|dc.description.abstracteng||Major histocompatibility complex (MHC) matching is essential to reduce the risk of graft versus host disease (GvHD) but minor histocompatibility antigens (mHags) also affect the outcome of hematopoietic stem cell transplantation (HSCT). In addition, single nucleotide polymorphisms (SNPs), which do not give rise to mHags, can influence the result of HSCT. In addition to genotyping, profiling of gene expression in tissues affected by GvHD might identify genes, which are important for the development of GvHD.
Previously, 11 non-class I/II MHC and 174 other genes were identified to be regulated during GvH reactions in rat skin explant assays, which provide an in vitro model of GvHD as well as in rat GvHD models. The expression of 27 of these genes was tested in human skin explant assays and in human GvHD skin biopsies and several genes were confirmed to be regulated also in the human skin biopsies. In this study, we aimed to validate the regulation of these candidate genes in tissues affected by acute GvHD (aGvHD) in two animal models and compare their regulation in 186 gastrointestinal biopsies from patients after HSCT. Notably, the candidate genes HCLS1, UBD and TGM2 were strongly upregulated in all the mice aGvHD tissues compared to controls. A similar trend in regulation was observed in the rat aGvHD tissues and human GI GvHD biopsies.
In addition several cytokines, chemokines and other molecules have been implicated in GvHD pathophysiology. In this study we selected several genes that are expected to be important in HSCT outcome in view of previous reports or known functions and determined their regulation in different tissues affected by aGvHD. We wanted to compare the regulation of these genes during aGvHD and after preconditioning and transplantation not leading to aGvHD. In addition, we analysed the regulation of these genes in human GI aGvHD. In the mouse model, an upregulation of Th1, Th2 and Th17 cytokines in the lung, small and large intestines was associated with aGvHD. Genes associated with Treg regulation and activation were increased in the liver, lung and both intestines during aGvHD. Overall, we observed that aGvHD in both the mouse tissues and human GI biopsies was associated with a marked chemokine regulation. Several chemokines Cxcl9, Cxcl10, Cxcl11 and their receptor Cxcr3, Ccl4, Ccl9 and their receptors Ccr5, Ccr1 were upregulated in mouse tissues affected by aGvHD compared to the healthy controls, whereas a strong downregulation was observed in the expression of the chemokines Ccl5, Cx3cl1 and chemokine receptors Ccr4 and Cxcr4. These genes showed a similar trend of expression in the human GI aGvHD biopsies as well.
In view of previous data of our group and others, we also studied the gene expression profiles of KLRK1, encoding NKG2D and CD226, encoding DNAM-1, and their ligands in the animal models of aGvHD and human GI GvHD. NKG2D and DNAM-1 are activating NK receptors on NK and CD8+ T cells. They control cytotoxicity and interferon-γ production by NK cells and serve as co-stimulatory molecules on CD8+ T cells. NKG2D and DNAM-1 ligands can be up-regulated in several pathological conditions. Blockade or deficiency of either DNAM-1 or NKG2D in donor cells, has been shown to reduce the intensity of aGvHD in mice. Both NKG2D and DNAM-1 have also been implicated in inducing aGvHD by co-stimulating allogeneic cytotoxic T cells (CTL) directly via upregulation of their ligands on non-professional antigen presenting cells (APCs). Moreover, we recently described that a polymorphism in the human NKG2D ligand MICA has a major impact on the risk of aGvHD after allogeneic hematopoietic stem cell transplantation (HSCT) by modulating NKG2D signalling. The mRNA expression of KLRK1 and CD226 was increased significantly in all the mouse tissues affected by aGvHD, and showed an increased expression trend in the rat and human GvHD biopsies. Additionally, we observed a strong upregulation of their ligands, Ulbp1, Pvr and Pvrl2 in mouse aGvHD and Rae1l in the rat. MICA was upregulated in patients with aGvHD that were treated with steroids. On the other hand, Rae1 was downregulated in the different aGvHD mice tissues and MICB was downregulated in human GI aGvHD. PVR and PVRL2 showed a trend of upregulation in rats, however they were not differentially regulated in the human aGvHD biopsies. Additionally, the expression of MICA was increased whereas PVRL2 was decreased in patients who died due to transplant related causes compared to patients who were still alive or died due to other causes including relapse of malignancy.
In conclusion, we confirmed that several candidate genes previously suggested to be regulated during aGvHD were indeed significantly regulated in the different tissues in both animal models and human GI biopsies, and a number of genes showed similar expression trends. Moreover, we successfully showed that the regulation of many of our focus genes was significantly altered due to preconditioning, and their regulation was frequently exacerbated due to aGvHD in the different mouse tissues. Conditioning regimens that are less likely to induce these genes could be beneficial for attenuating the aGvHD response. Ligands of the activating NK receptors NKG2D showed a complex expression pattern during GvHD that might be important for the activation of allogeneic CTL in target tissues of aGvHD.||de