STAT3-and STAT5-dependent pathways competitively regulate the pan-differentiation of CD34 pos cells into tumor-competent dendritic cells

The clinical outcomes of dendritic cell (DC)-based immunotherapy remain disappointing, with DCs often displaying a tenuous capacity to complete maturation and DC1 polarization in the tumor host. Surprisingly, we observed that the capacity for successful DC1 polarization, including robust IL12p70 production, could be regulated by STAT-dependent events even prior to DC differentiation. Exposure of CD34 pos cells to single agent GMCSF induced multilineage, STAT5-dependent differentiation, including DCs which failed to mature in the absence of further exogenous signals. In contrast, Flt3L induced nearly global differentiation of CD34 pos cells into spontaneously maturing DCs. IL-6 synergized with Flt3L to produce explosive, STAT3-dependent proliferation of phenotypically undifferentiated cells which nevertheless functioned as committed DC1 precursors. Such precursors not only resisted many tumor-associated immunosuppressants, but also responded to tumor contact or TGF β with facilitated DC maturation and IL12p70 production, and displayed a superior capacity to reverse tumor-induced T-cell tolerance. GMCSF preempted Flt3L or Flt3L+IL6 licensing by blocking STAT3 activation and promoting STAT5-dependent differentiation. Paradoxically, following overt DC differentiation, STAT5 enhanced whereas STAT3 inhibited DC1 polarization. Therefore, non-overlapping, sequential activation of STAT3 and STAT5, achievable by sequenced exposure to Flt3L+IL6, then GMCSF, selects for multilog expansion, programming and DC1 polarization of tumor-competent DCs from CD34 pos cells.


Introduction
Dendritic cells (DCs) are the most potent antigen-presenting cells in the body and are employed in many tumor vaccine immunotherapy trials, rarely with therapeutic impacts. 1;2 DC preparations display a wide range of characteristics in vitro and in vivo, and it remains uncertain which individual properties may best promote successful immunotherapy. [3][4][5][6] A variety of single agents, including CD40 ligand, Toll-like receptor (TLR) agonists, and calcium ionophore, can induce DC phenotypic maturation. 3;4;6-9 Such maturation includes pronounced expression of MHC and costimulatory molecules, CD40 and CCR7, and IL-8 secretion, but falls short of the DC's potential to achieve DC1-polarization, a highly effective state for promoting cell-mediated immunity. 4;6;7;9;10 DC1-polarization includes abundant production of IL12p70 heterodimer and IL-23, secretion of the chemokine MIP-1β, and preferential expression of Delta-4 Notch Ligand. 4;6;7 Such DC1 products are highly associated with chemoattraction and activation of T1-type CD4 + and CD8 + Tcells. 4;6;7 Furthermore, IL12p70 production is critical to sensitize high avidity T-cells which directly recognize and kill tumor targets. 4;6;7 Although desirable for anti-tumor immunity, DC1-polarization is more easily signaled by infectious agents than by tumor exposure. Immature DCs employ recognition of pathogen-associated molecular patterns to assess the likelihood of host infection and the appropriateness of DC1-polarization. 6;7 Individual TLRs signal DCs primarily through the MyD88 pathway (e.g., TLR7-9) or TRIF pathway (e.g.,TLR3), with TLR4 evidencing pathway duplicity. 7 While activation of either pathway can induce elements of phenotypic DC maturation, dual pathway activation, or single pathway activation potentiated by exposure to IFN-γ or CD40 ligation, is required for robust DC1-polarization. 6;7 3 Fresh mobilization of DC1 precursors has the theoretical potential to promote cross-presentation of tumor Ags within the tumor host. Recent studies confirm the capacity of stem cell-mobilizing treatments, notably granulocyte-macrophage colony stimulating factor (GMCSF), granulocyte colony stimulating factor, fms-like tyrosine kinase receptor-3 (Flt3 ligand, Flt3L), or combined Flt3L+GMCSF to mobilize DC precursors, 5;11-13 but the potential of such precursors to achieve DC1polarization is presently unclear. Flt3L+GMCSF mobilization was recently reported to induce abundant infiltration of DCs into mouse tumors, but such DCs also activated regulatory T-cells and promoted tumor tolerance. 5 Immunosuppressive factors including IL-10, TGF-β (transforming growth factor beta), vascular endothelial growth factor (VEGF) and prostaglandin E 2 (PGE 2 ) are often produced by tumors, and may impede mobilized DC precursors from attaining optimal maturation and DC1-polarization. [14][15][16][17] However, mobilizing treatments themselves can also influence the later differentiation responses of stem cells. 5;11-13 We therefore postulated that particular stem cell proliferative treatments might provide conditioning signals which licensed rather than limited responsiveness to DC1-polarization stimuli.
