Defining the GvHD Transcriptome: Network Analysis Identifies the Key Pathways Responsible for Primate GvHD Pathogenesis and the Mechanisms of Partial GvHD Control with Sirolimus

We have developed a systems-biology approach to studying GvHD, using whole-transcriptome analysis of pathogenic T cells.  Using computational methods, we have identified, for the first time, the transcriptional networks that drive primate GvHD, and that lead to its partial control through mTOR inhibition with sirolimus.

Methods:  CD3+/CD20- T cells were purified flow cytometrically from 4 cohorts: (1) Healthy Controls (HC n = 15); (2) Auto-HSCT recipients (n = 3); (3) Untreated Allo-HSCT recipients who developed severe GvHD, (GvHD n = 4); and (4) Allo-HSCT recipients receiving sirolimus alone (Sirolimus n = 4).  Purification of T cells and RNA was followed by primate-specific Affymetrix Gene Array analysis.

Computation: Gene array signals were processed using the Robust Multichip Averaging Method.  Principal Component Analysis (PCA) revealed that variation was primarily determined by the experimental cohort (Fig1A). This result was critical, and confirmed that transcriptomics could be applied to identify genes and pathways controlling GvHD.  Differentially expressed genes (DE) were defined, yielding unique and overlapping gene signatures, with 775 DE genes between GvHD and HC and 286 DE genes between Sirolimus and HC (Fig 1B). Finally, using Ingenuity Pathway Analysis (IPA) we characterized gene signatures according to molecular pathways (Fig 1C).

Results: T cells from animals with severe aGvHD showed transcriptional signs of rampant proliferation and cytotoxicity, but also of cell death.  IPA identified highly statistically significant upregulation of Cell Cycle as well as Cell Trafficking and Inflammatory Response networks (Fig 1C, p< 0.001) These networks contained some expected genes and some surprises. Thus, GvHD was associated with upregulation of JAK and IFN signaling (p < 0.001), but unexpectedly, was also associated with upregulation of the Sonic Hedgehog and Aurora Kinase A Pathways (p < 0.01). Both of these represent targetable pathways with clinically-available novel therapeutics.  

Sirolimus resulted in partial downregulation of proliferation and cytotoxicity pathways.  However, many genes and networks were shared between the Sirolimus and GvHD cohorts, indicating inadequately controlled activation. These prominently included upregulation of the FOXM1 and IRF8 transcription factors, involved in cell cycle progression and interferon signaling (p< 0.01), respectively. Both GvHD and Sirolimus also demonstrated upregulation of the CD28, CCR5, IL-12 and IL-17 pathways (p <0.05), all targetable with FDA-approved therapeutics (CTLA4-Ig, maraviroc, ustekinumab).

Conclusions:  This is the first description of the primate GvHD transcriptome. This network approach has identified previously unappreciated genes and pathways associated with GvHD, for which several novel therapeutic strategies are immediately available for pre-clinical and clinical evaluation.