Cell surface DCIR is internalized into pDCs after receptor triggering. (A) DCIR internalization is induced by receptor triggering with anti-DCIR antibodies. PDCs were labeled with anti-DCIR, anti-BDCA-2, or anti-MHC class I antibodies followed by secondary antibodies on ice. Incubation at 37°C induces receptor internalization leading to 50% reduced DCIR (MFI 9 vs 18) and 37% reduced BDCA-2 (MFI 89 vs 141) surface expression (thick line) compared with cells incubated at 4°C (shaded area, left). Under the same conditions, MHC class I expression is reduced by only 17% (MFI 710 vs 857). Matching isotype controls did not show any binding (dotted lines). FACS data are representative of 3 independent experiments (left). PDCs were labeled with anti-DCIR or anti–BDCA-2 antibodies, washed, and incubated on ice with fluorochrome-conjugated secondary antibodies (blue). To visualize the cell surface, pDCs were counterstained with murine anti-MHC class I antibodies and isotype-specific fluorochrome-conjugated secondary antibodies (green). Cells were analyzed by CLSM (right). DCIR can be detected at the cell surface after antibody labeling at 4°C and is clearly internalized after elevating the incubation temperature to 37°C. BDCA-2 endocytosis after receptor triggering is detected as described in the literature.15 (B) Incubation of pDCs in 450 mM sucrose inhibits DCIR endocytosis at 37°C, which is restored after washing out sucrose and incubating the cells in isotonic medium (sucrose washout). In each histogram, total surface protein (gray shaded area) is compared with internalized antibody (thick line) after stripping off surface-bound antibodies, as described in “Methods.” A matched isotype control did not bind to the cells (dotted lines). Data shown are representative of 3 independent experiments.
