Limits...
Decoration of T-independent antigen with ligands for CD22 and Siglec-G can suppress immunity and induce B cell tolerance in vivo.

Duong BH, Tian H, Ota T, Completo G, Han S, Vela JL, Ota M, Kubitz M, Bovin N, Paulson JC, Paulson J, Nemazee D - J. Exp. Med. (2009)

Bottom Line: Although mutations in CD22 and its signaling machinery have been associated with dysregulated B cell development and autoantibody production, previous analyses failed to identify a tolerance defect in antigen-specific mutant B cells.Our results support a role for siglecs in B cell self-/nonself-discrimination, namely suppressing responses to self-associated antigens while permitting rapid "missing self"-responses to unsialylated multimeric antigens.The results suggest use of siglec ligand antigen constructs as an approach for inducing tolerance.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Immunology and Microbial Science, The Scripps Research Institute, La Jolla, CA 92037, USA.

ABSTRACT
Autoreactive B lymphocytes first encountering self-antigens in peripheral tissues are normally regulated by induction of anergy or apoptosis. According to the "two-signal" model, antigen recognition alone should render B cells tolerant unless T cell help or inflammatory signals such as lipopolysaccharide are provided. However, no such signals seem necessary for responses to T-independent type 2 (TI-2) antigens, which are multimeric antigens lacking T cell epitopes and Toll-like receptor ligands. How then do mature B cells avoid making a TI-2-like response to multimeric self-antigens? We present evidence that TI-2 antigens decorated with ligands of inhibitory sialic acid-binding Ig-like lectins (siglecs) are poorly immunogenic and can induce tolerance to subsequent challenge with immunogenic antigen. Two siglecs, CD22 and Siglec-G, contributed to tolerance induction, preventing plasma cell differentiation or survival. Although mutations in CD22 and its signaling machinery have been associated with dysregulated B cell development and autoantibody production, previous analyses failed to identify a tolerance defect in antigen-specific mutant B cells. Our results support a role for siglecs in B cell self-/nonself-discrimination, namely suppressing responses to self-associated antigens while permitting rapid "missing self"-responses to unsialylated multimeric antigens. The results suggest use of siglec ligand antigen constructs as an approach for inducing tolerance.

Show MeSH
Binding analysis of various sialylated PA–glycan conjugates to wild-type compared with siglec mutant B cells. Results are representative of three independent experiments. (A–E) B cells of the indicated genotypes were stained with biotinylated PA conjugated with the following glycans: (A) NeuAcα2-6Galβ1-4GlcNAc, (B) NeuGc, (C) bNeuGc, (D) NeuAcα2-3Galβ1-4GlcNAc, and (E), NeuGcα2-3Galβ1-4GlcNAc. Binding was revealed using fluorescent streptavidin. Background staining by streptavidin alone is shown in gray. (F) Chemical structures of the NeuGc-containing sialosides.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC2812539&req=5

fig1: Binding analysis of various sialylated PA–glycan conjugates to wild-type compared with siglec mutant B cells. Results are representative of three independent experiments. (A–E) B cells of the indicated genotypes were stained with biotinylated PA conjugated with the following glycans: (A) NeuAcα2-6Galβ1-4GlcNAc, (B) NeuGc, (C) bNeuGc, (D) NeuAcα2-3Galβ1-4GlcNAc, and (E), NeuGcα2-3Galβ1-4GlcNAc. Binding was revealed using fluorescent streptavidin. Background staining by streptavidin alone is shown in gray. (F) Chemical structures of the NeuGc-containing sialosides.

