Immunosuppression Withdrawal in Liver Transplant Recipients on Sirolimus

Abstract

Background and aims: As conversion from calcineurin inhibitor to sirolimus (SRL), a mechanistic target of rapamycin inhibitor (mTOR-I), has been shown to enhance immunoregulatory profiles in liver transplant (LT) recipients (LTRs), mTOR-I therapy might allow for increased success of immunosuppression (IS) withdrawal. Our aim was to determine if operational tolerance could be observed in LTRs withdrawn from SRL and if blood/graft tolerance biomarkers were predictive of successful withdrawal.

Approach and results: We performed a prospective trial of SRL monotherapy withdrawal in nonimmune, nonviremic LTRs > 3 years post-LT. SRL was weaned over ~6 months, and biopsies were performed 12 months postweaning or at concern for acute rejection. Twenty-one LTRs consented; 6 were excluded due to subclinical acute rejection on baseline biopsy or other reasons, and 15 underwent weaning (age 61.3 ± 8.8 years; LT to SRL weaning 6.7 ± 3 years). Eight (53%) achieved operational tolerance (TOL). Of the 7 who were nontolerant (non-TOL), 6 had mild acute rejection on biopsy near the end of weaning or at study end; 1 was removed from the trial due to liver cancer recurrence. At baseline preweaning, there were statistically increased blood tolerogenic dendritic cells and cell phenotypes correlating with chronic antigen presentation in the TOL versus non-TOL groups. A previously identified biopsy gene signature accurately predicted TOL versus non-TOL in 12/14 LTRs before weaning. At study end, biopsy staining revealed statistically significant increases in antigen-presenting cell:leukocyte pairings, FOXP3+ /CD4+ T cells, Tbet+ /CD8+ T cells, and lobular dendritic cells in the non-TOL group.

Conclusions: This study evaluated IS withdrawal directly from mTOR-I therapy in LTRs and achieved > 50% operational tolerance. Preweaning gene expression and peripheral blood mononuclear cell profiling may be useful as predictors of successful mTOR-I therapy withdrawal. NCT02062944.

© 2019 by the American Association for the Study of Liver Diseases.

Figures

Figure 1:. Study design.

Timing of visits, SRL presence/absence, and clinical and mechanistic tests are displayed over the study period.

Figure 2:. Diagram of study enrollment.

The number of participants eligible for the study based on the inclusion/exclusion criteria is indicated.

Figure 3:. Real-time flow cytometry detects baseline (pre-weaning) differences in DC and B cell populations between TOL and non-TOL participants.

PBMC were processed and stained in real-time for surface and intracellular markers to detect cells with regulatory phenotypes. Panel A: Tolerogenic DC (HLA-DR+CD11c+ILT4+ILT3+); Panel B: Bregs (CD19+IgM+IgD+IL-10+); Panel C: Tregs (CD4+Foxp3+, panel C). Patient numbers: TOL (n = 8 at baseline and n = 7 at study end for Panel A; n=8 at baseline and at study end for Panels B and C) and non-TOL (n=7 at baseline; n=6 at study end). Symbols represent medians; bars represent IQR. Medians are presented as the percentage of the parent population (DC scatter, CD19+ B cells, or CD4+ T cells, as appropriate). *p<0.05; **p<0.01; ***p<0.001.

Figure 4:. Batched flow cytometry detects baseline (pre-weaning) differences in antigen non-specific memory cell populations between TOL and non-TOL participants.

PBMC were processed in real-time and frozen until staining for surface and intracellular markers to detect naïve and memory cell populations. Naïve CD8+ T cells (Panel A), CD8+ TEMRA T cells (Panel B), CD8+Eomes+ T cells (Panel C), and CD8+Eomes+T-bet+ T cells (Panel D) are depicted for TOL and non-TOL participants (n=8 and n=6 at baseline and end of study, respectively). Symbols represent medians; bars represent IQR. Medians are presented as the percentage of the parent population, CD8+ T cells. *p<0.05; **p<0.01; ***p<0.001.

Figure 5:. DSA presence, absence, or development did not correlate with IS withdrawal outcome.

Serum samples were tested for DSA from TOL (n=8) and non-TOL (n=6) participants at screening, TOL (n=8) and non-TOL (n=5) before their last dose of SRL, and TOL (n=8) and non-TOL (n=3) following completion or termination of SRL withdrawal. The maximum MFI detected (Panel A) and sum of all DSA MFI detected (Panel B) are depicted. The upper limit of the linear range of detection for this assay is an MFI of 20,000; changes in DSA concentration above this MFI may not be detectable due to bead saturation. MFI values below 1,000 are considered negative and imputed for representation only.

Figure 6:. Immunohistochemistry detects differences between biopsies at baseline and end of study.

Biopsies were obtained from TOL and non-TOL participants at baseline and end of study. P values and n values for each stain/group combination can be found in Supplemental Table 8. Panels show representative images from end of study biopsies for TOL (left column) and non-TOL (right column) participants stained for APC:lymphocyte pairings (Panel A: in yellow circles; PT = portal tract), Foxp3+ and T-bet+ T cells (Panel B), and MHCII+CD11c+ILT4+ DC (Panel C). Main image bar = 50 μm, inset image bar = 10 μm or 5μm as indicated.