() Effect of adding GM-CSF/IL-4 to THP-1 after three-day treatment of GL-PS as determined by XTT proliferation assay

The results represented the mean ± SD of three representative experiments. *< 0.05; ** < 0.01; ***< 0.001 versus that without GM-CSF/IL-4 added. () Trypan blue exclusion assay. THP-1 cells after adding either GM-CSF/IL-4 or GL-PS were counted. The results represented the mean ± SD of three representative experiments. *< 0.05; ** < 0.01; ***< 0.001 versus that of THP-1 cells.<p><b>Copyright information:</b></p><p>Taken from "polysaccharides can induce human monocytic leukemia cells into dendritic cells with immuno-stimulatory function"</p><p>http://www.jhoonline.org/content/1/1/9</p><p>Journal of Hematology and Oncology 2008;1():9-9.</p><p>Published online 21 Jul 2008</p><p>PMCID:PMC2517069.</p><p></p

Microscopic morphology of normal mature monocyte-derived DCs (Mo-mDCs) and THP-1 DCs (upper panel)

The round THP-1 cells changed to be adherent flatten cells (white arrows). Under forward scatter and side scatter analysis of flow cytometer (lower panel), the THP-1 DCs increased in size when compared with THP-1 cells alone. () Surface expression of antigen presentation and costimulation molecules on immature Mo-DCs (Mo-iDCs), THP-1 cells alone, THP-1 stimulated with GM-CSF/IL-4; GL-PS treated THP-1, THP-1 stimulated with both GL-PS and GM-CSF/IL-4. The expressions of DC maturation markers CD11c, HLA-DR, CD40, CD80 and CD86 on DCs were analyzed by flow cytometer after five-day differentiation. The results shown were from one representative experiment of triplicate independent experiments performed. () The average relative expression of the DC maturation markers. The results were presented as mean ± SD of three representative experiments. *< 0.05; ** < 0.01; ***< 0.001 versus that of THP-1 cells.<p><b>Copyright information:</b></p><p>Taken from "polysaccharides can induce human monocytic leukemia cells into dendritic cells with immuno-stimulatory function"</p><p>http://www.jhoonline.org/content/1/1/9</p><p>Journal of Hematology and Oncology 2008;1():9-9.</p><p>Published online 21 Jul 2008</p><p>PMCID:PMC2517069.</p><p></p

() Treated DCs were incubated with FITC-dextran for 1 h at 37°C and then washed for four times

Increase shift in fluorescence intensity (x-axis) was compared with the control, the one without FITC-dextran. The result was from one representative experiment of three independent repeats. () The increase in fluorescence intensity was calculated when compared with that of THP-1 cells alone after normalized with the background fluorescence, which was from the parallel experiments performed for all cells at 4°C. The results represented the mean ± SD from three independent experiments. ** < 0.01 versus that of THP-1 cells.<p><b>Copyright information:</b></p><p>Taken from "polysaccharides can induce human monocytic leukemia cells into dendritic cells with immuno-stimulatory function"</p><p>http://www.jhoonline.org/content/1/1/9</p><p>Journal of Hematology and Oncology 2008;1():9-9.</p><p>Published online 21 Jul 2008</p><p>PMCID:PMC2517069.</p><p></p

() The time-response curves for the effect of GL polysaccharides (GL-PS, -▪-) at 100 μg/mL and vincristine (-▫-) at 0

1 μM on THP-1 (Left panel) and U937 cells (right panel) determined by XTT cell proliferation assay. The results represent the mean ± SD of triplicate cultures of three representative experiments. *< 0.05; ** < 0.01; ***< 0.001 versus negative control (-•-). () Cell cycle analysis with PI staining. The THP-1 (left panel) and U937 cells (right panel) were treated with or without 100 μg/mL GL-PS for three days. The cell cycle analysis was then analyzed with PI staining and Cylchred Version 1.0.2 (Cardiff University, Wales, UK). The results shown were from one representative experiment of three independent experiments performed. () PCNA expression of GL-PS treated THP-1 (upper panel) and U937 cells (lower panel) in three-day incubation. The results shown were from one representative experiment of three independent experiments performed.<p><b>Copyright information:</b></p><p>Taken from "polysaccharides can induce human monocytic leukemia cells into dendritic cells with immuno-stimulatory function"</p><p>http://www.jhoonline.org/content/1/1/9</p><p>Journal of Hematology and Oncology 2008;1():9-9.</p><p>Published online 21 Jul 2008</p><p>PMCID:PMC2517069.</p><p></p

