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Originally published online as doi:10.1189/jlb.0607348 on September 25, 2007

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(Journal of Leukocyte Biology. 2008;83:173-180.)
© 2008 by Society for Leukocyte Biology

Aberrant dendritic cell differentiation initiated by the Mll-Een fusion gene does not require leukemic transformation

Q. Sun*, C. T. Kong*,{dagger}, F. P. Huang* and L. C. Chan*,1

* Departments of Pathology, S. H. Ho Foundation Research Laboratories, and
{dagger} Biochemistry, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong, China

1 Correspondence: Department of Pathology, Division of Hematology, The University of Hong Kong, University Pathology Building, Queen Mary Hospital, 102 Pokfulam Road, Pokfulam, Hong Kong, SAR, China. E-mail: chanlc{at}pathology.hku.hk


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ABSTRACT
 
Dendritic cells (DCs), as specialized APCs, play a key role in the induction of anti-tumor immunity. They originate from bone marrow (BM) progenitors, which are frequently the targets of chromosomal translocations leading to development of leukemia. Aberrant DC differentiation and functions have been observed and are widely reported in patients with leukemia. It is not clear, however, whether such defects are a direct effect of a leukemic fusion gene or simply an outcome of the clinical disease. In this study, we demonstrate for the first time that knockin of the Mll-Een fusion gene can affect myeloid DC differentiation and functions directly, independent of the leukemic disease activities. We showed that the Mll-Een-expressing BM cells [enhanced green fluorescent protein+ (EGFP+)] from leukemic and nonleukemic mice had similarly impaired DC differentiation capacities with functional abnormalities. In contrast, BM cells without Mll-Een expression (EGFP–) showed normal DC differentiation and functions. A reduction in the frequency of CD11c+ DCs was also observed within the EGFP+ population in spleen and lymph nodes, and these cells were dysfunctional. Taken together, our findings suggest that the Mll-Een fusion gene can affect myeloid DC differentiation directly and functions in a cell-autonomous manner, where fully leukemic transformation of the hematopoietic progenitors is not required exclusively. Therefore, the study provides evidence for a direct causal relationship between leukemic gene fusion and abnormal DC differentiation, possibly contributing to the development of leukemia.

Key Words: myeloid leukemia • development


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INTRODUCTION
 
Graft versus leukemia effect in allogeneic bone marrow (BM) transplantation and increased frequency of tumor in immunodeficiency states in nude mice provide strong evidence indicating the role of the immune system in the control of hematological malignancies and tumors [1 ]. Dendritic cells (DCs) as the truly professional APCs are considered as important elements in the induction and control of the quality of a specific antitumor immune response [2 ]. They originate from BM hematopoietic stem cells (HSCs) and progenitors (common myeloid progenitor, myelomonocytic progenitor, common lymphoid progenitor, and pro-T cell progenitor), which can give rise to two distinct lineages and functional discrete subsets of myeloid or lymphoid DCs [3 4 5 6 7 ]. These HSCs and their progenitors are the frequent targets of chromosomal translocations encoding fusion genes, leading to leukemic transformation [8 9 10 ]. In acute leukemia, the mixed lineage leukemia (MLL) gene is commonly fused to many partner genes. Een, a fusion partner gene of MLL, has been cloned previously in our laboratory [11 ]. We were able to demonstrate that the Mll-Een fusion gene leads to enhanced proliferation of myeloid progenitors in embryonic stem cells, self-renewal of myeloid progenitors, and the development of myeloid leukemia in chimeric mice [12 ].

