Figure 6. MHC class I–restricted cross-presentation is restricted to lymphoid DCs as early as 2 h after priming. Low density cells were iso- lated from mice 2 or 14 h after priming with OVA-loaded b2m2/2 cells. Myeloid and lymphoid DCs were FACS® sorted based on their differen- tial CD11b/CD11c expression and tested for their ability to stimulate na- ive OT-I cells in vitro. The different bars represent titered numbers of DCs in the well. Error bars indicate SEM of triplicate wells. MyDC, my- eloid DCs; LyDC, lymphoid DCs; n.t., not tested. stimulated by a titration of in vitro peptide-pulsed low density cells. This comparison suggested that 0.32% of lym- phoid DCs, or 0.06% of low density cells, presented OVA (average of three experiments, data not shown). This is pre- sumably an underestimate of the number of OVA-present- ing cells, as the amount of OVA acquired in vivo is proba- bly lower than the amount of OVA used for exogenous peptide pulsing. Together, these experiments suggest that only z1% of the lymphoid DC subset present OVA in vitro. Discussion Cross-presentation of exogenous antigens in association with MHC class I molecules has been studied extensively in vitro. Both macrophages and DCs have been shown to present peptides derived from soluble and particulate anti- gens in the context of MHC class I molecules (for a review, 1691 den Haan et al. see reference 7). In addition, antigens from apoptotic or necrotic cells can be processed and presented in MHC class I by both cell types in vitro (15, 19, 53, 54). In contrast, there is little information concerning the identity of cross- presenting cells in vivo. DCs have been shown to present soluble protein-derived and cell-associated antigens in MHC class II molecules (48, 51, 55), but the cell that cross-presents antigens in association with MHC class I molecules in vivo has remained elusive. In our study, we have isolated low density spleen cells from in vivo–primed mice and shown that they present exogenously derived an- tigens in association with MHC class I in a TAP-dependent manner. Further analysis of the cross-presenting cells iden- tified them as CD11c1 DCs. This is the first isolation and characterization of DCs in the central lymphoid organs that have taken up cell-associated antigens to present them to CD81 T cells in vivo. Further experiments showed that only the lymphoid DC subset cross-presents cell-associated antigens in MHC class I molecules in the spleen. The number of actual OVA-pre- senting cells is very low. We estimated that only 1% of lym- phoid DCs present OVA to T cells. The lymphoid DC subset–restricted presentation was evident as early as 2 h af- ter immunization, which strongly suggests that lymphoid DCs themselves take up the exogenous cell-associated anti- gen. Transfer of antigen between different DC subsets, as had been suggested, is therefore unlikely (41, 48, 49). In support of the notion of direct uptake of antigen by lym- phoid DCs, rat DCs containing apoptotic cell remnants have been found to migrate to the T cell areas of lymph nodes. These DCs were CD42 and may be the rat equiva- lent of the mouse lymphoid DCs (56). Our finding that only lymphoid DCs cross-present cell-associated antigens in the spleen does not preclude the presence of other types of APCs with cross-priming function in other tissues. APCs have been isolated from tumors and islets of Langerhans that could stimulate CD81 T cells specific for tumor and is- let antigens, respectively (57–59). One of these studies de- fined the phenotype of the APCs as macrophage like (57). Why do only lymphoid and not myeloid DCs cross- present cell-associated antigens to CD81 T cells? In princi- ple, it could be the result of differences in the uptake of cell debris, processing ability, or their T cell stimulatory capac- ity. We show that, in addition to lymphoid DCs, both au- Figure 7. Uptake of cell-associated beads by both DC subsets and autofluorescent macrophages. Low density cells were isolated from mice 14 h after priming with OVA/ bead-loaded b2m2/2 cells. (A) The cells were stained for CD11c and CD11b and analyzed by FACS®. MyDC, my- eloid DCs; LyDC, lymphoid DCs; AFC, autofluorescent cells, mostly macrophages. (B) CD11c and CD11b profile of the gated low density cells containing beads.