Belatacept and sirolimus prolong nonhuman primate renal allograft survival without a requirement for memory T cell depletion

Abstract

Belatacept is an inhibitor of CD28/B7 costimulation that is clinically indicated as a calcineurin inhibitor (CNI) alternative in combination with mycophenolate mofetil and steroids after renal transplantation. We sought to develop a clinically translatable, nonlymphocyte depleting, belatacept-based regimen that could obviate the need for both CNIs and steroids. Thus, based on murine data showing synergy between costimulation blockade and mTOR inhibition, we studied rhesus monkeys undergoing MHC-mismatched renal allotransplants treated with belatacept and the mTOR inhibitor, sirolimus. To extend prior work on costimulation blockade-resistant rejection, some animals also received CD2 blockade with alefacept (LFA3-Ig). Belatacept and sirolimus therapy successfully prevented rejection in all animals. Tolerance was not induced, as animals rejected after withdrawal of therapy. The regimen did not deplete T cells. Alefecept did not add a survival benefit to the optimized belatacept and sirolimus regimen, despite causing an intended depletion of memory T cells, and caused a marked reduction in regulatory T cells. Furthermore, alefacept-treated animals had a significantly increased incidence of CMV reactivation, suggesting that this combination overly compromised protective immunity. These data support belatacept and sirolimus as a clinically translatable, nondepleting, CNI-free, steroid-sparing immunomodulatory regimen that promotes sustained rejection-free allograft survival after renal transplantation.

Figure 1

Belatacept and sirolimus promote rejection-free renal allograft survival. (A) The immunomodulation regimen is shown. Rhesus macaques received belatacept and sirolimus maintenance therapy beginning the day prior to transplantation. Eight animals received adjuvant therapy with alefacept, and 3 of these animals were given a donor-specific whole blood transfusion (DST) 7 days after transplantation. Immunomodulation was discontinued sequentially, and animals were weaned off all therapies by post-transplant day 168. (B) Animals receiving belatacept and sirolimus maintained stable renal allograft function well past the discontinuation of all immunomodulation. Alefacept-treated animals demonstrated decreased rejection-free renal allograft survival. Two-tailed p-values comparing treatment groups to the belatacept and sirolimus cohort are shown.

Figure 2

Absolute T cell counts and T cell subset distribution after transplantation. Polychromatic flow cytometry was used to follow T cell counts over time. (A–C) Animals treated with belatacept and sirolimus alone maintained stable CD3, CD4 and CD8 absolute T cell counts throughout therapy (A). (B) The percentages of T_NT_CMand T_EM were analyzed. These animals maintained a high percentage of CD4 T_N cells above baseline throughout belatacept therapy, with a shift to a T_CM phenotype after belatacept was withdrawn. (C) CD8 T_N cells predominated early after transplantation with a slow increase in T_EM over time. (D–F) Animals receiving alefacept in addition to belatacept and sirolimus showed reduced absolute T cell counts during alefacept therapy (D). T cell counts began to rebound soon after alefacept was discontinued. (E) Alefacept-treated animals also experienced a more rapid conversion from CD4 T_N to T_CM and (F) CD8 T_N to T_EM compared to animals not receiving alefacept. Depletion of CD8 T_EM was transient and repopulated more rapidly relative to other T cell subsets.

Figure 3

Alefacept targets CD3⁺CD2^hi T cells. CD4 (A) and CD8 (B) T cells were segregated based on CD2 expression and evaluated for the proportion of CD2^hi T cells over time. Alefacept efficiently targeted CD4 and CD8 CD2^hi T cells (solid line). CD2^hi T cells were detected again after discontinuation of alefacept therapy. Animals receiving belatacept and sirolimus alone (dashed line) demonstrated a modest decline in the proportion of CD4 and CD8 CD2^hi T cells from baseline, which corresponded to an enrichment of both CD4 and CD8 T_N cells.

Figure 4

Belatacept, sirolimus and alefacept decrease the number of peripheral T_Reg cells. (A) The absolute number of circulating CD4⁺CD25^hiFoxP3⁺ T_Reg cells was followed over time. Belatacept and sirolimus caused a 2-fold reduction in circulating T_Reg cells that was maintained throughout belatacept therapy. The number of peripheral T_Reg cells rebounded and approached baseline once all therapies were discontinued. The addition of alefacept caused a profound 6-fold decrease in circulating T_Reg cells compared to baseline, which marginally recovered after all therapies were weaned. * p<0.01, ** p<0.001 (B) The ratio of CD2 mean fluorescence intensity (MFI) on T_Reg cells relative to all CD4 T cells was analyzed. T_Reg cells consistently expressed higher levels of CD2 compared to total CD4 T cells, regardless of treatment group.

Figure 5

Alefacept increases the frequency and rapidity of rhCMV reactivation. Animals were serially monitored for rhCMV reactivation via quantitative real-time polymerase chain reaction. Animals with rhCMV viral loads above 10,000 copies/mL were treated with ganciclovir. (A) rhCMV viral loads over time from 5 alefacept-treated animals with at least one severe rhCMV viremia (>50,000 copies/mL) are shown. Four animals had recurrent rhCMV reactivation despite treatment. (B) The time to first rhCMV reactivation is shown. Animals receiving alefacept demonstrated a strikingly earlier time to rhCMV reactivation, with the first episode occurring during alefacept treatment in all 6 animals that experienced reactivation. One animal in the belatacept and sirolimus group experienced delayed CMV reactivation on post-transplant day 175.