A novel monoclonal antibody against human B7-1 protects against chronic graft-vs.-host disease in a murine lupus nephritis model

Abstract

Lupus nephritis (LN) is the most common complication that causes mortality in patients with systemic lupus erythematosus. The B7-1/B7-2 and CD28/cytotoxic T-lymphocyte associated protein 4 co-stimulatory pathway serves a key role in autoimmune disease and organ transplantation. The aim of the present study was to generate and characterize a monoclonal antibody (mAb; clone 4E5) against human B7-1 and to investigate its potential use for the treatment of LN. The results demonstrated that the 4E5 mAb was successfully generated and able to recognize both human and mouse B7-1. After injection of this mAb into a mouse model with chronic graft-vs.-host disease (cGVHD)-induced lupus-like disease, the expression of CD21, CD23, CD80 and CD86 on B220⁺ B-cells in the spleen, and the concentrations of serum autoantibodies and urine protein, were decreased. Direct immunofluorescence analysis of the kidneys revealed that immunofluorescence of immune complex deposits was weaker in the 4E5-treated mice and electron microscopy analyses of renal tissues indicated that pathological injury of the kidneys of 4E5-treated mice was decreased compared with that in the model control mice. The results of the present study demonstrated that inhibition of the B7-1/CD28 co-stimulatory signaling pathway with the 4E5 mAb may represent a promising strategy to decelerate the progression of LN that is induced by cGVHD with potential for use in the treatment of other autoimmune diseases.

Keywords: anti-human B7-1 antibody; autoantibodies; chronic graft-vs.-host disease; electron microscopy; lupus nephritis.

Figure 1

Histograms of cells stained with 4E5. Characterization of mouse anti-human B7-1 monoclonal antibody 4E5 using flow cytometry. Analyses suggested that 4E5 was able to recognize B7-1 on the L929 (B7-1), Daudi, Raji and mouse spleen cells, but not the L929-mock cells. Representative frequency plots are presented.

Figure 2

Effects of 4E5 on the activation marker expression levels in antigen-presenting cells. Expression of CD11b, CD11c and GR1 on isolated splenocytes was evaluated using flow cytometry. The expression of each of the markers on cells from 4E5-treated mice was significantly lower than that on cells of the model control mice. Representative histograms are provided. Values in the bar graphs are expressed as the mean ± standard deviation (n=5 per group at 3 weeks). ^**P<0.01, ^***P<0.001. GR1, granulocyte receptor 1 antigen; Ig, immunoglobulin; MG, model group.

Figure 3

Effects of 4E5 on the expression of B220+ B-cells activation markers. Expression of CD21, CD23, CD80 and CD86 on B220+ B-cells from spleens were measured using flow cytometry. The expression of CD80 and CD86 on cells from 4E5 mice was significantly lower than on cells from MG mice. Representative frequency plots are provided. Values in the bar graphs are expressed as the mean ± standard deviation (n=5 per group at 3 weeks). ^*P<0.05, ^**P<0.01, ^***P<0.001. Ig, immunoglobulin; MG, model group.

Figure 4

Effects of 4E5 on serum ANA and anti-dsDNA levels at 4 months after initial inoculation. Murine sera were collected monthly for evaluation of ANA (magnification, x400) and anti-dsDNA (magnification, x100) levels. Representative fluorescent images from sera of mice at the 4-month time-point after the initial inoculation are provided. dsDNA, double-stranded DNA; ANA, anti-nuclear antibody; MG, model group.

Figure 5

Analysis of kidneys at 4 months after initiation of inoculations. (A) H&E staining was used to evaluated the kidneys. The glomeruli of MG mice exhibited glomerular volume compensatory enlargement and leukocytic infiltration in perivascular and internal areas, as well as endothelial and mesangial cell hyper-cellularity. Only minor changes were noted in the organs of 4E5 mice when compared with mock mice (magnification, x400). (B) Representative fluorescence microscopy images of kidney sections from MG mice exhibited a granular linear staining pattern of IgG deposits along glomerular capillary loops; tissues of 4E5 mice had little-to-no staining intensity, suggesting minimal deposits (magnification, x400). (C) Representative transmission electron microscopy images revealing electron-dense deposits localized in subepithelial lesions of glomerular basement membrane in kidneys of MG mice; in glomeruli of 4E5 mice, the basement membrane layer was clear and intact, the ‘humps’ were less and smaller than MG group (magnification, x2,000). ANA, anti-nuclear antibody; dsDNA, double-stranded DNA; Ig, immunoglobulin; MG, model group.