Establishment of Xenogeneic Lung Chronic Graft-Verus-Host Disease Mice Model

Abstract: NOD/SCID/IL2 receptor chain null (NSG) mice bearing human PBMCs can develop xenogeneic acute graft-versus-host disease (aGVHD), which mimics human aGvHD manifestations, including skin or liver involvement. In contrast little is known about the chronic GVHD (cGVHD) mouse model, although a recently described humanized cGVHD model using NSG mice with human thymocytes showed liver fibrosis 14 weeks after transplantation. Here, we established a humanized cGVHDmodel with chronic lung inflammation and fibrosis mimicking human cGvHD 8 weeks post transplantation.

Material and Method: NSG mice were recipients and treated with cyclophosphamide (Cy) at different doses (20-120mg/kg) at day -3 and -2 plus 200cGy X-ray at day-1 followed by injection of 1-5x10⁶ G-CSF mobilized human PBMCs or 1x10⁵ CD34⁺ cells at day 0. Samples of skin, lung, liver, spleen and small intestine were taken 8 weeks post transplantation, and fixed in 10% neutral-buffered formalin, embedded in paraffin, sectioned, slide mounted, and stained with hematoxylin and eosin and Masson's trichrome. Specimens were evaluated blinded by a pathologist. Engraftment was assessed in peripheral blood by flow cytometry and in each organ by immunohistochemistry (IHC) using anti-human CD4/8/20/68 antibodies.

Result: Mice that received over 60mg/kg of Cy lost weight and died earlier than those receiving 20mg/kg or less. Subsequently we used 20mg/kg Cy combined with 200cGy TBI as conditioning for the study. Mice receiving 5x10⁶ PBMCs exhibited acute illness including hunching or ruffled hair and died earlier compared with mice receiving 1x10⁶PBMCs. IHC analysis revealed engraftment of CD4, CD8 T cells, CD20+ B cells but not CD68+ cells in the lung, liver, skin or spleen (Figure 1). Analysis at 8 weeks post-transplantation showed that the mice receiving 1x10⁶PBMCs exhibited aggregation of inflammatory cells, collagen deposition and expansion of airways in the lung despite showing no sign of acute illness or weight loss. Masson's trichrome revealed increased fibrosis in the lung (Figure 2), but not in other organs.

Conclusion: We have shown that in the NSG mouse, a combination of Cy/TBI with a low number of G-CSF mobilized human PBMCs causes chronic lung inflammation and fibrosis that can serve as an important pre-clinic model of lung cGVHD.

Figure 1. Engraftment of human cells in NSG mice

Lung or liver from NSG mice 8 weeks post transplantation were examined for human CD4, CD8, CD20 and CD68 engraftment by IHC. Three randomly selected sections per   slide were observed under the x40 objective. Data were calculated as: % positive cells = positive nuclei cells Ú total cells nuclei x 100.

Figure 2. Lung fibrosis in NSG mice 8 weeks after transplantation

The lung from NSG mouse bearing PBMCs (A) or CD34+ cells (B) 8 weeks after transplantation was stained with Masson's trichrome. Fibrosis is shown in blue.

A                                                                       B