacobsen, 1998; Shurin et al., 1998; Antonysamy and Thomson, 1999). FL is expressed ubiquitously, but expression of Flt-3 receptor is restricted to progenitor cells of the hematopoietic system. FL is similar to SCF in that both proteins stimulate the proliferation of early progenitor or stem cells. Neither of these factors has much proliferative activity on its own, but each factor can synergize with a wide range of other colonystimulating factors and interleukins to stimulate proliferation. A major difference between the two factors appears to be their effect on mast cells and erythroid development, which SCF stimulates but FL does not. In addition, FL has the unique ability to stimulate the generation of multiple dendritic cell (DC) subsets, in vivo, in mice and in humans. As a result, FL has considerable clinical potential in the areas of stem cell regeneration and mobilization, and in the immunotherapy of cancer and infectious diseases (Lyman, 1998; Lyman and Jacobsen, 1998; Shurin et al., 1998; Antonysamy and Thomson, 1999).

Discovery

Cytokine Reference

Two independent groups cloned the FL gene by using a soluble form of the Flt-3 receptor. Lyman et al. (1993a) screened a variety of cell lines and identified a murine T cell line that specifically bound the Flt-3 receptor. The ligand was the cloned from a cDNA expression library made from these cells. Hannum et al. (1994) used an affinity column made with the mouse Flt-3 receptor extracellular domain to purify FL from medium conditioned by a thymic stromal cell line. N-terminal sequencing of the purified protein generated a short amino acid sequence, which was then used to design degenerate oligonucleotide primers to amplify a portion of the FL gene by PCR, subsequently leading to the cloning of the full-length murine cDNA.

Structure FL is a type 1 transmembrane protein with a short cytoplasmic domain, with structural similarities to SCF and CSF-1 (see Description of proteins for more details).

Main activities and pathophysiological roles FL is a growth factor for hematopoietic progenitors in vitro and induces hematopoietic progenitor and stem cell mobilization in vivo. FL alone can weakly stimulate the proliferation of mouse and human hematopoietic progenitors, or can syngergize with a

Copyright # 2001 Academic Press

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number of cytokines to exert more potent effects. In addition, when mice are injected with FL, there is a dramatic expansion of multiple DC subsets. Immature B cells, natural killer (NK) cells, and monocytes are also expanded, to a lesser extent, in vivo. Similarly, injections of FL into humans causes a dramatic expansion of DCs in the blood (Lyman, 1998; Lyman and Jacobsen, 1998; Shurin et al., 1998; Antonysamy and Thomson, 1999).

GENE AND GENE REGULATION

Accession numbers Mus musculus FMS-like tyrosine kinase 3 ligand (Flt3L) mRNA: NM_013520 Homo sapiens fms-related tyrosine kinase 3 ligand (FLT3LG) mRNA: NM_001459 Bos taurus flt3 ligand isoform-2 mRNA, complete cds: AF282986 Bos taurus flt3 ligand isoform-1 mRNA, complete cds: AF282985 Felis catus Flt3 ligand mRNA, complete cds: AF155149 Canis familiaris Flt3 ligand mRNA, complete cds: AF155148

Chromosomal location Mouse FL gene: proximal portion of mouse chromosome 7. Human FL gene: chromosome 19q13.3.

Relevant linkages No genetic diseases have been associated with the loci for FL (Lyman and Jacobsen, 1998).

PROTEIN

Accession numbers Mus musculus: FMS-like tyrosine kinase 3 ligand: NP_038548 Homo sapiens: fms-related tyrosine kinase 3 ligand: NP_001450 Human: Flt3 ligand alternatively spliced isoform: I39076 Felis catus: Flt3 ligand: AAF87089 Canis familiaris: Flt3 ligand: AAF87088

Sequence See Figure 1.

Description of protein FL is a noncovalently linked homodimer, and is biologically active both as a transmembrane form and as a soluble form that is generated by proteolytic cleavage of the extracellular domains of the transmembrane protein (reviewed by Lyman and Jacobsen, 1998). The relative importance of the two forms is unclear. The predominant isoform in humans is the transmembrane protein, which can be proteolytically cleaved to generate a soluble FL molecule (Lyman et al., 1993a, 1994, 1995a; Hannum et al., 1994; McClanahan et al., 1996). In mice, the predominant isoform is a 220 amino acid form that is membrane bound, but not transmembrane (Hannum et al., 1994; Lyman et al., 1995a; McClanahan et al., 1996). This form arises due to a failure to splice an intron from the mRNA. Other isoforms of both human and murine FL have been identified, but their biological significance is unknown (Lyman et al., 1993a, 1994, 1995a; Hannum et al., 1994; McClanahan et al., 1996).

Figure 1 Amino acid sequence for human and mouse Flt-3 ligand. Human 1 mtvlapawsp ttylllllll ssglsgtqdc sfqhspissd favkirelsd yllqdypvtv 61 asnlqdeelc galwrlvlaq rwmerlktva gskmqgller vnteihfvtk cafqpppscl 121 rfvqtnisrl lqetseqlva lkpwitrqnf srclelqcqp dsstlpppws prpleatapt 181 apqpplllll llpvglllla aawclhwqrt rrrtprpgeq vppvpspqdl llveh Mouse 1 mtvlapawsp nsslllllll lspclrgtpd cyfshspiss nfkvkfrelt dhllkdypvt 61 vavnlqdekh ckalwslfla qrwieqlktv agskmqtlle dvnteihfvt sctfqplpec 121 lrfvqtnish llkdtctqll alkpcigkac qnfsrclevq cqpdsstllp prspialeat 181 elpeprprql llllllllpl tlvllaaawg lrwqrarrrg elhpgvplps hp

Flt-3 Ligand 3 The type 1 transmembrane protein consists of a short cytoplasmic domain, with structural similarities to SCF and CSF-1. The genomic loci of the coding regions of mouse and human FL are approximately 4.0 kb and 5.9 kb, respectively. The coding region comprises eight exons (reviewed in Lyman and Jacobsen, 1998). The mouse and human proteins contain 231 and 235 amino acids, respectively. The first 27 (mouse) or 26 (human) amino acids constitute a signal peptide that is absent from the mature protein, followed by a 161 (mouse) or 156 (human) amino acid extracellular domain, a 22 (mouse) or 23 (human) amino acid transmembrane domain, and a 21 (mouse) or 30 (human) amino acid cytoplasmic tail. The mouse and human FL proteins each contain two potential sites for glycosylation.

Discussion of crystal structure The crystal structure of FL reveals that it is a homodimer of two short-chain -helical bundles (Savvides et al., 2000). Comparisons with the homologous cytokines SCF and CSF-1 suggest that they have a common receptor-binding mode that is distinct from the complex of growth hormone with its receptor. Furthermore, there are recognition features common to all helical and cystine-knot protein ligands that activate type III tyrosine kinase receptors, and the closely related type V tyrosine kinase receptors (Savvides et al., 2000).

CELLULAR SOURCES AND TISSUE EXPRESSION

Cellular sources that produce Flt-3 ligand FL is ubiquitously expressed in different tissues in both mice and humans; highest expression is found in human peripheral blood mononuclear cells (PBMCs), but expression is also found in most other tissues examined (Rosnet et al., 1991; Matthews et al., 1991; Brasel et al., 1995; DeLapeyriere et al., 1995). In contrast, Flt-3 receptor expression is largely restricted to the progenitor/stem cell compartment.

RECEPTOR UTILIZATION FL utilizes Flt-3 receptor.

