Entry of B cell receptor into signaling domains is inhibited in tolerant B cells

Abstract

Signal transduction through the B cell antigen receptor (BCR) is altered in B cells that express a receptor that recognizes self-antigen. To understand the molecular basis for the change in signaling in autoreactive B cells, a transgenic model was used to isolate a homogeneous population of tolerant B lymphocytes. These cells were compared with a similar population of naive B lymphocytes. We show that the BCR from naive B cells enters a detergent-insoluble domain of the cell within 6 s after antigen binding, before a detectable increase in BCR phosphorylation. This fraction appears to be important for signaling because it is enriched for lyn kinase but lacks CD45 tyrosine phosphatase and because the BCR that moves into this domain becomes more highly phosphorylated. Partitioning of the BCR into this fraction is unaffected by src family kinase inhibition. Tolerant B cells do not efficiently partition the BCR into the detergent-insoluble domain, providing an explanation for their reduced tyrosine kinase activation and calcium flux in response to antigen. These results identify an early, regulated step in antigen receptor signaling and self-tolerance.

Figure 1

BCRs accumulate in a subcellular compartment that is detergent insoluble and salt extractable after antigen stimulation of naive but not tolerant B cells. Spleen cells from anti-HEL BCR transgenic (naive B cells) or anti-HEL/soluble HEL double-transgenic mice (tolerant B cells) were mock stimulated or stimulated with 10 μg/ml HEL for 3 min and then fractionated to obtain the detergent-soluble supernatant and the detergent-insoluble salt extract. The salt extract lanes represent three times as many cell equivalents as the detergent-soluble lanes. (A) Unreduced SDS-PAGE Western blots were probed with anti–κ L chain to resolve IgM and IgD H chain–L chain BCR complexes. (B–D) Reduced SDS-PAGE Western blots were probed (B) with a mixture of anti-Igα, anti-Igβ, and anti-lyn; (C) with 4G10 antiphosphotyrosine; (D) with cholera toxin, which binds to GM1 sphingolipids running with the dye front. In B, numbers below each lane are the amount of lyn immunoreactivity, corrected for loading, and the amount of Igα and Igβ immunoreactivity normalized to the average amount of lyn in each fraction. Quantitation was determined by PhosphorImager analysis of ¹²⁵I–Protein A-developed blots. (E) Data from six independent experiments show that Igα in the salt extract fraction is more highly phosphorylated than Igα in the soluble fraction after stimulation of naive B cells. The degree of phosphorylation of Igα was obtained by dividing the intensity of the Igα-sized band on the antiphosphotyrosine blot by the intensity of the Igα band on the same blot reprobed with anti-Igα. The degree of Igα phosphorylation induced in the extract was significantly different from that induced in the supernatant to P < 0.1.

Figure 2

The salt extract fraction contains lyn and stimulated BCR but not syk, CD22, or CD45. Spleen cells from anti-HEL BCR transgenic mice were stimulated and separated as in Fig. 1 and probed with a mixture of anti-Igα, anti-Igβ, and anti-lyn or with anti-syk, anti-CD22, or anti-CD45. Three times as many cell equivalents were loaded in the salt extract fraction lanes as in the soluble fraction lanes. The CD45 blot is from a different experiment than the other blots, but BCR partitioning was similar in that experiment. ¹²⁵I quantitation shows >300-fold enrichment of CD45 in the soluble fraction.

Figure 3

BCR becomes detergent insoluble before Igα and Igβ phosphorylation increases. Spleen cells from anti-HEL BCR transgenic mice were stimulated for the indicated time at either 4 or 37°C. Detergent insolubility of BCR was monitored by following appearance of Igα and Igβ in the salt extract (top panels), whereas tyrosine phosphorylation was monitored by probing the same blots with antiphosphotyrosine (bottom panels).

Figure 4

BCR partitioning to the detergent-insoluble fraction is independent of src family kinase activity. Spleen cells from anti-HEL BCR transgenic mice were stimulated in the presence of varying concentrations of the src family–specific kinase inhibitor PP1. Tyrosine phosphorylation of Igα and Igβ in unstimulated and stimulated cells was measured by antiphosphotyrosine Western blotting (bottom panel). Partitioning of the BCR to the salt extract fraction was measured by probing the same blots for Igα and Igβ (top panel).