The finding that leukemic DCs may present a constellation of endogenously expressed known and unknown leukemia antigens to the host immune system [13 , 14 ] has led to the development of cellular vaccines for leukemia therapy using DCs derived from leukemic blasts. Previous studies have shown that myeloid leukemic blasts from selected leukemia patients are able to differentiate in vitro into cells with mature DC features. In some cases, these leukemic DCs are shown to have a potent capacity to induce an antileukemic T cell response and still retain the leukemic chromosomal abnormality of the original blasts [15 ]. Therefore, DCs generated from leukemia patients can facilitate an immune response, which might help in the induction of effective antileukemic T cell responses. However, studies to date have shown that DCs generated from patients with leukemia have impaired differentiation capacities and functional defects in their T cell stimulatory capacity, production of cytokines, and induction of Th1 versus Th2 responses [16 , 17 ]. Whether these abnormal DC phenotypes and functions observed in patients are a result of genetic lesions present in hematopoietic progenitors themselves or an expression per se of the transformed cells is poorly understood.

To address this issue, we analyzed BM DC differentiation potential as well as functional properties from BM and splenic Mll-Een-expressing cells from MllEen/+ chimeric mice (leukemic and nonleukemic). The BM Mll-Een-expressing cells from leukemic or nonleukemic mice showed abnormalities in DC differentiation and functions. In contrast, enhanced green fluorescent protein– (EGFP–) cells (without Mll-Een expression) from both groups of mice were unaffected. We next examined CD11c+ DC frequency with functions in peripheral lymphoid organs of the chimeric mice. Constistent with the in vitro findings, a significant reduction in the percentage of CD11c+ DCs was also observed within the EGFP+ but not the EGFP– population in the spleens and lymph nodes, and these cells were dysfunctional. Taken together, our findings show that the Mll-Een fusion gene can lead to aberrant DC differentiation in the chimeric mice in a cell-intrinsic manner, independent of disease activities, and thus, provide support for a causal link between primary defects of DCs and the subsequent development of leukemia.


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MATERIALS AND METHODS
 
Mice
MllEen/+ chimeric mice (129Sv/Ev, H-2d and C57BL/6, H-2d background) were generated as described previously through knockin of the Mll-Een fusion gene in mouse embryonic stem cells [12 ]. These mice were maintained in pathogen-free conditions in the animal units of the Li Ka Shing Faculty of Medicine, The University of Hong Kong (China). Four out of nine mice studied (Table 1 , L 1–4) had BM with increased blasts and/or enlarged organs as a result of leukemic cell infiltration, and no indications of the above signs of leukemia were found in the other five mice (Table 1 , NL 5–9). BM chimerism, i.e., contribution from Mll-Een-expressing cells, was assessed by EGFP expression. Age- and sex-matched 129Sv/Ev (H-2d) and C57BL/6 (H-2d) inbred mice were used as wild-type (WT) control mice. CBA mice (H-2k) were used as the source of allogeneic responder T cells in the MLR assay.


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  		Table 1. Clinical and Laboratory Findings in MllEen/+ Chimeric Mice

All studies were performed following strict institutional animal guidelines.

Cells and BM DC cultures
BM cells were flushed out from the femurs and tibiae of the mice by cold PBS, and a single cell suspension was made by repeated pipetting. Upon depletion of erythrocytes, EGFP+ cells were sorted out using an Epics-Altra cell sorter (Beckman Coulter, Fullerton, CA, USA), purity >95%. BM DCs were generated as described previously [18 ]. Briefly, EGFP+ cells and EGFP– cells were plated separately at a density of 106/ml in a 24-well plate in complete RPMI medium supplemented with 2% murine GM-CSF (kindly provided by Prof. A. Neil Barclay, MRC Cellular Immunology Unit, Sir William Dunn School of Pathology, Oxford, UK) and IL-4 (1 ng/ml, PeproTech EC Ltd., London, UK). Cells were cultured at 37°C in a humidified 5% CO2-containing atmosphere. When appropriate, LPS (TLR2 and -4 ligands), 1 µg/ml, was added for the final 24 h of culture. At Culture Day 8, nonadherent cells were harvested and enriched by centrifugation (600 g for 10 min at room temperature) over 14.5% metrizamide (Sigma-Aldrich, St. Louis, MO, USA) for further analysis. DC viabilities were tested by trypan blue exclusion and propidium iodide (PI) staining (>90%).

