Targeting antigen to CD16 on slanDCs enables efficient internalization and restimulation of antigen-specific CD4+ T cells. (A) Internalization of CD16-targeted antigen into MHC class II–containing compartments. slanDCs were preincubated with FITC–anti-CD16 at 4°C. Internalization of cross-linked CD16 was induced by incubation for 1 hour at 37°C. Control cells were stored at 4°C. Localization of cross-linked CD16 in incubated and control cells was then analyzed by confocal laser scanning microscopy. (i) Distribution of CD16 on the surface of control cells. Four sections of the same cells are shown. (ii) Distribution of cross-linked CD16 after incubation for 1 hour. Four sections of the same cells are shown. (iii-v) Costaining of incubated slanDCs for HLA-DR (red, iii) and internalized CD16 (green, iv) shows partial colocalization of both signals in the overlay (v). (B) Efficient restimulation of antigen-specific T cells after targeting CD16 on slanDCs. Targeted antigen presentation was measured with specific mAbs to either CD16 (clone 3G8, mouse IgG1) or CD32 (clone AT10, mouse IgG1) followed by coculture with mouse IgG1–specific B13 T cells. Antigen presentation after nonspecific antigen uptake was measured using an anti-CD8 mAb (clone RPA-T8, mouse IgG1) with no affinity to slanDCs. Proliferation rates of IgG1-specific B13 T cells were measured by determining the 3H-thymidine incorporation into the DNA. Results are representative of 3 experiments.
