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The Journal of Immunology, 2003, 170: 3554-3564.
Copyright © 2003 by The American Association of Immunologists

Murine Flt3 Ligand Expands Distinct Dendritic Cells with Both Tolerogenic and Immunogenic Properties 1

George Miller, Venu G. Pillarisetty, Alaap B. Shah, Svenja Lahrs and Ronald P. DeMatteo2

Hepatobiliary Service, Memorial Sloan-Kettering Cancer Center, New York, NY 10021


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Human Flt3 ligand can expand dendritic cells (DC) and enhance immunogenicity in mice. However, little is known about the effects of murine Flt3 ligand (mFlt3L) on mouse DC development and function. We constructed a vector to transiently overexpress mFlt3L in mice. After a single treatment, up to 44% of splenocytes became CD11c+ and the total number of DC increased 100-fold. DC expansion effects lasted for >35 days. mFlt3L DC were both phenotypically and functionally distinct. They had increased expression of MHC and costimulatory molecules and expressed elevated levels of B220 and DEC205 but had minimal CD4 staining. mFlt3L DC also had a markedly altered cytokine profile, including lowered secretion of IL-6, IL-10, IFN-{gamma}, and TNF-{alpha}, but had a slightly increased capacity to stimulate T cells in vitro. However, in a variety of in vivo models, DC expanded by mFlt3L induced tolerogenic effects on T cells. Adoptive transfer of Ag-pulsed mFlt3L splenic DC to naive mice actually caused faster rates of tumor growth and induced minimal CTL compared with control DC. mFlt3L also failed to protect against tumors in which human Flt3 ligand was protective, but depletion of CD4+ T cells restored tumor protection. Our findings 1) demonstrate that mFlt3L has distinct effects on DC development, 2) suggest an important role for mFlt3L in generating DC that have tolerogenic effects on T cells, and 3) may have application in immunotherapy in generating massive numbers of DC for an extended duration.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Dendritic cells (DC) 3 are the primary APC of the immune system. Although DC are commonly recognized for initiating potent T cell immune responses, they also serve the paradoxical role of inducing tolerance to self-Ags (1). The precise events or mediators that direct DC toward immunity or tolerance are not understood. Some have postulated that CD8{alpha}- (myeloid) DC are inherently immunogenic while CD8{alpha}+ (lymphoid) DC are tolerogenic (2, 3, 4). However, recent studies have indicated that CD8{alpha}+ and CD8{alpha}- DC are not distinct populations but simply represent different maturational stages of the same DC lineage (5). A number of cytokines produced by DC are important in directing DC toward immunogenicity or tolerance. IL-6 enhances the immunogenicity of CD8{alpha}+ DC and prevents induction of T cell tolerance by mediating the effects of CD40 ligand (CD40L) (6). IL-12 secretion from CD8{alpha}- DC acts in an autocrine fashion to increase their immunogenicity and enhance Ag presentation (7, 8). In contrast, IL-10 secretion by DC promotes T cell tolerance (9). Similarly, IFN-{gamma} induces CD8{alpha}+ DC to mediate tolerogenic effects on T cells (6, 10).

Flt3 ligand (Flt3L) (3) is a growth factor that plays a critical role in the differentiation of hematopoietic stem cells in both humans and mice (11, 12, 13, 14, 15). However, substantial differences exist between human Flt3L (hFlt3L) and mouse Flt3L (mFlt3L) on both the genetic and protein levels (16). The hFlt3L gene is 5.9 kb long and maps to chromosome 19q (17). In contrast, the mFlt3L gene is ~4 kb in size and is located on chromosome 7 (17, 18). The primary translational product of the Flt3L gene is a type 1 transmembrane protein, which is cleaved to generate the soluble and biologically active form of Flt3L (19). The human and mouse Flt3L proteins contain 235 and 231 aa, respectively, and have 72% homology. However, while the extracellular domains of mouse and human Flt3L are very similar, their cytoplasmic domains are only 52% identical. Furthermore, mice abundantly express an additional membrane-associated isoform of Flt3L that is resistant to proteolytic cleavage but is biologically active on the cell surface (19). Besides the differences between mouse and human Flt3L, the Flt3R is also structurally distinct between the two species (15). Furthermore, the Flt3R is expressed in mice on some mature B cell lines but not on early B cell lines, myeloid, macrophage, or megakaryocyte cell lines (20, 21). In stark contrast, the Flt3R is expressed on a high percentage of human myeloid and monocytic cell lines as well as on some human megakaryocytic and early B cell lines (20, 22, 23).

Despite the differences between mouse and human Flt3L and their receptors, administration of rhFlt3L to mice results in the expansion of both CD8{alpha}+ and CD8{alpha}- DC subsets in multiple organs (24, 25). hFlt3L not only expands DC but also enhances DC-mediated immunogenicity in mice in a variety of models. Pulendran et al. (26) demonstrated that hFlt3L administration prevents peripheral tolerance and induces a potent DC-dependent immune response to soluble Ag. Kremer et al. (27) reported that treatment with hFlt3L protected against parasitic infections in susceptible mice. hFlt3L also induces rejection of allogenic transplants (28, 29) and augments antitumor immunity via activation of CD8+ T cells or NK cells (30, 31, 32).

