The Journal of Immunology

Inhibition of Natural Type I IFN-Producing and Dendritic Cell Development by a Small Molecule Inhibitor with Flt3 Affinity1

Roxane Tussiwand,* Nobuyuki Onai,* Luca Mazzucchelli,† and Markus G. Manz*‡

In vivo steady-state type I natural IFN-producing and dendritic cell (DC) development is largely dependent on Flt3 signaling. Natural IFN-producing and DC progenitors and their respective downstream cell populations express the flt3 receptor, and Flt3 (Flt3L)؊/؊ mice have reduced while Flt3L-injected mice develop markedly increased numbers of both cell types. In the present study, we show that SU11657, a small multitargeted inhibitor with Flt3 affinity, suppressed in vitro natural IFN-producing and DC development in Flt3L-supplemented mouse whole bone marrow cell cultures in a dose- dependant manner, while DC development in GM-CSF-supplemented cultures was not affected. In vivo SU11657 application led .to a significant decrease of both natural IFN-producing and DCs, comparable to the reduction observed in Flt3L؊/؊ mice Conversely, Flt3L plasma levels increased massively in inhibitor-treated animals, likely via a regulatory feedback loop, without being able to compensate for pharmacological Flt3 inhibition. No obvious toxicity was observed, and hemopoietic progenitor cell and stem cell function remained intact as assessed by myeloid colony-forming unit activity and in vivo bone marrow repopulation assays. Furthermore, upon treatment discontinuation, IFN-producing and DCs recovered to normal levels, proving that treatment effects were transient. Given the importance of IFN-producing and DCs in regulation of immune responses, these findings might lead to new pharmacological strategies in prevention and treatment of autoimmune diseases and complications of organ or blood cell transplantation. The Journal of Immunology, 2005, 175: 3674–3680.

atural type I IFN-producing cells (IPCs,3 also called confined to Flt3 expressing hemopoietic progenitor cells along both plasmacytoid cells or plasmacytoid predendritic cells lymphoid and myeloid differentiation pathways (12, 13); Flt3L-defi- N (pre-DCs)) and DCs are important regulators of innate cient mice and mice with hemopoietic system confined deletions of and adaptive immune responses (1–5). IPCs and DCs are cells of Stat3, a activated in the Flt3 signaling cascade, the hemopoietic system and, with the exception of subpopulations show massively reduced DCs in lymphoid organs (16, 17); and fi- as Langerhans cells (6), are continuously replenished by new input nally, injection of Flt3L dramatically increases IPCs and DCs in both cells derived from bone marrow (BM) hemopoietic stem cells mice (18–21) and humans (22, 23), with up to 30% of mouse spleen (HSCs) and progenitor cells (7). cells expressing CD11c and MHC class II (18, 24). Flt3, a receptor tyrosine kinase with homology to c-Kit (the Given the above findings, we reasoned that targeted pharmacologic receptor for (SCF)), and c-fms (the receptor for disruption of Flt3 tyrosine kinase signaling should lead to inhibition of M-CSF) plays an important role in early hemopoietic development both IPC and DC development. To test this, in the present study we (reviewed in Ref. 8) and is a nonredundant for in vivo evaluated in vitro and in vivo effects of SU11657, a small molecule steady-state differentiation of IPCs and DCs: flt3 is expressed in multitargeted receptor tyrosine kinase inhibitor with Flt3 affinity that mouse short-term hemopoietic stem cells (9, 10), in lymphoid and has been proven to be efficient in treatment of in vivo Flt3-driven myeloid committed hemopoietic progenitor cell fractions (11–13), experimental (25, 26) but also acts on other tyrosine kinases and, as we have shown recently, also on steady-state IPCs and DCs as vascular endothelial receptor (VEGF-R), platelet de- (12); Flt3 ligand (Flt3L) as a single cytokine is capable to induce rived (PDGF-R), and c-Kit (27). differentiation of both IPCs and DCs in mouse whole BM cell cultures (14, 15); in vivo IPC and DC differentiation potential is Materials and Methods Animals *Institute for Research in Biomedicine, Bellinzona, Switzerland; †Institute of Pathol- ogy, Locarno, Switzerland; and ‡Eberhard-Karls-University Medical School, Depart- C57BL/Ka-Thy1.1 (CD45.1 and CD45.2) and Flt3LϪ/Ϫ (16) mice were ment of Medicine II, Tuebingen, Germany bred and maintained at the Institute for Research in Biomedicine animal Received for publication December 22, 2004. Accepted for publication July 5, 2005. facility, in accordance with institutional guidelines. 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. Abs and flow cytometry 1 This work is supported in part by a Swissbridge and a Swiss National Science Biotinylated, FITC-, PE-, or allophycocyanin-conjugated mAbs against the Foundation Grant (3100-102221) (to M.G.M.). following mouse Ags were used: CD3⑀ (145-2C11), CD11b (M1/70), 2 Address correspondence and reprint requests to Dr. Markus G. Manz, Institute for CD19 (MB19-1), CD45R/B220 (RA3-6B2), CD45.1 (A20), CD45.2 (104) Research in Biomedicine, Via Vincenzo Vela 6, CH-6500 Bellinzona, Switzerland. CD90.1 (HIS51), CD117/c- (ACK2), CD127/IL-7R␣ (A7R34) CD135/ E-mail address: [email protected] Flt3 (A2F10), MHC class II (M5/114.15.2), Gr-1 (RB6-8C5), Ter119 3 Abbreviations used in this paper: IPC, IFN-producing cell; DC, dendritic cell; BM, (TER119) (all eBioscience), CD11c (HL3), and NK1.1 (PK136) (all BD bone marrow; HSC, hemopoietic stem cell; SCF, stem cell factor; Flt3L, Flt3 ligand; Pharmingen). For visualization of biotinylated Abs, streptavidin-conju- VEGF-R, vascular endothelial growth factor receptor; PDGF-R, platelet-derived gated FITC, PE, or allophycocyanin was used (all BD Pharmingen). For all growth factor receptor; CML, chronic myelogenous leukemia. experiments, dead cells were excluded by propidium iodide staining, and

