Volume 25, Issue 6, Pages 885-894 (December 2006) Dendritic Cells Regulate Exposure of MHC Class II at Their Plasma Membrane by Oligoubiquitination  Guillaume van Niel, Richard Wubbolts, Toine ten Broeke, Sonja I. Buschow, Ferry A. Ossendorp, Cornelis J. Melief, Graca Raposo, Bas W. van Balkom, Willem Stoorvogel  Immunity  Volume 25, Issue 6, Pages 885-894 (December 2006) DOI: 10.1016/j.immuni.2006.11.001 Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 1 MHC II-β Is Oligoubiquitinated (A) MHC II was immunoprecipitated from 106 D1 cells with M5/114, Y3P, or an isotype control antibody for M5/114 (1st IP) as indicated. Immunoprecipitates were eluted from the beads and reprecipitated with ubiquitin antibodies (2nd IP). Those precipitates were analyzed by immunoblotting (WB) for ubiquitin or MHC II-β, with either monoclonal M5/114, an isotype control monoclonal antibody, or a polyclonal antibody. The positions of ubiquitinated MHC II-β, MHC-β, and background IgG from the first IP as indicated. The figure is representative for three independent experiments. (B) Ubiquitinated MHC II was immunoprecipitated from 109 D1 cells as described above. A small aliquot (0.1%) was analyzed by immunoblotting for ubiquitin as reference (WB), and the remainder was separated by SDS-PAGE and silver stained. The bands indicated in the middle lane were identified by mass spectrometry (data not shown) and contain both ubiquitin and MHC II-β. The figure is representative for two independent experiments. (C) D1 cells or BMDC from WT or Ab-β KO mice were lysed and MHC II was immunoprecipitated (IP). Immunoprecipitates were eluted at 100°C and immunoblotted (WB) for MHC II-β or ubiquitin as indicated. The figure is representative for three independent experiments. Immunity 2006 25, 885-894DOI: (10.1016/j.immuni.2006.11.001) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 2 MHC II-β Is Ubiquitinated after Processing of Associated Ii (A) MHC II was immunoprecipitated (IP) from D1 cell lysates as indicated by either M5/114, Y3P, or 15G4, eluted from the beads in SB at 20°C or 100°C, and immunoblotted (WB) for MHC II-β, Ii, or ubiquitin. The figure is representative for four independent experiments. (B) Summary of the results in (A). M5/114 precipitated all MHC II complexes, irrespective of Ii processing. Y3P precipitated all MHC II complexes except those containing full-length Ii (two splice variants, p41 and p31). 15G4 recognizes the CLIP domain of Ii and therefore precipitated all MHC II complexes except mature MHC II-peptide and ubiquitinated MHC II. (C) MHC II was immunoprecipitated with Y3P, eluted in SB at 20°C, and separated by SDS-PAGE in a first dimension. The lane was excised, incubated at 100°C in SB, separated in a second dimension by SDS-PAGE, and immunoblotted for ubiquitin. Appropriate immunoblots of in parallel prepared 1D lanes are projected above and to the right of the 2D blot. Ubiquitinated MHC II-β dissociated only after incubation at 100°C. The figure is representative for two independent experiments. Immunity 2006 25, 885-894DOI: (10.1016/j.immuni.2006.11.001) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 3 MHC II-β Ubiquitination Requires Ii Processing and Diminishes during DC Maturation (A) D1 cells were incubated for 3 hr in the absence or presence of MG132, leupeptin, or E64d before lysis. MHC II was immunoprecipitated (IP) with M5/114, eluted at 20°C in SB, and analyzed by immunoblotting for MHC II-β, Ii, or ubiquitin as indicated. (B) D1 cells were incubated for 0, 2, 4, or 24 hr in the presence of LPS before lysis. Samples of total cell lysates (TL) were immunoblotted for ubiquitin (left) or MHC II-β (middle bottom). MHC II was immunoprecipitated (IP) with M5/114, eluted at 20°C or 100°C in SB, and analyzed by immunoblotting for MHC II-β (right) and ubiquitin (middle top), respectively. The figures are representative for three independent experiments. Immunity 2006 25, 885-894DOI: (10.1016/j.immuni.2006.11.001) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 4 Ak-βK225A Is Not Ubiquitinated and Targeted to the Plasma Membrane (A) BMDC from either WT or Ab-β-deficient mice were transduced for either Ak-βWT or Ak-βK225A as indicated. Fixed cells were immunodouble labeled for DM (red) and Ab or Ak (green) as indicated and analyzed by CSLM. The figures are representative for three independent experiments. (B) Nontransduced D1 cells (control) or D1 transduced for either Ak-βWT or Ak-βK225A were lysed. Endogenous MHC II was immunoprecipitated (IP) with M5/114 and Ab-α–Ak-β complexes with 10.2.16. Precipitates were eluted as indicated at 100°C and immunoblotted (Odorizzi et al., 1994) for MHC II-β or ubiquitin. The figure is representative for two independent experiments. (C) D1 cells were transduced for either Ak-βWT (left) or Ak-βK225A (right) and after fixation, immunodouble labeled for Ak (green) and DM (red) (top) or for Ak (green) and endogenous Ab (red) (bottom). The figures are representative for four independent experiments. Immunity 2006 25, 885-894DOI: (10.1016/j.immuni.2006.11.001) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 5 K225 at MHC II-β Is Required for Sorting of MHC II to LV in Immature DCs (A) Ultrathin cryosections of either nontransduced (control) or Ak-β or Ak-βK225A-transduced D1 were double immunogold-labeled for endogenous Ab-β (10 nm gold particles) and Ak-β (15 nm gold particles; indicated by arrows). Examples of MVB are shown with LV and LM indicated. Scale bars represent 200 nm. (B) Gold particles on the LM versus LV of MVB were counted, and the ratios (LM/LV) of the totals from >50 MVB profiles for each condition are plotted. Immunity 2006 25, 885-894DOI: (10.1016/j.immuni.2006.11.001) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 6 K225 at MHC II-β Is Required for Efficient Internalization of MHC II from the Plasma Membrane D1 cells were transduced for either Ak-βWT or Ak-βK225A. (A) Cells were allowed to bind and endocytose PE-conjugated 10-3.6 for transduced Ak-β (green) at indicated temperatures, fixed, and costained for the early endosomal marker EEA1 (red) and DM (blue). The figure is representative for three independent experiments. (B) Cells were allowed to bind and endocytose PE-conjugated 10.3-6 for transduced Ak-β (green) and APC-conjugated M5-114 (red) for endogenous Ab-β for 1 hr at 37°C or 20°C as indicated. Cells were washed and fixed, and images were acquired by CSLM. Areas representative for plasma membrane (asterisk) or intracellular structures (dot) were encircled (see indicated examples), and the integrated densities of fluorescence within these areas was determined for 10 distinct samples for each condition. (C) The relative efficiencies of uptake were then determined by comparing the ratio of fluorescence at the plasma membrane (PE/APC) with that at the internal membranes (SD are indicated in the bar diagrams and are given in the text). Immunity 2006 25, 885-894DOI: (10.1016/j.immuni.2006.11.001) Copyright © 2006 Elsevier Inc. Terms and Conditions