Impact of Antibodies That React With Liver Tissue and Donor-Specific Anti-HLA Antibodies in Pediatric Idiopathic Posttransplantation Hepatitis

Abstract

Background: The cause of late graft dysfunction has not been elucidated. Although an antibody-mediated reaction is suspected as a potential mechanism, the target antigens have not been clarified.

Methods: To clarify the etiology of idiopathic posttransplantation hepatitis (IPTH), we simultaneously examined the presence of antibodies that react with liver tissue (ARLT) by means of indirect immunofluorescence staining, as well as the presence of donor-specific anti-human leukocyte antigen antibodies (HLA-DSA). A subanalysis of the IPTH group was also performed. Within the IPTH group, the correlation between ARLT titer and clinical data were analyzed.

Results: In the sera of patients with IPTH (30 patients), ARLT were found at a significantly higher frequency than in patients without IPTH (42 patients; P < 0.001). Moreover, the ARLT titer appeared to be correlated with the severity of hepatitis or hepatic injury. In contrast, the frequency of HLA-DSA was significantly lower in patients with IPTH than in patients without IPTH (P = 0.001).

Conclusion: Our findings indicate that ARLT, and not HLA-DSA, profoundly influence the etiology of IPTH.

FIGURE 1

Patient classification. Thirty IPTH patients and 42 control patients with matching background, including age at LTx, follow-up period, and gender, were enrolled. The control group was classified into 3 subgroups according to the pathological findings (inflammation, fibrosis, and normal). In a subanalysis of the IPTH patients, the IPTH group was classified into 3 subgroups according to the immunofluorescence staining results (negative, positive, and strongly positive).

FIGURE 2

Immunofluorescence staining patterns. (1) Strongly positive, (2) positive, (3) negative, and (4) negative control (healthy volunteer). Each patient’s serum served as the primary antibody, while anti-human IgG antibody conjugated to CF488A Dye served as the secondary antibody. There was no cross-reactivity of the secondary antibody to rat liver tissue. All images were obtained at 100× magnification. Staining of the hepatocyte cytoplasm was evaluated. Kupffer cells and the vascular endothelium showed higher staining intensity than hepatocytes, even with the serum of the healthy volunteer, and intense staining of Kupffer cells or the vascular endothelium was considered nonspecific.

FIGURE 3

Fluorescence intensity of ARLT and HLA-DSA. (1) Fluorescence intensity of ARLT in the 4 groups classified according to the pathological findings. Patients with IPTH showed higher fluorescence intensity than other patients, and a significant difference was noted between patients with IPTH and the other groups. *P < 0.001. (2) MFI of HLA-DSA. The IPTH group showed a significantly lower MFI than the other groups. **P = 0.001.

FIGURE 4

IgG subclass immunofluorescence staining of IPTH patients.(1) IgG1, (2) IgG2, (3) IgG3, (4) IgG4. Twenty-four of 26 ARLT-positive patients were IgG1-positive.

FIGURE 5

Time course of the IPTH relapse patients. Panels 1, 2, and 3 show the time course of the pathological findings, AST level, ALT level, and the fluorescence intensity of ARLT. Three patients experienced relapse and remission of IPTH. The fluorescence intensity of ARLT was correlated with the AST level, ALT level, and pathological findings. The R² between AST and ARLT and between ALT and ARLT for patients 1, 2, and 3 were 0.74, 0.76, and 0.72 and 0.64, 0.99, and 0.80 respectively. The X-axis represents months after LTx. The left Y-axis shows the levels of AST and ALT, and the right Y-axis shows the intensity of immunofluorescence staining. A1 and A2 illustrate the hepatitis activity score according to the METAVIR scoring system. Panel 4 shows the immunofluorescence staining during IPTH progression in a patient. The 4 figures (A), (B), (C), and (D) correspond to the time points (A), (B), (C), and (D), respectively, in panel 2.