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Activation of the Flt3 signal transduction cascade rescues and enhances type I interferon–producing and dendritic cell development

Flt3 ligand (Flt3L) is a nonredundant cytokine in type I interferon–producing cell (IPC) and dendritic cell (DC) development, and IPC and DC differentiation potential is confined to Flt3+ hematopoietic progenitor cells. Here, we show that overexpression of human Flt3 in Flt3− (Flt3−Lin−IL-7Rα−Thy1.1−c-Kit+) and Flt3+ (Flt3+Lin−IL-7Rα−Thy1.1−c-Kit+) hematopoietic progenitors rescues and enhances their IPC and DC differentiation potential, respectively. In defined hematopoietic cell populations, such as Flt3− megakaryocyte/erythrocyte-restricted progenitors (MEPs), enforced Flt3 signaling induces transcription of IPC, DC, and granulocyte/macrophage (GM) development–affiliated genes, including STAT3, PU.1, and G-/M-/GM-CSFR, and activates differentiation capacities to these lineages. Moreover, ectopic expression of Flt3 downstream transcription factors STAT3 or PU.1 in Flt3− MEPs evokes Flt3 receptor expression and instructs differentiation into IPCs, DCs, and myelomonocytic cells, whereas GATA-1 expression and consecutive megakaryocyte/erythrocyte development is suppressed. Based on these data, we propose a demand-regulated, cytokine-driven DC and IPC regeneration model, in which high Flt3L levels initiate a self-sustaining, Flt3-STAT3– and Flt3-PU.1–mediated IPC and DC differentiation program in Flt3+ hematopoietic progenitor cells.

Diff erentiation of hematopoietic stem cells (HSCs) to mature hematopoietic cells is characterized by progressive loss of developmental options and restriction to one lineage (1). In both mice and men, HSCs as well as multiple developmental intermediates with limited cellular expansion and restriction to specifi c mature cell types have been identifi ed. These include myeloid progenitors, as clonal common myeloid progenitors (CMPs) that give rise to either granulocyte/macrophage progenitors (GMPs) or megakaryocyte/erythrocyte progenitors (MEPs; references 2 and 3), and clonal common lymphoid progenitors (CLPs; references 4 and 5), which robustly produce the respective mature cell types. Hematopoietic diff erentiation is regarded as a multi-linear, unidirectional process, and regeneration and expansion of specifi c lineages are largely regu-lated extrinsically by diff erent hematopoietic cytokines. However, it is unclear whether under physiologic conditions cytokines are capable to instruct HSCs and subsequent progenitors to diff erentiate to lineage-restricted progenitors (extrinsic lineage determination). Alternatively, HSCs and subsequent progenitors commit to lineage-restricted progenitors by intrinsic differentiation programs (intrinsic lineage determination), and restricted progenitors are consecutively stimulated by hematopoietic cytokines produced upon demand (6,7).
On the molecular level, access to lineage developmental options and readiness to receive lineage-permissive and -instructive signals might be determined by graded, relative expression levels of diverse transcription factors and cytokine receptors (8,9). Indeed, it has been demonstrated that genetic deletion or overexpression of diff erent single transcription factors is sufficient to reprogram committed progenitors or mature cells to alternative hematopoietic lineages (10): Pax5-defi cient pre-B cells lose B cell diff erentiation potential and mature into T and myelomonocytic cells, but reexpression of Pax5 restores B cell commitment (11,12); ectopic expression of GATA-1 instructs HSCs and CMPs and converts CLPs and GMPs to the megakaryocyte/erythrocyte lineage, respectively (13); and enforced expression of C/EBPα and C/EBPβ in B cells leads to macrophage diff erentiation (14). Furthermore, it has been shown that artifi cial expression of GM-CSF receptor and stimulation with the cognate ligand redirect CLPs and early T cell progenitors to myeloid lineage outcomes (15)(16)(17). This proves that hematopoietic lineage instruction can be mediated extrinsically by cytokines, at least in these experimental settings.