Copyright © 2005 by The American Association of Immunologists, Inc. 0022-1767/05/$02.00 The Journal of Immunology 3675

appropriate isotype-matched, irrelevant control mAbs were used to deter- Results mine the level of background staining. Analysis was performed on a FAC- SU11657 inhibits both IPC and DC development in Flt3L- SCalibur and FACSVantage SE (BD Biosciences Immunocytometry Systems). supplemented, whole BM cultures in a dose-dependant manner DC and IPC cultures It was shown that both IPCs and DCs can be generated from Flt3L- supplemented mouse whole BM cultures (15). As expected, addi- To generate IPCs and DCs, mouse whole BM cells (1 ϫ 106/ml) were cultured in flat-bottom 48- or 96-well plates in RPMI 1640 medium, sup- tion of increasing doses of SU11657- to Flt3L-supplemented plemented with 10% FCS, 10Ϫ4 M 2-ME, sodium pyruvate, antibiotics, whole BM cultures resulted in a dose-dependent suppression of ␮ and either 20 ng/ml murine GM-CSF (R&D Systems) or 100 ng/ml re- both IPC and DC development (functional IC50, 0.02 M) (Fig. 1, combinant human Flt3L-Fc fusion . Flt3L-Fc was A and B). Although tyrosine kinase inhibitors are designed to have produced in Drosophila cells as described previously (28). Increasing doses of SU11657, a small molecule multitargeted receptor tyrosin kinase high affinity to one selected target kinase, most inhibitors are not