IN VITRO ACTIVITIES

In vitro findings A number of studies have investigated the effect of FL on the expansion of hematopoietic cells in vitro. FL alone weakly stimulates the growth of hematopoietic stem and progenitor cells as well as lineages in the lymphoid and myeloid pathways, but has a much more potent effect when used in combination with other growth factors (Table 1; Lyman, 1998; Lyman and Jacobsen, 1998; Shurin et al., 1998; Antonysamy and Thomson, 1999). For example, FL induces the growth of colony-forming cells and progenitors from murine bone marrow cells when used in combination with IL-3, IL-6, IL-11, IL-12, granulocyte±macrophage colony-stimulating factor (GMCSF), granulocyte colony-stimulating factor (G-CSF), thrombopoietin (TPO), CSF-1, or SCF (Hudak et al., 1995; Jacobsen et al., 1995; Ramsfjell et al., 1996). The strongest synergy was observed when FL was used in combination with the ligands that did not signal through the tyrosine kinase receptor. FL is a weak proliferative stimulus for Sca-1‡Linlo cells isolated from mouse fetal liver, but synergizes with IL-7 or SCF to promote growth (Lyman et al., 1993b). FL has a weak proliferative capacity on human bone marrow, cord blood, and peripheral blood cells, but synergizes with other growth factors such as GMCSF, IL-3, SCF, IL-6, G-CSF, IL-1 , TPO and erythropoietin (EPO) to induce the ex vivo expansion and colony growth of human hematopoietic cells (Lyman et al., 1994; Broxmeyer et al., 1995; Gabbianelli et al., 1995; McKenna et al., 1995; Brashem-Stein et al., 1996; Koller et al., 1996; Shapiro et al., 1996; Piacibello et al., 1998). Human cord blood cells expanded for 12 weeks in culture with FL, SCF, TPO, and IL-6 have been successfully engrafted into sublethally irradiated nonobese diabetic severe combined immunodeficient mice and the addition of IL-3 greatly reduced the repopulating potential of these cultures (Piacibello et al., 1999, 2000). It has been postulated that the increased recruitment of CD34‡CD38ÿ cells into the cell cycle is the basis for the ex vivo expansion potential of FL (Haylock et al., 1997). FL acts synergistically with G-CSF to enhance expansion of colony growth from human fetal liver (Muench et al., 1997). FL acts in synergy with various combinations of other growth factors and cytokines in the early stages of thymopoiesis (Freedman et al., 1996; Moore and Zlotnik, 1997), NK cell development (Miller et al., 1998) and B cell development (Hirayama et al., 1995;

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Table 1 Flt-3 ligand acts in synergy with other growth factors to induce the expansion of various hematopoietic progenitor cell populations Starting population

Combination of growth factors

Reference

Expansion and colony formation of human stem/progenitor cells CD34‡ BM and CB

FL ‡ PIXY321, IL-3, or GM-CSF

Lyman et al., 1994

Primitive progenitors

FL ‡ SCF, IL-3, IL-6 or GM-CSF

Hannum et al., 1994

CD34‡ BM

FL ‡ PIXY321; FL ‡ EPO ‡ PIXY321; FL ‡ IL-1 ‡ IL-3 ‡ IL-6 ‡ EPO

McKenna et al., 1995

CD34‡ Linÿ CD38ÿ

FL ‡ IL-3, IL-6, G-CSF, GM-CSF or SCF; FL ‡ IL-3 ‡ SCF

Brashem-Stein et al., 1996

CD34‡ BM and CB

FL ‡ GM-CSF, G-CSF, CSF-1; FL ‡ IL-3  SCF, FL ‡ EPO ‡ SCF

Broxmeyer et al., 1995

CD34‡ BM and PB

FL ‡ IL-3, GM-CSF, TPO

Gabbianelli et al., 1995

BM and PB

FL ‡ GM-CSF, IL-3

Koller et al., 1996

CD34‡ CD38ÿ BM

FL ‡ IL-3, SCF

Petzer et al., 1996

CD34‡ CD38ÿ BM

FL ‡ IL-3, IL-6, SCF

Shah et al., 1996

CD34‡ CB

FL ‡ TPO

Piacibello et al., 1998

CD34‡‡ fetal liver

FL ‡ IL-3, GM-CSF, SCF

Muench et al., 1995

CD34‡ BM, CB, PB, fetal liver

FL ‡ IL-3 ‡ IL-6 ‡ G-CSF

Shapiro et al., 1996

Generation of human T cells using irradiated thymic stromal layers CD34‡ BM

FL ‡ IL-12

Freedman et al., 1996

Proliferation of human CD34‡ CD19‡ pro-B cells Fetal BM

FL ‡ IL-7

Namikawa et al., 1996

Frequency of human NK cell progenitors using stroma noncontact culture CD34‡ Linÿ DRÿ BM

FL ‡ IL-2 ‡ IL-7 ‡ SCF ‡ stromal factors

Miller et al., 1998

Expansion of human megakaryocyte colony-forming units CD34‡ PB stem cells

FL ‡ IL-3, IL-6, IL-11, SCF, TPO, MIP-1

Halle et al., 2000

CD34‡ CB

FL ‡ TPO

Li et al., 2000

Expansion and colony formation of murine stem/progenitor cells Linÿ Ly-6A/E‡ BM

FL ‡ IL-6, IL-11, G-CSF

Hirayama et al., 1995

Thylo Sca-1‡ Linÿ BM

FL ‡ IL-3, IL-6, SCF, GM-CSF, G-CSF, IL-10

Hudak et al., 1995

Linÿ Sca-1‡ BM

FL ‡ IL-11, IL-12, G-CSF, IL-3, IL-6, SCF

Jacobsen et al., 1996

Linÿ Sca-1‡ BM

FL ‡ TPO; FL ‡ SCF ‡ TPO; FL ‡ IL-11 ‡ TPO; FL ‡ IL-12 ‡ TPO

Ramsfjell et al., 1996

Maintenance of murine progenitor and stem cells with reconstituting ability Linÿ Sca-1‡ c-kit‡ BM

FL ‡ IL-11

Yonemura et al., 1997

Proliferation and differentiation of murine T cells Thymic CD4lo cells

IL-3 ‡ IL-6 ‡ IL-7

Moore and Zlotnik, 1997

FL ‡ SF ‡ IL-7

Hirayama et al., 1995

Proliferation of murine B cells Linÿ Ly-6A/E‡ BM

BM, bone marrow; CB, cord blood; PB, peripheral blood; PIXY-321, GM-CSF/IL-3 fusion protein.

Flt-3 Ligand 5 Namikawa et al., 1996; Jayne et al., 1999). FL, again acting in synergy with other growth factors, can increase the expansion of megakaryocyte progenitors in human cord blood (Li et al., 2000) and peripheral blood stem cells (Halle et al., 2000). Transforming growth factor (TGF ) and tumor necrosis factor  (TNF) inhibit the growth of murine and human hematopoietic cells induced by FL (Jacobsen et al., 1996; Ohishi et al., 1996; Ramsfjell et al., 1997). FL does not appear to have any effects on murine (Hudak et al., 1995; Jacobsen et al., 1995) or human (Hannum et al., 1994; McKenna et al., 1995) erythroid development in vitro. Similarly, FL does not have any effect on mast cell or eosinophil development (Lyman et al., 1995b; Hudak et al., 1995; Hjertson et al., 1996). This is in contrast to SCF which does have potent effect on these two lineages (Costa et al., 1996).

The potential of FL to expand various cell populations in vitro The ability of FL to expand hematopoietic cell populations has been utilized in a number of different in vitro culture systems to provide increased numbers of cells, particularly DCs, for either further analysis or for further manipulation (Table 2).

Bioassays used The quantification of FL has been performed on a Ba/F3 cell line expressing murine Flt-3/flk-2 receptor (Hannum et al., 1994). Additionally, the WWF7, a murine Pro-B cell line, is used as a bioassay for FL (Brasel et al., 1995).

Table 2 Flt-3 ligand can be used as a growth factor to expand cell populations, particularly DCs, in vitro Cell type

In vitro treatment

Result

Reference

Human PB CD34‡ cells

FL, GM-CSF, TNF, SCF, IL-4

More efficient than SCF to generate functional CD1a‡ DCs

Guardiola et al., 1997

Human PBMCs

FL, IL-4; maturation by TNF or CD40L

Generation of DCs

Brossart et al., 1998

Human CB cells

FL, TNF, GM-CSF, SCF

Generation CD1a‡ CD14ÿ and CD1aÿ CD14‡ DCs

Canque et al., 1998

Human BM CD34‡ cells

FL, GM-CSF, IL-4, TNF,  SCF

Increase in numbers of functionally mature DCs

Maraskovsky et al., 1996

Human BM CD34‡ cells

FL, G-CSF, EPO, MGDF, IL-6, IL-3, SCF

Preferential expansion of precursor cells, CD34‡ cells, CFU-GM, BFU-E, LTC-IC

Kobari et al., 1998

Human BM CD34‡ cells

FL, GM-CSF, TNF, TGF , SCF

Maximum expansion and purity of DCs

Soligo et al., 1998

Human BM CD34‡ cells

FL, SCF, TNF, TGF 1, GM-CSF

FL further enhanced LC development

Strobl et al., 1998

Murine BM and spleen GM-CSF, IL-4 (prior in vivo administration of FL)

Large numbers of functionally mature DCs

Shurin et al., 1997

Murine BM

FL; activation induced by LPS or IFN

Generation of functional CD8‡ and CD8ÿ DCs

Brasel et al., 2000

Murine livers (prior in vivo administration of FL)

GM-CSF, IL-4

Large numbers of functional DCs Drakes et al., 1997

Bovine BM

FL, GM-CSF, IL-4

Generation of functional DCs exclusively of the myeloid origin

Baboon BM (prior in vivo administration of G-CSF and SCF)

FL and other hematopoietic Increase in gene transfer growth factors into hematopoietic cells

Hope et al., 2000 Kiem et al., 1998

PB, peripheral blood; PBMC, peripheral blood mononuclear cells; CB, cord blood; BM, bone marrow; LC, Langerhans cells; MGDF, megakaryocyte growth and development factor; CFU-GM, colony-forming unit±granulocyte macrophage; BFU-E, burst-forming unit erythroid; LTC-IC, long-term culture-initiating cell.