Whereas the effects of exogenously administered hFlt3L on mouse DC development and immunogenicity have been carefully studied, the effects of mFlt3L on mouse DC generation, phenotype, and immunostimulation are less certain. In a landmark report using transgenic mice lacking Flt3L, McKenna et al. (14) showed that these animals had fewer DC in their spleen, lymph nodes, and thymus. In contrast, O’Keeffe et al. (33) demonstrated that daily administration of 10 µg of mFlt3L to mice resulted in far weaker DC expansion than comparably administered hFlt3L. For this study, we constructed a gene transfer vector to endogenously overexpress mFlt3L in mice. The goals of this study were 1) to elucidate the role of mFlt3L on murine DC development and population expansion, 2) to determine the effect of mFlt3L on DC induction of immunity, and 3) to assess the potential use of our vector in immunotherapy regimens against tumors. We found that a single treatment with our vector resulted in DC expansion that was superior to rhFlt3L administration. Moreover, mFlt3L DC were distinct from controls in terms of their phenotype, cytokine secretion, and immunostimulatory function. Most notably, they expressed high levels of CD8{alpha}, B220, and DEC205, produced lowered levels of a variety of cytokines, and induced robust T cell stimulation in vitro. However, in vivo, they variably induced tolerance or immunity to Ag and tumor.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
DC isolation and culture

Spleens were perfused with collagenase (Sigma-Aldrich, St. Louis, MO) and mechanically disrupted. DC were then purified (>90%) using anti-CD11c immunomagnetic microbeads and LS separation columns (Miltenyi Biotec, Auburn, CA). DC were cultured in complete medium (RPMI with 10% heat-inactivated FBS, 2 mM L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, and 0.05 mM 2-ME) supplemented with GM-CSF (20 ng/ml; from supernatant of a GM-CSF-transduced J558 cell line; gift of R. Steinman (Rockefeller University, New York, NY)).

Flow cytometry

Flow cytometry was performed on an EPICS-XL flow cytometer (Beckman Coulter, Fullerton, CA) after incubating 5 x 105 splenocytes or DC with 1 µg of Fc block (anti-CD16/CD32 (2.4G2); BD PharMingen, Franklin Lakes, NJ) and then labeling with 1 µg of PE- or FITC-conjugated Ab. Cells were stained for B cells (B220 (RA3-6B2) and CD19 (SJ25C1)), NK cells (NK1.1 (PK136)), macrophages (Mac-3 (M3/84)), T cells (CD4 (GK1.5) and CD8{alpha} (53-6.7)), DC (CD11c (HL3)), MHC class I (H-2Kb) and class II (I-Ab), CD40 (3/23), CD54 (ICAM-1 (3E2)), CD80 (B7-1 (1G10)), CD86 (B7-2 (GL1)), CD11b (M1/70), Ly-6G (Gr-1 (RB6-8C5)) (all BD PharMingen), and DEC205 (NLDC-145) (Cedarlane Laboratories, Hornby, Ontario, Canada).

Recombinant adenovirus and reagents

A recombinant adenovirus carrying the mFlt3L gene (AdmFlt3L) was constructed as follows: a plasmid containing the mFlt3L cDNA was obtained from the National Gene Vector Laboratories (Ann Arbor, MI). The SalI fragment was ligated into the SalI site of the shuttle plasmid pDC316 (Microbix, Toronto, Ontario, Canada) that contains a portion of the adenoviral genome and the murine cytomegalovirus promoter (34). Cre-mediated homologous recombination at specific loxP sites was then performed on 293 human embryonic kidney cells (American Type Culture Collection, Manassas, VA) with pBHGlox (Microbix) that contains the adenovirus type 5 genome with E1 and E3 region deletions. DNA was isolated from candidate viral plaques and screened with restriction enzymes to confirm the presence of the mFlt3L transgene. Results were compared with the predicted banding pattern using OMIGA DNA analysis software (Omiga, San Diego, CA). Virus was quantitated by plaque assay and stored at -80° in PBS with 10% glycerol. Adenovirus encoding green fluorescent protein (AdGFP; Quantum Biotechnologies, Montreal, Quebec, Canada) is a recombinant adenovirus under the control of a cytomegalovirus promoter. All adenoviruses were propagated and purified as previously described (35). For selected experiments, 10 µg of Chinese hamster ovary cell-derived hFlt3L (gift of Immunex, Seattle, WA) was injected i.p. for 10 consecutive days.

Cytokine measurement and Ag uptake assays

For in vitro cytokine assays, freshly isolated DC were cultured at a concentration of 1 x 106 cells/ml for 24 h. Supernatant was then harvested for ELISA. For selected experiments, LPS (10 ng/ml; Sigma-Aldrich), TNF-{alpha} (100 ng/ml; R&D Systems, Minneapolis, MN), or an agonistic CD40 Ab obtained from the clone FGK45 (36) (100 ng/ml; Monoclonal Antibody Core, Sloan-Kettering Institute) were used to stimulate DC. Serum or cell culture supernatant was tested by ELISA for IL-2, IL-4 (both BD PharMingen), IL-6, IL-10, IL-12 (p70), IFN-{gamma}, TNF-{alpha}, and Flt3L (all R&D Systems) according to the respective manufacturers’ protocols. For in vitro Ag uptake assays, freshly isolated DC (2 x 105) were incubated with FITC-albumin (1 mg/ml; Sigma-Aldrich) at 37°. For in vivo Ag uptake assays, mice were injected i.v. with 2.5 mg of FITC-dextran or DQ-OVA (Molecular Probes, Eugene, OR). After 30 min, splenic DC were harvested for flow cytometry.

T cell proliferation and cytotoxicity assays

For MLR, DC were irradiated (3000 rad) and added in various ratios to 1 x 105 syngeneic or allogenic T lymphocytes (purified using CD90 (Thy1.2) microbeads (Miltenyi Biotec)) in 96-well plates. T cells were pulsed with thymidine (0.5 µCi/well) on day 3 for 20 h. For Ag-specific T cell stimulation assays, DC were incubated with either OVA257–264 peptide (10 µg/ml; Protein Synthesis Core, Sloan-Kettering Institute) or OVA protein (2 mg/ml; Sigma-Aldrich) for 90 min before being plated for 2 days in 96-well plates with an H-2Kb-restricted CD8+ T cell hybridoma specific for OVA257–264 peptide (37). T cell activation was determined by measuring supernatant IL-2 levels by ELISA. CTL assays were performed as described with modifications (38). Briefly, splenocytes from experimental animals were cultured at 5 x 106 cells per well in 24-well plates with OVA257–264 peptide (10 µg/ml) for 5 days. Afterward, effectors were plated against 1 x 104 51Cr-labeled target cells for 4 h in 96-well plates. For some experiments, splenocytes were restimulated with 1 x 106 irradiated (20,000 rad) EG7 cells which express OVA (American Type Culture Collection). Targets included EG7 cells, parental EL4 cells (American Type Culture Collection), and EL4 that had been loaded in vitro with OVA257–264 peptide (10 µg/ml). Spontaneous release (no effectors) and maximum release (2% Triton-X; Sigma-Aldrich) were also assayed. Percent lysis was calculated as follows: ((experimental - spontaneous release) x 100)/(maximum release - spontaneous release). For NK cytotoxicity assays, NK cells were isolated from splenocytes using anti-NK (DX5) microbeads and LS separation columns (Miltenyi Biotec). The purity of isolated NK cells was >85% by flow cytometry. NK cells were then plated in 96-well plates against 5 x 103 51Cr-labeled Yac-1 cells (American Type Culture Collection). Percent lysis was calculated in the same manner as for the CTL assays.

