Effect of TfR internalization blockade in iron-induced internalization of IFN-γR2. (A) ST4 cells were cultured for 24 hours without serum, then incubated for 30 minutes at 4°C with isotype-negative control mouse IgG1 (i-ii), nonblocking anti-TfR mAb (iii-iv), or blocking anti-TfR mAb (v-vi). Cells were further cultured in the absence (i,iii,v) or presence (ii,iv,vi) of 10 μM FeSO4 at 37°C. The histogram represents the expression of IFN-γR2 (gray histogram) or background of mouse IgG1 negative control (open histogram) in the ST4 T-cell line in 1 representative experiment of 3 independently performed. Boxed results show the percentage of positive cells calculated by subtracting nonspecific fluorescence detected with isotype-matched control IgG1 from that obtained with specific fluorescence. (B) ST4 cells were cultured as described in legend of Figure 3A with nonblocking anti-TfR mAb (□) or blocking anti-TfR mAb (▪). Cells were further cultured in the absence or presence of 10 μM FeSO4 or 50 μg/mL Holo-Tf. After 1 hour, IFN-γR2 cell-surface expression was evaluated by flow cytometry. Percentage of IFN-γR2 expression was calculated as described in legend of Figure 3A. The percentage of IFN-γR2 expression was similar in cells cultured in isotype control mAb (data not shown) and nonblocking anti-TfR mAb.