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IN VIVO BIOLOGICAL ACTIVITIES OF LIGANDS IN ANIMAL MODELS

Normal physiological roles The in vivo effects of FL have been thoroughly investigated with many reflecting the observations made in vitro (Table 3). Chinese hamster ovary (CHO)-expressed FL (rather than yeast-derived FL or E.coli-derived FL) has profound hematopoietic effects when injected into mice for 2 weeks (Brasel et al., 1996). There is a large increase in the peripheral blood leukocyte count comprising of an approximate 3-fold increase in lymphocytes, a 10-fold increase in granulocytes, and a 78-fold increase in monocytes. Leukocyte counts also rise in bone marrow and spleen. In addition, a profound increase of hematopoietic progenitor cells in the spleen (100-fold), blood (200- to 500-fold) and bone marrow (5-fold) occurs. FL administration has no apparent effect on mast cell proliferation or activation. FL synergistically enhances the G-CSF-induced in vivo expansion of peripheral blood progenitors in mouse blood, increasing the levels of monocytes and basophils and mobilizing blood stem cells with long-term repopulating potential (Sudo et al., 1997; Molineux et al., 1997; Ashihara et al., 1998). Similar results were found when FL was injected into primates (Papayannopoulou et al., 1997). Furthermore, in mice, the numbers and functional activity of CD3ÿ NK1.1‡ NK cells is increased in bone marrow, thymus, blood, spleen, and liver (Shaw et al., 1998). In contrast, an increase in numbers of CD3‡ NK1.1‡ T cells occurred mainly in the spleen. Perhaps the most striking, and clinically relevant effect of FL injections is the dramatic increase in DC numbers (Table 3; discussed below).

Effects on DC development Daily FL injections into mice causes a dramatic increase in the numbers of mature myeloid (CD11c‡ CD11b‡ CD8ÿ MHC class II‡) and putative lymphoid-related (CD11c‡ CD11bÿ CD8‡ MHC class II‡) DC subsets in various lymphoid and nonlymphoid tissues such as the spleen, lymph nodes, Peyer's patch, thymus, bone marrow, blood, lungs, liver, gut-associated lymphoid tissues, and the peritoneal cavity (Maraskovsky et al., 1996; Pulendran et al., 1997). Maximal numbers of DCs are obtained when FL is injected for 9±11 days, with

DC numbers plateauing with continued injection. Following cessation of treatment however, there is a dramatic decline in the numbers of DCs, with baseline levels being reached within a week. Phenotypically and functionally, and in their microenvironmental localization, FL-generated DCs appear identical to the corresponding DC subsets in normal mice (Maraskovsky et al., 1996; Pulendran et al., 1997; Shurin et al., 1997). When freshly isolated they possess numerous dendrites, a feature of mature DCs. They express significant levels of CD86, and efficiently stimulate CD4 and CD8 T cells in vitro and in vivo. FL-generated myeloid and lymphoidrelated DCs induce TH2 and TH1 responses, respectively, in vivo (Pulendran et al., 1999) in a similar fashion to responses observed with these DC subsets from normal mice (Maldanado et al., 1999). This ability to differentially modulate T helper responses may, in part, be explained by differences in their ability to secrete IL-12 (Maldanado et al., 1999; Pulendran et al., 1999). Recently, the intravenous injection into mice of naked DNA that encoded secreted human FL dramatically increased the number of both DCs and NK cells (He et al., 2000). Mature DCs do not express Flt-3 receptor and do not proliferate when cultured in FL alone. Thus the potent effects of FL on DC development are most likely due to its ability to induce the expansion and differentiation of DC precursors or progenitors. Indeed, in addition to elevated numbers of mature DCs, FL injections also increase the numbers of precursor and immature DCs, which express much lower levels of MHC class II, CD11c, and costimulatory molecules (Maraskovsky et al., 1996; Pulendran et al., 1997).

Species differences The effects of FL in vitro are not species-specific, with recombinant human FL and recombinant murine FL active on human cord blood and bone marrow and on mouse bone marrow (Lyman et al., 1994; Broxmeyer et al., 1995). Additionally, recombinant human FL has been used to expand hematopoietic cells and DCs in vivo in the mouse (Brasel et al., 1996; Maraskovsky et al., 1996). In fact, human FL can stimulate mouse, cat, rabbit, nonhuman primate, and human cells (Lyman and Jacobsen, 1998).

Knockout mouse phenotype In mice lacking FL (FLÿ/ÿ mice, generated by targeted gene disruption), leukocyte cellularity was

Flt-3 Ligand 7 Table 3 In vivo activities of Flt-3 ligand Description of FL

Administration

Effect

Reference

CHO cell-hFL

Daily injections into mice, i.p. or s.c., 15 days

Expansion of CFU in BM and mobilization of hematopoietic progenitors/stem cells into PB and spleen

Brasel et al., 1996

CHO cell-hFL

Daily injections into mice, s.c., 9 days

Increase in DC number in multiple tissues; identified two major DC subsets

Maraskovsky et al., 1996

CHO cell-hFL

Daily injections into mice, s.c., 9 days

Detailed analysis of DC populations expanded by FL

Pulendran et al., 1997

CHO cell-hFL

Daily injections into mice, s.c., up to 10 days

Time-dependent, reversible accumulation of DCs in spleen, BM, lymph nodes and liver and extramedullary hematopoiesis

Shurin et al., 1997

CHO cell-hFL

Daily injections into mice, i.p., 10 days

Increase in nonparenchymal cells in liver. Propagation of DCs when cultured with GM-CSF and IL-4

Drakes et al., 1997

CHO cell-hFL and rmGM-CSF

Daily injections into mice, s.c., 3±9 days

Synergistic effect of mobilization of hematopoietic stem and progenitor cells

Sudo et al., 1997

E. coli expressed rhFL and rhG-CSF

Continuous s.c infusion into mice

Synergistic effect to increase monocytes and basophils in blood and to mobilize cells that facilitate long-term hematopoietic engraftment

Molineux et al., 1997

Yeast- or CHO cell-hFL and rhG-CSF

Daily injections into primates, s.c, 7±12 days

Synergistic effect in mobilization of progenitors into blood

Papayannopoulou et al., 1997

CHO cell-hFL

Daily injections into mice, s.c., 9 days

Enhancement of B and T cell responses in vivo to soluble protein and prevention of establishment of peripheral tolerance

Pulendran et al., 1998

CHO cell-hFL

Daily injections into mice, i.p, up to 18 days

Increase in number of functional NK cells in multiple tissues

Shaw et al., 1998

FL

Daily injections into mice, s.c, 5±6 days

Mobilization of primitive and committed progenitors into PB, dose-dependent

Ashihara et al., 1998

CHO cell-hFL

Daily injections into mice, s.c., 9 days

Expansion of DC subsets with functional differences

Pulendran et al., 1999

DNA encoding human FL (hFlex)

Single injection into mice, i.v.

Expansion of functional DCs and NK cells

He et al., 2000

CHO cell-hFL

Daily injections into humans, s.c., 14 days

Phase 1 clinical study; Expansions of DC precursors and subsets

Maraskovsky et al., 2000

CHO cell-hFL or G-CSF

Daily injections into humans, s.c., 10 days (FL) or 5 days (G-CSF)

FL mobilized CD11c‡ DCs and CD11cÿ DCs into blood; G-CSF only increased levels of CD11cÿ DC subset.

Pulendran et al., 2000

CHO cell-hFL, Chinese hamster ovary cell-derived human FL; rm, recombinant murine; rh, recombinant human; CFU, colony-forming unit; BM, bone marrow; PB, peripheral blood.

