Summary B cell activation leads to proliferation and antibody production that can protect from pathogens or promote autoimmunity. Regulation of cell metabolism is essential to support the demands of lymphocyte growth and effector function and may regulate tolerance. Here, we tested the regulation and role of glucose uptake and metabolism in the proliferation and antibody production of control, anergic, and autoimmune-prone B cells. Control B cells had a balanced increase in lactate production and oxygen consumption following activation, with proportionally increased glucose transporter Glut1 expression and mitochondrial mass upon either LPS or BCR stimulation. This contrasted with metabolic reprogramming of T cells, which had lower glycolytic flux when resting but disproportionately increased this pathway upon activation. Importantly, tolerance greatly affected B cell metabolic reprogramming. Anergic B cells remained metabolically quiescent, with only a modest increase in glycolysis and oxygen consumption with LPS stimulation. B cells chronically stimulated with elevated B cell Activating Factor (BAFF), however, rapidly increased glycolysis and antibody production upon stimulation. Induction of glycolysis was critical for antibody production, as glycolytic inhibition with the pyruvate dehydrogenase kinase (PDHK) inhibitor dichloroacetate (DCA) sharply suppressed B cell proliferation and antibody secretion in vitro and in vivo. Further, B cell-specific deletion of Glut1 led to reduced B cell numbers and impaired antibody production in vivo. Together, these data show that activated B cells require Glut1-dependent metabolic reprogramming to support proliferation and antibody production that is distinct from T cells and that this glycolytic reprogramming is regulated in tolerance.

SUMMARY Germinal center (GC) B cells evolve towards increased affinity by a Darwinian process that has been studied primarily in genetically restricted, hapten-specific responses. We explored the population dynamics of genetically diverse GC responses to two complex antigens – Bacillus anthracis protective antigen and influenza hemagglutinin – in which B cells competed both intra- and interclonally for distinct epitopes. Preferred VH rearrangements among antigen-binding, naïve B cells were similarly abundant in early GCs but, unlike responses to haptens, clonal diversity increased in GC B cells as early “winners” were replaced by rarer, high-affinity clones. Despite affinity maturation, inter- and intraclonal avidities varied greatly, and half of GC B cells did not bind the immunogen but nonetheless exhibited biased VH use, V(D)J mutation, and clonal expansion comparable to antigen-binding cells. GC reactions to complex antigens permit a range of specificities and affinities, with potential advantages for broad protection.

Highlights d Human B cells specific for a novel epitope on influenza A groups 1 and 2 d Crystallography locates the epitope at the interface of the hemagglutinin head domains d Robust protection by antibodies to this epitope, dependent on IgG subclass d Protective, cross-group antibodies are encoded by diverse sets of Ig gene segments

Human B cell antigen-receptor (BCR) repertoires reflect repeated exposures to evolving influenza viruses; new exposures update the previously generated B cell memory (Bmem) population. Despite structural similarity of hemagglutinins (HAs) from the two groups of influenza A viruses, cross-reacting antibodies (Abs) are uncommon. We analyzed Bmem compartments in three unrelated, adult donors and found frequent cross-group BCRs, both HA-head directed and non-head directed. Members of a clonal lineage from one donor had a BCR structure similar to that of a previously described Ab, encoded by different gene segments. Comparison showed that both Abs contacted the HA receptor-binding site through long heavy-chain third complementarity determining regions. Affinities of the clonal-lineage BCRs for historical influenza-virus HAs from both group 1 and group 2 viruses suggested that serial responses to seasonal influenza exposures had elicited the lineage and driven affinity maturation. We propose that appropriate immunization regimens might elicit a comparably broad response.

Highlights d Hyperglycosylation of hemagglutinin (HA) does not dampen serum and GC responses d Hyperglycosylation of HA changes patterns of immunodominance d Glycan addition restricts antibody repertoire to a subdominant epitope d Antibodies targeting this occluded epitope on HA protect against viral challenge