Animals procedures and tumor models

Male C57BL/6 (H-2Kb) and BALB/c (H-2Kd) mice (6–10 wk old) were purchased from Taconic Farms (Germantown, NY). For adenoviral treatments, mice were given single tail vein injections of 8 x 1010 particles of adenovirus unless otherwise specified. For DC adoptive transfer experiments, mice were given two i.p. immunizations on days 0 and 7 of 5 x 105 DC that had been pulsed for 90 min in vitro with OVA257–264 peptide (10 µg/ml). For some experiments, mice were immunized s.c. with 500 µg of OVA 8 days after treatment with AdmFlt3L. Mice were challenged at various time points with a s.c. injection of 3 x 105 EG7 or EL4 cells, 1 x 105 CT26 colorectal carcinoma cells (American Type Culture Collection), or 2 x 105 B16 (American Type Culture Collection) or B16.OVA melanoma cells (gift of D. Brown, University of Rochester, Rochester, NY). Alternatively, mice were administered an intrasplenic injection of 2 x 104 B16 cells or 5 x 104 CT26 cells via a flank incision followed by splenectomy. T cell depletions were performed using three i.p. injections of 0.25 mg of GK1.5 (anti-CD4+) or 53-6.72 (anti-CD8+; both Monoclonal Antibody Core, Sloan-Kettering Institute) within 5 days before treatment and then once weekly. To deplete NK cells, 100 µl of anti-asialo GM1 (Wako Chemical, Richmond, VA) was administered i.p. every 5 days. All procedures were approved by the Institutional Animal Care and Use Committee.

Statistical analysis

Survival and time to tumor development were analyzed by the log rank test. Flow cytometry results were assessed using the {chi}2 test. All other comparisons were tested using ANOVA. SPSS statistical software (version 10) was used (SPSS, Chicago, IL).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
AdmFlt3L administration results in sustained elevations of serum mFlt3L

To determine the serum levels of mFlt3L after treatment with AdmFlt3L, we inoculated mice with a range of viral doses from 5 x 109 to 2 x 1011 particles. At doses below 1 x 1010 particles, minimal increases in serum mFlt3L were noted. However, at higher viral doses (>=4 x 1010 particles), mFlt3L levels rose sharply. By day 3 after administration of 2 x 1011 particles of AdmFlt3L, serum mFlt3L levels reached 10 µg/ml. However, sporadic toxicity including death was noted at this dose. Therefore, we used 8 x 1010 particles for further studies which produced day 3 serum levels of 1.5 µg/ml and no toxicity. We have previously shown that this dose transiently infects ~90% of hepatocytes (34). To determine the time course and duration of mFlt3L secretion, we injected mice with 8 x 1010 viral particles and measured serum mFlt3L levels at serial time points (Fig. 1). Serum levels peaked between days 3 and 7 after injection and remained elevated for >4 wk after a single viral administration. Administration of AdGFP did not produce elevations in serum mFlt3L over saline treatment (not shown).



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  		FIGURE 1. AdmFlt3L produces marked and sustained levels of serum mFlt3L. The time course and duration of mFlt3L production was determined by injecting mice with 8 x 1010 viral particles and measuring serum protein levels at serial time points. mFlt3L levels were highest between days 3 and 7 after treatment and dropped sharply by day 14. However, mFlt3L levels were still elevated by >30-fold over controls 28 days after treatment. Assays were done in triplicate and averages of three mice per data point are shown. SEM was <5%.

 
Endogenous secretion of mFlt3L results in massive and sustained DC expansion

To assess the effect of endogenous secretion of mFlt3L on DC population expansion, we injected mice with a range of doses of AdmFlt3L and determined the number of splenic DC 1 wk later using flow cytometry. At viral doses between 5 x 109 and 4 x 1010 particles, both the total number of splenocytes and the number of splenic DC increased only ~2-fold. However, at doses of 8 x 1010 particles or above, the total number of splenocytes increased by 5-fold to 250 million cells, and there was an up to 70-fold increase in the number of splenic DC by day 7.

To define the time course and duration of DC expansion, we injected mice with 8 x 1010 particles of AdmFlt3L and measured the number and percentage of splenic DC at various intervals (Fig. 2). The DC expansive effects of mFlt3L peaked on day 10 after viral administration at which time 44% of all splenocytes were CD11c+ and the total number of splenic DC approached 1 x 108 cells which was a ~100-fold increase over baseline (Fig. 2). DC infiltrated the entire spleen except the germinal centers (not shown). After day 10, effects waned. Nevertheless, DC populations remained elevated over controls for >35 days after a single treatment. In contrast, after 10 consecutive daily injections of 10 µg of rhFlt3L, only 15–20% of splenocytes become CD11c+ and DC levels return to baseline <1 wk later (24, 25). AdGFP treatment alone (8 x 1010 particles) resulted in a 2-fold increase in both the number and percentage of splenic DC. DC population expansion by AdGFP continued for 28 days (not shown).