DCs are important regulators of innate and adaptive immune responses and are involved in initiation of immunity as well as in maintenance of self-tolerance (18)(19)(20). DCs are cells of the hematopoietic system and are continuously replenished from hematopoietic stem and progenitor cells (1). In mice, multiple DC subsets that diff er in maturation state, phenotype, location, and in some functions were identifi ed (21). Here, for simplicity, these will grossly be divided into CD11c + B220 + natural type I interferon-producing cells (IPCs; also called plasmacytoid cells or plasmacytoid pre-DCs) and CD11c + B220 − " conventional" DCs, consisting of (21). Although it was suggested that IPCs as well as conventional CD11c + CD8α + DCs are derived from lymphoid-committed progenitors (21), it was demonstrated recently that any of the IPCs and conventional DCs can be generated via lymphoid and myeloid progenitors (22)(23)(24)(25)(26)(27). Specifi cally, all IPCs and conventional DCs are generated by mouse CMPs, GMPs, CLPs, and pro-T1 cells, whereas IPC and DC diff erentiation potential is lost once defi nitive MEP or B cell commitment occurs (22)(23)(24)(25)(26)(27). Thus, in contrast to other hematopoietic lineages, IPC and DC potentials are conserved along lymphoid and myeloid developmental pathways.
Flt3 is a receptor tyrosine kinase with homology to c-Kit (the receptor for stem cell factor) and c-fms (the receptor for M-CSF; reference 28) that has a nonredundant role in steadystate diff erentiation of IPCs and DCs in vivo: Flt3 ligand (Flt3L)-defi cient mice and mice with hematopoietic systemconfi ned deletions of STAT3, a transcription factor activated in the Flt3 signaling cascade, as well as mice that are treated with fl t3 tyrosine kinase inhibitors, show massively reduced IPCs and DCs (29)(30)(31). Conversely, injection or conditional expression of Flt3L in mice increases IPCs and DCs, with up to 30% of mouse spleen cells expressing CD11c (32)(33)(34). Furthermore, Flt3L as a single cytokine is capable of inducing diff erentiation of IPCs and DCs in mouse bone marrow cell cultures (35).
Flt3 is expressed in mouse short-term HSCs and multipotent progenitors (36,37) in most CLPs and CMPs, at lower levels on fractions of GMPs and pro-T1 cells, as well as on mature steady-state IPCs and DCs. It is down-regulated on pro-B cells, further downstream T cell progenitors, and absent on MEPs (33,38). To determine what might defi ne IPC and DC developmental potential in lymphoidand myeloid-committed cells, we and others showed that in vitro and in vivo IPC, DC, and Langerhans cell diff erentiation potential is confined to Flt3-expressing hematopoietic progenitors (33,38,39). Furthermore, we demonstrated that injection of Flt3L expands Flt3 + but not downstream Flt3 − progenitors and drives IPC and DC development along both lymphoid and myeloid diff erentiation pathways (33).
Based on these data, we postulate that high environmental Flt3L levels and consecutive Flt3 signaling might be both the earliest event and a continuous regulator that determines IPC and DC developmental outcomes in bone marrow hematopoietic progenitor cells. Thus, we were interested to test whether enforced Flt3 expression and signaling would be suffi cient to instruct IPC and DC development from Flt3 − progenitor cells and enhance IPC and DC development from Flt3 + progenitor cells, respectively.
Next, we evaluated IPC-and DC-associated surface antigen expression and function of CD11c + B220 + and CD11c + B220 − cells derived from diff erent progenitor cell populations at day 8 of culture. Consistent with typical mouse IPC and DC phenotypes, CD11c + B220 + cells expressed Gr-1, Ly6C, and CD45RA, whereas CD11c + B220 − cells expressed CD11b and intermediate levels of CD80 and CD86, respectively ( Fig. 2 A). Furthermore, CD11c + B220 + cells produced substantial amounts of IFN-α upon stimulation with either infl uenza virus or CpG, whereas CD11c + B220 − cells did not (Fig. 2 B). CD11c + B220 − cells, but not CD11c + B220 + cells, displayed typical DC morphology (Fig. 2 C) and were effi cient stimulators in allogeneic MLR cultures, as evaluated by thymidine incorporation (Fig. 2 D). Therefore, CD11c + B220 + cells phenotypically and functionally were typical IPCs, whereas CD11c + B220 − cells were typical conventional DCs. Collectively, these results indicate that huFlt3 signaling rescues and enhances the development of functional IPCs and DCs from Flt3 − and Flt3 + hematopoietic progenitor cell populations, respectively. In addition, it suggests that strong Flt3 signaling slightly skews development toward an IPC phenotype.
