Figure 1.
IgG protects NK sufficient, but not NK depleted mice from EAE and lowers associated immunological responses.
EAE was induced in C57BL/6N mice as described in Materials and Methods. 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. (A) EAE development. NK depleted or non-depleted mice were followed for EAE and disease was scored as described in Methods (n = 16–20). (B) Cytokine production. On day 10, cells were isolated from draining lymph nodes. 2.5×106 isolated cells were re-stimulated with MOG35–55 (20 µg/ml) for 72 hours. Supernatant was collected and cytokine production was determined (n = 4–7). Data are pooled from 4 independent experiments (A) and 2 independent experiments (B) and displayed as mean ± SEM. *: p<0.05; **: p<0.01, Krusaki-Wallis test.
Figure 2.
Adoptive transfer of IgG-NK cell suppresses EAE.
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. (A) 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) At day 10, cells were isolated from draining LNs. 2.5×106 isolated cells were re-stimulated with MOG35–55 for 72 hours. Cytokine productions were determined (n = 4–7). Data are pooled from 3 independent experiments (A) and 2 independent experiments (B) and displayed as mean ± SEM. *: p<0.05; **: p<0.01, Krusaki-Wallis test.
Figure 3.
IgG could not induce CD4+Foxp3+ Treg cells in NK cell-depleted EAE mice.
(A, B) EAE was induced in C57BL/6N mice. 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 day after the immunization. Cells were isolated from draining lymph nodes of EAE mice 10 days after EAE induction. Anti-CD4 antibody was used to stain the surface expression of CD4. Foxp3 expression on gated CD4 cells was determined by intracellular staining. Mice: n = 6 per group. Data are representative of at least 2 independent experiments and displayed as the mean ± SEM. Krusaki-Wallis test was used for comparison. *: p<0.05.
Figure 4.
IgG-NK cells suppress EAE by inducing CD4+Foxp3+ Treg cells with stronger inhibitory effect.
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. (A, 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. (C, D) 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+CD25hi T cells from EAE mice (TregEAE), NK treated EAE mice (TregNK-EAE) or IgG-NK treated EAE mice (TregIgG-NK-EAE) were added according to indicated ratios with MOG35–55. The intensities of CFSE were determined at day 5 by FACS. CD4+CD25hi Treg cells from EAE mice with IgG-NK cell treatment displayed stronger suppressive effect (p< 0.05). Mice: n = 4 per group. (E) 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.
Figure 5.
IgG-NK cells induce CD4+Foxp3+ Treg cells and antigen specific T cells to express higher CD25 and IL-2 respectively.
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. (A) 10 days after EAE induction, cells were isolated from draining lymph nodes and 2.5×106 isolated cells were re-stimulated with MOG35–55 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) Cells were isolated from draining lymph nodes 10 days after EAE induction and studied by FACS. CD4+Foxp3+ Treg cells from EAE mice (TregEAE) and IgG-NK treated EAE mice (TregIgG-NK-EAE) were gated and the expression of CD25 was shown. TregIgG-NK-EAE expressed significantly higher CD25 when compared with TregEAE (n = 4). Data are pooled from 2 independent experiments and displayed as the mean ± SEM. *: p<0.05. Mann-Whitney-U-Test.
Figure 6.
IgG-NK cells induce naïve CD4+CD62L+ T cells to convert into CD4+Foxp3+ Treg cells in the presence of IL-2 and TGF-β1.
(A, B) 1×106 naïve CD4+CD62L+ T cells purified from spleens of naïve mice were cultured with plate-bound anti-CD3 and soluble anti-CD28 antibodies. 1×106 isolated NK cells or IgG-NK cells were added to the culture with or without low dose of TGF-β (0.1 ng/ml) for 4 days. (B) 10 µg/ml of anti-IL-2 antibody or isotype control was added to neutralize IL-2. Data displayed as the mean ± SEM from 4 independent experiments. *: p<0.05. Mann-Whitney-U-Test.
Figure 7.
CD4+CD25hi Treg cells induced by IgG-NK cells suppresses CD4+ T cells proliferation.
(A, B) CD4+CD25hi Treg cells was induced by coculturing IgG-NK cells and CD4+CD62L+ T cells and sorted by FACS. 2×104 CFSE-labeled CD4+CD62L+ T cells were cocultured with the sorted CD4+CD25hi T cells according to the indicated ratios. Plate bound anti-CD3 and soluble anti-CD28 antibodies were used to stimulate T cell proliferation. The intensities of CFSE were determined at day 4 by flow cytometry. CD4+CD25hi Treg cells significantly suppressed CD4+ T cells proliferation at 1 to 1 ratio (p<0.01). Representative of 4 independent experiments (A); Data pooled from 4 independent experiments (B).