RNA Sequencing of Intestinal Biopsies Expands the T Cell-Centric Paradigm of Steroid-Refractory Acute Graft-Versus-Host Disease

Abstract

Background Steroid-refractory acute GVHD (aGVHD) is a fatal disease with little known regarding its pathophysiology at the tissue level. We studied the gene expression profiles of onset and steroid-refractory aGVHD intestinal biopsies, including both supervised analyses of tolerance (indoleamine 2,3 dioxygenase [IDO1]), repair factors (amphiregulin [AREG]), and immune cell composition, plus unsupervised analyses to define the transcriptome profile. Patients and Methods We performed ribonucleic acid sequencing (RNA-seq) on archival rectal biopsies from 22 patients with clinical stage 3-4 GI aGVHD (a) at the onset of GI aGVHD and (b) in the same patients at the diagnosis of steroid-refractory GI aGVHD, compared to 10 healthy controls at the University of Minnesota. We compared differential gene expression using Limma package, analyzed pathways using Enrichr®, and estimated immune cell composition using CIBERSORT®. Results Several immune- and damage-relevant genes differed between GVHD and normal samples. The mostly highly differentially expressed gene in aGVHD compared to normal was chitinase 3-L-1 (CHI3L1, fold-change 3.4, adjusted p=0.003), with known pathogenic roles in neovascularization, macrophage recruitment, and bacterial adhesion. The most significantly decreased gene in aGVHD was aquaporin-8 (AQP8, fold change -5.0, adjusted p Discussion While we observed distinctions between normal and onset aGVHD samples (increased CHI3L1 and decreased AQP8), we did not identify differences in T-cell driven inflammation in onset versus steroid-refractory aGVHD. Steroid-refractory aGVHD may be characterized by increased stress-induced MT expression, loss of host tissue tolerance signals, and accumulation of M2 macrophages. Our results expand the paradigm of T cell-centric therapies for steroid-refractory GI aGVHD and highlight new mechanisms for therapeutic exploration.