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  		FIGURE 2. Endogenous secretion of mFlt3L massively expands splenic DC. The time course and duration of splenic DC expansion by AdmFlt3L were determined by injecting mice with 8 x 1010 viral particles and assessing the number and percentage of CD11c+ cells at serial intervals. DC expansion peaked at day 10 at which time 44% of splenocytes were CD11c+ and the total number of DC approached 100 million. Moreover, DC populations remained elevated for >35 days after a single treatment. Data are representative of experiments repeated more than five times.

 
mFlt3L does not differentially expand splenic NK populations

In contrast to the considerable expansion of splenic DC populations, the percentage of splenic NK cells did not change appreciably by overexpression of mFlt3L (Table I). This is notable because rhFlt3L injections have been reported to triple the percentage of splenic NK cells (39). Furthermore, to compensate for the differential expansion of DC, we observed a sharp decrease in the percentage of splenic B cells (from 66 to 45%) and T cells (from 31 to 14%). Nevertheless, because of the increase in total splenocytes, the absolute number of each subtype still increased. This resulted in a 6- to 7-fold increase in the splenic weight of AdmFlt3L-treated mice compared with saline controls and a ~3-fold increase compared with AdGFP-treated mice (Table I). Although NK populations were not differentially expanded by mFlt3L, they were activated. For example, when we harvested NK cells 1 wk after AdmFlt3L administration and plated them against Yac-1 cells, they induced considerably higher lysis compared with NK cells from saline-treated mice (Fig. 3). However, this marked NK activation was an effect of systemic adenovirus alone and was not further enhanced by mFlt3L production.


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  		Table I. Cellular composition of spleens 1 wk after treatment with saline or virus (8 x 1010 particles)

 


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  		FIGURE 3. Adenovirus alone activates NK cells in vivo. Mice were treated with saline or 8 x 1010 particles of AdmFlt3L or AdGFP. Splenic NK cells were then harvested 1 wk later and plated against chromium-labeled Yac-1 cells. Systemic adenovirus caused a profound increase in NK cell lytic activity (p < 0.001), whereas expression of mFlt3L did not confer additional activation. Triplicate wells were tested and data are representative of experiments repeated on three occasions.

 
mFlt3L DC express high CD8{alpha}, DEC205, and B220 and are more mature than controls

The DC population expanded by mFlt3L was phenotypically distinct from saline and AdGFP controls. mFlt3L DC were 55–80% CD8{alpha}+CD11blow/- compared with 20–35% CD8{alpha}+CD11blow/- staining in control DC (Fig. 4A). Furthermore, DEC205 was expressed in ~40% of DC from mFlt3L-treated mice compared with only 5–15% of saline or AdGFP controls (Fig. 4A). Other distinct characteristics of mFlt3L DC were their exceptionally low (<10%) surface expression of CD4 and high (up to 60%) B220 expression (Fig. 4B). The latter finding did not represent plasmacytoid DC because cross-staining of B220 and Gr-1 was negligible (not shown). Besides expanding distinct DC subsets, mFlt3L DC were also phenotypically more mature than controls. They expressed moderately higher levels of MHC class I, class II, CD40, CD54, and CD80 than mice treated with saline or AdGFP (Table II). These phenotypic findings were similar on days 7, 10, 14, and 21 after treatment. Consistent with their relative phenotypic maturity, mFlt3L DC were also morphologically more mature on cytospin analysis. They were larger and exhibited more abundant dendritic processes than DC from saline- or AdGFP-treated mice (not shown).



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  		FIGURE 4. mFlt3L expands phenotypically distinct DC. Freshly isolated splenic DC from mice treated with AdmFlt3L or controls were analyzed by flow cytometry on day 7 after injection. A, CD11c+ cells were stained for CD8{alpha}, DEC205, and CD11b. B, mFlt3L-expanded DC had low CD4 staining but high B220 expression compared with isotype controls which were set within the first decade (not shown). Data are representative of experiments repeated more than six times (p < 0.001).

 

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  		Table II. DC expression of MHC, integrins, and costimulatory molecules by flow cytometry 1 wk after treatment with saline or virus (8 x 1010 particles)

 
The phenotype of mFlt3L DC contrasts with reports of DC expanded by hFlt3L. Whereas mFlt3L generated predominantly lymphoid DC, hFlt3L reportedly attracts fairly equal numbers of CD8{alpha}+ and CD8{alpha}- DC (24). Furthermore, mFlt3L DC that expressed DEC205 were CD8{alpha}+CD11blow/-, whereas DEC205- DC were CD8{alpha}-CD11bhigh (Fig. 4A). In contrast, hFlt3L expands substantial numbers of DEC205+CD8{alpha}-CD11bhigh DC (33). In addition, whereas B220 is highly expressed on mFlt3L DC, it is essentially absent after hFlt3L injections (24).

mFlt3L DC secrete less IL-6, IL-10, IFN-{gamma}, and TNF-{alpha}

Because mFlt3L DC were phenotypically and morphologically distinct, we postulated that they would have altered function. We first tested their cytokine secretory profile and their response to activating stimuli. mFlt3L DC produced lower levels of a number of cytokines including 5-fold lower levels of IL-6 and 40% lower IL-10 compared with mice treated with saline (Fig. 5A). Furthermore, IFN-{gamma} and TNF-{alpha} were undetectable in the supernatant of mFlt3L DC (Fig. 5, A and B). The decreased cytokine production in mice overexpressing mFlt3L appeared to be partially an effect of systemic adenovirus alone because DC from AdGFP-treated mice produced intermediate levels of IL-6, IL-10, IFN-{gamma}, and TNF-{alpha}. Despite the lowered cytokine secretion from unstimulated mFlt3L DC, activation with CD40L or LPS increased their IL-6, IL-10, TNF-{alpha}, and IFN-{gamma} production albeit at lower levels than those of controls (Fig. 5, B and C, and not shown). The relative differences in cytokine production between groups was also unaltered when the DC were cultured in the absence of GM-CSF or when DC were further stimulated by TNF-{alpha} (not shown). IL-2, IL-4, and IL-12, and Flt3L were undetectable in the supernatant of all DC groups even after CD40 ligation or stimulation with LPS or TNF-{alpha}.



