Endogenous antigen tunes the responsiveness of naive B cells but not T cells

Abstract

In humans, up to 75% of newly generated B cells and about 30% of mature B cells show some degree of autoreactivity. Yet, how B cells establish and maintain tolerance in the face of autoantigen exposure during and after development is not certain. Studies of model B-cell antigen receptor (BCR) transgenic systems have highlighted the critical role of functional unresponsiveness or ‘anergy’. Unlike T cells, evidence suggests that receptor editing and anergy, rather than deletion, account for much of B-cell tolerance. However, it remains unclear whether the mature diverse B-cell repertoire of mice contains anergic autoreactive B cells, and if so, whether antigen was encountered during or after their development. By taking advantage of a reporter mouse in which BCR signalling rapidly and robustly induces green fluorescent protein expression under the control of the Nur77 regulatory region, antigen-dependent and antigen-independent BCR signalling events in vivo during B-cell maturation were visualized. Here we show that B cells encounter antigen during development in the spleen, and that this antigen exposure, in turn, tunes the responsiveness of BCR signalling in B cells at least partly by downmodulating expression of surface IgM but not IgD BCRs, and by modifying basal calcium levels. By contrast, no analogous process occurs in naive mature T cells. Our data demonstrate not only that autoreactive B cells persist in the mature repertoire, but that functional unresponsiveness or anergy exists in the mature B-cell repertoire along a continuum, a fact that has long been suspected, but never yet shown. These results have important implications for understanding how tolerance in T and B cells is differently imposed, and how these processes might go awry in disease.

Figure 1. Nur77-GFP Bac Tg reporter is responsive to antigen receptor signaling in vitro

(A) Histograms represent GFP and CD69 expression of GFP^LO Tg lymph node (LN) T cells treated with varying doses of plate-bound anti-CD3ε for 16 hours (0.00625-6.4μg/ml in a four-fold dilution series). (B) Histograms represent GFP and CD69 expression of IgHEL Tg / GFP^HI Tg LN B cells treated with varying doses of hen egg lysozyme (HEL) for 16 hours (0.125–16 ng/ml in a two-fold dilution series). Data are representative of at least three independent experiments.

Figure 2. Expression of Nur77-GFP Bac Tg reporter is up-regulated at specific checkpoints during B cell development

(A) Left: Plot of GFP^HI Tg BM CD19+ B cells stained for IgM and IgD to identify pre-B, immature, transitional, and mature recirculating subsets (counter-clockwise from bottom left corner). Middle: BM subsets are color-coded. Right: overlaid histograms representing GFP expression in these subsets. (B) Left: Plot of GFP^HI Tg Splenic CD19+ B cells stained for CD23 and CD21 expression to identify NF/T1 (CD23-CD21−), T2/FO (CD23+CD21int), and MZ (CD21hi) subsets. Middle: Splenic B cell subsets are color-coded. Right: overlaid histograms represent GFP expression in these subsets. (C) Left: Plot of GFP^HI Tg Splenic CD19+ B cells, excluding MZ compartment, stained for CD23 and AA4.1 expression to identify T1 (AA4.1+CD23−), T2/3 (AA4.1+CD23+), and FO (AA4.1-CD23+) subsets. Middle: Splenic B cell subsets are color-coded. Right: overlaid histograms represent GFP expression in these subsets. (D) Overlaid histograms represent GFP (left) and IgM (right) expression in T1, T2/3, and FO subsets as identified in 2C. (E) Left: T2/3 (AA4.1+CD23+) B cell subset subdivided by IgM expression into T2 (IgM^hi) and T3 (IgM^lo) stages. Right: overlaid histograms represent GFP expression T2 and T3 subsets. All data are representative of at least three independent experiments.

Figure 3. Expression of Nur77-GFP Bac Tg reporter is sensitive to genetic modulation of BCR signal strength and antigen

(A) CD45 allelic series (low to high CD45 expression: L/L, L/+, +/+, H/−) GFP^HI Tg splenic B cells were stained to identify B cell subsets as in Figure 2B, C. Overlaid histograms represent GFP expression in splenic subsets as gated in S5A. (B) CD45+/+ GFP^HI Tg and H/- (high CD45) GFP^HI Tg splenic B cells with unrestricted (IgHEL-) or restricted (IgHEL+) repertoire in the absence of sHEL antigen were analyzed as in 3A. Overlaid histograms represent GFP expression in splenic subsets as gated in S5C. (C) CD45+/+ GFP^HI Tg splenic B cells with unrestricted (IgHEL-) or restricted (IgHEL+) repertoire in the presence or absence of sHEL antigen were analyzed as in 3A. Overlaid histograms represent GFP expression in splenic subsets as gated in S5D. All animals in Figure 3 were generated through genetic crosses. All data are representative of at least five independent experiments.

Figure 4. GFP expression predicts functional responsiveness and autoreactivity of B cells

(A) Left: GFP^HI Tg FO (CD23+AA4.1-) splenic B cells were subdivided into color-coded bins on the basis of GFP expression. Right: GFP^HI Tg splenic B cells were loaded with Indo-1 dye and stimulated with 10μg/ml anti-IgM. Ratio-metric assessment of intra-cellular calcium was carried out by flow cytometry. Upper right panel represents calcium entry in total FO splenic B cells. Lower right panel represents basal and inducible intra-cellular calcium in GFP-specific bins. (B, C) Intra-cellular calcium entry was assessed in GFP^HI Tg FO splenic B cells upon anti-IgM (B) or anti-IgD (C) stimulation at varying doses. High and low GFP-expressing gates are overlaid. Data in 4A-C are representative of at least three independent experiments. (D) Overlaid histograms represent pre- and post-sort follicular mature CD23+ AA4.1- B cells as gated in S9. 15% lowest and highest GFP fractions from the follicular mature B cell compartment were selected for sorting. (E) High and low-expressing GFP B cells sorted as described in S9A, 4C were stimulated in vitro with LPS for 4 days. Supernatants were subjected to ANA IgM ELISA and total IgM ELISA. Graph represents quantification of ANA IgM normalized to total IgM from 4 independent sorting experiments +/− SEM. Significance was assessed via unpaired t-test. ** = p<0.005