Figure 3
From: Synergy between B cell receptor/antigen uptake and MHCII peptide editing relies on HLA-DO tuning
Low DMfree/CLIPfreq and high CLIPLAMP1/CLIPtot in memory B cells. (a) Naïve, memory and GC sub-populations sorted from human tonsil B cells and tested for purity. (b) Fixed/permeabilized tonsillar B cell subsets co-stained for DO, DM, DR, and CLIP, and analyzed by flow cytometry (n = 5). Mean FIs was normalized to that for GC B cells. (c) A comparison of DMfree/CLIPfreq determined by iFACS of individual B cells from different sub-populations. Data at each condition include a 96-well plate of single cells from the same donor. Experiments were repeated with equivalent results using cells from three donors. (d) 3D-SIM analysis (n = 3) of fixed/permeabilized B cells co-stained for LAMP1, DM, and DO. (e) Comparisons of %DO or %DM co-localized with the indicated endosomal marker in different subtypes of B cells. #Some GC B cells had almost no DO, although any detectable DO was co-localized with LAMP1 (see also Supplementary Fig. 4). Ncell (from left to right): 7, 13, 13, 21, 19, 15, 20, 14, 16. (f) %DO co-localized with DM in different subtypes of B cells. Ncell: 25, 21. (g) %DM co-localized with DO in different subtypes of B cells. Ncell: 52, 51, 69. (h) 3D-SIM analysis (n = 3) of fixed/permeabilized naïve or memory B cells co-stained for LAMP1 and CLIP. Right panel: an overlay view of the CLIP channel (red) and the calculated LAMP1/CLIP overlapping surface (yellow). (i) Comparisons of %CLIP or %DR co-localized with the indicated endosomal marker in different subtypes of B cells (see also Supplementary Fig. 5). Ncell: 24, 21, 26, 19, 18, 16, 52, 60, 40. (j) An inverse correlation between CLIPLAMP1/CLIPtot and DMfree/CLIPfreq among tonsil B cell sub-populations. Data are represented as mean ± SEM. ns: non-significant, p > 0.05; *p < 0.05, **p < 0.01; ***p < 0.001; ****p < 0.0001.
