Figure 2.
Figure 2. CCL22 mediates DC–T reg contacts. (A) T convs (green) and T regs (red) from OT-II mice were mixed at a 1:1 ratio and cultured with unlabeled Ccl22−/− or C57BL/6 WT CD11c+ splenic DCs in the presence of 1 µg/ml OVA323–339 peptide. After 12 h, cells were imaged by confocal microscopy (representative images are shown; bars, 15 µm), and the ratio of T regs to T convs per DC cluster was determined. (B) 106 T convs or T regs (both red) from C57BL/6 mice were mixed in a collagen gel with 105 BMDCs (green) from Ccl22−/− or WT mice, and DC–T cell interaction was analyzed by confocal microscopy over 8 h. Resulting videos were analyzed for DC–T cell contacts by a computer algorithm. Left: One dot represents the percentage of DCs in contact with T cells at one time point. Right: Triplicates of the respective conditions were cocultured in parallel. One dot represents the mean percentage of DCs in contact with T cells over the total culture time. (C) BMDCs were treated with either control or CCL22 siRNA and, as in B, mixed with T regs, and DC–T cell interaction was quantified. (D) DC–T cell interaction was analyzed and depicted as in B, and DC2.4-CCL22dox cells in which CCL22 is inducible by doxycycline (Dox) were used. (E) Differently labeled OVA323–339-pulsed BMDCs were pretreated with control or CCL22 siRNA 18 h before injection into the footpad of OT-II-Foxp3-GFP mice. The footpad-draining lymph node was imaged by two-photon microscopy (a representative image of a 1-h video is shown; bar, 30 µm), and the number of OT-II-Foxp3+–T reg contacts with DCs per hour as well as the contact time for each T reg–DC contact was quantified by two blinded independent investigators. 15 control DCs and 16 CCL22 knockdown DCs were examined. The instantaneous velocities were calculated from manually tracked cells using Imaris. All in vitro data are representative of three and all in vivo data of two independent experiments. Data are shown as mean ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, not significant (two-sided Student’s t test).

CCL22 mediates DC–T reg contacts. (A) T convs (green) and T regs (red) from OT-II mice were mixed at a 1:1 ratio and cultured with unlabeled Ccl22−/− or C57BL/6 WT CD11c+ splenic DCs in the presence of 1 µg/ml OVA323–339 peptide. After 12 h, cells were imaged by confocal microscopy (representative images are shown; bars, 15 µm), and the ratio of T regs to T convs per DC cluster was determined. (B) 106 T convs or T regs (both red) from C57BL/6 mice were mixed in a collagen gel with 105 BMDCs (green) from Ccl22−/− or WT mice, and DC–T cell interaction was analyzed by confocal microscopy over 8 h. Resulting videos were analyzed for DC–T cell contacts by a computer algorithm. Left: One dot represents the percentage of DCs in contact with T cells at one time point. Right: Triplicates of the respective conditions were cocultured in parallel. One dot represents the mean percentage of DCs in contact with T cells over the total culture time. (C) BMDCs were treated with either control or CCL22 siRNA and, as in B, mixed with T regs, and DC–T cell interaction was quantified. (D) DC–T cell interaction was analyzed and depicted as in B, and DC2.4-CCL22dox cells in which CCL22 is inducible by doxycycline (Dox) were used. (E) Differently labeled OVA323–339-pulsed BMDCs were pretreated with control or CCL22 siRNA 18 h before injection into the footpad of OT-II-Foxp3-GFP mice. The footpad-draining lymph node was imaged by two-photon microscopy (a representative image of a 1-h video is shown; bar, 30 µm), and the number of OT-II-Foxp3+–T reg contacts with DCs per hour as well as the contact time for each T reg–DC contact was quantified by two blinded independent investigators. 15 control DCs and 16 CCL22 knockdown DCs were examined. The instantaneous velocities were calculated from manually tracked cells using Imaris. All in vitro data are representative of three and all in vivo data of two independent experiments. Data are shown as mean ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, not significant (two-sided Student’s t test).

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