For analysis of primary CD11c+ DCs in the peripheral lymphoid organs, spleens and mesenteric lymph nodes were harvested from the mice in each group and minced into small pieces. Pooled, single cell suspensions were prepared by incubation with collagenase D (Roche Applied Science, Germany) at 37°C for 30 min and then gently pressing the tissue through a 70-µm plastic mesh with PBS containing 2% FBS and 2 mM EDTA (Sigma-Aldrich). EGFP+ and EGFP– cells were further sorted out from the cell suspensions by an Epics-Altra cell sorter (Beckman Coulter).

Purification of CD11c+ DCs from BM cultures or spleen single cell suspensions (sorted EGFP+ or EGFP– cells) was performed using magnetic anti-CD11c microbeads (Miltenyi Biotech, Germany), according to the manufacturer’s instructions (purity>95%) for further microscopy studies, endocytosis, MLR, and cytokine production assays.

Flow cytometry
Single cell suspensions from BM cultures or spleen/lymph nodes were analyzed by flow cytometry using FACSCalibur (BD Biosciences, San Jose, CA, USA) and CELLQuest software (BD Biosciences). DC immunophenotype was performed after staining with CD11c-PerCP, PE-conjugated I-Ab, CD80, CD86, and CD40, together with matched isotype controls (BD PharMingen, San Diego, CA, USA). Biotin-cocktail (CD3, CD5, B220, Gr-1, Ter119) and secondary-labeled streptavidin-allophycocyanin were used to exclude Lin+ cells (Miltenyi Biotech). Live cells were selected based on forward/side-scatter gating and/or PI exclusion. At least 104 gated cells were collected for analysis.

Microscopy studies
The development of DC morphology from Mll-Een-expressing cells was evaluated after 8 days in culture using an inverted microscope (Nikon TS100, Nikon, Japan). Purified CD11c+ cells were centrifuged onto microscope slides (Shandon Southern Products, Astmoor, UK), stained with May-Grünwald-Giemsa solution and analyzed by light microscopy.

For confocal microscopy study, purified CD11c+ cells were adhered to polylysine-coated glass slides for 30 min at room temperature, fixed in absolute ethanol for 2 min, and preincubated with 3% BSA for 30 min. Cells were then stained with primary anti I-Ab-biotinylated mAb and fluorochrome-conjugated streptavidin-Cy3 (BD PharMingen and Jackson ImmunoResearch, West Grove, PA, USA, respectively). Slides were then mounted using fluorescent mounting medium (Dako, Carpinteria, CA, USA). Confocal analysis was performed with a two-photon confocal microscope system (Zeiss LSM 510 Meta, Zeiss, Germany).

Endocytosis, MLR assay, and cytokine profile
To measure antigen uptake ability, CD11c+ DCs from BM cultures or splenocytes were incubated with Cy3 fluorescein-labeled Dextran (Invitrogen, Carlsbad, CA, USA) for 1 h at 4°C or 37°C. Fluorescence was measured by flow cytometry using a FACSCalibur.

Allogeneic T cell stimulation ability was conducted using 105 splenocytes from CBA mouse strain (H-2k) as the cell sources for responder T cells and irradiated (30 Gy) CD11c+ DCs purified from 8-day BM cultures as stimulators. On Day 3 of culture, the cells were pulsed with 0.5 µCi methyl-3H-thymidine for 16 h. Incorporated methyl-3H-thymidine was quantified by a scintillation counter. Results were presented as the mean cpm of triplicates.

For cytokine production assays, purified CD11c+ cells were replated in a 48-well plate at a density of 5 x 105/ml, followed by stimulation with 5 µg/ml LPS. Supernatants were collected 24 h after LPS stimulation, and ELISA was performed to determine the levels of IL-12p70 and IL-10 using murine OptEIATM sets (BD Biosciences).

