In order to assess how entry of surface-derived Kb molecules into endocytic compartments coincides with exogenous antigen loading, Kb surface-labeled DCs were pulsed with soluble OVA protein for 6 hours to allow simultaneous Kb-FITC internalization and OVA uptake and antigen processing.
Overnight incubation of B6.Tap−/− splenocytes with 0.2 10 µM Mulv peptide induced Kb cell surface expression of 10 to 80% levels as compared to Kb+/+ cells.
Notably, overlapping of EEA-1 and Kb-OVA complexes without surface Kb (indicated by pink color) was significantly higher (p = 0.002) in Δ7-derived DCs (Figure 5D).
Co-staining with the Golgi marker Giantin showed very little colocalization of surface Kb or Kb-OVA complexes with Golgi in DCs from KbWT and Δ7 mice.
Upon incubation at 37°C for 6 hrs, both surface Kb and Kb-OVA complexes showed abundant colocalization (white) with EEA1-positive intracellular compartments in KbWT-derived DCs (Figure 5A).
Moreover, the influence on the capacitance and internal resistance when KB containing a surface functional group is used as the conductive nanofiller of the polarized electrode was examined.
The viral infection slightly increased the surface Kb expression in both CMT.64 and CMT.1,2/Kb cells (Fig. 3 C right panel).
As little as 10 nM bruceantin inhibited peptide supply as judged by expression of surface Kb MHC I molecules (data not shown).
DCs from Δ7 mice showed an intermediate phenotype, with ∼20% of surface Kb molecules being internalized at 30 minutes of chase time (Figure 2A and B).
Surface Kb molecules that lack exon 7, by contrast, appear to traffic readily into early endosomal compartments, but are significantly delayed in their ability to traffic into late endosomes or lysosomes.