(The optical densities of incorporated BrdU from DC:T co-cultures were normalized with that from T cells alone

The results represented one experimental result of three independent experiments. The results represented the mean ± SD of three independent experiments. *< 0.05 versus that of THP-1 cells alone. () CD4/CD8 ratio of the co-cultured T cells. The co-cultured T cells so harvested were stained with CD4 and CD8 antibody and analyzed with flow cytometry. The results represented the mean ± SD of two independent experiments. **< 0.01 versus that of T cells alone.<p><b>Copyright information:</b></p><p>Taken from "polysaccharides can induce human monocytic leukemia cells into dendritic cells with immuno-stimulatory function"</p><p>http://www.jhoonline.org/content/1/1/9</p><p>Journal of Hematology and Oncology 2008;1():9-9.</p><p>Published online 21 Jul 2008</p><p>PMCID:PMC2517069.</p><p></p

Essential Role of NK Cells in IgG Therapy for Experimental Autoimmune Encephalomyelitis

<div><p>Intravenous immunoglobulin has long been used in treating autoimmune diseases, although mechanisms remain uncertain. Activating Fcγ receptors are receptors of IgG and reported to be essential in intravenous immunoglobulin (IVIG) therapy. Therefore, we hypothesized natural killer (NK) cells, which express abundant activating Fcγ receptors, are the potential cellular target. In experimental autoimmune encephalomyelitis (EAE), we demonstrated that IgG suppressed disease development in intact, but not in NK cell depleted mice. Adoptive transfer of IgG-treated NK cell could protect mice against EAE, and suppressed interferon γ and interleukin 17 production. The percentage of CD4⁺Foxp3⁺ regulatory T cells was significantly increased. The increase of regulatory T cells was also observed in IgG-treated EAE mice but not in NK cell depleted mice. In vitro experiments confirmed that IgG-treated NK cells enhanced regulatory T cell induction from naïve CD4⁺ T cells. Interestingly, cells from draining lymph nodes produced more interleukin 2 after the adoptive transfer of IgG-treated NK cells. We neutralized interleukin 2 and the induction of CD4⁺Foxp3⁺ T cells by IgG-treated NK cells was significantly reduced. To our knowledge, we identified for the first time the critical role of NK cells in the mechanism of IgG-induced induction of Treg cells in treatment of autoimmunity.</p> </div

IgG-NK cells suppress EAE by inducing CD4⁺Foxp3⁺ Treg cells with stronger inhibitory effect.

<p>EAE was induced in C57BL/6N mice and 1×10⁶ of IgG-NK cells or untreated-NK cells were injected intravenously at the day of EAE induction. (<b>A, B</b>) Cells were isolated from draining LNs 10 days after EAE induction and analyzed by Flow cytometry. Treg cells were identified by intracellular expression of Foxp3 on the gated CD4⁺ cells. Data are displayed as the mean percentage ± SEM of the combined data with 4–6 mice per group from 2 independent experiments. (<b>C, D</b>) 2×10⁴ CFSE-labeled CD4⁺CD25⁻ T cells from the spleen of immunized EAE mice were cocultured with 1×10⁴ irradiated CD4⁻ depleted splenocytes. CD4⁺CD25^hi T cells from EAE mice (Treg^EAE), NK treated EAE mice (Treg^NK-EAE) or IgG-NK treated EAE mice (Treg^IgG-NK-EAE) were added according to indicated ratios with MOG_35–55. The intensities of CFSE were determined at day 5 by FACS. CD4⁺CD25^hi Treg cells from EAE mice with IgG-NK cell treatment displayed stronger suppressive effect (p< 0.05). Mice: n = 4 per group. (<b>E</b>) Treg cells were depleted by injection of 100 µg of anti-CD25 antibody (PC61) intravenously 2 days before EAE induction. 1×10⁶ of IgG-NK cells were injected intravenously at the day of EAE induction. The mean clinical scores of EAE mice with IgG-NK cell treatment were significantly lower but it was reversed by anti-CD25 antibody treatment. Mice: n = 4 per group. Data are representative of at least 2 independent experiments and displayed as the mean ± SEM. *: p<0.05; **: p<0.01, Krusaki-Wallis test.</p

