Increased accumulation of pMHCII complexes in the absence of MHCII ubiquitination. (a) WT and I-A^b(K>R) HEL-specific Hy10 B cells were transferred, together with OT-II CD4⁺ T cells, into WT hosts. Recipient mice were subsequently immunized with HEL-OVA/Sigma (Ribi) adjuvant by s.c. injection. Mice later received HEL-EaGFP or HEL-BSA conjugates at the same sites at various time points before analysis. (b) Representative plots showing GFP florescence and I-A^b–Ea_52–68 staining on the two populations of IgD^low CD95⁺ GC B cells. (c, left) GFP florescent intensity on each genotype of GC B cell at each time point. (right) I-A^b(Ea_52–68) mean fluorescence intensity (MFI) with mean background staining from HEL-BSA treated mice (4.5-h time point) deducted to give the delta MFI for each condition and time point. The fold differences in delta MFI between I-A^b(K>R) and WT Hy10 cells are indicated above the plots. Each data point represents GC B cells from a single iLN, with two LNs analyzed per mouse. Data in c are from one experiment that is representative of two (48 h) and three (4.5 or 24 h) independent experiments. (d) The fold differences in delta MFI between WT and I-A^b(K>R) Hy10 GC B cells from all mice and experiments are shown. Horizontal lines in c and d indicate means. Analysis was performed using an unpaired two-tailed Student’s t test. *, P < 0.05; ***, P < 0.001.