Masking of CD22 by cis ligands does not prevent redistribution of CD22 to sites of cell contact

Abstract

CD22, a negative regulator of B cell signaling, is a member of the siglec family that binds to alpha2-6-linked sialic acids on glycoproteins. Previous reports demonstrated that binding of multivalent sialoside probes to CD22 is blocked, or "masked," by endogenous (cis) ligands, unless they are first destroyed by sialidase treatment. These results suggest that cis ligands on B cells make CD22 functionally unavailable for binding to ligands in trans. Through immunofluorescence microscopy, however, we observed that CD22 on resting B cells redistributes to the site of contact with other B or T lymphocytes. Redistribution is mediated by interaction with trans ligands on the opposing cell because it does not occur with ligand-deficient lymphocytes from ST6GalI-null mice. Surprisingly, CD45, proposed as both a cis and trans ligand of CD22, was not required for redistribution to sites of cell contact, given that redistribution of CD22 was independent of CD45 and was observed with lymphocytes from CD45-deficient mice. Furthermore, CD45 is not required for CD22 masking as similar levels of masking were observed in the WT and null mice. Comparison of the widely used sialoside-polyacrylamide probe with a sialoside-streptavidin probe revealed that the latter bound a subset of B cells without sialidase treatment, suggesting that cis ligands differentially impacted the binding of these two probes in trans. The combined results suggest that equilibrium binding to cis ligands does not preclude binding of CD22 to ligands in trans, and allows for its redistribution to sites of contact between lymphocytes.

Fig. 1.

CD22 redistributes to the site of cell-to-cell contacts in murine splenocytes. Representative immunofluorescent images of murine splenocytes are shown. Freshly isolated splenocytes were stained with Hoechst for nuclei (Left), Texas red-labeled anti-IgM (Center), and FITC labeled anti-CD22 (Right).

Fig. 2.

Redistribution of CD22 to the sites of lymphocyte contacts requires Siaα2-6Gal terminated glycans. CD22 redistributes to the sites of cell contact between B lymphocytes as well as T and B lymphocytes. (A) Representative immunofluorescent images of B-to-B lymphocyte contacts with splenocytes from WT (Upper) or ST6GalI-null (Lower) mice stained with Hoechst for nuclei (Left), Texas red-labeled anti-IgM (Center), and FITC-labeled anti-CD22 (Right). (B) B-to-T lymphocyte contacts with splenocytes isolated from WT (Upper) or ST6GalI-null (Lower) mice stained with Hoechst for nuclei (Left), Texas red-labeled anti-Thy 1.2 to mark T cells (Center), and FITC-labeled anti-CD22 (Right). (C) B-to-B lymphocyte contact with purified B cells isolated from WT and ST6GalI-null mice stained with Hoechst for nuclei (Left), FITC-labeled SNA to detect the product of ST6GalI (Siaα2-6Gal) on the WT B cell (Center), and Texas red-labeled anti-CD22 (Right).

Fig. 3.

CD22 masking and recruitment to the sites of lymphocyte contact in the absence of CD45. (A) Masking status of CD22 as measured by binding of NeuGcα2-6Gal-PAA probe (Upper) to WT (black traces) and CD45-null (red traces) splenocytes, pretreated with (thick trace) or without (thin trace) sialidase to remove cis ligands. Splenocytes from WT (Left Lower) or CD45 (Right Lower) mice were stained with anti-CD45 (B220) and anti-CD22. (B) Representative immunofluorescent images of splenocytes from WT mice stained with anti-CD45 (Left) and anti-CD22 (Right). (C) Representative immunofluorescent images of splenocytes from CD45-deficient mice stained with Hoechst for nuclei (Left) or anti-CD22 (Right).

Fig. 4.

Cis ligands differentially mask binding of two multivalent sialoside probes. Binding of NeuGcα2-6Gal-SAAP to native B cells does not require unmasking, as measured by a decrease in SNA receptors. (A) Splenocytes mock-pretreated or A. ureafaciens sialidase-pretreated were incubated with the NeuGcα2-6Gal-SAAP probe or the NeuGcα2-6Gal-PAA probe and anti-B220, as described in Materials and Methods. (B) Native (untreated) lymphocytes from bone marrow, spleen, and lymph node were incubated with anti-B220 and either NeuGcα2-6Gal-SAAP probe (Upper) or the SAAP carrier alone (Lower) without any pretreatment. The percentage of the B220hi cells that bound either the sialoside–SAAP probe or SAAP carrier is indicated in the upper right quadrant of each panel. (C) Native splenocytes were stained with SNA, anti-B220, and NeuGcα2-6Gal-SAAP. (Left) Binding of NeuGcα2-6Gal-SAAP to native B220+ cells. Gates were set for cells that exhibited background (R1) or high (R2) binding of the sialoside probe. (Right) SNA binding to gated B cells with background (R1, dotted line) or high (R2; solid line) levels of NeuGcα2-6Gal-SAAP probe.