Immunobiology of graft versus host disease in chemotherapy based conditioning : new mouse model

Abstract

Introduction: Graft versus host disease (GVHD) is a life threatening complication of allogeneic hematopoietic stem cell transplantation. The pathophysiology of GVHD is rather complex and several factors including; conditioning; MHC disparity and donor/host immune system influences both development and intensity of GVHD. Animal models have improved our knowledge about the different aspects of acute GVHD, however, these models should be closer to the clinical setting to increase the relevancy of the results.

Aims: The present thesis aimed to develop a new mouse model of acute GVHD based on chemotherapy conditioning to explore the immunobiological events in the pathogenesis of acute GVHD and to improve our knowledge about the underlying mechanisms in order to develop new treatment strategies and improve the clinical outcome.

Material and Methods: Female BALB/c mice were used as recipients while male C57BL/6 and female BALB/c were used as allogeneic and syngeneic donors. Busulfan (Bu) and cyclophosphamide (Cy) were used as conditioning agents. On the transplantation day, recipient mice were infused with either allogeneic or syngeneic bone marrow and spleen cells. GVHD was evaluated according to clinical manifestations, immunohistopathology and flow cytometry. Serum cytokines were measured using the Gyrolab Bioaffy technique and gene expression patterns in different tissues were examined utilizing the Affymetrix GeneChip technique.

Results: Similar treatment efficacy, engraftment and lower general toxicity were observed when the administration order of standard Bu-Cy was reversed to Cy-Bu. The use of Bu (80mg/kg) for four days, followed by Cy (200mg/kg) for two days induced lethal GVHD in the BALB/c (H-2kd) recipients transplanted with allogeneic graft from C57BL/6 (H-2kb); mice receiving a syngeneic transplantation recovered. Furthermore, seven days post allogeneic BMT, donor T cells (mainly CD8+ T cells) migrated from the spleen to target tissues (skin, liver and intestine) initiating organ damage. In the early phase of GVHD, host DCs transiently expanded and initiated the inflammatory response however, donor DCs expansion and activation was more prominent than the recipients DCs at early phase of GVHD (day+3). Naïve T cells (CD3+CD44lowCD62high) were primed by donor/host APCs in the spleen of the recipient to gain effector/memory cells (CD3+CD44highCD62low) phenotype. Subsequently, effector/memory cells migrate to peripheral tissues and induce tissue damage. Gene expression analysis at day 0, showed that STAT1 and STAT3 were increased in liver while the antigen presentation, IL-4 and Leukocyte extravasation pathways were over expressed in the intestine. Gene expression pattern at day +7, in the allogeneic setting showed that antigen presentation, IFNγ, apoptosis, IL-1, IL-6 signaling and leukocyte trafficking pathways were mostly affected in the liver, intestine and kidney compared to syngeneic group.

Conclusions: These studies have shown that dose optimization may be used to improve the transplantation outcome. The newly introduced mouse model of GVHD have brought experimental animal models close to the clinical settings for further investigations of the mechanisms underlying GVHD as well as for evaluation of conditioning related toxicity. We have also shown that naïve T cells undergo early allo-activation by host/donor DCs in the secondary lymphoid organs to produce effector/memory T cells that initiate tissue damage in GVHD. Prophylactic therapy should be used prior to donor T cell activation. Gene expression analysis was shown to be vastly different in allogeneic compared to syngeneic transplanted mice and was synchronized with the clinical and biochemical changes. Gene expression analysis may be utilized for early diagnosis of GVHD.