pDC precursors can mediate efficient internalization of anti–Siglec-H Abs. (A) Bone marrow cells enriched in pDC precursors were incubated with sheep anti–Siglec-H IgG or F(ab′)2 fragments or preimmune F(ab′)2 fragments for 1 hour on ice and then either incubated on ice or at 37°C for 3 hours. At the end of the incubation period, the remaining surface anti–Siglec-H IgG or F(ab′)2 was detected using donkey anti–sheep IgG-FITC. (B) To determine whether anti–Siglec-H IgG or F(ab′)2 was internalized or shed at 37°C, we treated bone marrow cells enriched in pDC precursors as in panel A, using Alexa 488–labeled sheep anti–Siglec-H IgG or F(ab′)2 fragments and total cell-associated Ab (surface + internalized) was measured after 3 hours at either 0°C or 37°C. (C) Time course of internalization. Bone marrow cells were incubated with biotinylated sheep anti–Siglec-H IgG for 1 hour on ice and then incubated either on ice for 3 hours or at 37°C for the indicated time points. After 3 hours, percent remaining surface anti–Siglec-H was measured using streptavidin-APC. Data show means ± 1 SD of triplicate measurements and are representative of 3 experiments performed. (D) Bone marrow cells enriched in pDC precursors were labeled with Alexa 488-labeled sheep anti–Siglec-H IgG or F(ab′)2 fragments treated as in panel B, counterstained with the plasma membrane marker cholera toxin subunit B-Alexa 594 (red) and the DNA marker DAPI (blue). Cells were examined on a DeltaVision deconvolution fluorescence microscope and images from the central z-section of representative cells are shown. Magnification: 1000×.
