Fig 2: B cell IgM is heavily sialylated and binds to HA.(A) Secreted B cell IgM was immunoisolated (silver stain, upper left) and the mu chain gel band was excised for MS analysis. The IgM LC MS/MS and analysis first confirmed the IgM protein sequence (lower left) and MALDI-TOF-MS was used to analysis the glycans cleaved using PNGase. The spectra for this carbohydrate analysis is presented along with a summary table of the glycan structures. Abbreviations: Hex = Hexose; HexNAc = N-acetylhexosamine; dHex= deoxyhexose; NeuNAc = N-acetylneuraminic acid (=sialic acid; bolded in the summary table). (B) SA-dependent binding to secreted IgM was confirmed by the interaction between non-cognate IgM and WT HA but not Y98F HA. This binding was assessed by ELISA and is presented is the mean and sem of three independent binding replicates.

Fig 3: Recombinant influenza HA activates primary B cells in the absence of pre-existing antigen specificity.Freshly purified PBMCs were purified and incubated with B cell stimulators, nanomolar levels of HA or its sialic acid binding mutant Y98F HA and lymphocyte activation was evaluated by surface expression of CD69, CD86 and CD80 on naïve B cells or T cells. (A) Flow cytometry scheme: naïve B cells were defined as CD19+/CD3−/CD14−/IgD+/IgM+/IgG−/CD27−; and T cells in same preparation were defined as CD3+/CD14−/CD19−. (B) Histograms for B cell and T cell surface levels of CD69, CD86 and CD80 following incubation with known B cell stimulators (Ig or CpGC). (C) Histograms for B cell and T cell surface levels of CD69, CD86 and CD80 following incubation with 50 nM HA, 300 nM HA or 300 nM Y98F HA. Presented are the results of stimulating PBMC from three different individuals.

Fig 4: HA co-precipitates with BCR in a Tyr98 dependent manner.(A) B cells were exposed to WT or Y98F HA and the BCR was immunoisolated using an antibody specific for the IgM mu chain. The silver stained SDS PAGE of HA inputs and subsequent BCR immunoisolation is presented. To assay for the presence of HA, the immunoisolate was blotted for reactivity to C179, an antibody specific for the conserved stem region of HA56. (B) To confirm that the immunoisolation procedure was capturing cell surface bound BCR and not immature BCR within the cell, we also performed the procedure after surface B cell shaving with proteinase K57. The BCR was not isolated following surface shaving indicating that the procedure largely captures surface trafficked receptor.