Calcineurin inhibitor withdrawal after renal transplantation with alemtuzumab: clinical outcomes and effect on T-regulatory cells

Abstract

To address the results of calcineurin inhibitor (CNI) withdrawal after alemtuzumab induction relative to CNI continuation, we performed a pilot randomized clinical trial in renal allograft recipients on CNI, a mycophenolic acid derivative and steroids after the first 2 months posttransplantation. Forty patients were randomized to taper off CNI or to maintain it, and followed for at least 1 year. Four patients in the withdrawal group were treated for acute rejection while no patient received antirejection treatment in the control group. Two control patients withdrew CNI due to nephrotoxicity. Estimated GFR was similar in both groups after 1 year. Flow cytometry of CD4(+)CD25(+)CTLA-4(+)FoxP3(+) regulatory T cells (Treg) demonstrated a significant increase in Treg percentages in the peripheral blood of alemtuzumab-treated patients on CNI early postransplant. Furthermore, the increased Treg percentages in the withdrawal cohort were unchanged at month 6 postenrollment, whereas they decreased significantly in those patients maintained on CNI. Patients withdrawn from CNI after alemtuzumab trend toward a higher rejection rate, but most patients can be weaned from a CNI using this regimen. With the exception of maintaining increased Treg levels, the benefits are not appreciable in this short follow-up, and a larger randomized trial is justified.

Figure 1:

Patient disposition during the study.

Figure 2:. T-regulatory cells in peripheral blood.

CD4⁺CD25⁺FoxP3⁺ T-regulatory cells were identified by six-color flow cytometry at the time of enrollment and 6 months thereafter for 8 withdrawal and 12 control patients. (A) Gating strategy for Tregs from frozen PBMC preparations. Cells were first gated on the live cells using the Hoechst stain, then gated on the lymphocyte gate by forward and side scatter, then gated on the CD4⁺ population and then on the CTLA-4⁺FOXP3⁺ cells. 100% of double positives in this latter gate were CD25⁺. Tregs are measured as a percentage of the total CD3+CD4+ population. (B) Comparison of 20 patients at the time of enrollment in the study (before CNI withdrawal) versus 5 healthy individuals. Statistical analysis was performed using an unpaired two-tailed t-test. (C) Comparison of Treg percentages in the CD4⁺ gate between eight patients in the withdrawal group versus 12 patients in the maintenance (control) group. Statistical analysis was performed using a paired two-tailed t-test.