Background Several studies in mice have suggested that the B cell receptor (BCR) stimulation in addition to CD40 was required for regulatory B cell (Breg) development. Fault of model in humans, the effect of an antigen-specific stimulation on the regulatory function of B cells has never been studied.

Objectives We took advantage of the influenza vaccination campaign to study the regulatory function of B cells in response to hemagglutinin (HA), the antigen contained in the vaccine.

Methods B and T cells were isolated from blood of 19 healthy donors before (W0), 3 weeks (W3), 3 months and 6 months after vaccination. To evaluate the functional regulatory properties of peripheral blood B cells in the presence or absence of rHA (antigen-specific stimulation) or a (Fab')2 anti-IgM Ab used to stimulate BCR (polyclonal stimulation), a co-culture assay was performed to assess the ability of pre-activated B cells with multimeric forms of CD40L to inhibit polyclonal anti-CD3 and anti-CD28 Ab-induced proliferation of autologous T cells. Antigen specific B cells (B HA+) have been phenotyped by FACS.

Results Influenza vaccination induces HA specific B cells (B HA+) (2.52±0.1% at W3 versus 0.95±0.1% at W0, p<0.001). Before vaccination, when B cells were added to the co-culture assay, the proliferative response of T cells was inhibited by 19.2±2.5%, confirming their regulatory function. The addition of rHA has no additive effect whilst BCR engagement enhanced the suppressive effect of B cells on T cell proliferation (21.1±2.6% vs 35.3±3.8%, p<0.005). Interestingly, when the same experiment was performed 3 weeks after influenza vaccination, the presence of rHA in the co-culture assay increased the regulatory function of B cells (32.7±3.5% of T cell proliferation inhibition with rHA versus 22.6±2.6% without rHA, p<0.001) without achieving the inhibition of T cell proliferation observed after BCR engagement (37.3±4.3%). The regulatory functions of B cells pass through the induction of regulatory T cells and the secretion of TGFβ in an IL-10-independent manner. We identified by FACS analysis after vaccination, a B cell subset specific for HA (CD19+ HA+ IgD+ CD38+ CD27- CD24- CD22+) that was positively correlated with the increase in the inhibition of T cell proliferation in response to HA.

Conclusions After vaccination, an antigen-specific B cell subset prone to be induced in Bregs is generated in addition to antigen-specific effector B cells.

Disclosure of Interest None declared