DC migration assay
In vitro migration of the BM EGFP+ cell-derived DCs in response to chemokines was performed in a double-chamber transwell system. Complete RPMI medium containing chemokines (100 ng/ml CCL4 or CCL5; R&D Systems, Minneapolis, MN, USA) was put in the lower chamber of a Costar 24-well transwell (Costar, UK), and RPMI medium without chemokines served as control. DCs were purified (CD11c+) from Day 8 BM cell cultures, washed, and plated into the upper compartment of a 5-µm pore-size transwell plate at 1 x 105 cells/well in 100 µl RPMI medium. After 4 h of incubation at 37°C, cells, which transmigrated to the bottom compartment, were collected and counted by flow cytometry.

DC-mediated antigen-specific T cell responses
DC-induced antigen-specific CD4+ and CD8+ T cell responses were performed in a TCR transgenic system [19 , 20 ]. The OT-I transgenic T cell (OT-I mice) expressing the OVA257–264/Kb-specific TCR was obtained from the Jackson Laboratory (Bar Harbor, ME, USA). OT-II mice expressing OVA323–339/Ad-specific TCR and OVA323–339 peptide were kindly provided by Prof. Xuetao Cao from the Second Military Medical University (Shanghai, China) [19 ]. Splenic CD4+ (OT-II) and CD8+ (OT-I) T cells from these transgenic mice were sorted out by magnetic-activated cell sorting (Miltenyi Biotech) and used as antigen-specific responders. To examine a T cell-proliferative response, purified CD11c+ DCs derived from the EGFP+ BM cell cultures as above were cocultured at various ratios with a fixed number (1x105) of CD4+ or CD8+ T cells in the presence of 0.2 µM OVA323–339 or OVA257–264 peptide (Sigma-Aldrich) in a round-bottomed, 96-well plate for 5 days. Cells were pulsed with 0.5 µCi 3H-thymidine for the last 16 h of coculture. Cell proliferation was measured by a scintillation counter.

To examine antigen-specific T cell activation, the purified CD11c+ DCs were cocultured with naïve CD4+ T cells at a ratio of 1:10 in the presence of OVA323–339 peptide. Cells at Culture Days 1, 3, and 5 were harvested, respectively, and stained with FITC-conjugated CD25 or CD69 or the isotype control mAb (BD Biosciences). The expression level of these T cell activation markers was then analyzed by flow cytometry.

To examine Th cell differentiation and the activation of CD8+ T cells by Mll-Een DCs, CD4+ and CD8+ T cells were also purified from the above cocultures (DC:T, 1:10) by magnetic-activated cell sorting at Day 3 and replated in a 96-well plate precoated with 2 µg/ml anti-CD3 mAb (R&D Systems). Cells were then incubated for 48 h, and the supernatants were collected for cytokine quantitation (IL-2, IL-4, IL-5, and IFN-{gamma}) using the murine OptEIATM sets (BD Biosciences).

Statistical analysis
Statistical analysis was done by Student’s t-test using the SPSS software package (SPSS, Chicago, IL, USA). Results were presented as the mean ± SEM unless otherwise indicated.


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RESULTS
 
Mll-Een-expressing cells showed abnormal DC differentiation with maturation arrest in Mll-Een DCs
We performed an in vitro DC differentiation assay from BM Mll-Een-expressing cells (purified EGFP+ cells) and cells without Mll-Een expression (EGFP– cells) from nonleukemic and leukemic groups, respectively. We observed that EGFP+ cells had similar impairment of DC differentiation and functions in mice from both groups, whereas EGFP– cells were unaffected and sustained the same capacity to generate functional DCs as WT BM cells. We first investigated the frequency of DCs generated from BM cells at Day 8. Although there was no significant difference with cells recovered from Day 8 cultures among each group, the proportion of DCs (CD11c+MHC-II+ cells) from the recovered cells showed that those generated from EGFP+ cells produced significantly less DCs (approximately threefold reduction, P<0.01) in nonleukemic and leukemic groups, with no considerable differences between EGFP– cells and WT cells (P>0.05; Table 2 ). In line with this observation, a reduction of CD11c+ DCs was observed in the EGFP+ population in the spleen and lymph nodes of nonleukemic and leukemic mouse groups (Table 3 ).


