Chemotherapy and Inflammation Induced Damage of Intestinal Epithelium Is Associated with Increased T Cell Chemotaxis

Abstract

Gastro-intestinal (GI) Graft-versus-Host Disease (GvHD) involves a complex interplay of conditioning-related injury and the pathologic immune response at the epithelial level. Enhanced tissue damage is a risk factor for GVHD. We demonstrated that donor T cell-derived IFNγ causes intestinal stem cell damage in GvHD. Recent studies have proposed that donor T cells directly interact with GI epithelium in GvHD <i>in vivo</i>, but the precise mechanisms remain incompletely understood. Here, we evaluate the influence of both cytokine- and chemotherapy-induced intestinal epithelial damage on T cell migration and direct epithelial cell-T cell interactions using GI organoids as a model system. We developed a human intestinal organoid-T cell interaction co-culture system and studied the role of T cells in intestinal damage during GVHD. We observed that polyclonally activated T cells accumulate in close proximity to intestinal organoids (Fig. 1). In this co-culture system T cell-derived IFNg- plays a key role in tissue damage. RNA-seq analyses of IFNg-treated intestinal organoids indicated significant upregulation of T cell chemotaxis associated genes including CXCL9, 10 and 11. Luminex analysis of conditioned media confirmed release of these chemokines in co-culture versus controls. In addition, stimulation of organoids with IFNγ led to upregulation of HLA-I and HLA-II at both the RNA and membrane protein expression level (Fig. 2A). These experiments place activated T cells at the cusp of intestinal damage during GVHD through the production of cytokines, the induction of T cell recruiting chemokines and mechanisms of antigen driven cytotoxicity. Next, we set out to characterize the effect of chemotherapy on epithelium by treating organoids with Busulfan (Bu), Fludarabine (Flu) or Clofarabine (Clo). Epithelial damage was confirmed by the presence of increasing levels of caspase activation (Fig. 3A), a decrease in the number of surviving organoids, a smaller organoid size and decreased proliferation. Bu, Flu, Clo treatment did not affect HLA-I-II mRNA expression, but a variable, organoid donor-dependent upregulation of membrane HLA-I upon exposure to Bu was observed (Fig. 2B). By using a migration assay, we established that medium that we obtained from chemotherapy-treated organoids exerts increased T cell chemotaxis compared to control medium (Fig. 3B). In conclusion, both (T cell derived) IFNγ- and chemotherapy induced intestinal epithelial damage can lead to increased T cell migration <i>ex vivo</i>, resulting in direct contact between allogeneic T cells and co-cultured human intestinal epithelium. Cytokine signaling and conditioning-related injury may contribute to GVHD pathogenesis <i>in vivo</i> by promoting recruitment to intestinal crypt epithelium.