A new mouse anti-mouse complement receptor type 2 and 1 (CR2/CR1) monoclonal antibody as a tool to study receptor involvement in chronic models of immune responses and disease

Abstract

Although reagents are available to block mouse complement receptor type 2 and/or type1 (CR2/CR1, CD21/CD35) function in acute or short term models of human disease, a mouse anti-rat antibody response limits their use in chronic models. We have addressed this problem by generating in Cr2−/− mice a mouse monoclonal antibody (mAb 4B2) to mouse CR2/CR1. The binding of murine mAb 4B2 to CR2/CR1 directly blocked C3dg (C3d) ligand binding. In vivo injection of mAb 4B2 induced substantial down regulation of CR2 and CR1 from the B cell surface, an effect that lasted six weeks after a single injection of 2 mg of mAb. The 4B2 mAb was studied in vivo for the capability to affect immunological responses to model antigens. Pre-injection of mAb 4B2 before immunization of C57BL/6 mice reduced the IgG1 antibody response to the T-dependent antigen sheep red blood cells (SRBC) to a level comparable to that found in Cr2−/− mice. We also used the collagen-induced arthritis (CIA) model, a CR2/CR1-dependent autoimmune disease model, and found that mice pre-injected with mAb 4B2 demonstrated substantially reduced levels of pathogenic IgG2a antibodies to both the bovine type II collagen (CII) used to induce arthritis and to endogenous mouse CII. Consistent with this result, mice pre-injected with mAb 4B2 demonstrated only very mild arthritis. This reduction in disease, together with published data in CII-immunized Cr2−/− mice, confirm both that the arthritis development depends on CR2/CR1 receptors and that mAb 4B2 can be used to induce biologically relevant receptor blockade. Thus mAb 4B2 is an excellent candidate for use in chronic murine models to determine how receptor blockage at different points modifies disease activity and autoantibody responses.

Foot-and-mouth disease virus localisation on follicular dendritic cells and sustained induction of neutralising antibodies is dependent on binding to complement receptors (CR2/CR1)

“…Up to 35 days post inoculation mAb 4B2 is still capable of blocking CR, with a significant reduction of anti-CR2/CR1 mAbs binding to splenocytes, as demonstrated not only by mAb 7G6, but mAbs 8C12, which is monospecific to CR1, and 7E9, which binds a different epitope on CR2/CR1 (Fig 1B). This is in alignment with previous data, whereby the blocking effect is not purely due to steric inhibition, but induces a substantial decrease in the expression level of receptors when mAb 4B2 is used in vivo (30).…”

“…The most notable reduction was the binding of mAb 7G6 to splenocytes from 2 days post inoculation (Fig 1A). This mAb binds a similar and overlapping epitope on CR1 and CR2 as mAb 4B2 as described previously (30).…”

Targeting the Immune Complex–Bound Complement C3d Ligand as a Novel Therapy for Lupus

“…with 200 ml of 1 3 10 7 SRBC per mouse. Detection of Ab to SRBC was measured by ELISA, as described previously (36,37). Bound Abs were detected by incubation with alkaline phosphatase (AP)-conjugated anti-mouse IgG1 Ab (Jackson ImmunoResearch), followed by washing and addition of p-nitrophenyl phosphate as the AP substrate (Sigma-Aldrich) at a concentration 1 mg/ml.…”

“…Altered levels of CR2 expression on B cells from patients with SLE and lower levels of CR2/CR1 expression on B cells from MRL/lpr mice are a hallmark of B cell changes in lupus (45-47). When we stained splenic B cells for the level of CR2/CR1 using mAb 4B2 (37) (Fig. 9A) or for the level of CR1 using anti-CR1-specific mAb 8C12 (46) (Fig.…”

Complement Properdin Determines Disease Activity in MRL/lpr Mice

“…The observed, ameliorated, phenotype in MRL/ lpr P KO compared with congenic MRL/ lpr P WT cannot be explained by just the absence of alternative pathway amplification caused by immune complex mediated complement activation. The reason for lower autoantibody production in MRL/ lpr P KO is not clear, but, based on current developments in the field [ 31 , 32 ], likely involves relative changes in signalling strengths via CR2/C3d, which may be exploitable for clinical application [ 33 ]. CR2 is of interest because CR2 is part of the B cell receptor complex and C3d binding leads to augmented BCR signalling.…”