Journal List > Immune Netw > v.18(2) > 1148256

Na, Sohn, Ryu, Choi, In, Shin, and Park: Extended Culture of Bone Marrow with Granulocyte Macrophage-Colony Stimulating Factor Generates Immunosuppressive Cells

Abstract

Bone marrow-derived dendritic cells (BM-DCs) are generated from bone marrow (BM) cells cultured with granulocyte macrophage-colony stimulating factor (GM-CSF) for a week. In this study we investigated the effect of duration on the BM culture with GM-CSF. Within several months, the cells in the BM culture gradually expressed homogeneous levels of CD11c and major histocompatibility complex II on surface, and they became unable to stimulate allogeneic naïve T cells in mixed lymphocyte reaction (MLR). In addition, when the BM culture were sustained for 32 wk or longer, the BM cells acquired ability to suppress the proliferation of allogeneic T cells in MLR as well as the response of ovalbumin-specific OT-I transgenic T cells in antigen-dependent manner. We found that, except for programmed death-ligand 1, most cell surface molecules were expressed lower in the BM cells cultured with GM-CSF for the extended duration. These results indicate that BM cells in the extended culture with GM-CSF undergo 2 distinct steps of functional change; first, they lose the immunostimulatory capacity; and next, they gain the immunosuppressive ability.

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Figure 1.
Cells from the extended cultures of BM with GM-CSF lose their capacity to stimulate allogeneic T cells. BM cells isolated from C57BL/6 mouse are cultured with GM-CSF, and harvested in 4-wk intervals. The 5×104 CFSE-labeled allogeneic T cells from BALB/c mouse are co-cultured with 1.7×104 BM cells derived respectively from (A) 4-, and (B) 8-wk old cultures in comparison with the 1-wk old culture. After 4 days, CFSE low T cells are analyzed by a flow cytometer and counted. Representative data are shown from at least 2 independent experiments in quadruplicate. Error bars indicate mean ± standard error of the mean across quadruplicate samples. ** p≤0.01, *** p≤0.001.
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Figure 2.
Long-term cultured BM cells retain homogeneous levels of CD11c and MHC II. BM cells cultured with GM-CSF for 1 wk versus (A) 4 wk or (B) 57 wk are compared for their MHC II and CD11c expression. Representative data are shown from at least 2 independent experiments.
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Figure 3.
The expression of surface molecules is evaluated on BM cells in the extended cultures with GM-CSF. BM cells in the 1-wk and 70-wk old cultures are stained side by side with antibodies against the indicated molecules. (A) In the 1-wk old BM culture, CD11c+ MHC II hi cells are gated as DCs in red tetragon and CD11c+ MHC II int cells are gated as macrophages in purple tetragon. The whole BM cells in the 70-wk old culture are gated for analysis. (B) Histograms illustrate the staining of a respective antibody (open red line) versus an isotype control (area filled with gray). The expression of each molecule is detected on cell surface but IDO-1 detected intracellularly. Mean fluorescent index is denoted in each histogram. Representative data are shown from at least 2 independent experiments.
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Figure 4.
Cells from the extended cultures of BM with GM-CSF are suppressive to MLR. (A) BM cells cultured for 1 wk (1W-BMC) were mixed with either control syngeneic splenocytes or BM cells cultured long-term (LT-BMC) for 16 or 32 wk and co-cultured with 5×104 CFSE-labeled allogeneic T cells isolated from BALB/c mice. After 4 days, CFSE low allogeneic T cells are assessed. Representative flow cytograms are shown from 2 independent experiments in quadruplicate. (B) The suppressor cell activity of BM cells in extended cultures at different time points is assessed as in (A). Assays are performed at the ratios of 1(+):1(+):3(+) and 1(+):3(+++):3(+) for 1W-BMC:LT-BMC/Splenocyte:Allogeneic T cell. The number of CFSE low T cells were counted and plotted in graphs. Data are generated from 2 independent experiments in quadruplicate. Error bars indicate mean ± standard error of the mean across quadruplicate samples. * p≤0.05, ** p≤0.01, *** p≤0.001.
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Figure 5.
Cells from the extended culture of BM with GM-CSF are suppressive to the proliferation of OT-I T cells in antigen-dependent manner. (A) BM cells cultured for 1 wk (1W-BMC), 60-wk old BM cells cultured long-term (LT-BMC), and control syngeneic splenocytes are pre-treated with OVA (100 μg/ml), mixed as indicated, co-cultured with 105 CFSE-labeled OT-I T cells for 3 days, and analyzed for the number CFSE low OT-I T cells. (B) The suppressor cell activity is assessed as in (A) except that 62-wk old LT-BMC and control syngeneic splenocytes are not pre-treated with OVA. Assays are performed at the ratios of 1(+):1(+):100(+), 1(+):3(++):100(+), and 1(+):10(+++):100(+) for 1W-BMC:LT-BMC/Splenocyte:OT-I T cell. The number of CFSE low T cells were counted and plotted in graphs. Data are generated from 2 independent experiments in triplicate. Error bars indicate mean ± standard error of the mean across triplicate samples. NS, not significant. * p≤0.05, ** p≤0.01, *** p≤0.001.
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Figure 6.
LPS stimulation does not affect the stimulatory function of BM cells cultured long-term (LT-BMC). BM cells in the 1-wk and 70-wk old cultures are stimulated side by side with LPS (100 μg/ml) for 18 h. After LPS stimulation, BM cells in the respective culture are stained with antibodies against the indicated molecules (A) or co-cultured with 5×104 CFSE-labeled allogeneic T cells for MLR (B). Data are generated from 2 independent experiments in quadruplicate. Error bars indicate mean ± standard error of the mean across quadruplicate samples. 1W-BMC, BM cells cultured for 1 wk; NS, not significant. * p≤0.05.
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