T-Cell Transcriptome Analysis Reveals the Mechanisms Controlling Synergy Between Costimulation Blockade and mTOR Inhibition during Gvhd Prevention

While the first-in-disease trials of GvHD prevention with costimulation blockade add CTLA4-Ig to standard tacrolimus/methotrexate, previous work has suggested that mTOR inhibition with sirolimus is more pro-tolerogenic when paired with costimulation blockade.  We have now investigated combination CTLA4-Ig + sirolimus to prevent GvHD in the NHP model, and have interrogated the outcomes using a systems-based approach. Our experiments provide strong clinical, immunologic and trancriptomic evidence for potent synergy when CTLA4-Ig and sirolimus are combined for GvHD prevention.  

We determined the impact of the following treatments on GvHD after haplo-identical HSCT: 1) no therapy (n=4) 2) CTLA4-Ig monotherapy (using belatacept, n=3) 3) sirolimus monotherapy (n=4) 4) combination belatacept and sirolimus (n=3).  To determine the relative impact of each therapeutic approach, we monitored clinical GvHD, GvHD-free survival, and flow cytometric signs of immune activation post-transplant. Moreover, to create a comprehensive molecular map of their impact on GvHD, we performed transcriptomic analysis, on CD3+/CD20- T cells that were purified on day +14.

The synergistic impact of combined belatacept +sirolimus was evidenced through all analyses techniques: Thus, GvHD-free survival with belatacept + sirolimus was prolonged compared to all other groups (MST belatacept + sirolimus = 33d, p < 0.02 compared to MST for untreated controls (7.5d), belatacept monotherapy (9d, p < 0.03) and sirolimus monotherapy (12d, p <0.05), with GvHD clinical scores as well as canonical flow cytometric signs of CD8+ T cell activation and excessive cytotoxicity mirroring the clinical survival.

Comparing the expression profile of T cells during acute GvHD allowed examination of treatment synergy at an unprecedented level of molecular detail.  Unsupervised analysis revealed clustering of principal components from belatacept + sirolimus to be strikingly similar to a large comparative healthy control cohort (n=28), underscoring the high degree of early control of alloreactivity with this regimen. Moreover, the comparison of differentially expressed genes from animals receiving belatacept + sirolimus revealed significant divergence from monotherapy with either belatacept or sirolimus, again underscoring the profound control of allo-activation that was observed with belatacept + sirolimus. Those genes for which expression was significantly normalized showed pathway enrichment prominently in T cell effector function (prominently including granzyme signaling), cytokine networks (prominently IL2, IL12 and IL-18), as well as in proliferation and cell cycle pathways. 

These data reveal a treatment synergy between T cell costimulation blockade with CTLA-4-Ig and mTOR inhibition and suggest that this combination of therapies will be useful for acute GvHD immunoprophylaxis in humans.