B cell gene signature with massive intrahepatic production of antibodies to hepatitis B core antigen in hepatitis B virus-associated acute liver failure

Abstract

Hepatitis B virus (HBV)-associated acute liver failure (ALF) is a dramatic clinical syndrome due to a sudden loss of hepatic cells leading to multiorgan failure. The mechanisms whereby HBV induces ALF are unknown. Here, we show that liver tissue collected at the time of liver transplantation in two patients with HBV-associated ALF is characterized by an overwhelming B cell response apparently centered in the liver with massive accumulation of plasma cells secreting IgG and IgM, accompanied by complement deposition. We demonstrate that the molecular target of these antibodies is the hepatitis B core antigen (HBcAg); that these anti-bodies display a restricted variable heavy chain (V(H)) repertoire and lack somatic mutations; and that these two unrelated individuals with ALF use an identical predominant V(H) gene with unmutated variable domain (IGHV1-3) for both IgG and IgM anti-HBc antibodies, indicating that HBcAg is the target of a germline human V(H) gene. These data suggest that humoral immunity may exert a primary role in the pathogenesis of HBV-associated ALF.

Fig. 1.

(A) Histopathologic features of HBV-associated ALF. Liver specimens were obtained from the native livers of patients 1 and 2 at the time of OLT. In patient 1, the parenchyma shows massive hepatic necrosis with the presence of vascular spaces. There are no viable hepatocytes; lobular lymphoid cell infiltrates and Kupffer cells are prominent. The collapse of the lobules has resulted in the proximity of the portal tracts, which show extensive lymphoid infiltrates and bile duct regeneration. In patient 2, the parenchyma shows submassive liver necrosis with prominent lobular lymphoid cell infiltrates and Kupffer cell hyperplasia. Portal areas show prominent lymphoid cell infiltrates. (hematoxylin and eosin; magnification: ×75.) (B) Gene expression profiling of HBV-associated ALF. Multidimensional-scaling plot showing the 3D projection of eight liver specimens from two patients with HBV-associated ALF (four specimens per patient) and eight from individual liver donors with the use of all 15,652 transcripts that passed the filtering criteria. In the scatterplot, each point represents a liver specimen, and the distance between points is proportional to the overall dissimilarity of gene expression profiles. The eight samples from patients with ALF are shown in red (dark red dots, patient 1; light red dots, patient 2). The eight samples from the control liver donors are shown in blue. This plot illustrates how the gene expression profiles differentiate between the two classes.

Fig. 2.

Supervised hierarchical cluster analysis showing the differential expression of immune response-related transcripts in HBV-associated ALF and control liver donors. Each row represents data for a particular human transcript, and each column the expression of transcripts in a single liver specimen. The color in each cell reflects the level of expression of the corresponding transcript in the corresponding sample, relative to its mean level of expression in the entire set of 16 samples. Mean-centered ratios of transcript expression are depicted by a log₂-transformed scale. According to the color scale, up-regulated transcripts are shown in shades of red and down-regulated transcripts in shades of green.

Fig. 3.

Immunohistochemical staining of B cell lineage markers in liver tissue from two patients with HBV-associated ALF and a representative control liver donor. Liver specimens obtained at the time of OLT from the native livers of patient 1 with massive hepatic necrosis and patient 2 with submassive hepatic necrosis, and from a control liver donor, were stained with monoclonal antibodies against CD20, MUM1/IRF4, IgM, and IgG with the use of immunoperoxidase. Sections from patient 1 and patient 2 show the presence of B cell clusters (CD20) around the portal areas; strong nuclear staining for MUM1/IRF4 is seen in a large number of plasmacytoid cells and plasma cells diffusely distributed in the lobules; IgM-secreting mature plasma cells are seen predominantly in the lobules; IgG-secreting plasma cells show a greater range of differentiation with plasmacytoid cells containing more open chromatin and often a prominent nucleolus (Fig. S5 A and B). In addition, a diffuse staining for IgG, and to a lesser extent IgM, is seen in the intercellular spaces and in patient 2 on the surface of residual hepatocytes. Sections from the control liver show few CD20-positive cells within the portal space and limited Ig deposits in the sinusoids. (Magnification: ×75.)

Fig. 4.

Immunohistochemical staining of T cell lineage markers in liver tissue from two patients with HBV-associated ALF and a representative control liver donor. Liver specimens obtained at the time of OLT from the native livers of patient 1 with massive hepatic necrosis and patient 2 with submassive hepatic necrosis, and from a control liver donor, were stained with monoclonal antibodies against CD3, CD4, CD8, granzyme B (GzB), and perforin with the use of immunoperoxidase. Liver sections from patient 1 and patient 2 show the presence of T cell clusters (CD3), with CD8-positive cells diffusely distributed within the lobules and few dispersed CD4-positive cells. Low-level diffuse CD4 staining reflects the extensive tissue infiltration by macrophages. Only a small proportion of the CD8-positive cells express granzyme B, and an even smaller proportion express perforin. Sections from the control liver show only rare cells expressing T cell markers, granzyme B, or perforin. (Magnification: ×65.)

Fig. 5.

Degree of liver necrosis in HBV-associated ALF and immunohistochemical staining of complement in liver tissue from two patients with HBV-associated ALF and a representative control liver donor. Liver sections were stained with hematoxylin and eosin. In patient 1, the parenchyma shows massive hepatic necrosis with no viable hepatocytes; in patient 2, the parenchyma shows submassive liver necrosis with residual hepatocytes (arrows and Inset). The control liver donor shows normal liver histology. Liver sections obtained from patient 1 and patient 2, and from a normal liver donor, were also stained with antibodies against C1q, C3, and C4d with the use of immunoperoxidase. In patient 1, C4d staining is seen on the endothelial surface of the central vein (Inset), as well as focally in a macrophage-like cell (arrowhead). Deposition of C1q and C3 is seen mainly within macrophage-like cells, focally with a granular pattern suggestive of immune complexes. No residual hepatocytes are visible. In patient 2, C4d staining is seen on the endothelial surface of the central vein (Inset), as well as in macrophage-like cells (arrowheads) and in residual hepatocytes (arrows). Deposition of C1q and C3 is seen within macrophage-like cells, focally with a granular pattern suggestive of immune complexes. There is also C1q staining along intercellular spaces (Inset), suggesting plasma membrane deposition, and focal C3 staining of residual hepatocytes. Liver tissue from the control liver donor shows no significant staining for any of these complement proteins. (Magnification: ×90; Insets, ×270.)