Stem cell proliferation is inducible via multiple signaling pathways, including receptor-linked tyrosine kinases (Flt3L and stem cell factor (SCF)), [18][19][20] gp130 (e.g., IL-6), 18;19 and the hematopoetin receptor superfamily (e.g., GMCSF), 20 During the systematic testing of such signaling agents we identified that combined exposure to Flt3L plus IL-6 (Flt3L+IL6) not only synergized for stem cell proliferation, but also licensed CD34 pos progenitor cells to forego multilineage differentiation in favor of STAT3-dependent, dedicated DC differentiation. Such STAT3-dependent DC differentiation proved highly conducive to DC1-type functional competence during later interactions with tumor. In contrast, exposure of CD34 pos cells to GMCSF dominantly promoted an alternative, STAT5-dependent pathway of DC differentiation which was less conducive to tumor interactions.
For personal use only. on August 20, 2017. by guest www.bloodjournal.org From 5 Intracellular staining for TLR was performed directly on fixed, permeabilized cells with conjugated mAb (TLR3, TLR4, TLR8, TLR9), or indirectly with unconjugated mAb (TLR7), and appropriate controls from Imgenix (San Diego, CA). Staining for intracellular IRF4 and IRF8 was performed with reagents from Santa Cruz Biotechnology (Santa Cruz, CA) and mouse adsorbed F (ab)' 2 fragments of donkey anti-goat Ab (Research Diagnostics, Inc, Concord, MA), following the method of Tamura et al. 30 To isolate CD34 pos and CD34 neg cells from freshly harvested BM, the latter were stained with FITC-rat anti-mouse CD34 (dialyzed to remove sodium azide), then sorted on a FACSAria, yielding >96% pure CD34 pos and CD34 neg subpopulations.
In addition to PCR STAT determinations (see above), pSTAT proteins was analyzed on the cellular level by FACS. 31 Nuclei of cultured BM cells were permeabilized by sequential exposure to formalin (CytoFix™) and 90% methanol. Cells were then stained with PE-or FITC-conjugated anti-pSTAT3 (pY705), anti-pSTAT5 (pY694) or isotype controls (BD-Biosciences).

T-cell co-cultures Prior to T-cell harvest, DCs were prepared under various Step 1 conditions in CM;
Step 2 was performed in 1% non heat-deactivated mouse serum (MS) instead of FCS, during which BM cultures were exposed to viable irradiated tumor cells, then CpG+LPS. On the day of DC harvest, T-cells were freshly harvested from tumor-draining lymph nodes (TDLN) of 12 day tumor-bearing mice. The L-selectin low (tolerized pre-effector) fraction of T-cells was isolated as previously described, 29;32 and cultured in CM-MS with immobilized anti-CD3 29;32 or variously conditioned DCs. Beginning on day 2 of T-cell culture, some groups also received rhIL-2 (24 I.U./ml), or rhIL-2, rhIL-7 (50 ng/ml) and rhIL-15 (5 ng/ml). T-cells were harvested for assays and adoptive therapy after 12 days of culture.

T-cell specificity assays (intracellular IFN-γ)
Cultured T-cells were replated in fresh CM-MS at 2 million T-cells/well in 24 well plates. Whole cell irradiated tumor digests (5000 cGy) were added at 2 million cells/well as stimulators. Co-culture proceeded for 18h, with monensin added the final 13 hours. At harvest, individual treatment groups were FcR-blocked, then stained with FITC-anti-CD4 and Cychrome-anti-CD8. Following fixation/permeabilization, cells were additionally stained with PE-anti-mouse IFN-γ or isotype controls, then analyzed.
Adoptive immunotherapy 1.5 million viable tumor cells were injected into into healthy syngeneic mice to establish intradermal tumors. 29 5 or 10 days later, mice received conventional nonmyeloablative whole body irradiation (WBI, 500 cGy), 29 followed by culture-activated T-cells intravenously. Perpendicular bi-dimensional tumor measurements were performed twice weekly. Mice were euthanized when bidimensional product exceeded 225 mm 2 .
