Accumulation of self-reactive naïve and memory B cell reveals sequential defects in B cell tolerance checkpoints in Sjögren's Syndrome

Sjö gren's syndrome (SS) is an autoimmune disease characterised by breach of self-tolerance towards nuclear antigens resulting in high affinity circulating autoantibodies. Although peripheral B cell disturbances have been described in SS, with predominance of naïve and reduction of memory B cells, the stage at which errors in B cell tolerance checkpoints accumulate in SS is unknown. Here we determined the frequency of self- And poly-reactive B cells in the circulating naïve and memory compartment of SS patients. Single CD272IgD+ naïve, CD27+IgD+ memory unswitched and CD27+IgD2 memory switched B cells were sorted by FACS from the peripheral blood of 7 SS patients. To detect the frequency of polyreactive and autoreactive clones, paired Ig V_H and V_L genes were amplified, cloned and expressed as recombinant monoclonal antibodies (rmAbs) displaying identical specificity of the original B cells. IgV_H and V_L gene usage and immunoreactivity of SS rmAbs were compared with those obtained from healthy donors (HD). From a total of 353 V_H and 293 V_L individual sequences, we obtained 114 rmAbs from circulating naïve (n566) and memory (n548) B cells of SS patients. Analysis of the Ig V gene repertoire did not show significant differences in SS vs. HD B cells. In SS patients, circulating naïve B cells (with germline V_H and V_L genes) displayed a significant accumulation of clones autoreactive against Hep-2 cells compared to HD (43.1% vs. 25%). Moreover, we demonstrated a progressive increase in the frequency of circulating anti-nuclear naïve (9.3%), memory unswitched (22.2%) and memory switched (27.3%) B cells in SS patients. Overall, these data provide novel evidence supporting the existence of both early and late defects in B cell tolerance checkpoints in patients with SS resulting in the accumulation of autoreactive naïve and memory B cells.