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  		FIGURE 5. Unstimulated mFlt3L-expanded DC secrete low IL-6, IL-10, IFN-{gamma}, and TNF-{alpha}. Freshly isolated DC were cultured for 24 h at a density of 1 x 106 cells/ml in 24-well plates. Supernatant was then harvested and tested by ELISA. mFlt3L-expanded DC secreted lower levels of IL-6 (p < 0.001) and IL-10 (p = 0.11) and undetectable IFN-{gamma} (p < 0.001) (A) and TNF-{alpha} (p < 0.001) (B). Stimulation with LPS (10 ng/ml) induced mFlt3L-expanded DC to produce low levels of TNF-{alpha}. C, Similarly, expanded DC increased their secretion of IL-6 and IL-10 upon CD40 ligation (100 ng/ml). Assays were done in triplicate and repeated three times. SEM was <5% for each ELISA shown.

 
mFlt3L DC have enhanced capacity to capture Ag

We next tested the ability of DC expanded by mFlt3L to capture Ag in vitro. Freshly isolated mFlt3L DC had a 30–40% increased uptake of albumin compared with saline and AdGFP controls (Fig. 6). To examine the relative ability of mFlt3L DC to capture Ag in vivo, we i.v. injected fluorescent dextran into mice and harvested their splenic DC 30 min after administration for flow cytometry. In consort with our in vitro data, nearly 50% of mFlt3L DC captured dextran compared with 7 and 22% for saline and AdGFP controls, respectively (Fig. 6). Next, to test the ability of DC expanded by AdmFlt3L to process Ag, we i.v. injected DQ-OVA which requires Ag processing to become fluorescent. We found that mFlt3L splenic DC had a similar capacity to process DQ-OVA compared with that of controls (not shown).



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  		FIGURE 6. DC expanded by mFlt3L have enhanced capacity to capture Ag in vitro and in vivo. Freshly isolated mFlt3L-expanded DC (2 x 105) had higher uptake of FITC-albumin (1 mg/ml) than controls after 5 min of incubation (p < 0.001). Similar relative differences were seen at 30, 45, and 60 min (not shown). To test DC Ag uptake in vivo, mice were inoculated with FITC-dextran 7 days after treatment with saline or virus (8 x 1010 particles). Mice were sacrificed 30 min after FITC-dextran injection, and splenic DC were isolated and assayed by flow cytometry. A far higher percentage of mFlt3L-expanded DC captured Ag in vivo compared with saline and AdGFP controls (p < 0.001). Averages of triplicates are shown. SEM was <5% for the in vitro and in vivo assays.

 
mFlt3L DC induce potent T cell stimulation in vitro

Because mFlt3L DC had modestly elevated expression of MHC and costimulatory molecules and increased Ag capture capabilities, we postulated that these DC would have an increased ability to stimulate T cells. We first tested their allostimulatory capacity in an MLR. We consistently observed that mFlt3L-expanded DC induced slightly higher allogenic T cell proliferation than DC from saline- or AdGFP-treated mice. However, results did not reach statistical significance (Fig. 7A). To test their Ag-restricted T cell stimulatory capacity, we loaded freshly isolated DC in vitro with OVA257–264 peptide or OVA before plating them with OVA257–264 peptide-restricted T cells. Consistent with the MLR data, peptide- or protein-pulsed mFlt3L DC induced slightly higher IL-2 production from OVA257–264 peptide-restricted T cells than controls at the highest concentrations of DC stimulators (Fig. 7, B and C). For example, 3 x 104 OVA257–264 peptide-pulsed mFlt3L DC induced 92 pg/ml IL-2 production from OVA257–264 peptide-restricted T cells compared with 68 pg/ml for saline and AdGFP controls (Fig. 7B).



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  		FIGURE 7. mFlt3L-expanded DC induce slightly higher stimulation of allogenic and Ag-restricted T cells in vitro. A, C57BL/6 splenic DC were isolated 1 wk after treatment with saline or virus (8 x 1010 particles) and plated in various ratios with 1 x 105 allogenic T cells from BALB/c mice. mFlt3L-expanded DC showed a trend of inducing higher alloproliferation than controls (p = 0.06). Syngeneic proliferation was <2000 cpm for all groups (not shown). To test their Ag-restricted T cell stimulation, we loaded freshly isolated DC in vitro for 1 h with OVA257–264 peptide (10 µg/ml) (B) or OVA (2 mg/ml) (C) before plating them for 2 days with 5 x 104 OVA257–264 peptide-restricted T cells. Both peptide- and protein-pulsed mFlt3L-expanded DC induced higher IL-2 production from the OVA257–264 peptide-restricted T cells. T cell stimulation assays were repeated at least three times with similar results. Averages of triplicate wells are shown. The value of p < 0.001 for the OVA257–264 peptide assay, and a value of p = 0.005 for the OVA assay at the highest concentrations of DC stimulators. Differences were not significant at lower concentrations of DC stimulators. Data are presented as mean ± SEM.

 
Adoptive transfer of Ag-loaded mFlt3L DC accelerates tumor growth

Because mFlt3L DC induced potent T cell stimulation in vitro, we postulated that they would protect against tumor in vivo. To test this, we loaded freshly isolated mFlt3L splenic DC or controls in vitro with OVA257–264 peptide and used them to immunize naive mice. After two weekly immunizations, recipient mice were challenged 1 wk later with a s.c. injection of EG7 lymphoma cells. Immunization with OVA257–264 peptide-loaded DC from mice treated with saline or AdGFP delayed or prevented tumor growth. However, surprisingly, adoptive transfer of OVA257–264 peptide-pulsed mFlt3L DC actually increased the rate of tumor growth (Fig. 8). For example, 60–70% of mice that were immunized with DC.OVA257–264 from saline- or AdGFP-treated donors were tumor free by day 21 and their mean tumor size was ~50 mm2. In contrast, only 8% of mice receiving mFlt3L DC.OVA257–264 were tumor free, and they had a mean tumor size of 400 mm2, which was even twice the size of unimmunized mice. These findings were consistent in four separate experiments. Sorting of the freshly isolated mFlt3L DC into CD8{alpha}+ and CD8{alpha}- fractions before immunization did not change the outcome of the tumor experiment (not shown). Furthermore, CD40 ligation in vitro only marginally improved the results of immunization using mFlt3L DC (Fig. 8B).