In vivo monitoring of BM cells Cultured BM cells were labelled with CFSE 21 and injected intravenously into syngeneic mice bearing 10 day s.c. MCA-203 or MCA-105 tumors. 48h later, mice were euthanized, tumors harvested and enzymatically digested to produce whole cell digests, with spleen cell suspensions prepared in parallel. 21 Preparations from individual mice were analyzed by FACS for CFSE pos cell frequencies. Groups were then co-stained with PE-conjugated mAb against DC-associated surface determinants, and FACS gated to analyze the CFSE pos subpopulation.
Statistics: Survival among treatment groups was compared by Fisher's exact test. Individual mice were scored for final treatment outcome (lethal tumor vs cure) and treatment groups compared. A two-tailed p value <0.05 was deemed significant. Proliferative synergy was assessed by Wilcoxon 6 signed rank test for paired data (proliferation following exposure to combined factors vs summed synchronous proliferations of the individual factors). Trafficking accumulation of CFSE-labelled cells and FACS pSTAT quantitations for cultured BM were assessed by Wilcoxon signed rank test for paired data. In all cases, a two-tailed p value <0.05 was deemed significant.
For personal use only. on August 20, 2017. by guest www.bloodjournal.org From

Results:
Later potentials for DC differentiation, DC maturation and DC1-polarization are determined by early CD34 pos cell conditioning We cultured fresh mouse BM in two steps, a 6-7 day proliferative culture (Step 1) and a 48-72 hour post-proliferative culture (Step 2), followed in some experiments by T cell co-culture (Fig 1a).
Consistent with previous reports, 18-20 three treatment pairings (Flt3L+IL6, SCF+IL6, or Flt3L+GMCSF) produced synergistic proliferation during Step 1 culture (Fig 1b). Such expansions represented a selective 35-80 fold numeric expansion of the CD34 pos cell subpopulation, with rapid dropout of initially CD34 neg cells (Fig 1c). CD34 pos cells continued brisk expansion for at least one additional week in culture if replenished with the same treatment pairings (not shown).
Even though Flt3L is myeloproliferative when administered to animals, 5;33 Step 1 culture with single agent Flt3L produced poor yields, even when dosing extended up to 300 ng/ml (Fig 1b and not  shown). This validated previous reports that single agent Flt3L is poorly proliferogenic ex vivo unless BM cultures are seeded at sufficiently high density to confer natural IL-6 supplementation. 34 Prior to initial culture, freshly harvested CD34 pos BM cells rarely displayed DC or other lineage markers (not shown). By the end of Step 1, however, cultures treated with either single agent Flt3L or single agent GMCSF displayed frequent differentiation into immature DCs, based on their dual positivity for CD11c and MHC Class II and low expression of CD40 and B7.2 (Fig 1d, also see Supplemental Fig S1a for full tested panel). Conventional DCs (positive for CD11b pos but negative for B220 neg ) predominated in both instances (Supplemental Fig S1b). Uniformly high MHC Class II expression was a hallmark of Flt3L-induced DC differentiation, whereas GMCSF-induced DCs were heterogeneous and predominantly low in regards to MHC Class II expression (Fig 1d).
Since Flt3L by itself was poorly proliferogenic (Fig 1b), we examined the superimposed impacts of IL-6 or GMCSF, since either agent produced proliferative synergy in conjunction with Flt3L (Fig 1b). BM proliferatively conditioned with Flt3L+IL6 failed to acquire either CD11c or MHC Class II expression, due to a pronounced anti-differentiative effect attributable to IL-6 (Fig 1d). In contrast, BM conditioned with either Flt3L+GMCSF or Flt3L+IL6+GMCSF developed heterogeneous differentiation which closely resembled treatment with GMCSF alone (Fig 1d). Therefore, even though IL-6 could exert a pronounced anti-differentiative effort upon Flt3L cultures, GMCSF could dominantly antagonize the conditioning impacts of both Flt3L and IL6 during Step 1 culture.

