min at room temperature. RP10/10 mM EDTA/20 mM Hepes (RP10/HE) was added and the cells were pelleted. Red blood cells were lysed with ACK lysis buffer. Cells were washed once with RP10 and the cells were resuspended in 10 ml/5 spleens of RP10/HE. Undigested material was removed by filtration through a wire mesh screen and the cell suspension was loaded on 14.5% Accudenz (Accurate Chemical & Scientific Corpora- tion) gradients in RP10/HE and centrifuged at 530 g for 20 min at room temperature (25). The low density fraction was recov- ered and washed once in RP10/HE and resuspended in RP10. Magnetic Bead Depletion. 5 3 106 low density cells were incu- bated with 300 ml 2.4G2 supernatant (anti-Fc receptor) for 15 min on ice. Cells were then incubated with 200–250 ml of bio- tinylated antibody specific for CD11c, CD11b, CD8a, or CD8b in PBS/0.1% BSA for 30 min at 48C under slow rotation. Deple- tion of antibody-bound cells with streptavidin-coated magnetic beads (Dynal) was performed in PBS/0.1% BSA according to the manufacturer’s instructions. Proliferation Assay. To detect OVA cross-priming, different DC preparations from injected mice were used as stimulators for naive OT-I cells in a [3H]thymidine incorporation assay. Indi- cated numbers of irradiated (2,250 rads) DCs were incubated with 105 OT-I RAG22/2 cells in flat-bottomed plates in 200 ml RP10. As a positive control, stimulator cells were coated with 1 mM OVA257–264 peptide for 1 h and washed three times. After 48 h, the plates were pulsed for 16 h with 1 mCi/well of [3H]thy- midine and harvested. Results Cross-presentation to CD81 T Cells In Vivo. To study the MHC class I–restricted cross-presentation of cell-asso- ciated antigens, we injected B6 mice intravenously with b2m2/2 cells that had been osmotically loaded with OVA. As expected, the injected cells are efficiently taken up and processed for MHC class I presentation by host APCs. This is demonstrated by the proliferative response of adoptively transferred, CFSE-labeled H2-Kb–restricted, OVA-spe- cific TCR transgenic OT-I cells that serve as an indicator for efficient presentation in vivo. 3 d after priming, the transferred OT-I cells had divided up to seven times, as re- vealed by the sequential loss of CFSE intensity (Fig. 1 A). In contrast, OT-I cells did not proliferate in mice that did not receive OVA-loaded cells (Fig. 1 B). Low Density Cells Cross-present Cell-associated Antigens in a TAP-dependent Manner. Because we immunized mice via the intravenous route, we focussed our attention on the Figure 1. CD81 T cell proliferation in vivo after priming with OVA- loaded b2m2/2 (b2m0/0) cells. Thy1.11 CFSE-labeled OT-I cells were transferred into B6 mice. 3 d later, mice were primed by injection with irradiated OVA-loaded b2m2/2 cells. Spleen cells were isolated 3 d after priming and stained for Thy1.1, Va2, and CD8. (A) CFSE profile of Thy1.11Va21CD81 OT-I cells from mice primed with OVA-loaded b2m2/2 cells. (B) The same profile from a mouse that received no OVA- loaded b2m2/2 cells. Figure 2. Low density spleen cells cross-present OVA in a TAP- dependent fashion. B6 (A) and TAP2/2 (TAP0/0) mice (B) were primed with OVA- or BSA-loaded b2m2/2 cells. Low density spleen cells were isolated 14 h after injection and analyzed for their ability to stimulate OT-I cells in vitro. As indicated, the different bars represent titered numbers of low density cells added per well. Error bars indicate SEM of triplicate wells. The stimulatory capacity of low density cells after pulsing with OVA257–264 peptide in vitro is shown in the insets. 1687 den Haan et al.