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reduced in the bone marrow, peripheral blood, lymph nodes, and spleen (McKenna et al., 2000). Thymic cellularity, blood hematocrit, and platelet numbers were not affected. Significantly reduced numbers of myeloid and B lymphoid progenitors were noted in the bone marrow of FLÿ/ÿ mice. In addition a marked deficiency of NK cells in the spleen was noted. DC numbers were also reduced in the spleen, lymph node, and thymus. Both myeloid-related (CD11c‡ CD11b‡ CD8ÿ MHC class II‡) and lymphoid-related (CD11c‡ CD11bÿ CD8‡ MHC class II‡) DC numbers were reduced (McKenna et al., 2000). This confirms that FL has an important role in the expansion of early hematopoietic progenitors and in the generation of mature peripheral leukocytes. Mice lacking the Flt-3 receptor exhibit a number of hematological defects but the phenotype is not as profound as for the FL knockout mice (Mackarehtschian et al., 1995; McKenna et al., 2000).

Transgenic overexpression The effect of chronic expression of FL on in vivo hematopoiesis was studied by retroviral vectormediated gene transfer in a mouse model of bone marrow transplantation to enforce expression of mouse FL cDNA in hematopoietic tissues (Juan et al., 1997). Peripheral blood white blood cell counts in FL-overexpressing recipients were significantly elevated compared with controls. With the exception of eosinophils, all nucleated cell lineages studied were similarly affected in these animals. Experimental animals also exhibited severe anemia and progressive loss of marrow-derived erythropoiesis. All of the FL-overexpressing animals, but none of the controls, died between 10 and 13 weeks posttransplantation. Upon histological examination, severe splenomegaly was noted, with progressive fibrosis and infiltration by abnormal lymphoreticular cells. Abnormal cell infiltration also occurred in other organ systems, including bone marrow and liver. In situ immunocytochemistry on liver sections showed that the cellular infiltrate was CD3‡/ NLDC145‡/CD11c‡, but B220ÿ and F4/80ÿ, suggestive of a mixed infiltrate of DCs and activated T lymphocytes. Infiltration of splenic blood vessel perivascular spaces resulted in vascular compression and eventual occlusion, leading to splenic necrosis consistent with infarction (Juan et al., 1997). These results confirm that FL can affect both myeloid and lymphoid cell lineages in vivo.

PATHOPHYSIOLOGICAL ROLES IN NORMAL HUMANS AND DISEASE STATES AND DIAGNOSTIC UTILITY

Normal levels and effects In normal individuals, serum levels of FL are relatively low (<100 pg/mL) when compared with levels of other structurally homologous growth factors, SCF and CSF-1, which are present at levels ranging from 2 to 6 ng/mL, and 2 to 4 ng/mL, respectively (Lyman et al., 1995; Hjertson et al., 1996).

Role in experiments of nature and disease states Enhanced levels of FL (<10 ng/mL) have been noted in certain stem cell or progenitor cell disorders such as Fanconi anemia and acquired aplastic anemia (Lyman et al., 1995b). Cancer patients treated with chemotherapy and/or radiation also have high levels of serum FL (Wodnar-Filipowicz et al., 1996). It has been postulated that the augmentation in FL in these situations is the result of a physiological homeostatic response to increase the level of FL to stimulate the proliferation of the remaining stem cells (Lyman et al., 1995b). A priori, the levels of FL are not elevated in disease states that affect the erythroid lineage (e.g.  thalassemia or red cell aplasia). Normal levels of FL result in an individual when hematological disorders are treated with immunosuppressive therapy, blood transfusions or a bone marrow transplant (Lyman et al., 1995b; Wodnar-Filipowicz et al., 1996).

IN THERAPY The potent effects of FL on hematopoiesis and DC development have prompted much interest in its therapeutic potential. A plethora of preclinical studies have suggested numerous applications for FL which are considered below. DC-based Immunotherapies DCs, by virtue of their unique ability to capture, process, and present antigens, and their potent immunoregulatory potentials, have been described as `Nature's adjuvants' (Steinman, 1991). However,

Flt-3 Ligand 9 their exploitation for clinical use has been hampered by their rarity. As such, FL has prompted much interest as a potential agent for tumor therapy or a vaccine adjuvant in infectious disease settings. FL injections have been observed to have antitumor activities in mouse models of fibrosarcoma (Lynch et al., 1997), breast cancer (Chen et al., 1997), leukemia (Pawlowska et al., 1998), ovarian cancer (Silver et al., 2000) and, when combined with IL-12, C3 sarcoma liver metastases (PeÂron et al., 1998). The effect appears to be mediated, in part, by CD8‡ T cells (presumably cytotoxic T cells), because tumorrejecting activity can be transferred by spleen cells, but not by CD8‡ or Thy1‡ depleted spleen cells (Lynch et al., 1997). NK cells also contribute since depletion of these cells leads to a greatly decreased antitumor effect by FL on C3 sarcoma liver metastases (PeÂron et al., 1998) and an absence of an FL-mediated delay in the growth of ovarian tumors engrafted in SCID mice (Silver et al., 2000). CD40L and FL act synergistically to promote the proliferation and survival of DCs and increase antitumor immunity against B10.2 and 87 sarcomas (Borges et al., 1999). Favre-Felix et al. (2000) recently showed that FL lessens the growth of tumors obtained after a colon cancer cell line was injected into rats, but it did not restore tumorsuppressed DC function. In addition, injection of B16 melanoma cells expressing FL resulted in the dramatic increase of CD11c‡ cells in the spleen and tumor infiltrate (Mach et al., 2000). Results from these studies suggest that FL can potentially be used in the treatment of human cancer patients. DCs generated by FL in mice can efficiently stimulate antigen-specific CD4‡ and CD8‡ T cells in vitro and in vivo (Maraskovsky et al., 1996; Pulendran et al., 1998; Daro et al., 2000). FL administration to mice dramatically enhances antigen-specific B and T cell responses against soluble antigens, injected subcutaneously or intraperitoneally, without any other adjuvants (Pulendran et al., 1998). Interestingly, FL injection is also able to abrogate the peripheral tolerance induced by systemic injections of soluble antigens (Pulendran et al., 1998). Injection of FL also markedly enhances the magnitude of polyomavirusspecific CD8‡ T cell responses during acute infection and the pool of memory anti-polyomavirus CD8‡ T cells (Drake et al., 2000). These findings suggest that virus-infected DCs induce polyoma virus-specific CD8‡ T cells in vivo and raise the potential for the use of FL as a vaccine adjuvant to promote CD8‡ T cell immunity against viral infections (Drake et al., 2000). In addition, FL was

recently shown to augment the immune response to an HIV peptide vaccine in mice (Pisarev et al., 2000). The HGP-30 peptide used in these studies is a synthetic peptide that corresponds to a highly conserved region of HIV-1 p17 gag. Mice were immunized with HGP-30 or HGP-30 conjugated to keyhole limpet hemocyanin and delayed-type hypersensitivity responses, antibody (IgG) amount and antigen-specific proliferative responses by spleen cells were used to monitor the immune response. Daily injections of FL prior to HGP-30 administration enhanced significantly the antigen-specific lymphocyte proliferation response when compared with FL, HGP-30 alone, or HGP-30 containing liposomes. In humans, FL causes a dramatic increase in the numbers of immature DCs in the peripheral blood (Maraskovsky et al., 2000; Pulendran et al., 2000). Experiments are now underway to assess whether healthy volunteers injected with FL exhibit increases in B and T cell responses towards a cocktail of antigens such as KLH, tetanus toxoid, and the hepatitis and influenza vaccines. In addition to FL acting as a potent immune adjuvant, studies have shown that the expansion of DCs by FL can augment the induction of oral tolerance (Viney et al., 1998). In contrast, a combination of FL and IL-1 induces a potent active response to fed soluble antigen rather than the tolerogenic response noted with FL alone (Williamson et al., 1999). The contrasting effects on T cell tolerance to orally versus systemically administered antigens may reflect functional differences in DC subsets or differences in microenvironments (Pulendran et al., 1998). It is possible that the tolerance-inducing capacity of FL could be utilized in transplantation. Donor microchimerism, the long-term persistence of donor hematopoietic cells in transplant recipients, has been postulated to be a vital prerequisite for the induction of immune tolerance to transplanted organs (Starzl et al., 1996). The use of FL to augment natural microchimerism is now being investigated as a new immunotherapeutic approach to allograft rejection. FL can increase microchimerism in allogenic bone marrow and heart transplant recipients in the presence of immunosuppression (Antonysamy et al., 1998; for a detailed review see Antonysamy and Thomson, 1999). On a cautionary note, there was an increase in the antidonor response and reduced graft survival when the immunosuppressive drug was withdrawn, highlighting the risks of creating an immunological imbalance between donor and host cells following the use of growth factors.