Following
Step 1 conditioning, Step 2 cultures were exposed to TLR agonists to examine their real-time potentials for DC1-polarization (Fig 1e). 6;7 Since both TLR9 and TLR4 were invariably expressed at the end of Step 1 mouse BM cultures (not shown), we standardly employed CpG (ODN 1826) and lipopolysaccharide (LPS) during Step 2 culture to elicit coordinate activation of MyD88 and TRIF pathways. 6;7 Step 1 conditioning with single agent GMCSF rendered a large proportion of cells hyporesponsive or unresponsive to subsequent TLR agonists, as evidenced by limited or absent upregulation of MHC Class II, CD40 and B7.2 expression (Fig 1e/Supplemental Fig S2a). Gr-1 co-expression was a common feature of poorly responsive subpopulation(s) (Supplemental Fig S2b). 35;36 In contrast, Step 1 conditioning with single agent Flt3L licensed consistently high TLR responsiveness, manifested by nearly global phenotypic DC maturation and only scant Gr-1 pos elements during subsequent Step 2 culture (Fig 1e/Supplemental Fig S2b). However, Flt3L's global licensing impacts were antagonized if GMCSF was also included during Step 1 culture (Fig 1e/Supplemental Fig S2a). Such 8 inhibition was not observed if initial exposure to GMCSF was deferred until Step 2 of culture (not shown).
In contrast to GMCSF, IL-6 not only produced proliferative synergy in conjunction with Flt3L (Fig  1b/1c), but also promoted Flt3L's global licensing impacts. Although an undifferentiated state persisted during Step 1 conditioning in Flt3L+IL6 (Fig 1d), nearly the entire expanded CD34 pos progenitor cell pool responded to subsequent TLR stimulation with DC differentiation, robust phenotypic maturation, and nearly uniform IL12p70 production (Fig 1e, Fig 2a). IFN-β was co-elicited rather than IFN-α by all tested TLR agonist combinations, indicating that conventional DC differentiation dominated during Step 2 culture (Supplemental Fig S2c). 7 However, as for Flt3L, Flt3L+IL6's global licensing impacts were abrogated if GMCSF was also included during Step 1 culture (Fig 1e/Supplemental Fig  2a).
The functional consequences of each of these conditioning regimens proved age-independent, with indistinguishable outcomes observed for BM obtained from mice aged 4-80 weeks (not shown). Similar responses were observed in all tested mouse strains.

Prior Flt3L or Flt3L+IL6 conditioning promotes spontaneous DC maturation and consistent TLR/IRF expression
Following lineage commitment, DCs typically remain immature unless they are exposed to exogenous signals such as CD40 ligation, calcium ionophore or TLR agonists. 3;4;6-9 It was observed, however, that BM cells conditioned in single agent Flt3L subsequently displayed spontaneous DC maturation, even when GMCSF was the only exogenous supplement provided during Step 2 culture (Fig  2b/Supplemental Fig S3a). Similar spontaneous DC maturation was also observed following Step 1 proliferative conditioning in Flt3L+IL6, even though the inclusion of IL-6 had delayed the onset of DC differentiation until the onset of Step 2 culture (Fig 1d vs Fig 2b/Supplemental Fig S3a). Spontaneous DC maturation following either Flt3L or Flt3L+IL6 conditioning was even more vigorous when IL-4 was also provided during Step 2 culture (Supplemental Fig S3b). Marked upregulation of the endocytic C-type lectin receptor DEC-205 was a conspicuous component of such spontaneous maturation (Supplemental Fig S3c).
For all conditioning treatments other than Flt3L or Flt3L+IL6, spontaneous Step 2 maturation was either highly attenuated or not observed. Importantly, Flt3L+IL6 was the only factor pairing which resulted both in proliferative synergy during Step 1 culture and in spontaneous DC maturation during Step 2 culture (Fig 2b/Supplemental Fig S3). Even when other conditioning treatments evoked limited elements of DC maturation during Step 1 culture (Fig 1d/Supplemental Fig S1a), such elements spontaneously reverted during Step 2 culture unless exogenous maturational stimuli such as TLR agonists were also provided (Fig 1e vs 2b, full panels in Supplemental Fig S2a vs S3). Flt3L or Flt3L+IL6's capacity to license spontaneous DC maturation was abrogated, however, if GMCSF was also included during Step 1 conditioning (Fig 2b/Supplemental Fig S3).
We characterized expression elements of the conventional DCs which dominated Step 2 culture following Flt3L+IL6 conditioning. Expression of TLR3, TLR4, TLR7, TLR8 and TLR9 remained uniformly detectable at all stages of culture, functionally confirmed by these cells' broad responsiveness to respective TLR agonists (Supplemental Fig S4a/S2c). Interferon-regulatory factors IRF4 and IRF8 were dually expressed both by DC precursors and polarized DC1 (Supplemental Fig S4b), demonstrating a largely homogeneous sequence of differentiation, maturation and polarization following Flt3L+IL6 conditioning.