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  		FIGURE 8. Immunization with peptide-loaded mFlt3L DC causes increased tumor growth. Mice were immunized twice with OVA257–264 peptide-pulsed DC derived from donor mice that had been treated 1 wk earlier with saline, AdGFP, or AdmFlt3L. After two immunizations, recipient mice were challenged with s.c. EG7. Time to tumor development (A) and tumor size measurements (presented as mean ± SEM) (B) were recorded. Mice immunized with peptide-pulsed DC from saline (n = 5)- or AdGFP (n = 6)-treated mice were partially protected from tumor development compared with unimmunized mice (n = 10). However, immunization with Ag-pulsed mFlt3L-expanded DC (n = 12) caused a faster rate of tumor growth. In vitro CD40 ligation (100 ng/ml) of mFlt3L-expanded DC during Ag loading only partially reduced this effect. For the time-to-tumor-development experiment, p < 0.001; for the tumor size analysis, p = 0.002.

 
To determine whether mFlt3L DC were actually deficient in generating a CTL response in vivo, we used a similar strategy of loading DC with OVA257–264 peptide and adoptively transferring these DC to naive mice. After two immunizations, we harvested splenocytes from the recipient animals, restimulated them in vitro with OVA257–264 peptide for 5 days, and plated them against chromium-labeled EG7 cells in a CTL assay. In consort with the results of the tumor experiment, immunization with OVA257–264 peptide-loaded mFlt3L DC produced considerably lower CTL activity than did controls (Fig. 9A). This was not related to an adenovirus effect alone (not shown). To determine whether the high CD8{alpha}+ DC fraction among mFlt3L-expanded DC was responsible for their reduced capacity to induce CTL, we again sorted the CD8{alpha}+ and CD8{alpha}- mFlt3L DC before peptide loading and adoptive transfer. However, CTL activity after immunization with either of the mFlt3L DC subsets was markedly lower than after immunization with peptide-pulsed DC from saline-treated animals (Fig. 9A). To confirm that CTL activity was OVA257–264 peptide specific, we plated splenocytes against EL4 cells. Percent lysis against this target was ~70% lower than against EG7 (not shown). Although T cells from animals immunized with Ag-pulsed mFlt3L DC had reduced lytic activity in CTL assays, they produced far higher levels of IFN-{gamma} in their spent supernatants than did controls (Fig. 9B). This was due to the high CD8{alpha}+ DC fraction expanded by mFlt3L and is consistent with previous reports which indicated that CD8{alpha}+ DC subsets generate predominantly IFN-{gamma}-secreting T cells in vivo (40, 41). Conversely, immunization with Ag-pulsed control DC resulted in higher splenocyte IL-4 production (Fig. 9C).



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  		FIGURE 9. Immunization with peptide-loaded mFlt3L-expanded DC induces weak CTL activity. Mice were either unimmunized or immunized twice with OVA257–264 peptide-pulsed DC from mice treated with saline or AdmFlt3L. After two immunizations, splenocytes were harvested from recipient mice, restimulated in vitro for 5 days, and plated against EG7 cells. A, Immunization with mFlt3L-expanded DC resulted in far weaker CTL activity than immunization with saline DC (p < 0.001). Neither the CD8{alpha}+ nor the CD8{alpha}- mFlt3L-expanded DC fraction induced potent CTL. B, Despite their lowered lytic activity, cytokine analysis of the spent CTL supernatant revealed that the CD8{alpha}+ DC fraction among mFlt3L-expanded DC caused increased secretion of IFN-{gamma} from the splenocytes of immunized mice (p < 0.001). C, IL-4 levels in the spent CTL supernatant were higher after adoptive transfer of OVA257–264 peptide-loaded DC from saline-treated mice (p < 0.001). Data are representative of experiments repeated on three separate occasions in triplicate and are presented as mean ± SEM.

 
Overexpression of mFlt3L can induce overwhelming CTL activity in vivo

To assess the capacity of mice treated directly with AdmFlt3L to generate a CTL response to Ag, we s.c. immunized AdmFlt3L-treated mice and controls against OVA. We then harvested the splenocytes from immunized mice, restimulated them with OVA257–264 peptide and plated them against EG7 targets in a CTL assay. Splenocytes from immunized mice that had been treated with saline or AdGFP induced no lysis of EG7. However, in contrast to our DC adoptive transfer experiments, splenocytes from mice that had been treated with AdmFlt3L induced considerable lysis (Fig. 10A). To confirm that CTL activity was OVA257–264 peptide specific, we also plated effectors against parental EL4 cells. None of the effector groups lysed these targets (Fig. 10B). Conversely, when we loaded EL4 targets in vitro with OVA257–264 peptide, splenocytes from mFlt3L-treated mice again induced >70% lysis while control splenocytes were nonactivated (Fig. 10C). To further assess the extent of Th1 activation, we measured IFN-{gamma} levels in the spent supernatant of the restimulated splenocytes. Splenocytes from mice overexpressing mFlt3L produced 50 ng/ml IFN-{gamma} compared with undetectable levels in controls (Fig. 10D). Conversely, they did not secrete any detectable IL-4 (not shown).



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  		FIGURE 10. Ag immunization of mice overexpressing mFlt3L results in a potent CTL response. C57BL/6 mice were treated with saline or 8 x 1010 particles of AdmFlt3L or AdGFP. On day 8 after treatment, mice were inoculated s.c. with 500 µg of OVA. Splenocytes from mice were then harvested 1 wk later, restimulated with OVA257–264 peptide, and plated in a CTL assay against EG7 cells (A), parental EL4 cells (B), or EL4 cells that had been pulsed for 1 h with OVA257–264 peptide (C). Splenocytes from AdmFlt3L-treated mice induced elevated OVA257–264 peptide-specific lysis of targets (p < 0.001 for EG-7 and EL4.OVA257–264 targets). In contrast, splenocytes from saline- or AdGFP-treated controls were completely nonactivated even in comparison to splenocytes from mice that had not been immunized against OVA (No OVA). Data are presented as mean ± SEM. D, In consort with the CTL lysis data, analysis of the spent CTL supernatant revealed that restimulated splenocytes from immunized mice overexpressing mFlt3L produced nearly 50 ng/ml IFN-{gamma} compared with <2 ng/ml from control mice. SEM was <5% for the ELISA; p < 0.001.

