Inhibition of the B cell by CD22: a requirement for Lyn

Abstract

Mice in which the Lyn, Cd22, or Shp-1 gene has been disrupted have hyperactive B cells and autoantibodies. We find that in the absence of Lyn, the ability of CD22 to become tyrosine phosphorylated after ligation of mIg, to recruit SHP-1, and to suppress mIg-induced elevation of intracellular [Ca2+] is lost. Therefore, Lyn is required for the SHP-1-mediated B cell suppressive function of CD22, accounting for similarities in the phenotypes of these mice.

Figure 1

Changes in [Ca²⁺]i caused by ligating mIg alone or together with CD22 on B cells from Lyn+/+ and Lyn−/− mice. (A) Splenic lymphocytes that had been loaded with Indo-1 were incubated with biotinylated Fab fragments of anti-κ and either anti-CD22 (Lyb-8.2; solid line) or isotype control antibodies (dashed line). The fluorescence ratio of the cells was measured by flow cytometry until the addition of avidin to cross-link the cell-bound Fab fragments, after which the measurement was continued for an additional 320 s. B cells were identified in this experiment by counterstaining with B220. (B) Analysis of these splenocytes showed that although there were fewer B220-positive cells in Lyn−/− mice (–5), all expressed CD22 at levels comparable to those of their Lyn+/+ littermates.

Figure 2

Tyrosine phosphorylation of CD22 in resting and mIg-activated B cells from Lyn+/+ and Lyn−/− mice. (A) Purified B cells were stimulated with the F(ab′)₂ fragment of anti-μ for timed intervals and then lysed. CD22 was immunoprecipitated (IP) from the lysates with anti-CD22 or control mouse IgG1. The precipitates were subjected to SDS-PAGE followed by sequential immunoblotting with ¹²⁵I-labeled monoclonal antiphosphotyrosine, and with rabbit anti-CD22 with detection by ECL. (B) The ratio of the intensity of the antiphosphotyrosine and anti-CD22 signals at each time point was determined by densitometric analysis, which permitted a comparison of the relative tyrosine phosphorylation of CD22 in B cells from the Lyn+/+ and Lyn−/− mice.

Figure 2

Tyrosine phosphorylation of CD22 in resting and mIg-activated B cells from Lyn+/+ and Lyn−/− mice. (A) Purified B cells were stimulated with the F(ab′)₂ fragment of anti-μ for timed intervals and then lysed. CD22 was immunoprecipitated (IP) from the lysates with anti-CD22 or control mouse IgG1. The precipitates were subjected to SDS-PAGE followed by sequential immunoblotting with ¹²⁵I-labeled monoclonal antiphosphotyrosine, and with rabbit anti-CD22 with detection by ECL. (B) The ratio of the intensity of the antiphosphotyrosine and anti-CD22 signals at each time point was determined by densitometric analysis, which permitted a comparison of the relative tyrosine phosphorylation of CD22 in B cells from the Lyn+/+ and Lyn−/− mice.

Figure 3

Association of SHP-1 with tyrosine phosphorylated CD22 in resting and mIg-activated B cells from Lyn+/+ and Lyn−/− mice. (A) Purified B cells were stimulated with the F(ab′)₂ fragment of anti-μ for timed intervals and lysed. The lysates were immunoprecipitated with monoclonal anti-SHP-1 or control mouse IgG1. The precipitates were subjected to SDS-PAGE, and proteins with molecular weights >90 kD were immunoblotted with ¹²⁵I-labeled antiphosphotyrosine, and proteins with lower molecular weights were immunoblotted with anti–SHP-1, which was detected by ECL. (B) The ratio of the intensity of the antiphosphotyrosine and anti–SHP-1 signals at each time point was determined by densitometric analysis, which permitted a comparison of the relative association of phosphorylated CD22 with SHP-1 in B cells from the Lyn+/+ and Lyn−/− mice.

Figure 3

Association of SHP-1 with tyrosine phosphorylated CD22 in resting and mIg-activated B cells from Lyn+/+ and Lyn−/− mice. (A) Purified B cells were stimulated with the F(ab′)₂ fragment of anti-μ for timed intervals and lysed. The lysates were immunoprecipitated with monoclonal anti-SHP-1 or control mouse IgG1. The precipitates were subjected to SDS-PAGE, and proteins with molecular weights >90 kD were immunoblotted with ¹²⁵I-labeled antiphosphotyrosine, and proteins with lower molecular weights were immunoblotted with anti–SHP-1, which was detected by ECL. (B) The ratio of the intensity of the antiphosphotyrosine and anti–SHP-1 signals at each time point was determined by densitometric analysis, which permitted a comparison of the relative association of phosphorylated CD22 with SHP-1 in B cells from the Lyn+/+ and Lyn−/− mice.

Figure 4

Demonstration that only those phosphotyrosyl peptides of CD22 that mediate SHP-1 binding and activation (Y783, Y843, and Y863; reference 15) share residues with the consensus sequence of a preferred model substrate for Lyn (30).