9
Flt3L+IL6 conditioned DC precursors are stimulated rather than inhibited by tumor interactions We investigated whether Flt3L+IL6 proliferative conditioning licensed uniform responsiveness to DC1-polarization stimuli even in the presence of putative immunosuppressive factors. We added factors at doses which equaled and exceeded those reported to inhibit the maturation of other DC preparations. 14-17 IL-10 and VEGF exposure had negligible impacts upon DC1-polarization, whereas TGFβ1 exposure paradoxically enhanced both phenotypic maturation and IL-12 secretion (Fig 2c/2d). Only early PGE 2 exposure detectably inhibited TLR agonist-induced phenotypic DC maturation and IL-12 production (Fig 2c/2d). Nonetheless, a large subpopulation of Flt3L+IL6 conditioned BM cells resisted PGE 2 inhibition even at the beginning of Step 2 culture (Fig 2c, note the bimodal B7.2 expression), and such resistance became increasingly prevalent within hours of Step 2 culture (Fig 2d and  Supplemental Fig S5).
We tested the impact of exposing DCs to voluminous tumor burdens at 16-24 hours of Step 2 culture. To each well containing 4 million preconditioned BM cells, we added either 4 million freeze-thawed (killed) tumor cells; 3 million irradiated (10,000 cGy), trypan-excluding apoptotic tumor bodies; or 2 million unirradiated, actively proliferating tumor cells.

After
Step 1 Flt3L+IL6 conditioning, contact with any of these tumor materials accelerated DC phenotypic maturation, mimicking the stimulatory impact of exposure to a single TLR agonist (Fig 3a). 6;7 Combining such tumor exposure with IFN-γ treatment induced IL12p70 production (Fig 3a), mimicking the impact of combined exposure to IFN-γ plus a single TLR agonist. 4;7 When other Step 1 conditioning conditions were compared, activating effects of tumor were either highly attenuated or completely absent (Fig 3a).
The capacity of Flt3L+IL6 conditioned DCs to be activated by tumor contact proved strainindependent, occurring even for BALB/c BM-derived DCs despite this strain's Th2-biasing tendency 37 (not shown). All tested tumor lines stimulated DC maturation after Flt3L+IL6 conditioning, including MCA-205 and MCA-203 sarcomas and B16 melanoma derived from C57BL/6N mice, CT26 colon adenocarcinoma derived from BALB/c mice, and 888mel from a melanoma patient, and was not attributable to endotoxin content (not shown). Both freshly harvested whole cell tumor digests and established tumor lines proved stimulatory, indicating that host stromal cells were unessential, and DC maturation was stimulated whether tumor was syngeneic, allogeneic or xenogeneic (not shown). While fully killed lysate was effective, viable tumor proved more effective. Sequestration of tumor from Flt3L+IL6 conditioned DC precursors by Transwell™ membranes abrogated the activating effects of tumor, underscoring a requirement for direct contact; in contrast, phagocytosis of latex beads did not accelerate maturation (not shown).

Flt3L+IL6 conditioned DCs promote superior reversal of tumor-induced T cell tolerance
We examined the capacities of previously conditioned DCs to reverse tolerance in T-cells harvested from mice bearing advanced tumors. L-selectin low T-cells from tumor-draining lymph nodes (TDLN) are naturally sensitized to the relevant tumor but are also tolerized, consequent to the progressive upstream tumor burden. 29 In vitro exposure to anti-CD3, followed by IL-2 stimulation, can reverse tolerance and numerically expand anti-tumor effector T-cells. 29 However, such polyclonal stimulation causes CD8 + T-cells to overgrow CD4 + T-cells, and lacks the element of antigen presentation needed to selectively promote the tumor-specific T-cell subset. 29;32 Conditioned DC precursors were transferred to Step 2 culture, exposed to viable irradiated tumor, optionally further activated with CpG+LPS, then co-cultured with tolerized TDLN T-cells from mice bearing the relevant tumor. Flt3L+IL6 conditioned DCs efficiently reversed tolerance and stimulated robust T-cell proliferation, even when exogenous cytokines such as IL-2 were not added to co-culture 10 (Fig 3b/4a). CpG+LPS treatment enhanced but was unessential for such efficacy (not shown). In contrast, DCs prepared after other conditioning treatments typically proved lethal to T-cells, and toxicity could not be prevented by exogenous IL-2, IL-15 and/or IL-7 (Fig 3b/4a). Co-cultures driven by Flt3L+IL6 conditioned DC displayed superior outgrowth of both CD4 + and CD8 + tumor-specific Tcells compared to anti-CD3 treatment (Fig 4b.1/4b.2), and were highly potent when provided as adoptive therapy against early or advanced established tumors (Fig 4c/Supplemental Fig S6).