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  		Table 2. Relative DC Frequency from BM Cultures at Day 8


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  		Table 3. CD11c+ DC Frequency in Spleen and Mesenteric Lymph Nodes

Immunophenotype analysis of DCs generated from BM EGFP+ cells (without LPS stimulation) showed a global reduction of all DC markers, i.e., MHC-II, CD80, CD86, and CD40 (gated on CD11c+ cells), in contrast to DCs derived from the EGFP– population and the WT control cells (Fig. 1 ). We have excluded the possibility that EGFP expression per se could influence DC differentiation and function, as we have shown in a previous study [12 ], and observed in the MlllacZ/+ mice (Mll-lacZ fusion) that EGFP expression in hematopoietic cells did not affect DC differentiation and function (unpublished data). These results confirmed that the Mll-Een fusion gene had conferred abnormal DC differentiation in a cell-autonomous manner.


Figure 1
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  		Figure 1. Immunophenotype and functional maturation of Mll-Een DCs. MHC-II, CD80, CD86, and CD40 expression on BM CD11c+ DCs after 8 days of culture from EGFP+ and EGFP– cells from each mouse group in the absence or presence of LPS stimulation. The data shown are the mean fluorescence intensity (MFI) ± SEM calculated from cells in each group. Black bars indicate WT DCs (n=5); shaded and open bars indicate DCs generated from nonleukemic (n=5) and leukemic group mice (n=4), respectively. EGFP+ versus WT, P < 0.01; EGFP– versus WT, P > 0.05.

DC functional activities are closely related to the maturation and activation stages of the cells [21 ]. We next investigated whether Mll-Een DCs could undergo functional maturation after stimulation by microbial product, LPS. BM DC cultures at Day 7 were stimulated with LPS at 1 µg/ml for 24 h, and DC immunophenotype analysis revealed that DCs generated from EGFP+ cells (nonleukemic and leukemic) following LPS treatment were refractory to up-regulation of MHC-II, CD40, and costimulatory molecules, indicating maturation arrest in these cells, as compared with the WT controls and EGFP– cells (Fig. 1) .

Aberrant cytologic features of DCs generated from Mll-Een-expressing cells
Cells with the classical cytologic features of DCs generated from WT BM cultures were observed from Day 2 onward. These show irregular cellular membrane and hypergranular cytoplasm with a number of long-length dendritic protrusions, and by Day 8, some loosely adherent cells aggregated with long dendrites were seen. In contrast, DCs generated from EGFP+ cells from nonleukemic and leukemic groups were smaller with regular cell shape, condensed nuclei, hypogranular cytoplasm, and few short-length cytoplasmic projections. There were also no obvious cell clusters with dendrites in the cultures (Fig. 2A and 2B ). These Mll-Een DCs also showed decreased staining intensity of MHC II and aberrant cytology, as demonstrated by confocal microscopy (Fig. 2C) . We counted 100 cells randomly from different views under fluorescence microscopy and concluded that over 90% of low MHC II staining cells from nonleukemic and leukemic mice groups displayed less dendrites as compared with the normal control group. DCs derived from the EGFP– cells, however, showed normal cytological features similar to the WT controls (data not shown).


Figure 2
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  		Figure 2. Morphological characteristics of Mll-Een DCs. Representative cytological sections showing purified CD11c+ DCs generated from WT and EGFP+ BM cells (mouse 5 and mouse 3). (A) Phase contrast of cells with cytologic features of DCs at x200 original magnification. (B) Cytospin appearances of May-Grünwald-Giemsa staining and (C) MHC II-Cy3 immunofluorescence under (B) light and (C) confocal microscopy at x400 and x63 original magnification, respectively.

