Thus, the cross-presentation pathways for particular and soluble receptor-targeted Ags use a similar though not entirely overlapping combination of endosomal proteases and the proteasome

Thus, the cross-presentation pathways for particular and soluble receptor-targeted Ags use a similar though not entirely overlapping combination of endosomal proteases and the proteasome. Open in a separate window Figure 5 An alternative model of cross-presentation pathways. both Ags. The kinetics of the effect of low-temperature preincubation on cross-presentation paralleled Homoharringtonine that on Kb expression; first small effects were notable after 2C4 h, while 16 h was required for full normalization (Fig 2C). Conversely, when DCs preincubated overnight at low temperature were shifted to 37C, incubation of 2 h was sufficient to reverse the effect of low-temperature incubation on cross-presentation capacity and on class I expression (Fig 2D). Open in a separate window Figure 2 Effect of temperature on direct and cross-presentation of phagocytosed antigens by TAP-deficient DCs. All results of antigen presentation experiments correspond to means of duplicate or triplicate wells. In (A), BM-DCs were infected with vaccinia viruses encoding epitope S8 L preceded by a signal peptide (left-hand panel), or full-length OVA. (BCD) In all cross-presentation experiments shown, wt and TAP ko BM-DC preincubated overnight at 37C or 26C were pulsed for 8 h with antigen, fixed, and added for 24 h to naive OT-I T cells. (B) The left-hand and center panels show representative experiments using OVA-coated beads or yeast cells expressing OVA at the surface, respectively. The right-hand panel shows the percentage stimulation relative to wt BM-DCs preincubated at 37C, set at 100, in 10 experiments (mean+s.e.m.). In (C), TAP ko BM-DCs cultured at 37C were Homoharringtonine shifted for different periods to 26C, followed by analysis of H-2 Kb expression (left-hand panel) and of cross-presentation capacity (right-hand panel). Wt cells were tested as reference. The panels in (D) show the inverse experiments, in which TAP ko BM-DCs were first incubated for 16 h at 26C before shifting for different periods to 37C, followed by analysis of Kb expression and cross-presentation. BM, bone marrow; DC, dendritic cell; IL, interleukin; ko, knockout; OD, optical density; OVA, ovalbumin; wt, wild Homoharringtonine type. Role of recycling MHC class I in cross-presentation Constitutive recycling of class I molecules through endocytic compartments has been described in a variety of cell types including DCs (Gromme et al, 1999; Basha et al, 2008). Recycling class I molecules have been reported to be required for cross-presentation of soluble OVA and for cross-presentation of bacterial Ags through the vacuolar pathway (Chefalo et al, 2003; Basha et al, 2008). To find out whether cell surface class I molecules were implicated in restored cross-presentation by TAP ko DCs preincubated at 26C, we treated DCs with acid and trypsin before adding yeast cells (Fig 3A). Removal of surface class I molecules compromised cross-presentation by TAP ko DCs, suggesting that cell surface class I molecules are recruited to present exogenous Ags in these cells. The same treatment had a much Homoharringtonine smaller but reproducible effect on wt DCs. Open in a separate window Figure 3 Distinct pathways for cross-presentation of phagocytosed PDGFRA and soluble receptor-targeted antigen. In (A), cells were treated with acid and/or trypsin before addition of OVA-expressing yeast cells. (B) BM-DCs were pretreated with acid followed by trypsinization or not before pulsing with OVA-expressing yeast cells in the presence of primaquine at indicated concentrations. (C) Phagosomes were prepared from wt or TAP ko BM-DCs preincubated at 37C or 26C, respectively, that had phagocytosed polystyrene beads in the presence or absence of 0.04 mM primaquine. Phagosomes were fixed, stained with a cocktail of antibody recognizing Kb and Db, and analysed by flow cytometry. (D) BM-DCs preincubated overnight at 37C or 26C were pulsed with graded amounts of CD11c-targeted OVA fusion protein. In (E), BM-DCs were pulsed Homoharringtonine similarly in the presence or absence of 0.04 mM primaquine with CD11c-targeted OVA fusion.