Flt3L+IL6 conditioned CD34 pos cells achieve spontaneous intratumoral DC maturation in vivo
Following various Step 1 culture treatments, BM cells were CFSE-labelled and administered to 10 day tumor-bearing mice. Cells injected immediately after Step 1 conditioning with Flt3L+GMCSF or Flt3L+GMCSF+IL6 displayed negligible trafficking into either tumor or spleen. In contrast, Flt3L+IL6 conditioned BM cells infiltrated both established tumors and spleen, achieving essentially uniform DC differentiation at either location (Fig 5a/5b). Moreover, accelerated DC maturation was observed following entry into tumor compared to spleen (Fig 5b), consistent with observed stimulatory impacts of tumor contact in vitro during Step 2 culture (Fig 3a).

Flt3L-vs GMCSF-conditioned DC programming reflects competing STAT3-vs STAT5dependent events
Knockout studies have demonstrated that Flt3 ligation transitions CD34 pos common precursors into committed DC precursors via a STAT3-dependent process, whereas GMCSF promotes STAT3-independent myeloid differentiation. 38 IL-6 signaling is known to induce gp130-mediated STAT3 activation in CD34 pos cells, 39 whereas GMCSF induces more complex STAT modulations, including STAT5 activation, at several stages of myeloid differentiation. 38;40;41 Because Step 1 GMCSF consistently abrogated the unique conditioning impacts of Flt3L or Flt3L+IL6, we examined whether STAT modulations played a pivotal role in GMCSF's apparently dominant regulation.
Step 1 IL-6 in the absence of GMCSF produced sustained activation of STAT3 but not STAT5, as evidenced by intranuclear staining for pSTAT3 (pY705) and pSTAT5 (pY694) (Fig 6a, "End Step 1" and Supplemental Fig S7). In contrast, inclusion of GMCSF as a component of any Step 1 regimen depressed STAT3 activation and produced biphasic upregulation of pSTAT5 (Fig 6a, "End Step 1" and Supplemental Fig S7). These distinctive pSTAT expression patterns modulated again during Step 2 culture, however, when cultures previously conditioned in Flt3L+IL6 now displayed the greatest capacity for STAT5 activation (Fig 6a, "End Step 2").
We next examined how STAT knockout BM preparations responded to Flt3L+IL6 conditioning. Consistent with STAT3's putative obligate role in Flt3L-induced DC differentiation, 38 we observed that STAT3 KO BM could not survive Flt3L+IL6 in vitro conditioning (not shown). In contrast, STAT5 KO BM responded to Flt3L+IL6 conditioning with intact robust proliferation (not shown) and nearly uniform DC differentiation during subsequent Step 2 culture (Fig 6b, row 2). Nonetheless, compared to wildtype littermates, Flt3L+IL6 conditioned STAT5 KO DCs displayed submaximal DC maturation and IL12p70 production (Fig 6b, row 2 vs 1, and Fig 6d left). Therefore, the proliferative and differentiative impacts of Flt3L+IL6 conditioning were absolutely STAT3-dependent, whereas subsequent phenotypic maturation and DC1-polarization were at least partially STAT5-dependent.
We also examined how STAT KO BM preparations responded to GMCSF-containing Step 1 regimens (GMCSF alone, Flt3L+GMCSF, or Flt3L+GMCSF+IL6). All of these GMCSF-based conditioning regimens were strikingly ineffective for generating DCs from STAT5 KO BM, instead yielding predominantly Gr-1 pos , MHC Class II neg cells which displayed morphologic features of mature neutrophils (e.g. , Fig 6b, row 4 and not shown). In contrast, STAT3 KO BM displayed normal proliferative kinetics (not shown) and typical heterogeneous differentiation, including DC differentiation, in response to all GMCSF-based regimens (Fig 6c, row 4). Following GMCSF-based conditioning, however, STAT3 KO 11 DCs displayed an abnormally heightened maturational and IL12p70 response to TLR stimulation (Fig  6c, row 4 vs 3 and Fig 6d right). Therefore, all tested GMCSF-containing regimens required intact STAT5 for DC differentiation, and later phenotypic maturation and DC1-polarization displayed the capacity for negative regulation through STAT3.

