Research Article

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

Figure 4

CD206-HLA-A2 complexes are efficiently bound and taken up by moDCs through CD206 receptor mediated endocytosis. (a) Diagrammatic representation of the targeting Ab. Avidin groups conjugated and their position depicted in the diagram are arbitrarily chosen. (b) moDCs were stained with the conjugated (CD206-A) or nonconjugated (CD206) Ab at different concentrations. Indicated is the mean florescence intensity (MFI). (c) Formation of the biotinylated HLA-A2/CD206-A immune-complex was investigated by setting up a sandwich ELISA system as explained in Materials and Methods. Briefly, the CD206/CD206-A was coated onto a 96-well plate, biotinylated HLA-A2 was added at different concentrations, and subsequently an anti-HLA-A2 antibody and a peroxidase targeting the secondary antibody were added. ABTS was used as substrate. (d) Cross-reactivity of the antibodies was ruled out by coating CD206-A (grey bars) or CD206 (dark bars) in the presence or absence of HLA-A2 and incubation of the Ab as depicted in C. CD206-A (grey bars) or CD206 (dark bars) were coated with or without HLA-A2. (e) To demonstrate that CD206-targeted complexes were taken up via CD206, we created complexes of APC-labeled streptavidine, biotinylated HLA-A2 monomer, and CD206 (CD206-A2-strep) at a molecular ratio of, respectively, 1 : 2 : 2. Uptake of CD206-A2-strep was determined by culturing moDC with the complex for 24 h at 4°C (light grey) or 37°C (dark grey). (f) To determine CD206-specific uptake, moDCs were cultured with the CD206-A2-strep complex, with competing MR/CD206 or irrelevant CD89 Ab. After overnight incubation, the APC fluorescence intensity was assessed by flow cytometry. Shown is one representative experiment.