Molecular underpinning of B-cell anergy

Abstract

A byproduct of the largely stochastic generation of a diverse B-cell specificity repertoire is production of cells that recognize autoantigens. Indeed, recent studies indicate that more than half of the primary repertoire consists of autoreactive B cells that must be silenced to prevent autoimmunity. While this silencing can occur by multiple mechanisms, it appears that most autoreactive B cells are silenced by anergy, wherein they populate peripheral lymphoid organs and continue to express unoccupied antigen receptors yet are unresponsive to antigen stimulation. Here we review molecular mechanisms that appear operative in maintaining the antigen unresponsiveness of anergic B cells. In addition, we present new data indicating that the failure of anergic B cells to mobilize calcium in response to antigen stimulation is not mediated by inactivation of stromal interacting molecule 1, a critical intermediary in intracellular store depletion-induced calcium influx.

Fig. 1

Signaling pathways operative in naive and anergic B cells.

Fig. 2. Stim1 phosphorylation in naive cells

Bal-17 B-lymphoma cells (5 × 10⁷ cells/ml) were treated with 50 nM Calyculin A or vehicle, and whole cell lysates were prepared using an NP-40 lysis buffer. 100 μl of lysate containing 5 × 10⁶ cell equivalents of Calyculin A-treated cells was incubated with λ PPase (400 u) for the indicated time. The lysate was fractionated on SDS-PAGE gels, transferred to PVDF membrane, and blotted using rabbit anti-Stim1 antibody followed by rat anti-rabbit-680 nm secondary antibody. Blots were analyzed using a LYCOR fluorescence analyzer.

Fig. 3. Stim1 is functional in anergic B cells

(A) Splenocytes isolated from κ tg (naive) and Ars/A1 (anergic) mice were CD43 depleted to yield B cells. Naive and anergic B cells were cell lysed using NP-40, separated on SDS-PAGE gels, transferred to PVDF membrane, and blotted with rabbit anti-Stim1 antibody followed by rat anti-rabbit-680 nm secondary antibody. (B) Naive and anergic splenocytes were loaded with Indo-AM and [Ca⁺²]_i measured by flow cytometry. EGTA (4 mM final concentration) was added to each sample immediately before recording to chelate calcium. After the baseline was established, cells were stimulated with ionomycin (1 μM final concentration) (solid arrow) and analysis continued. Calcium was then repleted (dashed arrow) by addition of calcium chloride (4 mM final concentration) and analysis resumed.