inhibitor with demonstrated Flt3 tyrosine kinase affinity (IC50, 0.05 mM to specific, and cross-reactivity to other tyrosine kinases will occur. mutated, constitutively active Flt3) (25), VEGF-R (VEGF-R1 IC50, 0.013 To rule out that in vitro inhibition of IPC and DC development was mM; VEGF-R2 IC50, 0.017 mM), PDGF (PDGF-R IC50, 0.005 mM), and due to SU11657-induced cross-inhibition of VEGF-R tyrosine ki- c-Kit (IC50, 0.04 mM) affinity (27) or SU10944, a small molecule receptor tyrosin kinase inhibitor with demonstrated VEGF-R affinity (VEGF-R1 nase, we in parallel tested effects of SU10944, a tyrosine kinase inhibitor with VEGF-R affinity (29). No inhibition of IPC and DC IC50, 0.006 mM; VEGF-R2 IC50, 0.096 mM) (29) were added as indicated (both provided by Pfizer Global Research and Development/Sugen). Cul- differentiation was observed (Fig. 1, A and B). Also, to rule out that tures were analyzed at day 9 by FACS as indicated. SU11657-induced inhibition of differentiation was due to effects Myeloid colony-forming unit and in vivo BM repopulation assay independent of Flt3 signaling, we added SU11657 in increasing doses to GM-CSF-supplemented whole BM DC cultures. DC dif- To test myeloid colony formation, whole BM cells were cultured for 10 days in IMDM (Invitrogen Life Technologies) based methylcellulose me- ferentiation was not affected (Fig. 1C). Thus, SU11657 inhibits dium (MethoCult H4100; StemCell Technologies) supplemented with 30% IPC and DC development in Flt3L-supplemented whole BM cul- FCS, 4% BSA, 2 mM L-glutamine, 50 ␮M 2-ME, antibiotics, and 10 ng/ml tures likely via flt3 receptor tyrosine kinase inhibition in a dose- IL-3, 10 ng/ml human IL-11, 10 ng/ml SCF, 10 ng/ml GM-CSF, 50 ng/ml dependent manner. human Tpo (all R&D Systems), 10 ng/ml Flt3L, and 4 U/ml human Epo (Roche Diagnostic Systems). To test gamma irradiation protective, hemopoietic repopulation capacity of BM cells, 8- to 12-wk-old CD45.1-recipient mice were lethally irradi- In vivo SU11657 tyrosine kinase inhibitor application leads to ated (2 ϫ 6.5 Gy in a 4-h interval from a cesium 137 source, Biobeam IPC and DC reduction in spleen and lymph nodes, resembling ϫ 6 8000; STS) and were i.v. transplanted with 2.5 10 whole BM cells from IPC and DC levels in Flt3LϪ/Ϫ mice vehicle- or SU11657-treated CD45.2 congenic mice. Animals were main- tained on antibiotic water (ciprofloxacin) for 3 wk. Survival and engraft- We first tested in vivo effects of SU11657 on cell numbers and ment was evaluated at 4 wk after transplantation. composition of the hematolymphoid system. Six- to 8-wk-old mice In vivo tyrosine kinase inhibitor treatment and analysis of mice were treated orally with 40 mg/kg body weight SU11657, a dose For oral application via gavage, SU11657 was resolved in 100 ␮l of car- previously determined as optimal for in vivo application (25, 26), boxymethyl-cellulose suspension. Six- to 8-wk-old mice received once over 14 days. Control mice received vehicle treatment only. daily 40 mg/kg SU11657, a dose previously determined to be sufficient for SU11657 treatment was well tolerated; mice showed no signs of in vivo treatment of a murine experimental leukemia carrying an activating sickness and did not lose weight (data not shown). Total cell num- Flt3 (25). Control animals received vehicle (100 ␮l of carboxy- methyl-cellulose suspension) alone. Because IPCs and DCs have a half-life bers in BM and spleen were significantly reduced in SU11657- of 4–11 days (30, 31) and early hemopoietic progenitor cells differentiate treated animals (Fig. 2, A and B). Reduction of BM and spleen in ϳ2 wk into IPCs and DCs in vivo (12, 32–34), mice were treated over cellular compartments was accompanied by a significant absolute 14 days, and organs were analyzed at day 15. Spleens and lymph nodes and relative reduction of CD11bϩ (Mac-1ϩ) and Gr-1ϩ myeloid were cut in small fragments and digested under repeated agitation for 30 ϩ ϩ cells and also spleen NK1.1 NK cells, while relative CD19 B min at 37°C in RPMI 1640 medium supplemented with 10% FCS, 1 mg/ml ϩ collagenase (collagenase D; Roche Diagnostic Systems), and 50 ␮g/ml cell and CD3 numbers were less, nonsignificantly affected DNase (DNase I from bovine pancreas grade II; Roche Diagnostic Sys- in BM and spleen (Fig. 2, A and B). Thus, cell numbers and cellular tems). Debris was removed by filtration, and red cells were lysed osmot- composition in SU11657 tyrosine kinase inhibitor-treated animals ically. Nucleated cells were stained as indicated with fluorochrome-con- jugated Abs and were analyzed by FACS. IPCs were identified as were changed similar as in Flt3L-deficient and, in a reverse direction CD11clowB220ϩ and DCs as CD11cϩMHC class IIϩ cells. as compared with, Flt3L-injected mice, respectively (12, 16). To evaluate if, similar to the in vitro findings, IPC and DC differ- Flt3-ligand and SCF measurement entiation was inhibited in vivo by SU11657, spleen and lymph nodes Serum concentrations of Flt3L and SCF were measured by ELISA, ac- of mice were analyzed for CD11clowB220ϩ and CD11cϩMHC class cording to the manufacturer’s instructions (R&D Systems). IIϩ cells. Indeed, DCs were significantly reduced in spleens and RT-PCR lymph nodes, and IPCs were significantly reduced in spleens, whereas RNA was isolated from BM cells of SU11657 and vehicle-treated mice using lymph node IPC reductions did not reach statistical significance. IPC TRIzol Reagent (Invitrogen Life Technologies), followed by DNase I (Invitro- and DC percentages compared very closely to the respective cell pop- gen Life Technologies) treatment. cDNA was synthesized using random hex- ulations in Flt3LϪ/Ϫ mice (Fig. 3, A–C). DCs in treated animals did amers and Superscript II reverse transcriptase (Invitrogen Life Technologies). not differ from that in vehicle-treated animals in terms of CD80 and Five-fold dilutions of cDNA products were amplified by PCR using specific primers (GAPDH, 5Ј-ACCACAGTCCATGCCATCAC-3Ј and 5Ј-TCCAC- CD86 costimulatory marker expression, indicating that SU11657 CACCCTGTTGCTGTA-3Ј; Flt3, 5Ј-ATGACGCCCAGTTCACCAAA-3Ј treatment did not induce activation of DCs (data not shown). Upon and 5Ј-GCAAGACGTGCCCATAGAATT-3Ј; and c-Kit, 5Ј-ACAGGAGCA- discontinuation of SU11657 treatment, IPC and DC numbers returned Ј Ј Ј GAGCAAAGGTG-3 and 5 -CGACCACAAAGCCAATGAGC-3 . PCR to high normal levels within 10 days (Fig. 3, A–C). Thus, in vivo products were electrophoresed on an ethidium bromide-stained agarose gel. SU11657 tyrosine kinase inhibition led to reduction of both IPCs and Statistical analysis DCs, comparable to IPC and DC levels observed in the absence of the Ϫ/Ϫ Differences between groups were evaluated for statistical significance us- cognate ligand (Flt3L mice), without irreversible blocking IPC ing the two-tailed paired Student’s t test, assuming equal variances. and DC progenitors or HSCs. 3676 INHIBITION OF IPC AND DC DEVELOPMENT BY SU11657

