The murine AML cell line, WEHI-3, was maintained in the laboratory of the Department of Clinical Hematology, Second Affiliated Hospital, Medical School of Xi'an Jiao Tong University (Xi'an, China) in DMEM medium supplemented with 10% fetal bovine serum (Biological Industries, Israel) and 1% penicillin-streptomycin (100 U/ml penicillin and 100 mg/ml streptomycin; cat no. 15140-122; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA). The following antibodies were used for flow cytometry (FCM): Fluorescein isothiocyanate (FITC) anti-mouse cluster of differentiation (CD)4 (cat no. 11-0042), allophycocyanin (APC) anti-mouse CD25 (cat no. 17-0251), phycoerythrin (PE) anti-mouse/rat Forheac box P3 (Foxp3) (cat no. 12-5773) and PE anti-mouse CD184 (CXCR4; cat no. 12-9991), and all were purchased from eBioscience (Thermo Fisher Scientific, Inc.). FITC anti-mouse CD11b (cat no. 557396), PE anti-mouse lymphocyte antigen 6G (Ly-6G) and Ly-6C (cat no. 561084) were purchased from BD Biosciences (San Jose, CA, USA). The mouse SDF-1α (cat. no. MCX120), transforming growth factor β (TGF-β; cat. no. MB100B) and interleukin 10 (IL-10; cat. no. M1000B) ELISA kits were purchased from R&D Systems, Inc. (Minneapolis, MN, USA), and the arginase 1 (Arg-1; cat. no. JL13668) ELISA kit was purchased from and Shanghai Jiang Lai Biotechnology Co., Ltd. (Shanghai, China; http://www.laibio.com/).

All animal experiments were reviewed and approved by the Ethics Committee of the Medical College, Xi'an Jiao Tong University (Xi'an, China). Male BALB/c wild-type mice (6–8 weeks old, weighed 21±2 g) were purchased from the Laboratory Animal Center, Medical College of Xi'an Jiao Tong University. The mice were maintained under specific pathogen-free conditions, in a 12 /12 h light/dark cycle at 21±2°C, with access to food and water ad libitum. A total of 21 mice were randomly divided into 3 groups of 7. For the syngeneic acute myelomonocytic leukemia model (AML-M4), BALB/c mice were injected intravenously into the tail vein on day 0 with 1×10⁶ WEHI-3 cells in 100 µl PBS as previously described (32–34). On day 15, all 7 mice in the AML (control) group were humanely sacrificed and the spleen, liver, femur and blood were collected and stored for further experiments. For the daunorubicin and cytarabine (DNR-AraC; DA) group, 7 mice were treated with 10 mg/kg DNR intravenously on days 15–17 and 50 mg/kg AraC intraperitoneally on days 15–21. Mice in DA group were sacrificed on day 22 and the spleen, liver, femur and blood were collected for further experiments. For the CAG group, 7 mice were treated with 6.25 mg/kg AraC via hypodermic injection (IH) on days 15–18, 3 mg/kg aclarubicin (ACR) intravenously on days 15–28 and 0.2 mg/kg G-CSF via IH on days 15–28. All mice in CAG group were sacrificed on day 29 and the spleen, liver, femur and blood were collected for further experiments.

SDF-1α, IL-10 and Arg-1 levels in the blood plasma were determined using the aforementioned commercially available mouse ELISA kits, according the manufacturers' protocols. Absorbance was measured at 450 nm on a microplate reader (Thermo Fisher Scientific, Inc.).

Single-cell suspensions of spleen cells and bone marrow mononuclear cells (BMMCs) were stained with FITC-conjugated anti-mouse CD4 (dilution, 1:100) and APC-conjugated anti-mouse CD25 (dilution, 1:100) for 30 min on ice. The cells were washed with flow cytometry staining buffer (eBioscience; Thermo Fisher Scientific, Inc.), resuspended in 1 ml fixation/permeabilization working solution (eBioscience; Thermo Fisher Scientific, Inc.) and incubated at 4°C, in the dark, overnight. The cells were washed twice with 1× permeabilization buffer (eBioscience; Thermo Fisher Scientific, Inc.) and incubated for 30 min at 4°C in 1× permeabilization buffer containing PE-conjugated anti-mouse Foxp3 (dilution, 1:40) antibody. Forward vs. side scatter was plotted for lymphocytes, followed by gating for CD4⁺ T cells. These cells were then analyzed for the expression of CD25 and FoxP3. For the detection of MDSCs and CXCR4, single-cell suspensions were stained with CD11b (dilution, 1:200), Gr-1 (Ly-6G and Ly-6C; dilution, 1:200) or CXCR4 (dilution, 1:20) antibodies. FCM was performed using a FACS flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA) and the data was analyzed using CellQuest Pro (version 6.0; BD Biosciences).

