spleen as the source of the cross-presenting APC. To iden- tify the cross-presenting cell, we isolated low density spleen cells from mice 14 h after injection of b2m2/2 cells loaded with BSA or OVA. These cells were then used to stimulate naive OT-I cells in an in vitro proliferation assay in the ab- sence of additional antigen. The low density cell prepara- tion was enriched for DCs and generally contained ,1% Db/Kb-negative cells, indicating the paucity of injected OVA-loaded b2m2/2 cells (data not shown). Low density spleen cells from mice injected with OVA/b2m2/2 cells, but not from mice injected with BSA/b2m2/2 cells, were able to stimulate naive OT-I cells in vitro (Fig. 2 A). Low density cells from both types of mice were able to stimulate proliferation after peptide pulsing in vitro (Fig. 2 A, inset). Processing of exogenous antigens can involve an endo- some to cytosol pathway in which the endocytosed antigen is transported to the cytosol and further processed and pre- sented via the “classical” endogenous pathway (20). The TAP transporter is essential in this pathway, in that it trans- ports peptides that are generated by the proteasome from the cytosol to the endoplasmic reticulum where they bind nascent MHC class I molecules. TAP has been shown to be essential for cross-priming MHC class I–restricted responses in vivo (3, 45). Alternatively, a TAP-independent pathway of class I MHC cross-presentation has also been described (7, 46, 47). To determine which pathway is used for the cross-presentation of cell-associated antigens in vivo, BSA- or OVA-loaded b2m2/2 cells were injected into TAP2/2 mice and low density spleen cells from these mice were evaluated for their capacity to stimulate OT-I in vitro. Low density cells from TAP2/2 mice could not stimulate OT-I cells, indicating that the endosome to cytosol pathway is employed for cross-presentation of cell-associated antigens in this system (Fig. 2 B). This result also shows that con- taminating APCs in the OT-I cell preparation did not contribute to the proliferation in vitro. The various low density spleen cell preparations did not differ in their pre- sentation capacity, as exogenous OVA peptide pulsing of all preparations showed similarly strong stimulatory capac- ity for OT-I T cells. Only Lymphoid DCs Cross-present OVA Antigen to CD81 T Cells. To investigate further which cell population within the low density cell preparation was responsible for cross-presentation, we first evaluated which cell types were present. Flow cytometric analysis of the low density cells revealed four cell populations as defined by CD11c and CD11b staining (Fig. 3). A population of CD11c2CD11b2 cells that consisted mainly of T and B cells made up 10– 20% of the preparation. Besides these residual T and B cells, a population of CD11c2 and CD11b2 cells were de- tected that were autofluorescent in the FL-1, FL-2, and FL-3 channels, which could be misinterpreted as low level 1688 CD81 Dendritic Cells Cross-prime CTLs In Vivo Figure 3. Phenotypic analysis of low density cell preparation. Low density spleen cell prepara- tions were isolated from B6 mice 14 h after priming with OVA- loaded b2m2/2 cells. The cells were stained for CD11c, CD11b, and the indicated third reagent, and analyzed by FACS®. Gated myeloid DCs (CD11bhighCD11chigh) and lym- phoid DCs (CD11blow CD11chigh) were further ana- lyzed for CD8a, DEC-205, CD4, and MHC class II expres- sion. MyDC, myeloid DCs; LyDC, lymphoid DCs; AFC, autofluorescent cells, mostly macrophages.