FIGURE 1. SU11657 but not SU10944 inhibits both IPC (CD11cϩB220ϩ) and DC (CD11cϩMHC class IIϩ) development in Flt3L-supplemented, whole BM cul- tures in a dose-dependent manner. A, Graph shows per- cent reduction of CD11cϩ cells in day 9 Flt3L whole BM cultures supplemented with increasing doses of SU11657 (F, solid line) or SU10944 (f, dashed line). Mean and SD of three independent experiments are shown. B, DC (CD11cϩMHC class IIϩ) and IPC (CD11cϩB220ϩ) reduction in 50 nM SU11657-supple- mented cultures; no reduction is observed in 50 nM SU10944-supplemented cultures. C, Both SU11657 and SU10944 have no influence on DC (CD11cϩMHC class IIϩ) development in GM-CSF-supplemented whole BM cultures. B and C, Representative dot plots of three in- dependent experiments, respectively.

FIGURE 2. Total BM and spleen cell numbers are reduced in 14 days SU11657-treated animals. A, Total BM cells are reduced in SU11657 (dark gray bar) compared with vehi- cle treated ( gray bar) animals (p ϭ 0.01) with a preferential relative reduction of CD11bϩ (Mac-1ϩ) and/or Gr-1ϩ myeloid cells (p Ͻ 0.01). B, Total spleen cells are re- duced in SU11657 (dark gray bar) compared with vehicle-treated (light gray bar) animals (p ϭ 0.01), with a preferential relative reduction of CD11bϩ (Mac-1ϩ) and/or Gr-1ϩ my- eloid cells (p Ͻ 0,01), and NK1.1ϩ cells (p ϭ 0.02). Mean and SD of three independent experiments (two vehicle- and three SU11657-treated animals for each experiment) (A and B). Statistical significant differences are indicated. The Journal of Immunology 3677

