Potentially autoreactive naturally occurring transitional T3 B lymphocytes exhibit a unique signaling profile

Abstract

B lymphocytes exhibit phenotypic differences that correlate with their developmental or functional stages and affect humoral immune responses. One recently described subset of naturally occurring immature transitional type 3 (T3) B lymphocytes is believed to consist of potentially autoimmune cells whose signaling properties have not been studied in detail. This study characterizes intracellular signaling in T3 B cells in wildtype C57BL/6 mice. Protein phosphorylation and Ca2+ responses upon B cell antigen receptor (BCR) engagement were measured by multicolor flow cytometry. We observed high baseline signaling activity and reduced BCR-mediated responses in T3 cells, which confirmed their anergy – a functional state in which lymphocytes recognize chronically present self-antigens but cannot produce immune response due to intrinsic signaling inhibition. Our results also revealed a previously unknown T3-specific phosphorylation pattern of 24 key signaling molecules involved in BCR signal transduction. These characteristics reflect the balance between stimulatory and inhibitory BCR signaling pathways in anergy. Results obtained in the collagen induced arthritis model demonstrate the loss of anergy in T3 B cells during the onset of the disease. Our findings provide rationale for further investigating alterations in B cell signaling patterns as earliest functional biomarkers of changes in the immune tolerance of autoreactive B cells.

Rheumatoid arthritis is associated with signaling alterations in naturally occurring autoreactive B-lymphocytes

“…Our recent findings in murine experimental arthritis model demonstrate the loss of B cell anergy during the onset of disease, and a distinct BCR signal inhibitory pathway that clearly distinguishes anergic cells from other B cell types [10]. This study expands our approach into human RA.…”

“…For intracellular phosphoprotein staining, purified PBMCs were stimulated with anti-BCR for 10 min (5µg/ml), then rapidly fixed and permeabilized in 20x volume of BD Cytofix/Cytoperm buffer and stained with a cocktail of surface marker Abs (as described above) and with phospho-specific Abs followed by FITC labeled secondary Ab. Experiments were performed as described [12] on BD LSR II flow cytometer (Beckton Dickinson, San Jose, CA). B cell subsets of interest were gated upon as indicated in Figure 2A, B and protein phosphorylation intensity (mean fluorescence intensity, MFI) or intracellular Ca 2+ influx (Indo-1AM Ex/Em ratio units) were analyzed as described [12].…”

“…Experiments were performed as described [12] on BD LSR II flow cytometer (Beckton Dickinson, San Jose, CA). B cell subsets of interest were gated upon as indicated in Figure 2A, B and protein phosphorylation intensity (mean fluorescence intensity, MFI) or intracellular Ca 2+ influx (Indo-1AM Ex/Em ratio units) were analyzed as described [12]. Fluorescence intensity of phospho-specific Ab staining within the gated subsets was compared between stimulated and nonstimulated cells.…”

General Approach for Tetramer-Based Identification of Autoantigen-Reactive B Cells: Characterization of La- and snRNP-Reactive B Cells in Autoimmune BXD2 Mice

“…T3 B cells generally are considered not strictly transitional but anergic B cells maintaining self-tolerance through rapid turnover in vivo (74, 75). Interestingly, self-Ag stimulation has been shown to promote regression of mature B cells into the T3 compartment (75, 76). Thus, expansion of these cells coupled with abnormal T-cell help (25, 77) may present a risk for B-cell tolerance loss to La 13–27 or snRNP 357–373 in BXD2 mice.…”

Regulatory CD4+ T Cells Promote B Cell Anergy in Murine Lupus

“…Autoreactive lupus B cells with an anergic phenotype could have an abnormally prolonged lifespan, being neither activated nor deleted (23). Of interest, a loss of anergy in T3 B cells has been observed in autoimmune diseases such as collageninduced arthritis and rheumatoid arthritis (24)(25)(26), suggesting that the generation and survival of anergic B cells requires a fine control to avert autoimmunity. 2+ flux was measured after BCR stimulation in NZB/W mice (dark gray) and in CW controls (light gray).…”