Figure 6
Supernatants from CCL21-DC stimulate chemotaxis of T2 cells. (A) The supernatants from DC, CCL21-DC and CV-DC from different individuals (n = 12) were utilized in a standard chemotaxis assay to assess their ability to induce chemotaxis of T2 cells. Briefly 2.0 × 105 T2 cells were loaded in the upper chamber of a 24-well transwell apparatus and allowed to migrate through a 3 μm insert for 2 and 1/2 h at 37°C. In A, the lower chambers of the transwell had been loaded with the supernatant from DC or CCL21-DC (supernatant #1–11) from the same individual, or DC or CV-DC (#12) from the same individual, as indicated. Recombinant CCL21 (600 ng/ml) and 10% AB medium were added to the wells as positive and negative controls, respectively. A summary of chemotaxis from 12 different samples is shown. The CCL21 protein concentration in ng/ml is indicated over each column. Migrated cells were analyzed by flow cytometry by counting the number of events per minute (expressed as migrated cell number) within a pre-determined T cell gate. One-way ANOVA p < 0.0001. In blocking experiments (B), the supernatants from CCL21-DC or recombinant CCL21 were pre-treated with a neutralizing concentration of anti-CCL21 antibody (1.5 μg/50 ng CCL21) or isotype immunoglobulin, prior loading in the lower chamber of the transwell apparatus. The supernatant from CV-DC and medium containing 10% AB were also assessed. To determine total T2 cell migration, T2 cells were loaded without inserts. A representative experiment of a total of seven is shown. The number of migrated cells was analyzed by flow cytometry as above, and is expressed as percentage of total T2 cell migration. Asterisks denote statistical significance (p < 0.05). The surface expression of CCR7 is analyzed by flow cytometry in T2 cells (C) and in immature DC (D) cultured for 6 days in GM-CSF and IL-4. Histograms are shown: dotted lines indicate isotype control, bold lines indicate marker expression. The percentage of positive cells and MFI are indicated. A representative experiment of two is shown.