FIGURE 3. SU11657 treatment reversibly reduces IPCs and DCs to levels comparable to that in Flt3LϪ/Ϫ mice. A, Bar graphs show percent- ages of CD11cϩMHC class IIϩ DCs and CD11cϩB220ϩ IPCs in spleen and lymph nodes of vehicle-treated mice (light gray bar), SU11657- treated mice (dark gray bar), Flt3LϪ/Ϫ mice (Ⅺ), as well as of mice 10 days after discontinuation of SU11657 treatment (o). Statistically significant differences are indicated. Mean and SD of six vehicle-treated, nine SU11657-treated, and five Flt3LϪ/Ϫ mice and mean value of two mice after treatment discontinu- ation. B and C, Contour plots show representative analysis and gating of CD11cϩMHC class IIϩ DCs and CD11cϩB220ϩ IPCs in spleen and lymph nodes of vehicle and SU11657-treated, as well as Flt3LϪ/Ϫ, mice and mice 10 days af- ter discontinuation of SU11657 treatment.

In vivo SU11657 application leads to massive increase of serum strated to expand early hemopoietic progenitors (8, 11, 12, 35). Flt3L levels and undetectable surface Flt3 and c-Kit expression Therefore, we were interested to evaluate in vivo effects of on lineage negative BM cells, while mRNA expression of both SU11657 on short-term HSCs (9, 10) as well as lymphoid (36) and Flt3 and c-Kit are not altered myeloid (37) progenitor cells. Surprisingly, within the lineage Ag- Ϫ Ϫ Ϫ Ϫ Ϫ Ϫ Flt3 was shown to be expressed on short-term HSCs (9, 10) and in negative (CD3 CD19 B220 CD11b Gr-1 TER119 ) BM cell lymphoid and myeloid committed hemopoietic progenitor cell fraction, both surface Flt3 (CD135) and c-Kit (CD117) receptors fractions (11–13), and stimulation of the receptor was demon- were not detectable by mAb staining in SU11657-treated animal 3678 INHIBITION OF IPC AND DC DEVELOPMENT BY SU11657

BM (Fig. 4A). Therefore, HSCs and both lymphoid and myeloid Table I. Myeloid colony formation is not inhibited in SU11657-treated progenitors could not be identified by phenotype. However, both animal bone marrow cellsa Flt3 and c-kit mRNA transcripts were expressed at similar levels in lineage-negative cells of SU11657-treated animals and in non- CFU-G/-M/-GM CFU-Mix CFU-E/Meg/MegE Total CFUs treated controls (Fig. 4B). Thus, failure of surface detection was Vehicle 19 1 4.5 24.5 not due to down-regulation of transcription but possibly to SU11657 23 1 4 28 receptor internalization upon ligation (38). Evaluation of Flt3L and a Table depicts mean numbers of colony-forming units granulocyte/macrophage/ SCF levels in sera of SU11657-treated animals revealed signifi- granulocyte-macrophage (CFU-G/M/GM), mixed colony-forming units (CFU-Mix), cantly ϳ10-fold elevated Flt3L and ϳ1.5-fold (nonsignificant) el- and colony-forming units erythrocyte/megakaryocyte/megakaryocyte-erythrocyte (CFU-E/Meg/-MegE) at day 10 of cultures. A total of 104 bone marrow cells was and evated SCF levels (Fig. 4C). Both surface Flt3 and c-Kit expres- plated each. Mean colony numbers of three independent experiments using two ve- sion as well as elevated cytokine levels returned to normal values hicle and two SU11657-treated mice each are shown. No significant differences were upon discontinuation of treatment (Fig. 4, A and C). observed. Thus, in vivo SU11657-mediated inhibition of tyrosine kinase activity led to a substantial increase of Flt3L and, to a somewhat lower extent, also of SCF, indicating that possibly regulatory feed- back loops were activated that, however, were not able to over- come SU11657 induced effects on IPC and DC development.