As reported previously for untransduced CMPs (22)(23)(24)(25)(26)(27)38), GFP + -CMPs generated both IPCs and DCs. The effi cacy of GFP + -CMPs to produce these off spring cells was about threefold higher than that observed from huFlt3 + -GMPs (Fig. 3 C). Thus, enforced huFlt3 expression is suffi cient to rescue IPC and DC developmental potential in MEPs to levels comparable to their developmental counterparts, GFP + -GMPs. Furthermore, huFlt3 expression enhances IPC and DC development from GMPs, but not to levels observed in the upstream CMP population.
Enforced expression of huFlt3 permits myelomonocytic development from huFlt3 + -MEPs but not megakaryocyte/ erythrocyte development from huFlt3 + -GMPs Because huFlt3 signaling in MEPs activated IPC and DC development, a diff erentiation option normally confi ned to Flt3 + progenitors as CMPs and GMPs, we were interested to test whether huFlt3 signaling in MEPs would also reestablish myeloid CFU activity. GFP + -transduced CMPs, GMPs, and MEPs gave rise to their respective colony types, but with somewhat lower plating effi cacy as compared with freshly isolated CMPs, GMPs, and MEPs (Fig. 4 A; reference 2). huFlt3 + -MEPs gave rise to not only erythroid-affi liated colonies but also granulocyte/macrophage (GM)-affi liated colonies, including CFU-G, CFU-M, and CFU-GM (Fig. 4 A). Compared with CMPs and GMPs, the myelomonocytic colonyforming effi ciency of huFlt3 + -MEPs was lower; however, the diversity of colony formation resembled that of CMPs with the exception that no CFU-Mix colonies developed. No substantial diff erence in CFU activity was observed in huFlt3 + -GMPs compared with GFP + -GMPs (Fig. 4 A). Thus, huFlt3 signaling in MEPs reestablishes myelomonocytic CFU activity, whereas huFlt3 signaling in GMPs does not aff ect their overall CFU activity and particularly does not reestablish megakaryocyte/erythrocyte read out.

Enforced expression of huFlt3 in MEPs is suffi cient to rescue IPC, DC, and myelomonocytic cell development in vivo
To test the robustness of Flt3 transduction-mediated eff ects observed in vitro, we compared in vivo reconstitution activity of GFP + -MEPs, huFlt3 + -MEPs, and GFP + -CMPs. 2 × 10 4 cells of each progenitor population combined with 2 × 10 5 cells of host bone marrow cells were transplanted into lethally irradiated mice, and spleen progeny cells were analyzed on day 7. As reported previously for MEPs, GFP + -MEPs produced ‫%7.0ف‬ of nucleated GFP + spleen cells that consisted mostly of Ter119 + erythroid cells, but no DCs, IPCs, or Gr-1 + myeloid cells (Fig. 4 B, top; references 2, 23, and 25). In contrast, huFlt3 + -MEPs gave rise to ‫%7.2ف‬ of nucleated GFP + spleen cells containing CD11c + MHC class II + conventional DCs, low numbers of CD11c + B220 + IPCs, as well as Ter119 + erythroid and Gr-1 + myeloid cells (Fig. 4 B, middle). As expected, GFP + -CMPs gave rise to ‫%0.6ف‬ of nucleated GFP + spleen cells that contained DCs, IPCs, as well as ARTICLE erythroid and myeloid cells (Fig. 4 B, bottom; references 2, 23, and 25). This formally demonstrates that enforced expression of huFlt3 is suffi cient to rescue in vivo IPC, DC, and myelomonocytic cell development from MEPs.

HuFlt3 signaling in MEPs induces activation of DC and myeloid development-associated genes
To evaluate the immediate consequences of huFlt3 signaling on gene transcription profi les that might be involved in the consecutive activation of IPC, DC, and myelomonocytic developmental options, we analyzed the transcription of a panel of IPC, DC, and myeloid development-associated genes by RT-PCR in huFlt3 + -MEPs. STAT3, an indispensable transcription factor for Flt3L-mediated DC development (30), was hardly detectable in GFP + -MEPs but was clearly upregulated in huFlt3 + -MEPs. Furthermore, huFlt3 + -MEPs but not GFP + -MEPs expressed myelomonocytic development-associated genes, such as the cytokine receptors for G-CSF, M-CSF, and GM-CSF, as well as the transcription factors C/EBPα and PU.1, similar to that found in GFP + -GMPs, huFlt3 + -GMPs, and normal CMPs (Fig. 5 A; references 2 and 40). However, transcription factors RelB, ICSBP, and Id2 could not be detected in any of the retrovirus-transduced MEPs and GMPs, whereas they were detectable in unmanipulated CMPs (Fig. 5 A). Real-time RT-PCR revealed low-level STAT3 expression in GFP + -MEPs and similar high levels of STAT3 expression in huFlt3 + -MEPs, GFP + -GMPs, huFlt3 + -GMPs, and CMPs (Fig. 5 B). Furthermore, PU.1 expression levels in huFlt3 + -MEPs increased to levels found in CMPs, whereas expression levels of this gene were somewhat higher in GM-committed GFP + -GMPs and huFlt3 + -GMPs (Fig. 5 B). CD11c + B220 + and CD11c + B220 − cells show typical phenotypes of IPCs and DCs, respectively. Results are from one representative experiment out of three. (B) Sorted CD11c + B220 + IPCs but not CD11c + B220 − DCs, both derived from either huFlt3-transduced Flt3 − progenitors or GFPtransduced Flt3 + progenitors, produce IFN-α upon infl uenza virus or CpG stimulation. Culture supernatants were collected after 24 h and analyzed by ELISA. Results are from one representative experiment out of three.
