Myeloid DCs Take Up Cell-associated Antigens. The lack of presentation by myeloid DCs could be explained if they do not have access to the injected, antigen-bearing cells. This could either be due to the failure of the injected cells to home to the marginal zone areas where myeloid DCs reside, or the myeloid DCs may lack specific surface recep- tors for the internalization of cell-associated antigen. To determine whether myeloid DCs take up cell-associated antigens, b2m2/2 cells were shocked with both OVA and yellow/green fluorescent beads and injected into B6 mice. Splenic low density cells were isolated 14 h after injection, stained for CD11c and CD11b, and analyzed by flow cy- tometry (Fig. 7 A). Cells containing one or more fluores- cent beads were detected in the FITC channel. The CD11c and CD11b FACS® profile of bead-positive cells clearly shows that in addition to lymphoid DCs, both my- eloid DCs and autofluorescent macrophages have taken up beads derived from b2m2/2 cells (Fig. 7 B). This result in- dicates that the restricted presentation by lymphoid DCs is not due to restricted uptake of cell-associated antigens. Only a Small Fraction of Lymphoid DCs Present OVA. We made two estimates of the fraction of cells within the isolated low density preparation that could present OVA to OT-1 after in vivo uptake of antigen. At 14 h after injec- Figure 5. Lymphoid DCs, but not myeloid DCs, cross- present OVA antigen. Low density cells were isolated from mice primed previously with OVA- or BSA-loaded b2m2/2 cells. Myeloid and lymphoid DCs from OVA/ b2m2/2-injected mice were (A) FACS® sorted based on their differential CD11b/CD11c expression and (B) tested for their ability to stimulate naive OT-I cells in vitro. The different bars represent titered numbers of DCs in the well. The proliferation of OT-I induced by peptide-pulsed cells is depicted in the inset. One of three experiments with similar results is shown. Error bars indicate SEM of triplicate wells. MyDC, myeloid DCs; LyDC, lymphoid DCs; AFC, autofluorescent cells, mostly macrophages; n.t., not tested. tion of cells loaded with OVA and fluorescent beads, 0.8% of the total low density cell preparation was bead-positive, as determined by FITC fluorescence (Table I). Within the lymphoid DC subset, which made up 27% of the cells, 1% were bead-positive. Therefore, only 0.27% of the total low density cell preparation was estimated to contain and present OVA to OT-1 cells. Second, the amount of OT-1 proliferation stimulated by in vivo–derived low density cells was compared with that Table I. In Vivo Uptake of Cell-associated Beads Low density cell preparation Myeloid DCs Lymphoid DCs Autofluorescent cells The percentage of different subsets in low density cell preparation and the percentage of each that contain beads. Percentage of total 100 41 27 13 Percentage of bead1 cells in each subset 0.8 0.6 1.0 1.5 1690 CD81 Dendritic Cells Cross-prime CTLs In Vivo