 
Overexpression of mFlt3L induces immunogenic or tolerogenic responses to tumor

Because endogenous overexpression of mFlt3L generated a potent CTL response in vivo, we postulated that treatment with AdmFlt3L would confer tumor protection. However, in a variety of tumor models, AdmFlt3L was nonprotective. For example, BALB/c mice treated with AdmFlt3L and then challenged 4 days later with an intrasplenic inoculation of CT26 colorectal carcinoma were not protected from tumor development (Fig. 11A). Conversely, daily treatment with 10 µg of rhFlt3L protein prolonged survival in 40% of mice. Moreover, even AdGFP injection conferred protection which we proved was due to nonspecific NK cell activation (Fig. 11, A and B). We have shown above (Fig. 3) that adenovirus alone has a strong NK activating effect. However, mFlt3L transgene expression abrogated this advantage. Extending the time interval between AdmFlt3L administration and tumor challenge to 10 days or injecting both the virus and tumor simultaneously did not change the outcome (not shown). Similarly, s.c. challenge with CT26, injection of AdmFlt3L into alternate sites such a thigh muscle, administering lower doses of AdmFlt3L (3 x 1010 particles), or even engineering the CT26 cells themselves to secrete mFlt3L (using AdmFlt3L infection) did not extend survival compared with that of controls (Table III and not shown). AdmFlt3L also failed to protect against B16 melanoma in hepatic metastases or s.c. models (Table III). Even direct injection of 8 x 1010 particles of AdmFlt3L into nascent B16 tumor flank nodules failed to delay growth (not shown). Similarly, mFlt3L was nonprotective against s.c. EL4 lymphomas (Table III).



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  		FIGURE 11. Endogenous overexpression of mFlt3L may have immunogenic or tolerogenic effects on tumor development. A, Mice were treated with saline, AdGFP, AdmFlt3L, or daily injections of hFlt3L and challenged with an intrasplenic inoculation of 5 x 104 CT26 cells 4 days later (n = 5 per group). Both AdGFP and hFlt3L conferred partial protection against tumor development (p = 0.04). However, AdmFlt3L was nonprotective. B, Mice were either nondepleted and treated with saline or AdGFP, or depleted of NK cells and treated with AdGFP (n = 5 per group). Mice were challenged 4 days later with an intrasplenic inoculation of CT26. In consort with the previous tumor experiment, AdGFP treatment prolonged survival (p = 0.002). The protection conferred by AdGFP was NK cell mediated. C, Mice were treated with saline (n = 10), AdGFP (n = 8), or AdmFlt3L (n = 7) and challenged s.c. with 3 x 107 EG7 cells. Endogenous overexpression of mFlt3L was partially protective in this model (p = 0.04). AdGFP conferred no protection. D, Subcutaneous immunization with OVA 1 wk before EG7 challenge eliminated the protection of AdmFlt3L-treated mice against EG7. However, CD4 depletion restored protection (n = 5 per group; p = 0.003).

 

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  		Table III. Outcome after intrasplenic or s.c. tumor challenge

 
Because the failure of direct administration of AdmFlt3L to protect against tumors may have resulted, in part, from insufficient DC uptake of tumor Ag, we tested its effects against tumors overexpressing a distinct Ag. We challenged mice with s.c. EG7 10 days after treatment with AdmFlt3L. Treated mice were, in fact, partially protected against tumor development (Fig. 11C). We next postulated that the addition of an OVA immunization before tumor challenge would produce even more protection because it led to CTL in vitro (Fig. 10). Therefore, we immunized mice against OVA on day 8 after treatment and then challenged the animals with EG7 or B16.OVA on day 15. Immunized mice overexpressing mFlt3L were not protected against B16.OVA (Table III). Moreover, immunization of AdmFlt3L-treated mice against OVA abrogated the partial protection against EG7 conferred by mFlt3L overexpression alone without immunization (Fig. 11D). We postulated that immunization against OVA in mFlt3L-treated mice resulted in the generation of tolerogenic T cells. To test this, we depleted mice of CD4+ T cells before OVA immunization. CD4+ T cell depletion restored protection against EG7 suggesting that immunization with a protein Ag in mice overexpressing mFlt3L can shift the balance from immunity to tolerance (Fig. 11D).


   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We have shown that a single treatment with a gene transfer vector encoding mFlt3L results in up to 44% of splenocytes becoming CD11c+ 10 days later. This represents up to a 100-fold increase in the total number of splenic DC. Our findings are a significant improvement over daily injections of hFlt3L which results in 15–20% of splenocytes becoming CD11c+ and an ~17-fold increase in the total number of DC (24). Furthermore, using mFlt3L, the DC expansion effects continued for >35 days after a single treatment. In contrast, the effects of hFlt3L reportedly last only for a few days after the cessation of injections (24). It is unlikely that the higher DC expansion we observed was simply due to the high serum Flt3L levels produced by our vector because daily administration of 10 µg of hFlt3L yielded similar serum Flt3L levels on days 3 and 10 (not shown). This suggests that mFlt3L is more potent in expanding DC in mice than xenogeneic hFlt3L. An alternative explanation is that endogenous secretion achieves higher local tissue concentrations of Flt3L. We have previously shown that 8 x 1010 particles of systemically administered adenovirus causes transgene expression in ~90% of hepatocytes and 5% of splenocytes (34). Continuous secretion of mFlt3L from the liver or spleen for >28 days may have an advantage in expanding DC in these organs. In fact, we have found an even greater relative increase in the number of liver DC (up to 300-fold, not shown). Nevertheless, systemic administration of protein may have certain advantages over viral vectors in terms of better control of dosing and Flt3L clearance. Juan et al. (42) noted that systemic overexpression of Flt3L may result in significant pathology and death. However, in the present study we did not observe toxicities at our standard dose of 8 x 1010 particles. Besides the differences in their DC expansive effects, the murine and human forms of Flt3L differ in their capacity to expand NK cells. Peron et al. (39) reported that up to 25% of mouse splenocytes became NK cells after daily treatments with hFlt3L. In contrast, we did not observe any differential expansion of splenic NK cells.