DCs generated from Mll-Een-expressing cells exhibited defective antigen uptake, allogeneic T cell-stimulating capacity, and cytokine production
We postulated that the developmental defects and maturation arrest of Mll-Een DCs may have adverse impacts on functions. Subsequent experiments were designed to evaluate their functional activities, including antigen uptake, allogeneic T cell stimulatory capacity, and cytokine expression. Purified CD11c+ DCs at Day 8 BM cultures were used in endocytosis assay to determine the efficiency of antigen uptake measured by MFI. A decreased Dextran uptake activity was evident in DCs derived from EGFP+ cells from nonleukemic and leukemic groups (P<0.01; Fig. 3A and 3B ). We showed further that DCs generated from BM Mll-Een-expressing cells were defective, with or without LPS stimulation, to elicit T cell proliferation in the MLR assay (P<0.05; Fig. 4 ). We next examined their ability to produce cytokines, i.e., IL-12p70 and IL-10, which are known to be important to stimulate or regulate T cell activation. Purified CD11c+ cells from BM cultures were replated at Day 7 in the absence or presence of LPS stimulation, and culture supernatants were collected at 24 h after LPS treatment. Figure 5 shows that Mll-Een DCs (nonleukemic and leukemic) also produce significantly reduced levels of IL-12p70 and IL-10 in response to LPS stimulation (P<0.01). These functional abnormalities (endocytosis, allogeneic T cell stimulatory ability, cytokine profile) were also found on splenic CD11c+ DCs purified from EGFP+ cells from both mouse groups (data not shown). These results demonstrated the profound functional abnormalities of Mll-Een DCs in antigen uptake, T cell stimulation, and cytokine production.


Figure 3
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  		Figure 3. Endocytosis activity of Mll-Een DCs. (A) Cy3 fluorescence-labeled Dextran uptake ability of BM Mll-Een DCs from representative mice in each group. The solid and open area indicate cells incubated at 4°C and 37°C, respectively. (B) Dextran uptake ability of CD11c+ cells derived from BM EGFP+ (gray bars) or EGFP– cells (open bars) was calculated by MFI in each group of mice. EGFP+ versus WT, P < 0.01; EGFP– versus WT, P > 0.05. Data represent two independent experiments.


Figure 4
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  		Figure 4. Allostimulatory T cell ability of Mll-Een DCs. Allogeneic T cell responses as measured by 3H-thymidine incorporation at Day 4. CD11c+ DCs derived from BM EGFP+ or EGFP– cells (with or without LPS stimulation) were {gamma}-irradiated and cocultured at a different stimulator:responder ratio with the allogeneic splenocytes. Data are representative of three independent experiments. EGFP+ versus WT, P < 0.05; EGFP– versus WT, P > 0.05.


Figure 5
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  		Figure 5. Cytokine profile of Mll-Een DCs. CD11c+ DCs generated from BM EGFP+ or EGFP– cells were stimulated with LPS for 24 h, and the IL-12p70 and IL-10 levels from the supernatants were determined by ELISA. Black bars indicate WT DCs (n=5), gray and open bars indicate DCs from nonleukemic (n=5) and leukemic group mice (n=4), respectively. EGFP+ versus WT, P < 0.01; EGFP– versus WT, P > 0.05. Data represent three independent experiments.

DCs generated from Mll-Een-expressing cells are defective in migration and in the induction of antigen-specific T cell responses
To assess further the functional activity of DCs, their migratory ability in response to chemokines was then determined by an in vitro cell migration assay previously established [22 ]. CCL4 (MIP-1β) and CCL5 (RANTES) chemokines were used in the chemotaxis assay to examine the migratory ability of Mll-Een DCs derived from BM EGFP+ cells in each mouse group. Significantly reduced frequencies of the Mll-Een DCs from the nonleukemic and leukemic mice group were found in the lower compartment, indicating a defect of the cells in migration responding to chemokine stimulation (P<0.01; Fig. 6 ).


Figure 6
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  		Figure 6. Mll-Een DC migration in response to chemokine stimulation. The migratory ability of Mll-Een DCs derived from BM EGFP+ cells in each mouse group was performed in a double-chamber transwell system. Cells migrated into the lower compartment in response to CCL4 (filled bars) and CCL5 (open bars) chemokine stimulation were enumerated. WT versus nonleukemic/leukemic, P < 0.01; nonleukemic versus leukemic, P > 0.05. Data are representative of two separate experiments.