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Mobilization of Stem Cells Injection of FL for 10±14 days into mice, primates, and humans produces a large increase in the number of colony-forming cells in peripheral blood (reviewed by Lyman and Jacobsen, 1998). Stem cells mobilized by FL results in long-term multilineage hematopoietic reconstitution in mice (Brasel et al., 1996). As observed in vitro, FL also synergizes with either GM-CSF or GCSF to mobilize colony-forming cells into peripheral blood in mice (Molineux et al., 1997; Brasel et al., 1996) and primates (Papayannopoulou et al., 1997). These properties make FL an attractive candidate for the treatment of patients who are hematopoietically compromised (for detailed reviews see Emerson, 1996; Lyman and Jacobsen, 1998). Gene Therapy The ability of hematopoietic stem cells to self renew and produce large numbers of progeny makes them ideal targets for gene therapy. FL can be used to stimulate stem cell proliferation, thereby providing the actively dividing cells that are necessary for retroviral transduction. The combination of FL and IL-3 was most potent in promoting the transduction efficiency of a retrovirus into human hematopoietic cells (Elwood et al., 1996). The potential to target gene therapy to DCs that have been expanded by FL is also appealing (Antonysamy and Thomson, 1999). Radioprotective Effects Support for the radioprotective clinical potential of FL has been demonstrated in vivo where FL alone or in combination with G-CSF protected rabbits against the effects of total body irradiation on the stem cell compartment (Gratwohl et al., 1998). FL in combination with SCF, TPO, and IL-3 reduces radiation-induced apoptosis in CD34‡ progenitor cells in vitro, further suggesting a potential therapeutic role in preserving functional hematopoietic progenitor and stem cells from in vivo radiation damage (Drouet et al., 1999). Toxicity FL administration to healthy human volunteers appears to be safe (Maraskovsky et al., 2000; Pulendran et al., 2000). Clinical Results Clinical trials are now underway to determine the safety and efficacy of transplanting cell populations

expanded ex vivo with FL. Hematopoietic reconstitution following high-dose chemotherapy was observed when small volumes of bone marrow aspirates from patients with breast cancer were expanded ex vivo with EPO, PIXY 321 (GM-CSF/IL-3 fusion protein) and FL (Bachier et al., 1999; Stiff et al., 2000). Trials have begun using FL to expand human DC populations in vivo. In a phase I clinical study, FL was administered to healthy human volunteers resulting in an expansion of circulating CD11c‡ and CD11cÿ DC subsets and precursors (Maraskovsky et al., 2000). Both FL and G-CSF enhance DC subset numbers in the peripheral blood of healthy human volunteers, with FL expanding both the precursor DC (CD11cÿ) subset and the immature DC (CD11c‡) subset, whereas G-CSF expands only the precursor subset (Pulendran et al., 2000). Studies are now underway to determine if FL and G-CSF elicit distinct types of immune responses in healthy individuals and thereby provide a novel strategy for the manipulation of immune responses in humans. The number of DCs mobilized into the peripheral blood of patients with colon cancer metastatic to the liver or lung increased following in vivo administration of FL, an observation that may be associated with increases in DC numbers at the periphery of the tumors (Morse et al., 2000).

ACKNOWLEDGEMENTS We thank Dr Hilary McKenna (Immunex) for her expert advice during the preparation of this manuscript. Supported by grants from Baylor Health Care Foundation and NIH (DK57665-01, AI48638-01 and 1R21AI/DE48154-01) to BP.

References Antonysamy, M. A., and Thomson, A. W. (1999). FLT3 ligand (FL) and its influence on immune reactivity. Cytokine 12, 87±100. Antonysamy, M. A., Steptoe, R. J., Khanna, A., Rudert, W. A., Subbotin, V. M., and Thomson, A. W. (1998). Flt-3 ligand increases microchimerism but can prevent the therapeutic effect of donor bone marrow in transiently immunosuppressed cardiac allograft recipients. J. Immunol. 160, 4106±4113. Ashihara, E., Shimazaki, C., Sudo, Y., Kikuta, T., Hirai, H., Sumikuma, T., Yamagata, N., Goto, H., Inaba, T., Fujita, N., and Nakagawa, M. (1998). FLT-3 ligand mobilizes hematopoietic primitive and committed progenitor cells into blood in mice. Eur. J. Haematol. 60, 86±92. Bachier, C. R., Gokmen, E., Teale, J., Lanzkron, S., Childs, D., Franklin, W., Shpall, E., Douville, J., Weber, S., Muller, T., Armstrong, D., and Le Maistre, C. F. (1999). Ex-vivo expansion of bone marrow progenitor cells for hematopoietic recon-

Flt-3 Ligand 11 stitution following high-dose chemotherapy for breast cancer. Exp. Hematol. 27, 615±623. Borges, L., Miller, R. E., Jones, J., Ariail, K., Whitmore, J., Fanslow, W., and Lynch, D. H. (1999). Synergistic action of fms-like tyrosine kinase 3 ligand and CD40 ligand in the induction of dendritic cells and generation of anti-tumor immunity in vivo. J. Immunol. 163, 1289±1297. Brasel, K., Escobar, S., Anderberg, R., de Vries, P., Gruss, H-J., and Lyman, SD. (1995). Expression of the flt3 receptor and its ligand on hematopoietic cells. Leukemia 9, 1212±1218. Brasel, K., McKenna, H. J., Morrissey, P. J., Charrier, K., Morris, A. E., Lee, C. C., Williams, D. E., and Lyman, S. D. (1996). Hematologic effects of flt3 ligand in vivo in mice. Blood 88, 2004±2012. Brasel, K., De Smedt, T., Smith, J. L., and Maliszewski, C. R. (2000). Generation of murine dendritic cells from flt3-ligandsupplemented bone marrow cultures. Blood 96, 3029±3039. Brashem-Stein, C., Flowers, D. A., and Berstein, I. D. (1996). Regulation of colony forming cell generation by Flt-3 ligand. Br. J. Haematol. 94, 17±22. Brossart, P., GruÈnebach, F., Stuhler, G., Reichardt, V. L., MoÈhle, R., Kanz, L., and Brugger, W. (1998). Generation of functional human dendritic cells from adherent peripheral blood monocytes by CD40 ligation in the absence of granulocyte-macrophage colony-stimulating factor. Blood 92, 4238± 4247. Broxmeyer, J. E., Lu, L., Cooper, S., Ruggieri, L., Li, Z. H., and Lyman, S. D. (1995). Flt3 ligand stimulates/costimulates the growth of myeloid stem/progenitor cells. Exp. Hematol. 23, 1121±1129. Canque, B., Camus, S., Yagello, M., and Gluckman, J. C. (1998). IL-4 and CD40 ligation affect differently the differentiation, maturation, and function of human CD34‡ cell-derived CD1a‡CD14ÿ and CD1aÿCD14‡ dendritic cell precursors in vitro. J. Leukoc. Biol. 64, 235±244. Chen, K., Braun, S., Lyman, S., Fan, Y., Traycoff, C. M., Wiebke, E. A., Gaddy, J., Sledge, G., Browmeyer, H. E., and Cornetta, K. (1997). Antitumor activity and immunotherapeutic properties of Flt3-ligand in a murine breast cancer model. Cancer Res. 57, 3511±3516. Costa, J. J., Demetri, G. D., Harrist, T. J., Dvorak, A. M., Hayes, D. F., Merica, E. A., Menchaca, D. M., Gringeri, A. J., Schwartz, L. B., and Galli, S. J. (1996). Recombinant human stem cell factor (kit ligand) promotes human mast cell and melanocyte hyperplasia and functional activation in vivo. J. Exp. Med. 183, 2681±2686. Daro, E., Pulendran, B., Brasel, K., Teepe, M., Pettit, D., Lynch, D. H., Vremec, D., Robb, L., Shortman, K., McKenna, H. J., Maliszewski, C. R., and Maraskovsky, E. (2000). Polyethylene glycol-modified GM-CSF expands CD11bhighCD11chigh but not CD11blowCD11chigh murine dendritic cells in vivo: a comparative analysis with Flt3 ligand. J. Immunol. 165, 49±58. DeLapeyriere, O., Naquet, P., Planche, J., Marchetto, S., Rottapel, R., Gambarelli, D., Rosnet, O., and Birnbaum, D. (1995). Expression of the Flt3 tyrosine kinase receptor gene in mouse hematopoietic and nervous tissues. Differentiation 58, 351±359. Drake D. R. III., Moser J. M., Hadley A., Altman J. D., Maliszewski C., Butz E., and Lukacher A. E. (2000). Polyomavirus-infected dendritic cells induce antiviral CD8‡ T lymphocytes. J. Virol. 9, 4093±4101. Drakes, M. L., Lu, L., Subbotin, V. M., and Thomson, A. W. (1997). In vivo administration of flt3 ligand markedly stimulates generation of dendritic cell progenitors from mouse liver. J. Immunol. 159, 4268±4278.

