T-helper signals restore B-cell receptor signaling in autoreactive anergic B cells by upregulating CD45 phosphatase activity

• Published in: Journal of allergy and clinical immunology 2016-09, Vol.138 (3), p.839-851.e8
• Main Authors: Szodoray, Peter, MD, PhD, Stanford, Stephanie M., PhD, Molberg, Øyvind, MD, PhD, Munthe, Ludvig A., MD, PhD, Bottini, Nunzio, MD, PhD, Nakken, Britt, PhD
• Format: Article
• Language: English
• Subjects: Adult; Allergy and Immunology; Antigens; Autoimmune diseases; B-cell anergy; B-Lymphocytes - immunology; Blood & organ donations; break of tolerance; Calcium - metabolism; CD45 phosphatase activity; Cells, Cultured; Clonal Anergy; Disease; Human subjects; Humans; Immunoglobulins; Kinases; Leukocyte Common Antigens - genetics; Leukocyte Common Antigens - immunology; Leukocyte Common Antigens - metabolism; Lupus Erythematosus, Systemic - immunology; Pathogenesis; Phosphatase; Population; Receptors, Antigen, B-Cell - immunology; RNA, Messenger - metabolism; Rodents; Signal Transduction; src-Family Kinases - genetics; Syk Kinase - genetics; systemic lupus erythematosus; T cell receptors; T-cell help; T-Lymphocytes - immunology; Up-Regulation
• ISSN: 0091-6749; 1097-6825
• DOI: 10.1016/j.jaci.2016.01.035

Background We recently identified a human B-cell population that is naturally autoreactive and tolerized by functional anergy (BND cells). Objective We sought to identify the molecular mechanism of how anergic autoreactive BND cells escape functional anergy and whether this process is altered in patients with lupus. Methods Isolated peripheral blood naive and BND cells were cultured with various stimuli, and their activation status was determined by using an intracellular Ca2+ mobilization assay. Lyn kinase and Syk activities were assessed by using phospho-flow analysis. CD45 phosphatase activity was determined by using a novel flow-based assay, which takes advantage of the fluorogenic properties of phosphorylated coumaryl amino propionic acid, an analog of phosphotyrosine, which can be incorporated into peptides. Real-time quantitative PCR was used to quantitate LYN , SYK , and CD45 mRNA. Results T-helper signals reversed the state of anergy, allowing BND cells to fully respond to antigenic stimulation by restoring signaling through the B-cell receptor (BCR). The mechanism was dependent on increased activity of the tyrosine phosphatase CD45 and CD45-dependent activation of Lyn and Syk. CD45 phosphatase activity was increased by T-cell help both in BND and naive B cells. Furthermore, we found that BND cells obtained from patients with systemic lupus erythematosus exhibited increased CD45 activity and BCR-signaling capacity, thus being less tolerized than BND cells from healthy control subjects. Conclusion Our findings suggest that CD45 is a key regulator of BCR-signaling thresholds mediated by T-cell help. This raises the possibility that BND cells could represent precursors of autoantibody-secreting plasma cells and suggests a role for these autoreactive B cells in contributing to autoimmunity if not properly controlled.