To determine the capacities of Mll-Een DCs in inducing and polarizing Th cell activation and differentiation, OVA-specific TCR-transgenic CD4+ T cells, purified from the OT-II mice, were used as the responder cells cocultured with Mll-Een or control DCs in the presence of OVA323–339 peptide. T cell proliferation was assayed by 3H-thymidine incorporation at Day 5. As shown in Figure 7A , the OVA323–339 peptide-pulsed Mll-Een DCs from nonleukemic and leukemic mice were similarly defective in stimulating T cell proliferation as compared with the WT control DCs (P<0.01). In addition, these Mll-Een DCs were unable to activate T cells, as no expression of activation-related markers (CD25 and CD69) was observed in Mll-Een DC-stimulated CD4+ T cells through 5 days of coculture. Representative data of expression of CD25 and CD69 on CD4+ T cells at Day 5 were shown in Figure 7B .


Figure 7
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  		Figure 7. The capacity of Mll-Een DCs to induce antigen-specific T cell responses. (A) T cell-proliferative responses. Mll-Een DCs derived from BM EGFP+ cells at Day 8 from the nonleukemic and leukemic mice group at the indicated numbers were cocultured with OVA-specific CD4+ T cells (1x105) in the presence of OVA323–329 peptide for 5 days. T cell proliferation was measured by 3H-thymidine incorporation. (B) T cell activation marker expression. The expression of CD25 and CD69 on the CD4+ T cells after 5 days of coculturing with DCs (DC:T, 1:10) was assessed by flow cytometry. (C and D) Cytokine production. The CD4+ T cells were sorted out from the above cocultures (DC:T, 1:10) at Day 3 and stimulated with an anti-CD3 mAb for a further 48 h. The culture supernatants were collected and assayed for IFN-{gamma} (C) and IL-4 and IL-5 cytokines (D) by ELISA. Controls were T cells cultured in the absence of DC-OVA. WT-DC/OVA versus nonleukemic/leukemic-DC/OVA, P < 0.01; nonleukemic-DC/OVA versus leukemic-DC/OVA, P > 0.05. Data represent two independent experiments.

Following activation, naïve T cells differentiate into effector cells and most commonly develop into Th1 or Th2 cells. In general, the type I responses are featured by T cells producing IFN-{gamma} and the type 2 responses, by T cells producing IL-4, IL-5, and IL-10 [23 ]. We therefore also examined the capacity of Mll-Een DCs to promote Th1 and Th2 responses. Purified CD4+ T cells were cocultured with OVA323–339 peptide-pulsed DCs, and Th1/Th2 cell differentiation was determined by cytokine production following stimulation by an anti-CD3 mAb. As shown in Figure 7C and 7D , the OVA323–339 peptide-pulsed WT DCs stimulated OT-II T cells to produce high levels of IFN-{gamma}, IL-4, and IL-5. In contrast, Mll-Een DCs failed to induce T cells to produce the Th1 (IFN-{gamma}) or Th2 (IL-4 and IL-5) cytokines (Fig. 7C and 7D) . The Mll-Een DCs pulsed with OVA257–264 failed to promote CD8+ T cell responses too, as evident by the cell proliferation assay and cytokine (IL-2 and IFN-{gamma}) production (data not shown). Taken together, these results showed Mll-Een DCs were defective as APCs to induce T cell proliferation and activation, owing to deficiencies in antigen uptake and presentation, and in providing the crucial costimulatory signals (CD80, CD86, CD40) and T cell-polarizing cytokines (IL-12 and IL-10).