In vivo SU11657 application does not decrease myeloid colony- forming unit activity and in vivo repopulation potential of BM hemopoietic cells To determine whether in vivo SU11657 treatment would have a lasting negative effect on biological functions of myeloid progen- itor cells and HSCs, we tested BM myeloid progenitor function in vitro and hemopoietic reconstitution potential in vivo. BM cells isolated from vehicle- or SU11657-treated animals were plated in myeloid colony-forming assays. As shown in Table I, no difference in colony formation was observed. Also, lethally irradiated ani- mals, reconstituted with total BM cells from vehicle- or SU11657- treated mice were protected from radiation-induced death, and no differences in repopulation of BM and lymphoid organs at 4 wk after transplantation were observed (Table II). Therefore, in vivo SU11657 treatment did not irreversibly im- pair myeloid progenitor cells and HSCs as assessed by myeloid colony forming, short-term in vivo repopulation, and radioprotec- tive capacity.

Discussion In vivo steady-state IPC and DC development largely depends on Flt3L as Flt3LϪ/Ϫ mice have substantially decreased, and Flt3L- injected mice and humans develop increased absolute and relative IPC and DC numbers (16, 18, 19, 21–23). Both IPCs and DCs develop from mouse whole BM cells (14, 15) and from human CD34ϩ hemopoietic progenitors (39, 40) in Flt3L-supplemented cell cultures. Thus, inhibition of Flt3 signaling should lead to de- creased differentiation of both cell populations. Following the find- ing that mutated, constitutively active Flt3 tyrosine kinase signal- ing is involved in human and experimental murine leukemia, small molecule tyrosine kinase inhibitors have been developed and are tested in preclinical and clinical settings (41–45). In the present study, we used SU11657, a multitargeted tyrosine kinase inhibitor FIGURE 4. SU11657 treatment leads to increased endogenous Flt3L levels and undetectable surface Flt3 and c-Kit receptors on lineage-nega- with affinity to Flt3 (25) to evaluate the effects of inhibition on IPC tive progenitor cells, while mRNA expression of Flt3 and c-Kit are not altered. A, Contour plots show representative c-Kit (CD117) and Flt3 (CD135) ex- pression on linϪ (CD3ϪCD19ϪB220ϪCD11bϪGr-1ϪTER119Ϫ) BM cells in Table II. In vivo reconstitution potential is not inhibited in a vehicle- and SU11657-treated animals, as well as in mice 10 days after dis- SU11657-treated animal bone marrow cells continuation of SU11657 treatment. Percentages of cells in respective gated Ϫ areas are given. B, Flt3, c-Kit, and GAPDH mRNA expression in lin BM BM Spleen Thymus Lymph Node cells of vehicle- and SU11657-treated mice. Numbers on top indicate decreas- ing cell number equivalents of which RNA was used. Representative experi- Vehicle 86 78 97 66 SU11657 89 75 98 63 ment of two. C, Bar graphs show serum Flt3L and SCF levels in vehicle (light gray bar)- and SU11657 (dark gray bar)-treated mice, as well as in mice 10 a Table depicts mean percentages of CD45.2ϩ donor derived cells in lethally ϩ days after discontinuation of SU11657 treatment. Mean and range of two in- irradiated CD45.1 recipient mice 4 wks after transplantation with 2.5 ϫ 106 whole dependent experiments (two vehicle- and three SU11657-treated and two an- bone marrow cells from vehicle or SU11657-treated animals. Mean values of two independent experiments are shown. In each experiment, three mice received com- imals 10 days after discontinuation of SU11657 treatment per experiment). bined bone marrow cells from two SU11657-treated animals, and two mice received Statistically significant differences are indicated. combined bone marrow cells from two vehicle-treated mice. The Journal of Immunology 3679 and DC development. Indeed, pharmacologic disruption of Flt3 tion, as well as on Flt3 regulation, are at least controversial, and the tyrosine kinase signaling lead to inhibition of both IPC and DC molecular basis of these observations remains to be determined. development in a dose-dependent manner in vitro (Fig. 1). This In contrast to this, it is known that Flt3L is a nonredundant effect was not due to cross-inhibition of VEGF-R tyrosine kinase cytokine for IPC and DC development (16, 21), and we demon- signaling by SU11657 because SU10944, a tyrosine kinase inhib- strated for the first time that application of a multitargeted receptor itor with VEGF-R affinity (29), showed no effect on development tyrosine kinase