Mentions: Because our goal was to design copolymers of siglec ligands and a TI-2 antigen that would recruit siglecs to the hapten-specific BCR, we characterized highly multivalent sialylated glycans linked to PA for their ability to compete with cis ligands and bind to CD22 and Siglec-G on native B cells. We and others previously showed that synthetic trans ligands of CD22 (NeuGcα2-6Gal) compete poorly with endogenous cis ligands unless they were displayed on highly multivalent polymers and included hydrophobic substituents at the 9 position that increase affinity (Razi and Varki, 1998; Collins et al., 2002; Collins et al., 2006a). Therefore, we evaluated binding of highly multivalent PA conjugates (1,000 kD) carrying sialosides (n ≈ 400) and biotin tags to facilitate their detection. Each was analyzed for its binding to B cells from wild-type, CD22-deficient, Siglec-G–deficient, and CD22/Siglec-G double-deficient mice (Fig. 1). The PA–sialosides with NeuAcα2-6Galβ1-4GlcNAc–containing glycans showed no binding to mouse B cells, consistent with the known preference of mouse CD22 for NeuGcα2-6Galβ1-4GlcNAc (NeuGc; Fig. 1, A and D; Crocker et al., 2007). The conjugate containing the preferred native ligand of CD22, NeuGc, was bound well by wild-type cells (Fig. 1 B). Surprisingly, however, binding to this glycan by both CD22−/− and Siglecg−/− single-deficient B cells was also significantly reduced, whereas CD22−/−;Siglecg−/− double-deficient B cells completely failed to bind, indicating that both siglecs contribute to binding (Fig. 1 B). Thus, the glycan previously described as the natural ligand for CD22 (Pezzutto et al., 1987; Collins et al., 2002; Blixt et al., 2003; Crocker et al., 2007) is also a ligand of Siglec-G, and both siglecs are required for optimal binding of this ligand. Similarly, the PA–9-biphenylacetyl-NeuGcα2-6Galβ1-4GlcNAc (PA–bNeuGc) conjugate, previously shown to have high affinity for mouse CD22 (Collins et al., 2006a), was bound strongly by B cells, and whereas binding to CD22 was dominant, both CD22 and Siglec-G contributed to the association (Fig. 1 C). B cells from CD22−/−;Siglecg−/− mice exhibited residual binding to PA–bNeuGc (∼6%), suggesting that an additional receptor might also recognize this ligand. Importantly, binding to wild-type B cells of sialoside with NeuGc in α2-3 linkage to Galβ1-4GlcNAc was also observed (Fig. 1 E). However, in this case binding was exclusively caused by Siglec-G, for there was little change in binding to CD22−/− B cells. We conclude that Siglec-G and CD22 have distinct and overlapping specificities, and together bind native sialosides containing both the NeuGcα2-6 and NeuGcα2-3 linkages to Galβ1-4GlcNAc found as terminal sequences on glycans of most, if not all, mouse cells.


Decoration of T-independent antigen with ligands for CD22 and Siglec-G can suppress immunity and induce B cell tolerance in vivo.

Duong BH, Tian H, Ota T, Completo G, Han S, Vela JL, Ota M, Kubitz M, Bovin N, Paulson JC, Paulson J, Nemazee D - J. Exp. Med. (2009)

Binding analysis of various sialylated PA–glycan conjugates to wild-type compared with siglec mutant B cells. Results are representative of three independent experiments. (A–E) B cells of the indicated genotypes were stained with biotinylated PA conjugated with the following glycans: (A) NeuAcα2-6Galβ1-4GlcNAc, (B) NeuGc, (C) bNeuGc, (D) NeuAcα2-3Galβ1-4GlcNAc, and (E), NeuGcα2-3Galβ1-4GlcNAc. Binding was revealed using fluorescent streptavidin. Background staining by streptavidin alone is shown in gray. (F) Chemical structures of the NeuGc-containing sialosides.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2812539&req=5