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DISCUSSION
 
Previous studies have suggested active immune responses to tumor-associated antigens to be initiated at some point during the development of tumors and in some later time course, suppressed, resulting in clonal expansion of the tumor. However, the specific cellular factors, which are responsible for anti-tumor effects, are not well understood [24 ]. Increasing interest is now focused on DCs as professional APCs, which play a key role in the initiation of immune responses, including anti-tumor immunity through stimulating T effector cells, especially tumor-specific cytotoxic T cells [25 ]. Therefore, it is hypothesized that aberrant DC development and functions could be involved in setting up a dysfunctioning immune environment in which tumor cells can clonally expand, leading to the overt development of leukemia.

To date, studies of fusion proteins encoded by chromosome translocation in human leukemia, including those involving the MLL gene, have focused on how fusion genes affect self-renewal and differentiation. We have previously shown that the Mll-Een fusion gene can lead to enhanced self-renewal of myeloid progenitors and development of leukemia in chimeric mice. Aberrant differentiation and functions of leukemic blast-derived DCs have been observed and widely reported in patients of different types of leukemia. It is not clear, however, whether such defects are a direct effect of hematopoietic reprogramming by a leukemic fusion gene or simply an outcome of the clinical diseases. In this study, we have investigated the differentiation and functional potential of DCs in the MllEen/+ chimeric mice and showed that there is an aberrant developmental pattern of DCs generated from Mll-Een-expressing cells in a cell-autonomous manner, independent of leukemic disease activities, where leukemic transformation in the hematopoietic progenitors is not exclusively required. We found that DCs generated from Mll-Een-expressing cells or the primary splenic Mll-Een DCs had exhibited several phenotypic and functional abnormalities. First, Mll-Een DCs had an immature DC phenotype with low levels of surface MHC-II, CD80, and CD86. Second, these DCs lacked the morphological changes of "dendrites," a signature of DC morphology, which are well-adapted for the localized cell surface presentation of peptide-MHC complexes and other molecules involved in DC-T cell interactions [26 , 27 ]. Third, Mll-Een DCs displayed profound functional defects, including maturation arrest, antigen uptake, cytokine production, migration, activation of T cells, and Th cell-polarization capacities.

Based on the present findings, we conclude that the abnormalities of Mll-Een DCs are most likely to be a result of a hematopoietic reprogramming in BM myeloid progenitors initiated by the Mll-Een fusion gene. MLL has been detected at high levels in more differentiated myeloid cells and macrophages at lower levels in earlier hematopoietic progenitor cells, T/B lymphocytes, and is not expressed in erythroid cells [28 ]. Gene-targeting experiments showed that loss of functional Mll results in marked reductions in the number of myeloid and macrophage colonies in yolk sac cultures [28 , 29 ]. More recent work found that the N terminus of the MLL proteins promotes cell cycle arrest and monocytic differentiation, a myelomonocytic origin of BM DCs [30 ]. Taken together, these data raise the possibility that MLL functions to skew hematopoietic differentiation away from erythroid differentiation and toward myelomonocytic differentiation. We believe that this latter program of myelomonocytic differentiation is disrupted in the Mll-Een-expressing cells and hence, contributes to abnormal DC differentiation and function.

In summary, the results presented in this study demonstrated for the first time that the Mll-Een fusion gene can affect the developmental pattern of myeloid DCs directly in a cell-autonomous manner. We believe our findings of abnormal morphology, phenotype, and functions in DCs, seen in MllEen/+ chimeric mice, underscore the importance of further studies about DC development and its role in the pathogenesis of leukemia. It will be important to examine in further details the cellular and molecular targets, which may determine the fate of DC lineage in view of the potential use of DCs for therapeutic intervention and management of human leukemia.


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ACKNOWLEDGEMENTS
 
This study is supported by research grants from the Research Grants Council of the Hong Kong Special Administrative Region, China (project nos. HKU 7269/02M, HKU 7291/02M, HKU 1/98C, HKU 2/02M). We thank Prof. Xuetao Cao for supplying OT-II cells, Cary So for excellent technical support, and Ms. Juliana Kwok for the manuscript preparation. We are grateful to Dr. Ricardo Dewey for valuable discussion and critical review of the manuscript.

Received June 5, 2007; revised August 1, 2007; accepted August 24, 2007.


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