Abstract
Sjögren’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 CD27−IgD+ naïve, CD27+IgD+ memory unswitched and CD27+IgD− 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 VH and VL genes were amplified, cloned and expressed as recombinant monoclonal antibodies (rmAbs) displaying identical specificity of the original B cells. IgVH and VL gene usage and immunoreactivity of SS rmAbs were compared with those obtained from healthy donors (HD). From a total of 353 VH and 293 VL individual sequences, we obtained 114 rmAbs from circulating naïve (n = 66) and memory (n = 48) 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 VH and VL 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.
Highlights
Sjogren’s syndrome (SS) is a chronic inflammatory/autoimmune disease characterised by immune cell infiltration in the salivary and lacrimal glands leading to the classical signs and symptoms of xerostomia and keratoconjuctivitis sicca [1]
Control Peripheral blood mononuclear cells (PBMC) samples were obtained from 7 healthy donors (HD) including fresh naıve B cells obtained at QMUL (SG and HJ) as well as previously characterised Ig variable heavy (VH) and VL plasmids and/or Ig VH and VL sequences and autoreactive profiles obtained from IgD+ naıve [19], IgM+ memory B cells [20] and IgG+ memory B cells [21] kindly provided by Prof Hedda Wardemann at Max Planck Institute for Infection Biology (Berlin, Germany)
This analysis confirmed that in our cohort of SS patients the frequency of circulating CD272 naıve B cells was increased compared to HD, whereas the frequency of CD27+ switched (6.0¡5.2% versus 17.5¡8.1%) and unswitched (4.9¡6.6% versus 20.6¡2.4%) memory B cells was significantly reduced (Fig. 1B, including p values)
Summary
Sjogren’s syndrome (SS) is a chronic inflammatory/autoimmune disease characterised by immune cell infiltration in the salivary and lacrimal glands leading to the classical signs and symptoms of xerostomia (dry mouth) and keratoconjuctivitis (dry eyes) sicca [1]. The hallmark of SS is the presence of circulating autoantibodies directed against organ- and non-organ-specific autoantigens. Several other autoantibody specificities, including those against alpha-fodrin, carbonic anhydrase II and the muscarinic acetylcholine receptor 3 (M3R) have been described in SS patients and suggested to be involved in salivary dysfunction, especially the latter [1, 3,4,5,6]. Besides the presence of autoantibodies, SS patients are characterised by profound disturbances in the frequency of different B cell subpopulations, both in the peripheral compartment and in the inflamed salivary glands. SS patients show a large predominance of circulating CD272 naıve B cells and a significant reduction of peripheral CD27+ memory B cells, in particular the memory unswitched CD27+IgD+ subpopulation [7]. A significant accumulation of both CD27+ memory and (to a lesser extent) CD272 naıve B cells have been described in the SS salivary glands [7,8,9], possibly as a result of increased migration/retention in the inflamed tissue, in the context of ectopic lymphoid structures which develop in ,30% of SS salivary glands [10,11,12]
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