fig1: Binding analysis of various sialylated PA–glycan conjugates to wild-type compared with siglec mutant B cells. Results are representative of three independent experiments. (A–E) B cells of the indicated genotypes were stained with biotinylated PA conjugated with the following glycans: (A) NeuAcα2-6Galβ1-4GlcNAc, (B) NeuGc, (C) bNeuGc, (D) NeuAcα2-3Galβ1-4GlcNAc, and (E), NeuGcα2-3Galβ1-4GlcNAc. Binding was revealed using fluorescent streptavidin. Background staining by streptavidin alone is shown in gray. (F) Chemical structures of the NeuGc-containing sialosides.
Mentions: Because our goal was to design copolymers of siglec ligands and a TI-2 antigen that would recruit siglecs to the hapten-specific BCR, we characterized highly multivalent sialylated glycans linked to PA for their ability to compete with cis ligands and bind to CD22 and Siglec-G on native B cells. We and others previously showed that synthetic trans ligands of CD22 (NeuGcα2-6Gal) compete poorly with endogenous cis ligands unless they were displayed on highly multivalent polymers and included hydrophobic substituents at the 9 position that increase affinity (Razi and Varki, 1998; Collins et al., 2002; Collins et al., 2006a). Therefore, we evaluated binding of highly multivalent PA conjugates (1,000 kD) carrying sialosides (n ≈ 400) and biotin tags to facilitate their detection. Each was analyzed for its binding to B cells from wild-type, CD22-deficient, Siglec-G–deficient, and CD22/Siglec-G double-deficient mice (Fig. 1). The PA–sialosides with NeuAcα2-6Galβ1-4GlcNAc–containing glycans showed no binding to mouse B cells, consistent with the known preference of mouse CD22 for NeuGcα2-6Galβ1-4GlcNAc (NeuGc; Fig. 1, A and D; Crocker et al., 2007). The conjugate containing the preferred native ligand of CD22, NeuGc, was bound well by wild-type cells (Fig. 1 B). Surprisingly, however, binding to this glycan by both CD22−/− and Siglecg−/− single-deficient B cells was also significantly reduced, whereas CD22−/−;Siglecg−/− double-deficient B cells completely failed to bind, indicating that both siglecs contribute to binding (Fig. 1 B). Thus, the glycan previously described as the natural ligand for CD22 (Pezzutto et al., 1987; Collins et al., 2002; Blixt et al., 2003; Crocker et al., 2007) is also a ligand of Siglec-G, and both siglecs are required for optimal binding of this ligand. Similarly, the PA–9-biphenylacetyl-NeuGcα2-6Galβ1-4GlcNAc (PA–bNeuGc) conjugate, previously shown to have high affinity for mouse CD22 (Collins et al., 2006a), was bound strongly by B cells, and whereas binding to CD22 was dominant, both CD22 and Siglec-G contributed to the association (Fig. 1 C). B cells from CD22−/−;Siglecg−/− mice exhibited residual binding to PA–bNeuGc (∼6%), suggesting that an additional receptor might also recognize this ligand. Importantly, binding to wild-type B cells of sialoside with NeuGc in α2-3 linkage to Galβ1-4GlcNAc was also observed (Fig. 1 E). However, in this case binding was exclusively caused by Siglec-G, for there was little change in binding to CD22−/− B cells. We conclude that Siglec-G and CD22 have distinct and overlapping specificities, and together bind native sialosides containing both the NeuGcα2-6 and NeuGcα2-3 linkages to Galβ1-4GlcNAc found as terminal sequences on glycans of most, if not all, mouse cells.

Bottom Line: Although mutations in CD22 and its signaling machinery have been associated with dysregulated B cell development and autoantibody production, previous analyses failed to identify a tolerance defect in antigen-specific mutant B cells.Our results support a role for siglecs in B cell self-/nonself-discrimination, namely suppressing responses to self-associated antigens while permitting rapid "missing self"-responses to unsialylated multimeric antigens.The results suggest use of siglec ligand antigen constructs as an approach for inducing tolerance.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Immunology and Microbial Science, The Scripps Research Institute, La Jolla, CA 92037, USA.

ABSTRACT
Autoreactive B lymphocytes first encountering self-antigens in peripheral tissues are normally regulated by induction of anergy or apoptosis. According to the "two-signal" model, antigen recognition alone should render B cells tolerant unless T cell help or inflammatory signals such as lipopolysaccharide are provided. However, no such signals seem necessary for responses to T-independent type 2 (TI-2) antigens, which are multimeric antigens lacking T cell epitopes and Toll-like receptor ligands. How then do mature B cells avoid making a TI-2-like response to multimeric self-antigens? We present evidence that TI-2 antigens decorated with ligands of inhibitory sialic acid-binding Ig-like lectins (siglecs) are poorly immunogenic and can induce tolerance to subsequent challenge with immunogenic antigen. Two siglecs, CD22 and Siglec-G, contributed to tolerance induction, preventing plasma cell differentiation or survival. Although mutations in CD22 and its signaling machinery have been associated with dysregulated B cell development and autoantibody production, previous analyses failed to identify a tolerance defect in antigen-specific mutant B cells. Our results support a role for siglecs in B cell self-/nonself-discrimination, namely suppressing responses to self-associated antigens while permitting rapid "missing self"-responses to unsialylated multimeric antigens. The results suggest use of siglec ligand antigen constructs as an approach for inducing tolerance.

Show MeSH