Discussion
Certain timely stimuli can condition DCs to adhere to an extended period of programming. For example, we recently reported that exposure of monocyte-derived human DCs to IFNγ+LPS not only stimulated an initial burst of IL12p70 secretion, but also licensed a second burst of IL12p70 secretion even days later if CD40 ligation was experienced. 6 We report here that durable DC programming can also be secured at remarkably early stages of hematopoeisis, even prior to discernible phenotypic DC lineage commitment. Exposure of fresh mouse BM to Flt3L+IL6 triggered multilog expansion of CD34 pos progenitor cells, and committed nearly all cells to subsequent DC differentiation. Such programming included subsequent spontaneous upregulation of MHC/costimulatory molecules, as well as nearly uniform responsiveness to DC1-polarization stimuli. Moreover, proliferative conditioning with Flt3L+IL6 conferred progressive resistance to many tumor-associated immunosuppressive factors, as well as the capacity to respond to either tumor contact or to TGFβ with facilitated DC1-polarization.
Flt3L+IL6 treatment produced identical licensing whether performed upon unfractionated BM or upon sorted CD34 pos cells, indicating that more primitive (CD34 neg /Sca-1 pos ) hematopoietic stem cells were not essential conditioning targets. Pronounced biasing towards ultimate DC differentiation was observed whether Step 1 conditioning was performed in Flt3L+IL6 or Flt3L alone. However, in the absence of co-conditioning by IL-6, Flt3L-induced DC precursors remained scant in number and displayed early maturation, whereas the inclusion of IL-6 produced an immense proliferative pool of CD11c neg /MHC Class II neg cells which were nonetheless already committed to subsequent DC differentiation. Such still phenotypically undifferentiated cells displayed the capacity both for wide distribution following i.v. injection and for spontaneous DC differentiation/maturation after entry into tumor.
IL-6 and its receptor transducing component gp130 have long been recognized to synergize for stem cell proliferation with receptor tyrosine kinase-activating stimuli, including both c-kit ligand (SCF) and Flt3-L, [18][19][20] but only combined Flt3L+IL6 also preconditioned for nearly global differentiation of expanded CD34 pos cells into spontaneously maturing DCs. Paradoxically, although GMCSF also synergized with Flt3L to induce Step 1 stem cell proliferation, GMCSF antagonized every DClicensing effect attributable to Flt3L.
Our evidence supports the existence of at least two discrete pathways for conventional DC differentiation with strikingly divergent functional outcomes, a Flt3L-promoted STAT3-dependent pathway and a GMCSF-promoted STAT5-dependent pathway (schematized in Fig 7). The Flt3L-promoted pathway was highly potentiated by IL-6, most likely due to the latter's capacity to confer sustained STAT3 activation during extended CD34 pos cell proliferation, and biased for differentiation into DC precursors which were uniformly facilitated for DC1-polarization, spontaneous DC maturation, and activation by tumor contact. In contrast, the GMCSF promoted, STAT5-dependent pathway gave rise to diverse myeloid elements, including DCs which failed to mature in the absence of further downstream driving signals such as TLR agonists.
We hypothesize that STAT5-dependent DC differentiation constitutes the normally dominant pathway, based on several observations: (1) the inclusion of GMCSF during CD34 pos progenitor cell proliferation blocked STAT3 activation and promoted STAT5-dependent myeloid differentiation regardless of whether Flt3L or Flt3L+IL-6 were also present; (2) recent studies indicate that STAT3-dependent DC differentiation is not normally detectable in adult mice.

13
Despite the competing roles played by STAT3 and STAT5 in DC differentiation, it is also apparent that their respective impacts change before and after DC differentiation occurs. Pronounced sequential STAT3/STAT5 activation (i.e., Flt3L+IL6 conditioned DCs in Fig 6a) corresponded to the greatest observed attainment of DC tumor-competence, and neither STAT3 nor STAT5 knockout BM responded optimally to Flt3L+IL6 conditioning. The preemptive impacts of GM-CSF during Step 1 culture appear to be mediated through early combined STAT3 inhibition and STAT5 activation (Fig  6). Paradoxically, however, appropriately delayed exposure to GMCSF may promote maximal DC1polarization through the identical STAT modulations (Fig 7).
