Even though there are mechanisms to eliminate developing B cells that recognize self-antigens in the bone marrow, a proportion of these cells are thought to escape elimination and enter the periphery. With their potential to contribute to autoimmune reactions, these self-reactive B cells must be kept in an anergic or tolerant state in the periphery. So, the authors of a recent report in Nature Immunology asked what might be required from the B-cell receptor (BCR) to maintain B-cell anergy. Using a mouse model of B-cell anergy, they showed that continuous BCR occupancy is required to ensure that self-reactive B cells remain unresponsive to encounter with additional antigens.

It had previously been suggested that, because some tissue-specific self-antigens might be encountered only rarely by patrolling B cells, a single, transient encounter with cognate self-antigen would be sufficient to induce the anergic state that is then 'memorized' for the lifetime of the B cell. But this hypothesis needed direct assessment. To this end, John Cambier and colleagues made use of immunoglobulin-transgenic mice in which B cells are specific for the hapten arsonate but crossreact with a self-antigen that induces anergy (probably single-stranded DNA). However, when these arsonate-specific B cells are cultured in the presence of high concentrations of a monovalent form of arsonate (arsonate-tyrosine, denoted ArsTyr), autoantigen is competitively dissociated, and anergy is lost.

To explore the kinetics of this reversal of anergy, the authors looked at intracellular concentrations of free calcium in the anergic B cells treated with or without ArsTyr. Anergic B cells are known to have higher basal concentrations of intracellular calcium than naive B cells, possibly because of either continuous BCR signalling or an altered physiological state that is triggered by a single exposure to self-antigen. Consistent with the former explanation, treatment of arsonate-specific B cells with ArsTyr led to a rapid reduction (within 2–4 minutes) in intracellular calcium concentrations. Washing the cells free of ArsTyr and then culturing them in the absence of ArsTyr rapidly restored intracellular calcium to the original high concentration. These findings indicate that at least some features of anergy are maintained by continued biochemical signals rather than by genetic reprogramming.

Treatment with ArsTyr also reversed other features of anergic B cells, including increased basal phosphorylation of extracellular-signal-regulated kinase (ERK) and increased expression of activation markers, such as CD80 and CD95. Importantly, the shortened lifespan that is characteristic of anergic B cells was markedly extended by treatment with ArsTyr, and the inability of these cells to upregulate expression of the activation marker CD86 after stimulation with IgM-specific antibody was restored by treatment with ArsTyr.

Using pharmacological inhibitors of protein kinases that are known to be involved in BCR signalling, the authors confirmed that maintenance of the anergic B-cell phenotype depends on signals from the BCR.

One implication of these observations is that transient loss of self-antigen-mediated BCR signalling might lead to a resetting of a threshold trigger such that activation now might contribute to autoimmunity. Indeed, pretreatment of arsonate-specific B cells with ArsTyr for as little as 2–3 minutes allowed these previously unresponsive cells to respond to stimulation with IgM-specific antibody, by mobilizing calcium.