expression of CD11c and CD11b. The autofluorescent cells expressed MHC class I, heterogeneous levels of MHC class II, and low levels of the macrophage marker F4/80, but no CD4, CD8a, or DEC205 (data not shown). This phenotype is consistent with recently described autofluo- rescent cells present in DC cell preparations that were iden- tified as macrophages (28). Our low density cell preparation contained z30–40% myeloid and 10–30% lymphoid DCs, both expressing high levels of CD11c but different levels of CD11b (Fig. 3). Analysis of the myeloid and lymphoid DC subsets revealed high MHC class II expression by both sub- sets, high CD8a and DEC205 expression by the lymphoid DCs, and heterogenous CD4 and low DEC-205 expres- sion by the myeloid DC subset, consistent with previously published studies (25, 26, 28). We asked whether cross-presentation is a specific charac- teristic of DCs and, if so, whether it is a feature of a partic- ular DC subset. Therefore, we depleted the low density cell preparation from OVA/b2m2/2-primed mice of spe- cific cell subsets using antibodies and magnetic beads and the remaining cells were evaluated for their capacity to stimulate OT-I cells in vitro (Fig. 4). Depletion of CD11c1 cells in the DC preparation resulted in the loss of both the myeloid and lymphoid DC subsets (66% CD11c1 cells Figure 4. OVA cross-presentation by low density cells depleted of dif- ferent cell subsets. Low density spleen cells were isolated from B6 mice 14 h after priming with BSA/b2m2/2 or OVA/b2m2/2 cells. The low density cells from OVA/b2m2/2-injected mice were depleted of CD11c1, CD11bhigh, CD8ahigh, and CD8b1 cells by magnetic beads and the de- pleted populations were tested (A) directly for their presenting activity in an in vitro OT-I proliferation assay (2 3 105 stimulator cells/well) or (B) after pulsing with OVA peptide in vitro (2 3 104 cells/well). Error bars indicate SEM of triplicate wells. 1689 den Haan et al. were present before depletion, and 8% after depletion, data not shown). The remaining cells consisted of autofluores- cent and CD11c2CD11b2 cells and showed almost no OVA presentation. These results demonstrate that cross- presentation of cell-associated OVA is mediated entirely by CD11c1 DCs. Depletion of F4/801 cells resulted in no loss of activity (data not shown). Therefore, CD11c2 macro- phages do not seem to play an important in vivo role in cross-presenting cell-associated antigens. Depletion of CD11b1 cells specifically removed myeloid DC (38% predepletion to 9% postdepletion) and concomi- tantly increased the percentage of lymphoid DCs (26% pre- depletion to 48% postdepletion). This resulted in a higher stimulatory activity of the DC preparation (Fig. 4). To spe- cifically deplete for lymphoid DCs, we used anti-CD8a antibodies. However, this depletion of the lymphoid DC subset was only z50% complete (22% predepletion to 13% postdepletion) with the preferential loss of the CD8ahigh- expressing lymphoid DCs (data not shown). The specific depletion of CD8ahigh lymphoid DCs resulted in significant loss of stimulatory activity (Fig. 4). Finally, depletion for CD8b1 cells did not lead to a significant change in num- bers of the different subsets or in OVA cross-presenting ac- tivity. These results suggest that the lymphoid DC was the main cross-presenting APC. To confirm our findings, we isolated low density cells from mice injected previously with OVA-loaded b2m2/2 cells and sorted myeloid and lymphoid DCs on the basis of their CD11b and CD11c expression. Sorting for myeloid and lymphoid DC populations resulted in 87 and 84% pure populations, respectively (Fig. 5 A). The sorted DC subsets were used as APCs for OT-I in an in vitro proliferation as- say. Whereas sorted myeloid DCs lacked stimulatory ca- pacity, sorted lymphoid DCs had stimulatory activity that was stronger than that of the starting DC preparation (Fig. 5 B). This result clearly demonstrates that only lymphoid DCs have the ability to cross-present antigen acquired in vivo from injected b2m2/2 cells. Lymphoid DCs Rapidly Acquire Antigen In Vivo. It has been suggested that antigens are transferred from migratory DCs to DCs in the T cell areas (41, 48, 49). Other studies clearly indicate a migration of DCs from the marginal zone where the myeloid DCs are localized into the central T cell areas of the PALS where lymphoid DCs can be found (50– 52). This could indicate that marginal zone DCs can trans- fer antigens to the lymphoid DCs or that marginal zone DCs may mature into lymphoid DCs. Both alternatives would predict that early after injection of OVA/b2m2/2 cells, the OVA stimulatory capacity would not be restricted to the lymphoid DC subset. We therefore isolated myeloid and lymphoid DCs soon after injection of OVA/b2m2/2 cells and evaluated their OT-I stimulatory capacity (Fig. 6). As early as 2 h after injection, all the OVA stimulatory ac- tivity was found in the lymphoid DC subset. Unless trans- fer of antigens between DCs or the maturation of myeloid DCs into lymphoid DCs is an extremely rapid process, this result suggests that the lymphoid DCs themselves take up and present the cell-associated antigens in vivo.