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. 2016 Oct 31:6:36298.
doi: 10.1038/srep36298.

Reconstituted B cell receptor signaling reveals carbohydrate-dependent mode of activation

Rina F Villar  1 , Jinal Patel  2 , Grant C Weaver  1 , Masaru Kanekiyo  3 , Adam K Wheatley  4 , Hadi M Yassine  5 , Catherine E Costello  6 , Kevin B Chandler  6 , Patrick M McTamney  7 , Gary J Nabel  8 , Adrian B McDermott  3 , John R Mascola  3 , Steven A Carr  2 , Daniel Lingwood  1
Affiliations

Reconstituted B cell receptor signaling reveals carbohydrate-dependent mode of activation

Rina F Villar et al. Sci Rep. .

Abstract

Activation of immune cells (but not B cells) with lectins is widely known. We used the structurally defined interaction between influenza hemagglutinin (HA) and its cell surface receptor sialic acid (SA) to identify a B cell receptor (BCR) activation modality that proceeded through non-cognate interactions with antigen. Using a new approach to reconstitute antigen-receptor interactions in a human reporter B cell line, we found that sequence-defined BCRs from the human germline repertoire could be triggered by both complementarity to influenza HA and a separate mode of signaling that relied on multivalent ligation of BCR sialyl-oligosaccharide. The latter suggested a new mechanism for priming naïve B cell responses and manifested as the induction of SA-dependent pan-activation by peripheral blood B cells. BCR crosslinking in the absence of complementarity is a superantigen effect induced by some microbial products to subvert production of antigen-specific immune responses. B cell superantigen activity through affinity for BCR carbohydrate is discussed.

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Figures

Figure 1
Figure 1. Tyr98-dependent binding of HA to SA provides a structurally defined multivalent affinity for this carbohydrate sequence.
(A) Both WT HA and Y98F HA are the correct size of the HA trimer as defined by size exclusion on a Sepharose 10/300 column and are electrophoretically identical. (B) HA trimer or Y98F HA were also antigenically identical as measured by reactivity to two conformational antibodies: the HA receptor binding site specific antibody CH65 and the HA stem-specific antibody CR6261. Reactivity of the HIV-specific antibody VRC01 is presented as an isotype control. The HA antigen in complex with CH65 and CR6261 is derived from 3SM5 and 3GBM entries in the PDB. Surface plasmon resonance has also shown that for CH65 and CR6261, the association/dissociation rates and affinities for this antigen are unaffected by removal of the oxygen atom at amino acid position 98. (C) The crystal structure displaying the HA loops with residues making hydrogen bond interactions with the SA (displayed by receptor analog LSTc l, PDB 1RVZ). Polar contacts are shown as dashed lines, a water molecule is in red, and Try 98 is in pink (hydroxyl group) and cyan, respectively. When the hydroxyl group is removed through Y98F, SA-binding is lost, as depicted by Tyr98 dependent adherence to 293F cells. (D) Both WT and Y98F HA can be assembled into nanoparticles of ferritin to display 8mers of the HA trimer, that have overlapping elution profiles following size exclusion on a Superose 6 10/300 column.
Figure 2
Figure 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.
Figure 3
Figure 3. 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 HA. (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 K. The BCR was not isolated following surface shaving indicating that the procedure largely captures surface trafficked receptor.
Figure 4
Figure 4
Reconstituted interactions between germline BCR and SA-binding HA reveal that the same BCR receptor can signal through both cognate and non-cognate/carbohydrate dependent interactions with antigen. Signaling as measured by Ca2+ flux (A) and tyrosine phosphorylation of p75 (B) in response to ferritin nanoparticle arrayed HA trimer or Y98F HA trimer was assessed through Ramos B cells expressing: VH1-69 germline BCR specific for the HA stem; I53A, F54A VH1-69 germline BCR which prevents binding to the HA stem; Ramos B cell negative for surface BCR expression; or VRC01 BCR specific for the HIV envelope. Calcium flux was measured using the ratiometric calcium dye Fura Red, wherein flux in response to stimulation and is presented as the average of two independent runs, and for tyrosine phosphorylation, mean +/− sem of p75 signal (presented as a fraction of total signal seen with crosslinking with anti-IgM; uncropped immunoblots are provided in Supplementary Fig. 2) is presented for three independent experiments (B) (****P < 0.0001, ***P < 0.001, *P < 0.01). In all cases empty ferritin nanoparticle was used as an additional control.
Figure 5
Figure 5. 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.
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