Consistent with their maintained Meg/E-developmental potential, Meg/E-related genes, such as EpoR, β-globin, GATA-1, and GATA-2, were still transcribed in huFlt3 + -MEPs, although the expression of NF-E2 decreased (Fig. 5 A). Interestingly, huFlt3 + -GMP showed some transcription of Meg/E development-associated genes as β-globin and GATA-1. However, GATA-2 and NF-E2 transcripts were not detectable, and hu-Flt3 + -GMPs were not able to give rise to Meg/E lineage colonies (Figs. 4 A and 5 A). Collectively, these results demonstrate on a molecular level that MEPs have a latent IPC, DC, and myelomonocytic lineage potential that is inducible by enforced Flt3 signaling.

HuFlt3 signaling in MEPs leads to downstream STAT3 phosphorylation
To test activation of STAT3 in huFlt3 + -MEPs, we performed intracellular phospho-STAT3 staining in retrovirustransduced MEPs that were Flt3L deprived and then stimulated with huFlt3L-Ig. Indeed, phosphorylation of STAT3 was detected in huFlt3 + -MEPs but not in GFP + -MEPs, indicating that as expected (30) STAT3 is a downstream activated transcription factor of enforced huFlt3 signaling (Fig. 6 A).

Enforced STAT3 or PU.1 expression instructs MEPs to differentiate into IPCs, DCs, and GM lineage cells
Given the importance of STAT3 for IPC and DC development, and the fi nding that STAT3 and PU.1 were upregulated in huFlt3 + -MEPs, we tested whether STAT3 and PU.1 could directly activate IPC, DC, and myelomonocytic development from MEPs. Mouse STAT3 and PU.1 were transduced into MEPs using retrovirus expression vectors, respectively (Fig. S2, available at http://www.jem.org/cgi/ content/full/jem.20051645/DC1). However, survival of cells was low when cultured in SCF alone or SCF and huFlt3L-Ig (not depicted). To possibly support the survival of MEPs, we fi rst added thrombopoietin (TPO), followed by TPO and huFlt3L-Ig to SCF in consecutive cultures. STAT3 + -MEPs
Finally, we evaluated the expression of GATA-1, a nonredundant transcription factor, for megakaryocyte/erythrocyte development by real-time RT-PCR. GATA-1 expression was down-regulated in STAT3 + -MEPs and PU.1 + -MEPs compared with that in GFP + -MEPs, huFlt3 + -MEPs, and CMPs (Fig. 6 D). Thus, enforced expression of STAT3 and PU.1 in MEPs reprogrammed them to diff erentiate into IPCs, DCs, and myelomonocytic cell lineages and inhibited Meg/E lineage potentials, indicating that strong Flt3 downstream signals were capable of inducing complete lineage conversion.

DISCUSSION
A standing question in early hematopoiesis is whether cytokine signaling is suffi cient to induce cell fate decisions. Here, we showed that enforced expression of huFlt3 in Flt3 − progenitors rescued their potential to diff erentiate into functional IPCs and DCs with comparable in vitro diff erentiation efficiency as Flt3 + progenitors (Figs. 1 and 2). We also demonstrated that enforced expression of huFlt3 in MEPs, which under normal conditions cannot give rise to IPCs and DCs (23)(24)(25)(26)(27) and are contained in Flt3 − progenitor cells, induced in vitro IPC and DC diff erentiation comparable to that observed from GFP + -GMPs (Fig. 3). Furthermore, huFlt3 + -MEPs diff erentiated into IPCs and DCs upon in vivo transfer, the most informative assay available to prove the robustness of in vitro observations (Fig. 4 B). Thus, these data demonstrate that enforced expression and signaling of huFlt3 in Flt3 − progenitors deliver an instructive signal to activate latent IPC and DC diff erentiation programs.
