Fig. 4.

Different populations of B cells express distinct levels of glycosyltransferases. (a) Sialylation and (b) galactosylation levels of all afucosylated (grey) and all fucosylated (black) anti-S IgG for naive (left, blue shading) and antigen-experienced (right, yellow shading) vaccinated participants over time of cohort 1 (n = 39) and 2 (n = 9) pooled. All afucosylated glycoforms were set to 100%, and all fucosylated glycoforms were set individually to 100% (c–n) Flow cytometry analysis of blood cells gated on single, living lymphocytes from naive and antigen-experienced vaccinees (subset of cohort 3 (n = 15), see Table S2) were analysed 7–14 days upon the first (naive: n = 6 and antigen-experienced: n = 5) or 5–8 days upon the second (naive: n = 15 and antigen-experienced: n = 4) dose (c–e) Gating strategy exemplified for a naive individual (c) pre-immunization and (d) after the first dose. S1-reactive B cells were gated and further gated for CD19int CD38⁺ PCs to analyse IgG⁺ PC subsets as defined by (e) CD27 and CD138 (f–g) Naive and (j–k) antigen-experienced vaccinees analysed according to the gating strategy (h, l) Relative fucosyltransferase 8 (FUT8; median fluorescence intensity (MFI)) expression per IgG⁺ PC subset compared by Mann–Whitney U test and (i, m) its correlation with relative α2,6-sialyltransferase (ST6GAL1) expression (n) Spearman correlation of relative FUT8 expression of CD27^low CD138^- IgG⁺ PCs correlated with anti-S IgG1 Fc fucosylation found in the corresponding serum and statistics of naive vaccinees (Fig. S9c). The median (MFI) of FUT8 or ST6GAL1 expression in CD138⁺ IgG⁺ S1-reactive PCs of each sample was set to 1 for inter-assay comparison. Dotted horizontal lines indicate corresponding values of total IgG⁺ PC subsets from untreated healthy controls (Fig. S11). ∗, ∗∗∗: p-value <0.05, 0.001, respectively. Each analysis in c–n was carried out within only cohort 3.