The femur, liver and spleen tissues were harvested and immediately fixed in 10% neutral-buffered formalin for 24 h. Liver and spleen tissues were embedded in paraffin and the femurs were decalcified in 10% EDTA for ~30 days at room temperature prior to being embedded in paraffin. A total of 54-µm-thick sections were cut from each paraffin block. The sections were stained at room 25°C using hematoxylin (H) for 6 min and eosin (E) for 1 min (cat no. C0105; Beyotime Institute of Biotechnology, Shanghai, China), according to standard histochemical procedures. Images were acquired using a light microscope (DP71; Olympus Corporation, Tokyo, Japan).

All data are expressed as the mean ± standard deviation. Differences between treatment groups were determined using analysis of variance followed by Fisher's Least Significant Difference post-hoc test. P<0.05 was considered to indicate a statistically significant difference. All analyses were performed using SPSS 18.0 software (SPSS Inc., Chicago, IL, USA).

To determine whether the CAG and DA treatment regimens affect AML immunosuppressive cells in the BMM, FCM was performed to investigate the percentage of CD4⁺CD25⁺FoxP3⁺ Tregs and CD11b⁺Gr-1⁺ MDSCs in BMMCs. It was revealed that the levels of Tregs and MDSCs in the DA and CAG groups were significantly lower than in the AML group (P<0.01; Fig. 1A). Furthermore, compared with mice in the DA group, mice in CAG group exhibited significantly decreased levels of Tregs and MDSCs in BMMCs (P<0.05; Fig. 1A).

TGF-β and IL-10 are anti-inflammatory and immunosuppressive cytokines secreted by Tregs (35,36), while Arg-1, which suppresses T-lymphocyte function, is expressed by MDSCs (37,38). The ELISA results demonstrated that the concentrations of TGF-β, IL-10 and Arg-1 in the bone marrow supernatant were significantly decreased in the DA (P<0.01) and CAG (P<0.01) groups compared with the AML group (Fig. 1B). The CAG group exhibited lower TGF-β1 (P<0.01), IL-10 (P<0.01) and Arg-1 (P<0.05) concentrations compared with the DA group (Fig. 1B). These results suggest that the CAG treatment regimen may rescue immune suppression in AML bone marrow.

The percentages of Tregs and MDSCs in mice spleen single cell suspensions were tested by FCM. As shown in Fig. 2, mice in the DA and CAG groups both exhibited lower percentages of Tregs and MDSCs in spleen than AML group (P<0.01). However, there were significantly higher levels of Tregs (P<0.01) and MDSCs (P<0.05) in the CAG group than the DA group.

The level of SDF-1α in bone marrow supernatant was detected by ELISA, and the results revealed that the concentration of SDF-1α in the CAG group was significantly lower than the AML and DA groups (P<0.01). However, there was no significant difference (in the concentration of bone marrow SDF-1α between AML and DA groups (P>0.05; Fig. 3).

ELISA results demonstrated that the CAG group exhibited the lowest levels of SDF-1α in the peripheral blood among the 3 groups of mice (Fig. 4). In consistence with the concentration of SDF-1α in the bone marrow, there was no significant difference in the concentration of SDF-1α in the peripheral blood between the AML and DA groups (P>0.05).

Macroscopically, the femurs of AML mice were pale (Fig. 5Aa) and the liver and spleen were swollen (Fig. 5Ab and c) compared with the DA and CAG mice. The DA (Fig. 5Ba) and CAG (Fig. 5Ca) treatment regimens reddened the femurs and decreased the size of the liver and spleen (Fig. 5Bb and c; Fig. 5Cb and c). Leukemic cells in the bone marrow cavity of mice from the AML group were visible under the light microscope (Fig. 6Aa). By contrast, the femur sections from mice of the DA and CAG groups exhibited a recovered hematopoietic system (Fig. 6Ba and Ca). In AML mice, leukemic cells infiltrated the liver and spleen (Fig. 6Ab and c). However, in the DA group, fewer leukemic cells infiltrated the liver and spleen and the pathological sections exhibited liver fibrosis and spleen trabecula fibrosis (Fig. 6Bb and c). In the CAG group, leukemic cells infiltrated the hepatic portal and central veins (Fig. 6Cb) indicated by the arrows. In the spleens, the boundary of red pulp widened and the white pulp was clear (Fig. 6Cc).