inhibitor with Flt3 affinity leads to decreased IPC of both IPCs and DCs in vitro. Although the data presented here and DC levels in vivo. Flt3 tyrosine kinase inhibitors used in clin- does not dissect to what extent the observed effects are mediated ical trials for the treatment of leukemia with mutated, constitu- by SU11657 cross-reactivity to other receptor tyrosine kinases be- tively active Flt3 (41–44) might therefore rather lead to suppres- yond VEGF-R, SU11657 effects were likely Flt3 mediated because sion then restoration of immune functions. We would like to 1) SU11657 addition to GM-CSF-supplemented BM cultures did suggest that this effect might be exploited in other clinical settings not affect in vitro DC development, and 2) in vivo SU11657 treat- where suppression of immune responses is a therapeutic goal, as in ment led to a significant decrease of relative and absolute IPC and the treatment of autoimmune diseases or complications of organ or DC numbers and a reduction of other hemopoietic cells as Mac- blood cell transplantation. For example, it has been shown by oth- 1ϩGr-1ϩ cells and NK cells, closely resembling the cellular com- ers and us that residual host DCs are involved in initiation of position found in Flt3LϪ/Ϫ mice (Fig. 3). Thus, SU11657-medi- graft-vs-host disease after allogeneic cell transplantation in animal ated inhibition of the Flt3 tyrosine kinase is sufficient to explain the models (57–61). It is conceivable that implementation of a Flt3 cellular alterations observed in vitro and in vivo. tyrosine kinase inhibitor during the conditioning regimen before Interestingly, SU11657 treatment led to a massive increase of allogeneic hemopoietic cell transplantation would diminish host endogenous Flt3L and moderate increase of SCF that, however, DC numbers, consecutively might prevent acute graft-vs-host dis- were not able to overcome the IPC and DC differentiation block ease, and thus could allow to reduce T cell immunosuppressive induced by SU11657 (Fig. 4). Increased endogenous Flt3L levels drugs in the posttransplantation period. were described during hemopoietic recovery following combined The data presented here provides the basis to evaluate Flt3 ty- gamma irradiation and conditioning in humans, and rosine kinase inhibitors as immunomodulators in autoimmune dis- these increases were positively correlated with DC recovery (46). eases and in immune system-mediated complications of organ or Thus, these findings point to a remarkable Flt3/Flt3L regulatory- blood cell transplantation. loop in hematopoiesis. It will be of interest to test how Flt3L ex- pression is induced in SU11657 tyrosine kinase inhibitor-treated Acknowledgments animals, if it is a direct result of decreased downstream Flt3 sig- Congenic C57BL/Ka-Thy1.1 CD45.1/CD45.2 mouse breeder pairs were naling, or if it is possibly caused by diminished hemopoietic stem kindly provided by I. L. Weissman (Stanford University, Stanford, CA). Ϫ/Ϫ and progenitor cell proliferation and differentiation. Similarly, it Flt3-ligand mice (originally generated by H. J. McKenna and J. J. Pe- will be important to determine whether observed, nonsignificant schon (16)) were kindly provided by E. Sitnicka (Stem Cell Research Cen- ter, University of Lund, Lund Sweden). We thank K. Karjalainen for help alterations in SCF levels are due to SU11657 cross-reactivity to the in production of recombinant human Flt3-ligand-Fc fusion protein, F. Sal- c-Kit tyrosine kinase. lusto for critical reading of the manuscript, and Pfizer Global Research and Of note, both 4-wk radiation protection and myeloid colony Development/Sugen for supplying SU11657 and SU10944. forming potential of in vivo SU11657-pretreated BM cells was not impaired, and IPC and DC numbers recovered within 10 days after Disclosures discontinuation of treatment, about the time it takes for HSCs and The authors have no financial conflict of interest. early progenitors to differentiate to these cell populations upon in vivo transfer (32, 33, 47). Therefore, stem cell and myeloid pro- References genitor function remained intact, at least as determined by these 1. Banchereau, J., and R. M. Steinman. 1998. Dendritic cells and the control of immunity. Nature 392: 245–252. short-term assays (Tables I and II; Fig. 3). 2. Shortman, K., and Y. J. Liu. 2002. Mouse and human dendritic cell subtypes. Nat. mesylate (also know as Glivec or STI571) is a small Rev. 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