Five-year histological and serological follow-up of operationally tolerant pediatric liver transplant recipients enrolled in WISP-R

Abstract

Pediatric liver transplant recipients arguably have the most to gain and the most to lose from discontinuing immunosuppression (IS). Whereas IS undoubtedly exerts a cumulative toll, there is concern that insufficient or no IS may contribute to allograft deterioration. Twelve pediatric recipients of parental living donor liver grafts, identified as operationally tolerant through complete IS withdrawal (WISP-R; NCT00320606), were followed for a total of 5 years (1 year of IS withdrawal and 4 years off IS) with serial liver tests and autoantibody and alloantibody assessments. Liver biopsies were performed 2 and 4 years off IS, and, at these time points, immunoglobulin G (IgG) subclass and C1q binding activity for donor-specific antibodies (DSAs) were determined. There were no cases of chronic rejection, graft loss, or death. Allografts did not exhibit progressive increase in inflammation or fibrosis. Smooth-muscle actin expression by stellate cells and CD34 expression by liver sinusoidal endothelial cells remained stable, consistent with the absence of progressive graft injury. Three subjects never exhibited DSA. However, 3 subjects showed intermittent de novo class I DSA, 4 subjects showed persistent de novo class II DSA, and 5 subjects showed persistent preexisting class II DSA. Class II DSA was predominantly against donor DQ antigens, often of high mean fluorescence intensity, rarely of the IgG3 subclass, and often capable of binding C1q.

Conclusion: Operationally tolerant pediatric liver transplant recipients maintain generally stable allograft histology in spite of apparently active humoral allo-immune responses. The absence of increased inflammation or progressive fibrosis suggests that a subset of liver allografts seem resistant to the chronic injury that is characteristic of antibody-mediated damage. (Hepatology 2017;65:647-660).

Figure 1. WISP-R timeline for transplant center visits, antibody assessments, and liver biopsies

Schematic showing timeline of IS withdrawal, assessment of primary endpoint, and long-term follow-up, including timing and frequency of transplant center visit and liver biopsies

Figure 2. ALT and GGT Profiles for Operationally Tolerant WISP-R Subjects

The 12 operationally tolerant subjects are divided into three groups as previously described (14). A. Six subjects exhibiting generally stable profile throughout study follow-up B. Three subjects with discrete spikes in ALT and GGT, reflecting the diagnosis of biliary obstruction made during the study C. Three subjects exhibiting persistent and/or recurrent elevation of predominantly GGT during the study

Figure 2. ALT and GGT Profiles for Operationally Tolerant WISP-R Subjects

The 12 operationally tolerant subjects are divided into three groups as previously described (14). A. Six subjects exhibiting generally stable profile throughout study follow-up B. Three subjects with discrete spikes in ALT and GGT, reflecting the diagnosis of biliary obstruction made during the study C. Three subjects exhibiting persistent and/or recurrent elevation of predominantly GGT during the study

Figure 3. Inflammation and Fibrosis Scores and Class II DSA MFIs (Single Antigen Bead, IgG 1–4 Subclasses, and C1q) for Operationally Tolerant WISP-R Subjects

For each operationally tolerant subject, data is shown at three time points: baseline (BL), prior to study entry and IS withdrawal; Yr 3, three years after study entry, corresponding to 2+ years after last dose of IS; and Yr 5, five years after study entry, corresponding to 4+ years after last dose of IS. The top two rows show inflammation and fibrosis scores (range 0 – 3) by compartment. The remaining rows show single antigen bead MFIs, IgG 1, 2, 3, and 4 MFIs, and C1q binding activity MFIs.

Figure 3. Inflammation and Fibrosis Scores and Class II DSA MFIs (Single Antigen Bead, IgG 1–4 Subclasses, and C1q) for Operationally Tolerant WISP-R Subjects

For each operationally tolerant subject, data is shown at three time points: baseline (BL), prior to study entry and IS withdrawal; Yr 3, three years after study entry, corresponding to 2+ years after last dose of IS; and Yr 5, five years after study entry, corresponding to 4+ years after last dose of IS. The top two rows show inflammation and fibrosis scores (range 0 – 3) by compartment. The remaining rows show single antigen bead MFIs, IgG 1, 2, 3, and 4 MFIs, and C1q binding activity MFIs.

Figure 4. Biopsies from two representative WISP-R subjects with consistently normal liver test profiles as shown in Figure 2A

Biopsies from two representative WISP-R participants (Subjects 4 and 5; liver test profiles shown in Figure 2A) are shown. A scale bar is in the upper left corner of the panels. Trichrome-stained sections of entire Yr 0 and Yr 5 biopsies captured at low magnification from Subject 4 (A) and 5 (C) show intact architecture. Higher magnification of representative areas of Yr 0 and Yr 5 biopsies are shown (B and D). Each inset shows higher magnification of representative portal tracts: all are devoid of inflammation. Finally, note that the thinner, 18 gauge Yr 0 biopsy is fragmented whereas the thicker, 16 gauge, Yr 5 biopsy is not (A).

Figure 5. Biopsies from three representative WISP-R subjects with abnormal liver test profiles as shown in Figures 2B and 2C

Biopsies from three representative WISP-R participants are shown. The scale bar is shown in the upper left corner of the panels. Two participants (Subjects 1 and 14; liver test profiles in Figure 2B) exhibited the most significant histopathological changes during the study had biliary obstruction secondary to anastomotic stricture that required intervention(s) during the five-year follow-up period. A–B: Subject 1: Trichrome-stained sections of Yr 0 and Yr 5 biopsies: Severe NRH changes and biopsy fragmentation were already evident at baseline, prior to IS withdrawal. The Yr 5 biopsy shows increased subsinusoidal fibrosis and architectural distortion. No portal inflammation was evident in either the Yr 0 or Yr 5 biopsy (A: H&E-stained upper right insets). C: Subject 14: Yr 0 and Yr 5 biopsies: This subject showed obvious obstructive cholangiopathic changes and increased portal/periportal fibrosis. The insets (40X magnification) show non-inflamed portal tracts. The third participant (Subject 9; liver test profile in Figure 2C) exhibited fluctuations in GGT. D: Subject 9: Yr 0 and Yr 5 biopsies showed neither inflammation nor noticeable change in fibrosis over five years.

Figure 6. Sum of Class II DSA MFIs and Biopsy C4d and MHC Class II Scores for Operationally Tolerant WISP-R Subjects

For each operationally tolerant subject, data is shown at two time points: baseline (BL), prior to study entry and IS withdrawal and Yr 5, five years after study entry, corresponding to 4+ years after last dose of IS. The sum of Class II DSA single antigen and C1q binding MFIs and at the specified time point is shown, aligned with the C4d and MHC Class II scores by compartment.