Complement Component C3d-Antigen Complexes Can Either Augment or Inhibit B Lymphocyte Activation and Humoral Immunity in Mice Depending on the Degree of CD21/CD19 Complex Engagement

Abstract

C3d can function as a molecular adjuvant by binding CD21 and thereby enhancing B cell activation and humoral immune responses. However, recent studies suggest both positive and negative roles for C3d and the CD19/CD21 signaling complex in regulating humoral immunity. To address whether signaling through the CD19/CD21 complex can negatively regulate B cell function when engaged by physiological ligands, diphtheria toxin (DT)-C3d fusion protein and C3dg-streptavidin (SA) complexes were used to assess the role of CD21 during BCR-induced activation and in vivo immune responses. Immunization of mice with DT-C3d3 significantly reduced DT-specific Ab responses independently of CD21 expression or signaling. By contrast, SA-C3dg tetramers dramatically enhanced anti-SA responses when used at low doses, whereas 10-fold higher doses did not augment immune responses, except in CD21/35-deficient mice. Likewise, SA-C3dg (1 μg/ml) dramatically enhanced BCR-induced intracellular calcium concentration ([Ca2+]i) responses in vitro, but had no effect or inhibited [Ca2+]i responses when used at 10- to 50-fold higher concentrations. SA-C3dg enhancement of BCR-induced [Ca2+]i responses required CD21 and CD19 expression and resulted in significantly enhanced CD19 and Lyn phosphorylation, with enhanced Lyn/CD19 associations. BCR-induced CD22 phosphorylation and Src homology 2 domain-containing protein tyrosine phosphatase-1/CD22 associations were also reduced, suggesting abrogation of negative regulatory signaling. By contrast, CD19/CD21 ligation using higher concentrations of SA-C3dg significantly inhibited BCR-induced [Ca2+]i responses and inhibited CD19, Lyn, CD22, and Syk phosphorylation. Therefore, C3d may enhance or inhibit Ag-specific humoral immune responses through both CD21-dependent and -independent mechanisms depending on the concentration and nature of the Ag-C3d complexes.

DENV-2 subunit proteins fused to CR2 receptor-binding domain (P28)-induces specific and neutralizing antibodies to the Dengue virus in mice

“…This evidence suggests that C3d does not always function as expected, i.e., the conjugation of certain antigens to C3d does not guarantee an adjuvant effect. 41 Experimental data suggest that the interaction of P28 with CR2 is complex and involves more than one mechanism, including electrostatic interactions. Owing to the small size of P28, the mechanisms by which it interacts with CR2 may be limited.…”

The ABCs (Antibody, B Cells, and Carbohydrate Epitopes) of Cholera Immunity: Considerations for an Improved Vaccine

“…Complement receptors on B cells can bind C3d, a proteolytic product of the complement cascade protein C3 which can be covalently bound to bacterial LPS or to outer membrane structures. The complement receptor, CD21, is associated with other membrane proteins including CD19 and CD81 that together form signaling complexes (136). Antigen-complement binding to B cell membrane receptors enhances the activation of B cells by lowering the threshold of cross linking required for B cell activation (148).…”

Defective B cell ontogeny and immune response in human complement receptor 2 (CR2, CD21) transgenic mice is partially recovered in the absence of C3

“…The down regulation of hCR2 expressions levels is consistent with this idea and suggests the B cell attempts to reduce the comparatively increased negative signal generated through hCR2 in the absence of CD19 ( Figure 1C). The potential for direct signaling through CR2 has not been totally ruled out (Delcayre et al, 1987;Frade et al, 1992) and despite overwhelming data confirming the positive signaling potential of CR2 (Fearon and Carroll, 2000;Fearon and Carter, 1995), negative signaling in response to CR2 ligation has also been demonstrated (Chakravarty et al, 2002;Lee et al, 2005). CR2 dependent BCR signal amplification has been shown to depend on CD19 (Lyubchenko et al, 2005), but CD19 independent effects could result from CR2 being free to associate and/or interfere with other signaling proteins or processes that it would not normally have access to or depend on CD19 during this phase of B cell development.…”