Drouet, M., Mathieu, J., Grenier, N., Multon, E., Sotto, J-J., and Herodin, F. (1999). The reduction of in vitro radiation-induced Fas-related apoptosis in CD34‡ progenitor cells by SCF, FLT-3 ligand, TPO, and IL-3 in combination resulted in CD34‡ cell proliferation and differentiation. Stem Cells 17, 273±285. Elwood, N. J., Zogos, H., Willson, T., and Begley, C. G. (1996). Retroviral transduction of human progenitor cells: use of granulocyte colony-stimulating factor plus stem cell factor to mobilize progenitor cells in vivo and stimulation by Flt3/Flk-2 ligand in vitro. Blood 88, 4452±4462. Emerson, S. G. (1996). Ex vivo expansion of hematopoietic precursors, progenitors, and stem cells: the next generation of cellular therapeutics. Blood 87, 3082±3088. Favre-Felix, N., Martin, M., Maraskovsky, E., Fromentin, A., Moutet, M., Solary, E., Martin, F., and Bonnotte, B. (2000). Flt3 ligand lessens the growth of tumors obtained after colon cancer cell injection in rats but does not restore tumorsuppressed dendritic cell function. Int. J. Cancer 86, 827±834. Freedman, A. R., Zhu, H., Levine, J. D., Kalams, S., and Scadden, D. T. (1996). Generation of human T lymphocytes from bone marrow CD34‡ cells in vitro. Nature Med. 2, 46±51. Gabbianelli, M., Pelosi, E., Montesoro, E., Valtieri, M., Luchetti, L., Samoggia, P., Vitelli, L, Barberi, T., Testa, U., Lyman, S., and Peschle, C. (1995). Multi-level effects of flt3 ligand on human hematopoiesis: expansion of putative stem cells and proliferation of granulomonocytic progenitors/monocytic precursors. Blood 86, 1661±1670. Gratwohl, A., John, L, Baldomero, H., Roth, J., Tichelli, A., Nissen, C., Lyman, S. D., and Wodnar-Filipowicz, A. (1998). FLT-3 ligand provides hematopoietic protection from total body irradiation in rabbits. Blood 92, 765±769. Guardiola, P., Lacassagne, M. N., Benbunan, M., Dal Cortivo, L., and Marolleau, J-P. (1997). FLT3-L versus SCF for the generation of dendritic cells from peripheral blood CD34‡ hematopoietic stem cells. Exp. Hematol. 25, 884 (abstract). Halle, P., Rouzier, C., Kanold, J., Boiret, N., Rapatel, Cl, Mareynat, G., Tchirkov, A., Berger, M., Travade, P., Bonhomme, J., and Demeocq, F. (2000). Ex vivo expansion of CD34‡/CD41‡ late progenitors from enriched peripheral blood CD34‡ cells. Ann. Hematol. 79, 13±19. Hannum, C., Culpepper, J., Campbell, D., McClanahan, T., Zurawksi, S., Bazan, J. F., Kastelein, R., Hudak, S., Wagner, J., Mattson, H., Luh, J., Duda, G., Martina, N., Peterson, D., Manon, S., Shanafelt, A., Muench, M., Kelner, G., Namikawa, R., Rennick, D., Roncarolo, M-G., Zlotnic, A., Rosnet, O., Dubreuil, P., Birnbaum, D., and Lee, F. (1994). Ligand for flt3/flk2 receptor regulates growth of hematopoietic stem cells and is encoded by variant RNAs. Nature 68, 643±648. Haylock, D. N., Horsfall, M. J., Dowse, T. L., Ramshaw, H. S., Niutta, S., Protopsaltis, S., Peng, L., Burrell, C., Rappold, I., Buhring, H-J., and Simmons, P. (1997). Increased recruitment of hematopoietic progenitor cells underlies the ex vivo expansion potential of FLT3 ligand. Blood 90, 2260±2272. He, Y., Pimenov, A. A., Nayak, J. V., Plowey, J., Falo, L. D., and Huang, L. (2000). Intravenous injection of naked DNA encoding secreted flt3 ligand dramatically increase the number of dendritic cells and natural killer cells in vivo. Hum. Gene Ther. 11, 547±554. Hirayama, F., Lyman, S. D., Clark S. C., and Ogawa, M. (1995). The flt3 ligand supports proliferation of lymphohematopoietic progenitors and early B-lymphoid progenitors. Blood 85, 1762±1768. Hjertson, M., SundstroÈm, C., Lyman, S. D., Nilsson, K., and Nilsson, G. (1996). Stem cell factor differentiation and activation of human mast cells. Exp. Hematol. 24, 748±754.

12

Bali Pulendran and Stephanie Dillon

Hope, J. C., Werling, D., Collins, R. A., Mertens, B., and Howard, C. J. (2000). Flt-3 ligand, in combination with bovine granulocyte-macrophage colony-stimulating factor and interleukin-4, promotes the growth of bovine bone marrow derived dendritic cells. Scand. J. Immunol. 51, 60±66. Hudak, S., Hunte, B., Culpepper, J., Menon, S., Hannum, C., Thompson-Snipes, L., and Rennick, D. (1995). Flt3/Flk2 ligand promotes the growth of murine stem cells and the expansion of colony-forming cells and spleen colony-forming units. Blood 85, 2747±2755. Jacobsen, S. E. W., Okkenhaug, C., Myklebust, J., Veiby, O. P., and Lyman, S. D. (1995). The Flt3 ligand potently and directly stimulates the growth and expansion of primitive murine bone marrow progenitor cells in vitro: synergistic interactions with interleukin 11, IL-12, and other hematopoietic growth factors. J. Exp. Med. 181, 1357±1363. Jacobsen, S. E., Veiby, O. P., Myklebust, J., Okkenhaug, C., and Lyman, S. D. (1996). Ability of flt3 ligand to stimulate the in vitro growth of primitive murine hematopoietic progenitors is potently and directly inhibited by transforming growth factor-beta and tumor necrosis factor-alpha. Blood 87, 5016±5026. Jayne, J. A., Reid, S., Braun, S. E., and Broxmeyer, H. E. (1999). Differential in vitro maturation of hematopoietic stem cells from wild-type and immunoglobulin transgenic mice. Cytokines Cell. Mol. Ther. 5, 195±204. Juan, TS., McNiece, IK., Van G., Lacey, D., Hartley, C., McELroy, P., Sun, Y., Argento, J., Hill, D., Yan XQ., and Fletcher, FA. (1997). Chronic expression of murine flt3 ligand in mice results in increased circulating white blood cell levels and abnormal cellular infiltrates associated with splenic fibrosis. Blood. 90(1), 76±84. Kiem, H. P., Andrews, R. G., Morris, J., Peterson, L., Heyward, S., Allen J. M., Rasko, J. E., Potter, J., and Miller, A. D. (1998). Improved gene transfer into baboon marrow repopulating cells using recombinant human fibronectin fragment CH-296 in combination with interleukin-6, stem cell factor, FLT-3 ligand megakaryocyte growth and development factor. Blood 92, 1878±1886. Kobari, L, Giarrantana, M. C., Poloni, A., Firat, H., Lobopin, M., Gorin, N. C., and Douay, L. (1998). Flt 3 ligand, MGDF, Epo and G-CSF enhance ex vivo expansion of hematopoietic cell compartments in the presence of SCF, IL-3 and IL-6. Bone Marrow Transplant. 21, 759±767. Koller, M. R., Oxender, M., Brott, D. A., and Palsson, B. O. (1996). Flt-3 ligand is more potent than c-kit ligand for the synergistic stimulation of ex vivo hematopoietic cell expansion. J. Hematother. 5, 449±459. Li, K., Yang, M., Lam, A. C., Yau, F. W., and Yuen, P. M. (2000). Effects of flt-3 ligand in combination with TPO on the expansion of megakaryocytic progenitors. Cell Transplant. 9, 125±131. Lyman, S. D. (1998). Biologic effects and potential clinical applications of Flt3 ligand. Curr. Opin. Hematol. 5, 192±196. Lyman, S. D., and Jacobsen, S. E. W. (1998). C-kit ligand and Flt3 ligand: stem/progenitor cell factors with overlapping yet distinct activities. Blood 91, 1101±1134. Lyman, S. D., James, L., Vanden Bos, T., de Vries, P., Brasel, K., Gliniak, B., Hollingsworth, L. T., Picha, K. S., McKenna, H. J., Splett, R. R., Cletcher, F. F., Maraskovsky, E., Farrah, T., Foxworthe, D., Williams, D. E., and Beckmann, M. P. (1993a). Molecular cloning of a ligand for the flt3/flk-2 tyrosine kinase receptor: a proliferative factor for primitive hematopoietic cells. Cell 75, 1157±1167.

