Sialylated multivalent antigens engage CD22 in trans and inhibit B cell activation

Abstract

CD22 is an inhibitory coreceptor on the surface of B cells that attenuates B cell antigen receptor (BCR) signaling and, therefore, B cell activation. Elucidating the molecular mechanisms underlying the inhibitory activity of CD22 is complicated by the ubiquity of CD22 ligands. Although antigens can display CD22 ligands, the receptor is known to bind to sialylated glycoproteins on the cell surface. The propinquity of CD22 and cell-surface glycoprotein ligands has led to the conclusion that the inhibitory properties of the receptor are due to cis interactions. Here, we examine the functional consequences of trans interactions by employing sialylated multivalent antigens that can engage both CD22 and the BCR. Exposure of B cells to sialylated antigens results in the inhibition of key steps in BCR signaling. These results reveal that antigens bearing CD22 ligands are powerful suppressors of B cell activation. The ability of sialylated antigens to inhibit BCR signaling through trans CD22 interactions reveals a previously unrecognized role for the Siglec-family of receptors as modulators of immune signaling.

Fig. 1.

Synthetic antigens used to investigate CD22 recognition. Polymers with a degree of polymerization of 250 (n ≈ 250) were used. The substituents include the dinitrophenyl (DNP group, R₁, blue), the CD22 ligand Neu5Acα2,6Galβ1,4Glc (CD22L, R₂, red), or the spacer unit derived from ethanolamine coupling. The level of substitution of each group (mole fraction χ) for compound 1 is R₁ χ = 0.33, R₃ χ = 0.67; for compound 2 is R₂ χ = 0.23, R₃ χ = 0.77; for compound 3 is R₁ χ = 0.36, R₂ χ = 0.24, R₃ χ = 0.40.

Fig. 2.

Antigenic polymers possessing CD22 ligands inhibit calcium flux. (A) Calcium flux was assayed by using the ratiometric dye indo-1 and flow cytometry (Left). After establishing the base line, cells were treated with polymers (DNP concentration of 5 μM; CD22L concentration of 3.5 μM) at t = 0. Peak Ca²⁺ flux from A20HL cells treated (Right) with both DNP homopolymer and DNP/CD22L copolymers at 1.25–10 μM DNP. (B) Cells also were treated with the CD22L homopolymer alone or CD22L homopolymer followed by DNP homopolymer (Left) and peak Ca²⁺ flux (Right) determined. All error bars represent 1 standard deviation from the mean of 3 independent experiments.

Fig. 3.

Antigenic polymers possessing CD22 ligands attenuate tyrosine phosphorylation. Stimulation of A20HL cells with DNP homopolymer results in an increase in cellular protein tyrosine phosphorylation as assessed by immunoblot of cellular lysates using anti-phosphotyrosine antibody. In contrast, DNP/CD22L copolymer-treated cells show little change in tyrosine phosphorylation after stimulation, whereas CD22L homopolymer-treated cells remain unchanged. Cells were stimulated by using a final concentration of 5 μM DNP and/or 3.5 μM CD22L.

Fig. 4.

Polymer stimulation results in Lyn and CD22 phosphorylation. (A) A20HL cells were stimulated with polymers and lysed, and the resulting samples were subjected to immunoprecipitation using anti-Lyn. Immunoblot analysis with a phosphospecific antibody was performed. Immunoprecipitation efficiency was monitored by probing with anti-Lyn. Treatment with either the DNP homopolymer or the DNP/CD22L copolymer resulted in Lyn phosphorylation (Y396). (B) Similarly CD22 was immunoprecipitated with anti-CD22. Both the DNP homopolymer and the DNP/CD22L copolymer stimulation resulted in CD22 phosphorylation; however, increased phospho-CD22 is observed at later time points with copolymer. Cells were stimulated by using a final concentration of 5 μM DNP (3.5 μM CD22L). See Fig. S6.

Fig. 5.

Antigen sialylation prevents Syk and PLCγ2 activation. (A) A20HL cells were stimulated with polymers and lysed, and the resulting samples were subjected to immunoprecipitation using anti-Syk. Immunoblot analysis with a phosphospecific antibody (Y519/520) was performed. Two isoforms are visible. (B) PLCγ2 activation was assessed by immunoblot analysis of lysate from polymer-treated cells using a phosphospecific antibody (Y1217). As with Syk, the DNP homopolymer treatment resulted in PLCγ2 activation as assessed by phosphorylation (Y1217). Cells were stimulated by using a final concentration of 5 μM DNP (3.5 μM CD22L). See Fig. S6.

Fig. 6.

Antigenic polymers displaying hapten (DNP) initiate activation of early B cell signaling events. Copolymers displaying DNP and a ligand for the coreceptor CD22 result in the attenuation of B cell activation.