Some of the phenotypic characteristics of the mFlt3L DC were quite remarkable. They expressed high B220. In contrast, B220 is minimally expressed in hFlt3L DC (24). Although the precise significance of B220 expression is unknown, Martin et al. (43) recently described a B220+ DC subpopulation that exerts tolerogenic effects in their steady state but differentiate into potent APC upon microbial stimulation. In contrast to their high B220 expression, only 5–10% of DC were CD4+ compared with 50% expression in controls. We also found that mFlt3L expanded a greater number of CD8{alpha}+CD11blow/- (lymphoid) DC than CD8{alpha}-CD11bhigh (myeloid) DC. Similarly, in Flt3L knockout mice, CD8{alpha}+ dendropoiesis was decreased by 20% more than was CD8{alpha}- DC development (14). We observed that mFlt3L DC were either CD8{alpha}+DEC205+ or CD8{alpha}-DEC205-. This further contrasts with hFlt3L treatment in which approximately one-half of CD8{alpha}- DC expressed DEC205 (33). mFlt3L DC were also slightly more mature than controls in terms of MHC and costimulatory molecule expression and morphology on cytospins. This observation may be attributed to their high CD8{alpha}+ fraction. Vremec and Shortman (44) reported that CD8{alpha}+ DC express higher levels of costimulatory molecules. The findings that systemic adenovirus alone results in minor alterations in DC phenotype (Table II) and cytokine profile (Fig. 5) highlight the fact that a portion of the changes seen in mice treated with AdmFlt3L may be related to an effect of systemic adenovirus. Although we have controlled for this effect by using AdGFP, it is possible that there may be unknown synergistic effects of adenovirus expression in an environment of a rapidly increasing number of DC.

In addition to their unique phenotype, we also found that mFlt3L DC had slightly enhanced capacity to stimulate allogenic and Ag-restricted T cells. The effect of Flt3L on DC capacity to stimulate T cells in vitro is a matter of current controversy. One report (45) indicated that both cultured and freshly isolated splenic DC expanded by hFlt3L induced higher alloproliferation at intermediate DC:T cell ratios. However, other reports using both daily injections of hFlt3L as well as studies in Flt3L knockout mice (14, 46) indicated that Flt3L does not enhance DC induction of alloproliferation. However, the differences in T cell stimulatory capacity between the current and past reports may simply reflect the distinct phenotype of the DC expanded by endogenous overexpression of mFlt3L.

Although mFlt3L DC had potent T cell stimulatory ability in vitro, they paradoxically exhibited either immunogenic and tolerogenic properties in vivo depending on the model used. Overexpression of mFlt3L induced potent OVA257–264 peptide-specific CTL activity in mice immunized against OVA (Fig. 10, A–C). AdmFlt3L also conferred partial protection against EG7 tumors (Fig. 10C). However, adoptive transfer of Ag-pulsed mFlt3L-expanded DC generated far weaker CTL activity than did controls (Fig. 9A) and actually induced faster growth of EG7 tumors (Fig. 8). Furthermore, immunization of AdmFlt3L-treated mice against OVA abrogated the protection against EG7 (Fig. 11D). However, tumor protection was restored by depletion of CD4+ T cells (Fig. 11D) suggesting that tolerogenic T cells were generated by immunizing against OVA in the context of the massive DC expansion of mFlt3L. In addition, AdmFlt3L failed in variety of models where daily injections of hFlt3L have been shown to induce considerable antitumor immunity (30, 31, 39, 47). For example, treatment with hFlt3L protected against B16 melanomas and EL4 lymphomas (47). However, AdmFlt3L failed against these tumors at a variety of sites (Table III). Similarly, AdmFlt3L failed to protect against CT26 colorectal tumors in both s.c. and hepatic metastases models (Fig. 11A). In contrast, we found a 40% extended survival rate using daily injections of hFlt3L in the same experiment. Moreover, even AdGFP conferred limited protection against CT26 (Fig. 11, A and B). This was shown to be a result of NK activation after administration of recombinant adenovirus. However, mFlt3L transgene expression eliminated this protection despite activating NK cells equally (Fig. 3).

Defining the conditions and factors that direct DC toward immunogenicity or tolerance remains one of the greatest conundrums in DC biology. Considerable recent data have implicated CD4+CD25+ suppressor T cells as the final arbiters of peripheral tolerance (48, 49, 50). An exciting future investigation would be to determine the role of endogenous Flt3L- or mFlt3L-expanded DC in the generation of CD4+CD25+ T cells. Another worthwhile endeavor would be to reverse the tolerogenic tendencies of mFlt3L DC. We attempted to activate mFlt3L DC using CD40L, but this had minimal effect (Fig. 9B). A recent report by Merad et al. (51) indicated that systemic administration of immunostimulatory DNA activates DC in vivo and potentiates the antitumor effects of otherwise nonprotective protocols. Application of in vivo DC activators to the AdmFlt3L model holds considerable promise for immunotherapy.


   Footnotes
 
1 This work was supported in part by CA94503. Back

2 Address correspondence and reprint requests to Dr. Ronald P. DeMatteo, Hepatobiliary Service, Memorial Sloan-Kettering Cancer Center, Box 203, 1275 York Avenue, New York, NY 10021. E-mail address: dematter{at}mskcc.org Back

3 Abbreviations used in this paper: DC, dendritic cell; CD40L, CD40 ligand; Flt3L, Flt3 ligand; hFlt3L, human Flt3L; mFlt3L, murine Flt3L; AdmFlt3L, adenovirus encoding the murine Flt3L transgene; AdGFP, adenovirus encoding green fluorescent protein. Back

Received for publication September 6, 2002. Accepted for publication January 27, 2003.


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 Introduction
 Materials and Methods
 Results
 Discussion
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