CD8(+) but not CD8(-) dendritic cells cross-prime cytotoxic T cells in vivo

Abstract

Bone marrow-derived antigen-presenting cells (APCs) take up cell-associated antigens and present them in the context of major histocompatibility complex (MHC) class I molecules to CD8(+) T cells in a process referred to as cross-priming. Cross-priming is essential for the induction of CD8(+) T cell responses directed towards antigens not expressed in professional APCs. Although in vitro experiments have shown that dendritic cells (DCs) and macrophages are capable of presenting exogenous antigens in association with MHC class I, the cross-presenting cell in vivo has not been identified. We have isolated splenic DCs after in vivo priming with ovalbumin-loaded beta2-microglobulin-deficient splenocytes and show that they indeed present cell-associated antigens in the context of MHC class I molecules. This process is transporter associated with antigen presentation (TAP) dependent, suggesting an endosome to cytosol transport. To determine whether a specific subset of splenic DCs is involved in this cross-presentation, we negatively and positively selected for CD8(-) and CD8(+) DCs. Only the CD8(+), and not the CD8(-), DC subset demonstrates cross-priming ability. FACS((R)) studies after injection of splenocytes loaded with fluorescent beads showed that 1 and 0.6% of the CD8(+) and the CD8(-) DC subsets, respectively, had one or more associated beads. These results indicate that CD8(+) DCs play an important role in the generation of cytotoxic T lymphocyte responses specific for cell-associated antigens.

Figure 1

CD8⁺ T cell proliferation in vivo after priming with OVA-loaded β2m^−/− (β2m^0/0) cells. Thy1.1⁺ CFSE-labeled OT-I cells were transferred into B6 mice. 3 d later, mice were primed by injection with irradiated OVA-loaded β2m^−/− cells. Spleen cells were isolated 3 d after priming and stained for Thy1.1, Vα2, and CD8. (A) CFSE profile of Thy1.1⁺Vα2⁺CD8⁺ OT-I cells from mice primed with OVA-loaded β2m^−/− cells. (B) The same profile from a mouse that received no OVA-loaded β2m^−/− cells.

Figure 2

Low density spleen cells cross-present OVA in a TAP-dependent fashion. B6 (A) and TAP^−/− (TAP^0/0) mice (B) were primed with OVA- or BSA-loaded β2m^−/− 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 OVA_257–264 peptide in vitro is shown in the insets.

Figure 3

Phenotypic analysis of low density cell preparation. Low density spleen cell preparations were isolated from B6 mice 14 h after priming with OVA-loaded β2m^−/− cells. The cells were stained for CD11c, CD11b, and the indicated third reagent, and analyzed by FACS^®. Gated myeloid DCs (CD11b^highCD11c^high) and lymphoid DCs (CD11b^low CD11c^high) were further analyzed for CD8α, DEC-205, CD4, and MHC class II expression. MyDC, myeloid DCs; LyDC, lymphoid DCs; AFC, autofluorescent cells, mostly macrophages.

Figure 4

OVA cross-presentation by low density cells depleted of different cell subsets. Low density spleen cells were isolated from B6 mice 14 h after priming with BSA/β2m^−/− or OVA/β2m^−/− cells. The low density cells from OVA/β2m^−/−-injected mice were depleted of CD11c⁺, CD11b^high, CD8α^high, and CD8β⁺ cells by magnetic beads and the depleted populations were tested (A) directly for their presenting activity in an in vitro OT-I proliferation assay (2 × 10⁵ stimulator cells/well) or (B) after pulsing with OVA peptide in vitro (2 × 10⁴ cells/well). Error bars indicate SEM of triplicate wells.

Figure 6

MHC class I–restricted cross-presentation is restricted to lymphoid DCs as early as 2 h after priming. Low density cells were isolated from mice 2 or 14 h after priming with OVA-loaded β2m^−/− cells. Myeloid and lymphoid DCs were FACS^® sorted based on their differential CD11b/CD11c expression and tested for their ability to stimulate naive OT-I cells in vitro. The different bars represent titered numbers of DCs in the well. Error bars indicate SEM of triplicate wells. MyDC, myeloid DCs; LyDC, lymphoid DCs; n.t., not tested.

Figure 5

Lymphoid DCs, but not myeloid DCs, cross-present OVA antigen. Low density cells were isolated from mice primed previously with OVA- or BSA-loaded β2m^−/− cells. Myeloid and lymphoid DCs from OVA/β2m^−/−-injected mice were (A) FACS^® sorted based on their differential CD11b/CD11c expression and (B) tested for their ability to stimulate naive OT-I cells in vitro. The different bars represent titered numbers of DCs in the well. The proliferation of OT-I induced by peptide-pulsed cells is depicted in the inset. One of three experiments with similar results is shown. Error bars indicate SEM of triplicate wells. MyDC, myeloid DCs; LyDC, lymphoid DCs; AFC, autofluorescent cells, mostly macrophages; n.t., not tested.

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 β2m^−/− cells. (A) The cells were stained for CD11c and CD11b and analyzed by FACS^®. MyDC, myeloid DCs; LyDC, lymphoid DCs; AFC, autofluorescent cells, mostly macrophages. (B) CD11c and CD11b profile of the gated low density cells containing beads.