MHC class I in vitro (for a review, see reference 7). Exog- enous antigens in the form of soluble proteins, particulate antigens, and cell-associated antigens derived from apop- totic or necrotic cells have been shown to be taken up, processed, and presented to CD81 T cells by both cell types (11–17). Other reports have indicated that cross-pre- sentation is a specific property of DCs (18–20). However, which bone marrow–derived APC is involved in MHC class I–restricted presentation of exogenous antigens in vivo has not been examined. DCs comprise populations of cells with heterogeneous phenotypes (21, 22). Murine splenic DCs can be divided into two subsets, both of which express high levels of CD11c. One DC population is further characterized by high expression of the myeloid marker CD11b and by absence of expression of CD8a and DEC-205, whereas the other is CD11blowCD8a1DEC-2051 (23–27). The CD82CD11bhigh DCs can be further divided into a CD41 and CD42 population (28). Some evidence suggests that the CD81 and CD82 DC subsets belong to different lin- eages. Several transcription factor knockout and mutant mice exhibited differential effects on the development of each subset in vivo (29–32). In addition, CD81 DCs were shown to be derived from a thymic progenitor that could not generate myeloid cells, whereas CD82 DCs can be grown from bone marrow using GM-CSF (33–35). There- fore, CD81 DCs are thought to relate to the lymphoid lin- eage, whereas CD82 DCs are considered myeloid related. For convenience, in this paper we will use the terms lym- phoid DC and myeloid DC to describe the CD81 and CD82 DC subsets. In addition to phenotypic differences, the lymphoid and myeloid DCs reside in different areas of the spleen. Whereas lymphoid DCs are localized in the T cell–rich ar- eas of the periarteriolar lymphatic sheaths (PALS), myeloid DCs can be found in the marginal zone (21, 36). Func- tional distinctions between lymphoid and myeloid DCs have also been reported. Initial in vitro studies indicated that lymphoid DCs could suppress T cell responses by caus- ing apoptosis of CD41 T cells and by limiting IL-2 produc- tion by CD81 T cells, whereas myeloid DCs were shown to be strong stimulators of primary T cell responses (37, 38). These findings led to the suggestion that myeloid and lymphoid DCs function as T cell stimulators and tolerizers, respectively. However, in a recent study, peptide-coated DCs of both subsets were shown to induce strong CD81 T cell responses, indicating no inherent tolerizing function for lymphoid DCs (39). Both DC subsets do appear to dif- fer in their ability to activate CD41 cells, as myeloid DCs preferentially stimulate Th2 responses, whereas lymphoid DCs preferentially induce Th1 responses (40–42). The role of both DC subsets in cross-priming of CD81 T cells in vivo has not been investigated. To determine the cell types responsible for cross-priming in vivo, we primed C57BL/6 (B6) mice with irradiated, b2-microglobulin2/2 (b2m2/2) splenocytes loaded with OVA in their cytoplasm. Such b2m2/2 splenocytes are un- able to present the OVA epitope in association with MHC class I molecules, but they elicit, when injected in B6 mice, OVA-specific, MHC class I–restricted CTL responses. We show that host splenic DCs take up and present cell-associ- ated OVA to CD81 T cells. Analysis of the OVA-present- ing DCs indicated that only lymphoid DCs, and not my- eloid DCs, cross-present antigens in vivo. This result points to an important and distinct role for lymphoid DCs in the generation of CD8 T cell responses directed towards cell- associated antigens. Materials and Methods Mice. b2m2/2 and B6 mice were purchased from Taconic Farms. OT-I recombination-activating gene (RAG)22/2 and Thy1.11 OT-I mice were bred in our specific pathogen-free fa- cility and have a transgenic Va2Vb5 TCR specific for the OVA257–264 epitope in the context of H2-Kb (43). Antibodies. CD11c-, CD11b-, CD8a-, CD8b-, Kb/Db-, and I-Ab–specific antibodies were purchased from BD PharMingen. Biotinylated DEC-205–specific antibodies were a gift from A. Rudensky (University of Washington, Seattle, WA). Flow cy- tometry was conducted on a FACSCalibur™ and analyzed using CELLQuest™ software (Becton Dickinson). Cell sorting was performed in HBSS with 25 mM Hepes using a FACS Vantage™ (Becton Dickinson). Cross-priming with OVA-loaded b2m2/2 Cells. Single cell sus- pensions were prepared in serum-free medium from spleen and cervical, axillary, brachial, inguinal, and mesenteric lymph nodes from female b2m2/2 mice. Cells were loaded with OVA by os- motic shock as described previously (44). In short, z15 3 107 cells were incubated in 1 ml of hypertonic medium (0.5 M su- crose, 10% wt/vol polyethylene glycol 1000, and 10 mM Hepes in RPMI 1640, pH 7.2) containing 10 mg/ml OVA (Calbio- chem) for 10 min at 378C. 13 ml of prewarmed hypotonic me- dium (40% H2O, 60% RPMI 1640) was added and the cells were incubated for an additional 2 min at 378C. The cells were centri- fuged, washed twice with cold PBS, and irradiated (1,350 rads). 20–35 3 106 OVA-loaded cells were injected in 200 ml of PBS into the tail vein. In one experiment, b2m2/2 cells were shocked with both OVA and yellow/green fluorescent 0.2 mm beads (Molecular Probes). The beads were washed two times in PBS by centrifugation for 10 min at 1,400 rpm and sonication for 10 min. The beads were resuspended to 0.004% (wt/vol) in hyper- tonic solution containing OVA before shocking. After osmotic shocking of the cells, free beads were removed by three washes in PBS. CFSE Labeling of OT-I Cells. OT-I cells from spleen and lymph nodes were washed twice in PBS containing 0.1% BSA. To label cells with the intracellular fluorescent dye carboxyfluo- rescein diacetate succinimidyl ester (CFSE; Molecular Probes) the cells were resuspended at 5 3 107 cells/ml in PBS/0.1% BSA with 10 mM CFSE for 10 min at 378C. Cells were washed twice with cold RPMI 1640/10% FCS (RP10) followed by two washes in PBS. 106 CD81Va21CFSE1 OT-I cells in 200 ml of PBS were injected into the tail vein. Low Density Cell Preparation. 14 h after injection with irradi- ated OVA-loaded b2m2/2 cells, spleens of 5–35 mice were cut into grain size pieces and incubated in 1 ml per spleen of 1 mg/ ml collagenase/dispase (Sigma-Aldrich) and 50 mg/ml DNase I (Boehringer) in PBS with continuous stirring at 378C for 30 min or until digested. EDTA was added to a 10 mM final concentra- tion, and the cell suspension was incubated for an additional 5 1686 CD81 Dendritic Cells Cross-prime CTLs In Vivo