Abstract
Background: Transplantation related complications such as graft versus host disease (GVHD) are limiting factors for allogeneic haematopoietic stem cell transplantation (HSCT). The majority of mice models of GVHD are based on the use of radiotherapy as a conditioning regimen. However, these models may not explain mechanisms of HSCT related complications following chemotherapy-based conditioning regimen.
Aims: The aim of this study was establishment of a mice model of GVHD using the chemotherapy as conditioning regimen.
Methods: Recipient female BALB/c (H-2Kd) were conditioned using busulfan (Bu) in total dose of 80 or 100 mg/kg followed by cyclophosphamide (Cy) in total dose of 200 or 300 mg/kg. Twenty millions of bone marrow (BM) cells and 30×106 spleen cells of male C57BL/6 (H-2Kb) were injected into the recipient mice in allogeneic setting. As a control, syngeneic HSCT using female BALB/c as recipients and donors and the same conditioning regimen and cell doses as in allogeneic setting was used. Acute GVHD manifestations and histo-pathological changes in skin, liver and intestine were evaluated at different time points after BMT. The chimerism and tissue mapping were studied by FACS, immunohistochemistry and FISH analysis. Affected skin was examined by FACS analysis to find donor T cells. Cytokines level were measured by using luminex assay.
Results: Infusion of 20×106 BM cells and 30×106 spleen cells from C57BL/6 mice to BALB/c mice conditioned with 80 or 100mg/kg Bu plus 200mg/kg Cy induced lethal acute GVHD between days 5–7 post transplantation. Histological and clinical signs of GVHD were found in 85% and 100% of the allogeneic transplanted mice based on the intensity of conditioning. About 35% of mice in allogeneic group died due to GVHD progression. The number of donor T cell in the recipient skin increased with GVHD progression and was correlated to the level of chimerism in the spleen but not in the BM. CD3+ CD8+ T cells infiltration and apoptosis were found in the skin, intestine and liver of mice suffering from GVHD. None of the syngeneic transplanted mice died or developed any sign of GVHD even in long term follow up, and CD3+CD8+ T cells were absent in their tissue samples. Increasing the dose of Cy in conditioning regimen in allogeneic transplanted mice increased mortality of animals.
Conclusion: We have established a new mouse model of acute GVHD using chemotherapy based conditioning. This model can be utilized to study the basic mechanisms underlying GVHD triggered by cytostatics used in the conditioning regimen and affecting different cell sub-populations and cytokine storm.