The mechanism(s) causing Flt3L+IL6 preconditioned DCs to respond to tumor as a maturational signal remains to be elucidated. A plausible mechanism involves DC activation via enhanced expression of lectin receptors such as asialoglycoprotein receptor and DEC-205 (Supplemental Fig S3c), 3;45 since identical or similar carbohydrate receptors are employed by tumoricidal macrophages to bind and kill tumor cells in an MHC-unrestricted and antigen-unrestricted manner. 46;47 Many tumor cells bind lectin more avidly than non-transformed cells, due to a chronically high density of exposed carbohydrates and a diminished presence of differentiation elements which normally mask such carbohydrates. 46;47 Similarly, we have observed that exposure to albumin crosslinked with mannose or N-acetyl-glucosamine accelerates the maturation of Flt3L+IL6 conditioned BM cells (not shown).
We are investigating why most proliferative conditioning treatments caused DC preparations to be highly toxic to T-cell cultures. Such lethality was unlikely attributable to activation of regulatory Tcells or indoleamine dioxygenase-expressing pDC, since toxicity was not remedied by adding exogenous IL-2 to the T-cell co-cultures. 48;49 There was, however, a correlation between the Gr-1 pos cell-inducing tendency of individual conditioning treatments and observed lethality. It is therefore possible that the Gr-1 pos subpopulation(s) mediate the untoward effects of many of the conditioning treatments. 35;36 Consistent with this possibility, heightened induction of Gr1 pos myeloid suppressor cells appears to be the basis of immune disruption by high GMCSF-producing vaccine formulations. 36 In contrast to GMCSF-conditioned cultures, Flt3L+IL6 conditioned DC cultures were exceptional for their low Gr1 pos content and consistent absence of toxicity, even when added to T-cells in high proportions.
The above experiments, as well as preliminary studies with human CD34 pos progenitor cells (not shown), demonstrate that Flt3L+IL6 may provide an effective means to proliferate, condition and mobilize highly therapeutic DC precursors for tumor therapy. It has long been appreciated that IL-6 has extremely potent therapeutic properties against established mouse tumors, even when administered as a single agent. 50 We postulate that the major mechanistic role and benefit of IL-6 therapy will be in tandem with Flt3L to proliferate and condition tumor-competent DC precursor populations in cancer patients.         Fig 1a). Fig 1d) Step 2 treatment ↓ D C  Fig 1e, except following Step 1 conditioning each group was replated in fresh medium for 72 hours solely with rGMCSF (no IL4 or TLR agonists), after which FACS analyses were performed (selected groups shown in Fig 2b; all groups shown in Supplemental Fig 3a  online). Number within each histogram is the calculated "mean fluorescence specificity index" at 72h. Results shown are representative of three comprehensive comparisons. A similar but even more pronounced pattern of spontaneous maturation was observed during 48-72 hour Step 2 culture in GMCSF+IL4 (Supplemental Fig  3b online). (c) Step 2 cultures performed as in Fig 1e except that tumor- Fig 6 online). This is representative of 4 experiments.         Figure 7: Schematic representation of postulated STAT-dependent DC differentiation pathways. Flt3L stimulates pan-differentiation of CD34 pos common myeloid and common lymphocyte precursors into CD11cpos committed DC precursors via a STAT3dependent process. 31;38 This is markedly potentiated by IL-6, but is dominantly suppressed by Step 1 exposure to GMCSF, due to inhibition of STAT3 activation and concomitant STAT5 activation (Fig 6a). Such Step 1 GMCSF exposure instead favors differentiation of CD34pos common myeloid precursors into granulocyte/monocyte progenitors (rather than committed DC precursors). The granulocyte/monocyte progenitors achieve subsequent multilineage differentiation, including STAT5-dependent differentiation into conventional DCs and macrophages, and STAT5-independent differentiation into neutrophils (Fig 6b). Phenotypically conventional DCs generated by STAT3-vs STAT5-dependent pathways differ in many critical characteristics (Fig 1-5). It should be emphasized that although Step 1 exposure to GMCSF blocks STAT3-dependent DC differentation, Step 2 exposure of committed DC precursors to GMCSF may instead promote maturation and DC1polarization, again by stimulating STAT5 and inhibiting STAT3 (Fig 6a).