Granulocyte colony-stimulating factor-induced immature myeloid cells inhibit acute graft-versus-host disease lethality through an indoleamine dioxygenase-independent mechanism

Abstract

Granulocyte colony-stimulating factor (G-CSF)-mobilized donor graft tissue used for peripheral blood stem cell transplantation contains a large number of immature myeloid cells that suppress alloreactive donor T cells, resulting in an inhibition of acute graft-versus-host disease (GVHD). However, the molecular mechanism underlying the suppressive function of immature myeloid cells is not fully understood. Here, we investigated whether indoleamine 2,3-dioxygenase (IDO) is related to the suppressive mechanism of G-CSF-induced immature myeloid cells (gMCs). We found that Gr-1(+) CD11b(+) cells were highly induced in G-CSF-injected donor graft tissue, which is a phenotype of immature myeloid cells, resulting in an inhibition of acute GVHD lethality by suppressing alloreactive donor T-cell expansion. IDO was not detected in primary isolated gMCs; however, this enzyme was markedly induced after treatment with interferon-gamma (IFN-gamma). This level was significantly higher in IFN-gamma-treated gMCs than in bone marrow myeloid cells, which promote alloreactive T-cell responses. We next investigated the functional role of IDO in gMC-mediated inhibition of acute GVHD lethality. We found no changes in gMC-mediated survival or alloreactive donor T-cell suppression when IDO activity was blocked using 1-methyl tryptophan. In addition, there was no difference in gMC-mediated survival rates between recipients transferred with either wild-type gMCs or IDO(-/-) gMCs. Taken together, our data suggest that gMC-mediated inhibition of lethal acute GVHD is through an IDO-independent mechanism.

Figure 1

Phenotypic characteristics of granulocyte colony-stimulating factor (G-CSF)-induced immature myeloid cells (gMCs). Naïve B6 mice were injected subcutaneously with control phosphate-buffered saline (PBS) or G-CSF (10 μg) daily for 5 days. Peripheral blood cells (PBCs) and plenocytes were obtained and the subpopulation of cells was analysed by flow cytometry. (a) The dot plot analysis shows the percentage of gated cells analysed for forward-scatter (FSC) and side-scatter (SSC) properties. (b) Splenocytes were obtained from G-CSF-injected B6 mice and stained with fluorscein isothiocyanate (FITC)-anti-Gr-1 and phycoerythrin (PE)-anti-CD11b or FITC-anti-linage markers and PE-anti-c-kit. Data represent the percentage of quadrant gated on R1. (c) Splenocytes (SP) and bone marrow cells (BM) were obtained from G-CSF-injected and naïve B6 mice, respectively. Cells were stained for Gr-1, CD11b, and each surface marker was investigated by three-colour fluorescence. The histogram shows the percentage of positive cells gated from the Gr-1⁺ CD11b⁺ cells. Representative data from one of three experiments are shown; all three experiments showed similar results.

Figure 2

Granulocyte colony-stimulating factor (G-CSF)-induced immature myeloid cells (gMCs) suppress alloreactive T-cell expansion, which results in inhibition of acute graft-versus-host disease (GVHD) lethality. (a) Mixed lymphocyte reaction (MLR) cultures were set up with 10⁵ carboxyfluorescein succinimidyl ester-labelled naïve B6 T-cell responder (R) and 10⁵ BALB/c splenic stimulators (S) per well. Both gMCs and bone marrow-derived myeloid cells (bmMCs) were isolated from the spleens of G-CSF-injected mice and the bone marrow of naïve B6 mice, respectively. MLRs were cocultured with no, 10³ or 10⁴ gMCs or bmMCs for 4 days. Cultures were harvested and stained with PE-Cy5-anti-H-2K^b with or without PE-anti-CD4 or CD8, and analysed by flow cytometry. (b) B6D2F1 recipient mice were lethally irradiated (950 cGy) and transferred with 2 × 10⁶ B6 donor T cells alone or plus each above-listed number of gMCs or bmMCs. Each group consisted of eight mice. Data represent the results from three similar experiments. *P < 0·05, gMCs versus bmMCs.

Figure 3

Indoleamine 2,3-dioxygenase (IDO) is induced in granulocyte colony-stimulating factor (G-CSF)-induced immature myeloid cells (gMCs) following interferon-γ (IFN-γ) treatment. (a) The gMCs and bone-marrow-derived myeloid cells (bmMCs) were isolated from the spleens of G-CSF-injected mice or the bone marrow of naïve B6 mice as described in the Materials and methods. Total RNA was extracted from freshly isolated cells (−) and reverse transcribed into complementary DNA. Polymerase chain reaction (PCR) was performed using IDO-specific primers, and the products were analysed by agarose gel electrophoresis. Isolated cells were stimulated with 200 U/ml IFN-γ for 24 hr (+). (b) IFN-γ-induced IDO expression levels were compared between gMCs and bmMCs using real-time PCR. Results are expressed as the mean ratio of IDO : GAPDH ± SD. *P < 0·05, gMCs versus bmMCs. (c) Cells were cultured in the absence (−) or presence (+) of IFN-γ. After 2 days of culture, the supernatant was harvested and the kynurenine concentration was measured by high-performance liquid chromatography as described in the Materials and methods. The positive control (PC) was prepared from IFN-γ-treated RAW cells. Levels are expressed as μm± SD. *P < 0·05, gMCs versus bmMCs.

Figure 4

Effect of indoleamine 2,3-dioxygenase (IDO) on granulocyte colony-stimulating factor (G-CSF)-induced immature myeloid cell (gMC)-mediated alloreactive T-cell suppression. Mixed lymphocyte reaction (MLR) cultures were set up as described in Fig. 2a (upper histogram). MLRs alone, MLRs/gMCs (10⁴) or MLRs/bone-marrow derived myeloid cells (bmMCs; 10⁴) were cultured in the presence of 1-methyl-dl-tryptophan (1-MT; 250 μm) or control vehicle (middle histogram). The gMCs and bmMCs were isolated from the spleens of G-CSF-injected mice and the bone marrow of naive IDO^−/− B6 mice, respectively and cocultured in MLRs (low histogram). Representative data from one experiment of three are shown.

Figure 5

Effect of indoleamine 2,3-dioxygenase (IDO) on granulocyte colony-stimulating factor (G-CSF)-induced immature myeloid cell (gMC)-mediated inhibition of acute graft-versus-host disease (GVHD). (a) B6D2F1-recipient mice were lethally irradiated (950 cGy) and transferred with 2 × 10⁶ B6 donor T cells alone or plus 2 × 10⁶ gMC. Recipient mice were orally administered with 1-methyl-dl-tryptophan (1-MT) or control vehicle daily from day − 1 until 4 days after the cells were transferred. (b) The gMCs were isolated from G-CSF-injected wild-type (WT) and IDO^−/− mice and cotransferred with donor T cells to irradiated recipient mice. Each group consisted of eight mice. Data represent the results from two similar experiments.