Overexpression of huFlt3 in total Flt3 + progenitors, in Flt3-expressing GMPs, and in CLPs (of which ‫%07ف‬ are Flt3 + ) led to a gain of higher relative and absolute (two-to threefold) numbers of IPCs and DCs in vitro (Figs. 1, D and E, and 3 C, and not depicted). Thus, beyond activation, increased Flt3 signaling also enhanced IPC and DC development. The gain in off spring cells was consistently higher for IPCs than for DCs (Figs. 1 E and 3 C), in line with our previous fi ndings that after 10 d of in vivo Flt3L injection, spleen IPCs and DCs were expanded on average 28-and 21-fold, respectively (33). This suggests that a continuous strong Flt3 signal might induce a shift toward relatively higher IPC levels.
We previously found that Flt3 is expressed in lymphoidand myeloid-committed progenitor cells, and in vivo Flt3L application mediates the expansion of both cell types without changing their biology (33). The enforced expression of huFlt3 in MEPs not only led to a gain of IPC and DC developmental capacity, but, with the exception of mixed colony formation, also to a gain of CFU activity of upstream myeloid progenitors as well as to diff erentiation of erythroid and myelomonocytic cells in vivo (Fig. 4 A). In contrast, huFlt3 signaling in GMPs did not activate megakaryocyte/ erythrocyte potential (Fig. 4 A). This implies that beyond activation and enhancement of IPC and DC development,

ARTICLE
Flt3 signaling is not immediately deterministic but primarily opens access to an IPC, DC, and myelomonocytic diff erentiation program. Thus, we propose that fi nal IPC and DC lineage outcome might be a gradual process, depending on continuous strong Flt3 signaling.
What are the Flt3 signaling-initiated downstream molecular events? It was shown that hematopoietic system-confi ned deletion of STAT3 transcription factor leads to the inhibition of Flt3-driven IPC and DC development (30). Furthermore, human Flt3 transfection and stimulation with Flt3L in mouse myeloid 32Dcl3 cells leads to the induction of PU.1 and C/EBPα expression (41). These transcription factors are indispensable for granulocyte and monocyte development (42), and it was shown that PU.1 cooperatively with C/EBPα activates myeloid development-associated cytokine receptor genes, including G-CSFR, M-CSFR, and GM-CSFR (42). Interestingly, PU.1-defi cient mice, in addition to other hematopoietic defects, lack either CD8α − or both CD8α − and CD8α + DCs, depending on the type of PU.1 deletion (43,44).
Here, we showed that enforced huFlt3 signaling in MEPs results in enhanced expression of IPC, DC, and GM lineage development related transcription factors STAT3, PU.1, and C/EBPα, as well as expression of G-, M-, and GM-CSF cytokine receptors (Fig. 5). Thus, at least in terms of these RNA transcripts, huFlt3 + -MEPs but not GFP + -MEPs resembled the gene expression profi les of CMPs (2,40).
Importantly, enforced expression of STAT3 or PU.1 in Flt3 − MEPs was again suffi cient to permit the development of both IPCs and DCs (Fig. 6 B). This, however, was only possible once TPO was added to SCF or SCF and Flt3L in cultures. Thus, TPO possibly substitutes for a survival signal otherwise delivered by Flt3. In addition, or alternatively, TPO might be involved in the phosphorylation of overexpressed STAT3 (45). Interestingly, enforced expression of STAT3 or PU.1 in MEPs led to the up-regulation of mouse Flt3 mRNA levels (Fig. S3). This in turn likely allowed culture-supplemented human Flt3L to cross-reactively stimulate STAT3-or PU.1-transduced cells via mouse Flt3. These results suggest a self-sustaining eff ect of Flt3 signaling-induced Flt3 transcription via downstream STAT3 and PU.1.