Lyman, S. D., James, L., Zappone, J., Sleath, P. R., Beckmann, M. P., and Bird, T. (1993b). Characterization of the protein encoded by the flt3 receptor like tyrosine kinase gene. Oncogene 8, 815±821. Lyman, S. D., James, L., Johnson, L., Brasel, K., de Vries, P., Escobar, S. S., Downey, H., Splett, R. R., Beckmann, M. P., and McKenna, H. J. (1994). Cloning of the human homologue of the murine Flt 3 ligand: a growth factor for early hematopoietic progenitor cells. Blood 83, 2795±2801. Lyman, SD., Stocking, K., Davison, B., Fletcher, F., Johnson, L., and Escobar, S. (1995a). Structural analysis of human and murine flt3 ligand genomic loci. Oncogene. 11, 1165±1172. Lyman, S. D., Seaberg, M., Hanna, R., Zappone, J., Brasel, K., Abkowitz, J. L., Prchal, J. T., Schultz, J. C., and Shahidi, N. T. (1995b). Plasma/serum levels of flt3 ligand are low in normal individuals and highly elevated in patients with Fanconi anemia and acquired aplastic anemia. Blood 86, 4091±4096. Lynch, D. H., Andreasen, A., Maraskovsky, E., Whitmore, J., Miller, R. E., and Schuh, J. C. L. (1997). Flt3 ligand induces tumor regression and antitumor immune responses in vivo. Nature Med. 3, 625±631. Mach N., Gillessen S., Wilson S. B., Sheehan C., Mihm M., and Dranoff G. (2000). Differences in dendritic cells stimulated in vivo by tumors engineered to secrete granulocyte-macrophage colonystimulating factor or Flt3-ligand. Cancer Res. 60, 3239±3246. Mackarehtschian, K., Hardin, J. D., Moore, K. A., Boast, S., Goff, S. P., and Lemischka, I. R. (1995). Targeted disruption of the flk2/flt3 gene leads to deficiencies in primitive hematopoietic progenitors. Immunity 3, 147±161. Maldanado, R., De Smedt, T., Michel, P., Godfroid, J., Pajak, B., Heirman, C., Thielemans, K., Leo, O., Urbain, J., and Moser, M. (1999). CD8‡ and CD8ÿ subclasses of dendritic cells direct the development of distinct T helper cells in vivo. J. Exp. Med. 189, 587±592. Maraskovsky, E., Brasel, K., Teepe, M., Roux, E. R., Lyman, S. D., Shortman, K., and McKenna, H. J. (1996). Dramatic increase in the numbers of functionally mature dendritic cells in Flt3 ligand-treated mice: multiple dendritic cell subpopulations identified. J. Exp. Med. 184, 1953±1962. Maraskovsky, E., Daro, E., Roux, E., Teepe, M., Maliszewski, C. R., Hoek, J., Caron, D., Lebsack, M. E., and McKenna H. J. (2000). In vivo generation of human dendritic cell subsets by Flt3 ligand. Blood 96, 878±884. Matthews, W., Jordan, C. T., Wiegand, G. W., Pardoll, D., and Lemischka, I. R. (1991). A receptor tyrosine kinase specific to hematopoietic stem and progenitor cell-enriched populations. Cell 65, 1143±1149. McClanahan, T., Culpepper, J., Campbell, D., Wagner, J., Franzbacon, K., Mattson, J., Tsai, S., Luh, J., Guimaraes, M. J., Mattei, M. G., Rosnet, O., Birnbaum, D., and Hannum, C. H. (1996). Biochemical and genetic characterization of multiple splive variants of the Flt3 ligand. Blood 88, 3371±3382. McKenna, H. J., de Vries, P., Brasel, K., Lyman, S. D., and Williams, D. E. (1995). Effect of flt3 ligand on the ex vivo expansion of human CD34‡ hematopoietic progenitor cells. Blood 86, 3413±3420. McKenna, H. J., Stocking, K. L., Miller, R. E., Brasel, K., De Smedt, T., Maraskovsky, E., Maliszewski, C. R., Lynch, D. H., Smith, J., Pulendran, B., Roux, E. R., Teepe, M., Lyman, S. D., and Peschon, J. J. (2000). Mice lacking flt3 ligand have deficient hematopoiesis affecting hematopoietic progenitor cells, dendritic cells, and natural killer cells. Blood 95, 3489± 3497.

Flt-3 Ligand 13 Miller, J. S., McCullar, V., and Verfaillie, C. M. (1998). Ex vivo culture of CD34‡Linÿ/DRÿ cells in stoma-derived soluble factors, interleukin-3 and macrophage inflammatory protein-1 maintains not only myeloid but also lymphoid progenitors in a novel switch culture assay. Blood 91, 4516±4522. Molineux, G., McCrea, C., Qiang Yan, X., Kerzic, P., and McNiece, I. (1997). Flt-3 ligand synergizes with granulocyte colony-stimulating factor to increase neutrophil numbers and to mobilize peripheral blood stem cells with long-term repopulating potential. Blood 89, 3998±4004. Moore, T. A., and Zlotnik, A. (1997). Differential effects of Flk-2/ Flt-3 ligand and stem cell factor on murine thymic progenitor cells. J. Immunol. 158, 4187±4192. Morse, M. A., Nair, S., Fernandez-Casal, M., Deng, Y., St Peter, M., Williams, R., Hobeika, A., Mosca, P., Clay, T., Cumming, R. I., Fisher, E., Clavien, P., Proia, A. D., Niedzwiecki, D., Caron, D., and Lyerly, H. K. (2000). Peroperative mobilization of circulating dendritic cells by Flt3 ligand administration to patients with metastic colon cancer. J. Clin. Oncol. 18, 3883±3893. Muench, M. O., Roncarolo, M. G., Menon, S., Xu, Y., Kastelein, R., Zurawski, S., Hannum, C. H., Culpepper, J., Lee, F., and Namikawa, R. (1995). FLK-2/FLT-3 ligand regulates the growth of early myeloid progenitors isolated from human fetal liver. Blood 85, 963±972. Muench, M. O., Roncarolo, M. G., Rosnet, O., Birnbaum, D., and Namikawa, R. (1997). Colony-forming cells expressing high levels of CD34 are the main targets for granulocyte colonystimulating factor and macrophage colony-stimulating factor in the human fetal liver. Exp. Hematol. 25, 277±287. Namikawa, R., Muench, M. O., de Vries, J. E., and Roncarolo, M-G. (1996). The FLKs/FLT3 ligand synergizes with interleukin-7 in promoting stromal-cell-independent expansion and differentiation of human fetal pro-B cells in vitro. Blood 87, 1881±1890. Ohishi, K., Katayama, N., Itoh, R., Mahmud, N., Miwa, H., Kita, K., Minami, N., Shirakawa, S., Lyman S. D., and Shiku, H. (1996). Accelerated cell-cycling of hematopoietic progenitors by the flt3 ligand that is modulated by transforming growth factorbeta. Blood 87, 1718±1727. Papayannopoulou, T., Nakamoto, B., Andrews, R. G., Lyman, S. D., and Lee, M. Y. (1997). In vivo effects of Flt3/Flk2 ligand on mobilization of hematopoietic progenitors in primates and potent synergistic enhancement with granulocyte colonystimulating factor. Blood 90, 620±629. Pawlowska, A. B., Hashino, S., McKenna, H. J., Taylor, P. A., and Blazar, B. R. (1998). The in vivo infusion of either Flt3 ligand protein or ex vivo generated BM-derived dendritic cells (DC) pulsed with leukemia antigen(s) protect naive or syngeneic BMT mice from a lethal dose of acute myeloid leukemia (AML) cells. Blood 92, 721a (abstract). PeÂron, J-M., Esche, C., Subbotin, V. M., Maliszewski, C., Lotze, M. T., and Surin, M. R. (1998). FLT-3 ligand administration inhibits liver metastases: role of NK cells. J. Immunol. 161, 6164±6170. Petzer, A. L., Zandstra, P. W., Piret, J. M., and Eaves, C. J. (1996). Differential cytokine effects on primitive (CD34‡ CD38ÿ) human hematopoietic cells: novel responses to Flt3ligand and thrombopoietin. J. Exp. Med. 183, 2551±2558. Piacibello, W., Sanavio, F., Garetto, L., Dane, A., Gammaitoni, L, and Aglietta, M. (1998). The role of c-Mpl ligands in the expansion of cord blood hematopoietic progenitors. Stem Cells 16, 243±248. Piacibello, W., Sanavio, F., Severino, A., Dane, A., Gammaitoni, L, Fabioli, F., Perissinotto, E., Cavalloni, G., Kollet, O., Lapidot, T., and Aglietta, M. (1999). Engraftment in nonobese

