Research Article

Uptake of HLA Alloantigens via CD89 and CD206 Does Not Enhance Antigen Presentation by Indirect Allorecognition

Figure 2

CD89 antibody and HLA-A2 complexes are efficiently bound and taken up by monocytes. (a) Diagram representing the targeting Ab. Positions of conjugated avidin groups depicted in the diagram are arbitrarily chosen. (b) Monocytes were stained with 1 μg/mL avidin conjugated (CD89-A (grey)) or nonconjugated (CD89 (white with black outline)) Ab. Staining was visualized with a secondary APC-labeled Ab. (c) Formation of the CD89-A HLA-A2 immune-complex (CD89-A2) was confirmed by an ELISA conjugated or nonconjugated (CD89-A/CD89) Ab which were coated on a 96-well plate. (d) Ab cross-reactivity was tested by removing HLA-A2 from the system and incubating the antibodies as was done in (c). Grey bars indicate CD89, and white bars are the avidinylated CD89 Ab (CD89-A). ((e) and (f)) To test whether avidinylation had an effect on receptor mediated endocytosis (RME) monocytes were incubated with either conjugated CD89 (CD89-A) or nonconjugated CD89 at 4°C (grey histograms) or at 37°C (white with dark outline histograms). After 2 h incubation CD89 on the cell surface was visualized with a secondary Ab. (g) Uptake was confirmed by formation of a complex with biotinylated-CD89 and HLA-A2 monomer incubated with APC-labeled streptavidine (CD89-A2-strep) at a ratio of 2 : 2 : 1, respectively. The complex was incubated for 24 h at 4°C (light grey) or 37°C (dark grey) in the presence of monocytes. Shown is one representative experiment. (h) To establish CD89 mediated uptake monocytes were cultured with CD89-A2-strep, in the presence of increasing amounts of competing CD89 Ab or MR. After overnight incubation, the APC fluorescence intensity was assessed by flow cytometry as a measure for uptake. Shown is one representative experiment.