As enforced expression of huFlt3 in MEPs did not terminate megakaryocyte/erythrocyte diff erentiation potential, whereas huFlt3 expression in GMPs did not lead to a gain of these diff erentiation potentials (Fig. 4 A), how can Flt3 signaling be integrated in megakaryocyte/erythrocyte versus IPC, DC, and GM lineage commitment? By using PU.1 gfp reporter mice, PU.1 expression was recently mapped in early hematopoietic progenitor cells (46). It was shown that PU.1 + Flt3 + CMPs contain high myelomonocytic developmental potential, whereas PU.1 − Flt3 − CMPs and PU.1 − MEPs have high megakaryocyte/erythrocyte potential (46). The data presented here suggests that Flt3 might be critical in PU.1 regulation, although this likely will not be an exclusive event. GATA-1 is a nonredundant transcription factor for megakaryocyte and erythrocyte development (47). DNA binding activity of GATA-1 can be suppressed by enforced PU.1 expression, resulting in a diff erentiation block and apoptotic cell death of an erythroid cell line (48). Conversely, GATA-1 inhibits the binding of PU.1 to c-Jun, a critical coactivator of myeloid gene transactivation by PU.1 (49). Furthermore, GATA-1 interferes with DNA binding activity of STAT3 and inhibits TPO-dependent growth of the Ba/F3 cell line (50). Thus, as suggested previously for PU.1 and GATA-1 (51,52), relative dosage of gene transcription and protein levels will likely determine lineage outcomes. Indeed, STAT3 and PU.1 expression levels in huFlt3 + -MEPs were increased to levels of normal CMPs and were somewhat lower than those observed in GFP + -GMPs or huFlt3 + -GMPs (Fig. 5). Thus, it is possible that MEPs with relatively lower huFlt3 and consecutive STAT3 and PU.1 expression do not fully inhibit GATA-1, whereas high Flt3 expressing and signaling cells develop to IPC, DC, or GM lineages. Of note, enforced expression of STAT3 and PU.1 in MEPs suppressed GATA-1 expression and inhibited megakaryocyte/erythrocyte development (Fig. 6, C and D). HuFlt3 overexpression in GMPs in turn induced some EpoR, β-globin, and GATA-1 mRNA expression; however, this was not suffi cient to reactivate megakaryocyte/erythrocyte development as demonstrated for enforced high-level GATA-1 expression in GMPs (Figs. 4 A and 5 A; reference 13).
Because overexpression of huFlt3 in Flt3 − progenitors does not occur under physiologic conditions, what do these fi ndings imply for normal hematopoiesis? Flt3 is expressed on short-term HSCs, multipotent progenitors, CLPs, CMPs, and GMPs, and in vivo injection of Flt3L resulted in increased numbers of these cells as well as IPCs and DCs, whereas MEPs and their progeny remained unchanged (32,33). The data presented here demonstrate that enforced Flt3 cytokine receptor signaling is suffi cient to activate as well as enhance IPC and DC diff erentiation programs, suggesting that instructive cytokine signaling might indeed occur in hematopoiesis. Thus, we speculate that once Flt3 + short-term HSCs and their off spring Flt3 + cells are exposed to Flt3L-rich environments, these cells will be instructed to diff erentiate into IPCs and DCs. This might be enhanced by a self-sustaining process in which Flt3 downstream transcription factors STAT3 and PU.1 in turn maintain Flt3 receptor expression. However, Flt3 signaling does not immediately silence other developmental options. It is likely that most Flt3-expressing progenitors will not continuously be stimulated via Flt3L but will receive and activate alternative signals, and thus consecutively acquire myeloid or lymphoid, but not IPC or DC, cell fates. Beyond previous studies, our data further emphasize that IPC and DC development does not fi t into a deterministic "lymphoid" nor "myeloid" lineage, but rather a "Flt3-permissive" developmental model, where Flt3-expressing progenitors maintain IPC and DC diff erentiation options in response to Flt3L as long as no competing signal shuts these down. It will be of interest to test whether downstream dividing Flt3 + common IPC and DC progenitors with silenced alternative developmental programs exist and which critical factors are involved in fi nal IPC or DC lineage termination.
In vitro IPC and DC diff erentiation assays. Retrovirus-transduced Flt3 + and Flt3 − progenitors as well as CMPs, GMPs, and MEPs were cultured in IMDM, supplemented with 10% FCS, 10 −4 M 2-ME, sodium pyruvate, and antibiotics, 100 ng/ml huFlt3L-Ig, and 10 ng/ml mSCF as indicated. Half of the media was replaced every 3 d and new cytokines were added. Human Flt3L-Ig fusion protein was produced in Drosophila cells as described previously (54).