Anergy and not Clonal Ignorance Determines the Fate of B Cells that Recognize a Physiological Autoantigen

Abstract

Autoantibodies to insulin arise spontaneously in the insulin autoimmune syndrome and in type I diabetes. In addition, administration of insulin to individuals without autoimmune disease routinely results in Abs that bind autologous hormone. These observations and findings in transgenic models of tolerance led to an inference that physiological levels of hormones and growth factors, such as insulin, are not sufficient to induce tolerance in B cells, a state termed clonal ignorance. In contrast, we have discovered that virtually all conventional B cells expressing a low affinity anti-insulin transgene interact with endogenous insulin and are effectively silenced for Ig production and for T cell-dependent immune responses. A fraction of transgenic B cells escapes silencing and functions autonomously to produce insulin Abs that may lower fasting blood sugars similar to an insulin autoimmune syndrome. These B cells have characteristics of a B1-like subset and are depleted by hypotonic peritoneal lysis. These findings question the concept of clonal ignorance and show that physiological concentrations of Ag may effectively silence conventional B cells even when the affinity for autoantigen is low. Self-reactivity may arise in the repertoire because of compartmental differences that govern the fate of B cells and not as a result of true clonal ignorance.

Enhanced responsiveness to T‐cell help causes loss of B‐lymphocyte tolerance to a β‐cell neo‐self‐antigen in type 1 diabetes prone NOD mice

“…2), as had been demonstrated previously when the expression of HEL was confined to another tissue specific site, the thyroid [23]. Ignorance in our model could not be explained simply in terms of Ag concentration since soluble cleaved HEL was present in the circulation at similar levels to insulin, which normally confers tolerance [24]. Moreover, insufficient affinity for self-Ag was an equally unlikely explanation given that the BCR on IgHEL-Tg B lymphocytes has an exceptionally high affinity for HEL (2 Â 10 10 M À1 , [25]).…”

Impaired Intracellular Calcium Mobilization and NFATc1 Availability in Tolerant Anti-Insulin B Cells

“…B cells in 125Tg mice show no signs of allelic inclusion (25) and maintain high levels of allelic exclusion for long periods of time (Ͼ30 wk); Ն 95% of B220 ϩ lymphocytes express only IgM a as revealed by staining with anti-IgM a vs anti-IgM b (27,28). Nontransgenic (non-Tg) C57BL/6 mice (Taconic Farms) were used as controls.…”

“…Anti-insulin 125Tg B cells mature normally but are maintained in an anergic state by autologous insulin occupying their BCR (25,27). To examine the BCR-derived responses of B lymphocytes that have been rendered tolerant by exposure to physiologic concentrations of insulin in vivo, protein p-Tyr was examined following stimulation with 10 g/ml anti-IgM.…”

“…The 125Tg mice used in this study harbor conventional anti-insulin Ig transgenes (Ig only) derived from mAb 125 on a C57BL/6 background (backcross Ͼ27) and were generated and maintained as described previously (27,28). B cells in 125Tg mice show no signs of allelic inclusion (25) and maintain high levels of allelic exclusion for long periods of time (Ͼ30 wk); Ն 95% of B220 ϩ lymphocytes express only IgM a as revealed by staining with anti-IgM a vs anti-IgM b (27,28).…”

“…Purified B cells were suspended in complete medium as described previously (25,27) and stimulated (where specified) with 10 g/ml F(abЈ) 2 goat anti-mouse -chain (Jackson ImmunoResearch Laboratories) or 1 M ionomycin (Sigma-Aldrich) for the indicated times. Total cell lysates were prepared in lysis buffer containing 50 mM HEPES (pH 7.5) (Invitrogen Life Technologies), 5% glycerol (Invitrogen Life Technologies), 1% Triton X-100 (Sigma-Aldrich), 1 mM sodium orthovanadate (Sigma-Aldrich), 100 mM sodium chloride (Sigma-Aldrich), and protease inhibitors (Roche Applied Science).…”