diabetic severe combined immunodeficient mice of human CD34(‡) cord blood cells after ex vivo expansion: evidence for the amplification after sefl-renewal of repopulating stem cells. Blood 93, 3736±3749. Piacibello, W., Gammaitoni, L., Bruno, S., Gunetti, M., Fagioli, F., Cavalloni, G., and Aglietta, M. (2000). Negative influence of IL-3 on the expansion of human cord blood in vivo long-term repopulating stem cells. J. Hematother. Stem Cell Res. 9, 945±956. Pisarev V. M., Parajuli, P., Mosley, R. L., Sublet, J., Kelsey, L., Sarin, P. S., Zimmerman, D. H., Winship, M. D., and Talmadge, J. E. (2000). Flt3 ligand enhances the immunogenicity of a gag-based HIV-1 vaccine. Int. J. Immunopharmacol 11, 865± 876. Pulendran, B., Lingappa, J., Kennedy, M. K., Smith, J., Teepe, M., Rudensky, A., Maliszewski, C. R., and Maraskovsky, E. (1997). Developmental pathways of dendritic cells in vivo. Distinct function, phenotype, and localization of dendritic cell subsets in FLT3 ligand-treated mice. J. Immunol. 159, 2222±2231. Pulendran, B., Smith, J. L., Jenkins, M., Schoenborn, M., Maraskovsky, E., and Maliszewski, C. R. (1998). Prevention of peripheral tolerance by a dendritic cell growth factor: Flt3 ligand as an adjuvant. J. Exp. Med 188, 2075±2082. Pulendran, B., Smith, J. L., Caspary, G., Brasel, K., Petitt, D., Maraskovsky, E., and Maliszewski, C. R. (1999). Distinct dendritic cells subsets differentially regulated the class of immune response in vivo. Proc. Natl Acad. Sci. USA 96, 1036±1041. Pulendran, B., Banchereau, J., Burkeholder, S., Kraus, E., Guinet, E., Chalouni, C., Caron, D., Maliszewski, C., Davoust, J., Fay, J., and Palucka, K. (2000). Flt3-ligand and granulocyte colony-stimulating factor mobilize distinct human dendritic cell subsets in vivo. J. Immunol. 165, 566±572. Ramsfjell, V., Borger, O. J., Veiby, O. P., Cardier, J., Murphy, M. J. Jr, Lyman, S. D., Lok, S., and Jacobsen, S. E. (1996). Thrombopoietin, but not erythropoietin, directly stimulates multilineage growth of primitive murine bone marrow progenitor cells in synergy with early acting cytokines: distinct interactions with the ligands for c-kit and FLT3. Blood 88, 4481±4492. Ramsfjell, V., Borge, O. J., Cui, L., and Jacobsen, S. E. (1997). Thrombopoietin directly and potently stimulates multilineage growth and progenitor cell expansion from primitive (CD34‡ CD38ÿ) human bone marrow progenitor cells: distinct and key interactions with the ligands for c-kit and flt3, and inhibitory effects of TGF-beta and TNF-alpha. J. Immunol. 158, 5169±5177. Rosnet, O., Marchetto, S., de Lapeyriere, O., and Birnbaum, D. (1991). Murine Flt3, a gene encoding a novel tyrosine kinase receptor of the PDGFR/CSF1R family. Oncogene 6, 1641±1647. Savvides, S. N., Boone, T., and Andrew Karplus, P. (2000). Flt3 Ligand structure and unexpected commonalities of helical bundles and cysteine knots. Nature Struct. Biol. 7(6), 486±491. Shah, A. J., Smogorzewska, E. M., Hannum, C., and Crooks, G. M. (1996). FL induces proliferation of quiescent human bone marrow CD34‡CD38ÿ cells and maintains progenitor cells in vitro. Blood 87, 3563±3570. Shapiro, F., Pytowski, B., Rafii, S., Witte, L., Hicklin, D. J., Yao, T. J., and Moore, M. A. (1996). The effects of Flk-2/flt3 ligand as compared with c-kit ligand on short-term and long-term proliferation of CD34‡ hematopoietic progenitors elicited from human fetal liver, umbilical cord blood, bone marrow and mobilized peripheral blood. J. Hematother. 5, 655±662. Shaw, S. G., Maung, A. A., Steptoe, R. J., Thomson, A. W., and Vujanovic, N. L. (1998). Expansion of functional NK cells in multiple tissue compartments of mice treated with Flt3-ligand: implications for anti-cancer and anti-viral therapy. J. Immunol. 161, 2817±2824.

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Shurin, M. R., Pandharipande, P. P., Zorina, T. D., Haluszczak, C., Subbotin, V. M., Hunter, O., Brumfield, A., Storkus, W. J., Maraskovsky, E., and Lotze, M. T. (1997). FLT3 ligand induces the generation of functionally active dendritic cells in mice. Cell. Immunol. 179, 174±184. Shurin, M. R., Esche, C., and Lotze, M. T. (1998). FLT3: receptor and ligand. Biology and potential clinical application. Cytokine Growth Factor Rev 9, 37±48. Silver, D. F., Hempling, R. E., Piver, M. S., and Repasky, E. A. (2000). Flt-3 ligand inhibits growth of human ovarian tumors engrafted in severe combined immunodeficient mice. Gynecol. Oncol. 77, 377±382. Soligo, D., Lambertenghi Deliliers, G., Quirici, N., Servida, F., Caneva, L., and Lamorte G. (1998). Expansion of dendritic cells from human CD34‡ cells in static and continuous cultures. Br. J. Haematol. 101, 352±363. Starzl, T. E., Demetris, A. H., Murase, N., Trucco, M., Thomson, A. W., and Rao, A. S. (1996). The lost chord: microchimerism and allograft survival. Immunol. Today 17, 577±584. Steinman, R. M. (1991). The dendritic cell system and its role in immunogenicity. Annu. Rev. Immunol. 9, 271±296. Stiff, P., Chen, B., Franklin, W., Oldenberg, D., Hsi, E., Bayer, R., Shpall, E., Douville, J., Mandalam, R., Malhotra, D., Muller, T., Armstrong, D., and Smith, A. (2000). Autologous transplantation of ex vivo bone marrow cells grown from small aliquots after high-dose chemotherapy for breast cancer. Blood 95, 2169± 2174.

Strobl, H., Riedl, E., Bello-Fernandez, C., and Knapp, W. (1998). Epidermal Langerhans cell development and differentiation. Immunobiology 198, 588±605. Sudo, Y., Shimazaki, C., Ashihara, E., Kikuta, T., Hirai, H., Sumikuma, T., Yamagata, N., Goto, H., Inaba, T., Fujita, N., and Nakagawa, M. (1997). Synergistic effect of FLT-3 ligand on the granulocyte colony-stimulating factor-induced mobilization of hematopoietic stem cells and progenitor cells into blood in mice. Blood 89, 3186±3191. Viney, J. L., Mowat, A. M., O'Malley, J. M., Williamson, E., and Fanger, N. A. (1998). Expanding dendritic cells in vivo enhances the induction of oral tolerance. J. Immunol. 160, 5815±5825. Williamson, E., Westrich, G. M., and Viney, J. L. (1999). Modulating dendritic cells to optimize mucosal immunization protocols. J. Immunol. 163, 3668±3675. Wodnar-Filipowicz, A., Lyman, S. D., Gratwhol, A., Tichelli, A., Speck, B., and Nissen, C. (1996). Flt3 ligand level reflects hematopoietic progenitor cell function in aplastic anemia and chemotherapy-induced bone marrow aplasia. Blood 88, 4493± 4499. Yonemura, Y., Ku, H., Lyman, S. D., and Ogawa, M. (1997). In vitro expansion of hematopoietic progenitors and maintenance of stem cells: comparison between Flt3/Flk-2 ligand and Kit ligand. Blood 89, 1915±1921.