IgG-NK cells induce CD4⁺Foxp3⁺ Treg cells and antigen specific T cells to express higher CD25 and IL-2 respectively.

<p>EAE was induced in C57BL/6N mice and 1×10⁶ of IgG-NK cells or untreated-NK cells were injected intravenously at the day of EAE induction. (<b>A</b>) 10 days after EAE induction, cells were isolated from draining lymph nodes and 2.5×10⁶ isolated cells were re-stimulated with MOG_35–55 for 72 hours. Supernatant was collected and the IL-2 levels were studied by ELISA. IL-2 production was significantly increased after adoptive transfer of IgG-NK cells (p<0.05) (n = 5–7). Krusaki-Wallis test. (<b>B</b>) Cells were isolated from draining lymph nodes 10 days after EAE induction and studied by FACS. CD4⁺Foxp3⁺ Treg cells from EAE mice (Treg^EAE) and IgG-NK treated EAE mice (Treg^IgG-NK-EAE) were gated and the expression of CD25 was shown. Treg^IgG-NK-EAE expressed significantly higher CD25 when compared with Treg^EAE (n = 4). Data are pooled from 2 independent experiments and displayed as the mean ± SEM. *: p<0.05. Mann-Whitney-U-Test.</p

IgG protects NK sufficient, but not NK depleted mice from EAE and lowers associated immunological responses.

<p>EAE was induced in C57BL/6N mice as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060862#s4" target="_blank">Materials and Methods</a>. IgG was injected intravenously at day 0 and 4. NK cell was depleted in EAE mice with or without IgG treatment by injecting anti-asialo GM1 antibody intravenously 1 day before and 3 days after the immunization. (<b>A</b>) <b>EAE development</b>. NK depleted or non-depleted mice were followed for EAE and disease was scored as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060862#s4" target="_blank">Methods</a> (n = 16–20). (<b>B</b>) <b>Cytokine production.</b> On day 10, cells were isolated from draining lymph nodes. 2.5×10⁶ isolated cells were re-stimulated with MOG_35–55 (20 µg/ml) for 72 hours. Supernatant was collected and cytokine production was determined (n = 4–7). Data are pooled from 4 independent experiments (<b>A</b>) and 2 independent experiments (<b>B</b>) and displayed as mean ± SEM. *: p<0.05; **: p<0.01, Krusaki-Wallis test.</p

Adoptive transfer of IgG-NK cell suppresses EAE.

<p>EAE was induced in C57BL/6N mice. NK cells were isolated from the spleen of naïve mice and incubated with IgG for 18 hours. 1×10⁶ of IgG-NK cells or untreated-NK cells were injected intravenously on the day of EAE induction. (<b>A</b>) The mean clinical scores were significantly decreased after injection of IgG-NK cell when compared with untreated EAE mice (p<0.01). No significant difference was observed between untreated EAE mice and EAE mice with adoptive transfer of untreated-NK cells (n = 10–11). (<b>B</b>) At day 10, cells were isolated from draining LNs. 2.5×10⁶ isolated cells were re-stimulated with MOG_35–55 for 72 hours. Cytokine productions were determined (n = 4–7). Data are pooled from 3 independent experiments (<b>A</b>) and 2 independent experiments (<b>B</b>) and displayed as mean ± SEM. *: p<0.05; **: p<0.01, Krusaki-Wallis test.</p